Quinoline functionalized schiff base silver (I) complexes: Interactions with biomolecules and in vitro cytotoxicity, antioxidant and antimicrobial activities

molecules

Article

Quinoline Functionalized Schiff Base Silver (I) Complexes:

Interactions with Biomolecules and In Vitro Cytotoxicity,

Antioxidant and Antimicrobial Activities

Adesola A. Adeleke ^1,2, Sizwe J. Zamisa ³, Md. Shahidul Islam ⁴, Kolawole Oloﬁnsan ⁴, Veronica F. Salau ⁴

,

Chunderika Mocktar ⁵and Bernard Omondi ^1,

*

1

School of Chemistry and Physics, Pietermaritzburg Campus, University of Kwazulu-Natal, Private Bag X01,

Scottsville 3209, South Africa; 217080311@stu.ukzn.ac.za

Department of Chemical Sciences, Olabisi Onabanjo University, P. M. B. 2002, Ago-Iwoye 120107, Nigeria

School of Chemistry and Physics, Westville Campus, University of Kwazulu-Natal, Private Bag X54001,

Westville 4001, South Africa; zamisas@ukzn.ac.za

Discipline of Biochemistry, School of Life Sciences, Westville Campus, University of Kwazulu-Natal,

Private Bag X54001, Durban 4000, South Africa; islamd@ukzn.ac.za (M.S.I.); 219017967@stu.ukzn.ac.za (K.O.);

218087036@stu.ukzn.ac.za (V.F.S.)

Discipline of Pharmaceutical Sciences, School of Health Sciences, Westville Campus,

University of Kwazulu-Natal, Private Bag X54001, Durban 4000, South Africa; mocktarc@ukzn.ac.za

Correspondence: owaga@ukzn.ac.za

2

3

4

5

*

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: A series of ﬁfteen silver (I) quinoline complexes Q1–Q15 have been synthesized and

studied for their biological activities. Q1 Q15 were synthesized from the reactions of quinolinyl

Schiff base derivatives L1–L5 (obtained by condensing 2-quinolinecarboxaldehyde with various

aniline derivatives) with AgNO₃, AgClO₄and AgCF₃SO₃. Q1 Q15 were characterized by various

–

Citation: Adeleke, A.A.; Zamisa, S.J.;

Islam, M.S.; Oloﬁnsan, K.; Salau, V.F.;

Mocktar, C.; Omondi, B. Quinoline

Functionalized Schiff Base Silver (I)

Complexes: Interactions with

–

spectroscopic techniques and the structures of [Ag(L1)₂]NO₃Q1, [Ag(L1)₂]ClO₄Q6, [Ag(L2)₂]ClO₄

Q7, [Ag(L2)₂]CF₃SO₃Q12 and [Ag(L4)₂]CF₃SO₃Q14 were unequivocally determined by single

crystal X-ray diffraction analysis. In vitro antimicrobial tests against Gram-positive and Gram-

negative bacteria revealed the inﬂuence of structure and anion on the complexes⁰moderate to

excellent antibacterial activity. In vitro antioxidant activities of the complexes showed their good

radical scavenging activity in ferric reducing antioxidant power (FRAP). Complexes with the ﬂuorine

substituent or the thiophene or benzothiazole moieties are more potent with IC₅₀between 0.95

and 2.22 mg/mL than the standard used, ascorbic acid (2.68 mg/mL). The compounds showed a

strong binding afﬁnity with calf thymus-DNA via an intercalation mode and protein through a static

quenching mechanism. Cytotoxicity activity was examined against three carcinoma cell lines (HELA,

MDA-MB231, and SHSY5Y). [Ag(L2)₂]ClO₄Q7 with a benzothiazole moiety and [Ag(L4)₂]ClO₄Q9

with a methyl substituent had excellent cytotoxicity against HELA cells.

Biomolecules and In Vitro

Cytotoxicity, Antioxidant and

Antimicrobial Activities. Molecules

2021, 26, 1205. https://doi.org/

10.3390/molecules26051205

Academic Editor: Antonella

Dalla Cort

Received: 29 December 2020

Accepted: 18 January 2021

Published: 24 February 2021

Keywords: Ag(I); quinolines; cytotoxicity; antimicrobial; antioxidant; CT-DNA; BSA

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

1. Introduction

Schiff bases are an extensively studied important class of ligands. They are commonly

synthesized by the condensation reaction of aromatic aldehydes or ketones with primary

amines resulting in an azomethine moiety with at least one aryl group attached to either

the carbon or nitrogen atom. The interest in Schiff base ligands is therefore as such due

Copyright:

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

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4.0/).

to their ease of synthesis, selectivity to the central metal ion [

structural diversity [3], and their broad biological applications [4].

Quinolines are derivatives of pyridine and are found in many biologically active

natural [ ] and synthetic [ ] compounds. Their Schiff base derivatives have also been

identiﬁed with various pharmaceutical activities [10 11]. Although some free Schiff base

ligands are known to be bioactive, literature has shown that incorporating metal in such

1], synthesis ﬂexibility [2],

5,6

7–9

,

Molecules 2021, 26, 1205. https://doi.org/10.3390/molecules26051205

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 1205

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ligands enhances their activities [12

,13]. This has lately driven research in metal-based

drugs. Transition metal ions regarded as potential biological agents are platinum, cobalt,

copper, zinc, and ruthenium [14 17]. However, there is an emerging curiosity in exploring

–

silver(I) and its complexes as antimicrobial and antioxidant agents as well as efﬁcient DNA

and protein binders [18] with potential cytotoxicity activity. Our interest in silver(I) ion is

due to its low toxicity to human at low concentration [19], high cytotoxicity [20], and good

potency compared to other bioactive metal complexes such as cisplatin, known to have

drawbacks which include nephrotoxicity, drug resistance, and cervical renal problems [21].

Silver(I) complexes have been reported to have signiﬁcant anti-tumor activity [22

with minimum side effects; this is in addition to their well-known antimicrobial activ-

ities [24 25] and their effective scavenging of free radicals and reactive oxygen species

(ROSs) [26].

The interaction of metal complexes with deoxyribonucleic acid (DNA) and plasma

,23]

,

proteins are important when designing effective bioactive drugs that target the DNA

since binding of small organic and inorganic molecules to DNA can inﬂuence numerous

biological processes in which DNA participate, like transcription and replication [27].

The serum albumins are also considered to be one of the main molecular targets in the

action of anticancer agents. Since proteins play an important role in the transportation and

deposition of various endogenous and exogenous substances in the blood, their interactions

with drugs lead to the formation of stable drug-protein complexes [28].

Inspired by the biological activities of pyridinyl imine reported previously by our

group [29], we decided to incorporate a quinoline moiety into an imine ligand to investigate

the effect of an additional phenyl ring in the ligand in biological systems. We have as such

explored some ligands derived from the condensation reaction of 2-quinolinecarboxaldehyde

with 2-fluoroaniline, 2-aminobenzenethiol, 4-chloroaniline, p-toluidine, and thiophene-2-

ylmethanamine, respectively. Report herein is the synthesis, characterization, and bio-

logical studies of N,N-bidentate quinolinyl Schiff base ligands, and their Ag(I) -nitrate,

-perchlorate, and -triﬂate complexes.

2. Results and Discussion

2.1. Synthesis and Characterization of L1–L5

The ligands were prepared by the condensation reaction of 2-quinolinecarboxaldehyde

with ﬁve substituted aromatic anilines (2-ﬂuoroaniline, L1; 2-aminobenzenethiol, L2; 4-

chloroaniline, L3; p-toluidine, L4; and 2-thiophenemethylamine, L5) in anhydrous ethanol

in excellent yields as per the method in literature [30]. All the physical and spectral data

for the known ligands [31–33] were "in agreement" with the ones previously reported.

2.2. Synthesis and Characterization of the Complexes

Complexes Q1–Q15 were obtained in good to excellent yields between 50 and 100%

by reacting two equivalents of the respective ligands (L1–L5) with one equivalent of the

silver(I) salts in anhydrous ethanol (Scheme 1). The air-stable solids are soluble in common

organic solvents such as dichloromethane, acetonitrile, tetrahydrofuran, dimethylsulfoxide,

dimethylformamide, and some in water.

The reaction of L1–L5 with silver(I) metal ion in a 1:2 (Ag: L) molar ratio in anhy-

1

drous ethanol afforded ﬁfteen complexes (Q1–Q15) and was followed using H NMR

spectroscopy in DMSO-d6. The solution behavior of the complexes differs in the solid-state

as observed in the crystal structures of Q7,

Q12, and Q14 and their ¹H NMR spectra peaks.

In the ¹H NMR spectra of Q1–Q15, three and four coordinate geometries were observed,

indicating the presence of two types of complex species in solution. In the ¹H-NMR spectra

of Q1–Q15 (except Q1, Q6, and Q11), the integration values and the signiﬁcant downﬁeld

shifts in the resonance of the protons in the vicinity of the -C=N- atoms and the alpha

proton on the quinoline ring relative to their free ligand resonance peaks (Table S1) point

to the formation of the complexes in 1:2 (Ag:L) stoichiometry. This suggests that L2

, L3,

L4, and L5 are four coordinates in solution coordinating to the silver metal center via the

Molecules 2021, 26, 1205

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N_imand N_qyatoms and in L2 via N_thiazoleand N_qyatoms. This mode of coordination is

consistent with other reported silver(I) complexes of similar ligand [34–36].

Scheme 1. Synthesis of Q1–Q15 under constant magnetic stirring in anhydrous ethanol.

Molecules 2021, 26, 1205

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The pattern of coordination is different in complexes Q1

,

Q6, and Q11. The integration

values of Q1, Q6, and Q11 showed that the complexes adopt a 3-coordinate geometry as

is evident from their ¹H-NMR spectra. A signiﬁcant downﬁeld shift in the azomethine

protons and upﬁeld shift in the alpha protons with respect to the N atom in the quinoline

ring was observed in comparison with their free ligand resonance. In addition to this,

protons on the carbon of aniline in Q1, Q6, and Q11 and a singlet of amine protons at δ

5.04 ppm was seen, which shows that the silver ion coordinated to one ligand L1 (through

the N_imand N_qyatoms) and the N atom of aniline from the fraction of the second ligand

resulting in a three-coordinate geometry. In the ¹H-NMR spectrum of Q11, the NH₂proton

is absent which could be attributed to the low concentration of the complex in solution.

The tricoordinate observed in Q1

between the two Schiff bases resulting in the hydrolysis of one of the ligands. Further, the

electron-withdrawing substituent in Q1 Q6, and Q11 could have inﬂuenced this type of

, Q6, and Q11 are likely due to the steric repulsions

,

coordination because electron-withdrawing substituent is known for their ability to reduce

electron density around the metal center and thus affects binding interaction between the

metal and the coordination donor [37]. The effects of solvent on the Schiff base hydrolysis

can also not be overlooked as this type of Schiff base hydrolysis during coordination has

been reported [38,39].

Coordination of L1–L5 to the Ag(I) centers were conﬁrmed by comparing the FT-IR

spectroscopy (Table S1). Bands observed between 1613 and 1636 cm⁻¹associated with

the -C=N- bond stretching frequencies shifted to higher frequencies between 1624 and

1684 cm⁻¹in the spectra of the L4 and L5 upon coordination to Ag(I) while in L1 and L3

the -C=N- band shifted to lower frequencies from 1618–1693 cm⁻¹to 1613–1624 cm⁻¹

respectively, on coordination to Ag(I).

,

In the spectrum of L2 with thiazole moiety, the -C=N- bond shifted from 1452 cm⁻¹

to higher frequency on coordination to Ag(I) in the spectra of Q2

ca. 1456 and 1457 cm⁻¹. The absorption bands associated with the C=N quinolinyl ring

in the range 1591–1593 cm⁻¹in the spectra of L1, L2

L4, and L5 shifted to the lower

frequency on coordination to 1585–1592 cm⁻¹. In L3 spectra, the C=N quinolinyl ring at

1588 cm⁻¹blue-shifted to 1591–1592 cm⁻¹on coordination in the spectra of Q3

Q8, and

Q13. Coordination of L2 L3 L4, and L5 to Ag(I) can thus be proposed to be via either N_im

, Q7, and Q12 between

,

or N_thiazoleand N_quidonor atoms [34,40,41], while for L1, coordination was via N_im, N_qui,

and N_aminedonor atoms.

The mass spectra of Q1

ion peaks m/z = 468 for Q1

their base peak at m/z 633 corresponding to [Ag(L2)₂]⁺while for Q3

Q4

at m/z 641 and m/z 601, respectively. The molecular formula [Ag(L5)₂]⁺corresponded

to a prominent molecular ion peak at m/z 613 for complexes Q5 Q10, and Q15. All

–Q15 were all obtained in the positive ion mode. Molecular

,

Q6, and Q11 arise from [Ag(L1)]⁺, Q2

,

Q7, and Q12 had

Q8, and Q13 and

,

Q9, and Q14 base peaks corresponding to [Ag(L3)₂]⁺and [Ag(L4)₂]⁺were observed

,

spectroscopic studies, along with the elemental analysis (see Supplementary Materials

Tables S1 and S2), conﬁrmed the formation and purity of the proposed structures in

Scheme 1.

Molar conductivity of Q1

the values range between 0.28 and 4.05

Table S2), indicating that the complexes act as electrolytes.

–

Q15 was measured in dimethylformamide at 25 ^◦C and

×

10⁴S m²mol⁻¹(see Supplementary Materials

Electronic absorption spectra recorded in acetonitrile at room temperature in the

UV-Vis region of the free L1–L5 ligands and their corresponding complexes Q1–Q15 are

presented in Figure 1. The absorption spectra of L1, L3, and L4 (Figure 1a,c,d) and their

respective complexes are similar having two major absorption bands in the UV region be-

tween 232–275 nm and 300–340 nm. In L2 and its complexes absorption spectra (Figure 1b),

ﬁve similar absorption bands in the UV region between 216–289 and 318–350 nm attributed

to intraligand (IL) π–π * and n–π * transitions respectively are displayed. L5 and its com-

plexes showed one major absorption spectra between 287 and 291, accompanied by two

shoulders between 316 and 337 nm. All the complexes⁰bands can be attributed to intra-

Molecules 2021, 26, 1205

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ligand n–

general slight red-shift in the spectra of L1

ciated with a decrease in energy at the excited states upon coordination to silver(I) [44

π

*,

π

–

π

*, and metal to ligand charge transfer transitions (MLCT) [42

,

43]. A

–L5 complexes was observed, which can be asso-

–

46].

The steric effects of the various substituents were observed to have inﬂuenced the mode of

coordination in the structures of L1, L2, and L4 complexes.

Figure 1.

and Complexes Q2

absorption spectra of L4 and Complexes Q4

and Q15.

(

a

): Electronic absorption spectra of L1 and Complexes Q1

,

Q6 and Q11, (

b

) Electronic absorption spectra of L2

Q8 and Q13, ( ) Electronic

) Electronic absorption spectra of L5 and Complexes Q5 Q10

,

Q7 and Q12, ( ) Electronic absorption spectra of L3 and Complexes Q3

c

,

d

,

Q9 and Q14, (

e

,

Molecules 2021, 26, 1205

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In the structures of L2 complexes, the involvement of anion in the coordination to the

Ag(I) center seems to be inﬂuenced by the less steric hindrance of the benzothiazole moiety

as seen in complexes Q7 and Q12 structures (Figure 2 c,d). These types of coordination by

triﬂuoromethanesulfonate anion have been reported [47]. In L1 complexes, the hydrolytic

cleavage of one molecule of the Schiff base ligands could be related to the steric repulsions

between the two Schiff bases.

Figure 2. The ORTEP diagrams showing the atom numbering scheme with the thermal ellipsoids drawn at the 50%

probability level for molecule of complexes (

for clarity.

a) Q1, (b) Q6, (c) Q7, (d) Q12, and (e) Q14. Hydrogen atoms have been omitted

Molecules 2021, 26, 1205

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2.3. Crystal Structures of Complexes Q1, Q6, Q7, Q12, and Q14

The crystals of Q1 Q6 Q7 Q12, and Q14 were each grown by slow evaporation of

toluene diffused into dichloromethane solution of the complexes. The structures of Q1

and Q6 conﬁrmed their solution behavior while that of Q7 Q12, and Q14 are different

,

in solution. The asymmetric units of Q1 and Q6 (Figure 2a,b) had a full molecule of

the ligand coordinated to the metal center in a bidentate manner, an amine (from the

hydrolytic cleavage of the second ligand molecule) and a molecule of the nitrate (Q1) or

perchlorate (Q6), kicked out of the coordination sphere. The coordination to the metal

center was via a N atom of the quinoline moiety, an imine N atom and a N atom of

amine forming a three-coordinate geometry. The silver(I) centers in Q1 and Q6 ﬁts in

Davis et al. 2015 [48] “Extended Y”geometry description (120

±

2^◦<

α

&

,

β

< 180

±

2^◦,

γ < 120 ± 2^◦; Σ]’s ≥ 354^◦) with the sum of their angles almost 360^◦(Q1

Σ]’s = 359.96^◦;

Q6

,

Σ]’s_◦= 359.31^◦). The bond angles around the Ag(I) center lie between 72.96(6) and

146.71 (6) (Table 1). The asymmetric unit of Q7 consists of a disordered ligand, one silver(I)

center with a perchlorate anion with coordination via the quinoline N atom, the imine N

atom in a bidentate manner, and to an O atom of the perchlorate anion making half of the

complex. The other unsymmetrically related half is generated through an Ag.Ag interaction

and in the process the Ag(I) centers adopt a pseudo tetrahedral geometry. The bond angles

around the Ag(I) center lie between 72.8 (4) and 105.49 (2)^◦. This type of coordination has

been observed in related silver(I) complexes [46,49]. Q12 has a molecule of the disordered

ligand, an Ag(I) metal center and a molecule of the triﬂuoromethanesulfonate anion in the

asymmetric unit. The other half is generated through a center of inversion lying in the

center of an eight-member metallacycle in which two triﬂuoromethanesulfonate anions

through two O atoms each bridge two metal centers. While the two sets of O atoms occupy

two coordination sites of each Ag(I) center, the other two sites are occupied by two N atoms

of the ligand in a bidentate manner. The resulting geometry is distorted pseudo tetrahedral

with the bond angles around the Ag(I) center being 99.86 (10), 105.57 (10), 162.01 (10), and

175.69 (9)^◦. A similar mode of coordination has been reported [50]. The asymmetric unit

of Q14 has a molecule of the ligand, an Ag(I) metal center, a triﬂuoromethanesulfonate

molecule and a molecule of water. Coordination in this complex is via a N atom of the

quinoline moiety, an imine N atom, an O atom of a triﬂuoromethanesulfonate, and an O

atom of a water molecule forming a pseudo tetrahedral with the bond angles around the

Ag(I) center lying between 93.18 (6) and 145.17 (6)^◦.

Table 1. Selected geometric parameters for complexes Q1, Q6, Q7, Q12, and Q14.

Q1

Q6

Q7

Q12

Q14

Ag–N_im

Ag–N_qy

Ag–N_am

Ag–N_th

2.398 (16)

2.240 (16)

2.192 (16)

2.400 (17)

2.246 (18)

2.208 (17)

2.317 (18)

2.321 (17)

2.37 (6)

2.346 (3)

2.37 (2)

2.66 (6)

3.06 (7)

2.465 (3)

2.587 (3)

2.397 (3)

2.609 (2)

2.233 (16)

Ag–O

Ag–Ag

146.71 (6)

140.28 (6)

72.97 (6)

145.25 (6)

140.93 (6)

73.12 (6)

N–Ag–N

72.8 (4)

70.42 (10)

73.08 (6)

105.57 (10)

162.01 (10)

175.69 (9)

99.86 (10)

83.56 (9)

145.17 (6)

134.92 (6)

95.22 (6)

93.18 (6)

101.85 (7)

105.49 (2)

83.23 (2)

N–Ag–O

O–Ag–O

All the complexes feature two sets of planes described by a ﬁve-member metallacycle

including the quinoline imine unit (Plane 1) and by substituted phenyl ring for Q1

, Q6,

and Q14 and by the thiazole moiety in the structures of complexes Q7 and Q12.

Molecules 2021, 26, 1205

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The Ag–N_imbond distances in Q1

,

Q6

,

Q7, and Q12 range between 2.317 (18) and

2.465 (3) Å and are generally longer than the Ag–N_qybond distance between the range

2.240 (16) and 2.346 (3) Å but shorter in Q14 (Table 1). The Ag–O bond distances range

from 2.233 (16) and 2.609 Å. The Ag–N and Ag–O bond distances in complexes Q1

, Q6,

Q7 Q12, and Q14 are within expected ranges and are comparable with those reported

,

in the literature [50

is 3.0563 (11) Å.

,51]. Complexes Q7 has Ag Ag interaction where the bond distance

2.4. In Vitro Antimicrobial Studies

All the ligands and complexes were tested against two Gram-positive bacteria, Methicillin-

resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (S. aureus), and four

Gram-negative bacteria, Escherichia coli (E. coli), Salmonella typhimurium (S. typhi), Klebsiella

pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa). The minimum

inhibitory concentrations (MIC) values were compared to Ciproﬂoxacin and the three

silver(I) salts as standards (Table 2). The aim was to investigate the effect of introducing

the silver metal to the ligands in the bacteria strains. Q1–Q15 showed better antibacterial

activity compared to L1–L5 in general an indication of enhanced activity of the ligands

on complexation to Ag(I). The general belief is that complexation of the ligands leads to

high lipophilicity which in turn can lead to the rupturing of the cell membranes of the

microorganisms [52]. In addition, silver(I) ion is well known for its bactericidal effects,

mechanistically acting by obstruction of bacteria cell functions through direct interaction

with the bacteria enzymes, DNA and the negatively charged peptidoglycan from the

bacterial cell wall. This interrupts cell division and replication and cell breathing leading to

cell death through the release of their ﬂuid and electrolyte content into the environment [53].

Table 2. Minimum inhibitory concentration of Silver complexes of L1–L5 (µg/mL).

Compounds

Gram + Ve Bacteria

Gram − Ve Bacteria

SA

MRSA

PA

ST

EC

KP

L1

L2

L3

L4

L5

Q1

Q2

Q3

Q4

Q5

Q6

Q7

100

NA

25

50

12.5

50

3.125

1.6

NA

1000

NA

1000

50

100

25

50

NA

100

NA

100

25

12.5

6.25

1.6

NA

1000

50

100

50

12.5

25

50

1000

NA

1000

NA

50

25

12.5

NA

100

50

NA

100

0.2

0.05

0.8

0.025

1.6

12.5

25

0.2

0.4

Q8

Q9

Q10

Q11

Q12

Q13

Q14

Q15

50

25

6.25

25

0.1

6.25

1.6

25

50

100

25

50

1000

0.2

12.5

0.4

1.6

12.5

6.25

50

25

0.8

25

50

0.0125

0.05

25

0.2

0.1

0.05

1000

1.6

1000

0.8

1000

50

12.5

0.8

12.5

0.8

50

1000

100

0.2

6.25

1000

25

12.5

3.125

NA

25

AgNO₃

AgClO₄

AgCF₃SO₃

Ciproﬂoxacin ^s[54]

1000

0.2

1.6

Only L1 showed some activity against S. aureus (100

activity against MRSA (1000 g/mL). Against the Gram-negative bacteria, the ligands

showed selective activity. L1 is active against E. coli with an MIC value of 1000 g/mL, L2

µg/mL) while L3 also had some

µ

Molecules 2021, 26, 1205

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and L3 against P. aeruginosa and K. pneumoniae with MIC values between 50 and 100

µg/mL,

L4 against S. typhi and E. coli and L5 against P. aeruginosa, S. typhi, and K. pneumoniae with

MIC values of 100 g/mL, 50 g/mL, and 100 g/mL, respectively. Q7 and Q8 showed

µ

the highest antimicrobial activity against all bacteria strains. The ligands of each of Q7

and Q8 possess either the benzothiazole moiety or the p-chloro substituent. Enhanced

antimicrobial activity of compounds containing either benzothiazole moiety or the chlorine

substituent in the para-position is in accordance with the literature report [55,56] making

them targets for drug design. In addition, both Q7 and Q8 have the perchlorate anion.

Enhance antimicrobial activity was also observed in other complexes with perchlorate

anion against most of the bacteria strains.

All the complexes with the exception of Q4

against K. pneumoniae (in comparison to the other strains) with MIC values between 0.0125

and 0.4 g/mL, lower than that of Ciproﬂoxacin (0.8 g/mL). The activity of Q7 against

S. aureus with an MIC value of 1.6 g/mL can be singled out as it was better than that of the

standard ciproﬂoxacin, AgNO₃, AgClO₄, or AgCF₃SO₃. Similarly, Q4 Q6 Q11 Q12, and

Q15 with an MIC value below 25 g/mL signiﬁed their better activity than ciproﬂoxacin

against S. aureus. Those of Q1 Q2 Q10, and Q13 have the same MIC value as the standard

reference against S. aureus. All the complexes are fairly active against MRSA with only

Q6 and Q8 with MIC value 25 g/mL showing the same activities as ciproﬂoxacin. Q6

, Q6, and Q10, were selectively active

µ

,

µ

,

µ

and Q8 have ﬂuorine and chlorine respectively, as their substituent and perchlorate as

their counter anion. This revealed the inﬂuence of electron-withdrawing functional groups

on antimicrobial activity. This type of compound with an electron-withdrawing group

has been reported to have excellent antimicrobial activity [57]. Against P. aeruginosa, Q8

having chlorine as its substituent, showed excellent activity higher than the reference

standards while Q7 has the same MIC value of 0.2

Q15 have similar MIC value as AgClO₄(0.8 g/mL), Q5

within 1.6–6.25 µg/mL lower than AgNO₃and AgCF₃SO₃while Q2

Q13 (6.25 g/mL) have similar MIC value as AgNO₃. Q6 Q10 Q14, and Q15 having

µ

g/mL as ciproﬂoxacin. Q14 and

Q6 Q9 Q10, have MIC values

Q3 Q4 Q12, and

µ

,

µ

,

either thiophene moiety, ﬂuorine, methyl substituent in their ligand performed better than

the standard against P. aeruginosa, these complexes also have either perchlorate or triﬂate

as their anion.

The MIC of all the complexes except Q15 and AgNO₃(1000

within 12.5–100 g/mL, these values are higher than the ciproﬂoxacin standard (0.4

only AgClO₄(0.2 g/mL) had better activity than the standard. Q5 having thiophene

moiety and nitrate ion as counter anion has the same MIC value as AgCF₃SO₃(12.5 g/mL).

This shows complexes Q1–Q15 fair activity against S. typhi. The activities of Q5 (with thio-

phene) and Q8 (with chlorine) against E. coli are the same as the standard (1.6 g/mL). Com-

plexes Q6 and Q9 (12.5 g/mL) were half of their salt (25 g/mL), while Q10 (6.25 g/mL)

µg/mL) against S. typhi is

µ

µg/mL),

µ

was four times lower than the salt against E. coli. The enhanced activity of Q8 with chlorine

substituent and Q5 (with thiophene) are 64 and 32 times higher than the standard respec-

tively against K. pneumoniae. All the complexes showed excellent antibacterial activities

against K. pneumoniae. Q4 has the same MIC value as the standard (0.8 µg/mL).

In summary, the antimicrobial activities of the complexes exhibited a structural activity

relationship because those with the chlorine substituents, benzothiazole, and thiophene

moieties and with perchlorate anion displayed a remarkable antimicrobial activity. En-

hanced antimicrobial activities of compounds containing similar electron-withdrawing

groups [58,59], benzothiazole [60], and thiophene [61] moieties have been reported. Reports

on the effects of different counter anions are also known [62].

A comparison bar chart representing each salt complex is shown in Figure 3a–c.

Compounds with 1000

µg/mL MIC value and those without antimicrobial activity are

omitted from the bar plot for clarity.

Molecules 2021, 26, 1205

10 of 34

Figure 3.

(

a) Minimum inhibitory concentration (µg/mL) of L1–L5 silver(I) complexes with nitrate as counter anion,

(

b) Minimum inhibitory concentration (µg/mL) of L1–L5 silver(I) complexes with perchlorate as counter anion, and

(c) Minimum inhibitory concentration (µg/mL) of L1–L5 silver(I) complexes with triﬂate as counter anion.

2.5. In Vitro Antioxidant Studies

Antioxidants are important in protecting the human body against damage by reactive

oxygen species (ROS). Accumulation of ROS in the cells may lead to damage of the

DNA, RNA, and proteins which subsequently leads to several physiological disorders

such as cardiovascular disease, diabetes, rheumatism, atherosclerosis, cancer, aging and

cell death [63

complexes [65] and pyridine derivatives complexes [66

the ability to slow down and stop damage to DNA, RNA, protein substrates and thereby

,64]. The antioxidant activity of some compounds such as dithiocarbamate

–

68] have been reported to possess

Molecules 2021, 26, 1205

11 of 34

prevent the occurrence of diseases. The free radical scavenging ability of all ligands

and complexes in this study were investigated using ferric reducing antioxidant power

(FRAP). In antioxidant studies, FRAP assay is often preferred because it is simple, fast,

economical, and direct in antioxidant measurement. FRAP assay is based on the electron-

donating ability of an antioxidant compound. This involves the reduction of a colorless Fe³⁺

tripyridyltriazine complex (Fe³⁺(TPTZ)) to an intense blue-colored Fe²⁺tripyridyltriazine

complex (Fe²⁺(TPTZ)) by the electron-donating antioxidants at low pH [69]. The reducing

power is to determine the reductive ability of the test compounds. Table 3 summarizes

the mean values from three independent experiments of in vitro antioxidant activities of

the ligands L1–L5 and complexes Q1–Q15 at different concentrations. The antioxidant

activities of the compounds are expressed as 50% inhibitory concentration (IC₅₀in mg/mL).

Ascorbic acid with an IC₅₀value of 2.68 mg/mL was used as a standard for the study.

Table 3. In vitro antioxidant activities of L1–L5 and Complexes Q1–Q15 done in triplicate.

Antioxidant (IC₅₀mg/mL)

COMPOUND

FRAP

L1

L2

L3

L4

L5

Q1

Q2

Q3

Q4

Q5

Q6

Q7

Q8

Q9

Q10

Q11

Q12

22.42 ± 0.0048

2.29 ± 0.0041

20.35 ± 0.0047

4.50 ± 0.0053

3.79 ± 0.0052

4.97 ± 0.002

1.10 ± 0.0044

4.75 ± 0.0042

2.14 ± 0.0035

2.04 ± 0.0032

0.95 ± 0.003

1.13 ± 0.044

3.27 ± 0.093

1.62 ± 0.078

0.98 ± 0.055

6.27 ± 0.038

1.86 ± 0.024

2.22 ± 0.0055

3.55 ± 0.022

3.35 ± 0.012

1.43 ± 0.03

Q13

Q14

Q15

AgNO₃

AgClO₄

AgCF₃SO₃

Ascorbic Acid

0.88 ± 0.051

0.88 ± 0.054

2.68 ± 0.11

All the compounds tested showed some antioxidant activity. Of the ligands, L2

had the highest activity with an IC₅₀value of 2.29 mg/mL even better than that of the

standard reference. The complexes and the metal salts had better activities than the ligands

on the general. The antioxidant activity of the silver salts was evaluated to study the

0

inﬂuence of the metal salts on the ligand s antioxidant activity. Interestingly, the silver salts

exhibit signiﬁcant antioxidant activity comparable to the standard, but the introduction

of an organic ligand to a metal ion is known to help with the regulation of metal ion

absorption, distribution and metabolism in the biological systems [70,71]. Hence, we focus

on the antioxidant activity of complexes Q1–Q15 as an alternative synthetic antioxidant in

this study.

Q2, Q4, Q5, Q6, Q7, Q9 Q10, and Q13 had IC₅₀values between 0.95 and 2.22 mg/mL

(Table 3) better than that of ascorbic acid (2.68 mg/mL) but not those of the free metal salts.

The implication is that the complexes had a good ability to reduce Fe³⁺to Fe²⁺(in FRAP)

was good. The common occurrence in these complexes is either a ﬂuorine substituent,

thiophene or a benzothiazole moiety. There have been reports of compounds with ﬂuorine

Molecules 2021, 26, 1205

12 of 34

substituent (C_{0.5 FRAP}= 424.5–502.6

µM) with potent antioxidant activities in FRAP [72].

Similarly, other reports have shown signiﬁcant antioxidant activities of benzothiazole

(553.5 mmol Fe²⁺/mmol Compound) [73] and thiophene (4.55 mmol TE/g compound) [74

]

derivatives. In this study, Q6 with ﬂuorine substituent and perchlorate counter anion

had the lowest ferric reducing power. It is assumed that the Ag(I) center in Q6 which is

tricoordinate, could be responsible for the enhanced activity. Q10 has the thiophene moiety

with a lone pair of electrons on its thiophene. However, despite a ﬂuoro substituent in

Q1 and a chloro substituent in Q3, the two complexes had the lowest activities, with IC₅₀

values of 4.97 and 4.75 mg/mL. The counter anions in both complexes is a nitrate and that

could be responsible for their low activities. The bar chart in Figure 4 represent Q1–Q15

free radical scavenging activity by FRAP assay.

Figure 4. FRAP % free radical scavenging vs. Concentration (mg/mL) of complexes Q1–Q15, silver

nitrate, silver perchlorate, silver triﬂate, and Ascorbic Acid. AgN = silver nitrate, AgC = silver

perchlorate, AgF = silver triﬂate and As = Ascorbic acid values represent mean

±

standard deviation

a–f

(n = 3).

Different alphabets over the bars for a given concentration for each complex represent

0

signiﬁcant difference (Tukey s-HSD multiple range post hoc test, p < 0.05).

Summarily, most of the complexes tested had signiﬁcant antioxidant activity. The

antioxidant data obtained for complexes Q1–Q15 clearly showed that complexes with

either a ﬂuorine substituent, thiophene or a benzothiazole moiety had good reducing

power. This is an evidence of their effective antioxidant activities. Thus, these complexes

are potential synthetic antioxidant supplements required to protect the body against free

radical damages.

2.6. DNA Binding Studies

Deoxyribonucleic acid (DNA) is a molecule possessing genetic information in living

cells. It is an important molecular target for anticancer agents because of its involvement in

gene expression and protein synthesis, which are fundamental steps in cell division and

growth [75]. DNA binding studies are signiﬁcant when developing novel therapeutic drugs.

Their interactions with potential drugs have been associated with the anticarcinogenic

activity of such drugs. This makes the determination of the type of interaction between

metal complexes and DNA important. In studying DNA interaction with metal complexes,

electronic absorption spectroscopy is one of the most convenient techniques usually used to

investigate the possible modes of binding. DNA can interact with metal complexes either

through the covalent or non-covalent bond. Non-covalent interaction of the DNA with

metal complexes is usually via three major modes namely: intercalations between base

Molecules 2021, 26, 1205

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pairs, binding into the groove of DNA, and electrostatic binding [76], while the covalent

interaction involves the replacement of the DNA N atom by the metal complex’s labile

ligand [77].

In this study, we set to examine the extent to which the ligands and the complexes in-

teract with calf thymus DNA (CT-DNA) by monitoring the hyperchromic and hypochromic

effects of the electronic absorption titration. Incremental additions of DNA to the test com-

pounds showed prominent hypochromic shifts with intraligand π–π * bands absorption

bands between 249 and 372 nm in the absorption spectra of the ligands and complexes

studied. The hypochromism is probably due to the formation of strong stacking interaction

between the quinoline planar aromatic chromophore and the base pairs of CT-DNA [27

]

which is an indication of intercalation binding behavior of the compounds to CT-DNA.

Intercalators are molecules that stack perpendicular to the DNA backbone without forming

covalent bonds or breaking up the hydrogen bonds between DNA bases. The p-orbital of

the compounds could be coupled with the p-orbital of the base pairs, which decreases the

transition energy and induces hypochromism.

In complexes Q1–Q15, a hypochromic shift between 6.27 and 80.49% in the absorption

bands with slight bathochromic between 1 and 3 nm was observed in all the compounds

(Supplementary Materials Figures S6–S19) except for Q1

tion bands are accompanied by a slight hypsochromic shift between 2 and 4 nm as the

concentration of the CT-DNA was increased. L1–L5 Q1 Q15 and AgNO₃, AgClO₄, and

, Q6, and Q11, whose absorp-

,

–

AgCF₃SO₃in the absence and presence of CT-DNA are shown in Figures S1–S22 (see

Supplementary Materials).

AgNO₃, AgClO₄, and AgCF₃SO₃were used as the standard in this study and the

titration of these metal salts against CT-DNA also showed notable hypochromic shifts

of 23.64–50.86% with absorption bands between 263 and 274 nm. The silver salts: A

hypochromic shift in the absorption bands with a slight bathochromic shift of ~2 nm was

observed in the spectra of the metal salts (Supplementary Materials Figures S20–S22).

The intrinsic binding constant of L1–L5, Q1–Q15 and AgNO₃, AgClO₄, and AgCF₃SO₃

were evaluated using the Wolfe–Shimer equation (Equation (5)) by determining the ratio

of the slope to the intercept from the plot of [DNA]/(ε − ε_f) versus [DNA]. The binding

a

afﬁnity of the free ligands and free metal salts are lower than those of complexes Q1–Q15

,

an indication that the ligands binding afﬁnities were enhanced on complexation of ligands

to Ag(I). The binding constant obtained lie between 3.33

×

10⁵and 3.0

×

10⁶M⁻¹for Q1–

Q15, between 7.50 L5, and between 2.00

×

10⁴and 1.38 10⁵in L1

×

–

×

10⁵and 3.30 × 10⁵for

AgNO₃, AgClO₄, and AgCF₃SO₃(Table 4). Q8 had the highest binding afﬁnity (Figure 5a)

with a calculated intrinsic binding constant (K_b) of 3.00

1.57

10⁶and 1.5 10⁶, respectively. The high binding afﬁnities of the three complexes

Q3, Q7, and Q8) are probably due to the -electron interactions of the quinolinyl with the

×

10⁶, followed by Q3 and Q7 with

×

(

π

base pairs of the DNA [78]. In addition, the presence of electron-withdrawing chlorine

atoms in para-position in Q3 and Q8 and the presence of benzothiazole moiety in Q7

probably also contributes to the binding afﬁnity to CT-DNA. High DNA interaction with

similar compounds has been reported for complexes with electron withdrawing group

and benzothiazole moiety [78–80]. This interaction favors the aromatic environment of

the CT-DNA base pairs resulting in strong structural perturbations in the DNA molecule.

These perturbations increase the distance between the adjacent base pairs [27]. The order

of the interaction of complexes Q1–Q15 and their metal salts with DNA are Q8 > Q3 >

Q7 > Q11 > Q9 = Q10 > Q12 > Q13 > Q1 = Q4 = Q5 = Q15 > Q6 > Q14 > Q2 > AgClO₄>

AgNO₃> AgCF₃SO₃. The binding mode needs to be proved by further studies, whereas

ligands L1–L5 were exempted since their binding afﬁnity to CT-DNA are lower than those

of the complexes.

Molecules 2021, 26, 1205

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Figure 5.

line) and the presence of different concentrations of CT-DNA (0–3.0

A Stern–Volmer plot of Q8 interaction with CT- DNA. ( ) Fluorescence spectra of EB-CT-DNA in

the absence (dashed line) and the presence of a different concentration of complex Q8. (inset) The

stern–Volmer plot of Q8 interaction with EB-CT- DNA. ( ) Stern–Volmer plot of Complexes Q1–Q15

interaction with EB-CT- DNA. (d) The double-logarithmic plot of Complex Q10.

(

a

) Electronic Absorption Spectra of complex Q8 at 3.0

×

10⁶M in the absence (dashed

×

10⁵M) at 303 nm

λ_max. (inset)

b

c

Molecules 2021, 26, 1205

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Table 4. The binding parameters of complexes Q1–Q15 interactions with CT-DNA, ethidium bromide, and bovine

serum albumin.

CT-DNA-

Cmp.

CT-DNA-EB-Complex

Protein-Complex

K_b

K_sv

(M⁻¹) ×

10⁴

K_q

K_bin

(M⁻¹) ×

10⁵

K_b

(M⁻¹) ×

10⁴

K_sv

K_q

K_bin

(M⁻¹) ×

10⁴

(M

)

R²

(M⁻¹S

)

n

(M

)

n

−1

(M⁻¹S

)

× 10⁵

× 10¹²

× 10⁴

× 10¹²

Q1

Q2

Q3

Q4

Q5

Q6

Q7

Q8

Q9

4.00

3.00

15.7

4.00

3.33

15.0

30.0

5.00

6.67

4.50

4.29

3.33

4.00

3.00

3.30

2.00

1.38

1.50

7.50

1.60

1.17

0.99

0.96

0.98

0.97

0.99

0.98

2.76

2.68

3.93

3.50

2.31

2.03

3.96

3.98

2.49

2.98

2.38

3.14

3.33

2.98

2.48

3.12

3.31

3.01

2.75

2.68

3.93

3.50

2.31

2.03

3.96

3.98

2.49

2.98

2.38

3.14

3.33

2.98

2.48

3.12

3.31

3.01

1.07

5.03

9.17

2.11

1.03

2.41

1.29

1.02

6.53

9.61

3.45

2.12

5.74

5.31

5.29

4.50

6.00

1.60

1.13

1.27

1.29

1.17

1.15

1.21

1.11

1.09

1.30

1.31

1.21

1.19

1.27

1.28

1.30

1.24

1.26

1.15

2.16

7.97

5.11

10.3

5.95

5.73

9.38

9.48

5.43

8.01

8.62

20.7

2.89

7.50

13.1

4.49

4.82

4.46

2.50

0.117

0.69

2.59

4.09

2.59

4.09

2.56

1.01

0.68

Q10

Q11

Q12

Q13

Q14

Q15

AgNO₃

AgClO₄

AgCF₃SO₃

L1

0.138

L2

L3

L4

L5

R is the correlation coefﬁcient.

Fluorescence Competitive Displacement Studies

Further studies on binding interactions of Q1–Q15 and the silver salts with CT-DNA

were done using ﬂuorescence competitive displacement assay. Although the complexes

absorb UV radiation, they do not ﬂuoresce. The ﬂuorescence competitive displacement

study is based on the displacement of ethidium bromide from the CT-DNA-EB adduct

by the test compounds. Ethidium bromide, a well-known intercalating agent, is a planar

cationic dye that ﬂuoresces weakly in phosphate buffer saline solution but emits intensely

in the presence of CT-DNA due to the strong intercalation between the DNA double

helix base pairs [81]. The intense ﬂuorescence of the ethidium bromide in the presence of

CT-DNA can be quenched by introducing another molecule that can displace the bound

ethidium bromide. A phenomenon known as ﬂuorescence quenching [80]. The incremental

addition of the complexes concentration to ethidium bromide pre-treated with CT-DNA

resulted in an appreciable decrease in the ethidium bromide ﬂuorescent intensity at 605 nm

(Figure 5b and Figures S23–S39). The gradual decrease in the intensity of the EB pre-treated

with CT-DNA upon addition of Q1–Q15 complexes could be as a result of the complexes

interacting with DNA via intercalation.

The relative binding interaction of the complexes with CT-DNA was determined

by the quenching strength, K_svcalculated from the slope of the linear quenching plot

of F_o/F versus [complex] (Equation (6)). The K_svvalues for Q1–Q15 were between 2.03

and 3.98 × 10⁴M⁻¹and those of the silver salts are between 3.01 and 3.31

×

10⁴M⁻¹

(Table 4). These K_svvalues indicated the displacement of ethidium bromide by Q1–Q15

and conﬁrmed binding via intercalation mode to CT-DNA. The complexes have high

quenching efﬁciency and a binding afﬁnity to CT-DNA. Complex Q8 (Figure 5b) had

the highest binding afﬁnity to CT-DNA which is consistent with the compound with the

Molecules 2021, 26, 1205

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highest intrinsic K_bconstant (Figure 5 a). The quenching of the ﬂuorescence intensity of

EB-CT-DNA adduct by the complexes was further used to determine the binding constant

(K_bin) and the number of binding sites (n).

Fluorescence quenching can take place via two mechanisms, namely, static quenching

and dynamic quenching. The static quenching involves the collision of the ﬂuorophore

and the quencher (complex) in the ground state while the dynamic quenching involves the

collision of the ﬂuorophore and the quencher in the excited state. In order to determine the

type of quenching taking place, two approaches are commonly used, namely: temperature-

dependent approach and the Stern–Volmer plot approach.

The Stern–Volmer plot approach was used based on the fact that the Stern–Volmer

plots for Q1–Q15 (Figure 5c), were linear an indication of only one mechanism for the

quenching ﬂuorescence, i.e., either dynamic or static. Evaluation of K_qusing Equation (1)

would, therefore, suggest the quenching mechanism through which the complexes quench

ethidium bromide emission.

K_sv= K_qτ_o

(1)

where K_sv, K_q, and τ_orepresent Stern–Volmer quenching constant, bimolecular quench-

ing constant, and an average lifetime of the ﬂuorophore in the absence of the quencher,

respectively.

were found to be between 2.03 and 3.98

τ

_onormally takes about 10⁻⁸s in biom₋a₁cromolecules [82]. The values of K_q

×

10^{12 −1}S (Table 4), which is greater than the

M

maximum diffusion collision quenching rate constant of various quenchers of biological

macromolecules (2

10^{10 −1}S⁻¹) [82]. These values showed that the quenching by

Q1 Q15 was not initiated by a dynamic mechanism but happened via a static quenching

×

M

–

mechanism; thus, at the ground state, complexes formed between quenching molecules

and ﬂuorescence molecules are stabilized [83].

The double-logarithmic Equation (2) was used in determining the binding constant of

complexes Q1–Q15 and CT-DNA.

F_o− F

log

= log K_b+ n log[Q]

(2)

F

A plot of log F_o

−

F/F vs. log [Q] of complexes Q1–Q15 gave a straight line with

a slope of n and y-axis intercept of log K_b(Figures S40–S56) (where F_oand F denote the

relative ﬂuorescence intensities of CT-DNA in the absence and presence of the quencher,

respectively, while [Q] is the concentration of the quencher, K_bis the binding constant and

n is the number of binding sites. The values of n for complexes Q1–Q15 are approximately

equal to 1 (Table 4), suggesting that all the complexes bind to one reactive site on the

CT-DNA. The corresponding results are shown in Table 4. Complex Q10 (Figure 5d)

showed the most binding afﬁnity to CT-DNA with a binding constant (K_bin) equal to

9.61 × 10⁵M⁻¹and the number of binding sites (n) approximately equal to 1.

Based on the spectroscopic studies of the interaction of L1–L5 and complexes Q1–Q15

with CT-DNA, it can be summarized that the test compounds in this study had a strong

binding afﬁnity to calf thymus DNA, especially those with either electron-withdrawing

substituent or benzothiazole moiety. The binding interaction of the compounds to CT-DNA

was via intercalation mode mainly due to the planarity of the chelating quinolinyl ligand

and the competitive study with EB shows that the complexes can displace EB from the

CT-DNA-EB adduct and compete for the DNA-binding sites with EB, which is usually

characteristic of an intercalative interaction of compounds with DNA.

2.7. Albumin Binding Studies Using Absorption Spectroscopy Titration

Interaction of potential therapeutic drugs with carrier proteins is very important

in bio-inorganic studies because the binding properties of the drug–protein reveal the

absorption, transportation, distribution, and metabolism of the drugs. For this reason,

the protein-binding study of Q1 to Q15 is an important way of studying the compounds

metabolism in vivo.

Molecules 2021, 26, 1205

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Bovine serum albumin (BSA) was used as the protein standard in this study because of

its close resemblance to human serum albumin (HSA), its stability, high solubility, neutrality

in many biochemical reactions and low cost [84]. In protein binding study, absorption

spectroscopy titration is a known method used in studying the conformational changes

in proteins, and this protein is characterized by an intense absorbance peak at 280 nm

depicting the microenvironment around tyrosine and tryptophan residue polarity [85].

On each addition of various concentrations of complexes Q1–Q15 and the silver salts to a

constant BSA concentration, the intensity of the BSA absorbance intensiﬁed between 260

and 300 nm with slight shifts (Figures S57–S73). This shows some binding interactions

between the complexes and BSA exist. K_bin this instance was determined from the intercept

to the slope ratio of the linear curve of a plot of 1/[A-A^◦] vs. 1/[complex]. The K_bof all

the complexes were in the range of 2.16

×

10⁴–2.07

×

10⁵M⁻¹(Table 4) which are in

conformation with reported related BSA binding constants, normally between 10⁴and

10⁶M⁻¹[86]. This range is a recognized range for drug-carrier complexes. The silver salts

with K_bvalue between 4.46 and 4.82

Q1 (Figure S57) and Q13 (Figure S68). This shows that the binding afﬁnity of the ligands

to BSA was enhanced on coordination. Complex Q12 had the highest binding afﬁnity

×

10⁴is lower than those of the complexes except

(Figure 6a), followed by Q4 (Figure S60) and Q15 (Figure S70). The inﬂuence of Q4

and Q15 ligand substituents on their binding afﬁnity to BSA can be related to similar

literature reports [87 89]. The order of binding constant for the complexes is: Q12 > Q15 >

, Q12,

–

Q4 > Q8 > Q7 > Q11 > Q10 > Q2 > Q14 > Q5 > Q6 > Q9 > Q3 > Q1 > Q13.

Fluorescence Quenching of Bovine Serum Albumin in the Presence of Complexes Q1–Q15

The ﬂuorescence quenching of protein can provide information on protein bind-

ing mechanisms, binding mode, binding sites and the rates of interaction with drugs or

complexes of study [86]. When BSA is excited at 280 nm, it ﬂuoresces strongly between

300 and 400 nm leading to excitation in tyrosine (Tyr) and tryptophan (Trp) residues in

proteins. In the presence of a small molecule, BSA has the possibility of undergoing

different types of interactions such as hydrophobic, hydrogen bonding, van der Waals,

and electrostatic interactions. The ability of a complex to quench BSA ﬂuorescence in-

tensity depends on the hydrophobic interaction between the Trp and the hydrophobic

ligand of a complex. The interaction is either enhanced or stabilized by the surrounding

amino acid residues [90]. The binding of BSA to compounds was studied by ﬂuorescence

measurements at room temperature.

The free BSA has strong ﬂuorescence emission around 350–360 nm when excited at

280 nm and on adding complexes Q1–Q15, the BSA ﬂuorescence intensity of only Q1

and Q11 decreased gradually as the concentration of these three complexes was increased

(Figure 6b and Figures S74–S75). The reduction in the ﬂuorescence intensity of Q1 Q6, and

Q11 is accompanied by a redshift of the emission wavelength between 2 and 15 nm, an

indication that the interaction of Q1 Q6, and Q11 with BSA causes a conformational change

, Q6,

,

in the protein structure [91]. Furthermore, an isosbestic point between 366 and 388 nm

in the spectra of the complex–BSA interaction was observed, implying the formation of a

stable complex–protein complex. Q11 (Figure 6b) quenched the intrinsic ﬂuorescence of

BSA more than Q1 and Q6.

The inability of all the complexes (with the exception of Q1

the intrinsic ﬂuorescence of BSA after the addition of between 1 and 2

,

Q6, and Q11) to quench

M concentration

µ

could be structurally related because from our previous report where pyridine moiety

was involved as against the quinoline moiety in this study, a contrary behavior regarding

the interaction of the protein with complexes of similar substituents and moieties were

reported. Complexes Q1

complexes mode of coordination which means their interaction with BSA was probably

via the aniline substituent while the quinoline ring of L2 L3 L4, and L5 complexes were

not planar enough and are not readily available for quenching the active site of the protein

, Q6, and Q11 mode of coordination are different from other

,

Molecules 2021, 26, 1205

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buried in the hydrophobic environment. This suggests no interaction of the complexes

with the BSA protein.

Figure 6.

different concentrations of complexes Q12. (inset) Plot of 1/(A_o

) Fluorescence emission spectra of BSA in the absence(dashed line) and the presence of a different

(a) Electronic Absorption Spectra of BSA in the absence (dashed line) and the presence of

−4

−1

−

A) vs. 1/[Complex]

×

10

M

.

(

b

concentration of complex Q11. Inset: A Stern–Volmer plot of the interaction of Q11 with BSA. (

double-logarithmic plot of BSA–Complex Q6 interactions.

c) The

Molecules 2021, 26, 1205

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The quenching mechanism of the Q1

,

Q6, and Q11 was evaluated using the Stern–

Volmer Equation (Equation (7)). K_svvalues for the complexes were obtained from the slope

of the plot of I₀/I vs. [Q] and found to be between 2.50

×

10⁴and 4.09 10⁴M⁻¹. The

×

calculated values of K_svand K_qfor the interaction of the complexes with BSA are given

in Table 4. The linearity of the Stern–Volmer plots (inset Figures S64–S71) is an indicator

of either a static or a dynamic quenching mechanism. The K_qvalues obtained for the

complexes are between 2.50

×

10¹²and 4.09

×

10^{12 −1}S^−1,which are greater than the

M

maximum scatter collision-quenching constant of biomacromolecules (2 × 10¹⁰M⁻¹

S

⁻¹).

This indicates that the interactions between the complexes and BSA are static. Therefore,

the binding constant (K_bin) and the number of binding sites (n) can be determined using

Scatchard Equation (3):

I_o− I

log

= log K_bin+ n log[Q]

(3)

I

The n and K_bincan be calculated from the slope and the intercept of the double

logarithm regression curve of log(I_oI)/I versus log[Q] (Figures S76–S77). The values of

−

the binding constant (K_bin) suggest BSA has a moderate binding afﬁnity to the complexes.

Complex Q6 (Figure 6c) has the highest binding afﬁnity to BSA. The number of the binding

site (n) for Q1, Q6, and Q11 are approximately one; this suggests that one molecule of the

complexes bound per bov₋in₁e_,serum albumin. The binding constant values of Q1

, Q6, and

Q11 fall within 10⁴–10⁶M

which is the recognized values of non-covalent interaction of

BSA with drugs [92].

In summary, the interaction of the complexes with BSA causes conformation changes

of both tyrosine and tryptophan environments due to the hydrophobic interactions of the

complexes’ ligand with BSA. In essence, the complexes possess a moderate binding afﬁnity

for protein. Thus, most of the complexes can be stored in protein and can be easily released

in desired target areas.

2.8. Anticancer Studies

Cancer is one of the major life-threatening diseases worldwide. The clinical application

of chemotherapy is still considered a major method in treating cancer, therefore, the

development of novel effective anticancer drugs is of importance. For this study, we

picked complexes Q6, Q7, and Q9 based on the signiﬁcant effect the ﬂuorine and methyl

substituent and benzothiazole moiety has on their complexes⁰biological activities. All

these complexes have perchlorate as their anion since perchlorate has been identiﬁed to

likely enhance biological activities as seen in the antimicrobial, antioxidant, activities, and

DNA and protein binding assays.

The in vitro cytotoxicity of metal complexes, Q6, Q7, Q9, and cisplatin toward human

embryonic kidney 293 (HEK293), cervical cancer cell line (HELA), breast cancer cell line

(MDA-MB231) and neuronal cancer cell line (SHSY5Y) were examined via standard method.

The EC₅₀and the selectivity index (SI) values obtained are summarized in Table 5. The

result obtained indicate that there is a structure activity relationship of the complexes in

terms of cytotoxicity of the complexes. Q7 and Q9 showed potent cytotoxicity activity

towards HELA cells with EC₅₀values of 22.80

which is lower than cisplatin (EC₅₀> 50). Q7 and Q9 also showed slight cytotoxicity against

±

3.11 and 22.34

±

4.86 µM, respectively

MDA-MB231 and SHSY5Y.

Table 5. EC₅₀and SI values of the cytotoxic activity of complexes Q6

,

Q7

,

Q9, and cisplatin against various human cell lines.

Compound

EC₅₀± Std Dev (µM)

Selectivity Index (SI)

HEK293

HELA

MDA-MB231

SHSY5Y

HELA

MDA-MB231

SHSY5Y

Cisplatin

Q6

14.2 ± 3.8

≥100

>50

22.68 ± 4.89

≥ 100

19.74 ± 3.77

≥100

0.28

1.00

4.39

4.48

0.63

1.00

2.40

2.57

0.72

1.00

3.30

2.12

≥100

Q7

Q9

≥100

22.80 ± 3.11

41.62 ± 4.25

30.33 ± 4.67

≥100

22.34 ± 4.86

38.84 ± 4.33

47.23 ± 3.34

EC₅₀value calculated as the compound concentration required to reduce cell viability by 50%. Std Dev = Standard deviation. n = 3 for all

samples. SI values: selectivity index; calculated as the quotient of the EC₅₀value obtained in HEK293 over the EC₅₀value obtained in the

respective cancer cell lines.

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The remarkable cytotoxicity recorded for Q7 and Q9 towards HELA cells might be

due to the presence of benzothiazole moiety and methyl substituent, respectively. In similar

reports, Saeed et al. 2010 [93] in their anticancer study of various benzothiazole derivatives

obtained IC₅₀values in the range 38–46 µM, and Sreelatha et al. 2014 [94], identiﬁed the

inﬂuence of methyl group in the anticancer activity of naphthoquinone amide derivatives

against HELA with IC₅₀value of 20 µM.

The positive charge of the metal is also known to inﬂuence the cytotoxic activity of

the complexes. The charge increases the acidity of the coordinated ligand that accept

protons [95]. Q6 had low cytotoxicity activity (EC₅₀> 100 µM) towards all the cancer cell

lines which could be attributed to their unusual mode of coordination. In comparison, the

cytotoxic activity of the complexes toward HeLa cell follows the order Q9 > Q7 > Q6.

In MDA-MB231, Q7 and Q9 with EC₅₀of 41.62

±

4.25

µ

M and 38.84

±

4.33 µM,

respectively, showed fair cytotoxicity when compared to cisplatin (EC₅₀= 22.68 ± 4.89 µM),

similarly, Q7 and Q9 had moderate anticancer activity when compared with cisplatin

(

EC₅₀= 19.74 ± 3.77 µM) against SHSY5Y with EC₅₀value of 30.33

3.34 µM respectively.

All the complexes are less toxic toward the normal cell (HEK293), with EC₅₀

and are signiﬁcantly higher than that of cisplatin with EC₅₀values of 14.2 3.8

±

4.67

µ

M and 47.23

±

≥

100

µ

M

±

µM. The SI

data shown in Table 5 demonstrates the differential activity of the complexes and cisplatin,

the greater the SI value is, the more selective it is. An SI value of less than two indicates the

general toxicity of the compound. Based on this, the SI values indicate that complexes Q7

and Q9 displayed excellent selectivity towards all the cancer cell lines, while complexes

Q6 had better selectivity towards all the cancer cell lines better than the Cisplatin standard.

In conclusion, the cytotoxicity activity of the studied complexes is coordination ge-

ometry, structural substituent, and moiety dependent. The introduction of benzothiazole

moiety and methyl substituent on the phenyl ring enhance the anticancer activity espe-

cially towards HELA, as a consequence, complexes Q7 and Q9 are promising anti-cervical

cancer agents. Notably, all the tested complexes are benign to the normal cell and they all

demonstrated a remarkable selectivity for cancer cells over normal cells.

3. Experimental

3.1. Materials and Instrumentation

Ethanol 99.5% (Aldrich, St. Louis, MO, USA), diethyl ether 99.8% (Aldrich, St. Louis,

MO, USA), DMSO–d6 99.8% (Merck, Darmstadt, Germany), 2-quinoline- carboxaldehyde

99% (Aldrich, St. Louis, MO, USA), 2-aminobenzenethiol 99.5% (Aldrich, St. Louis, MO,

USA), 2-thiophenemethylamine > 92% (MerckDarmstadt, Germany), p-toluidine 99.5%

(Aldrich, St. Louis, MO, USA), 2-ﬂuoroaniline 99.5% (Aldrich, St. Louis, MO, USA), 4-

chloroaniline 99.5% (Aldrich, St. Louis, MO, USA), dichloromethane 99% (Aldrich, St.

Louis, MO, USA), calf-thymus DNA (CT-DNA), ethidium bromide 95% (Aldrich, St. Louis,

MO, USA), bovine serum albumin 99% (Aldrich, St. Louis, MO, USA), and nitrogen gas,

5.0 technical grade (Air ﬂex Industrial Gases, Pietermaritzburg, Africa) were purchased

from local suppliers. All chemicals were in analytical grade and used as received, while

most of the solvents were dried using conventional techniques.

¹H NMR and ¹³C NMR spectra were recorded on a BRUKER 400 MHz spectrom-

eter in DMSO–d6 and acetone-d6. Chemical shift values are reported in parts per mil-

lion (ppm) relative to the solvent residual peaks in DMSO-d6 and acetone-d6; 2.5 and

2.05 ppm respectively for ¹H NMR and 39.5 and 29.4 ppm respectively for ¹³C NMR. The

splitting patterns in ¹H NMR spectra are reported as s for singlet, d for doublet, m for mul-

tiplet while J (the coupling constant is given in Hertz). The infrared spectra were recorded

using a PerkinElmer Spectrum 100 FT-IR spectrometer, and the data are reported as per-

centage transmittances at the respective wavenumbers (cm⁻¹), between 4000 and 650 cm⁻¹

.

The mass spectra were recorded using Shimadzu LCMS-2020 instrument with only molec-

ular ions (M⁺) and major fragmentation peaks being reported with intensities quoted as

percentages of the base peak. Elemental analyses were performed on Thermal-Scientiﬁc

Molecules 2021, 26, 1205

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Flash 2000 CHNS/O analyzer. All melting points were determined using the Stuart Scien-

tiﬁc melting point apparatus. Absorption studies of CT-DNA and protein were recorded

using Shimadzu UV-1800 UV-Vis Spectrophotometer. The photoluminescence studies of

CT-DNA and protein were recorded using PerkinElmer LS 45 Fluorescence spectrometer.

3.2. Synthesis of Quinolinyl Schiff Bases L1–L5

L1–L5 were synthesized using a modiﬁed method from the literature [96,97] for similar

ligands. The ligands were synthesized by the addition of a solution of 2-quinolinecarboxalde-

hyde (1 mmol, 0.16 g) in anhydrous ethanol (10 mL) to a solution of 2-ﬂuoroaniline

(1 mmol, 0.10 mL), 2-aminothiophenol (1 mmol, 0.10 mL), 4-chloroaniline (1 mmol, 0.13 g),

p-toluidine (1 mmol, 0.11 g), and 2-thiophenemethylamine (1 mmol, 0.10 mL), respectively,

in anhydrous ethanol (10 mL) in the presence of glacial acetic acid. The resulting reactions

for L1

constant stirring at ambient temperature for 6 h. L2

,

L3, and L4 were reﬂuxed at ca. 80 ^◦C for 4 h while L2 and L5 were subjected to

,

L3, and L4 solutions were evaporated

to one-third of their initial volume under reduced pressure after which 10 mL of ether

was added to each solutions to afford precipitates. The precipitates were isolated via

ﬁltration, washed twice with 10 mL cold ether, recrystallized from ethanol and dried over

anhydrous magnesium sulfate. L1 and L5 on the other hand were evaporated completely

under pressure. The oil obtained was recrystallized from ethanol and dried over anhydrous

magnesium sulfate.

3.3. Single-Crystal X-ray Analysis

Crystal evaluation and data collection of Q1, Q6, Q7, Q12, and Q14 were recorded on

a Bruker Apex Duo diffractometer equipped with an Oxford Instruments Cryojet operating

at 100 (2) K and an Incoatec microsource operating at 30 W power. The data were collected

with Mo Kα (λ = 0.71073 Å) radiation at a crystal-to-detector distance of 50 mm using

omega and phi scans. The data were reduced with the programme SAINT [98] using outlier

rejection, scan speed scaling, as well as standard Lorentz and polarization correction factors.

A SADABS [99] semi-empirical multi-scan absorption correction was applied to the data.

The structures of Q1, Q6, Q7, Q12, and Q14 were solved by the direct method using

the SHELXS [100] program and reﬁned. The visual crystal structure information was

performed using ORTEP-3 [101], system software. Non-hydrogen atoms were ﬁrst reﬁned

isotropically and then by anisotropic reﬁnement with a full-matrix least-squares method

based on F²using SHELXL [102]. All hydrogen atoms were positioned geometrically,

allowed to ride on their parent atoms, and reﬁned isotropically. The crystallographic data

and structure reﬁnement parameters for Q1, Q6, Q7, Q12, and Q14 are given in Table S3

(See Supplementary Materials).

In complex Q7, the perchlorate ion including the bidentate ligand was disordered over

two positions with each component having 50% site occupancy. The bridging ligand was

disordered over a special position with an equal site occupancy factor. PART-1 instruction

was used to model the disorder.

3.4. Synthesis of Q1–Q15

Q1

each of L1

ca. 0.09 g), silver perchlorate (0.5 mmol, ca. 0.10 g), and silver triﬂuoromethanesulfonate

0.5 mmol, ca. 0.13 g), respectively, under constant stirring in the dark. The reactions were

–Q15 were synthesized by dropwise addition of ethanolic solution (ca. 15 mL) of

–

L5 (1 mmol) to an ethanolic (ca. 10 mL) solution of silver nitrate (0.5 mmol,

(

carried out under nitrogen at ambient temperature for 6 h. The resulting precipitates were

isolated using a vacuum ﬁlter. Afterward, the precipitates were washed with cold ethanol

(10 mL

×

2) followed by cold ether (10 mL

×

2) and dried in vacuo. The obtained com-

plexes were recrystallized by dissolving the complexes in dichloromethane and layering

with toluene.

Molecules 2021, 26, 1205

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3.4.1. [Ag(L1)₂]NO₃Q1

◦

1

Yellow solid, 0.50 g, 94%, Melting point; 152–153 C. H-NMR (400 MHz, DMSO-

d6, δ ppm): 8.93 (1H, s, Hg-CH=N-), 8.62 (1H, d, J = 8.69 Hz, He-C₉H₆N), 8.31 (1H, d,

J = 8.55 Hz, Hf-C₉H₆N), 8.18 (1H, d, J = 8.46 Hz, Hd-C₉H₆N), 8.12 (1H, d, J = 8.46 Hz,

Ha-C₉H₆N), 7.89 (1H, m, Hb-C₉H₆N), 7.75 (1H, m, Hc-C₉H₆N), 7.55 (1H, m, Hj-C₆H₄), 7.34

(3H, m, Hk, i, h-C₆H₄), 6.95 (1H, m, Ho-C₆H₄), 6.86 (1H, m, Hm-C₆H₄), 6.75 (1H, m, Hn-

C₆H₄), 6.50 (1H, m, Hl-C₆H₄), 5.04 (2H, s, Hp-NH₂). ¹³C-NMR (400 MHz, DMSO-d6,

δ

ppm

25 ^◦C):

δ

= 162.97 (C10-C=N-), 156.35 (C22-C₆H₄), 153.11 (C16-C₆H₄), 149.43 (C1-C₉H₆N),

146.99 (C9-C₉H₆N), 137.74 (C7-C₉H₆N), 136.36 (C11-C₆H₄), 136.23 (C17-C₆H₄), 130.70

(C6-C₆H₄), 129.36 (C3-C₆H₄), 128.89 (C2-C₆H₄), 128.58 (C14-C₆H₄N), 128.37 (C5-C₉H₆N),

128.18 (C4-C₆H₄N), 125.18 (C13-C₉H₆N), 125.14 (C19-C₆H₄), 124.41 (C12-C₆H₄), 124.38

(C20-C₆H₄), 121.73 (C8-C₉H₆N), 119.60 (C15-C₆H₄), 116.45 (C18-C₆H₄), 116.17 (C21-C₆H₄).

FT-IR (cm⁻¹): (-C=N-) 1612, (C=N quinolinyl) 1588, (C-F) 1500. UV/Vis (CH₃CN): λ_max

234, 276, 326 nm. MS: m/z Calcd. For [C₂₂H₁₆AgF₂N₃]: 468; found [Ag(L1)–2F + Na +

CH₃CN]⁺: 495 (79%), 496 (26%), [Ag(L1) + 2EtOH]⁺: 583 (53%), 584 (18%). Anal. Calcd. (%)

for [C₂₂H₁₇AgF₂N₄O₃]: C, 49.74; H, 3.23; N, 10.55; found (%): C, 49.67; H, 3.14; N, 10.38.

3.4.2. [Ag(L2)₂]NO₃Q2

Brown solid, 0.39 g, 56%, Melting point; 266–267 C. ¹H-NMR (400 MHz, (DMSO,

◦

δ ppm): 8.62 (2H, d, J = 8.58 Hz, Hg-C₆H₄-), 8.46 (2H, d, J = 8.53 Hz, He-C₉H₆N-), 8.17 (8H,

Hj-C₆H₄, Hd-C₉H₆N, Ha-C₉H₆N, Hf-C₉H₆N), 7.88 (2H, t, J = 7.68 Hz, Hb-C₉H₆N), 7.72

(2H, d, J = 7.50 Hz, Hc-C₉H₆N), 7.58 (4H, m, Hh-C₆H₄, Hi-C₆H₄). ¹³C-NMR (400 MHz,

(DMSO, 25 ^◦C):

δ = 168.94 (C9-C₉H₆N), 153.60 (C16-C₆H₄), 150.38 (C10-C=N-), 147.04 (C1-

C₉H₆N), 138.04 (C7-C₉H₆N), 135.61 (C11-C₆H₄), 130.83 (C3-C₉H₆N), 128.94 (C2-C₉H₆N),

128.77 (C5-C₉H₆N), 128.23 (C4-C₉H₆N), 128.03 (C6-C₉H₆N), 126.76 (C14-C₆H₄), 126.35

(C13-C₉H₆N), 123.53 (C12-C₆H₄), 122.65 (C15-C₆H₄), 118.12 (C8-C₉H₆N). FT-IR (cm⁻¹):

(Ar-CH) 3051, (C-S-C) 750, (quinolinyl) 1588. UV/Vis (CH₃CN): λ_max217, 284, 318, 335,

350 nm. MS: m/z Calcd. For [C₃₂H₂₀AgN₄S₂]: 632,53; found [Ag(L2)₂+ H)]⁺: 633 (100%),

631 (90%), 634 (38 %). Anal. Calcd. (%) for [C₃₂H₂₀AgN₅O₃S₂]: C, 55.34; H, 2.90; N, 10.08;

found (%): C, 55.23; H, 2.85; N, 9.92.

3.4.3. [Ag(L3)₂]NO₃Q3

Brown solid, 0.67 g, 96%, Melting point; 182–183 ^◦C. ¹H-NMR (400 MHz, (CD₃)₂CO,

δ ppm): 9.08 (2H, s, Hg-CH=N-), 8.72 (2H, d, J = 8.47 Hz, He-C₉H₆N), 8.27 (2H, d,

J = 8.46 Hz, Hf-C₉H₆N), 8.17 (4H, m, Hd, a-C₉H₆N), 7.90 (2H, m, Hb-C₉H₆N), 7.77 (2H,

◦

m, Hc-C₉H₆N), 7.56 (8H, m, Hi, h-C₆H₄). ¹³C-NMR (400 MHz, (CD₃)₂CO, 25 C): δ = 152.20

(C10-C=N-), 148.00 (C1-C₆H₄), 146.81 (C11-C₉H₆N), 138.90 (C9-C₉H₆N), 132.52 (C7-C₉H₆N),

131.52 (C14-C₉H₆N), 131.56 (C6-C₆H₄), 129.79 (C13, C15-C₉H₆N), 129.75 (C3-C₆H₄), 129.68

(C2-C₆H₄), 128.94 (C5-C₆H₄), 128.61 (C4-C₆H₄), 124.16 (C12 & C16-C₉H₆N), 122.27 (C8-

C₉H₆N). FT-IR (cm⁻¹): (Ar-CH) 3063, (-C=N-) 1613, (C=N quinolinyl) 1584. UV/Vis

(CH₃CN): λ_max212, 257, 313, 331 nm. MS: m/z Calcd. for [C₃₂H₂₂AgCl₂N₄]: 641.32; found

[Ag(L3)₂]⁺: 641 (100%), 639 (54%), 643 (48%), 642 (31%), [Ag(L3) + H + CH₃CN + Na]⁺:

597 (56%), 595 (23%). Anal. Calcd. (%) for [C₃₂H₂₂AgCl₂N₅O₃]: C, 54.65; H, 3.15; N, 9.96;

found (%): C, 54.32; H, 3.15; N, 9.93.

3.4.4. [Ag(L4)₂]NO₃Q4

Yellow solid, 0.47 g, 72%, Melting point; 141–142 ^◦C. ¹H-NMR (400 MHz, DMSO-d6,

δ ppm): 9.21 (2H, s, Hg-CH=N-), 8.76 (2H, d, J = 8.26 Hz, He-C₉H₆N), 8.28 (2H, d, J =

8.46 Hz, Hf-C₉H₆N), 8.14 (4H, m, Hd, a-C₉H₆N), 7.86 (2H, m, Hb-C₉H₆N), 7.75 (2H, m,

Hc-C₉H₆N), 7.52 (4H, m, Hi-C₆H₄), 7.29 (4H, d, Hh-C₆H₄), 2.33 (6H, m, Hj-CH₃). ¹³C-NMR

◦

(400 MHz, DMSO-d6, 25 C):

δ = 159.06 (C10-C₉H₆N), 151.67 (C1-C₆H₄), 146.26 (C11-

C₉H₆N), 145.71 (C9-C₉H₆N), 138.88 (C15-C₉H₆N), 138.16 (C7-C=N-), 131.43 (C6-C₆H₄),

130.02 (C13 & C16-C₉H₆N), 129.35 (C3-C₆H₄), 129.19 (C2-C₆H₄), 128.58 (C5-C₆H₄), 128.33

(C4-C₆H₄), 122.75 (C12 & C17-C₉H₆N), 122.25 (C8-C₉H₆N), 20.64 (C14-C₉H₆N). FT-IR

Molecules 2021, 26, 1205

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(cm⁻¹): (Ar-CH) 3050, (-C=N-) 1684, (quinolinyl) 1586. UV/Vis (CH₃CN): λ_max243, 302,

339 nm. MS: m/z Calcd. For [C₃₄H₂₈AgN₄]: 600.49; found [Ag(L4)₂+ H)]⁺: 601 (100%), 599

(90%), 602 (33%), 600 (23%). Anal. Calcd. (%) for [C₃₄H₂₈AgN₅O₃]: C, 61.64; H, 4.26; N,

10.57; found (%): C, 61.54; H, 4.04; N, 10.38.

3.4.5. [Ag(L5)₂]NO₃Q5

Brown solid, 0.54 g, 78%, Melting point; 134–135 ^◦C, ¹H-NMR (400 MHz, DMSO-d6,

δ ppm): 9.03 (2H, t, J = 1.48 Hz, 1.51 Hz, Hg-CH=N-), 8.79 (2H, d, J = 8.22 Hz, He-C₉H₆N),

8.16 (2H, d, J = 7.36 Hz, Hd-C₉H₆N), 8.09 (2H, d, J = 8.41 Hz, Hf-C₉H₆N), 8.01 (2H, d,

J = 8.32 Hz, Ha-C₉H₆N), 7.87 (2H, m, Hb-C₉H₆N), 7.76 (2H, m, Hc-C₉H₆N), 7.45 (2H, m,

Hk-C₄H₃S), 7.06 (2H, m, Hi-C₄H₃S), 6.97 (2H, m, Hj-C₄H₃S), 5.20 (4H, s, Hh-CH₂). ¹³C-

NMR (400 MHz, DMSO-d6, 25 ^◦C):

δ = 162.60 (C10-C=N-), 149.61 (C9-C₉H₆N), 145.82 (C1-

C₉H₆N), 140.21 (C15-C₄H₃S), 139.53 (C7-C₉H₆N), 131.67 (C6-C₉H₆N), 129.40 (C3-C₉H₆N),

128.78 (C2-C₉H₆N), 128.32 (C5-C₉H₆N), 127.42 (C4-C₉H₆N), 127.33 (C13-C₄H₃S), 126.96

(C12-C₄H₃S), 126.24 (C14-C₄H₃S), 123.71 (C8-C₉H₆N), 57.40 (C11-CH₂). FT-IR (cm⁻¹):

(Ar-CH) 3059, (-C=N-) 1645, (C=N quinolinyl) 1589. UV/Vis (CH₃CN): λ_max288, 320sh,

334sh nm. MS: m/z Calcd. for [C₃₀H₂₄AgN₄S₂]: 612.54; found [Ag(L5)₂+ H]⁺: 613 (100%),

611 (85%), 614 (29%), 612 (18%), 615 (10%), [Ag(L5) + CH₃CN]⁺: 400 (30%), 402 (29%). Anal.

Calcd. (%) for [C₃₀H₂₄AgN₅O₃S₂]: C, 53.42; H, 3.59; N, 10.38; found (%): C, 53.14; H, 3.29;

N, 10.19.

3.4.6. [Ag(L1)₂]ClO₄Q6

◦

1

Brown solid, 0.56 g, 99%, Melting point; 198–199 C. H-NMR (400 MHz, DMSO-

d6, δ ppm): 8.98 (1H, s, Hg-CH=N-), 8.66 (1H, d, J = 8.46 Hz, He-C₉H₆N), 8.30 (1H, d,

J = 8.46 Hz, Hf-C₉H₆N), 8.18 (1H, d, J = 8.26 Hz, Hd-C₉H₆N), 8.13 (1H, d, J = 8.04 Hz,

Ha-C₉H₆N), 7.89 (1H, m, Hb-C₉H₆N), 7.76 (1H, m, Hc-C₉H₆N), 7.57 (1H, m, Hj-C₆H₄), 7.34

(3H, m, Hk, i, h-C₆H₄), 6.95 (1H, m, Ho-C₆H₄), 6.86 (1H, m, Hm-C₆H₄), 6.75 (1H, m, Hn-

C₆H₄), 6.50 (1H, m, Hl-C₆H₄), 5.04 (2H, s, Hp-NH₂). ¹³C-NMR (400 MHz, DMSO-d6,

δ

ppm

25 ^◦C):

δ

= 162.73 (C10-C=N-), 155.88 (C22-C₆H₄), 153.90 (C16-C₆H₄), 150.85 (C1-C₉H₆N),

150.28 (C9-C₉H₆N), 138.64 (C7-C₉H₆N), 137.72 (C11-C₆H₄), 137.46 (C17-C₆H₄), 128.71

(C6-C₆H₄), 128.64 (C3-C₆H₄), 128.42 (C2-C₆H₄), 127.50 (C14-C₆H₄N), 127.31 (C5-C₉H₆N),

126.38 (C4-C₆H₄N), 125.12 (C13-C₉H₆N), 125.09 (C19-C₆H₄), 124.41 (C12-C₆H₄), 124.38

(C20-C₆H₄), 122.16 (C8- C₉H₆N), 121.83 (C15-C₆H₄), 116.41 (C18-C₆H₄), 116.25 (C21-C₆H₄).

FT-IR (cm⁻¹): (Ar-CH) 3070, (-C=N-) 1624, (pyridyl) 1585, (C-F) 1490. UV/Vis (CH₃CN):

λ_max234, 276, 327 nm. MS: m/z Calcd. [C₂₂H₁₆AgF₂N₃]: 468; found [Ag(L1) + K]⁺: 507

(57%), 508 (54%), [Ag(L1)-F]⁺: 463 (37%). Anal. Calcd. (%) for [C₂₂H₁₇AgClF₂N₃O₄]: C,

46.46; H, 3.01; N, 7.39; found (%): C, 46.32; H, 2.89; N, 7.14.

3.4.7. [Ag(L2)₂]ClO₄Q7

Light brown solid, 0.46 g, 63%, Melting point; 242–243 C, ¹H-NMR (400 MHz, (DMSO-

◦

d6, δ ppm): 8.62 (2H, d, J = 8.54 Hz, Hg-C₆H₄-), 8.47 (2H, d, J = 8.54 Hz, He-C₉H₆N-),

8.17 (8H, Hj-C₆H₄, Hd-C₉H₆N, Ha-C₉H₆N, Hf-C₉H₆N), 7.89 (2H, t, J = 7.67 Hz, Hb-

C₉H₆N), 7.73 (2H, d, J = 7.50 Hz, Hc-C₉H₆N), 7.57 (4H, m, Hh-C₆H₄, Hi-C₆H₄). ¹³C-NMR

◦

(

400 MHz, (DMSO-d6, 25 C):

δ

= 169.27 (C9-C₉H₆N), 153.93 (C11-C₆H₄), 150.73 (C10-

C=N-), 147.37 (C1-C₉H₆N), 138.33 (C7-C₉H₆N), 135.94 (C16-C₆H₄), 131.14 (C3- C₉H₆N),

129.25 (C2-C₉H₆N), 129.08 (C5-C₉H₆N), 128.54 (C4-C₉H₆N), 128.34 (C6-C₉H₆N), 127.06

(C13-C₆H₄), 126.66 (C14-C₆H₄), 123.85 (C15-C₆H₄), 122.95 (C12-C₆H₄), 118.41 (C8-C₉H₆N).

FT-IR (cm⁻¹): (Ar-CH) 3062, (C-S-C) 751, (quinolinyl) 1586. UV/Vis (CH₃CN): λ_max217,

289, 318, 335, 350 nm. MS: m/z Calcd. For [C₃₂H₂₀AgN₄S₂]: 632,53; found [Ag (L2)₂+ H)]:

633 (100%), 631 (81%), 634 (30%), [Ag(L2) + H + CH₃CN)]⁺: 412 (38%), 410 (35%). Anal.

Calcd. (%) for [C₄₈H₃₀Ag₂ClN₆O₄S₃]: C, 52.31; H, 2.74; N, 7.63; found (%): C, 52.16; H,

2.64; N, 7.60.

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3.4.8. [Ag(L3)₂]ClO₄Q8

Yellow solid, 0.66 g, 89%, Melting point; 190–191 ^◦C. ¹H-NMR (400 MHz, (CD₃)₂CO,

δ ppm): 9.19 (2H, s, Hg-CH=N-), 8.80 (2H, d, J = 8.30 Hz, He-C₉H₆N), 8.25 (4H, q,

J = 8.48 Hz, J = 8.41 Hz, Hd, f-C₉H₆N), 8.18 (2H, d, J = 7.27 Hz, Ha-C₉H₆N), 7.94 (2H, m,

Hb-C₉H₆N), 7.80 (2H, m, Hc-C₉H₆N), 7.67 (4H, m, Hi-C₆H₄), 7.60 (4H, m, Hh-C₆H₄). ¹³C-

NMR (400 MHz, (CD₃)₂CO, 25 ^◦C):

δ = 151.31 (C10- C=N-), 147.64 (C1-C₆H₄), 146.62 (C11-

C₉H₆N), 139.73 (C9-C₉H₆N), 132.16 (C7-C₉H₆N), 131.40 (C14-C₉H₆N), 130.25 (C6-C₆H₄),

130.18 (C13 & C15-C₉H₆N), 130.01 (C3-C₆H₄), 128.96 (C2-C₆H₄), 128.88 (C5 & C8-C₆H₄),

124.69 (C4-C₆H₄), 124.13 (C12 & C16-C₉H₆N). FT-IR (cm⁻¹): (Ar-CH) 3065, (quinolinyl)

1592. UV/Vis (CH₃CN): λ_max242, 320 nm. MS: m/z Calcd. for [C₃₂H₂₂AgCl₂N₄]: 641.32;

found [Ag(L3)₂]⁺: 641 (81%), 639 (41%), 643 (39%), [Ag(L3) + H + CH₃CN]⁺: 416 (56%),

414 (44%), 418 (15%). Anal. Calcd. (%) for [C₃₂H₂₂AgCl₃N₄O₄]: C, 51.89; H, 2.99; N, 7.56;

found (%): C, 51.69; H, 2.85; N, 7.31.

3.4.9. [Ag(L4)₂]ClO₄Q9

◦

1

Brown solid, 0.55 g, 80%, Melting point: 149–150 C. H-NMR (400 MHz, DMSO-

d6, δ ppm): 9.20 (2H, s, Hg-CH=N-), 8.76 (2H, d, J = 8.37 Hz, He-C₉H₆N), 8.27 (2H, d,

J = 8.50 Hz, Hf-C₉H₆N), 8.15 (4H, m, Hd, a-C₉H₆N), 7.87 (2H, m, Hb-C₉H₆N), 7.76 (2H,

m, Hc-C₉H₆N), 7.52 (4H, m, Hi-C₆H₄), 7.29 (4H, d, J = 7.95 Hz, Hh-C₆H₄), 2.34 (6H, m,

Hj-CH₃). ¹³C-NMR (400 MHz, DMSO-d6, 25 C):

δ = 159.03 (C10-C₉H₆N), 151.62 (C1-

◦

C₆H₄), 146.25 (C11-C₉H₆N), 145.71 (C9-C₉H₆N), 138.89 (C15-C₉H₆N), 138.16 (C7-C=N-),

131.43 (C6-C₆H₄), 130.02 (C13 & C16-C₉H₆N), 129.37 (C3-C₆H₄), 129.18 (C2-C₆H₄), 128.58

(C5-C₆H₄), 128.33 (C4-C₆H₄), 122.79 (C12 & C17-C₉H₆N), 122.26 (C8-C₉H₆N), 20.64 (C14-

C₉H₆N). FT-IR (cm⁻¹): (Ar-CH) 3043, (-C=N-) 1682, (quinolinyl) 1587. UV/Vis (CH₃CN):

λ_max242, 301, 339 nm. MS: m/z Calcd. For [C₃₄H₂₈AgN₄]: 600.49; found [Ag (L4)₂+ H)]:

601 (100%), 599 (90%), 602 (29%), 600 (19%) [Ag(L4) + H + CH₃CN)]⁺: 396 (26%), 394 (21%).

Anal. Calcd. (%) for [C₃₄H₂₈AgClN₄O₄]: C, 58.34; H, 4.03; N, 8.00; found (%): C, 58.11; H,

3.98; N, 7.92.

3.4.10. [Ag(L5)₂]ClO₄Q10

Orange solid, 0.71 g, 100%, Melting point; 132–133 C. ¹H-NMR (400 MHz, (CD₃)₂CO,

◦

δ ppm): 9.02 (2H, s, Hg-CH=N-), 8.76 (2H, d, J = 8.41 Hz, He-C₉H₆N), 8.12 (4H, d,

J = 8.48 Hz, Hf, d-C₉H₆N), 7.71 (6H, m, Ha, b, c-C₉H₆N), 7.23 (2H, m, Hk-C₄H₃S), 6.98

(2H, d, J = 3.43 Hz, Hi-C₄H₃S), 6.77 (2H, m, Hj-C₄H₃S), 5.13 (4H, s, Hh-CH₂). ¹³C-NMR

◦

(400 MHz, DMSO-d6, 25 C):

δ = 162.47 (C10-C=N-), 150.07 (C9-C₉H₆N), 146.13 (C1-

C₉H₆N), 140.31 (C15-C₄H₃S), 136.65 (C7-C₉H₆N-), 132.09 (C6-C₉H₆N), 130.19 (C3-C₉H₆N),

129.24 (C2-C₉H₆N), 128.70 (C5-C₉H₆N), 127.92 (C4-C₉H₆N), 127.47 (C13-C₄H₃S), 127.35

(C12-C₄H₃S), 126.26 (C14-C₄H₃S), 124.00 (C8-C₉H₆N), 57.70 (C11-CH₂). FT-IR (cm⁻¹):

(Ar-CH) 3063, (-C=N-) 1644, (quinolinyl) 1589. UV/Vis (CH₃CN): λ_max287, 320sh, 334sh

nm. MS: m/z Calcd. for [C₃₀H₂₄AgN₄S₂]: 612.54; found [Ag(L5)₂+ H]⁺: 613 (100%), 611

(79%), 614 (30%), 612 (15%), 615 (11%). Anal. Calcd. (%) for [C₃₀H₂₄AgClN₄O₄S₂]: C, 50.61;

H, 3.40; N, 7.87; found (%): C, 50.32; H, 3.40; N, 7.83.

3.4.11. [Ag(L1)₂]CF₃SO₃Q11

◦

1

Brown solid, 0.60 g, 97%, Melting point; 154–155 C, H-NMR (400 MHz, DMSO-

d6, δ ppm): 9.17 (1H, s, Hg-CH=N-), 8.78 (1H, d, J = 8.36 Hz, He-C₉H₆N), 8.30 (1H, d,

J = 8.45 Hz, Hf-C₉H₆N), 8.17 (2H, m, Ha, d-C₉H₆N), 7.88 (1H, m, Hb-C₉H₆N), 7.77 (1H, m,

Hc-C₆H₄), 7.62 (1H, m, Hj-C₉H₆N), 7.34 (3H, m, Hk, i, h-C₆H₄), 6.95 (1H, m, Ho-C₆H₄), 6.86

(1H, m, Hm-C₆H₄), 6.76 (1H, m, Hn-C₆H₄), 6.50 (1H, m, Hl-C₆H₄). ¹³C-NMR (400 MHz,

DMSO-d6,

δ δ = 162.83 (C10-C=N-), 156.48 (C22-C₆H₄), 154.00 (C16-C₆H₄),

ppm 25 ^◦C):

151.17 (C1-C₉H₆N), 146.26 (C9-C₉H₆N), 139.02 (C7-C₉H₆N), 137.11 (C11-C₆H₄), 137.02

(C17-C₆H₄), 131.51 (C6-C₆H₄), 129.48 (C3-C₆H₄), 129.33 (C2-C₆H₄), 129.25 (C14-C₆H₄N),

128.84 (C5-C₉H₆N), 128.34 (C4-C₆H₄N), 125.24 (C13-C₉H₆N), 125.21 (C19-C₆H₄), 124.42

(C12-C₆H₄), 124.39 (C20-C₆H₄), 122.81 (C8-C₉H₆N), 122.11 (C15-C₆H₄), 116.57 (C18-C₆H₄),

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116.37 (C21-C₆H₄). FT-IR (cm⁻¹): (Ar-CH) 3061, (-C=N-) 1624, (C=N quinolinyl) 1585.

UV/Vis (CH₃CN): λ_max234, 276, 327 nm. MS: m/z Calcd. for [C₂₂H₁₆AgF₂N₃]: 468; found

[Ag(L1) + CH₃CN]⁺: 509 (98%), 507 (87%). Anal. Calcd. (%) for [C₂₃H₁₇AgF₅N₃O₃S]: C,

44.68; H, 2.77; N, 6.80; found (%): C, 44.62; H, 2.51; N, 6.73.

3.4.12. [Ag(L2)₂]CF₃SO₃Q12

Yellow solid, 0.68 g, 87%, Melting point; 245–246 ^◦C. ¹H-NMR (400 MHz, (CD₃)₂CO,

δ ppm): 8.62 (2H, d, J = 8.11 Hz, Hg-C₆H₄-), 8.48 (2H, d, J = 8.54 Hz, He-C₉H₆N-), 8.16

(8H, Hj-C₆H₄, Hd-C₉H₆N, Ha- C₉H₆N, Hf-C₉H₆N), 7.89 (2H, m, Hb-C₉H₆N), 7.72 (2H,

m, Hc-C₉H₆N), 7.58 (4H, m, Hh-C₆H₄, Hi-C₆H₄). ¹³C-NMR (400 MHz, (CD₃)₂CO, 25 ^◦C):

δ = 150.84 (C9-C₉H₆N), 138.27 (C11-C₆H₄), 135.97 (C10-C=N-), 131.10 (C1-C₉H₆N), 129.22

(C7-C₉H₆N), 129.06 (C16-C₆H₄), 128.54 (C3-C₉H₆N), 128.32 (C2 & C5-C₉H₆N), 127.03

(C4-C₉H₆N), 126.63 (C6 & C13-C₉H₆N), 123.85 (C14-C₆H₄), 122.94 (C12 & C15-C₆H₄),

118.30 (C8- C₉H₆N). FT-IR (cm⁻¹): (Ar-CH) 3059, (C-S-C) 754, (quinolinyl) 1588. UV/Vis

(CH₃CN): λ_max217, 285, 320, 335, 350 nm. MS: m/z Calcd. For [C₃₂H₂₀AgN₄S₂]: 632,53;

found [Ag (L2)₂+ H)]⁺: 633 (100%), 631 (81%), 634 (30%), [Ag(L2) + H + CH₃CN)]⁺: 412

(38%), 410 (35%). Anal. Calcd. (%) for [C₃₄H₂₀Ag₂F₆N₄O₆S₄]: C, 39.32; H, 1.94; N, 5.39;

found (%): C, 39.15; H, 1.78; N, 5.27.

3.4.13. [Ag(L3)₂]CF₃SO₃Q13

Brown solid, 0.40 g, 50%, Melting point; 193–194 ^◦C. ¹H-NMR (400 MHz, (CD₃)₂CO,

δ ppm): 9.16 (2H, s, Hg-CH=N-), 8.77 (2H, d, J = 8.30 Hz, He-C₉H₆N), 8.24 (4H, q,

J = 8.48 Hz, J = 8.41 Hz, Hd, f-C₉H₆N), 8.17 (2H, d, J = 7.27 Hz, Ha-C₉H₆N), 7.92 (2H,

m, Hb-C₉H₆N), 7.78 (2H, m, Hc-C₉H₆N), 7.63 (4H, m, Hi-C₆H₄), 7.58 (4H, m, Hh-C₆H₄).

¹³C-NMR (400 MHz, (CD₃)₂CO, 25 ^◦C):

δ = 151.20 (C10-C=N-), 147.32 (C1-C₆H₄), 146.24

(C11-C₉H₆N), 138.99 (C9-C₉H₆N), 132.44 (C7-C₉H₆N), 131.50 (C14-C₉H₆N), 129.59 (C6-

C₆H₄), 129.58 (C13 & C15-C₉H₆N), 129.47 (C3-C₆H₄), 128.79 (C2-C₆H₄), 128.34 (C5-C₆H₄),

124.05 (C4-C₆H₄), 123.00 (C12 & C16-C₉H₆N), 121.94 (C8-C₉H₆N). UV/Vis (CH₃CN): λ_max

244, 307, 335 nm. MS: m/z Calcd. for [C₃₂H₂₂AgCl₂N₄]: 641.32; found [Ag(L3)₂]⁺: 641

(100%), 639 (65%), 643 (52%), 642 (25%), [Ag(L3) + H + CH₃CN ]⁺: 416 (37%), 414 (29%).

Anal. Calcd. (%) for [C₃₃H₂₂AgCl₂F₃N₄O₃S]: C, 50.15; H, 2.81; N, 7.09; found (%): C, 50.15;

H, 2.70; N, 6.98. FT-IR (cm⁻¹): (Ar-CH) 3063, (quinolinyl) 1591.

3.4.14. [Ag(L4)₂]CF₃SO₃Q14

◦

1

Brown solid, 0.60 g, 80%, Melting point: 152–153 C. H-NMR (400 MHz, DMSO-

d6, δ ppm): 9.04 (2H, s, Hg-CH=N-), 8.66 (2H, d, J = 8.58 Hz, He-C₉H₆N), 8.28 (2H, d,

J = 8.50 Hz, Hf-C₉H₆N), 8.14 (4H, m, Hd, a-C₉H₆N), 7.89 (2H, m, Hb-C₉H₆N), 7.75 (2H,

m, Hc-C₉H₆N), 7.47 (4H, m, Hi-C₆H₄), 7.31 (4H, d, J = 7.92 Hz, Hh-C₆H₄), 2.36 (6H, m,

Hi-CH₃). ¹³C-NMR (400 MHz, DMSO-d6, 25 C):

δ = 159.01 (C10-C₉H₆N), 151.62 (C1-

◦

C₆H₄), 146.21 (C11-C₉H₆N), 145.66 (C9-C₉H₆N), 138.83 (C15-C₉H₆N), 138.11 (C7-C=N-),

129.97 (C6-C₆H₄), 129.43 (C13 & C16-C₉H₆N), 129.30 (C3-C₆H₄), 129.14 (C2-C₆H₄), 128.53

(C5-C₆H₄), 128.28 (C4-C₆H₄), 122.70 (C12 & C17-C₉H₆N), 122.20 (C8-C₉H₆N), 20.59 (C14-

C₉H₆N). UV/Vis (CH₃CN): λ_max245, 299, 340 nm. FT-IR (cm⁻¹): (Ar-CH) 3067, (-C=N-)

1626, (quinolinyl) 1584. MS: m/z Calcd. For [C₃₄H₂₈AgN₄]: 600.49; found [Ag (L4)₂+ H)]⁺:

601 (100%), 599 (99%), 602 (34%), 600 (22%), 603 (6%), [Ag(L4) + H + CH₃CN)]⁺: 396 (38%),

394 (34%). Anal. Calcd. (%) for [C₃₅H₂₈AgF₃N₄O₃S]: C, 56.08; H, 3.77; N, 7.47; found (%):

C, 55.96; H, 3.67; N, 7.23.

3.4.15. [Ag(L5)₂]CF₃SO₃Q15

◦

1

Brown solid, 0.66 g, 87%, Melting point; 148–149 C. H-NMR (400 MHz, DMSO-

d6, δ ppm): 8.84 (2H, s, Hg-CH=N-), 8.62 (2H, d, J = 8.62 Hz, He-C₉H₆N), 8.10 (4H, t,

J = 7.22 Hz, Hf, d-C₉H₆N), 7.99 (2H, d, J = 8.29 Hz, Ha-C₉H₆N), 7.82 (2H, m, Hb-C₉H₆N),

7.71 (2H, t, J = 7.49 Hz, Hc-C₉H₆N), 7.42 (2H, m, Hk-C₄H₃S), 7.09 (2H, m, Hi-C₄H₃S),

6.97 (2H, m, Hj-C₄H₃S), 5.15 (2H, s, Hl-CH₂). ¹³C-NMR (400 MHz, DMSO-d6, 25 C):

◦

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δ = 162.40 (C10-C=N-), 150.00 (C9-C₉H₆N), 146.06 (C1-C₉H₆N), 140.24 (C15-C₄H₃S), 136.58

(C7-C₉H₆N-), 132.03 (C6-C₉H₆N), 130.12 (C3-C₉H₆N), 129.47 (C2-C₉H₆N), 128.63 (C5-

C₉H₆N), 127.85 (C4-C₉H₆N), 127.40 (C13-C₄H₃S), 127.28 (C12-C₄H₃S), 126.19 (C14-C₄H₃S),

123.93 (C8-C₉H₆N), 57.63 (C11-CH₂). FT-IR (cm⁻¹): (Ar-CH) 3068, (-C=N-) 1646, (C=N

quinolinyl) 1591. UV/Vis (CH₃CN): λ_max291, 316 sh, 330 sh nm. MS: m/z Calcd. for

[C₃₀H₂₄AgN₄S₂]⁺: 612.54; found [Ag(L5)₂+ H]⁺: 613 (100%), 611 (85%), 614 (29%), 612

(18%), 615 (10%), [Ag(L5) + CH₃CN]⁺: 400 (30%), 402 (29%). Anal. Calcd. (%) for

[C₃₁H₂₄AgF₃N₄O₃S₃]: C, 48.89; H, 3.18; N, 7.36; found (%): C, 48.65; H, 3.11; N, 7.24.

3.5. In Vitro Antimicrobial Studies

3.5.1. Müeller–Hinton Agar Test Plates Preparation

Sterilized Nutrient agar medium was ﬁrst prepared by dissolving 38 g of Müeller–

Hinton agar (MHA) (Biolab, Midrand, Africa) in distilled water (1 L). The resulting Nutrient

agar medium was subjected to sterilization by autoclaving for 15 min at 121 ^◦C and then

◦

cooled down to 45 C in a water bath. The cooled Agar medium was poured in Petri

dishes while ensuring a uniform 4 mm depth of the medium and cooling further to ambient

temperature [25].

3.5.2. Inoculation Procedure

Complexes Q1–Q15 and their ligands were tested against four Gram-negative bacteria:

Salmonella typhimurium ATCC 14026, Pseudomonas aeruginosa ATCC 27853, Escherichia coli

ATCC 25922, and Klebsiella pneumoniae ATCC 31488; and two Gram-positive bacteria, viz.,

Staphylococcus aureus ATCC 700699 (methicillin-resistant) and Staphylococcus aureus ATCC

25923. These bacteria were inoculated in a sterilized Nutrient Broth (NB) (Biolab, Midrand,

Africa) through streak plate technique and incubated at 37 ^◦C for 18 h. The nutrient broth

was sterilized by dissolving 1.3 g in distilled water (100 mL). Ca. 10 mL of the nutrient broth

was transferred into a cotton wool-plugged test tubes and wrapped with an aluminium foil.

◦

The nutrient broth in the test tubes was then autoclaved for 15 min at 121 C and cooled to

◦

37 C. Following this was the isolation of a single colony of the bacteria and inoculated into

◦

a 10 mL sterile NB which was then incubated in a shaking incubator at 37 C for 18 h. Each

of the bacteria strain concentrations was adjusted with sterile distilled water to get a ﬁnal

concentration of 1.5

×

10⁸cfu/mL, i.e., 0.5 Mc Farland’s Standard using a densitometer

(Mc Farland Latvia) [62]. The bacteria were lawn inoculated on the set MHA Petri dishes

using a sterile cotton swab. Screening of L1–L5 and Q1–Q15 for antibacterial activity was

ﬁrst carried out by spotting 5 µL of their solution (prepared from the dissolution of 1000 µg

of the L1–L5 and Q1–Q15 in 1 mL dimethyl sulfoxide (DMSO)) on an MHA petri dish and

incubated at 37 ^◦C for 18 h. The antibacterial activity was determined by looking out for

a clear zone at the spotting point. The compounds with potential antimicrobial activity

were then tested for their minimum inhibitory concentration (MIC) against six bacterial.

Ten serial dilution of L1–L5 and Q1–Q15 was done to obtain 1000

concentrations, where the compounds showed lower MICs than 0.2

was further diluted 5 times to obtain 0.100 g/mL to 0.00625 g/mL concentrations.

Evaluation of the compounds MIC was determined by spotting 5 L of each concentration

of L1–L5 and Q1–Q15 on the MHA plates, and the plates were incubated at 37 ^◦C for 18 h

µg/mL to 0.2

µg/mL

µg/mL, the solution

µ

.

These procedures were done in triplicate to give the accurate lowest concentration of the

compounds where no visible bacterial growth was seen after incubation. Ciproﬂoxacin

was used as a standard and DMSO used as a negative control showed no bactericidal effect

against all the bacteria strains [50,62,103].

3.6. Antioxidant Assay

Antioxidant activity studies of L1–L5 and Q1–Q15 were done using ferric reducing

antioxidant power (FRAP) assay. The ferric reducing antioxidant power assay is often

used to evaluate the ability of an antioxidant to donate an electron. The reducing ability

of the compounds was measured using a previously published method [104] after slight

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modiﬁcations where different concentrations (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, and

2 mg/mL) of L1–L5 and Q1–Q15 and standard (Ascorbic acid) were mixed with 2.5 mL

of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 1% w/v potassium ferricyanide

respectively. The resulting mixture was incubated at 50 ^◦C for 20 min, and 2.5 mL of 10%

trichloroacetic acid was added to acidify the mixture. After that, 1 mL of the acidiﬁed

mixture was added to 1 mL of distilled water and 0.5 mL of 0.1% FeCl₃. The absorbance

of the resulting solution was then measured at 700 nm. The antioxidant power of the

compounds was expressed as a percentage of ferric reducing antioxidant power Ascorbic

acid equivalent, as shown in Equation (4).

Absorbance o f Sample

Absorbance o f Ascorbic Acid

% FRAP = Absorbance o f

× 100

(4)

3.7. DNA Binding Experiments

The interaction of the L1–L5 and complexes Q1–Q15 with calf thymus-DNA was

carried out in a Phosphate buffer saline solution pH 7.2 stored at 4 ^◦C. The DNA stock

solution was prepared by dissolving CT-DNA sodium salt in Phosphate buffer saline

solution with continuous stirring overnight. It was ﬁltered and stored at 4 ^◦C and used

within four days. The ﬁnal concentration of CT-DNA sodium salt was determined by

UV-visible absorption using the extinction coefﬁcient ε [105]. The

260 = 6600 M⁻¹cm⁻¹

ratio of CT-DNA at 260 and 280 nm UV absorbance was 1.8–1.93:1, indicating that it is pure

and sufﬁciently free of protein contamination. A ﬁxed amount of the L1–L5 and complexes

Q1–Q15 (50

µM) were prepared by dissolving an appropriate amount in DMSO. At a

low DMSO concentration, the stability of biomolecules is known to be retained [106

,

107].

Therefore, in all the experimental of the interaction of L1–L5 and complexes Q1–Q15 with

biomolecules, the ﬁnal concentration of DMSO in the incubation mixture was ~2% (v/v).

Thus, the effect of DMSO on the stability of the biomolecules are negligible.

3.7.1. DNA Absorption Spectral Study

The binding modes of L1–L5 and complexes Q1–Q15 to CT-DNA were characterized

through electronic absorption titration by varying concentrations of CT-DNA (0–30

µM)

in phosphate buffer saline solution against ﬁxed concentrations (20 M) of L1–L5 and

µ

complexes Q1–Q15. The compounds-CT-DNA mixture was incubated for 10 min before

measuring the absorbance using UV-vis absorption spectroscopy [108]. The absorbance

of CT-DNA is cancelled by adding equivalent amounts of CT-DNA to both the tested

compounds and the reference solutions.

The compounds intrinsic binding constant, K_bwere determined using Wolfe–Shimer

equation as follows:

[DNA]

ε_a− ε_f

[DNA]

ε_b− ε_f

1

=

(5)

ε_b− ε_f

where [DNA], ε_a, ε_b, and ε_fare DNA concentration, apparent, fully bound complex, and

free complex extinction coefﬁcients, respectively. The ratio of the slope to the intercept from

the plot of [DNA]/(ε − ε_f) against [DNA] gave the binding constant K_bof the compounds.

a

3.7.2. Luminescence Competitive Displacement Study

Ethidium bromide (EB) is one of the most sensitive ﬂuorescent probes which can bind

to DNA through intercalation. Competitive binding of complexes Q1–Q15 to DNA with

EB could provide information with regards to the DNA-binding afﬁnity.

A competitive displacement study was carried out using ﬂuorescence spectroscopy

between CT-DNA pre-treated with ethidium bromide (EB) and complexes Q1–Q15 to

establish the complexes⁰actual mode of binding to CT-DNA. Ethidium bromide in buffer

solution has low ﬂuorescence intensity due to ﬂuorescence quenching of the free ethidium

bromide by the solvent molecule, but on binding through intercalation to DNA, its emission

intensity is drastically enhanced [109]. The competitive luminescence displacement assay

Molecules 2021, 26, 1205

28 of 34

was done using a modiﬁed method [91]. Pre-treated 15

CT-DNA was prepared in a phosphate buffer of pH 7.2 and left to equilibrate for 30 min.

Keeping CT-DNA-EB concentrations constant, different concentrations (0–20 M) of the

µM solution of EB with 15 µM of

µ

complexes were added at 8 min incubation interval at room temperature. The ﬂuorescence

quenching ability of CT-DNA bound EB by the complexes were recorded in the wavelength

range of 530–700 nm with an excitation wavelength at 525 nm at 25 ^◦C.

F_o

F

= 1 + K_SV[Q] = 1 + K_qτ [Q]

(6)

o

A Stern–Volmer (Equation (6)) was used to evaluate the CT-DNA-EB ﬂuorescence

quenching of the complexes. Where F_oand F denote the relative ﬂuorescence intensities

of CT-DNA in the absence and presence of the quencher, K_sv, Stern–Volmer quenching

constant, [Q], quencher concentration, Kq, bimolecular quenching constant and τ_o, average

−8

lifetime of the ﬂuorophore in the absence of the quencher which is typically equal to 10

s

in biomacromolecules.

3.7.3. Albumin Binding Assay Using Absorption Spectroscopy Titration

The absorbance assay of albumin binding is a very simple method used in identifying

the conformational changes in protein. The studies aimed at the binding of bioactive

compounds with protein to provide useful information on their biodistribution, toxicity,

and mechanism of action [18]. A stock solution of bovine serum albumin (BSA) was

prepared by dissolving an appropriate amount of BSA in phosphate buf_◦fer saline so-

◦

lution (pH 7.2) under constant stirring for 1 h at 25 C. It was kept at 4 C and used

within four days. The BSA concentration was determined spectrophotometrically by us-

ing ε280 = 44300 M⁻¹cm⁻¹absorption coefﬁcient. The stock solution of the complex was

prepared by dissolving 1 mmol of the complexes in DMSO. The absorption titration assay

was done by adding different concentrations (0–10

concentration (6 M). The sample solution was incubated after each addition of the con-

centrations for 10 min at 25 ^◦C before recording the absorbance at

µM) of the complexes to a constant BSA

µ

λ

max 280 nm. Binding

◦

constant K_bwas calculated from the intercept to the slope ratio of 1/[A-A ] vs. 1/[complex]

linear curve where A and A^◦represent BSA absorbance in the presence and absence of

complexes, respectively [86].

3.7.4. Albumin Binding Studies Using Fluorescence Quenching Method

A solution of 1

(0–35

M) and incubated for 8 min at 25 ^◦C. BSA was excited at 280 nm and emitted at

346 nm wavelength [92]. K_svconstant was determined from the Stern–Volmer equation:

µM of BSA was titrated against various concentrations of the complexes

µ

I_o/I = 1 + Kq τ_o[Q] = 1 + Ksv[Q]

(7)

where I_oand I represent the intensities of the ﬂuorescence in the presence and absence

of the quencher respectively while K_q, K_sv, τ_o, [Q] represent the BSA quenching rate

constant, dynamic quenching constant, the average lifetime of BSA without the quencher

(τ_o= 10⁻⁸s) and concentration of quencher, respectively.

3.8. Cytotoxicity Evaluation

In vitro cell viability with cultured cells is a method commonly used for cytotoxicity

activities of potential anticancer compounds due to reduced cost speed, and potential for

automation, and tests using human cells other than some in vivo animal tests [110]. The

signiﬁcant effect the perchlorate anion has on the complexes’ biological activities, as seen

in the antimicrobial, antioxidant, DNA and protein binding assays prompted the choice

of complexes to be assayed for cytotoxicity activity. Therefore, the cytotoxicity of some

selected complexes; Q6, Q7, Q9, and cisplatin as reference standard was evaluated on four

Molecules 2021, 26, 1205

29 of 34

human cell lines, human embryonic kidney 293 (HEK293), cervical cancer cell line (HELA),

breast cancer cell line (MDA-MB231), and neuronal cancer cell line (SHSY5Y).

The cytotoxicity assay was conducted as per the standard method and as described

by Abrahams et al. 2018 [111]. Brieﬂy, 96-well microtiter plates were seeded with

HEK293 (non -cancerous cell line), HELA, MDA-MB231, and SHSY5Y at a concentra-

tion of 1 × 10⁵cells/mL and allowed to stabilize for 4 h at 37 ^◦C and 5% CO₂. Thereafter,

Q6, Q7, Q9, and cisplatin were added to the plate through twofold serial dilution to allow

for eight ﬁnal compound concentrations ranging from 100 to 0.781

µM in a total volume of

200

µ

L / well. The plate was then incubated for 72 h at 37 ^◦C and 5% CO₂. To each well,

20 µL CellTiter 96 AQ_ueousOne Solution (Promega, Madison, WI, USA) was added and the

plates were incubated for 4 h as previously described. Thereafter, absorbance was read at

490 nm on a multiplate reader (Molecular Devices, San Jose, CA, USA). EC₅₀values were

determined as the concentration of each compound required to reduce cell viability by 50%

and were calculated using GraphPad Prism 8 (2019) (GraphPad Software, San Diego, CA,

USA). The values recorded are averages of at least three separate experiments (n = 3).

4. Conclusions

Herein, we have described the synthesis and characterization of silver(I) complexes

of ﬁve quinolinyl imine. The inﬂuence of the quinolinyl imine variable substituents and

silver(I) anion on the interaction of the compounds with Ct-DNA, protein, and their an-

timicrobial, antioxidant, and cytotoxicity activities were evaluated. The antimicrobial

evaluation data revealed that all the complexes had moderate to excellent antibacterial

activity and complexes [Ag(L2)₂]ClO₄Q7 and [Ag(L3)₂]ClO₄Q8 showed remarkable an-

timicrobial activity compare to the standard ciproﬂoxacin against all the bacteria tested.

The ligands of each of [Ag(L2)₂]ClO₄Q7 and [Ag(L3)₂]ClO₄Q8 possess either the ben-

zothiazole moiety or the p-chloro substituent, and both complexes have perchlorate as the

anion, which could have enhanced their antimicrobial activity. Similarly, complexes with

notable antimicrobial activity also displayed good antioxidant activity than the ascorbic

acid (2.68 mg/mL) as seen in Q2, Q4, Q5, Q6, Q7, Q9, Q10, and Q13 with IC₅₀between

0.95 and 2.22 mg/mL. In the absorption and emission spectral of the interaction of the com-

pounds with CT-DNA and bovine serum albumin, the results indicate that the compounds

bind to CT-DNA via intercalation mode due to the planarity of the chelating ligand and

the competitive study with ethidium bromide shows that the complexes can displace EB

from the CT-DNA-EB adduct and compete for the DNA-binding sites with EB, which is

usually characteristic of the intercalative interaction of compounds with DNA.

Further study demonstrated that the complexes exhibit a moderate afﬁnity for protein

and the possible quenching mechanism between the complexes and bovine serum albumin

is static. Thus, [Ag(L1)₂]NO₃Q1, [Ag(L1)₂]ClO₄Q6 and [Ag(L1)₂]CF₃SO₃Q11 can be

stored in protein and can be easily released in desired target areas.

Complexes [Ag(L2)₂]ClO₄Q7 and [Ag(L4)₂]ClO₄Q9 with benzothiazole moiety and

Methyl substituent respectively displayed a remarkable cytotoxicity activity especially

towards Hela (cervical cancer cell line) ca. two times better than the cisplatin standard and

ﬁve times than [Ag(L1)₂]ClO₄Q6 with ﬂuorine substituent and a trigonal geometry.

Based on the remarkable biological activities recorded for silver(I) quinolinyl Schiff

base derivatives in this study, Ag(I) quinolinyl Schiff bases tend to be promising candidates

for the development of novel anticancer, antioxidant, and antibiotics drugs.

Supplementary Materials: The following are available online at https://www.mdpi.com/1420-3

049/26/5/1205/s1, Figures S1–S22: Electronic absorption spectra of L1–L5, silver salts and Q1–

Q15 at 5.0 × 10⁵M in the absence (dashed line) and the presence of difference concentrations of

CT-DNA, Figures S23–S39: The ﬂuorescence spectra of EB-CT-DNA in the absence (dashed line)

and the presence of different concentration of silver salts and Q1–Q15, Figures S40–S56: The double-

logarithmic plot of EB-CT-DNA–Complex Q1–Q15 and silver salts interaction, Figures S57–S73:

Electronic Absorption Spectra of BSA in the absence (dashed line) and the presence of different

concentrations of Q1–Q15 and silver salts, Figures S74 and S75: Fluorescence emission spectra of

Molecules 2021, 26, 1205

30 of 34

BSA in the absence(dashed line) and the presence of different concentration of complex Q1 and

Q6, Figures S76 and S77: The double-logarithmic plot of BSA–Complex Q1 and Q11 interactions,

Figures S78–S97: ¹H NMR spectra of L1–L5 and Q1–Q15, Figures S98–S117: ¹³C NMR spectra of

L1–L5 and Q1–Q15, Figures S118–S137: IR spectra of L1–L5 and Q1–Q5, Figure S138-S157: Mass

1

Spec. spectra of L1–L5 and Q1–Q15, Table S1: H-NMR chemical shifts of some protons in L1–L5

complexes (Q1–Q15) and the IR band of (C=N) and quinolinyl N for L1–L5 and complexes Q1–Q15

,

Table S2: Physical and chemical data of silver(I) complexes Q1–Q15, Table S3: Crystal data and

structure reﬁnement for complexes Q1, Q6, Q7, Q12 and Q14.

Author Contributions: B.O. conceived and designed the experiments; A.A.A. carried out the syn-

thesis, characterized the synthesized compounds, carried out the in vitro cytotoxicity, CT-DNA, and

protein binding studies; B.O. and S.J.Z. reported the crystal structures; M.S.I., K.O., and V.F.S. carried

out the in vitro antioxidant experiment; C.M. and A.A.A. performed the in vitro antimicrobial assay.

A.A.A. drafted the manuscript; B.O. revised the manuscript. All authors have read and agreed to the

published version of the manuscript.

Funding: This research was funded by the National Research Foundation of South Africa (Grant

number: 119342) and the APC was funded by National Research Foundation of South Africa.

Acknowledgments: Adesola A. Adeleke acknowledges Olabisi Onabanjo University, Ago-Iwoye, for

granting her study leave to pursue the programme at the University of KwaZulu-Natal. The authors

would like to appreciate the University of KwaZulu-Natal and the National Research Foundation of

South Africa (Grant number: 119342) for their ﬁnancial assistance for Adesola A. Adeleke.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Disclosure Statement: No potential conﬂict of interest was reported by the authors.

References

1.

2.

3.

Bine, F.K.; Nkungli, N.K.; Numbonui, T.S.; Numbonui Ghogomu, J. Structural Properties and Reactive Site Selectivity of Some

Transition Metal Complexes of 2,2’(1E,1’E)-(ethane-1,2-diylbis(azan-1-yl-1-ylidene))bis(phenylmethan-1-yl-1-yli dene)dibenzoic

Acid: DFT, Conceptual DFT, QTAIM, and MEP Studies. Bioinorg. Chem. Appl. 2018, 2018, 4510648. [CrossRef] [PubMed]

Manna, A.K.; Rout, K.; Chowdhury, S.; Patra, G.K. A dual-mode highly selective and sensitive Schiff base chemosensor for

ﬂuorescent colorimetric detection of Ni(2+) and colorimetric detection of Cu(2). Photochem. Photobiol. Sci. 2019, 18, 1512–1525.

[CrossRef] [PubMed]

Khandar, A.A.; Afkhami, F.A.; Hosseini-Yazdi, S.A.; White, J.M.; Kassel, S.; Dougherty, W.G.; Lipkowski, J.; van Derveer, D.;

Giester, G.; Costantino, F. Anion inﬂuence in the structural diversity of cadmium coordination polymers constructed from a

pyridine based Schiff base ligand. Inorg. Chim. Acta 2015, 427, 87–96. [CrossRef]

4.

5.

6.

Kajal, A.; Bala, S.; Kamboj, S.; Sharma, N.; Saini, V. Schiff Bases: A Versatile Pharmacophore. J. Catal. 2013, 2013, 1–14. [CrossRef]

Garrido Montalban, A. Quinolines and Isoquinolines. Heterocycl. Nat. Prod. Synth. 2011, 299–339. [CrossRef]

Kouznetsov, V.V.; Meléndez Gómez, C.M.; Peña, J.L.V.; Vargas-Méndez, L.Y. Natural and synthetic quinoline molecules against

tropical parasitic pathologies: An analysis of activity and structural evolution for developing new quinoline-based antiprotozoal

agents. In Discovery and Development of Therapeutics from Natural Products against Neglected Tropical Diseases; Elsevier: Amsterdam,

The Netherlands, 2019; pp. 87–164. [CrossRef]

7.

8.

9.

Rajesh, Y.B. Quinoline Heterocycles: Synthesis and Bioactivity. In Heterocycles-Synthesis and Biological Activities; IntechOpen:

London, UK, 2018. [CrossRef]

Casal, J.J.; Asís, S.E. Natural and synthetic quinoline derivatives as anti-tuberculosis agents. Austin Tuberc. Res. Treat. 2017

2, 1007–1010.

,

Kumar, S.; Bawa, S.; Gupta, H. Biological activities of quinoline derivatives. Mini Rev. Med. Chem. 2009, 9, 1648–1654. [CrossRef]

10. Adsule, S.; Barve, V.; Chen, D.; Ahmed, F.; Dou, Q.P.; Padhye, S.; Sarkar, F.H. Novel Schiff base copper complexes of quinoline-2

carboxaldehyde as proteasome inhibitors in human prostate cancer cells. J. Med. Chem. 2006, 49, 7242–7246. [CrossRef]

11. Beyzaei, H.; Moghadam, H.H.; Bagherzade, G.; Aryan, R.; Moghaddam-Manesh, M. Synthesis and In Vitro Antibacterial

Evaluation of Schiff Bases Derived from 2-Chloro-3-Quinolinecarboxaldehyde. Avicenna J. Med. Biochem. 2019, 7, 9–15. [CrossRef ]

12. Sakthi, M.; Ramu, A. Synthesis, structure, DNA/BSA binding and antibacterial studies of NNO tridentate Schiff base metal

complexes. J. Mol. Struct. 2017, 1149, 727–735. [CrossRef]

13. Shivakumar, L.; Shivaprasad, K.; Revanasiddappa, H.D. SODs, DNA binding and cleavage studies of new Mn(III) com-

plexes with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2013, 107, 203–212.

[CrossRef] [PubMed]

14. Ommenya, F.K.; Nyawade, E.A.; Andala, D.M.; Kinyua, J. Synthesis, Characterization and Antibacterial Activity of Schiff Base,

4-Chloro-2-{(E)-[(4-Fluorophenyl)imino]methyl}phenol Metal (II) Complexes. J. Chem. 2020, 2020, 1–8. [CrossRef]

Article Doi

Quinoline functionalized schiff base silver (I) complexes: Interactions with biomolecules and in vitro cytotoxicity, antioxidant and antimicrobial activities

DOI: 10.3390/molecules26051205

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Article abstract of DOI:10.3390/molecules26051205

Full text of DOI:10.3390/molecules26051205

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