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

Article abstract of DOI:10.1016/j.bmcl.2019.05.047

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.

Full text of DOI:10.1016/j.bmcl.2019.05.047

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Folic acid conjugates of a bleomycin mimic for selective targeting of folate
receptor positive cancer cells
⁎
⁎
Arjan Geersinga, Reinder H. de Vriesa, Gerrit Jansenb, Marianne G. Rotsc, , Gerard Roelfesa,
a Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
b Department of Rheumatology, Amsterdam Rheumatology and Immunology Center (ARC), VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The
Netherlands
c Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
A R T I C L E I N F O
A B S T R A C T
Keywords:
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.
Folate conjugate
Bleomycin mimic
Folate receptor
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 lym-
phomas3 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(2-
pyridyl)-methylamine) has been of particular value.6 Previous work by
our group has revealed the iron(II) complex of N4Py to be an excellent
structural7–9 and functional mimic10–13 of Fe(II)-BLM. In addition, in-
itial 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.
carriers,15,23 small molecule drug conjugates of FA are not substrates
for these transporters.24,25 The potential of folate conjugates is illu-
strated by the number of preclinical and clinical studies of various drug
delivery systems.26–30
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 dis-
ulphide 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,20,35–44 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.
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 There-
fore, 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.17,20–22
Even though FA can also be shuttled into the cell by other folate
Synthetic modification of FA can be accomplished by functionali-
zation 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
⁎ Corresponding authors.
E-mail addresses: M.G.Rots@umcg.nl (M.G. Rots), j.g.roelfes@rug.nl (G. Roelfes).
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
0960-894X/©2019TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/BY/4.0/).
Pleasecitethisarticleas:ArjanGeersing,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.05.047

A. Geersing, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
O
OH
O
O
H
N
N
N
H
OH
N
NH2
H2N
H
N
N
O
+
N
N
N
N
H
N
H2N
2
N
N
O
O
O
O
1
N
O
X
O
O
X
N
Et
DMSO,
o/n
3N,
r.t.,
O
O
=
=
X
X
3
2 ( )
( )
CH
4
S
N
OH
N
O
O
H
H
N
H
N
X
N
N
N
N
H
OH
X
N
H
N
N
N
O
O
N
N
O
H
X= CH2 (N4Py-C-C-FA)
X= S (N4Py-S-S-FA)
H2N
N
Scheme 1. Synthesis of N4Py conjugates N4Py-C-C-FA and N4Py-S-S-FA.
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,35–37,41,46–49 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).
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 de-
picted 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).
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-re-
gioselective method similar to the one described by Luo et al (Scheme
S2).50 Conjugation of N4Py to FA was then achieved using dis-
uccinimidyl 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 addi-
tion 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 ex-
periments.
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)53–55 in three different cell
lines, each with different FR surface expression levels: high FR ex-
pressing KB cells (30–50 pmol/106 cells), moderately FR expressing
IGROV-1 cells (∼1 pmol/106 cells) and low FR expressing MCF7 cells
(FR generally undetectable).56–59
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(ClO4)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 oxida-
tion chemistry does not negatively affect the disulphide bond cleavage
by GSH.
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 IC50 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 IC50 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
2

A. Geersing, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
(Fig. 3). KB (FR+) cells were not affected by treatment with N4Py-C-C-
FA within 48 h, and only little activity compared to DMSO control was
observed after 72 h (30 μM: 88.1
1.7; 50 μM: 62.4
2.5). However,
treatment with N4Py-S-S-FA had a large effect on the metabolic ac-
tivity, with relative metabolic activity values approaching those of Fe
(II)-N4Py after 72 h treatment (30 μM: 38.8
1.4, p < 0.01; 50 μM:
11.2
0.6, p < 0.05). In contrast, MCF7 (FR-) cells showed no sig-
nificant response to N4Py-C-C-FA over a period of 72 h and also little
effect upon treatment with N4Py-S-S-FA after 72 h treatment (30 μM:
78.9
2.4; 50 μM: 76.4
2.6). Interestingly, treatment of moder-
ately FR expressing IGROV1 cells, which contain physiologically re-
levant FR levels, with N4Py-S-S-FA for 72 h resulted in metabolic ac-
tivity values that differed only ∼10% from the results obtained in KB
cells (30 μM: 47.2
1.9; 50 μM: 22.0
0.9). A much smaller, albeit
significant, decrease in cell metabolism was observed after 72 h for
N4Py-C-C-FA (30 μM: 82.4
4.1; 50 μM: 58.1
2.3). The same ex-
periments carried out in regular cell medium (HF, 2.3 μM FA in RPMI
medium, 10% FCS) with KB and MCF7 cells, showed a similar trend
with generally slightly higher metabolic activities compared to LF
conditions (Figs. S8 and S9).
Fig. 3d summarizes the results of the MTS assays performed with
50 µM concentrations of N4Py-S-S-FA and Fe(II)-N4Py after 72 h
treatment in MCF7 (FR-), IGROV1 (FR+/−) and KB (FR + ) cell lines.
This graph illustrates the clear decrease in cell metabolism for N4Py-S-
S-FA with increased expression of the FR, while no FR expression level
dependence is observed for Fe(II)-N4Py under the indicated conditions.
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.
Fig. 1. Schematic representation of the proposed mode of action of the N4Py-
folic acid conjugate containing a cleavable linker: upon binding to the FR, the
conjugate is (a) internalized via endocytosis, followed by (b) reductive cleavage
of the linker, which allows (c) release of the active N4Py moiety.
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, non-
significant 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 FR-
mediated uptake and delivery of the conjugates. The 2-fold lower affi-
nity 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 hydro-
phobic 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 pro-
cess, 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 in-
cubation with excess FA, further confirming folate receptor-mediated
internalization (Figs. 4, S12, S13).
Fig. 2. FR affinity measurements by competition experiments of N4Py-CC-FA
(blue), N4Py-SS-FA (green) and FA (purple) at 25, 50, 100, 250, 500,
1000 pmol quantities in combination with 50 pmol Folate-FITC and 0.5·106 KB
cells per experiment. Results are measured as MFI on fluorescence channel PL-
1. Data are presented as the mean
SEM and N ≥ 5.
experiments in standard (High Folate, HF) RPMI-1640 cell culture
medium containing supraphysiological concentrations of FA (HF,
2.3 μM FA in RPMI medium, 10% FCS) showed only marginal changes
in metabolic activity compared to LF conditions (Fig. S9).
The presence of a cleavable disulphide linker in N4Py-S-S-FA in-
creased 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
Next, the effect of 30 μM and 50 μM concentrations of the N4Py-FA
conjugates on the metabolic activity was determined and compared to
the activity of Fe(II)-N4Py after incubation for 24 h, 48 h and 72 h
3

A. Geersing, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 3. MTS assay performed on KB cells. Metabolic activity of a) KB cells, b) MCF7 cells and c) IGROV1 cells, upon treatment with Fe(II)-N4Py (red), N4Py-C-C-FA
(blue) or N4Py-S-S-FA (green) under optimized (low folate) conditions. Cells treated for 24 h (no line), 48 h (single line) and 72 h (crossed lines) with 30 or 50 μM of
Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green). For each experiment, every treatment was performed in triplicate and the experiment was carried out
in triplo. Data are presented as the mean
SEM. ***p < 0.001, **p < 0.01, *p < 0.05 (only the most relevant statistics are shown); d) Summary of the results of
the MTS assays performed in MCF7 (FR−), IGROV1 (FR+/−) and KB (FR+) cells, treated for 72 h with 50 µM N4Py-S-S-FA (green) and 50 µM Fe(II)-N4Py (red).
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 ap-
proached 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 un-
selective.
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 sug-
gests the potential of this design for future in vivo studies.
Fig. 4. Metabolic activity in KB cells upon simultaneous treatment with FA and
Fe(II)-N4Py (red), FA and N4Py-C-C-FA (blue), FA and N4Py-S-S-FA (green)
and FA alone, in standard RPMI medium (high folate). KB cells treated for 24 h,
48 h and 72 h with 150 μM FA and 30 μM of: Fe(II)-N4Py (red), N4Py-C-C-FA
(blue) or N4Py-S-S-FA (green), or 150 μM FA alone. For each experiment, every
treatment was performed in triplicate and the experiment was carried out in
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 ap-
proaching 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 po-
tency and increased selectivity compared to compounds without tar-
geting moieties.
triplo. Data are presented as the mean
SEM.
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.
4

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Acknowledgements
The authors thank Ms. Jelleke M. Dokter-Fokkens for advice and
help with the cell experiments. Financial support from the Netherlands
Organisation for Scientific Research (NWO, GRANT no. 728.011.101),
the European Research Council (ERC Starting Grant 280010), the Ubbo
Emmius Foundation of the University of Groningen and the Netherlands
Ministry of Education, Culture, and Science (Gravitation Program No.
024.001.035) is gratefully acknowledged. In addition, the authors ac-
knowledge the H2020 COST Action CM1406 www.EpiCHemBio.eu.
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recurrent ovarian cancer: a review. Oncol Targets Ther. 2014;7:1223–1236.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://
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