ES2380864B1 - INHIBITORS OF THE DIVISION OF TUMOR CELLS. - Google Patents

Abstract

Inhibitors of tumor cell division # The present invention relates to new inhibitors of tumor cell division. Said inhibitors are semi-synthetic derivatives of neurostatin. Furthermore, a new method of synthesis of inhibitors and neurostatin is described in the present invention. In addition, the use of these inhibitors for the manufacture of a medicament for the treatment of brain tumors is described.

Description

Inhibitors of tumor cell division

The present invention relates to new inhibitors of tumor cell division. Said inhibitors are semi-synthetic derivatives of neurostatin. Furthermore, the present invention relates to a new method of synthesis of inhibitors and neurostatin. It also refers to the use of these inhibitors for the manufacture of a medicament for the treatment of brain tumors. The invention is part of the pharmaceutical sector and is applicable in the medical sector for the treatment of brain tumors.

STATE OF THE TECHNIQUE

Glioblastoma multiforme is the most common primary brain tumor in adults and also 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 metastasize to the brain), which is about 14,000 deaths. Brain tumors are 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 tumor growth or to make it shrink and chemotherapy destroys the remaining tumor cells after surgery and radiation therapy. The most common chemotherapy (BCNU, CCNU) does not seem to have a significant effect, although in some occasions it has been possible to increase survival a few months. Therefore, the development of new molecules capable of stopping the proliferation of gliomas is of great interest for the treatment of this disease.

Gangliosides are glycolipids formed by a ceramide chain (which is composed of an amino alcohol residue sphingosine bound to a fatty acid) linked to an oligosaccharide chain. Gangliosides have as their structural characteristic, the presence of one or more sialic acid residues in the oligosaccharide chain. Neurostatin is characterized as a ganglioside of the b-series, GD1b, with an O-acetylation modification in one of the hydroxyls of its terminal sialic (Romero-Ramirez L, Nieto-Sampedro M. Inhibiting human astrocytoma growth: structure -activity re lationships in neurost atin related glycolipids.Jurnal of Me dicinal Chemistry 2004; 47 (21): 4983-4.). The position of this O-acetylation is preferably in the hydroxyl of carbon 9, since although O-acetylations in carbons 7 and 8 have also been described, under physiological pH conditions the Oacetyls in those carbons migrate to position 9.

Neurostatin is a natural ganglioside that is expressed in very low concentration in the central nervous system of mammals (Abad-Rodriguez J, Bernabe M, Romero-Ramirez L, Vallejo-Cremades M, Fernandez-Mayoralas A, Nieto-Sampedro M. Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain. Journal of Neurochemistry 200 0; 74 (6): 2547-56.). The neurostatin purification procedure from mammalian brains is very laborious and the yield obtained makes its use unfeasible, both in animal and clinical experimentation.

Recently, a series of procedures have been developed for obtaining O-acetylated gangliosides by different methods of O-acetylation of the commercial ganglioside GD1b (Valle-Argos B, Gomez-Nicola 0, Nieto-Sampedro M. Synthesis and characterization of neurostatin- related compounds with high inhibitory activity of glioma growth. European Journal of Medicinal Chemistry; 2010, 45 (5): 2034-43.). Although chemical procedures reduce the steps for obtaining O-acetylated gangliosides, they are nonspecific reactions where neither the number nor the position of the O-acetylations are controlled. Consequently, a wide variety of products are obtained that hinder their purification to homogeneity and considerably reduce the yield of the reaction. Although this procedure reduces the steps necessary to produce neurostatin, the product obtained remains insufficient for possible clinical use.

On the other hand, the procedure described in the publication (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate O-acetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8linked sialic acid.The Journal of Biological Chemistry 2006; 281 (17): 11480-6) achieves O-acetylate glycolipid polysaccharides derived from the hexanoic acid ester 6- (5-Fluorescein-carboxamido) - (FCHASE) and glycoproteins, but not gangliosides

The procedure described in the patent (WO 2007016792) is quite general. The pH ranges he describes are quite wide (from 5 to 8), he talks about an acetyl group donor and an acetyl group acceptor, where the use of buffer substances is anticipated and the reaction is carried out between 0 and 40 ° C, preferably between 20 and 37 ° C, in aqueous medium. It also describes that the reaction mixture may contain divalent metal cations (such as magnesium or manganese) and may also contain solubilizing detergents or organic solvents, if necessary. This procedure does not detail any specific protocol for O-acetylate gangliosides and following it is not possible to obtain O-acetylate gangliosides.

ES 2 380 864 Bl

In the procedure described by Houliston, R. et al. (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate Oacetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8linked sialic acid. The Journal of Biological Chemistry 2006; 281 ( 17): 11480-6) which uses an O-acetyltransferase from the bacteria Campylobacter jejuni. This procedure was used to specifically O-acetylate the terminal sialic acids of the polysaccharide chains of glycolipids derived from the succinate ester of hexanoic acid 6- (5Fluorescein-carboxamide) (FCHASE) and also glycoproteins. The use of this enzyme for O-acetylated oligosaccharides is patented (WO 2007016792 20070215).

The procedure described by these researchers failed to successfully O-acetylate gangliosides. Gangliosides in aqueous solution are grouped in micelles that prevent the access of the O-acetyltransferase to the terminal sialic.

DESCRIPTION OF THE INVENTION

The present invention describes a new series of gangliosides with inhibitory activity (of the neurostatin family) of tumor cell division and glioma growth. In addition, the present invention describes a novel method of obtaining gangliosides and neurostatin which is characterized by its simplicity, specificity and high performance.

Using the same procedure as described, we have obtained new gangliosides from the O-propionylated neurostatin family, not described in nature and that have greater inhibitory activity on the proliferation of the C6 glioma line (Table 1). These compounds could be more resistant to hydrolysis by glycosidases, so their effect would be more lasting.

Therefore, these O-propionylated gangliosides are very interesting products for clinical use as antitumor agents.

Taking into account the severity of glioblastomas and the limited effectiveness of available treatments, the development of new molecules capable of stopping the proliferation of gliomas is of great interest.

Therefore a first aspect of the present invention relates to a ganglioside of the general formula (I):

OH

Y

X1

HO

X3

(I)

or any of its salts, isomers or solvates, where; And it is a ceramide; Ac is an acetyl group; X1 is a group –CH2-O-CO-X2 X2 is a C1-C2 alkyl group;

X3 is selected from H, OH, a compound of general formula (II) or a compound of general formula (III)

AcHN O X4O OH AcHN O

HO OR

HO

OR OR

OR HO O

H HO

HO OH

(II) (III)

X4 is selected from H and a compound of general formula (IV):

(IV)

or any of its salts, isomers or solvates, X5 is selected from an OH group or a –O-COX6 group; X6 is a C1-C2 alkyl group;

10 except when:

-

X1 is -CH2-O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) and X4 is hydrogen;

-

X1 is -CH2-O-CO-X2, X2 is methyl, and X3 is the compound (II); Y

-

X1 is -CH2-O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) where X4 is the compound of general formula (IV) where X5 is OH.

According to a preferred embodiment, Y is a ceramide comprising a sphingosine and a natural or synthetic fatty acid. Preferably, sphingosine comprises 18 to 20 carbon atoms and has an unsaturation number of 0 to 2. Preferably, the fatty acid comprises 8 to 24 carbon atoms and has an unsaturation number of 0 to 4;

According to another preferred embodiment, X2 is a methyl group.

According to another preferred embodiment, X2 is an ethyl group. According to another preferred embodiment, X3 is H. According to another preferred embodiment, X3 is OH. According to another preferred embodiment, X3 is a group of formula (II). According to another preferred embodiment, X3 is a compound of general formula (III).

According to another preferred embodiment, X4 is H.

According to another preferred embodiment, X4 is a compound of general formula (IV). According to another preferred embodiment, X5 is an OH group. According to another preferred embodiment, X5 is a group -O-COX6. According to another preferred embodiment, X6 is a methyl. According to another preferred embodiment the compounds of the present invention are selected from the following list:

OR H3C

C

HOCH2

OR COOH HO

OR OAcHN COOH

HO HO

OH OH OHO

OR

OO OHOAcHN O

Y

OH

HO

OH

OH

O-propionyl-GD3

O-propionyl-GD2

Or HO

H3C

C O COOH

CH2 HO

Or OAcHN

COOH HO HO

OH OH OHO OO

OOAcHN

HOO

Y

OH

HO

OHNHAc

OH HO

OR

OR OR

Or HO

HOOH OH

 O-propionyl-GD1b

OR

di-O-acetyl-GT1b

OR CH3 C HO

CH2 O

OH OR

OR OR

HO

Y

HO OH HO

HO AcHN HO

HOOH OH

O-propionyl-GT1b

5 where Y is any ceramide, which is composed of a sphingosine and a fatty acid. Preferably, sphingosine comprises 18 to 20 carbon atoms and has a number of unsaturations of 0 to 2. Preferably, the fatty acid may contain 8 to 24 carbon atoms and with a number of unsaturations of 0 to 4;

In the present invention the terms GD1b, GD3, GD2 or GT1b, refer to gangliosides of the same name known in the state of the art by any person skilled in the art.

10 In this same sense, these gangliosides comprise among other elements, a ceramide (Y) which in turn comprises a sphingosine and a fatty acid. Sphingosine is preferably 18 carbon atoms with an unsaturation or 20 carbon atoms with an unsaturation. Preferably, the fatty acid is 18 carbon atoms without any unsaturation or unsaturation.

Another aspect of the present invention relates to the compound of general formula (I) for use as a medicament.

A second aspect of the present invention relates to a pharmaceutical composition comprising at least the compound of general formula (I) and at least one pharmaceutically acceptable carrier, adjuvant and / or vehicle.

In a preferred embodiment, the pharmaceutical composition further comprises another active ingredient.

A third aspect of the present invention relates to ganglioside synthesis method of general formula (I):

(I)

or any of its salts, isomers or solvates, where;

5 Y, Ac, X1, X2, X3, X4, X5 and X6 are defined as defined above. which comprises the stages of: a) dissolve a ganglioside in a buffer solution; b) add an emulsifier to the mixture of stage a) c) add to the mixture of step b) the enzyme o-acetyltransferase; Y

10 d) stop the reaction by adding methanol to the mixture obtained in step c). According to a preferred embodiment, gangliosides of general formula (I) are selected from the following list:

OR H3C

C

HOCH2

OR COOH HO

Or OAcHN

COOH

HO HO

OH OH OHO

OR

OO OHOAcHN O

Y

OH HO

OH OH

O-propionyl-GD3

OR

H3C HO CH2 C

OR

OH OO O

HO

Y

OH

OH

O-propionyl-GD2

 O-propionyl-GD1b

di-O-acetyl-GT1b

Or CH3 CHO

CH2 O

OH OOO

HO

Y

HO OH HO

HO AcHN HO

O-propionyl-GT1b

OR

C

HOCH3 OR COOH HO OR OAcHN

COOH

HO HO

OH OH OR

HOOO O

OHOAcHN O

Y

OH HO

OH OH

O-acetyl-GD3

OR HO

CCH3

OH OH OO OHO

Y

OR

HO OH

OH

NHAc HO O O HO

OH

O-acetyl-GD2

OR

O-acetyl-GT1b

Or HO C

OR CH3

OH OR

OR OHO

Y

OH

HO OH OH

Neurostatin

5 According to a preferred embodiment, the ganglioside of step a) is selected from the group consisting of GD1b, GD3, GD2 or GT1b.

According to another preferred embodiment, an amount of ganglioside from step a) between 50 to 600 IM, preferably between 300 and 600 IM, and more preferably 500 IM is added.

According to another preferred embodiment, the buffer solution is added in a concentration from 1 to 250 mM,

10 preferably from 10 to 100 mM, more preferably at a concentration of 50 mM, at a pH from 6 to 9, preferably at a pH between 7 and 8, more preferably at a pH of 7.0. Preferably, the buffer solution is from TRIS (tris (hydroxymethyl) aminomethane), MES (2- (N-morpholino) ethanesulfonic acid), phosphate, etc., preferably from MES.

 According to another preferred embodiment, MgCl2 buffer (10 mM) and DTT (Dithiothreitol) (1mM) are added to the solution,

According to another preferred embodiment, Acetyl-Coenzyme A or Propionyl-Coenzyme A buffer is added to the solution at a concentration from 0.1 to 10 mM, preferably between 0.5 and 2 mM, more preferably at 1 mM.

According to another preferred embodiment, the emulsifier is a bile acid or bile salt at a preferable concentration between 0.05% and 0.2% weight / volume, more preferably 0.1% by weight / volume.

Preferably, the bile acid is selected from the group consisting of colic acid, deoxycholic acid, taurocolic, glycocolic, ursodexosolic, lithocolic, etc. as its sodium and potassium salts, (example: cholate, taurocholate,

20 deoxycholate, ursodeoxycholate, glycocholate, etc.). Preferably, the emulsifier is the sodium bile colata salt.

The emulsifier reduces the micellation of gangliosides, increasing the accessibility of terminal sialics to the enzyme O-acetyltransferase.

In another preferred embodiment, in step c), the enzyme O-acetyltransferase is added at a concentration of between 2 and 30 Ig / Il, preferably between 15 and 25 Ig / Il, more preferably at 20 Ig / Il.

The process of the present invention uses as an catalyst an O-acetyltransferase obtained from the bacteria Campylobacter jejunni, whose cloning, sequencing and activity have been patented (WO 2007016792 20070215) and

published by Houliston R. et al (Houliston RS, Endtz HP, Yuki N, et al. The Journal of Biological Chemistry 2006; 281 (17): 11480-6).

In another preferred embodiment, the reaction of step c) was performed between 30-40 ° C, preferably between 35-38 ° C, more preferably at a temperature of 37 ° C.

In another preferred embodiment, the reaction of step c) was carried out for a reaction time between 1 hour and 24 hours, preferably between 5 and 12 hours, more preferably for 7 hours, and with stirring (600 rpm).

After stopping the reaction with methanol in step d), the following steps were carried out:

-

 desalt the products obtained after step d) using a Sep-Pak C18 reverse phase cartridge (Waters),

-

 Dry the sample in a vacuum (Speed-Vac).

-

 Purify the different products by preparative TLC or preparative HPLC on an amino column.

-

 calculate the molecular weight of the products by means of flight time mass spectrometry (MALDITOF-MS).

-

 characterization of the position of the O-acetylations or O-propionylations by sequencing by electrospray mass spectrometry.

The procedure described in the present invention is a specific protocol for ganglioside O-acetylation. Unlike the procedure described in (Houliston RS, Endtz HP, Yuki N, et al. Identification of a sialate Oacetyltransferase from Campylobacter jejuni: demonstration of direct transfer to the C-9 position of terminalalpha-2, 8linked sialic acid. The Journal of Biological Chemistry 2006; 281 (17): 11480-6) and in the patent (WO 2007016792), in the process of the present invention an emulsifier, which can be a bile acid or a bile salt, is added to the reaction mixture, which in combination of the pH reduces the micellation of gangliosides, increasing the accessibility of terminal sialics to the enzyme O-acetyltransferase. The pH of the reaction must be equal to or greater than 6.5 in order for the sodium cholate to be soluble in the buffer and to act on the gangliosides.

The process described in the present invention is specific for terminal sialics and not for any O-acetylable hydroxyl group, as in the other prior art processes.

Therefore, the number of reaction products is reduced, favoring the purification of the products. In addition, the procedure has a much higher performance than those described above. In the case of the reaction to obtain neurostatin (O-acetylGD1b) from GD1b, the yield is 90%, compared to the scarce 10-15% of other procedures described in the prior art. Thanks to this performance, neurostatin can now be considered as an interesting product as an antitumor in patients and, therefore, with possible commercial use.

The process described in the present invention also allows obtaining natural O-acetylated products of the neurostatin family, such as O-acetyl-GD2 and O-acetyl-GT1b, which have not been described in the scientific literature as anti-tumor; and others, such as di-O-acetyl-GT1b, which have not been described either in nature or as antitumor agents. In addition to O-acetylated gangliosides, the process described in the present invention can be used to prepare O-propionylated gangliosides. These O-propionylated products, such as O-propionyl-GD3, O-propionyl-GD2, Opropionyl-GD1b and O-propionyl-GT1b, have never been described in the literature, nor are they found in nature. In addition, these O-propionylated products have greater inhibitory activity of the proliferation of the C6 glioma line (Table 1) and could be more resistant to hydrolysis by glycosidases, so that their effect would be more lasting.

A fourth aspect of the present invention relates to the use of the ganglioside of general formula (I) or any of its salts, isomers or solvates, where; And, Ac, X1, X2, X3, X4, X5 and X6 are defined as defined above for the preparation of a medicament.

A preferred embodiment refers to the use for the preparation of a medicament for the treatment or prevention of tumors, preferably for the treatment or prevention of astrocytomas, glioblastomas, oligodendrogliomas, neuroblastomas or meningiomas.

A fifth aspect of the present invention relates to the use of the ganglioside of the general formula (I) or any of its salts, isomers or solvates, where; Y, Ac, X1, X2, X3, X4, X5 and X6 are defined as defined above, as a reagent in biological assays.

The compound described in the present invention, its salts, prodrugs and / or solvates as well as the pharmaceutical, cosmetic and nutritional compositions containing them can be used together with other drugs, or active ingredients, additional to provide a combination therapy. Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising the compound described above, or a salt, prodrug or solvate. of the same.

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, as sterile solutions, suspensions, or lyophilized 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 to be treated and the weight of the patient. 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.

The term "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 3 carbon atoms or 1 to 2 carbon atoms, for example, methyl, ethyl, n-propyl, i- propyl, etc. Preferably the alkyl group has between 1 and 2 carbon atoms or preferably the alkyl group is a methyl. The alkyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido. alkoxide, thiol, amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester, aryl or heteroaryl or combinations of these groups. When the alkyl group is substituted, it is preferably substituted by one or more amine, amide or ether groups, which in turn may or may not be substituted by alkyl, amide, cycloalkyl or ethers groups and these, in turn, may also be substituted or no.

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 are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

A) Preparation of Neurostatin (O-acetyl-GD1b),

To a solution of the ganglioside GD1b (400 Ig) in MES buffer (2-morpholino ethanesulfonic acid), 50 mM, pH = 7.0, MgCl2 (10 mM), DTT (1 mM), Acetyl-Coenzyme A (1 mM) was added and 0.1% by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 µg / µl, in a total reaction volume of 432 µl. The reaction was carried out at 37 ° C for 7 hours with stirring (600 rpm). After time, the reaction was stopped by the addition of methanol.

The O-acetylation reaction of GD1b resulted in a reaction product whose mobility in thin layer chromatography (TLC) was different from GD1b. Through flight time spectrometry (MALDI TOF-MS) and Electrospray, it was characterized as Neurostatin, for presenting a single O-acetylation in the terminal sialic. The reaction yield was 90%.

B) Preparation of O-acetyl-GT1b and di-O-acetyl-GT1b

Following the same procedure as for neurostatin, but starting from ganglioside GT1b, two were obtained

reaction products whose mobility in thin layer chromatography (TLC) was different from that of GT1b. Both products were characterized by flight time spectrometry (MALDITOF-MS) and Electrospray. The compound with the greatest mobility in thin layer chromatography presented 2 O-acetylations, each in one of the two terminal sialics and was called di-O-acetyl-GT1b. The second product presented a single O-acetylation in the terminal sialic that is linked to another sialic and was called O-acetylGT1b. The reaction yield was 40% for both reaction products.

C) Preparation of O-propionyl-GD1b

Ganglioside GD1b (400 Ig) in a 50 mM buffer of MES (2- morpholino ethanesulfonic acid) pH = 7.0, to which was added MgCl2 (10 mM), DTT (1mM), Propionyl-Coenzyme A (1 mM) and 0.1 % by weight / volume of sodium cholate. Finally, the enzyme O-acetyltransferase was added at a concentration of 20 0g / 0l in a total reaction volume of 432 0l. The reaction was carried out at 37 ° C for 7 hours with stirring (600 rpm). After time, the reaction was stopped by the addition of methanol.

The O-propionylation reaction of GD1b resulted in a reaction product whose mobility in thin layer chromatography was different from that of GD1b. Through flight time spectrometry (MALDITOF-MS) and electrospray, it was characterized as an O-propionylated mono derivative in the terminal sialic we call O-propionyl-GD1b. The reaction yield was 10%.

D) Preparation of O-propionyl-GT1b

Following the same procedure as for O-propionyl-GD1b, but starting from the GT1b ganglioside, a reaction product was obtained whose mobility in thin layer chromatography (TLC) was different from that of GT1b. This product was characterized by flight time spectrometry (MALDITOF-MS) and Electrospray, such as the GT1b ganglioside with an O-propionylation in the terminal sialic which is attached to another sialic and was called O-propionyl-GT1b. The reaction yield was 20%.

E) Inhibitory activity of the compounds obtained above

The inhibitory activity of the ganglioside tumor cell division and its O-acetylated and Opropionylated derivatives obtained was determined by MTT assay measuring mitochondrial activity, in cultures of the C6 tumor line (rat glioma) for 48 hours of treatment. , using 10 ng / ml of basic FGF as a mitogen

GD1b

Neurost O-Prop-GD1b GT1b O-AcGT1b di-O-Ac-GT1b O-Prop-GT1b

C6

 - 270 120 550 380 190 150

Table 1. Inhibition of division in C6 cells (48 h) in average values of ID50 (nM). The symbol "-" indicates that the compound weakly inhibited proliferation at the maximum concentration tested (4 0M), but not enough to calculate the value of its ID50.

In table 1 we present the activity of gangliosides GD1b, GT1b, and their modified derivatives. The GD1b ganglioside has no inhibitory activity on the proliferation of the C6 glioma line. While the compounds derived from GD1b, Neurostatin (O-acetyl-GD1b), and O-propionyl-GD1b have activity in the nanomolar range. The ganglioside O-propionyl-GD1b is slightly more active than Neurostatin.

Ganglioside GT1b has a proliferation inhibitory activity. The compound O-acetyl-GT1b almost twice improves its inhibitory activity with respect to GT1b. When the ganglioside GT1b is modified with two O-acetylations (di-O-acetyl-GT1b) it also improves its activity twice as much as the O-acetyl-GT1b and 4 times with respect to the GT1b. The addition of Oacetyls to the ganglioside GT1b progressively increases its proliferation inhibitory activity in C6 cells.

O-propionylated gangliosides have greater inhibitory activity (O-propionyl-GD1b and O-propionyl-GT1b) than their respective mono-O-acetylated gangliosides (O-acetyl-GD1b and O-acetyl-GT1b). These O-propionylated compounds could be more resistant to hydrolysis by glycosidases, so their effect would be more lasting.

Claims (46)

1. A compound of general formula (I): Oy OHX1 OR HO       (I) 5 or any of its salts, isomers or solvates, where; And it's a ceramide, Ac is an acetyl group; X1 is a group –CH2-O-CO-X2; 10 X2 is a C1-C2 alkyl group; X3 is selected from H, OH, a compound of general formula (II) or a compound of general formula (III) X4O OH AcHN OH AcHN O HO OHO O o OR HO O H HO HO OH (II) (III) where X4 is selected from H or a compound of general formula (IV): HO OH (IV) where: X5 is selected from an OH group or a –O-COX6 group; Y X6 is a C1-C2 alkyl group; 5 except when:

-

X1 is -CH2-O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) and X4 is hydrogen;

-

X1 is -CH2-O-CO-X2, X2 is methyl, and X3 is the compound (II); Y

-

X1 is -CH2-O-CO-X2, X2 is methyl, X3 is the compound of general formula (III) where X4 is the compound of general formula (IV) where X5 is OH.

The compound according to claim 1, wherein Y is a ceramide comprising a sphingosine and a natural or synthetic fatty acid. 3. The compound according to claim 2, wherein the sphingosine comprises 18 to 20 carbon atoms and 0 to 2 unsaturations. 4. The compound according to any of claims 2 or 3, wherein the fatty acid comprises from 8 to 24 carbon atoms and with an unsaturation number from 0 to 4.

5.

 The compound according to any one of claims 1 to 4, wherein X2 is a methyl group.

6.

 The compound according to any one of claims 1 to 4, wherein X2 is an ethyl group.

7.

 The compound according to any one of claims 1 to 6, wherein X3 is H.

8.

 The compound according to any one of claims 1 to 6, wherein X3 is OH.

The compound according to any one of claims 1 to 6, wherein X3 is a group of formula (II).

10.

 The compound according to any one of claims 1 to 6, wherein X3 is a group of formula (III).

eleven.

 The compound according to claim 10, wherein X4 is H.

12.

 The compound according to claim 10, wherein X4 is a compound of general formula (IV).

13. The compound according to claim 12, wherein X5 is an OH group. 14. The compound according to claim 12, wherein X5 is a group -O-COX6.

fifteen.

 The compound according to claim 14, wherein X6 is a methyl.

16.

 The compound according to claim 1, which is selected from the following list:

 Or H3C C HOCH2 OR COOH HO OR OAcHN COOH HO HO OH OH OHO OO O OHOAcHN O Y OH HO OH OH O-propionyl-GD3 O-propionyl-GD2  O HO H3C C O COOH CH2 HO Or OAcHN COOH HO HO OH OH OHO OO OOAcHN HOO Y OHHO OHNHAc OH HO OR OR OR  Or HO HOOH OH O-propionyl-GD1b OR di-O-acetyl-GT1b  OR CH3 CHO   CH2 O   OH OO OR HO Y  HO OH HO   HO AcHN HO HOOH OH O-propionyl-GT1b 5 where Y is a ceramide.

17.

 The compound according to any one of claims 1 to 16, for use as a medicament.

18.

 Pharmaceutical composition comprising at least one compound according to any one of claims 1 to 16 and at least one pharmaceutically acceptable carrier, adjuvant and / or vehicle.

19. The pharmaceutical composition according to claim 18, further comprising another active ingredient. 10 20. Synthesis procedure of a compound of general formula (I): OH (I) or any of its salts, isomers or solvates, where; Y, Ac, X1, X2, X3, X4, X5 and X6 are defined as in claim 1, which comprises the stages of: a) dissolve a ganglioside in a buffer solution; b) add an emulsifier to the mixture of step a); c) add to the mixture of step b) the enzyme o-acetyltransferase; Y d) stop the reaction by adding methanol to the mixture obtained in step c).

twenty-one.

 The method according to claim 20, wherein in step a) the ganglioside is selected from the group consisting of GD1b, GD3, GD2 or GT1b.

22

 The method according to any of claims 20 or 21, wherein in step a) an amount of ganglioside between 50 to 600 IM is added.

2. 3.

 The process according to claim 22, wherein in step a) an amount of ganglioside between 300 and 600 IM is added.

24.

 The method according to claim 23, wherein in step a) an amount of ganglioside of 500 IM is added.

25.

 The process according to any of claims 20 to 24, wherein the buffer solution is in a concentration from 1 to 250 mM.

26.

 The process according to claim 25, wherein the buffer solution is in a concentration from 10 to 100 mM.

27.

 The process according to claim 26, wherein the buffer solution is in a concentration of 50 mM.

28.

 The process according to any of claims 25 to 27, wherein the buffer solution is selected from the group consisting of TRIS, MES, or phosphate.

29.

 The method according to claim 28, wherein the buffer solution is MONTH.

30

 The process according to any of claims 25 to 29, wherein the buffer solution has a pH from 6 to

9.

31.

 The method according to claim 30, wherein the pH of the buffer solution is between 7 and 8.

32

 The process according to claim 31, wherein the pH of the buffer solution is 7.0.

33.

 The process according to any of claims 25 to 32, wherein 10mM MgCl2 buffer and 1mM DTT, Acetyl-Coenzyme A or Propionyl-Coenzyme A are added to the solution at a concentration between 0.1 to 10 mM.

3. 4.

 The process according to claim 33, wherein the Acetyl-Coenzyme A or the Propionyl-Coenzyme A are at a concentration between 0.5 and 2 mM.

35

 The process according to claim 34, wherein the Acetyl-Coenzyme A or the Propionyl-Coenzyme A is at a concentration of 1 mM.

36.

 The process according to any of claims 20 to 35, wherein the emulsifier is a bile acid or bile salt at a concentration between 0.05% and 0.2% weight / volume.

37.

 The process according to claim 36, wherein the emulsifier has a concentration of 0.1% by weight / volume.

38.

 The method according to any of claims 36 or 37, wherein the bile acid is selected from the group consisting of colic acid, deoxycholic, taurocholic, glycocolic, ursodexosolic, lithocolic acid, or its sodium and potassium salts, within the group formed by colatos, Taurocolates, deoxycholates, ursodeoxycholates or glycocholates.

39.

 The method according to claim 38, wherein the emulsifier is the bile salt sodium cholate.

40

 The process according to any of claims 20 to 39, wherein in step c), the enzyme Oacetyltransferase is added at a concentration between 2 and 30 Ig / Il.

41.

 The process according to claim 40, wherein the enzyme is added at a concentration between 15 and 25 Ig / Il.

42

 The process according to claim 41, wherein the enzyme is added at a concentration of 20 Ig / Il.

The process according to any of claims 20 to 42, wherein the reaction of step c) is carried out at a temperature between 30-40 ° C. 44. The process according to claim 43, wherein the reaction of step c) is carried out at a temperature between 35-38 ° C. 45. The process according to claim 44, wherein the reaction of step c) is carried out at a temperature of 10 37 ° C. 46. The method according to any of claims 20 to 45, wherein additionally after step d) the following steps are carried out:

-

 desalt the products obtained after step d) using a Sep-Pak C18 reverse phase cartridge,

-

 dry the sample in vacuo; 15 - purify the different products by preparative TLC or preparative HPLC on an amino column;

-

 calculate the molecular weight of the products by flight time mass spectrometry;

-

 characterization of the position of the O-acetylations or O-propionylations by sequencing by electrospray mass spectrometry.

47. Use of a compound of general formula (I) or of a pharmaceutical composition according to any of the claims 18 or 19, for the preparation of a medicament. OH O OY HO OH X1 OR HO OH X3

(I)

or any of its salts, isomers or solvates,

where; Y, Ac, X1, X2, X3, X4, X5 and X6 are defined as in claim 1.

48.

 Use of the compound or composition according to claim 47, for the preparation of a medicament for the treatment or prevention of tumors.

49.

 Use of the compound or composition according to claim 48, wherein the tumor is selected from the list comprising: astrocytoma, glioblastoma, oligodendroglioma, neuroblastoma or meningioma.

30 50. Use of the compound of general formula (I), HO OH OH OR OR Oy HO OHX1 COOH OR HO OH COOH OR OR OO HO OH X3 AcHN HO HO OH AcHN OH

(I)

or any of its salts, isomers or solvates, where; Y, Ac, X1, X2, X3, X4, X5 and X6 are defined as in claim 1, as a reagent in biological tests.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201031552 SPAIN Date of submission of the application: 22.10.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

US 5766887 A (SCRIPPS RESEARCH INST.) 16.06.1998, the whole document, especially figures 2A and 4; column 3, lines 31-36. 1-5.8

X

US 5102663 A (SOLAN-KETTERING INSTITUTE FOR CANCER RESEARCH) 07.04.1992, the whole document, especially claims 1, 2, 6; column 2, lines 25-32. 1-5,8,17-19,47,48

X

FR 2662697 A (SNOW BRAND MILK PRODUCTS CO. LTD.) 06.12.1991, claims 1-3. 1-5.8

X

IGARASHI, M. et al. Characteristics of gangliosides including O-acetylated species in growth cone membranes at several developmental stages in rat forebrain. Developmental Brain Research. 1994, Vol. 78, No. 1, pages 17-24. 1-5.8

X

ROMERO-RAMIREZ, L. and NIETO-SAMPEDRO, M. Inhibiting Human Astrocytoma Growth: Structure-Activity Relationships in Neurostatin Related Glycolipids. J. Med. Chem., 2004, Vol. 47, No. 21, pages 4983-4984. Compound 6, table 2; compound 8, Table 1. 1-5,8,47-49

X

ABAD-RODRIGUEZ, J. et al. Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain. Journal of Neurochemistry. 2000, Vol. 74, No. 6, pages 2547-2556, ISSN 0022-3042. The whole document, especially page 2551, paragraph “Inhibition of glial cell proliferation”. Page 2553, paragraph "Neurostatin biological activity". 47-49

X

Page 2553, paragraph "Neurostatin biological activity", O-acetyl-GD3. 1-5.8

X

ARGOS VALLEY. B et al. Synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth. European Journal of Medicinal Chemistry. May 2010, Vol. 45, No. 5, pages 2034-43. The whole document, especially page 2036, table 3; page 2037, table 4, compounds 2 and 6. 47-49

TO

HOULISTON, R.S. et al. Identification of a Sialate O-Acetyltransferase from Campylobacter jejuni. Demonstration of direct transfer to the C-9 position of terminal -2, 8-linked sialic acid. J. Biol. Chem. 2006, Vol. 281, No. 17, pages 11480-11486. The entire document, especially page 11481, left column, paragraph: "In vitro O-Acetyltransferase Reaction"; page 11483, Figure 3 A and B. 20-46

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 09.02.2012

Examiner E. Albarrán Gómez Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201031552 CLASSIFICATION OBJECT OF THE APPLICATION C07H15 / 10 (2006.01) C12P19 / 26 (2006.01) C12N9 / 10 (2006.01) A61P35 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C07H, C12P, C12N, A61P Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, REGISTRY, BEILSTEIN, MEDLINE, EMBASE, BIOSIS, PUBMED, MESH State of the Art Report Page 2/5 WRITTEN OPINION Application number: 201031552 Date of Written Opinion: 09.02.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 6, 7, 9-16, 20-46, 50 1-5, 8, 17-19, 47-49 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 6, 7, 9-16, 20-46, 50 1-5, 8, 17-19, 47-49 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986). Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5 WRITTEN OPINION Application number: 201031552 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

US 5766887 A (SCRIPPS RESEARCH INST.) 16.06.1998

D02

US 5102663 A (SOLAN-KETTERING INSTITUTE FOR CANCER RESEARCH) 07.04.1992

D03

FR 2662697 A (SNOW BRAND MILK PRODUCTS CO. LTD.) 06.12.1991

D04

IGARASHI, M. et al. Characteristics of gangliosides including Oacetylated species in growth cone membranes at several developmental stages in rat forebrain. Developmental Brain Research. 1994, Vol. 78, No. 1, pages 17-24.

D05

ROMERO-RAMIREZ, L. and NIETO-SAMPEDRO, M. Inhibiting Human Astrocytoma Growth: Structure-Activity Relationships in Neurostatin Related Glycolipids. J. Med. Chem., 2004, Vol. 47, No. 21, pages 4983-4984.

D06

ABAD-RODRIGUEZ, J. et al. Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain. Journal of Neurochemistry. 2000, Vol. 74, No. 6, pages 2547-2556, ISSN 0022-3042.

D06

Page 2553, paragraph "Neurostatin biological activity", O-acetyl-GD3.

D07

ARGOS VALLEY. B et al. Synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth. European Journal of Medicinal Chemistry. May 2010, Vol. 45, No. 5, pages 2034-43.

D08

HOULISTON, R.S. et al. Identification of a Sialate O-Acetyltransferase from Campylobacter jejuni. Demonstration of direct transfer to the C-9 position of terminal -2, 8-linked sialic acid. J. Biol. Chem. 2006, Vol. 281, No. 17, pages 11480-11486.

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present invention relates to a series of gangliosides of the general formula (I) from which Neurostatin (9O-acetyl-GD1b), O-acetyl-GD2 and O-acetyl-GT1b are excluded and the gangliosides O-propionyl are explicitly described -GD3, O-propionyl-GD2, O-propionyl-GD1b, O-propionyl-GT1b, di-O-acetyl-GT1b and the pharmaceutical compositions thereof. It also has as its object the process of obtaining these compounds without excluding the products of the disclaimer that appear in claim 1, a method in which the enzyme O-acetyltransferase is used to transfer acetyl or propionyl groups to the base gangliosides from the acetyl donor groups -coenzyme A or propionyl-coenzyme, in the presence of an emulsifier. The invention also relates to the use of the compounds of general formula (I) in the treatment of brain tumors such as astrocytoma, glioblastoma, oligodendroglioma, neuroblastoma or meningioma. The inventors present in the application data on the inhibitory activity of these compounds against tumor cells, highlighting the greater inhibitory activity of the propionylated compounds (O-propionyl-DG1b and O-propionyl-GT1b) against mono-O-acetylated, and attribute this difference to the fact that the O-propionylates could be more resistant to hydrolysis by glycosidases, so their effect would be more lasting. The applicant's attention is drawn to the following points:

•

In the definition of the general formula (I) X3 cannot be H.

•

The wording of claim 47 as "use of a compound of general formula (I) ..." implies that the products of the disclaimer of claim 1 are not excluded.

State of the Art Report Page 4/5 WRITTEN OPINION Application number: 201031552 Document D01 refers to a process of regioselective acetylation in the 9-hydroxy group of compounds carrying an N-acetylneuraminic group, including gangliosides GD3, GD2, GD1a, GD1b, GM1, GT1b, GQ1b with the subtilisin BPN protease. This document (Figure 2A; column 3, lines 31-36) also briefly describes the in vivo synthesis of 9-O-acetyl-GD3 by enzymatic acetylation of GD3 with O-acetyltransferase using acetylcoenzyme A. The purpose of document D02 is a pharmaceutical composition to stimulate the production of antibodies in patients with melanoma against the ganglioside 9-O-acetyl-GD3 comprising an effective amount of the ganglioside 9-O-acetyl-GD3 and suitable excipients. Document D03 describes 9-O-acetyl-GD3 and its method of obtaining from cow's milk or other dairy products. Document D04 discloses the presence of various acetylated gangliosides such as O-acetyl-GD3, O-acetyl-GT1b and O-acetyl-GQ1b in different stages of rat brain development. Document D05 refers to a study in which the inhibitory activity on human glioma cells U-373 of several gangliosides GD1b, GM3, GD3, GM1a and their corresponding O-acetylated derivatives in terminal sialic acid is compared. The compound named 6 corresponds to 9-O-acetyl-GD3. The inhibition data corresponding to this compound are shown in Table 2. The synthesis of 9-O-acetyl-GD3 is carried out from GD3 by acetylation with the enzyme subtilisin and vinyl acetate as a donor of the acetyl group. In view of documents D01 to D05 of the state of the art in which the compound 9-O-acetyl-GD3 is described and its use as a medicament (D02), claims 1-5, 8, 17- are considered 19, 47 and 48 lack novelty and inventive activity (Art. 6.1. And 8.1. LP 11/1986). It is also disclosed in document D05 that the compound named 8, which corresponds to Neurostatin (9-Oacetyl-GD1b), inhibited the proliferation of human glioma cell lines U-373 and T98G (page 4983, table 1). Document D06 discloses a study on Neurostatin, its purification of extracts from brains of rats and cattle, its structure and its role as an inhibitor of astrocyte division in mammalian brains. The authors conclude that this compound inhibited the proliferation in culture of primary astroblasts and gliomas, both of rats and humans, at nanomolar concentrations. On page 2553, paragraph "Neurostatin biological activity" refers to the ganglioside O-acetyl-GD3. Document D07 discloses a study in which Neurostatin and different analogues with acetyl or butyryl groups are synthesized in one or more positions (page 2036, table 3), in particular O-butyryl-GD1b by chemical synthesis with trimethyl orthoacetate (TMOA) or trimethyl orthobutyrate (TMOB). Table 4 on page 2037 shows the data corresponding to the inhibitory activity of these compounds in human astrocytoma cells and rat glioma, highlighting the remarkable effect of monosubstituted derivatives (O-acetyl-GD1b and O-butyryl-GD1b ), especially the latter compound (Table 4, compound 6). Therefore, based on the documents of the prior art D05 to D07, it is considered that claims 47 to 49 of the present application lack novelty and inventive activity (Art. 6.1. And 8.1. LP 11/1986). Document D08 discloses a study on the identification of an O-acetyltransferase from Campylobacter jejuni and the procedure for O-acetylating the terminal sialic acids of polysaccharides derived from the succinate ester of hexanoic acid 6- (5-Fluorescein-carboxamide) (FCHASE) ( page 11481, left column, paragraph "In vitro O-Acetyltransferase Reaction"; page 11483, Figure 3 A and B) by this enzyme, with acetyl-CoA, Month, MgCl2 and dithiothreitol. The content of claims 6, 7, 9-16, 20-46, 50 has not been disclosed in the state of the art, consequently it is considered that these claims are novel and involve inventive activity (Art. 6.1. And 8.1 LP 11/1986). State of the Art Report Page 5/5