Folic acid conjugates of a bleomycin mimic for selective targeting of folate receptor positive cancer cells

A major challenge in the application of cytotoxic anti-cancer drugs is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. A particularly promising target for selective targeting are the folate receptors (FR) that are often over-expressed on cancer cells. Here, we report on a conjugate of the pentadentate nitrogen ligand N4Py to folic acid, via a cleavable disulphide linker, which shows selective cytotoxicity against folate receptor expressing cancer cells. The bleomycins (BLMs) are a group of antitumor antibiotics that have long been used for the treatment of various tumors such as squamous cell carcinomas,1 testicular carcinomas,2 malignant lymphomas and ovarian cancer. Their unique structures and ability to activate dioxygen, resulting in oxidative degradation of DNA,5 have been a major inspiration for the design of molecular mimics of its metal binding domain. Of these, N4Py (N,N-bis(2-pyridylmethyl)-N-bis(2pyridyl)-methylamine) has been of particular value. Previous work by our group has revealed the iron(II) complex of N4Py to be an excellent structural and functional mimic of Fe(II)-BLM. In addition, initial in vitro experiments have shown that Fe(II)-N4Py has a different mechanism of action compared to BLM: whereas BLM is a cytostatic reagent, Fe(II)-N4Py behaves as a cytotoxic reagent.14 Finally, it was shown that N4Py could also be used as free ligand; it proved capable of binding Fe(II), which is required for the activity, from the biological environment. A major challenge of cytotoxic anti-cancer agents is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. Rapidly dividing cancer cells, however, require various nutrients and vitamins such as, for example, folate.15 As a consequence, the receptors involved in folate uptake are often over-expressed on cancer cells.16–19 Therefore, the folate receptor (FR), to which folic acid (FA) binds with high affinity (∼0.1–1 nM), is a particularly promising target for selective drug delivery and imaging agents by using folate conjugates. Even though FA can also be shuttled into the cell by other folate carriers,15,23 small molecule drug conjugates of FA are not substrates for these transporters. The potential of folate conjugates is illustrated by the number of preclinical and clinical studies of various drug delivery systems. FA conjugates enter FR positive (+) cells by receptor-mediated endocytosis. Enhancement of intracellular release of a drug from the FA moiety can be obtained by either exploiting the decrease in pH upon endosomal uptake of the conjugate in the endosomes with a pH sensitive linker, or by using the reducing conditions of the intracellular environment in comparison with the extracellular milieu with a disulphide linked drug.16 However, since pH sensitive linkers often suffer from inefficient release of the cargo, a linker containing a redox sensitive disulphide bond is more attractive.34 Surprisingly, even though some folate-conjugated metal complexes have been used as tracer molecules for diagnostic purposes,20,35–44 to the best of our knowledge, no folate conjugated metal complexes have been reported as cytotoxic agents. Here, we report on a bleomycin mimic, the pentadentate ligand N4Py, conjugated to FA via a redox sensitive cleavable linker, N4Py-S-S-FA, which shows good activity and is selective towards to FR(+) cells. Synthetic modification of FA can be accomplished by functionalization at the αor γ-position of the glutamate moiety. Even though some studies have shown that α-functionalization can result in similar binding affinities to FR compared to γ-functionalization,38,45 others show that the γregioisomer can bind FR orders of magnitude better compared to the α-regioisomer.36,37 It was therefore decided to https://doi.org/10.1016/j.bmcl.2019.05.047 Received 9 April 2019; Received in revised form 21 May 2019; Accepted 23 May 2019 ⁎ Corresponding authors. E-mail addresses: M.G.Rots@umcg.nl (M.G. Rots), j.g.roelfes@rug.nl (G. Roelfes). Bioorganic & Medicinal Chemistry Letters 29 (2019) 1922–1927 Available online 24 May 2019 0960-894X/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). T

A major challenge in the application of cytotoxic anti-cancer drugs is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. A particularly promising target for selective targeting are the folate receptors (FR) that are often over-expressed on cancer cells. Here, we report on a conjugate of the pentadentate nitrogen ligand N4Py to folic acid, via a cleavable disulphide linker, which shows selective cytotoxicity against folate receptor expressing cancer cells.
The bleomycins (BLMs) are a group of antitumor antibiotics that have long been used for the treatment of various tumors such as squamous cell carcinomas, 1 testicular carcinomas, 2 malignant lymphomas 3 and ovarian cancer. 4 Their unique structures and ability to activate dioxygen, resulting in oxidative degradation of DNA, 5 have been a major inspiration for the design of molecular mimics of its metal binding domain. Of these, N4Py (N,N-bis(2-pyridylmethyl)-N-bis(2pyridyl)-methylamine) has been of particular value. 6 Previous work by our group has revealed the iron(II) complex of N4Py to be an excellent structural [7][8][9] and functional mimic [10][11][12][13] of Fe(II)-BLM. In addition, initial in vitro experiments have shown that Fe(II)-N4Py has a different mechanism of action compared to BLM: whereas BLM is a cytostatic reagent, Fe(II)-N4Py behaves as a cytotoxic reagent. 14 Finally, it was shown that N4Py could also be used as free ligand; it proved capable of binding Fe(II), which is required for the activity, from the biological environment.
A major challenge of cytotoxic anti-cancer agents is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. Rapidly dividing cancer cells, however, require various nutrients and vitamins such as, for example, folate. 15 As a consequence, the receptors involved in folate uptake are often over-expressed on cancer cells. [16][17][18][19] Therefore, the folate receptor (FR), to which folic acid (FA) binds with high affinity (∼0.1-1 nM), is a particularly promising target for selective drug delivery and imaging agents by using folate conjugates. 17,[20][21][22] Even though FA can also be shuttled into the cell by other folate carriers, 15,23 small molecule drug conjugates of FA are not substrates for these transporters. 24,25 The potential of folate conjugates is illustrated by the number of preclinical and clinical studies of various drug delivery systems. [26][27][28][29][30] FA conjugates enter FR positive (+) cells by receptor-mediated endocytosis. 25,31 Enhancement of intracellular release of a drug from the FA moiety can be obtained by either exploiting the decrease in pH upon endosomal uptake of the conjugate in the endosomes with a pH sensitive linker, or by using the reducing conditions of the intracellular environment in comparison with the extracellular milieu with a disulphide linked drug. 16 However, since pH sensitive linkers often suffer from inefficient release of the cargo, 32,33 a linker containing a redox sensitive disulphide bond is more attractive. 34 Surprisingly, even though some folate-conjugated metal complexes have been used as tracer molecules for diagnostic purposes, 20,[35][36][37][38][39][40][41][42][43][44] to the best of our knowledge, no folate conjugated metal complexes have been reported as cytotoxic agents. Here, we report on a bleomycin mimic, the pentadentate ligand N4Py, conjugated to FA via a redox sensitive cleavable linker, N4Py-S-S-FA, which shows good activity and is selective towards to FR(+) cells.
Synthetic modification of FA can be accomplished by functionalization at the α-or γ-position of the glutamate moiety. Even though some studies have shown that α-functionalization can result in similar binding affinities to FR compared to γ-functionalization, 38,45 others show that the γ-regioisomer can bind FR orders of magnitude better compared to the α-regioisomer. 36,37 It was therefore decided to prepare both N4Py-FA conjugates exclusively as their γ-regioisomers. Even though the synthesis of many FA conjugates have been reported by using simple carbodiimide chemistry, [35][36][37]41,[46][47][48][49] this method often results in varying mixtures of α-,γ-and bis-functionalization products together with starting material. In addition, purification can be tedious. Therefore, both N4Py-S-S-FA and N4Py-C-C-FA, which is the conjugate that contains a non-cleavable linker, were prepared in high-yielding synthesis via formation of a pyrofolic acid derivative (Scheme 1).
First, an N4Py derivative containing a propylamine linker (1) was synthesized using a method that was previously reported by our group (Scheme S1). 11 Subsequently, FA was aminated (2) using a γ-regioselective method similar to the one described by Luo et al (Scheme S2). 50 Conjugation of N4Py to FA was then achieved using disuccinimidyl suberate (DSS, 3), as a non-cleavable linker, or dithiobis (succinimidyl propionate) (DSP, 4), the redox-sensitive disulfide analogue of 3. Due to the fact that this coupling method is based on a homobifunctional linker and both 1 and 2 contain a primary amine functionality, it was anticipated that homocoupling of N4Py or the FA would lead to formation of byproducts. Indeed, stoichiometric addition of all three components gave the preferred product together with the homocoupled products as major impurities. Repeating the reaction with only 0.5 eq. of 2, followed by removal of the homocoupling product of N4Py by washing with MeOH, resulted in almost pure product. The resulting products N4Py-C-C-FA and N4Py-S-S-FA were subsequently purified by preparative reversed phase (RP)-HPLC to obtain pure compounds (Figs. S1 and S2) suitable for biological experiments.
The stability and in situ formation of the iron complexes of both conjugates were tested in tris-HCl buffer (pH 8.5) in presence of two equivalents of Fe(ClO 4 ) 2 and 5 mM of glutathione (GSH), since cellular levels of GSH are in the range of 1-10 mM. 51,52 Analysis by LC-MS showed that 5 min after addition of GSH, a large amount of N4Py-S-S-FA was already converted to the corresponding cleavage products (Figs. S3 and S4, Table S1, Chart S1), with complete disappearance of the signal for N4Py-S-S-FA after 20 min. After that, no changes in peak ratios were observed for another 3 h. In contrast, N4Py-C-C-FA was found to be intact after a period of 5 h. In addition, both conjugates are capable of coordinating iron. It therefore appears that the iron oxidation chemistry does not negatively affect the disulphide bond cleavage by GSH.
The DNA cleavage activities of the in situ generated iron complexes of N4Py conjugates were determined as a measure for their inherent oxidation activity. Supercoiled pUC18 plasmid DNA was incubated with 1 μM iron complex at 37°C in presence of DTT as reducing agent (Fig.  S5). Extensive DNA cleavage was observed for both N4Py-conjugates, with turnover frequencies of both conjugates similar to that of Fe(II)-N4Py. Notably, the cleavage of the disulphide bond of N4Py-S-S-FA under these conditions does not affect its ability to cut DNA. These results thus indicate that the intrinsic oxidation chemistry of the N4Py moiety in both conjugates is unchanged, resulting in DNA cleavage rates of both conjugates that are comparable to Fe(II)-N4Py (Fig. S6).
In vitro binding competition assays were performed with the FR expressing KB cell line to compare relative binding affinities of the N4Py-conjugates with that of FA itself. Affinities of FR for N4Py-C-C-FA and N4Py-S-S-FA were assessed by Fluorescence Assisted Cell Sorting (FACS) competition experiments with Folate-FITC and compared with FA. The folate receptor has a good affinity for both conjugates, as depicted by relative binding affinities of FR for N4Py-C-C-FA and N4Py-S-S-FA, which are approximately only 2-fold lower than for FA (Figs. 1 and 2, S7).
The effect N4Py-FA conjugates on the metabolic activity of cancer cells was evaluated by the MTS assay. The compounds were used as free ligand: It is assumed that they are able to bind an iron ion in the cellular environment, as shown before. 14 First, experiments were performed under low folate (LF) medium conditions (RPMI-1640 medium, 10% dialyzed FCS, supplemented with 2 nM FA) [53][54][55] in three different cell lines, each with different FR surface expression levels: high FR expressing KB cells (30-50 pmol/10 6 cells), moderately FR expressing IGROV-1 cells (∼1 pmol/10 6 cells) and low FR expressing MCF7 cells (FR generally undetectable). [56][57][58][59] The cellular response toward FA and Fe(II)-N4Py at concentrations varying between 1 and 150 μM was tested with incubation times of 24 h, 48 h and 72 h (Fig. S8). The IC 50 values for Fe(II)-N4Py are in the range of 30 μM for KB and MCF7 cell lines, while being slightly lower for IGROV1 cells (20 μM) after 24 h. KB and MCF7 cells were more strongly affected when the incubation times were increased to 48 h and 72 h with reduced metabolic activity and IC 50 values in the range of 20 μM, while the metabolic activity of IGROV1 was barely affected by longer incubation times. As control, all cell lines were incubated with FA, which did not cause any appreciable decrease in metabolic activity compared to solvent control over a period of 72 h. Repeating the The selective cytotoxicity of N4Py-conjugates towards FR positive cells was further investigated by simultaneous treatment of KB cells with a mixture of 30 μM N4Py-conjugate and an excess of FA (150 µM). This resulted in no appreciable changes in metabolic activities over a period of 72 h (Fig. 4). Moreover, similar results as with KB cells were obtained after treatment of MCF7 (Fig. S12) and IGROV1 (Fig. S13) cells with the same mixture.
The DNA cleavage activity of the Fe(II)-N4Py complex is the result of generation of highly Reactive Oxygen Species (hROS). 11,12 It was investigated whether this ability was retained for the N4Py-folate conjugates in KB cells and MCF7 cells. The amount of hROS in the cells was therefore measured with the hROS sensitive APF probe, as inferred from an increase in APF mean fluorescence intensity (MFI) as measured by FACS. 59 Importantly, hROS production as detected by APF for N4Py-S-S-FA in KB cells was about 2.5 times higher than for N4Py-C-C-FA and about 3.5 times higher compared to solvent control. Notably, nonsignificant increase in APF mean fluorescence intensity was observed for both FA and N4Py-C-C-FA (Fig. 5). Moreover, as expected, the hROS production for both conjugates was similar in the FR(−) MCF7 cells and only ∼1.6 times higher compared to solvent control, in this case.
Collectively, the results reported here are consistent with an FRmediated uptake and delivery of the conjugates. The 2-fold lower affinity for the folate receptor compared to FA is in agreement with other studies in which the pterin moiety of the folate or folate derivative was unmodified, 60-62 resulting in sufficient hydrogen bonding and hydrophobic interactions in the binding pocket of the folate receptor. 63 In addition, the change in metabolic activity of both conjugates in time shows the characteristic behaviour of a folate receptor mediated process, in which FR recycling between the cell surface and its intracellular compartments typically takes 8-12 h (Fig. 3a-c). 16,64 Furthermore, the selective cytotoxicity of N4Py-S-S-FA to KB (FR+) cells effectively disappeared when the FR receptor was blocked by simultaneous incubation with excess FA, further confirming folate receptor-mediated internalization (Figs. 4, S12, S13).
The presence of a cleavable disulphide linker in N4Py-S-S-FA increased the activity against cancer cells dramatically when compared to N4Py-C-C-FA, which has a non-cleavable linker. Cellular GSH most likely cleaves the N4Py moiety from N4Py-S-S-FA, which can then diffuse out of the endosomal compartment. Reduction of the disulphide linkage in folate-disulfide-drug conjugates following endocytosis  Bioorganic & Medicinal Chemistry Letters 29 (2019Letters 29 ( ) 1922Letters 29 ( -1927 typically occurs with a half-time of ∼6 h, 32 which explains the faster decrease in metabolic activity for N4Py-S-S-FA compared to N4Py-C-C-FA and thus, its higher activity in vitro. The MTS assay performed in KB cells showed a significant effect on metabolic activity by N4Py-S-S-FA after 48 h, which is increased after 72 h (Fig. 3a). In contrast, the effects observed upon treatment with N4Py-C-C-FA were limited on the same timescale. The decrease of metabolic activity in KB cells after 72 h by N4Py-S-S-FA approached that of the parent complex Fe(II)-N4Py, which indicates that the potency of the conjugate did not decrease significantly due to conjugation to folate. However, the initiation time of Fe(II)-N4Py is much shorter, which indicates a different influx pathway into the cell. In addition, the MTS and APF results for both conjugates from MCF7 (FR−) cells showed almost no activity and hROS production, while Fe(II)-N4Py proved to be lethal (Figs. 3b and 5). This further illustrates the selectivity of the N4Py-conjugates for FR+ cancer cells in comparison to the parent N4Py-iron complex, which is unselective.
Finally, the finding that N4Py-S-S-FA is also functionally active and selective for IGROV1 cells, which have a 30 to 50 times lower folate receptor density on the cell membrane compared to KB cells, 55,56 suggests the potential of this design for future in vivo studies.
Here we have presented a folate conjugate of a pentadentate iron binding ligand that is able to cause selective cell death of FR(+) cancer cells and that has improved efficiency due to the presence of a cleavable linker moiety containing a disulphide bond. The decrease in metabolic activity of FR(+) cells after treatment with N4Py-S-S-FA is approaching that of Fe(II)-N4Py after a period of 72 h, with a minimum effect observed in the same time period for FR(−) cells. These results emphasize the power of ligand-targeted therapeutics, with good potency and increased selectivity compared to compounds without targeting moieties.