Hybrid Molecules of Benzylguanidine and the Alkylating Group of Melphalan: Synthesis and Effects on Neuroblastoma Cells

The therapy of neuroblastoma relies, amongst other things, on administering chemotherapeutics and radioactive compounds, e.g., the (meta-iodobenzyl)guanidine [131I]mIBG. For special applications (conditioning before stem cell transplantation), busulfan and melphalan (M) proved to be effective. However, both drugs are not used for normal chemotherapy in neuroblastoma because of their side effects. The alkylating drug melphalan contains a (Cl-CH2-CH2-)2N- group in the para-position of the phenyl moiety of the essential amino acid phenylalanine (Phe) and can, therefore, be taken up by virtually all kinds of cells by amino acid transporters. In contrast, mIBG isotopologs are taken up more selectively by neuroblastoma cells via the noradrenaline transporter (NAT). The present study aimed at synthesising and studying hybrid molecules of benzylguanidine (BG) and the alkylating motif of M. Such hybrids should combine the preferential uptake of BGs into neuroblastoma cells with the cytotoxicity of M. Besides the hybrid of BG with the dialkylating group (Cl-CH2-CH2-)2N- bound in the para-position as in M (pMBG), we also synthesised mMBG, which is BG meta-substituted by a (Cl-CH2-CH2-)2N- group. Furthermore, two monoalkylating hybrid molecules were synthesised: the BG para-substituted by a (Cl-CH2-CH2-)NH- group (pM*BG) and the BG meta-substituted by a (Cl-CH2-CH2-)NH- group (mM*BG). The effects of the four new compounds were studied with human neuroblastoma cell lines (SK-N-SH, Kelly, and LS) with regard to uptake, viability, and proliferation by standard test systems. The dialkylating hybrid molecules pMBG and mMBG were at least as effective as M, whereas the monoalkylating hybrid molecules pM*BG and mM*BG were more effective than M. Considering the preferred uptake via the noradrenaline transporter by neuroblastoma cells, we conclude that they might be well suited for therapy.


Introduction
Neuroblastoma is a malignant tumour of the sympathetic nervous system in childhood [1] with a poor prognosis in stage IV patients [2]. Characteristically, most neuroblastoma cells are able to synthesise catecholamines (dopamine, noradrenaline) [3] and they can express the noradrenaline transporter (NAT) for the reuptake of released dopamine and noradrenaline [4]. Besides catecholamines, a few other compounds can be taken up by the NAT, among them (meta-iodobenzyl)guanidine (mIBG) [5]. From the 1980s until now [6], radio-iodine-labelled mIBG {first [ 131 I]mIBG [7,8], (Figure 1, line 1), soon replaced by [ 123 I]mIBG}, has been used for scintigraphic diagnosis [9]  For the treatment of high-risk neuroblastoma before a hematopoietic stem cell transplantation, the alkylating drugs melphalan (M) [14] and busulfan (B; traded as "myleran" (GlaxoSmithKline, Stevenage, UK) or "busulfex IV" (Otsuka America For the treatment of high-risk neuroblastoma before a hematopoietic stem cell transplantation, the alkylating drugs melphalan (M) [14] and busulfan (B; traded as "myleran" (GlaxoSmithKline, Stevenage, UK) or "busulfex IV" (Otsuka America Pharmaceutical, Inc, Rockville, MD, USA) [15] are used for conditioning; (Figure 1, line 2). Investigations by the Society for Pediatric Oncology (SIOP; HR-NBL1/SIOPEN) showed that, in such cases, the use of a combination of M and B is superior to the formerly used protocol using a combination of M, carboplatin, and etoposide [16]. Despite the high effectivity of M and B, they are not included in chemotherapy protocols for neuroblastoma without subsequent stem cell rescue because of their severe toxicities. The unspecific toxicity of M is not surprising since the carrier molecule of the dialkylating group is phenylalanine (Phe) (Figure 1, line 2), an essential aromatic amino acid, which can be taken up by almost every cell via amino acid transporters [17,18]. In contrast, the uptake of mIBG and related compounds in neuroblastoma cells is essentially restricted via the noradrenaline transporter. In some other cells, catecholamines and mIBG can be taken up by organic cationic transporters (OCTs) [19,20].
In M, the alkylating entity is bound to the para-position of the aromatic ring of Phe. In order to use the potential of M for neuroblastoma treatment in "conventional" chemotherapy, we synthesised and studied hybrid molecules of BG and M, combining the highly effective alkylating group(s) of the alkylating entity of M with BG (rather than with Phe), with the hope of specific uptake into neuroblastoma cells via the NAT. Conceptually related hybrids between BG und B (namely, mBBG and pBBG; Figure 1, line 3) were already synthesised and studied by our groups [21,22].
The aim of our study was (a) to synthesise the conceptualised four hybrid molecules and (b) to analyse their uptake via the noradrenalin transporter in vitro and their cytotoxic effects.

MTS-Test (Viability Test)
Substances were tested in the concentration range from 0.01 µmol/L to 1000 µmol/L using the CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay (MTS) from Promega (Walldorf, Germany). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt in combination with a reductant (phenazine methosulfate) was used. The test is a colorimetric assay for determining the viable cells by bioreduction to a formazan product. The influence of the respective substances was measured on cell viability. The assay was performed according to manufacturer's instruction in 96 flat bottom cell culture plates. In addition, 200 µL of cell suspension (SK-N-SH: 2.5 × 10 4 cells/well; Kelly: 1.5 × 10 4 cells/well; LS: 1 × 10 4 cells/well) in cell culture medium was transferred to the wells of a 96-well plate. After 24 h, 10 µL of the respective substances and concentrations was added in triplicates to the wells. The incubation was stopped after 72 h of incubation at 37 • C/5% CO 2 and the samples were analysed according to the test protocol. The viability of the cells after treatment with the respective substance concentrations was related to the control samples (incubation in cell culture medium), set as 100% cell viability. Each experiment was carried out in three independent runs.

CELLigence Assay
In order to gain detailed information concerning the effectivity of the hybrid molecules during the proliferation of the neuroblastoma cells, real-time neuroblastoma cancer cell proliferation was determined using the xCELLigence SP system (Roche Applied Science, Mannheim, Germany). In contrast to the MTS assay, the xCELLigence assay allowed us to follow the growth of cells continuously in the absence and presence of the added drugs.
Neuroblastoma cells were seeded in 96-well plates (E-Plate 96, ACEA Biosciences, San Diego, CA, USA), 20,000 cells/well. Real-time dynamic cell proliferation was monitored in 30 min intervals for 96 h at 37 • C. 24 h after seeding, and cells were treated with M and the MBG hybrids, as indicated. Cell index values were calculated using the RTCA Software (2.0). All curves were normalised to the time point after drug treatment was conducted (~24 h after seeding) applying the RTCA software [32,33].

Synthesis of Our Four MBG Hybrid Compounds
The syntheses of the four hybrid compounds are described in detail in Section 2.1. The syntheses of mMBG·TFA and pMBG·TFA comprised 3 steps in the longest linear sequence and succeeded in total yields of 38% and 27%, respectively. The syntheses of mM*BG·TFA and pM*BG·TFA comprised 6 steps in the longest linear sequence and succeeded in total yields of 7% and 11%, respectively.

Stability of the Newly Synthesised MBG Hybrid Compounds
Once any of our MBG hybrids, i.e., mMBG, pMBG, mM*BG, and pM*BG, had been synthesised as the respective trifluoroacetate mMBG·TFA, pMBG·TFA, mM*BG·TFA, or pM*BG·TFA, they were stored in the freezer at −20 • C. Trifluoroacetate (TFA) was chosen as a counter ion for each respective benzylguanidinium moiety because the corresponding salts were highly soluble in aqueous media. Storage at this temperature was possible for several to many weeks, neither affecting the integrity nor the homogeneity of the respective compound. In order to estimate these compounds' stabilities under the conditions of our cell culture experiments (see below), an aqueous solution of the compound of interest was incubated at 37 • C. After 1 h or 5 h of such exposure, 1 H-NMR spectroscopy (300.1 MHz) in DMSO-d 6 solution revealed to which extent the respective MBG hybrid had survived the heat stress (Table 1). The least stable compound was the dialkylating agent mMBG·TFA, whereas the most stable compounds were the monoalkylating agents mM*BG·TFA and pM*BG·TFA. Table 1. Fraction of originally intact MBG hybrid molecules (formulas: Figure 1) remaining unaltered after having been maintained at +37 • C in aqueous solutions a .  [34]. Figure 2 shows that the hybrids-in contrast to M-significantly inhibited the uptake of

Viability Analyses (MTS Assays) of MBG Hybrid Compounds
Cell viability assays measure the reduction of MTS by NADPH and/or NADH, which are generated by dehydrogenases in metabolically active cells. Potentially toxic drugs can lead to impaired activity, associated with reduced cell proliferation and finally to cell death.
In the experiments shown in Figure 3, the effects of monoalkylating MBGs (mM*BG and pM*BG) on cell viability were compared with the effects of mIBG and M (dialkylating MBGs were not obtainable at this time). In later experiments shown in Figure 4, the effects of dialkylating MBGs (mMBG and pMBG) were directly compared to the effects of monoalkylating MBGs (mM*BG and pM*BG).

Viability Analyses (MTS Assays) of MBG Hybrid Compounds
Cell viability assays measure the reduction of MTS by NADPH and/or NADH, which are generated by dehydrogenases in metabolically active cells. Potentially toxic drugs can lead to impaired activity, associated with reduced cell proliferation and finally to cell death. In the experiments shown in Figure 3, the effects of monoalkylating MBGs (mM*BG and pM*BG) on cell viability were compared with the effects of mIBG and M (dialkylating MBGs were not obtainable at this time). In later experiments shown in Figure 4, the effects of dialkylating MBGs (mMBG and pMBG) were directly compared to the effects of monoalkylating MBGs (mM*BG and pM*BG).  After an incubation period of 72 h, clear effects were observed from about 10 µmol/L, in SK-N-H cells already at 1 µmol/L. Both M*BGs were more effective than M and mIBG·HCl, the isomers mM*BG·TFA and pM*BG·TFA being similarly effective.  After an incubation period of 72 h, clear effects were observed from about 10 µmol/L, in SK-N-H cells already at 1 µmol/L. Both M*BGs were more effective than M and mIBG·HCl, the isomers mM*BG·TFA and pM*BG·TFA being similarly effective.
Taken together, the effects of BG hybrids with only one alkylating group (mM*BG/pM*BG) were similar or even a little bit better than those with two alkylating groups (mMBG/pMBG).

Proliferation Kinetics of Neuroblastoma Cells (xCELLigence Assay) of MBG Hybrid Compounds
In order to gain detailed information concerning the effectivity of the hybrid molecules during the proliferation of the neuroblastoma cells, real-time neuroblastoma cancer cell proliferation was determined using the xCELLigence SP system (Roche Applied Science, Mannheim, Germany). In contrast to the MTS assay, the xCELLigence assays allowed us to follow the growth of cells continuously in the absence and presence of the added drugs.
In screening experiments, the effects of M and the MBGs were first investigated in the concentration range of 0.1-100 µmol/L in order to estimate acceptable concentrations for further experiments: 1-10 µmol/L (final concentration in the cell cultures) proved to be well suitable. The following experiments shown in Figures 5 and 6 were carried out using a drug concentration of 10 µmol/L. incubations with the indicated compounds at 37 °C for 72 h: Effects of mM*BG·TFA (■) an pM*BG·TFA (■) (left), compared to mMBG·TFA (■) and pMBG· TFA(■) (right), administered i concentrations of 0.01-0.05-0.1-0.5-1-5-10 µmol/L (corresponding to near logarithmic horizonta scale). The extreme left of each diagram depicts the corresponding PBS ++ (+ 5.5 mmol/L glucose control (whose MTS assay served to define "100% viability"). Mean ± S.D; 3 independen experiments, each in triplicate.
Taken together, the effects of BG hybrids with only one alkylating grou (mM*BG/pM*BG) were similar or even a little bit better than those with two alkylatin groups (mMBG/ pMBG).

Proliferation Kinetics of Neuroblastoma Cells (xCELLigens Assay) of MBG Hybrid Compounds
In order to gain detailed information concerning the effectivity of the hybri molecules during the proliferation of the neuroblastoma cells, real-time neuroblastom cancer cell proliferation was determined using the xCELLigence SP system (Roch Applied Science, Mannheim, Germany). In contrast to the MTS assay, the xCELLigen assays allowed us to follow the growth of cells continuously in the absence and presenc of the added drugs.
In screening experiments, the effects of M and the MBGs were first investigated i the concentration range of 0.1-100 µmol/L in order to estimate acceptable concentration for further experiments: 1-10 µmol/L (final concentration in the cell cultures) proved t be well suitable. The following experiments shown in Figure 5 and Figure 6 were carrie out using a drug concentration of 10 µmol/L.     Figure 5 shows that the hybrids with one alkylating group (mM*BG·TFA an pM*BG·TFA) proved to be more effective than M to all three cell lines.  The hybrids with two alkylating groups (mMBG TFA and pMBG·TFA) were mor toxic than M in Kelly and LS cells, but less pronounced in SK-N-SH cells.

Discussion
To an important extent, the chemotherapy of cancer relies on the in vivo alkylation of biomolecules by alkylating agents [35,36]. The earliest used examples were th compounds (Cl-CH2-CH2-)3N and (Cl-CH2-CH2-)2N-Me [37][38][39]. The last-mentioned compound is the simplest representative of a class of compounds containing the structura motif (Cl-CH2-CH2-)2N-. They possess high alkylating power, which makes them akin to and possibly even superior to, the alkylating agent (Cl-CH2-CH2-)2S. The latter wa dubbed "mustard gas" during WWI warfare. This is why compounds containing the moti (Cl-CH2-CH2-)2Nare commonly referred to as "N-mustards". N-mustards-not only M  Figure 5 shows that the hybrids with one alkylating group (mM*BG·TFA and pM*BG·TFA) proved to be more effective than M to all three cell lines.
The hybrids with two alkylating groups (mMBG TFA and pMBG·TFA) were more toxic than M in Kelly and LS cells, but less pronounced in SK-N-SH cells.
As already described, the alkylating chemotherapeutics busulfan (B) and melphalan (M) have been successfully introduced into the therapy of high-risk neuroblastoma before stem cell transplantation [16]. Due to their toxicity, especially on hematopoietic stem cells, both substances are not used in conventional chemotherapy of neuroblastoma without stem cell transplantation. Therefore, we decided to synthesise hybrid molecules of benzylguanidine (with the aim of more specific uptake into neuroblastoma cells) and the alkylating groups of these cytostatic drugs B and M. In a preceding paper, we reported the effects of mBBG and pBBG (busulfan benzylguanidine hybrids) compared to B {busulfan, 5-[(methanesulfonyl)oxy]pentyl methanesulfonate} [21]. In the actual manuscript, we present our results concerning the effects of MBGs (melphalan benzylguanidine hybrids) on neuroblastoma cells.
As already mentioned in the introduction, it is obvious that the alkylating cytostatic drug M has a fundamental handicap: the carrier structure of its alkylating group is Phe, which can probably be taken up by almost all cell types by amino acid transporters [17,18]. This is probably the reason for the unspecific side effects of M. Nevertheless, M proved to be very effective in the conditioning regime in high-risk neuroblastoma for the destruction of neuroblastoma cells and hematopoietic cells before transplantation [16,52]. In order to reduce the side effects observed during treatment with M, our aim was to synthesise hybrid molecules consisting of the high effective alkylating group of M and a carrier group (benzylguanidine), which can be taken up by the NAT of neuroblastoma cells. However, their uptake is not limited to neuroblastoma cells: mIBG (and probably the MBGs) can also be incorporated by cells expressing organic cation transporters (OCTs), such as kidney and liver cells, but uptake via these transporters can be reduced by clinically approved corticosteroids [53]. Therefore, a combination of corticosteroids with [ 123 I]mIBG/[ 131 I]mIBG or with MBGs should enhance their preferred uptake via the NAT and thereby improve diagnosis and therapy of neuroblastoma.
For synthesis, the alkylating group (Cl-CH 2 -CH 2 -) 2 N-of M binds to benzylguanidine in the meta-(instead of iodine in mIBG) and para-position. Furthermore, hybrids with only one alkylating group, (Cl-CH 2 -CH 2 -)NH-, bound in the metaand para-position, were also synthesised. All four M hybrids were at least equally as effective, but in most cases more effective, on neuroblastoma cell cultures than M. Surprisingly, hybrids containing only one alkylating group were at least equally as effective as those with two alkylating groups. In M, the two activated alkylating, positively charged groups can crosslink the strands of DNA by reacting with the nucleophilic amino groups comprising guanine N-7 and adenine N-3 [54][55][56]. Concerning the M hybrids mM*BG and pM*BG with only one alkylating group, they are definitely unable to crosslink DNA. However, their alkylating power towards the mentioned nucleophilic amino groups containing guanine N-7 and adenine N-3 should be essentially the same as the monoalkylation power of their bifunctional analogues mMBG and pMBG. This allows us to speculate that the more pronounced effects of the hybrids mM*BG and pM*BG with only one alkylating group sometimes observed may be, at least partly, associated their higher thermal stability (Table 1).
Comparing the three neuroblastoma cell lines with different NAT expression, it was conspicuous that LS cells (very low NAT expression) were similarly sensitive against the M hybrids than SK-N-SH and Kelly cells. This aspect needs to be investigated in more detail with other cells lacking NAT expression. We suppose that the presence of OCTs in LS cells is responsible for the uptake of the M hybrids (not yet investigated).

Conclusions
Our newly synthesised M hybrids mMBG and pMBG containing the alkylating entity (Cl-CH 2 -CH 2 -) 2 N-of M-two 2-chloroethyl groups bound to one N-proved to be more effective against neuroblastoma cell lines as the equally (Cl-CH 2 -CH 2 -) 2 N-containing drug M, indicating their potential as an improved chemotherapeutic drug in neuroblastoma therapy. Although our original intention was to only synthesise molecules with strict homologies to M, we found that the less closely related, M-inspired (Cl-CH 2 -CH 2 -)NHcontaining benzylguanidine hybrids mM*BG and pM*BG-with only one 2-chloroethyl group bound to the N atom-were also effective in neuroblastoma cells. This may be due to-either altogether or in part-mM*BG and pM*BG being more thermally stable than mMBG and pMBG during incubation at 37 • C.
All four hybrid molecules were at least equally as cytotoxic, but in most cases even more cytotoxic against the neuroblastoma cells than melphalan in vitro. Based on these data, we conclude that all four hybrids should essentially be suitable for neuroblastoma therapy. However, further experiments in vitro and in vivo are necessary in order to support this concept. Next, in vitro experiments will be done with an expanded set of well characterised, commercially available neuroblastoma cell lines together with non-neuroblastoma cells (kidney, liver, fibroblast, and hematopoietic stem cells) in order to comparatively analyse the cytotoxicity and uptake of the MBGs and melphalan by the noradrenaline transporter, amino acid transporter, and organic cation transporter in detail. The influence of glucocorticoides for a more selective uptake via the noradrenaline transporter will also be analysed. Subsequently, mouse experiments (the toxicity and treatment of the neuroblastoma-bearing mouse) should be performed with melphalan and the most suitable MBGs. Funding: This research was funded by the Deutsche Forschungsgemeinschaft (grants HA 2386/2-1 und BR 881/9-1). We are indebted for this generous support.