WO2018100562A1 - Coordination compounds, syntheses, nanoformulation and use thereof in oncology - Google Patents
Coordination compounds, syntheses, nanoformulation and use thereof in oncology Download PDFInfo
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- WO2018100562A1 WO2018100562A1 PCT/IB2017/057594 IB2017057594W WO2018100562A1 WO 2018100562 A1 WO2018100562 A1 WO 2018100562A1 IB 2017057594 W IB2017057594 W IB 2017057594W WO 2018100562 A1 WO2018100562 A1 WO 2018100562A1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0046—Ruthenium compounds
- C07F15/0053—Ruthenium compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/003—Compounds containing elements of Groups 3 or 13 of the Periodic Table without C-Metal linkages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
Definitions
- the present invention regards mononuclear and dinuclear Ru-based and Ga-based coordination compounds, pharmaceutical formulations thereof, the related synthesis and encapsulation method in macromolecules, supramolecular aggregates or nanostructures, as well as their use in the diagnosis and/or the treatment of neoplasms.
- coordination compounds and/or their formulations are glycoconjugated with carbohydrates which act as cancer-targeting moieties, thus increasing the therapeutic selectivity.
- Cisplatin is a milestone among the anticancer compounds and, since its introduction in pharmacopoeia in 1978, has shown to be one of the most effective drugs, particularly in the treatment of testicular, ovarian, lungs, bladder and uterine cervix cancer, mesothelioma and head and neck carcinoma. Despite its therapeutic efficacy, the clinical use of cisplatin is characterized by severe side effects. The most serious one is the high toxicity, especially to kidneys, to auditory apparatus and to bone marrow. In addition, cisplatin demonstrates intrinsic or acquired resistance to certain tumors.
- a first strategy aims to synthesize new coordination compounds with metal centers other than platinum that can maintain the antitumor activity but, at the same time, are characterized by a better toxicological profile.
- Fregona et al. were able to synthesize a class of Au(lll) dithiocarbamato complexes, described in ITMI20030600 which proved intrinsically less toxic than the Pt(ll) counterparts.
- WO2010105691 A1 Fregona et al. have claimed a new class of Au(lll) dithiocarbamato compounds functionalized with a peptide moiety designed to be internalized by tumor cells by PEPT transporters.
- the ruthenium-based complexes represent another example of compounds reported in the literature. For instance, H. Fasal et al. report that (J. Coord. Chem., 2016, 70 (2), 279-295), ionic Ru(ll) mononuclear complexes are known, and they contain DMP (2,9-dimethyl-1 ,10- phenanthroline) and dithiocarbamates derived from piperazine (with the nitrogen atom in position 4 functionalized with a para- or meta-methoxyphenyl group) as ligands. Such complexes have been studied as DNA (both purified and derived from calf thymus) intercalants.
- Another class of coordination compounds with potential biomedical applications consists of mononuclear Ga(lll) complexes with alkyl- dithiocarbamato ligands, wherein the nitrogen atom is bound to a methyl and a 2-methyl-1 ,3- dioxolane or a CH 2 -CH-(OMe) 2 group, alternatively (Ferreira et al., J. Coord. Chem., 2014, 67 (6), 1097-1 109).
- glucose and other carbohydrates are potentially useful biomolecules to selectively target antitumor agents.
- FDG fluoro-2-deoxy-D- glucose
- glucose and other carbohydrates are potentially useful biomolecules to selectively target antitumor agents.
- glyco-conjugates made up of known cytotoxins or chemotherapeutics linked to glucose (or other carbohydrates) have been synthesized to increase their selectivity to and absorption by the tumor cells.
- the glyco-conjugate glufosfamide is currently in clinical trials for the treatment of ovarian and pancreatic cancer, glioblastoma multiforme and non-small cell lung carcinoma.
- oxalilplatin encapsulated in liposomes (known under the trade term of Lipoxal ® ) is currently in an advanced stage of development, while the same oxalilplatin, encapsulated in transferrin-conjugated liposomes (MBP-426), has completed the Phase I.
- a modified oxalilplatin (NC-4016) wherein a PEG is linked to the platinum(ll) metal center via a polyglutamic acid linker is about to start the phase I.
- the present inventors intend to overcome the existing limits in the state-of-the-art related to coordination compounds useful as antitumor agents.
- a first and main object of the present invention to design and synthesize Ru- based and Ga-based mononuclear and dinuclear coordination compounds to be used as antitumor agents. Furthermore, a second object of the present invention is to identify mononuclear and dinuclear Ru-based and Ga-based coordination compounds with carbohydrate-functionalized ligands which act as selective cancer-targeting moieties.
- the present invention intends to disclose the advantageous use of carbohydrates as selective cancer-targeting moieties.
- carbohydrates could provide the additional advantage of increasing the solubility of the final compound in aqueous media.
- a third important object of the present invention is to identify coordination compounds endowed with low or negligible toxicity with respect to other metal-based compounds, such as cisplatin and subsequent compounds.
- a fourth object of the present invention is to identify coordination compounds characterized by high solubility in aqueous media, high stability and bioavailability, inherent or achieved by encapsulation in macromolecules, nanostructures or supramolecular aggregates, such as micelles, liposomes, proteins or cyclodextrins. In particular, such structures may or not be functionalized with carbohydrates on the outer surface.
- a fifth object of the present invention is to provide a stable pharmaceutical formulation including one or more of said coordination compounds, usable as an antitumor agent and preferably administrable intravenously or orally.
- Such formulations may optionally include additional anticancer drugs.
- a further object of the present invention is to provide a method for synthesizing said mononuclear and dinuclear Ru-based and Ga-based coordination compounds, bioconjugated or not with carbohydrates, as well as a method for encapsulating said compounds in macromolecules, supramolecular aggregates made up of biocompatible polymers, functionalized or not with carbohydrates.
- a further object of the present invention is to disclose the use of said coordination compounds as antitumor agents, particularly in the treatment of "orphan tumors", such as the "triple negative” breast cancer (TNBC), the castration-resistant prostate cancer (CRPC), head and neck cancer, the NSCLC, the melanoma, the mesothelioma, the lung, pancreas and liver carcinoma.
- TNBC triple negative breast cancer
- CRPC castration-resistant prostate cancer
- head and neck cancer the NSCLC
- the melanoma the mesothelioma
- the lung pancreas and liver carcinoma.
- a final object of the present invention is to produce coordination compounds which, besides possessing the necessary chemical-biological properties, are stable and obtainable through a synthesis process both able to intrinsically yield high-purity compounds and being industrially applicable with known and cost-effective technologies compared to state-of-the-art solutions.
- M 2 , M 3 identify the metal center of the coordination compound which can be Ga(lll), Ru(ll), Ru(lll).
- the mononuclear and dinuclear coordination compounds herein disclosed are neutral or ionic complexes whose charge is neutralized by at least one counter- ion G and have different coordination geometries, for example regular octahedral or distorted octahedral.
- the coordination compounds of formula 1(a) or 1(b) are not bioconjugated with a cancer-targeting moiety and act as antitumor agents due to the inherent reactivity of the Ru and Ga metal centers.
- the coordination compounds of formula 1(a) or 1(b) are bioconjugated with a carbohydrate which acts as a selective cancer-targeting moiety, advantageously exploiting the so-called "Warburg effect".
- a carbohydrate which acts as a selective cancer-targeting moiety, advantageously exploiting the so-called "Warburg effect”.
- the presence of the cancer-targeting moiety, in combination with the physico-chemical properties of the final compound ⁇ e.g. ionic nature) can result in a very high solubility in physiological media.
- the coordination compounds of formula I (a) or l(b) are encapsulated in macromolecules, nanostructures or in supramolecular aggregates, such as liposomes or cyclodextrins, acting as carriers to carry out a passive- targeting mechanism mediated by the Enhanced Permeability and Retention (EPR) effect that characterizes tumor districts.
- Said compounds may be or not conjugated with carbohydrates.
- the coordination compounds described in the formula l(a) or l(b) may be addressed to the tumor site by encapsulating such compounds in macromolecules, nanostructures or supramolecular aggregates, for example micelles, in turn functionalized with carbohydrates to achieve an active-targeting approach ("Warburg effect"), which accompanies and strengthens the passive-targeting mechanism of the previous embodiment.
- the encapsulated compounds can be or not conjugated with carbohydrates.
- the present invention discloses Ru-based and Ga-based coordination compounds, glycoconjugated or not, which, according to experimental tests, possess remarkable anti-tumor properties. Furthermore, various synthesis processes of said coordination compounds have been disclosed, characterized by high yields: they allow the expert to choose the most advantageous scheme depending on the metal center and type of desired ligands, including those containing a cancer-targeting moiety represented by a specific carbohydrate. Said cancer targeting moiety and metal centers can in turn be chosen based on the molecular profile of the patient's neoplasm according to the paradigm of personalized medicine.
- the Ru-based and Ga-based coordination compounds according to the present invention can be loaded into supramolecular aggregates or macromolecules, consisting of a wide range of biocompatible polymers so to obtain stable nanoformulations.
- said compounds do not react nor establish interactions with other loaded complexes, and with the polymeric carrier neither.
- the coordination compounds and nanoformulations allow to realize a both active and passive cancer-targeting mechanism. These compounds are characterized by optimal LiPE values for a promising pharmaceutical development ⁇ "druglikeness").
- nanoformulations not only make the hydrophobic active principles soluble in the aqueous media but also mask the active principles herein described, both hydrophobic and hydrophilic, from reactions/interactions with the cellular and molecular components of the blood. Additional objects and advantages of the invention will be set forth in part in the detailed description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
- FIG. 1 depicts the levels of expression of the glucose transporter (Glutl ) in different cell lines.
- Glutl glucose transporter
- letter A Northern analysis of the Glutl mRNA levels evaluated in healthy human prostate samples and in three different human prostate cancer cell lines with decreasing differentiation.
- LNCaP is a hormone-sensitive cancer cell line, whereas the DU-145 and PC3 lines represent poorly-differentiated tumors (source: P. Effert et al., Anticancer Research 24: 3057-3064, 2004).
- Figure 2 shows the UV-Vis spectra of the coordination compound [Ru 2 (PipeDTC) 5 ]CI with (VII) general structure.
- said compound is dissolved in phosphate buffer (pH 7.4, 0.5% v/v DMSO, spectra recorded at 37° C over 24 hours), whereas in panel B), it is encapsulated in PF127 micelles (5 mg/mL) dissolved in phosphate buffer - cell culture medium 9:1 v/v (spectra recorded at 37 °C for 72 hours).
- FIG. 4 shows the DLS analysis of [Ru 2 (PipeDTC) 5 ]CI encapsulated in PF127 micelles with 10% of the polymer conjugated to glucose via glycosylation (C1 position).
- the chemical elements are defined by means of the respective symbols as reported in a common Periodic Table of Elements, such as that present in the "Handbook of Chemistry and Physics, 93rd ed ".
- the chemical symbol includes all isotopes and ions. Therefore, the chemical symbols C, F, Cu, Ga , Au and I include, as an example, their respective isotopes 11 C, 18 F, 64 Cu, 63 Cu, 65 Cu, 68 Ga, 198 Au, 131 1, 127 l, 129 l.
- the claimed and described structures are intended to include all possible isomers such as coordination isomers, structural isomers, and conformational isomers.
- the claimed and described structures are designed to include all possible optical isomers, such as enantiomers and/or diastereoisomers, their mixtures, either as racemic mixture or in various ratios.
- These chiral centers may be present in the coordination compound or already present in the involved chelating ligands, for instance in the molecular fragments comprising a carbohydrate T.
- the coordination compounds having formula l(a) and/or l(b) are bioconjugated to cancer-targeting moieties.
- cancer-targeting moieties This is a well-known term for an expert in the field, and it defines a molecular fragment of the compound (moiety), which has been ad hoc engineered for its recognition by cell-membrane proteins present in tumor cells.
- said "cancer-targeting moiety” is a carbohydrate, and according to this, the terms “glycoconjugation” and “glycoconjugates” will be also used to indicate the “bio- conjugation” and the "bio-conjugates", respectively.
- the synthetic processes of the first and second embodiments involve amino functional groups for the preparation of the dithiocarbamato ligand. These groups may be ad hoc introduced in a specific stage of the process, or alternatively they can be generated by subsequent reactions during the previous steps. On the other hand, a suitable amine reagent can be purchased and used as such. Again, in the context of the present patent, these amino groups and amine reagents will be defined "precursor amine(s)" for brevity.
- Some embodiments of the present invention involve the term "pharmaceutical formulations" to define preparations made up of a therapeutically-effective amount of the coordination compounds of Formula 1(a) and/or 1(b) together with "pharmaceutically acceptable” additives and/or diluents and/or excipients.
- pharmaceutically acceptable additives and/or diluents and/or excipients are intended to be compounds that, in contact with animal or human tissues, in light of their composition, dosage and administration route or anything else, do not cause any toxicity, irritation, allergy, and other complications considered excessive or unacceptable by a specialized physician based on a reasonable risk/benefit ratio.
- Said compounds may optionally be encapsulated in "pharmaceutically acceptable" macromolecules or supramolecular structures or nanostructures.
- encapsulation means a process by which a therapeutically-effective amount of the coordination compounds of Formula l(a) and/or l(b), establishes intermolecular bonds ⁇ e.g. van der Waals forces, hydrogen bonds) with the single units of a "nanocarrier” or with specific structural domains.
- the surface or the hydrophobic core of the nanocarrier e.g., micelles
- the hydrophilic core ⁇ e.g., liposomes
- the term "theranostic compound” or more generally “theranostic agent” will refer to the coordination compound according to this invention, the composition containing at least one of said coordination compounds, or also a pharmaceutical formulation made up of the said compound and/or composition, which combines therapeutic properties with regard to a pathology, preferably neoplastic pathologies, along with the property to be detectable by suitable devices/detectors, hence to be usable in association with diagnostic and imaging systems for patient follow-up in therapeutic treatments and, if possible, to take the necessary corrective action.
- a pathology preferably neoplastic pathologies
- Said mononuclear and dinuclear coordination compounds are neutral or ionic complexes whose charge is neutralized by at least one counter-ion G and have different coordination geometries, for example regular octahedral or distorted octahedral.
- Formula 1(a) or Formula 1(b) Mi , M 2 , M 3 , X, Y, W, Z, L 1 5 L 2 , L 3 and L 4 are independently chosen and have the following meaning.
- M 2 , M 3 identify the metal center of the coordination compound and they are selected from Ga(l ll), Ru(l l), Ru(l l l), also defined in short as Ru (11,111).
- the stylized arch connecting the two S- atoms represents a first ligand of dithiocarbamic nature (DTC);
- X, Y, W, Z, I_2, L 3 and L 4 are selected from the group consisting of: N, S, O, P, C and Se, and represent donor atoms being the same or different from each other and they belong to one or more bidentate chelating ligands.
- the G symbol identifies at least one counter-ion having the total charge d, being an integer ranging from -4 to +4.
- G is pharmaceutically acceptable ion, which, by way of example and not limitation, is selected among : CI “ , I “ , F “ , Br “ , CH 3 C0 2” , PF 6 “ , BF 4 “ , SbF 6 “ , [B ⁇ C 6 H 3 (CF 3 ) 2 ⁇ 4 ] “ , [B(C 6 F 5 ) 4 ] “ , OH “ , 0 3 SNH 2 " , nitrites and nitrates ⁇ e.g., N 2 0 “ , N0 3 " ), acetates, phosphates ⁇ e.g., hexafluorophosphate, H 2 0 4 P “ , P0 4 3” ), sulfates ⁇ e.g., triflate, HS0 4 " ), carbonates, perchlorates, acetylacetonates ⁇ e.g., hexafluoroacetylacetonate), propionat
- the stylized arch connecting the S- donor atoms indicates a bidentate chelating dithiocarbamato ligand (DTC), in compliance with the usual chemical notation.
- the number of chelating ligands in the structures l(a) and l(b) is 3 and 5, respectively.
- Said chelating dithiocarbamato ligands (DTC) may have a closed (cyclic) or a open ⁇ e.g., linear or branched) structure.
- said first or second DTC ligand has a closed structure such as that depicted below, which is presented by way of example but not limitation of the present invention:
- Such structure comprises groups R 1 5 R 2 , R 3 and R 4 and optionally one R 5 group.
- the group and - if present - the R 5 group are bound to the dithiocarbamic nitrogen atom and to the R 2 and R 4 groups, respectively; the remaining R, groups are bound to the next and the previous group in the cyclic structure. If R 5 is absent, R 4 is directly bound to the dithiocarbamic nitrogen atom. All the bonds involving the R, groups, where i is an integer ranging from 1 to 5, may be single or double.
- the DTC ligand with the closed structure herein described comprises a glucide or carbohydrate T, which is bound to at least one of said R, groups directly or through a unit A by respectively T-R, bonds or T-A-R, bonds, which can be single, double or triple.
- bonds may be: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C.
- other chemically-equivalent molecular fragments may be selected by the expert of the branch.
- said DTC ligands have an open structure of the type depicted below by way of example, but not limitation:
- the structure comprises a terminal group R linked to the dithiocarbamic nitrogen atom, and optionally one or more R 2 , R 3 , R 4 and R 5 groups.
- the R 4 group if present, is a terminal group and is bound to R 5 or to said nitrogen atom; alternatively, R 4 is bound to R 5 or to said nitrogen atom and is bonded to R 3 or R 2 , depending on whether the R 3 or R 2 groups are absent or not.
- the DTC ligand with the open structure herein described comprises at least one glucide or a carbohydrate T.
- Said unit T is linked to said dithiocarbamic nitrogen atom or to at least one of the said R, groups (where i is an integer from 1 to 5) directly with respectively T-N or T-R, bonds, or alternatively via a unit A, by T-A-N or T-A-R, bonds.
- said bonds may be single, double or triple, and of the type: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C.
- the unit A may be an atom or a functional group or a spacer, as well as the unit A is made up of other chemically-equivalent molecular fragments.
- said unit A or said R, group (where i is an integer ranging from 1 to 5) are preferably selected from: an atom such as H, C, O, N, S, P, Se or a group selected among -CH, -CH 2 , -CH 3 , -C(CH 3 ) 3 , -NH, -NH 2 , -NHR S , -NR s2 , -S-S-, -SH, -PH, -OH, -COOH, -CH-Br, -CHCH 2 NH 2 , -CHCH 2 OH, -CHCH 2 NH-, -CHCH 2 0-, (- CN), -CF 3 , -C 2 H 5
- unit A or groups R it is possible to use molecular fragments being chemically-equivalent to those herein reported by way of example, but not limitation of the present invention.
- said DTC ligands may contain in the unit A or in the R, groups chemical groups in salified form comprising pharmaceutically acceptable counterions ⁇ e.g., -NH 3 + CI " ).
- the unit A or the various R, groups may be variously substituted, in any combination and position, with one or more of said atoms or said groups.
- said DTC ligands may a priori have different isomeric forms.
- the herein disclosed formulae are intended to include any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, as racemic mixtures or in various ratios.
- said glucide or carbohydrate T is preferably a monosaccharide or a deoxy- monosaccharide, in particular a those, a tetrose, a pentose, an esose, an eptose.
- said glucide or carbohydrate T is selected from the group consisting of: glucose, galactose, mannose, xylose, rhamnose, arabinose, glucosamine, galactosamine, mannosamine, fructose, fructosamine, ribose, ribulose, sedoheptulose, erythrose, threose, erythrulose, allose, altrose, lixose, gulose, idose, talose.
- said glucide or carbohydrate T is a natural or synthetic polysaccharide, also in deoxy-carbohydrate form, for example maltose, cellobiose, lactose, trealose, sucrose, amylose, amylopectin.
- the T-R, or T-A-(R,) bond (where i is an integer ranging from 1 to 5) takes preferably place in at least one of the carbon positions of the T moiety, preferably in the positions C1 or C2 or C3 or C4 or C6, for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.
- the T-N or T-R, bond, or T-A-N or T-A-R, bond occurs preferably in the C1 or C2 or C3 or C4 or C6 positions for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.
- the ligand may have an overall neutral ⁇ e.g., zwitterionic), positive or negative charge.
- one or more functional groups can be suitably protected during the synthetic process.
- DTC dithiocarbamato
- one or more functional groups can be suitably protected during the synthetic process.
- Well-known groups are available for the protection of hydroxyl, amine, amide and carboxylic acid groups, as well as the conditions in which protection and deprotection can occur, even in so-called orthogonal conditions.
- protecting groups are introduced during the synthesis to avoid undesirable side-reactions and may be removed or not at the end of the synthetic procedure.
- the new herein disclosed compounds may contain protective groups, even mutually different, in order to modulate the in vivo therapeutic profile, for example in terms of reactivity, solubility, stability and bioavailability.
- one or more of the hydroxyl groups (also known as alcoholic) of one or more cancer-targeting moieties T of the second embodiment can be functionalized with equal or different protecting groups chosen, for example, from silyl ethers ⁇ e.g., trimethylsilyl ether or dimethyltertbutylsilylether) or esters ⁇ e.g., acetate, pivalate, propionate, carbonate, phosphate, butyrate).
- the abovementioned protecting groups are indicated here by the notation R 6 ;
- the substituent in position C1 may have an a or ⁇ conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C2 (as indicated by the black rectangle).
- the protecting groups are again indicated by the notation R 6 ; the substituent in position C1 may have an a or ⁇ conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C5 (as indicated by the black rectangle).
- the first and the second chelating dithiocarbamato (DTC) ligand may be either equal to or different from each other.
- the coordination compound is referred to as homoleptic, while in the latter it is heteroleptic.
- the coordination compounds having the general formula I (a) and l(b) may be conveniently synthesized by a process including at least the following steps:
- step c) Isolation of the coordination compound synthesized during step b).
- This process may optionally include a further step d) related to the purification and drying of the coordination compound obtained at the end of the step c).
- the synthesis of the dithiocarbamato ligands is carried out at a temperature ranging from -30 and 60 °C in water, or methanol, or dry THF via reaction between an amine precursor and carbon disulfide (CS 2 ), optionally in the presence of a base, such as KOH or sodium tert- butoxide, or an excess of said amine precursor.
- a base such as KOH or sodium tert- butoxide, or an excess of said amine precursor.
- the volume of the solvent is reduced and the DTC ligand can be isolated via precipitation or co- precipitation by adding diethyl ether, washed with diethyl ether and dried under vacuum in presence of P 2 0 5 .
- the synthesis of the coordination compound of general formula I (a) and/or l(b) is carried out in water or in an organic solvent, and it involves the coordination of the DTC, which has been optionally isolated in the previous step, to a selected metal center among Ru (II, III), and Ga (III). In the case of some compounds, it is preferable to work under inert atmosphere, using well- known equipment and techniques.
- the metal center is selected starting from the corresponding precursors, preferably chlorides or metal-halide salts, or complex salts.
- the precursors are metal derivatives with the metal center presenting a lower or higher oxidation state, such as organometallic-, amino-, thioether- precursors, or phosphine derivatives, or alternatively some of said coordination compounds can be themselves precursors, useful for the synthesis of other complexes.
- contrast agents can be advantageously integrated in the coordination compound according to the invention in the form, for example, of radioisotopes such as 11 C, 18 F, 68 Ga, 127 l, 129 l, 131 1.
- these isotopes can be included in the DTC ligand, or they may coincide with the metal center or also introduced into the counter-ion.
- these contrast agents allow to combine the treatment of the neoplastic diseases with the diagnosis.
- the isolation of the compound synthesized in the previous step b) proceeds via usual separation techniques, preferably filtration followed by reduction of the volume of water, or of said solvent, and by precipitation with ethyl ether. Alternatively, water or said solvent is evaporated under reduced pressure, in order to obtain the coordination compound of general formula 1(a) and/or 1(b), or a mixture containing said coordination compound.
- the purification is optionally carried out using techniques well-known to the skilled in the art, for instance by chromatography, precipitation from organic solvent, washing with water or organic solvents and drying of said coordination compound.
- step b describes further details of the process described in the step b), related to the coordination of the DTC ligand to a specific metal center.
- the resulting product is actually a mixture of the neutral mononuclear complex [Ru(DTC) 3 ] and the dinuclear ionic derivative of the general formula l(b).
- reaction mixture is stirred for at least 1 h at room temperature. Subsequently, the solvent is removed and the solid obtained is taken up with chloroform. After filtration, the obtained solution is precipitated with diethyl ether, leading to the isolation of a solid.
- This reaction leads to a mixture of products consisting of the ligand dimer (DTC) 2 , the neutral mononuclear derivative [Ru(DTC) 3 ] and the dinuclear ionic complex [Ru 2 (DTC) 5 ]CI (as a mixture of its a and ⁇ isomers).
- the [Ru(DTC) 3 ] and the ⁇ , ⁇ - [Ru 2 (DTC) 5 ]CI complexes are separated by chromatography.
- the mixture containing ⁇ , ⁇ - [Ru 2 (DTC) 5 ]CI is refluxed in DCM or isopropanol for 8 hours, in order to convert the residual a- isomer to the thermodynamically more stable ⁇ -[ ⁇ 2 ( ⁇ ) 5 ] ⁇ .
- the purified compounds are washed with n-hexane and dried under vacuum in presence of P 2 0 5 .
- the counter-ion G can be advantageously chosen or replaced, using techniques well-known to the skilled in the art, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target” toxicity, solubility, and stability.
- the first synthetic step is related to the preparation of the organometallic Ru(ll)-NBD complex, synthetized from RuCI 3 -3H 2 0 at reflux with the NBD ligand, according to a procedure reported in literature (J. Organomet. Chem., 1966, 5, 275-282). Successively, the Ru(ll)-NBD derivative is treated with 2 eq. of DTC to form the [Ru"(NBD)(DTC) 2 ] complex. This step leads to the formation of the "building blocks" of the final heteroleptic complex, and hence the introduced DTC ligands are prevalent in the dinuclear compound (4 units among the 5 ligands).
- the synthesis of the Ru(ll)-DTC derivative is conducted in hot DMF (100 °C) by adding a DMF solution of the dithiocarbamato ligand (2 eq.) to the Ru(ll)-NBD precursor.
- the precipitation with an appropriate solvent leads to the isolation of the precursor.
- the Ru(ll)-DTC is treated with bromine, to achieve the oxidation of the ruthenium center to Ru(lll), and, after the cleavage of the olefin ligand with hexane, the addition of the DTC * ligand results in the formation of the [Ru 2 (DTC) 4 (DTC * )]Br coordination compound.
- the final product is purified via silica gel chromatography with an appropriate eluent, according to the nature of the DTC ligands.
- the counter-ion G can be advantageously chosen or replaced, using well-known techniques, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target” toxicity, solubility, and stability.
- coordination compounds having formula 1(a) or 1(b) are reported below.
- said compounds do not contain a cancer-targeting moiety in the form of carbohydrate, and contain, for example, dithiocarbamato ligands derived from: piperidine, L-proline methyl ester, and L-proline ferf-butyl ester and which for brevity are named as PipeDTC, ProOMeDTC, and ProOtBuDTC, respectively.
- the compounds were characterized using several techniques, including elemental analysis, NMR spectroscopy, FT-IR spectrophotometry, and ESI-MS mass analysis.
- the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the first preferred embodiment of the present invention does not contain a "cancer-targeting moiety" in the form of carbohydrate.
- DTC PipeDTC
- DTC * DEDT
- the coordination compounds of Formula I (a) and Formula l(b) contain dithiocarbamato ligands functionalized with carbohydrates which act as a selective cancer-targeting moiety towards cancerous cells in vivo, advantageously exploiting the Warburg effect.
- the glycoconjugation process involves a number of synthetic steps for the functionalization of a specific amino precursor with an likewise specific carbohydrate. Such amino precursor is subsequently converted to the corresponding dithiocarbamato (DTC) ligand, through the phase a) of the procedure of the first embodiment.
- DTC dithiocarbamato
- the synthesis involves the protection of the hydroxyl groups of carbohydrate with protecting groups which are orthogonal to the reaction conditions of the subsequent synthetic steps.
- the carbohydrate ⁇ e.g., glucose, mannose) is functionalized ⁇ e.g., in position 1 or 2) with a molecular fragment containing an amine, preferably secondary, which is able to react with CS 2 to form a DTC ligand, ready for the subsequent complexation to the metal center (Ru, Ga).
- a molecular fragment containing an amine preferably secondary, which is able to react with CS 2 to form a DTC ligand, ready for the subsequent complexation to the metal center (Ru, Ga).
- amine group or other functional group, for example -OH is not directly used to prepare the dithiocarbamato ligand, but its reactivity could be exploited to bind a molecular fragment which, depending on the molecular design, may have different length and chemical composition.
- said molecular fragment must contain, after one or more synthetic steps, an amine group (that is, the precursor amine, as an example a proline) on which the CS 2 -based reaction will be carried out to form the DTC ligand.
- an amine group that is, the precursor amine, as an example a proline
- the deprotection of protecting groups can be performed by means of chemical or biochemical methods including the use of enzymes or pseudoenzymes.
- Pseudoenzymes are generally referred to proteins, such as the HSA (Human Serum Albumin), which may hydrolyze, for example, ester substrates due to the presence of several nucleophilic residues (such as lysine) on their surface, but which do not return to the native state after the hydrolysis reaction.
- HSA Human Serum Albumin
- the Scheme 2 reported below shows the synthesis of a dithiocarbamato ligand ⁇ c-g), being a glucose derivative functionalized in position C-2 (glucosamide, namely an amide derivative of theglucosamine).
- the schemes 3, 4 and 5 show the syntheses of carbohydrate derivatives having an amino function (-NH 2 ) in C3, C4 and C6 positions, respectively.
- Such amine function can in turn be functionalized with a molecular fragment on which the dithiocarbamato group is synthesized, or alternatively, such amine group can be directly converted to dithiocarbamate by reaction with CS 2 or may be advantageously modified in a different functional group, for example a secondary amine or an isothiocyanate, in order to conduct subsequent reactions for the bioconjugation of the metal.
- coordination compounds having Formula I (a) or l(b) containing glucides or carbohydrates in one or more dithiocarbamato ligands are reported below, by way of example, but not limitation.
- the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the second preferred embodiment of the present invention contains a "cancer-targeting moiety" in the form of carbohydrate.
- DTC PipeDTC
- the human tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) were cultured in RPMI-1640 and Hams-F12 medium, respectively.
- the cells (8x10 3 / mL) were seeded in 96-well plates in the growth medium previously mentioned (100 ⁇ _) and then incubated at 37 °C in a controlled atmosphere of carbon dioxide. After 24 hours, the cell culture medium was removed and replaced with fresh one containing the tested compound, previously dissolved in DMSO (0.1 % v/v, freshly prepared solution and keeping in the dark), or saline solution, at various concentrations.
- the cytotoxic activity of each compound was evaluated as a percentage of vital cells in the treated sample compared to the cells treated with the vehicle only (control). From the obtained dose-response curves, the IC 50 values for each compound were calculated ⁇ i.e. the concentration expressed in ⁇ , which inhibited 50% growth in tumor cells compared to the control).
- the above mentioned culture media and the human tumor cell lines can be easily purchased on the market.
- the following table collects the IC 50 data of some compounds of the first and second embodiments, the corresponding dithiocarbamato ligands (DTC) and metal precursors.
- Table 1 In vitro cytotoxic activity (IC 50, expressed in ⁇ ) of some compounds and DTC ligands against the h uman tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) after 24 hours of treatment; against the human tumor cell lines: cervix adenocarcinoma (HeLa), colon neoplasia (HCT116), HepG2 : epithelial cells of human liver hepatoma and its more aggressive cou nterpart HepG2/SB3, overexpressing the anti-apoptotic protein SerpinB3; NSC lung carcinoma (A-549); acute T cell leukemia (Jurkat); AGS: gastric adenocarcinoma; triple negative breast cancer (crl2335) after 72 hours of treatment.
- IC 50 In vitro cytotoxic activity (IC 50, expressed in ⁇ ) of some compounds and DTC ligands against the h uman tumor cell lines MeWo (malignant mel
- the inorganic reference-drug cisplatin (Sigma-Aldrich) was tested under the same experimental conditions .
- IC 50 concentration expressed in ⁇ able to inhibit the 50% of the cancer cell growth compared to the control (cells treated with the vehicle).
- the data represent the mean ⁇ SD of at least four independent experiments.
- the inventors have shown that such antitumor activity is due to the combination, in a single compound, of a properly-designed DTC ligand with a metal center endowed with specific chemical (oxidation state, reduction potential, geometry coordination, kinetic and thermodynamic properties) and biochemical properties.
- mice Provided by Charles River Laboratory Italy, Calco, Lecco; 6-week- old mice.
- This administration route has been chosen because it presents a reduced barrier to substance absorption, provides a more stringent toxicity measure compared to other in vivo assays and it is a typical route of administration in humans.
- the species/strain mouse/CD-1 ® was chosen because many regulatory authorities accept and indicate that preclinical acute toxicity tests are performed with this species/strain, in light of the large amount of related bibliography.
- the administered dose (10 mg/kg) was selected based on the dosages clinically used on humans ⁇ e.g., 2 mg/kg) in order to highlight potential signs of toxicity.
- the tested compound was: ⁇ -[ ⁇ 2 ( ⁇ ) 5 ] ⁇ .
- the chosen vehicle was DMSO-EtOH-RL 50:10:40 % v/v in which RL stands for Ringer lactate (Eurospital ® ).
- Each experimental group consisted of 6 mice + 4 control animals (vehicle-treated only).
- Each dose was prepared by dissolving a calculated amount of compound in DMSO; this volume was subsequently diluted with ethanol and Ringer lactate (RL) to obtain the final desired concentration.
- the administration of the antitumor compound under study was performed by intravenous injection into the caudal artery of each mouse. Each treated animal received an accurate injected volume of 100 ⁇ , containing the amount of test substance as described above, that is the equivalent of 10 mg/kg. All animals were treated with a single dose at T 0 after detecting the body weight of each mouse. Clinical observations were recorded at the time of injection, during the first hour and then in the following days for total 7 experimental days, the same for the body weight.
- Table 3 Clinical and behavioral observations during the experimental study .
- mice were sacrificed by C0 2 asphyxiation. All animals were subjected to autopsy examination including the opening of the cranial, thoracic and abdominal cavities.
- advantageous supramolecular systems encapsulating the active compound can be micelles, vesicles (liposomes), cyclodextrins, dendrimers, organic polymeric nanoparticles, for instance derived from proteins or peptides, and inorganic nanoparticles ⁇ e.g., silica, zirconia, titanium dioxide).
- Said supramolecular architectures can be in turn produced with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan, polyethylene glycol (PEG), mPEG acid, poly(lactic-co-glycolic acid) (PLGA), Pluronic®, cholesterol, derivatives of phosphatidylcholine and phosphatidylethanolamine, Cremophor®, pullulan, hyaluronic acid, ferritin, human serum albumin (HSA), heparin, dextran, polyaminoacids ⁇ e.g., a- poly-L-glutamic acid) and their derivatives ⁇ e.g., PHEA, PHEG), polyglycerol, polyacrylamide, polyvinylpyrrolidone, poly(2-oxazoline) and their derivatives.
- these polymers or molecules such as PEG-PLGA or PEG Pluronic®, may be used for the construction of appropriate supramolecular architecture
- block copolymers such as those with the A-B-A or A-B architecture, can be advantageously used to prepare supramolecular systems suitable for the delivery of the coordination compounds herein disclosed.
- said building blocks which can be equal or different each other, can be intended both as the monomer (e.g., ethylene oxide) of a polymer such as PEG or PF127, or of an inorganic nanostructure ⁇ e.g., Ti0 2 ), and the same polymer inside aggregates, for instance micelles.
- nanostructures encompasses also for example micelles, liposomes, protein- and peptide-based aggregates and carriers consisting of cyclodextrins or dendrimers.
- biocompatible polymers and oligomers natural or synthetic, identical or different ⁇ e.g., phospholipids, pullulan, PEG, PF127, cholesterol) can form micelles or liposomes, in order to modulate the final properties of the composition and therefore of the final formulation.
- These polymers, identical or different are the constituent units that aggregate during the formation of the supramolecular system.
- One of them or all of these polymers can be conveniently functionalized with carbohydrates (by using the functional groups exploitable for this purpose), and in an independent manner, with yields up to 100%.
- This/these polymer(s) can then be diluted with the same or other polymers to achieve, for example, a micelle or a liposome with a water-exposed surface having a variable percentage of functionalization, ranging from 1 to 50% mol/mol.
- the range 10 "5 ⁇ 10 "10 mol of compound/mg of formulation can be achieved when preparing compositions of said coordination compound.
- said compound can be encapsulated in the hydrophobic core of a micelle, or in the hydrophilic counterpart of a liposome, or in their lipidic/polymeric layer.
- Such supramolecular architectures can in turn be made with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan and polyethylene glycol.
- the nanocarrier ⁇ e.g., liposome, micelle
- the nanocarrier can be conveniently covered with a hydrophilic and biocompatible coating in order to improve the pharmacokinetic profile of the "naked" supramolecular aggregate itself.
- the surface of the supramolecular aggregate is hidden from blood components, including the opsonin proteins, which are responsible for the recognition and attack of the nanocarriers by phagocytes (monocytes, macrophages).
- This masking strategy allows the supramolecular systems to sidestep the natural processes of biotransformation/elimination of exogenous constituent entities or substances. Moreover, it determines an increased bioavailability and, hence, a prolonged circulation time of the compound loaded into the carrier, if compared to supramolecular aggregates incorporating the coordination compounds according to the present invention and non-covered with said hydrophilic and biocompatible coating.
- the application of the "effect stealth" to the coordination compounds according to the first and the second embodiment of the present invention may also limit or eliminate any problem at the physiological level, such as hemolysis or immunogenicity of the active compounds.
- hydrophilic coating of nanocarrier obtained with polymers such as PEG reduces the self- aggregation of the particles by means of steric stabilization, thus resulting in an immediate impact on the freshly-prepared formulation for the administration at the hospital level and storage.
- the surface of the nanocarrier can be coated, by adsorption or conjugation, with human serum albumin (HSA) in order to conveniently increase the stability, the circulation times, and the biocompatibility of the formulation.
- HSA human serum albumin
- the whole supramolecular aggregate reduces the off-target release of the drug by the so-called EPR effect (Enhanced Permeability and Retention).
- EPR effect is based on the anomalous and high permeability of blood vessels in the tumor region, characterized by not-adherent endothelial cell-to-cell junctions, if compared to normal tissue capillaries.
- Appropriate nanocarrier sizes ranging from 20 to 100 nm (as reported in the examples of this invention), result in a prolonged circulation of the supramolecular systems in the blood stream, and the consequent selective extravasation into tumor tissues, resulting in a passive targeted therapy, which positively influences the toxicological profile.
- the lack of lymphatic drainage of cancerous compartments promotes the selective accumulation of the nanocarriers in the tumor microenvironment.
- the functionalization of the nanocarriers with cancer-targeting moieties can also restrict the onset of side effects, thereby improving the chemotherapeutic index in terms of toxicity/activity ratio (active targeting).
- nanocarriers are functionalized with carbohydrates as cancer-targeting moieties
- these supramolecular or macromolecular systems give the possibility to increase the water-solubility and the stability of the encapsulated compound(s) as well as to improve the bioavailability, and to extend and facilitate the selective release towards cancer cells.
- the first cancer-targeting strategy, associated with the third embodiment, is related to the passive targeting mediated by EPR effect described above.
- the second strategy, presented in the fourth embodiment takes into account the active targeting via glycoconjugation of the supramolecular nanosystem, thus providing an additional way to achieve a high therapeutic selectivity.
- the encapsulation allows the loaded compounds to increase their blood circulation times, as well as to improve the chemotherapeutic index of the active compounds (ratio between the lethal dose LD 50 and the effective dose ED 50 ), in other words their pharmacokinetic and pharmacodynamic profiles.
- n-octanol/water P
- P partition coefficient
- the coordination compounds having a general Formula l(a) and/or Formula l(b) are encapsulated in supramolecular aggregates, in particular micelles, to achieve a passive targeting mechanism mediated by the EPR effect.
- Said encapsulated compounds may be those of the second embodiment, and hence conjugated to carbohydrates, or without a cancer-targeting moiety (first embodiment).
- the ideal size of supramolecular systems ranges from 10 nm to 100 nm.
- a fast drainage from the injection site and an effective distribution in vivo are obtained with particles characterized by a hydrodynamic diameter (D H ) ranging from 10 to 70 nm.
- the nanocarriers with D H ⁇ 100 nm at the cellular level can be absorbed by an endocytic process.
- the size of the aggregates can significantly affect the circulation times and the bioavailability of the encapsulated compound.
- n- octanol/water (P) expressed in its logarithmic form, logP
- logP logarithmic form
- this procedure is fundamental for the choice of the most suitable supramolecular system for a specific type of compound and/or application.
- concentration of the compounds in the two immiscible phases has been determined in n-octanol before and after mixing with a defined volume of water.
- n-octanol was pre-saturated with deionized water for 24 hours under stirring, then let to equilibrate for 6 h at 25 °C.
- the encapsulation of the complexes having Formula l(a) and/or Formula l(b) in PF127 micelles was obtained via a process comprising the following steps, described herein by way of example, but not limitation of the present invention: 1 ) Co-dissolution of the compound to be loaded and the polymer in the desired stoichiometric ratio ⁇ e.g., 0.5 mg and 500 mg, respectively) in an organic solvent, preferably chloroform; 2) Evaporation of the organic solvent, preferably under reduced pressure, followed by drying of the obtained powder, preferably under vacuum; 3) Hydration of the powder by addition of deionized water; 4) in order to remove the non- encapsulated compound, bacteria and other impurities, purification for instance by filtration with a membrane with a 0.20 ⁇ cut-off; 5) Freezing of the sample in a dry ice/acetone cooling bath at -78 °C and cryoesiccation to remove the residues of the aqueous solvent, thus obtaining
- the amount of the encapsulated coordination compound was assessed by UV-Vis analysis, after dilution in DCM of a defined amount of lyophilized micellar formulation.
- concentration of the compound was defined using the Lambert-Beer law, after experimental determination of the molar extinction coefficient ⁇ in DCM (based on the "matrix effect", in the presence of the same polymer used for the preparation of micelles) for some absorption bands. The obtained results are reported in Table 5.
- the encapsulation in micelles of the metal-based compounds according to the present invention increases their stability in physiological media and make them water-soluble for at least 72 hours.
- the collected electronic spectra surprisingly show no significant change over time for the considered formulations (if compared to the not encapsulated compound) once dissolved in a physiological environment consisting of phosphate buffer/cell culture medium 9: 1 v/v, and phosphate buffer/human serum 95: 5 v/v.
- compositions comprising the coordination compounds according to the present invention, characterized by a complete solubility and stability in physiological media compared to the corresponding non-encapsulated compounds, thus achieving a further object of the present invention.
- nanoformulations engineered and finely tailored, are unexpectedly stable also in a chemically aggressive medium, such as that charactering the tumor microenvironment, here mimicked with an acetate buffer (pH 5.5, Figure 3).
- micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus exploiting to the best the EPR effect.
- PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.
- micel la r systems here reported, determined via DLS ana lysis.
- PDI polydispersion index.
- micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus exploiting to the best the EPR effect.
- PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.
- the inventors have surprisingly demonstrated the possibility to encapsulate the coordination compounds of general Formula l(a) and l(b) in macromolecules or in a supramolecular system, thus overcoming in a non-trivial manner evident limits in the state of the art.
- the encapsulation techniques of the metal-based derivatives are still at the beginning, due to the inherent reactivity of the inorganic molecules.
- the encapsulation allows to protect said compounds from the biotransformation, avoiding interactions/reactions with blood stream biomolecules or cells.
- the encapsulation allows increasing the stability as well as the solubility of hydrophobic compounds in the physiological media, as the here-presented data demonstrate.
- the coordination compounds having general Formula l(a) and l(b) are advantageously encapsulated in supramolecular aggregates and functionalized with carbohydrates. Therefore, they are directed to the tumor site according to an active-targeting approach which is paralleled with (hence boosting it) the mechanism of passive targeting of the previous embodiment.
- the whole supramolecular aggregate acts as a cancer-targeting carrier, conveniently exploiting the Warburg effect, to further improve the therapeutic selectivity.
- the inventors aim to implement a mechanism wherein a coordination compound according to the invention is absorbed by the cell by endocytosis along with the whole supramolecular aggregate; alternatively, said compound can be released from the nanocarrier in the extracellular matrix, and consequently diffusing inside the cell if hydrophobic enough, or entering the cell through alternative mechanisms of uptake, or also triggering its antitumor activity at the cell membrane level.
- compounds of the first embodiment which by their chemical nature inherently exhibit affinity for the cell membrane, or compounds of the second embodiment that can exploit the presence of a cancer-targeting moiety T in the selective recognition by carbohydrate transporters ⁇ e.g., GLUT1 ), are advantageously used, alternately or in combination between them.
- supramolecular aggregates can be micelles, vesicles (liposomes), cyclodextrins , dendrimers, organic polymeric nanoparticles, for instance of protein or peptide nature, and inorganic nanoparticles ⁇ e.g., silica, zirconia, titania).
- Carbohydrates can be monosaccharides, polysaccharides, pentoses, hexoses, aldoses, ketoses.
- Such supramolecular aggregates may have a mixed structure, for example being made up of pullulan and PF127, or PLA and PEG.
- the biopolymer so functionalized with a carbohydrate after dilution with a non-functionalized biopolymer (equal or different), is used to form a supramolecular aggregate with a degree of functionalization, ranging from 1 % to 50% mol/mol.
- a degree of functionalization ranging from 1 % to 50% mol/mol.
- the polymer mPEG5000-Ts was refluxed in dry DMF for 5 hours in presence of 20 eq. of phthalimide potassium salt (Phta K). Successively, after filtration of byproducts and precipitation with diethyl ether, a light yellow solid was isolated.
- the functionalization degree was equal to 100%, in light of the integration values of the NMR signals corresponding to the aromatic protons of the phthalimide and methyl protons of the terminal methoxy group of mPEG5000 polymer.
- IR: v (cm 1 ) 2885, 1716, 1467, 1344, 1 1 14, 1060, 842, 724, 690.
- IR: v (cm 1 ) 2884, 1467, 1344, 1 1 13, 1060, 842.
- This reaction allows the conversion of the terminal hydroxyl group to a carbonate which is very reactive towards nucleophiles due to the presence of a succinimido leaving group.
- the reaction was performed in a Schlenk line. Briefly, mPEG5000 was dissolved in dry 1 ,4-dioxane and was added to a mixture of DMAP (6 eq) and ⁇ , ⁇ '-disuccinimmidil carbonate (DSC) in dry acetone. After 6 hours, the by-products were filtered and the solution volume was reduced to precipitate a white solid after the addition of diethyl ether.
- the functionalization degree is 100%, considering the integration values of NMR signals corresponding to the methylene protons of the succinimido group and the methyl protons of the terminal methoxy group of mPEG5000 polymer.
- the bio-conjugated mPEG5000-GluOAc was obtained via nucleophilic attack of the terminal hydroxyl group of the polymer to the anomeric C1 carbon of 1 ,2,3,4,6-penta-0-acetyl ⁇ - glucopyranose in the presence of BF 3 Et 2 0.
- 3 eq. of BF 3 Et 2 0 were added to a mixture of mPEG5000 and 1 ,2,3,4,6-penta-0-acetyl ⁇ -D-glucopyranose (3 eq.) in dry CH 2 CI 2 dry at 0 °C, and the mixture was stirred for 48 hours.
- the functionalization degree was equal to 20%, based on the integration values of NMR signals corresponding to the methyl protons of the acetate and the methyl protons of terminal methoxy group of mPEG5000 polymer.
- IR: v (cm 1 ) 2887, 1759, 1467, 1344, 1 1 1 1 1 , 1060, 842.
- the polymer mPEG5000 functionalized with ⁇ -D-glucopyranose was obtained from the product of the previous step (mPEG5000-GluOAc) via deprotection of the acetyl groups under basic conditions (NaOMe in dry methanol) under stirring for 15 hours. Then, the reaction mixture was neutralized with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After the filtration of the resin, the product was precipitated with diethyl ether and dried under vacuum.
- IR: v (cm 1 ) 3453, 2886, 1759, 1467, 1344, 1 1 12, 1060, 842.
- the obtained polymer results 20% functionalized as O-glycoside (in C1 position), and can be used as such to encapsulate the coordination compounds, thus forming micelles or aggregates.
- cancer-targeting nanoformulations such as, without loss of generality: mixed micelles, liposomes, HSA nanoparticles.
- the latter covered with a carbohydrate-functionalized mPEG, realize a "stealth effect", which is known to be associated with a decreased opsonization.
- the degree of oxidation to aldehyde (evaluated via 1 H-NMR in CD 2 CI 2 ) was equal to 80%, considering the integration values of NMR signals corresponding to the aldehyde proton and the methyl protons of the central PPO unit of the PF127 polymer.
- IR: v (cm 1 ) 2884, 1630, 146, 1344, 1 1 15, 1061 , 842.
- PF127-GlnOAc has been obtained by reductive amination between the PF127-CHO derivative described above and 1 ,3,4,6-tetra-0-acetyl-2-amino-2-deoxy ⁇ -D-glucopyranose HCI.
- the first step involves the nucleophilic attack of the glucosamine, reaction carried out in acetonitrile for 2 hours at room temperature. Subsequently, the reduction of the imine to amine was obtained by the addition of sodium cyanoborohydride.
- the reaction occurs in acetonitrile for 30 minutes at room temperature with a total stoichiometric ratio 1 :1 :3 between PF127-CHO, glucosamine- HCI and NaCNBH 3 , respectively. Subsequently the solvent was removed under reduced pressure and the residue is taken up in DCM, washed in a separatory funnel with "brine". The organic phase is anhydrified and the product precipitated with diethyl ether.
- the degree of functionalization (assessed via 1 H-NMR in CD 2 CI 2 ) was equal to 44%, considering the integration values of NMR signals corresponding to the acetate protons of glucosamine and the methyl protons of the central PPO unit of the PF127 polymer.
- IR: v (cm 1 ) 2883, 1757, 1467, 1344, 1 1 14, 1060, 842.
- the polymer PF127 functionalized with 2-amino-2-deoxy-D-glucose (glucosamine) was obtained from the PF127-GlnOAc of the previous step by deprotection of the acetyl groups under basic conditions (NaMeO in dry methanol) under stirring for 15 h. Subsequently, the mixture was treated with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After filtration, the product was precipitated with diethyl ether and dried under vacuum.
- an acid resin e.g., Amberlite® FT form
- IR: v (cm 1 ) 3432, 2885, 1467, 1344, 1 1 12, 1060, 842.
- the obtained polymer results 44% functionalized with glucosamine in C2 position and can be used as such to encapsulate the coordination compounds, forming micelles. Alternatively, it may be used in combination with other biocompatible polymers to obtain cancer-targeting nanoformulations.
- mixed micelles or supramolecular aggregates can be prepared starting from this derivative, after "dilution" with non-functionalized PF127, or another biocompatible polymer, in order to obtain different degrees of functionalization of the supramolecular aggregate, preferably in the range 2 ⁇ 18%.
- micellar systems herein described determined by DLS analysis. Surprisingly, all the synthetic procedures allow achieving a degree of functionalization equal or greater than 20%, as verified through 1 H-NMR analysis in CD 2 CI 2 .
- the purification is often troublesome due to the high chemical similarity between the polymeric reagents and their products, characterized by minimal changes in the molecular structure (in terms of molecular weight and introduced functional groups). Given the complexity of these systems, also the functionalization/bio-conjugation yields are often low ( ⁇ 50%), and the assessment of the degree of functionalization of the products results difficult because of the abovementioned purification problems and the instrument sensitivity.
- the coordination compounds according to the first and second embodiment of the present invention possess a remarkable anticancer activity.
- said compounds loaded into aggregates, according to the third and fourth embodiment maintain such antitumor capacity, thus achieving a further advantageous object of the present invention.
- these supramolecular aggregates play only the role of nanocarriers, in particular increasing the stability and solubility of said compounds in aqueous medium, and anyway allowing the release of the active compound within the cancer cell or in the extra-cellular matrix, or in general, in the bloodstream.
- Table 8 IC 50 values ( ⁇ ) for PF127-based formulations evaluated after a 72-h treatment. Values are calculated based on the concentration of the encapsulated ruthenium-DTC complex. Lyophilized micelles were dissolved in cell culture medium. Data represent the mean ⁇ SD of at least three independent experiments. THERANOSTIC USE OF THE COORDINATION COMPOUNDS AND THE RELATED COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS
- the coordination compounds, the compositions, and the pharmaceutical formulations according to the present invention can be used as "theranostic agents" i.e. not only for the treatment of human or animal diseases, in particular neoplastic diseases, but also for the diagnosis and the patient follow-up.
- such mechanism that combines therapy and diagnosis can be made in many ways, properly including one or more contrast agents in a coordination compound, or in a composition comprising at least one of said coordination compound, or also in a pharmaceutical formulation that includes said compound and/or composition.
- the contrast agent can be "intrinsic" to the compound, for example when the metal centers M M 2 , M 3 are radionuclides ⁇ e.g., 68 Ga, previously introduced).
- the donor atoms X, Y, W, Z, L L 2 , L 3 and L 4 are radioisotopes ⁇ e.g., 131 1 , 127 l, 129 l), as well as T can be 2-deoxy-2-[ 18 F]-fluoroglucose.
- the coordination compound, the unit A, the carbohydrate or glucide T and/or T is paramagnetic or diamagnetic.
- the contrast agent can be "exogenous" to the compound, and may be included in the composition that comprises at least one coordination compound according to the present invention, or also in a pharmaceutical formulation, that includes said compound and/or composition.
- this category comprises a carrier that encapsulates said compound, such as a up-converting bismuth oxide nanoparticle (e.g. Italian patent 0001419393, in the name of BEP Sri et al.).
- the contrast agent is a dual-mode contrast agent that take advantage of the X-ray absorption and the optical emission (through a NIR->NIR or NIR->VIS mechanism) exhibited by the doped bismuth oxide nanoparticles.
- the "exogenous" contrast agent may also be a component of the pharmaceutical formulation, for example, a known contrast agent.
- a “combined” contrast agent can be used and obtained mixing one or more "intrinsic" contrast agents with “exogenous” contrast agents.
- radionuclides such as 131 1, 127 l, 129 l may be previously introduced as donor atoms X, Y, W, Z, L L 2 , L 3 and L 4 in the general Formula l(a) or l(b) ; the coordination compound can be linked to a carrier consisting of a photoactivatable nanoparticle, such as a properly doped bismuth oxide nanoparticle.
- the coordination compound according to the invention can be advantageously used to obtain a compound or a theranostic agent
- the contrast agent is detectable by suitable devices or detectors, hence to be usable in association with diagnostic and imaging systems in the medical or biomedical field.
- said contrast agent must be able to emit a signal detectable spontaneously, or after interaction with, for instance, irradiation with external particles, or with an external magnetic/electric field.
- Contrast agents capable of spontaneously emit a detectable signal are, for example, radionuclides such as 1 1 C, 18 F, and 68 Ga (in this case the signal is a radiation and/or a particle), or radiopaque materials (the signal here is related to the X-ray absorption spectrum).
- contrast agents emit a signal which is detectable after an external stimulus, which is typically the irradiation with particles and suitable electromagnetic radiations (based on the needs).
- this category comprises: luminescent photoactivable contrast agents, such as nanoparticles, dye-molecules or luminophores, capable of emitting IR-Vis radiation as a result of laser irradiation with a suitable wavelength ; contrast agents capable of emitting a detectable signal due to the presence of dipoles or magnetic domains within their structure, which interact with an appropriate electromagnetic field; it is also possible to generate radionuclides inside of the compound and/or the composition according to the invention, treating them with particles and electromagnetic radiations with the suitable energy, using known techniques.
- theranostic applications of the coordination compounds having general Formula l(a) and/or l(b) are very promising in light of the high selectivity and bioavailability, being as above presented, related to the presence of the carbohydrate or glucide T and/or T', that acts as cancer-targeting moieties able to implement a passive and active targeting-mechanism towards the tumor cells.
- Pluronic® is a trademark of BASF AG
- Cremophor ® is a trademark of BASF AG
- CD-1 ® is a trademark of di Charles River Laboratories, Inc. Corp.
- Eurospital ® is a trademark of Eurospital Spa
- Lipoxal ® is a trademark of Regulon A.E;
- Amberlite ® is a trademark of Santa Cruz Biotechnology Inc.
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Abstract
The present invention relates to mononuclear and dinuclear coordination compounds of Ru and Ga, pharmaceutical formulations based thereof, the relative method of synthesis and encapsulation of the compounds in macromolecules, supramolecular aggregates or nanostructures, as well as their use for the diagnosis and/or treatment of neoplasia. Advantageously, such coordination compounds and/or formulations may contain carbohydrates which act as "cancer-targeting moieties" thereby increasing therapeutic selectivity. Said compounds and formulations are characterized by a promising toxicological profile, a remarkable and highly-selective anti-cancer activity, as well as stability and solubility in the physiological means.
Description
TITLE: COORDINATION COMPOUNDS, SYNTHESES, NANOFORMULA TION AND USE
THEREOF IN ONCOLOGY.
TECHNICAL FIELD
The present invention regards mononuclear and dinuclear Ru-based and Ga-based coordination compounds, pharmaceutical formulations thereof, the related synthesis and encapsulation method in macromolecules, supramolecular aggregates or nanostructures, as well as their use in the diagnosis and/or the treatment of neoplasms. Advantageously, such coordination compounds and/or their formulations are glycoconjugated with carbohydrates which act as cancer-targeting moieties, thus increasing the therapeutic selectivity.
BACKGROUND ART
Cisplatin is a milestone among the anticancer compounds and, since its introduction in pharmacopoeia in 1978, has shown to be one of the most effective drugs, particularly in the treatment of testicular, ovarian, lungs, bladder and uterine cervix cancer, mesothelioma and head and neck carcinoma. Despite its therapeutic efficacy, the clinical use of cisplatin is characterized by severe side effects. The most serious one is the high toxicity, especially to kidneys, to auditory apparatus and to bone marrow. In addition, cisplatin demonstrates intrinsic or acquired resistance to certain tumors. Finally, it is characterized by a very limited intrinsic selectivity, so that only a small percentage (around 2 - 5% in moles) of cisplatin reaches the tumor cell DNA, which is currently recognized as its most important target. Further limitations to the clinical use of cisplatin are the poor solubility and the high reactivity of the platinum(ll) center which limit the bioavailability and, therefore, the mode of administration.
The intense research activity carried out in the recent decades has attempted to moderate these effects and limitations but has only partially achieved the desired effects. In fact, the numerous cisplatin-like compounds, such as carboplatin and oxalilplatin, still have a lower antitumor activity or a poor toxicological profile. The main reason for this failure is due to the fact that this metal has a high affinity for sulfur-containing residues of biomolecules.
As mentioned above, the oncological research in recent years has considered alternative approaches that will be described briefly here.
A first strategy aims to synthesize new coordination compounds with metal centers other than platinum that can maintain the antitumor activity but, at the same time, are characterized by a better toxicological profile. For example, in a first patent Fregona et al., were able to synthesize a class of Au(lll) dithiocarbamato complexes, described in ITMI20030600 which proved intrinsically less toxic than the Pt(ll) counterparts. Moreover, in a second document, WO2010105691 A1 , Fregona et al. have claimed a new class of Au(lll) dithiocarbamato compounds functionalized with a peptide moiety designed to be internalized by tumor cells by PEPT transporters. However, the syntheses therein proposed do not permit to obtain the
compounds in pure form, but as a mixture of two different complexes having the same empirical formula. This result was not evident from the elemental analysis. This aspect represents a serious limitation, as in the oncology field, syntheses that lead to pure compounds or to mixtures of easily purified molecules are needed in order to favor both administration to humans and the industrial-scale production. In addition, for some types of cancer, PEPT transporters do not represent the optimal target to favor selectivity as they are not adequately overexpressed in tissues.
The ruthenium-based complexes represent another example of compounds reported in the literature. For instance, H. Fasal et al. report that (J. Coord. Chem., 2016, 70 (2), 279-295), ionic Ru(ll) mononuclear complexes are known, and they contain DMP (2,9-dimethyl-1 ,10- phenanthroline) and dithiocarbamates derived from piperazine (with the nitrogen atom in position 4 functionalized with a para- or meta-methoxyphenyl group) as ligands. Such complexes have been studied as DNA (both purified and derived from calf thymus) intercalants. Other authors reported on some Ru (II, III) complexes with cytotoxic properties: Nardon et al., (Future Med. Chem., 2016), Nagy et al. (Chem. Eur. J, 2012 and Dalt. Trans. 201 1 ), Giovagnini et al. (Dalt. Trans, 2008). In particular, these compounds are mono- and dinuclear complexes with DTCs of the type DMDT (dimethyl-DTC), PDT (pyrrolidine-DTC) and RSDT (DTCs derived from sarcosine; R = tert-butyl, ethyl, methyl). Another class of coordination compounds with potential biomedical applications consists of mononuclear Ga(lll) complexes with alkyl- dithiocarbamato ligands, wherein the nitrogen atom is bound to a methyl and a 2-methyl-1 ,3- dioxolane or a CH2-CH-(OMe)2 group, alternatively (Ferreira et al., J. Coord. Chem., 2014, 67 (6), 1097-1 109).
Along with the development of new coordination compounds, a second and promising strategy in the oncological research is associated with the exploitation of the so-called Warburg effect. In this regard, most cancer cells trigger an accelerated metabolism sustained by an high carbohydrate consumption, if compared to healthy cells. In particular, tumor cells produce energy mainly through glycolysis, followed by the reduction of pyruvate to lactic acid in the cytoplasmic matrix, rather than oxidation of pyruvate within the mitochondria, which is the favorite ATP-producing mechanism in almost all the normal cells. The enclosed Figure 1 clearly highlights that the expression levels of the GLUT1 transporter generally increase during the neoplasia development, as well as with the degree of malignancy of the tumor itself.
Nowadays, this phenomenon is exploited in imaging-based diagnostic techniques and in the oncological patient follow-up via the administration of the tracer FDG (2-[18F] fluoro-2-deoxy-D- glucose). Accordingly, glucose and other carbohydrates are potentially useful biomolecules to selectively target antitumor agents. According to this approach, a variety of glyco-conjugates made up of known cytotoxins or chemotherapeutics linked to glucose (or other carbohydrates) have been synthesized to increase their selectivity to and absorption by the tumor cells. For example, the glyco-conjugate glufosfamide is currently in clinical trials for the treatment of
ovarian and pancreatic cancer, glioblastoma multiforme and non-small cell lung carcinoma. Other examples of metal and non-metal glyco-conjugates derivatives are cited in various works published in recent years, including that of Emilia C. Calvaresi and Paul J. Hergenrother "Glucose conjugation for the specific targeting and treatment of cance , the paper of S. Tabassum and coworkers "Exploration of glycosylated-organotin(IV) complexes as anticancer drug candidates", and the work of P. M. Abeysinghe and M. M. Harding "Antitumour bis(cyclopentadienyl) metal complexes: titanocene and molybdocene dichloride and derivatives".
Concerning the patent literature, for example, Lippard et al. attempted to overcome the above- mentioned limits of platinum-based drugs through the conjugation of one or more metal- coordinated ligands with different cancer-targeting moieties, including carbohydrates. However, these documents {e.g., the patent US/ 2014/034313), claim but do not describe synthetic procedures useful for an expert in order to conjugate a coordination compounds to carbohydrates, to be used just as cancer-targeting moieties.
To sum up, according to the best knowledge of the present inventors, only a limited number of coordination compounds coniugated to sugars, their synthetic processes, optimized for a specific metal, are known up to date.
Lastly, a further strategy adopted to mitigate the side effects (toxicity) and the limitations (low solubility and bioavailability) of cisplatin, as well as of other drugs used in oncology, is the recourse to encapsulation techniques in supramolecular systems, such as liposomes. However, if these techniques are already mature in the oncological field for organic compounds, the encapsulation of coordination compounds is still in the early stages due to inherent difficulties when combining highly- reactive metal-based compounds with biocompatible polymers. For these reasons, the encapsulation of coordination compounds is a still poorly explored research field in literature, except for some studies related to platinum derivatives. Among these, at present the formulation of cisplatin in liposomes composed of cholesterol and phosphatidylcholine, known with the LipoPlatin® is in phase III clinical trials. In addition, cisplatin modified to bond by a linker (polyglutamic acid) the PEG (Nanoplatin®, NC -6004) has received the authorization to start the clinical phase III of test on pancreatic cancer in combination with gemcitabine. Among the formulations based on cisplatin substitutes, oxalilplatin encapsulated in liposomes (known under the trade term of Lipoxal®) is currently in an advanced stage of development, while the same oxalilplatin, encapsulated in transferrin-conjugated liposomes (MBP-426), has completed the Phase I. A modified oxalilplatin (NC-4016) wherein a PEG is linked to the platinum(ll) metal center via a polyglutamic acid linker is about to start the phase I.
TECHNICAL PROBLEM
The intense research activity conducted over the last few decades has attempted to mitigate the side effects (toxicity) and the limitations (low solubility and bioavailability) of cisplatin, but the strategies adopted so far have only partially achieved the desired results.
Due to the peculiar reactivity preferably against neoplastic cells, drugs based on coordination compounds still represent one of the most promising anti-cancer option, but there is a need to make available a large number of new compounds with metal center other than platinum, which may replace cisplatin in the future, considering that most candidate compounds do not pass the advanced preclinical or clinical tests and therefore do not become drugs.
There is also a need for other coordination compounds with metals other than platinum and conjugated with cancer-targeting moieties, represented by carbohydrates, potentially useful in oncology and characterized by high selectivity (limited or negligible "off-target activity") and effectiveness towards different types of tumors and ultimately low or negligible toxicity. Lastly, it is necessary to have coordination compounds with metals other than platinum encapsulated in supramolecular aggregates, such as micelles, liposomes or cyclodextrins, which are conjugated or not to cancer-targeting moieties represented by carbohydrates.
DISCLOSURE OF INVENTION OBJECT/SCOPE OF THE INVENTION
Through the present invention, the present inventors intend to overcome the existing limits in the state-of-the-art related to coordination compounds useful as antitumor agents.
Accordingly, it is a first and main object of the present invention to design and synthesize Ru- based and Ga-based mononuclear and dinuclear coordination compounds to be used as antitumor agents. Furthermore, a second object of the present invention is to identify mononuclear and dinuclear Ru-based and Ga-based coordination compounds with carbohydrate-functionalized ligands which act as selective cancer-targeting moieties.
Particularly, the present invention intends to disclose the advantageous use of carbohydrates as selective cancer-targeting moieties. In addition, carbohydrates could provide the additional advantage of increasing the solubility of the final compound in aqueous media.
Still, a third important object of the present invention is to identify coordination compounds endowed with low or negligible toxicity with respect to other metal-based compounds, such as cisplatin and subsequent compounds. Furthermore, a fourth object of the present invention is to identify coordination compounds characterized by high solubility in aqueous media, high stability and bioavailability, inherent or achieved by encapsulation in macromolecules, nanostructures or supramolecular aggregates, such as micelles, liposomes, proteins or cyclodextrins. In particular, such structures may or not be functionalized with carbohydrates on the outer surface.
Moreover, a fifth object of the present invention is to provide a stable pharmaceutical formulation including one or more of said coordination compounds, usable as an antitumor agent and preferably administrable intravenously or orally. Such formulations may optionally include additional anticancer drugs.
Within the above-mentioned main tasks, a further object of the present invention is to provide a method for synthesizing said mononuclear and dinuclear Ru-based and Ga-based coordination
compounds, bioconjugated or not with carbohydrates, as well as a method for encapsulating said compounds in macromolecules, supramolecular aggregates made up of biocompatible polymers, functionalized or not with carbohydrates.
In addition, a further object of the present invention, is to disclose the use of said coordination compounds as antitumor agents, particularly in the treatment of "orphan tumors", such as the "triple negative" breast cancer (TNBC), the castration-resistant prostate cancer (CRPC), head and neck cancer, the NSCLC, the melanoma, the mesothelioma, the lung, pancreas and liver carcinoma. Last but not least, a final object of the present invention is to produce coordination compounds which, besides possessing the necessary chemical-biological properties, are stable and obtainable through a synthesis process both able to intrinsically yield high-purity compounds and being industrially applicable with known and cost-effective technologies compared to state-of-the-art solutions.
TECHNICAL SOLUTION
In view of the above disadvantages or drawbacks of the prior art, the present inventors have made a lot of studies related to the preparation of coordination compounds useful as anti-cancer agents.
After long terms of practice the inventors found a new class of Ru-based and Ga-based mononuclear and dinuclear coordination compounds, which are described by one of the following eneral formulae I (a) or l(b):
1(a) 1(b)
wherein , M2 , M3 identify the metal center of the coordination compound which can be Ga(lll), Ru(ll), Ru(lll). The mononuclear and dinuclear coordination compounds herein disclosed are neutral or ionic complexes whose charge is neutralized by at least one counter- ion G and have different coordination geometries, for example regular octahedral or distorted octahedral.
Further features and advantages of the new coordination compounds and the corresponding synthesis and encapsulation methods thereof, as well as the use of said compounds, will become apparent to those skilled in the art via the detailed description of its four preferred embodiments, herein provided below by way of example but not limitation.
In a first preferred embodiment of the present invention, the coordination compounds of formula 1(a) or 1(b) are not bioconjugated with a cancer-targeting moiety and act as antitumor agents due to the inherent reactivity of the Ru and Ga metal centers.
In a second preferred embodiment, the coordination compounds of formula 1(a) or 1(b) are bioconjugated with a carbohydrate which acts as a selective cancer-targeting moiety, advantageously exploiting the so-called "Warburg effect". Surprisingly, the presence of the cancer-targeting moiety, in combination with the physico-chemical properties of the final compound {e.g. ionic nature), can result in a very high solubility in physiological media.
In the third preferred embodiment of the present invention, the coordination compounds of formula I (a) or l(b) are encapsulated in macromolecules, nanostructures or in supramolecular aggregates, such as liposomes or cyclodextrins, acting as carriers to carry out a passive- targeting mechanism mediated by the Enhanced Permeability and Retention (EPR) effect that characterizes tumor districts. Said compounds may be or not conjugated with carbohydrates. In a fourth preferred embodiment, the coordination compounds described in the formula l(a) or l(b) may be addressed to the tumor site by encapsulating such compounds in macromolecules, nanostructures or supramolecular aggregates, for example micelles, in turn functionalized with carbohydrates to achieve an active-targeting approach ("Warburg effect"), which accompanies and strengthens the passive-targeting mechanism of the previous embodiment. Even in this case, the encapsulated compounds can be or not conjugated with carbohydrates.
ADVANTAGEOUS EFFECTS OF INVENTION
The coordination compounds of structure I (a) and/or l(b) according to four preferred embodiments have a number of remarkable advantages which cannot be achieved by prior art compounds as it will be apparent to those skilled in the art.
First of all, the present invention discloses Ru-based and Ga-based coordination compounds, glycoconjugated or not, which, according to experimental tests, possess remarkable anti-tumor properties. Furthermore, various synthesis processes of said coordination compounds have been disclosed, characterized by high yields: they allow the expert to choose the most advantageous scheme depending on the metal center and type of desired ligands, including those containing a cancer-targeting moiety represented by a specific carbohydrate. Said cancer targeting moiety and metal centers can in turn be chosen based on the molecular profile of the patient's neoplasm according to the paradigm of personalized medicine.
The Ru-based and Ga-based coordination compounds according to the present invention can be loaded into supramolecular aggregates or macromolecules, consisting of a wide range of biocompatible polymers so to obtain stable nanoformulations. In other words, said compounds do not react nor establish interactions with other loaded complexes, and with the polymeric carrier neither.
Advantageously, the coordination compounds and nanoformulations allow to realize a both active and passive cancer-targeting mechanism. These compounds are characterized by optimal LiPE values for a promising pharmaceutical development {"druglikeness").
Moreover, said nanoformulations not only make the hydrophobic active principles soluble in the aqueous media but also mask the active principles herein described, both hydrophobic and hydrophilic, from reactions/interactions with the cellular and molecular components of the blood. Additional objects and advantages of the invention will be set forth in part in the detailed description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
DESCRIPTION OF PR A WINGS
The present invention will be more fully understood by reference to the following drawings which are for illustrative purposes only:
- Figure 1 depicts the levels of expression of the glucose transporter (Glutl ) in different cell lines. In letter A), Northern analysis of the Glutl mRNA levels evaluated in healthy human prostate samples and in three different human prostate cancer cell lines with decreasing differentiation. LNCaP is a hormone-sensitive cancer cell line, whereas the DU-145 and PC3 lines represent poorly-differentiated tumors (source: P. Effert et al., Anticancer Research 24: 3057-3064, 2004). In letter B), Western analysis of Glutl protein levels evaluated in samples derived from 4 different cell lines: cervix adenocarcinoma (HeLa), lymphoblast T (Jurkat), epidermoid skin cancer (A431 ), human embryonic kidney (HEK293) (from Abeam pic). Finally, in letter C), Western analysis of the Glutl protein levels evaluated (cell lysate 20 μg/lane, anti-Glut-1 ab 2 g/mL, anti-β- actin ab 0.5 g/mL, secondary antibody 1 :1000) in samples derived from 3 different human cell lines: triple negative breast cancer (MDA-MB-231 ), pulmonary adenocarcinoma (A549), colon carcinoma (Caco2).
Figure 2 shows the UV-Vis spectra of the coordination compound [Ru2(PipeDTC)5]CI with (VII) general structure. In panel A) said compound is dissolved in phosphate buffer (pH 7.4, 0.5% v/v DMSO, spectra recorded at 37° C over 24 hours), whereas in panel B), it is encapsulated in PF127 micelles (5 mg/mL) dissolved in phosphate buffer - cell culture medium 9:1 v/v (spectra recorded at 37 °C for 72 hours).
- Figure 3 displays the UV-Vis spectra of [Ru2(PipeDTC)5]CI encapsulated in PF127 micelles dissolved in acetate buffer (pH 5.5), recorded over a period of 72 hours at 25 °C.
- Figure 4 shows the DLS analysis of [Ru2(PipeDTC)5]CI encapsulated in PF127 micelles with 10% of the polymer conjugated to glucose via glycosylation (C1 position).
- Figure 5 collects TEM images of the formulations according to the third and fourth embodiments of the present invention, wherein PF127 micelles encapsulate the complex [Ru2(PipeDTC)5]CI.
These figures illustrate and demonstrate various features and embodiments of the present invention, and of the manufacturing method thereof, but are not to be construed as limiting the invention.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
For the purpose of understanding the specification and the appended claims, in the following description the chemical elements are defined by means of the respective symbols as reported in a common Periodic Table of Elements, such as that present in the "Handbook of Chemistry and Physics, 93rd ed ". In addition, unless otherwise indicated, the chemical symbol includes all isotopes and ions. Therefore, the chemical symbols C, F, Cu, Ga , Au and I include, as an example, their respective isotopes 11C, 18F, 64Cu, 63Cu, 65Cu, 68Ga, 198 Au, 1311, 127l, 129l. Moreover, the claimed and described structures are intended to include all possible isomers such as coordination isomers, structural isomers, and conformational isomers. In particular, wherever one or more chiral centers are present, the claimed and described structures are designed to include all possible optical isomers, such as enantiomers and/or diastereoisomers, their mixtures, either as racemic mixture or in various ratios. These chiral centers may be present in the coordination compound or already present in the involved chelating ligands, for instance in the molecular fragments comprising a carbohydrate T.
In the description and claims, the reference to classes of chemical compounds or functional groups, for example, an ester or an amide, has to be interpreted in the broadest meaning of the term in the chemical field, and, in particular it includes all possible types of substituents.
For the sake of brevity, the functional groups or solvents reported in the present invention may be indicated by acronyms or abbreviations widely used in the context of reference of the present invention (or defined in the aforementioned "Handbook"). Finally, for the sake of clarity, in the general formulae the chelating dithiocarbamato ligand (also indicated with the acronym DTC) is represented through a stylized arch that connects two sulfur atoms, in accordance with the usual chemical notation.
In some preferred embodiments, the coordination compounds having formula l(a) and/or l(b) are bioconjugated to cancer-targeting moieties. This is a well-known term for an expert in the field, and it defines a molecular fragment of the compound (moiety), which has been ad hoc engineered for its recognition by cell-membrane proteins present in tumor cells. Within the scope of the present invention, said "cancer-targeting moiety" is a carbohydrate, and according to this, the terms "glycoconjugation" and "glycoconjugates" will be also used to indicate the "bio- conjugation" and the "bio-conjugates", respectively. The synthetic processes of the first and second embodiments involve amino functional groups for the preparation of the dithiocarbamato ligand. These groups may be ad hoc introduced in a specific stage of the process, or alternatively they can be generated by subsequent reactions during the previous steps. On the
other hand, a suitable amine reagent can be purchased and used as such. Anyway, in the context of the present patent, these amino groups and amine reagents will be defined "precursor amine(s)" for brevity.
Some embodiments of the present invention involve the term "pharmaceutical formulations" to define preparations made up of a therapeutically-effective amount of the coordination compounds of Formula 1(a) and/or 1(b) together with "pharmaceutically acceptable" additives and/or diluents and/or excipients. These additives, diluents, and excipients are intended to be compounds that, in contact with animal or human tissues, in light of their composition, dosage and administration route or anything else, do not cause any toxicity, irritation, allergy, and other complications considered excessive or unacceptable by a specialized physician based on a reasonable risk/benefit ratio.
Said compounds may optionally be encapsulated in "pharmaceutically acceptable" macromolecules or supramolecular structures or nanostructures. For the sake of clarity, the term "encapsulation" means a process by which a therapeutically-effective amount of the coordination compounds of Formula l(a) and/or l(b), establishes intermolecular bonds {e.g. van der Waals forces, hydrogen bonds) with the single units of a "nanocarrier" or with specific structural domains. As an example, the surface or the hydrophobic core of the nanocarrier (e.g., micelles), or the hydrophilic core {e.g., liposomes), or also the lipidic/polymeric layer.
Finally, within the scope of the present invention, the term "theranostic compound" or more generally "theranostic agent" will refer to the coordination compound according to this invention, the composition containing at least one of said coordination compounds, or also a pharmaceutical formulation made up of the said compound and/or composition, which combines therapeutic properties with regard to a pathology, preferably neoplastic pathologies, along with the property to be detectable by suitable devices/detectors, hence to be usable in association with diagnostic and imaging systems for patient follow-up in therapeutic treatments and, if possible, to take the necessary corrective action.
COORDINATION COMPOUNDS
It is a first object matter of the present invention a new class of mononuclear and dinuclear Ru- based and Ga-based coordination compounds, whose features are defined in the appended independent claim. Such compounds are described by one of the following general formulae (here also referred to as "Formula 1 a)" or "Formula 1(b)"):
Said mononuclear and dinuclear coordination compounds are neutral or ionic complexes whose charge is neutralized by at least one counter-ion G and have different coordination geometries, for example regular octahedral or distorted octahedral. In light of the Formula 1(a) or Formula 1(b) Mi , M2 , M3 , X, Y, W, Z, L1 5 L2, L3 and L4 are independently chosen and have the following meaning.
, M2 , M3 identify the metal center of the coordination compound and they are selected from Ga(l ll), Ru(l l), Ru(l l l), also defined in short as Ru (11,111). The stylized arch connecting the two S- atoms represents a first ligand of dithiocarbamic nature (DTC);
X, Y, W, Z, I_2, L3 and L4 are selected from the group consisting of: N, S, O, P, C and Se, and represent donor atoms being the same or different from each other and they belong to one or more bidentate chelating ligands.
The integer n represents the charge of the complex, ranging from -4 to +4, where the case with n = 0 corresponds to a neutral complex.
The G symbol identifies at least one counter-ion having the total charge d, being an integer ranging from -4 to +4. Such counter-ion is present with a stoichiometric coefficient e, identified by the integer n/d (in absolute value) or zero (in the latter case of course the counter-ion is not present and also n=0). G is pharmaceutically acceptable ion, which, by way of example and not limitation, is selected among : CI", I", F", Br", CH3C02", PF6 ", BF4 ", SbF6 ", [B{C6H3(CF3)2}4]", [B(C6F5)4]", OH", 03SNH2 ", nitrites and nitrates {e.g., N20", N03 "), acetates, phosphates {e.g., hexafluorophosphate, H204P", P04 3"), sulfates {e.g., triflate, HS04 "), carbonates, perchlorates, acetylacetonates {e.g., hexafluoroacetylacetonate), propionate, glycolate, stearate, lactate, malate, pyruvate, tartarate, citrate, ascorbate, palmitate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulphonate, ethane-1 ,2-disulfonate, trifluoromethanesulfonate, oxalate, malonate, succinate, glutarate, adipate, pimelate or isethionate, Na+, K+, Mg2+. However, the text "Remington's pharmaceutical Sciences, 17 th ed." (Mack Publishing Company) reports other chemically equivalent salts that can be used.
For the sake of clarity, in the Formula l(a) and Formula l(b), the stylized arch connecting the S- donor atoms indicates a bidentate chelating dithiocarbamato ligand (DTC), in compliance with the usual chemical notation. The number of chelating ligands in the structures l(a) and l(b) is 3 and 5, respectively. Said chelating dithiocarbamato ligands (DTC) may have a closed (cyclic) or a open {e.g., linear or branched) structure.
In some embodiments, said first or second DTC ligand has a closed structure such as that depicted below, which is presented by way of example but not limitation of the present invention:
Such structure comprises groups R1 5 R2, R3 and R4 and optionally one R5 group. The group and - if present - the R5 group, are bound to the dithiocarbamic nitrogen atom and to the R2 and R4 groups, respectively; the remaining R, groups are bound to the next and the previous group in the cyclic structure. If R5 is absent, R4 is directly bound to the dithiocarbamic nitrogen atom. All the bonds involving the R, groups, where i is an integer ranging from 1 to 5, may be single or double.
Optionally, the DTC ligand with the closed structure herein described comprises a glucide or carbohydrate T, which is bound to at least one of said R, groups directly or through a unit A by respectively T-R, bonds or T-A-R, bonds, which can be single, double or triple. Depending on the situation, the nature of said bonds may be: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C. The unit A is made up of an atom or a functional group or a spacer, meaning with this term a molecular fragment properly designed to join the glucidic moiety (T) with the cyclic structure described above, for example -COOCH2CH20-CH2CH20-CH2CH20-CH2CH20- or - CH2CH=CHCH2CH2- or also -COOCH2CH2CH2CH2CH2CH2. However, for the unit A, other chemically-equivalent molecular fragments may be selected by the expert of the branch.
In other embodiments, said DTC ligands have an open structure of the type depicted below by way of example, but not limitation:
In this case, the structure comprises a terminal group R linked to the dithiocarbamic nitrogen atom, and optionally one or more R2, R3, R4 and R5 groups.
If R2 is present, it represents a terminal group which can be bound to R3 or R4 or R5 or directly to the dithiocarbamic nitrogen atom, depending on which groups R, (i = 3, 4 or 5) are absent or not. If R2 is present, it represents a terminal group which may be linked to R3, R4, or R5 or directly to the said dithiocarbamic nitrogen atom, depending on which R, groups (i = 3, 4 or 5) are absent or not. If present, the R3 group is bound to R2, or alternatively R3 is a terminal group when R2 is absent. In both cases, R3 is bound to R4 or R5, or to said nitrogen atom, according to
which group (among R4 or R5) is absent or not. Similarly, the R4 group, if present, is a terminal group and is bound to R5 or to said nitrogen atom; alternatively, R4 is bound to R5 or to said nitrogen atom and is bonded to R3 or R2, depending on whether the R3 or R2 groups are absent or not. Finally, the R5 group, when present in the structure, is a terminal group and is linked to said nitrogen atom or alternatively it is bound to R,, according to which R, (i = 2, 3 or 4) groups are absent or not. All the bonds involving the R, groups, where i is an integer from 1 to 5, can be single, double or also triple.
Optionally, the DTC ligand with the open structure herein described comprises at least one glucide or a carbohydrate T. Said unit T is linked to said dithiocarbamic nitrogen atom or to at least one of the said R, groups (where i is an integer from 1 to 5) directly with respectively T-N or T-R, bonds, or alternatively via a unit A, by T-A-N or T-A-R, bonds. Depending on the cases, said bonds may be single, double or triple, and of the type: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C. Also in this case, the unit A may be an atom or a functional group or a spacer, as well as the unit A is made up of other chemically-equivalent molecular fragments. In both types of structures (closed or open) of said DTC ligands, said unit A or said R, group (where i is an integer ranging from 1 to 5) are preferably selected from: an atom such as H, C, O, N, S, P, Se or a group selected among -CH, -CH2, -CH3, -C(CH3)3, -NH, -NH2, -NHRS, -NRs2, -S-S-, -SH, -PH, -OH, -COOH, -CH-Br, -CHCH2NH2, -CHCH2OH, -CHCH2NH-, -CHCH20-, (- CN), -CF3, -C2H5, -C2H4, -C4H9, -C3H7, -C3H6, -C3H6OH, -C4H8, -N02, -CH2OH, -C2H4OH, - C4H8OH, -C(OH), -C(OH)H2, -C(OH)H; -S02, -COO-, -N(CH)3-, -CHN(CH), -NN(CH),-NCHNCH- , -N(CH)2N-, -CHNHCH-, -NH(CH), -NHCHN-, -NHNCH-; -CONH-, -CONH2, -CONHRs, - CONRs2, (-O-C(O)-Rs), where Rs indicates substituent groups of the type: aliphatic, alkyl, halo- alkykl, cycloalkyl, alkenyl, alkynyl, aryl, etero-aryl, etero-aliphatic, aromatic, etero-aromatic, aliphatic-aromatic, eteroaliphatic-eteroaromatic, cycloaliphatic, eterocycloaliphatic, esteric, amidic, sulphonamidic, carbonylic, acetyl, ethyl, propylic, butilyc, isopropylic, acylic, ureidic, thioureidic, thiolate, imine, halogens, ethers, nitro groups, nitrile, aryl, benzyl, sulphonamidic, saturated linear or branched C1 -C18 alkylic optionally substituted with one or more Rs groups or including one or more unsaturated bonds, a group of the type -(CH2)m, -(CH20)m, -(CH2CH2S)m, (CH2CH2NH)m or (CH2CH20)m, (CH2CH2N)m where m represents an integer greater than 1 , or it is a combination thereof.
Depending on the needs, as unit A or groups R, it is possible to use molecular fragments being chemically-equivalent to those herein reported by way of example, but not limitation of the present invention. In addition, in some embodiments, combinations of the indicated species are possible. Furthermore, said DTC ligands may contain in the unit A or in the R, groups chemical groups in salified form comprising pharmaceutically acceptable counterions {e.g., -NH3 + CI"). It will be apparent to the skilled in the art that the unit A or the various R, groups may be variously substituted, in any combination and position, with one or more of said atoms or said groups. Similarly to the coordination compound, said DTC ligands may a priori have different isomeric
forms. Hence, the herein disclosed formulae are intended to include any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, as racemic mixtures or in various ratios.
Again with reference to both closed or open structures of said chelating dithiocarbamato (DTC) ligands, said glucide or carbohydrate T is preferably a monosaccharide or a deoxy- monosaccharide, in particular a those, a tetrose, a pentose, an esose, an eptose. By way of example, but not limitation, said glucide or carbohydrate T is selected from the group consisting of: glucose, galactose, mannose, xylose, rhamnose, arabinose, glucosamine, galactosamine, mannosamine, fructose, fructosamine, ribose, ribulose, sedoheptulose, erythrose, threose, erythrulose, allose, altrose, lixose, gulose, idose, talose. Alternatively, said glucide or carbohydrate T is a natural or synthetic polysaccharide, also in deoxy-carbohydrate form, for example maltose, cellobiose, lactose, trealose, sucrose, amylose, amylopectin.
In the case of the closed-structure DTC ligands, the T-R, or T-A-(R,) bond (where i is an integer ranging from 1 to 5) takes preferably place in at least one of the carbon positions of the T moiety, preferably in the positions C1 or C2 or C3 or C4 or C6, for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.
In the case of the open-structure DTC ligands, the T-N or T-R, bond, or T-A-N or T-A-R, bond (where i is an integer ranging from 1 to 5) occurs preferably in the C1 or C2 or C3 or C4 or C6 positions for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.
Said chelating DTC ligands are symmetrical bidentate ligands whose chelating function is generally characterized by a negative q charge {q = -1 ). In light of the choice of the R, groups, of the unit A and the glucide T, the ligand may have an overall neutral {e.g., zwitterionic), positive or negative charge.
Moreover, taking into account both closed and open structures of said chelating dithiocarbamato (DTC) ligands, and with reference to compounds of both the first embodiment and the second embodiment (i.e. containing carbohydrates T in the DTC ligand), one or more functional groups can be suitably protected during the synthetic process. Well-known groups are available for the protection of hydroxyl, amine, amide and carboxylic acid groups, as well as the conditions in which protection and deprotection can occur, even in so-called orthogonal conditions. In particular, protecting groups are introduced during the synthesis to avoid undesirable side-reactions and may be removed or not at the end of the synthetic procedure. In the second case, the new herein disclosed compounds may contain protective groups, even mutually different, in order to modulate the in vivo therapeutic profile, for example in terms of reactivity, solubility, stability and bioavailability. For example, one or more of the hydroxyl groups (also known as alcoholic) of one or more cancer-targeting moieties T of the second embodiment can be functionalized with equal or different protecting groups chosen, for example, from silyl ethers {e.g., trimethylsilyl ether or dimethyltertbutylsilylether) or esters {e.g.,
acetate, pivalate, propionate, carbonate, phosphate, butyrate). These concepts are in summarized in the structure of the hexose shown below only by way of example but limitation of the present invention.
In particular, the abovementioned protecting groups, identical or different, are indicated here by the notation R6; the substituent in position C1 may have an a or β conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C2 (as indicated by the black rectangle). For a pentose, depicted below only by way of example, but not limitation of the present invention, the protecting groups, equal or different, are again indicated by the notation R6; the substituent in position C1 may have an a or β conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C5 (as indicated by the black rectangle).
From the above description, it is clear that the first and the second chelating dithiocarbamato (DTC) ligand may be either equal to or different from each other. In the first case, the coordination compound is referred to as homoleptic, while in the latter it is heteroleptic.
SYNTHESIS AND CHARACTERIZATION OF THE COORDINATION COMPOUNDS
It is another subject matter of the present invention a process for the synthesis of the coordination compounds of general formula I (a) and l(b), whose features are set forth in the enclosed independent claim.
In the first preferred embodiment, herein described by way of example, but not limitation of the present invention, the coordination compounds having the general formula I (a) and l(b) may be conveniently synthesized by a process including at least the following steps:
a) Preparation and optional isolation of the dithiocarbamato ligand (DTC);
b) Coordination of the DTC ligand to one specific metal center;
c) Isolation of the coordination compound synthesized during step b).
This process may optionally include a further step d) related to the purification and drying of the coordination compound obtained at the end of the step c).
a) Preparation and isolation of the dithiocarbamato ligand (PTC)
The synthesis of the dithiocarbamato ligands (DTC) is carried out at a temperature ranging from -30 and 60 °C in water, or methanol, or dry THF via reaction between an amine precursor and carbon disulfide (CS2), optionally in the presence of a base, such as KOH or sodium tert- butoxide, or an excess of said amine precursor. In the case of some amines, it is preferable to work under inert atmosphere, using well-known equipment and techniques. Subsequently, the volume of the solvent is reduced and the DTC ligand can be isolated via precipitation or co- precipitation by adding diethyl ether, washed with diethyl ether and dried under vacuum in presence of P2 05.
By means of this synthetic strategy, which yields the isolation of pure DTC ligands (for example, as sodium salt, potassium salt or with the precursor amine under the form of ammonium), the present inventors have inventively achieved a number of remarkable advantages over the state of the art. First of all, this allows an improvement in the yield of the subsequent complexation reaction. Moreover, the DTC salt can be tested in vitro for its biological activity, in order to demonstrate that the antitumor activity is mediated by the coordination compound as a whole and not just by the ligand (or the metal center).
b) Coordination of the DTC ligand to a specific metal center
The synthesis of the coordination compound of general formula I (a) and/or l(b) is carried out in water or in an organic solvent, and it involves the coordination of the DTC, which has been optionally isolated in the previous step, to a selected metal center among Ru (II, III), and Ga (III). In the case of some compounds, it is preferable to work under inert atmosphere, using well- known equipment and techniques.
The metal center is selected starting from the corresponding precursors, preferably chlorides or metal-halide salts, or complex salts. Alternatively, the precursors are metal derivatives with the metal center presenting a lower or higher oxidation state, such as organometallic-, amino-, thioether- precursors, or phosphine derivatives, or alternatively some of said coordination compounds can be themselves precursors, useful for the synthesis of other complexes.
During this step, contrast agents can be advantageously integrated in the coordination compound according to the invention in the form, for example, of radioisotopes such as 11C, 18F, 68Ga, 127l, 129l, 1311. In particular, these isotopes can be included in the DTC ligand, or they may coincide with the metal center or also introduced into the counter-ion. As it will be more clearly described in following, these contrast agents allow to combine the treatment of the neoplastic diseases with the diagnosis.
c) Isolation of the coordination compound synthesized during the step b)
The isolation of the compound synthesized in the previous step b) proceeds via usual separation techniques, preferably filtration followed by reduction of the volume of water, or of
said solvent, and by precipitation with ethyl ether. Alternatively, water or said solvent is evaporated under reduced pressure, in order to obtain the coordination compound of general formula 1(a) and/or 1(b), or a mixture containing said coordination compound.
d) Purification and drying of the coordination compound
During this step, the purification is optionally carried out using techniques well-known to the skilled in the art, for instance by chromatography, precipitation from organic solvent, washing with water or organic solvents and drying of said coordination compound.
The following sections describe further details of the process described in the step b), related to the coordination of the DTC ligand to a specific metal center.
Preparation of the coordination compounds with M = Ga (III) or Ru (III)
For the mononuclear coordination compounds having the general formula l(a) with M1=Ru(lll) or Ga(lll), L1 = L2=S and X=Y=S, the coordination of the DTC ligand (3 eq., preferentially dissolved in methanol) to the metal, preferably ruthenium(lll) chloride or gallium (III) chloride, occurs in water or THF or methanol. In the case of Ru(lll), despite of the stoichiometric 1 :3 metal-to- ligand ratio, the resulting product is actually a mixture of the neutral mononuclear complex [Ru(DTC)3] and the dinuclear ionic derivative of the general formula l(b). The reaction mixture is stirred for at least 1 h at room temperature. Subsequently, the solvent is removed and the solid obtained is taken up with chloroform. After filtration, the obtained solution is precipitated with diethyl ether, leading to the isolation of a solid.
Preparation of the coordination compounds with M = Ru (II, III)
Concerning the ionic coordination compounds containing the cation [Ru2(DTC)5]+ having formula l(b) with M2=M3=Ru(lll), L^L^L^L^S and X=Y=W=Z=S, the synthesis is carried out in water where the ruthenium(lll) precursor RuCI3-3H20 is mixed with the selected DTC ligand according to a proper stoichiometric ratio. This reaction leads to a mixture of products consisting of the ligand dimer (DTC)2, the neutral mononuclear derivative [Ru(DTC)3] and the dinuclear ionic complex [Ru2(DTC)5]CI (as a mixture of its a and β isomers). The [Ru(DTC)3] and the α,β- [Ru2(DTC)5]CI complexes are separated by chromatography. The mixture containing α,β- [Ru2(DTC)5]CI is refluxed in DCM or isopropanol for 8 hours, in order to convert the residual a- isomer to the thermodynamically more stable β-[Ρυ2(ϋΤΰ)5]ΰΙ. Then, the purified compounds are washed with n-hexane and dried under vacuum in presence of P205.
The counter-ion G can be advantageously chosen or replaced, using techniques well-known to the skilled in the art, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target" toxicity, solubility, and stability.
Concerning the dinuclear coordination compounds obtained from the formula l(b) with M2=Ru(lll) and M3=Ru (II) or M2=Ru(ll) and M3=Ru(lll), it is possible to perform the reduction starting from the a or β isomer, or their mixture, using an appropriate reducing agent.
Preparation of heteroleptic coordination compounds with M = Ru (III)
Concerning the ionic heteroleptic coordination compounds containing the cation [Ru2(DTC)4(DTC*)]+ having formula 1(b) with M2=M3=Ru(lll),
and X=Y=W=Z=S, where four DTC ligands are the same whereas the fifth is different, the synthesis was performed according the following novel and inventive scheme.
1) Br2 CH2CI2 reflux, 10 min
2) hexane, 30 min
3) DTC*, l eq, CH2CI2 / MeOH
The first synthetic step is related to the preparation of the organometallic Ru(ll)-NBD complex, synthetized from RuCI3-3H20 at reflux with the NBD ligand, according to a procedure reported in literature (J. Organomet. Chem., 1966, 5, 275-282). Successively, the Ru(ll)-NBD derivative is treated with 2 eq. of DTC to form the [Ru"(NBD)(DTC)2] complex. This step leads to the formation of the "building blocks" of the final heteroleptic complex, and hence the introduced DTC ligands are prevalent in the dinuclear compound (4 units among the 5 ligands). The synthesis of the Ru(ll)-DTC derivative is conducted in hot DMF (100 °C) by adding a DMF solution of the dithiocarbamato ligand (2 eq.) to the Ru(ll)-NBD precursor. The precipitation with an appropriate solvent leads to the isolation of the precursor. Finally, the Ru(ll)-DTC is treated with bromine, to achieve the oxidation of the ruthenium center to Ru(lll), and, after the cleavage of the olefin ligand with hexane, the addition of the DTC* ligand results in the formation of the [Ru2(DTC)4(DTC*)]Br coordination compound. In most cases, the final product is purified via silica gel chromatography with an appropriate eluent, according to the nature of the DTC ligands.
The counter-ion G can be advantageously chosen or replaced, using well-known techniques, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target" toxicity, solubility, and stability.
Concerning the heteroleptic dinuclear coordination compounds related to the formula l(b) with M2=Ru(lll) and M3=Ru (II) or M2=Ru(ll) and M3=Ru(lll), it is possible to perform the reduction of the a or β dinuclear isomer, or their mixture, using an appropriate reducing agent.
EXAMPLES OF COMPLEXES ACCORDING TO THE FIRST EMBODIMENT
By way of example, but not limitation of the present invention, some specific examples of coordination compounds having formula 1(a) or 1(b) are reported below. In the first preferred embodiment of the present invention said compounds do not contain a cancer-targeting moiety in the form of carbohydrate, and contain, for example, dithiocarbamato ligands derived from: piperidine, L-proline methyl ester, and L-proline ferf-butyl ester and which for brevity are named as PipeDTC, ProOMeDTC, and ProOtBuDTC, respectively.
The compounds were characterized using several techniques, including elemental analysis, NMR spectroscopy, FT-IR spectrophotometry, and ESI-MS mass analysis.
Example 1 : [Ga(ProOM
Appearance: white solid; Yield: 75%
Anal. Calc. for C21 H30GaN3O6S6 (MW = 682.59 g-mol"1): C 36.95; H 4.43; N 6.16; S 28.19. Found: C 36.87; H 4.29; N 6.18; S 28.10.
1 H-NMFt (CDCI3, 300.13 MHz): δ (ppm) = 2.05-3.31 (m, 12H, H(3) + H(4)), 3.70 (s, 9H, + 0-CH3), 4.01 (m, 6H, H(5)), 5.02-5.06 (m, 3H, H(2)).
Medium FT-IR (KBr): v (cm 1) = 2965.1 1 , 2831 .24 (va, C-H); 1738.19 (v, C=0); 1 165.1 1 (va, N- CSS); 1 147.1 1 (va, C-OfBu); 999.54 (va, CSS).
Far FT-IR (nujol): v (cm 1) = 550.16 (vs, CSS); 336.29 (v, Ga-S).
Example 2: fi-[Ru2(PipeDTC)5]CI
R.f. (silica gel, CH2CI2/MeOH 9:1 ): 0.39
Anal. Calc. for C3oH5oCIN5Ru2Sio (MW = 1039.00 g-mol"1): C 34.68; H 4.85; N 6.74; S 30.86. Found: C 34.58; H 4.80; N 6.81 ; S 30.88.
1 H-NMFt (CDCI3, 300.13 MHz): δ (ppm) = 1 .50 (m, 10H, H(4)), 1 .83 (m, 20H, H(3) + H(5)), 3.03- 4.65 (m, 20H, H(2) + H(6)).
Medium FT-IR (KBr): v (cm 1) = 2933.43 (va, C-H); 1503.23, 1441 .03 (va, N-CSS); 1001 .63 (va, CSS).
Far FT-IR (nujol): v (cm 1) = 544.23 (vs, CSS); 406.62 (va, Ru-S); 321 .88 (vs, Ru-S).
ESI-MS m/z, [M-CI+] - found (calc): 1003.93 (1003.94)
Example 3: β -[Ru2(ProOtBuDTC)5]CI
Appearance: dark red solid; Yield: 32 %
R.f. (silica gel, CH2CI2/MeOH 94:6): 0.52
Anal. Calc. for C5oH8oCIN5Oi0Ru2Sio (MW = 1469.64 g-mol"1): C 40.87; H 5.49; N 4.77; S 21 .82. Found: C 40.80; H 5.39; N 4.60; S 21 .88.
1 H-NMR (CDCI3, 300.13 MHz): δ (ppm) = 1 .41 -2.26 (m, 65H, H(3) + H(4) + 0-C(CH3)3), 3.21 -4.02 (m, 10H, H(5)), 4.36-5.30 (m, 5H, H(2)).
Medium FT-IR (KBr): v (cm 1) = 2975.16 (va, C-H); 1735.39 (v, C=0); 1481 .10, 1450.06 (va, N- CSS); 1 148.87 (va, C-OfBu); 935.12 (va, CSS).
Far FT-IR (nujol): v (cm 1) = 551 .30 (vs, CSS); 471 .19 (va, Ru-S); 325.69 (vs, Ru-S).
ESI-MS m/z, [M-CI+] - found (calc): 1434.16 (1434.12).
Example 4: fi-[Ru2(PipeDTC)4(DEDT)]Br
By way of example, but not limitation, the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the first preferred embodiment of the present invention does not contain a "cancer-targeting moiety" in the form of carbohydrate. Such compound contains, for example, dithiocarbamato ligands derived from piperidine (DTC= PipeDTC), and ethylenediamine (DTC*= DEDT).
The compound was characterized using 1 H-NMR spectroscopy, and elemental and ESI-MS mass analysis.
Appearance: brown solid; Yield: 16 %
R.f. (silica gel, CH2CI2/MeOH 9:1 ): 0.31
Anal. Calc. for C29H5oBrN5Ru2Sio (MW = 1071 .45 g-mol"1): C 32.51 ; H 4.70; N 6.54; S 29.93.
Found: C 32.68; H 4.83; N 6.44; S 30.1 1 .
ESI-MS m/z, [M-Br+] - found (calc): 991 .92 (991 .94)
SYNTHESIS AND CHARACTERIZATION OF GLYCO-CONJUGATED COMPLEXES
It is another object of the present invention a process for the glyco-conjugation of the coordination compounds according to the present invention, whose features are set forth in the enclosed independent claim.
In the second preferred embodiment, the coordination compounds of Formula I (a) and Formula l(b) contain dithiocarbamato ligands functionalized with carbohydrates which act as a selective cancer-targeting moiety towards cancerous cells in vivo, advantageously exploiting the Warburg effect.
The glycoconjugation process involves a number of synthetic steps for the functionalization of a specific amino precursor with an likewise specific carbohydrate. Such amino precursor is subsequently converted to the corresponding dithiocarbamato (DTC) ligand, through the phase a) of the procedure of the first embodiment. In particular, the synthesis involves the protection of the hydroxyl groups of carbohydrate with protecting groups which are orthogonal to the reaction conditions of the subsequent synthetic steps. The carbohydrate {e.g., glucose, mannose) is functionalized {e.g., in position 1 or 2) with a molecular fragment containing an amine, preferably secondary, which is able to react with CS2 to form a DTC ligand, ready for the subsequent complexation to the metal center (Ru, Ga). Alternatively, such amine group or other functional group, for example -OH, is not directly used to prepare the dithiocarbamato ligand, but its reactivity could be exploited to bind a molecular fragment which, depending on the molecular design, may have different length and chemical composition. However, said molecular fragment must contain, after one or more synthetic steps, an amine group (that is, the precursor amine, as an example a proline) on which the CS2-based reaction will be carried out to form the DTC ligand. The deprotection of protecting groups can be performed by means of
chemical or biochemical methods including the use of enzymes or pseudoenzymes. Pseudoenzymes are generally referred to proteins, such as the HSA (Human Serum Albumin), which may hydrolyze, for example, ester substrates due to the presence of several nucleophilic residues (such as lysine) on their surface, but which do not return to the native state after the hydrolysis reaction.
Scheme 1. Example of functionalization in position CI: a) NaN3, acetone, 56°C, crystallization, 60%; b) H2/Pd, MeOH/EtOAc, 98%; c) Z-Sar-OH, N-methyl morpholine, isobutylchloroformiate, TH F-dry, -15°C, 83%; d) NaOMe, MeOH-dry, acid resin, rt, 100%; e) TMS-CI, Hexamethyldisilazane, pyridine, CH2CI2, 0°C, flash chromatography, 85%; f) H2/Pd, MeOH/EtOAc, 90%; g) CS2, KOH, MeOH, 100%.
During the synthetic process, in addition to the glyco-conjugation, various types of reactions can be conducted. For example: nucleophilic substitution, peptide coupling, protection or deprotection processes, redox reactions, formation of iminic or nitrile intermediates, reduction with H2, hydrolysis. By way of example, but not limitation, the Scheme 1 reported below depicts the synthesis of a dithiocarbamato ligand (a-g), bearing a glucose moiety functionalized in position 1 of the pyranosic ring (β-amido-glycoside).
Scheme 2. Example of functionalization in position C2 : c) Z-Pro-OH, N-methyl morpholine, isobutylchloroformiate, THF-dry, -15°C, 83%; d) NaOMe, MeOH-dry, acid resin, rt, 100%; e) TMS-CI, Hexamethyldisilazane, pyridine, CH2CI2, 0°C, flash chromatography, 85%; f) H2/Pd, MeOH/EtOAc, 90%; g) CS2, KOH, MeOH, 100%.
The Scheme 2 reported below shows the synthesis of a dithiocarbamato ligand {c-g), being a glucose derivative functionalized in position C-2 (glucosamide, namely an amide derivative of theglucosamine). By way of example, but not limitation, the schemes 3, 4 and 5 show the syntheses of carbohydrate derivatives having an amino function (-NH2) in C3, C4 and C6 positions, respectively. Such amine function can in turn be functionalized with a molecular fragment on which the dithiocarbamato group is synthesized, or alternatively, such amine group can be directly converted to dithiocarbamate by reaction with CS2 or may be advantageously modified in a different functional group, for example a secondary amine or an isothiocyanate, in order to conduct subsequent reactions for the bioconjugation of the metal.
Scheme 3. Example of fu nctionalization in position C3 : i) Ac20/py, -20°C; ii) Tf20/py, -15°C, iii) NaN3, rt; iv) H2/Pd/C, rt.
On the basis of the reaction Scheme 3-4 herein disclosed, it will be apparent to those skilled in the art that only obvious changes to the procedure are required in case the functionalization involves other carbohydrates, or other positions (depending on the type of carbohydrate), or also in case the synthesis is designed to obtain different amino precursors.
The synthesis of the organic part, the amino-precursor of the carbohydrate-functionalized dithiocarbamato ligand, is particularly challenging. In fact, some synthetic approaches have not been successful.
Some examples of coordination compounds having Formula I (a) or l(b) containing glucides or carbohydrates in one or more dithiocarbamato ligands are reported below, by way of example, but not limitation.
Scheme 4. Example of functionalization in C4: i) BnBr/py, rt; ii) NaCNBH3/TFA, 0°C; iii) MsCI, rt; iv) NaN3 60-7CTC; v) H2/Pd/C, rt.
Scheme 5. Example of functionalization in C6: i) Ph3CCI, BnBr, AICI3, rt; ii) MsCI, rt; iii) NaN3, 60°C; iv H2/Pd/C, rt.
EXAMPLES OF COMPLEXES ACCORDING TO THE SECOND EMBODIMENT
Example 5: [Tris(1,3,4,6-tetraacetyl-2-glucosammido-L-prolineDTC)ruthenium(lll)]
Appearance: dark green solid; Yield: 22%
Anal. Calc. for C60H81 N603oRuS6 (MW = 1659.77 g-mol"1): C 43.42; H 4.92; N 5.06; S 1 1 .59. Found: C 43.53; H 4.99; N 4.98; S 12.01 .
1 H-NMFt (CDCI3, 300.13 MHz): δ (ppm) = 0.65-2.24 (m, 12H, broad), 2.03, 2.08, 2.09, 2.17 (36H, s); 4.06-4.10 (3H, ddd); 4.26-4.30 (3H, dd); 4.48-4.51 (3H, dd); 4.91 (3H, t); 5.10 (3H, t); 5.18 (3H, t); 5.40 (3H, t); 6.79 (3H, m). 23.61 -33.1 (6H, broad), 40.07-44.32 (3H, broad).
Medium FT-IR (KBr): v (cm 1) = 1720 (v, C=0); 2975.22, 2872.34 (va, C-H); 1440.10 (va, N- CSS); 933.50 (va, CSS).
Far FT-IR (nujol): v (cm 1) = 547.60 (vs, CSS); 470.56 (va, Ru-S); 320.98 (vs, Ru-S).
By way of example, but not limitation, the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the second preferred embodiment of the present invention contains a "cancer-targeting moiety" in the form of carbohydrate. Such compound contain, for example, dithiocarbamato ligands derived from: piperidine (DTC= PipeDTC), and (DTC*= 1 -0-(A/-methylethanolamine)^-D-glucopyranoside dithiocarbamate = MAEGIuOHDTC) The compound was characterized using 1 H-NMR spectroscopy, and elemental and ESI-MS mass analysis.
Appearance: brown solid; Yield: 1 1 %
R.f. (silica gel, acetone/H20 1 :1 ): 0.56
Anal. Calc. for C34H57BrN5Ru2Sio (MW = 1234.55 g-mol"1): C 33.08; H 4.65; N 5.67; S 25.97. Found: C 32.90; H 4.73; N 5.54; S 25.81 .
EVALUATION OF THE BIOLOGICAL ACTIVITY
Antitumor activity in vitro
The human tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) were cultured in RPMI-1640 and Hams-F12 medium, respectively. The cells (8x103 / mL) were seeded in 96-well plates in the growth medium previously mentioned (100 μΙ_) and then incubated at 37 °C in a controlled atmosphere of carbon dioxide. After 24 hours, the cell culture medium was removed and replaced with fresh one containing the tested compound, previously dissolved in DMSO (0.1 % v/v, freshly prepared solution and keeping in the dark), or saline solution, at various concentrations. A total of four independent experiments were conducted and, after 24 hours of treatment, the medium was aspirated, and each well was treated with 100 μΙ_ of a MTT (Methylthiazolyldiphenyl tetrazolium bromide) solution (1 mg/mL in PBS) and the plate was incubated at 37 °C for 4 hours. The MTT solution was then removed and 100 μΙ_ of DMSO were added to each well. The plate was shaked for 10 minutes and then analyzed by spectrophotometry (560 nm).
The cytotoxic activity of each compound was evaluated as a percentage of vital cells in the treated sample compared to the cells treated with the vehicle only (control). From the obtained dose-response curves, the IC50 values for each compound were calculated {i.e. the concentration expressed in μΜ, which inhibited 50% growth in tumor cells compared to the control).
All the above mentioned culture media and the human tumor cell lines can be easily purchased on the market. By way of example, but not limitation of the present invention, the following table collects the IC50 data of some compounds of the first and second embodiments, the corresponding dithiocarbamato ligands (DTC) and metal precursors.
Table 1: In vitro cytotoxic activity (IC50, expressed in μΜ) of some compounds and DTC ligands against the h uman tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) after 24 hours of treatment; against the human tumor cell lines: cervix adenocarcinoma (HeLa), colon neoplasia (HCT116), HepG2 : epithelial cells of human liver hepatoma and its more aggressive cou nterpart HepG2/SB3, overexpressing the anti-apoptotic protein SerpinB3; NSC lung carcinoma (A-549); acute T cell leukemia (Jurkat); AGS: gastric adenocarcinoma; triple negative breast cancer (crl2335) after 72 hours of treatment. The inorganic reference-drug cisplatin (Sigma-Aldrich) was tested under the same experimental conditions . IC50: concentration expressed in μΜ able to inhibit the 50% of the cancer cell growth compared to the control (cells treated with the vehicle). The data represent the mean ± SD of at least four independent experiments.
From the exemplary and non-limiting data shown in Table 1 , it is clear that the inventors have demonstrated that metal precursors alone do not induce any significant antitumor activity. In fact, they show an IC50 value > 15 μΜ. Similarly, the dithiocarbamato ligands (DTC) alone do not possess remarkable cytotoxic activity. Conversely, the coordination compounds obtained from such precursors, in which the metal is covalently bound to specific DTC ligands, are characterized by a remarkable cytotoxic activity.
Thus, through the present invention, the inventors have shown that such antitumor activity is due to the combination, in a single compound, of a properly-designed DTC ligand with a metal center endowed with specific chemical (oxidation state, reduction potential, geometry coordination, kinetic and thermodynamic properties) and biochemical properties.
It will be apparent to the skilled in the art that a further important object of the present invention has been achieved.
From the data shown in Table 1 , it is also clear that the present inventors have obtained a number of coordination compounds according to the present invention which exhibit higher anti- tumor activity than the reference drug cisplatin. Indeed, as it is well known, the greater the
antiproliferative activity, the lower the IC50 value for a specific compound. In this way, another important aim of the present invention has been achieved.
Acute toxicity test in mouse model
One compound was tested in vivo to evaluate the acute toxicity by intravenous administration of a single dose to male mice (provided by Charles River Laboratory Italy, Calco, Lecco; 6-week- old mice). This administration route has been chosen because it presents a reduced barrier to substance absorption, provides a more stringent toxicity measure compared to other in vivo assays and it is a typical route of administration in humans. The species/strain mouse/CD-1® was chosen because many regulatory authorities accept and indicate that preclinical acute toxicity tests are performed with this species/strain, in light of the large amount of related bibliography.
The administered dose (10 mg/kg) was selected based on the dosages clinically used on humans {e.g., 2 mg/kg) in order to highlight potential signs of toxicity.
The tested compound was: β-[Ρυ2(ΡίρβΟΤΟ)5]ΟΙ. The chosen vehicle was DMSO-EtOH-RL 50:10:40 % v/v in which RL stands for Ringer lactate (Eurospital®). Each experimental group consisted of 6 mice + 4 control animals (vehicle-treated only).
Each dose was prepared by dissolving a calculated amount of compound in DMSO; this volume was subsequently diluted with ethanol and Ringer lactate (RL) to obtain the final desired concentration. The administration of the antitumor compound under study was performed by intravenous injection into the caudal artery of each mouse. Each treated animal received an accurate injected volume of 100 μί, containing the amount of test substance as described above, that is the equivalent of 10 mg/kg. All animals were treated with a single dose at T0 after detecting the body weight of each mouse. Clinical observations were recorded at the time of injection, during the first hour and then in the following days for total 7 experimental days, the same for the body weight.
Table 2. Body weights report of the treated and control mice.
2day 3day 4day 5day 6day 7day
Group ID sex 1 h 4h 1day
s s s s s s
1 M S P P P P P P P P
2 M P P P P P P P P P
Control
3 M P P P P P P P P P
4 M P P P P P P P P P
1 M s P P P P P P P P
2 M P P P P P P P P P p-[Ru2(PipeDTC)5]CI 3 M P P P P P P P P P 10 mg/kg/iv 4 M P P P P P P P P P
5 M s P P P P P P P P
6 M P P P P P P P P P
Legend:
ID: mice identification; Grade code: P = Present; S = low; M = Moderate; V = Severe; D = Death. Sex: F = Female; M = male.
Table 3: Clinical and behavioral observations during the experimental study .
At the end of the study period, animals were sacrificed by C02 asphyxiation. All animals were subjected to autopsy examination including the opening of the cranial, thoracic and abdominal cavities.
All animals survived until the end of the study, and during the post-treatment period they showed no sign of toxicity. The macroscopic necroscopy did not reveal any evident sign of toxicity on the examined organs: brain, lungs, spleen, testes, heart, liver and kidneys.
PREPARATION OF SUPRAMOLECULAR AGGREGATES ENCAPSULANTING ANTICANCER COORDINATION COMPOUNDS
It is another matter of the present invention a process for encapsulating in supramolecular aggregates the coordination compounds according to this invention, whose features are set forth in the enclosed independent claim. Particularly, in the third and fourth preferred embodiment of this invention, the coordination compounds having the general formula l(a) and l(b) are encapsulated in supramolecular aggregates.
By way of example, but not limitation, advantageous supramolecular systems encapsulating the active compound can be micelles, vesicles (liposomes), cyclodextrins, dendrimers, organic polymeric nanoparticles, for instance derived from proteins or peptides, and inorganic nanoparticles {e.g., silica, zirconia, titanium dioxide).
Said supramolecular architectures can be in turn produced with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan, polyethylene glycol (PEG), mPEG acid, poly(lactic-co-glycolic acid) (PLGA), Pluronic®, cholesterol, derivatives of phosphatidylcholine and phosphatidylethanolamine, Cremophor®, pullulan, hyaluronic acid, ferritin, human serum albumin (HSA), heparin, dextran, polyaminoacids {e.g., a- poly-L-glutamic acid) and their derivatives {e.g., PHEA, PHEG), polyglycerol, polyacrylamide, polyvinylpyrrolidone, poly(2-oxazoline) and their derivatives. Moreover, even combinations of
these polymers or molecules, such as PEG-PLGA or PEG Pluronic®, may be used for the construction of appropriate supramolecular architectures, usable as nanocarriers for the coordination compounds according to the present invention.
In addition, block copolymers, such as those with the A-B-A or A-B architecture, can be advantageously used to prepare supramolecular systems suitable for the delivery of the coordination compounds herein disclosed. In other words, said building blocks, which can be equal or different each other, can be intended both as the monomer (e.g., ethylene oxide) of a polymer such as PEG or PF127, or of an inorganic nanostructure {e.g., Ti02), and the same polymer inside aggregates, for instance micelles.
The terms "supramolecular aggregates", "macromolecules", nanostructures encompasses also for example micelles, liposomes, protein- and peptide-based aggregates and carriers consisting of cyclodextrins or dendrimers.
Moreover, as an example, biocompatible polymers and oligomers, natural or synthetic, identical or different {e.g., phospholipids, pullulan, PEG, PF127, cholesterol) can form micelles or liposomes, in order to modulate the final properties of the composition and therefore of the final formulation. These polymers, identical or different, are the constituent units that aggregate during the formation of the supramolecular system. One of them or all of these polymers can be conveniently functionalized with carbohydrates (by using the functional groups exploitable for this purpose), and in an independent manner, with yields up to 100%. This/these polymer(s) can then be diluted with the same or other polymers to achieve, for example, a micelle or a liposome with a water-exposed surface having a variable percentage of functionalization, ranging from 1 to 50% mol/mol.
Concerning the drug loading, the range 10"5÷10"10 mol of compound/mg of formulation can be achieved when preparing compositions of said coordination compound. Furthermore, said compound can be encapsulated in the hydrophobic core of a micelle, or in the hydrophilic counterpart of a liposome, or in their lipidic/polymeric layer.
Such supramolecular architectures can in turn be made with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan and polyethylene glycol. The nanocarrier {e.g., liposome, micelle) can be conveniently covered with a hydrophilic and biocompatible coating in order to improve the pharmacokinetic profile of the "naked" supramolecular aggregate itself. In other words, by exploiting the so-called "stealth effect", to date known for polymers such as PEG and poly-amino acids, the surface of the supramolecular aggregate is hidden from blood components, including the opsonin proteins, which are responsible for the recognition and attack of the nanocarriers by phagocytes (monocytes, macrophages). This masking strategy allows the supramolecular systems to sidestep the natural processes of biotransformation/elimination of exogenous constituent entities or substances. Moreover, it determines an increased bioavailability and, hence, a prolonged circulation time of the compound loaded into the carrier, if compared to
supramolecular aggregates incorporating the coordination compounds according to the present invention and non-covered with said hydrophilic and biocompatible coating. The application of the "effect stealth" to the coordination compounds according to the first and the second embodiment of the present invention may also limit or eliminate any problem at the physiological level, such as hemolysis or immunogenicity of the active compounds. In addition, the hydrophilic coating of nanocarrier obtained with polymers such as PEG, reduces the self- aggregation of the particles by means of steric stabilization, thus resulting in an immediate impact on the freshly-prepared formulation for the administration at the hospital level and storage.
In an alternative manner, and to achieve the same benefits, the surface of the nanocarrier can be coated, by adsorption or conjugation, with human serum albumin (HSA) in order to conveniently increase the stability, the circulation times, and the biocompatibility of the formulation. This approach "anticipates" indeed the adsorption of HSA present in the blood and, simultaneously, it avoids the absorption of the aforementioned opsonins.
The whole supramolecular aggregate reduces the off-target release of the drug by the so-called EPR effect (Enhanced Permeability and Retention). The EPR effect is based on the anomalous and high permeability of blood vessels in the tumor region, characterized by not-adherent endothelial cell-to-cell junctions, if compared to normal tissue capillaries. Appropriate nanocarrier sizes, ranging from 20 to 100 nm (as reported in the examples of this invention), result in a prolonged circulation of the supramolecular systems in the blood stream, and the consequent selective extravasation into tumor tissues, resulting in a passive targeted therapy, which positively influences the toxicological profile. In addition, the lack of lymphatic drainage of cancerous compartments promotes the selective accumulation of the nanocarriers in the tumor microenvironment.
Advantageously, the functionalization of the nanocarriers with cancer-targeting moieties can also restrict the onset of side effects, thereby improving the chemotherapeutic index in terms of toxicity/activity ratio (active targeting).
In addition, when the nanocarriers are functionalized with carbohydrates as cancer-targeting moieties, these supramolecular or macromolecular systems give the possibility to increase the water-solubility and the stability of the encapsulated compound(s) as well as to improve the bioavailability, and to extend and facilitate the selective release towards cancer cells.
In summary, by means of the encapsulation of one or more coordination compounds according to the present invention, the inventors have advantageously developed two distinct and complementary strategies for the selective delivery of said compounds in the tumor site. This will be clear from the description of some examples reported below by way of example, but not limitation of present invention.
The first cancer-targeting strategy, associated with the third embodiment, is related to the passive targeting mediated by EPR effect described above. On the other hand, the second
strategy, presented in the fourth embodiment, takes into account the active targeting via glycoconjugation of the supramolecular nanosystem, thus providing an additional way to achieve a high therapeutic selectivity.
Anyway, the encapsulation allows the loaded compounds to increase their blood circulation times, as well as to improve the chemotherapeutic index of the active compounds (ratio between the lethal dose LD50 and the effective dose ED50), in other words their pharmacokinetic and pharmacodynamic profiles.
In order to engineer the most suitable encapsulation system (e.g., micelle, liposome), the partition coefficient n-octanol/water (P) was evaluated (and expressed in its logarithmic form, logP) for some coordination compounds with general structure l(a) and l(b), below described as by way of example, but not limitation of the present invention. It is known that the value P expresses the ratio between the concentration of a compound in n-octanol and the concentration of the same compound in water, at equilibrium.
EXAMPLES OF NANOFORMULA TIONS FOR PASSIVE TARGETING
In the third preferred embodiment of the present invention, the coordination compounds having a general Formula l(a) and/or Formula l(b) are encapsulated in supramolecular aggregates, in particular micelles, to achieve a passive targeting mechanism mediated by the EPR effect. Said encapsulated compounds may be those of the second embodiment, and hence conjugated to carbohydrates, or without a cancer-targeting moiety (first embodiment). For pharmaceutical applications, the ideal size of supramolecular systems ranges from 10 nm to 100 nm. In particular, a fast drainage from the injection site and an effective distribution in vivo are obtained with particles characterized by a hydrodynamic diameter (DH) ranging from 10 to 70 nm. In addition, once reached the target cells, the nanocarriers with DH < 100 nm at the cellular level can be absorbed by an endocytic process. In fact, it is well known that the size of the aggregates can significantly affect the circulation times and the bioavailability of the encapsulated compound.
From the description provided above, by way of example but not limitation, it will be clear that, the inventors have advantageously showed the possibility to achieve nanoformulations characterized by different hydrodynamic diameters (DH) with an average value of about 25-30 nm, being of extreme interest for pharmaceutical applications .
LogP evaluation procedure
By way of example, but not limitation, the procedure to evaluate the partition coefficient n- octanol/water (P) (expressed in its logarithmic form, logP) of some coordination compounds having general structure l(a) and l(b) is here described. It is known that this procedure is fundamental for the choice of the most suitable supramolecular system for a specific type of compound and/or application. Going into detail, the concentration of the compounds in the two immiscible phases has been determined in n-octanol before and after mixing with a defined volume of water. In particular, n-octanol was pre-saturated with deionized water for 24 hours
under stirring, then let to equilibrate for 6 h at 25 °C. A defined amount of compound is weighted and dissolved in a determined volume of the organic phase, and then stirred at 25 °C for two hours after the addition of the same volume of deionized water. Subsequently, the mixture is equilibrated for 30 minutes. The concentration of compound in the organic phase before (C0) and after the separation (Ci) was evaluated by UV-Vis spectrophotometry. The obtained values were used in the calculation of the partition coefficient n-octanol/water (P) according to the formula logP= log[Ci / (C0- Ci)]. The results are reported for some compounds in Table 4.
Table 4. Experimental logP= log[Ci / (C0- Q)] va l ues of some coordination com pou nds.
Considering the logP values reported in Table 4 and the IC50 data recorded in vitro (Table 1 , with reference to the first and second embodiments), it appears evident to the skilled in the art that the here claimed coordination compounds are associated with different LiPE values. This parameter is related to the Lipophilic Efficiency and it is defined by the formula LiPE= - log IC50 - log P. Some of the analyzed derivatives are characterized by LiPE values ranging from 4 to 6, which are considered ideal for the purposes of the so-called "druglikeness".
Encapsulation in micelles
A procedure for the encapsulation (loading) in micelles of some compounds according to the invention having logP > 0 is described below. Without loss of generality, the inventors have developed a loading system using Pluronic® F127 (indicated also as "PF127") as a polymeric substrate. However, this procedure is basically similar for any amphiphilic polymer (e.g., mPEG), with trivial changes to the skilled person.
The encapsulation of the complexes having Formula l(a) and/or Formula l(b) in PF127 micelles was obtained via a process comprising the following steps, described herein by way of example, but not limitation of the present invention: 1 ) Co-dissolution of the compound to be loaded and the polymer in the desired stoichiometric ratio {e.g., 0.5 mg and 500 mg, respectively) in an organic solvent, preferably chloroform; 2) Evaporation of the organic solvent, preferably under reduced pressure, followed by drying of the obtained powder, preferably under vacuum; 3) Hydration of the powder by addition of deionized water; 4) in order to remove the non- encapsulated compound, bacteria and other impurities, purification for instance by filtration with a membrane with a 0.20 μηι cut-off; 5) Freezing of the sample in a dry ice/acetone cooling bath
at -78 °C and cryoesiccation to remove the residues of the aqueous solvent, thus obtaining a storable ready-to-use formulation.
The amount of the encapsulated coordination compound was assessed by UV-Vis analysis, after dilution in DCM of a defined amount of lyophilized micellar formulation. In particular, the concentration of the compound was defined using the Lambert-Beer law, after experimental determination of the molar extinction coefficient ε in DCM (based on the "matrix effect", in the presence of the same polymer used for the preparation of micelles) for some absorption bands. The obtained results are reported in Table 5.
Table 5. Amou nt of encapsu lated compou nd in terms of d rug loading (mol of encapsu lated compou nd per mg of formu lation) a nd Encapsu lation Ratio (ER), i. e., the percentage a mou nt of encapsu lated compou nd compa red to the amou nt of nomi nal ly-loaded compou nd .
Advantageously, the encapsulation in micelles of the metal-based compounds according to the present invention increases their stability in physiological media and make them water-soluble for at least 72 hours. In fact, with reference to the appended Figure 2, here illustrated by way of example, but not limitation, the collected electronic spectra surprisingly show no significant change over time for the considered formulations (if compared to the not encapsulated compound) once dissolved in a physiological environment consisting of phosphate buffer/cell culture medium 9: 1 v/v, and phosphate buffer/human serum 95: 5 v/v.
Hence, it is evident that the present inventors have obtained compositions comprising the coordination compounds according to the present invention, characterized by a complete solubility and stability in physiological media compared to the corresponding non-encapsulated compounds, thus achieving a further object of the present invention.
Similarly, these nanoformulations, engineered and finely tailored, are unexpectedly stable also in a chemically aggressive medium, such as that charactering the tumor microenvironment, here mimicked with an acetate buffer (pH 5.5, Figure 3).
The in vitro experiments highlight also that the high stability of these formulations does not prevent the release of the active compound, followed by its cellular uptake, or alternatively, it does not prevent the release of the active compound associated with the absorption of the entire load.
It is clear from the results presented in the following Table 6 that micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus
exploiting to the best the EPR effect. As already mentioned, the use of PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.
Table 6: Structura l parameters of the micel la r systems here reported, determined via DLS ana lysis. PDI= polydispersion index.
It is clear from the results presented in the Table 6 that micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus exploiting to the best the EPR effect. As already mentioned, the use of PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.
Through the non-limitative examples of the present invention described above, the inventors have surprisingly demonstrated the possibility to encapsulate the coordination compounds of general Formula l(a) and l(b) in macromolecules or in a supramolecular system, thus overcoming in a non-trivial manner evident limits in the state of the art. In fact, the encapsulation techniques of the metal-based derivatives are still at the beginning, due to the inherent reactivity of the inorganic molecules.
In this way, the encapsulation allows to protect said compounds from the biotransformation, avoiding interactions/reactions with blood stream biomolecules or cells. In addition, the encapsulation allows increasing the stability as well as the solubility of hydrophobic compounds in the physiological media, as the here-presented data demonstrate.
EXAMPLES OF NANOFORMULA TIONS FOR THE ACTIVE TARGETING
In a fourth preferred embodiment, herein illustrated by way of example, but not limitation of the present invention, the coordination compounds having general Formula l(a) and l(b), are advantageously encapsulated in supramolecular aggregates and functionalized with carbohydrates. Therefore, they are directed to the tumor site according to an active-targeting approach which is paralleled with (hence boosting it) the mechanism of passive targeting of the previous embodiment. In other words, in the fourth embodiment the whole supramolecular aggregate acts as a cancer-targeting carrier, conveniently exploiting the Warburg effect, to further improve the therapeutic selectivity. In particular, the inventors aim to implement a mechanism wherein a coordination compound according to the invention is absorbed by the cell by endocytosis along with the whole supramolecular aggregate; alternatively, said compound can be released from the nanocarrier in the extracellular matrix, and consequently diffusing inside the cell if hydrophobic enough, or entering the cell through alternative mechanisms of
uptake, or also triggering its antitumor activity at the cell membrane level. In the latter case, compounds of the first embodiment, which by their chemical nature inherently exhibit affinity for the cell membrane, or compounds of the second embodiment that can exploit the presence of a cancer-targeting moiety T in the selective recognition by carbohydrate transporters {e.g., GLUT1 ), are advantageously used, alternately or in combination between them. Such compounds are hence encapsulated exploiting the procedures disclosed by the inventors with reference to the third embodiment so to form a supramolecular aggregate with a variable degree of functionalization, for example ranging from 1 to 50%. By way of example, but not limitation, convenient supramolecular aggregates can be micelles, vesicles (liposomes), cyclodextrins , dendrimers, organic polymeric nanoparticles, for instance of protein or peptide nature, and inorganic nanoparticles {e.g., silica, zirconia, titania). Carbohydrates can be monosaccharides, polysaccharides, pentoses, hexoses, aldoses, ketoses. Such supramolecular aggregates may have a mixed structure, for example being made up of pullulan and PF127, or PLA and PEG.
FUNCTIONALIZA TION OF BIOCOMPA TIBLE POL YMERS
With reference to the scheme reported below, the following non-limitative examples illustrate some synthesis that demonstrate the possibility to functionalize with different chemical groups the hydrophilic terminal end of biocompatible polymers, such as the polyethylene glycol (PEG) and the family of polymers known under the trade name Pluronic® F127. These functionalized derivatives are subsequently bio-conjugated with specific carbohydrates, in turn suitably functionalized, advantageously exploiting known synthetic strategies.
The biopolymer so functionalized with a carbohydrate, after dilution with a non-functionalized biopolymer (equal or different), is used to form a supramolecular aggregate with a degree of functionalization, ranging from 1 % to 50% mol/mol. By way of example, but not limitation, the schemes and the descriptions of some reactions carried out starting from the methoxy polyethylene glycol (mPEG) 5000 polymer are reported.
Synthesis of mPEG5000-Ts
To a solution of mPEG5000 in dry CH2CI2 dry at 0 °C, 0.5 eq. of 4-dimethylaminopyridine (DMAP) and 10 eq. of triethylamine (Et3N) were added. Then, after the addition of 10 eq. of p- toluensulfonyl chloride (TsCI) the reaction was stirred for 24 hours. The mixture was washed with HCI 0.1 M and an aqueous solution saturated with NaCI ("brine"). After treatment of the organic phase with diethyl ether, a white product precipitated. The functionalization degree was equal to 100%, considering the integration values of NMR signals corresponding to the aromatic protons of the tosylate and the methyl protons of the terminal methoxy group of mPEG5000 polymer.
I : v (cm4) = 2885, 1467, 1344, 1113, 1060, 842, 664.
Synthesis of mPEG5000 -Phta
The polymer mPEG5000-Ts was refluxed in dry DMF for 5 hours in presence of 20 eq. of phthalimide potassium salt (Phta K). Successively, after filtration of byproducts and precipitation with diethyl ether, a light yellow solid was isolated. The functionalization degree was equal to 100%, in light of the integration values of the NMR signals corresponding to the aromatic protons of the phthalimide and methyl protons of the terminal methoxy group of mPEG5000 polymer.
IR: v (cm 1) = 2885, 1716, 1467, 1344, 1 1 14, 1060, 842, 724, 690.
Synthesis of mPEG5000-NH2
This Gabriel-like synthesis of amines was carried out according to a modified literature procedure. 50 eq. of hydrazine (NH2NH2) were added to a solution of mPEG5000-Phta in EtOH at reflux. After 16 hours the crude product was precipitated with diethyl ether, leading to a white solid. The functionalization degree (-NH2 terminal group) is equal to 45%, based on the integration values of the NMR signals corresponding to the a-methylene protons with respect to the amino group (CH2-NH2) and methyl protons of the terminal methoxy group of mPEG5000 polymer.
IR: v (cm 1) = 2884, 1467, 1344, 1 1 13, 1060, 842.
Synthesis of mPEG5000-SC
This reaction allows the conversion of the terminal hydroxyl group to a carbonate which is very reactive towards nucleophiles due to the presence of a succinimido leaving group. The reaction was performed in a Schlenk line. Briefly, mPEG5000 was dissolved in dry 1 ,4-dioxane and was added to a mixture of DMAP (6 eq) and Ν,Ν'-disuccinimmidil carbonate (DSC) in dry acetone. After 6 hours, the by-products were filtered and the solution volume was reduced to precipitate
a white solid after the addition of diethyl ether. The functionalization degree is 100%, considering the integration values of NMR signals corresponding to the methylene protons of the succinimido group and the methyl protons of the terminal methoxy group of mPEG5000 polymer.
Synthesis of mPEG5000-GluOAc
The bio-conjugated mPEG5000-GluOAc was obtained via nucleophilic attack of the terminal hydroxyl group of the polymer to the anomeric C1 carbon of 1 ,2,3,4,6-penta-0-acetyl^- glucopyranose in the presence of BF3 Et20. In particular, 3 eq. of BF3 Et20 were added to a mixture of mPEG5000 and 1 ,2,3,4,6-penta-0-acetyl^-D-glucopyranose (3 eq.) in dry CH2CI2 dry at 0 °C, and the mixture was stirred for 48 hours. Then it was concentrated under vacuum and precipitated with diethyl ether, leading to the isolation of a white solid. The functionalization degree was equal to 20%, based on the integration values of NMR signals corresponding to the methyl protons of the acetate and the methyl protons of terminal methoxy group of mPEG5000 polymer.
IR: v (cm 1) = 2887, 1759, 1467, 1344, 1 1 1 1 , 1060, 842.
Synthesis of mPEG5000-GluOH
The polymer mPEG5000 functionalized with β-D-glucopyranose was obtained from the product of the previous step (mPEG5000-GluOAc) via deprotection of the acetyl groups under basic conditions (NaOMe in dry methanol) under stirring for 15 hours. Then, the reaction mixture was neutralized with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After the filtration of the resin, the product was precipitated with diethyl ether and dried under vacuum. IR: v (cm 1) = 3453, 2886, 1759, 1467, 1344, 1 1 12, 1060, 842.
The obtained polymer results 20% functionalized as O-glycoside (in C1 position), and can be used as such to encapsulate the coordination compounds, thus forming micelles or aggregates.
Alternatively, it may be used in diluted form in combination with other biocompatible polymers to obtain cancer-targeting nanoformulations such as, without loss of generality: mixed micelles,
liposomes, HSA nanoparticles. The latter, covered with a carbohydrate-functionalized mPEG, realize a "stealth effect", which is known to be associated with a decreased opsonization.
Similarly, the reaction scheme related to the PF127 polymer herein illiustrated as non-limitative example of the present invention is reported.
Synthesis of PF127-CHO
PF127-CHO
80%
The oxidation of the terminal hydroxyl groups of the PF127 polymer to aldehyde was performed using the Parikh-Doering oxidation method. Briefly, a DMSO solution of 3 eq. of Py S03 (Py = pyridine) was reacted with a DMSO solution of PF127 (1 eq.) and Et3N (6 eq.) at 20 °C. After 24 hours, the mixture was diluted in CH2CI2 and washed with an aqueous solution saturated with NaCI ("brine"). The organic phase was then concentrated and treated with diethyl ether, thus isolating a white solid. The degree of oxidation to aldehyde (evaluated via 1 H-NMR in CD2CI2) was equal to 80%, considering the integration values of NMR signals corresponding to the aldehyde proton and the methyl protons of the central PPO unit of the PF127 polymer.
IR: v (cm 1) = 2884, 1630, 146, 1344, 1 1 15, 1061 , 842.
Synthesis of PF127-GlnOAc
According to the following reaction scheme:
PF127-CHO
PF127-GlnOAc has been obtained by reductive amination between the PF127-CHO derivative described above and 1 ,3,4,6-tetra-0-acetyl-2-amino-2-deoxy^-D-glucopyranose HCI. In particular, the first step involves the nucleophilic attack of the glucosamine, reaction carried out
in acetonitrile for 2 hours at room temperature. Subsequently, the reduction of the imine to amine was obtained by the addition of sodium cyanoborohydride. The reaction occurs in acetonitrile for 30 minutes at room temperature with a total stoichiometric ratio 1 :1 :3 between PF127-CHO, glucosamine- HCI and NaCNBH3, respectively. Subsequently the solvent was removed under reduced pressure and the residue is taken up in DCM, washed in a separatory funnel with "brine". The organic phase is anhydrified and the product precipitated with diethyl ether.
The degree of functionalization (assessed via 1 H-NMR in CD2CI2) was equal to 44%, considering the integration values of NMR signals corresponding to the acetate protons of glucosamine and the methyl protons of the central PPO unit of the PF127 polymer.
1 H-NMR (600 MHz, CD2CI2): δ (ppm) = 1 .10 (t, 241 H, CH3 PF127), 1 .99 (s, 3H, COCH3), 2.03
(s, 3H, COCH3), 2.12 (s, 3H, COCH3).
IR: v (cm 1) = 2883, 1757, 1467, 1344, 1 1 14, 1060, 842.
Synthesis PF127-GlnOH
The polymer PF127 functionalized with 2-amino-2-deoxy-D-glucose (glucosamine) was obtained from the PF127-GlnOAc of the previous step by deprotection of the acetyl groups under basic conditions (NaMeO in dry methanol) under stirring for 15 h. Subsequently, the mixture was treated with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After filtration, the product was precipitated with diethyl ether and dried under vacuum.
IR: v (cm 1) = 3432, 2885, 1467, 1344, 1 1 12, 1060, 842.
The obtained polymer results 44% functionalized with glucosamine in C2 position and can be used as such to encapsulate the coordination compounds, forming micelles. Alternatively, it may be used in combination with other biocompatible polymers to obtain cancer-targeting nanoformulations. By way of example, but not limitation, mixed micelles or supramolecular aggregates can be prepared starting from this derivative, after "dilution" with non-functionalized PF127, or another biocompatible polymer, in order to obtain different degrees of functionalization of the supramolecular aggregate, preferably in the range 2 ÷ 18%.
Through the synthetic schemes described above, the present inventors prepared formulations wherein the coordination compounds according to the invention are encapsulated in micelles which are functionalized with carbohydrates (Table 7).
Table 7: Structural parameters of the micellar systems herein described, determined by DLS analysis.
Surprisingly, all the synthetic procedures allow achieving a degree of functionalization equal or greater than 20%, as verified through 1 H-NMR analysis in CD2CI2.
Based on the syntheses herein disclosed, it will be apparent to the skilled in the art that he present inventors have overcome relevant issues in the state of the art in a novel and inventive manner. Indeed, although the functionalization of polyether derivatives such as Pluronic® F127 and mPEG is widely described in literature, the syntheses herein reported are often difficult to reproduce due to the high molecular weight of the involved surfactants as well as the viscosity of their solutions in organic media {e.g., DCM or THF). Consequently, all the synthetic strategies need to be adapted and optimized in every specific case. In addition, the purification is often troublesome due to the high chemical similarity between the polymeric reagents and their products, characterized by minimal changes in the molecular structure (in terms of molecular weight and introduced functional groups). Given the complexity of these systems, also the functionalization/bio-conjugation yields are often low (<50%), and the assessment of the degree of functionalization of the products results difficult because of the abovementioned purification problems and the instrument sensitivity.
Although the examples of functionalization herein described are referred to mPEG and PF127, it will be clear to the skilled in the art that the application of the usual synthetic techniques allows the functionalization of other types of biocompatible polymers.
EVALUATION OF THE BIOLOGICAL ACTIVITY
Antitumor activity in vitro
From the description and from the experimental data provided above, the coordination compounds according to the first and second embodiment of the present invention possess a remarkable anticancer activity. Likewise, said compounds loaded into aggregates, according to the third and fourth embodiment, maintain such antitumor capacity, thus achieving a further advantageous object of the present invention.
Indeed, by its nature the encapsulation does not alter the anticancer properties of said compounds, independently on the functionalization of the aggregate itself. In other words, these supramolecular aggregates play only the role of nanocarriers, in particular increasing the stability and solubility of said compounds in aqueous medium, and anyway allowing the release of the active compound within the cancer cell or in the extra-cellular matrix, or in general, in the bloodstream.
Therefore, the previously presented in vitro results concerning the antitumor activity of the coordination compounds, can be extended also to the described formulations, with reference to the third and fourth embodiments.
By way of example and not limitation, the antitumor activity of IC50 micelles loaded with Ru complexes is presented in the following table.
Formulation Nanoformulation HeLa HepG2/CTR HepG2/SB3 description
RuA Pure PF127 0.21 ± 0.6 1 .72 ± 0.1 2.4 ± 0.3
RuC PF127-GluOH 1 0% 0.12 ± 0.03 1 .1 1 ± 0.09 1 .4 ± 0.1
RuE PF127-p-(D)- 0.105 ± 0.009 0.89 ± 0.09 1 .07 ± 0.09 glucopyranose 10%
RuG PF127-GlnOH 1 0% 0.12 ± 0.03 0.8 ± 0.1 1 .3 ± 0.2
Rul PF127-p-maltose 0.12 ± 0.06 0.56 ± 0.07 0.99 ± 0,07
10%
Table 8IC50 values (μΜ) for PF127-based formulations evaluated after a 72-h treatment. Values are calculated based on the concentration of the encapsulated ruthenium-DTC complex. Lyophilized micelles were dissolved in cell culture medium. Data represent the mean ± SD of at least three independent experiments. THERANOSTIC USE OF THE COORDINATION COMPOUNDS AND THE RELATED COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS
Advantageously, the coordination compounds, the compositions, and the pharmaceutical formulations according to the present invention can be used as "theranostic agents" i.e. not only for the treatment of human or animal diseases, in particular neoplastic diseases, but also for the diagnosis and the patient follow-up.
Within the more general scope of the present invention, such mechanism that combines therapy and diagnosis can be made in many ways, properly including one or more contrast agents in a coordination compound, or in a composition comprising at least one of said coordination compound, or also in a pharmaceutical formulation that includes said compound and/or composition.
By way of example, but not limitation of the present invention, referring to the general Formula l(a) or l(b), the contrast agent can be "intrinsic" to the compound, for example when the metal centers M M2, M3 are radionuclides {e.g., 68Ga, previously introduced). Similarly, also the donor atoms X, Y, W, Z, L L2, L3 and L4 are radioisotopes {e.g., 1311 , 127l, 129l), as well as T can be 2-deoxy-2-[18F]-fluoroglucose. Finally, the coordination compound, the unit A, the carbohydrate or glucide T and/or T is paramagnetic or diamagnetic.
In addition, the contrast agent can be "exogenous" to the compound, and may be included in the composition that comprises at least one coordination compound according to the present invention, or also in a pharmaceutical formulation, that includes said compound and/or composition. Again, as non-limitative example of the present invention, this category comprises a carrier that encapsulates said compound, such as a up-converting bismuth oxide nanoparticle (e.g. Italian patent 0001419393, in the name of BEP Sri et al.). In this case, the contrast agent is a dual-mode contrast agent that take advantage of the X-ray absorption and the optical
emission (through a NIR->NIR or NIR->VIS mechanism) exhibited by the doped bismuth oxide nanoparticles. Other organic and inorganic luminescent systems, such as nanoparticles and compounds, as well as their use in the diagnosis and imaging of biological systems are well- known. Therefore, depending on the necessity, the "exogenous" contrast agent may also be a component of the pharmaceutical formulation, for example, a known contrast agent.
A "combined" contrast agent can be used and obtained mixing one or more "intrinsic" contrast agents with "exogenous" contrast agents. For example, radionuclides such as 131 1, 127l, 129l may be previously introduced as donor atoms X, Y, W, Z, L L2, L3 and L4 in the general Formula l(a) or l(b) ; the coordination compound can be linked to a carrier consisting of a photoactivatable nanoparticle, such as a properly doped bismuth oxide nanoparticle.
Other examples will appear or become evident to the skilled in the art, on the basis of the disclosure herein provided, or through practice of the same, and on the basis of the knowledge at the state of the art in the field of material science, devices and equipment for medical or biomedical imaging and diagnosis.
In the various non-limitative examples illustrated here, with the only aim to show that the coordination compound according to the invention can be advantageously used to obtain a compound or a theranostic agent, it is essential that the contrast agent is detectable by suitable devices or detectors, hence to be usable in association with diagnostic and imaging systems in the medical or biomedical field. In other words, said contrast agent must be able to emit a signal detectable spontaneously, or after interaction with, for instance, irradiation with external particles, or with an external magnetic/electric field.
Contrast agents capable of spontaneously emit a detectable signal are, for example, radionuclides such as 1 1C, 18F, and 68Ga (in this case the signal is a radiation and/or a particle), or radiopaque materials (the signal here is related to the X-ray absorption spectrum).
Other contrast agents emit a signal which is detectable after an external stimulus, which is typically the irradiation with particles and suitable electromagnetic radiations (based on the needs). As an example, this category comprises: luminescent photoactivable contrast agents, such as nanoparticles, dye-molecules or luminophores, capable of emitting IR-Vis radiation as a result of laser irradiation with a suitable wavelength ; contrast agents capable of emitting a detectable signal due to the presence of dipoles or magnetic domains within their structure, which interact with an appropriate electromagnetic field; it is also possible to generate radionuclides inside of the compound and/or the composition according to the invention, treating them with particles and electromagnetic radiations with the suitable energy, using known techniques.
It will be evident to those skilled in the art, based on the second and fourth embodiments, described above, by way of example and not limitation, the theranostic applications of the coordination compounds having general Formula l(a) and/or l(b) are very promising in light of the high selectivity and bioavailability, being as above presented, related to the presence of the
carbohydrate or glucide T and/or T', that acts as cancer-targeting moieties able to implement a passive and active targeting-mechanism towards the tumor cells.
CONCLUSIONS
In conclusion, it is apparent to those skilled in the art that the present invention fully achieved the intended aim and objects by means of the disclosure provided herein.
The invention thus conceived is susceptible of numerous modifications and variations, without departing from the basic concepts as disclosed herein. Moreover, all the details may be replaced with other technically equivalent elements. Furthermore, the order of the process steps described above is shown by way of example, but not limitation and can be changed according to convenience. The above description and drawings are only illustrative of preferred embodiments which achieve the objects, features and advantages of the present invention, and it is not intended that the present invention be limited thereto.
All structural, chemical, and functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. It will be appreciated that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art in view of this specification and which are all considered within the scope of the claimed invention
Although the description and examples above contains many details, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Therefore, any modification of the present invention which comes within the spirit and scope of the following claims is considered part of the present invention.
In the appended claims, reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." Where the characteristics and techniques mentioned in any claim are followed by reference signs, those reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly, such reference signs do not have any limiting effect on the interpretation of each element identified by way of example, but not limitation by such reference signs.
Trademarks
Pluronic® is a trademark of BASF AG;
Cremophor® is a trademark of BASF AG;
CD-1 ® is a trademark of di Charles River Laboratories, Inc. Corp.;
Eurospital® is a trademark of Eurospital Spa;
Lipoxal® is a trademark of Regulon A.E;
Amberlite® is a trademark of Santa Cruz Biotechnology Inc.
Claims
1 . A mononuclear or dinuclear coordination compound comprising a neutral or ionic complex and optionally at least one counter-ion G, said compound being represented by the general formulas I (a) and l(b):
1(a) 1(b)
wherein :
- the arc connecting the two sulfur atoms represents a first dithiocarbamato ligand (DTC) ;
- the arc connecting L and X represents a first bidentate chelating ligand;
- the arc connecting L2 and Y represents a second bidentate chelating ligand;
- the arc connecting and Y represents a third bidentate chelating ligand;
- the arc connecting L3 and X represents a fourth bidentate chelating ligand;
- the arc connecting L2 and L4 represents a fifth bidentate chelating ligand;
- the arc connecting W and Z represents a sixth bidentate chelating ligand;
any form of isomerism exhibited by said compound is included, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, either as racemes or in various ratios,
and wherein M M2, M3, X, Y, W, Z, L L2, L3 and L4 are independently selected in the following manner:
- M, , M2 e M3 are metal centers selected from Ru(l l) or Ru(l ll) or Ga(l ll);
- X, Y, Z, W, I_2, L3, l_4 are donor atoms, equal to or different from each other, selected from the group consisting of: S, N, O, P, Se.
and wherein :
- said at least one counter-ion G is selected between:
- a pharmaceutically acceptable ion, or;
- an ion produced during the synthesis of said compound;
d is the electric charge of said counter-ion represented by an integer number comprised between -4 and +4;
- e is the stoichiometric coefficient of said counter-ion G, represented by an integer number whose absolute value is equal to n/d or equal to zero;
- n is the electric charge of said complex represented by an integer number comprised between -4 and +4, wherein n=0 corresponds to a neutral complex having e=0.
2. The coordination compound according to claim 1 represented by the formula l(a), wherein:
- Mi=Ru(lll) or Ga(lll);
- said first bidentate chelating ligand is a second dithiocarbamato ligand (DTC) equal to or different from said first dithiocarbamato ligand (DTC);
- said second bidentate chelating ligand is a third dithiocarbamato ligand (DTC) equal to or different from said first dithiocarbamato ligand (DTC) or from said second dithiocarbamato ligand (DTC).
3. The coordination compound according to claim 1 represented by the formula l(b), wherein:
- M2=Ru(lll) or Ru(ll);
- M3=Ru(lll) or Ru(ll);
- X = Y = W= Z = U = L2 = l_3 = l_4 = S;
- said third bidentate chelating ligand is a second dithiocarbamato ligand (DTC);
- said fourth bidentate chelating ligand is a third dithiocarbamato ligand (DTC);
- said fifth bidentate chelating ligand is a fourth dithiocarbamato ligand (DTC);
- said sixth bidentate chelating ligand is a fifth dithiocarbamato ligand (DTC);
- said second, third, fourth and fifth dithiocarbamato ligands (DTC) are equal to or different from said first dithiocarbamato ligand (DTC);
- said second, third, fourth and fifth dithiocarbamato ligands (DTC) are equal or different from each other.
4. The coordination compound according to one or more of the claims 1 to 3 wherein said first or second or third or fourth or fifth dithiocarbamato ligand (DTC) comprises:
- a group R bound to the dithiocarbamic nitrogen atom;
- groups R2, R3 and R4, and optionally comprises
- a group R5, and optionally comprises
- at least one unit A consisting of an atom or a functional group or a spacer and optionally comprises
- at least a glucide or a carbohydrate T,
said first or second or third or fourth or fifth dithiocarbamato ligand (DTC) wherein:
- said group Ri is bound to said nitrogen atom and to said group R2;
- said group R2 is bound to said group Ri and to said group R3;
- said group R3 is bound to said group R2 and to said group R4;
- said group R4 is bound to said group R3 and to said group R5 or alternatively said group R4 is bound to said group R3 and to said nitrogen atom;
- said group R5 is bound to said group R4 and to said nitrogen atom;
- the bonds between said group R, and said group Rj (wherein i≠ j are integer numbers comprised between 1 and 5) are single or double bonds;
- said at least a glucide or a carbohydrate T is bound to at least one of said groups R1 5 R2, R3, R4 and R5, directly, with T-R, bonds, or through said at least one unit A, with T-A-R, bonds (wherein i is an integer comprised between 1 and 5), said bonds T-R, or T-A-R, being single or double or triple bonds and of the kind: C-C, C-O, O-C, C-N, N-C, C-S, S- C, C-P, P-C, C-Se, Se-C;
- said at least a glucide or a carbohydrate T is bound to at least one of said groups R1 5 R2, R3, R4 and R5, directly or through said at least one unit A, via at least one of the carbon positions of said T, preferably in the C1 or C2 or C3 or C4 or C6 positions for hexoses and preferably in the C1 or C2 or C3 or C5 positions for pentoses;
- said at least one unit A or said group R,, wherein i is an integer comprised between 1 and 5 is:
- an atom selected from: H, C, O, N, S, P, Se, F, CI, Br, I or is
- a first group selected from among: -CH, -CH2, -CH3, -C(CH3)3, -NH, -NH2, -NHRS, - NRs2, -S-S-, -SH, SeH, -PH, -OH, -COOH, -CH-Br, -CHCH2NH2, -CHCH2OH, - CHCH2NH-, -CHCH20-, (-CN), -CF3, -C2H5, -C2H4, -C4H9, -C3H7, -C3H6, -C3H6OH, - C4H8, -N02, -CH2OH, -C2H4OH, -C4H8OH, -C(OH), -C(OH)H2, -C(OH)H; -S02, -COO- , -N(CH)3-, -CHN(CH), -NN(CH),-NCHNCH-, -N(CH)2N-, -CHNHCH-, -NH(CH), - NHCHN-, -NHNCH-; -CONH-, -CONH2, -CONHRs2, -CONRs2, (-0-C(0)-Rs), or a combination thereof, wherein Rs indicates substituent groups of the type aliphatic, alkyl, halo-alkyl, cycloalkyl, alkene, alkyne, alkynyl, aryl, hetero-aryl, heteroaliphatic, aromatic, hetero-aromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic and hetero-cycloaliphatic, or is
- a second group selected from among: ester, amide, sulfonamide, carbonyl, acetate, ethyl, propyl, butyl, isopropyl, acyl, ureidic, thioureidic, thiolate, imine, halogen, ether, nitro groups, nitrile groups, aryl, benzyl, sulphonamide groups, alkyl C1 -C18 saturated linear or branched optionally substituted with one or more of said Rs groups, or comprising one or more unsaturated bonds, or a combination thereof, or is
- a third group of the kind -(CH2)m, -(CH20)m, -(CH2CH2S)m, (CH2CH2NH)m or of the kind (CH2CH20)m, (CH2CH2N)m (wherein m is an integer number higher than 1 ), or a combination thereof, or
- or a combination of said atom or of said first group or of said second group or of said third group,
said unit A or said R, group comprising single or double or triple bonds and being optionally in a salt form comprising a pharmaceutically acceptable counter-ion, said unit A or said R, group being optionally substituted with one or more of said atoms or said
groups in any combination and position, including any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, either as racemes or in various ratios.
5. The coordination compound according to one or more of the claims 1 to 3 wherein said first or second or third or fourth or fifth dithiocarbamato ligand (DTC) comprises:
- a terminal group R bound to the dithiocarbamic nitrogen atom, and optionally comprises
- one or more groups R2, R3, R4 and R5, and optionally comprises
- at least one unit A consisting of an atom or a functional group or a spacer and optionally comprises
- at least a glucide or a carbohydrate T,
said first or second or third or fourth or fifth dithiocarbamato ligand (DTC) wherein:
- said group R2 is a terminal group bound to R3, or R4, or R5 or to said nitrogen atom;
- said group R3 is bound to said group R2 or alternatively said group R3 is a terminal group and R3 is bound to R4 or R5, or to said nitrogen atom;
- said group R4 is bound to said group R5 or to said nitrogen atom and R4 is a terminal group, or alternatively R4 is bound to said group R5 or to said nitrogen atom and R4 is bound to said group R3 or to said group R2;
- said group R5 is bound to said nitrogen atom and it is a terminal group, or said group R5 is bound to R4 or R3 or R2;
- the bonds between said group R, and said group Rj (wherein i≠ j are integer numbers comprised between 1 and 5) are single or double or triple bonds;
- said at least a glucide or a carbohydrate T is bound to said dithiocarbamic nitrogen atom or to at least one of said groups R1 5 R2, R3, R4 and R5, directly, with T-N or T-R, bonds respectively (wherein i is an integer comprised between 1 and 5), or through said at least one unit A, with T-A-N or T-A-R, bonds respectively, said bonds T-N or T-R, or T-A-N or T-A-Ri being single or double or triple bonds of the kind: C C-C, C-O, O-C, C-N, N-C, C- S, S-C, C-P, P-C, C-Se, Se-C;
- said at least a glucide or a carbohydrate T is bound to said dithiocarbamic nitrogen atom or to at least one of said groups R1 5 R2, R3, R4 and R5, directly or through said at least one unit A, via at least one of the carbon positions of said T, preferably in the C1 or C2 or C3 or C4 or C6 positions for hexoses and preferably in the C1 or C2 or C3 or C5 positions for pentoses;
- said at least one unit A or said group R,, wherein i is an integer comprised between 1 and 5 is:
- an atom selected from: H, C, O, N, S, P, Se, F, CI, Br, I or is
- a first group selected from among : -CH, -CH2, -CH3, -C(CH3)3, -NH, -NH2, -NHRS, - NRs2, -S-S-, -SH, SeH, -PH, -OH, -COOH, -CH-Br, -CHCH2NH2, -CHCH2OH, -
CHCH2NH-, -CHCH20-, (-CN), -CF3, -C2H5, -C2H4, -C4H9, -C3H7, -C3H6, -C3H6OH, -
C4H8, -NO2, -CH2OH, -C2H4OH, -C4H8OH, -C(OH), -C(OH)H2, -C(OH)H; -S02, -COO-
, -N(CH)3-, -CHN(CH), -NN(CH),-NCHNCH-, -N(CH)2N-, -CHNHCH-, -NH(CH), -
NHCHN-, -NHNCH-; -CONH-, -CONH2, -CONHRs2, -CONRs2, (-0-C(0)-Rs), or a combination thereof, wherein Rs indicates substituent groups of the type aliphatic, alkyl, halo-alkyl, cycloalkyl, alkene, alkyne, alkynyl, aryl, hetero-aryl, heteroaliphatic, aromatic, hetero-aromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic and hetero-cycloaliphatic, or is
- a second group selected from ester, amide, sulfonamide, carbonyl, acetate, ethyl, propyl, butyl, isopropyl, acyl, ureidic, thioureidic, thiolate, imine, halogen, ether, nitro groups, nitrile groups, aryl, benzyl, sulphonamide groups, alkyl C1 -C18 saturated linear or branched optionally substituted with one or more of said Rs groups, or comprising one or more unsaturated bonds, or a combination thereof, or is
- a third group of the kind -(CH2)m, -(CH20)m, -(CH2CH2S)m, (CH2CH2NH)m or of the kind (CH2CH20)m, (CH2CH2N)m (wherein m is an integer number higher than 1 ), or a combination thereof,
- or a combination of said atom or of said first group or of said second group or of said third group,
said unit A or said R, group comprising single or double or triple bonds and being optionally in a salt form comprising a pharmaceutically acceptable counter-ion, said unit A or said R, group being optionally substituted with one or more of said atoms or said groups in any combination and position, including any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, either as racemes or in various ratios.
6. The coordination compound according to claim 4 or 5 wherein one or more of the hydroxyl groups of said at least one glucide or carbohydrate T are functionalized with protecting groups, equal to or different from each other, preferably silylethers or esters, or still more preferably, acetates, pivalates, propionates, carbonates, phosphates, butyrates.
7. A composition comprising:
- at least one coordination compound according to one or more of claims 1 to 6;
- one or more entities selected from: macromolecules, supramolecular aggregates, nanostructures, or a combination thereof, said entities having constituent units equal to or different from each other, and said supramolecular aggregates optionally consisting of molecules, polymers or oligomers, natural or synthetic, having composition equal to or different from each other, and
wherein at least one of said coordination compounds:
establishes intermolecular interactions with one or more of said entities, or
- is encapsulated in one or more of said entities.
8. The composition according to the preceding claim, wherein said macromolecules or said supramolecular aggregates or nanostructures:
- are carriers binding, preferably encapsulating, said at least one coordination compound to improve the pharmacological profile, preferably in terms of: solubility, stability, bioavailability or a combination thereof;
- have dimensions between 0.2 and 1200 nm, and have a neutral, positive or negative surface charge properly balanced by pharmaceutically-acceptable counter-ions; and
- are selected from the group consisting of: polymeric micelles, non-polymeric micelles, dendrimers, liposomes, cyclodextrins, proteins, peptides, organic nanoparticles, polymeric nanoparticles, inorganic nanoparticles, nanotubes, nanohorns, polymers, polymeric aggregates, metal oxides, semimetal oxides or a combination thereof, and
- optionally exhibit a stealth property.
9. The composition according to claim 7 or 8, wherein one or more of said constituent units or one or more of said molecules, polymers or oligomers are glycoconjugates with glucides or carbohydrates T having equal or different composition, said glycoconjugates being bound:
- directly or through spacer A' via at least one of the carbon positions of said glucides or carbohydrates T;
- through single or double or triple bonds with T or with A'-(T'), said bonds being of the kind: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C,
said spacer A' comprising at least:
- an atom selected from: H, C, O, N, S, P, Se, F, CI, Br, I, or
- a fourth group selected from: -CH, -CH2, -CH3, -C(CH3)3, -NH, -NH2, -NHRS, -NRs2, - S-S-, -SH, -PH, -SeH, -OH, -COOH, -CH-Br, -CHCH2NH2, -CHCH2OH, -CHCH2NH-, -CHCH20-, -CN, -CF3, -C2H5, -C2H4, -C4H9, -C3H7, -C3H6, -C3H6OH, -C4H8, -N02, - CH2OH, -C2H4OH, -C4H8OH, -C(OH), -C(OH)H2, -C(OH)H; -S02, -COO-, -N(CH)3-, - CHN(CH), -NN(CH),-NCHNCH-, -N(CH)2N-, -CHNHCH-, -NH(CH), -NHCHN-, - NHNCH-; -CONH-, -CONH2, -CONHRs2, -CONRs2, (-0-C(0)-Rs), or a combination thereof, wherein Rs indicates substituent groups of the type: aliphatic, alkyl, halo- alkyl, cycloalkyl, alkene, alkyne, alkynyl, aryl, hetero-aryl, heteroaliphatic, aromatic, hetero-aromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic and hetero-cycloaliphatic, or
- a fifth group selected from ester, amide, sulfonamide, carbonyl, acetate, ethyl, propyl, butyl, isopropyl, acyl, ureidic, thioureidic, thiolate, imine, halogen, ether, nitro groups, nitrile groups, aryl, benzyl, sulphonamide groups, alkyl C1 -C18 saturated linear or branched optionally substituted with one or more Rs groups, or comprising one or more unsaturated bonds, or a combination thereof, or
- a sixth group of the kind -(CH2)m, -(CH20)m, -(CH2CH2S)m, (CH2CH2NH)m or of the kind (CH2CH20)m, (CH2CH2N)m (wherein m is an integer number higher than 1 ), or a combination thereof,
- or a combination of said atom or of said fourth group or of said fifth group or of said sixth group, optionally substituted,
said spacer A' comprising single or double or triple bonds and comprising groups optionally substituted with one or more of said atoms or said groups in any combination and position,
said macromolecules or supramolecular aggregates or nanostructures:
including any form of isomerism, preferably structural isomers, coordination isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, either as racemes or in various ratios, and
- being functionalized with a total glycoconjugation percentage, referred to said composition, ranging from 1 to 50 % mol/mol, and
said composition optionally comprising said macromolecules or supramolecular aggregates or nanostructures having groups in a salt form.
10. A pharmaceutical formulation for the diagnosis or treatment of human and animal diseases including:
- at least one compound according to one or more of claims 1 to 6 in combination with pharmaceutically acceptable excipients and/or additives and optionally at least another active agent, or another drug, and optionally at least one contrast agent,
or
the composition according to one or more of claims 7 to 9 in combination with pharmaceutically acceptable excipients and/or additives and optionally at least another active agent, or another drug, and optionally at least one contrast agent,
said excipients and/or additives being selected from the group consisting of: diluents, solvents, bulking agents, rheology modifiers, stabilizers, pH stabilizers, binders, buffers, disaggregating, preservatives, elasticizing, emulsifiers, chelating agents, lubricants, top sweeteners, sweeteners, dyes, contrast agents and flavoring, alone or in combination thereof, said at least one contrast agent being able to emit a detectable signal for diagnostics applications, spontaneously or as a result of particle irradiation or electromagnetic irradiation.
1 1 . The pharmaceutical formulation according to the preceding claim for the diagnosis or treatment of cancer or the patient follow-up, characterized in that:
- said at least one compound or said composition is anticancer and provides a passive cancer cell-selective delivery, optionally associated with the emission of said signal by said at least one contrast agent; or
- said at least one compound is anticancer and comprises said glucide or carbohydrate T acting as a "cancer-targeting moiety" and wherein said compound provides an active cancer cell-selective delivery, optionally associated with the emission of said signal by said at least one contrast agent; or
- said glycoconjugated composition comprising said glucide or carbohydrate T' is anticancer and said T' acts as a "cancer-targeting moiety" and wherein said compound provides an active cancer cell-selective delivery, optionally associated with the emission of said signal by said at least one contrast agent; or
- said formulation provides a combination of active and passive cancer cell-targeting release, optionally associated with the emission of said signal by said at least one contrast agent,
and wherein
- said at least one contrast agent being selected from the group consisting of radioisotopes, radionuclides, luminescent particles, photoactivable particles, luminescent nanostructures, photoactivable nanostructures, dye molecules, photoactivable molecules, luminescent molecules, phosphors, luminophores, electric dipoles, magnetic dipoles, magnetic domains, including ferromagnetic, antiferromagnetic, paramagnetic, diamagnetic structures or compounds, or a combination thereof, and
- said contrast agent is optionally associated with one or more components of said compound or of said composition, said components being selected from : said first or second or third or fourth or fifth dithiocarbamato ligand (DTC), said first or second or third or fourth or fifth or sixth bidentate chelating ligand, said metal centers M M2, M3, said glucides or carbohydrates T or T', said unit A, said spacer A', said entity comprising macromolecules or supramolecular aggregates or nanostructures, or a combination thereof.
12. Use of a coordination compound according to any of the claims 1 to 6, in preparing pharmaceutical compositions or formulations for the diagnosis or treatment of cancer or follow-up in humans or animals.
13. A process for the preparation of the mononuclear or dinuclear coordination compound according to one or more of the preceding claims 1 to 6, comprising the following steps: a) synthesis of one or more of said dithiocarbamato ligand (DTC) in water or in an organic solvent, preferably methanol or tetrahydrofuran (THF), by reaction between carbon disulfide (CS2), an amine precursor and optionally a base. After at least 5 minutes, preferably in a protective atmosphere according to the choice of said precursor amine, optionally carrying out:
- reduction of the water volume or of said solvent volume and optionally,
- isolation of one or more DTC ligands by means of standard separation techniques, preferably by co-precipitation with addition of organic solvent and optionally washing and drying;
b) in organic solvent, preferably in a protected atmosphere, or in water, synthesis of the coordination compound of formula 1(a) and/or 1(b) through the coordination of one or more of said DTC, synthetized and optionally isolated in the step a), to a metal center selected from Ru(ll) or Ru(lll) or Ga(lll), starting from the corresponding precursors with the same oxidation state, preferably chlorides or halide salts, in anhydrous or hydrated form, or alternatively said corresponding precursors are derivatives wherein the metal center occurs in a lower or higher oxidation state, preferably being organometallic precursors, amines, thioethers, phosphine derivatives, or alternatively, said corresponding precursors may be some of said coordination compounds, said coordination being carried out for at least 5 minutes, according to a stoichiometric ratio metal-center/ligand in a range between 0.1 and 0.8, preferably said coordination being carried out for at least 20 minutes according to a stoichiometric ratio metal-center/ligand in a range between 0.2 and 0.7, even more preferably said coordination being carried out for at least 30 minutes according to a stoichiometric ratio metal-center/ligand in a range between 0.3 and 0.5, said process optionally including the following step:
c) isolation of the compound synthesized in the preceding step b) by means of standard separation techniques, so to obtain the coordination compound of formula l(a) and/or l(b) or a mixture containing at least one of said coordination compounds.
14. The process according to the preceding claim wherein said precursor amine is synthetized through a step including the glycoconjugation between said glucide or carbohydrate T, optionally functionalized with protecting groups or with said unit A, and an organic molecule, optionally functionalized with protecting groups or with said unit A, said organic molecule optionally containing at least an amine group.
15. The process according to claim 13 or 14 further comprising at least one of the following steps:
- a step wherein said at least one counter-ion G is exchanged;
- one or more steps to protect one or more functional groups of said glucide or carbohydrate T or of said precursor amine or of said unit A, via the introduction of protecting groups and optionally,
- one or more deprotection steps to deprotect one or more of said protecting groups present in said glucide or carbohydrate T or in said amine precursor or in said unit A, wherein said one or more deprotection steps are carried out by chemical or biochemical methods, including the use of enzymes or pseudo-enzymes.
16. The process according to one or more of claims 13 to 15, for preparing said dinuclear coordination compounds of formula l(b) with M2= M3= Ru(lll), further comprising the following phases:
a) carrying out one of the following schemes, optionally in combination thereof:
- coordination to the metal precursor of one or more of said dithiocarbamato ligands (DTC) dissolved in organic solvent, preferably methanol, said coordination being carried out in water or in an organic solvent, preferably methanol, ethanol, acetone, tetrahydrofuran (THF), so to obtain the a and β isomers of the ionic dinuclear complex [Ru2(DTC)5]+ in a first mixture containing also a coordination compound [Ru(DTC)3] having the general formula l(a);
- reaction between said coordination compound [Ru(DTC)3] and an excess of BF3 Et20 in organic solvent, preferably halogenated or aromatic, to obtain a second mixture containing the a or β isomers of the ionic dinuclear complex [Ru2(DTC)5]+,
and optionally comprising one or more of the following phases:
b) purification of said first mixture or said second mixture to isolate said a or β pure isomers or alternatively to isolate a binary mixture thereof;
c) converting said isomer a to said isomer β, thermodynamically more stable, by:
- heating said binary mixture, or alternatively the pure a isomer at reflux in an organic solvent, preferably isopropanol ((CH3)2CHOH), methanol (CH3OH) or dichloromethane (CH2CI2), for at least one hour, or alternatively by
- adding at least 1 eq. of dithiocarbamato ligand (DTC) to said pure a isomer or alternatively to said binary mixture in organic solvent, preferably halogenated or methanol.
17. The process according to one or more of claims 13 to 15, for preparing said dinuclear coordination compounds of formula l(b) with M2= Ru(ll) o Ru(lll), M3= Ru(ll) o Ru(lll), comprising the reaction between said pure isomers a or β, or a binary mixture thereof, with reducing agents, preferably NaBH4 in protected atmosphere.
18. A process for preparing the composition according to one or more of claims 7 to 9, comprising at least the following steps:
a) obtaining one or more coordination compounds according to one or more of claims 1 to 6;
b) optionally, evaluating the partition coefficient n-octanol/water (logP) of said one or more coordination compounds;
c) choosing one or more of said entities able to establish intermolecular interactions with one or more of said compounds or able to encapsulate one or more of said coordination compounds;
d) choosing said constituent units or said molecules, oligomers, polymers;
e) carrying out one of the following schemes:
- co-dissolving, in the desired stoichiometric ratio, one or more of said compounds and one or more of the elements selected in the step d) in at least one organic solvent, preferably dichloromethane (DCM), methanol or chloroform; removing said organic solvent, preferably at reduced pressure, and optionally drying, preferably in vacuum, so as to obtain a dry residue; hydrating said dry residue by addition of water or a saline solution or a buffer, preferably a phosphate buffer, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d),
or, alternatively,
co-dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent, or in a mixture thereof, preferably dichloromethane (DCM), methanol or chloroform; removing said organic solvent, preferably at reduced pressure, optionally drying, preferably in vacuum, so as to obtain a dry residue; addition of an aqueous solution of one or more of said compounds, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);
or, alternatively,
dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in water or in a mixture glycerol/ethanol, or a combination thereof, followed by addition of an aqueous solution of one or more of said compounds, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d),
or, alternatively,
dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent; removal of the solvent and optionally drying so as to obtain a film; hydration of said film with an aqueous solution and treatment of the obtained suspension by mechanical processes, preferably sonication and/or membrane extrusion, dialysis against aqueous solution; addition and incubation with the solution of said compound under continuous stirring, and optionally freeze-drying followed by hydration, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);
or, alternatively,
dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent; removal of the solvent and optionally drying so as to obtain a film; hydration of said film with an aqueous solution and treatment of the obtained suspension by mechanical processes, preferably
sonication and/or membrane extrusion, addition and incubation with the solution of said compound under continuous stirring optionally in the presence of cryopreservatives, and optionally freeze-drying followed by hydration, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);
or, alternatively,
dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d), in an organic solvent, for example chloroform, followed by addition of the aqueous solution of said compound and formation of an emulsion; stirring of the mixture and evaporation of said solvent; addition of an aqueous solution, preferably a buffer, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);
f) subjecting said mixture to at least one of the following treatments: sonication, stirring, freezing/thawing cycles preferably in liquid nitrogen and water bath; extrusion through a porous membrane, centrifugation;
g) optionally, purifying the mixture obtained at the end of the step f) by a process comprising filtration, dialysis and sterilization;
h) optionally, obtaining from the mixture obtained at the end of step f) or of step g) a powder consisting of one or more of said entities and one or more of said coordination compounds.
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