Synthesis, characterization, and anticancer activity of folate γ-ferrocenyl conjugates

Article abstract of DOI:10.1039/c9nj04954a

The quest for alternative therapeutic agents with safe and effective profiles is one of the biggest challenges in oncology. Folate-targeted therapy can deliver drugs selectively into malignant cells via the Frα receptor, whereas bioorganometallic chemistr

Full text of DOI:10.1039/c9nj04954a

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g
Synthesis, Characterization, and Anticancer Activity of Folate -
Ferrocenyl Conjugates†
Diego L. Bertuzzi,a Gabriel Perli,a Carolyne B. Braga,a and Catia Ornelasa*
Received 00th January 20xx,
Accepted 00th January 20xx
The quest for alternative therapeutic agents with safety and effective profiles is one of the biggest challenges in oncology.
The folate-targeted therapy can deliver drugs selectively into malignant cells via the Fra receptor, whereas the
bioorganometallic chemistry offers potential new drug candidates. Herein, novel folate g-ferrocene (g-Fc) conjugates were
synthesized through a regiospecific route by reacting amino-terminated glutamate (Glu) g-Fc residues with pteroyl azide (68-
78% yield), and their in vitro anticancer activities were evaluated on human cells. The Fc units were attached to protected
Glu residues through different linkers using amide coupling and/or “click” reactions. Cyclic voltammetry and UV-vis
measurements showed that the triazole group conjugated to the Fc donates electrons to the ferrous center, which facilitates
the oxidation of Fc that is responsible for the antiproliferative activity. These results were confirmed by the higher activity
of folate g-triazoleFc 21, with IC50 values at 25.4 µM (± 3.2) on HeLa and 21.7 µM (± 0.3) on MCF-7 cells. This compound was
up to 4-fold less toxic on healthy PNT2 cells and Fra-negative PC-3 cancer cells. Blocking experiments with excess of free
folic acid inhibited the folates activity suggesting their specific uptake via Fra. Glu residues analogs of folates but lacking the
pteroyl moiety were much less toxic on cancer cells than folates. Hence, folates g-Fc are potential anticancer drug candidates
due to their selectivity and enhanced cytotoxicity against Fra-positive malignant cells.
DOI: 10.1039/x0xx00000x
Toxicity of Fc compounds arises from the mild and reversible
oxidation of Fc units yielding ferrocenium (Fc+) cations.23, 24 This
cation generates reactive oxygen species (ROS) in physiological
Introduction
Bioorganometallic chemistry has brought new alternatives for
medicinal chemistry via application of organometallics as
therapeutic agents.1-3 Ferrocene (Fc) derivatives have played a
significant role in this area, being applied as antianemic drugs4
and antiproliferative agents against fungal and bacterial
infections,5-8 malaria,9 human immunodeficiency virus,10 and
cancer.7, 11-13 These therapeutical applications are enabled by
the exceptional electronic properties of Fc, relatively easy
synthesis of derivatives, and high stability in aqueous and
aerobic media.14-17
medium, which damage DNA and other essential biomolecules,
culminating in cell apoptosis.23, 24 Potent cytotoxic effects can
be observed when Fc is attached to biologically active
molecules.13, 16, 18, 25 Moreover, functional groups adjacent to
the Fc unit have a strong influence on the oxidation process by
reducing the redox potential (Ep).16, 26-28 These groups can also
interact with biomolecules through secondary interactions to
facilitate the charge transfer process and enhance its toxicity.16,
25
In this context, the triazole scaffold may be crucial in Fc-
bioconjugates for anticancer purposes. Triazole group can be
used as bioisostere in medicinal chemistry, it is highly stable,
and it can interact through hydrogen bonds mimicking amide
groups.29-32 Moreover, 1,4-disubstituted 1,2,3-triazole can be
easily obtained using copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) “click” reaction.32
Although we had decades of tremendous progress in cancer
research, conventional oncolytic agents are commonly
associated with significant side effects.33 In this context, the
folate-targeted therapy has induced high expectations to
improve the therapeutic index of drugs.33, 34 This approach relies
on the upregulated expression of the folate receptor alpha
Ferrocenyl compounds have shown remarkable in vitro and in
vivo anticancer activities,18, 19 enabling their use to treat certain
types of cancer, including lymphocytic leukemia P-388, breast
and colon cancer.16, 20 Ferrocifen is one of the most relevant Fc-
based anticancer drug, in which the b-phenyl ring of tamoxifen
– a well-known drug for the treatment of hormone-responsive
(ER+) breast cancer – is replaced by a Fc moiety.21, 22 Ferrocifen
has shown excellent results on ER+ and ER- breast cancer, with
activity 60-fold higher (IC50 = 0.5
line)22 than tamoxifen (IC50 = 29
µ
µ
mol against MDA-MB-231 cell
mol against MDA-MB-231).21
protein (Fra) in many epithelial tumor cells, including ovary,
uterus, endometrium, brain, kidney, lung and breast.35-37
Consequently, folate ligands can act as molecular Trojan horse
to deliver active molecules selectively into tumor cells.34 In
general, folate drug conjugates have shown improved cytotoxic
effects and higher drug accumulation in the tumor mass when
a. Institute of Chemistry, University of Campinas - Unicamp, Campinas 13083-861,
São Paulo, Brazil. *E-mail catiaornelas@catiaornelaslab.com
† Electronic Supplementary Information (ESI) available: Supplementary cytotoxicity,
experimental data on synthesis and structural characterization of all intermediates,
including 1H NMR spectra, 13C NMR spectra, 2D NMR contour maps, and mass
spectra. See DOI: 10.1039/x0xx00000x
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compared to the free drug.38 Moreover, they usually possess methodology,48 which starts with the commercial
View Article OGnllinue
DOI: 10.1039/C9NJ04954A
consistent pharmacokinetic behaviors.37-40
Folate drug conjugates can be easily prepared by attachment 5-(phenylmethyl) ester (Boc-Glu(OBzl)-OH). In the first step, the
of active molecules to folic acid (FA) via amidation.41 However,
-carboxylic acid group of Boc-Glu(OBzl)-OH was protected
FA has two available carboxyl groups yielding two regioisomers with 2-(trimethylsilyl)ethanol (TMSEtOH) via Steglich
after amidation: folate -conjugated and folate was obtained in 90% yield, after
-conjugated.41 esterification. Compound
It has been shown that both isomers can associate with Fr at purification with column chromatography on silica gel.
virtually identical levels, but with different in vivo Formation of compound was supported by the new signals in
derivative L-Glutamic acid, N-[(1,1-dimethylethoxy)carbonyl]-,
a
a
g
1
a
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pharmacological behaviors. 40, 42-47 In this context, the utilization the 1H NMR spectrum assigned to the silyl ester unit at 4.21 (m,
of regiospecific methods for the synthesis of folate conjugates COCH2, 2H), 1.00 (m, CH2Si(CH3)3, 2H) and 0.04 ppm (s, Si(CH3)3,
is crucial to the success of this folate-based therapy.
Herein, the synthesis, characterization and anticancer activity selectively removed via Pd/C-catalyzed hydrogenation reaction
of folates -Fc are reported for the first time. The Fc units were yielding compound in 83% yield, after purification with
9H) (SI, Figure S1). The g-benzyl ester of compound 1 was
g
2
connected to protected glutamate (Glu) residues via different column chromatography. Full deprotection was supported by
linkers using amide coupling and “click” reactions. Folates were 1H NMR spectrum due to the absence of the peaks at 7.34 and
synthesized via a regioselective route by reacting protected Glu 5.11 ppm assigned to the aromatic (Ar-H, 5H) and methylene
g
-Fc residues with pteroyl azide (Pte-N3). Three analogs were protons (OCOCH2, 2H) of the benzyl ester protecting group (SI,
synthesized varying the functional group close to the Fc: (i) one Figure S4). Compound has one protected amine group with
triazole group conjugated to the Fc, (ii) one triazole group distal tert-butyloxycarbonyl (Boc) group, one protected -carboxyl
and one amide group conjugated to the Fc, and (iii) only amide with a silyl ester, and one free -carboxylic acid that enables
groups conjugated and distal to the Fc. The effect of the triazole regiospecific -conjugation of linkers containing the Fc unit.
group was rationalized by comparing the electronic properties To obtain the Glu-Fc derivative bearing one triazole group
of each compound with their anticancer activity on human cells. conjugated to the Fc unit, compound was functionalized with
The cancer-targeting ability and enhanced cytotoxic effect of 3-azido-1-propanamine through HATU-mediated amide
folates -Fc were investigated by comparing their activity in coupling. Compound was obtained in 71% yield after
2
a
g
g
2
g
3
healthy and cancerous cells with Glu
Pte moiety.
g
-Fc residues lacking the purification with column chromatography on silica gel.
1
Compound
3
was identified by H NMR (SI, Figure S7) through
the presence of the multiplet peak at 3.40-3.33 ppm assigned to
the methylene groups adjacent to the azide (N3CH2, 2H), and the
signal of the new amide group (HNCH2, 2H). Subsequent CuAAC
Results and discussion
“click” reaction between compound
using sodium ascorbate and copper sulfate, afforded compound
in 84% yield. Compound was purified by flash
chromatography on silica gel, and its formation was supported
by 1H NMR spectrum (SI, Figure S10) through the
3 and ethynylferrocene,
Synthesis of folate
Regioselective synthesis of folate
performed by reacting a pteroyl intermediate with protected
glutamate residues containing a Fc unit at the position. The
g
-Fc and Glu
g
-Fc derivatives
g-Fc conjugates was
4
4
g
glutamate syntheses (Scheme 1) was adapted from a published
Scheme 1. Synthesis of protected glutamate g-ferrocene derivatives.
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presence of the peaks between 4.93-4.00 ppm assigned to the 4.02 ppm assigned to the Fc protons, as well as the signals at
Fc aromatic protons, as well as the singlet peak at 7.50 ppm 3.42-3.24 and 1.42 ppm assigned to the methylene (CH2NH) and
characteristic of 1 4-disubstituted 1,2,3-triazole proton. The Boc methyl protons of the Boc group, respectively. The Boc group of
group of compound
the amide-terminated compound
washing with diethyl ether. Full deprotection was supported by diethyl ether. Full deprotection was supported by 1H NMR
1H NMR spectrum (SI, Figure S14) due to the absence of the Boc spectrum (SI, Figure S35) through the absence of the Boc group
4
was removed with formic acid to afford compound 10 was removed after treatment with formic acid to
in quantitative yield, after afford compound 11 in quantitative yield after washing with
5
methyl protons signal at 1.45. The formation of compound
5
signal at 1.42 ppm. Subsequent condensation between
was further confirmed by mass spectrometry by its molecular compound
2 and compound 11 afforded compound 12 in 88%
ion peak [M+H]+ at 540.22 m/z (calculated for C25H37FeN5O3Si: yield, after purification with column chromatography on silica
1
540.21 m/z) (SI, Figure S17).
gel. Formation of compound 12 was supported by H NMR
The Glu-Fc derivative containing one conjugated amide group spectrum (SI, Figure S38) through the presence of signals from
to the Fc unit and one distal triazole group was obtained using both units (Fc and Glu), as well as the peak at 7.06 ppm assigned
a
similar approach. Compound
2
was reacted with to the newly formed amide group. The Boc group of compound
propargylamine via HATU-mediated amidation to afford 12 was removed with formic acid yielding compound 13 in
1
compound
chromatography. Compound
6
in 96% yield, after purification with column quantitative yield. Full deprotection was proven by H NMR
1
6
was identified by H NMR (SI, spectrum (SI, Figure S42) by the absence of the signal at 1.42
Figure S18) through the presence of the peaks at 6.38, 5.05, and ppm assigned to the Boc group. Mass spectrometry confirmed
2.23 ppm, which are assigned to the newly formed amide group the formation of compound 13 by its molecular ion peak [M+H]+
(CONH), methylene group adjacent to the amide (CONHCH2) at 502.18 m/z (calcd for C23H35FeN3O4Si: 502.18 m/z) (SI, Figure
and terminal alkyne proton, respectively. The Fc motif was S45).
prepared by coupling ferrocenecarboxylic acid (Fc-CO2H) with 3-
azido-1-propanamine using HATU as the coupling agent. following a methodology published by Jin Luo et al.49 Briefly,
Compound was obtained in 88% yield, after purification by folic acid (FA) was reacted with excess of trifluoroacetic
flash chromatography on silica gel. Compound was identified anhydride yielding N2,10-bis(trifluoroacetyl)pyrofolic acid 15
Pteroyl azide compound (Pte-N3) 14 was prepared (Scheme 2)
7
7
.
by 1H NMR (SI, Figure S21) through the presence of the multiplet This compound is a mixture of diacylated anhydride and
peaks at 3.49-3.40 ppm assigned to the methylene groups diacylated carboxylic acid. Subsequent addition of water to
adjacent to the azide (N3CH2) and newly formed amide group compound 14 afforded N10-(trifluoroacetyl)pyrofolic acid 16 in
(HNCH2). Compound
“click” reaction to afford compound
purification by flash chromatography on silica gel. The pteroyl hydrazide 17 in 98% yield. Pteroyl hydrazine 17 was
formation of compound
was supported by 1H NMR spectrum transformed into Pte-N3 14 via nitroso transfer reaction using
6
and
7
were combined using CuAAC quantitative yield from FA. N10-(trifluoroacetyl)pyrofolic acid
8
in 83% yield, after was reacted with hydrazine to eliminate the Glu moiety yielding
8
(SI, Figure S24) through the presence of the peaks assigned to tert-butyl nitrite and potassium thiocyanate, followed by
the Fc aromatic protons and the singlet peak at 7.66 ppm diazotization under acidic conditions. Pte-N3 14 was obtained in
assigned to the triazole proton. The Boc group of compound
was selectively removed after treatment with formic acid to
8
68% yield after purification by precipitation in 2-propanol. Pte-
3 14 can react with nucleophiles (e.g., the previous mentioned
N
afford compound
compound
9
in quantitative yield. Formation of amine-terminated Glu
g-Fc compounds) due to the presence of
1
9
was supported by H NMR spectrum (SI, Figure the acyl azide group.
S28) through the absence of the signal at 1.37 ppm assigned to
the methyl protons of the Boc group. Formation of compound
was further confirmed by mass spectrometry by its molecular Fc residues
ion peak [M+H]+ at 597.22 m/z (calcd for C27H40FeN6O4Si: 597.23 triazabicyclo[4.4.0]dec-5-ene (MTBD) as base (Scheme 2). All
m/z) (SI, Figure S31). three folates -Fc were obtained in reasonable reaction yields
Folate
coupling Pte-N3 14 with the respective amine-terminated Glu
and 13), using 7-methyl-1,5,7-
g-Fc conjugates 18, 19 and 20 were obtained after
9
g-
(
5,
9
g
In the third Glu-Fc derivative (13) both units were attached via (60% for folate 18, 56% for 19 and 58% for 20) after purification
amide bonds using an ethylenediamine linker. For this, Fc-CO2H by column chromatography on silica gel with a mixture of
reacted with N-Boc-ethylenediamine via HATU-mediated dichloromethane, methanol and triethylamine as eluent
coupling yielding compound 10 in 84% yield. Formation of (solvent mixture gradient from 99:0:1 to 79:20:1). Formation of
compound 10 was evidenced by the 1H NMR spectrum (SI, folates was confirmed by 1H NMR spectroscopy (SI, Figures S46,
Figure S32) through the presence of the peaks between 4.79- S51 and S55) through the presence of the signals from both
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Scheme 2. Synthesis of Pte-N3 intermediate and folates g-ferrocene conjugates.
pteroyl and Glu g-Fc moieties. Folates have characteristic signals
around 8.65, 7.66 and 6.64 ppm, which are assigned to the C(7)-
H of the pterin ring, and two aromatic protons Ar-H,
respectively. Fc presents its signals around 4.68-4.02 ppm. The
Glu moiety presents its signals near 4.35-4.32ppm (HNCH), and
between 2.23-1.92 ppm assigned to the methylene groups.
Subsequent deprotection of the
and 20 using tetrabutylammonium fluoride (TBAF) (solution 1.0
M in THF) afforded the final folates 21 22 and 23 (Scheme 2).
a-silyl ester of folates 18, 19,
,
Utilization of dried TBAF was crucial for these reactions.
Otherwise, degradation of the Fc moiety was observed probably
due to the formation of hydrofluoric acid. Folate
triazole group conjugated to the Fc unit, and it was obtained in
68% yield. Folate -Fc 22 has one conjugated amide group to the
Fc unit and one distal triazole group (72% yield). Folate -Fc 23
g-Fc 21 has one
g
g
has two amide groups distal and conjugated to the Fc unit (78%
yield). All folates were purified by precipitation in diethyl
ether:acetone (80:20), followed by washing with cold HCl
solution pH 2 (3×10 mL), cold water (3×10 mL) and cold
1
acetonitrile (3×10 mL). Full deprotection was supported by H
NMR spectrum (Figure 1) through the absence of the signals
assigned to the silyl ester unit near 4.36 (COCH2), 0.95-0.93
(CH2Si(CH3)3) and 0.00 ppm (Si(CH3)3), as well as the presence of
glutamine are essential bioactive compounds for the proper
functioning of both healthy and cancerous cells.50 Hence, their
derivatives may have antagonistic effects on cancers, being
used as antineoplastic agents or targeting groups.50 Some of
these compounds have great anticancer properties, and they
have already been employed in clinical trials, such as azaserine,
acivicin and thalidomide.51 Azaserine and acivicin are inhibitors
of key enzymes in the glutamine metabolism, but notorious side
effects limited their use.50
the signal around 11.5 ppm assigned to the
proton (see the expanded spectra in the SI, Figure S60, S65 and
S70). Formation of the folate -Fc conjugates was also
confirmed by high-resolution ESI-QTOF mass spectrometry.
Folate
-Fc 21 presented its molecular ion peak [M+H]+ at
734.2252 m/z (calcd for C34H35FeN11O5: 734.2245 m/z) (SI,
Figure S63), folate
-Fc 22 [M+H]+ at 791.2253 m/z (calcd for
-Fc 23
a-carboxylic acid
g
g
g
C36H38FeN12O6: 791.2259 m/z) (SI, Figure S68), and folate
g
Unprotected Glu g-Fc compounds were obtained in two
sequential deprotection reactions (Scheme 3): (i) silyl ester
removal with TBAF, and (ii) Boc deprotection with formic acid in
[M]- at 694.1859 m/z (calcd for C32H33FeN9O6: 694.1830 m/z) (SI,
Figure S73).
In order to investigate the targeting ability and enhanced
water. Glu
g-Fc compounds were obtained in good yields, being
cytotoxic effect of the folates
compounds , and 12 were removed (Scheme 3). The
resultant compounds 24 25 and 26 have analogs structures of
folates -Fc but lacking the Pte moiety. L-Glutamate and L-
g-Fc, the protecting groups of
84% for Glu
g
-Fc 24, 93% for Glu
g
-Fc 25, and 80% for Glu
g
-Fc
4, 8
1
26. Full deprotections were supported by H NMR spectra (SI,
,
g
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Table 1. Electrochemical and UV-vis data.
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Epc
(mV)
296
434
420
279
421
410
Epa
(mV)
252
386
381
222
368
368
Epc-Epa
(mV)
44
48
39
lmax
(nm)
Compound
EP (mV)
a
folate g-Fc 21
folate g-Fc 22
folate g-Fc 23
Glu g-Fc 24
Glu g-Fc 25
Glu g-Fc 26
274
410
401
251
394
389
a
a
57
53
42
442
411
410
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ad-d absorption of Fc is overlapped with p-p* of folates.
Figures S75, S79, and S83) due to absence of the peaks of the
Boc and silyl ester. Formation of Glu -Fc 24 25, and 26 were
confirmed by 13C NMR and mass spectrometry. All compounds
were fully characterized by 1H NMR, 13C NMR, two-dimensional
[1H-13C]-HSQC NMR, and mass spectrometry. The spectra and
assignments are detailed in the SI.
g
,
Electrochemical Characterization
Since the redox behavior of ferrocenyl compounds is
responsible for their biological activity, cyclic voltammetry (CV)
was employed to study the charge transfer process of such
compounds. Cyclic voltammograms of folates
g-Fc (Figure 2a)
and Glu
g
-Fc (Figure 2b) revealed reversible Fc/Fc+ redox waves
near the expected potential of Fc derivatives (Table 1). As
expected, compounds with triazole group conjugated to the Fc
(Folate g-Fc 21 and Glu g-Fc 24) exhibited lower Ep (274 and 251
mV, respectively) than the amide-Fc derivatives (22, 23, 25, and
30, 52
26).29,
These results confirm that the triazole group
adjacent to Fc donates electronic density to the redox center,
facilitating its oxidation. Moreover, the chemically reversible
(Ipc/Ipa=1 and Epc-Epa ≤ 60 mV) nature of the redox waves of all
compounds indicates their high stability in physiological
medium.25
Figure 2. Cyclic voltammetry of (a) folates g-Fc and (b) Glu g-Fc in PBS (0.1 M, pH 7.4).
Folates
(Figure 3b), assigned to
ring and
* transition of the pterin ring, respectively.56, 57 The
g-Fc showed two absorptions at 365 and 280 nm
UV-vis characterization
p-p
* transition localized on the pterin
Glu g-Fc derivatives exhibited two absorptions bands in the UV-
vis spectra around 265 and 400-450 nm (Figure 3a), which are
characteristic of Fc compounds.53-55 These bands are assigned
p-p
absorption from d-d transitions of Fc between 400-450 nm were
not observed because these low intensities bands are
overlapped with the broad absorption of the folate moiety
to the Fe(a1g)-Cp(e2g) charge transfer (or p-p* transitions) and
symmetry forbidden Fe(a1g)-Fe(e1g) transitions (or d-d
transitions), respectively.53-55 The absorption intensities around
400-450 nm were weak due to being symmetry forbidden
around 365 nm
.
Anticancer activity
Fr has been seen as a promising biomarker to deliver drugs
transitions.54, 55 The wavelength of maximum absorbance (lmax
)
a
from d-d transitions of compound 24 was observed at higher
wavelength (442 nm) when compared to compounds 25 and 26
(around 410 nm) (Table 1). Again, these results suggest that the
triazole group conjugated to the Fc unit is able to donate
electrons to the Fc complex, inducing a bathochromic shift in
the d-d absorption of Fc. These observations are in line with
those from CV in which lower Ep values were observed for
selectively into cancer cells with notorious avidity (KD = 1-10
nM).40 Folate-drug conjugates are internalized into malignant
cells via the natural and nondestructive endosomal route,
without loss of binding affinity.37, 40 Although Fr
a is also present
in healthy cells, its location is restricted to the apical surface of
the polarized epithelial cells, and the receptor is not exposed to
molecules in the bloodstream.40, 58, 59 On the other hand, Fr
a
compounds 21 and 24
.
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Competitive blocking experiments were carried out using 1
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mM of FA in the growth medium. In this cDoOnId: i1t0io.1n03,9th/Ce9aNcJt0i4v9it5i4eAs
of folates 21 and 22 against HeLa and MCF7 cells were much
lower (Table 2). However, the IC50 values on PNT2 and PC-3 cells
did not quantitatively change. These findings support the ability
of these compounds to bind selectively to Fra, with a low level
of unspecific uptake via the other folate protein carriers, such
as the reduced folate carrier (RFC). RCF is found in almost all
cells, but it may have low affinity for oxidized folates (Km around
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150-200
and damage cancer cells without harming healthy cells.
The Glu -Fc residues showed similar behavior of folates, being
Glu -Fc 24 the most active against cancer cells. Except on PC-3
cells where Glu -Fc 26 showed the lower IC50 (Table 2).
However, the IC50 values for Glu -Fc residues were much lower
µ g-Fc 21 and 22 can reach
M).36, 65 Therefore, folates
g
g
g
g
than the folates (Table 2). In this context, Lingling Shan et al.
described the synthesis of a bifunctional folate-paclitaxel (PTX)
prodrug conjugate and evaluated its anticancer properties. PTX
is a widely used oncolytic agent, but it is highly lipophilic with
poor pharmacokinetics. Attachment of the folate ligand to PTX
enhanced the tumor accumulation and efficacy. Folate-PTX was
five times more effective on MDA-MB-231 cells (human breast
adenocarcinoma) than the free drug. Moreover, the conjugate
was 2-fold less toxic on 293T normal cells (derivative of human
embryonic kidney), although it is still highly toxic on healthy
cells (IC50 = 4.4 nM).66 These observations are in line with the
results presented here, in which the folate targeting group
enhanced the antiproliferative effect of the ferrocenyl moieties.
Figure 3. UV-vis spectra of a) Glu g-Fc and b) folates g-Fc in PBS (0.1M, pH 7.4).
Interestingly, compound Glu
index against prostate cancer cells presenting a IC50 value of
31.6 M against PC-3 that is about 12 times higher than the IC50
value on PNT2 (371.8 M). This finding suggests that this
g-Fc 26 showed a high selectivity
can be found at very high levels in many epithelial tumors, with
expression up to 100 times greater.60-64
µ
In vitro cytotoxicity of folates
g-Fc and Glu g-Fc was
µ
investigated using cultured monolayers of human healthy or
cancerous cell lines: PNT2 (normal prostate epithelial cells),
HeLa (cervical cancer), MCF7 (breast adenocarcinoma), and PC-
compound might be a good candidate for selective prostate
cancer therapy and will be subject of further investigation.
As predicted by CV and UV-vis measurements, compounds
bearing one triazole group conjugated to the Fc are more toxic.
These compounds exhibited lower Ep’s (Figure 2, Table 1) and
bathochromic shifts in the UV-vis absorption above 400 nm
(Figure 3, Table 1). These data support the electron donating
effect of the conjugated triazole group on the Fc unit. Hence,
these compounds are readily oxidized, increasing the ROS
levels, and inducing cell death.
All these findings revealed the promising effects of donor
groups to the Fc on the anticancer activity. Although folate 21
showed great potential to be applied as an anticancer drug, it
has to be further studied to evaluate its pharmacokinetic
behavior. Overall, the combination of folate therapy with
bioorganometallic chemistry appears to be a relevant approach
to design new drugs with safety and effective profiles.
3 (prostate adenocarcinoma). PNT2 and PC-3 are Fr
a-negative,
whereas HeLa and MCF7 are Fr -positive. Cell viability was
a
determined by colorimetric assay using the Cell Counting Kit-8
(CCK-8), after incubation of the cells for 48 h with appropriate
concentrations of the ferrocenyl compounds.
Cell viability was plotted against the compounds concentration
(Figure 4), and the corresponding IC50 values are given in table
2. All folates exhibited substantial toxicity against cancer cells.
In general, folate
than the other folates. The IC50 values for compound 21 were
21.7 M (± 0.3) on MCF-7 and 25.4 M (± 3.2) on HeLa cells.
g-Fc 21 showed higher activity on cancer cells
µ
µ
These values were close to the IC50 of cisplatin (positive control,
Table 2), suggesting the ability of folate 21 to be applied as an
anticancer drug.
To verify the targeting ability of folates
cytotoxicity was assessed on normal PNT2 cells and Fr
negative PC-3 cells. Folates 21 and 22 were less active on such
cells. Folate -Fc 21 was up to 4-fold less toxic than in MCF-7
cells, with IC50 at 87.2 M (± 4.0) on PNT2 cells and 62.0 mM (±
4.9) on PC-3 (Table 2). Folate -Fc 23 was more toxic against PC-
3 cells rather than the Fr -positive HeLa and MCF7 cells,
g-Fc, their in vitro
a-
g
µ
g
a
suggesting a low affinity of this compound towards Fra.
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DOI: 10.1039/C9NJ04954A
ARTICLE
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Figure 4. Cell viability of folates on (a) HeLa cells, (b) MCF7 cells, (c) PC-3 cells, and (b) PNT2 cells.
Table 2. IC50 of ferrocenyl compounds
Folate g-Fc Folate g-Fc Folate g-Fc
21 + 1mM
FA
Folate g-Fc
Folate g-Fc
Folate g-Fc
Glu g-Fc
24
Glu g-Fc
25
Glu g-Fc
26
Cell line
Cisplatin
22 + 1mM
FA
76.2 µM
(± 8.2)
95.5 µM
(± 0.4)
72.6 µM
(± 8.2)
23 + 1mM
FA
235.0 µM
(± 18.4)
170.1 µM
(± 5.6)
53.6 µM
(± 2.3)
143.7 µM
(± 13.4)
21
22
23
17.3 µM
(± 3.7)
11.1 µM
(± 0.5)
6.9 µM
(± 0.15)
18.8 µM
(± 3.9)
25.4 µM
(± 3.2)
21.7 µM
(± 0.3)
62.0 µM
(± 4.9)
87.2 µM
(± 4.0)
56.2 µM
(± 6.7)
54.2 µM
(± 16.4)
66.8 µM
(± 6.8)
170.8 µM
(± 1.60)
80.6 µM
(± 12.2)
54.3 µM
(± 5.1)
60.2 µM
(± 5.2)
102.3 µM
(± 11.0)
72.5 µM
(± 5.0)
144.1 µM
(± 11.0)
84.3 µM
(± 1.7)
64.9 µM
(± 9.3)
169.6 µM
(± 6.4)
982.3 µM
(± 5.3)
313.2 µM
(± 18.0)
31.8 µM
(± 2.7)
HeLa
MCF7
PC-3
167.7 µM
(± 12.0)
461.5 µM
(± 24.2)
358.4 µM
(± 30.4)
101.3 µM
(± 15.0)
138.9 µM
(± 12.8)
80.0 µM
(± 6.3)
93.8 µM
(± 10.2)
324.0 µM
(± 35.5)
371.8 µM
(± 16.0)
PNT2
The IC50 values are the mean of two experiments done in triplicate.
electrode (SCE). Phosphate buffer (PBS) (0.1 M, pH 7.4) was used as
supporting electrolyte. Voltammograms were collected at a scan rate
of 100 mV.s-1, using a concentration of the test compounds at 10
mM. The Ep’s were calculated by the mean of Epc (cathodic potential)
and Epa (anodic potential).
Experimental
Information about the chemicals, experimental and
characterization techniques for all compounds are detailed in the SI.
All compounds were characterized by mass spectrometry, H, 13C,
1
and 2-D NMR spectroscopy. Supplementary cytotoxicity data are also
available in the SI.
Cell culture
Human cells were cultured in RPMI 1640 supplemented with
10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
For investigation of the folates cytotoxicity, cells were grown for
5 days in folate-free RPMI 1640 medium to induce the
expression of Fr
inactivated FBS as the only source of folates around 3 nM, a
Electrochemical measurements
CV’s were carried out in a VersaSTAT 3 potentiostat (Princeton
Applied Research) with three electrodes: glassy carbon working
electrode, Pt auxiliary electrode, and a reference saturated calomel
a. The folate-free medium contains heat-
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value at the low end of the physiological concentration in
human serum.45, 67 The cells were kept in 75 cm2 flasks, at 37 °C
in a humidified incubator with 5% CO2. Culture medium was
changed every 48 h. Cells were passaged using 0.25% trypsin-
0.02% EDTA solution with 80-90% of confluence.
Acknowledgements
View Article Online
This work was financially supported in DpOarI:t10b.y10t3h9e/CS9aNoJ0P49a5u4loA
Research Foundation (research grant for CO: 2018/02093-0;
PhD grant for DLB: 15/04929-0, fellowship #2017/06146-8 for
C.B.B.), the National Council for Scientific and Technological
Development
#307403/2018-1),
–
CNPq (productivity award for C.O.
and by the Coordenação de
In vitro cytotoxicity testing (CCK-8) calorimetric assay
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Cells were seeded into 96-well plates with 200 µL of culture
medium in a final density of 8×103 cells/well. After incubation
for 24 hours, solutions of the test compounds (in culture
Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES),
finance code 001.
medium) at concentrations ranging from 1 to 1000 µM were
added. Aqueous stock solutions of the folates were prepared by
adding equimolar amounts of sodium hydroxide yielding the
water-soluble carboxylate salts. After 48 hours, the growth
medium was removed and the cells were washed with PBS (0.1
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2013, 5, 961-978.
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J. Name., 2013, 00, 1-3 | 9
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New Journal of Chemistry
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Novel folate -ferrocene conjugates were synthesized through a regiospecific route and View Article Online
showed selectivity and enhanced cytotoxicity against Fr-positive malignant cells.
DOI: 10.1039/C9NJ04954A
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Page 11 of 11
New Journal of Chemistry
View Article Online
DOI: 10.1039/C9NJ04954A
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72x39mm (300 x 300 DPI)