JPH05238937A - Cell differentiation inducer - Google Patents

Abstract

PURPOSE: To obtain a new secosterol that is effective for inducing differentiation of malignant cells, such as differentiation of leukemic cells to normal macrophages, and useful as mammal cell differentiation inducer. CONSTITUTION: This secosterol of formula I or II (R is H, methyl, ethyl or propyl; X<1> and X<2> are each H or acyl), for example, 1-hydroxy-secosterol, is obtained through the processes of subjecting a compound of formula III to solvolysis in glacial acetic acid to convert the compound into a 5-enesteroid of formula IV, dehydrogenating the 5-enesteroid to a 5,7-dienesterol of formula V (X is CH3 CO), then opening the ring B of the steroid to form a compound of formula VI (X is H), tosylating the compound to a compound of formula VI in which X is tosyl, further subjecting the compound to solvolysis in methanol to form a compound of formula VII, and oxidizing the compound of formula VII to form a compound of formula III.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an agent effective for inducing differentiation of malignant cells such as differentiation of leukemia cells into normal macrophages.

[0002]

BACKGROUND OF THE INVENTION A useful treatment for malignant diseases is to administer a compound that stimulates the differentiation of malignant cells into normal cells, thereby preventing and / or reversing malignant transformation. is there. That is, according to Suda et al. (U.S. Pat. No. 4,391,802), 1α-hydroxyvitamin D compounds (eg, 1α, 25-dihydroxyvitamin D ₃ and 1α-hydroxyvitamin D ₃ ) are, for example, malignant cells ( In particular, it has been shown to have a strong anti-leukemic activity indicated by the effect of causing the differentiation of leukemia cells) into non-malignant macrophages (monocytes). Therefore, these compounds are useful in the treatment of certain malignancies, especially leukemia. However, when used in such treatments, these known 1α-hydroxyvitamin D compounds also have very strong calcemic (c
alcemic) agent, that is, it stimulates intestinal calcium absorption, raises blood calcium level, and causes bone calcium absorption. This calcemic activity indeed represents the well-known and typical function of these compounds. Moreover, the cell differentiation activity (and thus antileukemic activity) of these compounds correlates with their calcemic activity. For example, to rise to differentiation into macrophages of malignant cells is the most potent compound 1,25-dihydroxyvitamin D ₃ also stimulates calcium transport, i.e. the most potent vitamin D metabolite to increase the calcium level in the serum Is. In order to be practically used as a cell differentiation agent, this potent calcemic activity could lead to therapeutically too high serum calcium levels in the therapeutic body at the doses required to exert efficacy in the differentiation of malignant cells. Of course, it is an undesirable side effect.

[0003]

That effective cell differentiation (ie reversal of malignant transformation) can be achieved by a novel secosterol without the above-mentioned undesirable side effects (strong calcemic action). Was found here. The selectivity and specificity of this action makes this novel secosterol a useful and preferred agent for achieving malignant cell differentiation. Such unique and
Secosterol as an active ingredient used in the drug of the present invention showing an activity pattern which has hitherto been unknown is characterized by the following general structural formula I or II.

[0004]

[Chemical 2] Wherein R is hydrogen, methyl, ethyl or propyl group,
X ¹ and X ² each independently represent hydrogen or an acyl group.

The acyl groups used herein include alkanoyl groups having 1 to 6 carbon atoms or benzoyl groups including all isomers, or aroyl groups such as halo-, nitro- or alkyl-substituted benzoyl groups, or oxalyl, malonyl. A dicarboxylacyl group such as succinoyl, glutaroyl, or adipoyl. An alkyl group is a C1-C6 hydrocarbon group including all isomers. Purely structurally, this type of secosterol shares similarities with some known classes of vitamin D compounds. However, unlike known vitamin D compounds, this secosterol does not exhibit standard vitamin D activity in vivo, namely stimulation of intestinal calcium transport or bone calcium distribution activity. Therefore, it cannot be classified as a vitamin D derivative from a functional viewpoint. From the point of view of the prior art, it was found that these secosterols were found to be remarkably effective in causing the differentiation of leukemic cells into normal (non-malignant) macrophages. This is surprising and unexpected because the strong cell differentiation activity of related compounds has always been closely related to the strong calcemic activity. Thus, the secosterol of the present invention overcomes the above-mentioned drawbacks of the known vitamin D-related anti-leukemia agents and is a preferable agent for suppressing and treating malignant diseases such as leukemia. The finding is that these secosterols can be administered to patients in doses sufficient to cause the differentiation of malignant cells into normal cells without concomitantly leading to non-physiologically high and detrimental blood calcium levels. The drug of the present invention is extremely useful for treating malignant diseases.

In the drug of the present invention, as described above, and
As shown in the examples below, the general structural formula I
These secosterols (where R is hydrogen, methyl, ethyl, propyl) do not cause any significant biological response in the body regarding bone mineral transport and intestinal calcium transport activity in the living body, and thus, they are typical of the conventional living body. Does not perform the vitamin D function of. Therefore, the secosterols of the present invention have unexpected capabilities. That is, it shows a very large cell differentiation activity like some of the known vitamin D-related compounds, but does not show the typical calcemic activity of vitamin D derivatives. Therefore, the secosterol of the present invention is suitable as a drug for treating malignant diseases such as leukemia because it does not have an unfavorable calcemic action like known anti-leukemia vitamin D compounds.

The compounds of the invention can be formulated as pills, capsules or tablets, or as solutions, emulsions, dispersions or suspensions in pharmaceutically harmless and acceptable solvents or oils, The preparations may contain additional pharmaceutically harmless or beneficial ingredients such as antioxidants, emulsifiers, colorants, binders or coating substances. The agent of the present invention can be administered as an oral preparation or by injection or infusion of a suitable sterile solution. In the present invention, the secosterol compound as an active ingredient is advantageously administered in an amount effective enough to differentiate malignant cells into normal macrophages. Dosages of 2 μg to 1000 μg per day are suitable, but the doses to be administered can be adjusted according to the severity of the disease and the condition of the patient and the sensitivities, as is well understood by the person skilled in the art.

Preparation of secosterol compounds

[0009]

[Chemical 3]

The secosterol I (R) used in the present invention
= H) is the method of Ram et al. [Steroids 26, 422 (197
5)]. The secosterols of structural formulas I and II where R is methyl, ethyl or propyl are new compounds and can be prepared by the steps shown in Reaction Scheme I above. Suitable as starting materials are the i-ether steroids of general structural formula 1 , in which R can be methyl, ethyl or propyl depending on the desired end product. Conversion of the compound of structural formula 1 to the 5-ene steroid of structural formula 2 is accomplished by solvolysis in glacial acetic acid by known methods. Next 5-
The ensteroid is dehydrogenated by sequentially using allyl bromination and dehydrobromination reaction at C-7 to give a 5,7-diene steroid ( 3 ), and the acetic acid group of compound 3 is saponified to give the corresponding structural formula 4 Alcohol is obtained. When the solution of alcohol 4 was irradiated with ultraviolet rays, ring B of the steroid was opened to give the first secosterol derivative (5 (10), 6,8).
-Triene) is obtained. It can be purified and isolated if desired by standard gas chromatographic methods, but generally the most advantageous method is direct thermal isomerization to give 5,7,10 (19) -triene of structural formula 5. It can be a compound. Subsequent conversion of this intermediate to the desired 1α-hydroxylated analog is described in US Pat.
It can be carried out by the general method shown in 195,027 and 4,260,549. Intermediate 5 (Process Scheme I) is first tosylated to 3β-tosylate ( 6 ) which is then solvosolved in buffered methanol to give 6-methoxy-3,5-represented by Structural Formula 7. A cyclo-derivative is made. Solvolysis of tosylate in other alcoholic solvents (eg ethanol, propanol, butanol) yielded the analog, 6-O.
-Alkyl-3,5-cyclo intermediates are made. The alkyl group is derived from the alkyl portion of the alcohol used (eg ethyl, propyl, butyl, etc.).
Any of these 6-O-alkyl-homologs of compound 7 can be used for the next reaction in this step. Oxidation of intermediate 7 with selenium dioxide in the presence of hydroperoxide yields the desired 1α-hydroxy functionality, compound 8
Is obtained. Next, this intermediate is subjected to solvolysis in a medium containing a low molecular weight organic acid to give a compound of the structural formula 9 (X ¹ = acyl
3-acylate having X ² = H) and the corresponding structural formula 1
0 (X ¹ = acyl, X ² = H) trans isomer (each acyl group is derived from the organic acid used for the solvolysis) is obtained. These 5,6-cis and 5,6-
The trans 3β-acylates (compounds 9 and 10 ) are advantageously separated at this stage so that each is obtained in pure form. These can then be subjected to saponification (eg base in methanol) and the corresponding free hydroxy compound 9
(X ¹ = X ² = H) and 10 (X ¹ = X ² = H) are obtained. Apart from this, the monoacylates or free hydroxy compounds of 9 and 10 are acylated under usual known conditions (for example, acid anhydrides or acyl halides in a nitrogen-based solvent) to give C-1-monoacyl, C-3- Monoacyl or C
Either of the -1,3-diacyl derivatives can be made as desired. These are compounds of structures 9 and 10 , wherein X ¹ = acyl, X ² = H, or X ¹ = H, X ² = acyl, or X ¹ = acyl, X ² = acyl (even if the acyl groups are the same. May be different).

From the above description and the reaction step scheme it is clear that the nature of the side chains of the starting material determines the side chain structure of the final product. Thus, using a steroid of structural formula 1 in which R is methyl as the starting material, the products of structural formulas 9a and 10a in which R is methyl are obtained. Compound 1 where R is ethyl as starting material gives the products 9a and 10a where R is ethyl, and steroid 1 where R is propyl.
Are treated in the steps described above to give products 9a and 10a in which R represents propyl. The 5,6-trans compound of structural formula 10 in Reaction Step Scheme I has the corresponding structural formula
Although it has utility as a biologically active analog of the 5,6-cis-compound of 9 , it can also be converted to the 5,6-cis product of structural formula 9 by trans, cis isomerization steps known to those skilled in the art. it can.

Preparation of Starting Material Starting material of structural formula 1 (R
= CH _3, CH ₂ CH ₃ or CH ₂ CH ₃ CH ₃₎ may be prepared from stigmasterol by conventional methods. That is, conversion of stigmasterol into its i-ether derivative, ozonolytic cleavage of the side chain double bond, followed by hydride reduction gives the known 22-alcohol having the structure shown below.

[0013]

[Chemical 4]

The structure of R = CH ₃ was directly obtained by tosylation of the alcohol followed by hydride reduction of 22-tosylate.
1 compound is obtained. Treatment of the above 22-tosyl intermediate with sodium cyanide provides the corresponding 23-nitrile derivative. Two-stage hydride reduction of this nitrile gives the 23-alcohol, which is tosylated and then the hydride reduced again to the 23-tosyloxy intermediate to give the starting compound of structural formula 1 where R = ethyl. Similarly, if the corresponding 24-nitrile derivative obtained by treating the above-mentioned 23-tosyloxy intermediate with sodium cyanide is subjected to hydride reduction (to obtain 24-alcohol) and then tosylation and hydride reduction again sequentially, A compound of structural formula 1 is obtained in which R represents n-propyl.

[0015]

The agent for inducing cell differentiation of the present invention shows a very large cell differentiation activity like some of the known vitamin D-related compounds, but does not show the typical calcemic activity of vitamin D derivatives. Therefore, since it does not have an unfavorable calcemic action like the known anti-leukemia vitamin D compound, the secosterol of the present invention has no obstacle to the administration method for the treatment of malignant diseases such as leukemia, and is suitable as an anti-leukemic agent and the like. Is.

[0016]

EXAMPLES The present invention will be described in more detail based on examples. EXAMPLE Production of a compound of structural formula I for the differentiation of human leukemia cells
Physical Activity The chemicals and reagents required for the assay below were obtained from commercial sources as follows. 4-β-phorbol 12-myristate-13-acetate (PMA), nitroblu-tetrazolium (NBT) and α-naphthyl acetate-esterase assay reagents (α-naphthyl acetate, concentrated Trizmal)
^TM (TRIZMAL ^™ ) 7.6 buffer, Mayer's hematoxylin solution, Fast Bull-RR salt, concentrated citrate, ethylene glycol monomethyl ether) were obtained from Sigma Chemical Company of St. Louis, Missouri, and used as red and white spheres of sheep (cells in Alseverse solution). 50%), fetal calf serum and RP
M1-1640 medium was obtained from the Gibco Laboratory, Grand Island, NY, and rabbit antiserum against rabbit red blood cells (rabbit hemolysin) was obtained from the Flow Laboratory, MacLaine, VA. Cell culture : 10 human leukemia cells (HL-60 cells)
ml aliquot (RPM1 with 10% fetal calf serum
-1640 medium (about 2 × 10 ⁵ cells per ml) is spread in a plate on a Petri dish at 37 ° C. in a 5% CO ₂ atmosphere.
It was cultured in. After 16 hours, different amounts of the test compound (listed in Table 1 below) dissolved in 10-20 μl of ethanol were each applied to two dishes. Only ethanol was used for control culture. Cells were harvested 4 days after compound administration and aliquots of each were counted under a hemocytometer.
To assay the extent of differentiation produced by the test compounds, harvested cultures were resuspended in phosphate buffered saline (PBS) (about 10 ⁶ cells / ml) and aliquots of these cell cultures were used to Each of the three types of tests was carried out.

(A) Nitrobull-tetrazoli for differentiation
Um (NBT) Reduction Assay This assay is based on the fact that monocyte leukocytes can be stimulated by phorbol ester to produce superoxide. Superoxide can be detected by its ability to reduce soluble nitroblu-tetrazolium (NBT) to a black-blue precipitate, formazan. The NBT-reducing activity exhibited by HL-60 cells thus provides a measure of its differentiation into non-malignant monocytes. The assay was performed according to the method of En et al. Described in Journal of Cellular Physiology 118 , 277 (1984). NBT reagent is 50 mg of NBT and 5 μg of 4β-phorbol 12-myristate-13-acetate in 50 ml.
Was prepared by dissolving it in phosphate buffered saline.
This reagent (200 μl) was added to 200 μl of harvested cells (about 10 ⁶ cells / 1 ml of phosphate buffered saline). The mixture was incubated in a 37 ° C water bath for 30 minutes. The number of cells was then counted and the percentage of cells that reduced NBT to formazan blue was recorded. The results are shown in Table 1 below.

(B) Rosette formation assay This assay is based on the formation of a rosette (rosette rose flower) between differentiated monocytes and sheep red blood cells coated with rabbit antibodies and mouse complement. is there. Certificate is International Journal of Cancer 15, 7
31 (1975). Wash red and white sheep spheres three times with phosphate buffered saline,
Resuspended in 0.5% (v / v) suspension. Erythrocytes and an equal volume of a 1: 1500 solution of antiserum to rabbit red and white spheres were mixed and incubated at 37 ° C for 30 minutes. Antibody coated red blood cells (EA) were washed 3 times with phosphate buffered saline pH 7.0 and resuspended at 0.5% (v / v). Fresh mouse blood was spun down and serum was collected. EA was mixed with an equal volume of a 1:10 solution of mouse serum, incubated at 37 ° C. for 30 minutes, and then washed 3 times with phosphate buffered saline to obtain 1-fold red blood cells (EAC) coated with antibody and complement. Resuspend in RPM1 medium to a concentration of 100% (v / v) and add 100 μl EAC
Were mixed with an aliquot of HL-60 cells (about 10 ⁶ cells in 100 μl of RPM1), incubated at 37 ° C. for 30 minutes and then centrifuged at 500 × g for 3 minutes. The pellet was dispersed and the number of cells conjugated to EAC (ie, rosette number) was measured and expressed as% of total cells present. The "% rosette formation", which indicates the differentiation of HL-60 cells into monocytes, is shown in Table 1 below.

(C) α-Naphtyl acetate esterase activity
Assay α-Naphthyl esterase is an enzyme that exhibits monocyte characteristics. That is, the presence of this enzyme indicates the differentiation of HL-60 cells into monocytes. The assay is enzymatic hydrolysis of α-naphthyl acetate to separate free naphthol,
This is based on the fact that it binds to diazonium to form a highly colored precipitate at the position of enzymatic activity. Certification is Technical Bulletin No. 90 (Misley 6317)
8, Sigma Chemical Company of St. Louis). Cells were fixed on glass slides in citrate-acetone-methanol fixative for 30 seconds at room temperature. The slides are then washed with deionized water and at least 20
Air dried for a minute. Then slide the slides to a Trismal pH 7.6
25 mg of the Fast Blue RR salt was dissolved in 50 ml of a 1:10 solution of ^TM buffer concentrate and colored in a coloring solution prepared by adding 20 mg of α-naphthol (in 2 ml of ethylene glycol monomethyl ether). 37 slides
Incubation was carried out at 0 ° C for 30 minutes (in the dark). After that, it was washed, reverse-colored in Mayer's hematoxylin solution for 5 to 10 minutes, washed and air-dried. The percentage of cells with black particles showing α-naphthyl esterase activity was determined. The results are shown in Table 1 below.

[0020]

[Table 1]

The above results demonstrate the efficacy of secosterol of general structural formula I as a drug for the differentiation of human leukemia cells into macrophages (monocytes). This compound showed highly significant activity in all three differentiation assays used, achieving 50% differentiation at a concentration of approximately 10 ⁻⁶ M. For comparison, the above table shows two known vitamin derivatives having strong anti-leukemic effects, namely 1α-hydroxyvitamin D (1α-OH-D ₃ ) and 1α, 25-dihydroxyvitamin D (1,25- Also included is the cell differentiation activity exhibited by (OH) ₂ D ₃ ). The data shown is for secosterol (I) activity levels of 1,25-
Compounds known to be effective in treating leukemia in humans, although less than that exhibited by (OH) ₂ D ₃ (the most potent leukocyte differentiating vitamin D derivative) (Suda et al., US Pat. No. 4,391). , 802), which is almost the same as that exhibited by 1α-hydroxyvitamin D ₃ .

Calcium cost of secosterol of structural formula I
Xie and Calcium Transport Assays Low calcium, vitamins described by Suda et al. [Journal of Nutrition 100 , 1049 (1970)] in weanling male rats purchased from Holzman (Madison, Visconsin). A D-deficient diet was given ad libitum for 3 weeks.
The rats were then divided into 4 groups of 6 each. The first group (control group) was injected with 0.05 ml of 95% ethanol by jugular vein injection. For the second and third groups, secosterol I (R = CH) dissolved in 0.05 ml of ethanol was taken by the same route.
₃ , X ¹ = X ² = H) and 625 pmol and 6 respectively.
250 pmol was administered, and in the fourth group, 1α, 25-dihydroxyvitamin D ₃ was 625 pmol (ethanol: 0.
It was injected in the jugular vein (in 05 ml). Seven hours after administration, the rat was killed by decapitation, blood was collected and centrifuged to obtain serum.
Serum calcium concentrations were routinely measured by atomic absorption spectroscopy. The results are shown in Table 2 below. A small amount of the same rat intestine was taken out, and after washing, it was turned upside down and the calcium transport activity was measured according to the method of Martin and Delka [American Journal of Physiology 216 , 1351 (1969)]. Table 2 shows the measured values of intestinal calcium transport activity expressed by the concentration ratio of serosal calcium / mucosal calcium.
Shown in.

[0023]

[Table 2]

The above results show that secosterol I (R = CH
₃ , X ¹ = X ² = H) does not show any significant calcemic activity even at high doses. This compound does not elevate serum calcium levels and thus lacks significant bone calcium mobilizing activity. Furthermore, this compound is 1
No dose of 6250 pmol / animal stimulates intestinal calcium transport. Under the same conditions, the known vitamin D metabolite 1,25- (OH) ₂ D ₃ is 10 times more active at low doses as expected. Secosterols homologues of structural formula ^{I (R = H, X 1} = X 2 = H) above and similar conditions test at (same conditions except measured at 12 hours post compound injection administration sensitive) under the following As shown in Table 3, it gave very similar results.

[0025]

[Table 3]

[0026] of the data of Table 3 also structural formula I secosterols ^{(R = H, X 1 =} X 2 = H) is any of respect calcium circulation from the gut calcium transport or bone even in vivo at high doses It shows that it does not cause any sensitivity.

Reference Examples The preparation of the novel compounds of the present invention is described in more detail by the following examples. In these examples, the Arabic numbered products (eg compounds 1, 2, 3 etc.) correspond to the similarly numbered structures in the reaction scheme. Example 1 Preparation of compounds 9a and 10a (X ¹ = X ² = H) 3β-acetoxy-23,24-dinorcol-5-ene (2) Compound 1 (R = CH ₃ ) (1.26 g, 3.8 mmol) A solution of () in glacial acetic acid (35 ml) was heated to 70 ° C. for 4.5 hours. The reaction mixture was cooled, poured into ice water, neutralized with 10% aqueous sodium hydroxide solution, and chloroform (3 x 1
(00 ml). Chloroform extract was added to water (2 x 5
0 ml) and a saturated sodium chloride solution (2 × 50 ml), washed successively, dried over anhydrous magnesium sulfate, filtered and dried to obtain 0.86 g (63% yield) of compound 2 (R = CH ₃ ). It was Mass spectrum m / e (relative intensity), 358
(M ⁺ , 2), 298 (100), 283 (15), 255 (6), 190 (9), 177
(23); ¹ H-NMR (CDCl ₃ ), δ 0.66 (s, 18-H), 0.81 (d, J = 7
Hz, 22-H), 0.95 (d, J = 7.0 Hz, 21-H), 1.02 (s, 19-
H), 2.06 (s, 3-OCOCH ₃ ), 4.59 (m, 3-H), 5.38 (m, 6-H).

3β-acetoxy-23,24-dinorcola-5,7-diene ( 3 ) (R = CH ₃ ) in dry hexane (50 ml) containing finely ground sodium hydrogen carbonate (0.7 g, 8 mmol). 2 (R = CH ₃ )
(0.6 g, 8 mmol) was dissolved and heated under reflux at 80 ° C. under nitrogen atmosphere while stirring to give 1,3-dibromo-5,5.
5-Dimethylhydantoin (0.24 g, 0.85 mmol) was added. The reaction was allowed to proceed for 20 minutes. The reaction mixture was cooled, filtered, and concentrated under reduced pressure. The residue was immediately dissolved in 15 ml of dry xylene and mixed with xylene (25 m
l) and s-collidine (0.4 g, 3.3 mmol) were added dropwise. The mixture was flushed with nitrogen, cooled, diluted with benzene (50 ml), 3% aqueous hydrochloric acid solution (3 x 20 ml), saturated sodium hydrogen carbonate solution (1
X25 ml), water (1 x 25 ml), saturated aqueous sodium chloride solution (2 x 25 ml), washed successively, dried over anhydrous magnesium sulfate and filtered. The solvent was evaporated under reduced pressure. The oily residue was dissolved in dry dioxane (40 ml) and p-
Toluenesulfonic acid (0.076 g, 0.4 mmol)
Was added. Flush the mixture with nitrogen, 7
The mixture was refluxed at 0 ° C for 0.5 hour. After cooling, water (100m
l) and extracted with ethyl acetate (1 × 70 ml,
2 × 60 ml) and the extract was saturated sodium hydrogen carbonate solution (1 × 30 ml) and saturated sodium chloride solution (2 × 30 m).
l) sequentially, dried over anhydrous magnesium sulfate,
Filter and evaporate the solvent under reduced pressure to yield the desired 5,7-diene (3) (Rf 0.29 in 10% ethyl acetate-hexane) and 2,4,6-triene (10% ethyl acetate: hexane). An oil containing Rf 0.5) was obtained. 10% ethyl acetate - 3 with about 43% yield by preparative TLC using hexane (R = CH ₃₎ was obtained 0.26 g. UV (C ₂ H ₅ OH) λ _max 282, 293, 272, 262 nm; Mass spectrum: m / e (relative intensity), 356 (M ⁺ , 5), 296 (10
0), 281 (59), 253 (56), 211 (29), 158 (75), 143 (7
4); ¹ H-NMR (CDCl ₃ ): 0.62 (s, 18-H), 0.88 (d, J = 7.0
Hz, 22-H), 0.95 (s, 19-H), 0.96 (d, J = 7.0 Hz, 21-
H), 2.04 (s, 3-OCOCH ₃ ), 4.7 (m, 3-H), 5.4 (m, 7-H),
5.58 (m, 6-H).

3β-Hydroxy-23,24-dinorcola-5,7-diene ( 4 ) (R = CH ₃ ) 10% sodium hydroxide in methanol was added to ₃ (R = CH ₃ ).
₃ ) (259 mg: 25% ethyl acetate-hexane for Rf0.
56) in ether (15 ml) was added dropwise with stirring under a nitrogen atmosphere over a period of 5 minutes. The reaction was allowed to proceed at 23 ° C and was monitored by TLC. The reaction was completed within 35 minutes. The reaction mixture was diluted with ether (100 ml) and water (30 ml) was added. The layers were separated and the aqueous layer was extracted with ether (2 x 50 ml). The ether extracts were combined and water (2 x 30 ml), saturated sodium chloride solution (2 x
30 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure to give 25% ethyl acetate /
Preparative TLC with hexane gave 200 mg of 4 (77
% Yield) was obtained. UV (C ₂ H ₅ OH): λ _max 282, 293, 272, 2
62 nm; Mass spectrum: m / e (relative intensity), 314 (M ⁺ , 8
5), 296 (12), 281 (100), 261 (6), 255 (60), 211 (2
2), 171 (30), 143 (78), ¹ H-NMR (CDCl ₃ ): δ 0.62
(s, 18-H), 0.87 (d, J = 7.0 Hz, 22-H), 0.95 (s, 19-
H), 0.97 (d, J = 7.0 Hz, 21-H), 3.63 (sh, 3-H), 5.38
(m, 7-H), 5.58 (m, 6-H).

Secosterol 5 (R = CH ₃ ) 5,7-diene (4) (36 mg) dissolved in a benzene-ether (1: 4) mixture (100 ml) was added to a Vicol filter Hanobia 608A quartz-medium pressure mercury. It was equipped with the above UV lamp and placed in a jacket with double-walled water-cooled quartz immersion wells. The mixture was irradiated for 4.5 minutes. The system was continuously purged with nitrogen during irradiation. Then the solvent was removed under reduced pressure and the residue was redissolved in dry ethanol. After flushing with nitrogen, it was heated and refluxed at 70 ° C. for 3 hours in a nitrogen atmosphere. It was then cooled and concentrated under reduced pressure. T with 20% ethyl acetate-hexane
LC purification yielded 5 (R = CH ₃ ) in 31.7% yield (1
1.4 mg). UV (C ₂ H ₅ OH) λ _max 264 nm; Mass spectrum: m / e (relative intensity) 314 (M ⁺ , 14), 296 (21),
281 (4), 271 (2), 253 (3), 136 (82), 118 (100); ¹ H-
NMR (CDCl ₃ ): δ 0.54 (s, 18-H), 0.85 (d, J = 7.0 Hz,
22-H), 0.94 (d, J = 0.7 Hz, 21-H), 3.93 (m, 3-H), 4.8
2 (m (sharp), 19 (Z) -H), 5.04 (m (sharp),
19 (E) -H), 6.02 (d, J = 12.0 Hz, 7-H), 6.22 (d, J = 1
2.0 Hz, 6-H).

Tosylate 6 (R = CH ₃ ) 5 (15 mg, 47 mmol, 30% ethyl acetate-Rf 0.28 in hexane) in dry pyridine (0.5 ml) was added to p-toluenesulfonic acid at 5 ° C. under a nitrogen atmosphere. It was treated with chloride (22 mg, 117 mmol) for 24 hours.
The reaction product was quenched with ice water and the mixture was extracted with ether (3 x 30 ml). The extracts were combined and combined with a 3% aqueous solution of hydrochloric acid (2 x 30 ml), a saturated aqueous solution of sodium hydrogen carbonate (1 x 50 ml) and a saturated aqueous solution of sodium chloride (1 x 50 ml).
ml) and then dried over anhydrous magnesium sulfate,
By concentrating under reduced pressure, 20 mg of tosylate 6 (R
= CH ₃₎ (30% ethyl acetate - in hexanes Rf 0.
6) was obtained.

3,5-Cyclo-derivative 7 (R = CH ₃ ) Crude tosylate 6 (R = CH ₃ ) (20 mg, 0.41 mmol, Rf 0.5 in 25% ethyl acetate-hexane) was stirred. Finely ground sodium hydrogen carbonate (200 mg, 2.4
Mmol) in anhydrous methanol (20 ml). The mixture was heated under reflux at 55 ° C. under nitrogen atmosphere for 8 hours, cooled, diluted with ether (100 ml), water (3 × 30 ml), saturated aqueous sodium chloride solution (1 × 30 ml).
ml) and then dried over anhydrous magnesium sulfate,
After filtration, the solvent was evaporated under reduced pressure. The crude mixture of 10% ethyl acetate - hexane was chromatographed on preparative TLC using 7 in 50% yield _{(R = CH 3) (7mg} ,
Rf 0.66) in 10% ethyl acetate-hexane was obtained.
Mass spectrum: m / e (relative intensity), 328 (M ⁺ , 14), 296
(15), 281 (7), 253 (37), 159 (28), 135 (34), 145
(30); ¹ H-NMR (CDCl ₃ ): δ 0.55 (s, 18-H), 0.85 (d, J
= 7.0 Hz, 22-H), 0.93 (d, J = 7.0 Hz, 21-H), 3.26 (s,
6 (R) -OCH), 4.18 (d, J = 10 Hz, 6-H), 4.89 (m (sharp), 19 (Z) -H), 5.0 (d, J = 10 Hz, 7-H) , 5.06 (m
sharp)

1α-hydroxy-compound 8 (R = CH
₃ ) t-Butyl hydroperoxide (7 μl, 0.05 mmol) in selenium dioxide (SeO ₂ ; 1.1 mg, 10 μmol) in 1% dry pyridine-methylene chloride (5 ml).
The suspension was added under nitrogen atmosphere. The mixture was stirred at 23 ° C. for 0.5 hours and then diluted with an additional 10 ml of 1% dry pyridine-methylene chloride solution. The mixture was cooled on an ice bath and treated with intermediate 7 (R = CH) in dry methylene chloride.
₃ ) (7 mg, 21 μmol, 25% ethyl acetate-Rf 0.62 in hexane) was added. The reaction was monitored by TLC. After reacting at 23 ° C. for 16 minutes, 10% sodium hydroxide solution (20 ml) was added to quench the reaction. The mixture was diluted with ether (100 ml), the phases were separated and the ether phase was washed with 10% aqueous sodium hydroxide solution (2
(25 ml), water (2 × 20 ml), saturated aqueous sodium chloride solution (2 × 20 ml), and dried over anhydrous magnesium sulfate. After filtration the solvent was evaporated under reduced pressure. Preparative TLC of the residue using 25% ethyl acetate-hexane
Treated with 1α-hydroxy-derivative 8 (R = CH ₃ ).
(3 mg, Rf 0.15 in 25% ethyl acetate-hexane)
Was obtained in 41% yield. Mass spectrum: m / e (relative intensity): 344 (M ⁺ , 34), 312 (77), 271 (42), 177 (567),
135 (100), ¹ H-NMR (CDCl ₃ ): δ 0.55 (s, 18-H), 0.8
5 (d, J = 7.0 Hz, 22-H), 0.94 (d, J = 7.0 Hz, 21-H),
3.26 (s, 6 (R) -OCH ₃ ), 4.17 (m (sharp), 6-H), 4.22
(m (sharp), 1-H), 4.94 (d, J = 9 Hz, 7-H), 5.16
(d, J = 2.2Hz, 19 (Z) -H), 5.24 (d, J = 2.2 Hz, 19 (E)-
H).

5,6-cis and trans secosterol 3β-acetates 9a and 10a (X ¹ ═CH ₃ CO,
A solution of X ² = H) 8 (R = CH ₃ ) (3 mg) in glacial acetic acid (0.5 ml) was heated to 55 ° C for 20 minutes under a nitrogen atmosphere, cooled, and ice-cooled sodium hydrogen carbonate solution (15 ml). Poured on and extracted with ether (3 × 30 ml). The ether extracts were combined and saturated aqueous sodium hydrogen carbonate solution (1 x 20
ml), water (2 x 20 ml), saturated sodium chloride solution (1 x
20 ml), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed in 35% ethyl acetate-hexane (multiple elution, 4 times) with 0.8 mg of 5,6-cis-3β-acetate ( 9a ) and 0.45 g of 5,6-trans-. 3β-
Acetate ( 10b ) was obtained respectively.

Acetate 9a (X ¹ = CH ₃ CO, X ² = H): UV (C ₂
H ₅ OH): λ _max 264 nm, λ _min 228 nm, mass spectrum:
m / e (relative intensity) 372 (M ⁺ , 7), 312 (40), 269 (10), 1
89 (15), 134 (100); ¹ H-NMR (CDCl ₃ ): δ 0.54 (s,
18-H), 0.85 (d, J = 7.0 Hz, 22-H), 0.92 (d, J = 7.0 Hz,
21-H), 2.02 (s, 3-OCOCH ₃ ), 4.4 (broad, 1-H),
5.01 (m (Sharp), 19 (Z) -H), 5.21 (m, 3-H), 5.33
(m (sharp), 19 (E) -H, 6.0 (d, J = 12.0 Hz, 7-H),
6.34 (d, J = 12.0 Hz, 6-H). Acetate 10a (X ¹ = CH ₃ C
O, X ² = H): UV (CH OH): λ _max 273 nm, λ _min 228 nm;
Mass spectrum: m / e (relative intensity) 372 (M ⁺ , 3), 328
(4), 312 (14), 269 (6), 177 (37), 149 (58), 135 (1
00); ¹ H-NMR (CDCl ₃ ); δ 0.54 (s, 18-H), 0.85 (d, J =
7.0 Hz, 22-H), 0.92 (d, J = 7.0 Hz, 21-H), 2.02 (s,
3-OCOCH ₃ ), 4.4 (broad, 1-H), 4.99 (m (sharp),
19 (Z) -H), 5.13 (m (sharp), 19 (E) -H), 5.8 (d, J =
12.0 Hz, 7-H), 6.58 (d, J = 12.0 Hz, 6-H).

1α-hydroxy-cellosterol9a
(X¹ = X² = H) 3β-acetate obtained in the above experiment9aEther
The solution (10 ml) was added with 10% sodium hydroxide in methanol.
Using the solution, add 0.5 hours at 23 ℃ under nitrogen atmosphere.
Water decomposed. The mixture was diluted with water (10 ml) and ether
It was extracted with (3 x 50 ml). Ether extract combined
Water (2 x 10 ml), saturated sodium chloride solution (2 x 10 ml)
ml), then dried over anhydrous magnesium and filtered.
And evaporated to dryness under reduced pressure. Residue is 5% isoprop
Zolvacs at 265 nm with sol-hexane
HPL on Sill analytical column (4.6 mm x 25 cm)
Purified by C,9a(X¹ = X² = H) was obtained. High
Resolution mass spectrum analysis: Calculated value (C_{twenty two}H₃₄O₂ As) 33
0.2559, measured value 330.2541; UV (C₂H_FiveOH): λ_max 264 n
m, λ_min 227 nm; mass spectrumm / e (Relative strength) 330
 (M⁺, 55), 312 (71), 287 (7), 269 (7), 251 (5), 189
 (21), 152 (73), 134 (100);¹H-NMR (CDCl₃): δ 0.
54 (s, 18-H), 0.85 (d, J = 7.0 Hz, 22-H), 0.93 (d, J
= 7.0 Hz, 21-H), 4.23 (m, 3-H), 4.42 (m, 1-H), 5.0
(s, 19 (Z) -H), 5.34 (s, 19 (E) -H), 6.02 (d, J = 12.0H
z, 7-H), 6.39 (d, J = 12.0Hz, 6-H).

5,6-trans-analog 10a (X ¹ =
X ² = H) Hydrolyze 3β-acetate ( 10a ) in the same manner to give 5
Purification by HPLC at 273 nm using% isopropanol-hexane gave 10a (X ¹ = X ² = H). High-resolution mass spectrum analysis: Calculated value (C ₂₂ H ₃₄ O ₂ ).
330.2559, measured value 330.2532: UV (C ₂ H ₅ OH): λ _max 273
nm, λ _min 227 nm; Mass spectrum: m / e (relative intensity),
330 (M ⁺ , 69), 312 (48), 287 (21), 269 (18), 251
(15), 189 (31), 152 (76), 134 (100).

Example 2 Compound9bas well as10b(X¹ = X² = H) 3β-acetoxy-24-norcol-5-ene (Two, R
= Ethyl) 1 (R = ethyl) (0.5 g: 25% ethyl acetate-hex
Glacial acetic acid (7 ml) solution of Rf0.8) in sun to 70 ° C
Heated for 4.5 hours. The reaction mixture is cooled and poured into ice water.
Gigomi, neutralization with 10% sodium hydroxide solution
And extracted with chloroform (3 × 70 ml). Organic extraction
The product was water (2 x 25 ml), saturated sodium chloride solution (2 x 25 ml).
25ml) and then washed with anhydrous magnesium sulfate.
Dried. It is filtered and evaporated to dryness under reduced pressure,Two
(R = ethyl) (25% ethyl acetate-Rf in hexane
A residue containing 0.18) was obtained in a yield of about 90%. This
This material was used without further purification. Mass spec
Toll:m / e (Relative strength) 372 (M⁺, 1), 312 (100), 298 (3
8), 283 (7), 255 (12), 191 (30);¹H-NMR (CDCl₃ ):
δ 0.69 (s, 18-H), 0.82 (t, J = 7.5Hz, 23-H), 0.9
(d, J = 7.0 Hz, 21-H), 1.05 (s, 19-H), 2.04 (s, 3-OCOC
H₃), 4.58 (m, 3-H), 5.38 (d, J = 4 Hz, 6-H).

3β-acetoxy-24-norcola-5,
A solution of 7-diene ( 3 ) (R = ethyl) 2 (R = ethyl) (270 mg, 0.72 mmol) in finely divided sodium hydrogen carbonate (600 mg, 7.14 mmol) in hexane (50 ml) was stirred with nitrogen. The mixture was heated under reflux at 80 ° C. under an atmosphere, and then 1,3-dibromo-5,5-dimethylhydrantyne (125 mg, 0.
43 mmol) was added. The reaction was allowed to proceed at 80 ° C for 20 minutes, then cooled and filtered. The filtrate was vacuum dried. The residue was immediately dissolved in dry xylene (5 ml) and added dropwise to a mixture of xylene (30 ml) and S-collidine (174 mg, 144 mmol). The mixture was then flushed with nitrogen and refluxed at 145 ° C. for 1.5 hours. It is then cooled and diluted with benzene (100 ml), hydrochloric acid 3% solution (3 x 20 ml), saturated aqueous sodium hydrogen carbonate solution (1
X20 ml), saturated sodium chloride solution (2 x 20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was converted to dioxane (2
0 ml) and p-toluenesulfonic acid (60 mg,
0.32 mmol) was added. The mixture was flushed with nitrogen, refluxed at 70 ° C. for 30 minutes, cooled, diluted with water (10 ml) and extracted with ethyl acetate (1 × 1).
00 ml, 2 x 50 ml). The extracted organic phase is a saturated aqueous solution of sodium hydrogen carbonate (1 × 25 ml), water (1 × 25 ml),
It was washed successively with saturated sodium chloride solution (2 x 20 ml) and then dried over anhydrous magnesium sulfate. It was filtered and evaporated to dryness under reduced pressure. The residue was chromatographed by preparative TLC with 10% ethyl acetate-hexane to give 148 mg (54%) of 3 (R = ethyl). UV (C ₂ H ₅ OH): λ _max 282 nm, 293, 272, 262, mass spectrum: m / e (relative intensity) 370 (M ⁺ , 4), 328 (3), 310
(100), 296 (22), 253 (14), 158 (45), ¹ H-NMR (CDC
l ₃ ): δ 0.63 (s, 18-H), 0.84 (t, J = 7.5 Hz, 23-H),
0.95 (d, J = 7.0 Hz, 21-H), 0.96 (s, 19-H), 2.04
(s, 3-OCOCH ₃ ), 4.71 (m, 3-H), 5.4 (m, 7-H), 5.58
(m, 6-H).

3β-hydroxy-24-norcola-5,
7-Diene ( 4 ) (R = ethyl) 3% of sodium hydroxide solution in methanol (10% solution)
To a solution of (130 mg, 0.35 mmol, 15% Rf 0.4 in 15% ethyl acetate-hexane) in ether (20 ml) was added dropwise with stirring under a nitrogen atmosphere. The reaction was allowed to proceed for 40 minutes at 23 ° C. It was then diluted with ether (100 ml), water (20 ml) was added and the phases were separated. The aqueous phase is then extracted with ether (2 x 60 ml) and the ether extracts are combined,
Water (2 x 30 ml), saturated aqueous sodium chloride solution (2 x 3
0 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. 30% residue
Chromatography by preparative TLC in ethyl acetate-hexane afforded 103 mg (89%) of 4 (R = ethyl) (15% ethyl acetate-Rf 0.09 in hexane). UV (C ₂ H ₅ OH): λ _max 282, 293, 272, 262 nm; Mass spectrum: m / e (relative intensity), 328 (M ⁺ , 100), 314 (1
6), 310 (8), 295 (86), 281 (15), 269 (45), 255 (1
3), ¹ H-NMR (CDCl ₃ ): δ 0.63 (&, 18-H), 0.84 (t, J = 7.5 H
z, 23-H), 0.94 (d, J = 7.0 Hz, 21-H), 0.96 (s, 19-
H), 3.64 (m, 3-H), 5.38 (m, 7-H), 5.57 (m, 6-H).

Secosterol analog 5 (R = ethyl) 1: 4 5,7 in dry benzene-ether (150 ml).
Diene 4 (R = ethyl) (125 mg) was irradiated for 25 minutes in the same manner as in Example 1 above. The solvent was evaporated under reduced pressure. The crude residue was immediately dissolved in dry nitrogen saturated ethanol (30 ml). The solution was refluxed at 70 ° C. for 3 hours under nitrogen atmosphere, then cooled and concentrated under reduced pressure. Three
Purification by preparative TLC using silica gel plates with 0% ethyl acetate-hexane solution gave 5 (R = ethyl) (30
mg) was obtained in about 24% yield. UV (C ₂ H ₅ OH): λ _max 264 n
m, λ _min 227 nm; Mass spectrum: m / e (relative intensity)
328 (M ⁺ , 23), 310 (3), 295 (10), 271 (9), 253 (11),
136 (81), 118 (100); ¹ H-NMR (CDCl ₃ ): δ0.55 (s, 1
8-H), 0.83 (t, J = 7.5 Hz, 23-H), 0.92 (d, J = 7.0 Hz,
21-H), 3.93 (Broad, 3-H), 4.81 (m, (Sharp), 19 (Z) -H), 5.01 (m (Sharp), 19 (E) -H, 6.02
(d, J = 12.5 Hz, 7-H), 6.22 (d, J = 12.5Hz, 6-H).

Tosylate 6 (R = ethyl) 5 (R = ethyl) (15 mg, 0.045 mmol: 25)
% Ethyl acetate-Rf 0.23 in hexane) in dry pyridine (1 ml) was treated with p-toluenesulfonic acid chloride (20 mg, 0.105 mmol) for 24 hours under a nitrogen atmosphere at 5 ° C. The reaction was quenched with ice water and the mixture was extracted with ether (3 x 50 ml). The organic phases are combined and water (2 x 20 ml), hydrochloric acid 3% aqueous solution (2 x 20 m)
l), saturated sodium hydrogen carbonate solution (2 x 20 ml) and saturated sodium chloride solution (1 x 20 ml) successively,
Dry over anhydrous magnesium sulfate and concentrate under reduced pressure to 9
22 mg of tosylate 6 (R = ethyl) with a purity of more than 5%
Got Mass spectrum: m / e (relative intensity) 484 (M ⁺ ,
8), 310 (72), 296 (18), 295 (20), 281 (10), 253 (39),
158 (65), 143 (47), 118 (100).

3,5-Cyclo-derivative 7 (R = ethyl) Tosylate 6 (R = ethyl) (22 mg, 0.045 mmol; 25% ethyl acetate-Rf 0.54 in hexane) finely ground sodium hydrogen carbonate (60 mg) , 0.71 mmol) in anhydrous methanol (20 ml) was added with stirring. The mixture was heated to 55 ° C under a nitrogen atmosphere for 8 hours,
Cool, dilute with ether (150 ml) and water (2 x 30
ml) and saturated sodium chloride solution (2 × 30 ml). Dry over anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to give crude product 7 (E = ethyl) in about 68% yield.
(25% ethyl acetate-Rf 0.6 in hexane) was obtained.
Mass spectrum: m / e (relative intensity) 342 (M ⁺ , 10) 328 (1
2), 310 (11), 295 (10), 253 (8), 149 (41), 136 (6
7), 118 (100). 1α-hydroxy-3,5-cyclo-derivative 8 (R = ethyl) t-butyl hydroperoxide (14.0 μl, 0.1
1 mmol pyridine-methylene chloride (10 m
Selenium dioxide (2.4 mg, 0.022 mmol) in l) was added and cooled on an ice bath. Cyclovitamin 7 (R = ethyl) (1 in dry methylene chloride (5 ml)
5 mg, 0.044 mmol; 25% ethyl acetate-Rf 0.6 in hexane) was added. Monitor the reaction by TLC 4
Proceed for 0 minutes, 10% sodium hydroxide solution (10m
l) was added to quench the reaction. Mix the mixture with ether (1
50 ml) and the phases are separated, the ethereal phase is 10% sodium hydroxide solution (2 x 30 ml), water (2 x 30 m).
l), washed successively with sodium chloride (2 x 30 ml) and dried over anhydrous magnesium sulfate. It was filtered and concentrated under reduced pressure. Preparative TLC with 25% ethyl acetate-hexane gave 8 (R = ethyl) (8 m) in about 50% yield.
g, Rf 0.18) in 25% ethyl acetate-hexane. Mass spectrum: m / e (relative intensity) 358 (M ⁺ , 2
1), 326 (48), 285 (35), 269 (15), 191 (50), 135 (1
00); ¹ H-NMR (CDCl ₃ ): δ 0.55 (s, 18-H), 0.85 (t, J =
7.5 Hz, 23-H), 0.92 (d, J = 7.0 Hz, 21-H), 3.27 (s,
6R-OCH ₃ ), 4.18 (d, J = 10 Hz, 6-H), 4.22 (m, 1-H),
4.95 (d, J = 10 Hz, 7-H), 5.16 (d, J = 2.0 Hz, 19 (Z)-
H), 5.26 (d, J = 2.2 Hz, 19 (E) -H).

5,6-cis and trans secosterol 3β-acetate 9b and 10b (X ¹ = CH ₃ CO,
X ² ═H) 8 (R = ethyl) (8 mg) in glacial acetic acid (0.5 ml) was heated at 55 ° C. under nitrogen atmosphere for 15 minutes, cooled, and onto ice-cold sodium hydrogen carbonate solution (15 ml). Injected. The mixture was extracted with ether (3 x 30 ml) and the ether extract was extracted with saturated sodium hydrogen carbonate solution (1 x 20 ml), water (2 x 20 ml), saturated aqueous sodium chloride solution (1 x 20 ml).
ml) and then dried over anhydrous magnesium sulfate,
Filtered. The filtrate was concentrated under reduced pressure, 30% ethyl acetate - hexanes (× 2) was chromatographed by preparative TLC on 5,6 cis -3β- acetate 9b (X ¹ =
^{_{CH 3 CO, X 2 = H}} ) (25% ethyl acetate - hexane Rf0.13) and 5,6-trans -3β- Asete <br/> over preparative _{^{10b (X 1 = CH 3 CO}} , X 2 = H ) (Rf 0.11 with 25% ethyl acetate-hexane). The product was further HPLC using a Zorbax-Sill column (4.6 mm x 25 cm) in 1% isopropanol-hexane.
In purified, acetate 9b and 10b, respectively 28.
Obtained in 9% and 10.4% yields (holding volumes 39 ml and 4
6.5 ml).

3β-acetate 9b (X ¹ = CH ₃ C
O, X ² = H) UV (C ₂ H ₅ OH) λ _max 264 nm, λ _min 226 nm; mass spectrum m / e (relative intensity) 386 (M ⁺ , 17), 326 (45), 308 (8 ),
269 (16), 203 (17), 134 (100), ¹ H-NMR (CDCl ₃ ): δ
0.55 (s, 18-H), 0.84 (t, J = 7.5 Hz, 23-H), 0.92 (d,
J = 7.0 Hz, 21-H), 2.04 (s, 3-OCOCH ₃ ), 4.41 (m, 1-H),
5.02 (m (Sharp), 19 (Z) -H), 5.22 (m, 3-H), 5.35
(m (sharp), 19 (E) -H) 6.03 (d, J = 12.5 Hz, 7-H),
6.35 (d, J = 12.5 Hz, 6-H). 3β-acetate 10b (X ¹ = CH ₃ CO, X ² =
H) UV (C ₂ H ₅ OH) λ _max 273 nm, λ _min 226 nm; mass spectrum m / e (relative intensity): 386 (M ⁺ , 12), 326 (64), 312
(8), 297 (9), 279 (4), 269 (21), 203 (28), 134 (10
0); ¹ H-NMR (CDCl ₃ ): δ 0.56 (s, 18-H), 0.85 (t, J =
7.5 Hz, 23-H), 0.93 (d, J = 7.0 Hz, 21-H), 2.30 (s,
3-OCOCH ₃ ), 4.49 (m, 1-H), 5.0 (m (sharp), 19 (Z)
-H), 5.14 (m (sharp), 19 (E) -H), 5.26 (m, 3-
H), 5.82 (d, J = 12.5 Hz, 7-H), 6.58 (d, J = 12.5 Hz, 6-
H).

1α-Hydroxy-secosterol 9b and 10b (X ¹ = X ² = H) 3β-acetate 9b (X ¹ = CH ₃ CO, X ² = H)
(1.5 mg, Rf 0.3 in 20% ethyl acetate-hexane)
1) was hydrolyzed using a 10% methanol (2 ml) solution of sodium hydroxide under nitrogen at 23 ° C. for 0.5 hours. The mixture was diluted with ether (50 ml) and water (5 ml).
Was added. The phases were separated and the aqueous layer was washed with ether (2 x 30
ml). Combine the ether extracts and mix with water (2 x 1
0 ml), saturated sodium chloride solution (2 × 10 ml), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to yield 9b (X ¹ = X ² = H) (20% ethyl acetate- Rf 0.06) in hexane was obtained. Similarly, acetate 10b (X ¹ = CH ₃ CO, X ² = H) (0.75 mg,
40% ethyl acetate-Rf 0.26 in hexane) was hydrolyzed to give the 5,6-trans-isomer 10b (R = ethyl,
X ¹ = X ² = H) (40% ethyl acetate-Rf in hexane
0.06) was obtained. Both products are sequentially 60% ethyl acetate
Preparative TLC with hexane, Zorbax-Syl analytical column using 7% isopropanol-hexane (4.
HPLC on 6 mm x 2.5 cm) and 90% methanol-
Zorbax-ODS analytical column with water (4.6
Chromatography by reverse phase HPLC (RPHPLC) on mm x 25 cm).

Compound 9b (X ¹ = X ² = H) UV (C ₂ H ₅ OH): λ _max 264 nm, λ _min 227 nm; High resolution mass spectrometry: Calculated value (C ₂₃ H ₃₆ O ₂ ) 344. 2715; measured 344. 27
07; Mass spectrum: m / e ( relative intensity) 344 (M ⁺ , 23), 326
(12), 287 (7), 269 (6), 251 (5), 203 (9), 152
(7), 134 (100); ¹ H-NMR (CDCl ₃ ): δ 0.52 (s, 18-H),
0.82 (t, J = 7.5 Hz, 23-H), 0.90 (d, J = 7.0 Hz, 21-H),
4.29 (m, 3-H), 4.42 (m, 1-H), 4.99 (m (sharp), 19
(Z) -H), 5.31 (m (sharp), 19 (E) -H), 6.0 (d, J = 1
2.5 Hz, 7-H), 6.37 (d, J = 12.5 Hz, 6-H). 5,6-trans-compound 10b (X ¹ = X ² = H) UV (C ₂ H ₅ OH): λ _max 273 nm, λ _min 226 nm; high resolution mass spectrometry: calcd _{(C 23 H 36 O 2)} 344. 2715: measurement 3
44. 2705: Mass spectrum: m / e (relative intensity) 344 (M ⁺ ,
12), 326 (6), 287 (4), 269 (4), 251 (3), 203 (6),
152 (30), 134 (100): ¹ H-NMR (CDCl ₃ ): δ 0.56 (s, 1
8-H), 0.84 (t, J = 7.5 Hz, 23-H), 0.91 (d, J = 7.0 Hz, 2
1-H), 4.22 (m (sharp), 3-H), 4.48 (m, (sharp), 1-H), 4.96 (s, 19 (Z) -H), 5.12 (m (sharp), 1
9 (E) -H), 5.88 (d, J = 12.5 Hz, 7-H), 6.57 (d, J = 12.5
Hz, 6-H).

Example 3 Preparation of Compounds 9c and 10c All reaction steps shown in Example 2 above for 6β-methoxy-3α, 5-cyclo-5α-chorane (Compound 1, R = propyl) were run under similar experimental conditions. This provided the 1α-hydroxy-secosterol analog of (X ¹ = X ² = H) of structural formula 9c and the corresponding 5,6-trans-compound of structural formula 10c (X ¹ = X ² = H). Example 4 Starting Material Synthesis of Compound 1 (R = Methyl) (a) (22E) -6β-Methoxy-3α, 5-cyclo-5α-stigmast-22-ene (stigmasteryl i-methyl ether) Newly crystallized p-Toluenesulfonic acid chloride (20 g, 0.10 mol) was added to stigmasterol (25 g, 0.06 mol: 30% Rf 0.26 in ethyl acetate-hexane) in dry pyridine (250
ml) solution. The reaction mixture was stirred at 23 ° C. for 24 hours and then gradually poured into a saturated aqueous solution of sodium hydrogen carbonate. The precipitate was collected by filtration, washed several times with water until neutral and dried under reduced pressure overnight, 93.2%
Stigmasteryl-3β-tosylate (32.
06 g) was obtained. This tosylate i without purification
-Converted to ether. Tosylate (32g, 0.06
Chloroform (100 ml) in methanol was slowly added to a solution of finely ground sodium hydrogen carbonate (30 g, 0.36 mol) in methanol (400 ml) under reflux. The mixture was stirred under reflux for 14 hours, cooled and concentrated to about 100 ml. Hexane (300 ml) was added and the resulting mixture was washed with water (100 ml). The phases were separated and the aqueous layer was extracted again with hexane (2 x 100 ml, 1 x 100 ml). The organic layers were combined, washed sequentially with water (2 x 100 ml), saturated aqueous sodium chloride solution (2 x 100 ml), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to the desired i.
-Methyl ether (R in 10% ethyl acetate-hexane
24 g of crude oil containing f0.52) was obtained. Mass spectrum; m / e (relative intensity) 426 (M ⁺ , 87), 411 (34), 394
(54), 371 (59), 368 (19), 351 (21), 255 (61), 83 (1
00).

(B) 6β-Methoxy-3α, 5-cyclo-23,24-dinol-5α-cholan-22-ol The stigmasteryl i-ether (5.
0 g, 11.7 mmol, Rf 0.64) in 30% ethyl acetate-hexane 1% pyridine-methylene chloride (1
(00 ml) the solution was cooled to -69 ° C with stirring on a dry ice-acetone bath, with ozone generated using a Wellback type T816 ozone generator until the pale blue color persisted due to excess ozone. Processed. The mixture was purged with oxygen for 5 minutes and warmed to 23 ° C. A solution of sodium borohydride (0.7 g, 18.4 mmol) in ethanol was added. After 2 hours, the reaction mixture was diluted with ether (200 ml). Water (100 ml) was added, the phases were separated and the aqueous layer was extracted with ether (2 x 200 ml). The combined organics were washed with saturated aqueous sodium chloride solution (2 x 50 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was separated on a silica gel column and elution with 20% acetic acid-hexane gave 26 g of the desired 22-alcohol derivative in 65% yield. Mass spectrum: m / e (relative intensity)
346 (M ⁺ , 75), 331 (52), 314 (89), 291 (100), 288
(30); ¹ H-NMR (CDCl ₃ ): δ0.44 (m, 3-H), 0.65 (m, 4-
H), 0.75 (s, 18-H), 1.04 (s, 19-H), 1.08 (d, J = 7.0
Hz, 21-H), 2.77 (m, sharp, 6-H), 3.32 (s, 6-OCH
₃ ). (C) 6β-methoxy-3α, 5-cyclo-23,24
-Dinor-5α-cholan-22-yltosylate p-Toluenesulfonic acid chloride (2.0 g, 11 mmol) was obtained in the above experiment from the 22-alcohol (1.9).
g, 5.5 mmol) in a solution of dry pyridine (35 ml). The reaction mixture was stirred at 23 ° C. for 18 hours, poured into ice-cooled saturated aqueous sodium hydrogen carbonate solution (50 ml), and extracted with ethyl acetate (2 × 150 ml, 1 × 100).
ml). The organic extract was washed successively with water (3 x 50 ml) and saturated sodium chloride solution (2 x 50 ml) and dried over anhydrous magnesium sulfate. It was then filtered and evaporated to dryness under reduced pressure. The residue was dried under reduced pressure overnight to give 2.2 g of 22-tosylate in 91.0% yield. Mass spectrum: m / e (relative intensity) 500 (M ⁺ , 69), 485 (49), 468
(100), 445 (79), 442 (20), 296 (20), 273 (24).

(D) 6β-methoxy-3α, 5-cyclo-23,24-dinor-5α-cholane (Compound 1, R =
22-tosylate (2. Methyl) in anhydrous ether (100 ml).
15 g, 4 mmol) to 0.22 mg of lithium aluminum hydride (LiAlH, 0.22 mg, 6 mmol)
Was added. The reaction mixture was refluxed for 10 hours, cooled and the excess reaction product was decomposed with saturated aqueous sodium chloride solution. The mixture was filtered and the layers separated. The aqueous layer was back extracted with ether (2 x 100 ml). Combine the ether content,
Water (1 x 50 ml), saturated sodium chloride solution (2 x 50 ml)
ml) and then dried over anhydrous magnesium sulfate,
Evaporated to dryness and dried under vacuum to give compound 1 (R = methyl) in 96.0% yield. Mass spectrometry: m / e (relative intensity) 330 (M ⁺ , 41), 315 (46), 298 (100), 283 (21),
275 (81), 272 (18), 177 (45); ¹ H-NMR (CDCl ₃ ): δ: 0.
40 (m, 3-H), 0.64 (m, 4-H), 0.72 (s, 18-H), 0.85
(d, J = 7.0 Hz, 22-H), 0.92 (d, J = 7.0 Hz, 21-H), 2.7
7 (m (sharp) 6-H), 3.35 (m, 6-OCH ₃ ). Example 5

Preparation of Starting Material Compound 1 (R = Ethyl) (a) 6β-Methoxy-3α, 5-cyclo-24-nor-5α-cholan-23-nitrile 22-obtained in Example 4 (c) above Tosylate (10g, 2
2 mmol, Rf 0.5 in 25% ethyl acetate-hexane
Sodium cyanide (1.95 g, 40 mmol) was added to a solution of 3) in dimethylsulfoxide (200 ml). After stirring the mixture at 80 ° C. for 2 hours under nitrogen atmosphere,
It was cooled and stirred at room temperature for 1 hour. It was then poured onto ice-cold saturated ammonium chloride solution (250 ml) and extracted with ether (3 x 450 ml). The combined ether fractions are water (3 x 200 ml), saturated sodium chloride solution (2 x 20 ml).
0 ml), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give the desired 23-nitrile derivative (crude product 7.05 g, Rf 0.57 in 25% ethyl acetate-hexane). Got This crude product mixture was used in the next experiment without purification. Mass spectrum: m
/ e (relative intensity) 355 (M, 33), 340 (46), 323 (64), 308
(4), 297 (17), 300 (100), 218 (11), 149 (31); ¹ H-NMR
(CDCl ₃ ): δ 0.43 (m, 3-H), 0.65 (m, 4-H), 0.74 (s, 18-
H), 1.02 (s, 19-H), 1.15 (d, J = 7.0 Hz, 21-H), 2.77
(t, J = 2.5 Hz, 6-H).

(B) 6β-methoxy-3α, 5-cyclo-24-nor-5α-cholan-23-ol. The nitrile derivative obtained in (a) above (7.0 g, 19.
7 mmol, Rf in 10% ethyl acetate-hexane solution
A solution of 0.27) in dry benzene (150 ml) was cooled on ice under a nitrogen atmosphere, and diisobutylaluminum hydride [DIBAL-H, 1.5 mol toluene (20 ml, 30 mmol) solution] was gradually added to this solution. Was added. After the addition was complete, the ice bath was removed and the reaction was allowed to proceed at 23 ° C for 3 hours. Methanol (150 ml) was added to decompose the aluminum complex salt, and the mixture was poured onto ice water (200 ml). The mixture was filtered and the aqueous layer was washed with ether (3x
150 ml). The organic phases were combined, washed with saturated sodium chloride solution (2 × 60 ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 6.25 g of product. The product was a mixture of the starting 23-nitrile and the expected 23-aldehyde. This mixture was treated again with the same reducing agent. That is, the crude product was dissolved in dry benzene and DIBAL-H (17.5 ml,
26.25 mmol). Work-up as above and chromatography of the concentrated residue on silica gel, eluting with 10% ethyl acetate-hexane gave 0.85 g of the desired 23-alcohol (Rf 0.03 in 10% ethyl acetate-hexane). .. Mass spectrum: m / e (relative intensity) 360 (M ⁺ , 75), 345 (59), 328
(99), 305 (100), 302 (29), 281 (22), 255 (23); ¹ H-NM
R (CDCl ₃ ): δ 0.44 (m, 3-H), 0.65 (m, 4-H), 0.72
(s, 18-H), 0.93 (d, J = 7.0 Hz, 21-H), 1.02 (s, 19-H),
2.77 (m (Sharp) 6-H), 3.3 (s, 6-OCH ₃ ).

The remaining product (2 g) is the corresponding 23-aldehyde, which can be further reduced, if desired, under the above conditions to give some more 23-alcohol product. (C) 23-hydroxy-6β-methoxy-3α, 5-
Cyclo-24-nor-5α-cholan-23-tosylate. p-toluenesulfonic acid chloride (0.95 g, 5.0
23 mmol of the 23-alcohol obtained in (b) above (0.85 g, 2.36 mmol) in dry pyridine (2
5 ml) of the solution. The reaction mixture was allowed to stand at 0 ° C. for 24 hours and then ice-cooled, saturated aqueous sodium hydrogen carbonate solution (25 m
l), extracted with ethyl acetate (3 × 100 ml), water (3 × 40 ml), saturated sodium chloride solution (2 × 4).
(20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure to give 23-tosylate (1.15 g) in about 95% yield. Mass spectrum: m / e
(Relative intensity) 514 (M ⁺ , 42), 499 (26), 482 (100), 468
(14), 459 (42), 456 (12), 361 (13), 255 (29).

(D) 6β-methoxy-3,5-cyclo-
24-nor-5α-cholan (Compound 1 , R = ethyl) The above tosylate (1.15 g, 2.08 mmol, 1
LiAlH ₄ (0.12 g, 3 mmol) was added to a solution of 0% Rf 0.7) in ethyl acetate-hexane in anhydrous ether (100 ml). The reaction mixture was refluxed for 5 hours,
After cooling the excess reagent was destroyed by saturated sodium chloride solution. The mixture was filtered, the phases were separated and the aqueous phase was extracted with ether (2 x 100 ml). The ether extracts were combined, water (1 x 50 ml), saturated sodium chloride solution (2 x
50 ml), dried over anhydrous magnesium sulfate,
After evaporation to dryness under reduced pressure and vacuum drying, the desired 24-nor-cholan derivative (Compound 1, R = ethyl) was about 82%.
Yield (0.59 g, 10% ethyl acetate-Rf in hexane
0.64). Mass spectrum: m / e (relative intensity)
344 (M ⁺ , 9), 329 (12), 312 (19), 289 (22), 286 (4),
255 (7), 191 (11), 69 (90); ¹ H-NMR (CDCl ₃ ): δ0.44
(m, 3-H), 0.65 (m, 4-H), 0.72 (s, 18-H), 0.82 (t,
J = 7.5 Hz, 23-H), 0.9 (d, J = 7.0Hz, 21-H), 1.02 (s,
19-H), 2.77 (m, sharp, 6-H), 3.32 (s, 6-OCH ₃ ).

Example 6 Preparation of Starting Material Compound 1 (R = Propyl) The 23-tosylate obtained in Example 5 (c) above was used in Example 5
Treatment with sodium cyanide under similar conditions as described in (a) to give 6β-methoxy-3α, 5-cyclo-5.
α-Colane-24-nitrile is obtained. This nitrile was reduced using the method of Example 5 (b) above to give 6β-
Methoxy-3α, 5-cyclo-5α-cholan-24-ol is obtained. This alcohol is converted to the corresponding 24-tosylate in a manner similar to that described in Example 5 (C) above, and the tosylate is treated with the hydride reduction as described in Example 5 (d) above to give the desired compound. , 6β-methoxy-3α, 5-cyclo-5α-chorane (Compound 1 with R = propyl) is obtained.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Heinrich K. Siunose United States 53705 Wisconsin Madison Summit Avenyu 1806 (72) Inventor One Juan. Rho United States 77004 Texas Highstone Calhoun Road 4800 University of Texas Department of Chemistry

Claims (8)

[Claims] 1. Structural formula I or II: (Wherein R is hydrogen, methyl, ethyl or propyl,
A drug for inducing cell differentiation in mammals, which comprises a compound having X ¹ and X ² independently representing hydrogen or an acyl group as an active ingredient. 2. The drug according to claim 1, wherein the cells in which differentiation occurs are leukemia cells. 3. The drug according to claim 1 or 2, wherein R, X ¹ and X ² represent hydrogen, and the compound is a compound of structural formula I. 4. The agent according to claim 1 or 2, wherein R is methyl, X ¹ and X ² represent hydrogen, and the compound is a compound of structural formula I. 5. The drug according to claim 1 or 2, wherein R is ethyl, X ¹ and X ² represent hydrogen, and the compound is a compound of structural formula I. 6. R is propyl, X ¹ and X ² represent hydrogen, and the compound is a compound of structural formula I.
Or the drug according to 2. 7. The drug according to claim 1, which has a pharmaceutically acceptable excipient. 8. The drug according to claim 1, which is used for treating malignant cells.