Synthesis, antimicrobial evaluation, and in silico studies of quinoline—1H-1,2,3-triazole molecular hybrids

Molecular Diversity

https://doi.org/10.1007/s11030-020-10112-3

ORIGINAL ARTICLE

Synthesis, antimicrobial evaluation, and in silico studies

of quinoline—1H‑1,2,3‑triazole molecular hybrids

Paul Awolade¹· Nosipho Cele¹· Nagaraju Kerru¹· Parvesh Singh¹

Received: 3 March 2020 / Accepted: 27 May 2020

Abstract

Antimicrobial resistance has become a signifcant threat to global public health, thus precipitating an exigent need for new

drugs with improved therapeutic efcacy. In this regard, molecular hybridization is deemed as a viable strategy to aford

multi-target-based drug candidates. Herein, we report a library of quinoline—1H-1,2,3-triazole molecular hybrids syn-

thesized via copper(I)-catalyzed azide-alkyne [3+2] dipolar cycloaddition reaction (CuAAC). Antimicrobial evaluation

identifed compound 16 as the most active hybrid in the library with a broad-spectrum antibacterial activity at an MIC₈₀

value of 75.39 μM against methicillin-resistant S. aureus, E. coli, A. baumannii, and multidrug-resistant K. pneumoniae.

The compound also showed interesting antifungal profle against C. albicans and C. neoformans at an MIC₈₀value of 37.69

and 2.36 μM, respectively, superior to fuconazole. In vitro toxicity profling revealed non-hemolytic activity against human

red blood cells (hRBC) but partial cytotoxicity to human embryonic kidney cells (HEK293). Additionally, in silico stud-

ies predicted excellent drug-like properties and the importance of triazole ring in stabilizing the complexation with target

proteins. Overall, these results present compound 16 as a promising scafold on which other molecules can be modeled to

deliver new antimicrobial agents with improved potency.

Graphic abstract

A quinoline-based molecular hybrid has

been identified with broad-spectrum

antimicrobial activity

Compound 16-MraY complex

Keywords Antimicrobial resistance · Molecular hybridization · Quinoline · 1H-1,2,3-triazole · In silico

Introduction

Electronic supplementary material The online version of this

The world today is burdened with an increasing number and

article (https://doi.org/10.1007/s11030-020-10112-3) contains

widespread emergence of drug-resistant pathogens, which

supplementary material, which is available to authorized users.

puts man’s existence at risk. Despite the laudable progress

made in antimicrobial drug research, the witty evolutions

in the drug-resistance machinery of pathogenic microbes

have stayed abreast of man’s ingenuity. Infections that were

once curable with single or combination therapies are now

* Parvesh Singh

singhp4@ukzn.ac.za; parveshdurban@gmail.com

1

School of Chemistry and Physics, University of KwaZulu-

Natal, P/Bag X54001, Westville, Durban, South Africa

Vol.:(0123456789)

1 3

Molecular Diversity

insidiously elusive to control even as the likelihood of drug-

resistant infections compromises the outcome of invasive

surgeries [1]. The upsurge in the cost of medical care and the

rising rates of morbidity and mortality due to this menace

also stifes efective clinical practice [2, 3]. Therefore, the

development of new antimicrobial agents is indispensable to

efective disease control.

Quinoline is another prominent pharmacophore found in

many bioactive natural products and synthetic molecules.

A distinct class of this heterocycle, 8-hydroxyquinoline

(8-HQ), and its derivatives (Fig. 1) are privileged scafolds

with an excellent metal chelating property that confers desir-

able therapeutic potentials [18, 19]. Nevertheless, toxicity

burdens limit their clinical utility, for example the with-

drawal of the oral form of clioquinol, an antiparasitic agent.

The observed toxicity is partly due to the hydroxy unit’s

susceptibility to phase II metabolism via glucuronidation

pathway, subsequent enterohepatic circulation, and possible

β-glucuronidase-mediated release of the active form in ar eas

with high expression of the glycosyl hydrolase [20, 21].

Therefore, we envisage that the O-conjugation of 8-HQ will

attenuate its toxicity drawback while afording molecules

with desired antimicrobial activity.

Target-based approach has been widely employed in

drug discovery over the years [4]. However, in recent times,

multi-target-drug design (MTDD) strategies are favored

over conventional single-target-based drugs owing to the

propensity of pathogens to mutate or bypass susceptible

drug targets [5]. As a result, molecular hybridization (MH)

has gained increased attention as an MTDD strategy since

it allows the incorporation of two or more bioactive scaf-

folds into a molecular entity, which subsequently displays

multiple-receptor recognition and an improved therapeutic

efect [6].

Based on the foregoing, the present work describes

the synthesis of a library of quinoline—1H-1,2,3-triazole

hybrids as a continuation of our research on the development

of antimicrobial agents with an impro ved pharmacological

profle using the MH approach [22, 23]. The molecular

design incorporates an alkane spacer bear ing terminal O and

NH units to improve lipophilicity and H-b potential, respec-

tively. Potent compounds (% inhibition ≥ 80) were further

examined for their in vitro cytotoxicity and hemolytic activi-

ties against human embryonic kidney cells (HEK293) and

human red blood cells (hRBC), respectively. Also, in silico

techniques were used to predict the drugability of potent

compounds and their binding profles with selected drug

targets to rationalize their antimicrobial activities.

The preference of MH in drug design thrives on the easy

access to linkers and spacers explored in assembling the

hybrid molecules. In this regard, the 1H-1,2,3-triazole ring

has proven t o be exceptional thanks to its broad bioactivity

spectrum [7–9], resistance to biodegradation in vivo, and

bioisosterism of amide, trans-olefnic, carboxylic acid moi-

ety, and ester units [10]. The hydrogen bonding (H-b) ability

of the ring nitrogen and H-5’ atoms for multiple-binding to

drug targets also encourage its utility in drug design. For

instance, a quinoline-1H-1,2,3-triazole hybrid exhibited

909- and 364-fold superior anticandidal efcacy compar ed

to the parent 8-hydroxyquinoline and fuconazole [11, 12].

The antimicrobial profile of 1H-1,2,3-triazole hybrids

of ciprofoxacin-based drugs was also enhanced by up to

69-fold compared to the parent drugs [13–16]. Precisely,

the moxifoxacin-triazole hybrid showed tw o fold superior

inhibition of DNA-gyrase compared to moxifoxacin and

gatifoxacin [17].

Fig. 1 Antimicrobial agents

bearing 8-hydroxyquinoline and

1H-1,2,3-triazole units

1 3

Molecular Diversity

On the other hand, the alkyne fragment, compound

8, was synthesized in 93% yield from a base-promoted

O-alkylation of commercially available 8-HQ with propar-

gyl bromide (Scheme 2). Then, copper(I)-catalyzed azide-

alkyne [3 + 2] dipolar cycloaddition reaction (CuAAC)

aforded the target hybrids 9a–u, 10a–z, and 11a–n in

moderate to excellent yields. Compounds 14–18 were also

prepared for SAR studies to examine the infuence of O- or

NH-linked alkane spacer and triazole ring on antimicro-

bial activity (Scheme 3). The synthesized compounds were

characterized using spectroscopic techniques, i.e., FT-IR,

HRMS, and NMR (¹H, ¹³C, and 2D).

Results and discussion

Chemistry

The synthetic protocol began with the preparation of

required azide fragments according to their substrate

demands (Scheme 1). HCl/NaNO₂-mediated diazotiza-

tion of substituted anilines and subsequent treatment of

the resulting diazonium salt with NaN₃gave phenyl azides

1a–u in moderate to quantitative yields. (2-azidoethoxy)

benzenes 3a–z were obtained by refuxing phenols with

1,2-dibromoethane in 25% NaOH solution containing tert-

butylammonium bromide (TBAB), followed by heating the

bromoethyl intermediates (2a–z) with NaN₃to give azides

3a–z in over 70% overall yield. Notably, omitting TBAB

increased both the reaction time for 2a–z and the quantity

of undesired bis-products. The preparation of 7a–n akin

to 3a–z was, however, unsuccessful. Hence, azidation of

2-chloroethanol in water was performed frst, followed

by tosylation of the resulting 2-azidoethanol (5) to give

2-azido-O-ethyl tosylate (6). N-alkylation of substituted

anilines with compound 6 then aforded azides 7a–n in

over 50% yield. The phenol and aniline precursors of azide

3j , 3t–v, and 7g were prepared according to the literature

[24, 25].

Structural elucidation

The conversion of azides to triazoles was depicted in

the FT-IR spectra of target hybrids by the disappear-

ance of strong and sharp absorption bands for azido

(−N=N⁺=N⁻) and alkynyl (≡C–H) stretching vibrations

around 2090–2100 cm⁻¹and 3275 cm⁻¹, respectively. The

bands for C≡C stretching at 2121 cm⁻¹and ≡C–H defor-

mation at 700–600 cm⁻¹were also absent. Furthermore,

the exclusive formation of 1,4-disubstituted 1H-1,2,3-

triazole ring was confrmed by NMR spectroscopy. In

the ¹H NMR spectra, the terminal alkyne resonances and

allylic couplings, i.e., the triplet peak of −C≡CH at δ_H

2.54 (⁴J = 2.4 Hz) and doublet peak of −OCH₂at δ_H5.03

Scheme 1 Synthetic route to azide fragments. Reagents and con-

ditions: (i) HCl: H₂O (1:1), NaNO₂, 0 °C, 30 min, (ii) NaN₃, 0 °C

to RT, 2 to 3 h. 60–100% yield over two steps, (iii) 25% NaOH,

1,2-dibromoethane, TBAB, 80 °C, 5–12 h, (iv) NaN₃, DMF, 80 °C,

30 min to 1 h,>70% overall yield, (v) NaN₃, H₂O, 90 °C, 18 h, (vi)

p-TsCl, Et₃N, DCM, 0 °C to RT, 16 h, 66% yield, (vii) CaCO₃, KI,

acetone: H₂O (1:1), 50–70% yield

1 3

Molecular Diversity

Scheme 2 Synthetic route to target hybrids. Reagents and conditions: (viii) anhydrous K₂CO₃, DMF, RT, 4 h, 93%. (ix) appropriate azides,

DCM: H₂O (1:1), CuSO₄.5H₂O, sodium ascorbate, RT, 2 to 4 h

Scheme 3 Synthesis of compounds for SAR studies. Reagents and conditions: (iv) NaN₃, DMF, 80 °C, 30 min, quantitative yield, (ix) DCM:

H₂O (1:1), CuSO₄.5H₂O, sodium ascorbate, RT, 2 h, 50–90% yield, (x) anhydrous K₂CO₃, DMF, 80 °C, 1 h,~90% yield

(⁴J = 2.4 Hz), respectively, were absent, while a promi-

nent singlet peak for H-5’ of triazole ring appeared at δ_H

8.10 − 8.90 (9a–u), 7.85–8.37 (10a–z), and 7.71–7.85

(11a–n). The corresponding alkynyl −C≡CH signals at δ_C

78.30 and 76.08 were also absent in the ¹³C spectra, while

C-4’ and C-5’ resonances of triazole ring were observed at

δ_C140–145 and 121–125, respectively. Interestingly, in the

¹H NMR spectrum of phenyl hybrid 9b, the H-b ability of

triazole H-5’ atom was evidenced by a doublet peak at δ_H

8.29 (⁵J_HF= 2.6 Hz) due to ¹⁹F coupling. A corresponding

doublet signal at δ_C125.20 for C-5’ (⁴J_CF= 3.9 Hz) was

also observed in the ¹³C spectrum.

1 3

Molecular Diversity

The successful hybridization of quinoline and 1H-1,2,3-

triazole was further confrmed with 2D-NMR data. For

example, the heteronuclear multiple bond coherence

(HMBC) spectrum for compound 16 (depicted in Fig. 2)

showed HMBC correlations of H-5’ (δ_H7.85, 1H, s) to C-4’

(δ_C144.28) and C-7’ (δ_C49.09); H-6’ (δ_H5.51, 2H, s) to

C-8 (δ_C153.81), C-4’ (δ_C144.28) and C-5’ (δ_C124.39); as

well as H-7’ (δ_H4.60, 2H, t) to C-5’ (δ_C124.39) and C-8’

(δ_C67.32). Moreover, a prominent molecular ion peak at

m/z = (M + Na)⁺in the high-resolution mass spectrum of

target hybrids validated the established structures.

of phenoxy hybrids compared to anilino hybrids partly sug-

gests the preference of H-b acceptor over H-b donor.

The importance of O or NH-linked alkane spacer, triazole

core, and phenyl ring to antifungal activity was established

from an analysis of the structure–activity relationship (SAR)

of target hybrids and compounds 14–19. For instance, both

compounds 9a and 14, which lacks the alkane spacer, were

inactive compared to phenoxy and anilino hybrid 10a and

11a, respectively, with moderate activities. The activity of

compound 10a and inactivity of compound 15 also reveal

the signifcance of phenyl unit to lipophilicity.

Furthermore, the signifcance of 1H-1,2,3-triazole core

to antimicrobial activity and its synergy with alkane spacer

and sulfone unit is evidenced by the broad-spectrum antimi-

crobial potency of compound 16. The compound emerged

as the most potent antimicrobial agent overall. With an

MIC₈₀value of 75.39 μM, compound 16 displayed several-

fold superior antibacterial potency compared to compounds

15, 18, 19, and 8-HQ against the Gram-negative patho-

gens. Compound 16 is also the only promising inhibitor of

P. aeruginosa, with 64.8% inhibition at 75.39 μM (32 μg/

mL). Although the compound is less potent than the single-

spectrum antibacterial agents, colistin, and vancomycin,

the structure can serve as a good starting point to develop

broad-spectrum antibacterial agents with increased potency.

Moreover, compound 16 showed an MIC₈₀value of 2.36 μM

against C. neoformans, which is 11-fold stronger than fu-

conazole. This superior anticryptococcal activity might be

due to the compound’s increased lipophilicity, which will

favor cellular membrane penetration for efective inhibition

of fungal growth [26].

Antimicrobial studies

The antimicrobial potential of quinoline–1H-1,2,3-triazole

hybrids, compounds 14–18 and their precursor (8-HQ),

was examined against ESKAPE pathogens, viz. MRSA

(ATCC 43300), MDR K. pneumoniae (ATCC 700603), E.

coli (ATCC 25922), A. baumannii (ATCC 19606), and P.

aeruginosa (ATCC 27853) as well as two fungi: C. albicans

(ATCC 90028) and C. neoformans (ATCC 208821). A uni-

form test concentration of 32 μg/mL was used for the whole-

cell growth inhibition assays. Subsequently, potent hybrids,

i.e., those providing≥80% inhibition of pathogen’s growth,

were re-evaluated in a dose–response assay to determine

their minimum inhibitory concentration (MIC₈₀).

The results presented in Table 1 show that the molecular

hybrids exhibit activity profles similar to their precursor

8-HQ, i.e., stronger efcacy as antifungal agents. However,

moderate antibacterial activities were found with compound

9l (79.6% growth inhibition of A. baumannii at 101.15 μM;

32 μg/mL) and compound 10v (MIC₈₀=84.57 μM against

MRSA). Other compounds did not show≥80% inhibition of

selected bacteria strains at the maximum tested concentra-

tion (32 μg/mL). Antifungal activity was conserved with

methyl, hydroxy, and 2-chloro substituted hybrids. The most

active phenyl hybrid 9q (MIC₈₀=12.57 μM) exhibited two-

fold stronger anticryptococcal activity than the reference

drug fuconazole, whereas compounds 10d, 10v, and 10z

showed equipotent anticryptococcal activities with an MIC₈₀

value around 20 μM. Moreover, the superior activity profle

Cytotoxicity and hemolytic activity

Toxicity evaluation remains the crux of drug development

and the primary cause of failure for many drug candidates

in clinical trials [27]. The therapeutic safety of a drug is its

ability to exert the desired physiological response without

harmful efects on normal cellular structure and functions. A

signifcant diference between the efcacious and toxic dose

is therefore of uttermost importance.

The cytotoxicity and hemolytic profles of potent hybrids

were determined in vitro against HEK293 and hRBC. The

compounds were non-hemolytic at their active concentra-

tions, i.e., they did not lysis 10% of hRBCs at their MIC₈₀

values. However, well-defned, or partial cytotoxicity was

observed for some compounds. For instance, although com-

pound 9q bears a considerably safe margin as an anticrypto-

coccal agent (MIC₈₀=12.57 μM), its anticandidal potential

(MIC₈₀= 25.13 μM) is compromised by a CC₅₀value of

26.58 μM, lower than tamoxifen (CC₅₀= 35.15 μM) used

as the reference toxin. Compounds 9d, 10c, 10d, 10j, and

10z also share a similar fate with 9q, albeit with reduced

Fig. 2 Key HMBC correlations in compound 16

1 3

Molecular Diversity

Table 1 Antimicrobial potentials of quinoline—1H-1,2,3-triazole hybrids

Compound

Structure

Antimicrobial activity MIC₈₀(μM)

Cytotoxicity

(μM)

CC₅₀HC₁₀

Gram +ve Gram –ve

Fungi

MRSA

55.11

E. c. MDR K. p. P. a.

A. b.

C. a.

C. n.

a

8-HQ

9d

–

220.45 13.78 3.44

–

95.02 47.51 67.70

a

9 l

–

79.6%*

–

101.15

–

9q

25.13 12.57 26.58

a

9r

–

100.52 50.26

92.38 46.19

–

a

10a

–

10c

10d

10e

–

83.69 41.85 68.00

–

83.69 20.92 66.95

a

77.22 38.61

–

10j

–

63.47 31.74 54.55

–

10m

10n

88.79 88.79

88.79 44.39

–

1 3

Molecular Diversity

Table 1 (continued)

Compound

Structure

Antimicrobial activity MIC₈₀(μM)

Gram +ve Gram –ve

Cytotoxicity

(μM)

CC₅₀HC₁₀

Fungi

MRSA

–

E. c. MDR K. p. P. a.

A. b.

C. a.

C. n.

10q

10l

–

85.46

–

78.73

–

a

10v

84.57

–

42.29 21.14

–

10z

11a

11d

14

–

80.72 20.18 42.88

–

a

–

92.65 46.32

84.25 42.12

–

nd

–

15

–

a

16

75.39

75.39 75.39

64.8%* 75.39

37.69 2.36

–

17

–

nd

–

a

18

128.35

nd

128.35 32.09 16.04

–

c

19**

nd

1 3

Molecular Diversity

Table 1 (continued)

Compound

Structure

Antimicrobial activity MIC₈₀(μM)

Gram +ve Gram –ve

Cytotoxicity

(μM)

CC₅₀HC₁₀

Fungi

MRSA

E. c. MDR K. p. P. a.

A. b.

C. a.

C. n.

Colistin-Sulfate

Vancomycin-HCl

Fluconazole

Tamoxifen

–

0.67

0.09 0.18

0.18

–

0.18

–

0.41

26.12

35.15

Melittin

0.17

MIC₈₀: minimum concentration inhibiting≥80% growth of microbial cell; CC₅₀: concentration at 50% cytotoxicity to HEK293; HC₁₀: con-

centration at 10% hemolytic activity against hRBCs; MRSA: methicillin-resistant Staphylococcus aureus; E. c.: Escherichia coli; MDR K. p.:

multidrug-resistant Klebsiella pneumoniae; P. a.: Pseudomonas aeruginosa; A. b.: Acinetobacter baumannii; C. a.: Candida albicans; C. n.:

Cryptococcus neoformans; ‘–’: inactive at maximum tested concentration of 32 μg/mL; *: percentage growth inhibition at 32 μg/mL; ‘–^a’: com-

pounds showing partial toxicity at 32 μg/mL in a replicate but inactive in the other; **: values from Aneja et al. 2018. ‘–^c’: IC₅₀>400 μM

cytotoxicity compared to tamoxifen. Furthermore, the

most potent compound 16 showed partial cytotoxicity with

CC₅₀=68.56 μM akin to compounds 9l, 9r, 10a, 10e, 10v,

11a, and 18. In contrast, compounds 10 m, 10n, 10q, 10l,

and 11d were non-cytotoxic at their MIC₈₀values. There-

fore, further structural optimization of these hybrids, espe-

cially compound 16, is pertinent.

rule of fve (Ro5) and Jorgensen’s rule of three (Ro3). Pre-

cisely, high oral absorption is obtainable considering the

apposite balance of corresponding descriptors, i.e., molecu-

lar weight (MW), hydrogen bonding donor/acceptor (HB-

D/A), predicted total solvent accessible surface area (SASA),

aqueous solubility (QPlogS), and lipophilicity (QPlogP_o/w).

Favorable plasma distribution (bioavailability) for adequate

exposure to therapeutic targets is also expected, considering

the mid-ranged binding afnity to human serum albumin

(QPlogKhsa) [27, 29]. The predicted potential blockage of

hERG K⁺ion channel (QPlogHERG) for QT prolongation

and torsades de pointes (TdP) arrhythmia should be inter-

preted with caution, as not all hERG blockers pose a pro-TdP

arrhythmic risk [30]. Albeit, further optimization studies

should seek to reduce this property. The predicted sites of

metabolism of examined hybrids, together with the ADME

properties of other active hybrids, are given in the Online

Resource (Fig. S1 and Table S1, respectively).

In silico studies

ADME properties

Absorption, distribution, metabolism, and excretion

(ADME) are fundamental pharmacokinetic properties use-

ful for evaluation of the drugability of promising drug can-

didates as well as early identifcation of compounds that are

likely to fail clinical trials. Their evaluation through in silico

predictions also helps to fast-track the drug development

process while reducing cost. Thus, the ADME properties of

active hybrids in this study were computed with QikProp 6.0

[28] implemented in the Schrödinger molecular modeling

suite (version 2019-2).

Binding interactions with drug targets

The worthiness of compound 16 for further structural

optimization is supported by its excellent drug-like proper-

ties (Table 2), adjudged by the zero-violation of Lipinski’s

In order to rationalize the observed antimicrobial activi-

ties, potent hybrids were docked into the active sites of

antibacterial drug target: phospho-MurNAc-pentapeptide

Table 2 Predicted ADME properties of compound 16

MW

SASA

HBD HBA QPlogPo/w QPlogS

3.54 −5.39

2–20 −2.0 to 6.5 −6.5 to 0.5 >−5

QPlogHERG QPlogKhsa Oral abs Ro5 Ro3

Compound 16

424.47

780.47

0

8

−7.61

−0.04

–1.5 to 1.5

95%

0

Acceptable range 130.0–725.0 300.0–1000.0 0–6

>80% <4 <3

MW: molecular weight; SASA: total solvent accessible surface area in Å²; HBD: hydrogen bond donor; HBA: hydrogen bond acceptor;

QPlogPo/w: octanol/water partition coefcient; QPlogS: aqueous solubility S in mol dm⁻³; QPlogHERG: IC₅₀value for the blockage of “human

ether-a-go-go related gene” K⁺channels; QPlogKhSA: binding to human serum albumin; Oral abs: human oral absorption; Ro5: Lipinski’s rule

of fve; Ro3: Jorgensen’s rule of three

1 3

Molecular Diversity

translocase (MraY) [31], and anti fungal drug target: lanos-

ligand–receptor complex. These interactions induced criti-

cal conformational changes in the protein structure, as evi-

denced by the coordination of Asn190, Asp193, and Gly264

to Mg²⁺cofactor to foster a tight receptor binding (Table 3).

Furthermore, the similar binding modes of compound

10v (Fig. 3b), particularly H-b interactions with the cata-

lytic aspartic acid triad (Asp117, Asp118, and Asp265)

and pi-cation interaction with catalytic Mg²⁺ion, support

its activity against MRSA. However, the single-spectrum

terol 14α-demethylase (LDM) [32].

MraY

The molecular modeling of compound 16 in the cytoplas-

mic active site of MraY (PDB ID: 4J72) revealed that the

ligand accesses the receptor’s active site-cleft via the quino-

line fragment. The 8-O atom thus coordinates catalytically

essential Mg²⁺cofactor in the binding site, while the pyri-

dine N-atom is favorable to interact in like manner (Fig. 3a).

Subsequently, the triazole H-5’ atom forms an aromatic H-b

with Asp117 and Asp265 of the catalytic aspartic acid triad

residues, while the quinoline benzene ring is involved in

pi-pi stacking interaction with highly conserved His324

of the HHH motif. The oxygen atoms of the tosylate unit

also aforded H-b interaction with side-chain amino units of

Gln137, Lys133, and Asn190. Aromatic H-b interaction of

the tosyl ring with Gln137 and Asp193 as well as hydropho-

bic contacts with Phe134, Phe262, and Met263 stabilized the

Table 3 Molecular docking results of compound 16 and other potent

hybrids at the cytoplasmic active site of MraY

Compound Docking

score

Glide

Glide energy

gscore

emodel

16

−7.56

−4.99

−3.83

−3.86

−7.56

−7.51

−5.91

−3.86

−70.12

−72.48

−21.66

−44.36

−49.13

−58.15

−16.18

−38.59

10v

8-HQ

19

Fig. 3 Predicted binding modes of compound 16 (a), 10v (b), 8-HQ

(c) and 19 (d) in the cytoplasmic active site of MraY. Protein inter-

actions are displayed as dashed lines; hydrogen bond (yellow), aro-

matic H-bond (cyan), pi-pi stacking (light-blue) pi-cation (green)

and metal-coordination (red). Atoms: carbon (ligand: green; pro-

tein: gray), nitrogen (blue), oxygen (red), sulfur (yellow), hydrogen

(white), Mg²⁺cofactor (purple sphere)

1 3

Molecular Diversity

antibacterial activity can be partly justifed by the ligand’s

loose binding at the receptor’s active site, lack of interaction

with His324, and the inability to induce increased coordina-

tion to Mg²⁺ion. Similar rationalizations and the absence of

the 1H-1,2,3-triazole ring are valid for the activity profle of

8-HQ and compound 19 (Fig. 3c, d, respectively).

established the signifcance of molecular hybridization to

antimicrobial activity as each pharmacophore on the hybrid

concertedly contributed to the observed antimicrobial pro-

fle. These results suggest that further structural optimiza-

tion of the identifed compound is relevant to aford new

antimicrobial agents with stronger potencies.

LDM

Experimental

The binding profles of compounds 9q, 10d, 10v, 10z, 16,

18, and their precursor 8-HQ were simulated at the heme-

containing active site of LDM (PDB ID: 4WMZ). Analysis

of compound 16-LDM complex (Fig. 4) showed that the

1H-1,2,3-triazole ring is crucial to strong protein binding.

The ring assumed a conformation identical to the 1,2,4-tria-

zole unit of fuconazole near the heme cofactor, thus aford-

ing compound 16 the best binding profle overall (Table 4).

The docked complex features pi-pi stacking and pi-cation

interactions of the 1H-1,2,3-triazole with porphyrin ring and

Fe³⁺ion of heme cofactor, respectively, while the H-5’ atom

forms an aromatic H-b interaction with Tyr140. Also, the

sulfone unit presented H-b and aromatic H-b interactions

with Gly314 and Tyr140, respectively; the quinoline ring

interacted in like manner with Tyr126 and Ser382. The pre-

dicted binding modes of other hybrids (Table 4) corroborate

their antifungal activities.

General information

All reagents were used as purchased from Merck through

Capital Laboratory, South Africa. The progress of all reac-

tions was monitored with thin-layer chromatography (TLC)

on Merck alumina-backed TLC plates and visualized under

UV light. Product purifcation was achieved using column

chromatography with silica gel (0.063–0.200 mm) and

appropriate ratios of hexane–EtOAc or EtOAc-methanol as

eluant. Infrared (IR) spectra were recorded on a PerkinElmer

100 spectrophotometer with Universal ATR sampling acces-

sory; wavenumbers (ʋ) are expressed in cm⁻¹. NMR spectra

were obtained from Bruker Avance ^III400 or 600 Hz spec-

trometers at 400 or 600 Hz for H and 101 or 151 for ¹³C.

1

Chemical shifts (δ) are reported in parts per million (ppm)

relative to tetramethylsilane (TMS) as internal standard,

and the coupling constants (J) are reported in Hertz (Hz).

Splitting patterns are denoted as follows: singlet (s), doublet

(d), multiplet (m), triplet (t), quartet (q), doublet of doublets

(dd), doublet of triplets (dt), triplet of doublets (td), and dou-

blet of quartet (dq). Phenyl ring is denoted as “Ph,” while

quaternary carbons are distinguished with a “q.” The exact

molecular mass of target hybrids was obtained from Waters

Micromass LCT Premier TOF–MS spectrometer. Melting

points were determined with Stuart melting point instrument

Conclusion

A library of quinoline–1H-1,2,3-triazole molecular hybrids

was developed as antimicrobial agents using CuAAC. Com-

pound 16 was established as a promising scafold with a

broad-spectrum antimicrobial activity against the examined

pathogens. SAR analysis and molecular docking studies

Fig. 4 Binding modes of fuconazole and compound 16 around heme

cofactor in the active site of LDM. a Fluconazole. b Compound 16

Pose 1. c Pose 2. Protein interactions are displayed as dashed lines;

hydrogen bond (yellow), aromatic H-bond (cyan), pi-pi stacking

(light-blue) pi-cation (green) and metal-coordination (red). Atoms:

nitrogen (blue), oxygen (red), fuorine (lime green), carbon-com-

pound 16 (green); fuconazole (beige); protein (gray); heme cofactor

(brown tubes). Fe³⁺ion (brown sphere). Water (red balls)

1 3

Molecular Diversity

1 3

Molecular Diversity

(SMP-3) in open-end capillary tubes and were uncorrected.

The general synthetic procedures for azide precursors, com-

pounds 17 and 18, are given in Online Resource.

formula: C₁₈H₁₃ClN₄O; Mol. wt: 336.78 g/mol; R_f: 0.42

(EtOAc-hexane 80:20); mp: 112–114 °C.

FT-IR: 3057 (C-H_Ar), 2875 (OCH₂), 1497 (C=N), 1102

(C_Ar–Cl) cm⁻¹.

¹H NMR: (400 MHz, Chloroform-d) δ 8.96 (s, 1H, CH-

2), 8.24 (s, 1H, CH-5’ triazole), 8.18 (d, J = 8.2 Hz, 1H,

CH-4), 7.65–7.59 (m, 1H, CH-9’), 7.59–7.54 (m, 1H, CH-

12’), 7.53–7.48 (m, 1H, CH-6), 7.48–7.37 (m, 5H, CH-

3,5,7,10’,11’), 5.70 (s, 2H, OCH₂-6’).

Preparation of 8‑(prop‑2‑yn‑1‑yloxy)quinoline (8)

5.89 g (40.58 mmol, 1 eq.) of 8-HQ was added to a stir-

ring solution of anhydrous potassium carbonate (6.91 g,

50 mmol, 1.23 eq.) in 40 mL DMF at room temperature

(RT). After few minutes, the solution turned yellow, and

then, 3.62 mL (42 mmol, 1.04 eq.) of propargyl bromide

(80% in toluene) in DMF (5 mL) was added and stirring

continued at RT for 4 h. After that, the reaction mixture was

poured into a slurry of ice, stirred, and fltered in vacuo to

aford 6.90 g of deep brown solid.

¹³C NMR: (101 MHz, Chloroform-d) δ 153.83 (C-8),

149.36 (C–2), 143.86 (C-4’ triazole), 140.33 (C-8a), 136.01

(C-4), 134.80 (C-7’), 130.80, (10’) 130.73 (12’), 129.51 (C-

4a), 128.66 (C-8’), 127.88 (C-11’), 127.77 (C-9’), 126.70

(C-6), 125.47 (C-5’ triazole), 121.66 (C-3), 120.41 (C-5),

110.16 (C-7), 62.87 (OCH₂-6’).

HRMS: (ESI⁺-MS, m/z) calcd for C₁₈H₁₃ClN₄O

(M+Na)⁺: 359.0676, found: 359.0684.

Chemical formula: C₁₂H₉NO; Yield: 93%; Mol. wt:

183.21 g/mol; R_f= 0.51 (EtOAc-hexane 80:20). mp:

66–68 °C.

8‑((1‑(p‑tolyl)‑1H‑1,2,3‑triazol‑4‑yl)methoxy)qui-

noline(9 l) Brown solid; Yield: 66%; Chemical formula:

C₁₉H₁₆N₄O; Mol. wt: 316.36 g/mol; R_f: 0.43 (EtOAc-hex-

ane 80:20); mp: 126–128 °C.

FT-IR: 3275 (C≡C–H), 2121 (C≡C), 1501 (C=N), 1266

(C–O–C) cm⁻¹.

¹H NMR (400 MHz, Chloroform-d) δ 8.94 (dd, J=4.2,

1.7 Hz, 1H, CH-2), 8.13 (dd, J = 8.3, 1.7 Hz, 1H, CH-4),

7.53–7.39 (m, 3H, CH-6,5,3), 7.26 (dd, J=7.2, 1.7 Hz, 1H,

CH-7), 5.03 (d, J=2.4 Hz, 2H, OCH₂-9), 2.54 (t, J=2.4 Hz,

1H, CH-11).

FT-IR: 3078 (C-H_Ar), 2920 (Ar–CH₃), 1496 (C=N)

cm⁻¹.

¹H NMR: (400 MHz, Chloroform-d) δ 8.95 (dd, 4.3,

1.6 Hz, 1H, CH-2), 8.16 (s, 1H, CH-5’ triazole), 8.13 (dd,

J = 8.3, 1.6 Hz, 1H, CH-4), 7.58 (d, J = 8.2 Hz, 2H, CH-

9’,11’), 7.49–7.38 (m, 3H, CH-6,5,3), 7.36 (dd, J = 7.3,

1.5 Hz, 1H, CH-7), 7.28 (d, J=8.2 Hz, 2H, CH-8’,12’), 5.64

(s, 2H, OCH₂-6’), 2.39 (s, 3H Ar-CH₃-13’).

¹³C NMR (101 MHz, Chloroform-d) δ 153.09 (Cq-8),

149.45 (C-2), 140.34 (Cq-8a), 135.95 (C-4), 129.49 (Cq-

4a), 126.43 (C-6), 121.73 (C-3), 120.66 (C-5), 109.96 (C-7),

78.30 (Cq-10-), 76.08 (C-11), 56.52 (O-C-9).

¹³C NMR: (101 MHz, Chloroform-d) δ 153.83 (Cq-

8), 149.38 (C-2), 144.65 (Cq-4’ triazole), 140.33 (Cq-8a),

138.91 (Cq-7’), 135.98 (C-4), 134.68 (Cq-4a), 130.20 (C-

8’,12’), 129.51 (Cq-10’), 126.73 (C-6), 121.66 (C-3), 121.48

(C-5’ triazole), 120.46 (C-9’,11’), 120.30 (C-5’), 109.93 (C-

7), 62.88 (O-C-6’), 21.07 (Ar-C-13’).

General synthetic procedure

for quinoline‑1H‑1,2,3‑triazole molecular hybrids

To a 15 mL DCM solution of O-propargylated quinoline 8

(0.2 g, 1.09 mmol, 1 eq.) in a round-bottom fask (100 mL),

appropriate azide (1 eq.) was dissolved, then a solution of

10 mol% CuSO₄.5H₂O and 22 mol% sodium ascorbate in

water (15 mL) was added, and the fask’s contents were

stirred at RT for 2–4 h. After the reaction was complete, as

evident by the consumption of alkyne (8) on TLC, the reac-

tion mixture was diluted with water, fltered, and extracted

with DCM. Then, the combined DCM extracts were dried

over anhydrous sodium sulfate, fltered, and concentrated

in vacuo. Finally, the target hybrids were obtained in good

yields after column chromatography using hexane–EtOAc or

EtOAc–MeOH as eluant. The spectral data of active hybrids

shown in Table 1 are given below, while those of other com-

pounds along with the FT-IR, NMR, and HRMS spectra of

all compounds are presented in the Online Resource.

HRMS: (ESI⁺-MS, m/z) calcd for C₁₉H₁₆N₄O (M+Na)⁺:

339.1222, found: 339.1212.

3‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑triazol‑1‑yl)

phenol (9q) Brown solid; Yield: 62%; Chemical formula:

C₁₈H₁₄N₄O₂; Mol. wt: 318.33 g/mol; R_f: 0.27 (EtOAC-Hex-

ane 80:20); mp: 248–249 °C.

¹H NMR (400 MHz, DMSO-d₆): δ 10.07 (s, 1H, OH-

13’), 8.96 (s, 1H, CH-5’ triazole), 8.83 (d, J=3.8 Hz, 1H,

CH-2), 8.32 (dd, J=8.3, 1.7 Hz, 1H, CH-4), 7.58–7.50 (m,

3H, CH-6,5,3), 7.45 (dd, J=5.7, 3.3 Hz, 1H, CH-7), 7.39

(t, J=8.3 Hz, 1H, CH-11’), 7.35–7.29 (m, 2H, CH-8’,10’),

6.89 (dd, 1H, CH-12’), 5.43 (s, 2H, OCH₂-6’).

¹³C NMR (101 MHz, DMSO-d₆): δ 158.45 (Cq-9’),

153.76 (Cq-8), 148.98 (C-2), 143.55 (Cq-4’ triazole),

139.71 (Cq-8a), 137.52 (Cq-7’), 135.79 (C-4), 130.76 (C-

11’), 129.06 (Cq-4a), 126.72 (C-6), 123.17 (C-5’ triazole),

8‑((1‑(2‑chlorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)methoxy)

quinoline (9d) Dark brown solid; Yield: 73%; Chemical

1 3

Molecular Diversity

121.87 (C-3), 120.16 (C-5), 115.70 (C-12’), 110.50 (C-10’),

110.17 (C-7), 107.06 (C-8’), 61.67 (O-C-6’).

¹³C NMR (151 MHz, Chloroform-d): δ 157.26 (dd,

J=243.5, 10.4 Hz, Cq-2’’), 152.80 (dd, J=249.8, 12.1 Hz,

Cq-4’’), 144.32 (Cq-4’ triazole), 142.36 (dd, J = 10.9,

3.6 Hz, Cq-1’’), 135.86 (C-4), 126.79 (C-6), 124.62 (C-5’

triazole), 121.58 (C-3), 120.28 (C-5), 117.04 (d, J=8.5 Hz,

C-6’’), 110.63 (dd, J=22.6, 3.9 Hz, C-5’’), 110.07 (C-7),

105.07 (dd, J=26.8, 22.2 Hz, C-3’’), 69.02 (O-C-8’), 62.85

(O-C-6’), 49.86 (N–C-7’). Not observed (C-2,8,8a,4a).

HRMS: (ESI⁺-MS, m/z) calcd for C₁₈H₁₄N₄O₂

(M+Na)⁺: 341.1014, found: 341.1008.

4‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑triazol‑1‑yl)

phenol (9r) Brown solid; Yield: 44%; Chemical formula:

C₁₈H₁₄N₄O₂; Mol wt: 318.33 g/mol; R_f: 0.36 (EtOAC-Hex-

ane 80:20); mp: 277–279 °C.

¹³H NMR (400 MHz, DMSO-d₆): δ 9.95 (s, 1H, OH-13’),

8.91–8.76 (m, 2H, CH-5’ triazole, CH-2), 8.32 (dd, J=8.3,

1.7 Hz, 1H, CH-4), 7.69 (dd, J= 8.8 Hz, 2H, CH-8’,12’),

7.63–7.50 (m, 3H, CH-6,5,3), 7.45 (dd, J=5.5, 3.5 Hz, 1H,

CH-7), 6.94 (dd, J = 8.8 Hz, 2H, CH-9’,11’), 5.42 (s, 2H,

OCH₂-6’).

8‑((1‑(2‑(3,4‑difluorophenoxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10d) Cream solid; Yield:

54%; Chemical formula: C₂₀H₁₆F₂N₄O₂; Mol. wt: 382.36 g/

mol; R_f: 0.15 (EtOAc-hexane 80:20); mp: 110–112 °C.

¹H NMR (400 MHz, Chloroform-d): δ 8.95 (s, 1H,

CH-2), 8.17 (s, 1H, CH-4), 7.94 (s, 1H, CH-5’ triazole),

7.63–7.17 (m, 4H, CH-6,5,3,7), 7.00 (q, J=9.3 Hz, 1H, CH-

5’’), 6.60 (ddd, J = 11.7, 6.5, 2.9 Hz, 1H,, CH-2’’), 6.47

(dq, J=8.5, 2.7 Hz, 1H,, CH-6’’), 5.60 (s, 2H, O-CH₂-6’),

4.72 (t, J=5.0 Hz, 2H, N–CH₂-7’), 4.29 (t, J=5.0 Hz, 2H,

O-CH₂-8’).

¹³C NMR (101 MHz, DMSO-d₆): δ 157.78 (Cq-10’),

153.79 (Cq-8), 148.96 (C-2), 143.30 (Cq-4’ triazole),

139.71 Cq-8a), 135.78 (C-4), 129.05 (Cq-4a), 128.69 (Cq-

7’), 126.73 (C-6), 123.09 (C-5’ triazole), 122.03, 121.85

(C-3), 120.11 (C-5), 116.02 (C-9’,11’), 110.12 (C-7), 61.75

(O-C-6’).

¹³C NMR (101 MHz, Chloroform-d): δ 153.94 (dd,

J=8.5, 2.3 Hz, Cq-1’’), 150.39 (dd, J=248.6, 13.9 Hz, Cq-

3’’), 145.50 (dd, J=242.9, 11.3 Hz, Cq-4’’), 144.37 (Cq-4’

triazole), 126.81 (C-6), 124.40 (C-5’ triazole), 117.33 (dd,

J = 18.6, 1.4 Hz, (C-5’’), 109.72 (dd, J = 5.8, 3.5 Hz, C-

7,6’’), 104.50 (d, J=20.4 Hz, C-2’’), 66.97 (O-C-8’), 62.79

(O-C-6’), 49.60 (N–C-7’). Not observed (C-2,3,4,5,8,8a,4a).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₆F₂N₄O₂

(M+Na)⁺: 405.1139, found: 405.1149.

HRMS: (ESI⁺-MS, m/z) calcd for C₁₈H₁₄N₄O₂

(M+Na)⁺: 341.1014, found: 341.1008.

8‑((1‑(2‑phenoxyethyl)‑1H‑1,2,3‑triazol‑4‑yl)methoxy)

quinoline (10a) Light brown solid; Yield: 59%; Chemi-

cal formula: C₂₀H₁₈N₄O₂; Mol. wt: 346.38 g/mol; R_f: 0.20

(EtOAc-hexane 80:20); mp: 108–110 °C.

¹H NMR (400 MHz, Chloroform-d): δ 8.94 (s, 1H, CH-

2), 8.16 (s, 1H, CH-4), 7.98 (s, 1H, CH-5’triazole), 7.41 (s,

1H, CH-6), 7.35 (s, 3H, CH-5,3,7), 7.27–7.20 (t, J=7.8 Hz,

2H, CH-3’’,5’’), 6.95 (t, J=7.4 Hz, 1H, CH-4’’), 6.81–6.79

(d, J = 7.8 Hz, 2H, CH-2’’,6’’), 5.60 (s, 2H, O-CH₂-6’),

4.72 (t, J=5.0 Hz, 2H, N–CH₂-7’), 4.33 (t, J=5.0 Hz, 2H,

O-CH₂-8’).

8‑((1‑(2‑(4‑(trifuoromethyl)phenoxy)ethyl)‑1H‑1,2,3‑tri-

azol‑4‑yl)methoxy)quinoline (10e) Dark brown crystal-

line solid; Yield: 37%; Chemical formula: C₂₁H₁₇F₃N₄O₂;

Mol. wt: 414.38 g/mol; R_f: 0.17 (EtOAc-hexane 80:20);

mp: 150–152 °C.

¹³C NMR (101 MHz, Chloroform-d): δ 157.69 (O-Cq-

1”), 153.29 (C-2), 144.22 (Cq-4’ triazole), 129.59 (C-3”,5”),

126.88 (C-6), 124.48 (C-5’ triazole), 121.65 (C-4”), 114.52

(C-2”,6”), 110.49 (C-7), 66.12 (O-C-8’), 62.83 (O-C-6’),

49.81 (N–C-7’). Not observed (C-3,4,5,8,8a,4a).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₈N₄O₂

(M+Na)⁺: 369.1335, found: 369.1327.

¹H NMR (600 MHz, Chloroform-d): δ 8.93 (s, 1H,

CH-2), 8.12 (d, J = 8.2 Hz, 1H, CH-4), 7.92 (s, 1H, CH-

5’ triazole), 7.48 (d, J=8.4 Hz, 2H, CH-3’’,5’’), 7.44–7.38

(m, 3H, CH-6,5,3), 7.30 (d, J = 6.7 Hz, 1H, CH-7), 6.84

(d, J = 8.4 Hz, 2H, CH-2’’,6’’), 5.58 (s, 2H, O-CH₂-6’),

4.74 (t, J=5.1 Hz, 2H, N–CH₂-7’), 4.38 (t, J=5.1 Hz, 2H,

O-CH₂-8’).

¹³C NMR (151 MHz, Chloroform-d): δ 160.12 (Cq-

1’’), 153.82 (Cq-8), 149.35 (C-2), 144.42 (Cq-4’ triazole),

140.40 (Cq-8a), 135.94 (C-4), 129.52 (Cq-4a), 127.02 (q,

J=3.8 Hz, C-3’’,5’’), 126.69 (C-6), 124.37 (C-5’ triazole),

125.44–122.80 (m, Ph-Cq-7’’), 121.64 (C-3), 120.25 (C-5),

114.51 (Cq-2’’,6’’), 110.02 (C-7), 66.40 (O-C-8’), 62.87

(O-C-6’), 49.56 (N–C-7’).

8‑((1‑(2‑(2,4‑difluorophenoxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10c) Cream solid; Yield:

52%; Chemical formula: C₂₀H₁₆F₂N₄O₂; Mol. wt: 382.36 g/

mol; R_f: 0.15 (EtOAc-hexane 80:20); mp: 136–138 °C.

¹H NMR (600 MHz, Chloroform-d): δ 8.93 (s, 1H, CH-

2), 8.14 (s, 1H, CH-4), 7.98 (s, 1H, CH-5’triazole), 7.50–

7.30 (m, 4H, CH-6,5,3,7), 6.81–6.72 (m, 2H, CH-5’’,3’’),

6.68 (dddd, J=9.2, 7.8, 3.0, 1.6 Hz, 1H, CH-6’’), 5.60 (s,

2H, O-CH₂-6’), 4.72 (t, J=5.0 Hz, 2H, N–CH₂-7’), 4.35 (t,

J=5.0 Hz, 2H, O-CH₂-8’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₁H₁₇F₃N₄O₂

(M+Na)⁺: 437.1201, found: 437.1211

1 3

Molecular Diversity

8‑((1‑(2‑(2,4‑dibromophenoxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10j) Brown solid; Yield:

88%; Chemical formula: C₂₀H₁₆Br₂N₄O₂; Mol. wt: 504.17 g/

mol; R_f: 0.26 (EtOAc-Hexane, 80:20); mp: 129–131 °C.

¹H NMR (600 MHz, Chloroform-d): δ 8.92 (s, 1H, CH-

2), 8.18–8.08 (m, 1H, CH-4), 8.04 (s, 1H, CH-5’ triazole),

7.56–7.51 (m, 2H, CH-5’’,3’’), 7.49–7.21 (m, 5H, CH-

6,5,3,7,6’’), 5.62 (s, 2H, O-CH₂-6’), 4.77 (t, J=4.9 Hz, 2H,

N–CH₂-7’), 4.28 (t, J=4.9 Hz, 2H, O-CH₂-8’).

(C-4’’), 110.84 (C-6’’), 109.92 (C-7), 66.12 (O-C-8’), 62.80

(O-C-6’), 49.96 (N–C-7’), 16.15 (Ph-C-7’’). Not observed

(C-2,4a,5,3).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₁H₂₀N₄O₂

(M+Na)⁺: 383.1484, found: 383.1491.

8‑((1‑(2‑(m‑tolyloxy)ethyl)‑1H‑1,2,3‑triazol‑4‑yl)meth-

oxy)quinoline (10n) Beige solid; Yield: 75%; Chemical

formula: C₂₁H₂₀N₄O₂; Mol. wt: 360.41 g/mol; R_f: 0.22

(EtOAc-Hexane, 80:20); mp: 90–92 °C.

¹³C NMR (151 MHz, Chloroform-d): δ 153.53 (Cq-8),

151.46 (Cq-1’’), 144.23 (Cq-4’ triazole), 135.63 (C-4),

135.05 (C-3’’,5’’), 131.24 (C-6’’), 126.75 (C-6), 124.97 (C-

5’ triazole), 121.85 (C-3), 120.37 (C-5), 118.71 (Cq-2’’),

118.21 (Cq-4’’), 110.00 (C-7), 70.70 (O-C-8’), 62.85 (O-C-

6’), 50.20 (N–C-7’). Not observed (C-2,8a,4a).

¹H NMR (600 MHz, Chloroform-d): δ 8.93 (s, 1H, CH-

2), 8.13 (s, 1H, CH-4), 7.95 (s, 1H, CH-5’ triazole), 7.47–

7.36 (m, 3H, CH-6,5,3), 7.35–7.29 (m, 1H, CH-7), 7.11 (t,

J=7.9 Hz, 1H, CH-5’’), 6.76 (d, J=7.5 Hz, 1H, CH-4’’),

6.63 (t, J=2.0 Hz, 1H, CH-2’’), 6.59 (dd, J=8.2, 2.5 Hz,

1H, CH-6’’), 5.58 (s, 2H, O-CH₂-6’), 4.70 (t, J = 5.1 Hz,

2H, N–CH₂-7’), 4.31 (t, J=5.0 Hz, 2H, O-CH₂-8’), 2.28 (s,

3H, Ph-C-7’’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₆Br₂N₄O₂

(M+Na)⁺: 524.9538, found: 524.9545.

8‑((1‑(2‑(3,5‑dimethoxyphenoxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10 l) Beige solid; Yield: 69%;

Chemical formula: C₂₂H₂₂N₄O₄; Mol. wt: 406.43 g/mol; R_f:

0.17 (EtOAc-Hexane, 80:20); mp: 138–140 °C.

¹³C NMR (151 MHz, Chloroform-d): δ 157.74 (Cq-1’’),

153.90 (Cq-8), 149.24 (C-2), 144.21 (Cq-4’ triazole), 140.43

(Cq-8a), 139.70 (Cq-3’’), 135.93 (C-4), 129.48 (Cq-4a),

129.30 (C-5’’), 126.78 (C-6), 124.45 (C-5’ triazole), 122.47

(C-4’’), 121.46 (C-3), 120.26 (C-5), 115.48 (C-2’’), 111.33

(C-6’’), 110.08 (C-7), 66.09 (O-C-8’), 62.94 (O-C-6’), 49.82

(N–C-7’), 21.42(Ph-C-7’’).

¹H NMR (600 MHz, Chloroform-d): δ 8.95 (s, 1H,

CH-2), 8.14 (s, 1H, CH-4), 7.96 (s, 1H, CH-5’ triazole),

7.41–7.45 (m, 3H, CH-6,5,3), 7.34 (s, 1H, CH-7), 6.10

(t, J = 2.0 Hz, 1H, CH-4’’), 6.01 (d, J = 2.1 Hz, 2H, CH-

2’’,6’’), 5.59 (s, 2H, O-CH₂-6’), 4.71 (t, J=5.1 Hz, 2H, N–

CH₂-7’), 4.31 (t, J = 5.0 Hz, 2H, O-CH₂-8’), 3.74 (s, 6H,

O-CH₃-7’’,8’’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₁H₂₀N₄O₂

(M+Na)⁺: 383.1484, found: 383.1489.

8‑((1‑(2‑(3,5‑dimethylphenoxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10q) Brown solid; Yield:

50%; Chemical formula: C₂₂H₂₂N₄O₂; Mol. wt: 374.44 g/

mol; R_f: 0.20 (EtOAc-Hexane, 80:20). mp: 92–94 °C.

¹H NMR (600 MHz, Chloroform-d): δ 8.95 (s, 1H, CH-

2), 8.14 (s, 1H, CH-4), 7.96 (s, 1H, CH-5’ triazole), 7.50–

7.36 (m, 3H, CH-6,5,3), 7.34 (s, 1H, CH-7), 6.61 (s, 1H,

CH-4’’), 6.45 (s, 2H, CH-2’’,6’’), 5.59 (s, 2H, O-CH₂-6’),

4.70 (t, J=5.0 Hz, 2H, N-CH₂-7’), 4.31 (t, J=5.0 Hz, 2H,

O-CH₂-8’), 2.25 (s, 6H, Ph-CH₃-7’’,8’’).

¹³C NMR (151 MHz, Chloroform-d): δ 161.61 (Cq-

3’’,5’’), 159.60 (Cq-1’’), 149.10 (C-2), 136.03 (C-4), 126.83

(C-6),124.39 (C-5’ triazole), 121.57 (C-3), 120.26 (C-5),

110.19 (C-7), 93.93 (C-4’’), 93.47 (C-2’’,6’’), 66.18 (O-C-

8’), 62.96 (O-C-6’), 55.41 (O-C-7’’,8’’), 49.74 (N–C-7’).

Not observed (Cq-8, 8a,4a)

HRMS: (ESI⁺-MS, m/z) calcd for C₂₂H₂₂N₄O₄

(M+Na)⁺: 429.1539, found: 429.1542.

8‑((1‑(2‑(o‑tolyloxy)ethyl)‑1H‑1,2,3‑triazol‑4‑yl)meth-

oxy)quinoline (10 m) Beige solid; Yield: 69%; Chemi-

cal formula: C₂₁H₂₀N₄O₂; Mol. wt: 360.41 g/mol; R_f: 0.22

(EtOAc-Hexane, 80:20); mp: 98–100 °C.

¹³C NMR (151 MHz, Chloroform-d): δ 157.76 (Cq-1’’),

153.92 (Cq-8), 149.26 (C-2), 144.21 (Cq-4’ triazole), 140.27

(Cq-8a), 139.41 (Cq-3’’,5’’), 135.98 (C-4), 129.50 (Cq-

4a), 126.81 (C-6), 124.45 (C-5’ triazole), 123.40 (C-4’’),

121.63 (C-3), 120.19 (C-5), 112.31 (C-2’’,6’’), 110.16 (C-

7), 66.03 (O-C-8’), 62.97 (O-C-6’), 49.85 (N-C-7’), 21.35

(Ph-C-7’’,8’’).

¹H NMR (400 MHz, Chloroform-d): δ 8.93 (s, 1H, CH-

2), 8.13 (s, 1H, CH-4), 7.94 (s, 1H, CH-5’ triazole), 7.50–

7.26 (m, 4H, CH-6,5,3,7), 7.15–7.01 (m, 2H, CH-5’’,3’’),

6.85 (t, J = 7.4 Hz, 1H, CH-4’’), 6.70 (d, J = 8.0 Hz, 1H,

CH-6’’), 5.60 (s, 2H, O-CH₂-6’), 4.74 (t, J= 5.0 Hz, 2H,

N–CH₂-7’), 4.31 (t, J=5.0 Hz, 2H, O-CH₂-8’), 2.02 (s, 3H,

Ph-CH₃-7’’).

5‑(2‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑triazol‑1‑yl)

ethoxy)benzene‑1,3‑diol (10v) Beige solid; Yield: 50%;

Chemical formula: C₂₀H₁₈N₄O₄; Mol. wt: 378.38 g/mol; R_f

: 0.07 (EtOAc-Hexane, 80:20). mp: 197–199 °C

¹³C NMR (101 MHz, Chloroform-d): δ 155.81 (Cq-

1’’), 153.90 (Cq-8), 144.22 (Cq-4’ triazole), 140.32 (Cq-

8a), 135.85 (C-4), 130.90 (C-3’’), 126.84 (C-5’’), 126.79

(C-6), 126.69 (Cq-2’’), 124.48 (C-5’ triazole), 121.28

¹H NMR (600 MHz, DMSO-d₆): δ 9.21 (s, 2H, OH-

7’’,8’’), 8.84 (d, J=4.3 Hz, 1H, CH-2), 8.35 (s, 1H, CH-5’

triazole), 8.32 (dd, J=8.2, 1.7 Hz, 1H, CH-4), 7.57–7.50 (m,

1 3

Molecular Diversity

3H, CH-6,5,3), 7.42 (dd, J=6.5, 2.4 Hz, 1H, CH-7), 5.86 (t,

J=2.0 Hz, 1H, CH-4’’), 5.81 (d, J=2.0 Hz, 2H, CH-2’’,6’’),

5.37 (s, 2H, O-CH₂-6’), 4.76 (t, J=5.1 Hz, 2H, N-CH₂-7’),

4.29 (t, J=5.2 Hz, 2H, O-CH₂-8’).

2‑chloro‑N‑(2‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑tri-

azol‑1‑yl)ethyl)aniline (11d) Brown solid; Yield: 53%;

Chemical formula: C₂₀H₁₈ClN₅O; Mol. wt: 379.84 g/mol;

R_f: 0.24 (EtOAc-Hexane, 80:20); mp: 118–120 °C.

¹H NMR (600 MHz, Chloroform-d): δ 8.85 (d, J=4.7 Hz,

1H, CH-2), 8.04 (dd, J= 8.1, 1.7 Hz, 1H, CH-4), 7.70 (s,

1H, CH-5’triazole), 7.41–7.30 (m, 3H, CH-6,5,3), 7.22

(dd, J = 7.2, 1.6 Hz, 1H, CH-7), 7.15 (dd, J = 7.9, 1.5 Hz,

1H, CH-3’’), 7.02 (td, J = 7.8, 1.5 Hz, 1H, CH-5’’), 6.56

(td, J=7.7, 1.4 Hz, 1H, CH-4’’), 6.52 (dd, J=8.2, 1.4 Hz,

1H, CH-6’’), 5.46 (s, 2H, O-CH₂-6’), 4.55 (t, J = 6.4 Hz,

1H, NH-9’), 4.45 (t, J = 5.9 Hz, 2H, N-CH₂-7’), 3.63 (q,

J=5.9 Hz, 2H, N-CH₂-8’).

¹³C NMR (151 MHz, DMSO-d₆): δ 159.57 (Cq-1’’),

159.03 (Cq-3’’,5’’), 153.87 (Cq-8), 148.96 (C-2), 142.63

(Cq-4’ triazole), 139.77 (Cq-8a), 135.75 (C-4), 129.06 (Cq-

4a), 126.70 (C-6), 125.21 (C-5’ triazole), 121.81 (C-3),

120.03 (C-5), 110.15 (C-7), 96.01 (C-4’’), 93.28 (C-2’’,6’’),

65.80 (O-C-8’), 61.90 (O-C-6’), 49.08 (N-C-7’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₈N₄O₄

(M+Na)⁺: 401.1226, found: 401.1229.

8‑((1‑(2‑(naphthalen‑2‑yloxy)ethyl)‑1H‑1,2,3‑tria-

zol‑4‑yl)methoxy)quinoline (10z) Beige solid; Yield:

50%; Chemical formula: C₂₄H₂₀N₄O₂; Mol. wt: 396.44 g/

mol; R_f: 0.20 (EtOAc-Hexane, 80:20); mp: 123–125 °C.

¹H NMR (400 MHz, Chloroform-d): δ 8.92 (s, 1H, CH-

2), 8.12 (s, 1H, CH-4), 7.99 (s, 1H, CH-5’ triazole), 7.73

(d, J=8.1 Hz, 1H, CH-5’’), 7.70–7.64 (m, 2H, CH-4’’,8’’),

7.47–7.29 (m, 6H, CH-7’’,6,5,3,7,6’’), 7.04 (d, J=2.3 Hz,

1H, CH-1’’), 7.01 (dd, J= 8.8, 2.5 Hz, 1H, CH-3’’), 5.58

(s, 2H, OCH₂-6’), 4.75 (t, J=5.0 Hz, 2H, NCH₂-7’),4.41 (t,

J=4.9 Hz, 2H, OCH₂-8’).

¹³C NMR (151 MHz, Chloroform-d): δ 153.80 (Cq-8),

149.21 (C-2), 144.10 (Cq-4’ triazole), 142.62 (Cq-1’’),

140.26 (Cq-8a), 135.93 (C-4), 129.46 (Cq-4a), 129.42 (C-

3’’), 127.84 (C-5’’), 126.69 (C-6), 124.14 (C-5’ triazole),

121.60 (C-3), 120.22 (C-5), 119.54 (Cq-2’’), 118.11 (C-4’’),

110.91 (C-6’’), 109.93 (C-7), 62.75 (O-C-6’), 49.16 (N-C-

7’), 43.30 (N-C-8’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₈ClN₅O

(M+Na)⁺: 402.1098, found: 402.1102.

4.2.18 8‑((1‑phenethyl‑1H‑1,2,3‑triazol‑4‑yl)methoxy)

quinoline (14) Brown solid; Yield: 51%; Chemical for-

mula: C₂₀H₁₈N₄O; Mol. wt: 330.38 g/mol; R_f: 0.23 (EtOAc-

Hexane, 80:20); mp: 70–72 °C.

¹³C NMR (101 MHz, Chloroform-d): δ 155.63 (Cq-2’’),

144.24 (Cq-4’ triazole), 135.84 (C-4), 134.25 (Cq-8’’a),

129.68 (C-4’’), 129.27 (Cq-4’’a), 127.64 (C-5’’), 126.81

(C-8’’), 126.57 (C-7’’), 124.50 (C-5’ triazole), 124.07 (C-

6’’), 118.37 (C-3’’), 109.86 (C-7), 106.99 (C-1’’), 66.15

(O-C-8’), 62.83 (O-C-6’), 49.74 (N-C-7’). Not observed

(C-8,2,8a,4a,6,5,3).

¹H NMR (600 MHz, Chloroform-d): δ 8.90 (s, 1H, CH-2),

8.11 (d, J=8.2 Hz, 1H, CH-4), 7.48 (s, 1H, CH-5’ triazole),

7.43–7.35 (m, 3H, CH-6,5,3), 7.25 (d, J=6.8 Hz, 1H, CH-7),

7.15–7.09 (m, 3H, CH-11’,13’,12’), 6.99 (dd, J=7.7, 1.7 Hz,

2H, CH-10’,14’), 5.51 (s, 2H, O-CH₂-6’), 4.52 (t, J=7.3 Hz,

2H, N-CH₂-7’), 3.14 (t, J=7.3 Hz, 2H, Ph-CH₂-8’).

¹³C NMR (151 MHz, Chloroform-d): δ 153.78 (Cq-

8), 149.22 (C-2), 143.86 (Cq-4’ triazole), 140.29 (Cq-8a),

136.83 (Cq-9’), 136.00 (C-4), 129.48 (Cq-4a), 128.70 (C-

11’,13’), 128.56 (C-10’,14’), 127.00 (C-12’), 126.76 (C-6),

123.47 (C-5’ triazole), 121.59 (C-3), 120.15 (C-5), 109.99

(C-7), 62.87 (O-C-6’), 51.66 (N-C-7’), 36.61 (Ph-C-8’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₈N₄O (M+Na)⁺:

353.1378, found: 353.1384.

HRMS: (ESI⁺-MS, m/z) calcd for C₂₁H₁₈N₄O₃

(M+Na)⁺: 419.1484, found: 419.1494.

N‑(2‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑triazol‑1‑yl)

ethyl)aniline (11a) Of-white solid; Yield: 51%; Chemi-

cal formula: C₂₀H₁₉N₅O; Mol. wt: 345.40 g/mol; R_f: 0.20

(EtOAc-Hexane, 80:20); mp: 96–98 °C

¹H NMR (600 MHz, Chloroform-d): δ 8.93 (s, 1H,

CH-2), 8.15 (s, 1H, CH-4), 7.74 (s, 1H, CH-5’ triazole),

7.49–7.39 (m, 3H, CH-6,5,3), 7.31 (s, 1H, CH-7), 7.13 (dd,

J=8.5, 7.2 Hz, 2H, CH-3’’,5’’), 6.71 (t, J=7.3 Hz, 1H, CH-

4’’), 6.54 (dd, J=8.5, 1.2 Hz, 2H, CH-2’’,6’’), 5.56 (s, 2H,

O-CH₂-6’), 4.52 (dd, J=6.5, 4.9 Hz, 2H, N-CH₂-7’), 3.65 (t,

J=5.7 Hz, 2H, N-CH₂-8’). Not observed (NH-9’).

¹³C NMR (151 MHz, Chloroform-d): δ 153.82 (Cq-8),

149.24 (C-2), 146.59 (Cq-1’’), 144.15 (Cq-4’ triazole),

140.18 (Cq-8a), 136.08 (C-4), 129.62 (Cq-4a), 129.45 (C-

3’’,5’’), 126.82 (C-6), 124.12 (C-5’ triazole), 121.68 (C-3),

120.26 (C-5), 118.30 (C-4’’), 113.00 (C-2’’,6’’), 110.00 (C-

7), 62.89 (O-C-6’), 49.33 (N-C-7’), 43.66 (N-C-8’).

HRMS: (ESI⁺-MS, m/z) calcd for C₂₀H₁₉N₅O (M+Na)⁺:

368.1487, found: 368.1486.

4.2.19 2‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑tria-

zol‑1‑yl)ethanol (15) Brown solid; Yield: 60%; Chemi-

cal formula: C₁₄H₁₄N₄O₂; Mol. wt: 270.29 g/mol; R_f: 0.05

(EtOAc-Hexane, 80:20); 0.17 (EtOAc–MeOH, 98:2); mp:

140–142 °C

¹H NMR (400 MHz, Chloroform-d): δ 8.82 (dd, J=4.4,

1.7 Hz, 1H, CH-2), 8.11 (dd, J = 8.3, 1.7 Hz, 1H, CH-4),

7.94 (s, 1H, CH-5’ triazole), 7.47–7.35 (m, 3H, CH-6,5,3),

7.27 (dd, J=7.4, 1.6 Hz, 1H, CH-7), 5.44 (s, 2H, O-CH₂-6’),

4.45 (dd, J=5.7, 3.9 Hz, 2H, N–CH₂-7’), 4.00 (t, J=5.0 Hz,

2H, CH₂-8’), Not observed (OH-9’).

1 3

Molecular Diversity

¹³C NMR (101 MHz, Chloroform-d): δ 153.76 (Cq-

8), 149.12 (C-2), 143.69 (Cq-4’ triazole), 140.00 (Cq-8a),

136.16 (C-4), 129.48 (Cq-4a), 126.80 (C-6), 124.66 (C-5’

triazole), 121.67 (C-3), 120.16 (C-5), 109.74 (C-7), 62.72

(O-C-6’), 61.00 (C-8’), 52.82 (N–C-7’).

128.50 (Cq-4a), 128.07 (C-12,16), 127.56 (C-13,15), 125.62

(C-6), 125.56 (C-14), 120.55 (C-3), 118.67 (C-5), 107.81

(C-7), 68.75 (C-9), 34.61 (C-10).

HRMS: (ESI⁺-MS, m/z) calcd for C₁₇H₁₅NO (M+Na)⁺:

272.1051, found: 272.1046.

2‑(4‑((quinolin‑8‑yloxy)methyl)‑1H‑1,2,3‑triazol‑1‑yl)

ethyl 4‑methylbenzenesulfonate (16) Pale-yellow oil;

Yield: 71%; Chemical formula: C₁₄H₁₄N₄O₂; Mol. wt:

424.47 g/mol; R_f: 0.20 (EtOAc-Hexane, 80:20).

In vitro bioassays

Sample preparation

¹H NMR (600 MHz, Chloroform-d): δ 8.94 (d, J=3.2 Hz,

1H, CH-2), 8.15 (d, J=8.2 Hz, 1H, CH-4), 7.85 (s, 1H, CH-

5’ triazole), 7.65 (d, J=8.3 Hz, 2H, CH-3’’,5’’), 7.49–7.40

(m, 3H, CH-6,5,3), 7.31 (d, J=7.5 Hz, 1H, CH-7), 7.24 (d,

J=8.0 Hz, 2H, CH-2’’,6’’), 5.51 (s, 2H, O-CH₂-6’), 4.60 (t,

J=5.2 Hz, 2H, N-CH₂-7’), 4.40 (t, J=5.2 Hz, 2H, O-CH₂-

8’), 2.38 (s, 3H, Ph-CH₃-7’’).

Samples of examined hybrids were stored frozen at −20 °C

and then prepared in DMSO and water to a fnal concen-

tration of 32 μg/mL in 384-well plates for the preliminary

screening phase. For the hit-confrmation phase, they were

further diluted serially 1:2-fold for eight times. The concen-

tration was prepared in a non-binding surface plate (NBS)

for bacterial and fungal strains, tissue-culture-treated black

wall/clear bottom for mammalian cell types (cytotoxicity

assay), and polypropylene 384-well for hemolysis assays.

The preparation was done in duplicates (n=2) while keep-

ing the fnal concentration of DMSO to 1% DMSO for pre-

liminary antimicrobial screening phase and 0.5% for the

hit-confrmation phase, cytotoxicity, and hemolytic assays.

¹³C NMR (151 MHz, Chloroform-d): δ 153.81 (Cq-8),

149.25 (C-2), 145.38 (Cq-1’’), 144.28 (Cq-4’ triazole),

140.16 (Cq-8a), 136.16 (C-4), 132.11 (Cq-4’’), 130.01 (C-

3’’,5’’), 129.54 (Cq-4a), 127.83 (C-2’’,6’’), 126.79 (C-6),

124.39 (C-5’ triazole), 121.67 (C-3), 120.35 (C-5), 110.16

(C-7), 67.32 (O-C-8’), 62.84 (O-C-6’), 49.09 (N-C-7’),

21.59 (Ph-C-7’’).

8‑(2‑(p‑tolyloxy)ethoxy)quinoline (17) Beige solid; Yield:

91%; Chemical formula: C₁₈H₁₇NO₂; Mol. wt: 279.33 g/

mol; R_f: 0.60 (EtOAc-hexane 80:20); mp: 132–134 °C

¹H NMR (400 MHz, Chloroform-d) δ 8.98 (dd, J=4.3,

1.7 Hz, 1H, CH-2), 8.17 (dd, J = 8.3, 1.7 Hz, 1H, CH-4),

7.54–7.40 (m, 3H, CH-6,5,3), 7.20 (dd, J = 7.5, 1.5 Hz,

1H, CH-7), 7.09 (d, J = 8.7 Hz, 2H, CH-13,14), 6.88

(d, J = 8.6 Hz, 2H, CH-12,16), 4.63 (t, J = 5.3 Hz, 2H,

O-CH₂-9), 4.54 (t, J=5.3 Hz, 2H, O-CH₂-10), 2.29 (s, 3H,

Ph-CH₃-17).

Antibacterial assay

Selected bacteria strains were cultured in Cation-adjusted

Mueller–Hinton broth at 37 °C overnight, followed by dilut-

ing each culture sample by 40-fold in fresh broth and incu-

bating at 37 °C for 1.5–3 h. After that, the resultant mid-log

phase cultures were diluted (CFU/mL measured by OD₆₀₀

)

and added to each well plate containing the compounds to

give a cell density of 5×10⁵CFU/mL and a total volume of

50 μL. The plates were then covered and incubated at 37 °C

for 18 h without shaking.

¹³C NMR (101 MHz, Chloroform-d) δ 156.46 (Cq-11),

154.48 (Cq-8), 149.35 (C-2), 140.27 (Cq-8a), 136.12 (C-4),

130.34 (Cq-14), 129.96 (C-13,15), 129.60 (Cq-4a), 126.71

(C-6), 121.68 (C-3), 120.23 (C-5), 114.58 (C-12,16), 109.55

(C-7), 67.41 (O-C-9), 66.37 (O-C-10), 20.50 (Ph-C-17).

LRMS: (ESI⁺-MS, m/z) C₁₈H₁₇NO₂(M + Na)⁺:

302.1315.

To determine the inhibition of bacterial growth, a Tecan

M1000 Pro monochromator plate reader was used to meas-

ure the absorbance at 600 nm (OD₆₀₀) and subsequently the

percentage growth inhibition was calculated for each well

using the negative control (media only) and positive control

(bacteria without inhibitors) on the same plate as references.

The MIC was then determined as the lowest concentration

causing full growth inhibition, i.e., ≥80% inhibition.

8‑phenethoxyquinoline (18) Pale-yellow oil; Yield: 90%;

Chemical formula: C₁₇H₁₅NO; Mol. wt: 249.31; R_f: 0.61

(EtOAc-hexane 80:20).

Antifungal assay

¹H NMR (400 MHz, Chloroform-d) δ 8.88 (dd, J=4.2,

1.7 Hz, 1H, CH-2), 8.03 (dd, J = 8.3, 1.7 Hz, 1H, CH-4),

7.40–7.21 (m, 8H, CH-6,5,3,12,13,14,15,16), 6.96 (dd,

J=7.5, 1.4 Hz, 1H, CH-7), 4.34 (t, J=7.7 Hz, 2H, CH-9),

3.28 (t, J=7.7 Hz, 2H, CH-10).

Antifungal activity was determined by culturing the fungi

strains on a Yeast Extract-Peptone Dextrose (YPD) agar for

three days at 30 °C. Then, a yeast suspension of 1×10⁶to

5×10⁶CFU/mL was prepared from fve colonies, and the

suspension was diluted and added to each well plate con-

taining the compound to give a fnal cell density of fungi

¹³C NMR (101 MHz, Chloroform-d) δ 153.49 (Cq-8),

148.28 (C-2), 139.30 (Cq-8a), 136.78 (Cq-11), 134.90 (C-4),

1 3

Molecular Diversity

suspension of 2.5×10³CFU/mL and a total volume of 50

μL. All plates were covered and incubated at 35 °C for 36 h

without shaking.

In silico procedures

The X-ray crystal structure and experimental density of

selected proteins were refned using PrimeX 5.6 [33],

before docking simulations. Subsequently, the refned

protein structure was prepared with the Protein Prepara-

tion Wizard [34]. This approach allowed the controlled

minimization o f protein structure for high energy contacts,

improper binding site packing, and other shortfalls inher-

ited from the experimental crystal data. Moreover, con-

sidering the importance of accounting for protein fexibil-

ity to accurate prediction of protein–ligand interactions,

induced-ft docking protocol [35] was employed to study

the behavior of compound 16 and other potent hybrids

in the active sites of selected antimicrobial targets. 3D

structures of all ligands were prepared with LigPrep [36],

while their ionization states were assigned at pH 7.0 2.0

using Epik [37]. The overall workfow adopted for the in

silico studies is provided in the Online Resource (Fig. S2).

Analysis of fungi growth inhibition was achieved using

Biotek Multifo Synergy HTX plate reader to measure the

absorbance at 630 nm (OD₆₃₀) for C. albicans, while the dif-

ference in absorbance between 600 and 570 nm (OD_600-570

)

was determined for C. neoformans, both after the addition

of resazurin (0.001% fnal concentration) and incubation at

35 °C for 2 h. The percentage inhibition was determined as

in antibacterial assay.

Cytotoxicity assay

HEK293 cells (ATCC CRL-1573) were counted manually

in a Neubauer hemocytometer and then plated in the well

plates containing the hit compounds (≥80% inhibition) to

give a density of 5000 cells/well in a fnal volume of 50 μL.

The utilized growth media was a Dulbecco’s modifed eagle

medium supplemented with 10% fetal bovine serum (from

GE Healthcare Australia; cat #SH30084.03). The cells and

the compounds were incubated at 37 °C for 20 h in 5% CO₂.

Subsequently, cytotoxicity was determined by measur-

ing the fuorescence intensity at 560/10 nm and 590/10 nm

(F_560/590) as excitation and emission wavelengths, respec-

tively, using a Tecan M1000 Pro monochromator plate

reader and automatic gain calculation, after the addition of

5 μL of 25 μg/mL resazurin (2.3 μg/mL fnal concentration)

and another 3-h incubation at 37 °C in 5% CO₂. The con-

centration at 50% cytotoxicity (CC₅₀) was then calculated

by curve ftting the inhibition values vs. log(concentration)

using a sigmoidal dose–response function implemented in

Pipeline Pilot’s dose–response component.

Acknowledgements PS thanks the National Research Foundation of

South Africa for a Competitive Grant for unrated Researchers (Grant

No. 121276). We also appreciate the Centre for High-Performance

Computing (CHPC), Cape Town for computational resources, and the

Community for Antimicrobial Drug Discovery (CO-ADD), funded by

the Wellcome Trust (UK) and the University of Queensland (Australia)

for antimicrobial screening.

Compliance with ethical standards

Conflict of interest The authors declare that they have no confict of

interest.

Ethics approval Ethics approval for the hemolysis assay is The Uni-

versity of Queensland Institutional Human Research Ethics Approval

approved by the Medical Research Ethics Committee.

Hemolysis assay

References

Human whole blood (from Australian Red Cross Life Blood)

was washed three times with 3 volumes of 0.9% NaCl and

resuspended in the same to a concentration of 0.5×10⁸cells/

mL using manual cell count in a Neubauer hemocytometer.

Subsequently, the washed cells were transferred to the well

plates containing the compounds for a fnal volume of 50

μL, followed by shaking the plates for 10 min on a plate

shaker. After this, the plates were incubated at 37 °C for 1 h,

centrifuged at 1000 g for 10 min to pellet cells and debris,

and then, 25 μL of the supernatant was transferred to a poly-

styrene 384-well assay plate.

1. Brown ED, Wright GD (2016) Antibacterial drug discovery

in the resistance era. Nature 529(7586):336–343. https://doi.

org/10.1038/nature17042

2. Morens DM, Fauci AS (2013) Emerging infectious diseases:

threats to human health and global stability. PLoS Pathog

9(7):e1003467. https://doi.org/10.1371/journal.ppat.1003467

3. Lesho EP, Laguio-Vila M (2019) The slow-motion catastrophe

of antimicrobial resistance and practical interventions for all

prescribers. In: Mayo Clinic proceedings. Elsevier

4. Croston GE (2017) The utility of target-based discovery. Expert

Opin Drug Dis 12(5):427–429. https://doi.org/10.1080/17460

441.2017.1308351

Hemolysis was determined by measuring the absorbance

of the resulting supernatant at 405 mm (OD₄₀₅) with a Tecan

M1000 Pro monochromator plate reader. HC₁₀and HC₅₀

(concentrations at 10% and 50% hemolysis, respectively)

were calculated as in cytotoxicity assay above.

5. Morphy JR, Harris CJ (eds) (2012) Designing multi-target

drugs, vol 21. Royal society of chemistry. RSC Drug Discovery,

Cambridge

1 3

Molecular Diversity

21. CLH (2014) CLH report for 8-hydroxyquinoline

6. Shaveta MS, Singh P (2016) Hybrid molecules: the privi-

leged scafolds f or various pharmaceuticals. Eur J Med Chem

124:500–536. https://doi.org/10.1016/j.ejmech.2016.08.039

7. Zhang B (2019) Comprehensive review on the anti-bacterial activ -

ity o f 1,2,3-triazole hybrids. Eur J Med Chem 168:357–372. https

://doi.org/10.1016/j.ejmech.2019.02.055

22. Ebenezer O, Awolade P, Koorbanally N, Singh P (2020) New

library of pyrazole-imidazo[1,2-α]pyridine molecular conjugates:

synthesis, antibacterial activity and molecular docking stud-

ies. Chem Biol Drug Des 95:162–173. https://doi.org/10.1111/

cbdd.13632

23. Ebenezer O, Singh-Pillay A, Koorbanally NA, Singh P (2020)

Antibacterial evaluation and molecular docking studies of pyra-

zole–thiosemicarbazones and their pyrazole–thiazolidinone con-

jugates. Mol Divers. https://doi.org/10.1007/s11030-020-10046-w

24. Das B, Venkateswarlu K, Majhi A, Siddaiah V , Reddy KR (2007)

A facile nuclear bromination of phenols and anilines using

NBS in the presence of ammonium acetate as a catalyst. J Mol

Catal A Chem 267(1–2):30–33. https://doi.org/10.1016/j.molca

ta.2006.11.002

8. Xu Z, Zhao SJ, Liu Y (2019) 1,2,3-Triazole-containing hybrids

as potential anticancer agents: current developments, action

mechanisms and structure-activity relationships. Eur J Med Chem

183:111700. https://doi.org/10.1016/j.ejmech.2019.111700

9. Aneja B, Azam M, Alam S, Perwez A, Maguire R, Yadava U,

Kavanagh K, Daniliuc CG, Rizvi MMA, Haq QMR (2018) Natu-

ral product-based 1, 2, 3-triazole/sulfonate analogues as poten-

tial chemotherapeutic agents f or bacterial in f ections. ACS Omega

3(6):6912–6930. https://doi.org/10.1021/acsomega.8b00582

10. Bonandi E, Christodoulou MS, Fumagalli G, Perdicchia D, Ras-

telli G, Passarella D (2017) The 1,2,3-triazole ring as a bioisostere

in medicinal chemistry. Drug Discov Today 22(10):1572–1581.

https://doi.org/10.1016/j.drudis.2017.05.014

25. Lim JY, Marques I, Thompson AL, Christensen KE, Felix V, Beer

PD (2017) Chalcogen bonding macrocycles and [2]rotaxanes f or

anion recognition. J Am Chem Soc 139(8):3122–3133. https://doi.

org/10.1021/jacs.6b12745

26. Helsel ME, White EJ, Razvi SZA, Alies B, Franz KJ (2017)

Chemical and functional properties of metal chelators that mobi-

lize copper to elicit fungal killing o f Cryptococcus neoformans.

Metallomics 9(1):69–81. https://doi.org/10.1039/C6MT00172F

27. Kerns EH, Di L (2008) Drug-like properties: concepts, structure

design and methods: from ADME to toxicity optimization, 1st

edn. Elsevier, Amsterdam

11. Ir f an M, Alam S, Manzoor N, Abid M (2017) Efect of quinoline

based 1,2,3-triazole and its structural analogues on growth and

virulence attributes o f Candida albicans . PLoS ONE 12(4):1–23.

https://doi.org/10.1371/journal.pone.0175710

12. Ir f an M, Aneja B, Yadava U, Khan SI, Manzoor N, Daniliuc

CG, Abid M (2015) Synthesis, QSAR and anticandidal evalua-

tion of 1,2,3-triazoles derived from naturally bioactive sca f olds.

Eur J Med Chem 93:246–254. https://doi.org/10.1016/j.ejmec

h.2015.02.007

28. QikProp (2019) QikProp, version 6.0. Schrödinger, LLC, New

York

29. Hall ML, Jorgensen WL, Whitehead L (2013) Automated ligand-

and structure-based protocol for in silico prediction of human

serum albumin binding. J Chem Inf Model 53(4):907–922. https

://doi.org/10.1021/ci3006098

13. Xu Z, Zhang S, Song X, Qiang M, Lv Z (2017) Design, synthesis

and in vitro anti-mycobacterial evaluation of gatifoxacin-1H-1,

2, 3-triazole-isatin hybrids. Bioorg Med Chem Lett 27(16):3643–

3646. https://doi.org/10.1016/j.bmcl.2017.07.023

30. Vandenberg JI, Perry MD, Perrin MJ, Mann SA, Ke Y, Hill AP

(2012) hERG K(+) channels: structure, function, and clinical sig-

nifcance. Physiol Rev 92(3):1393–1478. https://doi.org/10.1152/

physrev.00036.2011

14. Xu Z, Lv ZS, Song XF, Qiang M (2017) Ciprofloxacin-

isatin-1H-1,2,3-triazole hybrids: design, synthesis, and in vitro

anti-tubercular activity against M. Tuberculosis. J Heterocycl

Chem 55(1):97–102. https://doi.org/10.1002/jhet

31. Hering J, Dunevall E, Ek M, Branden G (2018) Structural basis

for selective inhibition of antibacterial target MraY, a membrane-

bound enzyme involved in peptidoglycan synthesis. Drug Dis-

cov Today 23(7):1426–1435. https://doi.org/10.1016/j.drudi

s.2018.05.020

15. Kant R, Kumar D, Agarwal D, Gupta RD, Tilak R, Awasthi SK,

Agarwal A (2016) Synthesis of newer 1,2,3-triazole linked chal-

cone and favone hybrid compounds and evaluation of their anti-

microbial and cytotoxic activities. Eur J Med Chem 113:34–49.

https://doi.org/10.1016/j.ejmech.2016.02.041

32. Sagatova AA, Keniya MV, Wilson RK, Monk BC, Tyndall

JD (2015) Structural insights into binding of the antifun-

gal drug fuconazole to Saccharomyces cerevisiae lanosterol

14α-demethylase. Antimicrob Agents Chemother 59(8):4982–

4989. https://doi.org/10.1128/AAC.00925-15

16. Gao F, Ye L, Kong F, Huang G, Xiao J (2019) Design, synthesis

and antibacterial activity evaluation of moxifoxacin-amide-1,2,3-

triazole-isatin hybrids. Bioorg Chem 91:103162. https://doi.

org/10.1016/j.bioorg.2019.103162

17. Gao F, Chen Z, Ma L, Fan Y, Chen L, Lu G (2019) Synthesis

and biological evaluation of moxifoxacin-acetyl-1,2,3-1H-tria-

zole-methylene-isatin hybrids as potential anti-tubercular agents

against both drug-susceptible and drug-resistant Mycobacterium

tuberculosis strains. Eur J Med Chem 180:648–655. https://doi.

org/10.1016/j.ejmech.2019.07.057

33. PrimeX (2019) PrimeX version 5.6. Schrödinger, LLC, New York

34. Protein Preparation Wizard (2019) Protein Preparation Wiz-

ard; Epik, Schrödinger, LLC, New York, NY, 2019; Impact,

Schrödinger, LLC, New York, NY, 2019; Prime, Schrödinger,

LLC, New York, NY, 2019

35. Induced Fit Docking Protocol (2019) Induced Fit Docking pro-

tocol; Glide, Schrödinger, LLC, New York, NY, 2019; Prime,

Schrödinger, LLC, New York, NY. 2019

18. Song Yn X, Chen W, Zhan P, Liu X (2015) 8-Hydroxyquinoline: a

privileged structure with a broad-ranging pharmacological poten-

tial. MedChemComm 6(1):61–74. https://doi.org/10.1039/c4md0

0284a

36. LigPrep, Schrödinger, LLC, New York, NY. 2019. (2019)

37. Epik, Schrödinger, LLC, New York, NY. 2019. (2019)

19. Oliveri V, Vecchio G (2016) 8-Hydroxyquinolines in medicinal

chemistry: a structural perspective. Eur J Med Chem 120:252–

274. https://doi.org/10.1016/j.ejmech.2016.05.007

Publisher’s Note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional afliations.

20. Awolade P, Cele N, Kerru N, Gummidi L, Oluwakemi E, Singh P

(2020) Therapeutic signifcance of β-glucuronidase activity and

its inhibitors: a review. Eur J Med Chem 187:111921. https://doi.

org/10.1016/j.ejmech.2019.111921

1 3

Article Doi

Synthesis, antimicrobial evaluation, and in silico studies of quinoline—1H-1,2,3-triazole molecular hybrids

DOI: 10.1007/s11030-020-10112-3

Source and publish data:

Authors:

Article abstract of DOI:10.1007/s11030-020-10112-3

Full text of DOI:10.1007/s11030-020-10112-3

Products guided by the article

R&D Labs maybe for 85262-44-8

Relevant to this article

Hot Product