WO2011110723A1

WO2011110723A1 - N-acetylhexosamine sulfur derivatives and use of same as cell tumor inhibitors

Info

Publication number: WO2011110723A1
Application number: PCT/ES2011/070169
Authority: WO
Inventors: Alfonso FERNÁNDEZ-MAYORALAS ÁLVAREZ; Manuel Nieto Sampedro; Josefina Casas Brugulat; Isabel GARCÍA ÁLVAREZ; Lorenzo ROMERO RAMÍREZ
Original assignee: Consejo Superior De Investigaciones Científicas (Csic); Hospital Nacional De Paraplejicos De Toledo
Priority date: 2010-03-12
Filing date: 2011-03-11
Publication date: 2011-09-15
Also published as: ES2365314B1; ES2365314A1

Abstract

The present invention relates to a series of thioglycosides and to the oxidized sulfoxide and sulfone derivatives thereof, which provide better solubility in aqueous media, and to the use thereof as anti-tumor agents, principally against glioma and pulmonary adenocarcinoma cell lines. The novel thioderivatives are resistant to hydrolysis catalyzed by N-acetylhexosaminadases and reduce the ganglioside content of tumor cells. The invention likewise relates to a process for obtaining said compounds and to the use thereof in the manufacture of a medication for treating tumors.

Description

Sulfur derivatives of N-acetylhexosamines and their use as inhibitors of tumor cell division.

The present invention relates to thioglycosides, glycosylsulfoxides and glycosylsulfones derived from N-acetylhexosamines, their method of obtaining and their use as inhibitors of tumor cell division.

STATE OF THE PREVIOUS TECHNIQUE Glioblastoma multiforme is the most common primary brain tumor in adults and at the same time it is the most deadly. In the United States, only half of the patients receiving standard treatment survive one year after diagnosis. Less than one in ten survives more than five years. It usually occurs in people over forty, with a maximum incidence between 50 and 55 years and is more common among men. In adults, there are about 17,000 new cases of brain tumors each year (in addition, other types of cancer can cause brain metastases), which means about 14,000 deaths. The brain tumor is the leading cause of cancer death in children and children under 20 years.

Treatment depends on the location and grade of the tumor; Surgery is applied when the tumor is accessible and there is no danger of damaging vital structures. Radiation therapy is used to stop the growth of the tumor or to decrease its size and chemotherapy destroys the remaining tumor cells after surgery and radiation. The most common chemotherapy (BCNU, CCNU) does not seem to have a significant effect, although in some cases it has been possible to prolong survival for a few months. Gangliosides are a type of glycolipids formed by a residue of the ceramide sphingolipid and an oligosaccharide chain. Gangliosides have as their structural characteristic the presence of one or more acid residues sialic in the oligosaccharide chain. It is known that dramatic changes in the composition of certain gangiosides and their metabolism are associated with oncogenic transformation. Thus, there are several studies that suggest that tumor-associated gangiosides play an important role in its development and it has been observed that reducing the content of gangiosides reduces the ability of cells to form tumors (Birkle et al., Biochemie 2003, 85 , 455-463). Spanish patent 200000982 describes a process for obtaining glycosides derived from the N-acetyl-D-glucosamine monosaccharide and the investigation of its inhibitory and cytotoxic activity in gliomas.

Taking into account the severity of this type of tumors and the poor effectiveness of treatments, the development of new molecules capable of stopping the proliferation of gliomas is of great interest for the treatment of this disease.

DESCRIPTION OF THE INVENTION

The present invention describes a series of thioglycosides and their oxidized sulfoxide and sulfone derivatives, which confer greater solubility in aqueous media, and their use as antitumor against cell lines of glioma and lung adenocarcinoma. The new thioderivatives are resistant to hydrolysis catalyzed by N-acetylhexosaminidases enzymes and reduce the content of gangiosides in tumor cells.

In a first aspect, the present invention relates to a compound of formula (I)

Formula (I) where

X is selected from S, S (O) or S (0) 2

R-i, R2 and R3 are independently selected from H or C1-C6 acyl,

R4 is a C16-C24 alkyl or a C8-C24 alkenyl,

or any of its isomers, salts or solvates. The term "acyl" refers, in the present invention, to radicals of linear or branched carboxylic acids, having 1 to 6 carbon atoms, and which are attached to the rest of the molecule by an ester bond.

The term "alkenyl" refers to hydrocarbon chain radicals of 1 to 25 carbon atoms, preferably 8 to 24, containing one or more double carbon-carbon bonds, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1, 3-butadienyl etc. Alkenyl radicals may be optionally substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The compounds of the present invention represented by the formula (I) may include isomers, depending on the presence of multiple bonds (by example, Z, E), including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemic, etc., even their optically isomers. assets or mixtures in different proportions thereof. The individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques.

In a preferred embodiment, Ri to R ₃ are H. In another preferred embodiment, X is S. In another preferred embodiment, X is S (O). In another preferred embodiment, X is S (0) 2.

In another preferred embodiment, R ₄ is an alkenyl

In an even more preferred embodiment, R ₄ is an octadec-9-enyl (oleyl) group.

In a second aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as described above.

In a preferred embodiment, said composition comprises another active ingredient.

Some examples of the pharmaceutical compositions are solids (tablets, pills, capsules, granulated solid, etc.) or liquids (solutions, suspensions or emulsions) prepared for oral, nasal, topical or parenteral administration. In a preferred embodiment of the present invention, the pharmaceutical compositions are suitable for oral administration, in solid or liquid form. Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.

Compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion. The tablets can be coated following methods known in the pharmaceutical industry.

The pharmaceutical compositions can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dose. Suitable excipients, such as pH buffering agents or surfactants, can be used.

The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts. The administration of the compounds or compositions of the present invention can be performed by any suitable method, such as intravenous infusion and oral, intraperitoneal or intravenous routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.

The amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease at Treat and patient weight. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.

The compounds and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.

In a third aspect, the present invention relates to the use of a compound of formula (I) for the manufacture of a medicament.

In a fourth aspect, the present invention relates to the use of a compound of formula (I) for the manufacture of a medicament for the treatment of tumors. In a preferred embodiment, the tumor is selected from brain, melanoma, lymphoma, lung or uterine adenocarcinoma, from brain or lung adenocarcinoma.

Experiments have been performed in vivo, using immunosuppressed mice (nude mice) as experimental animals carrying a tumor. Animals (n = 6 for each treatment) were treated with compound 6a at two different doses (1 and 10 mg / kg / day) by subcutaneous injection for a period of fourteen days. For comparative purposes, another group of animals was treated with glycoside 9 (see Figure 2), analogous to compound 6α but having an oxygen atom instead of the sulfur atom. Tumor volume analysis shows that compound 6a is able to reduce tumor volume with respect to control in a dose-dependent manner. However, glycoside 9 does not produce an apparent reduction. of the tumor volume with respect to the control, which demonstrates the need to have sulfur derivatives stable to the hydrolysis catalyzed by giicosidases enzymes, as it is the case of 6 a, so that they present efficacy in the treatment of tumors.

In a fifth aspect the present invention relates to the use of a compound of formula (I) as a reagent in biological assays.

In a sixth aspect, the present invention relates to a process for obtaining a compound of formula (I) comprising the following steps: a. Reaction of a compound of formula (II)

Formula (II) with a chloride of formula CI-R5 where Rs is a C1-C6 acyl.

b. Reaction of the compound obtained in the previous stage with thiourea.

C. Reaction of the compound obtained in the previous step with a compound of formula (III) OR

SOR ₆

OR

Formula (III)

where Re is a C16-C24 alkyl or a Cs-C24 alkenyl. In a preferred embodiment, an oxidation step is also performed.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES

Fig. 1. Amount of lactosylceramide (LacCer; black bars) and gangliosides GM3 (white bars) and GM2 (striped bars) present in extracts of A549 cells treated with thioglycosides 6a and 6β at concentration 15 μΜ for 48 h, relative to control (untreated A549 cells).

Fig. 2. Tumor growth curves in mice treated (n = 6 per group) with the control solution (PBS + 5 mg / ml bovine serum albumin free of fatty acids + 5% DMSO), with the compound 9 at the dose of 10 mg / kg / day, and with compound 6 q at the doses of 1 and 10 mg / kg / day.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the compounds of the present invention. General procedure for preparing the compounds of formula (1)

Thioglycosides, glycosylsulfoxides and glycosyl sulfones of general formula (I) have been prepared from the following N-acetyl-D-glucosamine monosaccharide:

X = S, S (O), S (0) ₂

where the R moiety is a hydrocarbon chain, with or without unsaturations, linear or branched, preferably with an oleyl moiety. The configuration of the C-1 anomeric carbon in the glucosamine residue may be alpha (a) or beta.

(β)

The preparation of the compounds of general formula (I) begins by the reaction of N-acetyl-glucosamine with acetyl chloride to give the corresponding chloride. By reacting this chloride with thiourea in acetone under reflux, the intermediate 2,3,4,6-tetra-0-acetyl-D-glucosamine isothiouronium is obtained. Alkylation of this compound with a hydrocarbon chain mesylate, previously synthesized by treatment of the corresponding alcohol with mesyl chloride, gives thioglycosides as a mixture of anomers. The anomers are fractionated by silica gel column chromatography, obtaining the alpha α and beta β anomer, which are subjected to deacetylation to obtain the thioglycosides object of the present invention. The controlled oxidation of these thioglycosides with hydrogen peroxide catalyzed by zirconium tetrachloride gives rise to the glycosylsulfoxides and glycosylsulfones object of this invention. Preparation of 2,3,4,6-tetra-Q-acetyl-BD-glucosamine isothiouronium hydrochloride (3)

A mixture of the following compound 1:

Compound 2

(6.25 g, 0.028 mol) and acetyl chloride (18.0 mi, 0.253 mol) under argon was stirred at room temperature for 24 h. After diluting with CH2CI2 (40 mL) the organic phase was washed with cold H ₂ 0 (20 mL x 2) and with a saturated solution of NaHC0 ₃ . The organic phase was dried over Na ₂ S0 ₄ and the volume of solvent was reduced by evaporation under reduced pressure to 25 mL. To this solution was added 75 mL of Et ₂ 0 and allowed to crystallize at room temperature for 12 h, to give the following compound 2 as a white solid (6.8g, 66%): A solution of thiourea (0.44 g, 5.84 mmol) and this compound (2 g, 5.32 mmol) in anhydrous acetone (28 ml_) was heated at reflux (56 ° C) under argon atmosphere for 30 min. Compound 3:

precipitated during the reaction as a white solid. The reaction mixture was cooled to 0 ° C and the supernatant was removed. It was washed with acetone at 0 ° C and recrystallized from acetone-methanol and said compound 3 was obtained as a white solid (1.55 g, 65%). Compound 3: (400 MHz, D ₂ 0, COZY): δ 1.99 (s, 3H, NAc), 2.07, 2.09, 2.12 (3 s, 9H, OAc), 4.19 (ddd, 1 H, J = 10.1, J = 3.9, J = 1.8 Hz, H-5), 4.27 (dd, 1 H, J = 12.5, J = 1.7 Hz, H-6a), 4.33 (t, 1 H, J = 10.4 Hz, H-2) , 4.39 (dd, 1 H, J = 12.8, J = 4.4 Hz, H-6b), 5.17 (dd, 1 H, J = 9.9, J = 9.7 Hz, H-4), 5.35 (dd, 1 H, J = 9.9, J = 9.7 Hz, H-3), 5.45 (d, 1 H, J = 10.7 Hz, H-1). ³ CRN (100 MHz, D ₂ 0, HSQC): δ 20.1, 20.2, 20.3, 22.0 (NAc), 51.9 (C2), 62.1 (C6), 68.1 (C4), 73.1 (C3), 75.9 (C5), 82.2 (C1), 167.9 (SC (NH ₂ ) ₂ ), 172.8, 173.1, 173.7, 174.8. Analysis calculated for C15H24CI 3O8S (%): C, 40.77; H, 5.47; N, 9.51; S, 7.26; Cl, 8.02. Found (%): C, 40.55; H, 5.36; N, 9.43; S, 7.08; Cl, 7.97. Preparation of (Z) -octadec-9-enyl methanesulfonate (4, MsO-oleyl)

Oleic alcohol (4ml, 10.8mmol) was dissolved in anhydrous CH2CI2 (108ml) and Et3IM (4.5ml, x3) was added. Next, mesyl chloride (2.3ml, x3) was added and stirred at room temperature for 23 hours. After that time it was purified by chromatography on silica gel (hexane-AcOEt, 3: 1) and a yellow liquid was obtained (3.13 g, 83.91%).

Compound 4: ¹ H NMR (300 MHz, CDCI ₃ ): δ 5.3-5.2 (m, 2H, -CH = CH-), 4.19 (t, 2H, J = 6.6 Hz, CH3SO2O-CH2-) _, 3.0 (s , 3H, CH3SO2O-), 2.0-1.9 (m, 4H, -

1.9-1.6 (m, 2H, CH3SO2O-CH2-CH2-), 1.3-1.1 (m, 22H, CH ₃ S0 ₂ 0-CH ₂ -CH ₂ -C] i (CH¿l ₁ -CH2CH = CHCH2 (CH2 _] 6CH3), 0.85 (t, 3H, J = 6.6 Hz, CH ₃ S0 ₂ 0- (CH2) 7-CH ₂ CH = CH (CH _{2) 7} CH¿). ¹³ C NMR (75 MHz, CDCI ₃ ) : δ 130.2 (-CH = CH-), 129.9 (-CH = CH-), 70.5 (CH ₃ S020-CH2-), 37.5 (CH3SO2O- CH ₂ -), 32.8, 32.1, 30.0, 29.9, 29.7, 29.5 , 29.3, 29.3, 29.3, 29.2, 27.4, 27.4, 25.6, 22.9 and 21.2 (-CH ₂ -), 14.4 (-CH2-CH3).

Preparation of oleyl 2-acetamido-3A6-tri-Q-acetyl-2-deoxy-1-thio-aD-qlucopyranoside 5a and oleyl 2-acetamido-3,4.6-tri-0-acetyl-2-deoxy-1- thio-aD- qlcopyranoside 5β.

3 (2.9 g, 6.5 mmol) was dissolved in anhydrous DMF (9 ml_) and Et ₃ N (2.7 ml_) was added. Then 4 (2.3 g, 6.5 mmol) was added and the mixture was heated at 60 ° C for 8 h. It was concentrated in vacuo to remove the solvent and the mixture was purified by silica gel column chromatography (hexane-AcOEt 5: 1 → 2: 1) to obtain 5a (0.9 g 23%) and 5β (0.6 g, 15%).

Product 5a: [a] _D = + 112.4 ° (C 0.5, MeOH). ¹ H NMR (400 MHz, CDCI ₃ , COZY): δ 5.71 (d, 1 H, J = 8.7 Hz, NH), 5.39 (d, 1 H, J = 5.4 Hz, H-1a), 5.4-5.3 ( m, 1 H, H- 3), 5.1-5.0 (m, 2H, -CH = CH-), 4.5-4.4 (m, 1 H, H-5), 4.4-4.3 (m, 2H, H-6a , H-2), 4.2-4.0 (m, 2H, H-4, H-6b), 2.6-2.5 (m, 2H, SCH2-), 2.1-1.9 (m, 16H, OAc NHAc, -CH¿CH = CHCH¿-), 1.7-1.4 (m, 2H, -SCH2CH2-), 1.4-1.2 (m, 22H, SCH ₂ CH ₂ (CH2) ₅ CH ₂ CH = CHCH2 (CH ₂ ) 6CH3), 0.88 (t , 3H, J = 5.7 Hz, -CH ₂ CH3_). 3C NMR (75 MHz, CD ₃ OD): δ 171.6, 170.7, 169.8, 169.3 (CO), 130.0, 129.7 (CH = CH), 84.6 (C1), 71.4, 68.3, 68.1, 62.8, 52.3 (C5, C4 , C3, C6, C2), 36.5, 32.5, 31.9, 31.5, 29.7, 28.8, 27.2, 23.2, 22.7, 20.7, 14.6 (NAc, OAc, -CH ₂ -), 14.1 (-CH2CH3). MS (ES) m / z (caled 613.85): 614.5 (M +1). Anal, caled, for C ₃₂ H ₅ 5NO ₈ S (%): C, 62.61; H, 9.03; N, 2.28; S, 5.22. Found: C, 62.48; H, 8.95; N, 2.51; S, 5.36.

Product 5β: [a] _D = -13.1 ° (C 1.5, MeOH). ¹ H NMR (400 MHz, CDCI ₃ ): δ 5.46 (d, 1H, J = 9.3Hz, NH), 5.4-5.3 (m, 2H, -CH = CH-), 5.25.0 (m, 2H, H -3, H-4), 4.57 (d, 1H, J = 10.5 Hz, Η1β), 4.24 (dd, 1H, J = 4.9, J = 12.3 Hz, H-6a), 4.2-4.0 (m, 2H, , H-6b), 4.1-4.0 (m, 1H, H-2), 3.7-3.6 (m, 1H, H-5), 2.7-2.6 (m, 2H, SCH2-), 2.0-1.9 (m, 16H, OAc. NHAc, -CH¿CH = CHCH ₂ -), 1.7-1.5 (m, 2H, SCH2CH2-), 1.4-1.2 (m, 22 H, SCH ₂ CH2 (CH ^ CH ₂ CH = CHCH ₂ ( CH2j £ CH ₃ ), 0.88 (t, 3H, J = 6.3 Hz,

³ C NMR (100 MHz, CD ₃ OD): δ 171.1, 170.7, 170.0, 169.3 (CO), 130.0, 129.8 (-CH = CH-), 84.6 (C1), 76.7 (C5), 75.9 (C3), 73.8 (C4), 68.3 (C6), 62.30 (C2), 53.3 (SCH ₂ -), 32.6, 31.9, 30.1, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.9, 27.2, 27.1, 23.3, 22.7, 20.8, 20.7, 20.6 (NAc, OAc, -CH ₂ -), 14.1 (-CH ₂ CH ₃ )

MS (ES) m / z (caled 613.85): 614.5 (M +1).

Preparation of oleyl 2-acetamido-2-deoxy-1-thio-a-D-qlucopyranoside 6α.

5α was treated with a solution of MeONa in 0.1 M MeOH with stirring for one hour at room temperature. The mixture was neutralized with Amberlite 120-IR and concentrated. It was purified by silica gel column chromatography (AcEt-MeOH) 10: 1. 0.47 g of the 6a anomer (73%) was obtained.

[aj _D = + 132.0 ° (C 1.0, MeOH). ¹ H NMR (400MHz, CD ₃ OD): δ: 5.45 (d, 1H, J = 5.4 Hz, H-1), 5.4-5.3 (m, 2H, -CH = CH-), 4.00 (dd, 1H, J = 5.4, J = 11.1 Hz, H-2), 4.0-3.9 (m, 1H, H-5), 3.81 (dd, 1H, J = 2.0, J = 12.0 Hz, H6a), 3.70 (dd, 1H , J = 5.4, J = 12.0 Hz, H-6b), 3.60 (t, 1H, J = 9.0 Hz, H-3), 3.3 (t, 1H, J = 9.3 Hz, H- 4), 2.6-2.5 (m, 2H, SChb-), 2.1-2.0 (m, 4H, -CH ₂ CH = CHCH ₂ -), 2.0 (s, 3H, NHAc), 1.6-1.5 (m, 2H, SCH ₂ CH2-), 1.3-1.2 (m, 22H, SCH ₂ CH ₂ (CH2 ^ CH ₂ CH = CHCH ₂ (CH2jgCH3), 0.9 (t, 3H, J = 6.6 Hz, S (CH ₂ ) ₈ CH = CH (CH ₂ ) ₇ CH¿) ¹³ C NMR (75 MHz, CD ₃ OD): δ 173.6 (CO), 130.9 (CH = CH), 130.8 (CH = CH), 85.1 (C-1), 74.2 (C-5), 72.7 , 72.6 (C-3, C-4), 62.6 (C-6), 55.9 (C-2), 33.6 (S-CH ₂ ), 33.1, 31.6, 30.9, 30.8, 30.6, 30.6, 30.5, 30.3, 29.9, 28.2, 28.1 (SCH ₂ (CH ₂ ) ₇ CH = CH (CH ₂ ) ₆ CH ₂ CH ₃ ), 23.8 (CH ₂ CH ₃ ), 22.6 (COCH ₃ ), 14.9 ((CH ₂ ) ₇ CH ₃ ). MS (ES) m / z (caled 487.5): 488.5 (M +1). Anal, caled, for C ₂₆ H ₄ 9N0 ₅ S (%): C, 64.03; H, 10.13; N, 2.87; S, 6.57. Found: C, 63.99; H, 9.99; N, 3.13; S, 6.54.

Preparation of oleyl 2-acetamido-2-deoxy-1-thio-B-D-qlucopyranoside 6β.

Following the same procedure as for the preparation of 6a, from 5β 6β (0.21 g, 54%) was obtained.

[aJ _D = -18.3 ° (C 1.1, MeOH). H NMR (300 MHz, CD ₃ OD): δ 5.4-5.3 (m, 2H, - CH = CH-), 4.47 (d, 1 H, J = 10.3 Hz, H-1), 3.86 (dd, 1 H , J = 2.2, J = 12.0 Hz, H- 6a), 3.73 (t, 1 H, J = 10.1 Hz, H-2), 3.67 (dd, 1 H, J = 5.7, J = 12.0 Hz, H- 6b), 3.43 (t, 1 H, J = 9.7 Hz, H-3), 3.3-3.2 (m, 2H, H-4, H-5), 2.7-2.6 (m, 2H, SCH ^ -), 2.1- 2.0 (m, 4H, -CH ₂ CH = CHCj ± r), 2.00 (s, 3H, NHAc), 1.6-1.5 (m, 2H, SCHsCJi-), 1.4-1.2 (m, 22H, SCH ₂ CH ₂ (CH ₂ ) ₅ CH ₂ CH = CHCH ₂ (CH ₂ ) 6CH3), 0.90 (t, 3H, J = 6.6 Hz, S (CH ₂ ) ₈ CH = CH (CH ₂ ) ₇ CH ₃ ). ¹³ NMR (100 MHz, CD ₃ OD): δ 173.5 (CO), 130.9 (CH = CH), 130.8 (CH = CH), 85.7 (C-1), 82.1 (C-5), 77.4 (C-3 ), 71.9 (C-4), 62.9 (C-6), 56.3 (C-2), 33.6, 33.1, 30.9, 30.8, 30.8, 30.6, 30.5, 30.3, 30.2, 30.0, 28.2, 28.1, (SCH ₂ (CH ₂ ) ₇ CH = CH (CH ₂ ) ₆ CH ₂ CH ₃ ), 23.8 (CH ₂ CH ₃ ), 23.0 (COCH ₃ ), 14.5 ((CH ₂ ) ₇ CH ₃ ). MS (ES) m / z (caled 487.3341): 488.3415 (M + 1).

Preparation of oleyl 2-acetamido-2-deoxy-1-sulfinyl-a-D-qlucopyranoside 7a

6a (150 mg, 0.31 mmol) was dissolved in methanol (3mL) and treated with 33% hydrogen peroxide (2.2 mmol, x7) and ZrCI ₄ (17.0 mg, 0.77 mmol) and The mixture was stirred at room temperature for 3 h. The mixture was concentrated in vacuo and purified by silica gel column chromatography (AcOEt-MeOH 5: 1) to give 7a as a white solid (30.9 mg, 20%). [a] _D = +1 12.7 ° (C 1.5, MeOH). H NMR (400 MHz, CD ₃ OD): δ 5.3 - 5.2 (m, 2H, CH = CH), 4.81 (d, <1 H, J = 5.4 Hz, H1), 4.77 (d, <1 H, J = 5.9 Hz, H1), 4.34 (dd, 1 H, J = 10.8, 5.0 Hz, H2), 4.3.4.2 (m, 1 H, H5), 3.95 (dd, 1 H, J = 10.8, J = 8.5 Hz, H3), 3.82 (dd, 1 H, J = 12.2, J = 2.1 Hz, H6a), 3.61 (dd, 1 H, J = 12.3, J = 6.1 Hz, H6b), 3.38 (dd, 1 H , J = 9.5, J = 8.5 Hz, H4), 3.1-3.0 (m, 1 H, SCH ₂ ), 2.9-2.8 (m, 1 H, SCH ₂ ), 2.1-2.0 (m, 4H, CH¿CH = CH = CH¿), 1.98 (s, 3H, NHAc), 1 .8-1.7 (m, 2H, SCH ₂ CH¿-), 1.5-1.3 (m, 22H, SCH ₂ CH ₂ (CH ^) ₅ CH ₂ CH = CHCH ₂ (CH ₂ ) ₆ CH ₃ ), 0.90 (t, 3H, J = 6.8 Hz, -CH ₂ CH¿).

¹³ C NMR (101 MHz, CD ₃ OD): δ 174.1 (CO), 131.0 (CH = CH), 130.9 (CH = CH), 91.7, 80.7, 73.0, 72.0, 62.8, 54.8, 50.6 (C1, C2, C3, C4, C5, C6, SCH ₂ -), 49.7, 49.5, 49.3, 49.1, 48.9, 48.7, 48.5, 33.2, 31.0, 30.7, 30.6, 30.5, 30.4, 29.9, 28.3, 23.9, 23.0, 22.8

COCH ₃ ), 14.6 (-CH ₂ CH ₃ ). MS (ES) m / z (caled 503.74): 504.74 (M + 1), 526.32 (M + Na). Anal, caled, for C ₂ 6H ₄₉ N0 ₆ S (%): C, 61.99; H, 9.80; N, 2.78; S, 6.37. Found: C, 59.83; H, 9.65; N, 2.77; S, 6.12.

Preparation of oleyl 2-acetamido-2-deoxy-1-sulfinyl-B-D-qlucopyranoside 7β.

Following the same procedure as for the preparation of 7a, from 6β 7β (30 mg, 19%) was obtained. [a] _D = -7.2 ° (C 1.5, MeOH). ¹ H NMR (400 MHz, CD ₃ OD): δ 5.4 - 5.3 (m, 2H, CH = CH), 4.43 (d, <1 H, J = 10.8 Hz, H1), 4.27 (d, <1 H, J = 10.5 Hz, H1), 3.98 (t, 1 H, J = 10.3 Hz, H2), 3.90 (dd, 1 H, J = 12.4, J = 1.4 Hz, H6a), 3.8 - 3.7 (m, 1 H , H6b), 3.7-3.6 (m, 2H, H3, H5), 3.5-3.4 (m, 1 H, H4), 3.3 - 3.1 (m, 1 H, SOCH2-), 2.8 - 2.7 (m, 1 H , SOCH ₂ -), 2.1 - 1.9 (m, 7H, - CH¿CH = CHCH ₂ .-, NHAc), 1 .7 - 1.6 (m, 2H, SCH ₂ CH ₂ -), 1.5-1.2 (m, 22 H), 0.90 (t, 3H, J = 6.7 Hz, -CH ₂ CH ¿). ¹³ C NMR (101 MHz, CD ₃ OD) δ 130.9 (CH = CH), 130.8 (CH = CH), 88.8, 82.6, 78.0, 76.2, 70.8, 62.2, 52.4 (C1, C2, C3, C4, C5, C6, SCH ₂ -), 37.0, 33.6, 33.1, 30.8, 30.8, 30.6, 30.5, 30.4, 30.3, 30.3, 30.3, 29.9, 28.1, 24.0, 23.8, 22.9

COCH ₃ ), 14.5 (-CHzCHs). MS (ES) m / z (caled 503.74): 526.32 (M + Na).

Preparation of oleyl 2-acetamido-2-deoxy-1-sulfonyl-aD-glucopyranoside 8a 6α (100 mg, 0.20 mmol) was dissolved in methanol (1 mL) and treated with 33% hydrogen peroxide (2.05 mmol, x10) and ZrCI ₄ (1 1.3 mg, 0.51 mmol, x2.5) and the mixture stirred at room temperature for 3 h. The mixture was concentrated in vacuo and purified by silica gel column chromatography (AcOEt-MeOH 10: 1) to give 8a as a white solid (30.9 mg, 29%).

[aj _D = + 74.4 ° (C 3, MeOH). H NMR (400 MHz, CD ₃ OD): δ 5.3-5.2 (m, 2H, CH ₂ CH = CH-CH ₂ -), 5.2-5.1 (m, 1 H, H-1), 4.2-4.1 (m , 2H, H-2, H-5), 4.1 -4.0 (m, 1 H, H-6a), 3.9-3.6 (m, 2H, H-6b, H4), 3.3-3.0 (m, 1 H, H-3), 3.2-3.1 (m, 2H, - S0 ₂ -CH¿-), 2.0-1.8 (m, 7H, CH¿CH = CH-CH¿-, CH3CONH-), 1.7-1.6 (m, 2H, - S0 ₂ -CH2-CH2-CH2-), 1.2-1.0 (m, 20H, -CH ^ -), 0.88 (t, 3H, J = 6.8 Hz, -CH ₂ - CH¿). ³ C NMR (101 MHz, CD ₃ OD) δ 173.5 (CO), 130.3 (CH = CH), 129.7 (CH = CH), 86.3, 77.6, 70.6, 69.9, 61.6, 52.6, 51.2 (C1, C2, C3 , C4, C5, C6, SCH ₂ -), 32.4, 31.9, 29.6, 29.6, 29.4, 29.3, 29.2, 29.1, 29.1, 29.0, 28.9, 28.4, 26.9, 26.9, 22.5, 21.3, 21.2

COCH ₃ ), 13.3 (- CH ₂ CH ₃ ). MS (ES) m / z (caled 519.73): 520.33 (M +1). Anal, caled, for C ₂₆ H ₄₉ N0 ₇ S (%): C, 60.08; H, 9.50; N, 2.69; S, 6.17. Found: C, 59.87; H, 9.44; N, 2.91; S, 6.40. In Vitro Compound Activity

Inhibition of cell division by synthesized compounds was tested in vitro by analyzing mitochondrial activity by MTT assay in cultures of C6 rat glioma cells and A549 human lung adenocarcinoma. As a concrete example, the IC ₅ or (inhibitor concentration that produces 50% inhibition) values found for compounds 6α, 6β and 8a in four independent experiments at different concentrations of the compound (each performed in triplicate) are shown in table 1:

Table 1. Inhibition of division into C6 and A549 cells (48 h). The average values of IC ₅ or (μΜ) were: 6α 8α 6β cell line

C6 8 12 3

A549 35 41 30

To perform the MTT assays, the A549 and C6 cells were seeded in 96-well plates at a concentration of 2.5x10 ⁵ and 5x10 ⁴ cells / ml respectively and left 24 hours at 37 ° C with 5% CO2 to be adhere to the plate. The medium was removed and 100 μΙ of fresh medium was added along with the different concentrations of compounds to be tested. The products were allowed to incubate for 2 days, at 37 ° C with 5% CO2. After the incubation time, the medium was aspirated and 100 μΙ of fresh medium was added with the substrate 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazoyl bromide (MTT, 1 ml of medium and 200 μΙ of MTT whose initial concentration is 5mg / ml in 10x PBS). The plate was left for 3 hours in the incubator at 37 ° C with 5% CO2. After this time the medium was aspirated and 100 μΙ of DMSO was added to dissolve the crystals of formazan formed, and the absorbance at a wavelength of 590 nm was determined. The equipment used was Spectramax Plus (Molecular Devices Corporation) for 96-well plates.

Effect on the production of qlicoesfinqolipidos. The compounds of the present invention have been tested on the A549 lung cancer cell line (American Type Culture Collection), which is maintained in the middle of HAM F12 supplemented with 2 mM glutamine, 10% of bovine fetal serum and antibiotic (penicillin and streptomycin), at 37 ° C in an atmosphere of 5% C02 / 95% air.

The cells were seeded in 6-well plates at a density of 2.5x10 ⁵ cells / ml and after 24 h, the medium was removed and fresh medium (1 milliliter per well) was added, the products containing a concentration of 15 micromolar After 2 days of incubation, the cells were washed with PBS and transferred to glass vials, where lipid extracts were prepared following the procedure described (Merrill et al Methods, 2005, 36, 207). The analyzes were carried out by ultra-liquid liquid chromatography coupled to an accelerated flight time mass detector, which allows the identification of the compounds based on their exact mass, by electrospray ionization in positive mode. The chromatographic and analytical conditions were those described in Canals et al Bioorg. Med. Chem., 2009, 17, 235.

The amount of lactosylceramide (LacCer) and gangliosides GM3 and GM2 present in extracts of A549 cells treated with thioglycosides 6a and 6β are shown in Fig. one . Enzyme hydrolysis resistance test.

The compounds of the present invention were resistant to hydrolysis catalyzed by N-acetylhexosaminidases enzymes. As an example, the thioglycoside 6β (7.5 mM) in a 1: 5 mixture of methanol and sodium phosphate buffer (20 mM, pH = 6) was incubated in the presence of jack bean β-Ν-acetylglucosaminidase (0.6 units) at 37 ° C. After 30 min of incubation, HPLC analysis showed that thioglycoside 6β remains unchanged and no hydrolysis or methanolysis products are observed. In contrast, the corresponding 6β glycoside (with an oxygen atom instead of the sulfur atom) under the same conditions gave rise to 7% of hydrolysis and methanolysis products after 30 min. The HPLC analysis was performed in the reverse phase, using a Licrosorb RP18 column (5 μΜ, 4.6 x 250 mm) and as a mobile phase a mixture of water-methanol (95-5 (from 0 to 4 minutes) → 0-100 ( from 8 to 20 minutes) → 95-5 (from 25 to 30 minutes)). The flow rate was 1.0 mL / min and the detection wavelength of the hydrolysis products (retention time: 2.9 minutes) and methanolysis (retention time: 4.1 minutes) was 205 nm. Live activity of the compounds

The C6 rat glioma line was grown in DMEM medium with 10% fetal bovine serum in a constant temperature incubator of 37 ° C and 5% C02. Glioma cells were injected subcutaneously into one of the flanks of female immunosuppressed mice Foxn1 nu / nu at a rate of 3 x 106 cells in 00 µΙ of PBS. Once the tumors acquired an approximate size of 225-250 cm2, treatment with the compounds of the different groups was initiated by daily intra-tumor injections. The animals were segregated into 4 groups at the rate of 6 animals per group according to the treatments: Group 1 or control in which the control solution was injected in which the compounds were dissolved (PBS + 5 mg / ml of free serum bovine albumin of fatty acids + 5% DMSO); Group 2, animals treated with 10 mg / kg of compound 9 animal; Group 3, animals treated with 1 mg / kg of animal of compound 6 α and Group 4, animals treated with 10 mg / kg of animal of compound 6 a. The treatments were carried out for 2 weeks and the animals were sacrificed on day 15. The animals were weighed and the tumors were measured daily with a calimeter. The formula used to calculate tumor volume (V) was, V = TT / 6X (LXW2), where L is the length and W is the width of the tumor.

Claims

one . Compound of formula (I)

Formula (I) where

X is selected from S, S (O) and S (O) 2

Ri, _R2 and R3 are independently selected from H and C1-C6 acyl, R ₄ is a C2-Ci6 ₄ alkyl or Cs-C2 ₄ alkenyl,

or any of its isomers, salts or solvates.

2. Compound according to claim 1 wherein R1 to Rs are H.

3. Compound according to any of claims 1 or 2 wherein X is S.

4. Compound according to any of claims 1 or 2 wherein X is S (O).

5. Compound according to any one of claims 1 or 2 wherein X is S (O) ₂ .

6. Compound according to any one of claims 1 to 5 wherein R ₄ is a C6-C-2- alkenyl

7. Compound according to claim wherein R ₄ is an octadec-9-enyl group.

8. Pharmaceutical composition comprising a compound defined in any one of claims 1 to 7.

9. Composition according to claim 8 further comprising another active ingredient.

10. Use of a compound defined in any one of claims 1 to 7 for the manufacture of a medicament.

eleven . Use of a compound defined in any one of claims 1 to 7 for the manufacture of a medicament for the treatment of tumors.

12. Use according to claim 1 wherein the tumor is selected from brain, melanoma, lymphoma, lung or uterine adenocarcinoma, between brain or lung adenocarcinoma.

13. A compound defined in any one of claims 1 to 7 for use in the treatment of tumors.

14. Use of a compound defined in any one of claims 1 to 7 as a reagent in biological assays.

15. Process for obtaining a compound of formula (I) defined in any one of claims 1 to 7 characterized in that it comprises the following steps: to. Reaction of a compound of formula (II)

Formula (II) with a chloride of formula Cl-R ₅ where R ₅ is a C1-C6 acyl.

reaction of the compound obtained in the previous stage with thiourea. reaction of the compound obtained in the previous step with a compound of formula (III) or

Formula (III)

where R6 is a C6-C2 ₄ alkyl or a Cs-C2 ₄ alkenyl.

16. The method according to claim 14 wherein an oxidation step is also performed.