Design, Synthesis, and Biophysical Studies of Novel 1,2,3-Triazole

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Article Cite This: ACS Omega 2019, 4, 7213−7230

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Design, Synthesis, and Biophysical Studies of Novel 1,2,3-TriazoleBased Quinoline and Coumarin Compounds Sandip Paul,† Pritam Roy,‡ Pinki Saha Sardar,*,§ and Anjoy Majhi*,† †

Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700073, India Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India § The Department of Chemistry, The Bhawanipur Education Society College, Kolkata 700020, India ‡

ACS Omega 2019.4:7213-7230. Downloaded from pubs.acs.org by 5.62.157.201 on 04/23/19. For personal use only.

S Supporting Information *

ABSTRACT: A novel triazole-coumarin compound 2-oxo-N-((1-(6-oxo-6-(ptolylamino)hexyl)-1H-1,2,3-triazol-4-yl)methyl)-2H-chromene-3-carboxamide (compound 6) and quinoline compound N-((1-(6-oxo-6-(p-tolylamino)hexyl)-1H1,2,3-triazol-4-yl)methyl)quinoline-6-carboxamide (compound 9) are designed and synthesized using Cu(I)-catalyzed cycloaddition of alkyne and azide reaction. The binding interaction of coumarin and quinoline compounds with bovine serum albumin and human serum albumin has been explored using different photophysical studies. To determine the feasible binding sites and change in the microenvironment of the binding sites of the tryptophan (Trp)/tyrosine (Tyr) of the serum albumins exploiting Trp/Tyr emission in proteins, steady state and time-resolved fluorescence at room temperature (298 K) and low-temperature phosphorescence studies at 77 K have been carried out. The fluorescence spectroscopic study of Trp residue in both complexes showed that the strong quenching with the blue shift of the emission peak occurs through static quenching mechanism. The thermodynamic parameters obtained from isothermal titration calorimetry study clearly established the involvement of van der Waals force and hydrogen bonds during complexation. Molecular docking studies further predict the experimental findings of the protein-quinoline and coumarin complexes.

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INTRODUCTION Quinoline and coumarin are heterocyclic compounds with nitrogen and oxygen as the heteroatom, respectively. Quinoline and its derivatives are important because of their wide presence in natural products, and they are well known for their enormous biological activities. It has significant uses in a wide variety of medicinal applications such as anticancer,1,2 antimicrobial,3 antioxidant,4 antifungal,5 and antitubercular agents.6,7 Quinoline-based compounds are also known to exhibit excellent antiHIV8 and antihypertensive9 activities. Several quinoline derivatives are used as a fluorescent probe 10−12 since fluorescence probing is a powerful technique that can be used to visualize the activity of living cells. The interaction of fluoroquinolone and bovine serum albumin (BSA) was reported by fluorescence spectroscopy.13 Antagonistic activities of quinoline derivatives, viz., ciprofloxacin, ofloxacin, and lomefloxacin, in the presence of bovine serum albumin are studied.14 Recently, quinoline appended chalcone derivative has been reported for its antihypertensive activity and binding interaction with BSA.15 One of the most important strategies for cycloaddition reaction is the “click chemistry” reaction, i.e., Cu(I)-catalyzed cycloaddition of alkyne and azide (CuAAC) reaction, which was first introduced by Sharpless.17,18 By the CuAAC reaction, 1,2,3triazole moiety-containing compounds can be synthesized conveniently.16−18 Very recently, novel triazole-quinoline derivatives have been synthesized by the CuAAC reaction, © 2019 American Chemical Society

which can be used as acetyl cholinesterase inhibitor for treatment of Alzheimer’s disease.16 Coumarins, oxygen heterocyclic compounds, are also widely used for their biological activities.19−22 Several 1,2,3-triazole-based coumarin compounds have been synthesized earlier by the CuAAC reaction, which show diverse applications, including pharmaceuticals, and are available as drugs today.19−22 Recently, 1,2,3-triazolecoumarin derivative has been reported for its enormous biological activity.23−25 Hence, the present study is focused on the design and synthesis of two click compounds, i.e., 1,2,3-triazole-coumarin compound (6) (Figure 1) and quinoline compound (9) (Figure 1), by a suitable procedure and investigation of their interaction with the model transport protein, BSA, and human serum albumin (HSA) with the expectation that coumarin/quinoline alkyne and p-tolylhexanamide azide, which produce the desired triazole moiety, together can enhance the fluorescence property as well as interact efficiently with the serum albumins or ptotylhexanamide azide linker part that can go inside the pocket of serum albumins (SAs) for better binding. Serum albumin is the most abundant soluble plasma carrier protein, which is responsible for transporting many endogenous and exogenous agents,26 and it plays a key role in many Received: February 13, 2019 Accepted: April 10, 2019 Published: April 22, 2019 7213

DOI: 10.1021/acsomega.9b00414 ACS Omega 2019, 4, 7213−7230

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studies of protein−ligand or other molecules’ interaction with different spectroscopic techniques monitoring protein emission maxima enhance our knowledge in the binding phenomena and might be helpful for the study of advanced biophysical research.53,56,58a,61 The present work is mainly categorized into two parts, namely, the synthesis of compound 6 and compound 9 following a click reaction and the investigation of the interaction of these two synthesized compounds with both the serum albumins BSA/HSA by different photophysical studies and molecular docking studies, which reveal the following observations:

Figure 1. 1,2,3-Triazole-based coumarin (6) and quinoline (9) compounds.

1. In continuation of our previous work58a,b and further drug development, we have newly designed and synthesized 1,2,3-triazole-based coumarin (6) and quinoline (9) compounds by the CuAAC reaction. 2. Steady-state absorption and fluorescence studies at 298 K determine the binding mechanism of the newly synthesized compounds 6 and 9 with BSA/HSA by monitoring the fluorescence quenching of Trp emission for both the serum albumins. 3. Time-resolved fluorescence studies at 298 K visualize the nature or mode of quenching operating in the binding process of both the compounds 6 and 9 with the serum albumins. 4. Isothermal titration calorimetry (ITC) experiments for the complexes of serum albumins with compound 6 and compound 9 have been carried out to unravel the binding mechanism and nature of binding forces in these interactions by determining different thermodynamic parameters of binding. 5. Low-temperature phosphorescence studies at 77 K in a suitable cryosolvent forming a glassy matrix find any perturbation or alteration of microheterogeneous environment around Trp or Tyr residue(s) in the serum albumins occurring due to complexation with the compounds 6 and 9. 6. Protein−ligand docking studies are also exploited to identify the probable binding sites of the compounds 6 and 9 into the cavity of the serum albumins along with the change in the immediate environment of Trp residue(s) due to binding and to compare the experimental observations. 7. The aim of this study was to find the perturbation of the Trp environment and probable location of the compounds 6 and 9 in the core of the serum albumins (with different microenviroments) due to complexation by different photophysical experiments and theoretical calculations, which are clear from experimental results.

biological systems and processes.27−29 Serum albumins (BSA and HSA) are synthesized by the parenchymal cells of the liver and exported as a nonglycosylated protein. Therefore, the drugbinding ability of both the serum albumins is a crucial factor that should be carefully considered in drug research.30a−d The molecular weights of BSA and HSA are 66 and 66.5 kDa, respectively, and both display approximately 80% sequence homology. Analysis of the crystal structure reveals that BSA and HSA contain 582 and 585 amino acid residues, respectively. BSA is a protein containing two tryptophan (Trp) residues located at positions 213 and 134, whereas HSA contains only one tryptophan located at position 214.31 The steady-state and time-resolved fluorescence technique often provides useful information about the structural and functional aspects of a single- or multi-Trp protein exploiting the intrinsic fluorescence of Trp/tyrosine (Tyr) residue of a protein in the interaction of a protein molecule with other molecules, viz., ligand/drug/nucleic acids/lipid, etc. Several works regarding the binding and environmental sensitivity of protein using the above conventional studies are reported.32−35 Quantum mechanics and molecular mechanics simulation by Callis and co-workers helps us to understand the correlation between the spectral shift and multiexponential decay for a broad fluorescence spectrum arising from the Trp residue(s) of different environments in a single- or multi-Trp protein.36−39 The low-temperature phosphorescence (LTP) spectrum at 77 K, on the contrary, provides the well-resolved (0,0) band of the individual Trp residues situated in different electrostatic environments of a multi-Trp protein.40−57,58a Phosphorescence measurements at 77 K in a glassy matrix are executed to acquire knowledge about the entire structural and conformation aspects of Trp residue(s) for a biopolymer or biopolymer−substrate complexes. The narrow bandwidth reflected in the phosphorescence spectra of Trp residue owing to smaller dipole moment in the excited state of the triplet state is indicative of homogeneous and uniform environment. The well-resolved LTP bands correspond to buried tryptophan residue in the hydrophobic environment usually red-shifted, and a more homogeneous environment is observed,59 whereas blue-shifted bands for buried Trp residue might appear due to specific interaction with nearby polar residue.49,60 Hence, photophysical

Hence, an attempt is made for the binding study of the newly synthesized compounds 6 and 9 with biomolecules to investigate the role of a novel compound in the development of biomedicines for the drug design or drug delivery process at very preliminary stage of the modern research.

Scheme 1. Synthesis of 6-Azido-N-(p-tolyl)hexanamide

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show a peak at ∼280 nm, and in the complexes, the absorbance values increased with increasing concentration of compounds 6 and 9, and are also slightly blue-shifted in their maximum absorption wavelength (Figure 2). The results help us to understand the formation of the drug−protein complexes in their ground state along with the alteration of the microenvironment of tryptophan residues on BSA/HSA with the addition of compounds 6 and 9.62 The intrinsic fluorescence spectra of free BSA and HSA are commonly characterized by the presence of the tryptophan (Trp) residue. Although tyrosine and phenylalanine are natural fluorophores in proteins, due to their great differences in quantum yield, lifetimes, and energy transfer from phenylalanine to tyrosine and from tyrosine to tryptophan, the fluorescence spectrum of a protein consisting of these three amino acid residues generally appears similar to that of tryptophan.32,53,56,58a,61,63 The fluorescence spectra of inherent Trp residue(s) of free BSA (Trp 213) and free HSA (Trp 214) upon excitation at 290 nm appear at ∼343 and ∼339 nm, respectively, which implies that a relatively solvent-exposed environment of the emitting Trp residue(s) in the free protein is responsible for the total fluorescence spectra, whereas Trp 134 situated in a more polar environment contributes little to the fluorescence spectra for Trp of BSA.32,64,65 Depending on this well-documented sensitivity of tryptophan fluorescence of protein to its microenvironment, the modulation of the photophysics of Trp residue(s) in protein has been utilized for the interaction with a ligand or drug or any other exogenous molecule.32,53,56,58a The fluorescence spectra of two Trp residues (Trp 134 and Trp 213) in free BSA (5 μM) are progressively quenched with gradual addition of varying concentration of both the synthesized compounds 6 and 9 at 298 K in aqueous phosphate buffer of pH 7 at λexc = 290 nm (Figure 3A,C).66−68 The λmax value of the emission of Trp residue(s) is shifted toward blue from 342.8 nm (in free BSA) to 340.2 nm for compound 6 and from 342.5 nm (in free BSA) to 340.4 nm for compound 9 (Figure 3A,C). The occurrence of blue shift for both compounds 6 and 9 indicates that the emitting Trp residue(s) in the complex with compounds 6 and 9 is in less polar or more hydrophobic environment compared to that in free BSA. The decrease of fluorescence intensity along with the blue shift indicates that both the compounds 6 and 9 bind with BSA and may induce the microenvironment alterations around the BSA protein. The emission spectra of Trp residue in free HSA (5 μM) containing single Trp residue (Trp 214) have been quenched upon addition of both the synthesized compounds 6 and 9 like BSA at 298 K with excitation at 290 nm is shown in Figure 3B,D. The λmax value of the emission of Trp residues in HSA is also blue-shifted from 338.6 nm (in free HSA) to 334 nm (∼4.6 nm) in compound 6 and from 339.1 nm (in free HSA) to 335.4 nm (∼3.7 nm) in compound 9 (Figure 3B,D). This suggests that Trp 214 (free HSA) is in somewhat buried region when it is bound with compounds 6 and 9. The decrease of fluorescence intensity and blue shift indicates that both the compounds bind with HSA, like BSA. However, the interaction of both the compounds with HSA reveals somewhat more intriguing phenomena, which are further considered using low-temperature phosphorescence and docking studies. To get insight into the binding phenomena of compounds 6 and 9 with the protein molecules, the intensity of the fluorescence emission for serum albumins is conventionally analyzed by the following Stern−Volmer equation (eq 1)32

RESULTS AND DISCUSSION Chemistry. 6-Azido-N-(p-tolyl)hexanamide 3 was synthesized according to the procedure described in Scheme 1, which is one of the major required components for the Cu(I)-catalyzed cycloaddition of alkyne and azide (CuAAC). To get compound 3, 6-bromo hexanoic acid 1 was treated with NaN3 to afford 6azidohexanoic acid 2, which underwent coupling reaction with p-toluidine in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt) in dichloromethane (DCM). Target compound 6 was synthesized using the method described in Scheme 2. Coumarin-3-carboxylic acid was Scheme 2. Synthesis of Triazole-Coumarin Compound (6)

converted to its alkyne derivative 5 by coupling with propargyl amine in the presence of EDC, HOBt, and N-methylmorpholine (NMM) in dichloromethane. The CuAAC reaction was conducted between coumarin alkyne derivative 5 and azide molecule 3 in the presence of Cu(I) iodide in dimethylformamide (DMF), which afforded novel triazole-based coumarin compound 6. Scheme 3 describes the synthesis of triazole-based quinoline derivative 9. To get the target compound 9, quinoline alkyne Scheme 3. Synthesis of Triazole-Quinoline Compound (9)

derivative 8 was synthesized from quinoline-6-carboxylic acid 7 and propargyl amine, which on treatment with 3 in the presence of Cu(I) iodide in DMF afforded the triazole-based novel quinoline compound 9. Steady-State Absorption and Emission Studies at 298 K. The interactions between compounds 6 and 9 with BSA/HSA have been investigated by steady-state absorption spectra. Figure 2 shows the absorption spectra of serum albumins with compounds 6 and 9. The absorption spectra of BSA/HSA 7215


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Figure 2. Absorption spectra of (A) free BSA (5 μM) and its varying concentration with compound 6, (B) free HSA (5 μM) and its varying concentration with compound 6, (C) free BSA (5 μM) and its varying concentration with compound 9, and (D) free HSA (5 μM) and its varying concentration with compound 9 in aqueous phosphate buffer (pH 7) at 298 K.

Figure 3. Room-temperature fluorescence spectra of (A) free BSA (5 μM) and its varying concentration of compound 6, (B) free HSA (5 μM) and its varying concentration of compound 6, (C) free BSA (5 μM) and its varying concentration of compound 9, and (D) free HSA (5 μM) and its varying concentration of compound 9 in aqueous phosphate buffer (pH 7). Excitation wavelength = 290 nm; excitation and emission band pass = 10 and 5 nm, respectively, for each case.

F0/F = 1 + KSV[L] = 1 + kqτ0[L]

compounds 6 and 9, respectively, KSV is the Stern−Volmer quenching constant, [L] is the concentration of quencher compounds 6 and 9, kq is the bimolecular quenching rate constant, and ⟨τ0⟩ is the average fluorophore lifetime in the

(1)

where F0 and F are the fluorescence intensities of serum albumins in the absence and presence of the quencher 7216


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Time-Resolved Fluorescence Studies Monitoring Tryptophan Emission of BSA/HSA at 298 K. The lifetime measurement by time-resolved fluorescence decay technique monitoring the emission maxima of Trp emission was performed at 298 K to explore the nature of the observed fluorescence quenching. The fluorescence decay profile of the free protein HSA and the complexes of HSA with compounds 6 and 9 are displayed in Figure 5A,B, and the relevant lifetime data are given in Table 2. Free protein shows biexponential decay with good χ2 values and is consistent with several literature reports.32,53,56 The data collected in Table 2 provide that both the intensityweighted and amplitude-weighted average lifetimes of the protein are slightly perturbed with increasing concentration of the compounds 6 and 9. Thus, from the comparatively constant lifetime values of the HSA complexes (similar phenomena observed for BSA, data not shown) obtained from the fluorescence decay, it can be analyzed that the static quenching process is predominant over the dynamic quenching process in these cases. However, an insignificant deviation has been observed for the HSA−compound 9 complex, where the average fluorescence lifetime of protein is found to slightly decrease on gradual addition of compound 9 (Table 2 and Figure 5B). The slight variation in both the components and also the percent contribution resulted in decrease of the average lifetime values in HSA−compound 9 complexes (Table 2). This accounts for the perturbation of the microenvironment of Trp residue of HSA near the binding site of compound 9 to a greater extent compared to compound 6 as well as the complexes BSA− compound 6 and BSA−compound 9. Docking study and phosphorescence spectra also support to analyze this phenomenon.

absence of quencher compounds 6 and 9. F0 and F in Figure 5 are calculated using the area under the emission curve. The KSV values for different systems of serum albumins are summarized in Table 1. Table 1. Stern−Volmer Quenching Constant (KSV) and Bimolecular Quenching Constant (kq) of the Complex of Compounds 6 and 9 with Both the Serum Albumins in Aqueous Buffer (pH 7) at 298 K system

KSV (M−1)

kq (M−1 s−1)

R2

BSA−compound 6 BSA−compound 9 HSA−compound 6 HSA−compound 9

9.36 × 105 3.52 × 105 8.08 × 105 6.65 × 105

2.017 × 1014 0.759 × 1013 1.741 × 1014 1.433 × 1014

0.981 0.949 0.997 0.984

The occurrence of fluorescence quenching of serum albumins due to binding with compounds 6 and 9 can be investigated through static quenching and dynamic quenching processes. The static quenching process usually occurs from the formation of a stable complex between the protein and quencher, while the dynamic quenching process usually occurs from the collisional encounters between the protein and quencher.32 Both the static and dynamic quenching processes can be described depending upon the linearity of the Stern−Volmer equation.69−71 A linear Stern−Volmer plot is observed in Figure 4 for all of the complexes of both the serum albumins, indicating that the static quenching part is predominant over the dynamic quenching part of the emitting Trp residues of the proteins32 (Table 1 and Figure 4). In this context, the time-resolved fluorescence studies could be helpful to realize the nature of fluorescence quenching operating in all of the complexes (see next section)

Figure 4. Stern−Volmer plots for fluorescence quenching of (A) BSA−compound 6 complex, (B) HSA−compound 6 complex, (C) BSA−compound 9 complex, (D) HSA−compound 9 complex; λexc = 290 nm, [BSA] = [HSA] = 5 μM; excitation band pass = 10 nm; and emission band pass = 5 nm. 7217


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Figure 5. Representative time-resolved fluorescence decay profile of (A) free HSA (5 μM) and its complex with compound 6 (0.9 μm) and (B) free HSA (5 μM) and its complex with compound 9 (1 μm) in aqueous phosphate buffer (pH 7). λexc = 290 nm, λmonitored = 335 nm. The excitation and emission band passes are 10 nm each.

Table 2. Singlet-State Lifetime of HSA (5 μM) and Its Complexes with Compounds 6 and 9 in Aqueous Phosphate Buffer (pH 7) at 298 Ka [compound]

τ1b (ns)

τ2b (ns)

α1 (%)

0 μm 0.125 μm 6 0.250 μm 6 0.500 μm 6 0.625 μm 6 0.750 μm 6 0.900 μm 6 0.2 μm 9 0.4 μm 9 0.5 μm 9 0.6 μm 9 0.8 μm 9 1.0 μm 9

6.47 6.56 6.42 6.30 6.27 6.40 6.42 6.54 6.45 6.53 6.46 6.48 6.41

2.43 2.54 2.42 2.23 2.22 2.52 2.42 2.37 2.22 2.41 2.43 2.37 2.24

54.72 53.10 55.71 54.82 57.20 50.64 54.89 55.06 58.02 51.96 50.80 48.71 47.09

α2 (%)

⟨τa0⟩c (ns)

⟨τi0⟩c (ns)

χ2

45.28 46.90 44.29 45.18 42.80 49.36 45.11 44.94 41.98 48.04 49.20 51.29 52.91

4.64 4.67 4.64 4.46 4.53 4.48 4.61 4.66 4.67 4.55 4.47 4.37 4.20

5.51 5.53 5.49 5.38 5.42 5.32 5.47 5.59 5.60 5.48 5.38 5.34 5.23

1.14 1.06 1.19 1.08 1.29 1.34 1.48 0.94 1.17 1.14 1.05 1.16 1.01

Table 3. Binding Parameters of Serum Albumin−Compound 6 and 9 Complexes at 298 K Kb (M−1)

R2

n

ΔG (kJ mol−1)

BSA−compound 6 BSA−compound 9 HSA−compound 6 HSA−compound 9

11.5 × 10 11.0 × 105 8.91 × 105 5.56 × 105

0.935 0.960 0.998 0.982

1.373 1.361 1.123 0.894

−34.58 −34.45 −33.95 −32.78

5

where F0 and F represent the corrected fluorescence intensities of BSA and HSA in the absence and presence of quencher molecule (compounds 6 and 9), respectively, Kb is the binding constant of all of the complexes of BSA and HSA, n is the number of binding sites, F0 and F in all of the cases of Figure 6 are calculated considering the area under the emission curve of the corrected fluorescence spectra. The double-logarithmic plots of log[(F0 − F)/F] versus log[Q] of the complexes BSA and HSA with compounds 6 and 9 are presented in Figure 6. The values of Kb and n along with the correlation coefficients (R2) are listed in Table 3. There are several reports of the fluorescence quenching technique (using eq 2) for the determination of binding parameters.32,73,74 The emission study is also carried out to find the binding constant of the complexes of serum albumins and the parent compounds/key intermediates 5 and 8 of compounds 6 and 9, respectively, as a control. It has been observed that in both the serum albumins, the binding constant shows much higher values for compounds 6 and 9 compared to the parent compounds/key intermediates 5 and 8, respectively (Figure S1 and Table S1, Supporting Information). Isothermal Titration Calorimetry (ITC). Isothermal titration calorimetry (ITC) has been utilized extensively in various biomolecular interactions. The mechanism of ITC is the measurement of the heat released or absorbed throughout biomolecular interactions. ITC provides thermodynamic parameters such as K, ΔH, ΔS, and n of molecular interaction. The top section provides the heat changes generated during the titration of 2 μL of compounds 6 or 9 into HSA in 28 successive injections. The bottom portion denotes the normalized fit into independent model of binding to BSA/HSA and the derived thermodynamic energetics. The binding interactions between protein and synthesized compounds are known to be influenced by different factors such as electrostatic force, hydrophobic interaction, van der Waals interactions, hydrogen-bonding interaction, and others.75−78 Theoretically, when ΔH < 0 or ΔH ≈ 0 and ΔS > 0, it has been considered that hydrophobic and electrostatic forces serve as the

λexc = 290 nm, λmonitored = 335 nm. bError in the measurements is ±0.1 ns. c⟨τa0⟩ and ⟨τi0⟩ represent the amplitude-weighted and intensity-weighted average lifetimes, respectively.

a

Using the average lifetime values of HSA (Table 2) and the respective KSV values (Table 1), the bimolecular quenching constant (kq) values are found to be ca. 1013−1014 M−1 s−1 for BSA and HSA, respectively (Table 1). This observation again supports the occurrence of static quenching mechanism in these cases as the kq values are some orders of magnitude higher than the maximum threshold for a diffusion-controlled process (1010 M−1 s−1).32 Hypothetically, a specific electrostatic interaction may also play a key role in the overall binding phenomenon if the evaluated bimolecular quenching constant (kq) values are higher than those of the diffusion-controlled process.32,56,72 This static quenching phenomenon also agrees with the thermodynamic parameters of binding and the molecular docking studies discussed in the next section. Binding Data from Fluorescence Spectra. The interactions of the serum albumins with compounds 6 and 9 are quantitatively analyzed by evaluating the binding constants (Kb) and the associated free-energy change (ΔG) for the interaction processes (Table 3). The quantitative assessment of Kb and ΔG by the following equation (eq 2) portrays the equilibrium between free and bound units when they bind independently to a set of equivalent sites in a macromolecule32,73 log[(F0 − F )/F ] = log Kb + n log[Q]

system

(2) 7218


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Figure 6. Representative double-logarithmic plot of log[(F0 − F)/F] vs log[Q] of (A) BSA−compound 6 complex, (B) HSA−compound 6 complex, (C) BSA−compound 9 complex, and (D) HSA−compound 9 complex for determination of the binding constant of the complexes; λexc = 290 nm, [BSA] = [HSA] = 5 μM; excitation band pass = 10 nm; and emission band pass = 5 nm.

main forces of interaction; when ΔH < 0 and ΔS < 0, hydrogen bond and van der Waals forces serve as the main forces of interaction; and when ΔH > 0 and ΔS > 0, hydrophobic interaction plays the main role.75−78 For complexes of BSA with compounds 6 and 9 and the complex of HSA with compound 9, negative ΔH values and negative ΔS indicate that the binding interaction is strongly dominated by hydrogen bond and van der Waals force of interaction (Figure 7 and Table 4). In the case of interaction of compound 6 with HSA, both positive ΔH and ΔS values indicate that the interaction is strongly hydrophobic in nature (Figure 7B and Table 4). The binding and thermodynamic data observed in ITC experiment for all of the cases differ from fluorescence measurements. This could be due to the fact that ITC considers global change during binding, whereas fluorescence spectroscopy is observed only due to the changes in local environment surrounding Trp residues in BSA and HSA.79 Generally, in protein−ligand/drug-binding study, ITC experiment is a valuable tool for determining the different forces such as electrostatic interaction, hydrophobic interaction, H-bonding interaction, and van der Waals force. As in our experiment of ITC study, it was designed to perform each set at 25, 30, and 37 °C to understand details of specific heat changes during complexation of coumarin and quinoline derivatives with BSA and HSA. But in our case, we found that the experiment is favorable at 303 K. The probable reason is that to consider the experiment as entropically driven at lower temperature than at higher temperature, which is enthalpically driven. It is well known that the experimental temperature is an important parameter to consider a particular reaction whether it is enthalpically or entropically driven.79b

Low-Temperature Phosphorescence Study of Trp Residues of the Complexes of Serum Albumins with the Compounds 6 and 9 at 77 K. The perturbation of the microenvironment around the Trp residues, viz., Trp 134 and Trp 213 in BSA and Trp 214 in HSA, in the complexes of serum albumin with compounds 6 and 9 has been invoked by lowtemperature phosphorescence measurement at 77 K. The phosphorescence spectra of free BSA and its complex with both the compounds (6 and 9) in 40% ethylene glycol matrix at 77 K are compared in Figure 8A,C with λexc = 290 nm. Free BSA shows a distinct (0,0) band at 413.0 nm with a shoulder at 405.6 nm (Figure 8A,C and Table 5). The phosphorescence spectra in both the complexes (compounds 6 and 9) are quenched compared to those of the free proteins, and the (0,0) band is slightly red-shifted to 413.8 nm for compound 6, whereas for compound 9, the position of the (0,0) band remains unaltered. For both the compounds 6 and 9, the shoulder at ∼405 nm disappeared. The position of the (0,0) bands at 405.6 and 413.0 nm of the Trp residues in BSA correspond to Trp 134 and Trp 213 in BSA, which clearly indicates that Trp 134 is in a comparatively more polar environment while Trp 213 is in a comparatively more hydrophobic environment. Hence, this signifies that for compound 6, the microenvironment around Trp 213 of BSA is slightly perturbed, and the disappearance of band at ∼405 nm corresponding to Trp 134 for both the cases exhibiting only single (0,0) band in the phosphorescence spectra is probably due to the occurrence of either electron transfer from the excited state or energy transfer to other amino acid residue(s) or any other compound or ligand or the formation of a charge-transfer complex by interaction with other neighboring residue(s) (see next section). Conversely, a thorough surveillance on several multi-Trp proteins helps to anticipate the occurrence of multiple 7219


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Figure 7. ITC of (A) BSA−compound 6 complex, (B) HSA−compound 6 complex, (C) BSA−compound 9 complex, (D) HSA−compound 9 complex for determining binding and thermodynamic parameters at 303 K. The top panel shows power required to achieve isothermal calorimetry at 30 °C against each individual injection of 28 injections. The bottom panel shows total interaction, and solvation heat quantity reflects each individual injection in terms of kcal mol−1.

following the similar experimental conditions used for BSA. Free HSA exhibit a single (0,0) band at 410.8 nm, which corresponds to single Trp residue at 214 in HSA (Figure 8B,D and Table 5). The interactions of HSA with compounds 6 and 9 provide the quenched spectra compared to the free HSA (Figure 8B,D) with a small blue-shifted band for compound 6, but for compound 9, a well-distinct blue-shifted band appeared (Figure 8B,D and Table 5). This can be attributed to the fact that the microenvironment of Trp 214 in HSA is perturbed and is shifted to more polar or hydrophillic environment for both the compounds. Still compound 9 experiences a greater shift in the λmax value toward blue end than compound 6 during complexation with HSA. Steady-state, time-resolved, and docking studies (next section) support this contention. The phosphorescence spectra with λexc = 280 and 295 nm for free BSA, free HSA, and their complexes with compounds 6 and 9 are similar to those observed with λexc = 290 nm (data not

Table 4. Thermodynamic and Binding Parameters of the Complexes of Serum Albumins with Compounds 6 and 9 Obtained from TIC at 303 K system

ΔH (J mol−1)

ΔS (J mol−1 deg−1)

BSA−compound 6 HSA−compound 6 BSA−compound 9 HSA−compound 9

−8.86 × 10 10.67 × 104 −25.49 × 105 −5.72 × 105

−2.80 × 103 4.77 × 102 −8.28 × 103 −1.76 × 103

5

(0,0) bands in the phosphorescence spectra perhaps due to widely different microenvironments of Trp residue(s) in a multiTrp protein, and an inefficient photoinduced energy transfer among the emitting Trp residues is observed.40,41,43−45,48,51,61,80−82 The phosphorescence spectra for free HSA and its complexes with both compounds 6 and 9 are also presented in Figure 8B,D 7220


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Figure 8. Phosphorescence spectra of (A) free BSA (5 μM), BSA−compound 6 (1:1) complex, and free compound 6; (B) free HSA (5 μM), HSA− compound 6 (1:1) complex, and free compound 6; (C) free BSA (5 μM), BSA−compound 9 (1:1) complex, and free compound 9; and (D) free HSA (5 μM), HSA−compound 9 (1:1) complex, and free compound 9 at 77 K, λexc = 290 nm; excitation band pass = 10 nm; and emission band pass = 1 nm in each case.

during complexation with compounds 6 and 9. This phenomenon is more prominent in the case of the HSA− compound 9 complex as it is clear from the blue shift of the (0,0) band to a larger extent in the phosphorescence spectra (Table 5). Docking study also supports this observation (see next section). The phosphorescence spectra of all of the complexes of serum albumins exhibit a well-resolved spectra with (0,0) band at ∼465 nm (Figure 8). This is supposed as the phosphorescence spectra of compounds 6 and 9 as we have measured the phosphorescence spectra at 77 K in aqueous buffer containing 40% ethylene glycol matrix using λexc = 290 nm (Figure 8). Several reports51,53,57,58a,61,80 of protein−drug/ligand interaction demonstrated the correlation between position of (0,0) band and solvent exposure of Trp residue. In polar solvents, the phosphorescence spectra of free Trp residue appeared as a blueshifted (0,0) band having an environment of lower polarizability, and hence triplet state is stabilized to a lower extent due to the presence of rigid solvent geometry. Conversely, the Trp residue with red-shifted (0,0) band is in a buried polarizable hydrophobic environment that stabilizes the triplet state more than the ground state.40,49,83−85 In addition, the local charges and the strong rigidity of the microenvironment around Trp residue might be responsible to determine the position of the (0,0) band. Molecular Docking Studies of the Synthesized Compounds 6 and 9 with BSA and HSA. Molecular docking studies have been carried out to substantiate the experimental findings as docking study helps to envisage probable location of the compounds in the microenvironment of the Trp residue(s) of the serum albumins by the interaction between the serum albumin and compounds 6 and 9. The docked poses of the

Table 5. Phosphorescence Data for Serum Albumins and Their Complexes with Compounds 6 and 9 in a 40% Ethylene Glycol Matrix at 77 K (λexc = 290 nm)a

system

position of the phosphorescence (0,0) band (nm)

width of the phosphorescence (0,0) band at half-maxima (cm−1)

free BSA (5 μM) BSA−compound 6 (1:1) BSA−compound 9 (1:1) free HSA (5 μM) HSA−compound 6 (1:1) HSA−compound 9 (1:1)

405.6 (sh), 413.0 413.8 413.0 410.8 410.2 408.4

291 280 266 306 411 475

Error in the measurements is ±0.1 nm.

a

shown). An unstructured broad band appearing below 400 nm observed in the spectrum of serum albumins λexc = 290 nm (Figure 8) is assigned as tyrosine (Tyr). The contributions of tyrosine phosphorescence are reduced in the phosphorescence spectra for all of the complexes of serum albumins. The bandwidths (obtained by using a curve-fitting method)51,80 of the (0,0) band of all of the complexes of compounds 6 and 9 with serum albumins are also calculated and given in Table 5. The decrease in the bandwidths of the (0,0) band in both the complexes of BSA indicates that the microenvironments of the perturbing Trp residue of BSA (Trp 134 and Trp 213 for BSA) in the complexes move to more or less homogeneous environment compared to that of free BSA. On the other hand, HSA experiences a significant increase in their bandwidths of the (0,0) band of the phosphorescence spectra for both the complexes (Table 5). This could be due to the appearance of heterogeneous environment surrounding Trp residue of HSA 7221


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Figure 9. Docked poses of (A) compound 6 and (D) compound 9 in the complexes with BSA. Distances (in angstrom) obtained from docked poses of different Trp residues of BSA from the different atoms of (B) compound 6 and (E) compound 9. H-bonding distances between (C) compound 6 and (F) compound 9 of different polar and nonpolar residues around tryptophan of the docked complexes.

complexes of compounds 6 and 9 with serum albumins are provided in Figures 9 and 10.

Docking studies of two compounds 6 and 9 with BSA shows that these two compounds bind preferentially in the site II 7222


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Figure 10. Docked poses of (A) compound 6 and (D) compound 9 in the complexes with HSA. Distances (in angstrom) obtained from docked poses of different Trp residues of HSA from the different atoms of (B) compound 6 and (E) compound 9. H-bonding distances between (C) compound 6 and (F) compound 9 of different polar and nonpolar residues around tryptophan of the docked complexes.

region, which is away from the Trp residues (Figure 9A,D). Both the compounds are almost at the same distance from the nearby

Trp residue (Trp 213), whereas compound 6 is situated slightly closer to Trp 134 compared to compound 9 (Figure 9B,E and 7223


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Table 8. Changes in Accessible Surface Area (ΔASA, Å2) of the Amino Acid Residues of Docked Complexes of BSA with Compounds 6 and 9

Table 6). The residues close to both of these compounds (within 3−4 Å) are mainly polar residues (Arg, Glu) with a few Table 6. Distance of Tryptophan Residues of Serum Albumins from Compounds 6 and 9 Obtained from the Docked Complexes protein

residue

compound

BSA

Trp 213 Trp 134 Trp 213 Trp 134 Trp 214 Trp 214 Trp 214 Trp 214

compound 6

HSA

compound 9 compound 6 compound 9

atom/group of compound

distance (Å)

ring CO amide CO ring N amide N His (N) coumarin (CO) C (CH3−Ph) ring N

13.05 18.30 13.96 20.49 9.66 12.60 3.88 7.93

ΔASA (Å2)

hydrophobic residues like Leu and Ala (Figure 9C,F). Both these compounds contain amide bond in the molecular backbone, which promotes electrostatic interaction with the nearby polar residues and can involve in H-bonding interaction (Figures 9C,F and 10C,F, Table 7), thereby stabilizing the serum albumin−compound complexes. This is a clear signature of the disappearance of the band at ∼405 nm (corresponding to Trp 134) in the phosphorescence spectra for both the complexes of BSA with compounds 6 and 9 (Figure 8A,B). In addition, due to the presence of phenyl moiety, the nonpolar residues interact with the compounds through hydrophobic interaction. Comparing the change in accessible surface area (ΔASA) of the entire BSA, it can be seen that change in accessible surface area (ΔASA, Å2) is higher in the presence of compound 9 (440.71 Å2) in comparison to compound 6 (431.49 Å2), which indicates that compound 9 supports the decrease in bandwidth of (0,0) band in the phosphorescence spectra of BSA− compound 9 complexes to a greater extent compared BSA− compound 6 complexes (Tables 5 and 8) to interact with more number of residues in the site II region compared to compound 6. Moreover, Arg 458 shows the highest change in accessible surface area, followed by Leu 189, which signifies that the protein−compound 6 and 9 complexes for BSA are stabilized by both electrostatic and hydrophobic interactions (Table 8), which supports our data obtained from steady-state and timeresolved studies. The changes in the ASA values obtained from the docking studies are consistent with those of the shift of the emission maxima obtained from fluorescence and phosphorescence studies. HSA has almost similar domains like BSA; however, it has only one Trp residue near site I (Figure 10A,D). Docking studies show that both the compounds bind close to Trp 214 near site I (Figure 10A,D). Compound 9 is situated at a much closer

amino acid residues in BSA

compound 6

compound 9

Asp 108 Pro 110 Asp 111 Leu 112 Lys 114 Arg 144 His 145 Arg 185 Leu 189 Ala 193 Arg 196 Glu 424 Ser 428 Lys 431 Arg 435 Ile 455 Arg 458 total

1.35 11.18 10.2 10.42 25.54 34.49 39.01 30.57 39.7 27.04 9.11 24.36 23.86 19.15 13.19 13.64 65.79 431.49

4.53 11.18 4.47 7.16 31.3 30.41 37.49 23.63 45.73 31.26 16.32 39.1 24.14 14.53 14.42 14.39 66.22 440.71

proximity to the Trp residue compared to compound 6 (Figure 10B,E and Table 6). This is reflected in the calculation of ΔASA for both the complexes where the change in accessible surface area for Trp 214 (Table 9) is much higher upon binding with compound 9, whereas for compound 6, this Trp residue is mostly exposed to the solvent medium. However, the overall change in accessible surface area of HSA is higher upon binding with compound 6 compared to that of compound 9, which supports the phosphorescence spectra of HSA−compound 6 complex, where the (0,0) band is shifted to comparatively solvent-exposed region unlike the HSA−compound 9 complex (Tables 5 and 9). The heterogeneous microenvironment in HSA owing to larger bandwidth of the phosphorescence (0,0) band in the HSA−compound 9 complex might be evidenced by this change in accessible surface area obtained from docking studies (Tables 5 and 9). Amino acid residues like Arg 209, Ala 213, and Lys 351 in HSA are shielded to a greater extent upon binding compound 6, whereas the solvent exposure of Lys 195, Ala 291, and Glu 292 decreases significantly in the presence of compound 9 (Table 9). Similar to BSA, here also the ligands are expected to be involved in electrostatic as well as hydrophobic interactions (Figure 9). Additional stabilization is obtained from the hydrogen bonds formed between ligands and Arg/Lys residues (Figures 9C,F and 10C,F, Table 7).

Table 7. Hydrogen-Bonding Distances between Compounds 6 and 9 and Different Polar and Nonpolar Residues around Tryptophan Residue of the Serum Albumins Docked Complexes protein

residue

compound

atom/group of compound

distance (Å)

BSA

Arg 185 (NH2) Ser 428 (O−H) Arg 144 (CO) Ser 428 (O−H) Arg 209 (NH2) Arg 257 (NH2) Lys 199 (NH2)

compound 6

amide CO amide N−H amide N−H amide CO coumarin (CO) amide CO Ph ring N

2.04 2.23 1.80 2.48 3.32 2.79 2.28

HSA

compound 9 compound 6 compound 9

7224


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Table 9. Changes in Accessible Surface Area (ΔASA, Å2) of the Amino Acid Residues of Docked Complexes of HSA with Compounds 6 and 9

S3, Figures S2C and S3C, Supporting Information). As a result, electrostatic interaction is not very effective in promoting proper binding. In addition, for both the cases, Phe residue appeared after interaction with compound 8, which might interact through hydrophobic (or π−π interaction) interaction, thereby enabling compound 8 to bind in that position (Figures S2C and S3C, Supporting Information). However, in comparison to this key intermediate, compounds 8 and 9 are found to be involved in appreciable H-bonding interaction due to closer proximity of the polar residues, which subsequently resulted in greater stabilization of compound 9 (Table 7, Figures 9 and 10). For positive control, two different ligands were selected for BSA and HSA, which have been previously reported to have very high binding constant values (∼106 M−1) in comparison to our compound 9. BSA is being reported to show very high affinity with naproxen as found from experimental studies.86a For this reason, docking studies have been carried out, which showed that the ligand binds close to Trp 213 (Table S4 and Figure S4A,B, Supporting Information) and is involved with hydrogen bonding with Arg 208 and Lys 211 mainly through the carboxylic group present in the ligand (Table S5 and Figure S4C, Supporting Information). On the other hand, Liu et al.86b studied the binding of isofraxidin with HSA and showed that it is stabilized by Arg 257 residue situated near it. The docking result here shows that the ligand binds very close to Trp 214 (Table S4 and Figure S5A,B, Supporting Information), and Arg 222 and Lys 199 also stabilize the ligand through hydrogen bond in addition to Arg 257 (Table S5 and Figure S5C, Supporting Information). For both HSA and BSA, it can be seen that for proper interaction of the ligand with proteins, extensive hydrogen-bonding network plays an important role since it enables the ligand to bind strongly with the protein amino acid residues. This in turn increases the binding constant value for the ligand as obtained from the docking studies.

ΔASA (Å2) amino acid residues in HSA

compound 6

compound 9

Tyr 150 Glu 153 Glu 188 Ala 191 Ser 192 Lys 195 Lys 199 Phe 206 Arg 209 Ala 210 Lys 212 Ala 213 Trp 214 Arg 218 Arg 222 Ala 291 Glu 292 Asp 324 Leu 327 Gly 328 Leu 347 Ala 350 Lys 351 Lys 436 Asp 451 Tyr 452 Val 455 Ser 480 Val 482 total

0 0 0 0 0 0 0 29.14 67.05 24.88 11.72 56.3 8.15 0 0 0 0 18.12 15.54 15.71 11.08 17.44 43.71 0 0 0 0 25.3 21.26 365.4

13.41 11.35 13.15 15.7 20.99 51.92 18.33 0 0 0 0 0 16.8 23.27 18.93 28.49 35.68 0 0 0 0 0 0 17.31 26.69 19.9 10.67 0 0 342.59

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CONCLUSIONS 1,2,3-Triazole-based coumarin (6) and quinoline (9) compounds were designed and synthesized by CuAAC reaction, and later on, the photophysical modulation of these two compounds with the model transport protein serum albumins, viz., BSA and HSA, was carried out along with the molecular docking studies. The structures of compounds 6 and 9 were confirmed by NMR and mass spectroscopy. Room-temperature absorption band fluorescence studies revealed that the microenvironment of the Trp residue(s) has been altered due to complexation with these compounds. Steady-state and time-resolved fluorescence quenching study provides binding constant values as well as number of binding sites and the static quenching mechanism operating for both complexes of BSA and HSA with compounds 6 and 9. The emission experiment was carried out to find the binding constant of the complexes of serum albumins and the parent compounds/key intermediates 5 and 8 of 6 and 9, respectively, as a control. The result shows much lower binding constant for parent compounds/key intermediates 5 and 8 compared to compounds 6 and 9, respectively. ITC experiment for all of the cases helps to estimate the overall energetics and nature of forces responsible for complexation. The extensive study on the low-temperature phosphorescence spectroscopy at 77 K in a suitable glassy matrix helps to understand the microenvironment around Trp residue(s) in the proteins due to complexation. The perturbation of microenvironment in the HSA−compound 9 complex is somewhat different from that in the HSA−compound 6 complex. Molecular docking studies

It is true that the actual protein−ligand binding site can be obtained from protein−ligand crystallization techniques. However, in our study, we have used photophysical properties to study the changes in the behavior of protein and ligand after binding, using spectroscopic techniques (steady-state absorption and fluorescence studies at 298 K, low-temperature phosphorescence studies at 77 K, etc.), and we have used docking study only to predict the probable binding location of our compounds, which can give a better insight into our experimental findings. Also, we have carried out positive and negative control using these two proteins (BSA and HSA) through docking analysis and compared it with our reported compounds (compounds 6 and 9). The key intermediate compound 8 (quinoline alkyne) used as control to bind with serum albumins shows much lower affinity (∼104 M−1) than compound 9 (Table S1, Supporting Information). As a result, we have considered compound 8 as negative control. Docking experiments have been carried out in the same procedure as our ligand with HSA and BSA, and it shows that compound 8 binds in two different locations in two different proteins (Figures S2A and S3A, Supporting Information). In BSA, it binds somewhat close to Trp 213 whereas in HSA, it binds away from Trp 214 (Table S2, Figures S2B and S3B, Supporting Information). Further investigation in the nearby residues around compound 8 for both BSA and HSA shows that the ligand is located somewhat away from the polar residues (Arg and Lys) (Table 7225


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mmol) was added. Propargyl amine (prop-2-yn-1-amine) (0.173 g, 3.15 mmol) was added dropwise after 15 min of stirring. The stirring was continued for 12 h. The reaction was monitored by TLC. The reaction mixture was poured into water and extracted with EtOAc. The layer was washed with saturated bicarbonate solution followed by brine solution. It was dried under anhydrous Na2SO4 and the solvent was evaporated to yield solid products. The product was then purified through column chromatography using 50% EtOAc in PET ether solution. The pure solid product was then collected. Isolated yield, 66.8%; mp, 139−142 °C. 1 H NMR (400 MHz, CDCl3), δ: 2.208 (s, 1H), 4.189 (d, 2H, J = 2.8 Hz), 7.323 (d, 1H, J = 7.6 Hz), 7.355 (dd, 1H, J = 5.6, 3.6 Hz), 7.595 (dd, 1H, J = 8.0, 4.0 Hz), 7.644 (d, 1H, J = 4 Hz), 8.851 (s, 1H), 8.947 (s, 1H); 13C NMR (400 MHz, CDCl3), δ: 29.47, 71.65, 77.37, 116.71, 117.95, 118.51, 125.39, 129.91, 134.32, 148.83, 154.50, 161.28, 161.35. Preparation of 2-Oxo-N-((1-(6-oxo-6-(p-tolylamino)hexyl)-1H-1,2,3-triazol-4-yl)methyl)-2H-chromene-3-carboxamide (6). In a 50 mL two-neck round-bottom flask, compound 5 (0.100 g, 0.44 mmol), compound 3 (0.086 g, 0.37 mmol), and CuI (10 mol %) were dissolved in 1.5 mL of DMF under inert atmosphere at room temperature. Triethylamine (Et3N) (0.08 mL) was added dropwise and then stirred for 5 h. The progress of the reaction in monitored by TLC (solvent: 1:9 methanol (MeOH)/DCM). The mixture was filtered through a celite slurry of EtOAc. Cu was trapped in it. Then, eluted solution of EtOAc was washed with ammonium chloride (NH4Cl) (two times) carefully (never shake too hard), then washed with water, saturated sodium bicarbonate (NaHCO3), and brine solution. It was dried under anhydrous Na2SO4, and the solvent was removed at reduced pressure. The crude product was purified by silica gel column chromatography (solvent: 1.5:8.5 MeOH/ DCM). Isolated yield, 67%; mp, 145−147 °C. 1 H NMR (400 MHz, (CD3)2SO), δ: 1.244−1.337 (m, 2H), 1.638−1.674 (m, 2H), 1.863−1.899 (m, 2H), 2.277 (s, 3H), 2.316 (t, 2H, J = 7.4 Hz), 4.395 (t, 2H, J = 7 Hz), 4.636 (d, 2H, J = 5.6 Hz), 7.11 (d, 2H, J = 8.4 Hz), 7.49 (dd, 1H, J = 8.2, 8.6 Hz), 7.564 (d, 1H, J =8.4 Hz), 7.574 (s, 1H), 7.804 (dd, 1H, J = 3.8, 4.6 Hz), 8.077 (s, 1H), 8.050 (d, 2H, J = 7.6 Hz), 8.954 (s, 1H), 9.177 (t, 1H, J = 5.2), 9.791 (s, 1H); 13C NMR (400 MHz, (CD3)2SO), δ: 20.87, 24.98, 26.00, 30.02, 35.39, 36.55, 49.64, 116.61, 118.90, 119.20, 119.48, 123.25, 125.62, 129.45, 130.77, 132.22, 134.63, 137.25, 144.44, 148.14, 154.36, 160.82, 161.54, 171.24; MS-ESI (+): calcd for C26H28N5O4 [M + H]+ 474.210; found 474.1809. Preparation of N-(Prop-2-yn-1-yl)quinoline-6-carboxamide (8). Quinoline-6-carboxylic acid (0.4 g, 2.10 mmol), HOBT (0.283 g, 2.10 mmol), and EDC (0.402 g, 2.10 mmol) were dissolved in DCM (10 mL) in a 100 mL round-bottom flask. After 10 min stirring, NMM (0.318 g, 3.15 mmol) was added. Propargyl amine (prop-2-yn-1-amine) (0.173 g, 3.15 mmol) was added dropwise after 15 min stirring. The stirring was continued for 12 h. The reaction was monitored by TLC. The reaction mixture was poured into water and extracted with EtOAc. The layer was washed with saturated bicarbonate solution followed by brine solution. It was dried under anhydrous Na2SO4, and the solvent was evaporated to yield solid products. The product was then purified through column chromatography using 60% EtOAc in PET ether solution. The pure solid product was then collected. Isolated yield, 72.5%; mp, 151−154 °C.

support the complexation of compounds 6 and 9 with the serum albumins and the location of these compounds inside the proteins. Hence, the present study provides an attempt to investigate photophysically the interaction of the two newly synthesized compounds with serum albumins, which might be a footstep toward present-day drug binding or drug delivery research.

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EXPERIMENTAL SECTION Materials and Methods. All of the reagents were of analytical grade, and the chemicals and serum albumins (BSA and HSA) were purchased from Sigma-Aldrich Chemicals Pvt. Ltd. All of the solvents used were of spectral grade and further dried by standard procedures. Phosphate buffer of pH 7 was prepared in triple distilled water and used for making experimental solutions. Ethylene glycol was purchased from Alfa Aesar. The concentrations used in the photophysical studies are the physiological concentrations. The concentrations of serum albumin (SA) and compounds 6 and 9 are in the micromolar (μM) range, which are very dilute and physiologically active concentrations. The complexations occur at very low concentrations between SA and compounds 6 and 9. Analytical thin-layer chromatography (TLC) was performed on precoated (0.25 mm) silica gel plates (Merck, TLC Silica Gel 60 F254, Cat. No. 1.05554.0001). 1H and 13C NMR spectra were recorded on AVANCE 400 MHz FT-NMR spectrometer, Bruker Biospin AG using chloroform-d1 (CDCl3) containing 0.05% tetramethylsilane (TMS) (99.8%D, Cambridge Isotope Laboratories, Inc., Cat. No. DLM-7) and dimethyl sulfoxide-d6 (99.8%D, Cambridge Isotope Laboratories, Inc., Cat. No. DLM10-S-25). Synthetic Procedure. Preparation of 6-Azidohexanoic Acid (2). Compound 2 was synthesized following the literature procedure,87 and a 1H NMR value matches well with the literature value. Preparation of 6-Azido-N-(p-tolyl)hexanamide (3). In a 100 mL round-bottom flask, 6-azidohexanoic acid (0.5 g, 3.26 mmol) dissolved in 5 mL of DCM, NMM (0.43 mL, 3.91 mmol), HOBt (0.484 g, 3.26 mmol), and EDC (0.624 g, 3.26 mmol) were added and stirred for 10 min at room temperature. p-Toluidine (0.30 g, 3.26 mmol) was then added in one portion and stirred for 18 h. The reaction progress was monitored by TLC. The reaction mixture was poured into water and extracted with ethyl acetate (EtOAc). The layer was washed with saturated bicarbonate solution followed by brine solution. It was dried under anhydrous sodium sulfate (Na2SO4), and the solvent was evaporated to yield solid products. The product was then purified through column chromatography using 20% EtOAc in petroleum (PET) ether solution. The pure solid product was then collected. Isolated yield, 61.3%; mp, 137 °C. 1 H NMR (400 MHz, (CDCl3)), δ: 1.294−1.369 (m, 2H), 1.546−1.475 (m, 2H), 1.605−1.680 (m, 2H), 2.234 (s, 3H), 2.277 (t, 2H, J = 7.4 Hz), 3.161 (t, 2H, J = 6.6 Hz), 7.014 (t, 1H, J = 7.2 Hz), 7.213 (t, 2H, J = 7.8 Hz), 7.440 (d, 1H, J =7.6 Hz), 7.739 (s, 1H); 13C NMR (400 MHz, (CDCl3)), δ: 21.3, 25.08, 26.34, 28.62, 37.33, 51.21, 120.04, 124.29, 128.95, 137.97, 171.43; electrospray ionization mass spectra (MS-ESI (+)): calcd for C13H19N4O [M + H]+ 247.15; found 247.1350. Preparation of 2-Oxo-N-(prop-2-yn-1-yl)-2H-chromene-3carboxamide (5). Coumarin-3-carboxylic acid (0.4 g, 2.10 mmol), HOBT (0.283 g, 2.10 mmol), and EDC (0.402 g, 2.10 mmol) were dissolved in DCM (10 mL) in a 100 mL roundbottom flask. After 10 min of stirring, NMM (0.318 g, 3.15 7226


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H NMR (400 MHz, (CD3)2SO), δ: 3.151 (s, 1H), 4.15 (d, 2H, J = 2.4 Hz), 7.620 (dd, 1H, J = 4.0, 4.4 Hz), 8.104 (t, 1H, J = 7.2 Hz), 8.174 (d, 1H, J = 2 Hz), 8.195 (d, 1H, J = 2 Hz), 8.475 (d, 1H, J = 7.0 Hz), 8.515 (d, 1H, J = 8 Hz), 8.964 (s, 1H), 9.212 (d, 1H, J = 5.2 Hz); 13C NMR (400 MHz, (CD3)2SO), δ: 29.15, 73.52, 81.65, 122.68, 127.55, 128.09, 128.66, 129.54, 132.08, 137.61, 149.17, 152.63, 165.99. Preparation of N-((1-(6-Oxo-6-(p-tolylamino)hexyl)-1H1,2,3-triazol-4-yl)methyl)quinoline-6-carboxamide (9). In a 50 mL two-neck round-bottom flask, compound 8 (0.100 g, 0.44 mmol), compound 3 (0.086 g, 0.37 mmol), and CuI (10 mol %) were dissolved in 1.5 mL of DMF under inert atmosphere at room temperature. Et3N (0.08 mL) was added dropwise and then stirred for 5 h. The progress of the reaction was monitored by TLC (solvent: 2:8: MeOH/DCM). The mixture was filtered through a celite slurry of EtOAc. Cu was trapped in it. Then, eluted solution of EtOAc was washed with NH4Cl (two times) carefully (never shake too hard), then with water, saturated NaHCO3, and brine solution. It was dried under anhydrous Na2SO4 and the solvent was removed at reduced pressure. The crude product was purified by silica gel column chromatography (solvent: 3:7 MeOH/DCM). Isolated yield, 70.2%; mp, 160− 162 °C. 1 H NMR (400 MHz, (CD3)2SO), δ: 1.292−1.346 (m, 2H), 1.642−1.678 (m, 2H), 1.869−1.906 (m, 2H), 2.245 (s, 3H), 2.320 (t, 2H, J = 7.6 Hz), 4.395 (t, 2H, J = 7.2 Hz), 4.36 (t, 2H, J = 5.6 Hz), 7.113 (d, 2H, J = 8 Hz), 7.49 (d, 2H, J = 8 Hz), 7.666 (dd, 1H, J = 4.4, 4.0 Hz), 8.088 (s, 1H), 8.142 (d, 1H, J = 8.8 Hz), 8.257 (d, 1H, J = 2 Hz), 8.524 (d, 1H, J = 8 Hz), 8.607 (d, 1H, J = 1.6 Hz), 9.042 (t, 1H, J = 4 Hz), 9.327 (d, 1H, J = 5.6 Hz), 9.816 (s, 1H); 13C NMR (400 MHz, (CD3)2SO), δ: 20.87, 24.99, 26.03, 30.07, 35.50, 36.57, 49.61, 119.50, 122.64, 123.36, 127.53, 128.21, 128.56, 129.45, 132.23, 132.43, 137.25, 137.57, 145.27, 149.13, 152.54, 166.13, 171.24; MS-ESI (+): calcd for C26H29N6O2 [M + H]+ 457.23; found 457.2005. Instrumentation. Steady-State Measurements. UV−vis absorption spectra were recorded on a Hitachi U-4100 spectrophotometer at 298 K. Steady-state emission measurements were carried out in Hitachi model F-7000 spectrofluorimeter equipped with a 150 W xenon lamp, at 298 K using a stopper cell of 1 cm path length. Emission studies at 77 K were conducted using a Dewar system having a 5 mm outer diameter quartz tube. Freezing of the samples at 77 K was done at the same rate for all of the samples. Phosphorescence was measured in a Hitachi F-7000 spectrofluorimeter equipped with phosphorescence accessories. All of the samples were made in 40% ethylene glycol for measurements at 77 K. The samples were excited at different wavelengths (280, 290, and 295 nm) using a 10 nm band pass, and the emission band pass was 1 nm. Time-Resolved Fluorescence Studies. Singlet-state lifetimes were measured by a TimeMaster fluorimeter from Photon Technology International (PTI). The system consists of a pulsed laser driver PDL-800-B with interchangeable subnanosecond pulsed light-emitting diodes (LEDs) and picodiode lasers (PicoQuant, Germany) with a time-correlated single photon counting setup (PTI, USA). The software Felix 32 controls all acquisition modes and data analysis of the TimeMaster system.88 Decay measurement using “magic angle” detection with an emission polarizer set at 55 °C was carried out and no detectable difference in the fitted τ values from those obtained from normal decay measurements was observed. The lifetimes of all of the compounds are measured by 1

using PLS-290 (pulse width, 600 ps) at a repetition frequency of 10 MHz. Instrument response functions were measured at the respective excitation wavelengths of 290 nm (for LED source) using slits with a band pass of 3 nm using Ludox silica as scatterer. The decay of the sample was analyzed by nonlinear iterative fitting procedure based on the Marquardt algorithm. The deconvolution technique used can determine the lifetime up to 200 ps with subnanosecond pulsed LED. Intensity decay curves were fitted as summation of exponential terms32 F (t ) =

∑ αi exp(t /τi)

(3)

where αi represents the pre-exponential factor to the timeresolved decay of the component with lifetime τi. The decay parameters were recovered using a nonlinear iterative fitting procedure based on the Marquardt algorithm.32 A deconvolution technique was used to determine the lifetime up to 150− 200 ps with LED, while the time resolution was 100 ps with diode laser. The quality of fit was assessed over the entire decay, including the rising edge, and tested with a plot of weighted residuals and other statistical parameters, e.g., the reduced χ2 ratio and the Durbin−Watson parameter.88 The intensityweighted average lifetime ⟨τi0⟩ to be defined as32 ⟨τi0⟩ =

∑i αiτi2 ∑i αiτi

(4)

and an amplitude-weighted average lifetime ⟨τa0⟩ as ⟨τa0⟩ =

∑i αiτi ∑i αi

(5)

Isothermal Titration Calorimetry (ITC) Studies. The thermodynamics of the interaction of compounds 6 and 9 with both the serum albumins was studied using ITC model-VPITC (Make-MicroCal, now Malvern Instruments, U.K.).89 ITC is a reliable sophisticated tool to precisely study the thermodynamic parameters of molecular interactions. All of the buffer and samples were degassed extensively prior to titration, and protein was kept into the cell while the molecule was taken in the syringe. There was 10 μL injection and 180 seconds of spacing for all 28 injections in phosphate-buffered saline buffer at pH 7.4. The experiment was made isothermal at 30 °C. Heat of dilution observed due to the dilution was neglected from the integrated heat of reaction attained due to interactions of serum albumins and compound 6 or 9.77,78 Data were then fitted into the independent model of Nanoanalyze software to determine the different parameters. The enthalpy (ΔH) and entropy (ΔS) values are calculated according to the following the van’t Hoff equation79b Ln Kb =

−ΔH ΔS + RT R

(6)

Molecular Docking Studies. The crystal structures of BSA (PDB ID: 4F5S) and HSA (PDB ID: 1AO6) were obtained from Protein Data Bank.90 Sybyl 6.92 (Tripos Inc., St. Louis) was used to generate the three-dimensional structures of the two ligand molecules, which were further optimized using Tripos force field and Gasteiger−Hückel charges with 1000 iterations. These two ligands were separately docked with BSA and HSA using FlexX software, which is a part of the Sybyl suite. A series of structures were generated with different score values, which were based on the free energy of binding (ΔG) of the protein− ligand complex.91 For both the proteins, the structure 7227


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(4) Li, G. X.; Liu, Z. Q.; Luo, X. Y. Dichloro-4-quinolinol-3-carboxylic acid: Synthesis and antioxidant abilities to scavenge radicals and to protect methyl linoleate and DNA. Eur. J. Med. Chem. 2010, 45, 1821− 1827. (5) Kategaonkar, A. H.; Pokalwar, R. U.; Sonar, S. S.; Gawali, V. U.; Shingate, B. B.; Shingare, M. S. Synthesis, in vitro antibacterial and antifungal evaluations of new α-hydroxyphosphonate and new αacetoxyphosphonate derivatives of tetrazolo [1, 5-a] quinolone. Eur. J. Med. Chem. 2010, 45, 1128−1132. (6) Thomas, K. D.; Adhikari, V.; Telkar, S.; Chowdhury, I. H.; Mahmoode, R.; Pal, N. K.; Rowd, G.; Sumesh, E. Design, synthesis and docking studies of new quinoline-3-carbohydrazide derivatives as antitubercular agents. Eur. J. Med. Chem. 2011, 46, 5283−5292. (7) Eswaran, S.; Adhikari, A. V.; Kumar, R. A. New 1,3-oxazolo[4,5-c] quinoline derivatives: Synthesis and evaluation of antibacterial and antituberculosis properties. Eur. J. Med. Chem. 2010, 45, 957−966. (8) Strekowski, L.; Mokrosz, J. L.; Honkan, V. A.; Czarny, A.; Cegla, M. T.; Patterson, S. E.; Wydra, R. L.; Schinazi, R. F. Synthesis and quantitative structure-activity relationship analysis of 2-(aryl or heteroaryl)quinolin-4-amines, a new class of anti-HIV-1 agents. J. Med. Chem. 1991, 34, 1739−1746. (9) Muruganantham, N.; Sivakumar, R.; Anbalagan, N.; Gunasekaran, V.; Leonard, J. T. Synthesis, Anticonvulsant and Antihypertensive Activities of 8-Substituted Quinoline Derivatives. Biol. Pharm. Bull. 2004, 27, 1683−1687. (10) Li, G.; Zhu, D.; Xue, L.; Jiang, H. Quinoline-Based Fluorescent Probe for Ratiometric Detection of Lysosomal pH. Org. Lett. 2013, 15, 5020−5023. (11) Mao, Z.; Hu, L.; Dong, X.; Zhong, C.; Liu, B. F.; Liu, Z. Highly Sensitive Quinoline-Based Two-Photon Fluorescent Probe for Monitoring Intracellular Free Zinc Ions. Anal. Chem. 2014, 86, 6548−6554. (12) Lu, H. L.; Wang, W. K.; Tan, X. X.; Luo, X. F.; Zhang, M. L.; Zhang, M.; Zang, S. Q. A new quinoline-based fluorescent probe for Cd(2+) and Hg(2+) with an opposite response in a 100% aqueous environment and live cell imaging. Dalton Trans. 2016, 45, 8174−8181. (13) Kamat, B. P. Study of the interaction between fluoroquinolones and bovine serum albumin. J. Pharm. Biomed. Anal. 2005, 39, 1046− 1050. (14) Liu, B.; Zhao, F.; Xue, C.; Wang, J.; Lu, Y. Studies on the antagonistic action between chloramphenicol and quinolones with presence of bovine serum albumin by fluorescence spectroscopy. J. Lumin. 2010, 130, 859−864. (15) Kumar, H.; Devaraji, V.; Joshi, R.; Jadhao, M.; Ahirkar, P.; Prasath, R.; Bhavana, P.; Ghosh, S. K. Antihypertensive activity of a quinoline appended chalcone derivative and its site specific binding interaction with a relevant target carrier protein. RSC Adv. 2015, 5, 65496−65513. (16) Mantoani, S. P.; Chierrito, T. P. C.; Vilela, A. F. L.; Cardoso, C. L.; Martínez, A.; Carvalho, I. Novel triazole-quinoline derivatives as selective dual binding site acetylcholinesterase inhibitors. Molecules 2016, 21, 193−204. (17) Hein, C. D.; Liu, X. M.; Wang, D. Click chemistry, a powerful tool for pharmaceutical sciences. Pharm. Res. 2008, 25, 2216−2230. (18) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (19) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q. A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3Azidocoumarins and Acetylenes. Org. Lett. 2004, 4, 4603−4606. (20) Shi, Y. A.; Zhou, C. H. Synthesis and evaluation of a class of new coumarintriazole derivatives as potential antimicrobial agents. Bioorg. Med. Chem. Lett. 2011, 21, 956−960. (21) Stefani, H. A.; Gueogjan, K.; Manarin, F.; Farsky, S. H.; Schpector, J. Z.; Caracelli, I.; Rodrigues, S. R. P.; Muscara, M. N.; Teixeira, S. A.; Santin, J. R. Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)-coumarinderivatives: Effect on inducible nitric oxide synthase. Eur. J. Med. Chem. 2012, 58, 117− 127.

corresponding to the minimum score was selected and used for further studies. UCSF Chimera software was used to visualize the docked conformations and evaluate the protein−ligand interaction.92 Accessible Surface Area Calculations. NACCESS software93 was used to study the change in the accessible surface area (ASA) of the residues by using the following equation ΔASA i = ASA free BSA/free HSA − ASABSA − ligand/HSA − ligand complex

(7)

The change in accessible surface area of the ith residue of BSA or HSA is represented by eq 1.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00414. 1

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H NMR, 13C NMR, and mass spectra for new compounds 6 and 9 along with 1H NMR and 13C NMR spectra of compounds 5 and 8; fluorescence emission study of compounds 5 and 8 and docking studies for control experiment (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (P.S.S.). *E-mail: [email protected]. Mob: +91-7596900172. Tel/Fax: +91-33-2241-3893 (A.M.). ORCID

Anjoy Majhi: 0000-0003-0142-4940 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors sincerely acknowledge the funding agencies SERBDST Fast Track Research Grant for Young Scientist (SB/FT/ CS-188/2012 dated 30.06.2014) and UGC Start-Up Research Grant (F. 30-19/2014 (BSR), F.D. Dy. No. 2161 dated 19.06.2014) for the financial support. SP acknowledges CSIR for his junior research fellowship (Award Letter no-08/ 155(0051)/2017-EMR-1, Dated-4/12/2017). The authors are grateful to Faculty Research and Professional Development Fund (FRPDF), Presidency University, Kolkata, and Presidency University authority for providing the instrument and laboratory facilities. The authors thank Mr. Jishu Mandal, Indian Institute of Chemical Biology, for ITC experiment. The authors are also indebted to the editors and the reviewers of the journal for their valuable comments and suggestions.

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REFERENCES

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