Synthesis and Biological Properties of Ferrocenyl and Organic
Methotrexate Derivatives
Karolina Rózga, Andrzej Błauz,̇ Daniel Moscoh Ayine-Tora, Ernest Pusćion, Christian G. Hartinger,
Damian Plazu̇k,* and Błazėj Rychlik*

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ABSTRACT: Synthesis and biological activity of two series of modified side chain methotrexate (MTX) derivatives are presented,
one with a ferrocenyl moiety inserted between the pteroyl and glutamate portions of the molecule and the other with glutamate
substituted for short chain amino acids. Ferrocenyl derivatives of MTX turned out to be rather moderate inhibitors of dihydrofolate
reductase (DHFR) although molecular modeling suggested more effective interactions between these compounds and the target
enzyme. More interestingly, ferrocene-decorated MTX derivatives were able to impede the proliferation of four murine and human
cell lines as well as their methotrexate-resistant counterparts, overcoming the multidrug resistance (MDR) barrier. They were also
able to directly interact with Abcc1, an MDR protein. Of the amino acid pteroyl conjugates, the γ-aminobutyric acid derivative was
an efficient inhibitor of DHFR but had no effect on cell proliferation in the concentration range studied while a taurine conjugate
was a poor DHFR inhibitor but able to affect cell viability. We postulate that modification of the methotrexate side chain may be an
efficient strategy to overcome efflux-dependent methotrexate resistance.

■ INTRODUCTION
The era of cancer chemotherapy started in 1947 when Sidney
Farber purposely used synthetic folic acid analogues amino-
pterin and methotrexate (MTX, amethopterin) for the
treatment of acute leukemia in children.1 The latter compound
is still widely used in medicine. It is currently indicated not
only for the treatment of acute lymphoblastic leukemia or
other types of cancer but it is also used in autoimmune and
inflammatory diseases such as rheumatoid arthritis and
psoriasis or for the management of ectopic pregnancy.2

Methotrexate binds to and practically irreversibly inhibits
dihydrofolate reductase (DHFR), a key enzyme in folic acid
metabolism reducing dihydrofolate to tetrahydrofolate at the
expense of β-NADPH,3 thus impeding DNA replication by
decreasing the available nitrogen base pool. Due to high
structural similarity to folic acid, MTX undergoes poly-
(glutamylation)4,5 catalyzed by folylpolyglutamate synthetase
(FPGS), a process that originally evolved to retain folic acid
within the cell. Poly(glutamylated) forms of methotrexate are
recognized by and also inhibit other folate-dependent enzymes,
i.e., thymidylate synthase6 and phosphoribosylaminoimidazo-
lecarboxamide formyltransferase,7 thus increasing the pharma-

codynamic potency of the drug. The clinical usefulness of
MTX is, however, limited not only by its high systemic toxicity
but, more importantly, by the reduced sensitivity of target cells
toward this antifolate. The resistance to methotrexate may
originate from different processes, including amplification and
elevated DHFR gene expression,8 reduced MTX uptake rate,9

impaired poly(glutamylation),10 resulting from genetic alter-
ations of FPGS,11 and augmented efflux of MTX and/or its
active metabolites.12 The last of these mechanisms is of special
clinical importance as it is usually mediated by low-specific
membrane transporters of the ATP-binding cassette (ABC)
superfamily and may lead to multidrug resistance (MDR), i.e.,
decreased susceptibility of a target cell to a number of
structurally different compounds of variable modes of action. It
was demonstrated that MTX and its oligo(glutamylated) forms

Received: April 14, 2024
Revised: June 12, 2024
Accepted: July 3, 2024
Published: July 23, 2024

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are recognized and actively exported outside the cell by
ABCC1 and ABCC3,13 ABCC414 (MTX and MTX-Glu but
not MTX-Glu2) and ABCC515 (MTX, MTX-Glu, and MTX-
Glu2 but not MTX-Glu3) as well as wild type and some
variants of ABCG216−18 (MTX, MTX-Glu1−3). The practical
importance of MDR in cancer therapy can be exemplified by
the study of Jaramillo et al. demonstrating that elevated
expression of ABCC4 and ABCG2 negatively impacted the
MTX response in pediatric patients with acute lymphoblastic
leukemia.19

Although modern cancer treatment is focused more on
targeted therapies and less on chemotherapy, the widespread
use of methotrexate as an immunomodulatory compound
prompted us to seek MTX derivatives that can overcome
multidrug resistance. We wanted to preserve the active pteroyl
fragment of the molecule and concentrate on the glutamate
side chain as a factor important for the recognition of MTX
and its glutamylated forms by MDR transporters. An appealing
possibility was to introduce a metallocenyl moiety into a
methotrexate molecule. Such a strategy turned out to be
fruitful in the case of modification of many natural products
(for a review, see e.g., ref 20) and�except for a single
study21�was not employed for folates so far. We demon-
strated previously that inserting an organometallic component
in a biologically active molecule may dramatically alter its
properties, e.g., turn a vitamin into a toxin22 or change the
molecular target of the parental compound.23 Therefore, we
investigated if the introduction of a ferrocenyl moiety affected
the biological properties of MTX (Figure 1, compounds 1−3).
We also substituted the glutamate moiety in methotrexate for
another unbranched amino acid (Figure 1, compounds 4−7)

as this kind of modification was also not widely studied despite
extensive work done in this field by Rosowsky and colleagues
in the 1980s and 1990s.24−26

■ RESULTS AND DISCUSSION
Synthesis. The ferrocenyl conjugates 1−3 were synthe-

sized from rac-N-Fmoc-2-aminomethyl-1-ferrocenecarboxylic
acid 14. The latter compound was prepared in multistep
reactions according to Scheme 1. First, ferrocenecarboxalde-
hyde 8 reacted with 1,3-propanediol and TsOH produced
acetal 9 in 88% yield.27 The latter compound was lithiated with
sec-BuLi, followed by gaseous carbon dioxide to afford acid rac-
10. Then, the acetal moiety of crude 10 was removed by acidic
hydrolysis with 1 M hydrochloric acid in THF, affording rac-2-
formyl-1-ferrocenecarboxylic acid 11 in 83% overall yield. The
further reaction of 11 with an excess of hydroxylamine
hydrochloride and triethylamine afforded oxime 12 in 92%
yield (using sodium hydroxide instead of triethylamine gave 12
in only 54% yield), isolated as a mixture of the anti- and syn-
isomers (87:13 based on the 1H NMR spectra of crude
product). The formation of oxime 12 was confirmed by 1H,
13C{1H} and 15N NMR spectroscopy. In the 1H−15N HMBC
spectrum of pure 12, isolated as the anti-isomer, we observed a
15N signal at 364 ppm that correlated to a proton resonating at
8.45 ppm which was assigned to the vinyl proton in the CH�
N−OH moiety (Figure S13). To reduce the oxime moiety to
the aminomethyl group, we investigated various reducing
reagents, however, the best results were obtained when 8 equiv
of ammonium formate and 6 equiv of zinc dust were used in
boiling methanol, which gave rac-2-aminomethyl-1-ferrocene-
carboxylic acid 13. The obtained acid was directly transformed

Figure 1. Methotrexate (MTX) and its derivatives 1−7 studied herein. AA = amino acid.

Scheme 1. Synthesis of rac-N-Fmoc-2-Aminomethylferrocene-1-carboxylic Acid 14

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into Fmoc-protected 14 in a reaction with an excess of Fmoc-
Cl and sodium carbonate as a base in a mixture of water−
acetonitrile. Precursor 14 was isolated in 13% overall yield after

chromatography on silica. The formation of the desired
compound was confirmed by 1H and 13C{1H} NMR
spectroscopy.

Scheme 2. Synthesis of Functionalized Resins 21−26

Scheme 3. Synthesis of Functionalized Resins 27−29 and the Ferrocenyl Conjugates 1−3

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The target compounds 1−7 were synthesized from di- and
tripeptides bound to the resin 24−29 or amino acid 30
(Schemes 2 and 3). The required resins were prepared starting
from 2-chlorotrityl resin 20 using the same procedure (Scheme
2) that involved first the reaction of 2-chlorotrityl resin with
Fmoc-protected amino acids 15−18 and diisopropylethyl-
amine (DIPEA) in dichloromethane for 16 h. Next, free
positions on 2-chlorotrityl resin were blocked with a capping
solution for 1 h, and the Fmoc group was removed in reaction
with 20% piperidine in DMF for 20 min, producing
monosubstituted resins 21 and 24−26. Further reaction of
resin 21 with Fmoc-Glu(OtBu)−OH 15 and HOBt and DIC
as a coupling agent at RT for 5 h, followed by deprotection
with 20% piperidine in DMF, resulted in the formation of
resin-dipeptide 22. Repeating the latter steps of conjugation of
22 with 15 allowed obtaining tripeptide-functionalized resin
23. The conjugation of resins 21−23 with 14 in the presence
of HOBt and DIC at RT for 5 h, followed by deprotection with
20% piperidine in DMF, resulted in the formation of the
ferrocenyl peptides 27−29 bound to resin. The target
compounds 1−6 were synthesized by conjugation of acid
MT−OH with resins 24−29 and HOBt and DIC at RT for 5
h. Products 1−6 were cleaved from the resin with trifluoro-
acetic acid:triisopropylsilane and water (9:0.5:0.5) for 1 h
(Scheme 3). Compound 7 was synthesized directly according
to a modified procedure25 in the reaction of acid MT−OH
with taurine 30 and HOBt and DIC as coupling agents
(Scheme 4). The obtained products 1−7 were isolated by
preparative high-performance liquid chromatography (HPLC).
The structures of the methotrexate conjugates were confirmed
by NMR and HPLC-mass spectrometry (HPLC-MS) analysis.
The LC−MS data collected for the ferrocenyl conjugates
showed singly and doubly charged ions. For compound 1 these
ions were detected at m/z 696.2 and 348.1 and assigned to [M
+ H]+ and [M + H]2+, respectively (Figure S1). Similar results
were obtained for 2, with ions detected at m/z 825.2 and 412.7
(Figure S2), and for 3, with ions detected at m/z 954.5 and
477.3 (Figure S3), assigned to [M + H]+ and [M + H]2+,
respectively. The organic methotrexate conjugates 4−6 gave
only singly charged ions attributed to [M + H]+ (Figures S4−
S6). HPLC-MS analysis also confirmed a purity higher than
95% for all target compounds. The formation of amide bonds
with the amino acids was confirmed by 1H−15N HSQC NMR
spectroscopy. In all cases, we observed the 15N signals at ca.

112−121 ppm indicative of amide bond formation (for the
1H−15N HSQC spectra see Supporting Information (SI)).
Dihydrofolate Reductase Inhibitory Activity. We

started the evaluation of the biological properties of the
investigated MTX derivatives by the assessment of their
inhibitory activity toward dihydrofolate reductase. All com-
pounds were applied at concentration of 0.01, 0.1, 1, and 10
μM and methotrexate was used as a reference (Figure 2 and

Table 1). Although the enzyme is quite abundant in human
cancer cell lines (Pusćion, Master of Science thesis), we
decided to use the recombinant human DHFR to avoid any
concurrent enzymatic activities (the assay is based on β-
NADPH oxidation in the presence of dihydrofolic acid and β-
NADPH is a cofactor of numerous oxidoreductases).

The ferrocenyl conjugates of methotrexate were significantly
less active (approximately 1 order of magnitude) than the
parent compound and even at the maximum concentration, the
DHFR inhibition was incomplete (the residual activity was
between 9 and 12%). The length of the poly(glutamate) chain
did not correlate with the inhibitory potential of the ferrocenyl
MTX derivatives. These results are contrary to observations
that the number of glutamate residues enhances the inhibitory

Scheme 4. Synthesis of the Conjugates 4−7

Figure 2. Inhibitory activity of the investigated methotrexate
derivatives 1−7 toward recombinant hDHFR in comparison to
MTX. No modulator was present in the control samples. Data are
presented as mean ± SD, n = 9 in the case of MTX, 6 for 1−4, and 3
in the case of the other compounds.

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potential of MTX, as it was previously demonstrated for
various animal liver DHFRs.28

To investigate the underlying reason for the reduced activity,
the methotrexate derivatives 1−7 were docked into the
methotrexate binding site of human dihydrofolate reductase
(PDB ID: 1U72) in comparison to methotrexate (Tables S1
and S2). The binding site comprises an inner hydrophobic
binding site and a peripheral hydrophilic binding pocket with
the substituted pteroyl moiety of methotrexate buried deep in
the pocket. The docking experiments for methotrexate
demonstrated that ChemPLP and Goldscore gave good
overlaps with the cocrystallized molecule (Table S1).

Compounds 1−3 can exist as two diastereomers (1-PR, 1-PS;
2-PR, 2-PS; and 3-PR, 3-PS) due to planar chirality about the
substituted ferrocenyl Cp ring, while the chiral centers at the
substituents are locked. Modeling showed that a variety of
binding modes are possible for the compounds. The two
diastereomers of 1, i.e., 1-PR and 1-PS featured in poses where
the ferrocenyl moiety was buried deep in the binding pocket
and largely overlapped. All docking scores showed a higher
affinity for the former, as was found for the diastereomers of 2.
However, their binding was significantly different with the
ferrocene moieties sitting on the protein surface and the
pteroyl moieties in the pocket. For compound 3, isomer 3-PS
gave slightly higher docking scores which were however lower
than those for the highest-scoring diastereomers of 1 and 2.
The pteroyl substituent of the 3-PR diastereomer showed
overlap with that of methotrexate, while that of 3-PS was found
on the protein surface. The ferrocenyl groups of both were
largely in the same position with the interactions between the
compounds and the protein established through π- and
hydrogen bonding networks (Figure 3). For example, the 3-
PR diastereomer showed π-interactions between the Phe34
phenyl substituent and the aromatic groups of the pteroyl and
benzamide moieties of the compound. Also, the pteroyl amino
groups of the compound formed hydrogen bonds with the
backbone carbonyl oxygen atoms of Val115 and Ile7 as well as
the side chain carboxylic oxygen atom of Glu30. In addition, a
carbonyl oxygen atom of 3-PR was involved in hydrogen bonds
with the side chain and backbone amino groups of Arg32 and
Asn64, respectively. Moreover, the hydroxyl groups were part
of hydrogen bonds with the backbone amine of Gln35 and the
backbone amine and carbonyl moieties of Asn64. Overall, it
can be seen that the introduction of the ferrocenyl moieties
had a substantial impact on the interaction with the protein
which may explain the effects seen, although the docking
scores suggest a more effective interaction.

The inhibition pattern of purely organic MTX derivatives
was more complex. The shortest side chain derivative 4
significantly reduced the enzymatic activity of DFHR even at
the lowest concentration used. On the other side, compounds
5 and 6 were less active, exhibiting inhibitory properties in the
micromolar range (Table 1). Rosowsky et al. demonstrated
that the distance between the pteroyl and α-carboxyl group of
the glutamyl residue is important for MTX interactions with
DHFR as increasing the number of γ-aminobutyryl inserts in
the side chain resulted in a gradual decrease of inhibitory
potency of such “stretched” methotrexate derivatives.24 This
effect was explained by the reduced availability of the carboxyl
group for the invariant arginine residue in the enzyme
molecule. However, the introduction of 1 or 2 GABA units
increased the DHFR IC50 value two- to 3-fold while the effects
observed here were at least 2 orders of magnitude more
pronounced for much shorter spacers. It can be, however,
hypothesized that the astonishing activity of 4 results from the
beneficial geometry of this molecule and stronger inhibitor-
enzyme interaction. Docking studies showed that the organic
molecules 4−7 gave similar or lower docking scores compared
to methotrexate and they were particularly outperformed by
the ferrocenyl derivatives in CHEMPLP and GS. This is
surprising given the prominent activity of 4 over the other
analogs and crystallization studies may be needed to eventually
elucidate this phenomenon. Additionally, poor activity of
taurine conjugate 7 indicates the importance of side chain
negative charge density and distribution.
Antiproliferative Activity. To explore the antiproliferative

potential of the synthesized compounds we used a set of four
cell lines previously employed to study the patterns of
multidrug resistance development in the presence of selected
chemotherapeutics,29 i.e., the colon cancer cell lines CT26.WT
(murine) and SW620 (human) and the skin cancer cell lines
B16−F10 (murine) and A-431 (human). Along with parental,
drug-sensitive cells, we also employed their MTX-resistant
variants (denoted with M). To avoid competition of folic acid
with the investigated compounds, we decided to use a folate-
free medium. However, our attempts to precondition the cells
by culturing them in such medium for 2−3 passages before the
actual experiment failed as such conditions turned out to be
harmful, especially for B16−F10 cells. Therefore, we limited
the preconditioning to washing off the normal culture medium
and cultivating the cells in folate-free medium for the duration
of the experiment (72 h). The results are presented in Table 2.

The investigated compounds exerted at best moderate
antiproliferative effects. Of the ferrocenyl MTX derivatives,

Table 1. DHFR Inhibitory Potential of Methotrexate and its
Derivativesa

IC50 [μM]

MTX 0.05
1 0.38
2 0.59
3 0.43
4 0.01
5 1.21
6 1.01
7 3.03

aIC50 values (expressed in μM) were determined based on data
presented in Figure 2.

Figure 3. Docked configuration of 3 in the methotrexate binding site
of human dihydrofolate reductase as predicted by ChemPLP (A),
with 3 occupying the pocket. The protein surface is rendered. Blue
depicts a hydrophilic region on the surface, a brown hydrophobic
region, and gray shows neutral areas. (B) Hydrogen bonds are shown
as green lines between 3 and the amino acids Arg32, Glu30, Gln35,
Phe34, Ile7, Asn64, and Val115.

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only 1 was potent enough to significantly influence the
proliferation of all cell lines studied although it was 5- to 10-
fold less active than the parent compound. Effects of its
diglutamyl derivative 2 were even less pronounced while
triglutamyl 3 was effective toward the most MTX-sensitive
CT26.WT and CT26.WTM cells only. These effects are
contrary to those obtained in the enzymatic assay, suggesting
that the bulkiness of the compound may limit its entry into the
cell. This is not unexpected as it is known that poly-
(glutamylated) folates have to be converted to the respective
monoglutamyl forms before being absorbed by the cell.30

Surprisingly, the most powerful DHFR inhibitor 4 completely
lacked antiproliferative potential in the cell-based assay, and of
the purely organic series only 6 and 7 had some mild effects on
cell viability. However, it must be emphasized, that despite the
relatively low activity, the observed antiproliferative effects
were virtually independent of the MTX-resistance status of the
cells. The differences in IC50 values for active compounds and
sensitive and resistant cells were not statistically significant as
corresponding confidence intervals overlapped or the direction
of effect was reverted (as in the case of CT26.WT and
CT26.WTM cells and compounds 6 and 7) which was also
accentuated by resistance factor values close to 1.

The cell lines differed significantly in terms of response to
the investigated compounds with CT26.WT is the most
susceptible and SW620 is the least sensitive. Therefore, we
focused on CT26.WT and its MTX-resistant counterpart as
research models in further studies.

Cell Cycle Modulatory Effects. To confirm the mode of
action of the most active ferrocenyl MTX derivatives, we
incubated CT26.WT cells for 24 and 72 h with the investigated
compounds at the low concentration (equal to IC75 for MTX)
and determined cell cycle phase distribution (Figure 4). In
control samples, over 64% of cells remained in the G1 phase,
about 16% of the cells were found in the S phase and 18−19%
were dividing (G2/M). The effect of solvent (DMSO) was
negligible. MTX exposure resulted in the reduction of the G1
cell fraction to 35% after 24 h and 32% after 72 h, and an
increase in the S phase fraction to, respectively, 38 and 43%,
with approximately 22−23% of mitotic cells. A similar situation
was observed for investigated MTX derivatives, with
approximately 40−49% of G1 cells, 30−40% of S cells, and
11−24% of G2/M cells, irrespective of the compound. These
results demonstrate that the antifolate mode of action
manifesting as stopping the cell division in the S phase due
to lack of DNA building blocks is preserved in MTX analogues.
Direct Effects on Abcc1 Activity. ABCC1/Abcc1

mediates drug resistance by eliminating drugs or their
metabolites from the cytoplasm, thus diminishing their
effective concentration. To inspect whether the effects of the
investigated compounds observed in the proliferation assay are
indeed transporter-dependent, we performed a real-time efflux
assay (Figure 5). The cells were loaded with a nonfluorescent
acetoxymethyl ester of 2′,7′-bis(2-carboxyethyl)-5-(and-6)-
carboxyfluorescein (BCECF-AM) which is intracellularly
hydrolyzed to brightly fluorescent BCECF. The latter, as a

Table 2. Antiproliferative Potential of Methotrexate and its Derivatives towards a Set of MTX-Sensitive and Resistant Cell
Lines, as Determined by the Neutral Red Uptake Assaya

A-431 A-431M B16−F10 B16−F10M CT26.WT CT26.WTM SW620 SW620M

MTX 11.1 22.1 2.0 5.3 0.2 0.7 8.0 32.6
8.7−14.1 19.6−24.9 1.6−2.5 3.8−7.5 0.1−0.3 0.5−1.0 5.8−11.0 25.3−43.2

1.99 2.65 3.50 4.08
1 62.2 ∼79.7 69.0 68.9 20.9 14.7 83.6 ≫100

57.3−68.2 66.0−72.9 65.6−72.7 14.7−30.4 9.6−23.5 78.3−91.2 N/A
1.28 1.00 0.70 N/A

2 ∼85.0 ∼93.8 ∼98.1 95.2 19.5 16.5 ≫100 ≫100
� � � 90.6−99.1 15.1−24.1 13.4−20.5 N/A N/A

1.10 0.97 0.85 N/A
3 ≫100 ≫100 ≫100 ≫100 61.6 47.2 ≫100 ≫100

N/A N/A N/A N/A 51.7−75.0 38.3−59.6 N/A N/A
N/A N/A 0.77 N/A

4 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100
N/A N/A N/A N/A N/A N/A N/A N/A

N/A N/A N/A N/A
5 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100 ≫100

N/A N/A N/A N/A N/A N/A N/A N/A
N/A N/A N/A N/A

6 ∼98.6 ≫100 ≫100 ≫100 80 67.5 ≫100 ≫100
� N/A N/A N/A 77.5−82.6 63.5−71.9 N/A N/A

N/A N/A 0.84 N/A
7 ∼85.8 ∼84.2 ∼86.7 ∼75.9 ∼86.9 65.6 ∼83.0 ∼96.9

� � � � � 60.7−71.0 � �
0.98 0.87 0.75 1.17

aIC50 values (expressed in μM) were determined based on three independent experiments. 95%-confidence intervals are given in middle rows
(please note that due to the log-transformation of the data required to perform IC50 calculations, these are asymmetrical) and resistance factors
(resistance factor is a ratio of IC50 for a given compound for resistant and sensitive line) for respective pairs are reported in lower rows. ≫100
denotes a situation in which IC50 values and corresponding confidence intervals could not be determined (<50% viability was not achieved in the
concentration range used). Tilde is used to indicate situations when the best-fit value could be found using the software but the confidence intervals
could not be precisely calculated (which is indicated by “�” sign).

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large organic anion, is unable to cross the plasma membrane by
diffusion and is retained in the cytoplasm. If ABCC1/Abcc1 is
active in the cell, the dye is actively exported to the external
milieu and the cell fluorescence decreases with time.31 If
ABCC1/Abcc1 activity is impeded by an inhibitor (or a
competing substrate), the rate of intracellular fluorescence
reduction is much lower. Thus, this assay allows for the
detection of direct interactions between a potential modulator
and the transporter molecule.

To ensure the intracellular concentration of MTX
derivatives, the cells were preincubated with 100 μM of the

investigated compounds for 1 h before the actual experiment.
The compounds were also present during the measurement.
Despite the high concentration used, the exposure time was
too short for the compounds to exert any detrimental effects
on the cells taking into account their mode of action.
Ferrocenyl MTX derivatives inhibited Abcc1 activity to an
extent comparable with MTX (approximately 55−73 vs 62% of
control, respectively). The GABA conjugate 4 was almost
inactive in terms of modulatory effects on the protein.
Considering the combined results of the proliferation and
transport assays, it may be inferred that the direct interaction
of the investigated compounds with the multidrug transporter
may contribute to overcoming the MTX-resistance phenom-
enon.

■ CONCLUSIONS
We report two series of methotrexate derivatives differing in
their side chain composition. Of the ferrocenyl conjugates, the
number of glutamyl moieties had no evident effect on DHFR
inhibitory potency, while was inversely proportional to their
antiproliferative effects. Of the amino acid conjugates, the
GABA-decorated pteroyl conjugate was an even more effective
DHFR inhibitor than MTX, while it had no effects on intact
cells. The ferrocenyl derivatives of methotrexate were able to
cross the drug resistance barrier formed by the ABCC1/Abcc1
protein. They were also able to directly interact with the
transporter molecule. The combined results emphasize the
importance of cellular folate uptake/export systems for the
biological activity of antifolates.

■ MATERIALS AND METHODS
General. All reactions were performed under an argon

atmosphere using the standard Schlenk technique. Solvents
were dried before use by distillation from sodium-benzophe-
none (tetrahydrofuran) or calcium hydride (dichloromethane).
Reagents (Sigma Aldrich or Fluorochem) were used as
received. One-dimensional (1D) and two-dimensional (2D)
NMR spectra were recorded on a Bruker Avance III 600 MHz
spectrometer at 294 K with spectrometer frequencies of 600.26
MHz for 1H and 150.94 MHz for 13C or Bruker Avance Neo
600 MHz spectrometer equipped with N2-cryoprobe with
spectrometer frequencies of 600.1 MHz for 1H and 150.0 MHz
for 13C. Chemical shifts were referenced to residual peaks in
DMSO-d6 δ = 2.51 ppm for 1H and δ = 39.5 ppm for 13C.
Chemical shifts are given in ppm and coupling constants in
Hertz (Hz).

The target compounds 1−7 were isolated using a Shimadzu
HPLC system on a semiprep column (Phenomenex PFP 150 ×
21.6 mm, 5 μm, flow rate 25 mL·min−1) with a diode array
detector (SPD-M20A) and a gradient of A:B starting from 0 to
100% of B within 30 min, where A is 0.1% TFA in H2O and B
is 0.1% TFA in MeCN. The purity of most of the compounds
was ≥95% as measured by HPLC-MS analysis on an analytical
Phenomenex XB-C18 column (50 × 4.6 mm, 1.7 μm) with a
mobile phase flow of 0.4 mL·min−1 using a Shimadzu Nexera
XR system equipped with SPD-M40 and LC−MS-2020
detectors using a gradient of phase A (water + 0.01% of
HCOOH) and phase B (methanol + 0.01% of HCOOH)
starting from 5% of B (t = 0 min), then 90% of B (t = 3 min),
hold time (t = 4 min), 5% of B (t = 4.5 min), stop analysis time
at t = 7 min. MT−OH was synthesized according to a reported

Figure 4. Effects of the investigated ferrocenyl methotrexate
derivatives 1−3 on cell cycle phase distribution in CT26.WT cells.
Upper panel −24 h exposure, lower panel −72 h exposure. Data are
presented as mean ± SD, n = 3.

Figure 5. Effects of the investigated methotrexate derivatives 1−4
(100 μM) on Abcc1 activity measured as the BCECF efflux. Data of a
representative experiment. The initial efflux rate is presented along
with standard error.

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procedure.32 Compound 7 was synthesized according to a
modified procedure.25

Synthesis of rac-2-Formyl-1-ferrocenecarboxylic Acid
11. This compound was synthesized according to a modified
known procedure.33 17.8 mL (24.9 mmol) of sec-BuLi (c = 1.4
M in cyclohexane) was added at −78°C within 10 min to a
solution of 6.15 g (22.6 mmol) of 9 in 90 mL of anhydrous
THF. The reaction mixture was warmed up to −10°C and
stirred for 1 h. The resulting suspension was cooled down to
−78°C and dry CO2 was bubbled through the reaction mixture
for 1.5 h. During the bubbling of CO2, the reaction mixture
was allowed to warm up to room temperature. After finishing
bubbling, a few pieces of dry ice (ca. 50 g) were added to the
reaction mixture very carefully to complete the reaction. The
resulting slurry was dissolved in 50 mL of THF and 100 mL of
1 M solution of hydrochloric acid was carefully added and
stirred for 30 min. The reaction mixture was basified using
sodium carbonate until pH = 10 and diluted with dichloro-
methane. The organic phase was washed twice with dichloro-
methane (2 × 100 mL) and the combined organic layers were
discarded. An aqueous layer was acidified to pH = 1 using
concentrated hydrochloric acid and the product was extracted
with dichloromethane (3 × 100 mL). The combined organic
layers were washed with brine, dried over anhydrous sodium
sulfate, filtered, and evaporated to dryness, giving 11 as a dark
red solid in 83% yield (4.85 g). 1H NMR (CDCl3) δ = 12.98
(br. s, 1H, COOH), 9.95 (s, 1H, CHO), 5.63 (s, 1H, Cp(H-
3)), 4.98−4.96 (m, 2H, Cp(H4,5)), 4.44 (s, 5H, Cp′).
13C{1H} NMR (CDCl3) δ = 197.2 (CHO), 169.5 (COOH),
80.5 (C3), 77.9 (C4 or 5), 76.4 (C4 or 5), 75.4 (C1), 73.3
(C2), 72.4 (Cp′). The NMR spectra were identical to those
reported in the literature.27

Synthesis of 2-(Hydroxyiminomethyl)-1-ferrocene-
carboxylic Acid 12. 6.40 g (92.1 mmol) of hydroxylamine
hydrochloride followed by 17.1 mL (101 mmol) of triethyl-
amine were added to a solution of 7.93 g (30.7 mmol) of 11 in
154 mL of absolute ethanol at room temperature under argon
atmosphere and the resulting mixture was stirred at RT for 16
h. Then, the reaction mixture was quenched by adding 100 mL
of 1 M hydrochloric acid and additionally acidified using
concentrated hydrochloric acid to pH = 1. The crude product
was extracted with dichloromethane (3 × 50 mL) and
combined organic solutions were washed with brine, dried
over anhydrous sodium sulfate, filtered, and evaporated to
dryness. The crude product was purified by flash chromatog-
raphy on silica gel using a gradient of methanol in
dichloromethane (starting from 2 to 10% methanol) and
afforded the desired 12 as a dark red solid in 92% yield (7.65
g) which was used in the next step. An analytically pure sample
of 12 was obtained by chromatography on silica using a
gradient of methanol in dichloromethane starting from 1 to 3%
of methanol. ESI-MS calculated for C12H12FeNO3 m/z = 274.0
[M + H]+ found m/z = 274.1 [M + H]+. Elemental analysis
calculated for C12H11FeNO3 C 52.8, H 4.1, N 5.1%, found C
52.9; H 4.3, N 5.2%. 1H NMR (DMSO-d6) δ = 12.52 (br. s,
1H, COOH), 10.82 (s, 1H, CH�NOH), 8.45 (s, 1H, CH�
NOH), 4.862 (s, 1H, Cp), 4.858 (s, 1H, Cp), 4.59 (t, 1H, J =
2.4 Hz, Cp), 4.23 (s, 5H, Cp′). 13C{1H} NMR (DMSO-d6) δ
= 172.1 (COOH), 146.8 (CH�NOH), 79.1 (s, Cpipso), 72.1
(Cp), 71.2 (Cp), 70.6 (Cp′), 70.4 (Cpipso), 68.8 (Cp); 15N
(DMSO-d6) δ = 364 (based on 1H−15N HMBC).
Synthesis of rac-2-(((((9H-Fluoren-9-yl)methoxy)-

carbonyl)amino)methyl)-1-ferrocenecarboxylic Acid

14. To a solution of 2.05 g (7.51 mmol) of oxime 12 in 15
mL of anhydrous methanol at RT under argon atmosphere
2.95 g (45.1 mmol) of powdered zinc followed by 3.79 g (60.1
mmol) of ammonium formate were added and the resulting
mixture was refluxed for 16 h. Next, the reaction mixture was
filtered through Celite and evaporated to dryness giving crude
13. Next, 2.33 g (9.01 mmol) of Fmoc chloride followed by 4.0
g (37.5 mmol) of sodium carbonate was added to the solution
of crude 13 in 50 mL of water and 50 mL of acetonitrile and
the resulting solution was stirred at room temperature for 20 h.
Then, 1.0 mL of DIPEA and the second portion of 1.0 g (3.86
mmol) of Fmoc chloride were added and the resulting mixture
was stirred for an additional 20 h. The reaction was quenched
with 50 mL of 1 M hydrochloric acid solution and the crude
product was extracted with dichloromethane (3 × 50 mL).
Chromatography on silica gel using the gradient of methanol in
dichloromethane (starting from 2% up to 10% methanol)
afforded 9 in 13% yield (0.485 g) as a yellow solid. ESI-MS
calculated for C27H24FeNO4 m/z = 482.1 [M + H]+ found m/z
= 482.3 [M + H]+. 1H NMR (DMSO-d6) δ = 12.24 (br. s, 1H,
COOH), 7.89 (d, J = 7.5 Hz, 2H, CHAr), 7.73 (t, J = 6.7 Hz,
2H, CHAr), 7.54 (t, J = 5.9 Hz, 1H, NH), 7.42 (t, J = 7.4 Hz,
2H, CHAr), 7.35−7.32 (m, 2H, CHAr), 4.64 (s, 1H, Cp), 4.42−
4.32 (m, 5H, CH2NHCOOCH2 (3H) and Cp (2H)), 4.24 (t, J
= 6.8 Hz, 1H, CH2CHFmoc), 4.18−4.13 (m, 1H, CH2NH)
overlapped with 4.16 (s, 5H, Cp′); 13C{1H} NMR (DMSO-d6)
δ = 172.8 (COOH), 156.2 (NHCO), 143.9 (2 × CAr), 140.7
(CAr), 127.6 (CHAr), 127.0 (2 × CHAr), 125.2 (2 × CHAr),
120.1 (CHAr), 88.8 (Cpipso), 72.1, 71.4 (Cp), 70.5 (Cp), 70.1
(Cp′), 70.0, 69.2 (Cpipso), 68.9 (Cp), 65.3 (CH2NH), 46.8
(CH), 38.6 (OCOCH2).
General Procedure A�Conjugation of the Resin 20

with Fmoc-Protected Amino Acids 15−18�Synthesis
of Resins 21, 24−26. 2.5 equiv of DIPEA was added to a
slurry of the amino acid (2.5 equiv) in DCM and the obtained
mixture was stirred until the amino acid was completely
dissolved. The resulting solution was immediately added to the
2-chlorotrityl resin, pretreated with DCM for 30 min, and the
mixture was reacted for 16 h. After removal of the reagents by
suction, the obtained resin was washed with DCM and treated
with the capping mixture (DCM-MeOH-DIPEA, 17:2:1) for 1
h. Then the solution was removed, the resin was washed with
DCM and DMF and the Fmoc group was removed by treating
the resin with 20% piperidine in DMF (twice, each cycle 20
min). The obtained resin was washed 3 times with DMF,
DCM, and methanol and dried under vacuum for 1 h.

Resin 21 was synthesized according to General Procedure
A starting from 1.0 g (1.6 mmol) of 2-chlorotrityl resin 20, 1.7
g (4.0 mmol) of 15, 517 mg (700 μL, 4.0 mmol) of DIPEA,
and 15 mL of DCM.

Resin 24 was synthesized according to General Procedure
A starting from 0.25 g (0.4 mmol) of 2-chlorotrityl resin 20,
0.325 g (1.0 mmol) of 16, 129 mg (175 μL, 1.0 mmol) of
DIPEA, and 5 mL of DCM.

Resin 25 was synthesized according to General Procedure
A starting from 0.50 g (0.8 mmol) of 2-chlorotrityl resin 20,
0.706 g (2.0 mmol) of 17, 258 mg (350 μL, 2.0 mmol) of
DIPEA, and 10 mL of DCM.

Resin 26 was synthesized according to General Procedure
A starting from 0.25 g (0.4 mmol) of 2-chlorotrityl resin 20,
0.381 g (1.0 mmol) of 18, 129 mg (175 μL, 1.0 mmol) of
DIPEA, and 5 mL of DCM.

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General procedure B�Synthesis of Peptides: Con-
jugation of Resin 21 with Amino Acids�Preparation of
Resins 22−23 and 27−29. DMF solutions containing 2.5
equiv of conjugated amino acids, HOBt and DIC were added
to resin 21 (pretreated with DCM for 30 min) and the
obtained mixture was reacted at RT for 4 h. Then, the excess
reagents were removed by suction, the resin was washed 3
times with DMF and the Fmoc group was removed by treating
the resin with 20% piperidine in DMF (twice, each cycle 20
min). The obtained resin was washed 3 times with DMF,
DCM, and methanol and dried under vacuum for 1 h.

Resin 22 was synthesized according to General Procedure
B starting from 3.18 g of resin 21, 5.11 g (12.0 mmol) of 15,
1.62 g (12.0 mmol) of HOBt, and 1.52 g (1.88 mL, 12.0
mmol) of DIC in 10 mL of DMF.

Resin 23 was synthesized according to General Procedure
B starting from 1.62 g of resin 22, 2.72 g (6.4 mmol) of 15,
0.865 g (6.4 mmol) of HOBt, and 0.808 g (1.0 mL, 6.4 mmol)
of DIC in 10 mL of DMF.

Resin 27 was synthesized according to General Procedure
B starting from 0.22 g of resin 21, 0.385 g (0.80 mmol) of 14,
0.108 g (0.80 mmol) of HOBt, and 101 mg (125 μL, 0.80
mmol) of DIC in 1.0 mL of DMF.

Resin 28 was synthesized according to General Procedure
B starting from 0.22 g of resin 22, 0.385 g (0.80 mmol) of 14,
0.108 g (0.80 mmol) of HOBt, and 101 mg (125 μL, 0.80
mmol) of DIC in 1.0 mL of DMF.

Resin 29 was synthesized according to General Procedure
B starting from 0.22 g of resin 23, 0.385 g (0.80 mmol) of 14,
0.108 g (0.80 mmol) of HOBt, and 101 mg (125 μL, 0.80
mmol) of DIC in 1.0 mL of DMF.
General Procedure C�Conjugation of Resins 24−29

with MT−OH�Preparation of Methotrexate Deriva-
tives 1−6. The respective resin was pretreated with DCM for
30 min, the solvent was removed by suction and the resin was
washed with DMF and DMSO. Then, a solution of 2.5 equiv of
MT−OH and 2.5 equiv of HOBt in DMF followed by 2.5
equiv of DIC were added to the obtained resin. The obtained
mixture was reacted at RT for 48 h and the excess of the
reagents was removed by suction. The obtained resin was
washed with DMF, DCM, and methanol and dried under
vacuum for 1 h. Next, the products were cleaved off the resin
by treatment with a mixture of TFA-TIPS-H2O (9:0.5:0.5) for
1 h. Next, the solution was evaporated, the residue dissolved in
DMSO and the compounds were purified by HPLC. Fractions
containing the product were combined and freeze-dried.
Synthesis of 1. Compound 1 (31 mg) was synthesized

according to General Procedure B starting from resin 27 and
286 mg (0.88 mmol) of MT−OH, 119 mg (0.88 mmol) of
HOBt, and 111 mg (137 μL, 0.880 mmol) of DIC in 2.00 mL
DMF. Rf (HPLC) τ = 4.62 min, purity 98.4%; ESI-MS (m/z)
calculated for C32H34FeN9O6 m/z 696.2 [M + H]+, found m/z
696.2 [M + H]+ and 348.1 [M + H]2+. 1H NMR (600 MHz,
DMSO-d6) δ = 13.02−12.18 (br m, COOH), 9.20 (br s, 1H,
NH), 9.0 (br s, 1H, NH), 8.72 (s, 1H, CHAr), 8.56 (t, J = 5.9
Hz, 1H, NH), 8.47 (t, J = 6.1 Hz, 1H, NH), 8.18 (d, J = 7.8
Hz, 0.5H, NH), 8.00 (d, J = 7.8 Hz, 0.5H, NH), 7.71 (t, J = 8.4
Hz, 2H, 1,4-C6H4), 7.52 (br. s, 1H, NH), 6.84−6.83 (m, 2H,
1,4-C6H4), 4.94 (br s, 0.5H, Cp), 4.91 (br s, 0.5H, Cp), 4.86
(s, 2H, CH2−N(CH)3), 4.53 (dt, J = 6.3, 15.1 Hz, 1H, CH2),
4.45−4.34 (m, 3H, CH2 and CH), 4.27−4.26 (m, 1H, Cp),
4.22 (s, 2.5H, Cp′), 4.10 (s, 2.5H, Cp′), 3.24 (s, 3H, CH2−

N(CH)3), 2.43 (t, J = 7.3 Hz, 1H, CH2), 2.37−2.34 (m, 1H,
CH2), 2.17−2.21 (m, 1H, CH2), 1.99−1.93 (m, 1H, CH2).

Synthesis of 2. Compound 2 (56 mg) was synthesized
according to General Procedure B starting from resin 28 and
286 mg (0.88 mmol) of MT−OH, 119 mg (0.88 mmol) of
HOBt and 111 mg (137 μL, 0.880 mmol) of DIC in 3.00 mL
DMF. Rf (HPLC) τ = 4.7 min, purity 98.8%; ESI-MS (m/z)
calculated for C37H41FeN10O9 m/z 825.2 [M + H]+, found m/
z 825.2 [M + H]+ and 412.7 [M + H]2+. 1H NMR (600 MHz,
DMSO-d6) δ = 13.00−12.16 (br m, COOH), 9.23 (br s, 1H,
NH), 9.03 (br s, 1H, NH), 8.72 (s, 1H, CHAr), 8.56 (t, J = 6.0
Hz, 1H, NH), 8.47 (t, J = 5.9 Hz, 1H, NH), 8.28 (d, J = 7.5
Hz, 0.5 H, NH), 8.18 (d, J = 7.8 Hz, 0.5 H, NH), 8.16 (d, J =
7.8 Hz, 0.5 Hz, NH), 8.10 (d, J = 7.6 Hz, 0.5 Hz, NH), 7.72 (d,
J = 8.8 Hz, 1H, 1,4-C6H4), 7.71 (d, J = 8.8 Hz, 1H, 1,4-C6H4),
7.51−7.45 (m, 1H, 1,4-C6H4), 6.84 (d, J = 9.1 Hz, 1H, 1,4-
C6H4), 6.84 (d, J = 9.1 Hz, 1H, 1,4-C6H4), 4.95−4.94 (m, 0.5
H, Cp), 4.90−4.89 (m, 0.5H, Cp), 4.86 (s, 2H, CH2−
N(CH)3), 4.54−4.49 (m, 1H, CH2), 4.41−4.31 (m, 3H, CH2
and CH), 4.27−4.22 (m, 5 H, Cp′ and CH), 4.16 (s, 2H, Cp′),
3.24 (s, 3H, CH2−N(CH)3), 2.38−2.32 (m, 4H, CH2), 2.11−
2.07 (m, 1 H, CH2), 2.00−1.92 (m, 2H, CH2), 1.78−1.74 (m,
1H, CH2).

Synthesis of 3. Compound 3 (15 mg) was synthesized
according to General Procedure B starting from resin 29 and
286 mg (0.88 mmol) of MT−OH, 119 mg (0.88 mmol) of
HOBt and 111 mg (137 μL, 0.880 mmol) of DIC in 3.00 mL
DMF. Rf (HPLC) τ = 4.87 min, purity 90.1%; ESI-MS (m/z)
calculated for C42H48FeN11O12 m/z 954.3 [M + H]+, found m/
z 954.5 [M + H]+ and 477.3 [M + H]2+. 1H NMR (600 MHz,
DMSO-d6) δ 12.52 (br s, COOH), 9.22 (br s, 1H, NH), 9.02
(br s, 1H, NH), 8.72 (s, 1H, CHAr), 8.55 (t, J = 6.0 Hz, 0.5H,
NH), 8.46 (t, J = 5.9 Hz, 0.5H, NH), 8.31 (d, J = 7.5 Hz, 0.5H,
NH), 8.22−8.14 (m, 1.5 H, NH), 8.10 (d, J = 7.6 Hz, 1H,
NH), 8.09 (d, J = 8.5 Hz, 1H, NH), 7.72 (d, J = 8.8 Hz, 1H,
1,4-C6H4), 7.71 (d, J = 8.8 Hz, 1H, 1,4-C6H4), 7.42 (br s, 1H,
NH), 6.85−6.83 (m, 2H, 1,4-C6H4), 4.94 (br s, 0.5H, Cp),
4.90 (br s, 0.5H, Cp), 4.86 (s, 2H, CH2−N(CH)3), 4.55−4.50
(m, 1H, CH2), 4.41−4.31 (m, 3H, CH2 and CH), 4.27−4.26
(m, 2H, Cp), 4.22 (s, 3H, Cp′), 4.21−4.17 (m, 6H, Cp′ and
CH), 3.24 (s, 3H, CH2−N(CH)3), 2.39−2.17 (m, 6H, CH2),
2.10−2.07 (m, 1H, CH2), 2.00−1.91 (m, 4H, CH2), 1.78−1.72
(m, 2H, CH2).

Synthesis of 4. Compound 4 (56 mg) was synthesized
according to General Procedure B starting from resin 24 and
324 mg (1.00 mmol) of MT−OH, 135 mg (1.00 mmol) of
HOBt, and 126 mg (156 μL, 1.00 mmol) of DIC in 10.00 mL
DMF. Rf (HPLC) τ = 3.52 min; purity 95.2%; ESI-MS (m/z)
calculated for C19H23N8O3 m/z 411.2 [M + H]+, found m/z
411.1 [M + H]+. 1H NMR (600 MHz, DMSO-d6) δ 12.94 (br
s, 1H), 12.01 (s, 1H), 9.12 (br s, 1 H, NH), 8.93 (br s, 1H,
NH), 8.70 (s, 1H, CHAr), 8.11 (t, J = 5.6 Hz, 1H, NHCO),
7.69 (d, J = 8.9 Hz, 2H, 1,4-C6H4), 7.38 (br s, 1H, NH), 6.80
(d, J = 9.0 Hz, 2H, 1,4-C6H4), 4.85 (s, 2H, CH2−N(CH)3),
2.23 (s, 3H, CH2−N(CH)3), 3.21 (q, J = 6.3 Hz, 2H, CH2),
2.23 (t, J = 7.4 Hz, 2H, CH2), 1.72−1.68 (m, 2H, CH2).

Synthesis of 5. Compound 5 (33 mg) was synthesized
according to General Procedure B starting from resin 25 and
648 mg (2.00 mmol) of MT−OH, 270 mg (2.00 mmol) of
HOBt, and 252 mg (313 μL, 2.00 mmol) of DIC in 7.0 mL
DMF. Rf (HPLC) τ = 3.92 min; purity 98.0%; ESI-MS (m/z)
calculated for C21H27N8O3 m/z = 439.2 [M + H]+, found m/z
= 439.3 [M + H]+. 1H NMR (600 MHz, DMSO-d6) δ = 11.91

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(br s, 1H), 8.64 (s, 1H, CHAr), 8.61 (br s, 1H, NH),. 8.40 (br
s, 1H, NH), 8.07 (t, J = 5.6 Hz, 1H, NHCO), 7.68 (d, J = 8.9
Hz, 2H, 1,4-C6H4), 7.26 (br s, 1H, NH), 6.80 (d, J = 9.0 Hz,
2H, 1,4-C6H4), 4.82 (s, 2H, CH2−N(CH)3), 3.22 (s, 3H,
CH2−N(CH)3), 3.18 (q, J = 6.6 Hz, 2H, CH2), 2.19 (t, J =
7.38 Hz, 2H, CH2), 1.52−1.44 (m, 4H, CH2), 1.30−1.24 (m,
2H, CH2).
Synthesis of 6. Compound 5 (65 mg) was synthesized

according to General Procedure B starting from resin 26 and
324 mg (1.00 mmol) of MT−OH, 135 mg (1.00 mmol) of
HOBt, and 126 mg (156 μL, 1.00 mmol) of DIC in 10.00 mL
DMF. Rf (HPLC) τ = 5.10 min; purity 94.7%; ESI-MS (m/z)
calculated for C23H31N8O3 m/z = 467.2 [M + H]+, found m/z
= 467.4 [M + H]+. 1H NMR (600 MHz, DMSO-d6) δ 11.09
(br s, 1H), 9.19 (br s, 1H, NH), 8.99 (br s, 1H, NH), 8.70 (s,
1H, CHAr), 8.49 (br s, 1H, NH), 8.05 (t, J = 5.6 Hz, 1H,
NHCO), 7.68 (d, J = 9.0 Hz, 2H, 1,4-C6H4), 7.44 (br s, 1H,
NH), 6.80 (d, J = 9.0 Hz, 2H, 1,4-C6H4), 4.85 (s, 2H, CH2−
N(CH)3), 3.23 (s, 3H, CH2−N(CH)3), 3.18 (q, J = 6.6 Hz,
2H, CH2), 1.50−1.45 (m, 4H, CH2), 1.26 (br s, 6H, CH2).
Dihydrofolate Reductase Assay. The recombinant

human dihydrofolate reductase (DFHR) was purchased from
R&D (Bio-Techne, Minneapolis, Minnesota) and its activity in
the presence of the synthesized compounds was assayed as per
the manufacturer’s instructions. The assay buffer consisted of
50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 25 mM
tris(hydroxymethyl)aminomethane (Tris), 100 mM NaCl, 25
mM ethanolamine, 2 mM dithiotreitol, 0.1 mM dihydrofolic
acid and 0.125 mM β-NADPH. The final enzyme concen-
tration was 1 μg/mL. The absorbance at 340 nm was
monitored over 15 min at 37°C. The initial rate of absorbance
decrease over time (up to 5 min) was considered as the
measure of DHFR activity as per the manufacturer’s
instructions.
Molecular Modeling. Compounds 1−7 were docked into

the crystal structure of human dihydrofolate reductase (PDB
ID: 1U72, resolution 1.90 Å),34 which was obtained from the
Protein Data Bank (PDB).35,36 For 1−3, both diastereomers
with regard to the difference in planar chirality about the
substituted ferrocenyl Cp ring were investigated, i.e., 1-PR, 1-
PS, 2-PR, 2-PS, 3-PR, 3-PS. Scigress version FJ 2.637 was used to
prepare the crystal structure for docking, i.e., hydrogen atoms
were added and the cocrystallized methotrexate was removed.
The center of the binding site was defined as the carbon atom
on the phenyl ring adjacent to the carbonyl carbon of
methotrexate (x = 32.139, y = 18.3040, z = 0.049) with a
radius of 10 Å. The scoring functions GoldScore (GS)38 and
ChemScore (CS),39,40 ChemPLP41 and Astex statistical
potential (ASP)42 were implemented to validate the predicted
binding modes and relative energies of the ligands using the
GOLD v5.4 software suite. The cocrystallized ligand
methotrexate was first docked, and root-mean-square deviation
(RMSD) values were calculated for the heavy atoms. ASP
obtained an average RMSD of 1.8757, ChemPLP of 0.8191,
CS of 3.8814, and GS of 0.8149 which indicate the strong
prediction power of the ChemPLP and GS scoring functions.
The RMSD and docking scores are given in Tables S1 and S2,
respectively.
Cell Culture. Parent cell lines: A-431 (human epidermoid

carcinoma), SW620 (human colorectal adenocarcinoma),
B16−F10 (murine melanoma), and CT26.WT (murine
colon carcinoma) were purchased from the American Type
Culture Collection and cultured in standard cell culture

conditions (37°C, 5% CO2, 95% relative humidity). The
methotrexate-resistant sublines were obtained as previously
described.29 The cells were grown in high-glucose Dulbecco’s
Modified Eagle’s Medium (DMEM) buffered with HEPES and
supplemented with Glutamax-I (Thermo Fisher Scientific Inc.,
Waltham, MA) with the addition of 10% v/v fetal bovine
serum (EURx, Gdanśk, Poland). Care was taken to avoid
cross-contamination between the cell lines (handling one cell
line at a time, using antiaerosol tips and single-use equipment
were the minimum safety conditions). The cells were tested
every 3 months for Mycoplasma contamination with a
MycoProbe Mycoplasma Detection Kit by R&D (Bio-Techne,
Minneapolis, Minnesota).
Proliferation Assay. The antiproliferative potential of the

investigated compounds was determined using the neutral red
uptake assay.43 Cells were washed (100 × g, 10 min, RT) and
suspended in a custom-ordered folate-free DMEM supple-
mented with L-alanyl-L-glutamine and sodium pyruvate
(Biowest, Nuaille,́ France) with the addition of 10% v/v fetal
bovine serum (FBS; EURx, Gdanśk, Poland). Then they were
seeded in 96-well plates at densities of 104/well (human cell
lines) or 5 × 104/well (murine cell lines) in a final volume of
100 μL in the folate-free complete medium and allowed to
attach for 24 h. Stock solutions of synthesized compounds
were freshly prepared in dimethyl sulfoxide (DMSO) and
appropriately diluted in folate-free complete medium to the
desired concentration. 100 μL of such solutions were added to
the respective wells of the 96-well plate. The final DMSO
concentration was equal in all samples, including controls, and
did not exceed 0.1% v/v as it was determined to be nontoxic to
the cells. After 70 h of incubation, neutral red was added to the
medium to a final concentration of 1 mM. After another 2 h of
incubation, the cells were washed twice with phosphate-
buffered saline (PBS), destained in 200 μL extraction solution
(1% acetic acid (v/v) in 50% ethanol (v/v)), and shaken for 10
min, until the dye was released from the cells. The absorbance
was measured at 540 nm with an EnVision Multilabel Plate
Reader (PerkinElmer, Waltham, Massachusetts). The results
were calculated as the percentage of the controls and the IC50
values for each cell line and substance were calculated with
GraphPad Prism 10.2.1 software (GraphPad Software, LLC,
San Diego, California) using a four-parameter nonlinear
logistic regression.
Cell Cycle Analysis. CT26.WT were seeded in 6-well

plates at a density of 105 cells per well in a complete culture
medium. On the next day (the time necessary for the cells to
attach to the surface), the medium was replaced with folate-
free DMEM supplemented with 10% FBS, and the test
compounds were administered at a concentration equal to the
IC75 value of MTX in CT26.WT cells (30 nM). After 24 or 72
h, the cells were trypsinized and fixed with ice-cold 70% v/v
ethanol. Following rehydration in PBS, the cells were stained
with 75 μM propidium iodide and 50 Kunitz unit/mL of
RNase A in PBS for 30 min at 37°C and stored at 4°C until
measurement. All samples were analyzed using a BD
FACSymphony A1 cell analyzer (Becton Dickinson) at 561
nm excitation and 570/30 nm emission. Doublet discrim-
ination and final analysis of the cellular DNA content
distribution were determined using a built-in cell cycle module
(Watson pragmatic algorithm) by FlowJo 7.6.1 software.
BCECF Extrusion Assay. Following trypsinization,

CT26.WT and CT26.WTM cells were suspended in the
folate-free FBS-free DMEM supplemented with 100 μg/mL

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soybean trypsin inhibitor. The suspension was then centrifuged
(100 × g, 10 min, RT) and the cell pellet was resuspended in
folate-free DMEM to a final cell concentration of 2 × 106/mL.
To 750 μL of cell suspension, MTX or one of its derivatives
(DMSO in a control sample) was added to a final
concentration of 100 μM, and the samples were incubated at
37°C with continuous shaking at 500 rpm for 50 min. Then,
250 μL of 4 μM BCECF-AM was added to each sample, and
the incubation was continued for another 10 min. The samples
were then centrifuged (100 × g, 10 min, 4°C) and the
supernatant was carefully aspirated. 750 μL of ice-cold, folate-
free DMEM supplemented with 100 μM MTX or its
derivatives (DMSO in the control sample) was used to
resuspend the cells. Intracellular BCECF fluorescence was then
immediately measured at 488 nm excitation and 530/30 nm
emission (time 0) on a BD FACSymphony A1 cell analyzer
(Becton Dickinson). Further measurements were performed at
approximately 20-min intervals (an exact measurement time
was recorded by the instrument). All samples were incubated
at 37°C between measurements. Extrusion curves were plotted
and the rate of dye efflux was assessed with the GraphPad
Prism 10.2.1 software (GraphPad Inc.).

■ ASSOCIATED CONTENT

*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.4c03602.

HPLC-MS analysis data; NMR spectra and molecular
docking parameters (PDF)

■ AUTHOR INFORMATION

Corresponding Authors
Damian Plazu̇k − Department of Organic Chemistry, Faculty

of Chemistry, University of Lodz, 91-403 Łódź, Poland;
orcid.org/0000-0002-2898-6604;

Email: damian.plazuk@chemia.uni.lodz.pl
Błazėj Rychlik − Cytometry Lab, Department of Oncobiology

and Epigenetics, Faculty of Biology and Environmental
Protection, University of Lodz, 90-236 Łódź, Poland;

orcid.org/0000-0001-8928-5900; Email: blazej.rychlik@
biol.uni.lodz.pl

Authors
Karolina Rózga − Department of Organic Chemistry, Faculty

of Chemistry, University of Lodz, 91-403 Łódź, Poland
Andrzej Błauz ̇ − Cytometry Lab, Department of Oncobiology

and Epigenetics, Faculty of Biology and Environmental
Protection, University of Lodz, 90-236 Łódź, Poland

Daniel Moscoh Ayine-Tora − School of Chemical Sciences,
University of Auckland, Auckland 1142, New Zealand;
Department of Chemistry, University of Ghana, LG 56
Legon-Accra, Ghana

Ernest Pusćion − Cytometry Lab, Department of Oncobiology
and Epigenetics, Faculty of Biology and Environmental
Protection, University of Lodz, 90-236 Łódź, Poland

Christian G. Hartinger − School of Chemical Sciences,
University of Auckland, Auckland 1142, New Zealand;

orcid.org/0000-0001-9806-0893
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.4c03602

Author Contributions
K.R. synthesized the compounds. A.B. performed the cell cycle
assay and analyzed the data. E.P. performed the DHFR assay.
D.M.A.T. and C.H. performed the molecular modeling. D.P.
planned the chemical portion of the study, analyzed the results,
and coprepared the manuscript. B.R. planned the biological
portion of the study, performed proliferation, DHFR and
transport assays, analyzed the results, and coprepared the
manuscript. All authors contributed to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This study was financially supported by the Polish National
Science Centre (NCN) grant No. 2015/17/B/NZ7/03011.

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