DPD-Inspired Discovery of Novel LsrK Kinase Inhibitors: An Opportunity to Fight Antimicrobial Resistance

Article abstract of DOI:10.1021/acs.jmedchem.9b00025

Antibiotic resistance is posing a continuous threat to global public health and represents a huge burden for society as a whole. In the past decade, the interference with bacterial quorum sensing (QS) (i.e., cell-cell communication) mechanisms has extensively been investigated as a valid therapeutic approach in the pursuit of a next generation of antimicrobials. (S)-4,5-Dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule that modulates QS in both Gram-negative and Gram-positive bacteria, is phosphorylated by LsrK, and the resulting phospho-DPD activates QS. We designed and prepared a small library of DPD derivatives, characterized by five different scaffolds, and evaluated their LsrK inhibition in the context of QS interference. SAR studies highlighted the pyrazole moiety as an essential structural element for LsrK inhibition. Particularly, four compounds were found to be micromolar LsrK inhibitors (IC₅₀ ranging between 100 μM and 500 μM) encouraging further exploration of novel analogues as potential new antimicrobials.

Full text of DOI:10.1021/acs.jmedchem.9b00025

Subscriber access provided by WEBSTER UNIV
Article
DPD-inspired discovery of novel LsrK kinase inhibitors:
an opportunity to fight antimicrobial resistance
Silvia Stotani, Viviana Gatta, Prasanthi Medarametla, Mohan Padmanaban, Anna Karawajczyk,
Fabrizio Giordanetto, Päivi Tammela, Tuomo Laitinen, Antti Poso, Dimitros Tzalis, and Simona Collina
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00025 • Publication Date (Web): 20 Feb 2019
Downloaded from http://pubs.acs.org on February 21, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
DPD-inspired discovery of novel LsrK kinase
inhibitors: an opportunity to fight antimicrobial
resistance
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,2
3
4
2
Silvia Stotani , Viviana Gatta , Prasanthi Medarametla , Mohan Padmanaban , Anna
5†
2,§
3
4
4
Karawajczyk , Fabrizio Giordanetto , Päivi Tammela , Tuomo Laitinen , Antti Poso ,
2
2,*
Dimitros Tzalis and Simona Collina
1Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section,
University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy
2Medicinal Chemistry, Taros Chemicals GmbH & Co. KG, Emil-Figge-Straße 76a, 44227
Dortmund, Germany
3Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, FI-00014 Helsinki, Finland
4School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box
1627, FI-70211 Kuopio, Finland
5Selvita S.A., Park Life Science, Bobrzyňskiego 14, 30-348 Krakow, Poland
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
KEYWORDS: Antibiotic resistance; Quorum Sensing; LsrK inhibition; DPD-related
compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ABSTRACT: Antibiotic resistance is posing a continuous threat to global public health and
represents a huge burden for society as a whole. In the last decade, the interference with
bacterial Quorum Sensing (QS) (i.e., cell-cell communication) mechanisms has extensively
been investigated as a valid therapeutic approach in the pursuit of a next generation of
antimicrobials. (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small
signaling molecule that modulates QS in both Gram-negative and Gram-positive bacteria, is
phosphorylated by LsrK and the resulting phospho-DPD activates QS. We designed and
prepared a small library of DPD derivatives, characterized by five different scaffolds and
evaluated their LsrK inhibition in the context of QS interference. SAR studies highlighted the
pyrazole moiety as an essential structural element for LsrK inhibition. Particularly, four
compounds were found to be micromolar LsrK inhibitors (IC ranging between 100 µM and
50
500 µM) encouraging further exploration of novel analogues as potential new antimicrobials.
ACS Paragon Plus Environment

Page 3 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.
INTRODUCTION
Quorum Sensing (QS) is a cell-cell communication strategy that allows bacteria to act as a
population by coordinating their gene expression.1–5 QS regulates different pathogenic
processes such as biofilm formation,6–8 susceptibility to antibiotics and virulence factor
production. It is therefore not surprising that QS modulation has emerged in the last decade
as a potential tool to fight antibiotic resistance.10–13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
QS is mediated by the exchange of small signaling molecules termed autoinducers (AIs).
Oligopeptides and N-acyl homoserine lactones (AHLs) are the most common QS mediators in
Gram-positive and Gram-negative bacteria, respectively, while Autoinducer-2 (AI-2) features
in both classes. Targeting AI-2-mediated QS would therefore result in broad spectrum
antimicrobial activity. The precursor of AI-2, (S)-DPD, is biosynthesized intracellularly in a
three steps pathway. S-adenosylmethionine (SAM) is demethylated by a methyltransferase to
generate the toxic intermediate S-adenosylhomocysteine (SAH). In LuxS-containing
organisms, the enzyme Pfs removes adenine from SAH to form S-ribosylhomocysteine
(SRH). Lastly, LuxS breaks down SRH into adenine and (S)-DPD. LuxS synthase is found in
more than 70 evolutionary different bacterial species indicating that AI-2 mediates both inter-
ACS Paragon Plus Environment

Page 5 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and intraspecies communication.14 The neo-synthetized (S)-DPD is released extracellularly
with an unclear mechanism which might involve the membrane protein YdgG (Figure 1)15,16
.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Outside the cell membrane, linear (S)-DPD spontaneously rearranges into the two cyclic
isomers S-DHMF and R-DHMF (Figure 1).17 The aqueous environment allows for their
hydration at C to form the two cyclic tetrahydrated isomers S-THMF and R-THMF (Figure 1).
3
X-ray crystallography revealed that S-THMF, in the form of the borate ester S-THMF-borate,
18
binds to the periplasmic protein LuxP activating QS in V. harveyi. In enteric bacteria, when a
threshold level of AI-2 in the extracellular medium is reached, R-THFM is imported via the Lsr
19
(
LuxS regulated) ACBFG transporter and its linear form (i.e., S-THP) is phosphorylated by
LsrK. The resulting S-THP-phosphate, commonly known as phospho-DPD (P-DPD, Figure 1),
20
binds to the transcriptional repressor LsrR that dissociates from the promoter region of the
lsr operon, initiating the operon transcription. As a result, the expression of the transporter on
the cell surface is increased as well as the internalization of AI-2 and the QS response (Figure
21
1). Ultimately, P-DPD is processed by LsrG and LsrF to close the AI-2 signaling cycle. LsrG
catalyzes P-DPD isomerization to 3,4,4-trihydroxy-2-pentanone-5-phosphate (P-TPO, Figure
22
1) while LsrF acts as a thiolase and transfers an acetyl group from the hydrated form of P-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TPO to coenzyme A to form dihydroxyacetone phosphate (DHAP, Figure 1) and acetyl-CoA
22
(
Figure1). Considering the complexity of the mechanism and its regulation, there is a great
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
interest of the pharmaceutical community in identifying new compounds able to modulate QS
to control bacterial virulence and reduce its negative effects, including mortality.23–25 To lay
the foundation for the rational design/discovery of QS modulators/inhibitors, several attempts
to crystallize QS receptors have been performed. LuxP from V. harveyi has been co-
18
crystallyzed with S-THMF-borate (PDB ID: 1JX6 ) while its isomer, R-THMF, has been
19
solved in complex with the LsrB transporter (PDB ID: 1TJY ). Lastly, the linear hydrated form
20
of (S)-DPD has been shown to bind to the transcriptional repressor LsrR (PDB ID: 4L4Z ).
ACS Paragon Plus Environment

Page 7 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1: Biosynthesis, transport and degradation of (S)-DPD in enteric bacteria
To date, several structure-activity relationship (SAR) studies have been carried out around
26–33
the DPD backbone and the effects of substitution at the C position have been reported.
1
However, the exploration of new compound classes that are structurally distinctive from native
DPD is needed to discover new antibacterial agents. In this study, we report on the design,
synthesis and SAR of novel DPD-related compounds (Figure 2) with different “core
structures”, involving modifications at the DPD diketo moiety. In particular, we explored five
small libraries of DPD-inspired heterocyclic derivatives (Het-DPD derivatives, Figure 2).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Lastly, the in vitro inhibitory effect of the new compounds against LsrK has been evaluated.
Results obtained suggested that Het-DPD derivatives act as LsrK inhibitors and their analogs
may be considered as useful templates for the discovery of new effective DPD-based
antimicrobial agents. To the best of our knowledge, these are the first heterocyclic-based
LsrK inhibitors reported to-date.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2: DPD-inspired heterocyclic compounds
2.
RESULTS AND DISCUSSION
2.1 Compounds design
ACS Paragon Plus Environment

Page 9 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
LsrK belongs to a family of carbohydrate kinases called “FGGY family” whose members
34
transfer a phosphate group from ATP to several sugars ranging from trioses to heptoses. In
enteric bacteria, phosphorylation of DPD by LsrK represents the prerequisite activating step
for AI-2 signaling. Modulation/inhibition of LsrK could therefore potentially attenuate AI-2-
related pathogenesis and LsrK could be a new and attractive anti-infective target to further
explore.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Previous studies evidenced that both the hydroxyl groups at C and C of DPD (Figure 2) are
4
5
essential for phosphorylation. It has been demonstrated that DPD is phosphorylated at
position five and that phosphorylation occurs only when the hydroxyl group at C is not
4
derivatized.35 Starting from these considerations, we herein designed new DPD-related
compounds where the portions essential for LsrK-mediated phosphorylation (i.e., the two
hydroxyl groups at C and C ) are kept constant while the diketo moiety of DPD is embedded
4
5
in heteroaromatic rings. The strategy of incorporating the carbonyl groups at C2 and C3 in
heteroaromatic moiety may allow to obtain compounds stable in solution, and to avoid the
open/closed equilibrium typical of the majority of the DPD-analogs reported so far. e focused
our attention on different heterocyclic scaffolds decorated with different aliphatic and/or
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aromatic moieties (Schemes 1 – 4), with the final aim to investigate the chemical space of
compounds structurally related to DPD. Among the wide variety of heterocycles, we selected
annulated pyridines, pyridines, pyrimidines and pyrazoles, given their high frequency of
occurrence in several natural or synthetic drugs used for their antibacterial36,37, antifungal38–41
anticancer42–45 and anti-inflammatory46–48 properties, just to cite a few.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.2 Synthesis
Target compounds (Schemes 1 – 4) can be easily prepared applying our recently developed
protocol, starting from intermediate 1 bearing an ethyne group, suitable for further chemical
49
diversification. The synthesis of monosubstituted annulated pyridines-Het-DPD derivatives (Figure
2
, series A) was accomplished via a one pot, microwave-assisted, palladium-catalyzed strategy,
coupling alkyne 1 with o-bromoaldehydes. The resulting Sonogashira products were reacted with
ammonium acetate as imination reagent and then deprotected (Scheme 1). Briefly, alkyne 1 was
dissolved in DMF and then Pd(OAc) , PPh , KOAc and the appropriate o-bromoaldehyde were added.
2
3
The reaction mixtures were irradiated at 80 °C for 1 – 2 hours, ammonium acetate was added and the
resulting mixtures were irradiated again at 150 °C, thus affording the desired protected products 3a-g
(Scheme 1). Heteroaromatic o-bromoaldehydes (i.e., 3-bromofuran-2-carbaldehyde 2e, 3-
bromothiophene-2-carbaldehyde 2f and 5-bromo-2-methyl-4-thiazolecarboxaldehyde 2g) led to
isolation of the corresponding furopyridine, thienopyridine and thiazolopyridine derivatives 3e-f. Final
ACS Paragon Plus Environment

Page 11 of 98
Journal of Medicinal Chemistry
1
2
3
acidic treatment yielded the monosubstituted annulated pyridines-Het-DPD derivatives 4a-g (Scheme
).
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
O
OH
X
n
Y
X
Y
a,a'
O
X
n
b
HO
O
H
1
O
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
n
Y
R1
N
Br
R1
N
1
2a-g
3a-g
4a-g
a, n = 2, X = H, Y = H, R1 = H, 90%
b, n = 2, X = H, Y = H, R = p-CH , 54%
c, n = 2, X = H, Y = H, R = m-OH, 67%
d, n = 2, X = H, Y = H, R1 = o-F, 42%
e, n = 1, X = O, Y = H, R1 = H, 48%
f, n = 1, X = S, Y = H, R1 = H, 73%
1
3
1
1
g, n = 1, X = S, Y = N, R = CH , 70%
3
Scheme 1: Synthesis of monosubstituted annulated pyridines-Het-DPD derivatives 4a-g.
Reagents and conditions: (a) 2a-g (0.9 eq), Pd(OAc) (1.8% mol), PPh (3.6% mol), KOAc
2
3
(1.8 eq), DMF, mw, 80 °C, 1h – 2h; (a’) NH OAc (1.8 eq), MW, 150 °C, 2h – 3h; (b) 12M HCl
4
(
cat.), 1,4-dioxane, 0 °C to rt, 1h – 3h
Sonogashira coupling43 of terminal alkyne 1 with acyl chlorides, followed by addition of
amidinium salts to the corresponding ynones and final acidic removal of the acetal protecting
group provided rapid access to 2,4,6-trisubstituted pyrimidines-Het-DPD derivatives (Figure 2,
series B). Benzoyl chloride 5a was selected to screen three different conditions for the
synthesis of ynone 6a. Surprisingly, copper-, ligand- and solvent-free acylation51 as well as
26
the use of a mixture of PdCl (CH CN) , Sphos and Cs CO
did not furnish the desired
2
3
2
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
product. Good results were obtained with the copper- and palladium-catalyzed system
50
52
proposed by Karpov et al. which allowed us to obtain compound 6a with a yield of 87%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Three different ynones (6a-c) were reacted with six different amidinium salts and the resulting
products 7a-f treated under acidic conditions (i.e., Scheme 2, conditions c) to afford the 2,4,6-
trisubstituted pyrimidines-Het-DPD derivatives 8a-f with moderate to excellent yields (i.e.,
43% – 82%).
HO
HO
R2
O
N
O
O
a
O
b
O
R2
c
O
N
O
R1
N
Cl
O
N
R1
R1
R1
1
5a-c
6a-c
7a-f
8a-f
a, R1 = Ph, R2 = CH , 54%
3
b, R1 = Ph, R2 = Cyp, 53%
c, R1 = Cyp, R2 = Ph, 46%
d, R1 = Cyp, R2 = p-Pyr, 67%
e, R1 = 2-thiophene, R2 = n-propyl, 71%
f, R1 = 2-thiophene, R2 = m-F-Ph, 82%
Scheme 2: Synthesis of 2,4,6-trisubstituted pyrimidines-Het-DPD derivatives 8a-f. Reagents
and conditions: (a) 5a-c (1.5 eq), PdCl (PPh ) (9% mol), CuI (3% mol), Et N (1.25 eq), THF,
2
3 2
3
rt, overnight; (b) R NHNH *HCl (1.2 eq), Na CO *10 H O (3.0 eq), 40 °C, overnight; (c) 12M
2
2
2
3
2
HCl (cat.), 1,4-dioxane, 0 °C to rt, 1h – 3h
The 2,3,4,6-tetrasubstituted pyridines-Het-DPD derivatives (Figure 2, series C) were easily obtained
following the one-pot, three-component and acid-free methodology developed by Bagley et al. This
ACS Paragon Plus Environment

Page 13 of 98
Journal of Medicinal Chemistry
1
2
3
heteroannulation combines a Michael acceptor with a nucleophile in the presence of an excess (10.0 eq)
of ammonium acetate (to generate, in situ, the enamine).53 Ethyl acetoacetate was selected as
nucleophile to have an additional diversification point (i.e., the carboxylic ester moiety) and reaction
with 6a (i.e., the Michael acceptor) in refluxing ethanol provided intermediate 9a with 92% yield
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Scheme 3). Tetrasubstituted pyridines 9a and 9b (from ynone 6b), were hydrolyzed in basic conditions
and then treated with hydrochloric acid to give target compounds 11a-b with excellent yields (i.e., 80%
and 72%, respectively).
HO
O
O
O
O
HO
O
O
O
O
O
a
b
c
O
O
O
OH
O
O
OH
R1
N
R1
R1
R1
N
N
6a-b
Ethyl acetoacetate
9a-b
10a-b
11a-b
a, R1 = Ph, 80%
b, R1 = Cyp, 72%
Scheme 3: Synthesis of 2,3,4,6-tetrasubstituted pyridines-Het-DPD derivatives 11a-b.
Reagents and conditions: (a) Ethyl acetoacetate (10.0 eq), EtOH, 50 °C, overnight; (b) 1M
NaOH (3.0 eq), EtOH, 0 °C to rt, overnight; (c) 12M HCl (cat.), 1,4-dioxane, 0 °C to rt, 1h – 3h
The synthesis of 3,5-disubstituted (series D) and 1,3,-5-trisubstituted pyrazoles-Het-DPD
derivatives (series E) started with the production of three additional aliphatic (i.e., isopropyl,
cyclopentyl and adamantane 6d-f) and one aromatic (i.e., furanyl 6g) ynones. Each of the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
seven ynones (6a-g) was reacted with two different hydrazines (i.e., hydrazine and
methylhydrazine) to generate, respectively, 3,5-disubstituted (12a-g) and 1,3,5-trisubstituted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(13a-g) protected pyrazoles. Final acidic deprotection afforded the targeted products 14a-g
and 15a-g with excellent yields (i.e., 67% – 92%, Scheme 4).
OH
a, R1 = Ph, 88%
H
O
HO
N
b, R1 = 2-furanyl, 82%
O
H
c
N
N
c, R1 = 2-thiophene, 83%%
d, R1 = i-Pr, 87%
e, R1 = Cyp, 89%
N
R1
R1
f, R1 = Cyclopentyl, 82%
g,R1 = Adamantane, 75%
O
a
O
b
O
12a-g
14a-g
O
O
R1
a, R1 = Ph, 79%
OH
b, R1 = 2-furanyl, 85%
O
HO
N
1
6a-g
O
c
N
c, R1 = 2-thiophene, 76%
d, R1 = i-Pr, 77%
e, R1 = Cyp, 92%
f, R1 = Cyclopentyl, 78%
g,R1 = Adamantane, 67%
N
N
R1
R1
13a-g
15a-g
Scheme 4: Synthesis of 3,5-disubstituted and 1,3,5-trisubstitted pyrazoles-Het-DPD
derivatives 14a-g and 15a-g. Reagents and conditions: (a) R COCl (1.5 eq), PdCl (PPh )
1
2
3 2
(9% mol), CuI (3% mol), Et N (1.25 eq), THF, rt, overnight; (b) R NHNH (1.3 eq), EtOH, rt,
3
2
2
overnight; (c) 12M HCl (cat.), 1,4-dioxane, 0 °C to rt, 1h – 3h
2.3 Screening of LsrK inhibitors
All the prepared compounds were assayed for inhibitory activity against LsrK at 200 µM
concentration. Unexpectedly, only four compounds (belonging to series D and series E)
ACS Paragon Plus Environment

Page 15 of 98
Journal of Medicinal Chemistry
1
2
3
among the 29 prepared, resulted effective in inhibiting LrsK. Compounds 12a, 12b (series D)
and 13a, 13b (series E) showing an inhibition percentage higher than 40%, were retested at
different concentrations (Table 1) and their IC50 values calculated. In Figure 3 the dose-
response curves together with the IC values of compounds 12a, 12b, 13a, 13b are reported.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
Figure 3: Dose-response curves against LsrK for compounds 12a, 12b, 13a, 13b. Data points
represent the mean ±SD of two independent experiments (n = 4)
To evaluate the specificity of compounds 12a, 12b, 13a, 13b, these were further tested
against glycerokinase, which has high sequence similarity (i.e., 41.2%) to LsrK. None of the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds showed activity against glycerokinase (Figure S1, SI). To sum up, pyrazole-
containing DPD derivatives emerged as the most interesting LsrK inhibitors among the series
under investigation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Page 17 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Inhibition
(%)
Series
Structure
Compound
R1
R2
X
Y
N
SD
3
a
H
H
H
H
H
O
S
H
H
H
H
H
H
N
H
H
H
H
H
H
N
H
2
2
2
1
1
1
2
2
2
2
1
1
1
14.91
1.53
1.22
3.83
0.94
1.41
4.02
2.35
0.63
0.39
1.55
0.41
1.00
0.42
0.42
1.41
4.39
0.74
3.23
0.85
0.33
0.69
3.32
2.37
1.11
2.94
1.42
3
b
p-CH3
1.66
28.55
1.76
5.75
11.06
6.32
0.48
0.40
1.67
0.65
2.07
0.17
0.10
9.41
15.18
0.76
21.22
0.96
0.20
0.22
24.48
21.65
2.94
47.76
19.79
Annulated
pyridines and
derivatives
Series A
3
c
m-OH
O
X
X
n
Y
R
3d
3e
o-F
―
O
1
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
N
3
f
H
3g
CH3
S
4
a
H
p-CH3
m-OH
o-F
H
H
H
H
O
S
4
b
Annulated
pyridines and
derivatives
Series A
OH
n
Y
R
4
c
HO
4d
―
1
4
e
H
N
4
f
H
4
g
CH3
S
7a
Ph
CH3
Cyp
7
b
Ph
2
,4,6-
O
7
c
Cyp
Ph
trisubstituted
pyrimidines
Series B
O
R2
N
―
―
―
―
―
―
7d
Cyp
p-Pyr
n-propyl
m-F-Ph
N
7
e
2-thiophene
2-thiophene
Ph
R1
7f
8
a
CH3
HO
8
b
Ph
Cyp
2
,4,6-
HO
R2
N
trisubstituted
pyrimidines
Series B
8c
8d
8e
Cyp
Ph
N
Cyp
p-Pyr
R1
2-thiophene
2-thiophene
n-propyl
m-F-Ph
8
f
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Inhibition
(%)
Series
Structure
Compound
R1
R2
X
Y
N
SD
9
a
Ph
Cyp
Ph
CH CH
0.10
0.07
0.66
1.69
3
2
2
9
b
CH CH
0.36
0
3
O
2
,3,4,6-
tetrasubstituted
pyridines
1
0a
H
O
O
O
―
―
―
Series C
OR2
10b
11a
Cyp
Ph
H
6.26
0.81
0.65
0.59
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
R1
N
HO
2
,3,4,6-
HO
tetrasubstituted
pyridines
―
―
―
―
―
―
―
―
11b
Cyp
1.33
0.49
OH
Series C
R1
N
12a
Ph
53.60
43.48
26.50
7.14
1.47
0
5.67
26.98
5.52
1.78
0.26
0.21
0.81
1
2b
2c
2d
12e
2f
2g
2-furanyl
2-thiophene
i-Pr
3
,5-
1
O
disubstituted
pyrazoles
Series D
H
O
1
N
N
Cyp
1
Cyclopentyl
Adamantane
R1
1
0
ACS Paragon Plus Environment

Page 19 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Inhibition
(%)
Series
Structure
Compound
3a
3b
3c
13d
3e
3f
13g
R1
R2
X
Y
N
SD
1
Ph
78.05
3.44
11.91
0.22
0.67
0.57
0.82
0.11
0.91
0.51
2.14
1.11
1.74
2.39
5.13
1.50
0.02
0.05
1.16
0.06
0.08
0.96
0.44
0.46
0.66
0.05
0.24
0.47
0.32
0.72
1
2-furanyl
2-thiophene
i-Pr
51.63
1
0
0
1
Cyp
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
Cyclopentyl
Adamantane
0
1
,3,5-
0
O
trisubstitted
pyrazoles
Series E
O
1
3h
m-CH -Ph
―
―
―
―
1.07
0
N
3
N
1
3i
m-CN-Ph
1
3j
m-Cl-,m-F-Ph
p-N(CH ) -Ph
4.42
0.19
9.83
4.38
4.92
1.51
0
R1
13k
3l
3m
3n
13o
4a
4b
3
2
1
3-isoxazole
2-indole
m-Pyr
1
1
CyHex
1
Ph
1
2-furanyl
2-thiophene
i-Pr
0
OH
3
,5-
H
N
14c
14d
0
HO
disubstituted
pyrazoles
Series D
N
―
―
―
―
―
―
―
―
0
1
4e
4f
14g
5a
5b
5c
15d
5e
5f
15g
Cyp
0
R1
1
Cyclopentyl
Adamantane
Ph
0
0
1
0
1
2-furanyl
2-thiophene
i-Pr
0
OH
1
,3,5-
1
0
HO
N
trisubstitted
pyrazoles
Series E
N
0
1
Cyp
0
R1
1
Cyclopentyl
Adamantane
0
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Inhibition
Series
Structure
Compound
6a
6b
16c
6d
6e
R1
R2
X
Y
N
SD
(%)
0
1
CH CH
―
0.62
0.96
0.70
0.83
0.59
3
2
1
i-Pr
0
0
0
0
3
,5-
O
R1
Cyp
O
disubstituted
pyrazoles
New pyrazole
derivatives
N
1
(CH ) -CN
2 2
―
―
―
N
1
Cyclopentyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
6f
Ph
0
0.53
3
,5-
N
disubstituted
pyrazoles
New pyrazole
derivatives
N
18
―
―
―
―
―
―
―
―
2.01
0.78
22a
H
H
O
0
2
1
5.56
0
2.34
0.64
1.83
n
X
2
2b
2c
3
,5-
O
2
31.13
O
disubstituted
pyrazoles
New pyrazole
derivatives
N
N
2
2d
N-Ac
1
2.07
1.65
Table 1: Activities of the synthesized DPD-IHs
ACS Paragon Plus Environment

Page 21 of 98
Journal of Medicinal Chemistry
1
2
3
4
2.
Structure-Activity Relationship (SAR) studies of pyrazole-containing DPD derivatives
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The results of the first LsrK screening highlighted the pyrazole moiety as the most promising
scaffold. Accordingly, we further explored the 1,3,5-trisubstituted pyrazole series (series E),
applying our synthetic protocol which permits an easy access to molecular diversity at three
different diversification points, as outlined in Figure 4. Based on synthetic feasibility and
commercial availability of the starting materials, nineteen new 1,3,5-trisubstituted pyrazole
derivatives have been prepared by installing different aliphatic and aromatic substituents at
position one (Scheme 5) and three (Scheme 6) as well as by replacing at position five the
cyclohexylidene protecting group with aliphatic rings of different sizes (Scheme 7). Moreover,
the dioxolane ring was removed (compound 18, Scheme 7).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4: Further diversification of 1,3,5-trisubstituted pyrazolic compounds
For modification at N the effects of aliphatic (linear, branched and cyclic) and aromatic
1
substituents on bioactivity were investigated. The synthesis of this second set of 1,3,5-
trisubstituted pyrazoles-Het-DPD derivatives (16a-f) started from ynone 6a to which was
added an excess (1.3 eq) of the corresponding hydrazine.
ACS Paragon Plus Environment

Page 23 of 98
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
O
R1
O
a
O
N
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6a
16a-f
a, R1 = Et, 44%
b, R1 = i-Pr, 60%
c, R1 = Cyp, 54%
1
d, R = (CH ) -CN, 41%
2
2
e, R1 = Cyclopentyl, 70%
f, R1 = Ph, 55%
Scheme 5: Synthesis of the first new set of 1,3,5-trisubstituted pyrazoles-Het-DPD derivatives
16a-f. Reagents and conditions: (a) R NHNH (1.3 eq), EtOH, rt, overnight
1
2
Several analogues where the phenyl at position three of the pyrazole nucleus was replaced
with other aliphatic, aromatic and heteroaromatic rings were then studied. Compounds 16h-o
(
Scheme 6) were readily prepared by condensation of eight new ynones synthetized by us
with methylhydrazine.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
a
b
O
N
O
N
O
R1
R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6h-o
13h-o
1
h, R = m-CH -Ph, 54%
3
1
i, R = m-CN-Ph, 65%
1
j, R = m-Cl-, m-F-Ph, 61%
1
k, R = p-NH -Ph, 41%
2
1
l, R = 3-isoxazole, 55%
1
m, R = 2-indole, 75%
1
n, R = m-Pyr, 46%
1
o, R = CyHex, 55%
Scheme 6: Synthesis of the second new set of 1,3,5-trisubstituted pyrazoles-Het-DPD
derivatives 13h-o. Reagents and conditions: (a) R COCl (1.5 eq), PdCl (PPh ) (9% mol), CuI
1
2
3 2
(
3% mol), Et N (1.25 eq), THF, rt, overnight; (b) MeNHNH (1.3 eq), EtOH, rt, overnight
3
2
Lastly, to study the relevance of the dioxolane ring on biological activity, we prepared
compound 18, where it was completely removed. Additionally, derivatives 22a-d, bearing
acetal substituents with different steric properties were also synthesized. Compound 18 was
prepared reacting benzoyl chloride 5a with 1-propyne 17 and the resulting ynone 6p with
methylhydrazine as previously described (Scheme 7A). By reaction of the diol 19 with an
excess (3.0 eq) of the corresponding ketone and a catalytic amount of p-TSA in neat
conditions, further acylation with 5a and condensation with methylhydrazine the desired five
ACS Paragon Plus Environment

Page 25 of 98
Journal of Medicinal Chemistry
1
2
3
new pyrazoles-Het-DPD 22a-d (Scheme 7B) were obtained. It has to be noted that 4-
piperidone was found not to be suitable for our purpose as the free basic nitrogen impeded
the Sonogashira coupling with benzoyl chloride while N-acetylpiperidone did not show any
reactivity issue and compound 22d could be isolated with 44% yield.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
O
N
A)
a
b
Cl
5a
17
6p
18
n
n
X
X
X
n
O
O
O
O
B)
O
N
OH
O
HO
c
O
a
O
b
N
Cl
19
20a-d
5a
21a-d
22a-d
a, X = H, n = 0, 69%
b, X = H, n = 2, 57%
c, X = O, n= 1, 85%
d, X = N-Ac, n = 1, 44%
Scheme 7: A) Synthesis of compound 18. Reagents and conditions: (a) 5a (1.5 eq), 1-
propyne (1.0 eq), PdCl (PPh ) (9% mol), CuI (3% mol), Et N (1.25 eq), THF, rt, overnight; (b)
2
3 2
3
MeNHNH (1.3 eq), EtOH, rt, overnight. B) Synthesis of the third new set of 1,3,5-
2
trisubstituted pyrazoles-Het-DPD derivatives 22a-d. Reagents and conditions: (c) Ketone (3.0
eq), p-TSA (cat.), rt, overnight
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
All the prepared 1,3,5-trisubstituted pyrazoles-Het-DPD were assayed for inhibitory activity
54
against LsrK at 200 µM concentration, according to the protocol previously described. The
obtained results confirmed that the presence of the dioxolane ring at position 5 of the pyrazole
moiety is essential for the binding to LsrK and that acetals derivatives have a better profile
respect to the corresponding diols. Overall, the results indicate that the most active LsrK
inhibitor is compound 13a, bearing a methyl group at N , a phenyl group at C and an acetal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
moiety at position five.
2.5 Molecular modeling studies
To support the SAR analysis and to inspect the molecular basis of their binding to LsrK, all
the compounds were docked into the binding site of the LsrK crystal structure (PDB ID: 5YA1)
using Glide, a molecular docking program reliable for predicting the binding mode of small
molecules in protein complexes. Hence, docking poses were analyzed for both interactions
and geometry. At first, we considered the modification at the diol moiety of DPD, our inspiring
molecule, since in vitro experiments suggested the presence of the dioxolane ring in the Het-
DPD derivatives as essential for the binding to LsrK. We observed that the non-polar
ACS Paragon Plus Environment

Page 27 of 98
Journal of Medicinal Chemistry
1
2
3
cyclohexane ring of acetal compounds can form electrostatic interactions with the surrounding
environment. Compounds bearing the diol moiety showed an opposite orientation in the
binding site (Figure S4, SI). The orientation of the diol groups towards the negative
electrostatic potential surface of the binding site (Glu 454 and Thr 456) resulted in repulsion
effects, thus explaining the weaker binding of diols compared with acetals.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We then focused on pyrazole Het-DPD derivatives (series D and series E) since the in vitro
results of the first LsrK screening highlighted the pyrazole moiety as the most promising
scaffold. In line with experimental data, compound 13a gave the best interactions. Although
compounds 12a and 13a, which differ only in the presence of N -CH , showed similar binding
1
3
poses, we observed that the most active inhibitor 13a formed a hydrogen bond interaction
with Thr 275, whereas 12a with Phe 276 and Gln 278 (Figure S2, SI). Hence, we speculated
that the interaction with Thr 275 may be involved in the substrate phosphorylation reaction.
The binding poses of 12a and 13a (Figure S3, SI and Figure 5) are similar to those of the less
active furanyl-substituted derivatives 12b and 13b, with the only exception of the “pyrazole
ring flipping”, caused by the bulky N -CH group. The conformation of the pyrazole in 12a and
1
3
13a resulted in a better methyl stabilization energy of N -CH , thus favoring the interaction
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 98
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with the target. Moreover, the oxygen of the furan ring of 13b (Figure 5) originated a negative
electrostatic potential and the local binding site environment, for example the Thr 456 residue
contributed to the electrostatic repulsion.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Starting from these observations, further Het-DPD derivatives potentially able to better
interact with LsrK will be designed and synthetized in the next future.
Figure 5. Compound 13a (left) and 13b (right) binding pose in the LsrK binding site (PDB ID:
5YA1). Hydrogen bond interactions (red dashed line) are shown along with active site
residues. Electrostatic potential surfaces were presented as mesh (blue-positive potential and
red-negative potential)
3.
CONCLUSIONS
ACS Paragon Plus Environment

Page 29 of 98
Journal of Medicinal Chemistry
1
2
3
Resistance to antibiotics is constantly increasing and it is estimated that, by 2050, multidrug
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
resistant bacteria will kill more people than cancer. In the last decade, modulation of QS has
become an attractive therapeutic strategy to fight bacterial resistance. Two different DPD-
related compounds (i.e., isobutyl-DPD and phenyl-DPD) have already shown their activity in
combination with gentamicin and small molecules able to modulate/inhibit QS are therefore
considered interesting “non-conventional” tools to be used in combination with classical
antibiotics.10–13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As a continuation of our recent findings, where, applying a virtual screening approach we
were able to identify 2-substituted amino benzoic acids as LsrK inhibitors, in this
communication, we report for the first time on the “ad hoc” design and synthesis of potential
LrsK inhibitors structurally related to DPD. The medicinal chemistry efforts presented in this
work provided 48 Het-DPD derivatives, characterized by different heterocyclic scaffolds and
led to the identification of effective representatives characterized by in vitro LsrK enzymatic
activity (IC ) in the micromolar range. All the target compounds have been easily prepared in
50
maximum four synthetic steps, thus allowing to easily explore the chemical space of DPD.
Remarkably, four compounds displayed IC values between 100 µM and 500 µM, evidencing
50
ACS Paragon Plus Environment