CELLULAR SENESCENCE ACTIVATING COMPOUNDS

Abstract

The present invention describes a naturally occurring chemical compound, specifically guayulins A, B, C and D used in medicine having null cytotoxicity and genotoxicity on healthy lymphocyte cells. Said active compounds interfere with the inflammatory process and have antitumoral activity in human cancer cell lines, since they inhibit their growth through a senescence process.

Claims

1. A guayulin compound A, B, C and D called: Argentatines A and C, Isoargentine B and Argentatine D, of formula (I) and (I′) that present null cytotoxicity or genotoxicity on healthy lymphocytic cells in an In vivo animal model, characterized in that it presents anti-inflammatory activity, inhibits the growth of cancer cells through a senescence process; wherein the compound of formula (I):
(A)-(B)-(C) wherein: A is a group that is selected from one of: ##STR00044## B is ##STR00045## and C is a group that is selected from one of: ##STR00046## and where: R.sup.1 represents a group that is selected from: ##STR00047## R.sup.2 represents a group that is selected from: ##STR00048## R.sup.3 represents a group that is selected from: ##STR00049## R.sup.4 represents a group selected from: —OH, ##STR00050## and wherein: R.sup.1 and R.sup.3 can be at the same time ; R.sup.3 and R.sup.4 can be at the same time —OH or —OAc; R.sup.1 and R.sup.2 are not at the same time ##STR00051## and —Br, respectively; R.sup.5 is a group that is selected from one of: H, CH.sub.3, or an alkyl chain, and wherein when A is ##STR00052## then R.sup.1 is not ##STR00053## and R.sup.2 is not —Br, and R.sup.3 and R.sup.4 are at the same time a group selected from —OH or —OAc; when C is ##STR00054## then R.sup.1 and R.sup.3 can be at the same time ═O and R.sup.2 is not —Br; or when A is ##STR00055## then R.sup.1 and R.sup.2 can together form a group ##STR00056## and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof, And the compound of formula (I′):
(A)-(B)-T wherein: A and B have the meanings as defined above, R.sup.1 represents a group that is selected from: ═O, ##STR00057## R.sup.2 represents a group that is selected from: ##STR00058## R.sup.3 represents a group that is selected from: ##STR00059## and wherein: R.sup.1 and R.sup.3 can be at the same time ═O; R.sup.1 and R.sup.2 are not at the same time ##STR00060## and —Br, respectively; R.sup.5 is a group that is selected from one of: H, CH.sub.3, or an alkyl chain, and wherein when A is ##STR00061## then R.sup.1 is not ##STR00062## and R.sup.2 is not —Br; when A is ##STR00063## then R.sup.1 and R.sup.2 can together form a group ##STR00064## and where T represents a group ##STR00065## and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

2. A guayulin compound of formula (I):
(A)-(B)-(C) wherein A, B, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 is defined in accordance with claim 1, characterized in that: R.sup.3 and R.sup.4 cannot be —OH at the same time; and R.sup.1 and R.sup.2 are not at the same time ═O and —H respectively; and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

3. A guayulin compound of formula (I):
(A)-(B)-(C) according to claim 1, characterized in that: A is a group ##STR00066##  B is a group ##STR00067##  C is a group ##STR00068##  and wherein:  R.sup.1 represents a group: ═O,  R.sup.2 represents a group: —H;  R.sup.3 represents a group selected from: —OH;  R.sup.4 represents a group selected from: —OH;  and/or the enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compound and pharmaceutically acceptable salts thereof.

4. A guayulin compound of formula (I′) according to claim 1:
(A)-(B)-T wherein: A and B have the meanings as defined above, characterized in that: R.sup.1 represents a group that is selected from: ═O, ##STR00069## R.sup.2 represents a group that is selected from: ##STR00070## R.sup.3 represents a group that is selected from: ##STR00071## and wherein: R.sup.1 and R.sup.3 can be at the same time ═O; R.sup.1 and R.sup.2 are not at the same time ##STR00072## and —Br, respectively; R.sup.5 is a group that is selected from one of: H, CH.sub.3, or an alkyl chain, and wherein when A is ##STR00073## then R.sup.1 is not ##STR00074## and R.sup.2 is not —Br; when A is ##STR00075## then R.sup.1 and R.sup.2 can together form a group ##STR00076## and where T represents a group ##STR00077## and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

5. A guayulin compound of formula (I) and (I′) according to claim 1, characterized in that the compounds of formula (I) and (I′) are selected from: ##STR00078## ##STR00079## ##STR00080## ##STR00081##

6. A guayulin compound of formula (I) and (I′) according to claim 1, characterized in that the compound In is the compound ##STR00082##

7. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Ia) is carried out according to the following steps: 100 mg of In were dissolved in 5 ml of glacial acetic acid reacting with 0.4 ml of a 1 M solution of bromine in acetic acid; the reaction is carried out under stirring at 3° C. for 1.25 h, the reaction mixture is poured into an Erlenmeyer flask containing 50 g of ice, washed with a 5% NaHCO.sub.3 solution and subsequently recrystallized: ##STR00083##

8. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Ib) is carried out in accordance with the following steps: a solution of In (200 mg) and phenylselenium chloride (120 mg) in EtOAc (4.6 mL) stirring at room temperature for 2 h; subsequently 1 ml of water is added to the reaction mixture while stirring; the aqueous phase is separated and 2 mL of THF and 0.2 ml of 30% H.sub.2O.sub.2 were are added; the resulting mixture is stirred at room temperature for 1 h ##STR00084##

9. A guayulin compound according to claim 8, characterized in that the synthesis of the compound (Ic) is carried out according to the following steps: a mixture of 25.5 mg of derivative Ib, 21 mg of sodium acetate and 2 ml of acetic anhydride is heated at reflux temperature for one hour; subsequently, the mixture is poured into an Erlenmeyer flask containing 5 g of ice and stirred for 3 minutes; the contents of the flask are extracted with AcOEt (3×); the organic phase is dried and concentrated under reduced pressure to obtain a semi-solid; recrystallize and the acetate Ic is obtained ##STR00085##

10. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Id) is carried out according to the following steps: 301 mg of compound In in 4.5 ml of pyridine are reacted with 99 mg of NH.sub.20H.HCl stirring at reflux temperature for one hour; subsequently, the reaction mixture is poured into a flask containing 100 g of ice and extracted with AcOEt (3×); the organic phase is washed repeatedly with a 10% HCl solution followed by water and subsequently dried and concentrated under reduced pressure; the residue obtained after evaporation is purified by column chromatography, to obtain the oxime Id ##STR00086##

11. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Ie) is carried out in accordance with the following steps: a solution of 100 mg of In in 4 ml of acetic acid is treated at 0-5 ° C. with chromium trioxide (100 mg) in 0.3 ml of water; after 1 hour, the mixture is left at room temperature and subsequently extracted with AcOEt (3×); the organic phase is processed in a conventional way to obtain the product Ie ##STR00087##

12. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (If) is carried out in accordance with the following steps: a mixture of 200 mg of In, 60.2 mg of sodium acetate and 5 ml of acetic anhydride are heated at reflux temperature for 18 hours; subsequently, the mixture is poured into an Erlenmeyer flask containing 50 g of ice and stirred for 15 minutes; extracted with AcOEt (3×); the organic phase is dried and concentrated under reduced pressure to obtain a semi-solid; the product is recrystallized (hexane/AcOEt) to obtain acetate If ##STR00088##

13. A guayulin compound according to claim 12 characterized in that the synthesis of the compound (Ig) is carried out in accordance with the following steps: 200 mg of diacetate In in 6.5 ml of pyridine are reacted with 95 mg of NH.sub.20H.HCl stirring at reflux temperature for 1.5 hours; subsequently, the reaction mixture is poured into a flask containing 50 g of ice and extracted with AcOEt (3×); the organic phase is washed 3 times with a 10% HCl solution and then with water; recrystallization of the organic phase purifies the oxime Ig ##STR00089##

14. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Ih) is carried out in accordance with the following steps: a solution of 200 mg of In in 6 ml of dry pyridine, contained in Inert atmosphere, is added with 1 ml of ethyl formate (freshly distilled), 0.8 ml of a sodium solution in absolute MeOH (0.44 g/6 ml); the reaction is kept under stirring at room temperature for 8 to 12 hours until the appearance of an ocher color or the formation of an insoluble precipitate; subsequently the reaction mixture is placed in a cold solution of 3 ml of acetic acid in 27 ml of water; the precipitate is extracted with methylene chloride; the organic phase is washed with water and extracted with a 2% potassium hydroxide solution; the basic extract is washed with ether, acidified with glacial acetic acid, and finally extracted with methylene chloride; the final methylene chloride phase is dried and concentrated under reduced pressure to obtain an impure semi-solid product, purifying by preparative layer chromatography to obtain the formylated derivative Ih ##STR00090##

15. A guayulin compound according to claim 14, characterized in that the synthesis of compound (Ii) is carried out according to the following steps: a solution of 60 mg of the formylated derivative Ih in 5 ml of glacial acetic acid, with stirring, it is reacted for 2 hours with 30 mg of hydroxylamine hydrochloride at reflux temperature; subsequently the reaction mixture is poured into an Erlenmeyer flask containing 50 g of ice and extracted with AcOEt; the organic phase is washed with a 5% sodium bicarbonate solution (3×) and with water; the organic phase is dried and concentrated under reduced pressure to obtain an impure semi-solid, subsequently the semi-solid is purified by means of column chromatography to obtain isoxazole Ii ##STR00091##

16. A guayulin compound according to claim 10, characterized in that the synthesis of the compound (Ij) is carried out in accordance with the following steps: to 100 mg of In oxime dissolved in CHCl.sub.3, 0.5 mL of trifluoroacetic anhydride is added slowly at 0° C., the reaction mixture is stirred constantly at 25° C. for 18 min; subsequently the reaction mixture is evaporated under reduced pressure from this reaction, 16-trifluoroaxetoxy-lactam of In is obtained, a solution of potassium carbonate in methanol is added and stirred for 15 min. at room temperature, the solution is subsequently filtered, and the solvent is evaporated under reduced pressure; the reaction product is purified by column chromatography with polarity of 2:1 Hex:AcOEt, obtaining Ij ##STR00092##

17. A guayulin compound/argentatines according to claim 5, characterized in that the synthesis of the compound (Ik) is carried out in accordance with the following steps: 200 mg of In are treated with 170 mg m-chloroperoxybenzoic acid for 3 hours, to obtain 180 mg of a white solid Ik ##STR00093##

18. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Il) is carried out in accordance with the following steps: 100 mg of In are treated with 80 mg sodium borohydride (Na8H.sub.4) obtaining a white solid Il ##STR00094##

19. A guayulin compound according to claim 5, characterized in that the synthesis of the compound (Im) is carried out in accordance with the following steps: 0.15 mmol of hexadecanoyl chloride, previously obtained and in inert atmosphere, is added with 0.22 mmol of In, dissolved in 4 ml of dry dichloromethane; the reaction is stirred for 30 minutes; later, 15 mL of ethyl acetate was is added, and it was is placed in a separatory funnel; the organic phase is washed three times with distilled water and three times with a saturated solution of NaHCO.sub.3, dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure; the reaction mixture is subject to silica open and packed column chromatography; the elution mixture used is hexane-ethyl acetate (7:3); from fractions 3-8 a white amorphous solid (Im) is obtained ##STR00095##

20. A guayulin compound according to claim 11, characterized in that the synthesis of the compound (I′a) is carried out in accordance with the following steps: a solution of 100 mg of Ie in EtOH is kept at reflux with 289 mg of potassium hydroxide for 40 min; subsequently the reaction is neutralized and 3,16-dioxo-25-nor-cycloartan-17-en-24-oic acid [I′a] is obtained ##STR00096##

21. A guayulin compound according to claim 1, characterized in that they are active compounds that interfere with the inflammatory process and have antitumor activity in human cancer cell lines.

22. A guayulin compound according to claim 1, characterized in that the mechanism by which they exert an antitumor effect without generating cytotoxicity on healthy cells is through the induction of cellular senescence.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1. Astragloside compounds used to increase telomerase activity (U.S. Pat. No. 8,759,304 B2).

[0029] FIG. 2. Results of the apoptosis induction using a double labeling with annexin V and IP, due to the effect of compound In.

[0030] FIG. 3. Evaluation of the effect of compound In in mice xenotransplanted with HCT-116 cells. The arrows indicate the days of compound administration, in a regimen of 1 administration per week for 3 weeks, each point represents the average and SD of 6 mice. A significant difference between the treated groups was found with In (250 and 500 mg/kg) and the group of mice treated with cisplatin (4 mg/kg) versus the group treated with vehicle (****p <0.0001, Student's t-test).

[0031] FIG. 4. Images of the size of mouse tumors treated for three weeks with A) vehicle, B) cisplatin 4 mg/kg once a week, C) In 250 mg/kg once a week, D) 500 mg/kg of In once a week. The tumor was induced with the HCT-116 colon cancer cell line.

[0032] FIG. 5. Evaluation of the effect of compound In in mice xenotransplanted with HCT-116 cells. The arrows indicate the days of administration, with 3 administrations per week for 3 weeks, each point representing the mean±SD of 6 mice. Significant differences were observed between the In- and cisplatin-treated groups versus the vehicle-treated group (****p <0.0001, Student's t-test).

[0033] FIG. 6. Images of the size of the tumors treated for three weeks with A) vehicle, B) 250 mg/kg of In three times a week, and C) cisplatin 2 mg/kg three times a week.

[0034] FIG. 7. A) Weight variation of nu/nu mice treated with In at doses of 500 mg/kg, 250 mg/kg or cisplatin 4 mg/kg, administered intraperitoneally once a week for 3 weeks. B) Weight variation of nu/nu mice treated with In at doses of 250 mg/kg or cisplatin 2 mg/kg, administered intraperitoneally 3 times a week for 3 weeks. Each point in the graphs represents the mean±SD of the weight of 3 mice per experimental group. A statistical difference was observed between the weight of the cisplatin-treated mice versus the vehicle group mice (****p <0.0001, Student's t-test).

[0035] FIG. 8. Representative photomicrographs of HCT-116 cell xenotransplantation stained with hematoxylin-eosin. A) Vehicle, B) 500 mg of In 1 time per week, C) 250 mg/kg of In 1 time per week, D) 250 mg/kg of In 3 times per week. Images were taken with an Olympus IX71 inverted microscope using Qcapturepro 5 software from the Qlmaging company with a 20x microscopic scale.

[0036] FIG. 9. Representative photomicrographs of HCT-116 cell xenotransplantation stained with DAPI. A) Vehicle, B) 500 mg/kg of In 1 time per week, C) 250 mg/kg of In 1 time per week, D) 250 mg/kg of In 3 times per week. The images were taken with an Olympus IX71 inverted microscope using Qcapturepro 5 software from the Qlmaging company with a U-mwu2 filter, 330-420 nm excitation band, 400 dichroic mirror with a Fluorite plan 20× NA0.45 objective.

[0037] FIG. 10. Representative photomicrographs of PCNA immunolabelling. A) Vehicle, B) 500 mg of In 1 time per week, C) 250 mg/kg of In 1 time per week, D) 250 mg/kg of In 3 times per week. Images were taken with an Olympus IX71 inverted microscope using Qcapturepro 5 software from the Qlmaging company with a 20x microscopic scale. Brown color indicates PCNA positive cells.

[0038] FIG. 11. Antiproliferative effect of compound In observed with the cell proliferation marker PCNA in HCT-116 xenotransplanted cells. The results are shown in percentage of PCNA±SD. The fiji.sc software was used to calculate the average number of antigen-positive cells in 10 randomly selected microscopic fields from 3 xenotransplantation tissues per experimental group, leaving a total of 30 measurements. Significant differences were found between the different treatments with In and the control group (****p<0.0001, Student's t test).

[0039] FIG. 12. A) β-galactosidase activity in HCT-116 cells, B) β-galactosidase activity in HCT-15 cells, C) Cell control of the HCT-116 cell line, D) HCT-116 cells treated with In 30 pM for 72 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to a relevant and novel antitumor activity of a compound of formula (I) and (I′) with a null toxicity due to the fact that it acts on mechanisms of regulation of the cell cycle that indirectly has been shown not to cause an anti-proliferative effect on healthy cells.

Compound (1) has the formula:

(A)-(B)-(C)

where:

[0041] A is a group that is selected from one of:

##STR00001##

[0042] B is

##STR00002##

and

[0043] C is a group that is selected from one of:

##STR00003##

[0044] and where:

[0045] R.sup.1 represents a group that is selected from:

##STR00004##

[0046] R.sup.2 represents a group that is selected from:

##STR00005##

[0047] R.sup.3 represents a group that is selected from:

##STR00006##

[0048] R.sup.4 represents a group selected from: —OH,

[0049] and where:

##STR00007##

[0050] R.sup.1 and R.sup.3 can be at the same time ;

[0051] R.sup.3 and R.sup.4 can be at the same time —OH or —OAc;

[0052] R.sup.1 and R.sup.2 are not at the same time

##STR00008##

and —Br, respectively;

[0053] R.sup.5 is a group that is selected from one of: H, CH.sub.3, or an alkyl chain.

[0054] and wherein

when A is

##STR00009##

then R.sup.1 is not

##STR00010##

and R.sup.2 is not —Br, and R.sup.3 and R.sup.4 are at the same time a group selected from —OH or —OAc;

[0055] when C is

##STR00011##

then R.sup.1 and R.sup.3 can be at the same time ═O and R.sup.2 is not —Br; or

[0056] when A is

##STR00012##

then R.sup.1 and R.sup.2 can together form a group

##STR00013##

and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

[0057] In one embodiment, a compound of formula (1) is preferred:

(A)-(B)-(C)

[0058] wherein

[0059] A, B, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 have the meanings as defined above, provided that:

[0060] R.sup.3 and R.sup.4 cannot be at the same time —OH; and

[0061] R.sup.1 and R.sup.2 are not at the same time ═O and —H respectively;

[0062] and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

[0063] In one embodiment, the compound of formula (1) is a compound wherein:

[0064] A is a group

##STR00014##

[0065] B is a group

##STR00015##

[0066] C is a group

##STR00016##

[0067] and where:

[0068] R.sup.1 represents a group: ═O,

[0069] R.sup.2 represents a group: —H;

[0070] R.sup.3 represents a group selected from: —OH;

[0071] R.sup.4 represents a group selected from: —OH;

[0072] and/or the enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compound and pharmaceutically acceptable salts thereof.

[0073] In one embodiment, a compound of formula (I′) is preferred:

(A)-(B)-T

[0074] where:

[0075] A and B have the meanings as defined above,

[0076] R.sup.1 represents a group that is selected from: ═O,

##STR00017##

[0077] R.sup.2 represents a group that is selected from:

##STR00018##

[0078] R.sup.3 represents a group that is selected from:

##STR00019##

[0079] and where:

[0080] R.sup.1 and R.sup.3 can be at the same time ═O;

[0081] R.sup.1 and R.sup.2 are not at the same time

##STR00020##

and —Br, respectively;

[0082] R.sup.5 is a group that is selected from one of: H, CH.sub.3, or an alkyl chain.

[0083] and wherein

[0084] when A is

##STR00021##

then R.sup.1 is not

##STR00022##

and R.SUP.2 .is not —Br;

[0085] when A is

##STR00023##

then R.sup.1 and R.sup.2 can together form a group

##STR00024##

and where T represents a group

##STR00025##

and enantiomers, diastereoisomers, mixtures of enantiomers, mixtures of diastereoisomers, anomers, hydrates, solvates, polymorphs, of the aforementioned compounds and pharmaceutically acceptable salts thereof.

[0086] In one embodiment, compounds of formula (I) and (I′) are preferred, which are selected from:

##STR00026## ##STR00027## ##STR00028## ##STR00029##

[0087] The compound of formula (I) and (I′) have an antitumor activity due to the fact that they present null toxicity when acting on mechanisms of regulation of the cell cycle that indirectly has been shown not to cause an anti-proliferative effect on healthy cells in an in vivo animal model. Likewise, the compounds of the present invention refer to a method that favors the induction of a senescence process of tumor cells to inhibit the proliferation of this type of cells and induce the death of the same, through the arrest of the cell cycle.

[0088] Firstly, the cytotoxic activity of the compounds of formula (I) and (I′) was evaluated in a special way of compound In against different colon cancer cell lines (Table 1) where it was observed that the compound exhibits a moderate cytotoxic effect and that the HCT-116 colon cancer cell line was more sensitive to triterpene administration. Likewise, it was noted that despite the cytotoxic activity of compound In (Argentantine A) on cell lines, it only induced apoptosis in the HCT-116 line (30 μM) at 72 hours of treatment (FIG. 2). This indicates that the compound In can induce apoptosis in prolonged incubation intervals, however, this is an edge that shows that the compound induces a process of cellular senescence that eventually leads to cell death as will be explained later.

TABLE-US-00001 TABLE 1 Cytotoxicity of compound In against different colon cancer cell lines under treatment for 24, 48 and 72 hours. Cisplatin was used as a positive control. The presented values correspond to the average of 3 independent experiments performed in triplicate ± standard deviation. Composite, IC.sub.50 (μM) ± S.D. In Cisplatin Incubation Cell cancer line Cell cancer line time (h) HCT-15 HCT-116 SW-620 HCT-15 HCT-116 SW-620 24 95.43 ± 0.6  87.83 ± 0.7  115.62 ± 0.4   12.50 ± 0.5  10.81 ± 0.7  18.03 ± 0.5  48 59.67 ± 1.3  53.33 ± 0.2  72.46 ± 0.2  10.72 ± 0.6  8.37 ± 0.5  13.87 ± 0.4  72 44.83 ± 0.9  43.17 ± 0.4  61.33 ± 1.2  4.68 ± 0.3  3.09 ± 0.3  9.09 ± 0.3 

[0089] The National Cancer Institute and some scientific journals indicate that compounds isolated from medicinal plants should be considered cytotoxic agents only when they show ED.sub.50≤values 4 μg/mL (mean effective dose). Therefore, the compound In would be considered as an inactive compound according to this reference, however, what is observed in the state of the art is that the compounds of the triterpene type show a low cytotoxicity but an important anti-inflammatory activity that it can exert an important antitumor activity.

[0090] In this sense, the antitumor activity of compound In was demonstrated in a xenotransplantation study using mice inoculated with HCT-116 cancer cells. A first study was conducted in which In was administered at doses of 250 to 500 mg/kg of body weight once a week for 21 days and a reduction in tumor size was observed in 49.1% and 48.8%, respectively, compared to the tumor developed by the mouse that did not receive the treatment (FIGS. 3 and 4).

[0091] Subsequently, a study was carried out in which it was shown that with the administration of In at a dose of 250 mg/kg of weight, three times a week, for 21 days, a reduction of 78.1% of the tumor is achieved compared to the tumor of the untreated mice. This tumor reduction was equivalent to the effect exerted with the administration of 2 mg/kg of cisplatin in the same regimen of administration of compound In (FIGS. 5 and 6).

[0092] In addition to the fact that In is shown to possess an antitumor effect comparable to that of cisplatin, the triterpene-type compound has a very different toxicological profile from that of the drug cisplatin. Firstly, it is shown that the administration of In at doses of 500, 250 or 125 mg/kg, once a week, for 3 weeks does not show toxicity in nu/nu mice, and the calculated mean lethal dose (LD.sub.50) was very high greater than 500 mg/kg. Furthermore, the administration of In at doses of 250 and 500 mg/kg does not cause weight loss in mice, contrary to what happened with mice treated with cisplatin at doses of 4 mg/kg once weekly or at doses of 2 mg/kg three times a week for 21 days (FIG. 7).

[0093] Likewise, In-treated mice (500 mg/kg once a week and 250 mg/kg three times a week for three weeks) show no physical or behavioral changes compared to the control group. However, mice treated with cisplatin (4 mg/kg once a week and mg/kg three times a week for 3 weeks) show evidence of hepatotoxicity (increased alanine aminotransferase and aspartate aminotransferase values) and decreased leukocytes (Table 2).

TABLE-US-00002 TABLE 2 Blood parameters of the treated mice. Blood In 500 In 250 Cisplatin Cisplatin parameters Reference mg/Kg.sup.A mg/Kg.sup.B 2 mg/Kg.sup.B 4 mg/Kg.sup.A Leukocytes 3.2-7.0 × 10.sup.9 l 6.5 × 10.sup.9 l 7.2 × 10.sup.9 l 1.6 × 10.sup.9 l** 2.1 × 109 l** Lymphocytes 3.16- 6.9 × 10.sup.9/l 7.3 × 10.sup.9/l 0.52 × 0.52 × 109/l **** 7.8 × 10.sup.9/l 10.sup.9/l **** Erythrocytes 7.1- 7.29 × 10.sup.12/l 7.4 × 10.sup.12/l 7.14 × 10.sup.12/l 7.36 × 1012/l 10.2 × 10.sup.12/l Hemoglobin 149-170 g/l 158 g/l 164 g/l 113 g/L** 115 g/l** Glucose 6.6-8.5 mmol/l 7.1 mmol/l 8.0 mmol/l 7.80 mmol/l 8.56 mmol/l Urea 2.6-3.5 mmol/l 3.2 mmol/l 2.9 mmol/l 9.06 mmol/l**** 7.57 mmol/l**** Creatinine 8.8-26.5 μM/l 20.16 μM/l 14.2 μM/l 43.3 μM/l **** 29.06 μM/l * Alanine 46-55 UL 49.6 UL 53.3 UL 75.21 UL* 62.89 UL* aminotransferase Aspartate 85-101 UL 98.96 UL 91.07 UL 197.32 UL**** 151.25 UL **** aminotransferase .sup.AAdministration once a week, .sup.BAdministration 3

[0094] Additionally, studies were carried out to evaluate the effect of In administration on cell morphology in tumor tissues stained with hematoxylin-eosin. This shows that the tissues treated with the compound show slightly elongated nuclei and greater space between them, which suggests an expansion of the cytoplasmic area. This finding shows that the cells are suffering some type of damage compared to the tissues of the control group (FIG. 8).

[0095] To evaluate whether these nuclei are fragmented as a signal of cell death, the tissues were labeled with the fluorescent marker (CAPI (4′,6-diamino-2-phenylindole) which binds strongly to regions rich with adenine and thymine in DNA sequences, which is excited with ultraviolet light and detected with a blue filter through fluorescence microscopy (absorption maximum at 358 nm in the ultraviolet range, and its emission maximum is at 461 nm in the blue color spectrum). In this case, it was observed that the treated groups and the control group present similar patterns, that there are no fragmented nuclei that indicate cell death, however, they are noticed more elongated in the tissues from the groups treated with In (FIG. 9).

[0096] Meanwhile, the inhibition of cell proliferation caused by the compound In was demonstrated by a decrease in cells positive for the expression of proliferating cell nuclear antigen (PCNA) both in the tissues of the mice treated with In at doses of 500 mg/kg once a week as in the tissues of mice treated with the compound at a dose of 250 mg/kg 3 times a week (FIG. 10).

[0097] The quantification of PCNA confirmed that in the groups treated with compound In, at different doses, there is a decrease in cell proliferation compared to the control group 10-20% vs 54.81%±15.63) (FIG. 11).

[0098] Now, as already mentioned above, the mechanism by which In exerts an antitumor effect without generating cytotoxicity on healthy cells is through the induction of cellular senescence. This process is demonstrated by the evaluation of the activity of the β-galactosidase enzyme in cells of colon cancer cell lines. The activity of this enzyme is observed predominantly in cells of the HCT-116 line treated with In for 72 hours at a concentration of 30 μM (FIG. 12).

[0099] Therefore, with the results obtained, it can be established that compound In has an antitumor effect similar to that exerted by cytotoxic drugs currently on the market but with a marked safety advantage by not producing the same side effects as the available drugs, since this is done through the induction of a process of cellular senescence, where the tumor cell remains in a state of arrest of the cell cycle that consequently leads to cell death, without causing direct damage to healthy cells. The induction of the cellular senescence process in tumor cells can also be associated with derivatives of compound In (Ia-Ii) which are described below.

[0100] On the other hand, a study of the effect of compounds Ia-Ii on different cell lines is reflected in the following table.

TABLE-US-00003 TABLE 3 CI.sub.50 ± EE, μM Com- HCT-15 K562 PC-3 U251 pound (colon) (leukemia) (prostate) (CNS.sup.&) In 31.70 ± 1.10 38.61 ± 4.47 20.22 ± 3.44 27.34 ± 1.00 Ia  3.23 ± 1.10  4.34 ± 0.75 11.06 ± 0.66 13.89 ± 0.44 Ib 16.66 ± 1.50 16.84 ± 3.46 15.26 ± 1.70 18.88 ± 0.36 Ic >100 >100  13.93 ± 0.324 58.44 ± 4.00 Id 35.80 ± 1.98 44.43 ± 5.57 46.91 ± 2.75 26.79 ± 2.39 Ie >100 >100 >100 >100 If 40.68 ± 2.49 68.39 ± 8.30 22.69 ± 2.50 30.51 ± 3.45 Ig 28.87 ± 3.17 15.18 ± 2.35 12.56 ± 2.42 21.37 ± 1.67 Ih  9.82 ± 0.21 14.38 ± 0.78  5.69 ± 0.05  5.88 ± 1.03 Ii 10.24 ± 0.77 11.02 ± 1.05 13.00 ± 3.56 11.30 ± 1.55

[0101] While in the case of compound Im, the % of growth inhibition in different cell lines such as colon (HCT-15), breast (MCF-7), CNS glial cells (U-251), prostate (PC-3), lung (SKUL) and promyelocytic leukemia (K-562), yields the following results:

TABLE-US-00004 TABLE 4 % growth inhibition (50 μM) Compound PC-3 U251 HCT-15 K562 SKUL MCF7 In 20.2 27.3 31.70 38.6 0.0 26.6  Im 14.7 54.0 37.69 36.9 19.2   2.98

Synthetic Approach:

Synthesis of the Compound (Ia)

[0102] ##STR00030##

[0103] 100 mg of In was dissolved in 5 mL of glacial acetic acid and reacted with 0.4 mL of a 1 M solution of bromine in acetic acid. The reaction was carried out under stirring at 3° C. After 1.25 h the reaction mixture was poured into an Erlenmeyer flask containing 50 g of ice. The presence of an abundant precipitate was observed, which was washed with a 5% NaHCO.sub.3 solution and subsequently recrystallized to obtain the product Ia, (94%). P. f. 116-118° C. IR (film)ν.sub.max cm.sup.−1: 3380.48 (O—H), 2972.39-2872.9 (C—H), 1721.94 (C═O), 1463.42, 1382.14. EMIE m/z (%): 550 (M.sup.+, 0.77), 498 (15), 496 (15), 351 (5), 349 (5), 143 (100), 125 (30), 107 (31), 81 (20), 71 (32), 43 (30). RMN.sup.1H (200 MHz, CDCl.sub.3) δ ppm: 0.60 (d, J=4.3, 1 H, H-19), 0.73 (d, J=4.3, 1H, H-19′), 0.89 (s, 3H, CH.sub.3), 1.14 (s, 3H, CH.sub.3), 1.16 (s, 6H, 2CH.sub.3), 1.25 (s, 3H, CH.sub.3), 1.29 (s, 3H, CH.sub.3), 2.70 (sa, 2H, 2O—H), 3.86 (t, J=7.7, 1 H, H-24), 4.63 (m, 1H, H-16), 5.11 (dd, J=6.5, J=12.8, 1H, H-2β). RMN.sup.13C (75 MHz, CDCl.sub.3) δ ppm: 32.9 (C-1), 37.6 (C-2), 201.4 (C-3), 50.9 (C-4), 47.7 (C-5), 21.01 (C-6), 25.6 (C-7), 43.5 (C-8), 20.8 (C-9), 25.9 (C-10), 26.6 (C-11), 31.8 (C-12), 46.2 (C-13), 46.5 (C-14), 48.3 (C-15), 73.2 (C-16), 55.4 (C-17), 21.6 (C-18), 30.2 (C-19), 87.0 (C-20), 21.1 (C-21), 37.3 (C-22), 27.3 (C-23), 84.5 (C-24), 70.9 (C-25), 27.3 (C-26), 26.1 (C-27), 20.4 (C-28), 20.7 (C-29), 21.0 (C-30).

Compound Synthesis (Ib)

[0104] ##STR00031##

[0105] A solution of In (200 mg) and phenylselenium chloride (120 mg) in EtOAc (4.6 ml) was stirred at room temperature for 2 h. Then, 1 mL of water was added to the reaction mixture with stirring. The aqueous phase was separated and 2 mL of THF and 0.2 mL of 30% H.sub.2O.sub.2 were added. The resulting mixture was stirred at room temperature for 1 hr. After that time, the reaction was processed by conventional methods and an impure solid was obtained. The solid was purified by column chromatography to obtain 140 mg (70%) of the desired product as a crystalline solid of melting point 196-198° C. IR (Film)ν.sub.max cm.sup.−1: 3380.47 (O—H), 2967.73, 2873.35, 1667.21 (C═O), 1463.32, 1379.72. EM-IE m/z (%): 470 (M.sup.+, 1), 452 (12), 434 (7), 411 (2), 143 (100), 125 (25), 107 (10), 59 (10). RMN.sup.1H (200 MHz, CDCl.sub.3) δ ppm: 0.81 (d, J=4.6, 1H, H-19), 0.90 (s, 3H, CH.sub.3), 0.97 (s, 3H, CH.sub.3), 1.11 (s, 3H, CH.sub.3), 1.15 (s, 3H, CH.sub.3), 1.26 (s, 3H, CH.sub.3), 1.30 (s, 3H, CH.sub.3), 1.42 (s, 3H, CH.sub.3), 3.86 (t, J=7.6, 1 H, H-24), 4.61 (m, 1 H, H-16), 5.94 (d, J=10.0, 1 H, H-2), 6.77 (d, J=10.0, 1H, H-1). RMN.sup.13C (75.4 MHz, CDCl.sub.3) δ ppm: 153.7 (C-1), 126.7 (C-2), 205.1(C-3),47.0 15 (C-4), 44.9 (C-5), 19.7 (C-6), 27.6 (C-7), 44.5 (C-8), 24.1 (C-9), 29.9 (C-10), 24.0 (C-11), 32.8 (C-12), 46.1 (C-13), 46.3 (C-14), 47.6 (C-15), 73.2 (C-16), 55.5 (C-17), 21.5 (C-18), 30.8 (C-19), 87.2 (C-20), 25.5 (C-21), 37.5 (C-22), 23.8 (C-23), 84.5 (C-24), 70.9 (C-25), 27.3 (C-26), 26.1 (C-27), 19.8 (C-28), 20.1 (C-29), 19.1 (C-30).

Synthesis of the Compound (Ic)

[0106] ##STR00032##

[0107] A mixture of 25.5 mg of derivative Ib, 21 mg of sodium acetate and 2 mL of acetic anhydride was heated at reflux temperature for one hour. Subsequently, the mixture was poured into an Erlenmeyer flask containing 5 g of ice and stirred for 3 minutes. The contents of the flask were extracted with AcOEt (3×). The organic phase was dried and concentrated under reduced pressure to obtain a semi-solid. Said product was recrystallized (hexane/AcOEt) to obtain 27.4 mg of acetate Ic (99%) with a melting point of 158-160° C. IR (Film) cm.sup.−1: 2971.64, 2937.91, 2873.33, 1734.33 (C═O), 1668.83 (C═O), 1460.83, 1367.44, 1241.0, 755.46. RMN.sup.1H (300 MHz, CDCl.sub.3) δ ppm: 0.95 (s, 3H, CH.sub.3), 1.10 (s, 3H, CH.sub.3), 1.22 (s, 3H, CH.sub.3), 1.25 (s, 6H, 2CH.sub.3), 1.47 (s, 3H, CH.sub.3), 1.55 (s, 3H, CH.sub.3), 1.99 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3), 2.54 (d, J=8.49, 1H), 3.74 (t, J=7.9, 1 H, H-24), 5.40 (m, 1H, H-16), 5.95 (d, J=10, 1H, H-2), 6.77 (d, J=10, 1H, H-1). RMN.sup.13C (75.4 MHz, CDCl.sub.3) δ ppm: 153.5 (C-1), 126.9 (C-2), 205.1 (C-3), 47.0 (C-4), 44.6 (C-5), 19.6 (C-6), 27.8 (C-7), 44.2 (C-8), 24.1 (C-9), 30.1(C-10),23.9 (C-11), 31.8 (C-12), 46.0 (C-13), 46.9 (C-14), 44.6 (C-15), 74.8 (C-16), 56.8 (C-17), 19.4 (C-18), 29.7 (C-19), 85.1(C-20), 22.7 (C-21), 35.1(C-22),25.7 (C-23), 81.7 (C-24), 82.3 m(C-25), 28.6 (C-26), 22.9 (C-27), 19.1 (C-28), 19.1 (C-29), 19.1 (C-30), 21.6 and 22.5 (methyl of acetate groups), 170.4 and 170.3 (carbonyl of acetate groups).

Synthesis of Compound (Id)

[0108] ##STR00033##

[0109] 301 mg of compound In in 4.5 mL of pyridine was reacted with 99 mg of NH.sub.2O.HCl under stirring at reflux temperature for one hour. Subsequently, the reaction mixture was poured into a flask containing 100 g of ice and extracted with AcOEt (3×). The organic phase was repeatedly washed with a 10% HCl solution followed by water and subsequently dried and concentrated under reduced pressure. The residue obtained after evaporation was purified by means of column chromatography, to obtain 277 mg of the oxime Id (90%). P. f. 200-205° C. IR (KBr)ν.sub.max cm.sup.−1: 3379.8, 2968.9, 2870.9, 1638.5, 1460, 1380.1, 1103.9. EM-IE m/z (%): 487 (M.sup.+, 13), 470 (9), 452 (9), 286 (8), 143 (100), 125 (21), 59 (10). RMN.sup.1H (300 MHz, CDCl.sub.3) δ ppm: 0.54 (d, J=4.2, 1H, H-19), 0.74 (d, J=4.2, 1H, H-19′), 0.88 (s, 3H, CH.sub.3), 1.1 (s, 6H, 2CH.sub.3), 1.3 (s, 3H, CH.sub.3), 1.3 (s, 3H, CH.sub.3), 1.4 (s, 3H, CH.sub.3), 3.38 (dq, 1H), 3.9 (t, J=1.8, J=7.8, 1 H, H-24), 4.6 (m, 1 H, H-16). RMN.sup.13C (75.4 MHz) δ ppm: 32.7 (C-1), 20.0 (C-2), 167.1 (C-3), 43.4 (C-4), 48.8 (C-5), 21.7 (C-6), 26.1 (C-7), 47.6 (C-8), 20.9 (C-9), 27.3 (C-10), 26.1 (C-11), 33.1 (C-12), 46.3 (C-13), 46.6 (C-14), 48.7 (C-15), 73.4 (C-16), 55.6 (C-17), 21.2 (C-18), 30 (C-19), 87.2 (C-20), 25.7 (C-21), 37.3 (C-22), 23.7

[0110] (C-23), 84.5 (C-24), 70.8 (C-25), 27.3 (C-26), 26.3 (C-27), 20.3 (C-28), 20.9 (C-29), 20.9 (C-30). A 0.40×0.32×0.26 mm crystal was used to carry out an X-ray diffraction analysis. Said analysis was performed in a Siemens P4 diffractometer at temperature of 293 K. The compound crystallographic data are shown in Table 5 The experimental conditions and the results of the X-ray diffraction analysis were deposited in the CCDC under the code CCDC 254670.

TABLE-US-00005 TABLE 5 Id crystallographic information Empirical formula Formula weight 487.7 Crystal system Orthorhombic Space group P 2.sub.1 2.sub.1 2.sub.1 Unit cell dimensions A 27.458 (2) α 90° B 7.9900 (10) β 90° C 13.0030 (10) γ 90° Volume [Å.sup.3] 2852.9 (2) Z 4 Density (calculated) [g/cm.sup.3] 1.135 Absorption coefficient [mm.sup.−1] 0.577 F (000) 1072 θ Range for data collection 3.0 to 110.0 [°] Index ranges 0 ≤ h ≤ 29 0 ≤ k ≤ 8 0 ≤ l ≤ 13 Completeness to θ = 25.00° [%] Data/ restraints/ parameters Goodness-of-fit on F.sup.2 1.51 Final R indices [/ > 2σ(/)] R.sub.1 = 5.71 WR.sub.2 = 7.32 R indices (all data) R.sub.1 = 6.16 WR.sub.2 = 7.32 Largest diff. Peak and hole 0.39, −0.26 [e. Å.sup.3]

Synthesis of Compound (Ie)

[0111] ##STR00034##

[0112] A solution of 100 mg of In in 4 mL of acetic acid was treated at 0-5° C. with chromium trioxide (100 mg) in 0.3 mL of water. After 1 hour, the mixture was left at room temperature and subsequently extracted with AcOEt (3×). The organic phase was conventionally processed to obtain the desired product Ie (40%). P. f. 138-140° C. IR (Film)ν.sub.max cm.sup.−1: 2972.73, 2876.01, 1768.64 (C═O), 1737.46 (C═O), 1703.84 (C═O), 1462.38, 1385.8. EM-IE m/z (%): 426 (M.sup.+, 100), 411 (23), 313 (35), 288 (34), 270 (15), 99 (42), 43 (27). RMN.sup.1H (200 MHz, CDCl.sub.3) δ ppm: 0.68m (d, J=4.5, 1H, H-19), 0.88 (d, J=4.5, 1H, H-19′), 1.07 (s, 3H, CH.sub.3), 1.12 (s, 3H, CH.sub.3), 1.13 (s, 3H, CH.sub.3), 1.34 (s, 3H, CH.sub.3), 1.49 (s, 3H, CH.sub.3). RMN.sup.13C (75.5 MHz) δ ppm: 33.1(C-1),37.3 (C-2), 215.1 (C-3), 50.1 (C-4), 48.5 (C-5), 21.2 (C-6), 26.2 (C-7), 47.0 (C-8), 20.2 (C-9), 26.5 (C-10), 26.1 (C-11), 33.4 (C-12), 45.7 (C-13), 46.2 (C-14), 50.6 (C-15), 215.8 (C-16), 65.1 (C-17), 28.3 (C-18), 30.1 (C-19), 85.5 (C-20), 22.1 (C-21), 42.4 (C-22), 27.8 (C-23), 177.2 (C-24), 19.7 (C-28), 20.7 (C-29), 19.9 (C-30).

Synthesis of Compound (If)

[0113] ##STR00035##

[0114] A mixture of 200 mg of In, 60.2 mg of sodium acetate and 5 mL of acetic anhydride was heated at reflux temperature for 18 hours. Subsequently, the mixture was poured into an Erlenmeyer flask containing 50 g of ice and stirred for 15 minutes. Contents of the flask were extracted with AcOEt (3×). The organic phase was dried and concentrated under reduced pressure to obtain a semi-solid. Said product was recrystallized (hexane/AcOEt) to obtain the corresponding acetate If (99%) with a melting point of 200-206° C. IR (Film) cm.sup.−1 2971.49, 2943.41, 2872.45, 1734.68 (C═O), 1706.33 (C═O), 1459.92, 1368.46, 1241.62. EM-IE m/z, (%): 556 (M.sup.+), 496 (24), 436 (27), 395 (28), 185 (52), 143 (42), 125 (100), 43 (57). RMN.sup.1H (300 MHz) CDCl.sub.3 δ ppm: 0.53 (d, J=4.2, 1 H, H-19), 0.83 (d, J=4.2, 1H, H-19′), 0.96 (s, 3H, CH.sub.3-21), 1.05 (s, 3H, CH.sub.3-29), 1.09 (s, 3H, CH.sub.3-18), 1.23 (s, 3H, CH.sub.3-26), 1.40 (s, 3H, CH.sub.3-28), 1.47 (s, 3H, CH.sub.3-27), 1.55 (s, 3H, CH.sub.3-21), 2.00 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 3.74 (t, J=7.7, 1H, H-24), 5.39 (m, 1H, H-16). RMN.sup.13C (300 MHz, CDCl.sub.3): 33.2 (C-1), 37.3 (C-2), 214.9 (C-3), 50.1 (C-4), 47.7 (C-5), 21.2 (C-6), 25.9 (C-7), 48.4 (C-8), 20.7

[0115] (C-9), 26.5 (C-10), 26.8 (C-11), 32.1(C-12), 46.6 (C-13), 46.9 (C-14), 45.5 (C-15), 75.04 (C-16), 57.16 (C-17) 20.7 (C-18), 30.3 (C-19), 85.0 (C-20), 22.6 (C-21), 35.2 (C-22), 25.7 (C-23), 81.6 (C-24), 82.3 (C-25), 28.5 (C-26), 22.9 (C-27), 19.9 (C-28), 22.2 (C-29), 20.07 (C-30), 21.5, 22.4, 170.13, 170.29.

Synthesis of Compound (Ig)

[0116] ##STR00036##

[0117] 200 mg of the In diacetate in 6.5 mL of pyridine was reacted with 95 mg of NH.sub.2OH.HCl under stirring at reflux temperature for 1.5 hours. Subsequently, the reaction mixture was poured into a flask containing 50 g of ice and extracted with AcOEt (3×). The organic phase was washed 3 times with a 10% HCl solution and then with water. Recrystallization of the organic phase allowed the Ig oxime purification (90%) from p. f. 140-143° C. IR (film)ν.sub.max cm.sup.−1: 3318.57, 2972.66, 2942.16, 2872.86, 1734.27, 1456.29, 1368.98, 1242.09. EM-IE m/z (%): 571 (Mt), 511 (12), 434 (15), 185 (67), 143 (49), 125 (100), 43 (60). RMN.sup.1H (300 MHz, CDCl.sub.3) δ ppm: 0.44 (d, J=4.2, 1 H, H-19), 0.72 (d, J=4.2, 1 H, H-19′), 0.94 (s, 3H, CH.sub.3), 1.08 (s, 3H, CH.sub.3), 1.14 (s, 3H, CH.sub.3), 1.22 (s, 3H, CH.sub.3), 1.38 (s, 3H, CH.sub.3), 1.47 (s, 3H, CH.sub.3), 1.54 (s, 3H, CH.sub.3), 2.00 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 2.52 (d, J=8.4, 1H, H-17), 3.36 (dc, 1H), 3.73 (t, J=7.5, 1H, H-24), 5.38 (m, 1H, H-16), 6.62 (sa, 1 H, O—H). RMN.sup.13C (75.5 MHz) δ ppm: 32.6 (C-1), 20.0 (C-2), 166.9 (C-3), 42.7 (C-4), 48.7 (C-5), 21.0 (C-6), 25.8 (C-7), 47.5 (C-8), 20.4 (C-9), 26.8 (C-10), 26.5 (C-11), 32.0 (C-12), 46.6 (C-13), 46.9 (C-14), 45.4 (C-15), 75.1 (C-16), 57.0 (C-17), 21.6 (C-18), 30.2 (C-19), 85.1 (C-20), 22.6 (C-21), 35.0 (C-22), 25.6 (C-23), 81.7 (C-24), 82.3 (C-25), 28.6 (C-26), 22.9 (C-27), 19.8 (C-28), 23.6 (C-29), 20.0 (C-30), 21.5 and 22.5 (carbons of the methyls from acetate groups), 170.4 and 170.3 (carbons corresponding to carbonyls in acetate groups).

Synthesis of Compound (Ih)

[0118] ##STR00037##

[0119] To a solution of 200 mg of In in 6 mL of dry pyridine, contained in inert atmosphere, 1 mL of ethyl formate (freshly distilled) were added, as well as 0.8 mL of a sodium solution in absolute MeOH (0.44 g/6 ml). The reaction was kept stirring at room temperature overnight. The appearance of an ocher color and/or the formation of an insoluble precipitate were considered as evidence of the reaction. After the relevant time, the reaction mixture was placed in a cold solution of 3 mL of acetic acid in 27 mL of water. As a result of the above action, the appearance of a precipitate was observed, which was extracted with methylene chloride. The aqueous phase was discarded. The organic phase was washed with water and extracted with a 2% potassium hydroxide solution. The basic extract was washed with ether, acidified with glacial acetic acid and finally extracted with methylene chloride. The final methylene chloride phase was dried and concentrated under reduced pressure to obtain an impure semi-solid product. Said product was purified by preparative layer chromatography to obtain the formylated derivative Ih (89%). IR (KBr)ν.sub.max cm.sup.−1: 3403.7, 2974.0, 2941.9, 2874.8, 1635.1, 1586.9, 1464.6, 1355.9. EM-IE m/z (%): 500 (Mt, 2), 482 (6), 441 (M.sup.+-59, 6), 143 (100), 125 (22), 107 (13), 85 (12), 71 (12), 59 (8), 43 (13). RMN.sup.1H (200 MHz, CDCl.sub.3) δ ppm: 0.49 (d, J=4.4, 1 H, H-19), 0.70 (d, J=4.4, 1 H, H-19′), 0.92 (s, 3H, CH.sub.3), 1.1 (s, 3H, CH.sub.3), 1.15 (s, 3H, CH.sub.3), 1.22 (s, 3H, CH.sub.3), 1.26 (s, 3H, CH.sub.3), 1.3 (s, 3H, CH.sub.3), 1.46 (s, 3H, CH.sub.3), 2.60 (d, J=15, 1H), 3.46 (sa, 2H, 2 O—H), 3.86 (t, J=7.4, 1H, H-24), 4.63 (m, 1H, H-16), 8.7 (s, 1H, H-31), 14.8 (sa, 1H, O—H). RMN.sup.13C (50 MHz) δ ppm: 33.1 (C-1), 106.6 (C-2), 190.5 (C-3), 42.7 (C-4), 48.5 (C-5), 21.4 (C-6), 26.1 (C-7), 44.7 (C-8), 19.3 (C-9), 29.7 (C-10), 25.5 (C-11), 31.8 (C-12), 46.3 (C-13), 46.6 (C-14), 48.6 (C-15), 73.5 (C-16), 55.6 (C-17), 21.2 (C-18), 30.2 (C-19), 87.2 (C-20), 25.2 (C-21), 37.3 (C-22), 23.7 (C-23), 84.5 (C-24), 70.9

[0120] (C-25), 27.3 (C-26), 26.1(C-27), 20.6 (C-28), 24.4 (C-29), 21.6 (C-30), 188.9 (C-31).

Synthesis of Compound (Ii)

[0121] ##STR00038##

[0122] A solution of 60 mg of the formylated derivative Ih in 5 mL of glacial acetic acid, under stirring, was reacted for two hours with 30 mg of hydroxylamine hydrochloride at reflux temperature. The reaction mixture was then poured into an Erlenmeyer flask containing 50 g of ice and extracted with AcOEt. The organic phase was washed with a 5% sodium bicarbonate solution (3×) and with water. The aqueous phases were discarded. The organic phase was dried and concentrated under reduced pressure to obtain an impure semi-solid. Said semisolid was purified by means of column chromatography to obtain the isoxazole Ii (80%). P. f. 125-129° C. IR (Film)ν.sub.max cm.sup.−1: 3377.57, 2970.67, 2940.45, 2875.63, 1640, 1564.63, 1463, 1379.57. EM-IE m/z (%): 497 (Mt, 4), 479 (8), 439 (10), 420 (10), 337 (10), 296 (9), 143 (100), 125 (22), 107 (15), 43 (15). RMN.sup.1H (300 MHz, CDCl.sub.3) δ ppm: 0.47 (d, J=4.5, 1 H, H-19), 0.74 (d, J=4.5, 1 H, H-19′), 0.94(s, 3H, CH.sub.3), 1.15 (s, 15 3H, CH.sub.3), 1.21 (s, 3H, CH.sub.3), 1.26 (s, 3H, CH.sub.3), 1.30 (s, 3H, CH.sub.3), 1.36 (s, 3H, CH.sub.3), 1.46 (s, 3H, CH.sub.3), 2.66 (d, J=15.6, 1H), 3.87 (t, J=7.2, 1H, H-24), 4.63 (m, 1H, H-16), 7.98 (s, 1H). RMN.sup.13C (75.4 MHz) δ ppm: 28.3 (C-1), 109.9 (C-2), 174.8 (C-3), 37.4 (C-4), 48.5 (C-5), 20.8 (C-6), 26.4 (C-7), 46.3 (C-8), 19.7 (C-9), 24.8 (C-10), 25.3 (C-11), 33.2 (C-12), 46.6 (C-13), 46.8 (C-14), 48.8 (C-15), 73.4 (C-16), 55.7 (C-17), 21.2 (C-18), 30.4 (C-19), 87.2 (C-20), 25.7 (C-21), 37.3 (C-22), 23.8 (C-23), 84.5 (C-24), 70.9 (C-25), 27.3 (C-26), 26.1 (C-27), 20.6 (C-28), 25.5 (C-29), 22.2 (C-30), 149.4 (C-31).

Synthesis of Compound (Ij)

[0123] ##STR00039##

[0124] To 100 mg of In oxime dissolved in CHCl.sub.3 0.5 mL of trifluoroacetic anhydride were added slowly at 0° C. Once the addition was complete, the reaction mixture was kept under constant stirring at 25° C. for 18 min. The reaction mixture was evaporated under reduced pressure. From this reaction, a product identified as In 16-trifluoroaxetoxy-lactam was obtained. A solution of potassium carbonate in methanol was added to said product and it was kept stirring for 15 min. at room temperature, subsequently the solution was filtered, and the solvent was evaporated under reduced pressure. The reaction product was purified by column chromatography with polarity of 2:1 Hex:AcOEt, obtaining 38 mg of Ij. IR (CHCl.sub.3): ν.sub.max cm.sup.−1: 3612, 3395, 2963, 2871, 1644 cm.sup.−1. EIMS m/z (%): 487 (Mt, 29.27), 429 (M.sup.+−58, 5.4), 428 (11.5), 413 (64.86), 58 (100). HRMS: found m/z 488.3726, [M+H].sup.+; C.sub.30H.sub.50NO.sub.4 required 488.3739. .sup.1H NMR (CDCl.sub.3, 300 MHz): d 0.61 (1 H, d, J=6 Hz, H-190), 0.68 (1 H, d, J=6 Hz, H-19), 0.86 (3H, s, H-29130)*, 1.11 (3H, s, H-18), 1.21 (3H, s, H-21), 1.26 (3H, s, H-27), 1.30 (3H, s, H-26), 1.33 (3H, s, H-29/30)*, 3.1 (1 H, m, H-2), 3.83 (1 H, t, J=7 Hz, H-24), 4.60 (1 H, m, H-16), 7.5 (1 H, s, NH). .sup.13C NMR (75 MHz, CDCl.sub.3): d 20.2 (C-18), 20.3 (C-28), 21.0 (C-6), 21.0 (C-9), 22.1 (C-29), 23.9 (C-23), 24.4 (C-30), 25.3 (C-21), 26.5 (C-7), 26.5 (C-10), 26.7 (C-11), 27.0 (C-26), 27.5 (C-27), 29.5 (C-1), 29.9 (C-2), 30.8 (C-19), 33.0 (C-12), 36.2 (C-22), 45.5 (C-14), 45.9 (C-13), 48.2 (C-5), 48.5 (C-8), 50.08 (C-15), 55.5 (C-4), 56.3 (C-17), 69.4 (C-25), 71.5 (C-16), 83.4 (C-24), 85.6 (C-20), 175.6 (C-3).

Synthesis of Compound (Ik)

[0125] ##STR00040##

[0126] 200 mg of In were treated with 170 mg of m-chloroperoxybenzoic acid for 3 hours, to obtain 180 mg of a white solid identified as Ik.

Synthesis of the Compound (I1)

[0127] ##STR00041##

[0128] At 100 mg of In, was treated with 80 mg sodium borohydride (NaBH.sub.4), obtaining a white solid identified as Il, with a melting point of 125-128° C. and a molecular weight of 474 g/mol.

Synthesis of the Compound (Im)

[0129] ##STR00042##

[0130] To 0.15 mmol of hexadecanoyl chloride, previously obtained and in inert atmosphere, 0.22 mmol of In was added, dissolved in 4 mL of dry dichloromethane. The reaction was allowed to stir for 30 minutes. After this time, 15 mL of ethyl acetate was added to the reaction mixture, and it was placed in a separatory funnel. The organic phase was washed three times with distilled water and three times with a saturated solution of NaHCO.sub.3, dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The reaction mixture was subject to silica packed and open column chromatography and. AS elution mixture was used the hexane-ethyl acetate mixture (7:3). From fractions 3-8, was obtained 50 mg of a white amorphous solid with p.f. 246° C. with a yield of 70%. IR (CHCl.sub.3 solution) cm.sup.−1: 2920.70, 2851.35 (CH), 1732.60 (C═O), 1707.92 (C═O), 1463.77, 1379.20, 1270.56, 1153.83. EM-FAB.sup.+ m/z (%): 948 (1), 933 (5), 437 (77), 381 (34), 125 (100), 43 (93). EM-IE (70 eV), m/z, (%): 692 [M.sup.+-palmitic acid] (6), 436 (24), 381 (22), 256 (22), 125 (100), 43 (54). RMN.sup.1H (300 MHz, CDCl.sub.3) δ ppm: 5.44 (1 H, ddd J=5.4, J=2.7 y J=12.3 Hz, H-16), 3.66 (1 H, dd, J=6.9 Hz and J=8.51 Hz, H-24), 1.38 (3H, s), 1.25 (m, for two groups CH.sub.3 and several CH.sub.2 of the palmitate residue), 1.14 (3H, s), 1.09 (3H, s), 1.04 (3H, s), 0.87 (3H, s), 0.81 (1H, d, J=4.3 Hz, H-19a), 0.59 (1 H, d, J=4.3 Hz, H-19b). RMN.sup.13C (75 MHz, CDCl.sub.3) δ ppm: 215.68 (C3), 175.46 and 172.91 (carbonyls of palmitate esters), 81.91 (C24), 81.89 (C25), 74.45 (C16), 56.35 (C17), 49.58 (C4), 47.84 (C8), 47.15 (C5), 46.15 (C13), 46.02 (C14), 44.91 (C15), 35.97 (C2), 36.80 (C22), 33.64 (C1), 32.64 (C12), 29.56 (C19), 29.54 (methylenes from the palmitate residue), 29.01 (C20),26.30 (C11), 25.88 (C7), 27.90 (C26), 27.36 (C27), 24.40 (C23), 21.61 (C21), 21.61 (C18), 20.19 (C29), 19.63 (C30), 14.07 (palmitate methyls).

Synthesis of the Compound (I′a)

[0131] ##STR00043##

[0132] A solution of 100 mg of Ie in EtOH was reflux maintained with 289 mg of potassium hydroxide for 40 min.

[0133] After the reaction was neutralized and 70 mg of 3,16-dioxo-25-nor-cycloartan-17-en-24-oic acid [I′a] was obtained. P.f. 182-184° C. IR (KBr): v.sub.max cm.sup.−1: 3327, 1741, 1703, 1615 cm.sup.−1. EIMS m/z (%): 426 (M.sup.+, 39.69), 411 (100), 143 (4.5), 125 (3). HRMS: found m/z 427.2864, [M+H].sup.+; C.sub.27H.sub.39O.sub.4 required 427.2848. .sup.1H NMR (300 MHz, CDCl.sub.3): δ ppm: 0.64 (d, J=4.2 Hz, 1 H, H-19), 0.86 (d, J=4.8 Hz, 1 H, H-190), 0.99 (s, 3H, CH.sub.3), 1.06 (s, 3H, CH.sub.3), 1.11 (s, 3H, CH.sub.3), 1.35 (s, 3H, CH.sub.3), 1.92 (s, 3H, CH.sub.3). .sup.13C NMR (75 MHz, CDCl.sub.3): δ ppm: 20.2 (C-18), 20.7 (C-28), 20.8 (C-30), 20.9 (C-9), 21.2 (C-29), 22.1 (C-21), 22.6 (C-6), 26.0 (C-7), 26.3 (C-11), 26.3 (C-10), 29.2 (C-23), 30.6 (C-19), 30.8 (C-22), 32.6 (C-12), 33.1 (C-1), 37.2 (C-2), 42.3 (C-13), 45.7 (C-14), 47.9 (C-8), 48.2 (C-5), 50.20 (C-4), 51.0 (C-15), 141.7 (C-17), 149.5 (C-20), 177.8 (C-24), 207.3 (C-16), 216.0 (C-3).