New 1-(Spiro[chroman-2,1′-cycloalkan]-4-yl)-1H-1,2,3-Triazoles: Synthesis, QTAIM/MEP analyses, and DNA/HSA-binding assays

Article abstract of DOI:10.1016/j.molliq.2020.114729

This study synthesized and characterized the DNA/HSA properties of a new series of 12 examples of 4-(alkyl/aryl)-1-(spiro[chroman-2,1′-cycloalkan]-4-yl)-1H-1,2,3-triazoles (SCTz), in which alkyl = CH₃(CH₂)₅ and aryl = C₆H₅, 4-NH₂C₆H₄, 3-OCH₃C₆H₄ (47–95% yield) by regioselective Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC) reaction. Additioanally, single crystal X-ray diffraction (SC-XRD) analysis was carried out and the SC-XRD data enabled a broad molecular analysis using QTAIM and MEP analysis. Absorption UV–Vis properties of SCTz series were investigated and all derivatives absorb in the UV region and no are observed emission fluorescence in solution. The DNA-binding assays (UV–Vis analysis) showed that the all spiroderivatives demonstrated strong binding forces to CT-DNA. The Stern-Volmer quenching constants (K_SV) of SCTz were calculated and derivatives containing the OCH₃ group competed more strongly with DNA minor grooves, followed by the increasing general order of (K_SV): EB-DNA < DAPI-DNA. Additionally, the high values observed for the bimolecular quenching constant rate (k_q) indicated a static interaction between spiroderivatives and DNA in all cases. Finally, the HSA-binding experiments and molecular docking studies for the new spiroderivatives were determined and quenching (K_SV) and quenching rate (k_q) constants values obtained for the SCTz suggest good interaction between HSA and derivatives, probably by static mechanism, forming HSA:compound adduct in the ground state.

Full text of DOI:10.1016/j.molliq.2020.114729

Journal Pre-proof
New
1-(Spiro[chroman-2,1′-
cycloalkan]-4-yl)-1H-1,2,3-Triazoles: Synthesis, QTAIM/MEP
analyses, and DNA/HSA-binding assays
Felipe S. Stefanello, Yuri G. Kappenberg, Alex Ketzer, Steffany F.
Zorzin, Paulo R.S. Salbego, Thiago V. Acunha, Pablo A. Nogara,
João B.T. Rocha, Marcos A.P. Martins, Nilo Zanatta, Bernardo A.
Iglesias, Helio G. Bonacorso
PII:
S0167-7322(20)36971-3
DOI:
https://doi.org/10.1016/j.molliq.2020.114729
MOLLIQ 114729
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
20 August 2020
1 October 2020
5 November 2020
Please cite this article as: F.S. Stefanello, Y.G. Kappenberg, A. Ketzer, et al., New
1-(Spiro[chroman-2,1′-cycloalkan]-4-yl)-1H-1,2,3-Triazoles: Synthesis, QTAIM/MEP
analyses, and DNA/HSA-binding assays, Journal of Molecular Liquids (2020),
https://doi.org/10.1016/j.molliq.2020.114729
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1
New 1-(Spiro[chroman-2,1'-cycloalkan]-4-yl)-1H-1,2,3-
Triazoles: Synthesis, QTAIM/MEP Analyses, and DNA/HSA-
Binding Assays
Felipe S. Stefanelloa, Yuri G. Kappenberga, Alex Ketzera, Steffany F. Zorzina, Paulo R.
S. Salbegoa, Thiago V. Acunhab, Pablo A. Nogarac, João B. T. Rochac, Marcos A. P.
Martinsa, Nilo Zanattaa, Bernardo A. Iglesiasb,* bernardopgq@gmail.com and Helio G.
Bonacorsoa,* helio.bonacorso@ufsm.br
aNúcleo de Química de Heterociclos (NUQUIMHE), Departamento de Química,
Universidade Federal de Santa Maria, 97105-900 – Santa Maria, RS – Brazil.
bLaboratório de Bioinorgânica e Materiais Porfirínicos, Departamento de Química,
Universidade Federal de Santa Maria, 97105-900 – Santa Maria, RS – Brazil.
c Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Santa
Maria, Santa Maria - RS, Brazil.
*Corresponding Authors.
Abstract
This study synthesized and characterized the DNA/HSA properties of a new series
of 12 examples of 4-(alkyl/aryl)-1-(spiro[chroman-2,1'-cycloalkan]-4-yl)-1H-1,2,3-
triazoles (SCTz), in which alkyl = CH3(CH2)5 and aryl = C6H5, 4-NH2C6H4, 3-
OCH3C6H4 (47-95% yield) by regioselective Copper-Catalyzed Azide–Alkyne
Cycloaddition (CuAAC) reaction. Additioanally, single crystal X-ray diffraction (SC-
XRD) analysis was carried out and the SC-XRD data enabled a broad molecular
analysis using QTAIM and MEP analysis. Absorption UV-Vis properties of SCTz
series were investigated and all derivatives absorb in the UV region and no are observed
emission fluorescence in solution. The DNA-binding assays (UV-Vis analysis) showed
that the all spiro-derivatives demonstrated strong binding forces to CT-DNA. The Stern-
Volmer quenching constants (KSV) of SCTz were calculated and derivatives containing
the OCH3 group competed more strongly with DNA minor grooves, followed by the
increasing general order of (KSV): EB-DNA < DAPI-DNA. Additionally, the high
values observed for the bimolecular quenching constant rate (kq) indicated a static
interaction between spiro-derivatives and DNA in all cases. Finally, the HSA-binding
experiments and molecular docking studies for the new spiro-derivatives were
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determined and quenching (KSV) and quenching rate (kq) constants values obtained for
the SCTz suggest good interaction between HSA and derivatives, probably by static
mechanism, forming HSA:compound adduct in the ground state.
Keywords: CuAAC reactions; Triazoles; DNA-Binding; HSA-binding.
Introduction
The 1,2,3-triazole is an important class of compounds that performs numerous
biological activities and is widely used in organic synthesis, medicinal chemistry, and
material science.[1–6] Molecules with this isomeric chemical compound are known to
exhibit antimicrobial,[7] anti-HIV,[8] antitumoral,[9] anti-inflammatory,[10]
antitubercular activities.[11,12]
Furthermore, it is well known that the chromenone framework is present in the
synthetic components and a wide variety of natural compounds.[13–19] In this context,
spirochromonones have attracted significant interest as a privileged structure in the
development of several bioactive compounds with numerous biological activities.[20–
28] [29–32] Recently, our research group showed that the use of spirochromones in
cyclization with 2-aminobenzonitrile enables the production of tacrine hybrids. The
tacrine hybrids synthesized in this method also presented moderate acetylcholinesterase
(AChE) inhibitory effects.[22,33] Moreover, the same compounds were studied
regarding the cytotoxic activity of tacrine derivates using glioblastoma cell line
(SF295).[22]
Deoxyribonucleic acid is one of the main compositions of living organisms. The
DNA biomacromolecule regulates many biochemical processes that occur in the cellular
system. Therefore, understanding and investigating the interactions and mechanisms of
biological-target molecules binding with nucleic acids may be a more robust finding at
the molecular level and particularly important for developing new drugs in the
biological field.[34–36] In this context, it is necessary to design different compounds
that may fit well in DNA binding (between nucleobases or by grooves), cleavage, and
interactions with certain proteins, which is the main cellular target for anticancer
derivatives. Investigating the interactions of molecules with human serum albumin
(HSA) by spectroscopic methods provides important information on the binding or
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interactions of a determined site.[37–39] As serum proteins perform transport,
distribution, accumulation, and excretion properties of medical drugs in tumor tissues,
special consideration must also be given to investigating the binding of spiro-derivatives
to proteins.
In this manuscript, new triazole systems bounded to benzopyrans with spiro
carbons of 5, 6, and 7-membered rings (SCTz) are presented. The 1,2,3-triazole moiety
is linked to the spirochromanes via click chemistry reaction, which allowed the
production of a novel series of 4- (alkyl/aryl)-1-(spiro[chroman-2,1'-cycloalkan]-4-yl)-
1H-1,2,3-triazoles (SCTz 10-13) (Scheme 1). The efficiency of these new compounds
and a variety of substituents on the spectroscopic and electrochemical behavior of the
synthesized molecules was evaluated and the results presented and discussed.
Additionally, DNA-binding, HSA-binding, and molecular docking analyses were
conducted to better explain this biomolecule interaction type.
Scheme 1. Summary of this study. Reaction conditions: (i) Pyrrolidine; (ii) NaBH4; (iii) (a) MsCl (b)
NaN3; (iv) CuAAC reaction.
Results and Discussion
Synthesis and characterization
The spiro[chromane-2,1'-cycloalkyl]-4-ones (3a-c) were prepared by adapting a
common multi-component methodology developed by Kabbe et al.31 and further
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improved in the absence of solvent.[20,26] This resulted in a thermal condensation of 5-
substituted-2-hydroxyacetophenones (1a-c) in the 5-position with cycloalkanones (2a-c)
caused by the pyrrolidine, which produced the products in similar yields as described in
the literature.[26]
Then, spirochromanones (3a-c) were converted to spirochroman-4-ols (4a-c)
using sodium borohydride as the reducing agent in ethanol.[27] The products (4a-c)
were obtained in the form of yellow, brown, and white viscous oils and white solids in
yields of 67-85%, such as racemic mixtures with configuration 4R/4S and similar to
those described in the literature,[27] and used in the synthesis of new 4-
azidospiro[chroman-2,1'-cycloalkanes] (5a-c) without isolation (Scheme 2).
Scheme 2. Synthesis of spirochroman-4-ols. Reaction conditions: (i) Pyrrolidine, 110 ºC, 6 h; (ii) NaBH4,
EtOH, 80 ºC, 1h (67-85%).
The series of compounds (5a-c) were obtained from a nucleophilic substitution
reaction. For this, spirochroman-4-ols (4a-c) and mesyl chloride were used as they lead
to the formation of spiro[chroman-1'-cycloalkan]-4-yl methanesulfanates, followed by a
substitution reaction with sodium azide in order to form organic azides 5. The obtained
products were yellow oils and used in the next step also without purification (Scheme
3). From the 4-azidospiro[chroman-2,1'-cycloalkanes] (5a-c), it was then possible to
obtain a series of SCTz (10-13) via CuAAC reaction.
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Scheme 3. Synthesis of 4-(alkyl/aryl)-1-(spiro[chroman-2,1'-cycloalkan]-4-yl)-1H-1,2,3-triazoles.
Reaction conditions: (iii) (a) 4a-c (1 eq), MsCl (4 eq), CH2Cl2 dry, 0 ºC, 15 min; (b) NaN3 (2.5 eq), DMF,
80 ºC, 12h. (iv) Terminal alkynes, sodium ascorbate (10 mol%)/CuSO4.5H2O (20 mol%), THF/H2O (1:1),
room temperature, 24 h (47-95% yield).
Firstly, in order to obtain the triazole 10b as the target, a methodology previously
described by the NUQUIMHE group[26] was tested, starting with 5b. Employing the
conditions previously described,[26] factors such as time, proportions of copper iodide,
azide, and alkyne were optimized. The reaction conditions considered in the
optimization process are shown in Table 1.
Table 1. Optimization of the reaction for the 1,2,3-triazole-spirochromane 10b.
Entry Catalyst
(mol %)
Azide
(5b)
(mmol)
Alkyne Solvent (v.v.v)
(6)
(mmol)
Temp
(ºC)
Time Yield
(h)
(10b)
(%)
1
2
CuI (50)
CuI (25)
CuI (50)
CuI (50)
CuI (25)
1.5
1.5
1.5
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.5
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
80
80
80
80
80
12
12
24
12
12
5
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
5
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
16
19
24
3
4
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
5
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
6
7
8
1.5
1.1
1.5
THF/t-BuOH/H2O
3:2:1 (2.5 mL)
80
25
25
28
66
56
CuI (25)
24
24
Cu source (20/20) 1.0
Cu source (20/20) 1.0
THF/H2O 1:1
(5.0 mL)
THF/H2O 1:1
(5.0 mL)
24
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9
Cu source (20/20) 1.0
Cu source (20/20) 1.0
Cu source (30/30) 1.0
1.1
1.1
1.1
THF/H2O 1:1
(5.0 mL)
65
25
25
24
24
24
66
37
46
10
11
t-BuOH/H2O 1:1
(5.0 mL)
THF/H2O 1:1
(5.0 mL)
12
Cu source (20/10) 1.0
1.1
THF/H2O 1:1
(5.0 mL)
25
24
70
13
14
Cu source (30/10) 1.0
Cu source (50/10) 1.0
1.1
1.1
THF/H2O 1:1
(5.0 mL)
25
25
24
24
47
40
THF/H2O 1:1
(5.0 mL)
Cu source: CuSO4. 5H2O) : NaAsc (mol%). NaAsc = Sodium Ascorbate.
In entry 1, the reaction conditions previously described by Ketzer et al.[26] were
reproduced for this system. In entries 2-6, some parameters were changed in order to
increase the yield of the triazole, such as decreasing the concentration of copper(I)
iodide,[40] the reaction time, the proportions of the azide, and the terminal alkyne,
although there was no change in the yield.
Lastly, copper iodide as a catalyst for click chemistry reactions was not
considered satisfactory due to the low yields obtained. This is because the catalyst used
may have formed oxidative coupling products.[5] Another disadvantage of using Cu(I)
salts is their thermodynamic instability, which can be oxidized to Cu(II) and/or
disproportionate to copper and Cu(II) in the course of the reaction.[29]
Therefore, another strategy was used to obtain SCTz (10a-c), (11a-c), (12a-c),
and (13a-c), as it was observed in the literature[11] that heterocycles similar to those
proposed here used a different copper source than the one previously tested. Thus,
copper sulfate pentahydrate combined with sodium ascorbate was used and the catalyst
prepared in situ by reducing Cu (II) salts. Ascorbic acid or sodium ascorbate proved to
be excellent reducing agents to prepare several 1,4-triazoles in high yields and
purity.[4,6] According to the literature, a small excess of reducing agent is generally
added to prevent the formation of oxidative coupling products, which are often observed
when a source of Cu(I) is added directly.[5] The preparation of 1,2,3-triazoles-1,4-
disubstituted was carried out, on average, from 6 to 36 h at room temperature.
In entry 7, the reaction conditions previously described by Sajja[11] were
reproduced and generated a yield of 66% of the final product. During this reaction
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condition, we sought to improve the yield of the final product (10b) by varying its
reaction conditions. The variation of the proportion of alkyne of entries 8 and 9 were
initially studied by increasing their quantity of azide and temperatures, respectively,
while maintaing the other paramaters the same, resulting in a smaller yield for entry
7.[9] Many methodologies[6] report the use of solvent mixtures in the reactions, such as
tetrahydrofuran and water or tert-butanol and water, although these variations of the
reaction solvent proved to be inefficient in entry 10. After establishing that the
conditions of entry 7 were even more efficient, they varied for proportions of sodium
ascorbate and copper sulfate pentahydrate[30] in entries 11-14. Thus, it can be noted
that the increased proportions of the catalysts proved ineffective for entry 11.
Furthermore, the proportions of sodium ascorbate relative to copper sulfate
pentahydrate varied in entries 12, 13, and 14, showing that as the proportion of sodium
ascorbate increased, the yield also decreased proportionally.
In this way, it was possible to observe that entry 12 was the most efficient form
to obtain the triazole 10b, which was extended and used to synthesize the series SCTz
(10a-c), (11a-c), (12a-c), (13a-c), and yields ranging from 47 to 95%. The SCTz (10a-
c), (11a-c), (12a-c), and (13a-c) were characterized by NMR experiments. Depending
on the compound, gas chromatography coupled to mass spectrometry (GC/MS), high-
resolution mass spectrometry (LC-HRMS) or both were performed. Moreover, it was
possible to characterize some compounds by the crystal structure (SC-XRD).
No major differences in the NMR spectra were observed through the compound
series (10, 11, 12, and 13), which is mainly due to the nature of the spiro substituents.
Therefore, 10b will be used as an example for the proper signal assignment. In the 1H
NMR spectrum performed on 400 MHz, in CDCl3 as solvent for triazole 10b (see
Supporting Information, Fig. S15), it was possible to observe the signals of the
hydrogens of the phenyl in the aromatic region in which the most disjointed ones were
H-4c/H-4c', appearing in the form of a doublet in δ 7.83 ppm with 1JHH = 7 Hz. Then,
the aromatic H-4b/H-4b' hydrogens appeared in the aromatic region as the multiplet δ
7.46-7.37 ppm and H-4d appeared as a triplet at δ 7.33 ppm 1JHH = 7.5 Hz and also
hydrogen H-5 (triazole ring) appears as a singlet at δ 7.66 ppm.The two aromatic
hydrogens relative to the chromane moiety are in the form of multiplets at δ 7.31-7.21
ppm for H-7 and 7.01-6.94 ppm for H-5. The other two H-6 and H-8 aromatic
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hydrogens are in oxygen shielding zones (O1) and were overlapped at δ 6.90- 6.80 ppm
in the form of a multiplet.
It was also possible to observe a doublet of doublets at δ 6.14 ppm with 1JHH
11.5 Hz and 2JHH = 6.5 Hz referring to the H-4 of spirochromane moiety. The
=
multiplicity of H-4 is present due to the interaction with the diastereopic hydrogens H-
3a and H-3a, which are doublets of doublets in the 2.52 ppm and 2.29 ppm regions with
1JHH = 13 Hz and 2JHH = 6.5 Hz and 1JHH = 13 Hz and 2JHH = 11.5 Hz, respectively. The
multiplet at δ 1.99-1.37 ppm refers to signals from the ten diastereotopic hydrogens that
make up the spirocarbocyclic moiety.
For the 13C NMR spectrum was performed on 100 MHz in CDCl3 as solvent for
compound 10b (see Supporting Information, Fig. S16). The signal of the C-8a
quaternary carbon was bound to the chromane oxygen at δ 153.6 ppm and C-4a for the
triazole ring at δ 148.3 ppm. The aromatics bound to C-4, C-4c/C-4c', C-4d, C-4b/C-4b',
and C-5 of triazole are shown in δ 130.6, 128.8, 128.1, 125.7, and δ 117.8 ppm,
respectively. Signals of the chromane ring C-7, C-8, C-6, C-4a, and C-5 aromatic
carbons appear at δ 130.1, 127.5, 120.7, 119.2, and δ 118.0 ppm, respectively. The
signal for C-2 appears almost superimposed on CDCl3 at δ 76.1 ppm. Spiro carbon C-4
exhibited chemical shifts at δ 54.2 ppm and C-3 showed a higher field signal at δ 40.1
ppm. The signals relating to C-2', C-3', C-4', C-5', and C-6' of the spirocarbocyclic
moiety appeared in the range δ 37.8 – 21.5 ppm.
Molecular Insights
Single crystal X-ray diffraction (SC-XRD) analysis was carried out to confirm the
molecular structure of the obtained the SCTz series of compounds in the solid state.
Additional structures were obtained to furnish molecular insights on the influence of
different substituents in these novel structures. Complete crystallographic data and
molecular diagrams are shown in the Supporting Information - Table S1, Fig. S1 for
compounds 10b, 11b, 12b-c, and 13c. A racemic mixture was observed in the
crystalline solid state in all obtained structures.
From the molecular overlay and by using the triazole as reference, it is possible to
observe qualitatively the two main torsion angles that occur in these models in all five
structures (Table 2). Torsion angle θ1 changed and ranged from 17.41 to 30.5° in
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absolute values for 10b, 11b, and 12b-c. The highest difference occurred by the
presence of the MeO substituent (12b) compared with 10b (around 12°). The addition
of one carbon to the spiro portion in 12c did not affect torsion angle θ1 when compared
with 12b, although it significantly affected torsion angle θ2. The addition of the alkyl
chain in R1 (13c) echoed in the molecule, affecting the value of torsion angle θ2 and
overlapping the additional carbon in the spiro portion.
Table 2. Angle between planes (θ), torsion angles (ϕ), and molecular overlaya of the obtained
crystalline structures.
Torsion Angles (ϕ)b
Angle Between Planes (θ)
Structure
ϕ1 (°)
ϕ2 (°)
θ1 vs θ2 (°)
18.65
θ1 vs θ3 (°)
17.41
-62.59
-64.32
72.96
81.65
82.30
87.95
88.98
88.07
10b
11b
12b
12c
13c
-19.51
29.37
20.23
28.74
-30.51
-125.72c
-79.40
-71.90
31.21
84.27d
aMolecular overlay using triazole atoms (N1-N2-N3-C11-C10). bϕ1: C17-C12-C11-C10; ϕ2: C4-
C3-N1-C10. cϕ1: C13-C12-C11-C10. dAlkyl portion (C12-C13-C14-C15-C16-C17) used for
plane θ2.
The angles between planes (θ) were calculated to observe the geometric influence
of the substituents on the molecular structure (Table 2). The angle values between
planes θ1 and θ2 increased through the different substituents. These changes are likely
due to the higher influence of the intermolecular environment, that is, the result of the
intermolecular interactions with the neighboring molecules. Compound 13c is a good
example of this intermolecular influence, as it showed a value of almost 90° between
the planes. This may have occurred because of the topology adjustment of the alkyl
chain to achieve higher complementarity and energetic stabilization. The values
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between θ1 and θ3 were similar and ranged from 81.65 to 88.98°, which probably due to
a subtle intermolecular influence and another consequence of molecular changes.
This geometric discussion (Table 2) provides a superficial analysis of the nature
of these models. Additionally, this geometric approach (distances and/or angles between
atoms or planes) can sometimes result in misleading interpretations, especially when
dealing with ‗weaker‘ interactions, as already reported.[41] Therefore, a broader
molecular analysis of the context of intramolecular interactions must be performed.
Quantum Theory of Atoms in Molecules (QTAIM) analysis was performed in
order to better understand these models in an intramolecular interaction context. The
QTAIM provides a quantum description of the location and behavior of the atoms in a
chemical bond.[42] It also furnishes the electron density (ρ) of each bond critical point
(BCP), which is the minimum electron density of/in the bond path.[42] From these data,
the interaction between atoms is obtained by the BCP and may provide additional
information on the nature of the interactions and description of the chemical bond and
interactions.[43]
Nevertheless, only compound 10b showed some interaction (Figure 1), which
was revealed by C-H∙∙∙H-C intramolecular interactions between the hydrogen atom of
the chromane portion (atoms C3-H3) and a hydrogen atom from the spyro portion
(atoms C22-H22B). The observed C-H∙∙∙H-C interaction presented a considerable HBCP
(V/2) value of -2.82 kcal mol-1. The molecular QTAIM analysis for structures 11b, 12b-
c, and 13c are described in the ESI.
Figure 1. BCP of the intramolecular interaction observed by QTAIM for compound 10b.
Molecular electrostatic potential analysis (MEP)[44,45] was performed in order
to obtain information on the main molecular regions in which these structures interact,
either by self-assembly or possible molecular docking (Figure 2). This analysis
furnishes regions with negative and positive electrostatic potential in the considered
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molecule.[44,45] The MEPs of compounds 10b, 11b, 12b-c, and 13c, with their values
of higher potential regions highlighted, are illustrated in Figure 2.
Figure 2. Molecular electrostatic potential (MEP) maps of SCTz 10b, 11b, 12b-c, and 13c.
In general, the structures showed increased negative regions in the nitrogen
atoms of the triazole that may be involved in intermolecular interactions (binding)
(Figure 2). The analysis also showed the expected negative regions of the oxygen and
nitrogen atoms of CH3O (12b-c) and NH2 (13c) substituents, respectively. Moreover, it
is possible to observe a considerable portion of the molecule with positive regions,
especially the spiro portion. Aditionally, compound 13c presented a ‗blue wall‘ of
positive electrostatic potential region of the hydrogen atoms from the alkyl portion
(Figure 2). This portion may be important in the intermolecular context,
complementarity between molecules or binding situations, to best accommodate the
components.
The atoms involved in the observed C-H∙∙∙H-C intramolecular interaction
(Figure 1) in compound 10b did not demonstrate complementarity of electrostatic
potential regions in the MEP analysis (see Supporting Information, Fig. S2). This
indicates that this intramolecular interaction occurred due to high proximity between the
hydrogen atoms, which is the result of its molecular topology and likely caused by the
intermolecular environment.
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Absorption analysis by UV-Vis spectroscopy
The comparative UV-Vis absorption spectra of the compounds using dimethyl
sulfoxide (DMSO) as solvent are shown in Figure 3 and the absorption properties are
listed in Table 3. All SCTz showed absorption maxima around the 250–450 nm range in
the ultraviolet region. The values for the molar absorptivity coefficient (ε) for these
compounds indicated that spin and symmetry allowed electronic transitions, which may
be related to π→π* transitions (Figure 3a-c). Slightly changes were osberved in the
absorption maxima location or wavelength shift associated with the different
stereochemistry in the SCTz. Moreover, for all spiro-derivatives, no fluorescence
emission in DMSO solution was observed.
1.0
(a) five-member
0.8
0.6
0.4
10a
11a
0.2
12a
13a
0.0
250
300
350
400
450
Wavelength (nm)
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1.0
0.8
0.6
0.4
0.2
0.0
(b) six-member
10b
11b
12b
13b
250
300
350
400
450
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
(c) seven-member
10c
11c
12c
13c
250
300
350
400
450
Wavelength (nm)
Figure 3. Electronic UV–Vis absorption spectra of SCTz containing different structures (a) five-member,
(b) six-member and (c) seven-member spiro-ring, all in DMSO solutions ([ ] = 1.2 – 1.35 x 10-4 M).
Table 3. Absoption UV-Vis transitions values of SCTz.
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Derivative
λ, nm (ε; M-1 cm-1)
257 (2,641); 366 (bb)*
258 (4,808); 286 (sh)**
258 (3,900)
10a
10b
10c
282 (3,658)
11a
283 (5,192)
11b
256 (2,968); 281 (4,072)
258 (6,115); 288 (2,992); 298 (sh)**
258 (2,816); 288 (1,491); 298 (850)
259 (6,245); 288 (3,010); 298 (sh)**
278 (3,360); 286 (3,240)
278 (2,341); 286 (2,250)
279 (3,211); 285 (3,052)
11c
12a
12b
12c
13a
13b
13c
*Broad band; **Shoulder.
DNA-binding assays by UV-Vis analysis
The interaction of SCTz series with CT-DNA was also studied by absorption UV-
vis spectroscopy at the 250–600 nm range in DMSO (2%)/Tris-HCl buffer solution (pH
7.4). The SCTz interact with CT-DNA and give a transition band change in the
ultraviolet range. The effects of different concentrations of CT-DNA on the electronic
absorption spectra of the compounds are presented in Figure 4. Generally, as DNA
concentrations increased, the SCTz had characteristic changes (hyperchromicity and
bathochromic shift) in the absorption electronic spectra (Table 4).
The addition of various concentrations of CT-DNA (0 to 100 μM) increased
absorbance in the π→π* transition band at the ultraviolet range. Blue or red-shifts were
observed depending on the substituent, which is an indication of possible intercalation
modes of the spiro molecules between DNA nitrogeneous base pairs (e.g. compound
10a; Figure 4).[32] The other interactive possibility of these derivatives is probably H-
bonding forces with the DNA bases or the presence of the triazole moiety in the
structure that may interact with DNA bases via covalent interactions, as previously
reported for other triazoles derivatives.[46–48]
Moreover, the intrinsic binding constants (Kb) of the SCTz were calculated (Table
4). In general, all 1,2,3-triazoles derivatives demonstrated strong binding forces to CT-
DNA (Kb ≈ 106 M–1) following the increasing order of Kb values: 12c < 13c < 11a <
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10b < 10a ≈ 13a < 13b < 11c < 10c < 12b < 11b < 12a. The CT-DNA UV-vis titration
spectra of all derivatives are listed in Supporting Information (Figs. S60–S70).
Figure 4. UV-Vis titration absorption spectra of derivative 10a in a DMSO (2%)/Tris-HCl buffer (pH
7.4) mixture. The concentration of CT-DNA ranged from 0 to 100 μM. Insert graph: plot of [DNA]/(εa –
εf ) vs. [DNA].
Table 4. CT-DNA-binding results by UV-Vis absorption analysis for the SCTz series.
Derivative
10a
H%a
30.90
22.55
33.50
39.90
25.60
34.20
19.50
44.70
11.45
38.60
49.20
38.00
Δλ (nm)b
6.0
Kb (M-1)c
1.00 x 106
0.94 x 106
1.28 x 106
0.77 x 106
1.86 x 106
1.09 x 106
3.70 x 106
1.80 x 106
0.66 x 106
1.02 x 106
1.05 x 106
0.70 x 106
ΔGº (kcal mol-1)
−8.18
4.0
−8.14
10b
4.0
−8.32
10c
9.0
−8.02
11a
7.0
−8.55
11b
8.0
−8.23
11c
4.0
−8.95
12a
6.0
−8.53
12b
4.0
−7.93
12c
1.0
−8.19
13a
3.0
−8.21
13b
1.0
−7.97
13c
aHyperchromicity of Soret band: H(%) = (Absinitial – Absfinal) / (Absinitial) × 100;
bRed shift: Δλ = λinitial – λfinal
cBinding constant by absorption UV-Vis analysis.
;
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Dye-DNA competition assays by steady-state fluorescence emission
Competitive-binding experiments using the quenching method to determine the
displacement of the intercalating ethidium bromide (EB) or minor groove binder 4′,6-
Diamidine-2′-phenylindole (DAPI) dyes from CT-DNA may provide further
confirmation on the binding affinity of the complexes.
Emission spectra were monitored as increasing concentrations of SCTz series
were added by titration to a fixed concentration of DNA pretreated with dyes (EB or
DAPI). Interpretations of the changes observed in the dye spectra followed by binding
to CT-DNA are often used for interaction studies between DNA and other compounds
such as heterocyclic molecules.[49] The EB-DNA assays show strong emissions at 636
nm when excited at 510 nm (Figure 5). Moreover, Figure 5 also depicts the emission
spectra of EB bound to CT-DNA in both the absence and presence of compound 10a.
When spiro-derivatives were added to the DNA pre-treated with EB, the nucleic acid-
induced emission intensity of EB decreased (Figure 5). The results demonstrate a slight
quenching of the fluorescence intensity of the EB-DNA complex followed by the
addition of increasing concentrations of SCTz series, which may be attributed to the
competition of spiro molecules with EB over binding to the grooves of DNA. The inset
graphs in Figure 5 represent the Stern-Volmer plot, which was obtained as the
relationship between F0/F and [CT-DNA]. The EB-DNA emission spectra of the other
spiro-derivatives are listed in Supporting Information (Figures S71-S81).
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Figure 5. Steady-state emission fluorescence spectra of EB-DNA assays in the presence of compound
10a in a DMSO (2%)/Tris-HCl buffer solution (pH 7.4) at exc = 510 nm. The arrow indicates the changes
in fluorescence at increasing concentrations of samples. Insert graph: plot of F0/F versus [DNA].
In the next experiment, steady-state emission spectra were monitored as
increasing concentrations of SCTz were added by the addition to a fixed concentration
of CT-DNA pre-treated with DAPI dye as a minor groove binder molecule. The DAPI-
DNA interaction with compound 10a shows a strong emission at λem = 465 nm when
excited at λexc = 360 nm (Figure 6). Moreover, Figure 6 also depicts the emission
spectra of DAPI bound to CT-DNA in both the absence and presence of the selected
compound. When spiro-molecules were added to the DNA in the presence of the DAPI
dye, the DNA emission intensity of DAPI decreased.
Moderate quenching behavior was observed in the fluorescence intensity of the
DAPI-DNA adduct following the addition of increasing concentrations of SCTz.
According to the quenching constant (KSV) values found for the derivatives, this
occurred due to greater competition of the molecules for the minor groove when
compared to the EB intercalator dye (Table 5).
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In general, the Stern-Volmer quenching constants (KSV) of SCTz were calculated
and summarized in Table 5. Derivatives containing the CH3O group compete more
strongly with DNA minor grooves, following the increasing general order of (KSV): EB-
DNA < DAPI-DNA. In addition, the high values observed for the bimolecular
quenching constant rate (kq) indicated a static interaction between spiro-derivatives and
DNA in all cases (Table 5). The CT-DNA interactive emission spectra of all derivatives
are presented in Supplementary Information (Figs. S82-S92).
Figure 6. Steady-state emission fluorescence spectra of DAPI-DNA assays in the presence of compound
10a in a DMSO (2%)/Tris-HCl buffer solution (pH 7.4) at exc = 359 nm. The arrow indicates the changes
in fluorescence at increasing concentrations of samples. Insert graph: plot of F0/F versus [DNA].
Table 5. Competitive dye-DNA values by steady-state fluorescence emission analysis for SCTz.
EB-DNA emission
Compound
10a
Q%a
4.70
5.15
6.90
3.80
6.55
7.25
KSV (M-1)b
5.16 x 102
6.09 x 102
6.32 x 102
4.76 x 102
4.20 x 102
7.43 x 102
kq (M-1s-1)c
2.24 x 1010
2.64 x 1010
2.74 x 1010
2.07 x 1010
1.82 x 1010
3.23 x 1010
10b
10c
11a
11b
11c
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5.55
4.50
5.60
4.35
5.60
4.20
6.71 x 102
5.65 x 102
5.70 x 102
4.68 x 102
6.01 x 102
4.00 x 102
2.92 x 1010
2.45 x 1010
2.48 x 1010
2.03 x 1010
2.61 x 1010
1.74 x 1010
12a
12b
12c
13a
13b
13c
DAPI-DNA emission
Compound
10a
Q%a
11.20
6.30
KSV (M-1)b
kq (M-1s-1)d
6.68 x 1011
3.50 x 1011
3.41 x 1011
3.09 x 1011
6.18 x 1011
3.27 x 1011
6.04 x 1011
6.22 x 1011
2.57 x 1012
2.59 x 1011
3.04 x 1011
3.72 x 1011
1.47 x 103
0.77 x 103
0.75 x 103
0.68 x 103
1.36 x 103
0.72 x 103
1.33 x 103
0.49 x 103
5.65 x 103
0.57 x 103
0.67 x 103
0.82 x 103
10b
12.80
6.15
10c
11a
11.15
5.20
11b
11c
11.05
4.80
12a
12b
12.83
5.90
12c
13a
8.25
13b
7.85
13c
aQuenching: Q(%) = (Max. initial emission – Max. final emission) / (Max. initial emission) × 100;
bStern-Volmer quenching constant by steady-state fluorescence emission;
cBimolecular quenching rate EB-DNA constant by steady-state emission spectra (η0 = 23 ns);
dBimolecular quenching rate DAPI-DNA constant by steady-state emission spectra (η0 = 2.2 ns).
HSA-binding properties by steady-state fluorescence emission
Steady-state emission fluorescence spectroscopy can be used to monitor damage
to human serum albumin (HSA) upon spiro-derivative binding. In principle, these
interactions can produce changes in the position or orientation of the tryptophan residue,
altering its exposure to solvents and leading to alterations in relative fluorescence
properties.[50,51]
The Stern-Volmer quenching (KSV), bimolecular quenching rate (kq), and
association (Ka) constants were evaluated from the linear plots of emission intensities
versus spiro-derivative concentration and quenching values (Table 6). The significant
Stern-Volmer quenching (103 - 104 M−1 range) and association constant values (103 M−1
range) obtained for the SCTz suggest there is good interaction between the compounds
and HSA proteins.[52]
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Higher values of quenching rate constants (kq) were observed for spiro-
derivatives (1011 - 1012 M−1 s−1 range), indicating that steady-state fluorescence
quenching probably occurs via static mechanism with the formation of a complex
HSA:compound in the ground state (Table 6).43 Moreover, the number of binding sites
(n) in all compounds between 0.7 to 1.60 suggests the presence of possible double
binding sites for the compounds in the HSA subunits. All HSA fluorescence emission
spectra are shown in Supplementary Information (Figs. S93-S103).
Figure 7. Steady-state emission fluorescence quenching spectra of HSA in the absence and presence of
spiro-compound 10a in a DMSO (2%)/Tris-HCl buffer solution (pH 7.4) at exc = 290 nm. The arrow
indicates the changes in fluorescence at increasing concentrations of SCTz samples. Insert graph: plot of
F0/F versus [compound].
Table 6. Stern–Volmer quenching constant (KSV), quenching rate constant (kq), association constant (Ka),
and the number of binding site (n) values for the interactions of SCTz with human serum albumin (HSA).
Compound
10a
Q(%)a
33.40
19.00
33.30
39.50
46.15
KSV (M-1)b
5.00 x 103
2.55 x 103
3.65 x 103
6.70 x 103
8.26 x 103
kq (M-1s-1)c
8.81 x 1011
4.50 x 1011
6.44 x 1011
1.18 x 1012
1.45 x 1012
Ka (M-1)d
3.93 x 103
2.65 x 103
2.63 x 103
4.08 x 103
3.30 x 103
ne
1.06
0.88
0.76
1.07
0.84
10b
10c
11a
11b
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41.20
59.80
57.80
57.90
39.40
43.60
43.15
7.34 x 103
1.29 x 1012
2.50 x 1012
2.34 x 1012
2.18 x 1012
1.14 x 1012
1.36 x 1012
1.36 x 1012
4.07 x 103
4.88 x 103
6.30 x 103
6.33 x 103
4.38 x 103
4.58 x 103
4.62 x 103
1.04
1.19
1.53
1.54
1.13
1.16
1.18
11c
12a
12b
12c
13a
13b
13c
1.42 x 104
1.33 x 104
1.24 x 104
6.50 x 103
7.75 x 103
7.72 x 103
aQuenching = (Intinitial – Intfinal) / Intinitial x 100; bStern–Volmer quenching constant; cStern–Volmer
quenching rate constant (HSA – η0 = 5.67 ns); dBinding constant; eBinding site values.
Molecular docking calculations with DNA and HSA
The computational docking simulation suggested that SCTz10-13a-c molecules
interact in the minor groove region of the DNA (Figures 8 and S108), which is
consistent with the experimental CT-DNA binding assay. In general, all molecules
presented similar binding poses, interacting in the groove between dC9, dG10, and
dC11 residues from DNA strand A and the dG14, dC15, and dG16 residues from DNA
strand B. Van der Waals (vdW) and H-bond are the main interactions (Table S2), with
their distances ranging from 3.3 to 4.1 Å and from 1.9 to 2.3 Å, respectively. The OCH3
group from the 12a-c derivatives may assist in the interaction with the sugar-phosphate
backbone of dC9 and dG10 and, perhaps, may be linked to the strong competition with
DNA minor groove. Here, we highlight that the 1,2,3-triazole moiety has an important
role as an H-acceptor in the binding of 10-13a-c molecules with the DNA due to its H-
bond with the hydrogen from the amino group of dG10 and dG16 (Figure 8). The
involved triazole region in the interaction can be observed in the MEP analysis (Figure
2). Hemamalini[53] and Saini[54] reported that 1,2,3-triazole derivatives interact in the
minor groove of DNA and make H-bonds with the nitrogenated base pairs.
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Figure 8. Overview of the best docking pose between DNA and SCTz compounds 10-13a-c. DNA rings
in red, blue, pink, and green indicate deoxyadenosine (dG), deoxythymidine (dT), deoxycytidine (dC),
and deoxyguanosine (dG), respectively. dC9, dG10, and dC11 correspond to DNA strand A, while dG14,
dC15, and dG16 correspond to DNA strand B. As an example, the 10a and 12a H-bonds are highlighted
by green-dotted lines. Only the polar hydrogen atoms are shown. More details can be found in Supporting
Information.
The blind-docking simulations with the HSA protein indicate that SCTz
compounds 10a-c, 11a-c, 12b, and 13a-c interact with HSA subunit IB, while 12a and
12c bind in the region between subunits IB and IIA (Figures 9 and S109). SCTz series
10a-c, 11a-c, 12b, and 13a-c interact mainly with Leu115, Tyr138, Ile142, Tyr161, and
Arg186 residues by hydrophobic interactions (π-alkyl, π-π, π-alkyl, and alkyl-alkyl). In
addition, the 1,2,3-triazole moiety makes an H-bond with the hydroxyl group of Tyr161
(Table S2). The molecules 12a and 12c interact with Lys195, Cys245, and His242 by
hydrophobic interactions and with the Arg257 and His242 by H-bond. Notably, the
carbon atoms from the side chain of the Lys, Cys, and Arg residues showed
hydrophobic interactions. These observations are in accordance with the experimental
data. Previous studies have indicated that the interactions of triazole derivatives in the
IB (Leu115, Tyr138, and Tyr161) and IIA subunits (His242 and Arg257) may be
involved in HSA fluorescence quenching.[55–57] In addition, these regions correspond
to the FA1 and FA2 sites where the heme and ibuprofen bind, respectively.[58] The
predicted theoretical thermodynamic data (ΔG) indicated that 10-13a-c present
spontaneous interactions with DNA and HSA (Table S2). In general, the aromatic rings
and spirocarbocyclic moiety are essential for hydrophobic interactions and the triazole
group as a role of H-bond acceptor.
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Figure 9. Overview of the best docking pose between HSA and SCTz compounds 10-13a-c. As an
example, 10a and 12a interactions are highlighted. The H-bonds, hydrophobic interactions (π-alkyl, π-π,
and alkyl-alkyl), and electrostatic interactions (π-anion and π-cation) are shown in green, purple, and
orange dotted lines, respectively. Only the polar hydrogen atoms are shown. The Trp214 residue is
indicated by the red arrow. HSA domains IA (1−112), IB (113–195), IIA (196–303), IIB (304–383), IIIA
(384–500), and IIIB (501–584) are represented by different colors. More details can be found in
Supporting Information.
Conclusions
In summary, we described a useful and convenient route to prepare novel 1,2,3-
triazoles-spirochromanes, namely, 4-(alkyl/aryl)-1-(spiro[chroman-2,1'-cycloalkan]-4-
yl)-1H-1,2,3-triazoles (SCTz 10-13) in satisfactory yields. This was easy carried out via
Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC) reaction involving 4-
azidospiro[chroman-2,1'-cycloalkanes] (5a-c) and some selected alkynes as precursors.
The optimal reaction medium was using copper catalysts (CuSO4.5H2O/Sodium
Ascorbate), THF:H2O as solvent at room temperature for 24 h. The QTAIM analysis
showed intramolecular interactions only in compound 10b, which is due to the high
proximity between the hydrogen atoms rather than an electrostatic potential
complementarity. In the case DNA-binding assays by UV-Vis analysis, blue or red-
shifts were observed depending on the substituent and all SCTz demonstrated strong
binding forces to CT-DNA (Kb ≈ 106 M–1) following the increasing order of Kb values:
12c < 13c < 11a < 10b < 10a ≈ 13a < 13b < 11c < 10c < 12b < 11b < 12a. Besides that,
the Stern-Volmer quenching constants (KSV) of SCTz series were calculated and
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derivatives showed that the compounds (12a-c) compete more strongly with DNA
minor grooves, following the increasing general order of (KSV): EB-DNA < DAPI-
DNA. The Stern-Volmer and association constant values obtained for the SCTz series
imply there is good interaction between the compounds and albumin pocket.
This initial study showed a very accessible synthesis for a new heterocyclic
scaffold, which presents interesting photophysical and biointeraction properties that can
be contribute to the development of multi– and trans–disciplinary for integrated
investigations already in progress.
Experimental
Materials and methods
Unless otherwise indicated, all common reagents and solvents were used as
obtained from commercial suppliers, without further purification. The melting points
were determined using glass capillary tubes in an Electrothermal MEL-TEMP 3.0
apparatus, and are uncorrected. 1H and 13C NMR spectra were acquired either in a
Bruker DPX 200 (1H at 200 MHz), Bruker AVANCE NANOBAY V3-I-400 MHz (1H
at 400 MHz and 13C at 100 MHz) or Bruker Avance III (1H at 600 MHz and 13C at 150
MHz) with 5 mm sample tubes, 298 K, digital resolution of 0.01 ppm, in CDCl3, and
using TMS as an internal reference. The 19F NMR spectra were acquired on a Bruker
Avance III DPX 600, at 564.68 MHz, with 5 mm sample tubes at 298 K, digital
resolution of 0.01 ppm, in CDCl3, and using CFCl3 (d 0.0 ppm) as the external
reference. The 2D spectra were acquired on a Bruker Avance III DPX 600 (1H at 600
MHz and 13C at 150 MHz), with 5 mm sample tubes, solutions at 298 K, digital
resolution of 0.01 ppm, in CDCl3, and using TMS as internal reference. Mass spectra
were performed in a HP 5973 MSD spectrometer connected to a HP 6890 GC
spectrometer. The GC spectrometer was equipped with a split-splitless injector, auto
sampler, cross-linked HP-5 capillary column (30 m, 0.32 mm internal diameter), and
helium was used as the carrier gas.
Single Crystal X-ray Diffraction (SC-XRD)
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Suitable single crystals were obtained by slow evaporation of EtOH at 25 °C.
Diffraction measurements were performed using a Bruker D8 Venture diffractometer
equipped with a Photon 100 CMOS detector, using Mo Kα radiation (λ = 0.71073 Å),
for compounds 10b, 11b, 12b-c, and 13c. Absorption corrections were performed using
multi-scan methods. The structures were solved using SHELXS[59] or olex2.solve,[60]
and refinements were carried out using SHELXL 2018/3[61] with the aid of the
software Olex2 1.3.[62] Anisotropic displacement parameters for non-hydrogen atoms
were applied. The molecular diagrams were generated using the software Mercury.[63]
Crystal structure parameters of compound 10b, 11b, 12b-c, and 13c are listed in Table
S1 in Supporting Information. Crystallographic information file (CIF) for compounds
10b, 11b, 12b, 12c, and 13c were deposited at the Cambridge Crystallographic Data
Centre (CCDC) under identification number 1996495, 2013986, 1996330, 1996331, and
1996496, respectively.
Quantum Mechanical Calculations
The performed calculations were performed with geometries obtained from SC-
XRD. Density functional theory (DFT) calculations were performed using the Gaussian
09 software package[64] with a ωB97x-D/cc-pVDZ theory level. The counterpoise
method of Boys and Bernardi was employed to minimize the basis set superposition
error (BSSE).[65] QTAIM analyses were performed with the aid of the AIMALL
software package.[66] The wavefunctions used in the QTAIM and MEP analyses were
generated at the ωB97x-D/cc-pVDZ level of theory. MEP maps were built on the
electron density 0.01 a.u. isosurface with GaussView.[67]
Absorption UV-Vis analysis
UV-Vis absorption spectra were recorded using Shimadzu UV2600
spectrophotometer (data interval, 1.0 nm) using dimethyl sulfoxide (DMSO) as solvent.
CT-DNA interactions by absorption and fluorescence emission
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For DNA-binding assays, compound interactions with calf-thymus DNA (CT-
DNA) were performed by UV-vis absorption measurements at room temperature in
Tris-HCl buffer at pH 7.4 using DMSO stock solution of derivatives (~10-4 M range) at
the range of 250 to 600 nm. The DNA pair base concentrations of low molecular weight
DNA from calf thymus (ct-DNA) was determined by absorption spectroscopy using the
molar extinction coefficients 6,600 M−1cm−1 (per base pair) at λmax = 260 nm,
respectively. Derivative solutions in DMSO with Tris-HCl buffer were titrated with
increasing concentrations of DNA (ranging from 0-100 μM). The intrinsic binding
constants (Kb) of derivatives were calculated according to the decay of the absorption
bands of compounds using the following equation (1) through a plot of [DNA]/(εa − εf )
versus [DNA]:
[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/Kb(εb − εf )
(1)
where [DNA] is the concentration of DNA in the base pairs, εa is the extinction
coefficient (Aobs/[compound]), and εb and εf are the extinction coefficients of free and
fully bound forms, respectively. In plots of [DNA]/(εa − εf ) versus [DNA], Kb is given
by the ratio of the slope for the interception.
The standard Gibbs free-energy (ΔGº) of compound-DNA adduct was calculated
from the values of binding constant (Kb) using the following equation (2):
ΔGº = ‒RT ln Kb
(2)
where R and T are the gas constant (1.987 kcal K-1 mol-1) and temperature (298 K),
respectively.
Steady-state emission fluorescence analyses of SCTz series were measured with a
Varian Cary 50 fluorescence spectrophotometer (Exc./Em; slit 2.0 mm/2.0 mm) and
corrected according to the manufacturer‘s instructions. In competition dye-DNA assays,
steady-state emission fluorescence analysis was recorded and derivatives dissolved in
DMSO solutions. Competitive studies were performed through the gradual addition of
the stock solution of the spiro-derivatives to the quartz cuvette (1.0 cm path length)
containing ethidium bromide (EB, 2.0 x 10-7 M) or 4′,6-diamidino-2-phenylindole
(DAPI, 2.0 x 10-7 M) and DNA (2.0 x 10-5 M) in a Tris-HCl pH 7.4 buffer solution. The
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concentration of derivatives ranged from 0 to 100 μM. Samples were excited at λexc
=
510 nm (for EB dye) and λexc = 359 nm (for DAPI dye). Emission spectra were recorded
at the range of 350-850 nm, 3.0 min after each addition of the complex solution in order
to allow incubation to occur. The fluorescence quenching Stern-Volmer constants (KSV)
of compounds were calculated according to the decay of the emission bands of dye-
DNA using the following Stern-Volmer equation (3) through a plot of F0/F versus
[DNA],
F0/F = 1+ kqη0[Q] = 1 + KSV[Q]
(3)
where F and F0 are the fluorescence intensities in the presence and absence of a
quencher, respectively. KSV, kq, η0 and [Q] denote Stern–Volmer quenching constant,
quenching rate constant, lifetime of EB-DNA adducts (η0 = 23.0 ns),[68] lifetime of
DAPI-DNA adducts (η0 = 2.20 ns),[69] and quencher concentration, respectively.
According to equation (3), the Stern–Volmer constants (KSV) were calculated from the
slope and kq is equal KSV/η0.
HSA-binding properties by steady-state emission fluorescence
For emission HSA experiments, SCTz interactions with human serum albumin
(HSA) were performed by steady-state emission fluorescence measurements at room
temperature in Tris-HCl buffer at pH 7.4 using DMSO stock solution of derivatives
(~10-4 M range) at 300 to 500 nm range. It is well known that quenching occurs through
the static or dynamic quenching process, both of which can result in a linear Stern–
Volmer plot. To analyze the data from the quenching assays, we used the same Stern–
Volmer equation (3) above, where F and F0 are the fluorescence intensities in the
presence and absence of a quencher, respectively. KSV, kq, η0 and [Q] denote Stern–
Volmer constant, quenching rate constant, the original lifetime of HSA (5.67 ns),[70]
and the concentration of quencher, respectively. According to Equation (3), the Stern–
Volmer constants (KSV) were calculated from the slope and kq is equal KSV/η0. It is
generally accepted that the maximum Stern–Volmer constant allowed for the dynamic
quenching of biomolecules is 1.0 x 102 M-1. Therefore, to conclude that the quenching is
not a dynamic but static process occurring in the HSA-compound complex, Scatchard
equation (4) predicted the Ka (association constant) and n (binding site):
log (F0 – F)/F = log Ka + n log [Q]
(4)
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where F0 and F represent fluorescence intensities in the absence and presence of
chalcogen derivatives, respectively, and [Q] the quencher concentration. According to
equation (3), the association constants (Ka) were calculated from the interception and
the number of binding sites (n) for the slope, respectively.
Molecular Docking
Molecular docking is a computational method that predicts the binding-
conformation of ligands when interacting with a target. For this purpose, the AutoDock
Vina 1.1.1 program was used.[71] The crystallographic structures of DNA and HSA
were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/) with the codes
1BNA[72] and 3B9L,[73] respectively. The Chimera 1.8 software[74] was used to
remove water, ions, and other molecules and add hydrogen atoms to the DNA and HSA.
The compounds 10-13a-c were built in the software Avogadro 1.1.1,[75] followed by
semi-empirical PM6 geometry optimization[76] using the software MOPAC2016[77]
(the isomers R were considered as a representative model). The optimized structures of
SCTz 10b, 11b, 12c, and 13c were compared with their crystallography data and the
root-mean-square deviation (RMSD) values (0.184, 0.109, 0.228, and 0.125 Å,
respectively) indicated that the PM6 geometry optimization could reproduce the
experimental data. The ligands and macromolecules in the pdbqt format were generated
by AutoDockTools, where the ligands were considered flexible (with PM6 charges) and
the DNA/HSA rigid (with Gasteiger charges).[78] The blind docking was used for both
targets using the grid box size of 50 x 50 x 50 Å (coordinates: x = 14.8, y = 21.0, z =
12.3) and 90 x 50 x 90 Å (coordinates: x = 29.3, y = 0.4, z = 19.2) for DNA and HSA,
respectively, with 1.0 Å of grid spacing. As a model of interactions, the conformer of
the lowest binding free energy was selected. The results from the docking simulations
were analyzed using the Accelrys Discovery Studio 3.5 software.[79]
Synthesis
General procedure for the synthesis of 4-(alkyl/aryl)-1-(spiro[chroman-2,1'-
cycloalkan]-4-yl)-1H-1,2,3-triazoles (SCTz 10a-c, 11a-c, 12a-c, 13a-c)
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To a 25 mL flask containing a THF/H2O solution 1:1 v/v (5 mL) used as the
reaction solvent at room temperature and under magnetic stirring, the 4-
azidospirochromanes (5a-c) (1mmol) and terminal alkynes (6-9) (1.1 mmol) were added
after sodium ascorbate (20 mol%) and copper sulfate pentahydrate (10 mol%). After the
reaction completation (24 h), the mixture was extracted into ethyl acetate (2 x 10mL)
and distilled water (5mL). The organic phase was dried over sodium sulfate and filtered.
The solvent was then removed in the rotary evaporator under reduced pressure and the
SCTz compounds purified by column chromatography employing a 10% solution of
ethyl acetate in hexane (10a-c), (12a-c), (13a-c), and 30% solution of ethyl acetate in
hexane (11a-c) as eluent.
4-Phenyl-1-(spiro[chroman-2,1'-cyclopentan]-4-yl)-1H-1,2,3-triazole 10a
White solid, yield 70%, m.p. 175–180 °C. 1H NMR (400 MHz, CDCl3) Triazole: δ 7.84
(d, J = 7.5 Hz, 2H, H-4c/H-4c‘), 7.67 (s, 1H, H-5), 7.43 (t, J = 7.5 Hz, 2H, H-4b/H-4b‘),
7.35 (t, J = 7.5 Hz, 1H, H-4d). Chromane: δ 7.30-7.22 (m, 1H, H-7), 6.95-6.88 (m,
1H, H-5), 6.90-6.81 (m, 1H, H-6/H-8), 6.13 (dd, 1JH-H = 10.5 Hz 2JH-H = 5.5 Hz, 1H, H-
4), 2.64 (dd, 1JH-H = 13 Hz 2JH-H = 10.5 Hz, 1H, H-3a), 2.49 (dd, 1JH-H = 13.0 Hz 2JH-H
=
5.5 Hz, 1H, H-3a‘), 2.08-1.63 (m, 8H, 4CH2). 13C NMR (100 MHz, CDCl3) Triazole: δ
148.1 (C-4a), 130.5 (C-4), 128.8 (C-4c/C-4c‘), 128.2 (C-4d), 125.6 (C-4b/C-4b‘), 117.9
(C-5). Chromane: δ 154.0 (C-8a), 130.1 (C-7), 127.8 (C-8), 120.8 (C-6), 119.1 (C-4a),
118.1 (C-5), 86.2 (C-2), 55.4 (C-4), 39.1 (C-3), 39.0 (CH2), 36.3 (CH2), 23.8 (CH2),
23.7 (CH2). HRMS Calcd. for C21H21N3O: 332.1763. Found: 332.1757.
4-Phenyl-1-(spiro[chroman-2,1'-cyclohexan]-4-yl)-1H-1,2,3-triazole 10b
White solid, yield 70%, m.p. 164–166 °C. 1H NMR (400 MHz, CDCl3) Triazole: δ 7.83
(d, J = 7 Hz, 2H, H-4c/H-4c‘), 7.66 (s, 1H, H-5), 7.46-7.37 (m, 2H, H-4b/H-4b‘), 7.33
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