Tuning a modular system – synthesis and characterisation of a boron-rich s -triazine-based carboxylic acid and amine bearing a galactopyranosyl moiety

Introduction of a bis(isopropylidene)-protected galactopyranosyl moiety in s -triazine-based boron-rich carboxylic acids and amines results in soluble and suitable coupling partners for tumour-selective biomolecules with applications as selective agents for boron neutron capture therapy (BNCT). Bearing either a carboxylic acid or primary amine as a functional group, these compounds are highly versatile and thus largely extend the possible coupling strategies with suitable biomolecules. Modi ﬁ cation of the gastrin-releasing peptide receptor (GRPR) selective agonist [ D -Phe 6 , β -Ala 11 , Ala 13 , Nle 14 ]Bn(6 – 14) with the carboxylic acid derivative yielded a bioconjugate with an optimal receptor activation and internalisation pro ﬁ le. This demonstrates the great potential of this approach for the development of novel boron delivery agents.


Introduction
Since the first report by Locher in 1936, boron neutron capture therapy (BNCT) has been developed as a very promising approach for cancer treatment. 1 It combines two non-toxic components to produce cytotoxic species, which are able to destroy malignant tissue. Boron-10 containing drugs bearing a tumour-selective moiety to address the infected site are highly advantageous. [2][3][4][5][6][7][8] Ideally, these bioconjugates accumulate selectively in the malignant tissues in a required amount of 10-30 µg g −1 tumour tissue and can then be irradiated with thermal or epithermal neutrons. 2,9 Depending on the biomolecule, the BNCT agent is either just accumulated in the tumour tissue or internalised into the cancer cells, which of course increases the efficacy of this treatment. 10,11,[12][13][14][15][16][17][18][19][20] The particles which are generated upon neutron capture are lithium and helium nuclei (α particles). 2,21,22 These particles with a high linear energy transfer (LET) have a mean free path of about 5 to 10 µm, and thus, a limited radius of destructive action. 2,5,6,21,22 The combination of suitable boron-rich molecules with tumour-selective biomolecules opens up a very selective tumour therapy which only affects malignant tissue and spares normal tissue. 10,12,[14][15][16][17]23 However, there are still some major challenges, including the selectivity of the chosen biomolecules for a specific type of tumour, the required high concentration of boron-10 in the cancer cell, the water solubility of the final bioconjugate, and the neutron beam quality, 24 which are also the focus of current research. 13,[17][18][19]25,26 Recently, we reported the synthesis of s-triazine-based boron-rich carboxylic acids. 27 Preliminary studies showed that the incorporation of more than one s-triazine-based bis(carboranyl) derivative into the breast tumour-selective peptide [F 7 ,P 34 ]-neuropeptide Y leads to a decrease or even total loss of the hY 1 receptor activation potency. It was assumed that this was caused by the strong hydrophobic character of the carborane clusters attached to the peptide producing a hydrophobic collapse of the bioconjugate. 28 Here, we describe the incorporation of an α-D-galactopyranosyl-substituted glycine derivative to reduce the hydrophobicity and improve the water solubility, which are important features for successful BNCT. [5][6][7]29 We also demonstrate the expansion of potential receptor targets for tumour addressing by utilising the recently developed gastrin-releasing peptide receptor (GRPR, BB2) selective peptide agonist [D-Phe 6 , β-Ala 11 , Ala 13 , Nle 14 ]Bn (6)(7)(8)(9)(10)(11)(12)(13)(14) (sBB2L). 30 The GRPR is commonly used as a drug shuttle system because of its frequent overexpression in breast and prostate cancer and its ability to repetitively internalise together with the peptide drug conjugate leading to an intracellular drug accumulation. 31 Furthermore, inclusion of either a carboxylic acid or an amine moiety as functional group further extends the scope for potential coupling partners (Fig. 1).
The only comparable compound using an s-triazine scaffold in combination with carboranes, monosaccharides and carboxylic acids was reported by Panza and co-workers. 32 In this case, ortho-carborane is used, which is a potential problem as this isomer is prone to undergo deboronation reactions if good nucleophiles associated with basic or even neutral conditions are present. 33 This may cause problems with bioavailability or side effects when employed in therapy. 17,34 Using meta-carborane assures the integrity of the carborane cluster.
Since the carboxylate could act as a nucleophile towards triflates (Scheme 1, step d) and the directly attached secondary amine at the s-triazine backbone is a weak nucleophile and undergoes no substitution reaction (Scheme 2), these observations led to a third approach in the synthesis of compound 5 (Scheme 3).
All isolated compounds were fully characterised by NMR and IR spectroscopy, mass spectrometry and melting point determination. Additionally, compounds 6, 11 and 12 were characterised by single crystal X-ray diffraction.
The final product 5 was characterised by high-resolution mass spectrometry and NMR spectroscopy. A very broad signal in the 1 H NMR spectrum at 6.34 ppm was assigned to a protonated tertiary amine indicating the presence of a zwitterion (NHR 3 + /CO 2 − ). In comparison, typical carboxylic acid protons have chemical shifts around 9 to 13 ppm. 39 The characterisation of 6 was very challenging, as the obtained spectroscopic data were not unambiguous for the proposed structure. However, colourless crystals suitable for X-ray diffraction were obtained from an acetone solution confirming the formation of (1′,2′:3′,4′-di-O-isopropylidene-6′deoxy-α-D-galactopyranos-6′-yl)[4,6-bis(1,7-dicarba-closo-dodecaboran-9-ylthio)-1,3,5-triazin-2-yl]glycinate (6). Two independent molecules of 6 are linked by hydrogen bonds between the hydrogen atom of the secondary amine group and one nitrogen atom of the s-triazine ring (Fig. 2). The observation of hydrogen bonds was already described for compounds of this substance class. 27 Compound 11 crystallised from chloroform solution with two molecules in the asymmetric unit (Fig. 3).
In the case of compound 12, mass spectrometry was a very informative characterisation method, as the characteristic isotopic pattern for molecules with 20 boron atoms was observed. 42,43 Single crystals of 12 could be obtained from n-hexane/ethyl acetate solution. The molecular structure is depicted in Fig. 4.
The carbamate 14 and the following products 15 and 16 were characterised by NMR and mass spectrometry confirming the successful synthesis. For compound 15, the mass spectrum was very characteristic, because the isotopic pattern clearly showed the replacement of two chloro substituents by two carborane clusters. 42,43 In summary, the two target molecules 5 and 16 were obtained in good to excellent yield. Due to their different functional groups (R-COOH in 5 and R-NH 2 in 16), coupling with a large variety of different biomolecules as tumour-selective carriers can be envisaged for application in BNCT. 4,8,12,15,17,20 Biological studies The galactopyranosyl derivative 5 was incorporated into the recently developed GRPR selective peptide [D-Phe 6 , β-Ala 11 , Ala 13 , Nle 14 ]Bn(6-14) (sBB2L) 30 to investigate, whether the ratio of one galactopyranosyl unit per two carborane clusters is sufficient to counterbalance the hydrophobicity of the carborane clusters yielding biologically active bioconjugates. The necessity for increased hydrophilicity was concluded from previous studies concerning the introduction of meta-carboranes as single clusters 17 or s-triazine-based derivatives bearing no monosaccharide group. 27 Three different peptide conjugates were synthesised (Fig. 5A) by a combination of automated and manual solid phase peptide synthesis (SPPS). 30,44 The s-tri-

Dalton Transactions Paper
This journal is © The Royal Society of Chemistry 2020 azine derivative 5 as well as the previously published derivative without any galactopyranosyl moiety (compound #4 in Kellert et al.,ref. 27) were coupled manually in three-fold molar excess to an N-terminally introduced three ethylene glycol-unit spacer (EG 3 ). The reaction was carried out overnight at room temperature in dimethylformamide (DMF) containing three equivalents 1-hydroxybenzotriazole (HOBt) and N,N′-diisopropylcarbodiimide (DIC), yielding conjugates 17 and 18. To enhance the boron loading per molecule a branching step was introduced using (2S)-2,3-diaminopropanoic acid (Dap) allowing the incorporation of two carborane building blocks 5 as deprotected moieties 5* (Fig. 5A). This strategy facilitated the generation of conjugate 19 bearing 40 boron atoms. Analyses of all three conjugates were performed with analytical reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionisation mass spectrometry (ESI-MS) as well as with MALDI-TOF-MS ( Fig. 5B and C). The exemplary RP-HPLC chromatogram of 18 displayed the main peak at 17.2 min retention time and a small shoulder on the right side. Since this shoulder had the same mass as the desired product and conjugate 17 did not exhibit any shoulders, the deoxygalactosyl moiety is suggested to cause this shoulder by mutarotation (Fig. 5B).
The three bioconjugates were tested in receptor activation and internalisation studies. For this purpose, the human GRPR was selected due to its remarkable overexpression on various tumour tissues like small cell lung, 45 breast 46 and prostate cancer. 47 The neuromedin B receptor (NMBR) and the bombesin receptor subtype 3 (BRS-3), which are also part of the bombesin receptor family are only occasionally expressed in these tumours. 48 The use of the recently published GRPR selective ligand (sBB2L) allows the specific targeting of cancer cells while the accumulation in healthy tissues is kept to a minimum. 30 Thus, the risk of side effects is reduced. Compared to the unmodified ligand sBB2L, which displayed an EC 50 value of 0.12 nM at the GRPR, conjugate 17 exhibited a ca. 20-fold reduced potency (EC 50 2.2 nM; Fig. 6A). This can be explained by the insufficient solubility of derivate #4 which was recently demonstrated by the incorporation into the even longer and more hydrophilic peptide [F 7 ,P 34 ]-NPY. 27 However, conjugate 18 bearing an additional deoxygalactopyranosyl moiety regained the activity at the GRPR and, with an EC 50 value of 0.17 nM, demonstrated wild type like potency. In addition, fluorescence microscopy studies revealed that 17 and 18 induce internalisation of the GRPR at a peptide concentration of 100 nM. After one hour of stimulation, the membrane bound receptor was completely translocated into intracellular vesicles, as was observed for the unmodified sBB2L (Fig. 6B).
Due to the improved receptor activation of conjugate 18, Dap was introduced to allow the incorporation of more than one carborane-based building block per peptide molecule. The resulting conjugate 19, bearing two deprotected molecules 5*  (40 boron atoms), displayed a strongly reduced potency at the GRPR and showed nearly no internalisation after 1 h of stimulation with 100 nM peptide. This indicates that building block 5, featuring a ratio of one deoxygalactopyranosyl unit per two carborane clusters, is not optimal for the generation of highly carborane-loaded peptide conjugates. Nevertheless, the incorporation of the deoxygalactopyranosyl unit improved the hydrophilicity, which allowed the synthesis of a double modified conjugate (19). This was previously not possible with building block #4 using standard peptide purification methods (data not shown). These observations demonstrate the necessity of hydrophilicity providing moieties to generate highly boron-loaded bioconjugates for tumour delivery. Therefore, conjugate 18 can be considered as a promising selective boron delivery agent in BNCT.

Conclusions
By modifying s-triazine derivatives with a galactopyranosyl moiety the modular system based on readily available building blocks like 9-mercapto-1,7-dicarba-closo-dodecaborane (1) and cyanuric chloride was further extended to compounds with reduced hydrophobicity. The final products, carboxylic acid 5 and amine 16, are highly suitable for the synthesis of bioconjugates, shown here exemplarily for 5 being introduced to the GRPR selective peptide [D-Phe 6 , β-Ala 11 , Ala 13 , Nle 14 ]Bn(6-14) (18 and 19). Receptor activation and internalisation studies revealed an improved performance in comparison to the conjugate without monosaccharide moiety (17). This study corroborates the advantageous influence of galactopyranosyl moieties for the development of highly carborane-loaded biomolecules.
For chromatography, silica gel (60 Å) with a particle diameter in the range of 0.035 to 0.070 mm, the Biotage® Isolera 1 or the Biotage® Isolera 4 automatic purification system with SNAP ( particle diameter: 0.040 to 0.065 mm) and SNAP Ultra (spherical particle, diameter: 0.025 mm) cartridges were used. The triazine and carborane species were detected by an integrated UV/Vis detector (Isolera 1, Biotage) or evaporative light scattering detector (ELSD) A-120 (Isolera 4, Biotage). For chromatography, solvents were distilled before use. NMR measurements were carried out on a Bruker AVANCE III HD spectrometer with an Ascend™ 400 magnet at room temperature. Tetramethylsilane was used as internal standard for 1 H and 13 C{ 1 H} NMR spectra, and 11 B and 11 B{ 1 H} NMR spectra were referenced to the Ξ scale. 49 NMR spectra were recorded at the following frequencies: 1 H: 400. 16 MHz, 13 C: 100.63 MHz, 11 B: 128.38 MHz; chemical shifts are reported in ppm.
Assignment of the 1 H and 13 C signals was based on 2D NMR spectra (H,H-COSY, HSQC, HMQC, HMBC). Identification of the boron atom attached to sulfur was possible by comparison of the proton-coupled and -decoupled 11 B NMR spectra. NMR data were interpreted with MestReNova. 50 NMR signals that appear as broad overlapping signals with the shape of a multiplet in either 1 H, 11 B{ 1 H} or 11 B NMR spectra are described as 'br' (broad). In this case, the superscript a is added (br a ). The numbering schemes of the entire assignments of all chemical shifts from selected synthesised compounds are given in the ESI. † IR data were obtained with a PerkinElmer FT-IR spectrometer Spectrum 2000 as KBr pellets and on a Thermo Scientific Nicolet iS5 with an ATR unit in the range from 4000 to 400 cm −1 . Electrospray ionisation mass spectrometry was performed with an ESI ESQUIRE 3000 PLUS spectrometer with an IonTrap analyser from Bruker Daltonics or on a MicroTOF spectrometer from Bruker Daltonics with a ToF analyser in negative or positive mode. Dichloromethane, acetonitrile, methanol or mixtures of these solvents were used for the measurements. Melting points were determined with a Gallenkamp MPD350·BM2.5 melting point device. Melting points are not corrected.

Cell culture
The generation and cultivation of stably transfected HEK293_GRPR-tGFP cells was described before. 30 Briefly, cells were cultivated in T75 cell culture flasks and were grown in DMEM/HAM's F12 (1 : 1, v/v) containing 15% FBS (v/v) and 1.0 mg mL −1 G-418 under standard conditions in an incubator (37°C, 5% CO 2 , 95% humidity). After cells reached full confluency, they were split in desired ratios from 1 : 2 to 1 : 12 into new cell culture flasks, filled with fresh medium for further cultivation or seeded into cell culture vessels for assays.

Live cell microscopy
Receptor internalisation was investigated by using stably transfected HEK293 cells, which were seeded into 8-well µ-slides (ibiTreat, ibidi, Martinsried, Germany) and incubated for two days at standard conditions. At the assay day, cells were starved for 30 min with 200 µL OptiMEM® under standard conditions and OptiMEM® was subsequently replaced by 200 µL OptiMEM® containing 10 −7 M peptide. Stimulation was carried out for 1 h and nuclei visualisation was achieved by addition of 1 µL Hoechst 33342 (Sigma-Aldrich, 0.5 mg mL −1 ) 30 min before image recording. Subsequently, cells were washed twice with OptiMEM® to remove non-internalised conjugates. Images were taken directly after washing while cells were maintained in OptiMEM®, using an Axio Observer·Z1 microscope equipped with an ApoTome Imaging System and a Heating Insert P Lab-Tek S1 unit (Zeiss, Oberkochen, Germany). Image processing was performed with AxioVision 3.1.

Conflicts of interest
There are no conflicts to declare.