Chromene-triazole-pyrimidine based chemosensor therapeutics for thein vivoandin vitrodetection of Fe3+ions

Article abstract of DOI:10.1039/d0nj05697a

The development of a series of chromene-pyrimidine triad molecules for the selective detection of Fe³⁺is described. The new chemosensors were synthesized through the sequential use of multicomponent reactions (MCRs) and click chemistry marking its ease and cost effectiveness. The sensor molecules showed turn-off fluorescence towards Fe³⁺with an extremely low limit of detection (LOI) and a strong affinity to cancer causing CDK2 proteins with binding energy closer to -9.8 kcal mol^-1as indicated by the molecular docking studies. The free hydrophilic handles present on the sensors are advantageous for binding with cellular Fe³⁺demonstrating the potential of these molecules as therapeutics for iron overloading diseases. The therapeutic potential is further illustratedviacytotoxicity studies against the human cervical cancer cell line HeLa, which showed a very low IC50 value of 15 μg mL^-1

Full text of DOI:10.1039/d0nj05697a

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Chromene–triazole-pyrimidine based
chemosensor therapeutics for the in vivo and
in vitro detection of Fe ions†
Cite this: New J. Chem., 2021,
3
+
4
5, 6760
Aranhikkal Shamsiya
and Damodaran Bahulayan
*
3+
The development of a series of chromene–pyrimidine triad molecules for the selective detection of Fe
is described. The new chemosensors were synthesized through the sequential use of multicomponent
reactions (MCRs) and click chemistry marking its ease and cost effectiveness. The sensor molecules
3+
showed turn-off fluorescence towards Fe with an extremely low limit of detection (LOI) and a strong
À1
Received 24th November 2020,
Accepted 13th March 2021
affinity to cancer causing CDK2 proteins with binding energy closer to À9.8 kcal mol as indicated by
the molecular docking studies. The free hydrophilic handles present on the sensors are advantageous
3+
for binding with cellular Fe demonstrating the potential of these molecules as therapeutics for iron
DOI: 10.1039/d0nj05697a
overloading diseases. The therapeutic potential is further illustrated via cytotoxicity studies against the
À1
rsc.li/njc
human cervical cancer cell line HeLa, which showed a very low IC50 value of 15 mg mL
.
assembled via multistep synthesis. Although the majority of
the iron sensors developed so far show promising applications,
Introduction
Iron is one of the important elements that regulate many there is still a need for the development of more biocompatible
1
3+
metabolic processes in the human body. Iron can act as both Fe sensors/therapeutic agents for the complexation induced (i)
electron donor and acceptor by changing the oxidation states therapy for diseases like iron-loading anemia, (ii) drinking water
2
+
3+
4+
from Fe(II)–Fe , Fe(III)–Fe , and Fe(IV)–Fe . Such electron purification, etc.
transfer properties enable it to be a catalyst for many biochemical We have recently developed novel fluorescent probes based
3
+
11
12
reactions. Among the various oxidation states of iron, Fe plays a on coumarin and pyrimidine for the selective and sensitive
3
+
vital role in cellular events such as DNA replication, oxygen detection of Fe and azide ions respectively. In continuation of
transport, etc. However, deficiency or excess of iron in the our research to develop more biocompatible fluorescent probes, here
human metabolism may cause either iron-deficiency anemia or we report the design and synthesis of an array of new Fe sensors
iron-loading anemia leading to various forms of cancers, heart and their cytotoxicity against the human cervical cancer cell line
ailments, diabetes, liver disease, osteoarthritis, osteoporosis,
neurodegenerative disorders, Alzheimer’s disease and Parkinson’s tion of the biological base pyrimidine with a naturally occurring
disease. Consequently, metabolic iron has become an important chromene moiety using efficient and economic synthetic protocols
target in drug discovery for a broad spectrum of chronic diseases. like multicomponent reactions
A variety of small molecules based fluorescent probes have been
developed in the recent past as Fe sensors based on either
2,3
3+
4–6
(HeLa). The new sensor/probes were assembled through the integra-
7
8,9
1
3,14
15,16
and click chemistry.
3+
10
fluorescence turn on or quenching mechanisms. These probes
were featured with a hydrophilic iron binding handle, receptor
binding pocket, and a fluorophore for signaling. Common fluoro-
phores include coumarin, anthracene, naphthalene, fluorescein,
pyrene, rhodamine, etc. and the receptor binding pocket can be a
Experimental section
General procedure for the Cu(I) catalyzed (3+2) azide–alkyne
,3-dipolar cycloaddition reactions for the synthesis of
chromene-pyrimidinone triazole CP1
1
polypeptide, polyamine, or calixarene type supermolecules. The An equimolar mixture of azide P1 (1 mmol) and the alkyne
binding handle, receptor binding unit, and fluorophores were (C1, 1 mmol) was mixed with 0.2 equiv. of CuSO and 0.4 equiv.
4
of sodium ascorbate in a mixed solvent system containing tert-
butanol, water, and DMSO (4 : 2 : 1) at room temperature. After
Department of Chemistry, University of Calicut, Malappuram 673635, Kerala,
12 h, the reaction mixture was diluted with cold water to afford
India. E-mail: bahulayan@yahoo.com, cue2994@uoc.ac.in
Electronic supplementary information (ESI) available: Experimental details and
compound characterization. See DOI: 10.1039/d0nj05697a
the substituted triazoles in the solid form. The solid obtained
was washed with water to obtain CP1 in the pure form.
†
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Brown solid. Mp 145–147 1C, yield 85%; H NMR (500 MHz, electron transfer from the HOMO to the LUMO of the molecules.
DMSO): dH (ppm): 8.57 (s, 2H), 7.85–7.22 (m, 14H), 7.58 (s, 1H), The HOMO of the molecule is located in the coumarin core and
6
.21 (s, 2H), 5.96 (s, 1H), 5.52 (s,1H), 5.23–5.11 (d,1H), 5.02 it acts as an electron donor (D) whereas the LUMO of the
1
3
(s, 2H), 4.30 (s, 2H), 4.15–4.09 (q, 2H), 1.35–1.31 (t, 3H);
C
molecule is located in the dihydropyrimidinone moiety and it
NMR (DMSO-(d6), 500 MHz): 35.80, 39.50, 39.67, 39.83, 40.00, acts as electron acceptor (A). Hence there is an electron donor–
4
8
1
1
1
7
0.17, 40.26, 40.33, 40.42, 40.50, 40.59, 55.79, 71.25, 79.45, acceptor interaction possible in the molecule due to intra-
0.32, 81.00, 104.50, 114.73, 115.79, 116.21, 116.42, 123.78, molecular charge transfer (ICT). The electron is excited to the
24.43, 128.15, 131.83, 132.15, 133.64, 152.76, 158.16, 165.13, LUMO and subsequently relaxes to the ground state causing blue
À1
71.23; FT-IR (KBr) (nmax, cm : 3383, 2928, 2227, 1697, 1552, emission from the molecule. The normalized absorption and
504, 1512, 1487, 1457, 1369, 1307, 1227, 1175, 1097, 1030, emission spectra of CP1–CP6 are present in Fig. 1.
+
56); HRMS m/z calcd for C H N O [M] : 669.23358, found:
3
7
31 7 6
+
Solid-state fluorescence
[M] : 669.23352.
The fluorescence of these molecules showed a redshift in the
solid (aggregated) state. A variety of variables are believed to
contribute to the generation of aggregation induced emission
Results and discussion
Design strategy
(AIE) and aggregation induced enhanced emission (AIEE).
Our probe design strategy consists of mainly three synthetic These variables include the restriction of charge transfer (CT),
stages: (i) synthesis of the chromene with an alkyne functionality intramolecular rotation (RIR), intermolecular interaction,
1
7,18
(
ESI,† Scheme S1), (ii) synthesis of the pyrimidines with an azide etc.
The restriction of the intramolecular motion of the
functionality (ESI,† Scheme S2) and (iii) the final assembly of the compound in the aggregated state could be the reason for the
probe molecules by connecting the chromenes with the pyrimi- aggregation-induced emission enhancement (AIEE) for weakly
1
9–28
dines through a spacer triazole via the alkyne-azide (2+3) click fluorescent molecules.
The solid-state absorption and
cycloaddition (Scheme 1). Scheme S1 (ESI†) presents a base emission of the molecules are CP1–CP6 depicted in Fig. 2.
catalyzed three component reaction between naphthalene, an
alkyne functionalized aldehyde, and malononitrile at 80 1C
Sensor properties
which afforded the chromene alkynes in a quantitative yield. Since CP1–CP6 have similar photophysical properties with
The azide functionalized pyrimidines were synthesized via the comparable Stokes shifts and quantum yields, the variation
classical Biginelli multicomponent reaction of benzaldehydes in the fluorescence intensity of these probes in the presence of
3
+
2+
2+
2+
2+
2+
2+
2+
with urea and an active methylene compound and the sub- various analytes (Fe , Cu , Cd , Hg , Ca , Mg , Mn , Zn ,
2
+
2+
+
2+
sequent chlorine substitution with an azide functionality as Co , Pb , Na , Ni ) were evaluated using CP1 as the model
shown in Scheme S2 (ESI†). Both steps involved in Scheme S2 compound.
3
+
(ESI†) have proceeded nicely with the formation of the chloro
Among the various metal ions tested, the addition of Fe to
and azido pyrimidines in excellent yield and purity. The chromene the CP1 in DMSO showed significant variations in the absor-
and pyrimidine fragments were then assembled through copper bance and fluorescence intensity, unlike other analytes and a
catalyzed azide–alkyne (3+2) cycloaddition (CuAAC), as shown in significant fluorescence turn-off behavior as shown in Fig. 3 and 4.
Scheme 1 to obtain the probes CP1–CP6 as listed in Scheme 2.
The fluorescence titration was carried out by adding an aqueous
3+
À4
solution of Fe ions (0.01 mM) into CP1 (1.0 Â 10 M) in DMSO.
The fluorescence intensity of CP1 showed a gradual decrease upon
Solution state fluorescence
3+
The photophysical properties of these new probes were evaluated increasing the concentration of Fe from 0 to 0.5 equivalents.
by measuring the absorption, emission, and fluorescence quantum From the Stern–Volmer emission spectral profile (concentration
yields in the solution state, and the values are summarized in versus fluorescence intensity) given in the inset of Fig. 4b, the limit
3+
Table 1.
of detection (LOD) of the chemosensor CP1 for Fe was deter-
All 6 compounds showed fluorescence in the blue region mined to be 0. 14 mM. The association constant (K ) was also
a
with a large Stokes shift and high quantum yield. The emission obtained from the fluorescence titration. The low value obtained
properties of the molecules were related to the presence of for the LOD reveals that the new probe CP1 can be used for the
Scheme 1 Cu(I) catalyzed (2+3) alkyne-azide cycloaddition (click chemistry) assisted synthesis of chromene–pyrimidinone triads.
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Scheme 2 List of chromene–triazole-pyrimidine chemosensors prepared.
3
+
Table 1 Absorption maxima (nm), emission maxima (nm), and the quantum CP1 with Fe . This 1 : 1 interaction was also confirmed from the
yield of CP1–CP6
29,30
Benesi–Hildebrand (B–H) plot.
B–H plot is a statistical
method used for the determination of the equilibrium constant
K and stoichiometry of non-bonding interactions in the com-
plexes. The Benesi–Hildebrand (B–H) plot, in this case, was
obtained as a linear plot validating the assumption of 1 : 1
stoichiometry. The data obtained from multiple spectrofluoro-
metric titrations were used for the B–H calculations and the
Entry
Absmax
Emismax
Stokes shift
Quantum yield
CP1
CP2
CP3
CP4
CP5
CP6
371
370
352
369
370
360
472
463
459
465
471
459
101
93
107
96
101
99
0.316
0.440
0.235
0.370
0.327
0.315
6
equilibrium constant was obtained as a 5.36 Æ 0.09 Â 10 unit
(
Fig. 5b). The higher value obtained for the equilibrium constant
3
+
in vitro/in vivo detection of micromolar level iron present in various reveals the strong binding of CP1 with Fe to form a stable
entities.
complex.
3
+
To determine the stoichiometric ratio between CP1 and Fe ,
the method of continuous variation (Job’s plot) was adopted.
3
+
Mechanism of the turn off fluorescence
The total concentration of the receptor CP1 and ions Fe were
À4
kept constant (1.0 Â 10 M) with continuous variation of the To derive a possible mechanism of fluorescence quenching, we
mole fraction of CP1 ([receptor]/[receptor] + [ion]). Fig. 5a shows have carried out the Density Functional Theory (DFT) calcula-
3
+
31
the Job’s plots of CP1 with Fe . When the mole fraction of CP1 tions using the Gaussian 09 programme. For this, the CP1
3
+
was 0.5, the receptors CP1–Fe complex concentration attained was optimized with DFT/B3LYP-6-31+G(d,p) while the CP1–Fe
3
2
a maximum. This indicated the formation of a 1.1 complex of complex was analyzed by DFT/B3LYP-LANL2DZ.
Fig. 1 Normalized absorbance and emission spectra of CP1–CP6 in DMSO.
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Fig. 2 Solid-state absorption and fluorescence of the compounds CP1–CP6; Insets: The fluorescence emissions of the solid for CP1 under excitation at
62 nm.
3
À4
3+
Fig. 3 (a) UV-Vis spectra of CP1 in the presence of 2 equiv. of various cations. (b) Changes in UV spectra of CP1 (1 Â 10 M) as a function of Fe
(
1 Â 10À5 M) concentration.
3
+
Fig. 4 (a) Emission spectra of CP1 in the presence of 2 equiv. of various cations. (b) Changes in fluorescence spectra of CP1 as a function of Fe
3+
concentration; Inset: Stern–Volmer emission profile with the addition of Fe to determine the limit of detection (LOD); [CP1] = 1 Â 10À4 M, [Fe3+] = 1 Â 10À5 M.
The ESP (electrostatic potential) map of both the structures triazole, and oxygen of the carbonyl group connected to the
3
+
has been drawn to determine the nucleophilic and electrophilic pyrimidinone ring, and that constitutes the site for Fe
behavior of the molecule. The region red in Fig. 6c is the interaction.
electron-rich area, where the electrophilic attack occurs and
The HOMO–LUMO energy gap calculations revealed that the
the blue area indicates the electron-deficient region for the maximum stability for the complex was obtained through the
3
+
nucleophilic attack. The optimized structures and the ESP map interaction of Fe with the N of the CN group which is 3.2 eV,
of both the ligand and the complex are shown in Fig. 6. From which is 1 eV less than that of the ligand CP1 which is 4.2 eV.
3
+
Fig. 6, it is clear that the electronegative region is centered The energy gap obtained for the interaction of Fe through the
around the –CN nitrogen of the coumarin, nitrogen of the oxygen of the pyrimidinone is 6.68 eV, which is higher than that
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3+
3+
Fig. 5 (a) Job’s plot for receptor Fe , (b) Bensei–Hildebrand plot of the sensor CP1 in the response of Fe
.
Fig. 6 (a and b): Optimized structures of ligand CP1 and complex respec-
tively; (c and d) ESP map of ligand and complex respectively.
Fig. 7 (a) Resonance stabilization of the lone pair of electrons on the
NH group with the ring. (b) Frontier molecular orbital and energy gap
of the parent ligand and the complex becomes unstable. We (DE) of CP1 with Fe , if the interaction of CP1 is through NH
–
2
3
2
3+
2
group.
3
+
didn’t get any stable structures through the interaction of Fe
with the nitrogen of the triazole ring due to the absence of the
lone pair of electrons on the nitrogen atoms for the interaction the electronic distribution in the complex as the HOMO spreads
3
+
with Fe , since it is preoccupied for conjugation in the triazole over the whole coumarin part excluding amino and cyano
ring to make it aromatic. These observations were further groups, whereas LUMO spreads over the iron atom, as shown
3
3–35
3+
validated by natural bond orbital (NBO) analysis
in which in Fig. 8. The complexation of the ligand CP1 with Fe inhibits
the lone pair of electrons on the nitrogen atom is found to be the ICT resulting in the quenching of the fluorescence. This
more spatially distributed than oxygen and also the energy of makes CP1 an efficient turnoff fluorescence sensor for the
3
+
the former is higher than that of the latter. Similarly, the non- detection of Fe . These computational explanations were also
3
+
interaction of Fe with amine group in the pyran was explained experimentally validated from the Fourier transform infrared
by theoretical studies as shown in Fig. 7. As shown in Fig. 7a, (FT-IR) spectrum of CP1 obtained in the presence and absence of
3
+
the lone pair of electrons in the –NH group is in resonance Fe (Fig. 9). From Fig. 9, it is clear that the CN band has a peak
2
À1
À1
stabilization with the –CN group. Hence, this lone pair is also at 2227 cm in CP1 which is shifted to 2195 cm due to its
3
+
3+
not available for interaction with Fe . The confirmation of this interaction with Fe to form a stable complex.
hypothesis was obtained from the calculation of the HOMO– In order to prove the selective nature of the probe in sensing
LUMO energy gap of such an imaginary CP–Fe complex Fe , the fluorescence of the probe CP1 in the presence of 0.5
3
+
3
+
(Fig. 7b) proposed through the interaction of Fe with NH
2
.
equivalent of Fe and an excess amount of (25 equivalents)
The binding energy of this complex is obtained as 4.72 eV, other competing metal ions was observed (Fig. 10). As shown in
3
+
which is higher than that of the ligand CP1. Hence the Fig. 10, the tested metal ions show no interference for Fe .
formation of such complex is not possible and the interaction
of Fe is only through the –CN group.
3
+
Demonstration of iron sensing for practical applications
Before the attachment of the metal atom, the HOMO is Real sample experiments were carried out with distilled water and
primarily concentrated over the coumarin unit including cyano normal drinking water to check the practical applicability of the
3+
and amino groups, and the LUMO is spread over pyrimidinone sensor CP1 towards the fluorescence detection of Fe ions. For
and triazole rings. The interaction of the Fe with CP1 changes this, we used a 1.0 Â 10 M solution of CP1 and a 1.0 Â 10À5
3
+
À4
M
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Fig. 8 Frontier molecular orbital and energy gap (DE) of (a) probe CP1
3+
3+
Fig. 11 Fluorescence signaling of Fe ions by CP1 in distilled water and
normal drinking water.
and (b) CP1 + Fe
.
times and the error obtained was within the acceptable limit
(
1–8%). The results indicate that our sensor molecule can be used
3+
for selective fluorescence detection (sensing) of Fe ions in
practical and industrial applications.
Binding modes of CP1–CP6 with biological protein CDK2
W
The drug property descriptors (molecular weight (M ), log P,
topological surface area, and the number of H bond donor and
acceptor sites) of CP1–CP6 were calculated using an online
3
6
W
service, www.molinspiration.com. The M of these molecules
are in between 688.14 and 718.17, log P is in the range 4.5–6.02,
and the topological surface area is between 166.43 and
2
12.25(for descriptor data: see ESI,† Table S1). According to
3+
37,38
Fig. 9 FT-IR spectrum of CP1 before and after the addition of Fe ion.
Kihlberg’s classification,
these molecules are in the
extended rule of 5 (eRo5) class with the ability to bind with
challenging or difficult to druggable biological targets. In the
present study, cyclin-dependent kinase 2 (CDK2) was selected for
studying the binding modes of CP1–CP6 using AutoDockVina
1.1.2. The overexpression of CDK2 is one of the causes for
different types of cancers such as cervical, colon, breast, ovarian,
3
9,40
etc.
CP1–CP6 showed strong hydrophobic interaction with
CDK2 by embedding on its active sites through hydrogen bond-
ing, van der Waals, and pi–cation interactions. The compound
CP1 showed one hydrogen bonding interaction between ASN132
and oxygen of the hydroxyl group in the benzene ring near the
À1
pyrimidine ring with a docking score of À9.8 kcal mol . CP2 fit
in the active site of CDK2 (no H bonding) with a docking score of
À1
À9.6 kcal mol whereas CP3 interacts with CDK2 by pi–cation
interaction between LYS89 and naphthalene ring with a docking
À4
Fig. 10 Metal ion selectivity profiles of CP1 (10 M): black bars, fluore-
À1
2+
score of À9.4 kcal mol . CP4 does not form any hydrogen bonds
scence of CP1 in the absence and the presence of 25 equivalent Cd
,
2+
2+
2+
2+
2+
2+
2+
2+
+
2+
À5
Ba , Hg , Ca , Zn , Co , Ni , Fe , Pb , Na and Mg ions (10 M); with CDK2 but is also well fitted in its active site with a binding
À1
red bars, fluorescence of CP1 in the presence of various metal ions, score of À9.6 kcal mol . CP5 and CP6 are also showed binding
3+
À5
followed by 0.5 equivalents of Fe (10 M).
interactions with the protein by one hydrogen bonding in addition
to the hydrophobic interaction. The hydrogen bonding interaction
in CP5 is between ASP145 and oxygen of the NO on the pyrimi-
solution of Fe in normal drinking water as well as in distilled dinone part with a high binding energy of À9.6 kcal mol
water, and the results are shown in Fig. 11. From Fig. 11, it is clear whereas in CP6 the bonding is between THR14 and oxygen of
2
3+
À1
3+
that the sensing ability of CP1 towards Fe ions in distilled water the carbonyl group in the pyrimidinone ring with a binding score
À1
shows good agreement with the same property obtained from of À9.5 kcal mol . The docking scores are listed in Table S2 (see
normal drinking water. These experiments were repeated multiple ESI†) and the binding modes are presented in Fig. S1(see ESI†).
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chromene and pyrimidine, which also illustrate their bio- and
environmental compatibility. The sensor molecules showed
3
+
selective and sensitive turn-off fluorescence with Fe by forming
a stable complex with the chemosensor with an extremely low
limit of detection. The docking studies with biological protein
CDK2 showed the potential of these new molecules as therapeutic
sensors. This was further illustrated with the cytotoxicity studies
against the human cervical cancer cell line HeLa which showed a
À1
very low IC50 value of 15 mg mL . These promising results can be
further explored for the development of efficient chemosensor
therapeutics for a broad spectrum of applications.
Fig. 12 (a) Hydrophobic interaction of CP1 with CDK2 protein, b; its
enlarged portion, c; its cocrystal structure. (b) Changes in UV spectra of
CP1 (1 Â 10À4 M) as a function of Fe3+ (1 Â 10À5 M) concentration.
Author contributions
All authors conceived and planned the experiments. A. S. per-
formed the experiments, contributed to the analysis, and drafted
the manuscript. D. B. contributed to the analysis and edited the
manuscript. All authors read and approved the final manuscript.
The hydrophobic interaction of CP1 (having the highest docking
score) with CDK2 and its cocrystal structures are presented in
Fig. 12. From this discussion, it is clear that after the docking of
the probe molecules on the active sites of CDK2 through hydro-
phobic interactions, their hydrophilic binding sites are intact and
Conflicts of interest
3+
are available for complexation with endogenous Fe . This shows
the potential of the new probe molecules as efficient Fe sensors The authors declare no conflict of interest.
and therapeutic agents for addressing cancers or other diseases
caused by iron overloading (iron-loading anemia).
Acknowledgements
Cytotoxicity of CP1 against the human cervical cancer cell line
A. S. thanks the University Grants Commission (UGC), New
Delhi, for the research fellowship. The authors wish to thank
the Central Instrumentation Facility (CIF), University of Calicut,
Kerala, India for providing the computational facilities.
(
HeLa)
As a proof of concept, the molecule CP1 with the highest docking
score has been selected for evaluating its cytotoxicity against the
human cervical cancer cell line (HeLa) to show the potential of
this sensor as an efficient therapeutic too. The cytotoxic evalua-
tion has been carried out by MTT assay. Fig. S2a (ESI†) shows the
Notes and references
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1
2
3
4
5
6
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À1
18, 21, 27, 30 mg mL concentrations, in PBS, pH 7.4), against
the HeLa cells. The IC50 values of CP1 against HeLa cells were
À1
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using inverted phase-contrast microscopy as shown in Fig. S2b
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evidence for apoptotic activation.
Conclusion
7
In summary, an array of chromene–pyrimidine triad based
efficient chemosensor therapeutics have been developed for a
3
+
broad spectrum of applications such as detection of Fe
8 S. Devaraj, Y. Tsui, C.-Y. Chiang and Y.-P. Yen, Spectrochim.
Acta, Part A, 2012, 96, 594–599.
9 E. C. Theil and D. J. Goss, Chem. Rev., 2009, 109, 4568–4579.
present in biological environments, targeting and annihilation
of iron loaded anemia, and water purification. The therapeutic
sensors were designed and synthesized through the sequential 10 S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and
use of green synthetic methodologies marking its ease and cost J. F. Callan, Chem. Soc. Rev., 2012, 41, 7195–7227.
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