Tacrine-pyrimidine photoactive molecular hybrids: Synthesis, photophysics, docking and BSA interaction study

Article abstract of DOI:10.1016/j.molliq.2019.110983

This study reports the synthesis of two photoactive molecular hybrids containing Tacrine and pyrimidine moieties achieved by multicomponent reaction. The hybrids 11a and 11b present absorption maxima close to the Tacrine absorption (~320 nm). The hybrids present fluorescence emission around 400 nm, as observed for the separate Tacrine and pyrimidine structures. Time-resolved fluorescence indicates for 11a and 11b a monoexponential time decay around 1.5 ns. In this sense, the variation on the size of the aliphatic linker between both fluorophores seems not to affect the observed photophysical properties of the hybrids. In addition, the interaction of the compounds with bovine serum albumin (BSA)in phosphate buffer solution (PBS)was investigated, where a suppression mechanism was observed for both molecular hybrids. Docking was also performed to better understand the observed suppression mechanism.

Full text of DOI:10.1016/j.molliq.2019.110983

Accepted Manuscript
Tacrine-pyrimidine photoactive molecular hybrids: Synthesis,
photophysics, docking and BSA interaction study
João Paulo Bizarro Lopes, Viktor Saraiva Câmara, Dennis
Russowsky, Pablo Andrei Nogara, João Batista Teixeira da Rocha,
Fabiano da Silveira Santos, Fabiano Severo Rodembusch, Marco
Antonio Ceschi
PII:
DOI:
Article Number:
Reference:
S0167-7322(19)31802-1
https://doi.org/10.1016/j.molliq.2019.110983
110983
MOLLIQ 110983
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
28 March 2019
7 May 2019
15 May 2019
Please cite this article as: J.P.B. Lopes, V.S. Câmara, D. Russowsky, et al., Tacrine-
pyrimidine photoactive molecular hybrids: Synthesis, photophysics, docking and BSA
interaction study, Journal of Molecular Liquids, https://doi.org/10.1016/
j.molliq.2019.110983
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ACCEPTED MANUSCRIPT
Tacrine-pyrimidine
photoactive
molecular
hybrids:
synthesis,
photophysics, docking and BSA interaction study
João Paulo Bizarro Lopes,a,* Viktor Saraiva Câmara,a Dennis Russowsky,b
Pablo Andrei Nogara,c João Batista Teixeira da Rocha,c Fabiano da Silveira
Santos,d Fabiano Severo Rodembusch,d,* Marco Antonio Ceschia
aInstituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento
Gonçalves, 9500, Campus do Vale, 91501-970, Porto Alegre, RS, Brazil. E-
mail: joaopauloblopes@terra.com.br
bLaboratório de Sínteses Orgânicas K210, Instituto de Química, Universidade
Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Porto Alegre, Rio
Grande do Sul, Brazil.
cDepartamento de Bioquímica e Biologia Molecular, Centro de Ciências
Naturais e Exatas, Universidade Federal de Santa Maria - UFSM, RS, Brazil
dGrupo de Pesquisa em Fotoquímica Orgânica Aplicada, Instituto de Química,
Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Porto
Alegre, Rio Grande do Sul, Brazil. E-mail: rodembusch@iq.ufrgs.br
Abstract
This study reports the synthesis of two photoactive molecular hybrids containing
Tacrine and pyrimidine moieties achieved by multicomponent reaction. The
hybrids 11a and 11b present absorption maxima close to the Tacrine absorption
(~320 nm). The hybrids present fluorescence emission around 400 nm, as
observed for the separate Tacrine and pyrimidine structures. Time-resolved
fluorescence indicates for 11a and 11b a monoexponential time decay around
1.5 ns. In this sense, the variation on the size of the aliphatic linker between
both fluorophores seems not to affect the observed photophysical properties of
the hybrids. In adittion, the interaction of the compounds with bovine serum
albumin (BSA) in phosphate buffer solution (PBS) was investigated, where a
suppression mechanism was observed for both molecular hybrids. Docking was
also performed to better understand the observed supression mechanism.
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1. Introduction
The use of fluorescent molecular probes has enabled to understand the
functioning of the human body at a molecular level and to monitor biochemical
processes of specific interactions [1]. Exhibiting a high level of sensitivity,
fluorescence has emerged as a substitute for radioactivity on the generation of
images from biological materials [2,3]. Different small fluorescent compounds
have been developed for medical diagnostic imaging such as fluorescein and
rhodamine [4]. Recently, a study in patients showed that the fluorescence of
acridine orange, a quinoline derivative dye, can be used on a staining method
for screening of circulating tumour cells [5].
Quinoline is known as a fluorophore unit which is present in small molecular
fluorescent compounds such as acridine orange (Figure 1) [6]. Quinoline
derivatives are also well known for their good metal affinity and are emerging
from fluorescent probes for zinc and their applications in more complex
biological research [7]. Recently, Wang et al. synthesized acridine-based
fluorescence chemosensors with selective sensitivity for Fe3+ and Ni2+ ions [8].
Tacrine is an important quinoline structural derivative which was the first drug
approved by the FDA, in 1993, for the treatment of Alzheimer's disease (AD).
Although its use has been discontinued for a decade, Tacrine still have boosted
the search of novel therapeutic compounds for AD [9,10,11,12,13]. On the other
hand, dihydropyrimidin-2-ones (DHPMs, Figure 1), knows as Biginelli adducts,
are heterocyclic compounds with a broad spectrum of biological activity, such
as antibacterial, anti-inflammatory, antiviral and specially anticancer [14].
DHPMs have been reported to exhibit fluorescence and photophysical
properties, making them potential candidates for molecular probes [15,16].
Recently, we synthesized two lophine-pyrimidine hybrids which exhibit
fluorescence properties that probably experience photoinduced electron transfer
(PET) in the excited state. Also, they presented BSA suppression probably due
to the pyrimidine moiety, which presented a higher Stern-Volmer constant in
comparison to lophine [17].
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Figure 1. Quinoline derivatives and Biginelli adducts DHPMs.
Bovine serum albumin (BSA) is often selected as a model of drug-albumin
interaction due to its low cost, ready availability and the homology with human
serum albumins (HSA) [18,19]. Serum albumins are carrier proteins soluble in
plasma that play an essential role in the transport and disposition of
endogenous and exogenous compounds in blood, such as metabolites, drugs
and other organic compounds [20]. The study of the drug–albumin interaction
contributes to information on the absorption, transportation, metabolism,
distribution and efficacy of drugs in the body [21]. The potential application of
fluorescence spectroscopy to study the binding of small molecules to serum
albumins have been widely recognized for allowing non-intrusive measurements
of substances under physiological conditions [22]. In 2011, Chen and co-
workers showed that intrinsic fluorescence of HSA was quenched by 9-
aminoacridine derivatives [21]. Also, some studies have demonstrated that
acridine orange dye can interact with BSA quenching their fluorescence and
increase the degree of damage to protein molecules [20,23].
In this study, it was synthesized novel hybrids containing both Tacrine and
pyrimidine from the Biginelli DHPM moiety, connected by a methylene chain as
an aliphatic linker. The two novel Tacrine-pyrimidine hybrids were evaluated by
UV-Vis and fluorescence emission in order to improve the understanding of
their photophysical properties. Their potential application as fluorescent probes
to detect proteins in phosphate buffer solution (PBS) was also explored using
BSA as a model. Also, molecular modelling studies were performed to
understand the binding mode of ligand in the BSA.
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2. Experimental
2.1. Materials and methods
Purification of products by column chromatography was carried out on silica gel
60 Å (70-230 mesh). Analytical thin layer chromatography (TLC) was conducted
on aluminium plates with 0.2 mm of silica gel 60F-254 (Macherey-Nagel).
Solvents were obtained from Tedia and Nuclear, and reagents were purchased
from Sigma-Aldrich, Acros Organics and TCI. The following compounds were
preparing according to previous literature protocol: 3 [24], amines 5a and 5b
[25], DHPM 9 and pyrimidine 10 [26,27,28]. All infrared spectra were recorded
1
13
on a Varian 640-IR spectrometer in KBr disks. H NMR and C NMR spectra
were recorded in CDCl3 solution on a Varian VNMRS 300 MHz spectrometer.
The assignment of chemical shifts is based on standard NMR experiments (1H;
1
1
1
13C; H, H-COSY; H, 13C-HMQC). Chemical shifts (δ are in part per million
1
from the peak of tetramethylsilane (δ = 0.00 ppm as internal standard in H
NMR or from the solvent peak of CDCl3 (δ = 77.23 ppm in 13C NMR); and
multiplicities are given as s (singlet), d (doublet), dd (double doublet), t (triplet),
q (quartet), m (multiplet) or br (broad); coupling constants (J) are given in Hz.
High Resolution Mass Spectrometry with Eletrospray Ionisation (HRMS-ESI)
data on the positive mode was collected on a UHPLC-QTOF/MS Bruker Impact
II instrument. The instrument settings were the following: capillary voltage 4500
V, End Plate Offset Voltage 500 V, dry gas temperature 200 ºC. Nitrogen was
used as the desolvation gas. Methanol (Tedia HPLC grade) was used as
solvent for the analyzed samples and filtered prior to injection.
2.2. General procedure for the preparation of hybrids 11a and 11b
A mixture containing the ethyl 2-chloro-4-methyl-6-phenylpyrimidine-5-
carboxylate (10) (0.6 mmol) and N1-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-
diamine (5a) or N1-(1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine (5b) (0.4
mmol) was diluted in n-pentanol (2.0 mL) and refluxed during 48h. After this
time, the solvent was removed under vacuum at 120 ºC for an hour and the
mixture was diluted in CH2Cl2 (10 mL). The organic layer was washed with 10
mL of aqueous solutions of NaOH (10%) and brine, dried (Na2SO4) and
concentrated under vacuum. The crude oil was purified by column
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chromatography (eluting with hexane- ethyl acetate- triethylamine, 95:4:1 until
80:19:1) to give the desired product.
2.2.1.
Ethyl
4-methyl-6-phenyl-2-((6-((1,2,3,4-tetrahydroacridin-9-yl)amino)
hexyl)amino)pyrimidine-5-carboxylate (11a). Intermediate 10 and amine 5a
were reacted according to general procedure to give the desired product as a
yellow oil (75% yield). IR (KBr) νmax/cm-1: 3389, 3261, 2926, 2850, 1710, 1556,
1
1253, 1074, 754, 692; H NMR (CDCl3, 300 MHz) δ 7.99-7.87 (m, 2H), 7.55
(ddd, J = 8.3, 6.8 and 1.3 Hz, 3H), 7.44-7.36 (m, 3H), 7.34 (ddd, J = 8.3. 6.8
and 1.3 Hz, 1H), 5.31 (t, J = 5.7 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.54-3.36 (m,
4H), 3.12-2.98 (m, 2H), 2.75-2.61 (m, 2H), 2.49 (s, 3H), 1.97-1.83 (m, 4H), 1.72-
13
1.51 (m, 4H), 1.49-1.33 (m, 4H), 0.94 (t, J = 7.1 Hz, 3H); C NMR (CDCl3, 75
MHz) δ 169.0, 166.2, 161.3, 158.3, 151.0, 147.3, 139.3, 129.5, 128.5, 128.5,
128.3, 128.0, 123.7, 123.0, 120.2, 115.8, 115.2, 61.2, 49.5, 41.2, 33.9, 31.8,
29.6, 26.8, 26.7, 24.9, 23.1, 22.8, 13.7; HRMS-ESI: calcd for [M-H]+ 538.3177,
found 538.3186.
2.2.2.
Ethyl
4-methyl-6-phenyl-2-((8-((1,2,3,4-tetrahydroacridin-9-yl)amino)
octyl)amino)pyrimidine-5-carboxylate (11b). Intermediate 10 and amine 5b were
reacted according to general procedure to give the desired product as a yellow
oil (67% yield). IR (KBr) νmax/cm-1: 3389, 3262, 2922, 2850, 1712, 1562, 1255,
1
1074, 756, 692; H NMR (CDCl3, 300 MHz) δ 7.93 (dd, J = 15.2 and 8.5 Hz,
2H), 7.59-7.49 (m, 3H), 7.43-7.36 (m, 3H), 7.36-7.30 (m, 1H), 5.47 (s, 1H), 4.12-
3.97 (m, 2H), 3.54 – 3.37 (m, 4H), 3.05 (s, 2H), 2.69 (s, 2H), 2.48 (s, 3H), 1.90
(d, J = 2.9 Hz, 4H), 1.70-1.50 (m, 4H), 1.32 (s, 8H), 0.94 (t, J = 7.1 Hz, 3H); 13C
NMR (CDCl3, 75 MHz) δ 169.0, 166.3, 161.4, 158.3, 151.1, 147.3, 139.4, 129.5,
128.5, 128.3, 128.1, 123.8, 123.0, 120.2, 115.8, 115.2, 61.1, 49.6, 41.4, 33.9,
31.9, 29.7, 29.4, 29.3, 27.0, 26.9, 24.9, 23.1, 22.8, 13.7; HRMS-ESI: calcd for
[M-H]+ 566.3490, found 566.3491.
2.3. Photophysical Studies
UV-Vis absorption spectra in solution were performed on a Shimadzu UV-2450
spectrophotometer at a concentration of 10-5 M. Steady state fluorescence
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spectra were taken using a Shimadzu spectrofluorometer model RF-5301PC.
The maximum absorption wavelength was used as the excitation radiation for
emission spectra. Slits of 5.0 nm/5.0 nm were used for emission/excitation,
respectively. The quantum yields of fluorescence (ΦFL) were measured at 25ºC
using solutions with absorbance intensity lower than 0.05 (optical dilute regime).
In these experiments the emission spectra was obtained using slits of 1.5
nm/1.5 nm (Exc/Em). Quinine sulfate (exc=320 nm) in H2SO4 1N and
naphthalene in cyclohexane (exc=270 nm) was used as the quantum yield
standards [29,30]. Fluorescence time decay curves were performed in 1,4-
dioxane and acetonitrile with an EasyLife V spectrophotometer (OBB). All
measurements were performed at room temperature (25 °C). The fluorescence
decay curves were analysed using the software EasyLife V from OBB. A
nonlinear least square method was employed for the fit of the decay to a sum of
exponentials. The value of 2, residuals and the autocorrelation function were
used to determine the quality of the fit.
2.4. BSA interaction studies
The BSA association study was performed keeping the dye concentration
constant (2 μM in PBS). In this study different amounts of a previously prepared
BSA solutions (0-12 μM in PBS, pH 7.2) were added. The final solution was
kept to rest for 1h. The fluorescence spectra were obtained at 25oC and under
excitation at 320 nm (11a and 11b), 345 nm (1) and 277 nm (7) using Exc/Em
slits of 5.0 nm/5.0 nm, respectively. The BSA suppression study was performed
keeping the BSA concentration constant (11 μM in PBS, pH 7.2) and amounts
of a previously prepared dye solutions (0-20 μM in DMSO) were added. The
fluorescence spectra were obtained at 25 ºC and under excitation at 277 nm
using Exc/Em slits of 5.0 nm/5.0 nm, respectively.
2.5. Docking
The BSA structure was obtained from the Protein Data Bank
(http://www.rcsb.org/pdb/) with the ID: 4f5s [31]. The Chimera 1.8 software was
used to remove the chain B [32], waters and other molecules, and added
hydrogen atoms to the BSA protein. The ligands were built in the software
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Avogadro 1.1.1 [33], following the semi empirical PM6 [34] geometry
optimization using the program MOPAC2012 [35]. The ligands and protein in
the pdbqt format were generated by AutoDockTools, where the ligands were
considered flexible (with PM6 charges), and the BSA rigid (with Gasteiger
charges) [36]. AutoDock Vina 1.1.1 program was used for the blind docking
[37], using a gridbox of 94 x 62 x 86 and the coordinates x = 9.46, y = 23.36, z =
98.15, with an exhaustiveness of 150. As a model of binding pose, it was
selected the compound's conformer with lowest binding free energy (ΔG). The
results from docking were analyzed using the Accelrys Discovery Studio 3.5
software [38].
3. Results and Discussion
3.1. Synthesis
The classical Niementowski reaction between anthranilic acid (1) and
cyclohexanone (2) mediated by POCl3 provided the quinoline derivative 9-
chloro-1,2,3,4-tetrahydroacridine (3) [24]. In sequence, treatment of the chloride
with an excess of 1,6-diaminohexane (4a) or 1,8-diaminooctane (4b) under
reflux for 18 h provided 9-amino-1,2,3,4-tetrahydroacridines 5a and 5b, as
depicted in Scheme 1. This step was carried out in the presence of catalytic
amount of KI [25].
Scheme 1. Preparation of Tacrine derivative precursors 5a-b.
The Biginelli one-pot three component reaction between benzaldehyde (6),
ethyl acetoacetate (7) and urea (8) in the presence of catalytic SnCl2·2H2O
gave the DHPM 9 (Scheme 2) [26]. The following step was the selective
oxidation of 9 with potassium persulfate followed by chlorination with POCl3,
giving the pyrimidine ring in 10 [27,28]. Finally, the chloropyrimidine 10 was
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reacted with amine 5a and 5b through an ipso-substitution giving the desired
hybrids 11a and 11b, respectively (Scheme 2). The yields were 56% (11a) and
52% (11b).
Scheme 2. Preparation of Tacrine-pyrimidine hybrids from Biginelli reaction,
where (i) SnCl2·2H2O (10%), Ethanol, 100 ºC, 6 h. (ii) K2S2O8, CH3CN, H2O, 73
ºC, 80 min. (iii) POCl3, 115 ºC, 30 min.
3.2. Photophysical properties
The photophysical investigation in solution (10-5 M) was carried out using
different organic solvents with a wide range of dielectric constants (1,4-dioxane
dichloromethane, ethanol, acetonitrile and dimethylsulfoxide). The relevant data
from the electronic ground and excited states characterization in both steady-
state and time-resolved spectroscopies are summarized in Tables 1-3.
Figure 2 shows the UV-Vis spectra of the isolated Tacrine (Figure 2a) and
Biginelli DHPM 10 (Figure 2b), as well as the respective molecular hybrids 11a
(Figure 2c) and 11b (Figure 2d). Tacrine presents absorption maxima below
350 nm depending on the solvent but with any clear tendency on the
environment polarity, which can be related to an almost absent charge transfer
character in the ground state. The Biginelli pyrimidine precursor present
absorption maxima located between 275-278 nm, as expected due to its higher
HOMO-LUMO energy gap, due to the lower electronic conjugation if compared
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to the Tacrine. The respective molecular hybrids 11a and 11b present
absorption maxima, similar to that observed for Tacrine, located in the range
319-334 nm. In this sense, it seems that the molecular hybrids keep mostly the
electronic properties of Tacrine in the ground state. In addition, as already
observed for similar molecular hybrids, the aliphatic chain length seems not
affect at all the photophysics of these compounds in the ground state [17].
Figure 2. UV-Vis absorption spectra in solution of different organic solvents [ca.
10-5 M] of (a) Tacrine, (b) Biginelli DHPM 10 and molecular hybrids (c) 11a and
(d) 11b.
In this study, the UV-Vis spectroscopy was also used to obtain the experimental
extinction coefficient () and the respective theoretical rate constant for
0
emission (ke ), as well as the respective oscillator strengths (fe) using Equation
(1) [39]:
푓 ≈ 4.3푥10−9 ∫ 휀푑푣
̅ (1)
푒
For an electronic transition, Equation (1) relates the area under the absorption
curve from a plot of the molar absorptivity coefficient  (M-1·cm-1) against
9

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wavenumber 푣
̅
(cm-1) with its oscillator strength (fe). Moreover, from Equation
0
(2) and using the same integral, the theoretical rate constant for emission (ke )
0
can be obtained, where the pure radiative lifetime 0 is defined as 1/ke [40].
푘푒0 ≈ 2.88푥10−9푣02
̅ ∫ 휀푑푣̅ (2)
In this regard, the molar absorptivity coefficient values (), as well as the
0
calculated radiative rate constants (ke ) present in Table 1 indicate for all
studied compounds, spin and symmetry allowed electronic transitions, which
1
could be related to * transitions. In addition, an almost constant radiative
lifetime indicates that after the radiation absorption the molecular hybrids
populate the same excited state.
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Table 1. Photophysical data of compounds from UV-Vis absorption spectroscopy, where

is the molar extinction
0
coefficient (x104 M-1·cm-1),

abs is the absorption maxima (nm), fe is the calculated oscillator strength, ke is the calculated
radiative rate constant (108 s-1) and 0 is the calculated pure radiative lifetime (ns).
풌풆ퟎ
(108 s-1)
1.42
1.25
1.14
1.15
1.02
2.81
1.36
1.77
2.50
2.25
1.54
1.11
1.05
1.08
1.82
1.41
0.89
1.13
0.97
1.17

(ns)
7.05
7.98
8.80
8.72
9.84
3.55
7.36
5.65
4.00
4.44
6.50
9.00
9.49
9.23
5.50
7.07
11.15
8.82
10.29
8.53

abs
Compound
Solvent
1,4-Dioxane
Dichloromethane
Ethanol
풇풆
x104 M-1·cm-1)
1.40
(nm)
323
314
325
314
326
275
278
277
275
278
334
319
336
321
327
334
321
328
320
324
0.22
0.18
0.19
0.17
0.16
0.32
0.16
0.20
0.28
0.26
0.25
0.17
0.18
0.16
0.28
0.23
0.13
0.18
0.15
0.18
1.03
1.37
0.69
0.81
1.80
0.62
1.13
1.71
1.30
1.84
0.91
1.37
0.97
1.43
1.67
0.63
Tacrine
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
10
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
11a
11b
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
Acetonitrile
Dimethylsulfoxide
1.10
1.11
1.21
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The fluorescence emission curves were obtained by exciting the compounds at
the absorption maxima (Table 1). The Tacrine precursor presents in all studied
solvents a main fluorescence emission in the UV-A region (~375 nm). Biginelli
DHPM 10 shows a very weak emission in the 250-500 nm region (see
fluorescence quantum yields, Table 2). The molecular hybrids 11a and 11b
present fluorescence emission around 380 nm, quite similar to the emission
observed to pure Tacrine. Once more, the aliphatic chain length seems not
affect at all the photophysics of these compounds. The fluorescence quantum
yield of the Tacrine when compared to the respective hybrids, probably
indicates that the vibrational relaxation seems to play a fundamental role on the
deactivation of the excited state, different than observed for similar molecular
hybrids [17].
Figure 3. Steady-state fluorescence emission spectra in solution of different
organic solvents [ca. 10-5 M] of (a) Tacrine, (b) Biginelli DHPM 10 and molecular
hybrids (c) 11a and (d) 11b.
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Table 2. Photophysical data from steady-state fluorescence emission
spectroscopy of precursors Tacrine and Biginelli DHPM 10 and respective
molecular hybrids 11a-b, where em is the emission maxima, ST is the
Stokes shift (nm/cm-1) and
FL is the fluorescence quantum yield.
em
(nm)
359
374
362
368
379
378
378
306
378
334
378
378
371
380
385
380
380
372
382
388
ST
Compound
Solvent
FL
(nm/cm-1)
36/3105
60/5109
37/3145
54/4673
53/4290
103/9909
100/9516
29/3421
103/9908
56/6031
44/3485
59/4893
35/2808
59/4837
58/4607
46/3624
59/4837
44/3606
62/5072
64/5091
1,4-Dioxane
Dichloromethane
Ethanol
0.02
0.04
0.23
0.02
0.23
<0.01
<0.01
<0.01
<0.01
<0.01
0.02
0.01
0.01
<0.01
0.01
<0.01
<0.01
<0.01
<0.01
<0.01
Tacrine
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
10
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
11a
11b
Acetonitrile
Dimethylsulfoxide
1,4-Dioxane
Dichloromethane
Ethanol
Acetonitrile
Dimethylsulfoxide
Based on the steady-state results, time-resolved fluorescence was also
employed to better understand the photophysics of the molecular hybrids 11a
and 11b (Figure 3). The relevant data are summarised in Table 3 and the
respective residuals are presented in the ESI. The fluorescence decay profile of
Tacrine could be fitted with good 2 values, showing bi-exponential decay with
higher contribution (> 90%) from the shorter time decay (0.115-0.207 ns) in
despite the longer one (2.468-2.543 ns) alloying the calculation of an averaged
time decay below 0.3 ns. On the other hand, the DHPM precursor 10 presented
mono-exponential decays with values ranging 0.212-0.601 ns. Similarly, the
molecular hybrids showed mono-exponential decays, being closer to the longer
lifetime of Tacrine. In addition, it could also be observed that the solvent does
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not affect significantly the fluorescence lifetimes of the precursors and
respective hybrids. The individual curves, as well as the respective residuals
are presented in the supporting information.
Figure 4. Time-resolved fluorescence decays of molecular hybrids 11a and 11b
in (a) 1,4-dioxane and (b) acetonitrile (~10-5 M). The precursors Tacrine and
Biginelli DHPM 10 were also presented for comparison. IRF=instrument
response factor.
Table 3. Selected data from the time resolved fluorescence emission of
Tacrine, DHPM 10 and molecular hybrids 11a and 11b, where
 is the
experimental fluorescence lifetime (in ns), av is the fluorescence lifetime
average (in ns) and 2 represents the quality of the fit.
Compound
Tacrine
Solvent
1 (ns) (%)
0.115 (97.6)
0.207 (93.6)
0.601 (100)
0.212 (100)
1.851 (100)
1.642 (100)
1.389 (100)
1.261 (100)
(ns) (%)
av (ns)
0.173
0.351
0.601
0.212
1.851
1.642
1.389
1.261
2
1,4-Dioxane
Acetonitrile
1,4-Dioxane
Acetonitrile
1,4-Dioxane
Acetonitrile
1,4-Dioxane
Acetonitrile
2.543 (2.4)
1.147
1.050
1.092
1.039
1.073
1.199
1.145
1.193
2.468 (6.4)
-
-
-
-
-
-
10
11a
11b
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3.3 BSA interaction study
It is described in the literature to the BSA in buffered solution, an
absorption and an intrinsic fluorescence emission maxima located at 280
nm and 340 nm, respectively, with latter due to tryptophan residues [19].
In this sense, and based on the photophysical properties of the molecular
hybrids, BSA interaction with the molecular hybrids 11a and 11b was
studied from the fluorescence quenching of BSA, focusing on the
suppression experiment and also based on the increase on the
fluorescence emission of the fluorophore, related to the association study.
In this investigation, the photophysics of both precursors Tacrine and
DHPM 10 were also evaluated for comparison.
The first set of experiments regarding the BSA suppression was
performed to the pure precursor Tacrine as showed in Figure 5. The
Tacrine and DHPM 10 present in PBS media and in absence of BSA
absorption bands around 325 and 275 nm, respectively (data not shown,
see Supporting information). It can be observed that upon addition of the
Tacrine into the solution containing BSA, the UV-Vis absorption band
around 275 nm spectra seems almost constant (BSA absorption band),
with significative increase on the absorption intensities at ~325 nm
ascribed to the Tacrine absorption were observed, as expected (Figure
5a). The fluorescence emission spectra of BSA (Figure 5b) shows an
almost constant intensity with an F0/F ratio close to unity in relation to the
dye concentration (see Figure 5b, insert). This result indicates that
contrary to that observed for DHPM 10, tacrine shows no significant
interaction with BSA that would result in the suppression of its
fluorescence. It is worth mentioning that the interaction between the
precursor DHPM 10 and BSA was already studied in the literature, where
it could be observed that the absorption between 250 and 300 nm
increases after addition of the precursor 10, as expected, with significant
decrease in the fluorescence intensity of BSA [17]. In addition, any
association of the precursors with BSA, related to increase of the
fluorescence intensity of the precursors could be observed (data not
shown, see ESI Figures S7-S10).
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Figure 5. (a) UV-Vis absorption and (b) fluorescence emission spectra
(exc=277 nm) of BSA in PBS solution in the presence of different
amounts of Tacrine 1. The blank sample is the pure BSA. The inset
presents the Stern-Volmer relation.
On the other hand, as observed in Figure 6, the addition of the
molecular hybrids 11a and 11b increases the absorption intensity in the
region between 300-375 nm, as expected. However, different than
observed to the Tacrine, a significant decrease on the fluorescence
intensity of the BSA takes place after addition of 11a and 11b
.
16

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Figure 6. UV-Vis absorption and fluorescence emission spectra (exc=277
nm) of BSA in PBS solution in the presence of different amounts of 11a
(top) and 11b (bottom). The blank sample is the pure BSA. The inserts
present the respective Stern-Volmer relations.
Based on the photophysical results of the BSA in presence of the
molecular hybrids, the fluorescence quenching of the BSA could be
evaluated using the Stern-Volmer relation presented in Equation (3) [41].
F0/F = 1 + KSV[Q] = 1 + Kq0[Q] (3)
In Equation (3), F0 and F, are the fluorescence intensities of the
BSA in the absence and presence of the quencher, respectively, KSV is
17

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the Stern-Volmer constant and [Q] is the quencher concentration. In
addition, KSV can be related to the product of a bimolecular suppression
constant Kq and the fluorophore lifetime in absence of the quencher 0
,
with the latter presenting usually values around 10-8 s to biomolecules
(Figure 6, inserts) [41]. The molecular hybrids presented values to the KSV
of 6.26x104 M-1 (11a) and 5.00x104 M-1 (11b), which are quite close to the
values observed to the pure DHPM 10 (6.63x104 M-1) [17], indicating that
the pyrimidine moiety is playing an important role on the efficient BSA
quenching. Yet, the methylene chain seems not to affect the interaction
between BSA and quencher. As already discussed in this study, due to
the fluorophore lifetime (
0 ~10−8 s), the bimolecular suppression constant
Kq of the molecular hybrids for BSA were calculated to be 6.26x1012 M-1∙s-
1
(
11a) and 5.00x1012 M-1∙s-1 (11b). The calculated values for Kq exceed
the maximum values to the diffusional mechanism (2.0x1010 M-1∙s-1), it
can be concluded that the nature of quenching is not dynamic but
probably static, resulting in forming molecular hybrids-BSA complexes in
the ground state [42]. Taking the static approach into account, it is also
possible to calculate the binding constant (Ka) and the respective number
of binding sites (n) between the BSA and the suppressor by applying the
Scatchard relation presented in Equation (4) [43].
log [(F0-F)/F] = log Ka + n log [Q] (4)
In Figure 7, it is presented the double logarithmic plot relating the
fluorescence intensities from the BSA and the quenchers concentration.
For both molecular hybrids, it can be observed a linear plot correlation
with comparable binding constants (Ka) of 2.95x104 (11a) and 3.63x104
(
11b) with only one binding site for molecular hybrids 11a (n=0.94) and
11b (n=0.95). Once again, this result indicates that the methylene linker
does not plays a significant role on the molecular hybrids interaction with
the BSA.
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Figure 7. Log-log plot of BSA fluorescence intensities vs. molecular
hybrids 11a (a) and 11b (b) concentrations [Q] used to obtain the binding
constant (Ka) and binding sites (n) using Equation (4).
3.4 Molecular Modelling of BSA with hybrids
To better understand the binding modes of Tacrine, DHPM 10 and hybrids 11a
and 11b in the BSA protein, molecular docking simulations were carried out.
According to the docking, the molecules interacted in different binding sites. The
Tacrine bound in the IIA domain, 10 and 11a molecules interacted in the IB
domain, while the 11b compound bound between the IB and IIIA domains of
BSA (Figure 8a). The Tacrine interacted close with the Trp213 residue,
presenting H-bonds with the Arg256 and hydrophobic interactions with Leu237,
His241 and Ile289 residues (Figure 8b). The compound 10 bound in the region
close to the Trp134 residue, interacts with the Pro117, Ile181, Tyr160 and
Ile141 residues by hydrophobic interactions (Figure 8c). The compound 11a
demonstrated hydrophobic interactions with the Pro117, Leu122, Leu115 (in the
pyrimidine moiety) and Leu189 (in the Tacrine group) residues and an H-bond
with the Arg185 (in the Tacrine moiety) (Figure 8d). The 11b hybrid showed
hydrophobic interactions with the Ile181 (in the Tacrine moiety) and the Leu189
(in the pyrimidine group) residues, H-bonds with the Arg458 and Ser192 (in the
pyrimidine group), and electrostatic interactions with the Glu424, Arg458 (in the
pyrimidine moiety) and Arg158 (in the Tacrine group) residues (Figure 8e).
19

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Despite the hybrids molecules (11a and 11b) presented a binding pose
practically in the same site of 10, the pyrimidine moiety showed different
conformations and interactions. The insertion of the carbon chain (linker) and
the Tacrine moiety in 11a and 11b caused a change in the binding pose of
them. The compounds 10, 11a and 11b are 11.5-13.5 Å of distance from the
Trp134, while the Tacrine is 3.8 Å from the Trp213 residue. The interaction of
the compounds in these regions could lead a conformational change in BSA
and consequently affect the Trp fluorescence quenching. [44,45,46,47,48,49]
Moreover, the predicted binding energy (ΔGbind) obtained from molecular
docking indicated that Tacrine, 10, 11a and 11b, interacts spontaneously with
the BSA (ΔG= -8.2; -6.9; -9.3; and -9.7 kcal·mol-1, respectively). These results
could indicate that the molecular hybrids 11a and 11b may have a better affinity
for BSA than the Tacrine and DHPM 10. Previous studies have indicated that
these BSA regions are binding sites of molecules and could be involved in the
fluorescence quenching [44,45,46,50,51].
In addition, is important to mention that despite the short distance between the
Trp213 residue and the tacrine, obtained from the molecular docking
simulations, this molecule does not show significant suppression of the BSA
fluorescence. Probably, the orientation between the donor (Trp residue) and the
acceptor (Tacrine) is interfering in the fluorescence quenching.[48] In fact, a π-
π interaction, which could indicate a good orientation,[52,53] was not observed
here. In relation to the hybrids molecules, the distances between them and the
Trp134 (11.5-13.5 Å) are in accordance with previous studies (9-24
Å),[54,55,56] where also were observed the BSA fluorescence quenching.
20

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Figure 8. Docking simulations between the BSA protein (a) and the Tacrine (b),
DHPM 10 (c) and molecular hybrids 11a (d) and 11b (e). The H-bonds,
hydrophobic (π-alkyl, π-π T-shaped) and electrostatic (π-anion and π-cation),
interactions are shown in green, orange and purple dot lines, respectively. Red
dot lines represent the distances (in Å) between the molecules and the Trp
residues. The BSA domains I A (1-112); I B (113-195); II A (196-303); II B (304-
383); III A (384-500) and III B (501-583), were based on reports of Ramezani et
al. [57] and Wang et al. [58].
4. Conclusion
In summary, two new molecular hybrids containing Tacrine and Biginelli
pyrimidine connected by a methylene linker were synthesized by
multicomponent reaction in good yields (67-75%). The molecular hybrids
presented absorption around 320 nm, close to the Tacrine absorption. The
compounds also presented fluorescence emission around 400 nm, as observed
21

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for the separate Tacrine and pyrimidine structures. The photophysical results
showed an almost absent influence of the solvent on absorption or emission
maxima location. Although pure Tacrine did not presented any interaction with
BSA, both molecular hybrids showed BSA suppression properties due to the
pyrimidine moiety, with relative large values to the Stern-Volmer (~104 M-1) and
bimolecular suppression (~1012 M-1∙s-1) constants. Docking indicated that the
molecular hybrids presented better affinity for BSA than the isolated precursor
Tacrine and Biginelli pyrimidine. Finally, photoactive molecular hybrids seems to
be a powerful tool to design new optical sensors for biomolecules in solution,
where the synergy between their photophysical properties and specific
functional groups can be found.
Conflict of interests
The authors have declared no conflict of interest.
Acknowledgements
The authors would like to acknowledge FAPERGS (17/2551-0000968-1), CNPq
(409855/2018-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001 for the financial support.
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Graphical Abstract
29