BR102021024217A2 - B-CYCLODEXTRIN DERIVATIVE COMPOUND, COMPOSITION, PROCESS FOR OBTAINING AND USES - Google Patents

Abstract

The present invention describes the synthesis of cyclodextrin derivatives through the addition of polyhydroxy acid substituents (lactobionic, maltobionic, gluconic and galacturonic acid). Specifically, the present invention comprises preferably monosubstituted β-cyclodextrin derivatives of general structure (I), compositions comprising such derivatives, their process for obtaining and uses. The objective is to obtain cyclodextrin derivatives that have greater water solubility and compatibility with blood cells, when compared to natural β-cyclodextrin. The present invention lies in the fields of organic, pharmaceutical, cosmetic, food, polymer chemistry and drug delivery.

Description

B-CYCLODEXTRIN DERIVATIVE COMPOUND, COMPOSITION, PROCESS FOR OBTAINING AND USES Field of Invention

[0001] The invention refers to new compounds derived from natural cyclodextrin, compositions comprising the derivatives, their process of obtaining and uses, being located in the area of organic chemistry, pharmaceuticals, cosmetics, food, polymer chemistry, drug vectorization.

Background of the Invention

[0002] In the search for the state of the art in scientific and patent literature, the following documents were found that deal with the subject:

[0003] Document KR20180033862, Hangover-alleviating beverage and method of preparing same, the formulation has specificity for asialoglycoprotein receptors (ASGP) due to the presence of a surfactant with affinity for this receptor. Cyclodextrin is added in order to solubilize fat-soluble compounds

[0004] In patent application CN108743787 A, Throat lozenge for relieving sorethroat to restore voice and preparation method thereof, a pharmaceutical preparation is reported that may contain lactobionic acid, as a flavoring agent, and cyclodextrin as a complexing agent. The application of lactobionic acid in this formulation is different from that proposed by the present invention.

[0005] In patent CN101045063B, Clarithromycin water soluble preparation for injection use, the preparation of a water-soluble clarithromycin formulation for injection is reported. The formulation is prepared with clarithromycin or analogue and β-CD or pharmacologically acceptable water-soluble derivatives. β-CD derivatives are: hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, glucosyl-β-cyclodextrin, or sulfoalkyl-β-cyclodextrin. Derivatives synthesized by the present invention are different from those described in this patent. Still, in CN101045063B the synthesis of these derivatives is not reported.

[0006] In patent application US20180207193 A1, Compositions and methods for improving enzyme replacement therapy for lysosomal storage diseases, reports a formulation to improve the effectiveness of enzyme replacement therapy for lysosomal storage diseases. The formulation contains a cyclodextrin derivative (cyclodextrin polymer (CDP)-based nanoparticle), or another polymer, as a gene carrier. Furthermore, the carrier can be chemically conjugated to one or more moieties that bind to the asialoglycoprotein (ASGPR), apolipoprotein or glycosaminoglycan receptor.

[0007] In patent application CN108187059 A, Stimuli-responsive amphipathic cyclodextrin polymer medicine carrier and preparation and application thereof in preparing controlled release medicine refers to the pharmaceutical preparation of a polymeric drug carrier. The cyclodextrin-containing polymer comprises: cyclodextrin as a core, addition of a hydrophilic chain at position 6 of at least one glucose unit of the cyclodextrin, and replacement by a hydrophobic molecule at position 2 or 3 of at least one glucose unit. The hydrophobic chain corresponds to: C1-C20 alkyl chain, C1-C20 hydrocarbyl chain segment substituted by at least one halogen substituent, C1-C6 alkyl or C1-C6 alkoxy and benzyl. The hydrophobic chain corresponds to: C5-C20 straight or branched chain saturated alkyl chain segment. Polymers are synthesized for controlled and targeted release to tumor cells. However, the cyclodextrin derivatives synthesized by the present invention are different from those reported in this patent.

[0008] In the patent application CN110801433 A, Targeted drug composition for co-loading amphotericin B and adriamycin and application of targeted drug composition, a pharmaceutical composition for the treatment of leishmaniasis is reported, composed of β-cyclodextrin propionate modified with mannose, amphotericin B and doxorubicin. Mannose modified β-cyclodextrin propionate is prepared from 1 part of β-cyclodextrin, 7 parts of the mannose residue and 14 parts of propionyl carbon chain. The cyclodextrin derivative synthesized in this patent is different from that proposed by the present invention. The application of this derivative is for the treatment of leishmaniasis, different from the product of the present invention.

[0009] In patent CN105106969B, Novel saccharide nanomicelle and preparation method and application thereof, the synthesis of nanomicelles in which the building block is composed of cyclodextrins is reported. Cyclodextrin molecules are modified into primary and secondary hydroxyls. The primary hydroxyl of cyclodextrin is modified by monosaccharide or oligosaccharide molecules (such as mannitose and galactose) with hydrophilic and vectoring properties. The secondary hydroxyl is modified by aliphatic chains in order to be used as a hydrophobic moiety. The prepared nanomycella can be used as a vectorized carrier for all types of anticancer drugs. In this patent the substitutions of the cyclodextrin are made in the primary hydroxyls and in the secondary hydroxyls. Also in this patent, the synthesized cyclodextrin derivatives are interconnected to form a nanomycell, a structure different from that proposed by the present invention.

[0010] Patent JP3934705 B2, Cyclodextrin composition reports the obtainment of soluble cyclodextrins with low hemolytic activity. The cyclodextrin derivatives protected in this patent are: methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, glucosyl-β-cyclodextrins, Maltosyl-β-cyclodextrin, ether-β-cyclodextrin and sulfonated disulfidecanoic-β-cyclodextrin. Derivatives are different from those proposed by the present invention. In this patent, targeting to liver cells expressing the ASGP receptor is not reported.

[0011] Patent JP6468777 B2, Improved type folic acid modified cyclodextrin derivative, reports the synthesis of a derivative of folic acid-β-cyclodextrin linked to polyethylene glycol, obtaining a more water-soluble derivative compared to folic acid-β-cyclodextrin. The synthesized derivative has tumor accumulating ability and is available as a drug delivery system applicable in various formulations. The cyclodextrin derivative synthesized in this patent is different from the derivative of the present invention.

[0012] Patent CN102220128B B, Low-toxicity functionalized quantum dot modified by amination beta-cyclodextrin and preparation method thereof, refers to obtaining a quantum dot functionalized by amino-β-cyclodextrin. When folic acid is coupled to the amine group, the quantum dot can be used for tumor detection by fluorescence. However, the final product is different from that proposed by the present invention.

[0013] Patent CN103212091B B, Preparation method of hepatocarcinoma target CT (Computed Tomography) contrast medium based on Au DENPs-LA (Dendrimer-entrapped Gold Nanoparticles-Lactobionic acid), refers to a method of preparing a contrast, for computed tomography, directed to hepatocellular carcinoma cells. The contrast medium is composed of dendrimers loaded with lactobionic acid functionalized gold nanoporticles. However, the transport system is different from that employed by the present invention.

[0014] In the patent application WO201759547 A1, Multifunctional polyanionic cyclodextrin dendrimers the synthesis of a multifunctional cyclodextrin dendrimer is reported. The derivative comprising at least one anionic residue and at least one non-ionic residue attached to the cyclodextrin ring structure. The use of the dendrimer is for the transport of drugs. The dendrimer may contain a functional group aimed at vectorizing drug transport. The groups described are: biotin, folic acid, lactose, N-acetyllactosamine, D-glucose, D-mannose, "N-acetyl-D-glucosamine, L-fucose, N-acetylD-glucosamine, N-acetylneuraminic acid. The structure of the dendrimer is different from that proposed by the present invention.

[0015] The patent application CN104208703 A, Polyethylene glycol-lactobionic acid modified aminated hectorite nano particle as well as preparation method and application thereof defines a method of preparation and application of the namomaterial composed of polyethylene glycol-lactobionic acid. The nanomaterial has specific affinity for cells that express the asialoglycoprotein receptor (ASGP-R) and can be used as a carrier for antitumor drugs. Lactobionic acid is used as a vector for cells with ASGP-R. However, the transport system is different from that proposed by the present invention.

[0016] Patent CN105902521B B, Sodium alginate-phytosterol-lactobionic acid targeting nanoparticles, preparation method and application of targeting nanoparticles and drug-carrying nanoparticles reports the synthesis and application of sodium alginate-phytosterol-lactobionic acid nanoparticles. These nanoparticles are used as drug carriers. Lactobionic acid confers targeting to specific cells. What differs from the one proposed by the present invention is the drug transport system.

[0017] Patent CN104264479B B, Preparation method of lactobionic acid functional nano fibers for capturing cancer cells, reports a method of preparing nanofibers functionalized with lactobionic acid to capture cancer cells. The presence of lactobionic acid on the surface of the nanofibers confers specific capture by asialoglycoprotein receptors expressed in liver cancer cells. However, the transport system is different from that proposed by the present invention.

[0018] The patent application CN102863557 A, Preparation method and application of fatty acid-trimethyl chitosan polymer modified by lactobionic acid describes a method of preparation and application of a polymer of trimethyl chitosan fatty acid modified with lactobionic acid. Trimethyl chitosan is used as a carrier and the presence of lactobionic acid as a vector for tumor cells. The transport system of this patent application is different from the one proposed by the present invention.

[0019] The patent application CN104888223 A, Preparation method of modified graphene oxide (GO) composite material containing lactobionic acid (LA), reports a method for preparing a composite material containing graphene oxide (GO) and lactobionic acid (LA) . This material can be applied for sustained and vectorized release of drugs or specific molecules for cell imaging. However, the transport system is different from that proposed by the present invention.

[0020] The patent application CN104353087 A, Preparation method of polyethyleneimine based CT/MR (computed tomography/magnatic resonance imaging) bimodal imaging nano contrast agent with lactobionic acid targeting function refers to a method of preparing a nano contrast agent for computed tomography and magnetic resonance imaging. The contrast is composed of polyethyleneimine (carrier) and lactobionic acid (vectoring). Thus, the transport system differs from that proposed by the present invention.

[0021] In patent application CN104490772 A, Liver targeting taxol nanometer suspension and preparation method thereof, a method of preparation and application of a nanometer suspension of taxol is reported. The suspension consists of taxol (active principle) and poloxamer F127 modified with lactobionic acid (stabilizer). Asialoglycoprotein receptors on parenchymal cells on the surface of the liver recognize the galactose residue of lactobionic acid, and thus the drug is targeted to the liver. This compound reports vectoring using lactobionic acid.

[0022] The patent application CN102949728 A, Meso-porous silicon nano-drug carrier with both reduction responsiveness and targeting ability and preparation method thereof reports a method of production and application of a mesoporous silicon nano-drug with carrier and targeting capacity . The lactobionic acid molecule added on the meso-porous surface is used as a vector by affinity with the cell membrane surface receptor of liver cancer cells (ASGP-R). The transport system is different from the one proposed by the present invention.

[0023] The patent application CN111000826 A, Chemical photothermal therapy and targeting collaborated drug for treatment of liver cancer and preparation method thereof reports a method of preparation and application of a chemical photothermal therapy and drug targeting for the treatment of liver cancer. The product is composed of graphene oxide, lactobionic acid and curcumin. The galactose residue of lactobionic acid specifically recognizes the asialoglycoprotein receptor that is overexpressed on the surface of liver cancer cells. However, the patent application uses graphene oxide as a carrier of molecules.

[0024] Patent CN102302782B B, Preparation method of hepatoma carcinoma cell-targeted polyamido-amine dendrimer support reports a method of preparation and application of a polyamido-amine dendrimer support targeted to hepatocarcinoma cells. Dendrimers were functionalized with lactobionic acid for vectorization. The support can be used as a targeted carrier of drugs, genes or diagnostic probe molecules. However, the transport system is different from that proposed by the present invention.

[0025] Patent CN104146946B B, Oxidized graphene medicine-carried composite material for liver cancer targeting and preparation method of composite material refers to a method of preparation and application of a drug carrier directed to liver tumor cells based on oxide of graphene. However, the difference in relation to the present invention is in the transport system.

[0026] Patent application CN104689334 A, Hepatoma targeting carbon nano tube loaded with doxorubicin hydrochloride and preparation method thereof describes a method of preparing carbon nanotube loaded with doxorubicin targeted to hepatocarcinoma cells. The nanotube is functionalized with chitosan-lactobionic acid. The direction is given by the affinity of the galactose residue of lactobionic acid to the asialoglycoprotein receptors present in the cellular membrane of hepatic tumor cells. However, the transport system is different from that proposed by the present invention.

[0027] Document CN1210310C C, N-lactose acyl chitosan, reports the synthesis of a chitosan derivative functionalized with lactobionic acid. The product has a high degree of substitutions and vectorization property for liver cells. In the same way as proposed for the product lactobionic acid-β-cyclodextrin, lactobionic acid is used by selective affinity to liver cell receptors. However, the transport system is different from that proposed by the present invention.

[0028] Patent KR101357899 B1, Galactosylated polyethylene glycolchitosan-graft-spermine copolymer as a hepatocyte targeting gene carrier and gene therapy using the same reports the obtainment and application of a lactosylated polyethylene glycol-chitosan-g-spermine copolymer. Lactobionic acid bound to polyethylene glycol provides targeting to hepatocytes. This pharmaceutical composition is applied for gene transport for gene therapy. However, the transport system and application as gene therapy differ from the present invention.

[0029] Patent CN107875158B B, Preparation method of carrier free nano drug for chemotherapy/light treatment describes a method of preparing a carrier free nano drug for chemotherapy. The carrier is composed of a hydrophobic anticancer drug (ursolic acid), a water-soluble vector (lactobionic acid), and an amphipathic photosensitizer (indocyanine green). However, the transport system is different from that proposed by the present invention.

[0030] The patent application IN201611023040 A, Dipeptide based nanostructures for targeted delivery of mirna for hcc treatment reports the synthesis and application of nanostructures based on dipeptide, containing miRNA, targeted for the treatment of hepatocarcinoma (HCC). The transport of drugs/genes to the HCC is processed through asialoglycoprotein receptor (ASGP-R) mediated endocytosis using galactosylated nanocarriers. The lactobionic acid linked to the nanocarriers provides the galactose moiety for vectorization. However, the transport system is different from that proposed by the present invention.

[0031] Thus, depending on the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity compared to the state of the art.

[0032] Considering this panorama of the potential applicability of CDs in several areas, the problem of water solubility and limited biocompatibility of β-CD and the possibility of drug vectorization. The present invention proposes new compounds derived from CD.

Summary of the Invention

[0033] In this way, the present invention solves the problems of the state of the art from new compounds derived from cyclodextrin with the replacement of at least one hydroxyl by a substituent, in which the substituent comprises R1 linked to R2, with R1 being a spacer and R2 a hydroxy acid, polyhydroxy acid or aldonic acid and wherein R1 is linked to cyclodextrin.

[0034] This new derivative of substituted CD has greater water solubility and biocompatibility when compared to non-substituted CD, enabling its use in aqueous formulations for parenteral administration. Additionally, by binding the lactobionic acid (LA) molecule as an R2 substituent, the resulting derivative would be vectorized to the hepatic tissue where there is expression of asialoglycoprotein receptors (ASGP-R), since this receptor has affinity for LA.

[0035] Therefore, the greater water solubility and compatibility with blood cells also makes the new derivative described here as an alternative to derivatives already on the market. For the national market, this may represent an advance in autonomy in the production of this pharmaceutical ingredient.

[0036] A first object of the present invention comprises compounds derived from cyclodextrin with substitution of at least one hydroxyl by a substituent, in which the substituent comprises R1 linked to R2, R1 being a spacer and R2 a hydroxy acid, polyhydroxy acid or acid aldonic and where R1 is linked to cyclodextrin.

[0037] In a second object, the present invention comprises the composition (inclusion or non-inclusion complexes with drugs or other molecules, nanometric systems) comprising the compounds derived from cyclodextrin with the substitution of at least one hydroxyl by a substituent, wherein the substituent comprises R1 linked to R2, where R1 is a spacer and R2 is a hydroxy acid, polyhydroxy acid or aldonic acid, and where R1 is linked to cyclodextrin.

[0038] In a third object, the present invention comprises the process for obtaining the compound, which comprises the following steps: i) obtaining an intermediate compound β-cyclodextrin (β-CD) with at least one hydroxyl substituted by a leaving group ; ii) replacement of the leaving group of the intermediate compound of βcyclodextrin obtained by step i) by the spacer group; and iii) reacting the spacer group of the compound obtained in step ii) with at least one of hydroxy acid, polyhydroxy acid or aldonic acid in a reaction medium comprising a mixture of organic solvents.

[0039] In a fourth object, the present invention comprises the use of the compound derived from cyclodextrin in the preparation of chemical compositions, cosmetics, pharmaceuticals or food products.

[0040] In a fifth object, the compound is used to prepare a medicine in which the compound is a carrier for bioactive molecules or marker molecules.

[0041] These and other objects of the invention will be immediately appreciated by those skilled in the art and will be described in detail below.

Brief Description of Figures

[0042] The following figures are presented:

[0043] Figure 1 shows the GC-MS chromatogram of 1-(p-Toluenesulfonyl)imidazole.

[0044] Figure 2 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of 6-OToluenesulfonyl-β-cyclodextrin (6-Ts-β-CD).

[0045] Figure 3 shows: [A] 6-O-Toluenesulfonyl-β-cyclodextrin (6-Ts-β-CD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/MS by direct infusion.

[0046] Figure 4 shows the HPLC-HRMS chromatogram of 6-O-Toluenesulfonylβ-cyclodextrin (6-Ts-β-CD) with 94.2% purity. Retention time of 6-Tsβ-CD in 7.4 min and β-cyclodextrin (β-CD) in 6.3 min.

[0047] Figure 5 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of mono6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD).

[0048] Figure 6 shows the 13C NMR spectrum (300 MHz, DMSO-d6) of mono6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD).

[0049] Figure 7 shows: [A] Mono-6-ethylenediamine-β-cyclodextrin (6-EDA-βCD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/MS, for direct infusion.

[0050] Figure 8 shows the chromatogram (HPLC-HRMS) of Mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD). Retention time of 6-EDA-βCD (m/z 1177) in 4.5 min and β-CD (m/z 1135) in 6.1 min.

[0051] Figure 9 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of Mono6-lactobionic acid-β-cyclodextrin (6-LA-β-CD).

[0052] Figure 10 shows the 13C NMR spectrum (300 MHz, DMSO-d6) of Mono-6-lactobionic acid-β-cyclodextrin (6-LA-β-CD).

[0053] Figure 11 shows: [A] Mono-6-lactobionic acid-β-cyclodextrin (6-LAβ-CD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/MS, by direct infusion.

[0054] Figure 12 shows the chromatogram (HPLC-HRMS) of Mono-6-lactobionic acid-β-cyclodextrin (6-LA-β-CD). Mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD) retention time (m/z 1177) in 4.6 min, 6-LA-β-CD (m/z 770) in 5, 0 min and lactobionic acid (m/z 381) in 6.5 min.

[0055] Figure 13 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of Mono6-Maltobionic acid-β-cyclodextrin (6-MA-β-CD).

[0056] Figure 14 shows the 13C NMR spectrum (300 MHz, DMSO-d6) of Mono-6-maltobionic acid-β-cyclodextrin (6-MA-β-CD).

[0057] Figure 15 shows: [A] Mono-6-maltobionic acid-β-cyclodextrin (6-MAβ-CD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/MS, by direct infusion.

[0058] Figure 16 shows the HPLC-HRMS chromatogram of Mono-6-maltobionic acid-β-cyclodextrin (6-MA-β-CD). Mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD) retention time (m/z 1177) in 4.6 min, 6-MA-β-CD (m/z 770) in 5, 0 min and maltobionic acid (m/z 381) in 6.5 min.

[0059] Figure 17 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of Mono6-gluconic acid-β-cyclodextrin (6-GLU-β-CD).

[0060] Figure 18 shows the 13C NMR spectrum (300 MHz, DMSO-d6) of Mono-6-gluconic acid-β-cyclodextrin (6-GLU-β-CD).

[0061] Figure 19 shows: [A] Mono-6-gluconic acid-β-cyclodextrin (6-GLU-βCD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/MS, by direct infusion.

[0062] Figure 20 shows the HPLC-HRMS chromatogram of Mono-6-gluconic acid-β-cyclodextrin (6-GLU-β-CD). Retention time of mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD) (m/z 1177) in 4.6 min, (6-GLU-βCD). (m/z 1355) in 5.1 min and gluconic acid (m/z 219) in 6.7 min.

[0063] Figure 21 shows the 1H NMR spectrum (300 MHz, DMSO-d6) of Mono6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD).

[0064] Figure 22 shows the 13C NMR spectrum (300 MHz, DMSO-d6) of Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD).

[0065] Figure 23 shows: [A] Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD), [B] Mass spectrum (HRMS-ESI-QTOF) and [C] MS/ MS, by direct infusion.

[0066] Figure 24 shows the chromatogram (HPLC-HRMS) of Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD). Retention time of mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD) (m/z 1177) in 4.6 min, (6-GAL-β-CD) (m/z 1353) in 5.0 min and galacturonic acid (m/z 217) in 6.7 min.

[0067] Figure 25 shows the cell viability (%) in vitro by the MTT method, of HepG2 cells (48 h) [A] and Hek 293 cells (24 h) [B], incubated with Mono6-ethylenediamine-β- cyclodextrin (6-EDA-β-CD), Mono-6-lactobionic acid-β-cyclodextrin (6-LA -β-CD), Mono-6-maltobionic acid-β-cyclodextrin (6-MA-βCD), Mono-6 -gluconic acid-β-cyclodextrin (6-GLU-β-CD) and Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD) (Cell viability expressed as mean ± SD, n=9).

[0068] Figure 26 shows: [A] hemolytic activity of β-cyclodextrin (β-CD), Mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD), Mono-6-lactobionic acid-β -cyclodextrin (6-LA -β-CD), Mono-6-maltobionic acid-β-cyclodextrin (6-MA-β-CD), Mono-6-gluconic acid-β-cyclodextrin (6-GLU-βCD) and Mono- 6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD) in human red blood cells (HRBCs) at 37°C. Each point represents the mean ± SD. [B] Expanded graph up to 5.2% hemolysis (Hemolysis expressed as mean ± SD, n=3).

Detailed Description of the Invention

[0069] The present invention solves the problems of the state of the art from new compounds derived from cyclodextrin with the replacement of at least one hydroxyl by a substituent, in which the substituent comprises R1 linked to R2, with R1 being a spacer and R2 a hydroxy acid, polyhydroxy acid or aldonic acid and where R1 is linked to cyclodextrin. More specifically the compounds are β-cyclodextrin derivatives, wherein the substituent is composed of a spacer group and a hydroxy acid, polyhydroxy acid or aldonic acid, preferably polyhydroxy acid selected from the group comprising lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) or galacturonic acid (GAL).

[0070] Cyclodextrins (CD) are cyclic polymers of oligosaccharides, formed by glucose units linked by α-1,4 bond. There are CD molecules that occur naturally as fermentation products, as is the case of α-CD, β-CD and γ-CD, these differ by the number of glucose units that make up the macromolecule, 6, 7 and 8, respectively .

[0071] The CDs form a structure with a hydrophilic outer surface and a hydrophobic inner central cavity, allowing to encapsulate desired poorly water-soluble molecules within this central cavity. Due to this property, they are widely used to change physicochemical and kinetic parameters of a variety of compounds by forming inclusion complexes, and may have different applicability, including in the food, chemical, cosmetic and pharmaceutical industries.

[0072] In the chemical and food industry, for example, cyclodextrins are used to protect lipophilic chemical or food components against oxidation or degradation; improve the water solubility of substances such as fat-soluble vitamins, dyes. They can also be used for masking substances with an unpleasant odor and/or taste; improve the physical and chemical stabilization of volatile oils.

[0073] At the same time, in the pharmaceutical industry, cyclodextrins are used to improve the apparent water solubility of lipophilic drugs, making it possible to prepare aqueous solutions of these drugs, for oral, parenteral, ocular or other routes. Protection of active molecules or pharmaceutical adjuvants against thermal degradation, photodegradation, molecular arrangements by enzymes, among others. Molecules or functional groups that have undesirable odors or flavors can be hidden from sensory receptors by encapsulating them within the DC cavity. The properties of CDs are being explored for drug delivery systems.

[0074] Additionally, in the pharmaceutical area, there is an interest in functionalizing the DCs to achieve a vectorized carrier system, in which it is possible to bind molecules on the external surface of the DCs with affinity for receptors abundantly expressed in a certain specific site, reducing dosage and effects adverse effects of treatments with drugs included by complexation inside the cavity. Still, there is the possibility of applying DCs in diagnostic methods, in which they can carry fluorescent compounds that help in diagnostic imaging.

[0075] Thus, asialoglycloprotein receptors (ASGP-R) are expressed primarily on the sinusoidal surface of hepatocytes, the main role of these receptors is to bind and internalize glycoprotein molecules with terminal residue containing galactose or N-acetylgalactosamine. Some liver diseases can cause increased expression of ASGP-R, especially hepatocellular carcinoma (HCC), hepatitis C and hepatitis B. There are reports in the literature of several vectoring systems using lactobionic acid, which has affinity for ASGP- R.

[0076] Although CDs are hydrophilic on their surface, they have limited solubility in water, particularly β-CD. Being a problem in the development of aqueous solutions containing this oligomer. In this sense, there are efforts to increase the water solubility of β-CD, in which derivatives are developed. Among the existing derivatives for use in formulations are hydroxy-propyl-beta-cyclodextrin (HPβCD), sulfobutylether-betacyclodextrin sodium salt (SBEβCD) and randomly methylated-betacyclodextrin βCD (RMβCD). However, current options are scarce and expensive.

[0077] A first object of the present invention comprises compounds derived from cyclodextrin with substitution of at least one hydroxyl by a substituent, in which the substituent comprises R1 linked to R2, R1 being a spacer and R2 a hydroxy acid, polyhydroxy acid or acid aldonic and where R1 is linked to cyclodextrin.

[0078] In one embodiment, the cyclodextrin may be selected from the group comprising α-CD, β-CD or γ-CD.

[0079] In one embodiment, the cyclodextrin is β-CD.

[0080] In one embodiment the cyclodextrin is monosubstituted.

[0081] In one embodiment, the CD-derived compound has the general structure

[0082] In one embodiment, the R1 spacer is a primary carbon chain diamine, saturated or unsaturated, linear or branched, which may have between C2 and C6, which contains the amine group in at least two chain ends.

[0083] In one embodiment the R1 spacer is a primary diamine linked to the cyclodextrin by one of the -NH2 groups.

[0084] In one embodiment the spacer R1 is selected from the group comprising propane-1,3-diamine, butane-1,4-diamine, pentane-1,5-diamine and ethylenediamine.

[0085] In one embodiment the spacer R1 is ethylenediamine.

[0086] In one embodiment, R2 is selected from the group comprising glycolic acid, xylonic acid, retinoic acid, lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL).

[0087] In one embodiment, R2 is preferably selected from polyhydroxy acids, which are carboxylic acids that have a carbon chain of at least 6 carbons, preferably more than C2, more preferably from C2 to C6 saturated or unsaturated, branched or linear, and may contain cyclic or heterocyclic groups, comprising at least 2 –OH groups.

[0088] In one embodiment, R2 is preferably a polyhydroxy acid selected from the group comprising lactobionic (LA), maltobionic (MA), gluconic (GLU) and galacturonic (GAL) acid.

[0089] In one embodiment, β-CD-LA has affinity for asialoglycoprotein receptors (ASGP-R) and has the potential to be used as a vectorized carrier of bioactive molecules or marker molecules for liver cells.

[0090] In one embodiment, the bioactive molecule can be selected from doxorubicin, lamivudine, zidovudine, sorafenib.

[0091] In one embodiment, the marker molecule fluoresces and is preferably selected from fluorescein isothiocyanate.

[0092] In a second object, the present invention comprises the composition comprising the compounds derived from cyclodextrin with the substitution of at least one hydroxyl by a substituent, in which the substituent comprises R1 linked to R2, R1 being a spacer and R2 a hydroxy acid , polyhydroxy acid or aldonic acid and where R1 is linked to cyclodextrin.

[0093] In one embodiment the composition is a pharmaceutical, cosmetic or food composition.

[0094] In one embodiment the composition is a pharmaceutical composition comprising a bioactive molecule or marker molecule; wherein the molar ratio between bioactive molecule or marker molecule:cyclodextrin derivative ranges from 1:2 to 2:1, and may also be 1:1.

[0095] In one embodiment the bioactive molecule of the pharmaceutical composition can be selected from the group comprising doxorubicin, lamivudine, zidovudine and sorafenib.

[0096] In one embodiment, the marker molecule of the pharmaceutical composition fluoresces and is preferably selected from fluorescein isothiocyanate.

[0097] In one embodiment the composition is an aqueous, semi-solid or solid pharmaceutical composition.

[0098] In one embodiment the composition is a pharmaceutical composition administered by routes comprising oral, cutaneous, nasal, pulmonary, ocular and/or parenteral.

[0099] In a third object, the present invention comprises the process for obtaining the compound, which comprises the following steps: i) obtaining an intermediate compound β-cyclodextrin (β-CD) with at least one hydroxyl substituted by a leaving group ; ii) replacement of the leaving group of the intermediate compound of βcyclodextrin obtained by step i) by the spacer group; and iii) reacting the spacer group of the compound obtained in step ii) with at least one of hydroxy acid, polyhydroxy acid or aldonic acid in a reaction medium comprising a mixture of organic solvents.

[0100] In one embodiment, the present invention comprises the process for obtaining the compound, which comprises the following steps: i) tosylation of at least one hydroxyl of the cyclodextrin to obtain the intermediate compound; ii) replacement of the tosyl group of the βcyclodextrin intermediate compound obtained by step i) with the spacer group; and iii) reacting the spacer group of the compound obtained in step ii) with at least one of hydroxy acid, polyhydroxy acid or aldonic acid in a reaction medium comprising a mixture of organic solvents.

[0101] In one embodiment, step i) tosylation occurs primarily, but not only, in the C6 hydroxyl of β-cyclodextrin (β-CD) to obtain the intermediate compound.

[0102] In one embodiment of the process, the spacer group of step ii) is a primary diamine selected from the group comprising propane-1,3-diamine, butane-1,4-diamine, pentane-1,5-diamine and ethylenediamine.

[0103] In one embodiment, the primary diamine is ethylenediamine.

[0104] In one embodiment, step iii) occurs with hydroxy acid, polyhydroxy acid or aldonic acid selected from the group comprising glycolic acid, xylonic acid, retinoic acid, lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL).

[0105] In one embodiment, step iii) occurs with polyhydroxy acid selected from the group comprising lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL).

[0106] In one embodiment of the process, this comprises the additional step iv) of precipitating the compound obtained in step iii) by adding an organic solvent.

[0107] In one embodiment of the process, the mixture of organic solvents from step iii) comprises N,N'-dicyclohexylcarbodiimide (DCC) and dimethylformamide (DMF).

[0108] In a fourth object, the present invention comprises the use of the compound derived from cyclodextrin in the preparation of cosmetic, pharmaceutical, chemical compositions or food products.

[0109] In a fifth object, the compound is used to prepare a drug in which the compound is a carrier for bioactive molecules.

[0110] For clarity of terms, "natural cyclodextrin (CD)" is understood in the present patent application as equivalent to the term "unsubstituted cyclodextrin (CD)", that is, cyclic polymers of oligosaccharides, formed by glucose units linked by α-1,4 binding, obtained by enzymatic fermentation of starch.

[0111] Polyhydroxy acids are understood in the present invention as carboxylic acids that have a carbon chain of at least 6 carbons, saturated or unsaturated, branched or linear, and may contain cyclic or heterocyclic parts, comprising at least 2 -OH groups, the poly -hydroxy acids can be mono- or disaccharide carboxylic acids.

[0112] One of the problems with the application of natural β-CD, which is formed in greater abundance in the fermentation of starch, the lowest cost and suitable cavity size for the inclusion of numerous bioactive molecules, is that it cannot be used by parenterally, due to incompatibility with blood cells, causing hemolysis and also due to its nephrotoxicity. The cyclodextrin derivatives described here allow parenteral use, as they solve these toxicological limitations. Showing up more tolerant with better toxicological profile.

[0113] In addition to this feature, the CD derivatives of the present invention have a better water solubility profile when compared to unsubstituted CD, similar to the few hydrophilic derivatives on the market.

[0114] The new derivatives are structurally different from those on the market, which are few and expensive, due to market dominance by a few companies. The new derivatives may constitute an alternative, increasing the variety of CD derivatives for use in aqueous compositions, such as, for example, aqueous compositions for oral, cutaneous, nasal, ocular, parenteral administration, among others.

[0115] Considering the vectorization of drugs, there is an advantage of the possibility of decreasing the dosage and adverse effects caused by the treatment due to targeting the site of action, in which there is a greater accumulation of drug in the target tissue and consequent less distribution to other tissues . There are also other potentialities for the derivatives synthesized here, such as the formation of nanostructured systems. It can also be used for the diagnosis of liver diseases by complexing the β-CD-LA derivative with marker compounds that show fluorescence.

[0116] Therefore, the present invention refers to obtaining substituted cyclodextrin derivatives, in which the substitution comprises at least one of hydroxy acid, polyhydroxy acid or aldonic acid, which may be selected from the group comprising glycolic acid, xylonic acid , retinoic acid, lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL), preferably the replacement comprises a polyhydroxy acid selected from lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU ) and galacturonic (GAL). The objective is to obtain derivatives that present greater water solubility and compatibility with blood cells, when compared to natural cyclodextrin. Furthermore, the CD-LA derivative, due to its affinity for asyloglycoprotein receptors (ASGP-R), has the potential to be used as a vectorized carrier of bioactive molecules or markers for liver cells.

EXAMPLES

[0117] The examples shown here are intended only to exemplify one of the many ways to carry out the invention, but without limiting its scope.

Synthesis of β-cyclodextrin-polyhydroxyacid derivatives Synthesis of 6-O-toluenesulfonyl-β-cyclodextrin (6-Ts-β-CD)

[0118] For the synthesis of 6-Ts-β-CD, the method described by Byun and coauthors (BYUN; ZHONG; BITTMAN, 2000) was used, with modifications. The tosylation of C6 hydroxyl of β-Cyclodextrin (β-CD) is a reaction for the formation of a more reactive intermediate, from this compound, several derivatives can be obtained (ŘEZANKA, 2019; SZEJTLI, 1998). Tosylation consists of monosubstitution of the C6 hydroxyl group by the addition of a leaving group, 1-(p-Toluenesulfonyl)imidazole.

[0119] Initially, 1-(p-Toluenesulfonyl) imidazole (p-TsIm) was synthesized (Reaction 1 [A] ) as described by Byun and coauthors (BYUN; ZHONG; BITTMAN, 2000), with some modifications. Imidazole (10 g, 146.89 mmol) and dry dichloromethane (38 mL) were kept under N 2 and in an ice bath. Then a solution of p-toluenesulfonyl chloride (12.30 g, 64.51 mmol) in dry dichloromethane (38 mL) was added dropwise. The resulting mixture was warmed to room temperature and after 2h the reaction was filtered and washed with ethyl acetate: hexane (80 mL, 1:1). The filtrate was concentrated (about 70% by volume) under reduced pressure. The product was precipitated with ethyl acetate and washed with hexane. The resulting suspension was filtered and the product, 1-(p-toluenesulfonyl)imidazole, was lyophilized (BYUN; ZHONG; BITTMAN, 2000). For the synthesis of 6-Ts-β-CD (reaction 1B), β-CD (12.97 g, 11.42 mmol) and water (290 mL) were heated until solubilization (60°C). Then the solution was cooled to room temperature, 1-(p-toluenesulfonyl)imidazole was added (10.15 g, 45.66 mmol) and then stirring was continued for 2 h. Then sodium hydroxide (5.83 g, 145.70 mmol/16 mL water) was added dropwise. After 10 min, the unreacted precipitate (1-(p-toluenesulfonyl)imidazole) was filtered off. To stop the reaction, ammonium chloride (15.62 g, 291.01 mmol) was added to the solution. The solution was concentrated by blowing in compressed air. The resulting suspension was filtered and the solid washed twice with ice water (33ml) and twice with acetone (65ml). The final precipitate was lyophilized (BYUN; ZHONG; BITTMAN, 2000). Reaction 1 [B] shows the synthetic route to obtain 6-Ts-β-CD.

[0120] The synthesized 1 (p-toluenesulfonyl) imidazole was characterized by GC-MS (Figure 1). The GC-MS spectrum of 1-(p-Toluenesulfonyl)imidazole (Figure 1) shows a peak at 27.276 min corresponding to m/z of 222.1.

[0121] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-Ts-β-CD shows signals at δ (ppm) 7.75 (d, J = 8.2 Hz, 2H, H 8), 7.43 (d, J = 8.1 Hz, 2H, H 9), 5.87 – 5.62 (m, 14H, OH 2 and OH 3), 4.84 (s, 5H, H 1), 4.77 (d, J = 3.0 Hz, 2H, H 1* ), 4.56 – 4.42 (m, OH 6), 4.41 – 4.27 (m, 2H, H 6*), 4.24 – 4.12 (m, 1H, H 5*), 3.72 – 3.44 (m, H 3, H 5 and H 6), 3.43 – 3.16 (m, H2 and H4 overlapped with H2O), 2.43 (s, 3H, H 11) (Figure 2). The 1H NMR spectrum of 6-Ts-β-CD differs from β-CD by the presence of new signals at δ 7.75 and 7.43 corresponding to the aromatic ring and δ 2.43 of the methyl group, both of the tosyl group. The confirmation of monosubstitution was confirmed by the values of the integrals, where the signs in δ 7.75 and 7.43 correspond to two H per doublet and δ 2.43 corresponds to three H. The integral values H1 and H1* correspond to seven H , confirming the relationship of a tosyl group per cyclodextrin.

[0122] In the mass spectrum (HRMS-ESI-QTOF) of the 6-Ts-β-CD product (Figure 3 [B] ) two characteristic signals were identified. The most intense peak, with m/z of 667.1785 [M+Na] +2 (experimental error 0 ppm), was identified as 6-Ts-β-CD (C49H76O37S, 1288.38 g mol-1) and the signal with m/z of 590.1752 (experimental error 1.86 ppm) referring to β-CD (C42H70O35, 1134.37 g mol-1). The sample was also analyzed by HPLC-HRMS, where β-CD was quantified in 5.8% and 6-Ts-β-CD in 94.2% (Figure 4). In the analysis of fragmentation by direct infusion (MS/MS, 100 eV), a loss of six units of 162 Da was observed, corresponding to the breaking of the glycosidic bond between the glucose units. The fragment with the smallest m/z detected (m/z 325.1150) corresponds to a glucose unit linked to the tosyl group (Figure 3 [C] ).

Synthesis of mono-6-ethylenediamine-β-cyclodextrin (6-EDA-β-CD)

[0123] The addition of a spacer containing NH2 groups between β-CD and lactobionic acid is essential for obtaining an amide bond. Direct bonding between the β-CD hydroxyl and the carboxylic acid of lactobionic acid would result in the formation of an ester. This group is much more susceptible to hydrolysis, resulting in lower stability of the lactobionic acid-β-CD derivative.

[0124] In this step, the tosyl group can be easily replaced by the primary amine ethylenediamine (-NH2) (PUGLISI et al., 2014). 6-EDA-β-CD was synthesized from the reaction of 6-Ts-β-CD and excess ethylenediamine (Reaction 2). 6-Ts-β-CD (4 g, 3.10 mmol) and ethylenediamine (20 mL, 299 mmol) were heated at 80 °C for 6 h. Then the solution was cooled to room temperature and part of the unreacted ethylenediamine was removed by rotary evaporator. To this solution was added water and then precipitated with acetone (repeated 4 times). Residual acetone was removed by rotary evaporator and the precipitate was lyophilized (LIU; FAN; GAO, 2003).

[0125] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-EDA-β-CD (Figure 5) shows signals in δ (ppm) 5.73 (m, 14H, OH 2 and OH 3), 4.88 – 4.77 (m, 7H, H 1 and H1*), 4.64 - 4.20 (m, OH 6), 3.74 - 3.49 (m, H 3, H 5 and H 6), 3.43 - 3.20 (m, H2 and H4 overlaid with H2O ), 2.86 (d, J = 11.4 Hz, 1H, H6*) 2.79 – 2.65 (m, 2H, H6* and H8) 2.64 – 2.54 (m, 2H, H9). The 13C NMR spectrum (75 MHz, DMSOD6) (Figure 6) shows signals in δ (ppm) 102.06 (C1), 83.37 (C4*), 81.71 (C4), 73.21 (C3), 72.52 (C2), 72.19 ( C5), 70.48 (C5*), 60.09 (C6), 49.93 (C6*), 49.13 (C8). In the 1H NMR spectrum of 6-EDA-β-CD, the signals at δ (ppm) 7.75, 7.43 and 2.43 characteristic of the tosyl group were not detected. New signals were also observed, at δ 2.88-2.78, 2.77 - 2.55 and 2.54, corresponding to H6 *, H7 + H6 * and H8, respectively. In the 13C NMR spectrum, new peaks were detected in comparison with the β-CD spectrum. The peaks at 83.37, 70.48 and 49.93 ppm correspond to C4*, 5* and 6* of the glucose unit to which the ethylenediamine group is attached. Furthermore, the peak at δ 49.13 ppm corresponds to C bound to NH (C8).

[0126] In the mass spectrum (HRMS-ESI-QTOF) of 6-EDA-β-CD (Figure 7 [B] ) three characteristic signals were observed. 6-EDA-β-CD (C44H76N2O34, 1176.43 g mol-1) was identified with m/z of 600.2127 [M+Na+H] +2 (experimental error 0.83 ppm) and m/z of 1177 .4265 [M+H] +1 (experimental error -7.38 ppm) and β-CD was detected with m/z of 590.1766 [M+2Na] +2 (experimental error 4.23 ppm). The presence of the starting material, 6-Ts-β-CD was not detected. βCD was quantified at 3.1% by HPLC-HRMS (Figure 8). In the MS/MS spectrum (75 eV) the same pattern of fragmentation of 6-Ts-β-CD was observed and the fragment with the lowest m/z was identified as a unit of glucose linked to ethylenediamine (m/z of 205 .1198) (Figure 7 [C] ).

Synthesis of polyhydroxy acids -β-cyclodextrin

[0127] In this step, the reaction of the NH2 group of 6-ETN-β-CD with the carboxylic acid of the polyhydroxy acid was performed. Lactobionic, maltobionic, gluconic or galacturonic acid (1.02 mmol) was dissolved in DMF (15 mL) under N2 atmosphere. Then DCC (0.21 g, 1.02 mmol) in DMF (5 mL) was added to the solution and after 15 min NHS (0.12 g, 1.02 mmol) in DMF (5 mL) was also added (1:1:1). The reaction was maintained at 30°C, under magnetic stirring, for 24 hours. Then the by-product dicyclohexylurea (DCU) was removed by filtration.

[0128] 6-EDA-β-CD (0.60 g, 0.51 mmol) and DMF (10 mL) were added to a round bottom flask with two openings and the filtrate resulting from the previous step was added dropwise drop (1:2). The reaction was maintained at 30°C, with magnetic stirring, under N2 atmosphere for 48h (Reaction 3). Finally, the product was precipitated in ice-cold acetone. The solid was collected by filtration, dissolved in water and then precipitated again in acetone. The resulting solid was collected by filtration and washed with acetone. Residual solvent was removed by rotary evaporator and the precipitate lyophilized. The products obtained were mono-6-lactobionic acid-β-cyclodextrin (6-LA-β-CD), mono-6-maltobionic acid-β-cyclodextrin (6-MA-β-CD), mono-6-gluconic acid -βcyclodextrin (6-GLU-β-CD) or mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD) when lactobionic, maltobionic, gluconic or galacturonic acids were used, respectively.

[0129] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-LA-β-CD (Figure 9) shows signals in δ (ppm) 7.66 (t, J = 5.9 Hz, 1H, H10), 5.84 - 5.67 (m, OH 2 and OH 3), 5.23 (1H, OH 12), 5.17 (s, 1H), 4.89 - 4.77 (m, 7H, H 1 and H 1*), 4.69 (1H), 4.57 - 4.42 (m, 6H, OH 6), 4.26 (d, J = 6.6 Hz, 1H, AL), 4.14 - 3.97 (m, 2H, AL), 3.73 - 3.47 (m, H 3, H 5 and H 6), 3.45-3.22 (m, H2 and H4) overlapped with H2O, 2.88-2.78 (H6*), 2.77 - 2.55 (m, H6*, H7), 2.54 (s, H8). The 13C NMR spectrum (75 MHz, DMSO-d6) shows signals in δ (ppm) 172.65 (C11), 104.74 (C17), 102.10 (C1), 83.10 (C4*), 81.68 (C4), 75.82 (C21) , 73.21 (C3), 72.55 (C2), 72.18 (C5), 71.50 (C14), 71.26 (C15), 70.59 (C5*), 68.39 (C20), 62.43 (C16), 60.83 (C22), 60.09 (C6) ), 48.82 (C6* or C8), 25.37 (Figure 10). In the 1H NMR spectrum of 6-LA-β-CD, the peak at δ (ppm) 7.66 of the amide bond between lactobionic acid and 6-EDA-β-CD is observed. At 4.26 ppm the anomeric hydrogen of lactobionic acid (H14). In the 13C NMR spectrum the signal at 172.65 ppm is identified as the C of the amide bond and at 102.39 ppm it is attributed to the carbon of the glycosidic bond between the glucose units.

[0130] In the mass spectrum (HRMS-ESI-QTOF) of the product 6-LA-β-CD (Figure 11 [B] ) the major peak is observed with m/z of 770.2591 [M+H+Na /2] +2 (experimental error -4.41 ppm) corresponding to 6-LA-β-CD (C56H96N2O45). The peaks corresponding to the starting material 6-EDA-β-CD with m/z of 600.2086 [M+Na+H] +2 (experimental error -5.99 ppm), lactobionic acid with m/z were also identified. of 381.0969 [M+Na] +1 (experimental error -8.92 ppm) and β-CD with m/z of 590.1752 (experimental error 1.86 ppm). In the MS/MS spectrum (Figure 11 [C] ) a consecutive loss of seven units of 162 Da was observed, corresponding to the breaking of the glycosidic bond between the glucose units. The last m/z peak of 383.1625 corresponds to the amine group lactobionic acid. The HPLC-HRMS results (Figure 12) indicated 6-LA-β-CD in a percentage of 86.6%, 6-EDA-β-CD in 4.3%, β-CD in 4.0% and acid lactobionic in 5.1%.

Mono-6-Maltobionic acid-β-cyclodextrin (6-MA-β-CD)

[0131] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-MA-β-CD (Figure 13) shows signals in δ (ppm) 7.66 (t, J = 5.3 Hz, 1H, H10), 5.90 - 5.65 (OH 2 and OH 3), 5.58 (1H), 5.45 (1H), 5.00 - 4.93 (1H), 4.90 - 4.78 (7H, H 1 and H 1*), 4.72 (1H), 4.60 - 4.40 (6H , OH 6), 3.99 (2H), 3.73 - 3.50 (m, H 3, H 5 and H 6), 3.47 - 3.22 (m, H2 and H4) superimposed with H2O, 2.81 (H6*), 2.76 - 2.60 ( H6*, H7) 2.57 (H8). The 13C NMR spectrum (75 MHz, DMSO-d6) shows signals in δ (ppm) 172.53 (C11), 102.13 (C1), 100.89 (C17), 83.16 (C4*), 81.70 (C4), 73.24 (C3) , 72.55 (C2), 72.21 (C5), 69.97 (C5*), 62.75 (C16), 60.81 (C22), 60.09 (C6), 48.92 (C6* or C8), 25.25 (Figure 17). In the 1H NMR spectrum, the binding between maltobionic acid and 6-EDA-β-CD was identified by the presence of the signal corresponding to the amide at δ 7.64 ppm. In the 13C NMR spectrum (Figure 14), the signal corresponding to the C of the amide group (C11) was identified at 172.53 ppm and the C of the glycosidic bond of maltobionic acid (C17) at 100.86 ppm.

[0132] In the characterization of 6-MA-β-CD (C56H96N2O45) by HRMS-ESI-QTOF (Figure 15 [B] ) the peak corresponding to the product with m/z of 770.2642 [M+H+ Na]+2 (experimental error 2.20 ppm). The peak of the starting material 6-EDA-β-CD with m/z of 600.2106 [M+Na+H] +2 (experimental error -2.66 ppm) and maltobionic acid with m/z of 381.0981 [M+Na] +1 (experimental error -5.77 ppm). In the MS/MS spectrum (90 eV) the same fragmentation pattern as the other derivatives was observed (Figure 15 [C] ). In the analysis by HPLC-HRMS (Figure 16) the percentage of 6-MA-β-CD was determined in 89.00%, 6-EDA-β-CD in 4.43% and maltobionic acid in 6.57%. β-CD was detected below the limit of quantification (5 μg/mL).

Mono-6-gluconic acid-β-cyclodextrin (6-GLU-β-CD)

[0133] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-GLU-β-CD (Figure 17) shows signals in δ (ppm) 7.66 (t, J = 5.9 Hz, 1H, H10), 5.84 - 5.66 (14H, OH 2 and OH 3), 5.43 (1H, OH 12),4.88 - 4.79 (7H, H 1 and H 1*),4.59 - 4.43 (OH 6), 3.98 (1H), 3.90 (1H) , 3.70 - 3.51 (H 3, H 5 and H 6), 3.49 - 3.24 (H2 and H4) superimposed with H 2 O, 2.84 - 2.78 (H6*). The 13C NMR spectrum (75 MHz, DMSO-d6) shows signals in δ (ppm) 172.78 (C11), 102.13 (C1), 83.22 (C4*), 81.71 (C4), 73.23 (C3), 72.56 (C2), 72.19 (C5), 71.60 (C14), 63.48 (C16), 60.08 (C6), 56.24, 48.83 (C6* or C 8) (Figure 18). In the 1H NMR spectrum, the signal at δ (ppm) 7.65 was identified, characteristic of the amide bond between 6-EDA-β-CD and gluconic acid (H10). In the 13C NMR spectrum the signal corresponding to the C of the amide group (C11) was identified at 172.78 ppm. Unlike 6-LA-β-CD and 6-MA-β-CD, the C17 (glycosidic bond) and C22 signals were not identified, as expected. C16 of gluconic acid was identified at 63.48 ppm.

[0134] In the mass spectrum of the 6-GLU-β-CD derivative (Figure 19 [B] ) the major peak is observed with m/z of 689.2382 [M+H+Na] +2 (experimental error 3.19 ppm) corresponding to 6-GLU-β-CD (C50H86N2O40, 1354.47 g mol-1). Peaks corresponding to 6-EDA-β-CD with m/z of 600.2137 [M+Na+H] +2 (experimental error 2.49 ppm) and β-CD with m/z of 590.1755 are still detected [M+2Na] +2 (experimental error 2.37 ppm). In the MS/MS spectrum (80 eV) the same fragmentation pattern as the other derivatives is observed (Figure 19 [C] ). The m/z with the lowest value 222.0957 corresponds to gluconic acid linked to the amine group. The purity of 89.85% of 6-GLU-β-CD was determined by HPLC-HRMS, still 8.10% of 6-EDA-β-CD and 2.05% of glyconic acid were detected (Figure 20).

Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD)

[0135] The 1H NMR spectrum (300 MHz, DMSO-d6) of 6-GAL-β-CD (Figure 21) shows signals in δ (ppm). 7.60 (H10), 5.92-5.88 (14H, OH 2 and OH 3), 5.08, 4.99, 4.83 (7H, H 1 and H 1*), 4.59-4.32 .59 - 4.43 (OH 6), 4.19-3.85, 3.83-3.49 (H 3, H 5 and H 6), 3.48-3.18 (H2 and H4) overlaid with H2O. The 13C NMR spectrum (75 MHz, DMSO-d6) shows signals in δ (ppm) 174.76 (C11), 105.12, 102.06 (C1), 84.40 (C4*), 81.62(C4), 75.69, 73.24 (C3), 72.44 (C2), 72.16 (C5), 71.25(C14), 68.19, 62.31(C16), 60.48 (C6), 60.01 (Figure 22). In the 1H NMR spectrum, the signal at δ (ppm) 7.60 of the H of the amide bond between 6-EDA-β-CD and galacturonic acid was identified. In the 13C NMR spectrum the signal at 174.76 ppm corresponds to the carbonyl group.

[0136] In the mass spectrum (HRMS-ESI-QTOF) two signals were observed in greater intensity (Figure 23 [B] ). The most intense corresponding to the product 6-GAL-β-CD (C50H84N2O40, 1352.46 g mol-1) with m/z of 688.2307 [M+H+Na] +2 (experimental error 3.63 ppm) and at a lower intensity, 6-EDA-β-CD with m/z of 600.2149 [M+Na+H] +2 (experimental error 4.49 ppm). The MS/MS spectrum (75 eV) showed the same fragmentation pattern as the other derivatives. The m/z with the lowest value detected was 222.0779, corresponding to galacturonic acid linked to the amine group (Figure 23 [C] ). The purity of 6-GAL-β-CD was determined by HPLC-HRMS, and the percentage of 6-GAL-β-CD was 96.26%, 1.86% of 6-EDA-β-CD and 1 .88% galacturonic acid (Figure 24).

intrinsic solubility

[0137] The solubility in water was determined by the small-scale shaken flask method (BERGSTRÖM et al., 2002). Excess of the compounds 6-LAβ-CD, 6-MA-β-CD, 6-GLU-β-CD and 6-GAL-β-CD were added to 100 μL of Milli-Q water in an Eppendorf. The samples were kept in a shaker at 300 rpm, 30°C for 24 h. Samples were centrifuged at 10000 rpm for 10 min. The supernatant was separated, diluted and analyzed by HPLC-HRMS. The solubility results are described in Table 1, which shows data for Mono-6-lactobionic acid-β-cyclodextrin (6-LA-β-CD), Mono-6-maltobionic acid-β-cyclodextrin (6-MA- β-CD), Mono-6-gluconic acid-β-cyclodextrin (6-GLU-β-CD) and Mono-6-galacturonic acid-β-cyclodextrin (6-GAL-β-CD).

[0138] Comparing the water solubility value of β-CD (18.5 mg mL-1 at 25°C) (SZEJTLI, 1998) with the synthesized derivatives, an increase of about 40 times is observed. The solubility observed for the derivatives was, respectively, 820, 860, 760 and 780 mg mL-1 for 6-LA-β-CD, 6-MA-β-CD, 6-GLU-β-CD and 6-GAL -β-CD.

[0139] The water solubility of these derivatives is similar to hydroxy-propyl-betacyclodextrin (HPβCD) >600 mg mL-1 at 25°C, Sulfobutylether-beta-cyclodextrin sodium salt (SBEβCD) >500 mg mL-1 at 25 °C and randomly methylated-betacyclodextrin βCD (RMβCD) > 600 mg ml-1 at 25°C, which are some of the most soluble derivatives used (JANSOOK; OGAWA; LOFTSSON, 2018).

In vitro cytotoxicity assays

[0140] Cell viability was determined by the MTT assay ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide), which is based on the conversion of MTT into formazan crystals (E ,Z-1-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan) by mitochondrial dehydrogenases (ALLEY, M.C., SCUDIERO, D.A., MONKS, P.A., HURSEY, M.L. et al., 1988; MOSMANN, 1983). HepG2 cells (human liver carcinoma cells) and Hek 293 cells (human embryonic kidney 293 cells) were seeded at a density of 8x10 4 cells ml-1 in 100 µl of supplemented culture medium (10% fetal bovine serum). After 48h (HepG2) and 24h (Hek 293), the cells were treated with different concentrations (5, 50, 100 and 200 µg mL-1) of 6-EDA-β-CD, 6-LA-β-CD, 6 -MA-β-CD, 6- GLU-β-CD, 6- GAL-β-CD and lactobionic acid and then incubated at 37°C in 5% CO2 for 48 and 24 h, respectively. The medium and solvent controls (negatives) were treated with culture medium and culture medium with water (solvent), respectively. Then the medium was removed and 100 μL of MTT (1 mg mL-1) was added to the wells. The plates were incubated at 37°C for 2h in an atmosphere with 5% CO2. Subsequently, the MTT solution was dissolved in 100 µL of DMSO. Absorbance was measured using a microplate reader at 570 nm (Spectra Max190, Molecular Devices, USA). All readings were compared to the control.

[0141] In Figure 25, the cell viability of HepG2, treated with 100 and 200 μM of 6-GLI-β-CD, was 82.5±13.1 and 50.4±10.4%, respectively, showing low cytotoxicity at high concentration. However, the cell viability of Hek 293 treated with 100 and 200 μM of 6-GLI-β-CD was 100.2±12.7 and 90.8±12.5, respectively, not showing cytotoxicity.

[0142] The viability of HepG2 and Hek 293 cells treated with 6-EDA-βCD, 6-LA-β-CD, 6-MA-β-CD, 6-GAL-β-CD at concentrations 5, 50, 100 and 200 μM remained above 95% at the time tested. These results indicate that the synthesized derivatives, 6-EDA-β-CD, 6-LA-β-CD, 6-MA-β-CD, 6-GLI-β-CD and 6-GAL-β-CD, are not cytotoxic and are biocompatible with cells of the human liver carcinoma cell line (HepG2) and human embryonic kidney 293 cells (Hek 293) under the conditions tested.

Hemolysis assays

[0143] In order to evaluate the hemocompatibility of in vitro derivatives of cyclodextrins, the hemolysis assay was performed. Fresh human blood was collected in a 4 mL vacuum tube with EDTA, commercially available from the brand Vacutainer®, in which the tubes contain EDTA K2 blasted coating the inner wall of the tube in a concentration of 1650 g/L in dry form. Human red blood cells (HRBCs) were separated by centrifugation at 10,000 rpm for 10 min. Subsequently, the suspension of HRBCs was washed five times with sterile PBS solution (pH 7.4) and diluted 1/10 in PBS solution. Aliquots of 0.4 mL of the diluted HRBCs were mixed with 0.4 mL of solution of β-CD, 6-EDA-β-CD, 6-LA-β-CD, 6-MA-β-CD, 6-GLU -β-CD or 6-GAL-β-CD (1, 5, 10, 20 and 40 mM) in PBS. Sterile deionized water was added to the suspension of HRBCs as a positive control (100% hemolysis) and PBS as a negative control (0% hemolysis). The samples were incubated by maintaining agitation in a shaker at 50 rpm, 37°C for 4 hours. After this period, the samples were centrifuged at 1000 rpm for 10 min and 200 μl of the supernatants were transferred to microplates and the absorbance was measured at 540 nm (Spectra Max190, Molecular Devices, USA) (KHELGHATI et al., 2020). The percentage of hemolysis was calculated using the following formula:

[0144] Blood compatibility is essential when a molecule is used intravenously. Therefore, testing for hemolytic activity is of paramount importance. Figure 26 shows the hemolytic effect of different concentrations of CD derivatives synthesized in human red blood cells (HRBCs).

[0145] Natural cyclodextrin β-CD, used here as a reference, due to its well-known hemolytic effect, induced 11.3±2.8% hemolysis at a concentration of 10 mM and 100% at 20 mM. Values of 50% and 100% of hemolysis in rabbit blood cells were reported for β-CD at a concentration of 5.7 mM (TAVORNVIPAS et al., 2002) and 12 mM (ANNO et al., 2011). Also, in human red blood cells, 100% hemolysis was observed at a concentration of 0.015 M (STELLA; RAJEWSKI, 2020). These results show that β-CD causes extensive hemolysis of red blood cells.

[0146] The hemolytic activity of 6-EDA-β-CD, 6-LA-β-CD, 6-MA-β-CD, 6-GLUβ-CD and 6-GAL-β-CD at concentrations of up to 20 mM remained below 5%, evidencing low hemolytic activity when compared to β-CD. However, 100% hemolysis was achieved in 40 mM of the intermediate compound 6-EDAβ-CD. At the concentration of 40 mM, 6-LA-β-CD, 6-MA-β-CD and 6-GLU-β-CD, the percentage of hemolysis remained around 25% and 6-GAL-β-CD reached 49.51±1.84%. The results suggest that changes in the structure of β-CD resulted in a significant reduction in its hemolytic effect.

[0147] Those versed in the art will value the knowledge presented here and will be able to reproduce the invention in the presented modalities and in other variants and alternatives, covered by the scope of the following claims.

Claims (15)

Compound characterized by having the general structure (I):

Compound, according to claim 1, characterized in that R1 is a primary diamine preferably selected from the group comprising propane-1,3-diamine, butane-1,4-diamine, pentane-1,5-diamine and ethylenediamine, even more preferably the primary diamine is ethylenediamine. Compound, according to claim 1, characterized in that R2 is selected from the group comprising glycolic acid, xylonic acid, retinoic acid, lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL) , with maximum preference for polyhydroxy acid selected from the group comprising lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL). Composition characterized in that it comprises the compound as defined in any one of claims 1 to 3. Composition according to claim 4, characterized in that it is a pharmaceutical, cosmetic or food composition. Composition according to claim 4 or 5, characterized in that it is a pharmaceutical composition comprising a bioactive molecule or marker molecule; wherein the bioactive molecule or marker molecule:compound molar ratio, as defined in any one of claims 1 to 3, ranges from 1:2 to 2:1. Composition according to claim 6, characterized in that the bioactive molecule is selected from the group comprising doxorubicin, lamivudine, zidovudine and sorafenib. Composition according to claim 6, characterized in that the marker molecule exhibits fluorescence, preferably selected from fluorescein isothiocyanate. Composition according to any one of claims 5 to 8, characterized in that it is an aqueous pharmaceutical composition administered by routes including oral, ocular and/or parenteral. Process for obtaining the compound, as defined in any one of claims 1 to 3, characterized in that it comprises the following steps: i) obtaining an intermediate compound β-cyclodextrin (β-CD) with at least one hydroxyl substituted by a leaving group; ii) replacement of the leaving group of the intermediate compound of βcyclodextrin obtained by step i) with a primary diamine; and iii) reacting the -NH2 group of the primary diamine of the compound obtained in step ii) with at least one of hydroxy acid, polyhydroxy acid or aldonic acid in a reaction medium comprising a mixture of organic solvents. Process, according to claim 10, characterized by step iii) occurring with polyhydroxy acid selected from the group comprising lactobionic acid (LA), maltobionic acid (MA), gluconic acid (GLU) and galacturonic acid (GAL). Process according to claim 10, characterized in that it comprises the additional step iv) of precipitating the compound obtained in step iii) by adding an organic solvent. Process according to claim 10, characterized by the mixture of organic solvents from step iii) comprising N,N'-dicyclohexylcarbodiimide (DCC) and dimethylformamide (DMF) Use of the compound, as defined in any one of claims 1 to 3, characterized in that it is in the preparation of cosmetic, pharmaceutical compositions or food products. Use of the compound according to claim 14, characterized in that it is to prepare a medicine in which the compound as defined in any one of claims 1 to 3 is a carrier of bioactive molecules.

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