Design, Synthesis and Biological Evaluation of Novel Anthraniloyl-AMP Mimics as PQS Biosynthesis Inhibitors against Pseudomonas aeruginosa Resistance

Article abstract of DOI:10.3390/molecules25133103

The Pseudomonas quinolone system (PQS) is one of the three major interconnected quorum sensing signaling systems in Pseudomonas aeruginosa. The virulence factors PQS and HHQ activate the transcription regulator PqsR (MvfR), which controls several activiti

Full text of DOI:10.3390/molecules25133103

molecules
Article
Design, Synthesis and Biological Evaluation of Novel
Anthraniloyl-AMP Mimics as PQS Biosynthesis
Inhibitors Against Pseudomonas aeruginosa Resistance
1
2
, Theerthankar Das , David StC. Black 1 , Scott A. Rice 2
3
Shekh Sabir , Sujatha Subramoni
1
,
and Naresh Kumar *
1
School of Chemistry, Faculty of Science, The University of New South Wales, Sydney, NSW 2052, Australia;
s.sabir@student.unsw.edu.au (S.S.); d.black@unsw.edu.au (D.S.B.)
Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University,
Singapore 637551, Singapore; subramoni@ntu.edu.sg (S.S.); rscott@ntu.edu.sg (S.A.R.)
Department of Infectious Diseases and Immunology, School of Medical Sciences, The University of Sydney,
Sydney, NSW 2006, Australia; das.ashishkumar@sydney.edu.au
2
3
*
Correspondence: n.kumar@unsw.edu.au; Tel.: +61-2-9385-4698; Fax: +61-2-9385-6141
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Ivo Piantanida, René Csuk, Claus Jacob and Marilisa Leone
Received: 27 May 2020; Accepted: 2 July 2020; Published: 7 July 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The Pseudomonas quinolone system (PQS) is one of the three major interconnected quorum
sensing signaling systems in Pseudomonas aeruginosa. The virulence factors PQS and HHQ activate the
transcription regulator PqsR (MvfR), which controls several activities in bacteria, including biofilm
formation and upregulation of PQS biosynthesis. The enzyme anthraniloyl-CoA synthetase (PqsA)
catalyzes the first and critical step in the biosynthesis of quinolones; therefore, it is an attractive target
for the development of anti-virulence therapeutics against Pseudomonas resistance. Herein, we report
the design and synthesis of novel triazole nucleoside-based anthraniloyl- adenosine monophosphate
(AMP) mimics. These analogues had a major impact on the morphology of bacterial biofilms and
caused significant reduction in bacterial aggregation and population density. However, the compounds
showed only limited inhibition of PQS and did not exhibit any effect on pyocyanin production.
Keywords: Pseudomonas quinolone system; quorum sensing; Pseudomonas aeruginosa; virulence
factors; biofilm; anthraniloyl-AMP mimics; pyocyanin production
1
. Introduction
Antimicrobial resistance (AMR) is a growing threat to global public health. The Organization for
Economic Co-operation and Development (OECD) has predicted that resistant microbial infections
would be the leading cause of mortality in the next 30 years, and that around 2.4 million people will
die in Europe, North America, and Australia alone. The problem of AMR is even more acute in low
and middle-income countries, such as India, Brazil, and Indonesia, where 40–60% of infections are
already caused by multidrug-resistant microbes [1,2].
The rapid rise of AMR has created fears that the medical system will soon become overwhelmed
with “superbugs”, which are pathogenic bacterial strains that fail to respond to any antibiotic
treatment [
3
]. Recently, the World Health Organization (WHO) has submitted a list of pathogenic
]. One of the
bacterial strains that require the most urgent attention for therapeutic intervention [
4
bacteria in the critical priority class is Pseudomonas aeruginosa. Multidrug-resistant P. aeruginosa strains
are a leading cause of acute and chronic infections, particularly in immunocompromised patients such
as those suffering from other illnesses including cancer, burns and cystic fibrosis [
5]. Furthermore,
P. aeruginosa can switch from a planktonic (free-living) form to a sessile (community) form by the
Molecules 2020, 25, 3103; doi:10.3390/molecules25133103
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3103
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formation of a biofilm, which greatly contributes to drug resistance. Bacteria in biofilms are about
1
000-fold more resistant to antibiotics than planktonic bacteria [6,7].
Only a few classes of antibacterial agents like Oxazolidinones and Lipopeptides with new modes
of action have been approved for clinical use since the 1970s. Most of the antibacterial drugs introduced
over the last several decades have been obtained by structural modification of existing drugs like
Amoxicillin and Azithromycin. As these analogues possess similar mechanisms of action as their
predecessors, they are highly susceptible to AMR [8,9]. In this context, there is an urgent need for the
development of drugs that inhibit the colonization and virulence of bacteria without exerting selective
pressure on bacteria to develop resistance.
Quorum sensing (QS) is an intercellular communication system in bacteria. It is a chemical process
which involves the synthesis and release of diffusible quorum sensing signaling molecules (QSSMs),
also known as autoinducers (AIs). When the bacterial population grows, the concentration of these
AIs in the surrounding environment increases. After a certain threshold is passed, the AIs will activate
multiple genes that regulate the collective behavior of bacteria, including activities related to virulence,
motility, swarming, biofilm formation and antibiotic resistance [10–12].
Inhibition of QS-mediated bacterial communication by small molecules is now considered to
be contemporary approach to overcome AMR. The main idea is to inhibit bacterial virulence factors
and attenuate biofilm formation without affecting bacterial growth. QS inhibitors (QSIs) do not kill
bacteria, and therefore, exert minimum selective pressure on bacteria to develop AMR. Furthermore,
QSIs can be used as a combination therapy with conventional antibiotics to give synergistic effects,
particularly when the infection is caused by resistant bacteria [13–15].
P. aeruginosa employs las, rhl and pqs as its major QS systems. Although these QS systems use
different kinds of AIs, they are interconnected and modulate the activities of each other. For example,
las upregulates the rhl and pqs systems, while rhl downregulates pqs [16,17]. The las and rhl systems use
the structurally related chemical class of N-acyl homoserine lactones (AHLs) as AIs. These molecules
bind and activate their corresponding LuxR-type cognate receptor proteins LasR and RhlR, which leads
to the upregulation of the expression of genes associated with virulence and biofilm formation [18].
In contrast, the Pseudomonas quinolone signaling (pqs) system is based on the
chemical class of 2-alkyl-4(1H)-quinolones (AQs) as signal molecules. In P. aeruginosa,
2
2
5
-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and its immediate biosynthetic precursor,
-heptyl-4-hydroxyquinoline (HHQ), are the most common QSSMs. However, there are more than
0 other AQs, such as 2-alkyl 4-hydroxy quinoline N-oxide and other C-9 congeners of PQS and
HHQ. All these molecules activate the protein receptor PqsR (also known as MvfR), leading to the
upregulation of the pqsABCDE operon, which further promotes the biosynthesis of PQS through
positive feedback as well as increasing the expression of genes associated with bacterial defense
mechanisms, including biofilm formation and production of virulence factors [17]. Furthermore,
PQS-mediated bacterial aggregation and biofilm formation in P. aeruginosa are triggered through
different mechanisms, including chemical signaling among bacterial cell population, production of
iron-chelating molecules (siderophore), virulence factor pyocyanin biosynthesis and regulation of
extracellular DNA (eDNA) release through prophage induction, vesicles production and pyocyanin
mediated eDNA release [19–22].
Biosynthesis of AQs involves several different enzymatic reactions. The initial substrate
is anthranilic acid, which is converted to anthranilate–CoA by the enzyme PqsA (Figure 1).
Anthranilate–CoA is transformed through multiple steps into HHQ by enzymes PqsD, PqsE and
PqsBC. Finally, the enzyme PqsH catalyzes the hydroxylation of HHQ to form PQS. Hence, the two
main ways to block the PQS QS system are to inhibit PQS biosynthesis by targeting its biosynthetic
enzymes, and prevention of signal transduction by PqsR receptor antagonists [23–25].

Molecules 2020, 25, 3103
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Figure 1. Schematic diagram of PQS biosynthesis, leading to activation of PqsR (MvfR) and expression
of the pqsABCDE operon.
PqsA is an acyl-CoA synthetase enzyme belonging to the ANL superfamily of structurally
related adenylating enzymes. PqsA catalyzes the first and key step in the biosynthesis of PQS,
which involves transformation of anthranilic acid to anthranilate–CoA. Mechanistically, PqsA first
catalyzes the ATP-dependent adenylation of anthranilic acid to form an anthranilate-AMP intermediate,
which subsequently reacts with CoA-SH in a thioesterification reaction to yield anthranilate–CoA
(
Figure 2). PqsA possesses some attractive features that validates it as a promising target for antimicrobial
drug discovery. For example, PqsA-deficient mutant strains show reduced HHQ and PQS biosynthesis
and poor biofilm formation in cell culture, as well as highly attenuated virulence in animal infection
models. PqsA-deficient mutants also exhibit increased sensitivity towards antimicrobial therapy
in vivo. PqsA inhibitors decrease PQS production and reduce mortality in a mice infection model [26].
Importantly, PqsA is exclusively present in bacterial cells, therefore, selective PqsA inhibitors are
unlikely to affect host cell viability.
Figure 2. PqsA catalyzes the conversion of anthranilic acid to anthraniloyl-CoA—the first step of
PQS biosynthesis.
Despite its essential role in the biosynthesis of PQS, only few attempts to inhibit the PqsA enzyme
have been reported so far (Figure 3). Halogenated anthranilic acids have been reported as competitive
inhibitors of PqsA, however, they showed only weak inhibition, as millimolar concentrations are
required to show any effect [23]. Recently, Ji et al. reported sulfonyl adenosine-based small molecule

Molecules 2020, 25, 3103
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inhibitors mimicking the anthraniloyl-AMP intermediate for the competitive inhibition of PqsA.
While these inhibitors showed high binding affinity (K < 0.2 M) for the PqsA enzyme, they did
not exhibit satisfactory antibacterial potency in vitro (IC50 > 300 M). Compound accumulation
µ
i
µ
studies showed poor bacterial cell permeability, which possibly limited their cellular activity [26].
Recently, the crystal structures of PqsA complexed with the ligand anthraniloyl-AMP with the inhibitor
6
-fluoroanthraniloyl-AMP (6-FABA-AMP) were reported by Witzgall et al. [27]. The structures revealed
that the N-terminal domain of PqsA contains the active site that recognizes and binds the natural
ligand anthraniloyl-AMP as well as the competitor inhibitor, 6-FABA-AMP.
Figure 3. Previously reported inhibitors of PqsA.
2
. Design and Synthesis of Novel PqsA Inhibitors
Previously reported PqsA inhibitors based on anthranilate-AMP have shown high binding affinity
and promising activity in enzymatic assays. However, due to their highly polar nature, these inhibitors
cannot penetrate the bacterial cell membrane, and are therefore, devoid of any cellular activity in
P. aeruginosa, which limits their further development as novel antimicrobial agents.
Nucleotide analogues can be generated by modifications of the phosphate backbone, ribose sugar
or base [28]. To develop nonpolar nucleotide analogues of anthraniloyl-AMP, we first used the neutral
triazole linker as an isostere for the charged phosphodiester linkage. One advantage of using triazole
is that it is metabolically more stable than the phosphodiester linkage, because of its resistance to
degradation by nuclease. The second modification was to replace the adenine base of anthraniloyl-AMP
0
0
with pyrimidine bases, such as thymidine, 2 -deoxyuridine and 2 -deoxycytidine [29]. Finally, the ribose
0
sugar of the natural ligand was replaced with 2 -deoxyribose (Figure 4).
The synthetic route for the synthesis of the novel triazole-linked anthranilamide deoxynucleoside
compounds began with the ring-opening of 5-substituted isatoic anhydrides (1a–e) using
propargylamine (2) under mild reaction conditions. Different terminal alkyne-containing
anthranilamide intermediate compounds (3a–e) were synthesized in excellent yields (Scheme 1).
Next, we synthesized nucleoside azides following a reported protocol [30]. Selective tosylation
◦
of the primary hydroxy group of thymidine nucleoside (
4
) using pyridine and tosyl chloride at 0 C
generated tosylate (
substitution reaction with sodium azide in DMF at 70 C (Scheme 2).
5
), which was further converted to the corresponding azide (
6) by a nucleophilic
◦
The final step involved an azide–alkyne cycloaddition (click chemistry) reaction between alkynes
3a–e) and thymidine azide ( ). The reaction was performed in the presence of CuSO catalyst
(
6
4
and sodium ascorbate as reducing agents in t-BuOH:H O (1:1) medium [31]. The desired triazolo
2
nucleosides (7a–e) were synthesized between 59–74% yields (Scheme 3).
0
Furthermore, 2 -deoxyuridine (
8) was converted to the corresponding triazole nucleoside
analogues (9a–c) and (9e) by using the same three-step reaction sequence in moderate to good
isolated yields (Scheme 4).

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Figure 4. Ligand-based design of novel PqsA inhibitors.
O
O
R
R
O
N
+
THF
NH2
H
N
H
a-e
O
0 °C - rt, 2h
NH2
2
3a-e
1
8
9-99%
Comp.
R
Yield %
3
a
H
OCH
F
Cl
Br
96
99
89
94
92
3
b
3
3
c
3
3
d
e
Scheme 1. Synthesis of anthranilamide intermediates.
Scheme 2. Synthesis of thymidine azide intermediate 6.
Scheme 3. Synthesis of thymidine nucleotide analogues 7a–e.

Molecules 2020, 25, 3103
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0
Scheme 4. Synthesis of 2 -deoxyuridine nucleotide analogues 9a–c and 9e.
In the case of deoxycytidine (10), selective tosylation of the primary alcohol did not give the
desired product, most likely due to presence of the free amino group. We attempted to protect
the amino group via acetyl and trifluoroacetyl protecting groups, however, both were found to be
unstable and did not survive the subsequent tosylation reaction. After conducting a literature search,
we found that a benzoyl group could be used for the protection of the amino group of deoxycytidine [32].
Therefore, selective benzoyl protection of deoxycytidine (10) was achieved by using TMSCl and benzoyl
◦
chloride in pyridine at 0 C, giving N-benzoylated deoxycytidine (11) in excellent yields (Scheme 5).
Next, compound (11) was converted to the benzoyl-protected deoxycytidine triazole analogues (12a–e
)
following the same reaction pathway as above. Finally, the benzoyl group was deprotected by mild
hydrolysis using aqueous NH (28%):MeOH (1:1) at room temperature to give the desired deoxycytidine
3
analogues (13a–e) in excellent yields, from 81–92%.
NHBz
OH
OH
O
N
N
OH
OH
i) TsCl, Py, 0 °C, 16 h
N
O
N
TMSCl, BzCl
Py, 0 °C
N
O
ii) NaN3,DMF, 70 °C, 6 h
HO
N
O
O
N
NH
NH2
iii) 3a-e, CuSO .5H O
4
2
H2N
N
O
BzHN
N
O
sodium ascorbate
t-BuOH:H2O (1:1),
rt, 16 h
R
11
1
2a-e
10
95%
rt, 16 h
O
Aq. NH3 (28%) : MeOH
1:1)
NH2
(
N
N
N
O
N
HO
O
N
NH
NH2
8
1-92%
1
3a-e
R
0
Scheme 5. Synthesis of 2 -deoxycytidine nucleotide analogues 13a–e.
2D NMR experiments including COSY, HSQC and HMBC were performed to confirm the
regioselectivity of the compounds, particularly the connectivity between the anthranilamide unit and
the pyrimidine nucleoside via the 1,4-disubstituted triazole ring. In the COSY spectrum of compound
0 0
13a, the CH protons at C-17 (5 ) of the sugar showed correlation with the proton at C-18 (4 ) of the
2
sugar (Figure 5). Meanwhile, in the HMBC spectrum of 13a, the C-17 protons showed correlation with
0
C-18 and C-19 (3 ) carbons of the sugar, and with C-13 of the triazole ring. Similarly, the CH protons
2
at C-11 showed a COSY correlation with the N-H proton of the anthranilamide and HMBC correlations
with C-1, C-12, and C-13 carbons (Figure 5).

Molecules 2020, 25, 3103
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Figure 5. Key COSY and HMBC correlations in compound 13a.
. Results and Discussion
Biological Activity
3
For PQS inhibition assay, all the new triazole nucleotide analogues were screened for their ability
to inhibit the pqs QS system using the PAO1 (a P. aeruginosa reporter strain) pqsA:gfp reporter assay.
This assay measures the PqsR (MvfR) regulated expression of the pqsABCDE operon. It is expected
that the PqsA inhibitors will block the biosynthesis of PQS and HHQ, which leads to lower activation
of the PqsR (MvfR) receptor and ultimately, reduction in the expression of the pqsABCDE reporter.
The expression of the reporter in the untreated control reached its maximum level after 6–8 h which
then fell to basal expression levels at later time points. A known inhibitor of PQS was employed as a
positive control which prevented this expression [33]. Preliminary assays revealed that concentrations
of DMSO above 1.25% inhibited GFP expression of the reporter, even without the presence of any of the
test compounds, although growth was unaffected. Therefore, the final DMSO concentration was kept
at 1.25%. Based on the concentration of our compound stock solutions, this corresponded to 125 µg/mL
as the highest concentration tested for each compound. Then, two-fold lower serial dilutions of the
compound were tested whilst keeping the DMSO concentration constant. All compounds except one
showed no or very slight inhibition of the pqsA reporter at all concentrations tested. Only 13e at
1
25
to the negative control (Figure 6, Table 1). The LogP value of the compound plays a critical role in
bacterial cell penetration and the cLogP value of compound 13e 0.55) is highest among cytidine
µg/mL (400 µM approx.) showed approximately 30% inhibition of the reporter when compared
(
−
triazoles, which indicates that the compound 13e could be more permeable to bacterial cells compared
to all other derivatives.
Figure 6. PQS inhibition by compound 13e using PAO1 pqsA:gfp reporter-based assay.

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Table 1. PQS inhibition by synthesized compounds using PAO1 pqsA:gfp reporter-based assay.
Biological Activity
Inhibition of pqsA:gfp)
Compound Name
Inhibition (%)
(
1
2
3
4
5
6
7
8
9
7a
7b
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
Inhibition
-
-
7c
-
7d
-
7e
-
9a
-
9b
-
9c
-
9e
-
1
0
1
2
3
4
13a
13b
13c
13d
13e
-
1
1
1
1
-
-
-
30
For inhibition of biofilm formation, some of the representative compounds (7a
and 13e) were tested for their potential ability to inhibit P. aeruginosa biofilm formation. PAO1 planktonic
cultures were incubated with 400 M of compounds and added to microtiter wells to initiate bacterial
, 7e, 9a, 9e, 13d
µ
adhesion and biofilm growth. After 24 h, biofilms were visualized using phase contrast microscopy [34].
The compounds exerted a major impact on the morphology of bacterial biofilms. A drastic reduction in
bacterial aggregation and population density was observed in the presence of compounds 13e and 13d
as compared to both untreated and DMSO controls (Figure 7). Since the PQS system mediates bacterial
aggregation and biofilm formation in P. aeruginosa, this result could indicate that the compounds
interfere with PQS biosynthesis to reduce bacterial aggregation. However, given the weak PQS
inhibitory activity of the compounds in the in vitro reporter assay, we do not exclude the possibility
that the compounds might be acting through alternative pathways to inhibit biofilm formation.
20 µm
Figure 7. Phase-contrast microscopy images showing morphology of P. aeruginosa PAO1 grown in the
presence of ( ) control/untreated; ( ) Dimethyl sulfoxide (DMSO) only; ( ) 400 M 13e; ( ) 400 M13d.
Scale bar = 20 µm
A
B
C
µ
D
µ

Molecules 2020, 25, 3103
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For the pyocyanin inhibition assay, to investigate the activity of these compounds against bacterial
virulence factors, some of the derivatives (7a 7e 9a 9e 13d and 13e) were evaluated for their ability to
inhibit the production of pyocyanin from P. aeruginosa PA01 at 400 M concentration. However, out of
,
,
,
,
µ
six compounds tested, none of them showed any significant decrease in the production of pyocyanin.
The findings in this study match with previously published data showing compounds were effective in
inhibiting PQS biosynthesis without any impact on pyocyanin production [26]. This is an indication of
the complex relationship between the PQS biosynthesis and pyocyanin production.
Regarding effect on growth, none of the compounds had any effect on growth at concentrations
up to 256
µg/mL except for 7a. For 7a, even the 32
µg/mL concentration was enough to affect growth
and increase the lag phase. For all compounds, growth was inhibited at 512
µg/mL, although this
could be due to the amount of DMSO present, since 5.12% DMSO alone affected growth (for details,
see Supporting Information).
4
. Materials and Methods
4
.1. Synthesis
All the reagents and solvents were purchased from commercial sources (Combi-Blocks,
Oakwood Chemicals, Sigma-Aldrich, Sydney, Australia) and used without further purification.
Reactions were performed using oven-dried glassware under an atmosphere of nitrogen (if required).
◦
Room temperature refers to the ambient temperature (25 C). Yields refer to compounds isolated after
flash column chromatography unless otherwise stated. Progress of the reactions were monitored by
thin layer chromatography (TLC) precoated with Merck silica gel 60 F254 plates and visualization
using UV light (254 nm). Flash column chromatography was carried out using Grace Davison
LC60A 40–63 µm silica gel as the stationary phase and solvent gradient (methanol in ethyl acetate)
used as the mobile phase. High-resolution mass spectrometry (HRMS) was performed at Bioanalytical
Mass Spectrometry Facility, UNSW by electrospray (ESI) ionization using a Thermo LTQ Orbitrap XL
1
13
instrument (Thermo Scientific, Waltham, MA, USA). H- and C-NMR was recorded in deuterated
solvent (DMSO-d6) using Bruker Avance 300, 400 or 600 MHz instruments (Bruker Pty Ltd., Preston,
◦
NSW, Australia) at 24 C. Chemical shifts (
δ) are reported as relative to the corresponding solvent peak,
with tetramethylsilane as the internal standard and quoted in parts per million (ppm). Multiplicities
in the NMR spectra are described as: s—singlet; d—doublet; t—triplet; q—quartet; m—multiplet;
b—broad; coupling constants (J) are reported in hertz (Hz).
General procedure (A) for the ring opening of isatoic anhydrides (Synthesis of compounds 3a–3e):
To a solution of isatoic anhydride (3.0 mmol) in THF (20 mL), propargyl amine (4.5 mmol) was added
◦
at 0 C, and reaction stirred at rt for 2 h. After completion of the reaction as indicated by TLC,
the solvent was evaporated and residue was dissolved in minimum DCM, then, excess hexane was
added, and precipitate was collected and dried, which gives desired ring opened terminal alkyne
products from 89–99% yields.
2
-Amino-5-methoxy-N-(prop-2-yn-1-yl)benzamide (3b): Following the general procedure A, the title
1
product was obtained as brown solid (607mg, 99%); H-NMR (400 MHz, DMSO-d ):
δ 8.65 (t, J = 5.6 Hz,
6
1H, NH), 7.06 (d, J = 2.9 Hz, 1H, Ar-H), 6.85 (dd, J = 8.9, 2.9 Hz, 1H, Ar-H), 6.66 (d, J = 8.9 Hz, 1H,
Ar-H), 6.03 (s, 2H, NH ), 4.00 (dd, J = 5.6, 2.5 Hz, 2H, CH ), 3.68 (s, 3H, CH ), 3.08 (t, J = 2.5 Hz, 1H,
2
2
3
CH); 13C-NMR (101 MHz, DMSO-d6):
δ 168.32, 149.31, 144.17, 120.20, 117.92, 113.80, 111.61, 81.62,
+
7
2.60, 55.58, 28.12; HRMS (ESI) m/z calcd. for C H N O [M + H] 205.0972, found 205.0971.
11 12 2 2
2
-Amino-5-fluoro-N-(prop-2-yn-1-yl)benzamide (3c): Following the general procedure
A, the title
1
product was obtained as off-white solid (515 mg, 89%); H-NMR (400 MHz, DMSO-d ):
δ
8.70 (t, J = 5.4
,
6
1
H, NH), 7.34 (dd, J = 10.3, 3.0 Hz, 1H, Ar-H), 7.06 (td, J = 9.0, 8.1, 3.0 Hz, 1H, Ar-H), 6.72 (dd, J = 9.1
,
5
.0 Hz, 1H, Ar-H), 6.35 (s, 2H, NH ), 3.99 (dd, J = 5.5, 2.5 Hz, 2H, CH ), 3.09 (t, J = 2.5, 1H, CH);
2
2
1
3
C-NMR (101 MHz, DMSO–d6):
δ 167.60, 167.57, 153.75, 151.46, 146.66, 119.67, 119.44, 117.78, 117.71,

Molecules 2020, 25, 3103
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1
13.58, 113.36, 113.30, 81.37, 72.71, 28.20; HRMS (ESI) m/z calcd. For C H FN O [M + H]+ 193.0772,
10 9 2
found 193.0771.
2
-Amino-5-chloro-N-(prop-2-yn-1-yl)benzamide (3d): Following the general procedure A, the title
1
product was obtained as white solid (591 mg, 94%); H-NMR (400 MHz, DMSO-d ):
δ 8.77 (t, J = 5.3,
6
1
1
H, NH), 7.55 (d, J = 2.5 Hz, 1H, Ar-H), 7.17 (dd, J = 8.8, 2.5 Hz, 1H, Ar-H), 6.73 (d, J = 8.8 Hz,
13
H, Ar-H), 6.59 (s, 2H, NH ), 3.98 (dd, J = 5.5, 2.5 Hz, 2H, CH ), 3.09 (t, J = 2.5 1H, CH); C-NMR
2
2
(
101 MHz, DMSO-d6):
δ 167.41, 148.78, 131.76, 127.39, 118.17, 117.75, 114.46, 81.32, 72.72, 28.21;
+
HRMS (ESI) m/z calcd. for C H ClN O [M + H] 209.0476, found 209.0476.
9
10
2
General procedure (
azide (0.2 mmol) and corresponding alkyne (10a–e) (0.2 mmol) in (1:1) tert-butanol: water (4 mL),
sodium ascorbate (0.04 mmol) and CuSO .5H O (0.02 mmol) were added. The reaction mixture was
B) for the azide–alkyne cycloaddition reaction: To a mixture of nucleoside
4
2
stirred at rt for 16 h. After completion of the reaction as indicated by TLC, solvent was evaporated in
vacuo. The crude was purified by silica gel column chromatography (silica gel was pre-neutralized with
1
% triethylamine in ethyl acetate) using 0–20% methanol in ethyl acetate gradient system. The polarity
was gradually increased after every 100 mL of eluent. Desired compounds were eluted with 15–20%
methanol in ethyl acetate system as off-white to light brown solid between 52–74% yields.
2
-Amino-N-((1-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (7a): Following the general
1
procedure
B
, the title product was obtained as off-white solid (54 mg, 61%); H-NMR (400 MHz,
DMSO-d6):
δ
11.24 (bs, 1H, NH), 8.72 (t, J = 5.7 Hz, 1H, NH), 7.93 (s, 1H, Ar-H), 7.49 (dd, J = 8.0,
1
1
1
.5 Hz, 1H, Ar-H), 7.37 (d, J = 1.4 Hz, 2H, NH ), 7.12 (td, J = 8.5, 7.1, 1.5 Hz, 1H, Ar-H), 6.68 (dd, J = 8.3,
2
.2 Hz, 1H, Ar-H), 6.48 (td, J = 8.1, 7.1, 1.2 Hz, 1H, Ar-H), 6.42 (s, 2H, NH ), 6.17 (t, J = 7.7, 6.3 Hz,
2
H, CH), 5.48 (d, J = 4.3 Hz, 1H, OH), 4.67–4.51 (m, 2H, CH ), 4.44 (d, J = 5.7 Hz, 2H, CH ), 4.30–4.23
2
2
13
(m, 1H, CH), 4.09–4.05 (m, 1H, CH), 2.20–2.04 (m, 2H, CH ), 1.78 (d, J = 1.2 Hz, 3H, CH ); C-NMR
2 3
(101 MHz, DMSO-d6):
δ 168.80, 163.64, 150.41, 149.77, 145.45, 136.01, 131.79, 128.11, 123.59,
1
16.35, 114.50, 114.15, 109.88, 84.07, 84.02, 70.79, 51.13, 37.86, 34.46, 12.04; HRMS (ESI) m/z calcd.
+
for C H N O [M + H] 442.1833, found 442.1832.
20
23
7
5
2
-Amino-N-((1-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methoxybenzamide (7b): Following the
1
general procedure
DMSO-d6):
Ar-H), 7.10 (d, J = 2.9 Hz, 1H, Ar-H), 6.85 (dd, J = 8.8, 2.9 Hz, 1H, Ar-H), 6.67 (d, J = 8.9 Hz, 1H, Ar-H),
B, the title product was obtained as light brown solid (56 mg, 59%) H-NMR (300 MHz,
δ
11.31 (bs, 1H, NH), 8.79 (t, J = 5.7 Hz, 1H, NH), 7.95 (s, 1H, Ar-H), 7.38 (d, J = 1.5 Hz, 1H,
6
4
2
.17 (t, J = 7.0 Hz, 3H, CH, NH ), 5.49 (d, J = 4.3 Hz, 1H, OH), 4.65 (dd, J = 15.7, 6.0 Hz, 2H, CH ),
2 2
.46 (d, J = 5.6 Hz, 2H, CH ), 4.31–4.22 (m, 1H, CH), 4.13–4.05 (m, 1H, CH), 3.68 (s, 3H, OCH ),
2
3
13
.23–2.04 (m, 2H, CH ), 1.79 (s, 3H, CH ); C-NMR (76 MHz, DMSO-d6):
δ
168.47, 163.63, 150.40,
2
3
1
3
49.43, 145.35, 136.01, 123.64, 119.82, 117.92, 114.58, 111.85, 109.87, 84.06, 84.02, 70.79, 55.57, 51.14, 37.87,
+
4.47, 12.02; HRMS (ESI) m/z calcd. for C H N O [M + Na] 494.1759, found 494.1755.
2
0
25
7
6
2
-Amino-5-fluoro-N-((1-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (7c): Following the general
1
procedure
B
, the title product was obtained as off-white solid (60 mg, 65%) H-NMR (400 MHz,
DMSO-d6):
δ 11.29 (bs, 1H, NH), 8.80 (t, J = 5.6 Hz, 1H, NH), 7.96 (s, 1H, Ar-H), 7.40–7.30 (m, 2H,
Ar-H), 7.04 (td, J = 8.5, 2.9 Hz, 1H, Ar-H), 6.70 (dd, J = 9.0, 5.0 Hz, 1H, Ar-H), 6.31 (s, 2H, NH2),
6
2
.17 (t, J = 7.0 Hz, 1H, CH), 5.48 (d, J = 4.2 Hz, 1H, OH), 4.72–4.55 (m, 2H, CH ), 4.45 (d, J = 5.6 Hz,
2
H, CH ), 4.30–4.22 (m, 1H, CH), 4.11–4.04 (m, 1H, CH), 2.19–1.99 (m, 2H, CH ), 1.78 (s, 3H, CH );
2
2
3
1
3
C-NMR (101 MHz, DMSO-d6):
δ 166.72, 163.64, 150.41, 146.20, 136.00, 123.85, 119.58, 119.12, 117.56,
1
13.66, 113.06, 109.88, 84.04, 70.80, 51.16, 37.88, 34.52, 12.02; HRMS (ESI) m/z calcd. for C H FN O
20 22 7 5
+
[M + Na] 482.1559, found 482.1556.

Molecules 2020, 25, 3103
11 of 17
2-Amino-5-chloro-N-((1-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (7d): Following the general
1
procedure
B
, the title product was obtained as off-white solid (70 mg, 74%) H-NMR (400 MHz,
DMSO-d6):
δ 11.30 (bs, 1H, NH), 8.87 (t, J = 5.7 Hz, 1H, NH), 7.97 (s, 1H, Ar-H), 7.57 (d, J = 2.4 Hz,
1
6
4
H, Ar-H), 7.36 (s, 1H, Ar-H), 7.16 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 6.71 (d, J = 8.8 Hz, 1H, Ar-H),
.57 (s, 2H, NH ), 6.17 (t, J = 7.0 Hz, 1H, CH), 5.50 (d, J = 4.3 Hz, 1H, OH), 4.74–4.53 (m, 2H, CH ),
2
2
.45 (d, J = 5.5 Hz, 2H, CH ), 4.30–4.25 (m, 1H, CH), 4.12–4.04 (m, 1H, CH), 2.22–2.02 (m, 2H, CH ),
2
2
1
1
.78 (s, 3H, CH3); 13C-NMR (101 MHz, DMSO-d6):
δ
167.61, 163.65, 150.42, 148.68, 136.01, 131.56,
27.43, 123.66, 118.06, 117.73, 114.96, 109.90, 84.05, 70.81, 51.17, 37.90, 34.52, 31.31, 12.04; HRMS (ESI)
+
m/z calcd. for C H ClN O [M + H] 476.1444, found 476.1443.
20
22
7
5
2-Amino-5-bromo-N-((1-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (7e): Following the general
1
procedure
B
, the title product was obtained as light orange solid (68 mg, 65%) H-NMR (400 MHz,
DMSO-d6):
δ
11.29 (bs, 1H, NH), 8.87 (t, J = 5.5 Hz, 1H, NH), 7.96 (s, 1H, Ar-H), 7.67 (s, 1H, Ar-H),
.36 (s, 1H, Ar-H), 7.26 (d, J = 8.8 Hz, 1H, Ar-H), 6.67 (d, J = 8.8 Hz, 1H, Ar-H), 6.58 (s, 2H, NH2),
.17 (t, J = 7.0 Hz, 1H, CH), 5.48 (d, J = 4.2 Hz, 1H, OH), 4.87–4.52 (m, 2H, CH ), 4.44 (d, J = 5.5 Hz,
7
6
2
2
H, CH ), 4.32–4.22 (m, 1H, CH), 4.15–4.04 (m, 1H, CH), 2.32–2.01 (m, 2H, CH ), 1.78 (s, 3H, CH );
2
2
3
1
3
C-NMR (101 MHz, DMSO-d6):
δ 167.52, 163.65, 150.42, 149.00, 136.01, 134.25, 130.24, 128.36, 126.47,
1
23.73, 118.47, 115.58, 109.89, 104.90, 84.04, 70.80, 51.16, 37.90, 34.52, 12.05; HRMS (ESI) m/z calcd.
+
for C H BrN O [M + H] 520.0938, found 520.0939.
20
22
7
5
2-Amino-N-((1-(((2R,3S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (9a): Following the general procedure
B
, the title
1
product was obtained as off-white solid (60 mg, 70%); H-NMR (400 MHz, DMSO-d6):
δ 11.32 (bs, J = 2.2 Hz, 1H, NH), 8.72 (t, J = 5.7 Hz, 1H, NH), 7.92 (s, 1H, Ar-H), 7.56 (d, J = 8.1 Hz, 1H,
Ar-H), 7.49 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.13 (td, J = 8.4, 7.0, 1.5 Hz, 1H, Ar-H), 6.69 (dd, J = 8.3, 1.2 Hz,
1
H, Ar-H), 6.56–6.44 (m, 1H, Ar-H), 6.41 (s, 2H, NH ), 6.15 (t, J = 6.9 Hz, 1H, CH), 5.62 (dd, J = 8.1,
2
2
.2 Hz, 1H, Ar-H), 5.51 (s, 1H, OH), 4.67 (dd, J = 14.2, 4.5 Hz, 1H, CH ), 4.57 (dd, J = 14.3, 7.7 Hz, 1H,
2
CH ), 4.45 (d, J = 5.7 Hz, 2H, CH ), 4.25–4.22 (m, 1H, CH), 4.10–4.06 (m, 1H, CH), 2.22–2.04 (m, 2H,
2
2
13
CH ); C-NMR (101 MHz, DMSO-d ):
δ
168.85, 163.02, 150.41, 149.74, 145.46, 140.72, 131.82, 128.15,
2
6
1
23.59, 116.40, 114.59, 114.26, 102.11, 84.42, 84.28, 70.78, 51.20, 38.08, 34.49; HRMS (ESI) m/z calcd.
+
for C H N O [M + H] 428.1677, found 428.1673.
19
21
7
5
2-Amino-N-((1-(((2R,3S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methoxybenzamide (9b): Following the general procedure
the title product was obtained as off-white solid (55 mg, 60%) H-NMR (400 MHz, DMSO-d ):
B
,
1
6
δ 11.34 (bs, J = 2.2 Hz, 1H, NH), 8.79 (t, J = 5.7 Hz, 1H, NH), 7.94 (s, 1H, Ar-H), 7.57 (d, J = 8.1 Hz, 1H,
Ar-H), 7.08 (d, J = 2.9 Hz, 1H, Ar-H), 6.83 (dd, J = 8.9, 2.8 Hz, 1H, Ar-H), 6.65 (d, J = 8.9 Hz, 1H, Ar-H),
6
.15 (t, J = 6.9 Hz, 1H, CH), 6.03 (s, 2H, NH ), 5.62 (dd, J = 8.1, 2.0 Hz, 1H, Ar-H), 5.51 (d, J = 4.3 Hz,
2
1
4
H, OH), 4.72–4.64 (m, 1H, CH ), 4.58 (dd, J = 14.2, 7.7 Hz, 1H, CH ), 4.45 (d, J = 5.7 Hz, 2H, CH ),
2 2 2
13
.25–4.23 (m, 1H, CH), 4.10–4.05 (m, 1H, CH), 3.67 (s, 3H, OCH ) 2.21–2.10 (m, 2H, CH ); C-NMR
3
2
(101 MHz, DMSO-d6):
δ
168.56, 163.01, 150.41, 149.33, 145.39, 144.00, 140.74, 123.64, 119.90,
1
17.84, 114.33, 111.82, 102.10, 84.40, 84.29, 70.78, 55.58, 51.22, 38.08, 34.49; HRMS (ESI) m/z calcd.
+
for C H N O [M + H] 458.1783, found 458.1781.
20
24
7
6
2-Amino-N-((1-(((2R,3S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-fluorobenzamide (9c): Following the general procedure
the title product was obtained as off-white solid (64 mg, 72%); H-NMR (400 MHz, DMSO-d ):
B
,
1
6
δ
11.32 (bs, 1H, NH), 8.80 (t, J = 5.7, 1H, NH), 7.95 (s, 1H, Ar-H), 7.55 (d, J = 8.1 Hz, 1H, Ar-H),
7
.36 (dd, J = 10.3, 3.1 Hz, 1H, Ar-H), 7.03 (dt, J = 8.5, 4.3 Hz, 1H, Ar-H), 6.70 (dd, J = 9.1, 5.0 Hz, 1H,
Ar-H), 6.31 (s, 2H, NH ), 6.14 (t, J = 6.9 Hz, 1H, CH), 5.62 (d, J = 8.1 Hz, 1H, Ar-H), 5.51 (s, 1H, OH),
2

Molecules 2020, 25, 3103
12 of 17
4
.67 (dd, J = 14.2, 4.5 Hz, 1H, CH ), 4.57 (dd, J = 14.3, 7.7 Hz, 1H, CH ), 4.67–4.44 (d, J = 5.7 Hz, 2H,
2
2
13
CH ), 4.28–4.19 (m, 1H, CH), 4.12–4.02 (m, 1H, CH), 2.25–2.04 (m, 2H, CH ); C-NMR (101 MHz,
2
2
DMSO-d6):
δ
167.81, 163.02, 153.79, 151.53, 150.41, 146.52, 145.18, 140.74, 123.64, 119.43, 119.20, 117.68,
1
17.62, 113.96, 113.71, 113.48, 102.10, 84.44, 84.27, 70.79, 51.23, 38.10, 34.55; HRMS (ESI) m/z calcd.
+
for C H FN O [M + Na] 468.1402, found 468.1401.
19
20
7
5
2-Amino-5-bromo-N-((1-(((2R,3S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran
-
2-yl) methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (9e): Following the general procedure
B,
1
the title product was obtained as off-white solid (53 mg, 52%); H-NMR (400 MHz, DMSO-d ):
6
δ 11.32 (bs, 1H, NH), 8.87 (t, J = 5.7, 1H, NH), 7.95 (s, 1H, Ar-H), 7.67 (d, J = 2.4 Hz, 1H, Ar-H),
7.56 (d, J = 8.1 Hz, 1H, Ar-H), 7.26 (dd, J = 8.8, 2.3 Hz, 1H, Ar-H), 6.67 (d, J = 8.9 Hz, 1H, Ar-H),
6.58 (s, 2H, NH2), 6.14 (t, J = 6.9 Hz, 1H, CH), 5.62 (d, J = 8.0 Hz, 1H, Ar-H), 5.50 (d, J = 4.3 Hz,
1
4
δ
H, OH), 4.61 (d, J = 4.5 Hz, 1H, CH ), 4.49 (d, J = 7.6 Hz, 1H, CH ), 4.43 (d, J = 5.6 Hz, 2H, CH ),
.26–4.21 (m, 1H, CH), 4.14–4.02 (m, 1H, CH), 2.23–2.04 (m, 2H, CH2); C-NMR (101 MHz, DMSO-d6):
2 2 2
13
168.00, 163.45, 150.85, 149.44, 145.57, 141.18, 134.74, 134.71, 130.71, 124.05, 118.94, 116.10, 105.38,
+
1
02.55, 84.87, 84.72, 71.24, 38.54, 34.96; HRMS (ESI) m/z calcd. for C H BrN O [M + Na] 528.0602,
19
20
7
5
found 528.0600.
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (12a): Following the general procedure
B
, the title
1
product was obtained as off-white solid (65 mg, 61%); H-NMR (400 MHz, DMSO-d ):
δ 11.24 (bs, 1H,
6
NH), 8.73 (t, 1H, NH), 8.12 (d, J = 7.5 Hz, 1H, Ar-H), 8.05–7.98 (m, 3H, Ar-H), 7.62 (t, J = 7.4 Hz, 1H,
Ar-H), 7.56–7.46 (m, 3H, Ar-H), 7.37 (s, 1H, Ar-H), 7.13–7.07 (m, 1H, Ar-H), 6.67 (dd, J = 8.2, 1.2 Hz, 1H,
Ar-H), 6.48 (t, J = 1.0 Hz, 1H, Ar-H), 6.41 (s, 2H, NH ), 6.16 (t, J = 6.5 Hz, 1H, CH), 5.54 (d, J = 4.2 Hz,
2
1
H, OH), 4.76–4.61 (m, 2H, CH ), 4.46 (d, J = 5.7 Hz, 2H, CH ), 4.30–4.22 (m, 1H, CH), 4.22–4.14 (m, 1H,
2 2
CH), 2.37–2.09 (m, 2H, CH2); 1 C-NMR (151 MHz, DMSO-d6):
3
δ
168.82, 149.76, 145.52, 132.76, 131.77,
1
28.46, 128.13, 123.66, 116.35, 114.52, 114.19, 87.18, 84.94, 70.77, 51.22, 34.47; HRMS (ESI) m/z calcd.
+
for C H N O [M + H] 531.2099, found 531.2098.
26
26
8
5
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl) methyl)-5-methoxybenzamide (12b): Following the general procedure
the title product was obtained as light brown solid (70 mg, 62%); H-NMR (400 MHz, DMSO-d ):
B
,
1
6
δ
11.25 (bs, 1H, NH), 8.79 (t, J = 5.7 Hz, 1H, NH), 8.13 (d, J = 7.6 Hz, 1H, Ar-H), 8.06–7.96 (m, 3H, Ar-H),
.68–7.59 (m, 1H, Ar-H), 7.53 (t, J = 8.3, 7.0 Hz, 2H, Ar-H), 7.38 (s, 1H, Ar-H), 7.09 (d, J = 2.9 Hz, 1H,
7
Ar-H), 6.82 (dd, J = 8.9, 2.8 Hz, 1H, Ar-H), 6.65 (d, J = 8.9 Hz, 1H, Ar-H), 6.16 (t, J = 6.5 Hz, 1H, CH),
6
4
.00 (s, 2H, NH ), 5.56 (d, J = 4.3 Hz, 1H, OH), 4.78–4.62 (m, 2H, CH ), 4.47 (d, J = 5.7 Hz, 2H, CH ),
2 2 2
13
.34–4.23 (m, 1H, CH), 4.24–4.12 (m, 1H, CH), 3.66 (s, 3H, CH ) 2.38–2.09 (m, 2H, CH ); C-NMR
3
2
(
1
151 MHz, DMSO-d6): δ 168.56, 149.31, 145.46, 144.04, 132.78, 128.50, 128.47, 123.71, 119.89, 117.81,
14.32, 111.81, 86.53, 84.95, 70.79, 55.56, 51.26, 34.49; HRMS (ESI) m/z calcd. for C H N O [M + H]
27 29 8 6
+
5
61.2205, found 561.2204.
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-fluorobenzamide (12c): Following the general procedure
the title product was obtained as light yellowish solid (80 mg, 73%); H-NMR (400 MHz, DMSO-d ):
B
,
1
6
δ
11.24 (bs, 1H, NH), 8.81 (t, J = 5.7 Hz, 1H, NH), 8.12 (d, J = 7.5 Hz, 1H, Ar-H), 8.05–7.98 (m, 3H, Ar-H),
.67–7.59 (m, 1H, Ar-H), 7.52 (t, J = 7.7 Hz, 2H, Ar-H), 7.36 (dd, J = 10.3, 3.0 Hz, 2H, Ar-H), 7.06–6.92
7
(
m, 1H, Ar-H), 6.69 (dd, J = 9.0, 5.0 Hz, 1H, Ar-H), 6.31 (s, 2H, NH ), 6.16 (t, J = 6.5 Hz, 1H, CH),
2
5
.54 (d, J = 4.3 Hz, 1H, OH), 4.69 (dd, J = 8.9, 6.1 Hz, 2H, CH ), 4.46 (d, J = 5.6 Hz, 2H, CH ), 4.30–4.23
2 2
13
(
m, 1H, CH), 4.26–4.15 (m, 1H, CH), 2.38–2.10 (m, 1H, CH2); C-NMR (101 MHz, DMSO-d6):
δ
167.85,
1
8
53.71, 151.86, 148.73, 146.50, 145.22, 132.74, 128.43, 123.68, 119.48, 119.14, 117.61, 113.67, 113.45, 86.76,
+
4.92, 70.77, 50.80, 34.52; HRMS (ESI) m/z calcd. for C H FN O [M + Na] 571.1824, found 571.1823.
25
26
8
5

Molecules 2020, 25, 3103
13 of 17
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-chlorobenzamide (12d): Following the general procedure
1
B
, the title product was obtained as off-white solid (70 mg, 62%); H-NMR (400 MHz, DMSO-d ):
6
δ
11.22 (bs, 1H, NH), 8.90 (t, 1H, NH), 8.12 (d, J = 7.3 Hz, 1H, Ar-H), 8.06–7.94 (m, 4H, Ar-H),
7
.62 (t, 1H, Ar-H), 7.57 (d, J = 2.5 Hz, 1H, Ar-H), 7.52 (t, J = 7.7 Hz, 2H, Ar-H), 7.13 (dd, J = 8.8, 2.5 Hz,
1
H, Ar-H), 6.71 (d, J = 8.8 Hz, 1H, Ar-H), 6.56 (s, 2H, NH ,), 6.15 (t, 1H, CH), 5.63 (d, J = 4.3 Hz, 1H,
2
OH), 4.78–4.62 (m, 2H, CH ), 4.45 (d, J = 5.6 Hz, 2H, CH ), 4.30–4.22 (m, 1H, CH), 4.26–4.15 (m, 1H,
2
2
13
CH), 2.41–2.10 (m, 1H, CH2); C-NMR (151 MHz, DMSO-d6):
δ 167.63, 148.69, 145.20, 132.78, 131.53,
1
28.50, 128.47, 127.47, 123.72, 118.06, 117.72, 114.97, 96.44, 84.93, 70.73, 51.26, 34.52; HRMS (ESI) m/z
+
calcd. for C H ClN O [M + H] 565.1709, found 565.1709.
26
26
8
5
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-bromobenzamide (12e): Following the general procedure
B
,
1
the title product was obtained as off-white solid (65 mg, 53%); H-NMR (600 MHz, DMSO-d ):
δ 11.26
6
(bs, 1H, NH), 8.89 (t, J = 5.9 Hz, 1H, NH), 8.12 (d, J = 7.6 Hz, 1H, Ar-H), 8.06–7.98 (m, 3H, Ar-H),
7.68 (d, J = 2.4 Hz, 1H, Ar-H), 7.63 (t, J = 7.3 Hz, 1H, Ar-H), 7.52 (t, J = 7.7 Hz, 2H, Ar-H), 7.40–7.34
(m, 1H, Ar-H) 7.24 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 6.67 (d, J = 8.8 Hz, 1H, Ar-H), 6.59 (s, 2H, NH2),
6
.17 (t, J = 6.5 Hz, 1H, CH), 5.56 (d, J = 4.3 Hz, 1H, OH), 4.81–4.64 (m, 2H CH ), 4.45 (d, J = 5.8 Hz, 2H,
2
13
CH ), 4.29–4.24 (m, 1H, CH), 4.24–4.17 (m, 1H, CH), 2.32–2.02 (m, 2H, CH ); C-NMR (151 MHz,
2
2
DMSO-d6):
δ
167.98, 149.46, 145.65, 134.69, 130.72, 128.95, 128.90, 124.14, 118.92, 116.06, 105.36, 85.39,
+
7
9.66, 79.44, 79.22, 71.24, 51.72, 34.97; HRMS (ESI) m/z calcd. for C H BrN O [M + Na] 631.1023,
26
25
8
5
found 631.1022.
General procedure (
0.1 mmol) in methanol (6 mL), Aq. NH (28%) (6 mL) was added. The resulting mixture was stirred at
C
) for the deprotection of benzoyl group: To a solution of N-benzoyl triazole
(
3
rt for 16 h. After completion of the reaction, solvent was evaporated in vacuo, crude was purified by
dissolving in methanol (1 mL), then, excess diethyl ether was added, and solid precipitate was collected
and dried, which gives desired compound as off-white to light brown solid between (81–92% yields).
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (13a): Following the general procedure
C
, the title
1
product was obtained as off-white solid (38 mg, 88%); H-NMR (600 MHz, DMSO-d ): δ 8.73 (t, J = 5.8
,
6
1
7
6
H, NH), 7.94 (s, 1H, Ar-H), 7.52 (d, J = 7.5 Hz, 1H, Ar-H), 7.50 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H),
.14 (s, 2H, NH ), 7.13 (td, J = 8.4, 7.0, 1.5 Hz, 1H, Ar-H), 6.69 (dd, J = 8.2, 1.2 Hz, 1H, Ar-H),
2
.50 (td, J = 8.1, 7.0, 1.2 Hz, 1H, Ar-H), 6.42 (s, 2H, NH ), 6.18 (t, J = 7.5, 6.2 Hz, 1H, CH), 5.75 (d, J = 7.4
,
2
1
1
2
H, Ar-H), 5.45 (d, J = 4.3 Hz, 1H, OH), 4.66 (dd, J = 14.3, 4.4 Hz, 1H, CH ), 4.58 (dd, J = 14.2, 7.7 Hz,
2
H, CH ), 4.45 (d, J = 5.7 Hz, 2H, CH ), 4.22–4.21 (m, 3.6 Hz, 1H, CH), 4.09–4.06 (m, 4.2 Hz, 1H, CH),
2
2
13
.14–2.08 (m, 1H, CH ), 2.05–1.98 (m, 1H, CH ); C-NMR (101 MHz, DMSO-d6):
δ
168.84, 165.55,
2
2
1
5
54.99, 149.76, 145.42, 141.12, 131.83, 128.17, 123.60, 116.39, 114.60, 114.25, 94.41, 85.17, 84.17, 70.98,
1.38, 39.55, 34.48. HRMS (ESI) m/z calcd. for C H N O [M + H] 449.1656, found 449.1655.
19 22 8 4
+
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methoxybenzamide (13b): Following the general procedure
the title product was obtained as light brown solid (37 mg, 81%); H-NMR (400 MHz, DMSO-d ):
C
,
1
6
δ
8.77 (t, J = 5.7 Hz, 1H, NH), 7.95 (s, 1H, Ar-H), 7.53 (d, J = 7.4 Hz, 1H, Ar-H), 7.28–7.12 (m, 2H,
NH ), 7.09 (d, J = 2.9 Hz, 1H, Ar-H), 6.84 (dd, J = 8.9, 2.9 Hz, 1H, Ar-H), 6.66 (d, J = 8.9 Hz, 1H),
2
6
.18 (t, J = 6.8 Hz, 1H, CH), 6.01 (s, 2H, NH ), 5.75 (d, J = 7.4 Hz, 1H, Ar-H), 5.47 (d, J = 4.2 Hz, 1H,
2
OH), 4.62 (dd, J = 21.4, 6.1 Hz, 2H, CH ), 4.46 (d, J = 5.6 Hz, 2H, CH ), 4.23–4.22 (m, 1H, CH), 4.12–4.04
2
2
13
(
m, 1H, CH), 3.67 (s, 3H, OCH ), 2.23–1.96 (m, 2H, CH ); C-NMR (101 MHz, DMSO-d6):
δ
169.00,
3
2
1
7
65.99, 155.42, 149.78, 145.78, 144.47, 141.57, 124.08, 120.35, 118.27, 114.83, 112.30, 94.83, 85.62, 84.61,
+
1.43, 56.04, 51.84, 34.94; HRMS (ESI) m/z calcd. for C H N O [M + Na] 479.1762, found 479.1760.
20 24 8 5

Molecules 2020, 25, 3103
14 of 17
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-fluorobenzamide (13c): Following the general procedure
C
,
1
the title product was obtained as off-white solid (41 mg, 92%); H-NMR (400 MHz, DMSO-d ):
δ 8.82
6
(
t, J = 5.6 Hz, 1H, NH), 7.97 (s, 1H, Ar-H), 7.52 (d, J = 7.5 Hz, 1H, Ar-H), 7.37 (dd, J = 10.3, 3.0 Hz, 1H,
Ar-H), 7.17 (d, J = 13.1 Hz, 2H, NH ), 7.09–7.00 (m, 1H, Ar-H), 6.70 (dd, J = 9.0, 5.0 Hz, 1H, Ar-H),
2
6
.33 (s, 2H, NH ), 6.17 (t, 1H, J = 6.8 Hz, CH), 5.73 (d, J = 7.4 Hz, 1H, Ar-H), 5.45 (d, J = 4.3 Hz, 1H,
2
OH), 4.75–4.53 (m, 2H, CH ), 4.44 (d, J = 5.7 Hz, 2H, CH ) 4.24–4.16 (m, 1H, CH), 4.11–4.03 (m, 1H,
2
2
13
CH), 2.16–1.95 (m, 2H, CH2); C-NMR (101 MHz, DMSO-d6):
δ
167.78, 165.54, 154.98, 153.80, 151.48,
1
8
46.51, 145.13, 141.13, 123.62, 119.42, 119.19, 117.66, 117.58, 113.96, 113.90, 113.72, 113.50, 94.39, 85.19,
+
4.15, 70.98, 51.40, 34.52; HRMS (ESI) m/z calcd. for C H FN O [M + Na] 567.1562, found 567.1564.
19
22
8
4
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-chlorobenzamide (13d): Following the general procedure
C
,
1
the title product was obtained as off-white solid (38 mg, 82%); H-NMR (600 MHz, DMSO-d ):
δ 8.88
6
(
t, J = 5.7 Hz, 1H, NH), 7.96 (s, 1H, Ar-H), 7.57 (d, J = 2.5 Hz, 1H, Ar-H), 7.52 (d, J = 7.4 Hz, 1H, Ar-H),
7
.26–6.99 (m, 3H, NH , Ar-H), 6.72 (d, J = 8.8 Hz, 1H, Ar-H), 6.57 (s, 2H, NH ), 6.17 (t, J = 7.5, 6.2 Hz,
2 2
1
4
H, CH), 5.74 (d, J = 7.4 Hz, 1H, Ar-H), 5.46 (d, J = 4.0 Hz, 1H, OH), 4.66 (dd, J = 14.2, 4.4 Hz, 1H, CH2),
.57 (dd, J = 14.2, 7.7 Hz, 1H, CH ), 4.44 (d, J = 5.6 Hz, 2H, CH ), 4.25–4.19 (m, 1H, CH), 4.08–4.05
2
2
13
(
m, 1H, CH), 2.12–2.07 (m, 1H, CH ), 2.03–1.99 (m, 1H, CH ); C-NMR (151 MHz, DMSO-d6):
δ
168.06,
2
2
1
8
65.98, 155.41, 149.12, 145.53, 141.57, 132.02, 127.92, 124.06, 118.51, 118.19, 115.45, 108.11, 94.83, 85.61,
4.60, 71.42, 51.85, 34.96; HRMS (ESI) m/z calcd. for C H ClN O [M + H] 483.1267, found 483.1264.
19 21 8 4
+
2
-Amino-N-((1-(((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)
methyl)-1H-1,2,3-triazol-4-yl)methyl)-5-bromobenzamide (13e): Following the general procedure C,
1
the title product was obtained as off-white solid (43 mg, 85%); H-NMR (400 MHz, DMSO-d ): δ 8.87
6
(t, J = 5.5 Hz, 1H, NH), 7.96 (s, 1H, Ar-H), 7.68 (d, J = 2.4 Hz, 1H, Ar-H), 7.52 (d, J = 7.4 Hz, 1H,
Ar-H), 7.26 (dd, J = 8.9, 2.3 Hz, 1H, Ar-H), 7.17 (s, 2H, NH ), 6.67 (d, J = 8.8 Hz, 1H, Ar-H), 6.58 (s, 2H,
2
NH ), 6.17 (t, J = 6.8 Hz, 1H, CH), 5.76 (s, 1H, Ar-H), 5.45 (d, J = 4.3 Hz, 1H, OH), 4.65– 4.58 (m, 2H,
2
CH ), 4.43 (d, J = 5.5 Hz, 2H, CH ), 4.24–4.16 (m, 1H, CH), 4.11–4.03 (m, 1H, CH), 2.10–1.95 (m, 2H,
2
2
13
CH2); C-NMR (151 MHz, DMSO-d6):
18.47, 115.62, 104.92, 85.18, 84.15, 70.97, 69.79, 51.39, 34.50; HRMS (ESI) m/z calcd. for C H BrN O
4
M + H] 505.0942, found 505.0942.
δ 167.52, 165.54, 148.99, 145.07, 141.12, 134.26, 130.27, 123.61,
1
[
19
21
8
+
4
.2. Biological Activity
For the pqs:gfp reporter assay, the PAO1 pqsA-gfp P. aeruginosa strain carries the PqsR-regulated pqsA
promoter fused to gfp [35]. The compounds were dissolved in DMSO at a concentration of 20 mg/mL
(
approximately 40 mM based on molecular weights). The assays with pqsA reporters were carried
◦
out in MHB (Mueller Hinton Broth) in 96-well plates, at 37 C with intermittent shaking. Readings
were taken every 15 min for both OD600 and fluorescence; the fluorescence values shown in the graph
were normalized with respect to OD600. All the reporters tested peak in promoter activity after 6–8 h
growth. At intervals of 15 min, both GFP fluorescence and OD600 were recorded. Negative control
refers to medium containing the same concentration of DMSO as the highest concentration of the
test compound.
For microscopy imaging to evaluate adhesion and biofilm formation, planktonic cultures of
PAO1 were grown for 24 h followed by re-suspension in TSB media when a bacterial density of
OD600 = 0.10 ± 0.02 was reached. Then, 600
µ
L of the re-suspended cells was added to 12-well plates
◦
(
Corning Corp. USA), and then, incubated at 37 C and 100 rpm to initiate bacterial adhesion and
biofilm growth. Samples contained either compounds at 400
µM, or TSB medium or DMSO as negative
controls. After 24 h, wells were washed twice with 1 PBS to remove any loosely adhered bacteria and
×
imaged using phase contrast microscopy (Zeiss, Axio, Germany), for bacterial adhesion, aggregation
and biofilm formation as described [36].

Molecules 2020, 25, 3103
15 of 17
For pyocyanin quantification, planktonic cultures of PAO1 were grown in 12-wells plate for 24 h in
presence of plain TSB, solvent (DMSO) and compounds 400
were transferred into sterile 2 mL Eppendorf tubes and centrifuged at 5000 g for 10 min at 21 C.
µ
M. After growth, the planktonic cultures
◦
After centrifugation, the supernatant was removed by pipetting and transferred into new Eppendorf
◦
tubes, followed by re-centrifuging at 5000
.4
absorbance at OD695 using a plate reader (Tecan infinite M1000 pro, Sydney, Australia).
×
g for 10 min at 21 C. The supernatants were filtered using
µM filters and added to 96-well plates, and pyocyanin concentration was determined by measuring
0
5
. Conclusions
Inhibition of the PQS quorum sensing system is an attractive target for the development
of alternative therapies against multidrug-resistant P. aeruginosa. Anthraniloyl-AMP mimics are
competitive antagonists of the enzyme PqsA, and block production of virulence factors PQS and
HHQ without affecting bacterial growth. In this study, we have designed and synthesized 14 novel
anthraniloyl-AMP mimics containing a triazole linker as potential inhibitors of Pseudomonas quinolone
biosynthesis. However, most of these analogues did not show PQS inhibitory activity against a
P. aeruginosa reporter chain except for deoxycytidine analogue (13e), which inhibited PQS activity
by 30% at 125 µM, possibly due to limited cell penetration. Interestingly, these compounds induced
significant morphological changes in biofilms as observed by microscopy, suggesting that they could
inhibit PQS or other signaling systems responsible for bacterial aggregation. Our future efforts will
focus on modifying these analogues to increase bacterial cell penetration.
Supplementary Materials: The H- and 13C-NMR spectra for all the new compounds and growth inhibition data
1
are provided in the supplementary file.
Author Contributions: N.K., D.S.B. planned and designed the experiments. S.S. (Sujatha Subramoni), S.A.R.
and T.D. provided the data from biology screening. S.S. (Shekh Sabir) performed the chemistry experiments,
analyzed, and interpreted the data and wrote the paper. N.K. supported financially. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by a Discovery Project from Australian Research Council grant (DP180100845).
Acknowledgments: We thank the NMR and BMSF facility, UNSW Sydney. The authors would like to acknowledge
the Nanyang Technological University, Singapore and the University of Sydney, Australia. S. Sabir would like to
acknowledge UNSW TFS scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).