Design and Evaluation of New Quinazolin-4(3 H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa

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Design and Evaluation of New Quinazolin-4(3H)‑one Derived PqsR

Antagonists as Quorum Sensing Quenchers in Pseudomonas

aeruginosa

Fadi Soukarieh, Alaa Mashabi, William Richardson, Eduard Vico Oton, Manuel Romero,

Shaun N. Roberston, Scott Grossman, Tomas Sou, Ruiling Liu, Nigel Halliday, Irena Kukavica-Ibrulj,

Roger C. Levesque, Christel A. S. Bergstrom, Barrie Kellam, Jonas Emsley, Stephan Heeb, Paul Williams,

Michael J. Stocks,* and Miguel C á mara *

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ABSTRACT: P. aeruginosa (PA) continues to pose a threat to

global public health due to its high levels of antimicrobial resistance

(AMR). The ongoing AMR crisis has led to an alarming shortage

of eﬀective treatments for resistant microbes, and hence there is a

pressing demand for the development of novel antimicrobial

interventions. The potential use of antivirulence therapeutics to

tackle bacterial infections has attracted considerable attention over

the past decades as they hamper the pathogenicity of target

microbes with reduced selective pressure, minimizing the

emergence of resistance. One such approach is to interfere with

the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal

molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and

host−microbe interactions. In this study, we report the hit identiﬁcation and optimization of PqsR antagonists using virtual screening

coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA P_pqsApromoter controlled by PqsR with an IC₅₀

of 1 μM. Using isothermal titration calorimetry, a K_dof 10 nM for the PqsR ligand binding domain (PqsR^LBD) was determined for

61. Furthermore, the crystal structure of 61 with PqsR^LBDwas attained with a resolution of 2.65 Å. Compound 61 signiﬁcantly

reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this

compound potentiated the eﬀect of ciproﬂoxacin in early stages of bioﬁlm treatment and in Galleria mellonella infected with PA.

Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identiﬁcation of a PA

QS inhibitor which can be advanced into preclinical development.

KEYWORDS: Pseudomonas aeruginosa, PqsR, Pseudomonas quinolone signal (PQS), bioﬁlms, quorum sensing inhibition

past decades, the focus of the preclinical antibacterial projects

was shifted toward new targets, mostly in Gram-negative

pathogens, as in the case of antivirulence approaches, adjuvant

therapies, and antibiotic potentiators.⁶One of the extensively

investigated approaches to target virulence is the development

of drugs which can interfere with bacterial quorum sensing

systems (QSSs). These are cell-to-cell communication

mechanisms used by many bacterial pathogens to control the

he rising, wide spread of antimicrobial and multidrug-

T_{resistant bacterial strains at an unprecedented rate places}

a major burden on global public health predominantly with

regard to the shortage of eﬀective therapeutic options.¹

Alarmingly, within the antibacterial pipelines currently under

clinical development, there is no novel antibacterial class that

could actively treat any of the WHO top priority pathogens

also known as ESKAPE (Enterococcus faecium, Staphylococcus

aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudo-

monas aeruginosa, and Enterobacter spp).^2,3P. aeruginosa (PA)

is a Gram-negative opportunistic bacterium and a common

cause for nosocomial infections particularly in cystic ﬁbrosis

and immunocompromised patients.⁴Consequently, there is an

urgent need for the discovery of novel approaches to combat

bacterial infections with mechanisms of action that do not

impose strong selective pressure on bacteria.⁵In fact, over the

Received: March 31, 2021

American Chemical Society

https://doi.org/10.1021/acsinfecdis.1c00175

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A

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Figure 1. (A) Chemical structure of pqs hit with site of modiﬁcations and (B) improved hit molecule 22, with improved activity.

a

Scheme 1. Synthetic Route for 22 and its Analogues 23−41

a

Reagents and conditions: (a) Cs₂CO₃, TBAI, CH₃CN, reﬂux, yields 21−80%.

disease process.^7,8PA possesses three types of QSSs that

employ various diﬀusible chemical signals called quorum

sensing signaling molecules (QSSMs) or autoinducers (AIs).

The las and rhl systems use N-acylhomoserine lactone derived

signals while the pqs system relies on alkyl quinolone (AQ)

derived molecules.⁹Using these systems, PA can coordinate

the production of virulence determinants in a coordinated

population-dependent manner in response to external stimuli

or threats.^10,11While the las and rhl systems have been studied

extensively, the pqs system has been shown in recent years to

play a substantial role in the virulence of PA.^7,12The

biosynthesis of the AQ signals 4-hydroxy-2-heptylquinoline

(HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS) is ini-

tiated from anthranilate through a sequence of enzymes (PqsA,

PqsBC, PqsD, PqsH) encoded by pqsABCDEphnAB operon

and the pqsH gene.^13,14The resulting AQs then interact with

the C-terminal ligand binding domain of the LysR type

regulator PqsR (also known as MvfR) resulting in a

conformational change leading to the activation of the pqs

operon, likely by binding to the pqsA promoter (P_pqsA).¹³To

date, there have been several attempts to pharmacologically

inhibit the pqs system by either interfering with the

biosynthesis of AQs targeting the biosynthetic enzymes

(such as PqsD¹⁵and PqsA¹⁶) or preventing the signal

reception (through antagonists of PqsR).¹⁷Several chemical

series have been described as PqsR/pqs inhibitors including

quinazolinone, quinolinone, thioacetamide, and hydroxy-

benzamide derived molecules, but none have yet been taken

forward to clinical trials.^7,18,19In this study, we report a new

series of quinazolinone derived pqs inhibitors with low

micromolar potency in PA PAO1-L. Compound 61, one of

the most potent inhibitors in this series, was evaluated using

PA phenotypic analysis including pyocyanin and determination

of AQ signal levels in various clinical and lab strains. The study

also investigated the eﬀect of 61 on PA bioﬁlms, as a sole

treatment or in combination with ciproﬂoxacin. Moreover, the

cocrystal structure of two inhibitors (61, 40) with the PqsR

ligand binding domain was obtained revealing the main

interactions governing binding. To conclude this study, 61

was evaluated in a G. mellonella PA infection model alone and

in conjunction with ciproﬂoxacin.

RESULTS

■

Identiﬁcation of PqsR Hits through “in silico”

Docking and Bioreporter Assays. A virtual screening of

an in-house drug-like compound library composed of 85 061

curated molecules was performed using the PqsR ligand

̈

binding domain (PDB 4JVI) employing the Schrodinger Suite

for molecular docking.^20,21The 583 top scored compounds

were selected after introduction of a Glide docking score

threshold of −9.0. The selected compounds were then

validated for their ability to inhibit the pqs QS system at a

concentration of 10 μM using the bioluminescence-based

bioreporter strain PAO1-L harboring the chromosomal

transcriptional fusion mCTX::P_pqsA-lux. For a molecule to

score as active, a reduction of at least 50% of bioluminescence

had to be achieved without aﬀecting bacterial growth. This

process led to the discovery of multiple hits,^18,19among them

was 2-(4-((3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropyl)amino)phenyl)acetonitrile 1 with an IC₅₀of

13.1 0.6 μM (Figure 1).

Synthetic Chemistry Exploration for the Structure−

Activity Relationship Study. To investigate the structure−

activity relationship (SAR) of 1, our synthetic strategy focused

on three sites of modiﬁcation: the quinazoline headgroup, the

B

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a

Scheme 2. Synthetic Route of Compounds 42−51

a

Reagents and conditions: (a) Ph₃P, DIAD, DPPA, THF, 45 °C, overnight; then Ph₃P, NH₄OH, THF: H₂O, rt, overnight; 25%. (b) 46−48 react

corresponding anhydrides, 30 °C, DCM, 3 h; 90−95%. (c) 49−51 react corresponding (dimethyl amino) substituted acids, PyBrop,

trimethylamine, DMF, rt, 4 h; 54−49%. (d) Iodoethane, Cs₂CO₃, CH₃CN, 60 °C, overnight; 65%. (e) Dess−Martin periodinane, DCM, rt,

overnight; 76%. (f) Dimethylamino-sulfur triﬂuoride, 45 °C, DCM, 0.5 h; 68%.

a

Scheme 3. Synthetic Route of Compounds 54 and 55 to Explore the Inﬂuence of the Secondary Alcohol Present in 22

a

Reagents and conditions: (a) Cs₂CO₃, TBAI, CH₃CN, 100 °C, overnight; yields (52−53: 70−72%; 54−55: 40−49%).

a

Scheme 4. Synthetic Route for 22 Derivatives with Various Nitrile Replacements

a

Reagents and conditions (a) NH₂OH (50% in water), NaOH, EtOH, 80 °C, 16 h; then Ac₂O, toluene, 110 °C, 2 h; 63%. (b) Acetylhydrazine,

sodium methoxide, CH₃OH, 80 °C, overnight; 15%. (c) NaN₃, Et₃N·HCl, DMF, 130 °C, overnight; 75%. (d) Cs₂CO₃, DMSO: 30% H₂O₂, rt, 0.5

h; 96%. (e) HCl, H₂O reﬂux, overnight; 44%.

substituted aniline tail, and the β-amino alcohol linker (Figure

1, A). To expedite the synthesis, 22 (Figure 1, B) was designed

with a modiﬁed tail group bearing a phenyl ether linker in

place of the substituted aniline present in 1. Compound 22

showed a 4-fold increase in potency compared to 1 (IC₅₀3.2

1.0 μM) and was therefore considered as a new starting point

for development of this series. A robust synthetic strategy was

employed to synthesize a series of analogues (22−41) with

modiﬁed head or tail groups through an epoxide ring opening

reaction between substituted quinazolin-4(3H)-ones 2−12 and

2-(methylphenoxy)oxirane derivatives 13−21 to aﬀord com-

pounds 22−41 (Scheme 1).²²

Subsequently to further explore the SAR, the secondary

alcohol present in 22 was modiﬁed with isosteric replacements

(Scheme 2). The amino derivative 42 was obtained from a

Mitsunobu conversion of the hydroxyl group to azide²³which

was subsequently reduced to the primary amine using the

Staudinger reaction.²⁴Compound 42 was further function-

alized either through a direct alkylation with iodoethane to give

45 or through conventional amide coupling reaction with

either the corresponding anhydrides (46−48) or using PyBrop

and triethylamine to aﬀord 49−51.²⁵Oxidation of the

secondary alcohol by the Dess−Martin reagent aﬀorded

ketone 43.²⁶Finally, the ﬂuorine derivative 44 was obtained

from 22 using dimethylamino-sulfur triﬂuoride.²⁷

To further explore the pharmacological requirement of the

secondary alcohol, the alcohol was either removed 55 or

replaced with a methyl group 54. 6-Chloroquinazolin-4(3H)-

one 2 was reacted with either 1-bromo-3-chloro-2-methyl-

propane or 1-bromo-3-chloropropane to aﬀord 52 and 53

respectively, which were reacted with 2-(4-hydroxyphenyl)-

acetonitrile to provide 54 and 55 (Scheme 3).

The cyanomethyl group on the aromatic tail was also subject

to replacement with 5-membered ring heterocycles. The nitrile

group was reacted with hydroxylamine which was cyclized in

the presence of acetic anhydride to yield 5-methyl-1,2,4

oxadiazole derivative 56.²⁸While the triazole derivative 57 was

obtained by reacting 22 with acetylhydrazine and sodium

methoxide.²⁹The tetrazole derivative 58 was prepared through

the reaction of 22 with NaN₃in DMF. Acid hydrolysis of the

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a

Scheme 5. Synthetic Route of Single Enantiomers 61 and 62

a

Reagents and conditions: (a) Cs₂CO₃, MeCN, reﬂux, overnight; yields 58−64%.

nitrile to the corresponding carboxylic acid gave 60 and the

carboxamide derivative 59 was prepared through hydrolysis by

aqueous hydrogen peroxide in the presence of cesium

carbonate (Scheme 4).³⁰

Table 1. Structures and Activities of 22 Analogues with

Modiﬁed Head Groups 23−32

a

Finally, the single enantiomers of 22 were prepared through

reacting commercially available (R)- and (S)-oxiran-2-ylmethyl

3-nitrobenzenesulfonates with 2-(4-hydroxyphenyl)acetonitrile

to provide the corresponding epoxides that were subsequently

reacted with 2 to aﬀord (R)- and (S)-enantiomers 61 and 62,

respectively (Scheme 5).³¹The percentages of enantiomeric

excess (ee) of the products were 93.5% and 92.1% for the (R)-

isomer 61 and (S)-isomer 62 respectively, as determined

through chiral phase HPLC analysis.

Pharmacological Evaluation and Structure Activity

Relationship. Screening of Commercial Compound Library.

To provide initial insights into the exploration of the structure

activity relationship within this new hit series, a SAR by

catalogue study was initiated where a selection of commercially

available close analogues sharing structural similarity of over

90% with 22 were purchased and screened. The compounds

bear slight modiﬁcation of the quinazolin-4(3H)-one sub-

stituents and the aromatic tail of 22. Despite bearing close

structural similarity to 22, these compounds proved inactive

when screened at a 10 μM spot test concentration (Table S1−

Supporting Information). However, these results provided an

early indication of the tight SAR within this hit series and a

design-make-test strategy to introduce preserved and minimal

modiﬁcations was therefore implemented.

Compounds with Modiﬁed Headgroups. The SAR study

started by introducing simple and conserved modiﬁcations in

the quinazolinone ring (QZN) (Table 1). The chlorine atom

on the QZN ring had a variable eﬀect on biological activity,

with the optimal position being substitution at the 6-position

22 as the 7-substituted chlorine analogue 23 had a 2-fold loss

of activity. Analogues with chlorine atoms at the 5- or 8-

position (26 and 27, respectively) were inactive, likewise the

disubstituted compounds 24 (6, 7-diCl) or 25 (6, 8-diCl).

Replacement of the 6-substituted chlorine atom with the

sterically more demanding bromine atom 28 improved the

potency by 3-fold (IC₅₀0.8 μM) and a similar eﬀect was

obtained with an electron withdrawing group such as NO₂29

(IC₅₀0.6 μM) or CF₃31 (IC₅₀0.6 μM). It is noteworthy, that

the 7-nitro substituted derivative 30, as opposed to 23, was

completely inactive which is likely due to the size of the nitro

group compared to the chlorine substituent. Branching oﬀ the

QZN at the 2-position with a methyl group, 32 abolished

remaining activity in PAO1-L

ID

R¹

R²

X¹

at 10 μM (RA%)

IC₅₀(μM)

23

24

25

26

27

28

29

30

31

32

7-Cl

H

Me

CH

41.2 1.1

NA

22.3 4.7

17.2 0.6

NA

14.0 0.9

84.1 3.2

6.7 0.3

6,7-Cl

6,8-Cl

8-Cl

5-Cl

6-Br

6-NO₂

7-NO₂

6-CF₃

6-Cl

0.8 0.2

0.6 0.1

a

NA denotes no activity at a 10 μM concentration.

activity, demonstrating the limited substitution possibility

around the headgroup.

Compounds with Modiﬁed Tail Groups. As the manipu-

lation of the headgroup of 22 provided a slight improvement

for potency, a series of modiﬁcations were introduced at the

level of the aromatic phenyl ring of 22 to explore the SAR of

this region (Table 2). The cyanomethylene group proved

important for biological activity at the para-position while the

meta-substituted analogue 33 lacked biological activity. More-

over, aromatic nitriles, e.g. compounds 34 and 35 with para-

and meta-benzonitrile respectively, exhibited signiﬁcant loss of

activity. Further, modiﬁcation to the cyanomethylene group

included the introduction of a cyclopropyl moiety 37 resulted

in reduced activity, while extending the chain length by an

extra methylene group 36 preserved the potency. Introduction

of an ether bond between the phenyl and the cyanomethylene

group 40 caused a slight reduction of activity. In some cases,

disubstitution of the phenyl ring retained activity, in particular,

ortho-substituted ﬂuorine analogue 39 (IC₅₀2.2 μM) proved

active when compared to meta-position 38, which suﬀered a 2-

fold loss in potency (IC₅₀6.4 μM). Extending the para-

position of the phenyl ring with a bulky phenoxy group 41 led

to a 30% inhibition of pqs activity at 1 μM test concentration.

One of the advantages of having the aliphatic nitrile is the

versatility to convert it to various heterocycles to further

explore SAR. While the oxadiazole replacement 56 retained

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a

Table 2. Exploration of the Tail Group of 22

a

NA denotes no activity at a 10 μM concentration.

a

activity, triazole containing compound 57 demonstrated

signiﬁcant loss in activity and the tetrazole derivative 58 was

totally inactive. Finally, hydrolysis of the nitrile group to its

corresponding carboxamide led to inactive compound 59 while

the carboxylic acid derivative 60 reduced activity (35%

inhibition at 10 μM). Despite all these modiﬁcations on the

tail of 22, the para- substituted acetonitrile group proved to be

the optimal substituent with a ﬂuorine atom substituted at the

ortho-position 39 having a comparable level of activity.

Analogues with Modiﬁed Linker Groups. It became

apparent from the initial SAR investigation of the head and

tail groups of 22 was limited to conservative modiﬁcations,

therefore the focus of the evaluation moved to the linker group

and particularly the secondary alcohol (Table 3). In this series

of analogues, the head and tail moieties were ﬁxed

corresponding to those of 22, while the linker was varied

and substituted. Oxidation of the alcohol to ketone 43 retained

biological activity with IC₅₀of 4.3 μM. Conversely,

substitution of hydroxyl with a ﬂuorine atom 44 signiﬁcantly

reduced biological activity. Interestingly, replacing the alcohol

with an amine 42 gave a compound with comparable activity

(IC₅₀2.4 μM). However, alkylating the amine with an ethyl

group 45 or acylation with small carboxylic acids (46−50)

Table 3. Modiﬁcation of the Linker Group

remaining activity in

PAO1-L at 10 μM (RA%) IC₅₀(μM)

ID

R

42

43

44

45

46

47

48

49

50

51

54

55

61

62

NH₂

=O

F

NHCH₂CH₃

NHCOCH₃

NHCOEt

10.5 1.2

16.6 1.7

67.8 11.9

69.2 15.0

86.6 3.8

81.6 5.0

89.5 6.3

NA

2.4 0.2

4.3 1.3

NHCOPr

NHCOCH₂N(CH₃)₂

NHCO(CH₂)₂N(CH₃)₂

NH(CH2)₃N(CH₃)₂

H

CH₃

(R)-OH

NA

87.40 5.3

88.6 8.5

NA

11.0 0.8

43.5 4.9

1.1 0.4

7.3 1.4

(S)-OH

a

NA denotes no activity at a 10 μM concentration.

E

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Figure 2. Isothermal titration calorimetry analysis. (a) ITC thermogram of 61 binding to PqsR^LBD; (b) thermodynamic proﬁles of 61 titrations into

WT. Titrations for WT constructs performed in at least two separate experiments.

Figure 3. Crystal structures of PqsR^LBDto various ligands: (a) Ribbon representation of PqsR^LBDcrystallized with 61; (b) Surface representation of

PqsR^LBDwith 61 (green sticks) showing (A and B) pockets (PDB 7O2T); (c) Surface representation of PqsR^LBDwith 40 (cyan sticks) showing

(PDB 7O2U); (d) Overlay of NHQ (represented as orange sticks, PDB 4JVD³²) and 61 in the ligand binding domain; (e) Overlay of NHQ

(represented as orange sticks) and 61 (represented as green sticks) in the B pocket of the ligand binding domain; (f) Overlay of 61 and 40 in the

ligand binding domain.

resulted in a loss of activity. In a similar fashion, removing the

hydroxyl group 54 or substituting it with a methyl group 55

removed all biological activity.

To conclude the SAR study, the chiral resolution of the

alcohol was examined through the synthesis of the (R)- (61)

and (S)-enantiomers (62) (Scheme 4). The (R)-enantiomer

61 proved more active (IC₅₀of 1 μM) over the (S)-

enantiomer, which was 7-fold less active (IC₅₀of 7 μM).

In conclusion, the extensive SAR study demonstrated the

very tight structural requirements for activity within this hit

series. It is noteworthy that 61, the (R)-enantiomer of 22,

proved to be one of the most potent inhibitors and the

remainder of our study centered on this compound.

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Figure 4. Pyocyanin quantiﬁcation assay. Bar chart representing pyocyanin production in laboratory and clinical strains: blue bars refer to untreated

controls, gray bars represent treatment with 3 μM of 61. The data was normalized to a negative control of DMSO in each strain. The error bars

represent the standard deviation of biological triplicates. The statistical signiﬁcance was performed using paired t test analysis (Graphpad 9.0).

Isothermal Titration Calorimetry Assay. As the

bioreporter assay provides data about the inhibition levels of

the pqs system but does not provide direct evidence that the

compounds are binding to PqsR, it was paramount to conﬁrm

the binding using an orthogonal approach. For this purpose,

we used isothermal titration calorimetry (ITC) which also

enables the measurement the binding thermodynamics

parameters between PqsR^LBDand 61. This analysis revealed

that 61 has strong PqsR^LBDaﬃnity with dissociation constant

(K_dof 10 nM) (Figure 2) and a stoichiometry of 1:1.

antagonists. For this purpose, the expression of pyocyanin in

the presence of 61 at 3 μM (3-fold the IC₅₀value) was

measured on PAO1-L and PA14 lab strains showing a

reduction of 80% and 95% of pyocyanin levels respectively

compared to untreated controls (Figure 4). The study was

further extended to validate the eﬀect of 61 on a selection of

clinical isolates with diﬀerent genomic backgrounds where

PALESB58, PAK6085, and PA748 clinical isolates were

selected as representatives (Figure 4).³⁶The study concluded

that these isolates were less sensitive to the eﬀect of 61 with

only the PAK6085 strain showing a signiﬁcant reduction of

27%.

Alkyl Quinoline Quantiﬁcation Assay. To further inves-

tigate the impact of the PqsR inhibitor 61 on the expression of

the pqsABCDE operon, production of HHQ and PQS signals

was quantiﬁed using LCMS/MS analysis. Similar to the

pyocyanin assay, the eﬀect of 61 on alkylquinoline signal

production was quantiﬁed in lab strains as well the three

clinical isolates mentioned previously using a concentration

that equals 3 times the corresponding IC₅₀. The results

demonstrated that 61 signiﬁcantly inhibited HHQ and PQS

production in PAO1-L (>90%) and PA14 (∼50%) in

agreement with the pyocyanin data, although to a lesser extent

in PA14 (Figure 5). Once more the PAK6085 clinical isolate

was the only one to respond to 61 although to a lesser extent

than the lab strains whereas PALESB58 and PA748 did not

show a signiﬁcant response.

Eﬀect of 61 on PA Bioﬁlm Formation. Bioﬁlms are highly

resilient to antibiotic treatments. Quorum sensing has been

shown to play a critical role in bioﬁlm development.³⁷To

examine whether 61 could aﬀect bioﬁlm formation and

potentiate antibiotic activity, pre-established PAO1-L bioﬁlms

(48 h) grown in the presence of 61 were treated with the

broad-spectrum antibiotic Ciproﬂoxacin (Cip) and their

viability investigated using LIVE/DEAD BacLight bacterial

viability staining and confocal laser scanning microscopy

(CLSM). The eﬀect of 61 on bioﬁlm viability as a single

treatment or in combination with Cip was measured at two

diﬀerent time points (5, 16 h) to establish whether this PqsR

antagonist could enhance the eﬀect of the antibiotic. It was

evident that 61 on its own had no signiﬁcant eﬀect on bioﬁlm

viability, however, when combined with Cip it showed

signiﬁcant potentiation of the antibiotic eﬀect at 5h of

incubation (Figure 6). However, this was not observed at 16

Crystal Structure of PqsR Antagonists with PqsR^LBD

.

To gain a detailed insight into the molecular interaction of the

antagonists with PqsR, the X-ray cocrystal structures

complexed with 61 and 40 were obtained by ligand soaking

(Figure 3a, b, c, and f). As previously reported,³²PqsR^LBD

compromises a deep pocket (B pocket) which usually

accommodates the quinolone part of the autoinducer, as

exempliﬁed in the case of 2-nonyl-4-quinolone (NHQ), while

the superﬁcial pocket (A pocket) contains the aliphatic side

chain (Figure 3d, e).³²The structures revealed that the

quinazolinone moiety of 61 and 40 reside in the B pocket, in a

similar manner to NHQ, surrounded by aliphatic residues

(Ala¹⁰², Ala¹⁶⁸, Ile²⁰⁷, Ile²⁰⁸) while the hydroxyl group

establishes hydrogen bonding with glutamine (Glu¹⁹⁴) and

arginine (Arg²⁰⁹) (Figure 3b and c). The aromatic tail adopted

a similar conformation to NHQ sustaining hydrophobic

interaction while the nitrile group is oriented toward the

tyrosine Tyr²⁵⁸. It was noted that there was a slight shift of the

nitrile group position between 61 and 40 likely to the presence

of the ether bond in the latter with elevated B factors

suggesting the nitrile is thermally mobile within pocket A.

However, both compounds maintained the same conformation

overall and shared the electrostatic interaction with the same

residues.

Pharmacological Evaluation of PqsR Inhibitors. Pyocya-

nin Quantiﬁcation Assay. PA produces a range of redox active

phenazine derived molecules, among these pyocyanin is one of

the most studied compounds which plays various roles in

virulence, gene expression, bioﬁlm formation as well as host-

tissue damage.³³Pyocyanin biosynthesis is achieved through

the activation of the phenazine phz operons which are under

the regulation of the pqs system.^13,34,35Consequently, the

attenuation of pqs system leads to a reduction of pyocyanin

production, and hence quantiﬁcation of pyocyanin levels serves

as an indirect readout for the inhibition caused by PqsR

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Figure 6. Bioﬁlm viability assay. Bar chart showing PAO1-L bioﬁlm

viability quantiﬁed after treatment with diﬀerent conditions for 5 or

16 h. The concentrations of the drugs used were ciproﬂoxacin 60 μg/

mL (CIP), 61 10 μM. The statistical signiﬁcance was performed using

paired t test analysis (Graphpad 9.0).

Cytotoxicity Study of 61 on Lung Epithelial Cells. The

eﬀect of 61 on A549 lung epithelial cell cytotoxicity assay was

examined to evaluate the suitability of this inhibitor for further

development. This study concluded that PqsR inhibitor 61 did

not exhibit any cytotoxic eﬀect up to a concentration of 300

μM (LD₅₀> 300 μM) which is around 300-fold greater than

the corresponding IC₅₀in PAO1-L (1 μM).

Figure 5. Alkyl quinolone quantiﬁcation assay. Relative quantiﬁcation

of AQs produced by diﬀerent PA strains. Blue bars represent control

while gray bars indicate treated samples. Error bars represent the

standard deviation of biological triplicates. Data was normalized

against the DMSO control group. The statistical signiﬁcance was

performed using paired t test analysis (Graphpad 9.0).

DISCUSSION

■

In this study, we reported the discovery of (R)-2-(4-(3-(6-

chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)-

acetonitrile 61 as a novel PqsR inhibitor. The starting hit 1,

which originated from a hit identiﬁcation campaign, demon-

strated weak pqs inhibition in the bioreporter whole cell assay

with an IC₅₀of 13 μM. To facilitate the synthesis of analogues,

compound 22 was proposed and shown to be with 4-fold more

active than 1 (IC₅₀= 3.1 μM). To progress this hit to a lead

stage, an extensive structure activity relationship was

performed on 22 and concluded that even minor structural

alterations could lead to obliteration of potency. The key

ﬁndings of SAR can be summarized as follows: (I) The

acetonitrile group at the para-position of the phenyl ring is

important for activity and the overall potency can be

marginally improved with a meta-ﬂuorine atom, (II)

substitution at the 6-position of the quinazolin-4(3H) was

preferred for a halogen or an electron withdrawing group, (III)

the hydroxyl group of the linking group is preferred and the

(R)-enantiomer 61 possesses the improved activity compared

to the (S)-enantiomer. Nevertheless, inhibitors of the P_pqsA-lux

reporter could potentially be targeting the AQ biosynthetic

enzymes rather than PqsR. To further conﬁrm that 61 is acting

as a PqsR inhibitor, an ITC analysis was used to determine the

thermodynamic parameters of binding and to aid the

understanding of the biomolecular interactions. This showed

that 61 was able to bind to PqsR^LBDwith a K_dof 10 nM. These

outcomes were further conﬁrmed by the X-ray crystal

structures of 61 and 40 with PqsR^LBD. These structures

revealed that the binding is mainly governed by hydrophobic

h where Cip activity matched that of the combined therapy

(Cip and 61).

In Vivo Eﬃcacy of PqsR Antagonist 61. To evaluate the

impact 61 in attenuating disease, an in vivo infection model

using Galleria mellonella was used. The study started by

evaluating the toxicity of 61 on larvae at 10 and 100 μM

concentrations over 3 days. Neither of the concentrations

tested had an impact on larvae survival (Figure 7A). The eﬀect

of 61 as a single treatment and in combination with Cip was

then evaluated. Larvae inoculated with PAO1-L without

treatment showed a median survival time of 12 h, and addition

of 61 did not result in a change in mortality rate (Figure 7B).

There was a reduction in the overall mortality and mortality

rate with the addition of Cip compared to PAO1-L, and this

was further reduced with addition of 61 in combination with

Cip. Therefore, 61 was able to potentiate the action of Cip in

the treatment of wax worm PA infections. To determine the

eﬀect of strain variation PAO1-N was utilized, and the eﬀect of

61 as a single treatment and in combination with Cip was

evaluated (Figure 7B). Larvae inoculated with PAO1-N had a

median survival time of 19 and 20.5 h when treated with 61. A

signiﬁcant reduction (p < 0.0001) was observed with the

treatment with Cip with a median survival time of 37 h; this

was further enhanced with a combination of 61 and Cip with

21 larvae surviving at the end point.

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Figure 7. Galleria mellonella survival assays. (A) Three-day survival monitoring with 10 μM and 100 μM 61. DMSO controls of 0.05% and 0.5%

performed to determine any inﬂuence on survival of larvae as this is the ﬁnal concentration of DMSO in 10 and 100 μM of 61. (B) Galleria

mellonella survival with/without 10 μM of 61 treatment and with/without Cip treatment A median survival time of 12 h was observed with and

without 61 treatment alone. A small signiﬁcant trend in mortality rate and ﬁnal end point was observed for the combination of Cip + 61 compared

to Cip treatment only. ** p = 0.0085. (C) Galleria mellonella survival with/without 10 μM of 61 treatment and with/without Cip treatment. A

median survival time of 19 h was observed with PAO1-N and 20.5 h with PAO1-N + 61. A signiﬁcant improvement in survival rate was observed

with Cip treatment **** p < 0.0001, further enhanced with addition of 61*** p = 0.0002. Statistical analysis performed by Kaplan−Meier estimate

and Gehan−Breslow−Wilcoxon test, n = 30, over three independent biological replicates.

interactions and supported with electrostatic interactions at the

level of the side chains of Gln¹⁹⁴and Arg²⁰⁹. Despite the

interaction with Gln¹⁹⁴being reported previously in the

literature, the interaction with Arg²⁰⁹was new and unique to

these structures. It is worth mentioning that the quinazolinone

head groups in 61 and 40 reside in diﬀerent orientation to our

previously reported quinazolinone derived pqs inhibitors.¹⁹

The latter compounds were positioned in a conformation that

superior activity in suppressing PA phenotypes in lab strains

(PAO1-L and PA14), it failed to maintain this eﬀect in clinical

isolates. The sequence alignment of PqsR in these strains was

performed (see the Supporting Information) and suggested

that the sequence is mostly preserved apart from Ala³¹⁴to Val

mutation in PA14 which is distant from the ligand binding site.

The reasons behind the diﬀerence in activity of strains was not

pursued in this study, but it could be due to variation in the

level of activity of pqs operon in these strains or variation in

permeability issues through the bacterial membrane and the

presence of eﬄux pumps on the bacterial cell membrane.⁴⁰It is

noteworthy that all the clinical strains originated from cystic

ﬁbrosis patients which reﬂects the multidrug resistant nature of

the microbiome associated with this disease.^36,41

Another important phenotype of PA is the ability to form

antimicrobial resistant bioﬁlms. To this extent, PA bioﬁlms

were treated with 61 as sole agent and also in combination

with ciproﬂoxacin. While 61 demonstrated a slight eﬀect as

single treatment, it was able to potentiate ciproﬂoxacin

treatment at 5 h but not at 16 h time points. The reason

behind the diﬀerence in eﬃcacy was not investigated, but it

could be potentially due to ciproﬂoxacin reaching maximal

eﬃcacy at the experimental end point (16 h) shadowing the

eﬀect of 61. This suggests that this PqsR inhibitor may be

allows the carbonyl group to be in a close proximity to Thr²⁶⁵

.

Further investigation is needed to pinpoint whether the

diﬀerent ligand conformation in the receptor contributed to

the enhanced potency.

These ﬁndings along with previously reported structures can

be employed to ﬁne-tune potency further.^18,19,32,38Subse-

quently, the work moved to examine the eﬀect of 61 in a series

of phenotypic assays, such as pyocyanin and AQ signal

quantiﬁcation. Most importantly, these assays were performed,

unlike the majority of published work in relation to pqs

inhibition, in a selection of PA isolates with diﬀerent genetic

backgrounds including lab and clinical strains. This is of

exceptional importance in antibacterial drug discovery as the

potency of inhibitors may vary signiﬁcantly in a strain

dependent manner which could lead to an inevitable and

costly pitfall if not discovered early.³⁹Indeed, while 61 showed

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̈

more eﬀective as an adjuvant for the initial stages of treatment.

Further work is needed to establish the impact of treatment at

diﬀerent time points and concentrations, but this is beyond the

scope of the current study. Previous reports have shown that

PqsR inhibition can increase the sensitivity of mature PA

bioﬁlms to antibiotics; however, the bioﬁlm extracellular matrix

is a major barrier to the transport of these inhibitors.⁴²As an

analogy, our previously published thioacetamide derived pqs

inhibitors failed to show any inhibitory eﬀect on PA bioﬁlms

despite the high potency in planktonic growth.¹⁸Small

molecule penetration to bioﬁlms can be improved applying

drug delivery approaches such as bioﬁlm responsive nano-

particles.⁴³Indeed, the use of pH responsive nanoparticles has

been shown to increase the penetration and eﬃcacy of QS

inhibitors combined with ciproﬂoxacin in mature bioﬁlms.⁴⁴

Despite no measures being applied in our study to improve 61

penetration through PA bioﬁlms, our results indicate that this

QS inhibitor could improve conventional antibiotic therapy

against recalcitrant bacterial-bioﬁlm centered infections.

To conclude this work, a G. mellonella infection model was

employed to investigate the eﬀect of 61 on mortality rate due

to PA infections.⁴⁵In agreement with the previous observation

in bioﬁlms, 61 showed no eﬀect on mortality rate as a sole

agent; however, when combined with ciproﬂoxacin, 61 was

able to improve the survival compared to ciproﬂoxacin alone in

PAO1-L and PAO1-N inoculated wax worms. In this study the

quorum sensing inhibitor 61 was unable to signiﬁcantly alter

the survival of wax worms in both PAO1-L and PAO1-N, this

is in contrast to the study performed by D’angelo and

colleagues⁴⁶who showed that an approved FDA drug

Clofoctol that is a probable PqsR inhibitor was able to reduce

the mortality of PAO1 infected wax worms. It must be noted

that the wax worms used in the D’angelo study (500 mg) were

signiﬁcantly larger than those used in this study (250 mg) and

both PA strains used in this study caused 100% mortality. It is

noteworthy that 61 showed a low cytotoxic proﬁle when tested

on human lung carcinoma epithelial cells A549 (LD₅₀> 300

μM) supporting its suitability for further preclinical develop-

ment. In summary, this study delivered a robust evaluation of

61 as a PqsR antagonist using chemical, biophysical, structural

and biological means and provided a solid foundation to

enhance the potency and spectrum of this series in future lead

optimization studies.

(Small-Molecule Drug Discovery Suite 2017-4, Schrodinger,

LLC, New York, NY, USA) was then used for ﬁnal preparation

of ligands into their lowest energy 3D conformations. The

partial atomic charges were assigned to the molecular

structures, using the 2005 implementation of the OPLS-AA

force ﬁeld. These energy-minimized structures were used for

Glide docking.

Molecular Docking. The “Extra Precision” (XP) mode of

Glide docking (Small-Molecule Drug Discovery Suite 2017-4,

Schrodinger, LLC, New York, NY, USA) was used to perform

all docking calculations, using the OPLS-AA 2005 force ﬁeld.

The scale factor of 1.0 for van der Waals radii was applied to

atoms of protein with absolute partial charges of less than or

equal to 0.25. The number of positions per ligand was set to

ﬁve, after energy minimization. The best docked structures

were chosen using an XP Glide Score (XP Gscore) function as

well as visual observations.

̈

Chemistry Procedures and Compound Character-

ization. Reagents and solvents were purchased from Sigma-

Aldrich, Alfa Aesar, Fluorochem, and Fisher Scientiﬁc. Nuclear

magnetic resonance was performed at ambient temperature

using a Bruker AV400 spectrometer (400 MHz for H¹, 101

MHz for C¹³). The samples were prepared in deuterated

solvent: DMSO-d₆and CDCl₃. Chemical shifts (δ) were

recorded in ppm, and coupling constants (J) were recorded in

Hz. The spectra were analyzed using MestReNova 12.0.1

software. Mass spectrometry: Analytical HPLC were per-

formed on a Shimadzu UFLCXR system coupled to an Applied

Biosystems API2000. Three columns thermostated at 40 °C

were used. Column one: Phenomenex Gemini-NX 3 μm C18,

50 × 2 mm. Column two: Phenomenex Luna 3 μm (PFP2)

110A, 50 × 2 mm. Column three: Waters X terra MS C8 2.5

m, 4.6 × 30 mm. Flow rate: 0.5 mL/min. UV detection at 220

(channel 2) and 254 nm (channel 1). Analytical HPLC for

ﬁnal compounds were performed on a Shimadzu UFLCXR

system coupled to a mass spectrometer (Applied Biosystems

API2000). Three columns thermostated at 40 °C were used

(column 1: C8-110A, 50 × 2 mm (method 1); column 2: Luna

3u (PFP2) 110A, 50 × 2 mm (method 2); column 3: C18 50

× 2 mm (method 3)). The ﬂow rate was set to 0.5 mL·min⁻¹.

UV detection was performed at 254 (channel 1) and 220 nm

(channel 2). The eluting gradient was as follows: pre-

equilibration run for 1 min at 10% B, 10 to 98% B in 2 min,

98% for 2 min, 98 to 10% B in 0.5 min, then 10% B for 1 min

(where solvent A is 0.1% formic acid in water and solvent B is

0.1% formic acid in MeCN). All compounds reported here are

with purity over 95% unless otherwise mentioned. Chromatog-

raphy: Thin-layer chromatography (TLC) was performed, UV

light and standard TLC stains were used to visualize the Merck

Silica gel 60 Å F₂₅₄plates. Compounds were puriﬁed via

column chromatography using either a Thompson pump or

normal phase Interchim Puriﬂash prepacked cartridges ﬁlled

with 50 μM silica, or a glass column using Sigma-Aldrich silica

gel 60 Å (230−240 μm).

METHODS

■

Molecular Docking. Preparation of Protein and

Receptor Grid Generation. The X-ray crystal structure of

PqsR ligand binding domain in complex with the QZN

inhibitor (PDB ID 4JVI) was used as a protein template. The

protein was prepared using the protein preparation wizard

̈

(Small-Molecule Drug Discovery Suite 2017-4, Schrodinger,

LLC, New York, NY, USA), where hydrogen atoms were

added, water molecules were removed, and the correct bond

order was assigned to the amino acid residues. Afterward, a

receptor grid generation was performed, based on deﬁned

residues around the ligand binding sites: (Ala102, Pro129,

Ile149, Thr166, Ala 168, Val 170, Ile186, Ie189, Gln194,

Ser196, Leu196, Leu197, Leu207, Leu208, Pro210, Val211,

Trp234, Gly235, Pro238, Ser255). The inner grid box was set

to 10 Å, while the outer box was 20 Å.

Chiral HPLC was performed on Dionex ICS-3000 with an

Ultimate-3000 detector equipped with Lux 5 μm Cellulose-4,

LC column 250 × 4.6 mm column.

General Procedure (1) for preparation of epoxides 12, 18,

19, 21. To a stirred solution of diﬀerent phenols (1 equiv) in

MeCN (50 mL) was added Cs₂CO₃(2 equiv) and then

epichlorohydrin (1.2 equiv). The reaction mixture was reﬂuxed

at 85 °C for 16 h. The mixture was then ﬁltered and puriﬁed

Ligand Preparation. The chemical structures of the

inhibitors were sketched using ChemDraw (Version 16.0.1.4,

PerkinElmer informatics) via an SDF ﬁle. The LigPrep module

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+

using column chromatography with PE:EtOAc 8:2 as the

eluent.

calculated for C₁₉H₁₇ClN₃O₃[M + H]⁺: 370.1 found 370.3,

HRMS calculated for C₁₉H₁₇ClN₃O₃[M + H]⁺: 370.0953

+

2-(4-Oxiran-2-ylmethoxy)phenyl)acetonitrile 12. The title

compound was prepared according to general procedure (1)

using 4-hydroxyphenylacetonitrile (20 g, 0.15 mol) as the

starting material. White solid (7g, 25%). ¹H NMR (400 MHz,

DMSO-d₆) δ 7.30−7.24 (m, 2H), 7.02−6.95 (m, 2H), 4.33

(dd, J = 11.3, 2.7 Hz, 1H), 3.95 (s, 2H), 3.83 (dd, J = 11.3, 6.5

Hz, 1H,), 3.33−3.30 (m, 1H), 2.85 (t, J = 4.7 Hz, 1H), 2.71

(dd, J = 5.1, 2.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

158.17, 129.78, 129.78, 123.86, 119.92, 115.41, 115.41, 69.46,

50.14, 44.21, 22.04.

found 370.0956.

2-(4-(3-(7-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 23. The title compound

was prepared according to general procedure (2) using 3 (1g,

5.5 mmol) and 12 (1.25 g, 6.6 mmol). Oﬀ white solid (0.45 g,

1

24%). H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.15

(d, J = 7.5 Hz, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.67 (dd, J = 7.5,

1.9 Hz, 1H), 7.35−7.26 (m, 2H), 7.10−6.97 (m, 2H), 5.51 (d,

J = 5.0 Hz, 1H), 4.74−4.65 (m, 1H), 4.25 (dd, J = 12.3, 1.7

Hz, 1H), 4.11 (dd, J = 12.4, 4.9 Hz, 1H), 3.82 (dd, J = 12.4,

9.9 Hz, 1H), 3.75−3.62 (m, 2H), 3.48 (dd, J = 12.4, 4.9 Hz,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.18, 158.57,

149.72, 138.91, 127.78, 127.12, 124.54, 120.40, 117.53, 115.32,

71.22, 68.18, 51.16, 25.31. LCMS calculated for

2-(2-Fluoro-4-(oxiran-2-ylmethoxy)phenyl)acetonitrile

18. The title compound was prepared according to general

procedure (1) from 2-(2-ﬂuoro-4-hydroxyphenyl)acetonitrile

(4.1 g, 0.02 mol) as the starting material. White solid (2 g,

1

+

36%). H NMR (400 MHz, DMSO-d₆) δ 7.37 (t, J = 8.8 Hz,

C₁₉H₁₇ClN₃O₃[M + H]⁺: 370.1 found 370.5, HRMS

+

1H), 6.96 (d, J = 2.6 Hz, 1H), 6.86 (dd, J = 8.8, 2.6 Hz, 1H),

4.38 (dd, J = 11.5, 2.6 Hz, 1H), 3.96 (s, 2H), 3.86 (dd, J =

11.5, 6.6 Hz, 1H), 3.37−3.30 (m, 1H), 2.90−2.81 (m, 1H),

2.71 (dd, J = 5.1, 2.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 161.19 (d, J = 226.9 Hz), 159.78 (d, J = 8.7 Hz), 131.40

(d, J = 5.1 Hz), 118.81, 111.71 (d, J = 3.6 Hz), 110.91 (d, J =

15.3 Hz), 102.85 (d, J = 24.5 Hz), 69.98, 49.95, 44.18, 16.67.

2-(3-Fluoro-4-(oxiran-2-ylmethoxy)phenyl)acetonitrile

19. The title compound was prepared according to general

procedure (1) using 2-(3-ﬂuoro-4-hydroxyphenyl)acetonitrile

(3.2 g, 0.01 mol) as the starting material. White solid (1.5 g,

35%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.27−7.17 (m, 2H),

7.16−7.10 (m, 1H), 4.42 (dd, J = 11.4, 2.6 Hz, 1H), 3.97 (s,

2H), 3.92 (dd, J = 11.4, 6.6 Hz, 1H), 3.43−3.36 (m, 1H), 2.87

(h, J = 5.6, 5.2 Hz, 1H), 2.77−2.68 (m, 1H). ¹³C NMR (101

MHz, DMSO-d₆) δ 151.91 (d, J = 245.8 Hz), 146.01 (d, J =

10.5 Hz) 124.95 (d, J = 4.2 Hz), 119.56, 116.63 (d, J = 21.0

Hz), 116.01 (d, J = 1.5 Hz), 70.65, 49.99, 44.16, 21.91.

2-((4-Phenoxyphenoxy)methyl)oxirane 21. The title com-

pound was prepared according to general procedure (1) using

4-phenoxyphenol (2 g, 0.01 mol) as a starting material. White

solid (0.6 g, 23%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.32 (t, J

= 9.6, 8.7 Hz, 2H), 7.06 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 6.6,

3.8 Hz, 4H), 6.92 (t, J = 8.7, 7.2 Hz, 2H), 4.27−3.75 (m, 4H),

3.44 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.46,

155.42, 150.00, 130.32, 130.29, 123.03, 121.15, 121.09, 117.83,

117.75, 116.34, 116.19, 68.76, 51.11, 44.21.

General Procedure (2) for Preparation of 22−41. To a

stirred solution of QZN (2−11) (1 equiv) in MeCN, an

epoxide derivative (12−21) (1.2 equiv), Cs₂CO₃(2 equiv),

and TBAI (0.1 equiv) were added. The reaction mixture was

reﬂuxed at 100 °C for 16 h. The mixture was then ﬁltered,

concentrated in vacuo, and puriﬁed using column chromatog-

raphy with EtOAc:PE 80:20 as the eluent.

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 22. The title compound

was prepared according to general procedure (2) using 2 (7.3

g, 0.041 mol) and 12 (9.5 g, 0.05 mol). Oﬀ white solid (3.2 g,

21%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.19−

8.08 (m, 1H), 7.74 (dd, J = 10.6, 2.1 Hz, 1H), 7.58 (dd, J =

8.5, 2.1 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 6.99−6.92 (m, 2H),

5.51 (d, J = 5.6 Hz, 1H), 4.33 (dd, J = 13.4, 3.7 Hz, 1H), 4.21−

4.13 (m, 1H), 4.06−3.90 (m, 2H), 3.94 (s, 2H), 3.92−3.84

(m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.37, 158.30,

150.66, 147.46, 139.34, 129.78, 128.66, 127.66, 126.74, 123.78,

120.93, 119.99, 115.47, 70.73, 66.46, 49.95, 22.01. LCMS

calculated for C₁₉H₁₇ClN₃O₃[M + H]⁺: 370.0953 found

370.0957.

2-(4-(3-(6,7-Dichloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 24. The title compound

was prepared according to general procedure (2) using 4 (1g,

4.6 mmol) and 12 (1.05 g, 5.5 mmol). Oﬀ white solid (0.63 g,

28%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 8.19 (s,

1H), 7.66 (s, 1H), 7.30−7.21 (m, 2H), 6.96−6.88 (m, 2H),

5.59 (d, J = 5.0 Hz, 1H), 4.71 (dqd, J = 10.1, 5.0, 1.7 Hz, 1H),

4.35 (dd, J = 12.3, 9.9 Hz, 1H), 4.17 (dd, J = 12.4, 5.0 Hz,

1H), 3.89 (dd, J = 12.5, 1.7 Hz, 1H), 3.75−3.62 (m, 2H), 3.50

(dd, J = 12.4, 5.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d6) δ

159.47, 158.37, 148.88, 148.72, 129.96, 128.36, 126.12, 116.52,

115.31, 71.22, 68.18, 50.32, 24.30. LCMS calculated for

C₁₉H₁₆Cl₂N₃O₃[M + H]⁺: 404.1 found 404.5

+

2-(4-(3-(6,8-Dichloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 25. The title compound

was prepared according to general procedure (2) using 5 (1g,

4.6 mmol) and 12 (1.05 g, 5.5 mmol). Oﬀ white solid (0.78 g,

1

35%). H NMR (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 8.24

(d, J = 1.9 Hz, 1H), 7.88 (d, J = 2.1 Hz, 1H), 7.30−7.21 (m,

2H), 6.95−6.87 (m, 2H), 5.18 (d, J = 5.1 Hz, 1H), 4.64−4.73

(m, 1H), 4.24 (dd, J = 12.3, 9.6 Hz, 1H), 4.05 (dd, J = 12.3,

5.0 Hz, 1H), 3.83 (dd, J = 12.3, 1.7 Hz, 1H), 3.75−3.62 (m,

2H), 3.49 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

160.39, 157.57, 150.26, 149.46, 133.92, 133.38, 128.34, 126.74,

126.32, 126.12, 124.86, 117.52, 115.32, 71.32, 67.18, 50.16,

24.30. LCMS calculated for C₁₉H₁₆Cl₂N₃O₃⁺[M + H]⁺: 404.1

found 404.7

2-(4-(3-(5-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 26. The title compound

was prepared according to general procedure (2) using 6 (1g,

5.5 mmol) and 12 (1.25 g, 6.6 mmol). Oﬀ white solid (0.84 g,

41%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 7.77−

7.66 (m, 2H), 7.65 (dd, J = 6.5, 2.7 Hz, 1H), 7.27−7.21 (m,

2H), 6.95−6.87 (m, 2H), 5.04 (d, J = 5.1 Hz, 1H), 4.79 4.73

(m, 1H), 4.27 (dd, J = 12.3, 1.8 Hz, 1H), 4.12 (dd, J = 12.4,

4.9 Hz, 1H), 3.81 (dd, J = 12.3, 9.3 Hz, 1H), 3.74−3.62 (m,

2H), 3.47 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

158.57, 157.77, 147.27, 142.87, 134.82, 129.99, 127.74, 126.51,

126.12, 122.56, 118.98, 117.52, 115.32, 70.22, 67.18, 51.16,

24.25. LCMS calculated for C₁₉H₁₇ClN₃O₃[M + H]⁺: 370.1

found 370.3

2-(4-(3-(8-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 27. The title compound

was prepared according to general procedure (2) using 7 (1g,

+

K

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5.5 mmol) and 12 (1.25 g, 6.6 mmol). Oﬀ white solid (0.64 g,

29%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.26−

8.17 (m, 1H), 7.76 (dd, J = 7.5, 2.1 Hz, 1H), 7.65 (t, J = 7.4

Hz, 1H), 7.30−7.21 (m, 2H), 6.96−6.88 (m, 2H), 5.17 (d, J =

5.0 Hz, 1H), 4.78−4.69 (m, 1H), 4.24 (dd, J = 12.5, 1.7 Hz,

1H), 4.09 (dd, J = 12.5, 4.9 Hz, 1H), 3.84 (dd, J = 12.5, 9.9

Hz, 1H), 3.75−3.62 (m, 2H), 3.54−3.47 (m, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 158.96, 158 = 7.57, 147.46, 147.41,

131.40, 127.74, 126.53, 125.37, 122.75, 116.52, 115.31, 70.22,

4.0 Hz, 1H), 4.11−3.98 (m, 3H), 3.95 (s, 2H), 2.68 (s, 3H).

¹³C NMR (101 MHz, DMSO-d₆) δ 160.95, 158.35, 157.21,

146.37, 134.81, 130.71, 129.77, 129.27, 125.46, 123.73, 121.75,

119.99, 115.48, 70.94, 66.59, 39.68, 24.12, 22.02. LCMS

+

calculated for C₂₀H₁₉ClN₃O₃[M + H] : 384.1 found 384.8,

+

HRMS calculated for C₂₀H₁₉ClN₃O₃[M + H]⁺: 384.1110

found 384.1109.

2-(3-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 33. The title compound

was prepared according to general procedure (2) using 2 (1g,

5.5 mmol) and 13 (1.26 g, 6.6 mmol). Oﬀ white solid (1.4 g,

+

67.18, 50.16, 24.35. LCMS calculated for C₁₉H₁₇ClN₃O₃[M

+ H]⁺: 370.1 found 370.4

1

2-(4-(2-Hydroxy-3-(6-nitro-4-oxoquinazolin-3(4H)-yl)-

propoxy)phenyl)acetonitrile 29. The title compound was

prepared according to general procedure (2) using 8 (1g, 5.2

mmol) and 12 (1.19 g, 6.3 mmol). Yellow solid (1.1 g, 59%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (d, J = 1.9 Hz, 1H),

8.65−8.56 (m, 2H), 7.96 (d, J = 7.5 Hz, 1H), 7.30−7.21 (m,

2H), 6.96−6.88 (m, 2H), 5.08 (d, J = 5.0 Hz, 1H), 4.71 (ddd, J

= 9.9, 5.0, 1.7 Hz, 1H), 4.24 (dd, J = 12.5, 1.7 Hz, 1H), 4.17

(dd, J = 12.4, 4.9 Hz, 1H), 3.89 (dd, J = 12.3, 9.9 Hz, 1H),

3.75−3.62 (m, 2H), 3.54−3.48 (m, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 159.69, 158.56, 149.44, 148.14, 143.16, 127.78,

127.33, 126.50, 125.42, 117.52, 113.32, 71.22, 68.18, 51.16,

69%). H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.23

(d, J = 2.0 Hz, 1H), 7.72 (dd, J = 7.5, 1.9 Hz, 1H), 7.39−7.27

(m, 1H), 7.07 (t, J = 7.4 Hz, 1H), 7.01−6.87 (m, 3H), 5.12 (d,

J = 5.0 Hz, 1H), 4.61 (ddd, J = 9.9, 5.0, 1.9 Hz, 1H), 4.22 (dd,

J = 12.4, 1.8 Hz, 1H), 4.10 (dd, J = 12.4, 4.9 Hz, 1H), 3.94−

3.81 (m, 2H), 3.77−3.72 (m, 1H), 3.49 (dd, J = 12.3, 4.9 Hz,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.06, 157.99,

152.16, 149.43, 132.59, 128.09, 126.11, 125.59, 125.50, 123.69,

114.52, 113.80, 112.42, 71.34, 67.18, 50.16, 24.86.

4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)benzonitrile 34. The title compound was

prepared according to general procedure (2) using 2 (1g, 5.5

mmol) and 14 (1.15 g, 6.6 mmol). Oﬀ white solid (0.95 g,

24.30. LCMS calculated for C₁₉H₁₆N₄O₅[M + H]⁺: 381.1

+

found 380.2, HRMS calculated for C₁₉H₁₆N₄O₅[M + H]⁺:

1

48%). H NMR (400 MHz, DMSO-d₆) δ 8.29 (s, 1H), 8.09

381.1193 found 380.1198

(d, J = 2.5 Hz, 1H), 7.90−7.78 (m, 3H), 7.71 (d, J = 8.7 Hz,

1H), 7.54 (d, J = 8.3 Hz, 2H), 5.33 (d, J = 5.7 Hz, 1H), 4.62

(s, 2H), 4.28 (dd, J = 13.4, 3.6 Hz, 1H), 3.82 (dd, J = 13.4, 8.6

Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.93, 149.70,

147.22, 144.74, 134.81, 132.65, 131.60, 129.89, 128.31, 125.45,

123.30, 119.32, 110.53, 71.87, 66.97, 50.17. LCMS calculated

2-(4-(2-Hydroxy-3-(7-nitro-4-oxoquinazolin-3(4H)-yl)-

propoxy)phenyl)acetonitrile 30. The title compound was

prepared according to general procedure (2) using 9 (1g, 5.2

mmol) and 12 (1.19 g, 6.3 mmol). Yellow solid (1.3 g, 65%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 8.42 (d, J =

1.7 Hz, 1H), 8.32−8.23 (m, 2H), 7.30−7.21 (m, 2H), 6.96−

6.87 (m, 2H), 5.08 (d, J = 5.0 Hz, 1H), 4.69−4.58 (m, 1H),

4.23 (dd, J = 12.5, 1.7 Hz, 1H), 4.09 (dd, J = 12.4, 4.9 Hz,

1H), 3.86−3.83 (m, 1H), 3.75−3.62 (m, 2H), 3.50 (dd, J =

12.5, 4.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.97,

157.57, 148.43, 148.34, 147.16, 126.74, 126.33, 126.15, 125.42,

117.52, 115.32, 70.22, 67.18, 53.18, 24.19. LCMS calculated

for C₁₈H₁₅ClN₃O₃[M + H]⁺: 356.1 found 356.6.

+

3-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)benzonitrile 35. The title compound was

prepared according to general procedure (2) using 2 (1g, 5.5

mmol) and 15 (1.15 g, 6.6 mmol). Oﬀ white solid (1.1 g,

1

56%). H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.12

(d, J = 2.5 Hz, 1H), 7.90−7.78 (m, 3H), 7.69 (m, 1H), 7.57

(d, J = 8.3 Hz, 2H), 5.33 (d, J = 5.7 Hz, 1H), 4.62 (s, 2H),

4.28 (dd, J = 13.4, 3.6 Hz, 1H), 3.82 (dd, J = 13.4, 8.6 Hz,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.68, 159.48,

151.16, 148.43, 129.12, 127.11, 126.59, 126.50, 123.78, 120.61,

118.49, 110.75, 70.22, 67.18, 50.16. LCMS calculated for

for C₁₉H₁₆N₄O₅[M + H]⁺: 381.1 found 380.2

+

2-(4-(2-Hydroxy-3-(4-oxo-6-(triﬂuoromethyl)quinazolin-

3(4H)-yl)propoxy)phenyl)acetonitrile 31. The title compound

was prepared according to general procedure (2) using 10 (1g,

4.7 mmol) and 12 (1.06 g, 5.6 mmol). Yellow solid (0.9 g,

48%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.62 (s, 1H), 8.49−

8.43 (m, 1H), 8.13−7.91 (m, 1H), 7.74 (d, J = 7.5 Hz, 1H),

7.28−7.23 (m, 1H), 6.96−6.88 (m, 2H), 4.97 (d, J = 5.1 Hz,

1H), 4.81 (ddd, J = 9.9, 5.0, 1.7 Hz, 1H), 4.24 (dd, J = 12.3,

1.7 Hz, 1H), 4.12 (dd, J = 12.3, 4.9 Hz, 1H), 3.84 (dd, J =

12.4, 9.9 Hz, 1H), 3.74−3.62 (m, 2H), 3.49 (dd, J = 12.4, 4.9

Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.67, 157.57,

157.12, 148.43, 128.92, 128.40, 128.28, 127.74, 126.38, 126.28,

126.12, 124.78, 122.31, 117.63, 114.52, 112.31, 71.22, 67.18,

52.16, 23.95. LCMS calculated for C₂₀H₁₆F₃N₃O₃⁺[M + H]⁺:

404.1 found 403.0, HRMS calculated for C₂₀H₁₆F₃N₃O₃⁺[M +

H]⁺: 404.1217 found 404.1214.

2-(4-(3-(6-Chloro-2-methyl-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 32. The title compound

was prepared according to general procedure (2) using 11 (1g,

5.2 mmol) and 23 (1.17 g, 6.2 mmol). White solid (1.3 g,

65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (d, J = 2.5 Hz,

1H), 7.81 (dd, J = 8.7, 2.5 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H),

7.32−7.22 (m, 2H), 7.02−6.92 (m, 2H), 5.51 (d, J = 5.3 Hz,

1H), 4.37 (dd, J = 13.9, 3.6 Hz, 1H), 4.24 (ddd, J = 9.2, 5.5,

C₁₈H₁₅ClN₃O₃[M + H]⁺: 356.1 found 356.2.

+

3-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)propanenitrile 36. The title com-

pound was prepared according to general procedure (2) using

2 (1g, 5.5 mmol) and 16 (1.3 g, 6.6 mmol). Oﬀ white solid

(1.7 g, 80%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H),

8.09 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 8.7, 2.5 Hz, 1H), 7.71

(d, J = 8.7 Hz, 1H), 7.24−7.15 (m, 2H), 6.93−6.84 (m, 2H),

5.48 (d, J = 5.7 Hz, 1H), 4.33 (dd, J = 13.4, 3.7 Hz, 1H), 4.16

(tt, J = 8.9, 5.2 Hz, 1H), 3.97 (dt, J = 7.0, 3.2 Hz, 2H), 3.89

(dd, J = 13.4, 8.6 Hz, 1H), 2.85−2.70 (m, 4H). ¹³C NMR (101

MHz, DMSO-d₆) δ 159.97, 157.72, 149.72, 147.23, 134.86,

131.63, 131.58, 129.97, 129.91, 125.49, 123.34, 120.75, 115.01,

70.69, 66.50, 50.07, 30.22, 18.95. LCMS calculated for

C₂₀H₁₉ClN₃O₃[M + H]⁺: 384.1 found 383.9, HRMS

+

calculated for C₂₀H₁₉ClN₃O₃[M + H]⁺: 384.1109 found

+

384.1116.

1-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)cyclopropane-1-carbonitrile 37.

The title compound was prepared according to general

L

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procedure (2) using 2 (1g, 5.5 mmol) and 17 (1.4 g, 6.6

mmol). Oﬀ white solid (1.1 g, 53%). H NMR (400 MHz,

2H), 5.51 (d, J = 5.6 Hz, 1H), 4.35 (dd, J = 13.4, 3.7 Hz, 1H),

4.23−4.14 (m, 1H), 4.08−3.97 (m, 2H), 3.92 (dd, J = 13.4, 8.6

Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.99, 158.40,

155.23, 150.12, 149.75, 147.24, 134.88, 131.64, 130.37, 129.92,

125.49, 123.34, 123.11, 121.22, 121.12, 118.01, 117.84, 116.33,

116.20, 115.96, 71.11, 66.50, 50.05. LCMS m/z calc for

1

Methanol-d₄) δ 8.29 (s, 1H), 8.16 (s, 1H), 7.86−7.63 (m, 2H),

7.29−7.21 (m, 2H), 6.94 (m, 2H), 4.47 (dd, J = 13.6, 3.7 Hz,

1H), 4.31 (dq, J = 8.8, 4.7 Hz, 1H), 4.03 (m, 3H), 1.63 (m,,

2H), 1.38 (m, 2H). ¹³C NMR (100 MHz, methanol-d₄)

160.48, 158.28, 148.92, 145.38, 134.71, 132.63, 128.57, 127.33,

125.28, 125.08, 114.72, 70.38, 66.85, 49.75, 16.62, 16.57,

C₂₃H₁₉ClN₂O₄[M + H]⁺: 423.1, found 423.4.

+

Preparation of 2-(4-(2-Amino-3-(6-chloro-4-oxoquinazo-

lin-3(4H)-yl)propoxy)phenyl)acetonitrile 42. To a stirred

solution of 22 (2 g, 5 mmol) in anhydrous THF (100 mL),

Ph₃P (2 g, 8 mmol) was added at rt. After 5 min, the mixture

was cooled over ice bath before addition of DIAD (1.6 g, 8

mmol) which is followed by DPPA (2.2 g, 8 mmol). The

mixture was stirred for 5 min in ice bath, in addition to another

5 min at rt. This mixture was then allowed to stir at 45 °C

overnight. The reaction mixture was then reduced and puriﬁed

using column chromatography eluting the desired compound

with 40:60 EtOAc:PE. Yellow solid (1 g, 33%). ¹H NMR (400

MHz, DMSO-d₆) δ 8.40 (s, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.91

(dd, J = 8.8, 2.5 Hz, 1H), 7.76 (d, J = 8.8, Hz, 1H), 7.35−7.26

(m, 2H), 7.13−6.96 (m, 2H), 4.49−4.28 (m, 3H), 4.22−4.10

(m, 2H), 3.97 (s, 2H,). ¹³C NMR (101 MHz, DMSO-d₆) δ

160.02, 157.70, 149.02, 147.04, 135.11, 131.94, 130.03, 129.90,

129.90, 125.61, 124.37, 123.21, 119.96, 115.53, 68.80, 59.16,

12.67. LCMS calculated for C₂₁H₁₉ClN₃O₃[M + H]⁺: 396.1

+

found 396.4.

4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypro-

poxy)-2-ﬂuorophenyl)acetonitrile 38. The title compound

was prepared according to general procedure (2) using 2 (0.15

g, 0.8 mmol) and 18 (0.2 g, 1 mmol). White solid (93 mg,

1

25%). H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.11

(d, J = 2.4 Hz, 1H), 7.87 (dd, J = 8.7, 2.4 Hz, 1H), 7.72 (d, J =

8.7 Hz, 1H), 7.36 (t, J = 8.8 Hz, 1H), 6.95−6.88 (m, 1H), 6.83

(dd, J = 8.5, 2.5 Hz, 1H), 5.54 (d, J = 5.6 Hz, 1H, OH), 4.32

(dd, J = 13.4, 3.6 Hz, 1H), 4.18 (ddt, J = 13.9, 9.1, 5.6 Hz,

1H), 4.13−3.99 (m, 2H), 3.99−3.88 (m, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 161.18 (d, J = 226.9 Hz), 159.98, 159.78

(d, J = 8.7 Hz), 149.69, 147.22, 134.88, 131.40 (d, J = 5.1 Hz),

129.91, 125.31, 123.34, 118.83, 111.79 (d, J = 3.6 Hz), 110.91

(d, J = 15.3 Hz), 102.85 (d, J = 24.5 Hz), 71.13, 66.33, 49.90,

16.64. LCMS m/z calc for C₁₉H₁₅ClFN₃O₃⁺[M + H]⁺: 388.0,

found 388.3.

+

46.78, 22.01. LCMS m/z calc for C₁₉H₁₆ClN₆O₂[M + H]⁺:

395.1, found 395.0. The pure product was then added to a

solution of Ph₃P (1 g, 3.7 mmol) in THF (100 mL) and stirred

for 2 h at 65 °C. Subsequently, 10% ammonium hydroxide

solution in water was added and stirred overnight at rt. The

mixture was puriﬁed using column chromatography eluting the

desired compound with 90:10 EtOAc:MeOH with 1% NH₃

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxy-

propoxy)-3-ﬂuorophenyl)acetonitrile 39. The title compound

was prepared according to general procedure (2) using 2 (0.13

g, 0.7 mmol) and 19 (0.18 g, 0.8 mmol). White solid (90 mg,

24%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.15−

8.05 (m, 1H), 7.87 (ddd, J = 9.0, 6.5, 2.5 Hz, 1H), 7.72 (dd, J

= 9.0, 6.5 Hz, 1H), 7.27−7.19 (m, 2H), 7.14 (dd, J = 9.0, 1.9

Hz, 1H), 5.56 (d, J = 5.6 Hz, 1H), 4.34 (dd, J = 13.4, 3.7 Hz,

1H), 4.21 (d, J = 10.3 Hz, 1H), 4.10 (dd, J = 5.6, 2.6 Hz, 2H),

3.98 (s, 2H), 3.92 (dd, J = 13.4, 8.5 Hz, 1H). ¹³C NMR (101

MHz, DMSO-d₆) δ 160.00, 154 (d, J = 247.3 Hz), 147.24,

146.44, 134.93 (d, J = 10.2 Hz), 131.62 (d, J = 2.7 Hz),

129.91(d, J = 19.7 Hz), 125.31 (d, J = 1.5 Hz), 124.36, 123.35,

119.58, 116.49, 116.04, 72.12, 66.42, 50.12, 21.91. LCMS m/z

1

(0.7 N). White solid (100 mg, 11%). H NMR (400 MHz,

DMSO-d₆) δ 8.38 (s, 1H, C8H), 8.10 (d, J = 2.5 Hz, 1H,

C6H), 7.87 (dd, J = 8.8, 2.5 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H),

7.27 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.29 (dd, J =

13.3, 4.8 Hz, 1H), 3.94 (d, J = 10.9 Hz, 4H), 3.83 (dd, J = 13.3,

8.5 Hz, 1H), 3.38 (t, J = 4.2 Hz, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 160.04, 158.33, 149.77, 147.25, 134.76, 131.53,

129.88, 129.88, 125.49, 123.74, 123.40, 120.01, 115.44, 115.44,

+

71.86, 60.22, 49.49, 21.99. LCMS m/z calc for C₁₉H₁₈ClN₄O₂

calc for C₁₉H₁₅ClFN₃O₃[M + H]⁺: 388.0, found 388.3

[M + H]⁺: 369.1, found 369.1. HRMS m/z calc for

+

C₁₉H₁₈ClN₄O₂[M + H]⁺: 369.1113, found 369.1118.

+

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenoxy)acetonitrile 40. The title com-

pound was prepared according to general procedure (2)

using 2 (0.44 g, 2.4 mmol) and 20 (0.6 g, 2.9 mmol). White

solid (0.3 g, 26%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s,

1H), 8.10 (d, J = 2.5 Hz, 1H), 7.85 (dd, J = 8.7, 2.5 Hz, 1H),

7.71 (d, J = 8.7 Hz, 1H), 7.07−6.97 (m, 2H), 7.00−6.91 (m,

2H), 5.51 (d, J = 5.6 Hz, 1H), 5.10 (s, 2H), 4.40−4.31 (m,

1H), 4.23−4.12 (m, 1H), 3.99 (dd, J = 5.6, 1.8 Hz, 2H), 3.91

(dd, J = 13.5, 8.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

159.97, 154.34, 150.96, 149.68, 147.19, 134.82, 131.64, 129.86,

125.48, 123.30, 117.28, 116.68, 116.68, 116.10, 116.10, 71.17,

66.53, 54.69, 50.06. LCMS m/z calc for C₁₉H₁₆ClN₃O₄⁺[M +

H]⁺: 386.0, found 386.4.

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

oxopropoxy)phenyl)acetonitrile 43. To a mixture of 22 (0.5

g, 1.3 mmol) in DCM (50 mL), Dess−Martin periodinane (1g,

2.3 mmol) was added and stirred overnight at rt. The mixture

was washed with 1 M NaOH, washed again with H₂O, and

dried with anhydrous Na₂SO₄, and the mixture was reduced.

The residue was puriﬁed using column chromatography

1

EtOAc:PE 70:30 as eluent. White solid (0.4 g, 76%). H

NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.09 (d, J = 2.5

Hz, 1H), 7.88 (dd, J = 8.8, 2.5 Hz, 1H), 7.75 (d, J = 8.8 Hz,

1H), 7.30 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.5 Hz, 2H), 5.16

(s, 2H), 5.09 (s, 2H), 3.96 (s, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 200.35, 159.59, 157.48, 148.97, 147.17, 135.23,

132.07, 130.07, 130.07, 129.82, 125.50, 124.38, 122.97, 119.95,

115.53, 115.35, 71.57, 52.63, 22.01. LCMS m/z calc for

6-Chloro-3-(2-hydroxy-3-(4-phenoxyphenoxy)propyl)-

quinazolin-4(3H)-one 41. The title compound was prepared

according to general procedure (2) using 2 (0.36 g, 2 mmol)

and 21 (0.6 g, 2.4 mmol) as the starting material. White solid

C₁₉H₁₅ClN₃O₃[M + H]⁺: 368.1, found 367.7, HRMS m/z

+

calc for C₁₉H₁₅ClN₃O₃[M + H]⁺: 368.0796, found 367.7

1

(85 mg, 24%). H NMR (400 MHz, DMSO-d₆) δ 8.33 (s,

Preparation of 2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-

yl)-2-(ethylamino)propoxy)phenyl)acetonitrile 45. To a

stirred solution of 42 (20 mg, 0.054 mmol) in MeCN (20

mL), a mixture of iodoethane (40 uL, 0.54 mmol) and

1H), 8.11 (d, J = 2.5 Hz, 1H), 7.88 (dd, J = 8.7, 2.5 Hz, 1H),

7.73 (d, J = 8.7 Hz, 1H), 7.35 (t, J = 9.7, 6.0, 2.3 Hz, 2H), 7.08

(t, J = 7.4 Hz, 1H), 6.99 (d, J = 1.7 Hz, 4H), 6.95−6.90 (m,

M

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Cs₂CO₃(13 mg, 0.10 mmol) was added. The mixture was

stirred at 60 °C for 16 h. The mixture was puriﬁed using

column chromatography eluting the desired compound with

95:5 EtOAc:MeOH containing NH₃(0.7 N). White solid (13

47.15, 37.67, 22.01, 18.92, 13.82. LCMS m/z calc for

+

C₂₃H₂₄ClN₄O₃[M + H]⁺: 439.1, found 438.9.

General Procedure (4) for Compounds 49−51. To a

stirred solution of of the corresponding acetic acid (1 equiv) in

DMF (20 mL) was added a mixture of 42 (1.35 equiv),

PyBrop (2 equiv), and trimethylamine (3 equiv). The mixture

was stirred for 4 h at rt. The mixture was puriﬁed using column

chromatography eluting the desired compound with DCM:

MeOH 9:1 with 0.1% NH₃(0.7 N)

1

mg, 65%). H NMR (400 MHz, DMSO-d₆) δ 8.34 (s, 1H),

8.10 (d, J = 2.5 Hz, 1H), 7.87 (dd, J = 8.7, 2.5 Hz, 1H), 7.72

(d, J = 8.7 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.4

Hz, 2H), 4.23 (dd, J = 13.5, 5.5 Hz, 1H), 4.00 (dd, J = 10.0,

5.2 Hz, 3H), 3.94 (s, 2H), 3.23 (s, 1H), 2.69 (dq, J = 14.1, 7.1

Hz, 1H), 1.90 (s, 1H), 0.91 (t, J = 7.1 Hz, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 160.03, 158.27, 149.76, 147.20, 134.86,

131.60, 129.91, 129.75, 129.75, 125.51, 123.79, 123.22, 120.00,

115.44, 115.44, 68.72, 55.03, 48.62, 41.25, 21.99, 15.96. LCMS

Preparation of N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-

3 - ( 4 - ( c y a n o m e t h y l ) p h e n o x y ) p r o p a n - 2 - y l ) - 2 -

(dimethylamino)acetamide 49. The title compound was

prepared according to general procedure (4) using (dimethyl

amino) acetic acid (12 mg, 0.12 mmol), 42 (60 mg, 0.16

mmol), PyBrop (0.1 g, 0.24 mmol), and trimethylamine (46

+

m/z calc for C₂₁H₂₂ClN₄O₂[M + H]⁺: 397.1, found 397.0.

General Procedure (3) for synthesis of compounds 46−

49. To a solution of correspondent acid anhydride, 42 (50 mg,

0.1 mmol) was added and stirred at 30 °C for 3 h. The reaction

was concentrated to dryness and the residue was dissolved in

EtOAc (20 mL) and washed with saturated aqueous NaOH

(20 mL) and water (20 mL). The organic phase was

concentrated to aﬀord the desired product without further

puriﬁcation.

1

mg, 0.36 mmol). White solid (28 mg, 54%). H NMR (400

MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.10 (d, J = 2.5 Hz, 1H), 8.02

(d, J = 9.2 Hz, 1H), 7.87 (dd, J = 8.7, 2.5 Hz, 1H), 7.71 (d, J =

8.7 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H), 7.03−6.95 (m, 2H),

4.63 (d, J = 9.2 Hz, 1H), 4.39 (dd, J = 13.5, 4.1 Hz, 1H), 4.16−

4.04 (m, 3H), 3.96 (s, 2H), 2.76 (d, J = 1.3 Hz, 2H), 2.12 (s,

6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.53, 159.96,

158.10, 149.02, 147.07, 134.93, 131.77, 129.95, 129.84, 129.84,

125.50, 124.04, 123.18, 119.99, 115.57, 115.57, 68.33, 63.14,

47.54, 46.36, 45.82, 45.82, 22.00. LCMS m/z calc for

Preparation of N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-

3-(4-(cyanomethyl)phenoxy)propan-2-yl)acetamide 46. The

reaction was carried out in acetic anhydride (4 mL) according

to general procedure (3). White solid (53 mg, 95%). ¹H NMR

(400 MHz, DMSO-d₆) δ 8.28 (s, 1H), 8.17−8.07 (m, 2H),

7.87 (dd, J = 8.7, 2.5 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.28

(m, 1H), 7.02−6.94 (m, 2H), 4.55 (tt, J = 8.9, 4.8 Hz, 1H),

4.39 (dd, J = 13.6, 4.5 Hz, 1H), 4.07 (dd, J = 5.3, 1.6 Hz, 2H),

4.01−3.90 (m, 3H), 1.76 (s, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 170.04, 159.91, 158.09, 149.01, 147.06, 134.93,

131.76, 129.96, 129.81, 129.81, 125.52, 124.03, 123.17, 119.99,

115.55, 115.55, 68.45, 47.87, 47.33, 22.96, 22.00. LCMS m/z

C₂₃H₂₅ClN₅O₃[M + H]⁺: 454.1, found 453.9.

+

N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-3-(4-

(cyanomethyl)phenoxy)propan-2-yl)-3-(dimethylamino)-

propanamide 50. The title compound was prepared according

general procedure (4) using 3-(dimethylamino) propionic acid

(15 mg, 0.09 mmol), 42 (44 mg, 0.12 mmol), PyBrop (0.08 g,

0.18 mmol), and trimethylamine (35 mg, 0.27 mmol). White

1

solid (27 mg, 59.67%). H NMR (400 MHz, DMSO-d₆) δ

8.41−8.29 (m, 2H), 8.11 (dd, J = 11.0, 2.5 Hz, 1H), 7.89 (td, J

= 9.0, 2.6 Hz, 1H), 7.73 (dd, J = 10.9, 8.7 Hz, 1H), 7.30 (dd, J

= 7.8, 5.7 Hz, 2H), 7.03−6.94 (m, 2H), 4.59 (dq, J = 9.2, 4.5

Hz, 1H), 4.48−4.31 (m, 1H), 4.20 (s, 1H), 4.15−3.94 (m,

3H), 3.96 (s, 2H), 2.65−2.53 (m, 2H), 2.28 (t, J = 7.1 Hz,

1H), 2.25 (s, 4H), 2.10 (s, 1H), 1.19 (t, J = 7.1 Hz, 1H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 170.31, 161.94, 158.04, 149.09,

147.04, 135.06, 131.77, 130.06, 129.82 125.53, 124.41, 123.17,

120.00, 115.71, 68.32, 53.49, 47.56, 45.94, 43.09, 30.92, 29.16,

calc for C₂₁H₂₀ClN₄O₃[M + H]⁺: 411.1, found 411.1

+

N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-3-(4-

(cyanomethyl)phenoxy)propan-2-yl)propionamide 47. The

reaction was carried out in propionic anhydride (4 mL)

according to general procedure (3). White solid (50.32 mg,

87.12%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (s, 1H), 8.10

(d, J = 2.4 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.87 (dd, J = 8.7,

2.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.33−7.24 (m, 2H),

7.03−6.94 (m, 2H), 4.56 (tt, J = 9.7, 4.8 Hz, 1H), 4.40 (dd, J =

13.5, 4.4 Hz, 1H), 4.12−4.02 (m, 2H), 4.02−3.91 (m, 1H),

3.96 (s, 2H), 2.02 (q, J = 7.6 Hz, 2H), 0.85 (t, J = 7.6 Hz, 3H).

¹³C NMR (101 MHz, DMSO-d₆) δ 173.80, 159.96, 158.11,

149.07, 147.07, 134.91, 131.73, 129.92, 129.82, 125.52, 124.02,

123.18, 119.99, 115.57, 115.40, 68.49, 47.82, 47.24, 28.92,

25.92, 22.02. LCMS m/z calc for C₂₄H₂₇ClN₅O₃[M + H]⁺:

+

468.1, found 467.9.

N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-3-(4-

(cyanomethyl)phenoxy)propan-2-yl)-4-(dimethylamino)-

butanamide 51. The title compound was prepared according

general procedure (4) using 4-(dimethylamino) butyric acid

(16 mg, 0.09 mmol) 42 (44 mg, 0.12 mmol), PyBrop (0.08 g,

0.18 mmol), and trimethylamine (35 mg, 0.27 mmol). White

+

22.00, 10.19. LCMS m/z calc for C₂₂H₂₂ClN₄O₃[M + H]⁺:

424.1, found 424.0.

N-(1-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-3-(4-

(cyanomethyl)phenoxy)propan-2-yl)butyramide 48. Reac-

tion was carried out in butyric anhydride (4 mL) according

to general procedure (3). White solid (54.02 mg, 90.35%). ¹H

NMR (400 MHz, DMSO) δ 8.28 (s, 1H), 8.10 (d, J = 2.5 Hz,

1H), 8.04 (d, J = 8.9 Hz, 1H), 7.87 (dd, J = 2.5, 8.8 Hz, 1H),

7.71 (d, J = 8.6 Hz, 1H), 7.31−7.26 (m, 2H), 7.01−6.96 (m,

2H), 4.59 (dq, J = 4.6, 9.3 Hz, 1H), 4.39 (dd, J = 4.4, 13.6 Hz,

1H), 4.07 (d, J = 5.5 Hz, 2H), 3.98−3.93 (m, 3H), 1.99 (t, J =

7.2 Hz, 2H), 1.41−1.30 (m, 2H), 0.96−0.83 (m, 1H), 0.68 (t,

J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.85,

159.94, 158.12, 149.06, 147.09, 134.92, 131.73, 129.92, 129.82,

125.51, 124.01, 123.17, 120.00, 115.56, 115.22, 68.54, 47.82,

1

solid (27 mg, 58.76%). H NMR (400 MHz, DMSO-d₆) δ

8.38−8.25 (m, 2H), 8.11 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 8.7,

2.5 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.34−7.25 (m, 2H),

7.04−6.95 (m, 2H), 4.58 (tt, J = 9.7, 4.8 Hz, 1H), 4.41 (dd, J =

13.5, 4.2 Hz, 1H), 4.10 (d, J = 5.4 Hz, 2H), 4.04−3.93 (m,

3H), 3.18 (d, J = 4.5 Hz, 1H), 2.71 (d, J = 14.3 Hz, 2H), 2.56

(s, 5H), 2.13 (td, J = 7.3, 3.1 Hz, 2H), 1.75−1.57 (m, 2H). ¹³

C

NMR (101 MHz, DMSO-d₆) δ 172.03, 159.96, 158.08, 149.13,

147.03, 134.96, 131.79, 129.94, 129.84, 125.57, 124.08, 123.19,

120.01, 115.57, 75.48, 68.41, 56.92, 47.84, 47.39, 43.02, 39.70,

39.49, 32.42, 22.01. LCMS m/z calc for C₂₅H₂₉ClN₅O₃⁺[M +

H]⁺: 482.2, found 481.8.

N

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6-Chloro-3-(3-chloropropyl)quinazolin-4(3H)-one 52. To

a stirred solution of 2 (0.3 g, 1.6 mmol) in MeCN (50 mL), 1-

bromo-3-chloropropane (0.3 g, 1.9 mmol), Cs₂CO₃(1 g, 3.2

mmol), and TBAI (59 mg, 0.16 mmol) were added. The

formed mixture was reﬂuxed overnight. Subsequently, the

reaction was cooled down, ﬁltered and reduced. The residue

was chromatographed using PE:EtOAc 1:1 as an eluent. White

solid (0.3 g, 73%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (s,

1H), 8.09 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 8.7, 2.5 Hz, 1H),

7.71 (d, J = 8.7 Hz, 1H), 4.11 (t, J = 6.6 Hz, 2H), 3.72 (t, J =

6.6 Hz, 2H), 2.19 (p, J = 6.6 Hz, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 159.84, 148.92, 147.15, 134.84, 131.75, 129.94,

125.46, 123.33, 44.69, 43.04, 31.59. LCMS m/z calc for

C₁₁H₁₁Cl₂N₂O⁺[M + H]⁺: 257.0, found 259.1.

6-Chloro-3-(3-chloro-2-methylpropyl)quinazolin-4(3H)-

one 53. To a stirred solution of 2 (0.3 g, 1.6 mmol) in MeCN

(50 mL), 1-bromo-3-chloro-2-methylpropane (0.3 g, 1.9

mmol), Cs₂CO₃(1 g, 3.2 mmol), and TBAI (59 mg, 0.16

mmol) were added. The formed mixture was reﬂuxed

overnight. Subsequently, the reaction was cooled down,

ﬁltered, and reduced. The residue was chromatographed

using PE:EtOAc 1:1 as an eluent. White solid (0.3 g, 71%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 8.10 (d, J =

2.5 Hz, 1H), 7.86 (dd, J = 8.8, 2.5, 1H), 7.72 (d, J = 8.8 Hz,

1H), 4.08−3.88 (m, 2H), 3.73−3.60 (m, 2H), 2.49−2.41 (m,

1H), 0.99 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 160.02, 149.05, 147.08, 134.98, 131.81, 130.05, 125.63,

123.32, 50.79, 49.03, 34.73, 15.55. LCMS m/z calc for

C₁₂H₁₃C_l2N₂O⁺[M + H]⁺: 271.0, found 270.8.

General Procedure (5) for Preparation of 54−55. To a

stirred solution of 52 or 53 (0.3 g, 1 mmol), TBAI (37 mg, 0.1

mmol) and Cs₂CO₃(1 g, 1.3 mmol) in MeCN (100 mL) and

4-hydroxyphenyl acetonitrile (0.15 g, 1.1 mmol) was added.

The mixture was heated at 100 °C, overnight. The mixture was

ﬁltered and puriﬁed using column chromatography eluting the

desired compound with 40:60 PE:EtOAc.

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

methylpropoxy)phenyl)acetonitrile 54. The title compound

was prepared according to general procedure (5). White solid

(200 mg, 49%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.39 (s, 1H

C8H), 8.09 (d, J = 2.5 Hz, 1H), 7.87 (dd, J = 8.7, 2.5 Hz, 1H),

7.72 (d, J = 8.7 Hz, 1H), 7.24−7.20 (m, 2H), 6.86−6.79 (m,

2H), 4.12 (m, 2H,), 4.01 (m, 1H,), 3.92 (d, J = 5.5 Hz, 4H),

1.02 (d, J = 6.8 Hz, 3H). ¹³CNMR (101 MHz, DMSO-d₆) δ

160.02, 158.23, 149.28, 147.13, 134.87, 131.74, 129.95, 129.69,

129.69, 125.53, 123.63, 123.30, 119.99, 115.25, 115.25, 70.95,

50.05, 21.97, 14.96. LCMS m/z calc for C₂₀H₁₉ClN₃O₂⁺[M +

H]⁺: 368.1, found 367.7

EtOH and H₂O (20 mL) was added hydroxylamine HCl (22

mg, 0.32 mmol) and NaOH (13 mg, 0.24 mmol). The mixture

was reﬂuxed for 2 days before it was reduced and extracted

with EtOAc (10 mL × 3). The organic layer was then reduced

and dried over MgSO₄. The residue was then puriﬁed using

ﬂash chromatography using PE:EtOAc 7:3. White solid (95

mg, 81%).

¹H NMR (400 MHz, methanol-d₄) δ 6.75 (s, 1H), 6.65 (d, J

= 2.4 Hz, 1H), 6.26 (dd, J = 8.8, 2.5 Hz, 1H), 6.20−6.09 (m,

1H), 5.71−5.61 (m, 2H), 5.35 (d, J = 8.7 Hz, 2H), 2.94 (dt, J

= 13.6, 3.6 Hz, 1H), 2.81−2.60 (m, 1H), 2.56−2.35 (m, 5H),

0.99 (s, 3H). ¹³C NMR (100 MHz, methanol-d₄) δ 175.82,

168.04, 158.97, 156.32, 147.44, 144.96, 133.03, 131.12, 128.19,

127.06, 126.68, 123.77, 121.33, 112.93, 68.39, 65.39, 48.27,

+

29.03, 9.04. LCMS calculated for C₂₁H₂₀ClN₄O₄[M + H] :

427.1 found 427.2, HRMS calculated for C₂₁H₂₀ClN₄O₄⁺[M +

+

H] : 427.1168 found 427.1157.

6-Chloro-3-(2-hydroxy-3-(4-((3-methyl-1H-1,2,4-triazol-5-

yl)methyl)phenoxy)propyl)quinazolin-4(3H)-one 57. To a

stirred solution of 22 (0.3 g, 0.8 mmol) in methanol (50

mL), acetylhydrazine (0.17g, 2.4 mmol) and sodium

methoxide (0.1 g, 1.9 mmol) was added. The mixture was

reﬂuxed at 80 °C for 16 h. The mixture was diluted with

EtOAc (50 mL). Then, the EtOAc was washed with water (3 ×

50 mL), dry it with Na2SO4 anhydrous and concentrate the

mixture. The residue was puriﬁed using column chromatog-

raphy eluting the desired compound with 95:5 EtOAc:MeOH

containing NH₃(0.7 N). White solid (50 mg, 14%). ¹H NMR

(400 MHz, DMSO-d₆) δ 13.27 (s, 1H, NH), 8.31 (s, 1H,),

8.10 (d, J = 2.6 Hz, 1H), 7.87 (dd, J = 8.7, 2.6 Hz, 1H), 7.72

(d, J = 8.7 Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0

Hz, 2H), 5.50 (d, J = 5.7 Hz, 1H), 4.34 (dd, J = 13.4, 3.7 Hz,

1H), 4.21−4.12 (m, 1H), 3.97 (s, 2H), 3.93−3.80 (m, 3H,

C13), 2.25 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

162.77, 159.96, 157.26, 153.16, 149.73, 147.22, 134.87, 131.62,

130.11, 130.11, 129.91, 125.49, 123.32, 114.80, 114.80, 70.65,

+

66.47, 50.05, 33.63, 12.01. LCMS m/z calc for C₂₁H₂₁ClN₅O₃

[M + H]⁺: 426.1, found 426.2

3-(3-(4-((1H-tetrazol-5-yl)methyl)phenoxy)-2-hydroxy-

propyl)-6-chloroquinazolin-4(3H)-one 58. To a stirred

solution of 22 (0.3 g, 8 mmol) in DMF (50 mL), NaN₃

(0.15 g, 24 mmol) and Et₃N.HCl (0.3 g, 24 mmol) were

added. The reaction mixture was heated at 130 °C for 16 h.

The reaction was concentrated to dryness, and the desired

product was isolated using column chromatography eluting the

desired compound with 95:5 EtOAc:acetic acid. Brown solid

1

(250 mg, 75%). H NMR (400 MHz, DMSO-d₆) δ 8.32 (s,

1H), 8.11 (d, J = 2.5 Hz, 1H), 7.87 (dd, J = 8.7, 2.5 Hz,1H),

7.72 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.86−6.74

(d, J = 8.4 Hz, 2H), 5.64 (s, 1H), 4.34 (dd, J = 13.4, 3.6 Hz,

1H), 4.14 (dt, J = 9.0, 4.4 Hz, 1H), 3.95 (d, J = 5.2 Hz, 2H),

3.91 (s, 2H), 3.90−3.86 (m, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 175.44, 163.93, 160.30, 159.94, 156.85, 149.77,

147.23, 134.84, 133.58, 131.59, 129.98, 125.50, 123.32, 114.51,

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)propoxy)-

phenyl)acetonitrile 55. The title compound was prepared

according to general procedure (5). White solid (100 mg,

1

40%). H NMR (400 MHz, DMSO-d₆) δ 8.41 (s, 1H), 8.10

(d, J = 2.6 Hz, 1H), 7.87 (dd, J = 8.7, 2.6 Hz, 1H), 7.72 (d, J =

8.7 Hz, 1H), 7.28−7.19 (m, 2H), 6.91−6.83 (m, 2H), 4.18 (t,

J = 6.4 Hz, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.93 (s, 2H), 2.18 (p,

J = 6.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.85,

158.18, 149.12, 147.20, 134.84, 131.72, 129.95, 129.72, 129.72,

125.47, 123.63, 123.33, 120.00, 115.31, 115.31, 65.72, 44.57,

+

70.72, 66.52, 50.05, 31.27. LCMS m/z calc for C₁₉H₁₇ClN₆O₃

[M + H]⁺: 413.1, found 412.7.

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetamide 59. To a stirred solution

of 22 (0.1 g, 0.27 mmol) in 30% H₂O₂:DMSO (100 mL),

Cs₂CO₃(0.3 g, 0.54 mmol) was added, and the resulting

mixture was stirred for 30 min in rt. The mixture was puriﬁed

using column chromatography eluting the desired compound

28.25, 21.98. LCMS m/z calc for C₁₉H₁₇ClN₃O₂[M + H]⁺:

+

354.1, found 354.0

6-Chloro-3-(2-hydroxy-3-(4-((5-methyl-1,2,4-oxadiazol-3-

yl)methyl)phenoxy)propyl)quinazolin-4(3H)-one 56. To a

stirred solution of 22 (0.1 g, 0.27 mmol) in 1.1 mixture of

1

with 100% EtOAc. White solid (0.1 g, 96%). H NMR (400

O

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Table 4. Bacterial Strains and Plasmids Used in This Study

strain or plasmid

relevant characteristics

reference or origin

P. aeruginosa

PAO1-L

PAO1-L mCTX::P_pqsA

lux

wild type PAO1, Lausanne subline.

PAO1-L with chromosomal mini-CTX::P_pqsA-lux insertion; Tc^R

B. Holloway via D. Haas

47

-

PAO1-N

PA14

PALESB58

PAK6085

PA7-48

wild type PAO1, Nottingham subline

wild type UCBPP-PA14

cystic ﬁbrosis isolate

PA14 with chromosomal mini-CTX::P_pqsA-lux insertion; Tc^R

Plasmids

B. Holloway

48

49

36

47

PA14 mCTX::P_pqsA-lux

mini-CTX::P_pqsA-lux

R6K-based mini-CTX suicide plasmid for the chromosomal insertion of a P_pqsA-lux transcriptional reporter 50

fusion; Tc^R

pMMG

pME6032ΔlacI constitutively expressing GFP from the P_tacpromoter

51

MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.88

(dd, J = 8.6, 2.5 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.40 (s,

1H), 7.18 (d, J = 8.2 Hz, 2H), 6.91−6.86 (dd, 2H), 6.83 (s,

1H), 5.50 (d, J = 5.6 Hz, 1H), 4.35 (dd, J = 13.4, 3.6 Hz, 1H),

4.18 (m, 1H), 4.02−3.84 (m, 3H), 3.30 (s, 2H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 173.00, 159.98, 157.46, 149.74,

147.24, 134.87, 131.64, 130.52, 130.52, 129.92, 129.26, 125.50,

123.33, 114.76, 114.76, 70.68, 66.51, 50.07, 41.82. LCMS m/z

(m, 1H), 4.12−3.94 (m, 2H), 3.94 (s, 2H), 3.94−3.86 (m,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.48, 158.50,

149.61, 147.28, 134.89, 131.63, 129.91, 129.78, 125.57, 123.78,

123.39, 119.99, 115.48, 70.72, 66.98, 50.32, 22.01. LCMS

+

calculated for C₁₉H₁₇ClN₃O₃[M + H] : 370.1 found 370.5,

+

HRMS calculated for C₁₉H₁₇ClN₃O₃[M + H] : 370.0953

found 370.0950.

(S)-2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 62. White solid (0.53 g,

calc for C₁₉H₁₉ClN₃O₄[M + H]⁺: 388.1, found 387.9.

+

1

2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxy-

propoxy)phenyl)acetic acid 60. A suspension of 22 (0.1 g,

0.27 mmol) in water (5 mL) and HCl (1 mL, 10.8) was stirred

at 75 °C for 18 h. Afterward, the reaction was treated with

saturated NaHCO₃and then extracted with EtOAc (20 mL).

The aqueous layer was then acidiﬁed with HCl (36%), and the

resulting solid was ﬁltered and used without further

54%). H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.10

(d, J = 2.4 Hz, 1H), 7.86 (dd, J = 8.7, 2.5 Hz, 1H), 7.72 (d, J =

8.7 Hz, 1H), 7.26 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.3 Hz, 2H),

5.51 (d, J = 5.6 Hz, 1H), 4.33 (dd, J = 13.5, 3.7 Hz, 1H), 4.17

(m, 1H), 4.11−3.94 (m, 2H), 3.94 (s, 2H), 3.94−3.86 (m,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.98, 158.30,

149.71, 147.23, 134.86, 131.63, 129.91, 129.78, 125.49, 123.78,

123.34, 119.99, 115.48, 70.72, 66.47, 50.02, 22.01. LCMS

1

puriﬁcation. Oﬀ white solid (46 mg, 44%). H NMR (400

+

MHz, DMSO-d₆) δ 12.2 (br S, 1H), 8.32 (S, 1H), 8.23 (d, J =

2.2 Hz, 1H), 7.71 (dd, J = 7.4, 2.1 Hz, 1H), 7.52 (d, J = 7.5 Hz,

1H), 7.21−7.13 (m, 2H), 6.89−6.81 (m, 2H), 5.07 (d, J = 5.0

Hz, 1H), 4.91−4.81 (m, 1H), 4.23 (dd, J = 12.4, 1.7 Hz, 1H),

4.10 (dd, J = 12.4, 4.9 Hz, 1H), 3.82 (dd, J = 12.5, 9.9 Hz,

1H), 3.77−3.67 (m, 1H), 3.60−3.43 (m, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ 174.89, 159.87, 156.72, 150.16, 148.43,

133.56, 128.83, 127.11, 114.64, 71.22, 67.18, 50.16, 42.50.

calculated for C₁₉H₁₇ClN₃O₃[M + H] : 370.1 found 370.2,

+

HRMS calculated for C₁₉H₁₇ClN₃O₃[M + H] : 370.0953

found 370.0955.

Bacterial Strains and Growth Conditions. The P.

aeruginosa strains and plasmids used in this study are shown

in Table 4. Bacteria were grown in lysogeny broth (LB) at 37

°C, unless stated otherwise. Where required, tetracycline (Tc)

was added to the media at 125 μg/mL, to select for

recombinants. Synthetic alkylquinolones were added at the

concentrations indicated.

LCMS m/z calc for C₁₉H₁₇ClN₂O₅[M + H]⁺: 389.1, found

+

388.8.

General Procedure (6) for Preparation of 61−62. To a

stirred solution of 2-(4-hydroxyphenyl)acetonitrile (1 g, 5.3

mmol) in 100 mL MeCN was added Cs₂CO₃(3.3 g, 10.6

mmol) and either (R)-oxiran-2-ylmethyl 3-nitrobenzene-

sulfonate Or (S)-oxiran-2-ylmethyl 3-nitrobenzenesulfonate

(1.7 g, 6.4 mmol), and the mixture was reﬂuxed for 18 h.

Subsequently, the reaction mixture was ﬁltered and concen-

trated. The residue was puriﬁed using PE:EtOAc 9:1 as an

eluent. Either (R)-2-(4-(oxiran-2-ylmethoxy)phenyl)-

acetonitrile or (S)-2-(4-(oxiran-2-ylmethoxy)phenyl)-

acetonitrile (0.5 g, 3 mmol) was further reacted with 2 (0.47

g, 2.6 mmol) in the presence of Cs₂CO₃(1.7 g, 5.2 mmol) and

TBAI (110 mg, 0.3 mmol) according to general procedure 2.

(R)-2-(4-(3-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-2-

hydroxypropoxy)phenyl)acetonitrile 61. White solid (0.56 g,

Biosensor Reporter Assay. Strains PA14 mCTX::P_pqsA-

lux and PAO1-L mCTX::P_pqsA-lux were constructed using

plasmid mini-CTX::P_pqsA-lux, as previously described,⁵⁰and

the assay was performed according to a published method.⁵²

For initial screening, the compounds were tested at a

concentration of 10 μM, which was prepared from a 10 mM

stock, in DMSO.

Isothermal Titration Calorimetry (ITC). PqsR⁹⁴⁻³³²

(PqsR^LBD) was prepared by Ni-NTA (HisTrap HP) followed

by gel ﬁltration with a Superdex 75 16/60 and ﬂash frozen in

liquid nitrogen until required. For ITC, the sample was

thawed, clariﬁed by centrifugation, and dialyzed overnight

against a buﬀer of 50 mM tricine-NaOH (pH = 8.5), 250 mM

NaCl, 5% glycerol. The sample was quantiﬁed by A280 using

an extinction coeﬃcient of 22920 M⁻¹cm⁻¹. The sample was

diluted to 10 μM. Compound 61 was dissolved in DMSO and

diluted into assay buﬀer to 100 μM. An appropriate amount of

DMSO was added to the sample to prevent mismatch. ITC

measurements were conducted on a MicroCal PEAQ ITC

1

59%). H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.08

(d, J = 2.3 Hz, 1H), 7.91 (dd, J = 8.7, 2.3 Hz, 1H), 7.71 (d, J =

8.7 Hz, 1H), 7.24 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H),

5.51 (d, J = 5.6 Hz, 1H), 4.33 (dd, J = 13.5, 3.6 Hz, 1H), 4.17

P

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instrument using a sample volume of 280 μL with 19 ligand

injections of 2 μL. The cell temperature was kept at 25 °C and

stirring was carried out as 750 rpm. Reference power was set to

6 DP with spacing kept at 180 s. The same procedure was

carried out for the T265A mutant. Mutants were prepared

using the site directed mutagenesis protocol from Agilent and

the primer sequences: 5′-GCGGCATCGACGCGAAGGTG-

TATTGC-3′ and 5′-GCAATACACCTTCGCGTCGA-

TGCCGC-3′.

detected, a peak area was determined, and analyte peak area/

internal standard peak area calculated.

Bioﬁlm Assay Methodology. Mature P. aeruginosa

bioﬁlms were used to characterize the eﬀect of ciproﬂoxacin

and QSI treatment combinations. Bioﬁlms were grown on

round glass coverslips (8 mm, no. 1.5 thickness) under ﬂow

conditions (20 rpm) in FAB 10 mM glucose medium⁵⁴with or

without supplementation of 61 (10 μM), inoculated with

diluted (OD600 nm = 0.01) bacteria from overnight cultures

in LB.⁴⁴Bioﬁlms were cultivated at 30 °C for 2 days with

medium replacement after 24 h incubation. At this time,

bioﬁlms were washed in PBS to remove loosely attached cells

and incubated for further 5/24 h in fresh medium

supplemented with various treatments. These included free

ciproﬂoxacin 60 μg/mL, compound 61 at 10 μM, and

ciproﬂoxacin in combination with 61. Bioﬁlms exposed to

each treatment at the indicated concentrations were washed in

PBS and the viability of attached cells evaluated by ﬂuorescent

staining with the LIVE/DEAD BacLight bacterial viability kit

(Molecular Probes, Life Technologies) according to manu-

facturer instructions. Following staining, coverslips were rinsed

with distilled water and imaged using a LSM700 AxioObserver

(Carl Zeiss, Germany) confocal laser scanning microscope

(CLSM). Viable and nonviable bioﬁlm biomass quantiﬁcation

from image stacks of bioﬁlms was done with Fiji-ImageJ

software. Live/dead ratios were established for each treatment

and compared to untreated controls.

Protein Expression and Crystallization. Crystallization

and structural solution was performed as previously

described.^18,19

Pyocyanin Quantiﬁcation. The experiment was per-

formed following a published protocol with minor modiﬁca-

tions.⁵³PA strains were cultured into 5 mL fresh medium

overnight. Compounds were assayed at 3 × IC₅₀s concen-

tration, for 16 h, at 37 °C (Kuhner LT W Shaker, Adolf

Kuhner AG, Basel, Switzerland). Cells were centrifuged at

̈

10 000 RCF for 10 min (Allegra 64R centrifuge, Beckman

Coulter, High Wycombe, UK), and the supernatant was

transferred to 15 mL falcon tubes with a HSW 10 mL Soft-Ject

Syringe and a 0.22 μM Sartorius syringe-driven ﬁlter (Fisher

Brand, Loughborough, UK). Pyocyanin pigment was extracted

into chloroform by mixing 7.5 mL of supernatant with 4.5 mL

of chloroform. Pyocyanin was further extracted into 1.5 mL of

0.2 M HCL, which gave a pink/red solution, and the

absorbance was measured at 520 nm.

Galleria mellonella Survival Assay. Antibiotic free

Galleria mellonella were purchased from Livefoods Direct

Ltd. (Sheﬃeld, UK) maintained at room temperature in the

dark and used within 3 days. Larvae were selected in the ﬁnal

(7th) instar and were randomly assigned to groups after

weighing ∼250 mg. Ten larvae were assigned to each grouping

in 9 cm Petri dishes: Sham (injection, no infection), injection

(PBS), DMSO only (DMSO) inoculation only (PA),

inoculation plus ciproﬂoxacin (PA + CIP), inoculation plus

61 (PA + 61) and inoculation plus ciproﬂoxacin and 61 (PA +

CIP + 61). The PA strains PAO1-N and PAO1-L were selected

as these give diﬀering rates of mortality, chronic-like and acute-

like respectively. An inoculum was prepared separately for each

grouping at a ﬁnal size of 1 × 10⁴CFU/mL. The addition of

the quorum sensing inhibitor 61 was performed prior to

inoculation to a ﬁnal concentration of 10 μM. Inoculation of

wax worms was performed using a 50 μL Hamilton syringe

(Fisher, Leicestershire, UK) with 29 G 0.5 mm needle

attachment (Becton Dickinson U.K. ltd, Oxford, UK), brieﬂy

10 μL of inoculum was injected into the rear most left pro-leg

of the larvae. To the rear most right pro-leg, 10 μL of 0.025

μg/mL CIP treatment was injected post inoculum for Cip

treated wax worms. Wax worm larvae were incubated at 37 °C

in the dark. Larvae were monitored hourly and death event

recorded when no movement was observed and no response to

mechanical stimulus was elicited.

LCMS-MS Alkyl Quinoline Quantiﬁcation. For each test

sample, 100 μL of sterile ﬁltered supernatant (the same

preparation as for pyocyanin using the following concen-

trations: PAO1-L 3 μM, PA14 3 μM, PA7−48 15 μM, LESB58

5 μM, and PAK6085 9 μM) was spiked with 10 μL of an

internal standard solution (10 μM d4-PQS in MeOH) and

diluted with water, to a total volume of 500 μL. Samples were

then extracted three times with an 0.5 mL aliquot of ethyl

acetate, vortex mixing the aqueous/organic mix for 2 min, then

removing the organic phase once the layers had successfully

partitioned. For each sample, the combined organic extracts

were dried under vacuum and redissolved in 100 μL MeOH

prior to analysis. For the LC-MS/MS analysis of supernatant

extracts, the chromatography was achieved using a Shimadzu

series 10AD VP LC system (Columbia, MD, USA). The LC

column, maintained at 40 °C, was a Phenomenex Gemini C18

(3.0 μm, 100 × 3.0 mm) (Macclesﬁeld, Cheshire, UK) with an

appropriate guard column. Mobile phase A was 0.1% (v/v)

formic acid in water containing 2 mM 2-picolinic acid, and

mobile phase B 0.1% (v/v) formic acid in methanol. The ﬂow

rate throughout the chromatographic separation was 450 μL/

min. After an injection of a 2 μL/sample, a binary gradient,

beginning initially at 30% B, increased linearly to 99% B over 5

min. The composition remained at 99% B for 3 min, decreased

to 30% B over 1 min, and stayed at this composition for 4 min,

to allow for column equilibration. The MS system used for

analyte detection was an Applied Biosystems Qtrap 4000

hybrid triple-quadrupole linear ion trap mass spectrometer

(Foster City, CA, USA), equipped with an electrospray

ionization (ESI) interface. Instrument control, data collection

and analysis were conducted using Analyst software (Foster

City, CA, USA). The MS analysis was achieved with positive

electrospray (+ES) multiple reaction monitoring (MRM)

screening of the LC eluent for speciﬁc AQ analytes. Where

chromatographic peaks for HHQ, HQNO, and PQS were

Cytotoxicity Assay. The human carcinoma lung epithelial

cells A549 (ATCC CCL-185) are grown at 37 °C in 5% CO₂

for 24 h in RPMI medium. A cell concentration of 1 × 10⁴

cells/mL was used and dispensed in 96 well plates (100 μL in

each well). A serial dilution of 61 was then prepared in growth

media and added to the plate in triplicate. Potassium

dichromate at 100 μM was used as positive control, and a

solvent vehicle control was prepared as negative control. Cells

were then incubated for a further 16 h, and 20 μL of Alamar

blue added to each well. After 5 h incubation, ﬂuorescence was

Q

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Article

measured with excitation at 510 nm and emission at 590 nm.

Values were then normalized against the untreated control.⁵⁵

Data Management and Analysis. Instant JChem was used

for structure database management, Search and Prediction,

Instant JChem 16.2.15.0 2016, ChemAxon (http://www.

chemaxon.com).

Scott Grossman − School of Pharmacy, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.

Tomas Sou − Department of Pharmacy, Uppsala University,

Uppsala SE-751 23, Sweden

Ruiling Liu − School of Pharmacy, University of Nottingham

Biodiscovery Institute, University of Nottingham, Nottingham

NG7 2RD, U.K.

Sigmoidal dose−response curves and the representation of

all data were prepared using GraphPad Prism 9.0.2.

Nigel Halliday − School of Life Sciences, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00175.

■

sı

Irena Kukavica-Ibrulj − Institut de Biologie Integrative et des

Systemes, Universite Laval, Quebec G1 V 0A6, Canada

Roger C. Levesque − Institut de Biologie Integrative et des

Systemes, Universite Laval, Quebec G1 V 0A6, Canada

Christel A. S. Bergstrom − Department of Pharmacy,

Uppsala University, Uppsala SE-751 23, Sweden

Barrie Kellam − School of Pharmacy, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.; orcid.org/0000-0003-

0030-9908

Structures and activities of commercial analogues of 22,

representative isotherm generated from injection of 61

into the T265A PqsR mutant construct, and crystallo-

graphic data collection and reﬁnement statistics (PDF)

Special Issue Paper

Published as part of the Antibiotic Alternatives special issue.

Jonas Emsley − School of Pharmacy, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.

Stephan Heeb − School of Life Sciences, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.

Paul Williams − School of Life Sciences, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.

AUTHOR INFORMATION

Corresponding Authors

Michael J. Stocks − School of Pharmacy, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.; orcid.org/0000-0003-3046-137X;

■

Email: michael.stocks@nottingham.ac.uk

Miguel Cámara − School of Life Sciences, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.; Email: miguel.camara@nottingham.ac.uk

Complete contact information is available at:

https://pubs.acs.org/10.1021/acsinfecdis.1c00175

Authors

Fadi Soukarieh − School of Life Sciences, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.; orcid.org/0000-0002-6730-2543

Alaa Mashabi − School of Pharmacy, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.

Author Contributions

F.S. and A.M. contributed equally to this work. F.S. performed

in silico and in vitro screening, designed and performed

syntheses directed the microbiology experiments. A.M.

performed syntheses, microbiology experiments, and con-

tributed to writing. F.S., M.S., P.W., and M.C. designed and

supervised the study and led the writing of the manuscript.

E.V.O., M.R., S.N.R., S.G., R.L., and N.H. performed the

experimental microbiology. W.R. and J.E. performed and

designed crystallography and ITC experiments. B.K., R.L.,

S.H., T.S., C.A.S.B., I.K.-I., and R.C.L. contributed to

experimental design. We also thank Jeﬀ Gauthier for his

support in performing sequence alignment.

William Richardson − School of Pharmacy, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.

Eduard Vico Oton − School of Life Sciences, University of

Nottingham Biodiscovery Institute, University of Nottingham,

Nottingham NG7 2RD, U.K.; Present Address: E.V.O.:

́

Environmental Microbiology Laboratoy, Ecole

́

polytechnique federale de Lausanne, CH-1015, Lausanne,

Switzerland

Funding

This work was supported by the Engineering and Physical

Sciences Research Council [grant numbers EP/N006615/1

and EP/K005138/1, EP/N03371X/1] and JPI-AMR and

MRC for funding the SENBIOTAR program (ref. MR/

N501852/1). F.S., M.R., S.N.R., P.W., M.J.S., J.E., and M.C.

are funded by the National Bioﬁlms Innovation Centre

(NBIC) which is an Innovation and Knowledge Centre

funded by the Biotechnology and Biological Sciences Research

Council, InnovateUK and Hartree Centre [Award Number

BB/R012415/1]. S.G. and W.R. are funded via the Wellcome

Manuel Romero − School of Life Sciences, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.

Shaun N. Roberston − School of Life Sciences, University of

Nottingham Biodiscovery Institute and The National Bioﬁlms

Innovation Centre, University of Nottingham Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

U.K.

R

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Notes

The authors declare no competing ﬁnancial interest.

REFERENCES

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Design and Evaluation of New Quinazolin-4(3 H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa

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