Synthesis of Novel Carborane-Containing Derivatives of RGD Peptide

Short peptides containing the Arg-Gly-Asp (RGD) fragment can selectively bind to integrins on the surface of tumor cells and are attractive transport molecules for the targeted delivery of therapeutic and diagnostic agents to tumors (for example, glioblastoma). We have demonstrated the possibility of obtaining the N- and C-protected RGD peptide containing 3-amino-closo-carborane and a glutaric acid residue as a linker fragment. The resulting carboranyl derivatives of the protected RGD peptide are of interest as starting compounds in the synthesis of unprotected or selectively protected peptides, as well as building blocks for preparation of boron-containing derivatives of the RGD peptide of a more complex structure.


Introduction
The search for efficient pharmaceuticals for the diagnostics and treatment of tumor diseases is one of the most urgent problems of medicinal chemistry. Currently, molecular vectors-namely, short peptides, antibodies, aptamers, and other compounds that provide targeted delivery of the functional part of the molecule-are widely used in the constructs of targeted therapy agents. The mechanism of their selective accumulation is based on the interaction of the vector with a target molecule, typically a receptor protein located on the surface of tumor cells.
Today, the RGD peptide (L-arginyl-glycyl-L-aspartic acid, Arg-Gly-Asp) and structurally similar peptides ( Figure 1) are widely used as molecular vectors in the drug design of targeted agents for the diagnostics and therapy of tumor diseases [1][2][3][4][5][6]. The RGD amino acid sequence has a tropism for cell adhesion proteins, integrins, which are particularly overexpressed in tumor cells (namely, αvβ 3 and αvβ 5 integrins). Integrin inhibitors represent an important class of agents for the treatment of tumors, macular degeneration, acute coronary syndrome, and other diseases [7,8]. Among the derivatives and analogs of the RGD peptide, a number of integrin inhibitors have been found [9,10]. Cilengitide, a selective inhibitor of αvβ 3 and αvβ 5 integrins proposed for the treatment of recurrent glioblastoma [11,12], has not passed phase III clinical trials because of insufficient pharmacokinetic parameters [13]. At the same time, studies of a number of other integrin inhibitors related to the RGD peptide are currently ongoing [14][15][16][17].
One of the emerging approaches to tumor treatment is boron neutron capture therapy (BNCT). This method is based on the ability of the 10 B isotope to interact with thermal neutrons with the emission of 4 He and 7 Li nuclei, which locally damage cells containing boron compounds [40][41][42]. A crucial condition for the application of BNCT is the selective accumulation of boron-containing molecules by tumor cells. The design of low-toxic boron-containing tumor-targeting compounds is an urgent task of modern medicinal chemistry [43][44][45][46]. An important group of potential boron delivery agents are derivatives of 1,2dicarba-closo-dodecaborane (carborane), the molecule of which contains ten boron atoms and can be modified using various functional groups. Certain properties of carboranes such as stability under physiological conditions and low toxicity make them unique pharmacophores for the design of new biomimetics [47][48][49]. Carborane conjugates with natural amino acids and peptides are of particular interest from the point of view of drug design of BNCT agents, as well as theranostic agents [50]. In particular, carborane-containing derivatives of the c(RGDfK) peptide have been used for adhesion of cells expressing the αvβ3 integrin receptors [51], as well as for boron delivery to tumor cells [52,53]. The boroncontaining conjugate of the cyclic RGD peptide was able to selectively accumulate in murine SCCVII carcinoma cells but was highly toxic [53]. Boron-containing nanoparticles containing FITC-labeled RGD-K peptide residues [54] or internalizing RGD fragments [55,56] were selectively accumulated by ALTC1S1 glioma, GL261 glioma, and A549 adenocarcinoma cells. Modification of the sodium dodecaborate-loaded liposomes by c(RGDfK) [57,58] and c(RGDyC) [59] peptides made it possible to achieve their binding to human umbilical cord endothelial cells. The fact that RGD-functionalized closo-dodecaborate albumin conjugates are capable of accumulating in U87 MG xenografts has recently demonstrated the efficacy of BNCT in in vivo experiments [60]. The c(RGDfK) peptidebased theranostic agent containing both a dodecaborane residue and 67 Ga and 125 I isotope labels was highly stable and capable of accumulating in U87 MG glioblastoma cells [61].
Recently, we have demonstrated the possibility of obtaining carborane-containing derivatives and analogs of natural amino acids as a result of modifications of protected amino acids using classical methods of peptide chemistry (formation of an amide bond, selective introduction and removal of N-and C-protecting groups) [62][63][64][65][66][67]. One of the emerging approaches to tumor treatment is boron neutron capture therapy (BNCT). This method is based on the ability of the 10 B isotope to interact with thermal neutrons with the emission of 4 He and 7 Li nuclei, which locally damage cells containing boron compounds [40][41][42]. A crucial condition for the application of BNCT is the selective accumulation of boron-containing molecules by tumor cells. The design of low-toxic boron-containing tumor-targeting compounds is an urgent task of modern medicinal chemistry [43][44][45][46]. An important group of potential boron delivery agents are derivatives of 1,2-dicarba-closo-dodecaborane (carborane), the molecule of which contains ten boron atoms and can be modified using various functional groups. Certain properties of carboranes such as stability under physiological conditions and low toxicity make them unique pharmacophores for the design of new biomimetics [47][48][49]. Carborane conjugates with natural amino acids and peptides are of particular interest from the point of view of drug design of BNCT agents, as well as theranostic agents [50]. In particular, carborane-containing derivatives of the c(RGDfK) peptide have been used for adhesion of cells expressing the αvβ 3 integrin receptors [51], as well as for boron delivery to tumor cells [52,53]. The boroncontaining conjugate of the cyclic RGD peptide was able to selectively accumulate in murine SCCVII carcinoma cells but was highly toxic [53]. Boron-containing nanoparticles containing FITC-labeled RGD-K peptide residues [54] or internalizing RGD fragments [55,56] were selectively accumulated by ALTC1S1 glioma, GL261 glioma, and A549 adenocarcinoma cells. Modification of the sodium dodecaborate-loaded liposomes by c(RGDfK) [57,58] and c(RGDyC) [59] peptides made it possible to achieve their binding to human umbilical cord endothelial cells. The fact that RGD-functionalized closo-dodecaborate albumin conjugates are capable of accumulating in U87 MG xenografts has recently demonstrated the efficacy of BNCT in in vivo experiments [60]. The c(RGDfK) peptide-based theranostic agent containing both a dodecaborane residue and 67 Ga and 125 I isotope labels was highly stable and capable of accumulating in U87 MG glioblastoma cells [61].
Recently, we have demonstrated the possibility of obtaining carborane-containing derivatives and analogs of natural amino acids as a result of modifications of protected amino acids using classical methods of peptide chemistry (formation of an amide bond, selective introduction and removal of Nand C-protecting groups) [62][63][64][65][66][67].
The purpose of this work was to synthesize new Nand C-protected derivatives of the RGD peptide containing a closo-carborane residue linked to the arginine α-amino group via a short linker (compounds 1a-c, Scheme 1). We used a glutaric acid residue as a linker, which makes it possible to obtain conjugates of the RGD peptide with readily available 3-amino-ortho-carborane with a high boron content. The choice of protecting groups was due to the possibility of either selective deblocking of the guanidino group in the arginine residue and carboxyl groups in the aspartate residue (compound 2a), or removal of all protecting groups in one step (compounds 2b,c).
the RGD peptide containing a closo-carborane residue linked to the arginine α-amin group via a short linker (compounds 1a-c, Scheme 1). We used a glutaric acid residue a linker, which makes it possible to obtain conjugates of the RGD peptide with readi available 3-amino-ortho-carborane with a high boron content. The choice of protectin groups was due to the possibility of either selective deblocking of the guanidino group the arginine residue and carboxyl groups in the aspartate residue (compound 2a), or r moval of all protecting groups in one step (compounds 2b,c). Scheme 1. Synthetic routes to protected closo-carboranyl RGD peptide derivatives 1a-c.

Results and Discussion
We have carried out a comparative study of three synthetic routes for closo-carboran derivatives of the RGD peptide involving the use of different protecting groups.
The synthesis of peptides 1a,b was carried out starting from dimethyl and di-ter butyl esters 2a and 2b, which we had previously obtained, containing a 2,2,4,6,7-pentam thyldihydrobenzofuran-5-sulfonyl (Pbf) group in the arginine side chain and a glutar fragment at the arginine α-amino group [68][69][70]. The protecting groups of compounds 1 and 2a can be removed selectively: ester groups by alkaline hydrolysis; and the Pbf grou by the action of an acid, for example, TFA. Removal of the three protecting groups in com pounds 1b and 2b can be carried out in one step, by acid treatment.
To obtain conjugate 1c, it was necessary to synthesize a glutaryl derivative 2c of th protected RGD peptide containing a nitro group in the guanidine fragment and two be zyl ester groups, which can be simultaneously removed by hydrogenolysis. The synthes of derivatives of the RGD peptide containing benzyl aspartate and a nitro group protec ing the side chain of arginine has been described in the literature; however, informatio on the physicochemical characteristics of intermediate compounds is fragmentary [71][72][73][74][75][76] We synthesized glutaryl derivative 2c starting from dibenzyl (S)-aspartate ( (Scheme 2). Coupling of amino ester 3 to N-Boc-glycine using N,N'-dicyclohexylca bodiimide (DCC) as a coupling agent in the presence of N-hydroxysuccinimide (HOS and subsequent treatment of protected dipeptide 4 with hydrochloric acid in methan led to amino ester 5 in moderate yield after chromatographic purification. Coupling compound 5 to N α -Boc-N ω -nitro-(S)-arginine in the presence of TBTU gave the protecte tripeptide 6. Removal of the Boc group of compound 6 under acidic conditions an Scheme 1. Synthetic routes to protected closo-carboranyl RGD peptide derivatives 1a-c.

Results and Discussion
We have carried out a comparative study of three synthetic routes for closo-carboranyl derivatives of the RGD peptide involving the use of different protecting groups.
The synthesis of peptides 1a,b was carried out starting from dimethyl and ditert-butyl esters 2a and 2b, which we had previously obtained, containing a 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group in the arginine side chain and a glutaryl fragment at the arginine α-amino group [68][69][70]. The protecting groups of compounds 1a and 2a can be removed selectively: ester groups by alkaline hydrolysis; and the Pbf group by the action of an acid, for example, TFA. Removal of the three protecting groups in compounds 1b and 2b can be carried out in one step, by acid treatment.
To obtain conjugate 1c, it was necessary to synthesize a glutaryl derivative 2c of the protected RGD peptide containing a nitro group in the guanidine fragment and two benzyl ester groups, which can be simultaneously removed by hydrogenolysis. The synthesis of derivatives of the RGD peptide containing benzyl aspartate and a nitro group protecting the side chain of arginine has been described in the literature; however, information on the physicochemical characteristics of intermediate compounds is fragmentary [71][72][73][74][75][76].
We synthesized glutaryl derivative 2c starting from dibenzyl (S)-aspartate (3) (Scheme 2). Coupling of amino ester 3 to N-Boc-glycine using N,N -dicyclohexylcarbodiimide (DCC) as a coupling agent in the presence of N-hydroxysuccinimide (HOSu) and subsequent treatment of protected dipeptide 4 with hydrochloric acid in methanol led to amino ester 5 in moderate yield after chromatographic purification. Coupling of compound 5 to N α -Boc-N ω -nitro-(S)arginine in the presence of TBTU gave the protected tripeptide 6. Removal of the Boc group of compound 6 under acidic conditions and subsequent treatment of tripeptide 7 with glutaric anhydride gave compound 2c containing a free carboxyl group.
At each stage of the synthesis of glutaryl tripeptide 2c, the formation of side products was observed, so in order to obtain pure compounds 2c, 4-7, it was necessary to perform chromatographic purification. It is known that peptides containing an aspartic acid residue, including those in the RGD fragment, are prone to degradation, isomerization, and epimerization [77][78][79][80][81]. In our case, the total yield of compound 2c (Scheme 2) was only 9.2% relative to the starting amino ester 3. At the same time, the total yields of peptides 2a and 2b obtained from dimethyl and di-tert-butyl (S)-aspartates were about 20% [69]. At each stage of the synthesis of glutaryl tripeptide 2c, the formation of side products was observed, so in order to obtain pure compounds 2c, 4-7, it was necessary to perform chromatographic purification. It is known that peptides containing an aspartic acid residue, including those in the RGD fragment, are prone to degradation, isomerization, and epimerization [77][78][79][80][81]. In our case, the total yield of compound 2c (Scheme 2) was only 9.2% relative to the starting amino ester 3. At the same time, the total yields of peptides 2a and 2b obtained from dimethyl and di-tert-butyl (S)-aspartates were about 20% [69].
Coupling of compounds 2a-c to 3-amino-ortho-carborane (8) by the mixed anhydride method in the presence of ethyl chloroformate led to protected carboranyl peptides 1a-c in moderate yields (Scheme 3). Attempts to implement an alternative approach consisting in the acylation of amine 8 with glutaric anhydride followed by coupling to peptide 7 failed because of the low nucleophilicity of 3-aminocarborane. Conjugates 1a-c are colorless crystalline compounds that are stable during storage. Their 1 H NMR spectra contain characteristic signals of the 3-aminocarborane protons: singlets at δ 8.21-8.25 ppm (amino group) and δ 5.05-5.06 ppm (two CH groups in the cluster) as well as wide multiplets at δ 1.1-2.6 ppm (9 BH groups). The ratio of the integral At each stage of the synthesis of glutaryl tripeptide 2c, the formation of side products was observed, so in order to obtain pure compounds 2c, 4-7, it was necessary to perform chromatographic purification. It is known that peptides containing an aspartic acid residue, including those in the RGD fragment, are prone to degradation, isomerization, and epimerization [77][78][79][80][81]. In our case, the total yield of compound 2c (Scheme 2) was only 9.2% relative to the starting amino ester 3. At the same time, the total yields of peptides 2a and 2b obtained from dimethyl and di-tert-butyl (S)-aspartates were about 20% [69].
Coupling of compounds 2a-c to 3-amino-ortho-carborane (8) by the mixed anhydride method in the presence of ethyl chloroformate led to protected carboranyl peptides 1a-c in moderate yields (Scheme 3). Attempts to implement an alternative approach consisting in the acylation of amine 8 with glutaric anhydride followed by coupling to peptide 7 failed because of the low nucleophilicity of 3-aminocarborane. Conjugates 1a-c are colorless crystalline compounds that are stable during storage. Their 1 H NMR spectra contain characteristic signals of the 3-aminocarborane protons: singlets at δ 8.21-8.25 ppm (amino group) and δ 5.05-5.06 ppm (two CH groups in the cluster) as well as wide multiplets at δ 1.1-2.6 ppm (9 BH groups). The ratio of the integral Conjugates 1a-c are colorless crystalline compounds that are stable during storage. Their 1 H NMR spectra contain characteristic signals of the 3-aminocarborane protons: singlets at δ 8.21-8.25 ppm (amino group) and δ 5.05-5.06 ppm (two CH groups in the cluster) as well as wide multiplets at δ 1.1-2.6 ppm (9 BH groups). The ratio of the integral intensities of the signals of boron atoms in the 11 B NMR spectra of peptides 1a-c is 4:1:2:3 and corresponds to the symmetrical structure of 3-substituted closo-carborane.
To remove protecting groups in compounds 1a-c, rather mild conditions are usually suitable, in which, as a rule, cleavage of peptide bonds or degradation of the closo-carborane residue do not occur. Thus, these derivatives can be considered as convenient starting compounds for further modifications.

Conclusions
Thus, we synthesized several protected derivatives of the RGD peptide containing 3-amino-closo-carborane and glutaryl residue as a linker. The structural motif of the RGD peptide can be considered as a basis for the synthesis of potential boron delivery agents for BNCT; at the same time, the preparation of compounds of this group requires careful selection of reaction conditions. The derivatives obtained by us differ in the structure of the protecting groups; their removal can be carried out both in one stage (by hydrogenolysis or acidic treatment) and separately. This opens up prospects for further modification of the peptide fragment and the synthesis of carborane-containing peptides of a more complex structure.