Field of the Invention

The present invention concerns aminobenzylguanidine derivative ligands specifically targeting neuroblastoma cells, as well as their therapeutic and diagnostic use in neuroblastoma-related diseases.

Background of the Invention

In order to improve the selectivity of nanomedicines, specific targeting agents have been designed in recent years. These targeted nanosystems are able to transport the cytotoxic compounds specifically to the target cells and release their payload once there. This results in the destruction of the malignant cells while minimizing collateral damage in the surrounding tissue. For this reason, the development of new selective active targeting agents has become one of the main challenges in nanomedicine.

In the last few years, efficient ligand receptor active targeting motifs such as RGD and folate have been commonly grafted on the surfaces of many different kinds of nanosystems for vectorization purposes. Furthermore, new specific moieties with multiple functionalities, using several types of cell-penetrating peptides or vasculature targeting antibodies, have been reported recently.

The tumoral tissue accumulation characteristics of the nanoparticles are mainly possible due a specific passive targeting effect, which is based on the characteristic tumoral blood vessel architecture. These vessels present large pores and fenestrations with diameters of up to a few hundred nanometers. When the circulating nanoparticles reach a tumoral site, they pass through these pores and accumulate for longer periods within the site. Furthermore, tumoral tissue also exhibits poor lymphatic drainage. Both characteristics contribute to the well-known enhanced permeation and retention effect (EPR), which is one of the main advantages constituting the basis of the gold standard of the use of nanoparticles in oncology. However, not all of the cells that form a tumoral mass are malignant, due to the high heterogeneity of these tissues, which contain a large number of healthy supportive cells. The functionalization of the nanoparticle surface with molecules (targeting groups) that can be specifically recognized by tumoral cells constitutes the so-called active targeting. The presence of these groups is essential for enhancing the therapeutic efficacy of nanomedicines. Neuroblastoma (NB) is a type of cancer that forms in certain types of nerve tissue. It most frequently starts from one of the adrenal glands, but can also develop in the neck, chest, abdomen, or spine. Symptoms may include bone pain, a lump in the abdomen, neck, or chest, or a painless bluish lump under the skin. NB is one of the most challenging problems in pediatric oncology. This disease is the most frequent extracranial pediatric tumor, and it presents a dismal prognosis despite the combination of chemotherapy, radiotherapy, surgery and bone narrow transplants.

Thus, the development of nanocarriers that are able to detect and destroy only diseased cells would constitute a significant improvement in the efficacy of the treatment of neuroblastoma. There is a wide range of studies that analyze the vectorization effect of general cancer targeting agents in nanoparticles. Bulky targeting agents such as proteins, large peptides, aptamers, and antibodies are commonly used. However, these agents have several possible binding interaction points and can usually interact with unspecific proteins. Moreover, small molecules with fewer binding spots may go unnoticed in body fluids. This could increase the tolerance of these specific small moieties and preserve their selectivity toward the desired target. In special cases, some types of cells have intrinsic and particular receptors that make it possible to differentiate them more readily from each other.

Following this philosophy and seeking selective internalization in NB cells, meta- iodobenzylguanidine (MIBG) was developed as a suitable candidate for tuning the nanocarrier surface with vectorization purposes. The targeting properties of MIBG have been applied in the conventional medical diagnosis of NB in recent decades due to its affinity for the norepinephrine transporter (NET), which is expressed in 90% of NB tumors. Because of its high specificity for NB and other neuroendocrine tumors, the effectiveness of MIBG as a tumor-targeted vehicle has also been applied in therapeutic treatment by directed radiation using radioactive iodine ( ¹³¹ I). Moreover, a non- radioactive derivative has shown anticancer activity by itself. The conjugation of MIBG with antitumoral drugs not only enhances their selectivity against tumoral cells but also improves their cytotoxic capacity through a synergistic effect caused by tumor selective acidification due to the enhancement of glycolytic flux.

The possibility of performing structural modifications of MIBG in order to study and improve its uptake selectivity for the norepinephrine transporter (NET) has been widely discussed. Numerous variations have been incorporated in the para position of the aromatic ring, and the aromatic ring has even been replaced by a naphthyl or dibenzazepine moiety without a significant loss of selectivity for the NET receptor. However, it has also been suggested that the substitution of hydrogen in position 5 of the benzene ring is unfavourable for binding NET. On the other hand, the absence of the iodine atom in the meta-position does not affect

the uptake/selectivity properties.

Such structure-activity relationship studies (SAR) led to the synthesis of meta- aminobenzyl guanidine (MABG) as a MIBG analogue (G. Villaverde et al. _r A new targeting agent for the selective drug delivery of nanocarriers for treating neuroblastoma, Journal of Materials Chemistry B, 2015, 3, 4831-4842). MABG represents one of the simplest structurally accessible approximations to MIBG that maintains the same aromatic ring and the guanidine recognition moiety in the same position.

However, the need still exists for improved targeting of neuroblastoma tumors. The improved targeting agents should bind the receptors of the neuroblastoma cells with higher affinity and thus remain longer time in the neuroblastoma cells, which in turn will improve the usefulness of attaching a diagnostic or therapeutic agent to the targeting agent.

Summary of the Invention

Accordingly, in a first aspect, the present invention concerns a ligand compound having the formula :

wherein Xi is a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond;

X ₂ is independently a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond and, together with the benzylguanidine attached to it, is absent or present;

Ri is an optionally substituted chain containing stable linkages, such as amide bonds, thioamide bonds, carbamate bonds, thiocarbamate bonds, carbon-carbon bonds, ether bonds, sulfide bonds, and/or amine bonds, carbon, nitrogen and/or oxygen atoms in the chain, and having a chain length of 4 to 50, preferably 18 to 40 atoms, such as 21 to 35 atoms, e.g. 22 to 30 atoms;

Ri does not contain any conjugated double bonds; and

the benzylguanidine groups are in the meta and/or para position to Xi and X ₂ , respectively.

The ligand of the invention advantageously has a diagnostic agent or a therapeutic agent attached to it. Thus, in another aspect, the invention concerns a conjugate compound comprising the ligand compound according to the invention, wherein an Active is attached to Ri, optionally via a linker, the conjugate compound having the formula :

wherein

the linker may be absent or present and has a molecular weight in the range 50 to 10000 Da and is linked to Ri by a stable linkage, such as an amide bond, a carbamate bond, a carbon-carbon bond, an ether bond, or an amine bond; the Active is an active pharmaceutical ingredient, an immunostimulant, a contrast agent, a fluorophore, a nanoparticle loaded with an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore; and the Active is attached to the linker, if present, via a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond.

In a further aspect, the invention concerns the conjugate compound of the invention, wherein the Active is an active pharmaceutical ingredient, an immunostimulant, or a nanoparticle loaded with an active pharmaceutical ingredient or an immunostimulant for use in a method of treating neuroblastoma. In still a further aspect, the invention concerns the conjugate compound of the invention, wherein the Active is a contrast agent or a fluorophore, or a nanoparticle loaded with a contrast agent or a fluorophore for use in a method of diagnosis.

Brief Description of the Figures

Figures la to le show the structures of several ligand compounds according to the invention, as well as the structures of some comparative compounds, all of which have been tested for their cellular uptake in neuroblastoma cells.

Figure 2 shows the cellular uptake levels of the compounds shown in Figures la to le at various concentrations.

Detailed Description of the Invention

Definitions

Stable linkage

A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include but are not limited to the following : carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

Linkers

The linker linking the Active and the ligand-spacer molecule according to the present invention is not particularly limited and may be any linker known in the art useful for linking different parts of a conjugate in a biological environment. The length of the linker may vary and depends to some extent on the size of the Active to be linked to the ligand-spacer molecule. As an example, if the Active is a nanoparticle loaded with an active pharmaceutical ingredient, a contrast agent, and/or a fluorophore, the linker may be longer to create a suitable distance between the nanoparticle and the ligand- spacer molecule.

Compounds made of linear oligomers of ethylene glycol, so-called polyethylene glycol (PEG) linkers, enjoy particular popularity nowadays in biomolecular conjugation processes. PEG linkers are highly water soluble, non-toxic, non-antigenic, and lead to negligible or no aggregation. For this reason, a large variety of linear, bifunctional PEG linkers are commercially available from various sources, which can be selectively modified at either end with a (bio)molecule of interest. PEG linkers are the product of a polymerization process of ethylene oxide and are therefore typically obtained as stochastic mixtures of chain length, which can be partly resolved into PEG constructs with an average weight distribution centered around 1, 2, 4 kDa or more (up to 60 kDa). Homogeneous, discrete PEGs (dPEGs) are also known with molecular weights up to 4 kDa and branched versions thereof go up to 15 kDa. Several PEGylated proteins have been FDA-approved and are currently on the market.

By virtue of their polarity, PEG linkers are suitable for bioconjugation of small and/or water-soluble moieties under aqueous conditions.

Further linkers are known in the art, and disclosed in e.g. WO 2008/070291 , incorporated by reference. WO 2008/070291 discloses a linker for the coupling of targeting agents to anchoring components. The linker contains hydrophilic regions represented by polyethylene glycol (PEG) and an extension lacking chiral centers that is coupled to a targeting agent.

WO 01/88535, incorporated by reference, discloses a linker system for surfaces for bioconjugation, in particular a linker system having a novel hydrophilic spacer group. The hydrophilic atoms or groups for use in the linker system are selected from the group consisting of O, NH, C=0 (keto group), 0-C=0 (ester group) and CR ³ R ⁴ , wherein R ³ and R ⁴ are independently selected from the group consisting of H, OH, Ci-C ₄ alkoxy and Ci-C ₄ acyloxy.

WO 2014/100762, incorporated by reference, describes compounds with a hydrophilic self-immolative linker, which is cleavable under appropriate conditions and incorporates a hydrophilic group to provide better solubility of the compound. The compounds comprise a drug moiety, a targeting moiety capable of targeting a selected cell population, and a linker which contains an acyl unit, an optional spacer unit for providing distance between the drug moiety and the targeting moiety, a peptide linker which can be cleavable under appropriate conditions, a hydrophilic self-immolative linker, and an optional second self-immolative spacer or cyclization self-elimination linker. The hydrophilic self-immolative linker is e.g. a benzyloxycarbonyl group.

Immunostimulant

An "immunostimulant," or "immunostimulatory" molecule or domain or the like, herein refers to a molecule or domain, etc. which acts (or helps to act) to stimulate or elicit an immune response or immune action (either cellular or humoral or both) in a subject. Typical examples of such molecules include, but are not limited to, e.g., cytokines and chemokines. Cytokines act to, e.g., stimulate humoral and/or cellular immune responses. Typical examples of such include, e.g., interleukins such as IL-2, IL-12, etc. Chemokines act to, e.g., selectively attract various leukocytes to specific locations within a subject. They can induce both cell migration and cell activation. Common examples of chemokines include, e.g., RANTES, C-X-C family molecules, IL-8, mipla, Ghirΐb, etc. For further information, see, e.g., Arai, K. et al, 1990, "Cytokines: coordinators of immune and inflammatory responses" Annu Rev Biochem 59 :783 + ; Taub, 1996 "Chemokine-Leukocyte Interactions. The Voodoo That They Do So Well" Cytokine Growth Factor Rev 7: 355-76

Optionally substituted

In the present context, the term "optionally substituted" is intended to mean that the group in question may be substituted one or more times, such as 1-2 times with group(s) selected from hydroxy, Ci- ₆ -alkoxy, C _2-6 -alkenyloxy, carboxy, oxo, Ci _-6 - alkoxycarbonyl, Ci- ₆ -alkylcarbonyl, formyl, amino, mono- and di(Ci _-6 -alkyl)amino, carbamoyl, mono- and di(Ci _-6 -alkyl)aminocarbonyl, amino-Ci- ₆ -alkyl-aminocarbonyl, mono- and di(Ci- ₆ -alkyl)amino-Ci- ₆ -alkyl-aminocarbonyl, Ci- ₆ -alkylcarbonylamino, cyano, carbamido, nitro, Ci _-6 -alkylthio, benzylguanidino,

-C(0)-NH-benzylguanidino, -NH-C(0)-benzylguanidino, and halogen. Furthermore, the term "optionally substituted" may also mean that the group in question is unsubstituted. Chemotherapeutic agents

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin, a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Angew, Chem Inti. Ed. Engl., 33 : 183-186 (1994 )); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrol ino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin- engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid : pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

Ligand

In a first aspect, the present invention concerns a ligand compound having the formula : wherein

Xi is a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond;

X ₂ is independently a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond and, together with the benzylguanidine attached to it, is absent or present;

Ri is an optionally substituted chain containing stable linkages, such as amide bonds, thioamide bonds, carbamate bonds, thiocarbamate bonds, carbon-carbon bonds, ether bonds, sulfide bonds, and/or amine bonds, carbon, nitrogen and/or oxygen atoms in the chain, and having a chain length of 4 to 50, preferably 18 to 40 atoms, such as 21 to 35 atoms, e.g. 22 to 30 atoms;

Ri does not contain any conjugated double bonds; and the benzylguanidine groups are in the meta and/or para position to Xi and X ₂ , respectively.

It has been found that the ligands according to the present invention lead to a higher cellular uptake of a fluorophore attached to the ligand compared to compounds of the prior art, such as the MABG compounds. The Ri group serves as a spacer separating the benzylguanidine group attached to Xi from any moiety, such as a fluorophore or an active ingredient, that may be attached to Ri. In case X ₂ and the corresponding benzylguanidine group are present, the Ri spacer also serves to separate the two benzylguanidine groups. It has further been found that the Ri spacer provides the best cellular uptake when the spatial orientation of the bonds and atoms of Ri is flexible. Hence, in one embodiment, Ri does not contain any carbon-carbon double bonds. Ri should contain stable linkages so that it does not undergo hydrolysis under physiological conditions. Examples of stable linkages are amide bonds, thioamide bonds, carbamate bonds, thiocarbamate bonds, sulfide bonds and ether bonds. Thus, in one embodiment, Ri contains one or more stable linkages independently selected from the group consisting of amide bonds, thioamide bonds, carbamate bonds, thiocarbamate bonds, sulfide bonds and ether bonds. In a further embodiment, Ri contains one or more stable linkages independently selected from the group consisting of amide bonds and thioamide bonds.

It has also been found that alkanediyl moieties connected by stable linkages provide the desired flexibility. Hence, in one embodiment, Ri is a chain containing Ci to C alkanediyl moieties connected by amide bonds, thioamide bonds, carbamate bonds, thiocarbamate bonds, sulfide bonds and/or ether bonds. In a further embodiment, Ri is a chain containing Ci to C alkanediyl moieties connected by amide bonds and/or thioamide bonds, and wherein Ri is optionally substituted with amino groups, carboxylic acid groups, and/or thiocarboxylic acid groups. In still a further embodiment, Ri is a chain containing Ci to C ₄ alkanediyl moieties connected by amide bonds, and wherein Ri is optionally substituted with amino groups and/or carboxylic acid groups.

While it has been found that both ligands having a single benzylguanidine group and ligands having two benzylguanidine groups lead to improved cellular uptake compared to the prior art, it has also been found that ligands containing two benzylguanidine groups provide better cellular uptake than ligands containing a single benzylguanidine group. Accordingly, in one embodiment, X ₂ , together with the benzylguanidine attached to it, is present.

The guanidine groups may be positioned meta or para to Xi and/or X ₂ . In one embodiment, the guanidine groups are in the meta position to Xi and X ₂ . In another embodiment, the guanidine groups are in the para position to Xi and X ₂ . In yet another embodiment, one guanidine group is in the meta position and the other guanidine group is in the para position.

It has additionally been found that the chain length of the Ri spacer has an influence on the cellular uptake in neuroblastoma cells of the ligand according to the invention. Accordingly, Ri has a chain length of 3 to 50 atoms. In a preferred embodiment, Ri has a chain length of 18 to 40 atoms. In a further preferred embodiment, Ri has a chain length of 21 to 35 atoms. In a more preferred embodiment, Ri has a chain length of 22 to 30 atoms.

The ligand of the invention is useful for targeting various different agents, such as a diagnostic agent or a therapeutic agent, to neuroblastoma cells. Thus, in a further aspect, the invention concerns a conjugate compound comprising the ligand compound according to the invention, wherein an Active is attached to Ri, optionally via a linker, the conjugate compound having the formula:

Active— (linker)

wherein

the linker may be absent or present and has a molecular weight in the range 50 to 10000 Da and is linked to Ri by a stable linkage, such as an amide bond, a carbamate bond, a carbon-carbon bond, an ether bond, or an amine bond; the Active is an active pharmaceutical ingredient, an immunostimulant, a contrast agent, a fluorophore, a nanoparticle loaded with an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore; and the Active is attached to the linker, if present, via a stable linkage, such as an amide bond, a thioamide bond, a carbamate bond, a thiocarbamate bond, a carbon-carbon bond, an ether bond, a sulfide bond, or an amine bond. Ri, Xi, and X ₂ are as defined above.

Nanoparticles are known as possible carriers for active pharmaceutical ingredients that, due to their large internal surface area, are capable of holding a large amount of small molecules. Thus, in one embodiment, the Active is a nanoparticle loaded with an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore. Several different nanoparticles capable of holding small molecules are known in the art and any of these may be comprised in the conjugate compound of the invention. In one embodiment, the nanoparticle is a mesoporous silica nanoparticle, a polymeric nanoparticle, such as a biodegradable polymeric nanoparticle, a liposome, a metallic nanoparticle, or a viral nanoparticle. In a further embodiment, the nanoparticle is mesoporous silica nanoparticle.

The active pharmaceutical ingredient, immunostimulant, contrast agent, or fluorophore may also be attached to the conjugate without using a nanoparticle, optionally attaching it via a linker. Thus, in one embodiment, the Active is an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore.

Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Examples of chemotherapeutic agents useful in the treatment of neuroblastoma include cisplatin, carboplatin, cyclophosphamide, ifosfamide, melphalan, etoposide, doxorubicin, vincristine, topotecan, and irinotecan. Thus, in one embodiment, the Active is a chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, cyclophosphamide, ifosfamide, melphalan, etoposide, doxorubicin, vincristine, topotecan, and irinotecan.

The linker may be absent or present. It has been found that especially when the Active is a bulky component, such as a nanoparticle, it is advantageous if the linker is present. Furthermore, the length of the linker may be adjusted according to the bulkiness of the Active. Thus, in one embodiment, the linker is present. In a further embodiment, the linker is present and the Active is a nanoparticle loaded with an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore. The molecular weight of the linker is in the range 50 to 10000 Da. In one embodiment, the molecular weight of the linker is in the range 500 to 8000 Da, such as in the range 2000 to 4000 Da. Polyethylene glycol linkers are increasingly being used in conjugate compounds. Thus, in a further embodiment, the linker is polyethylene glycol. Conversely, when the Active is less bulky, the linker may be absent. Accordingly, in one embodiment, the linker is absent. In another embodiment, the linker is absent and the Active is an active pharmaceutical ingredient, an immunostimulant, a contrast agent, and/or a fluorophore. In this embodiment, the Active is therefore linked directly to Ri.

When the Active is an active pharmaceutical ingredient, an immunostimulant, or a nanoparticle loaded with an active pharmaceutical ingredient or an immunostimulant, the conjugate compound may be used in the treatment of neuroblastoma. Thus, in one embodiment, the invention concerns the conjugate compound, wherein the Active is an active pharmaceutical ingredient, an immunostimulant, or a nanoparticle loaded with an active pharmaceutical ingredient or an immunostimulant for use in a method of treating neuroblastoma.

The cellular uptake of the ligand of the invention may also be used for diagnostic purposes when attached to a marker molecule, such as a contrast agent or a fluorophore. Thus, in another embodiment, the invention concerns the conjugate compound, wherein the Active is a contrast agent or a fluorophore, or a nanoparticle loaded with a contrast agent or a fluorophore for use in a method of diagnosis.

Pharmaceutical formulations

The conjugate compounds of the present invention are intended for use as a medicament. The conjugate compounds of the invention may in principle be applied on their own, but they are preferably formulated with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is an inert carrier suitable for each administration method, and can be formulated into a conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.). As such a carrier there may be mentioned, for example, a binder, an excipient, a lubricant, a disintegrant and the like, which are pharmaceutically acceptable. When they are used as an injection solution or an infusion solution, they can be formulated by using distilled water for injection, physiological saline, an aqueous glucose solution.

The administration method of the conjugate compounds of the present invention is not particularly limited, and a usual oral or parenteral administration method (intravenous, intramuscular, subcutaneous, percutaneous, intranasal, transmucosal, enteral, etc.) can be applied. The dosage of the conjugate compounds or pharmaceutically acceptable salts thereof of the present invention may optionally be set in a range of an effective amount sufficient for showing a pharmacological effect, in accordance with the potency or characteristics of the compound to be used as an effective ingredient. The dosage may vary depending on administration method, age, body weight or conditions of a patient.

Preparation of ligand compounds

First step for affording the ligand compounds is the synthesis and modification of single targeting agents for further inclusion in double ligands. To this aim, benzyl guanidine analogues have been synthetized and functionalized with final amine or acid groups. Furthermore, other kinds of small molecules, such as triphenylphosphine (TPP), RGD, GABA or Choline, have also been synthetized or modified to be included in the ligand compounds as alternative vectorization agents able to interact with typical receptors of tumor cells.

To afford the ligand compounds' main structure, peptide solid phase synthesis was the chosen strategy. To this aim, classic approximation of Fmoc protection/coupling methodology has been applied using commercial Wang Resin and protected amino acids or small molecules as needed for each case. The protocol concerns the multi-step functionalization and coupling on the resin with the molecules needed in order to build the desired ligand structure. Finally, the coupling of the targeting agents has to be done before the release and purification steps.

The general methodology of each type of reactions in solid phase is described below:

I) Amino group Fmoc deprotection : A solution of piperidine (20%) in dimethylformamide (DMF) was added to the peptide functionalized resin. The suspension mixture was shaken in the solid phase reactor by wrist-shaker overnight. The obtained solid was filtered and washed with DMF.

II) Peptide amide bond formation : A solution of 1-Hydroxybenzotriazole hydrate (HOBt) (3 eq), N,N,N',N'-Tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU) (3 eq) and the corresponding amino acid (2 eq) in DMF (2 mL) was added to the washed resin, and after slight shaking N,N-Diisopropylethylamine (DIPEA) (6 eq) was finally added. The mixture was shaken in the reactor with a wrist- shaker overnight. The obtained solid was filtered and washed with DMF.

III) Final peptide release from the resin : Once the last amino acid of the chain was added, the solid was filtered, washed with DMF and dried under vacuum. A solution of trifluoroacetic acid (TFA) (95%), triisopropylsilane (TIPS) (2.5%) and water (2.5%) was added dropwise. The mixture was stirred in wrist-shaker for 4 hours, then filtered and washed with one milliliter of the same previous mixture. Both filtered solutions were then mixed.

The product was obtained in all cases by precipitation of the filtered solution with cold ether. The product was then added dropwise to cold ether. After one hour, the suspension was centrifuged at T = 4 °C; 14000 r.p.m. The obtained solid was dried overnight in a vacuum, at room temperature.

IV) Final peptide-like ligand isolation by chromatography: The obtained solid was dissolved in the minimum amount of water and purified by flash column for molecular exclusion chromatography (stationary phase: Sephadex® G-25; mobile phase: water). All phases were then frozen at -80 °C and lyophilized.

General methodology for fluorescent labeling for conjugates with linker.

For the fluorescent labeling of the ligand compounds, a polyethylene glycol (PEG) of approximately 3500 Da previously functionalized at the end of the chain with an amino and an acid group at the other end, was reacted with fluorescein isothiocyanate (FITC), forming a thiourea bond. In a second step, the already labeled PEG derivative binds with the ligand compound under study, through the formation of an amide bond between the amine of the ligand compound and the N-hydroxisuccinimide (NHS) ester of the fluorescein-labeled PEG. The general methodology follows the synthesis scheme below.

General methodology for grafting with linker on nanoparticle surface.

Mesoporous silica nanoparticles were synthesized, as a model nanocarrier, labeled with a fluorophore compatible with in vivo study by fluorescence, in this case the fluorophore Cy7, according to Villaverde et at. (G. Villaverde et at. , A new targeting agent for the selective drug delivery of nanocarriers for treating neuroblastoma, Journal of Materials C

To this aim, nanoparticles were functionalized with a primary amine group. On the other hand, ligand compounds were functionalized with b/s-NHS acid PEG chain in one end, in order to anchor the ligand to the particle surface by the formation of an amide bond between them.

Examples

Example 1 - (3-amino benzvIMN.N-bisftert-butoxycarbonvOauanidine

3-aminobenzylalcohol (1 g, 8.12 mmol) was dissolved in 10 ml. of tetrahydrofuran (THF) and stirred under inert atmosphere together with N,N-bis(tert- butyloxycarbonyl)guanidine (2.32 g, 8.93 mmol) and triphenylphosphine (TPP) (4.26 g, 16.24 mmol). Diisopropyl azodicarboxylate (DIAD) (3.20 ml_, 16.24 mmol) was added dropwise later. The resulting yellow mixture was stirred at room temperature overnight. The solvent was evaporated and the resulting crude was reconstituted in ethyl acetate. The organic phase was washed with water (3xl5ml_) and brine (2x20ml_). The organic phase was dried and the crude purified by silica column chromatography. Yield 60-70%. ^l H NMR (250 MHz, CDCH) d 9.41 and 9.35 (s, br, 2H, NH ₂ ), 7.18 - 7.04 (m, 1H, CH (Ar)), 6.64 - 6.52 (m, 3H, CH _(Ar) ), 5.09 (s, 2H, CH ₂ ), 3.62 (s, 2H, NH ₂ ), 1.48 (s, 9H, 3XCH ₃₍ BOC)), 1 -32 (s, 9H, 3XCH ₃₍ BOC)) . ¹³ C NMR (63 MHz, CDCI ₃ ) d 173.98 (C=0 _B oc), 173.93(C=0 _B O _C ), 155.42(C=N), 146.69(C _(Ar) ), 140.44(C _(Ar) ), 129.47 (CH _(Ar) ), 117.56 (CH _(Ar) ), 114.10 (CH _(Ar) ), 113.81 (CH _(Ar) ), 84.58 (2XC ₍ BOO), 48.11 (CH ₂ ), 28.70 (3XCH ₃₍ BO _C) ), 28.19 (3XCH _3(B O _C) ) . FTIR (cm ¹ ) : 3458; 3369; 3060; 2967; 2920; 1715; 1588; 1495; 1283; 1140; 1107; 977; 589. ESI(-) : 363m/z [M-l]

Example 2 - (4-amino benzvIMN.N-bisftert-butoxycarbonvnauanidine

A solution of 4-(aminomethyl)aniline (100 mg, 0.8 mmol) in anhydrous DMF (200 pl_) was treated with N, N-bis (tert-butyloxy-carbonyl) thiourea (249 mg, 0.9 mmol) and triethylamine (250 pL 1.8 mmol) in an inert atmosphere. Mukaiyama reagent (209 mg, 0.8 mmol) was dissolved in DMF (400 pl_) also in a nitrogen atmosphere and added over the previous mixture dropwise. The reaction was stirred at room temperature overnight. When the reaction came to an end, the mixture was partitioned with water and ethyl acetate. The organic phase was extracted and washed with brine (lx 5 ml_), dried and concentrated to dryness. The resulting crude was purified by silica column chromatography. Yield 60-70% .

¹ H NMR (250 MHz, CDCI3) d 11.52 (s, 1 H, NH, amide), 8.42 (s, broad, 1H, amide), 7.10 (d, J = 8.5 Hz, 2H, 2xCH (Ar)), 6.65 ( d, J = 8.5 Hz, 2H, 2xCH (Ar)), 4.48 (d, J = 4.9 Hz, 2H, CH ₂ ), 3.67 (s, 2H, NH ₂ ), 1.52 (s, 9H, 3xCH ₃ (BOC)), 1.46 (s, 9H, 3xCH ₃ (BOC)) . ¹³ C NMR (63 MHz, CDCI ₃ ) d 164.07 (C = O), 156.24 (C = O), 153.53 (C = N), 146.36 (NH ₂ -CAr), 129.72 (2xCHAr), 127.35 (CH ₂ -CAr), 115.67 (2xCHAr), 83.44 (C (BOC)), 79.75 (C (BOC)), 45.25 (CH2), 28.73 (3xCH ₃ (BOC)), 28.47 (3xCH ₃ (BOC)) . ESI (+) : 365.1 m / z [M + 1]

Example 3 - (Amino-halobenzylMN2. N3-bisitert-butoxycarbonyl ^' ) ^' )quanidines

(4-amino-3-iodobezyl) - (N2, N3-bis (tert-butoxycarbonyl) guanidine. (SI)

Iodination

As an example, the iodine compounds have been synthesized following this methodology: to a solution of 4-aminobenzonitrile (500 mg, 4.23 mmol) in methanol (50 ml_), I ₂ was slowly added (644.53 mg, 2.54 mmol) . The mixture was stirred for 30 minutes at room temperature and then half a milliliter of H ₂ 0 ₂ in 5 ml. of THF were slowly added over 20 minutes. The reaction was allowed to stir for three days at room temperature, until the total conversion of the starting product was confirmed by TLC. The reaction was terminated by the addition of an aqueous solution of Na ₂ S ₂ 0 ₃ and this mixture was left stirring for 20 minutes, and then extracted with dichloromethane. The organic phase was washed with brine and then dried in MgS0 ₄ and concentrated to dryness. The residue was purified by silica column chromatography. The resulting yellow solid was washed with heptane to a fine powder. Yield 250 mg, 24%.

4-amine-3-iodobenzonitrile

^ NMR (250 MHz, CDCI ₃ ) d 7.90 (d, J = 1.8 Hz, 1H, CH _ar ), 7.40 (dd, J = 8.4, 1.9 Hz, 1H, CH _ar ), 6.70 (d, J = 8.4 Hz, 1H, CH _ar ), 4.64 (s, 2H, NH ₂ ) .

¹³ C NMR (63 MHz, CDCI ₃ ) d 150.63 (C _ar -NH ₂ ), 142.93 (C _ar -H), 133.42 (C _ar -H), 113.64 (C _ar -H), 112.23 (CN), 101.89 (C _ar -CN), 81.99 (C _ar -I) .

Reduction

To a solution of 4-amino-3-iodobenzonitrile in THF (4 ml.) was added BH ₃ .THF (1 M solution in THF 2.5 ml.) drop-wise. The reaction mixture was refluxed for 3 h and 2 N HCI (1.0 ml.) was added . The resulting mixture was refluxed for an additional 1 h and then concentrated in vacuo in order to afford the crude, which was re-crystallized in MeOH for characterization to give 150 mg (74 %) of yellow powder.

4-amino-3-iodobenzylammonium chloride

^ NMR (250 MHz, DMF) d 8.90 (s, broad, 3H, ⁺ NH ₃ ), 7.88 (d, J = 2.0 Hz, 1H, CH _ar ), 7.42 (dd, J = 8.3, 2.1 Hz, 1H, CH _ar ), 6.93 (d, J = 8.3 Hz, 1H, CH _ar ), 5.81 (s, 6H, HCI), 4.06 (q, J = 5.6 Hz, 2H, CH ₂ ).

¹³ C NMR (63 MHz, DMF) d 151.64 (C _ar - ⁺ NH ₃ ), 149.11(C _ar ), 140.44 (CH _ar ), 131.17 (CH _ar ), 124.75 (C _ar ), 114.86 (CH _ar ), 82.93(C _ar -I), 42.30 (CH ₂ ).

Mitsunobu reaction for benzyl guanidine formation

To a mixture of the corresponding benzyldiamine hydrochloride (70 mg) and triethylamine (6 Eq) in DMF was added l,3-bis-(tert-butyloxycarbonyl)-2-methyl-2- thiopseudourea (77 mg, 1.5 Eq), and the mixture was stirred at room temperature overnight . The mixture was added in water and extracted by ethyl acetate; the combined organic layer was dried in NaS0 ₄ and concentrated . The crude mixture was chromatographed using 4: 1 Heptane/ethyl acetate to afford the corresponding product with yields around 50%. f4-amino-3-iodobenzylMN2.N3-bis(tert-butoxycarbonyl ^' )auanidine ^' )

NMR (250 MHz, CDCI ₃ ) d 11.51 (s, 1H, NH), 8.43 (s, 1H, NH), 7.59 (d, J = 2.0 Hz, 1H, CH _ar ), 7.10 (dd, J = 8.2, 2.0 Hz, 1H, CH _ar ), 6.71 (d, J = 8.2 Hz, 1H, CH _ar ), 4.44 (d, J = 5.1 Hz, 2H, CH ₂ ), 4.10 (s, 2H, NH ₂ ), 1.52(s, 9H, 3XCH _3(BO Q), 1-47 (s, 9H, 3XCH ₃₍ BOQ) . ¹³ C NMR (63 MHz, CDCI ₃ ) d 164.00 (C=0), 156.30 (N = C), 153.50 (C=0), 146.75(C _ar -NH ₂ ), 139.20 (C _ar -H) , 129.72 (C _ar) , 129.05 (C _ar -H), 115.09 (C _ar -H), 84.35(2XC ₍ BOO), 83.60 (C _ar -I), 79.83(C _(BOC) ), 44.28 (CH ₂ ), 28.72 (3xCH _3(BOC) ), 28.48 (3XCH _3(BOC) ) . ESI(+) : 491.1 m/z [M + l] f3-amino-4-chlorobenzylMN2.N3-bis(tert-butoxycarbonyl ^' )quanidine)

NMR (250 MHz, CDCI ₃ ) d 11.52 (s, 1H), 8.53 (s, 1H), 7.19 (d, J = 8.1 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.62 (dd, J = 8.1, 2.0 Hz, 1H), 4.50 (d, J = 5.2 Hz, 2H), 4.07 (s, 2H), 1.51 (s, 9H), 1.47 (s, 9H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 163.7, 156.2, 153.3, 143.2, 137.2, 129.8, 118.6, 118.5, 115.2, 83.38, 79.6, 44.6, 28.4 (3C), 28.2 (3C). MS (ESI+) Calc for C _I8 H ₂ 8 ³⁵ CIN ₄ 04 (M + H) ⁺ m/z = 399.1

f4-amino-3-chlorobenzylMN2.N3-bis(tert-butoxycarbonyl ^' )auanidine)

^ NMR (250 MHz, CDCI ₃ ) d 8.38 (s, 1H), 7.14 (d, J = 1.9 Hz, 1H), 6.94 (dd, J = 8.2, 1.9 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 4.40 (d, J = 5.1 Hz, 2H), 3.98 (s, 2H), 1.45 (s, 9H), 1.40 (s, 9H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 163.7, 156.0, 153.3, 142.6, 129.4, 128.0, 127.7, 119.3, 116.1, 83.3, 79.5, 44.3, 28.4 (3C), 28.2 (3C). MS (ESI+) Calc for C _I8 H ₂₈ ³⁵ CIN ₄ 0 ₄ (M + H) ⁺ m/z = 399.18

Example 4 - Synthesis of analogues with terminal acid group

A solution of succinic anhydride (1.1 Equivalent) in toluene (10 ml.) was treated with a solution of the corresponding analogue (100 mg) also in toluene. The resulting mixture was stirred at room temperature overnight. When the reaction is complete (followed by thin layer chromatography, TLC), the resulting powder was suspended in the mixture. The solid was filtered and washed with water (30 mL). The resulting powder was dried under reduced pressure at 40 ° C. The process was quantitative. 4-((3-((l,2-bis(tert-butoxycarbonyl)guanidino)methyl)phenyl) amino)-4-oxobutanoic acid.

^ NMR (250 MHz, CDCI ₃ ) d 9.38 and 9.08 (s, br, 2H, NH ₂ ), 8.09 (s, 1H, NH, amide), 7.42 (d, J = 7.8 Hz, 1H, CH _(Ar) ), 7.16 - 7.08 (m, 2H, CH _(Ar) ), 6.80 (d, J = 7.5 Hz, 1H, CH _(Ar) ), 5.03 (s, 2H, CH ₂ ), 2.67 - 2.51 (m, 4H, 2xCH ₂ , succinic), 1.40 (s, 9H, 3XCH3(BOC)), 1.25 (S, 9H, 3XCH _3(B OQ) ·

¹³ C NMR (63 MHz, CDCI ₃ ) d 175.85 (C=0), 175.56 (C=0), 170.56 (C=0), 154.95 (C=N), 143.16 (C=0), 139.56 (C _Ar ), 137.99 (C _Ar ), 128.99 (CH _Ar ), 122.30 (CH _Ar ), 118.60 (CH _AT ), 117.75 (CH _Ar ), 84.75 (2XC _(BOQ ), 47.62 (CH ₂ ), 31.81 (CH _2, succinico), 29.51 (CH ₂ succinic), 28.38 (3XCH _3(BOQ ), 27.89 (3XCH _3(BOQ ) ·

FTIR (cm ¹ ) : 3372; 3329; 2977; 2927; 1721; 1625; 1625; 1608; 1286; 1246; 1137; 1120; 981; 944; 615; 504.

ESI(-) : 462.9 m/z [M-l]

4-((4-((l,2-bis(tert-butoxycarbonyl)guanidino)methyl)phen yl)amino)-4-oxobutanoic acid

^ NMR (250 MHz, CDCI ₃ ) d 11.56 (s, 1H, NH, amide), 8.62 (t, J = 4.1 Hz, 1H, NH, amide), 8.01 (s, 1H, NH, amide), 7.42 (d, J = 8.5 Hz, 2H, 2xCH _(Ar) ), 7.17 (d, J = 8.0 Hz, 2H, 2xCH _(Ar) ), 4.54 (d, J = 4.7 Hz, 2H, CH ₂ ), 2.80 - 2.70 (m, 2H, CH ₂ , succinic), 2.69 - 2.59 (m, 2H, CH ₂ , succinic), 1.49 (s, 9H, 3XCH ₃₍ BOC))), 1.48 (s, 9H, 3COH ₃₍ BOO)) . ¹³ C NMR (63 MHz, CDCI ₃ ) d 170.56 (C=0), 169.63 (C=0), 163.52 (C=N), 156.38 (C=0), 153.27 (C=0), 137.18 (C _Ar ), 132.59 (C _Ar ), 128.20(2xCH _Ar ), 120.26 (2xCH _Ar ), 83.55 (C _(B oq), 79.84 56.50 (CH ₂ , guanidine), 31.94(CH ₂ ), 29.74(CH ₂ ), 28.37 (3XCH ₃ (BOC)) , 28.18. (3XCH ₃ (BOC)) ·

ESI(-) : 463.1 m/z [M-l] 4-((4-((l 2-bis(tert-butoxycarbonyl)guanidino)methyl)-2-iodophenyl)ami no)-4- oxobutanoic acid

^ NMR (250 MHz, CDCI ₃ ) d 11.43 (s, 1H, NH), 8.51 (t, br, J = 4.7 Hz, 1H, NH), 8.05 (d, J = 8.3 Hz, 1H, CH _ar ), 7.67 (d, J = 1.7 Hz, 1H, CH _ar ), 7.54 (s, 1H, NH), 7.24 (dd, J = 1.9 Hz, 1H, CH _ar ), 4.46 (d, J = 5.3 Hz, 2H, CH ₂ ), 2.73 (dd, J = 8.6, 4.6 Hz, 4H, 2XCH ₂ , succinic), 1.44 (s, 9H, 3xCH _3(B oc ₎ ), 1.41 (s, 9H, 3xCH _3(BOC) )·

¹³ C NMR (63 MHz, CDCI ₃ ) d 176.18 (C=0, succinic), 169.83(C=0, succinic), 164.21 (C=0), 156.19 (N=C), 153.17 (C=0), 138.36 (C _ar -H) , 138.22(C _ar -NH), 135.42 (C _ar) , 129.07 (C _ar -H), 122.04 (C _ar -H), 83.13 (2xC _(B0C) ), 83.0 (C _ar -I), 77.26 (C _(B0C) ), 43.42 (CH ₂ ), 31.34 (CH ₂ , succinic), 28.99 (CH ₂ , succinic), 28.02 (3xCH _3(BO c ₎ ), 27.89 (3XCH ₃ ( _B OC)) ·

ESI(-) : 589.1 m/z [M-l]

Example 5 - Synthesis of analogues with terminal amine group

To a solution of the protected aminobenzylguanidine according to Example 1 or according to Example 2 (50 mg) in DMF (2 ml_), diisopropylethylamine (100 pL) was added. 2,5-dioxopyrrolidin-l-yl (tert-butoxycarbonyl) glycinate (50 mg) dissolved in 1 rriL of DMF was added dropwise, over the reaction, which was allowed to react at 80 ° C overnight. The reaction ended with the addition of lOmL water and lOmL ethyl acetate, the organic phase was washed with NaHC0 ₃ (5%) 3x10 ml_, NaHS0 ₄ (20%) 3x10 ml_, and brine 3x10 mL, dried with anhydrous sodium sulfate and concentrated to dryness.

The crude was purified by column chromatography on silica gel. Then, on a solution of the corresponding aminobenzylguanidine analogue in dichloromethane (2mL), 2 mL of trifluoroacetic acid (TFA) were slowly added at room temperature over inert atmosphere. The resulting mixture was heated at 65 ° C for 48 hours and once the reaction was completed the mixture was concentrated to dryness. The resulting crude was washed with dichloromethane and dried in a vacuum oven.

It was found that preparing the ligands starting from an analogue with a terminal amine group was advantageous from a synthetic perspective compared to starting with a terminal acid group since it avoids the formation of a succinic amide byproduct:

2 (tert-Butoxycarbonyl) amino-N- (3- ( (1 ,2-bis (tert- butoxycarbonyl) guanidino) methyl -phenyl) acetamide

^ NMR (250 MHz, CDCI ₃ ) d 9.49 y 9.37 (s, br, 2H, NH ₂ ), 8.13 (s, 1H, NH), 7.46 (d, J = 7.7 Hz, 1H, CH _ar ), 7.26 - 7.18 (m, 2H, 2xCH _ar ), 6.94 (d, J = 7.8 Hz, 1H, CH _ar ), 5.24 (s, 1H, NH), 5.14 (s, 2H, CH ₂ ), 3.91 (d, J = 5.9 Hz, 2H, CH ₂ , glycine), 1.48 (s, 9H, 3XCH _3(BOC) ), 1-47 (s, 9H, 3XCH _3(BOC) ), 1-31 (s, 9H, 3XCH _3(BOC) ) ·

¹³ C NMR (63 MHz, CDCI ₃ ) d 167.97 (C=0), 164.09 (C=0, BOC), 161.33 (C=0, BOC), 155.24 (C=N), 151.70 (C=0, BOC), 140.29 (C _ar) , 137.90 (C _ar -NH), 129.31 (CH _ar) , 122.97(CH _ar) , 118.74 (CH _ar) , 118.22 (CH _ar ), 84.70 (C _(B oc ₎ ), 79.48 ( BOQ), 77.64 (C ₍ BOQ), 47.81 (CH ₂ ), 32.30 (CH ₂ , glycine), 28.71 (6xCH _3(BO c ₎ ), 28.19 (3xCH _3(BO c ₎ ).

2-Amino-N-(3-(guanidinomethyl)phenyl) acetamide

^ NMR (250 MHz, MeOD) d 7.87 (s, 1H, NH), 7.62 (s, 1H, CH _ar ), 7.45 (d, J = 7.8 Hz, 1H, CH _ar ), 7.32 (t, J = 7.9 Hz, 1H, CH _ar ), 7.06 (d, J = 7.6 Hz, 1H, CH _ar ), 4.36 (s, 2H, CH ₂ ), 3.82 (s, 2H CH ₂ , glycine).

¹³ C NMR (63 MHz, MeOD) d 178.91 (C=0), 158.51 (C=N), 148.72 (C _ar ), 147.98 (C _ar ), 130.33 (CH _ar ), 123.95 (CH _ar ), 120.03 (CH _ar ), 119.31 (CH _ar ), 45.51 (CH ₂ ), 41.84 (CH ₂ , glycine).

2-( tert-Butoxycarbonyl )amino-N-( 4-( ( l,2-bis( tert-butoxycarbonyl)guanidino )methyl phenyl) acetamide ^ NMR (250 MHz, CDCI ₃ ) d 12.50 (s, 1H, NH, amide), 8.37 (s, 1H, NH, amide), 8.09 (s, 1H, NH, amide), 7.50 (d, J = 8.4 Hz, 2xCH _ar ), 7.31 (d, J = 5.7 Hz, 2H, 2xCH _ar ), 5.28 - 5.12 (m, 1H, NH, amide), 4.54 (d, J = 5.7 Hz, 2H, CH ₂ ), 3.95 (d, J = 6.2 Hz, 2H, CH ₂ , glicine), 1.51 (s, 9H, 3XCH _3(BOQ ), 1 -48 (s, 9H, 3xCH _3(B oc ₎ ), 1 -28 (s, 9H, 3xCH _3(BOC) )· ethyl)phenyl) acetamide

d 7.68 (d, J = 8.6 Hz, 2H, 2xCH _ar ), 7.40 (d, J = 8.2 Hz, 2H, 2xCH _ar ), 4.51 (s, 1H, CH ₂ ), 3.83 (s, 1H, CH ₂ , glicine).

Example 6 - Synthesis of DR3 and DR4 precursor

To a solution of 25 g of the commercial 5-hydroxyisophthalic acid in lOOmL of MeOH, H ₂ SO ₄ was added in catalytic amount. The mixture was then refluxed for 24 hours to complete the esterification. After the reaction, which was monitored by TLC, it was poured into 250 ml_ of water, producing a precipitate. After filtration of the solid and drying, the corresponding dimethyl-6-hydroxyisophthalate was obtained with a 95% yield that was used without further purification . ( Tetrahedron Lett., 2007, 5899)

The transformation of the alcohol to triflate was carried out in an inert atmosphere starting from 840mg (4 mmol) of the diester, which was dissolved in 15 ml_ of anhydrous CH2CI2 and at a temperature of 0 °C. Then, and following this order, 0.4 ml_ (5 mmol, 1.2 equiv) of anhydrous pyridine and 4.4 ml_ of a 1.0 M solution of (CF ₃ SC>2)20 in CH2CI2 were added to the reaction . The reaction was maintained under stirring for 24 h. After that, the following general processing method was applied : dilution with lOOmL of ethyl acetate (AcOEt) washing with 2x50mL of a solution of NH4CI saturated in water, washing with 50mL of brine, drying over MgS0 ₄ , filtration and evaporation in a rotary evaporator. Finally, the crude obtained was purified by S1O2 column chromatography using Heptane/AcOEt (2 : 1) as a mobile phase, obtaining 1.14g (83%) of dimethyl 5 (trifluoromethane sulfonyloxy) isophthalate.

^X H NMR (250 MHz, CDCI3) d 8.71 (t, J = 1.4 Hz, 1H), 8.11 (d, J = 1.4 Hz, 2H), 3.99 (s, 6H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 164.5 (2C), 149.4, 133.2 (2C), 130.5, 126.5 (2C), 118.8 (q, J = 320.8 Hz) 53.1 (2C) .

Starting from 667 mg (1.95 mmol) of the triflate, the Sonogashira reaction was carried out. For the specific case of the reaction with the commercial N-Boc-propargylamine, 455 mg (2.93mmol, 1.5equiv) of the alkyne, 133mg of the catalyst Pd(PPh ₃ ) ₂ Cl2 (0.19mmol, O. lequiv), 36mg of Cul (0.19 mmol, O. lequiv) and 430 pL of NEt ₃ (3.12mmol, 1.6equiv) as base in 2mL of CH ₃ CN were used, under inert atmosphere at room temperature for 16h. Once the reaction was finished and after applying the general processing method, the crude obtained was purified by Si0 ₂ flash chromatography using Heptane/AcOEt (7: 3) as the mobile phase, obtaining the corresponding product. dimethyl 5-(3-(N-(tert-butoxyicarbonyl)amino)propyn-lyl)isophthalate

NMR (250 MHz, CDCI ₃ ) d 8.56 (t, J = 1.6 Hz, 1H), 8.21 (d, J = 1.6 Hz, 2H), 4.90 (bs N H, 1H), 4.15 (d, J = 5.5 Hz, 2H), 3.92 (s, 6H), 1.45 (s, 9H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 165.6 (2C), 155.4, 136.7 (2C), 131.0 (2C), 130.3, 123.9, 87.7, 81.2, 80.3, 52.6 (2C), 31.2, 28.5 (3C). MS (ESI+) Calc for: Ci ₈ H ₂₂ N0 ₆ (M + H) ⁺ : 348.14, found : 348.1

Hydrolysis of the ester groups was carried out starting from 1.44 mmol dissolved in 25 mL of methanol (MeOH), to which 29 mmol (20 equiv) of KOH was added, the reaction was maintained for 6h at room temperature. After the reaction was complete, 50mL of H ₂ 0 was added and the solution was brought to pH = 5 with HCI. Then, after applying the general processing method, the different isophthalic acids (96-99%) were obtained, which were used for the next step without further purification.

5-(3-(A/-(teft-butoxvcarbonvlamino)propvn-l-vl)isophthi alic acid

For the coupling reaction with bis-(tert-butoxycarbonyl)guanidinomethylanilines, 0.16 mmol of the isophthalic diacid, 0.35 mmol (2.2 equiv) of the corresponding protected aminobenzylmethylguanidine, 164mg (2.4 equiv) of (l-Cyano)-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) were used as coupling reagent and 83 pL of DIPEA (0.48 mmol, 3 equiv) as base. The reaction was carried out in anhydrous DMF, starting at 0 °C followed by heating at 50 °C for 18 h. Once the reaction was finished and after applying the general processing method, the crude obtained was purified by Si0 ₂ column chromatography using Heptane/AcOEt (1 : 1) as mobile phase, obtaining the different compounds provided as mixtures of isomers. (Yield : 64-79%)

1.3-Bis-fN3-f3-(Nl,N2-(Bis-tert-butoxycarbonyl ^' )ouan id inomethyQ phenyl l-S-fS-fN-tert- butoxycarbonyl-S-aminofpropyn-l-vn isophthalamide

^ NMR (250 MHz, CDCI ₃ ) d 9.65-9.38 (m, 4H), 9.06 (bs, 2H), 8.34 (bs, 1H), 8.01 (bs, 2H), 7.60 (d, J = 7.9 Hz, 2H), 7.47 (bs, 2H), 7.17 (t, J = 7.9 Hz, 2H), 6.83 (t, J = 8.7 Hz, 2H), 5.58 (bs, 1H), 5.18 - 4.88 (m, 4H), 4.16 (d, J = 4.9 Hz, 2H), 1.46 (s, 9H), 1.40 (s, 18H), 1.32 (s, 18H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 164.8, 163.9, 161.5, 155.3, 139.7, 138.7, 135.6, 133.8, 129.2, 122.6, 119.3, 118.1, 84.8, 79.9, 72.7, 70.7, 28.6, 28.2, 22.1. MS(TOF/TOF) Calc for: C52H69N9O12 (M + Na ⁺ ) : 1084.5114, found : 1084.687

1.3-Bϊ5-GN3-G4-(N1.N2-(Bϊ5-ί6Gί-duίocno3GdohnPou3h id inomethyll phenyl l-S-fS-fN-tert- butoxycarbonyl-S-aminofpropyn-l-vn isophthalamide

^ NMR (250 MHz, CDCI ₃ ) d 11.50 (s, 2H), 9.00 (bs, 1H), 8.58 (bt, J = 5.2 Hz, 2H), 8.18 (bs, 1H), 7.87 (s, 2H), 7.52 (d, J = 8.4 Hz, 4H), 7.06 (d, J = 8.4 Hz, 4H), 5.37 (bs, 1H), 4.45 (d, J = 4.8 Hz, 4H), 4.24 - 4.13 (m, 1H), 4.01 (d, J = 5.3 Hz, 2H), 1.42 (s, 18H), 1.39 (s, 18H), 1.38 (s, 9H). ¹³ C NMR (63 MHz, CDCI ₃ ) d 164.6, 163.5, 156.4, 153.3, 137.5, 135.1, 133.7, 133.4, 127.9, 123.8, 120.8, 83.5, 79.9, 28.5, 28.3, 28.2. MS(TOF/TOF) Calc for: C52H69N9O12 (M + Na ⁺ ) : 1084.5114, found : 1084.642

For the deprotection of the protective groups (Boc) of this family of compounds, 50 mg of the protected compound was dissolved in a mixture composed of CF ₃ COOH, (iPr) ₃ SiH and CH2CI2 (0.5 : 1 : 3ml_) maintaining the stirring for 12h. After that time, the reaction mixture was evaporated in a rotary evaporator, the obtained residue was triturated with Et ₂ 0 and the precipitate was filtered under vacuum. The solid obtained was used for the next step without further purification.

1.3-Bis-(Y3-auanidinomethv0phenyn-5-(3-aminopropyn-l-yl ^' ) isophthalamide

NMR (250 MHz, MeOD) d 8.56 (t, J = 1.7 Hz, 1H), 8.26 (d, J = 1.7 Hz, 2H), 7.83 (d, J = 1.6 Hz, 2H), 7.66 (dd, J = 8.2, 1.0 Hz, 2H), 7.43 (td, J = 7.9, 3.1 Hz, 2H), 7.18 (bd, J = 7.5 Hz, 2H), 4.47 (s, 4H), 4.12 (d, J = 2.3 Hz, 2H). ¹³ C NMR (63 MHz, MeOD) d 166.8, 158.9, 158.8, 140.2, 138.6, 137.2, 134.9, 130.4, 128.4, 124.5, 123.9, 121.6, 120.9, 85.9, 83.5, 73.3, 71.1, 45.8. MS(TOF/TOF) Calc for: C27H29N9O2 (M + H ⁺ ) : 512.2517, found : 512.453 1.3-Bis- uanidinomethv0phenyn-5-(3-aminopropyn-l-yl ^' ) isophthalamide

NMR (250 MHz, MeOD) d 8.56 (t, J = 1.5 Hz, 1H), 8.25 (d, J = 1.6 Hz, 2H), 7.79 (d, J = 8.5 Hz, 4H), 7.39 (d, J = 8.6 Hz, 4H), 4.43 (s, 4H), 4.13 (s, 2H) . ¹³ C NMR (63 MHz, MeOD) d 166.8, 158.7, 139.4, 137.3, 134.9, 134.0, 129.0, 128.3, 123.9, 122.4, 85.9, 83.5, 45.5. MS(TOF/TOF) Calc for: C ₂₇ H ₂₉ N ₉ O ₂ (M + H ⁺ ) : 512.2517, found : 512.425

Example 8 - Synthesis of DF3C and DF4C precursor

Starting from 1.5 g of the commercial amino acid Fmoc-Glu (OtBu) OH the deprotection of the tert-butyl group was carried out, dissolving it in a mixture of CF ₃ COOH, ('Pr) ₃ SiH and CH ₂ CI ₂ (3.0 : 1.5 : 7.0ml_) maintaining the stirring for 12 h . After that time, the reaction mixture was evaporated in a rotary evaporator, the obtained residue was triturated with Et ₂ 0 and the precipitate was filtered under vacuum. The solid obtained was used for the next step without further purification. ( Tetrahedron Lett., 1992, 5441- 4).

N-(9H-Fluoren-9-yl-methoxycarbonylMI-)-cilutamic acid

NMR (250 MHz, DMSO) d 12.37 (bs, 2H), 7.89 (d, J = 7.3 Hz, 2H), 7.73 (d, J = 7.0 Hz, 2H), 7.42 (t, J = 7.0 Hz, 2H), 7.33 (dt, J = 7.3, 3.7 Hz, 2H), 4.34 - 4.15 (m, 3H), 3.99 (td, J = 9.4, 4.7 Hz, 1H), 3.45 (bs, NH), 2.32 (t, J = 7.5 Hz, 2H), 1.99 (td, J = 12.7, 7.6 Hz, 1H), 1.88 - 1.68 (m, 1H).

Example 9 - Synthesis of DF3C and DF4C

For the coupling reaction with Bis-(tert-butoxycarbonyl)guanidinomethylanilines, 0.16 mmol of the Fmoc-protected glutamic acid, 0.35 mmol (2.2 equiv) of the corresponding protected aminobenzylmethylguanidine, 164 mg (2.4 equiv) of the coupling reagent (1- Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholi no-carbenium hexafluorophosphate (COMU) and 83 pL of DIPEA (0.48 mmol, 3 equiv) as base were mixed. The reaction was carried out in anhydrous DMF, starting at 0 °C followed by heating at 50 °C for 18 h. Once the reaction was finished and after applying the general processing method, the crude obtained was purified by Si0 ₂ column chromatography using Heptane/ AcOEt (1 : 1) as mobile phase, obtaining the different expected compounds.

For the deprotection of the Boc protective groups of this family of compounds, 50 mg of the protected compound were dissolved in a mixture composed of CF ₃ COOH, (iPr) ₃ SiH and CH2CI2 (0.5 : 1 : 3mL) maintaining the stirring for 12h. After that time, the reaction mixture was evaporated in a rotary evaporator, the obtained residue was triturated with Et ₂ 0 and the precipitate was filtered under vacuum. The solid obtained was used for the next step without further purification.

For the deprotection of the Fmoc protecting group in this series, the Boc-deprotected compound was deprotected in a mixture composed of 20% piperidine in DMF at room temperature for 2 h. After the partial evaporation of the solvent and applying the general processing method, the crude obtained was purified by Sephadex G-25 size exclusion column chromatography, using H ₂ 0 as eluent. Then, after lyophilization, the different completely unprotected compounds provided were obtained.

In the case of RGD, GABA and Choline amine-derivative moieties have been incorporated in the ligand compound following the same procedure employed in the case of aminobenzylguanidine analogues (ABG) analogues in order to afford the hetero ligand compounds. In the case of TPP, an amine-TPP derivative (see scheme below) has been incorporated in the ligand compound using the same procedure employed in the case of aminobenzylguanidine analogues (ABG).

iSl-2-amino-Nl.N5-bis(3-fauanidinomethylphenvnpentanodiamide . (isomer mixture! NMR (250 MHz, MeOD) d 7.73-7.62 (m, 1H), 7.57 - 7.29 (m, 4H), 7.23-7.17 (m, 1H), 7.08 (t, J = 7.0 Hz, 1H), 4.49-4.36 (m, 4H), 2.72-1.82 (m, 4H), 1.80 - 1.12 (m,

5H). ¹³ C NMR (63 MHz, MeOD) d 181.6, 176.9, 173.8, 163.4, 162.9, 158.8, 140.5, 140.1, 139.5, 138.5, 137.5, 131.0, 130.4, 129.6, 129.0, 128.3, 124.2, 123.8, 120.6, 120.0, 115.9, 59.0, 58.3, 45.9, 45.4, 34.2, 30.5, 28.7, 26.6, 24.5. 2-amino-Nl.N5-bis (4-(quanidinomethylphenyl ^' )pentanediamide. (isomer mixture ^' )

NMR (250 MHz, MeOD) d 7.55-7.42 (m, 2H), 7.40 - 7.29 (m, 2H), 7.23-7.06 (m, 4H), 4.43-4.19 (m, 4H), 2.71-1.72 (m, 4H), 1.71 - 1.11 (m, 5H). ¹³ C NMR (63 MHz, MeOD) d 176.9, 173.7, 163.4, 162.8, 158.8, 158.6, 139.8, 138.4, 136.6, 133.1, 130.7, 129.3, 128.9, 128.9, 121.5, 120.5, 58.9, 58.3, 46.0, 45.6, 45.4, 34.2, 28.6, 26.6, 24.3, 23.5.

Example 10 - Synthesis of S3-Chol, S4-Chol, S3-GABA, S4-GABA, S3-TPP and S3-RGD

Synthesis of heterogeneous flexible analogues were carried out following the general method of solid phase Fmoc/coupling strategy. 7.27 (m, 3H), 7.15 (d, J = 7.1 Hz, 1H), 4.40 (s, 4H), 4.34-4.21 (m, 2H), 3.20-3.04 (m, 2H), 3.00-2.81 (m, 4H),2.68 (s, 9H), 1.90 - 1.53 (m, 12H), 1.49 - 1.24 (m, 6H). MS(TOF/TOF) Calc for: [C ₃₃ H ₅₇ Nio0 ₇ ] ⁺ r (M ⁺ ) : 705.441, found : 705.460

S4-Chol

^ NMR (250 MHz, D ₂ 0) d 7.50 - 7.15 (m, 4H), 4.41 - 4.17 (m, 3H), 4.11 (bs, 1H), 3.68-3.54 (m, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.17 - 3.02 (m, 10H), 3.02 - 2.77 (m, 5H), 2.65 (bs, 4H), 2.52 (b, 4H), 1.94-1.51 (m, 8H), 1.49-1.21 (m, 6H) . MS(TOF/TOF) Calc for: [C ₃₃ H ₅₇ Nio0 ₇ ] ⁺ r (M ⁺ ) : 705.4406, found : 705.494

Asp coupled with water signal, 4.32 (s, 2H, CH ₂ , benzylguanidine), 4.10 - 3.99 (m, 2H, 2xCH , Lys), 3.75 (s, 1H, CH, Arg), 3.66 - 3.50 (m, 2H, CH _2, Gly), 3.49 - 3.40 (m, 1H, CH, Cys), 3.17 - 3.00 (m, 2H, CH ₂ , Arg), 2.95 - 2.73 (m, 6H, 2xCH ₂ , Cys, Asp, ), 2.60 (s, 4H, 2XCH ₂ succinic), 1.79 - 1.37 (m, 10H, 3xCH ₂ , Lys, 2xCH ₂ , Arg ), 1.34 - 0.98 (m, 6H, 3XCH ₂ , Lys). TOF/TOF z/m; 865 [M ⁺ -Fmoc-NH-C ₂ H ₃ COOH aspartic] (100%); 976 [M ⁺ -Asp+TFA] (51%); 1041 [M ⁺ -SH] (22%). Example 11 - Synthesis of DFL3 and DFL4.

Synthesis of DFL3 and DFL4 analogues were carried out following the general method of solid phase Fmoc/coupling strategy.

DFL3

^X H NMR (250 MHz, D ₂ 0) d ^X H NMR (250 MHz, D ₂ 0) d 7.41 - 7.11 (m, 6H), 7.04 (d, J = 7.3 Hz, 2H), 4.35 - 4.13 (m, 6H), 2.78 (t, J = 7.5 Hz, 4H), 2.59 (d, J = 4.4 Hz, 8H), 1.86 - 1.63 (m, 4H), 1.53 (dt, J = 15.0, 5.6 Hz, 6H), 1.40 - 1.21 (m, 2H). TOF/TOF m/z: 767 [M ⁺ + l] (100%).

DFL4

^X H NMR (250 MHz, D ₂ 0) d 7.40 (d, J = 8.5 Hz, 4H), 7.31 (d, J = 8.4 Hz, 4H), 4.37 (s, 4H), 4.25 (d, J = 4.7 Hz, 2H), 2.90 (d, J = 7.7 Hz, 4H), 2.67 (d, J = 5.4 Hz, 8H), 1.81

(d, J = 4.7 Hz, 6H), 1.61 (s, 4H), 1.39 (d, J = 7.3 Hz, 2H) . TOF/TOF m/z : 767 [M ⁺ +l]

(100%).

Example 12 - Preparation of ligands conjugated with fluorescein or nanoparticle containing fluorescein/Cv7

Step 1 - Conjugation with fluorescein

A solution of 0-(2-Aminoethyl)-0'-(2-carboxyethyl) polyethyleneglycol hydrochloride (3500 Da) (50 mg in 2 ml. of DMF) was treated with 18 pl_ of diisopropylethylamine. The mixture was stirred at room temperature for one hour in a nitrogen atmosphere. After that time, a solution of fluorescein isothiocyanate (65 mg in 1 ml_ of DMF) was added to the mixture slowly. The reaction mixture was allowed to stir overnight at room temperature.

The final product (F-PEG-COOH) was obtained by precipitation in cold ether and subsequent purification by molecular exclusion column (G-25 Sedaphex in water)

NMR (250 MHz, D ₂ 0) d 7.71-7.50 (m, 2H, CH _Ar , Fluorescein), 7.31 - 7.05 (m, 3H, CH _Ar , Fluorescein), 6.57 (m, 4H, CH _Ar , Fluorescein), 3.82 (s, broad, 2H, CH _2, PEG), 3.79 - 3.36 (s, broad, 248 H, CH ₂ , PEG), 3.29 (s, broad, 2H, CH _2, PEG-CH ₂ -COOH).TOF/TOF m/z: 3859 (100%).

Step 2 - Acid activation and ligand compound coupling

An equivalent of F-PEG-COOH obtained in the previous step was dissolved in 2 ml. of DMF together with 1.5 equivalents of N-hydroxysuccinimide (NHS). The mixture in an inert atmosphere was stirred together with 6 equivalents of diisopropylcarbodiimide (DIC) and 6 equivalents of diisopropylethylamine (DIPEA) for 4 hours to prepare the succinimide-activated acid.

The corresponding ligand compound with the available amino group (1.5 equivalents) is dissolved in 1 ml_ of DMF and activated by adding 6 equivalents of DIPEA in an inert atmosphere. The solution containing the analogue is added slowly to that containing the activated acid, the final mixture is allowed to stir at room temperature overnight. The final product was obtained by precipitation in cold ether and subsequent purification by molecular exclusion column (G-25 Sedaphex in water).

TOF/TOF m/z; 4551 [M]

Example 13 - in vitro cellular uptake

The in vitro cellular uptake was evaluated in neuroblastoma cells by attaching a fluorophore (Active = fluorescein) according to Example 12 to the ligand as prepared according to Examples 1 to 11. Bidimensional cell cultures of neuroblastoma cells (NB 1691-luc) were incubated in the presence of decreasing concentrations of the ligand compound of the invention (from 50 pg mL ¹ to 6.25 pg mL ¹ ). The cellular uptake was measured by flow cytometry observing the percentage of cells showing fluorescence at the wavelength of fluorescein (A _ex = 492 nm and A _em = 520 nm), as well as the intensity of median fluorescence per cell .

The tested conjugate compounds are shown in Figures la to le, and the cellular uptake results are summarized in Figure 2.

An increased cellular uptake in neuroblastoma cells was observed for both meta (DFC3, DF3, DLF3, DR3) and para (DFC4, DF4, DFL4, DR4) conjugates compared to the prior art compound S3 according to Villaverde et al. (G. Villaverde et al., A new targeting agent For the selective drug delivery of nanocarriers for treating neuroblastoma, Journal of Materials Chemistry B, 2015, 3, 4831-4842) . At 50 pg mL ¹ , the cellular uptake for most of the conjugates according to the invention was so high that they could not be distinguished . Therefore, some of the measurements were repeated at lower conjugate concentrations (25 pg mL ¹ , 12.5 pg mL ¹ , and 6.25 pg mL ¹ ).

The results demonstrate that the cellular uptake is generally increased when the Ri spacer is flexible rather than rigid, when the conjugate contains two benzylguanidine groups, and when the Ri spacer is longer.

Example 14 - in vivo cellular uptake

Neuroblastoma cells (cell line NB1691-luc) were implanted subcutaneously into the flanks of immunodeficient NSG mice (NOD/SCID gamma mice), originally obtained from Jackson Laboratories (Bar Harbor, ME). The recommendations of FELASA (Federation of Laboratory Animal Science Associations) and the Spanish and European laws and rules of animal experimentation were followed.

Three weeks after the inoculation, the presence of subcutaneous tumors was demonstrated by bioluminescence, as well as their degree of development. The mice were distributed homogenously in four groups (n=5), each group receiving the different preparations of the study (intravenous injection of 1 mg/mouse in 0.2 ml saline).

The preparations contained mesoporous nanoparticles labeled with Cy7 fluorophore (Aex = 750 / Aem = 773 nm). The distribution of particles and the quantification of the signal in the area of the tumor were observed through fluorescent image acquisition in an IVIS Lumina XRMS instrument (Perkin Elmer) at 24, 48 and 72 h after injection of the nanoparticles. The presence and localization of NB-Lucderived tumors was confirmed through the detection of a luminescent signal after i.p. injection of 1.25 mg per mice D-luciferin (Perkin Elmer). Image analysis was performed with Living Images 4.4 software (Perkin Elmer). Animal studies were in accordance with the guidelines of the EU on animal care (2010/63/EU) and approved by an institutional ethics committee (FELASA) of Hospital Universitario Nino de Jesus de Madrid. The animals were sacrificed 72 hours after the infusion.

The fluorescence results of the nanoparticles are summarized in Figure 3. The fluorescence level (relative to the control) of each group of treated mice is shown at 24, 48 and 72 hours.