ES2345809B1 - TRIPODAL COMPOUND BASED ESCUARAMIDE, PROCESS OF OBTAINING, SYSTEM CHEMICAL CONTAINER AND PROCEDURE FOR THE DETECTION OF POLYCARBYXYLATES. - Google Patents

Abstract

Squaramide based tripodal compound, process of obtaining, chemosensor system that contains it and procedure for the detection of polycarboxylates.

The present invention relates to a compound Squaramide-based tripodal along with its process of obtaining, when chemosensor system containing said compound as a receptor, and to a procedure that uses said chemosensor system for the qualitative and / or quantitative detection of polycarboxylates, specifically dicarboxylates and tricarboxylates, preferably folate and analogues thereof, in a fast, simple, effective way in Commercial samples in water.

one

Description

Squaramide based tripodal compound, process of obtaining, chemosensor system that contains it and procedure for the detection of polycarboxylates.

Field of the Invention

The present invention relates to a compound Squaramide-based tripodal along with its process of obtaining, when chemosensor system containing said compound as a receptor, and to a procedure that uses said chemosensor system for the qualitative and / or quantitative detection of polycarboxylates, specifically dicarboxylates and tricarboxylates, preferably folate and analogues thereof.

Background of the invention

Selective ion coordination in a medium highly competitive, like water, has captivated the chemist during years. The molecular recognition of biologically active anions, as phosphates or carboxylates, it has been a focus of attention priority in research with the aim of mimicking the enzymes or transport proteins. This type of research are directed towards establishing greater coordination and selectivity between receptor and substrate. The first aspect comes controlled by the type of functional groups and solvent used While the second aspect is subject to the introduction of complementary elements to geometry and proper characteristics of substrate coordination.

Despite the high degree of sophistication currently achieved in this type of research, the development of new receivers capable of establishing strong coordination selective with anions in a solvent, high constant Dielectric, like water, remains a very difficult task.

It is well known in the art that Squaramides are considered vinilogas to the amides and are a structural unit very suitable for link establishment of intermolecular hydrogen. This fact is based on the fact that a Squaramide dissecundaria offers two NH groups accepting linkers of hydrogen and two carbonyl oxygen atoms bond donors of intermolecular hydrogen, arranged so that they can act either separately, or in certain cases synergistically. To this we must add that theoretical calculations guarantee that the cycle of Squaramide when forming intermolecular hydrogen bonds with anions, or with cations, causes in the quantum cycle, and consequently throughout the molecule, an increase in stability close to aromaticity. All this together makes the unity of Squaramide dissecundaria is a promising link unit with carboxylate residues. It is solidly grounded in the bibliography, that the design of an anion receptor Polycarboxylates in water should gather a great preorganization, intermolecular hydrogen bonding capacity, establishment of different non-covalent intermolecular forces and complementary to hydrogen bonding, such as electrostatic, and be perfectly soluble in water. On the other hand, the ammonium groups have proven to be effective for training of ionic pairs that guanidinium group due to its high location of the positive charge, in this way, the nature shows preference for arginine rather than lysine for binding with this type of anions, possibly due to higher hydrogen bond accumulation.

We must not forget that the nature of solvent plays a critical role in controlling the strength of coordination and selectivity between receptor-analyte. These interactions are generally dominated by electrostatic type interactions. Usually anions they have a solvation-free energy greater than cations and require stronger interactions in water to compete so efficient with solid water molecules linked in the sphere of solvation of the anion. Examples of natural receptors for water anions are spermidine, the Carboxypeptidase A, and phosphate binding proteins.

The Frontera A. et al. , "Rational design, synthesis, and application of a new receptor for the molecular recognition of tricarboxylate salts in aqueous media". The Journal of Organic Chemistry. Sep 2006. ISSN 0022-3263 (Print). Vol. 71. Nr. 19. Page 7185-7195 describes tripodal receptors intended for the recognition of only tricarboxylate salts and in only partially aqueous media, in particular the use of a H2O: EtOH 1: 3 medium is mentioned.

The Prohens, R. et al. "Squaramido-based receptors: Molecular recognition of carboxylate anions in highly competitive media". Tetrahedron Letters Feb. 1998. ISSN 0040-4039. Vol. 39. Nr. 9. Page 1063-1066 describes ditopic receptors that show association values with dicarboxylates in acceptable non-aqueous medium, but that as the amount of water increases the association constant decreases dramatically, that is to say , dicarboxylates prefer to be associated with water (solvated) instead of forming a complex with the
receiver.

To date no one has been described Squaramide-based tripodal compound that is capable of interact with tricarboxylates and dicarboxylates, especially in samples with a completely aqueous medium.

On the other hand, within the biological scope the dicarboxylates are important species because they play different roles in numerous metabolic processes and in the biosynthesis of important intermediates, as well as in the generation of energy bonds with phosphates, and are a key structural piece in the composition of amino acids and proteins. In this way, the glutamate anion, an amino acid with two carboxylate groups, is the Major central nervous system stimulant.

Another important dicarboxylate is folate, which belongs to the family of B vitamins, with limited solubility in water at neutral pH, is only soluble in water at very acidic pH and in alkaline solutions, showing total insolubility in organic solvents. The major source of folate in the human diet comes from green leafy vegetables (spinach, chard, etc.), peas, legumes, egg yolk, wheat germ and yeasts. Folic acid is commercially very interesting because a deficiency in the human diet causes the massive incorporation of uracil into human DNA (4 million per cell) breaking the chromosome. This chromosomal rupture causes an increased risk of cancer and has added cognitive defects associated with this folate deficiency in the
diet.

For this reason, the lack of folic acid detected in the human diet worldwide has led to various manufacturers, to incorporate folic acid to a wide variety of food and especially those that are aimed at women pregnant women, children of growing age, and as a measure of Cancer prevention to the general population. Thus they find marketed foods enriched with folic acid, like cereals, cookies and dairy products. In cosmetic acid Folic is used in anti-aging creams. In addition, as a measure preventive against the appearance of babies with spina bifida is recommended during pregnancy by the mother to take Pharmacological preparations of folic acid.

Folate analogs are also compounds interesting, since, for example, methotrexate is used as antirheumatic, in the treatment of inflammatory diseases Chronic and polyartritic forms of juvenile idiopathic arthritis, as well as in the treatment of psoriasis, psoriatic arthritis and polyarthritis

Therefore, given the importance of folate and analogues thereof it is necessary to have methods for its detection and quantification in order to control quantities said compound in a fast and efficient manner.

Traditional methods are known in the art that use microbiological tests, or techniques chromatographic such as TLC or HPLC that require the use of devices specialized.

Microbiological tests require personnel expert in the management of cultures of microorganisms and an adequate control and maintenance of these microorganisms, but their greatest inconvenient is that these techniques are tedious and it takes a lot time to get an answer (24-48 h).

Another alternative used in the determination of folic acid are immunoassays (ELISA test), which are rapid (1-3 h) and that do not require expensive devices, nor neither highly specialized staff is required. However, Among its greatest limitations is the notorious variability in the results depending on the "kit" used. Other added disadvantage in this methodology is motivated by the rapid Expiration of these "kits".

At present there is no methodology for analysis of folic acid and analogs of it perfect and that stands out greatly, because folic acid is very sensitive to the physical environment in which it is found, and that it occurs in several forms. Although it should be noted that the microorganism test technique is the only official method of folate determination in foods established by the AOAC (Method 992.05 (2002)) and AACC (ACC Meted 86-47). In this technique the microorganism will be used is Lactobacillus rhamnosus that responds to a wide variety of folate analogs. Other microorganisms used are: Streptococcus faecalis, Pediococus cerevisae, Tetrahymena pyriformis and Bacillus coagulans . These determinations offer a high sensitivity (nanogram level) for most folate isomers. (Trends in Food Science & Technology, 2005, 16, 253-266). While the TLC and HPLC chromatographic techniques, and especially the latter, offer a high specificity towards the different folate isomers associated with an unequivocal identification of each isomer. It has been found that due to the limitation of the HPLC detectors used, in certain cases, a loss of sensitivity has been observed between 30 and 40% with respect to the tests carried out with microorganisms. Despite this, the HPLC method remains one of the most used methods in the literature.

The biological determination procedures of folic acid as the use of ligands bio-specific or immunoassays with antibodies (ELISA assay), are very sensitive and specific due to the strong interaction between antibody / folate ligand. And they are one clear alternative to the method by HPLC and microbiological especially due to the rapidity of obtaining results.

Although the biggest drawback they present these methods of determination by biospecific procedures is the absence of a measure of total folate.

In an exhaustive effort by the inventors to find compounds with solubility characteristics suitable for be used in chemosensing systems that allow detection qualitative and / or quantitative polycarboxylates (dicarboxylates, tricarboxylates), preferably folate and analogs thereof, the inventors of the present invention have surprisingly obtained and in a satisfactory way tripodal compounds based on squaramide that meet or allow to meet the following objectives mentioned below.

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An objective of the present invention is provide a tripodal compound with a squaramide base with some water solubility characteristics that allow its use in test methods with analytes hardly soluble in water.

Another objective of the present invention is provide a tripodal compound with a squaramide base with a design and a rigidity in its skeleton that allows its interaction with dicarboxylates.

Another objective of the present invention is provide a procedure for obtaining a compound Squaranide based tripodal according to the characteristics described in The above objectives.

Another objective of the present invention is provide a chemosensor system that allows interaction between the tripodal compound of squamide base indicated above and a fluorescence indicator.

Another objective of the present invention is provide a procedure that uses the chemosensor system mentioned above for qualitative detection and / or quantitative polycarboxylates (dicarboxylates, tricarboxylates), preferably folate for its logos, in a quick way, Simple, effective in commercial samples in water.

Another objective of the present invention is provide a kit that includes the indicated chemosensor system previously with which the procedure of detection indicated in the previous paragraph in a comfortable way and fast

Summary Description of the Invention

The present invention relates to a first aspect to a tripodal compound with a squared base of formula general (I)

2

in the that:

R1 is an alkyl ammonium salt C 3 -C 5 -N (CH 3) 3; Y

R2 is independently selected from yes, from an unsubstituted benzene ring; a benzene ring trisubstituted with radicals, the same or different from each other, selected from methyl or ethyl; and a polyamine.

In a second aspect, the present invention is refers to a procedure for obtaining compounds of general formula (I).

In a third aspect, the present invention is refers to a chemosensor system for qualitative detection and / or quantitative in aqueous samples of selected polycarboxylates from the group consisting of dicarboxylates and tricarboxylates, which understands:

to)

a receptor compound of general formula (I)

Y

b)

a fluorescent indicator

In a fourth aspect, the present invention is refers to a procedure for qualitative detection and / or quantitative of polycarboxylates selected from the group that consists of dicarboxylates and tricarboxylates, preferably the Dicarboxylates are folate and analogs thereof.

In a fifth aspect, the present invention is refers to a kit containing the mentioned chemosensor system previously.

Brief description of the figures

Figure 1A represents the thermogram corresponding to the valuation of the recipient of formula (I) where R_ {1} is an ammonium salt C 3 -N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and folic acid in a water and buffer system (TRIS / HCl) at pH = 9, at 294 K.

Figure 1B represents the curve that best fits fits the integrated values of the thermogram of Figure 1A to a 1: 1 stoichiometry model, once corrected with the solution of folic acid in a water and buffer system (TRIS / HCl) at pH = 9, a 294 K.

Figure 2A represents the thermogram corresponding to the valuation of the recipient of formula (I) where R_ {1} is an ammonium salt C 3 -N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and methotrexate in a water and buffer system (TRIS / HCl) at pH = 9, at 294 K.

Figure 2B represents the curve that best fits fits the integrated values of the thermogram of Figure 2A to a 1: 1 stoichiometry model, once corrected with the solution of folic acid in a water and buffer system (TRIS / HC1) at pH = 9, a 294 K.

Figure 3 represents the calibration line a 525 nm obtained for the system formed by the receptor of formula (I) where R_ {1} is an ammonium salt C 3 -N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and the carboxyfluorescein at different concentrations of folic acid, in a water and buffer system (TRIS / HCl) at pH = 9, at 294 K. In the inside the figure the adjustment results are shown mathematician of the line.

Figure 4 represents the calibration line a 525 nm obtained for the system formed by the receptor of formula (I) where R_ {1} is an ammonium salt C 3 -N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and the carboxyfluorescein at different concentrations of methotrexate, in a water and buffer system (TRIS / HCl) at pH = 9, at 294 K. In the inside the figure the adjustment results are shown matenatic of the straight.

Description of the invention

The present invention relates to a compound Squaramide-based tripodal of general formula (I)

3

in the that:

R1 is an alkyl ammonium salt C 3 -C 5 -N (CH 3) 3; Y

R2 is independently selected from yes, from an unsubstituted benzene ring; a benzene ring trisubstituted with radicals, the same or different from each other, selected from methyl or ethyl; and a polyamine.

In a preferred embodiment, R1 is a salt ammonium propyl-N (CH 3) 3.

The compounds of general formula (I) are obtained from the reaction between spacers of various nature, previously functionalized, of greater or lesser Molecular flexibility, with the starting sququaramides. These Squaramides are equipped with a quaternary ammonium salt terminal. The condensation reaction is carried out under very smooth and with good performance (> 80%). Schematically, the Synthesis process of compounds of general formula (I) comprises the stages of:

Stage TO

A1) reaction of ethyl squarate with N, N-dimethyl-C3-C5-alkylene diamine to obtain a product (a1) that is N, N-dimethyl-N '- (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -alkyl C 3 -C 5 amine;

A2) reaction of (a1) with methyl iodide to get iodide from N, N, N-trimethyl-N '- (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -alkyl C 3 -C 5 ammonium (a2).

Stage B

B1) reaction of 1,3,5-tris (bromomethyl) -benzene, where benzene can be substituted by equal radicals or different from each other selected from methyl or ethyl, with NaN_ {3}, to obtain the corresponding product (b1) which is 1,3,5-tris (azidomethyl) -benzene (or the corresponding substituted benzene);

B2) catalytic hydrogenation of (b1) for get the corresponding 1,3,5-tris (aminomethyl) -benzene (or the corresponding substituted benzene) (b2).

Stage C

C) reaction of the product obtained (a2) with the product obtained (b2) to produce the compound of general formula (I); or alternatively, reaction of the product (b2) with (a1) and subsequent reaction with CH 3 I to produce the compound of general formula (I);

in which if it is desired to obtain the compound of general formula (I) with R2 = polyamine, the product obtained (a2) reacts directly with tris (2-aminoethyl) amine without going through stage B and C.

Preferably, rent C 3 -C 5 and alkyl C 3 -C 5 correspond to propylene radicals and propyl, respectively.

The starting products or reagents used for the different reactions are available commercially or are easily obtainable from procedures described in the art.

It should be noted that the compound of the general formula (I) is usually obtained in the form of salt (in particular, triiodide).

Such compounds thus obtained are tripodal. which allows the predictable interaction with tricarboxylates. Do not However, surprisingly, the concrete structure of this compound makes it useful for interaction with dicarboxylates, since two of the arms of this compound form an association of electrostatic type (as a usual interaction with dicarboxylate) and the third arm interacts with the part hydrophobic dicarboxylate, which causes increased association exponentially. This structural feature allows the compounds of the present invention to be highly soluble in water at different pH values, and makes them optimal for assays with analytes that are hardly soluble in water.

Due to these characteristics, the compound Squaramide-based tripodal of general formula (I) is useful as receiver in a chemosensor system. Therefore, this invention also relates to a chemosensor system for the qualitative and / or quantitative detection in aqueous samples of polycarboxylates selected from the group consisting of dicarboxylates and tricarboxylates, comprising:

a) a receptor compound of the general formula (I)

4

in the that:

R1 is an alkyl ammonium salt C 3 -C 5 -N (CH 3) 3; Y

R2 is independently selected from yes, from an unsubstituted benzene ring; a benzene ring trisubstituted with radicals, the same or different from each other, selected from methyl or ethyl; and a polyamine;

Y

b) a fluorescent indicator.

In a preferred embodiment, R1 is a salt ammonium propyl-N (CH 3) 3.

In a preferred embodiment, the indicator fluorescent is selected from the group consisting of fluoresceins, Erythrosine, Eosin, Rose Bengal and Floxin.

In another preferred embodiment, the Dicarboxylates to be detected are folate and mime analogs.

"Folate" means acid anion folic acid or pteroylglutaic acid (PGA) which is a complex molecule formed by a bicyclic pterin moiety linked by a group methylene to paraaminobenzoic acid (pABA) which in turn and through of a peptide bond binds to an acid molecule L-glutamic. The rest of pteridine may exist both in oxidized and reduced form. The folic acid load, minus two, a dicarboxylate, is found in the rest of the acid L-glutamic.

5

When referring to "analogues of folate "in the present invention is understood to refer to compounds selected from the group consisting of:

- N- [4- [2- (2-Amino-4-methyl-7H-pyrrolo [2,3-d] pyrimidin-5-yl) ethyl] benzoyl] -L-Glutamic acid Registration Number
CAS: [306314-88-5].

- acid N- [4 - [[(2,4-diamino-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic. CAS Registry Number: [54-62-6];

- acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl) methyl] -2-propin-1-ylamino] benzyl] -L-Glutamic. CAS Registry Number: [76849-19-9];

- N- [4 - [[(1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl) methyl] amino] benzoyl] -L-Glutamic acid. Registration Number
CAS: [112887-79-3];

- acid (2S) - [[4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] amino] -4-sulfo- L-Glutamic. CAS Registry Number: [139347-01-6];

- (2S) - [5 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] -1,3-dihydro-1 -oxo-2H-
isoindol-2-yl] -pentanedioic. CAS Registry Number: [246545-24-4];

- acid N- [4 - [[(2-amino-1,4,5,6-tetrahydro-5-methyl-4-oxo-6-pteridinyl) methyl] amino] benzyl] -L-Glutamic acid.  CAS Registry Number: 28581-42-2;

- N- [4- (3-amino-1,2,5,6,6a, 7-hexahydro-1-oxoimidazo [1,5-f] pteridin-8 (9H) -yl) benzoyl] -L- acid Glutamic. Wildebeest-
mere CAS Registry: [3432-99-3];

- N- [4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] formylamino] benzoyl] -L-Glutamic acid No.
mere CAS Registry: [2800-34-2];

- dihydrogen phosphate N- [4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] -L-homoserine. CAS Registry Number: [866772-91-0];

- acid N - [[5- [2 - [(6R) -2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl] ethyl] -2-thienyl] carbonyl] -L-Glutamic. CAS Registry Number: [127228-54-0];

- calcium salt (1: 1) of the acid, N- [4 - [[(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic. CAS Registry Number: [1492-18-8];

- acid N- [4- [2 - [(6R) -2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl] ethyl] benzoyl ] -L-Glutamic. CAS Registry Number: [106400-81-1];

- 3-amino-8- [4 - [[[[(1S) -1,3-dicarboxypropyl] amino] carbonyl] phenyl] -1,2,5,6, (6aR), 7-hexahydro-1-oxo-imidazo [1,5-f] pteridinium. CAS Registry Number: [10360-12-0];

- acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl) methyl] methylamino] benzoyl] -L-Glutamic. CAS Registry Number: [2410-93-7];

- N- [4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic acid. Number
CAS Registry: [135-16-0];

- acid N- [4 - [[(1,4-dihydro-4-oxo-1,2,3-benzotriazin-6-yl) methyl] methylamino] benzoyl] -L-Glutamic. CAS Registry Number: [148315-60-0];

- acid (2S) - [[4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl ] amino] -4-sulfo-butanoic. CAS Registry Number: [866772-95-4];

- acid N- [4 - [[(2-amino-3,4,7,8-tetrahydro-4-oxo-6-pteridini1) methyl] amino] benzoyl] -L-Glutamic. CAS Registry Number: [4033-2 7-6];

- acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-? -Glutamyl-L-glutamic. CAS Registry Number: [19360-00-0];

- acid 2 - [[4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] amino] -4-phosphono-butanoic acid. CAS Registry Number: [170470-98-1];

- acid N- [4- [2- (2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-quinazolinyl) ethyl] benzoyl] -L-Glutamic. CAS Registry Number: [132014-31-4];

- acid N- (4-aminobenzoyl) -L-Glutamic. CAS Registry Number: [4271-30-1];

- Methotrexate;

- Pemetrexed; Y

- Raltitrexed.

Preferably, the "folate analogs" they are Methotrexate, Pemetrexed or Raltitrexed.

In the present invention, by "polycarboxylate" is strictly understood "dicarboxylates" and "tricarboxylates".

The present invention also relates to a procedure for the detection of selected polycarboxylates from the group consisting of dicarboxylates and tricarboxylates, which It comprises the stages of:

to)

dissolution of the sample in a pH 9 buffered solution containing the chemosensor system of the present invention with all its embodiments;

b)

fluorescence observation after of at least an hour.

In a particular embodiment, if said sample It is solid, the process further comprises a step of pretreatment of the sample consisting of solubilization of the solid sample in a pH 9 buffered solution by irradiation with ultrasound for at least 15 minutes, centrifugation and obtaining a supernatant liquid to be used in said stage to).

In another particular embodiment, the detection is quantitative and additionally includes the following stages:

C)

fluorescence measurement observed;

d)

correlation between said fluorescence observed and the concentration of said polycarboxylates in the analyte

In a preferred embodiment, the dicarboxylates to be detected are folate and analogues thereof, as they have been defined above.

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The present invention also relates to a kit comprising the chemosensor system described above in the present invention and the buffer in a suitable container. The kit preferably contains the instructions for using the system chemosensor for the detection of polycarboxylates (dicarboxylates, tricarboxylates).

The following are indicated below. examples for illustrative purposes only, and is not intended to in no way limit the scope of the present invention.

Examples

Synthesis of the recipient in which:

R_ {1} is an ammonium salt propyl-NR 3, wherein R is methyl; Y

R2 is a benzene ring not replaced

Synthesis of the N, N-dimethyl-N '- (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylamine

In a 100 mL round bottom flask, a solution of 2 g (11.8 mmol) of diethyl quarate in 10 mL of anhydrous diethyl ether is introduced. On it, a solution of 1 g of the N, N- dimethylpropylenediamine (9.8 mmol) in 10 mL of anhydrous diethyl ether is added dropwise under constant stirring and under argon. The mixture is left under stirring under an inert atmosphere for 12 hours. After this time, the appearance of a precipitate is observed. 20 mL of pentane are added and cooled in an ice-water bath, causing precipitation of the desired product. It is centrifuged and the liquid is removed by decantation. The resulting solid is washed three times with 10 mL of pentane and a few drops of diethyl ether. 2.21 g (9.78 mmol, 98%) of N, N -dimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylamine are obtained as a white solid which is dried on air.

Iodide synthesis of N, N, N-trimethyl-N '- (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylammonium

2.17 g (9.6 mmol) of the N, N -dimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylamine are dissolved in a 250 mL round bottom flask 60 mL of acetone. With the help of a syringe, 910 µL (14.6 mmol) of methyl iodide is introduced into the solution. The reaction mixture is allowed to reflux under an argon atmosphere for 12 hours. After this time, the crude oil is cooled to room temperature, 30 mL of pentane are added and placed in a water-ice bath. In this way the precipitation of a yellow powder is achieved, which is recovered by decantation and washed 3 times with 30 mL of pentane and a few drops of acetone. Air dried, obtaining 3.5 g (9.51 mmol, 99%) of N, N, N -trimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylammonium iodide .

Synthesis of 1,3,5-Tris (bromomethyl) -benzene

A solution containing 6 g (0.05 mol) of mesitylene, 27.5 g (0.15 mol) of N- bromosuccinimide (NBS), 0.27 g of benzoyl peroxide, and 150 mL of benzene is heated to reflux to start the reaction. The reaction is so exothermic that reflux is even maintained without heating. Later it is heated again to reflux for 1 hour you will go to complete the reaction. The solid is filtered, and the filtrate is washed with water and dried with anhydrous Na2SO4. Benzene is removed under reduced pressure to obtain a crystalline solid in the form of fine needles. The recrystallization of the crude is carried out in a mixture of ethanol and hexane (1: 1 by volume). Obtaining 16.2 g (0.046 mol, 92%) of 1,3,5-Tris (bromomethyl) -benzene pure.

Synthesis of 1,3,5-Tris (azidomethyl) -benzene

1 g dissolved in a round bottom flask (2.82 mmol) of 1,3,5-tris (bromomethyl) -benzene in 30 ml of anhydrous acetone. Then 1.48 g (22.77 are added mmol) of sodium azide. The mixture is refluxed for 8 hours, and is leave under stirring at room temperature 12 hours you will go. Past This time the solid is filtered on celite. The solvent is Eliminates under reduced pressure. The solid obtained is dissolved in CH 2 Cl 2, washed with 3 x 15 mL of water. It dries with sulfate anhydrous sodium And the solvent is distilled under reduced pressure. He product obtained, 0.68 g (2.80 mmol, 99%) of 1,3,5-tris (azidomethyl) -benzene It is a pale yellow oil.

Synthesis of 1, 3, 5-Tris (aminomethyl) -benzene

1.38 g (5.68 mmol) of tris (azidomethyl) -benzene in 40 mL of ethanol absolute in a hydrogenation flask. To this solution are added 170 mg of 10% Pd / C by weight to the mixture. The mixture is hydrogenated, to room temperature, for 4 hours at 30 mm Hg pressure. He Pd / C is filtered on celite and the filtrate is distilled under pressure reduced obtaining 0.63 g (3.82 mmol, 67%) of 1,3,5-tris (aminomethyl) -benzene as a pale yellow oil that does not need subsequent purification.

Synthesis of the triiodide of 1,3,5-Tris [methyl- [3-N ', N', N'-trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -benzene

In a round bottom flask, 368 mg (1 mmol) of the N, N, N -trimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylammonium iodide are dissolved in 20 ml of absolute methanol To this solution, under constant stirring and at room temperature, 54.4 mg (0.33 mmol) of 1,3,5-tris (aminomethyl) -benzene dissolved in 30 ml of absolute methanol is added dropwise. The addition is done in an inert atmosphere (Argon). Once finished, it is left at room temperature under stirring for 15 h. After this time, the formation of a spongy white precipitate is observed. 15 mL of pentane is added and left in a water-ice bath. The precipitate is recovered by centrifugation. The solid is washed three times with 10 ml of diethyl ether and a few drops of pentane. It is dried under vacuum for one hour, thus obtaining 280 mg (0.25 mmol, 75%) of the 1,3,5-tris [methyl- [3- N ', N', N ' -trimethylammoniumpropylamino] triiodide - 3,4-dioxo-1-cyclobutenyl] -benzene, as a white solid.

Synthesis of the recipient in which:

R_ {1} is an ammonium salt propyl-NR 3, wherein R is methyl; Y

R2 is a benzene ring trisubstituted with methyl

Synthesis of 1,3,5-Tris (azidomethyl) -2,4,6-trimethylbenzene

In a round bottom flask 2 g (4.6 mmol) of 1,3,5-Tris (bromomethyl) -2,4,6-trimethylbenzene are dissolved in 50 ml of N, N- dimethylformamide (DMF). Then 2 g (30.8 mmol) of sodium azide are added. The mixture is heated at 80 ° C for 8 hours, and then left under stirring at room temperature for an additional 12 hours. After this time, 40 ml of diethyl ether are added, to cause precipitation of BrNa. The precipitate is filtered on celite. The filtrate is washed with 5 x 40 mL of water. This is dried with anhydrous sodium sulfate. And the solvent is distilled under reduced pressure. The solid product obtained is recrystallized from ethyl acetate and hexane, obtaining 1.44 g (4.43 mmol, 97%) of 1,3,5-tris (azidomethyl) -2,4,6-trimethylbenzene.

Synthesis of 1,3,5-Tris (aminomethyl) -2,4,6-trimethylbenzene

286 mg (1 mmol) of tris (azidomethyl) -2,4,6-trimethylbenzene in 25 mL of absolute ethanol in a hydrogenation flask. Are added 30 mg of 10% Pd / C by weight at dissolution. The mixture is hydrogen, at room temperature for 4 hours at 30 mm Hg of Pressure. The Pd / C is filtered on celite and the filtrate is distilled at reduced pressure, obtaining 207 mg (1.73 mmol, 100%) of 1,3,5-tris (aminomethyl) -2,4,6-trimethylbenzene as a pale yellow solid that does not need subsequent purification.

Synthesis of the triiodide of 1,3,5-Tris [methyl- [3-N ', N', N'-trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -2,4,6-trimethylbenzene

In a round bottom flask, 368 mg (1 mmol) of the N, N, N -trimethyl- N ' - (2-ethoxy-3, 4-dioxo-1-cyclobutenyl) -propylammonium iodide are dissolved in 20 ml of ethanol. To this solution, under constant stirring and at room temperature, 69 mg (0.33 mmol) of 1,3,5-tris (aminomethyl) -2,4,6-trimethylbenzene dissolved in 20 ml is added dropwise of ethanol The addition is done in an inert atmosphere (Argon). Once finished, it is left at room temperature under stirring for 15 h. After this time, the formation of a white precipitate that is recovered by centrifugation is observed. The solid is washed twice with 10 mL of cold ethanol and then twice you will go with 10 mL of diethyl ether, dried under vacuum for 1 h, thereby obtaining 297 mg (0.254 mmol, 76%) of the triiodide of 1,3,5-tris [methyl- [3- N ', N', N ' -trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -2,4,6-trimethylbenzene as a pale brown solid.

Synthesis of the recipient in which:

R_ {1} is an ammonium salt propyl-NR 3, wherein R is methyl; Y

R2 is a benzene ring trisubstituted with ethyl.

Synthesis of 1,3,5-Tris (bromomethyl) -2,4,6-triethylbenzene

Slowly added at room temperature, 19.9 g (87.5 mmol) of zinc bromide to a mixture of paraformaldehyde (16.7 g, 556.3 mmol) and triethylbenzene (10 mL, 53.1 mmol) in 100 mL of HBr / AcOH (30% by weight). The mixture is heated to reflux at 90 ° C. for 17 hours, observing during this time the formation of white crystals The reaction crude is allowed to cool to room temperature, the solid formed by filtration separating. The solid is washed with water, and dried under vacuum overnight. From this form gives 22.79 g (51.7 mmol, 97%) of 1,3,5-Tris (bromomethyl) -2,4,6-triethylbenzene Like a white solid

Synthesis of 1,3,5-Tris (azidomethyl) -2,4,6-triethylbenzene

In a round bottom flask 0.2 g (0.45 mmol) of 1,3,5-Tris (bromomethyl) -2,4,6-triethylbenzene are dissolved in 12 ml of N, N- dimethylformamide (DMF). Then 0.2 g (3.08 mmol) of sodium azide are added. The mixture is heated at 80 ° C for 8 hours, and allowed to stir at room temperature an additional 12 hours. After this time, 20 ml of diethyl ether are added, to cause precipitation of BrNa. The crude is filtered on celite. The filtrate is washed with 5 x 40 mL of water. It is dried with anhydrous sodium sulfate. And the solvent is distilled under reduced pressure. The product obtained is recrystallized from ethyl acetate and hexane, obtaining 0.14 g, (0.43 mmol, 94.3%) of 1,3,5-tris (azidomethyl) -2,4,6-triethylbenzene.

Synthesis of 1,3,5-Tris (aminomethyl) -2,4,6-triethylbenzene

605 mg (1.8 mmol) of tris (azidomethyl) -2,4,6-triethylbenzene in 35 mL of absolute ethanol in a hydrogenation flask. Adding 61 mg of 10% Pd / C by weight to the mixture. The mixture is Hydrogen for 4 hours at 30 mm Hg pressure. The Pd / C is filter on celite and the filtrate is distilled under reduced pressure obtaining 430 mg (1.73 mmol, 95%) of 1,3,5-tris (aminomethyl) -2,4,6-triethylbenzene as a pale yellow solid that does not need subsequent purification.

Synthesis of 1,3,5-Tris - [(3-aminomethyl) -4- (2-N, N-dimethylaminopropylamino) -3-cyclobutene-1,2-dione] -2,4,6-triethylbenzene

In a round bottom flask 0.66 g (2.9 mmol) of N, N -dimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylamine are dissolved in 20 ml of ethanol. To this solution, under constant stirring, 0.22 g (0.89 mmol) of 1,3,5-tris (aminomethyl) -2,4,6-triethylbenzene dissolved in 20 ml of ethanol is added dropwise. The addition is done in an inert atmosphere (Argon). Once finished, it is left at room temperature under stirring for 15 h. After this time, the formation of a precipitate is observed, which is recovered by filtration. The solid is washed with 2 x 10 ml cold ethanol and dried under vacuum for one hour, thereby obtaining 0.44 g (0.56 mmol, 66%) of 1,3,5-tris - [(3-aminomethyl) -4- (2- N, N- dimethylaminopropylamino) -3-cyclobutene-1, 2-dione] -2,4,6-triethylbenzene as a white solid.

Synthesis of the triiodide of 1,3,5-Tris [methyl- [3-N ', N', N'-trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -2,4,6- triethylbenzene

In a round bottom flask, 758 mg (0.96 mmol) of 1,3,5-tris - [(3-aminomethyl) -4- (2- N, N- dimethylaminopropylamino) -3- are dissolved at room temperature cyclobutene-1,2-dione] -2,4,6-triethylbenzene in 40 mL of N, N -dime-
tilformamide (DMF). Then 60 mL of acetone are added. With the aid of a micro-syringe 162 microliters (2.6 mmol) of Methyl iodide are added. The solution is refluxed for 8 hours under argon. Observing that as the mixture heats up the solid dissolves. It is allowed to cool to room temperature and the flask is cooled in an ice-water bath to cause precipitation of the solid. The solid is isolated by centrifugation, and washed twice with 10 mL of acetone, and dried under vacuum for 1 hour, thus obtaining 456 mg (0.37 mmol, 39%) of 1,3,5 triiodide. -tris [methyl- [3- N ', N', N ' -trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -2,4,6-triethylbenzene as a white solid.

Synthesis of the recipient in which:

R_ {1} is an ammonium salt propyl-NR 3, wherein R is methyl; Y

R2 is a polyamine.

Synthesis of the triiodide of N, N, N-Tris [[3-N ', N', N'-trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -tris (2-aminoethyl) amine

In a round bottom flask, 368 mg (1 mmol) of the N, N, N -trimethyl- N ' - (2-ethoxy-3,4-dioxo-1-cyclobutenyl) -propylammonium iodide are dissolved in 20 ml of methanol To this solution, under constant stirring and at room temperature, 45.3 mg (0.33 mmol) of tris (2-aminoethyl) amine (train) dissolved in 15 ml of methanol is added dropwise argon. Then, it is left under stirring at room temperature for 12 h. After this time, the formation of a white precipitate is observed. The solvent is removed under reduced pressure. The solid is washed three times with 10 mL of diethyl ether, dried in vacuo, thereby obtaining 282 mg (0.253 mmol, 82%) of N, N, N -tris triiodide [[3- N ', N' , N ' -trimethylammoniumpropylamino] -3,4-dioxo-1-cyclobutenyl] -tris (2-aminoethyl) amine.

1.- Obtaining association isotherms by calorimetry ITC For folate

The isotherms have been obtained with a isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt C 3 -N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and folate (i.e. adding the folate on the receiver and not vice versa), to a stabilized temperature of 21 ° C. Both the receiver and the substrate they are dissolved in water containing 25% 1M buffer of TRIS: HC1 (pH = 9), at a concentration of approximately 2.06x10 -3 M receptor and 18.24x10 -3 M substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of the complexation, the parameter n shown in Table 1. Which correspond to Figures 1A and 1B.

For methotrexate

The isotherms (corresponding to figures 2A and 2B) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, by direct valuation between the receiver of formula (I) wherein R 1 is an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and methotrexate (it is say, adding methotrexate on the receptor and not the inverse), at a stabilized temperature of 21ºC. Both the receiver as the substrate are dissolved in water containing 25% of 1M TRIS buffer: HC1 (pH = 9), in a concentration approximately 1.03x10 -3 M of receptor and 8.76x10 -3 M of substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of the complexation, the parameter n shown in Table 1 plus ahead.

For pemetrexed

Isotherms (not shown) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and pemetrexed (it is say, adding the pemetrexed over the receiver and not to the inverse), at a stabilized temperature of 21ºC. Both the receiver as the substrate are dissolved in water containing 25% of 1M TRIS buffer: HC1 (pH = 9), in a concentration about 1.01x10 -3 M receptor and 8.85x10 -3 M of substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of complexation, parameter n. They are shown in Table 1 more ahead.

For raltitrexed

Isotherms (not shown) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and raltitrexed (it is say, adding the raltitrexed over the receiver and not to the inverse), at a stabilized temperature of 21ºC. Both the receiver as the substrate are dissolved in water containing 25% of 1M TRIS buffer: HC1 (pH = 9), in a concentration about 1.08x10 -3 M receptor and 9.45x10 -3 M of substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of complexation, parameter n. They are shown in Table 1 more ahead.

For citrate

Isotherms (not shown) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and citrate (it is say, adding citrate on the receiver and not vice versa), at a stabilized temperature of 21 ° C. Both the receiver and the substrate are dissolved in water containing 25% of 1M TRIS buffer: HC1 (pH = 9), at a concentration approximately of 1.91x10 <3> M receptor and 1.8x10 <2> M substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of complexation, parameter n. They are shown in Table 1 more ahead.

For tricarballate

Isotherms (not shown) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and tricarballate (it is say, adding the tricarballate on the receptor and not the inverse), at a stabilized temperature of 21ºC. Both the receiver as the substrate are dissolved in water containing 25% of 1M TRIS buffer: HCl (pH = 9), in a concentration approximately 1.05x10 -3 M receptor and 9.45x10 -3 M of substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you get the values of the process association constant, K_ {a}, the value of the reaction enthalpy, ΔH, and of the entropy, ΔS, as well as stoichiometry confirmation of complexation, parameter n. They are shown in Table 1 more ahead.

For trimesoate

Isotherms (not shown) have been obtained with an isothermal microcalorimeter (ITC) of the Microcal Inc. brand, for direct assessment between the recipient of formula (I) where R_ {1} it's an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and trimesoate (it is say, adding trimesoate over the receiver and not to the inverse), at a stabilized temperature of 21ºC. Both the receiver as the substrate are dissolved in water containing 25% of 1M TRIS buffer: HCl (pH = 9), in a concentration approximately 1.14x10 -3M of receptor and 9.84x10 -3M of substrate.

For this a total of 250 are added microliters of substrate solution to 1.8 ml of solution of receptor, in aliquots of 6 microliters each. The data obtained in this way are adjusted using the interaction model 1: 1 of the Microcal Origin (Delta H) program. With this setting you obtain the values of the process association constant, the value of the enthalpy of reaction, ΔH, and entropy, ΔS, as well as confirmation of stoichiometry of the complexation, parameter n. They are shown in Table 1 more ahead.

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TABLE 1 Association constants (K_asoc, M <-1>), Gibbs association free energy (\ cdotG, kJ / mol), enthalpy of association (\ cdotH, kJ / mol), association entropy (T \ cdotS, kJ / mol) for various polycarboxylates with a squaradic receptor in H2O at pH = 9.0 in a 1 M buffered solution TRIS-HCl, at 294 K

6

2.- Obtaining the concentration of folate in a acfol® sample

To make this determination they prepare three solutions "mother", the first with 4 (5) -carboxyfluorescein, the second with the receptor of formula (I) wherein R 1 is an ammonium salt propyl-N (CH 3) 3, and R 2 is a benzene ring trisubstituted with ethyl and finally another with Folate The three solutions are prepared with purified water with 25% HC1 / TRIS buffer (tris (hydroxymethyl) aminomethane)) 1M pH = 9. The solution containing carboxyfluorescein is 2.88x10-5 M. The solution containing the receptor is 6.26x10 -4 M and the solution containing folate is 444 ppm. From these stock solutions are prepared another four solutions of the Following way:

3 ml of the solution of carboxyfluorescein with 1 ml of purified water 25% buffer, obtaining a final concentration of 2.16x10-5 M. This dissolution is used to calibrate the emission intensity will go X-ray fluorescence, which is calibrated to 7.2 units of fluorescence.

A second solution is prepared from 3 ml of carboxif luorescein solution with 0.5 ml of solution of receiver and 0.5 ml of water with 25% buffer, obtaining final concentrations of 2.16x10-5 M carboxyfluorescein and of 6.26x10-5 M receptor.

The third solution is prepared from 3 ml of carboxyfluorescein solution, 0.5 ml of receptor and 0.5 ml of folate solution, obtaining final concentrations 2.16x10-5 M carboxyfluorescein, 6.26x10-5 M receptor and 55.5 ppm folate.

Finally, the fourth solution is prepared to from 3 ml of carboxyfluorescein solution, 0.5 ml of receptor solution and 0.5 ml sample solution unknown, obtaining final concentrations of 2.16x10-5 M of carboxy luorescein and 6.26x10-5 M receptor.

To prepare the sample solution Commercial (acfol®) is proceeded as follows:

A pill containing 5 mg of folic acid (acfol®) in 25 ml of a mixture of purified water with 25% TRIS / 1M HCl buffer. Treating the suspension in a bathroom ultrasound for 10 minutes. The resulting suspension is centrifuge at 7,000 rpm / min for 10 minutes. The liquid supernatant is transferred to a 25 ml volumetric flask flush with Millipore water with 25% TRIS / 1M HCl buffer.

The above solutions prepared in this way are let them age a minimum of 2 hours. After this time is measured the observed fluorescence, excitation wavelength 450 nm, emission wavelength 525 nm.

To obtain the calibration line, the solution containing only the 4 (5) -carboxyfluorescein and the receptor with the solution containing the 4 (5) -carboxyfluorescein and the receptor, and the folate The reading of each point is done three times, (see Figure 3).

Obtaining the concentration of the sample unknown is obtained from reading the intensity of the fourth solution, which contains the 4 (5) -carboxyfluorescein, receptor and sample unknown, calculating it from the calibration line (Figure 3) the concentration of folate in ppm. The value obtained, 4.6 mg, is the arithmetic mean of three independent measures being the error relative of 8%.

3.- Obtaining the concentration of methotrexate in a Metoiect® sample

To make this determination they prepare three solutions "mother", the first with 4 (5) -carboxyfluorescein, the second with the receptor of formula (I) wherein R 1 is an ammonium salt propyl-N (CH 3) 3 and R 2 is a benzene ring trisubstituted with ethyl and finally another with methotrexate The three solutions are prepared with purified water with 25% HCl / TRIS buffer (tris (hydroxymethyl) aminomethane)) 1M pH = 9. The solution containing carboxy luorescein is 2.72x10-5 M. The solution containing the receptor is 5.13x10 -4 M and the solution containing methotrexate is 438 ppm. From these stock solutions are prepared another four solutions of the Following way:

3 ml of the solution of carboxyfluorescein with 1 ml of purified water 25% buffer, final concentration 2.05x10-5 M. This solution is used. to calibrate the maximum fluorescence emission intensity, which It is calibrated to 7.2 fluorescence units.

A second solution is prepared from 3 ml of carboxyfluorescein solution with 0.5 ml of receiver and 0.5 ml of water with 25% buffer, obtaining final concentrations 2.05x10-5 M carboxy luorescein and 6.42x10-5 M receptor.

The third solution is prepared from 3 ml of carboxyfluorescein solution, 0.5 ml of receptor and 0.5 ml of methotrexate solution, obtaining final concentrations of 2.05x10-5 M carboxy luorescein, 6.42x10-5: M receptor and 54.7 ppm methotrexate.

Finally, the fourth solution is prepared to from 3 ml of carboxyfluorescein solution, 0.5 ml of receptor solution and 0.5 ml sample solution unknown, obtaining final concentrations of 2.05x10-5 M of carboxy luorescein and 6.42x10-5 M receptor.

To prepare the sample solution commercial (metoject®) proceed as follows:

120 microliters are diluted in a volumetric flask (1.2 mg) of the sample in 10 ml of purified water with 25% of 1M TRIS / HCl buffer.

The above solutions prepared in this way are let them age a minimum of 2 hours. After this time is measured the observed fluorescence, excitation wavelength 450 nm, emission wavelength 525 nm.

To obtain the calibration line, the solution containing only the 4 (5) -carboxyfluorescein and the receptor with the solution containing the 4 (5) -carboxyfluorescein and the receptor, and the methotrexate The reading of each point is done three times. (See Figure 4).

Obtaining the concentration of the sample unknown is obtained from reading the intensity of the fourth solution, which contains the 4 (5) -carboxyfluorescein, receptor and sample unknown, calculating it from the calibration line (Figure 4) the concentration of methotrexate in ppm. The value obtained, 1.37 mg, is the arithmetic mean of three independent measures being the relative error of 12%.

Claims (12)

1. Squaramide based tripodal compound of general formula (I)

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7 in the that: R1 is an alkyl ammonium salt C 3 -C 5 -N (CH 3) 3; Y R2 is independently selected from yes, from an unsubstituted benzene ring; a benzene ring trisubstituted with radicals, the same or different from each other, selected from methyl or ethyl; and a polyamine. 2. Squaramide based tripodal compound according to claim 1, wherein R1 is an ammonium salt propyl-NR 3. 3. Procedure for obtaining the compound according to claim 1 or 2 comprising the steps of:  Stage A A1) reaction of ethyl squarate with N, N-dimethyl-C3-C5-alkylene diamine to obtain a product (a1) that is N, N-dimethyl-N '- (2-ethoxy-3,4-dioxo-1- cyclobutenyl) -C 3 -C 5 alkyl amine; A2) reaction of (a1) with methyl iodide to get iodide from N, N, N-trimethyl-N '- (2-ethoxy-3, 4-dioxo-1-cyclobutenyl) -alkyl C 3 -C 5 ammonium (a2).  Stage B B1) reaction of 1,3,5-tris (bromomethyl) -benzene, where benzene can be substituted by equal radicals or different from each other selected from methyl or ethyl, with NaN_ {3} to obtain the corresponding product (b1) which is 1,3,5-tris (azidomethyl) -benzene (or the corresponding substituted benzene); B2) catalytic hydrogenation of (b1) for get the corresponding 1,3,5-tris (aminomethyl) -benzene (or the corresponding substituted benzene) (b2).  Stage C C) reaction of the product obtained (a2) with the product obtained (b2) to produce the compound of general formula (I); or alternatively, reaction of the product (b2) with (a1) and subsequent reaction with CH 3 I to produce the compound of general formula (I); in which if it is desired to obtain the compound of general formula (I) with R2 = polyamine, the product obtained (a2) reacts directly with tris (2-aminoethyl) amine without going through stages B and C. 4. Method according to claim 3, in the one that rent C 3 -C 5 and alkyl C 3 -C 5 correspond to propylene radicals and propyl, respectively. 5. Chemosensor system for detection qualitative and / or quantitative in aqueous samples of polycarboxylates selected from the group consisting of dicarboxylates and tricarboxylates, comprising: a) a receptor compound according to claim 1 or 2; Y b) a fluorescent indicator. 6. Chemosensor system according to claim 5 in which the fluorescent indicator is selected from the group that It consists of fluoresceins, Erythrosine, Eosin, Rose Bengal and Floxin 7. Procedure for the detection of polycarboxylates selected from the group consisting of dicarboxylates and tricarboxylates, comprising the steps of:

to)

dissolution of the sample in a pH 9 buffered solution containing the chemosensor system according to claim 5 or 6;

b)

fluorescence observation after of at least an hour.

8. Method according to claim 7 in the that said sample is solid, further comprising a step of pretreatment of the sample consisting in the solubilization of the solid sample in a pH 9 buffered solution by irradiation with ultrasound for at least 15 minutes, centrifugation and obtaining a supernatant liquid to be used in said stage to). 9. Method according to claim 7 or 8, wherein said detection is quantitative and additionally comprises the following stages:

C)

fluorescence measurement observed;

d)

correlation between said fluorescence observed and the concentration of said polycarboxylates in the analyte

10. Kit comprising the chemosensor system according to claims 5 or 6. 11. Chemosensor system according to claims 5 or 6, method according to claims 7 a 9 or kit according to claim 10, wherein the dicarboxylates they are folate or their logos selected from the group that consists in: - acid N- [4- [2- (2-amino-4-methyl-7H-pyrrolo [2,3-d] pyrimidin-5-yl) ethyl] benzoyl] -L-Glutamic; - acid N- [4 - [[(2,4-diamino-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic; - acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl) methyl] -2-propin-1-ylamino] benzyl] -L-Glutamic; - acid N- [4 - [[(1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl) methyl] amino] benzoyl] -L-Glutamic; - acid (2S) - [[4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] amino] -4-sulfo- L-Glutamic; - acid (2S) - [5 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] -1,3-dihydro-1-oxo -2H-isoindol-2-yl] -pentanedioic; - acid N- [4 - [[(2-amino-1,4,5,6-tetrahydro-5-methyl-4-oxo-6-pteridinyl) methyl] amino] benzyl] -L-Glutamic; - acid N- [4- (3-amino-1,2,5,6,6a, 7-hexahydro-1-oxoimidazo [1,5-f] pteridin-8 (9H) -yl) benzoyl] -L-Glutamic; - acid N- [4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] formylamino] benzoyl] -L-Glutamic; - dihydrogen phosphate N- [4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] -L-homoserine; - acid N - [[5- [2 - [(6R) -2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl] ethyl] -2-thienyl] carbonyl] -L-Glutamic; - calcium salt (1: 1) of the acid, N- [4 - [[(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic; - acid N- [4- [2 - [(6R) -2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl] ethyl] benzoyl ] -L-Glutamic; - 3-amino-8- [4 - [[[[(1S) -1,3-dicarboxypropyl] amino] carbonyl] phenyl] -1,2,5,6, (6aR), 7-hexahydro-1-oxo-imidazo [1,5-f] pteridinium; - acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl) methyl] methylamino] benzoyl] -L-Glutamic; - acid, N- [4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic; - acid N- [4 - [[(1,4-dihydro-4-oxo-1,2,3-benzotriazin-6-yl) methyl] methylamino] benzoyl] -L-Glutamic; - acid (2S) - [[4 - [[(2-amino-3,4,5,6,7,8-hexahydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl ] amino] -4-sulfo-butanoic; - acid N- [4 - [[(2-amino-3,4,7,8-tetrahydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L-Glutamic; - acid N- [4 - [[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl) methyl] amino] benzoyl] -L- γ-glutamyl-L-glutamic; - acid 2 - [[4 - [[(2-amino-1,4-dihydro-4-oxopyrido [2,3-d] pyrimidin-6-yl) methyl] amino] benzoyl] amino] -4-phosphono-butanoic acid; - acid N- [4- [2- (2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-quinazolinyl) ethyl] benzoyl] -L-Glutamic; - acid N- (4-aminobenzoyl) -L-Glutamic; - Methotrexate; - Pemetrexed; Y - Raltitrexed. 12. Chemosensor system according to claim 11, method according to claim 11 or kit according to the claim 11, wherein the dicarboxylates are folate, methotrexate, pemetrexed or raltitrexed.