Exploration of the Structural Space in 4(3 H)-Quinazolinone Antibacterials

Article abstract of DOI:10.1021/acs.jmedchem.0c00153

We report herein the syntheses of 79 derivatives of the 4(3H)-quinazolinones and their structure-activity relationship (SAR) against methicillin-resistant Staphylococcus aureus (MRSA). Twenty one analogs were further evaluated in in vitro assays. Subsequent investigation of the pharmacokinetic properties singled out compound 73 ((E)-3-(5-carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one) for further study. The compound synergized with piperacillin-tazobactam (TZP) both in vitro and in vivo in a clinically relevant mouse model of MRSA infection. The TZP combination lacks activity against MRSA, yet it synergized with compound 73 to kill MRSA in a bactericidal manner. The synergy is rationalized by the ability of the quinazolinones to bind to the allosteric site of penicillin-binding protein (PBP)2a, resulting in opening of the active site, whereby the β-lactam antibiotic now is enabled to bind to the active site in its mechanism of action. The combination effectively treats MRSA infection, for which many antibiotics (including TZP) have faced clinical obsolescence.

Full text of DOI:10.1021/acs.jmedchem.0c00153

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Article
Exploration of the Structural Space in 4(3H)-Quinazolinone Antibacterials
Yuanyuan Qian, Giuseppe Allegretta, Jeshina Janardhanan, Zhihong Peng, Kiran
V. Mahasenan, Elena Lastochkin, Melissa Malia N. Gozun, Sara Tejera, Valerie A.
Schroeder, William R. Wolter, Rhona Feltzer, Shahriar Mobashery, and Mayland Chang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00153 • Publication Date (Web): 28 Apr 2020
Downloaded from pubs.acs.org on April 29, 2020
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Exploration of the Structural Space in 4(3H)-
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Quinazolinone Antibacterials
Yuanyuan Qian†, Giuseppe Allegretta†, Jeshina Janardhanan†, Zhihong Peng†, Kiran V.
Mahasenan†, Elena Lastochkin†, Melissa Malia N. Gozun†, Sara Tejera†, Valerie A.
Schroeder†, William R. Wolter‡, Rhona Feltzer†, Shahriar Mobashery†*, and Mayland
Chang†*
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
Indiana 46556, United States
‡Freimann Life Sciences Center, University of Notre Dame, Notre Dame, Indiana 46556,
United States
KEYWORDS methicillin-resistant Staphylococcus aureus, (MRSA); quinazolinones;
antibacterials; antibacterial synergy
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ABSTRACT: We report herein the syntheses of 79 derivatives of the 4(3H)-quinazolinones
and their structure-activity relationship (SAR) against methicillin-resistant Staphylococcus
aureus (MRSA). Twenty-one analogs were further evaluated in in vitro assays. Subsequent
investigation of the pharmacokinetic properties singled out compound 73 ((E)-3-(5-
carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one) for further study. The
compound synergized with piperacillin-tazobactam (TZP) both in vitro and in vivo in a
clinically relevant mouse model of MRSA infection. The TZP combination lacks activity
against MRSA, yet it synergized with compound 73 to kill MRSA in a bactericidal manner.
The synergy is rationalized by the ability of the quinazolinones to bind to the allosteric site
of penicillin-binding protein (PBP)2a, resulting in opening of the active site, whereby the β-
lactam antibiotic now is enabled to bind to the active site in its mechanism of action. The
combination effectively treats MRSA infection, for which many antibiotics (including TZP)
have faced clinical obsolescence.
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INTRODUCTION
Antibiotic resistance is a major public health threat.1-3 A group of six bacteria, referred to
as the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia,
Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) pathogens,
account for the majority of the US hospital-acquired infections.4 Annually 35,000 deaths due
to antibiotic-resistant infections occur in the US.1 Infections caused by methicillin-resistant
S. aureus (MRSA) result in 10,600 of these deaths in the US according to the estimates.1 While
several antibiotics are clinically used for the treatment of MRSA infections, including
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ceftaroline and ceftobiprole (fifth-generation cephalosporins), linezolid and tedizolid
(oxazolidinone class), telavancin and dalbavancin (semisynthetic variants of the
glycopeptide antibiotic vancomycin), and daptomycin,5, 6 resistance to all these has been
documented and mortality and morbidity due to MRSA infections remain serious.1, 6-9
Discovery of novel classes of antibiotics for treatment of MRSA infections remains of high
urgency.10, 11
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Historically, -lactam antibiotics (penicillins, cephalosporins and carbapenems) were
agents of choice for treatment of S. aureus infections. With the emergence of MRSA over half
a century ago, the entire class of -lactam antibiotics became compromised as the pathogen
acquired a set of genes from a non-S. aureus source that conferred inducible resistance to all
known members of the -lactam antibiotics.12-16 One such gene is mecA, which encodes an
additional penicillin-binding protein (PBP), referred to as PBP2a.14, 17, 18 In a series of
publications from our labs, we have elucidated the mechanistic basis of how PBP2a is
impervious to inhibition by -lactam antibiotics, yet capable of performing its critical
physiological function of crosslinking of the cell wall in the face of the challenge by the
antibiotic.19-22 We also disclosed how the fifth-generation cephalosporin ceftaroline is
capable of inhibiting PBP2a by subverting the allosteric mechanism that the enzyme uses for
its physiological reaction of crosslinking the cell wall.17, 23, 24 Consequently, PBP2a is an
important target for the design of novel antibiotics.
We previously reported on the discovery of the quinazolinones as non-β-lactam
antibacterials active against MRSA, which target cell-wall biosynthesis.25 Quinazolinones, as
exemplified by compound 1, have oral bioavailability, low clearance, and show efficacy in
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mouse models of MRSA infection.25-27 We subsequently showed that the quinazolinones
inhibit both PBP2a and PBP1.25-27 We have documented that the quinazolinones bind to the
allosteric site of PBP2a by X-ray crystallography,25, 27 by competition binding assays25, 27 and
by antisense sensitization.25, 26 Binding at the allosteric site triggers conformational changes
that lead to opening of the active site.19 The active site of PBP2a is normally closed, does not
allow access to most -lactams for binding, and this accounts for the lack of activity of these
antibiotics against MRSA. To this effect, we have documented the ternary complex of a
quinazolinone bound to the allosteric site of PBP2a and the -lactam piperacillin at the active
site of PBP2a by X-ray crystallography.27
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In the present report, we define the chemical space of this antibacterial class by syntheses
of 79 quinazolinones, diversified structurally at sites highlighted as A, B and C in the
structure of 1. These compounds were screened for activity against the ESKAPE panel of
bacteria. Twenty-one compounds were further evaluated with four important MRSA strains
in in vitro toxicity and plasma protein binding assays. Selected compounds were then
evaluated for their pharmacokinetic (PK) properties.
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RESULTS AND DISCUSSION
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Synthesis. Two synthetic routes were devised (Scheme 1). One approach to the
quinazolinone scaffold was in three steps per methodology reported previously,26 with
minor modifications as needed for various derivatives. The synthesis started from
anthranilic acid (2), which was converted to 2-methyl-4H-3,1-benzoxazin-4-one (3) in
refluxed triethyl orthoacetate or acetic anhydride. Compound 3 was subsequently
transformed into the respective quinazolinone cores (4) by a condensation reaction with a
requisite amine (aniline derivatives) in refluxed pyridine, acetic acid or toluene
(intermediates 11–42 are of this type, Figure S1). Condensation of the quinazolinone
intermediates 4 and the selected aldehyde, catalyzed by acid, base or ZnCl2, would deliver
quinazolinone derivatives 5 (e.g., 48–110, 113) with limited further modifications.
Hydrogenation of compound 5 produced compound 6 (e.g., 114 and 115). The second
approach to the quinazolinone scaffold was also via the anthranilic acid (2). Conversion of 2
by the respective isothiocyanate(s) in refluxing acetic acid provided 2-thiocarbonyl 4(1H)-
quinazolinone 7 (intermediates 43, 44a, 45 are of this type, Figure S1). The thioether-linker
containing analogs 8 (final products 123 and 124) were directly obtained by substitution of
NaH-pretreated 2-thiocarbonyl 4(1H)-quinazolinones with the respective alkylbromides.
Various functionalizations of the 2-thiocarbonyl 4(1H)-quinazolinones, such as the
introduction of chloride (exemplified by compounds 9), provided intermediates 44b, 44, 46
and 47 (Figure S1) leading to derivatives of the general structure 10 by substitution in the
presence of a base (final products such as 116–123, 127–129).
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Scheme 1. General Synthetic Route for the Quinazolinones
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O
O
O
O
O
N
R1
S
R1
S
R1
N
f
O
b
a
e
N
OH
NH2
N
N
N
N
H
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R2
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2
g
c
O
N
O
N
O
O
R1
R1
R1
R1
d
N
N
N
h
N
Cl
N
N
NH
R2
R2
R2
5
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9
a Reagents and Conditions: (a) triethyl orthoacetate, reflux, 4 h; (b) R1NH2, AcOH or pyridine,
reflux or sealed tube at 130 °C, 8 h; or R1NH2, toluene, reflux, Dean-Stark, 8 h; (c) R2CHO,
AcOH, AcONa, reflux, 8 h; or R2CHO, pyridine, reflux, 8 h; or R2CHO, anhydrous ZnCl2, neat,
sealed tube at 130 °C, 1 h; (d) H2, Pd/C, rt, 12 h; (e) R1NCS, AcOH, reflux, 1 h; (f) R2CH2Br,
NaH, anhydrous DMF, ice-water to rt, 1 h; (g) PCl5, POCl3, reflux, 8 h; (h) R2NH2,
triethylamine, THF, sealed tube at 100 °C, 8 h.
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Structure–Activity Relationships. Structural modifications of the quinazolinones were
made in rings A and B, as well as in the linker C (Figure 1). Compounds were first screened
against S. aureus ATCC 29213; those with MIC ≤ 64 µg/mL were evaluated in the remaining
ESKAPE panel (Table S1) The quinazolinones in general exhibit selective activity against S.
aureus, with no activity against E. faecium,and Gram-negative bacteria. In SAR1 (Figure 1,
top panel), we altered the nature of ring A by substitutions that increased the hydrophilicity
of the moiety and by increasing the flexibility of the functionalities linked to the
quinazolinone core. Most of such replacements led to loss of activity (48–52 and 60–68). In
a few cases (53–56 and 58–59) for which the MIC was acceptable (MIC = 0.25–4 µg/mL),
the ring structure was incorporated with electron-withdrawing groups, such as halides or
nitrile.
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In SAR2 (Figure 1, middle panel), we modified ring B of the quinazolinone scaffold. Using
fluorine as a bioisostere for hydrogen,28 we started with incorporation of fluorine at every
position of ring B, while retaining the m-carboxylic acid (analogs 69–73). The addition of
fluorine at position 6 (73) improved the MIC 4-fold (from 2 µg/mL to 0.5 µg/mL). The basis
for this improvement might be a desirable dihedral angle between ring B and the
quinazolinone core. Based on this notion, we prepared different substitutions at ring
position 6 or 2, such as chloro, methyl, trifluoromethyl, hydroxyl and methoxyl groups. Only
smaller sized and hydrophobic substitutions, as in the chloro (74) or methyl (75) variants,
retained acceptable MIC values. Bulkier substituents (76 and 79) or hydrophilic ones (78,
81, 83 and 84) all led to loss of antibacterial property. Meanwhile, we replaced the m-
carboxylic acid with fused rings (85–87), hydrophilic aliphatic linkers (89–93), a few
bioisosteres (88, 94–99), ortho-/para-substitutions (100–104) and hydrogen-bond
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acceptors (105–108). We also attempted to eliminate the ring B structure by changing it to
aliphatic side chains (109–110). Compounds with the trifluoroacetamide group (88 and 99),
sulfonamide group (94 and 95), amide group (96) and ortho-hydroxyl substitution (100)
showed excellent MIC values (0.015–4 µg/mL). Compound 90 bearing an imidazole linker
exhibited MIC of 8 µg/mL against S. aureus. Yet, it also showed some activity against E.
faecium with MIC of 16 µg/mL (Table S1), in contrast to other quinazolinones, which target
S. aureus exclusively. As for the hydrogen-bond acceptor moieties, we tried pyridine (105),
pyridine N-oxide (106), nitrile (107) and azide substitution (108). Most of these compounds
showed no activity (MIC  16 µg/mL), with the pyridine-N-oxide (106) analog, a possible
electrostatic interactions partner, retaining activity (MIC of 2 µg/mL).
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In SAR3 (Figure 1, lower panel), we mainly focused on the modification of the alkenyl
linker. Compound 113, a variant of the potent derivative 94, was synthesized here for
comparison with 57. Like 57, compound 113 showed potent activity against S. aureus (MIC
= 0.06 g/mL). Meanwhile we replaced the alkenyl linker with saturated aliphatic (114 and
115), amine (116–123), sulfide (124 and 125), ether (126) and piperidine/piperazine
linkers (127–129). Except for the sulfides (0.25–2 µg/mL), all the other derivatives exhibited
either diminished or lost activity. Only 115, 123–125 sustained activity (0.125–2 µg/mL),
among which 123 stands out (MIC = 0.125 µg/mL). However, we had shown previously that
the mesylate group resulted in very high clearance.26
The best fragments of SAR1 (4-CN), SAR2 (CO2H), and SAR3 (amine linker) were combined
in compound 118, however activity was lost (MIC >128 g/mL).
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49,
50,
51,
MIC 128
>
X = S, Y= N, Z = CH
X = S, Y= CH, Z = N
X = CH, Y= CH, Z = S
X
SAR1:
O
N
MIC 128
>
X
Z
Y
Z
MIC 32
=
Y
N
CO2H
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55,
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MIC = 8
Z = CH, R = H
Z = CH, R = Cl
Z = CH, R = Br
Z = CH, R = CN
Z = C-Cl, R = H
60,
61,
62,
63,
64,
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66,
67,
68,
MIC = 64
MIC > 128
MIC > 128
MIC > 128
MIC = 64
X = CH, Y= CH, Z = N
X = CH, Y= CF, Z = N
X = CH, Y= CH, Z = NO
X = CH, Y= N, Z = CH
X = CH, Y= N, Z = C-CN
X = CF, Y= N, Z = CH
X = CH, Y= CO, Z = NH
X = CH, Y= NH, Z = CO
X = CO, Y= NH, Z = CH
S
MIC = 0.5
MIC = 0.25
MIC = 4
R
A
Z
MIC = 4
MIC > 128
1,
MIC = 2
R = C CH
R = CN
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MIC > 128
MIC > 128
MIC > 128
48, MIC > 128
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MIC = 0.03
MIC = 0.25
R = Cl
MIC = 0.5
R
Y
R = I
R
O
N
SAR2:
X
CO2R
X = C-OH, Y = CH, R = CH3
MIC > 128
SO2NHR
B
N
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84,
MIC >128
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104,
MIC > 128
MIC > 128
R = NO2
R = NH2
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95,
MIC = 0.25
R = H
R = Acetyl
X = C-OH, Y = CH, R = H
MIC >128
MIC = 4
X = N, Y = CH, R = CH3
MIC > 128
MIC > 128
X = N, Y = CH, R = H
X = CH, Y = N, R = H
X
CN
CONH2
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X
Y
N
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106,
MIC > 128
X = N
R
H
MIC = 2
X = NO
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87,
MIC >128
MIC >128
X = NH, Y = CO
X = NH, Y = CS
X = N, Y = CH
CO2H
R
MIC >128
R
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73,
74,
75,
76,
77,
78,
79,
MIC = 8
R = 2-F
R = 2-F, 4-F
R = 4-F
R = 5-F
R = 6-F
R = 6-Cl
R = 6-CH3
R = 6-CF3
R = 5-OH
R = 6-OH
R = 6-OCH3
MIC = 64
97,
MIC > 128
R = Phthalimide
107,
108,
MIC > 128
MIC = 16
R = CN
R = N3
NHCOR
MIC = 16
MIC > 128
MIC = 0.5
MIC = 2
98,
99,
MIC = 32
R = NH2
R = NHCOCF3
88,
89,
90,
91,
MIC = 2
R = 2-(methylamino)ethyl
R = (1H-imidazol-5-yl)ethyl
R = morpholinomethyl
MIC = 0.5
R = CF3
MIC > 128
MIC = 8
MIC = 32
R
CO2H
MIC = 2
109, MIC = 128
MIC > 128
MIC > 128
MIC > 128
CO2H
NHCONHR
100,
101,
102,
MIC = 0.015
MIC > 128
MIC = 32
R = OH
R = NO2
R = CO2H
MIC > 128
92,
93,
MIC > 128
R = (1H-imidazol-5-yl)methyl
R = (1H-imidazol-5-yl)ethyl
110, MIC > 128
MIC = 16
SAR3:
O
N
Y
=
S
R
N
N
X
Y
=
Y
=
114,
115,
MIC = 8
X = CO2H, R = CN
X = CO2H, R = F
R
MIC = 2
Y
C
124,
MIC = 0.25 127,
X = NO2
MIC = 2 128,
X = NH2
MIC > 128
MIC > 128
X = CO2H, R = H
X = CO2H, R = CN
125,
N
Y
=
H
R
Y
=
CN
MIC = 0.5
MIC = 0.015
116,
117,
118,
119,
120,
121,
122,
123,
MIC = 16
X = CO2Me, R = CN
X = CO2Me, R = F
X = CO2H, R = CN
N
111,
112,
X = NH2
X = NHMs
N
MIC > 128
MIC > 128
Y
=
O
Y
=
Cl
MIC > 128
X = NO2, R = CN
CN
MIC > 128
MIC > 128
MIC = 8
X = NO2, R = F
X = NH2, R = CN
126,
129,
MIC > 128
X = NH2
X = CO2H
Y
=
CH
MIC = 0.06
MIC = 64
X = NH2, R = F
113,
X = SO2NH2
MIC = 0.125
X = NHMs, R = CN
Figure 1. Antibacterial activities of the 4(3H)-quinazolinones derivatives. The MICs
(µg/mL) were determined with S. aureus ATCC 29213. The MICs for inactive (MIC ≥ 8
µg/mL) and active compounds (MIC ≤ 4 µg/mL) are in red and blue, respectively. The four
previously reported compounds, given here for comparison, are designated in underlined
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numbers.26 The MICs for the positive controls vancomycin and linezolid are 1 and 2 µg/mL,
respectively.
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52
53
54
55
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57
58
59
60
Three-dimensional QSAR of the quinazolinones. In light of the fact that the quinazolinones
target more than one PBP, the activities that we measured for these compounds as MIC
values are composites of the effects at both targets at the whole-organism level. This said,
the chemical structure and the corresponding MIC were computationally analyzed to find
broad features that influence the antibacterial activity. A total of 103 quinazolin-4(3H)-ones
reported herein and earlier26 were used for the computational analysis, which included 53
active (MIC ≤ 4 µg/mL) and 50 inactive (MIC ≥ 128 µg/mL) compounds. The compounds
were aligned computationally on the quinazolinone core (Figure S2A) and formed the basis
of a three-dimensional QSAR model computed by the Field-based Gaussian method.29-33 The
model identified the steric importance for sections of rings A and B (Figure S2B).
Substitution of the smaller three-membered cyclopropyl group (compound 48) in place of
ring A resulted in loss of activity. Compounds with moderately steric substitution in the
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meta-position of ring B (-NHCOCF3, -SO2NH2, -SO2NHAc, and -CH2NHCOCF3) were active
(compounds 88, 94, 95, and 99 respectively). However, extending the bulk further in the
region was detrimental to the activity (compounds 92 and 93). Additionally, the model
suggested that hydrophobicity plays a role in activity (Figure S2C). Ring A showed
pronounced preference for hydrophobic groups. For example, the chloro (53) and bromo
(54) substitution in the thiophene for ring A made the compounds more active than the
unsubstituted thiophene counterpart (compound 52). Similarly, compounds with 4-
acetylene (57), 4-Cl (58) and 4-I (59) substitutions in ring A were active. Compounds 60–68
with more polar ring A were inactive. While the hydrophobic nature in ring A appears to
impart a noticeable role in the activity, substitution in ring B demonstrated more complex
outcomes. Hydrophilic groups at position 3 of ring B were preferred as displayed by the
white contour map (Figure S2C). Compounds with substitution of the polar carboxylic acid
(1) and amine (111) at the position 3 demonstrated potent activity. On the other hand,
substitutions on the ring A at positions 5 and 6 revealed the importance of hydrophobicity
as shown in the contour map. Compounds with 6-F, 6-Cl, and 6-methyl substitutions on the
ring B (73, 74, and 75) were active, while polar hydroxyl substitution to either 5- or 6-
positions (77 and 78) rendered the compounds inactive. Similarly, introducing a nitrogen in
the ring (pyridine ring; 83 or 84 compared to 1) seemingly is sufficient in resulting in the
loss of activity. At the same time, compound 76 with hydrophobic trifluoromethyl
substitution at the 6 position was inactive, contrary to the above observation.
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Broader evaluation of quinazolinones with MRSA strains, Gram-positive organisms, in vitro
toxicity and plasma protein binding. Analogs with MIC of  4 µg/mL against the MSSA strain
ATCC 29213 were selected for further evaluation against four MRSA strains: NRS70, NRS119
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(linezolid-resistant), ATCC27660 and USA300 (the major cause of the community-
associated MRSA infections in the US),34 in vitro toxicity and plasma protein binding (Table
1). We also determined MIC against S. aureus ATCC29213 in the presence of bovine serum
albumin. Compounds 1, 58, 73, 94, 115, and 125 showed 4-fold or less increase in MIC with
added bovine serum albumin. Most of the analogs showed similar potency with the MRSA
strains, compared to the MSSA strain ATCC 29213 used for the data in Figure 1, except for
compounds 88 and 94, which proved more potent in their activity against the MRSA strains.
We also evaluated quinazolinones 1 and 73 for activity against several Gram-positive
organisms. Compound 73 was active (MIC ≤ 4 µg/mL) against S. epidermidis and S.
haemolyticus, while 1 showed decreased activity against these pathogens (Table 2).
We evaluated the cytotoxicity of selected analogs (IC50 evaluation) to HeLa cells by the XTT
assay (Table 1). Derivatives 55, 75, 95, 99, 115 and 124 had the ratio of IC50/MIC of  50 and
were not considered further. The MIC for determination of this ratio was that for the NRS70
strain. We also determined plasma protein binding for the compounds using equilibrium
dialysis with human plasma (Table 1). Derivatives 53–59, 74, 75, 88, 95, 99 and 115–124
had ≥ 99% plasma protein binding. High plasma protein binding decreases the in vivo
efficacy of an otherwise potent compound, as it limits the drug available for distribution from
blood to tissues; therefore, these compounds were not considered further. Derivatives with
IC50/MIC of > 50 and plasma protein binding  99% were 73, 94, 96, 100, 106 and 113.
Compounds 96, 100 and 106 had relatively poor water solubility and were not pursued
further. Compounds 73, 94 and 113 were selected for further analysis.
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Table 1. In vitro biological evaluation of selected active quinazolinones
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MIC against S. aureus strains (µg/mL)
XTT (IC50)
Compds
IC50/MIC
PPB (%)
ATCC29213a
2 (8)
NRS70b
2
NRS119c
1
ATCC27660d USA300e (µg·mL-1)
1 f
2
0.5
32
96.5 ± 0.7 g
173  1
99.6 ± 0.04
57 f
0.03 (0.25)
0.25
0.125
0.125
0.03
> 128
> 256
h
53
54
55
56
58
59
73
74
75
88
94
95
96
99
100
0.5 (4)
0.25 (2)
4 (32)
4 (32)
0.25 (1)
0.5 (16)
0.5 (2)
2 (16)
2 (16)
2 (32)
0.25 (0.5)
4
2 (16)
0.5 (8)
0.015
(0.125)
2 (32)
0.06 (0.5)
2 (8)
0.5
0.5
4
4
4
4
0.5
0.5
4
1
0.5
4
360
380
28
99.3 ± 1.0
> 99.9
99.2 ± 0.2
> 99.9
> 99.9
> 99.9
95.4  0.02
> 99.9
> 99.9
180  3
190  2
110  1
140  1
50  3
100  1
62  6
> 128
60  1
> 128
> 64
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2
1
0.125
2
0.25
2
2
4
70
0.25
0.5
0.5
2
0.25
0.5
0.25
2
0.125
0.25
0.25
1
200
200
124
> 64
15
> 2133
> 1067
50
4
4
4
1
0.06
0.06
4
1
0.06
0.06
4
0.06
0.06
4
1
4
> 99.9
0.125
2
97.1  0.6
99.2 ± 0.02
97.1 ± 0.3
> 99.9
200  4
> 128
150  6
2
64
32
2
16
64
> 64
2
0.06
0.125
0.25
0.03
667
98.1 ± 0.3
40  2
106
113
115
123
124
125
1
0.06
2
4
0.06
4
2
0.06
4
1
0.06
1
0.25
1
>128
61  1
79  4
62  4
> 64
> 128
1017
40
248
> 128
39
67.9 ± 2.8
98.2 ± 0.1
> 99.9
> 99.9
> 99.9
0.125 (1)
0.25 (4)
2 (8)
0.25
0.5
2
0.25
1
2
0.125
0.25
2
2
> 99.9
78  4
aMSSA strain for susceptibility testing; values in parenthesis are in the presence of bovine serum albumin. mec
(subtype II) positive, resistant to clindamycin, erythromycin and spectinomycin. cmecA positive, resistant to linezolid,
ciprofloxacin, gentamicin, oxacillin and penicillin. dmecA positive, resistant to oxacillin, tetracycline and penicillin. emec
(subtype IV) positive, pvl positive, resistant to oxacillin and erythromycin. fPreviously published compounds26 given here
b
for comparison; their MICs and XTT (IC50) were redetermined for this table. Data from reference.25 hData from
g
reference.26
Table 2. Activity of quinazolinones against Gram-positive organisms (MIC in µg/mL)
Bacteria
Compound 1
Compound 73
S. epidermidis ATCC 35547
S. haemolyticus ATCC 29970
S. pneumoniae ATCC 49619
C. difficile ATCC 43255
B. cereus ATCC 13061
B. licheniformis ATCC 12759
E. faecalis ATCC 29212
1
0.25
4
8
16
8
128
>128
>128
>128
128
>128
>128
>128
Pharmacokinetics (PK) Studies. The PK properties of compounds 73, 94 and 113 were
investigated after single intravenous (iv) dosing to mice and compared to those determined
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for compound 125 studied previously (Table 3). After a single 10 mg/kg iv dose of 73,
concentrations were above the MIC of 0.5 g/mL for 4 h. Systemic exposure, as measured by
area-under-the-curve (AUC), was 1290 g·min·mL-1, similar to that for compound 1.
Clearance of 73 was desirably low at 7.7 mL·min-1·kg-1, <10% of hepatic blood flow, and
volume of distribution was 0.19 L·kg-1. Compounds 94 and 113 showed low systemic
exposure and very high and moderate clearance of 98 mL·min-1·kg-1 and 28.2 mL·min-1·kg-1,
respectively, >10% hepatic blood flow; hence, both compounds were not considered further.
The absolute oral bioavailability (F%) of compound 73 was 65%. The elimination half-life
for 73 was 13.7 h and 11.2 h after iv and po administration, respectively. Comparisons of
these parameters with those of compound 1 are given in Table 3; plasma concentrations
after iv and po administration of compound 73 are shown in Figure 2.
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Table 3. Pharmacokinetic parameters of selected quinazolinone derivatives
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a
AUC0-last
b
AUC0-inf
dose
(mg/kg)
t½ elim
(h)
CL
Vd
Compds
F (%)
50
(g·min/mL
(mL·min-1·kg-1) (L·kg-1)
(g·min/mL)
)
1c
10 iv
10 po
10 iv
10 po
5 iv
1180
408
1290
840
51
1460
738
> 20
> 20
13.7
11.2
ND
6.9
7.7
0.30
0.19
73
1320
1010
ND
65
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94
98.0
28.2
NDd
NDd
113
5 iv
178
ND
ND
aArea under the curve from 0 min to the last quantifiable time point. bArea under the curve from 0 min extrapolated to
infinite time. cData reproduced from Bouley. dND, not determined.
25
Figure 2. Pharmacokinetics of compound 73 after a single 10 mg/kg dose administration
to mice (n = 3 per time point per route of administration, total n = 63 mice).
Time-kill assays. When the ratio of MBC/MIC is greater than 4, the compound is considered
bacteriostatic. In the case of compound 73, MBC against NRS70 was > 128 g/mL, hence the
compound is bacteriostatic. We evaluated the in vitro antibacterial activity of compound 73
as a function of time by time-kill assays and compared it to that of compound 1. At 0.5 to 16×
MIC, both compounds 1 and 73 were bacteriostatic, consistent with MBC of greater than 4×
MIC, but also by the observation that the compounds did not reduce bacterial count by >3-
log10 (Figure 3a and S3).25 Piperacillin is a fourth-generation penicillin and tazobactam is a
β-lactamase inhibitor. The clinical combination of piperacillin and tazobactam (abbreviated
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TZP) works against S. aureus strains that express the PC1 β-lactamase.16 However, the drug
combination is ineffective against MRSA (Figure 3a), as these strains express the gene mecA
for PBP2a. PBP2a is not inhibited by piperacillin, as the active site of the enzyme is closed
and it excludes β-lactam antibiotics. In light of the fact that the quinazolinones are known to
bind to the allosteric site of PBP2a, we evaluated the combination of 1 with TZP, as well as
that of 73 with TZP. At 12 h, >3-log10 reduction was observed for compound 1 + TZP,
however the combination was not synergistic at 24 h. On the other hand, compound 73 +
TZP inhibited growth of the MRSA strain with reduced bacterial count of >3-log10 at 24 h.
Compound 73 and the clinical TZP work well in combination by exhibiting bactericidal
behavior (Fig. 3a) in time-kill assays.
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Mouse neutropenic thigh-infection model. The efficacy of compound 73 was evaluated in
the mouse neutropenic thigh-infection model with strain NRS70, where bacteria in the
infected thighs were counted at 24 h after infection (Figure 3b). The dose of TZP used
consisted of 12 mg/kg piperacillin and 1.5 mg/kg tazobactam to maintain the
concentration of piperacillin below the Kd of 400 µM for binding to the allosteric site of
PBP2a.23 Compound 1 alone at 20 mg/kg resulted in 0.67 log10 reduction in bacterial count,
while compound 73 alone gave 0.47 log10 reduction at 20 mg/kg and 0.78 log10 reduction at
40 mg/kg. For comparison, TZP alone resulted in 0.25 log10 bacterial count reduction. Since
only compound 73 showed effective activity with TZP by time-kill assays (Figure 3a), this
compound was evaluated in combination with TZP in the mouse neutropenic thigh-
infection model (Figure 3b). Compound 73 + TZP gave 0.75 log10 reduction at 20 mg/kg
and 1.33 log10 reduction at 40 mg/kg. The results with the combination were statistically
significant compared to vehicle (p < 0.001) and to TZP (p < 0.001).
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Figure 3. (a) Time-kill assays for compounds 1 and 73 alone and in combination with TZP
with MRSA strain NRS70. Compounds 1, 73, and TZP were used at 0.5× MIC, with TZP in 8:1
clinical ratio. (b) Neutropenic thigh infection study in mice. Compound 73 in combination
with TZP shows significant reduction in colony counts. Mice (n = 8 per group, except
untreated mice, where n = 5) were infected in the right thigh with NRS70. Treatment was
started subcutaneously 1 h after infection; doses were given every 8 h (3 doses). The TZP
dose was at 12 mg/kg of piperacillin and 1.5 mg/kg of tazobactam. Bacteria were counted in
the infected thigh homogenates at 24 h after infection. Compound 73 was evaluated at 20
and 40 mg/kg. Data are mean ± SEM; statistical significance was determined by one-way
ANOVA with Tukey’s multiple comparison post hoc test. * p < 0.05, ** p < 0.01
CONCLUSION
The 4(3H)-quinazolinone antibacterials exhibit selective inhibitory activity against S.
aureus strains, inclusive of MRSA, which are clinically challenging pathogens. These agents
target cell-wall biosynthesis, and expressly PBPs, although a pleotropic activity cannot be
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discounted at present. In the present work we prepared 79 derivatives of these
antibacterials in exploring the SAR by syntheses of 20 analogs with modifications in ring A,
42 compounds with variations in ring B, and 17 derivatives with linker modifications. In light
of the fact that the compound class inhibits more than one PBP and the X-ray crystallographic
information for binding to PBPs currently is limited only to that of the quinazolinones to the
allosteric site of PBP2a, the SAR is largely activity-driven, rather than structure-driven. We
note that our initial discovery of the quinazolinone antibacterials was also followed up by
others.35-38 The present work led to compound 73 as a lead with potent MRSA activity and
good PK properties, including oral bioavailability of 65%. Notwithstanding good in vitro
activity in inhibition of growth of S. aureus, the in vivo activity of the quinazolinone alone
was limited, as the compound is bacteriostatic. However, quinazolinone 73 in combination
with TZP, a mixture of piperacillin and the -lactamase inhibitor tazobactam, which itself is
devoid of activity in treatment of MRSA infections, behaved synergistically and in a
bactericidal manner. We attribute this activity to allosteric activation of PBP2a by the
quinazolinone, whereby the active site becomes opened and available to covalent inhibition
by piperacillin. The quinazolinone 73 enabling of covalent inhibition of PBP2a by piperacillin
brings back from obsolescence the use of penicillins in treatment of MRSA infections.
EXPERIMENTAL SECTION
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60
Syntheses. Organic reagents and solvents were purchased from Sigma-Aldrich (St. Louis,
MO), Alfa Aesar (Tewksbury, MA), Oakwood Products, Inc. (Estill, SC) or Combi Blocks Inc.
(San Diego, CA) and used without further purification. Reactions were monitored by
analytical thin-layer chromatography (TLC) using pre-coated silica-gel aluminium foils (200
µm, 60 F254, Merck KGaA, Darmstadt, German) with UV light (254 nm). Flash column
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chromatography was performed on silica gel 60 Å (40–63 µm particle size) purchased from
Sigma-Aldrich. Preparative TLC was performed on pre-coated silica-gel plates (2000 µm, 20
× 20 cm; 60 F254, Miles Scientific, Newark, DE) using UV light (254 nm). NMR spectra (1H,
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1
13C) were recorded either on a Bruker AVANCE Ⅲ HD 400 Nanobay (400 MHz for H and
101 MHz for 13C, Bruker Biospin AG, Fällanden, France) or a Bruker AVANCE Ⅲ HD 500 (500
1
13
MHz for H and 126 MHz for C, Bruker Biospin AG, Fällanden, France) at 25 ºC and the
residual non-deuterated solvent signals were used as reference (DMSO-d6, 1H δ = 2.50 ppm,
13C δ = 39.5 ppm; DMF-d7, 1H δ = 2.75, 2.92 and 8.03 ppm, 13C δ = 29.8, 34.9 and 163.2 ppm;
acetone-d6, 1H δ = 2.05 ppm, 13C δ = 29.9 ppm; chloroform-d, 1H δ = 7.26 ppm, 13C δ = 77.2
1
13
ppm; methanol-d4, H δ = 3.31 ppm, C δ = 49.0 ppm). The purity of the compounds was
monitored by a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc., Waltham,
MA) with an Acclaim™ RSCL 120 C18 column (0.2 µm, 120 Å, 2.1 × 100 mm, Thermo Fisher
Scientific Inc., Waltham, MA) with monitoring at 254 nm. All the final compounds were 
95% pure. High-resolution mass spectra were recorded using a Bruker micrOTOF/Q2 mass
spectrometer (Bruker Daltonics Inc., Fremont, CA) with positive electrospray ionization
(ESI+).
Compounds 1, 3, 11, 30, 35 and 57 were made by the methods of Bouley et al.25 Compounds 111
and 115a were synthesized according to published protocols.26
The synthetic procedures and characterizations for representative intermediate
compounds are
detailed below. Detailed synthetic procedures or characterizations for the other compounds
are provided in the Supporting Information (S5). Spectra for representative compounds are
given in the Supporting Information (S35).
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General synthetic procedure A. The benzo-oxazinone derivative (typically 1.5 mL/mmol,
1.00 eq.) and the amine (1.05 eq.) are dispersed in glacial acetic acid and the mixture is
maintained under reflux for 8-12 h. After cooling down the mixture, the solvent is partially
removed under reduced pressure and the specific compound is isolated using the
appropriate purification method, as described in the detailed compound procedures.
General synthetic procedure B. The benzo-oxazinone derivative (typically 0.5 mL/mmol,
1.0 eq) and the amine (1.2 eq) are dissolved in anhydrous pyridine and the mixture is stirred
at 120 ºC for 8-12 h in a sealed vessel. On completion of the reaction, the mixture is cooled
down and diluted with brine. The resulting mixture is extracted by EtOAc (3×). The
combined EtOAc layer is dried over anhydrous Na2SO4 and the solvent is removed under
reduced pressure to dryness. The specific compound is isolated using the appropriate
purification method, as described in the detailed compound procedures.
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General synthetic procedure C. The quinazolinone (typically 20 mL/mmol, 1.0 eq.), the
aldehyde (1.3 eq) and sodium acetate (2.0 eq) are dispersed in glacial acetic acid, and the
mixture is stirred under reflux for 8-12 h. On completion of the reaction, the reaction mixture
is cooled down and the solvent is partially removed under reduced pressure. The specific
compound is isolated using the appropriate purification method, as described in the detailed
compound procedures.
General synthetic procedure D. The quinazolinone (typically 2.8 mL/mmol, 1.0 eq.), the
aldehyde (1.3 eq.) and sodium acetate (2.0 eq.) are dispersed in glacial acetic acid, and the
mixture was stirred at 135 ºC for 8 h in a sealed vessel. After cooling down the mixture, the
solvent is partially removed under reduced pressure. The specific compound is isolated
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using the appropriate purification method, as described in the detailed compound
procedures.
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General synthetic procedure E. Anthranilic acid (typically 0.5 mL/mmol, 1.0 eq.) and the
isothiocyanate (1.1 eq.) are dissolved in glacial acetic acid and the mixture is stirred at 130
ºC for 1 h. The mixture is cooled down to form a precipitate, which is filtered and washed
with acetic acid (3×) to give the pure product as a solid. The specific compound is isolated
using the appropriate purification method, as described in the detailed compound
procedures.
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General synthetic procedure F. 3-Aryl-2-thiocarbonyl 4(1H)-quinazolinone (20 mL/mmol,
1.0 eq.) and PCl5 (1.8 eq.) are dissolved in POCl3 in an oven-dried flask. The reaction mixture
was purged with dry nitrogen at rt and stirred for another 10 min. The resulting mixture is
heated to reflux for 8 h. The solution is then cooled down and concentrated under reduced
pressure to give an oil. The oil is dissolved in 20 mL of EtOAc and washed with water (10 mL,
3×). The organic layer is washed with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to give the given product. The specific compound is isolated using
the appropriate purification method, as described in the detailed compound procedures.
The detailed synthetic procedures for the preparation of compound 73 are given below.
3-(5-Carboxy-2-fluorophenyl)-2-methylquinazolin-4(3H)-one (16). The compound was
synthesized according to the general procedure A from compound 3. Water was poured into
the concentrated mixture, which resulted in a precipitate. The solid was filtered, washed
with water and diethyl ether to give the title compound (187 mg, 51%) as a yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 13.28 (br s, 1H), 8.26 (d, J = 6.1 Hz, 1H), 8.17–8.11 (m, 2H), 7.88
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(t, J = 6.9 Hz, 1H), 7.70–7.62 (m, 2H), 7.56 (t, J = 7.1 Hz, 1H), 2.19 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 167.7 (d, JCF = 255.9 Hz), 165.7, 160.8, 153.7, 147.2, 135.1, 128.6, 128.5, 126.89,
126.87, 126.4, 125.3, 125.1, 119.9, 117.1 (d, JCF = 21.3 Hz), 23.3.
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(E)-3-(5-Carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (73). The
compound was synthesized according to the general procedure C from compound 16. Water
was poured into the concentrated mixture, which resulted in a precipitate. The solid was
filtered, was washed with water and, then, with diethyl ether giving the title compound (642
mg, 58%). 1H NMR (400 MHz, DMSO-d6) δ 13.35 (br s, 1H), 8.24–8.15 (m, 3H), 7.99–7.91 (m,
2H), 7.84–7.80 (m, 3H), 7.73–7.70 (m, 2H), 7.65 (t, J = 9.0 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H),
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6.66 (d, J = 15.5 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 165.8, 160.7, 160.2 (d, J = 255.4
Hz), 150.6, 147.1, 139.1, 138.1, 135.3, 132.8, 132.7, 128.6, 128.5 (d, J = 2.9 Hz), 127.5, 127.4,
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126.6, 124.2, 124.1, 122.4, 120.2, 118.6, 117.1 (d, J = 21.3 Hz), 111.7. F NMR (376 MHz,
DMSO-d6) -115.69 (s, 1F). HRMS-ESI (m/z): [M + H]+ calcd for C24H15FN3O3, 412.1092; found,
412.1112.
Screening for PAINS. Active compounds were checked for pan-assay-interference structures
(PAINS) using the PAINS-remover.39
3D-QSAR. Field-based 3D-QSAR29 models were built by Gaussian (based on CoMSIA31, 32
)
methods. The MIC values for each of the compound were converted to pMIC value as previously
reported33 using equation pMIC = –ln (MIC/MW), where MW is the molecular weight of the
quinazolinone. For building the model, a subset of the dataset consisting of active (MIC  4
µg/mL) and inactive (MIC  128 µg/mL quinazolin-4(3H)-ones were used. The compound
structures were built with the program Maestro (v.11, Schrödinger LLC) and energy minimized
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with LigPrep program using the OPLS2005 forcefield. The structures were aligned on the
quinazolinone core (Figure S2A). The final model was built with 103 compounds after exclusion
of 11 outliers that emerged from the preliminary modeling. The dataset was randomly divided to
training (80%) and test set (20%).
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Bacterial strains. E. faecium NCTC 7171 (ATCC 19734), S. aureus ATCC 29213, K.
pneumoniae ATCC 700603, A. baumannii ATCC 17961, P. aeruginosa ATCC 17853, E.
aerogenes ATCC 35029, E. coli ATCC 25922, S. aureus ATCC 27660, S. epidermidis ATCC
35547, S. haemolyticus ATCC 29970, S. pneumonia ATCC 49619, C. difficile ATCC 43255, B.
cereus ATCC 13061, B. licheniformis ATCC 12759, and E. faecalis ATCC 29212 were
purchased from American Type Culture Collection (ATCC; Manassas, VA). Methicillin-
resistant S. aureus (MRSA) strains NRS70, NRS119, and USA300 were obtained from the
Network on Antimicrobial Resistance in Staphylococcus aureus (Chantilly, VA).
MIC determination. MICs were determined by the guidelines provided by the Clinical and
Laboratory Standards Institute using the microdilution method.40 Two-fold serial dilutions
of the quinazolinones were prepared in triplicate in cation-adjusted Mueller−Hinton broth
with 2% NaCl and inoculated with 5 × 105 CFU/mL bacteria. The 96-well plates were
incubated at 37 °C for 16−20 h. The MIC is defined as the lowest concentration of a
compound that inhibits visible growth of bacteria after incubation. MICs with S. aureus
ATCC29213 were also determined in the presence of 0.5% bovine serum albumin (Sigma-
Aldrich).
MBC determination. Following the MIC determination protocol, two-fold serial dilutions of
compound 73 were made in triplicate in 5 mL tubes and inoculated with 5 × 105 CFU/mL S.
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aureus NRS70. After 16–20 h of incubation, 100-L aliquots were delivered in triplicate onto
the surface of agar from 1× MIC to 128 g/mL concentration of 73. The MBC is determined
as the concentration of compound that enables 99.9% reduction of the initial inoculum (
500 CFU/mL).
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XTT Assay. Cytotoxicity assays were performed against HeLa cells (ATCC HB-8065) in
triplicate following the previously reported protocol.25 The IC50 values were determined
with GraphPad Prism 5 (San Diego, CA) using nonlinear regression.
Plasma protein binding. A rapid-equilibrium-dialysis device (Pierce Biotechnology,
Thermo Scientific, Waltham, MA) was used to determined plasma protein binding. Human
plasma (Innovative Research, MI) was thawed and centrifuged at 1200 g for 10 min to
remove particulates. A 200-µL aliquot of human plasma was added to the sample chamber
and 350 µL of 0.1 M phosphate buffered saline (pH 7.4) containing 0.15 mM sodium chloride
was added to the adjacent chamber. A 2-µL stock solution of the compounds at a
concentration of 1 mM in DMSO was diluted with human plasma to a final quinazolinone
concentration of 10 µM and added to the sample chamber. The compounds were dialyzed at
o
37 C in an orbital shaker for 6 h. Aliquots (50 µL) were taken from the buffer chamber
(representing the free concentration) and from the plasma chamber (representing the total
concentration) and mixed with 100 µL of internal standard in acetonitrile to a final
concentration of 5 µM. Samples were analyzed by ultraperformance liquid chromatography
(UPLC) with UV detection at 285 nm. The plasma protein-binding ratio (B%) was calculated
according to the following equation:
B% = (Cp-Cf)/Cp ×100
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where Cp and Cf are the total plasma concentration and the free concentration of compound,
respectively.
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Animals. Female ICR mice (6–8 weeks old, 20–25 g body weight) were purchased from
Envigo (Indianapolis, IN). Mice were housed in polycarbonate shoeboxes containing ¼ inch
of corncob (The Andersons Inc., Maumee, OH) and Alpha-Dri (Shepperd Specialty Papers,
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Inc., Richland, MI) bedding, at 72 F with a 12 h light/12 h dark cycle. Mice infected with
MRSA are housed in disposable cages manufactured by Innovive (San Diego, CA) with the
same bedding, temperature and light/dark cycle. Mice were fed Teklad 2918 irradiated
extruded rodent diet (Envigo, Indianapolis, IN) and water ad libitum. Animal studies were
conducted with approval by the University of Notre Dame Institutional Animal Care and Use
Committee.
Pharmacokinetic (PK) studies. Compounds 73, 94 or 113 were dissolved in 20%
DMSO/10% Tween-80/70% water at a concentration of 2 mg/mL or 4 mg/mL. The dosing
formulations were sterilized by filtration through a 0.2 µm, 13 mm diameter PTFE
membrane attached to an Acrodisc syringe filter (Pall Life Sciences, Ann Arbor, MI). Mice
(n=2 per time point) were given 50 µL of compounds 73, 94 or 113 iv (equivalent to 5 mg/kg
or 10 mg/kg) by tail-vein injection. Terminal blood was collected at 5 min, 40 min, 2 h, 4 h,
and 8 h. Blood was centrifuged at 1200 g for 10 min to harvest plasma. Plasma samples were
stored at -80 °C until analysis. A 50-µL aliquot of plasma was mixed with 100 µL of
acetonitrile containing internal standard (final concentration 8 µg/mL). After centrifugation
at 10,000 g for 10 min, the supernatant was analyzed by UPLC with UV detection at 285 nm.
A Waters Acquity UPLC System (Waters Corporation, Milford, MA), consisting of a binary
pump, an autosampler, a column heater, and a photodiode array detector was used. A
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Kinetex C18 2.6 µm, 2.1 mm i.d. × 75 mm column was used. Elution was at 0.5 mL/min with
80% A/20% B for 2 min, followed by a 10-min linear gradient to 10% A/90% B, then 80%
A/20% B for 2 min, where A = water and B = acetonitrile.
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Antibiotics. Tazobactam was purchased from Sigma-Aldrich (St. Louis, MO); piperacillin
was obtained from TCI (Portland, OR).
Time-kill assays. Time-kill assays were performed, as described previously.27 Compounds
1, 73 and TZP were maintained at 0.5× MIC concentration. The piperacillin-tazobactam
mixture was used at 8:1 ratio, which is used clinically. A control tube without antibiotic was
also included. At intervals of 3, 6, 12, and 24 h, aliquots of the cultures were plated on LB
agar media for colony counts. The limit of detection was >50 CFU/mL and a  3-log10
reduction of colonies from the starting inoculum was considered bactericidal. The
experiment was done in triplicate.
Dose tolerance studies. Compound 73 was evaluated for dose tolerance after single dose
intravenous and oral administration to mice (n = 4 per group) at 0 (vehicle control), 10, 20,
40, and 100 mg/kg. Compound 73 was dissolved in 5% DMSO/95% water at concentrations
of 0, 2.5, 5.0, 10, and 25 mg/mL, followed by sterilization by filtration. Mice were
administered 100 µL of solution via tail-vein injection or oral gavage. Mice were observed
for 3 days, body weights were measured every day (Figure S4), and clinical observations
were recorded.
Neutropenic thigh infection model. A neutropenic thigh infection study to evaluate the
efficacy of compound 73 was performed, as described previously.27 The mice (n = 8 per
group) were rendered neutropenic by intraperitoneal treatment with 200 mg/kg solution of
cyclophosphamide (Alfa Aesar, Haverhill, MA) at four days and one day prior to infection. An
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inoculum of 0.1 mL of MRSA strain NRS70 at a final concentration of 1 × 106 CFU/mL in brain
heart infusion broth was injected intramuscularly into the right thighs of all the animals. The
infected animals were treated with either vehicle (10% DMSO and 90% water), 20 mg/kg of
compound 1, 20 and 40 mg/kg of compound 73, 12 mg/kg of piperacillin with 1.5 mg/kg of
tazobactam or a combination of compound 73 at 20 mg/kg and 40 mg/kg with TZP. The
drugs were dissolved in 10% DMSO/90% water, sterile-filtered, and administered
subcutaneously (three doses) every 8 h starting 1 h after the infection. The animals were
euthanized 24 h later, and the infected thighs were harvested aseptically for bacterial counts.
The uninfected thighs were collected to measure drug levels.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Experiment procedures and characterization of synthesized compounds; chemical
structures of synthesized intermediates; MIC of reported compounds against the
ESKAPE organisms; information of compounds used in the QSAR study; computational
analysis of quinazolinones; representative NMR spectra/LCMS results. Molecular
formula strings for synthesized molecules (CSV)
AUTHOR INFORMATION
Corresponding Author
* E-mail: mchang@nd.edu. Phone: 574-631-2965
* E-mail: mobashery@nd.edu. Phone: 574-631-2933
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ORCID
Mayland Chang 0000-002-4333-3775
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Shahriar Mobashery 0000-002-7695-7883
Author Contributions
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Y.Q., G.A., and S.T., synthesized the quinazolinones. JJ conducted the time-kill assays and the
mouse neutropenic thigh infection studies. Z.P. and M.M.N.G. determined plasma protein
binding and conducted the PK studies. K.V.M performed computational analyses. E.L.
determined MIC. E.L. and R.F. conducted XTT assays. V.A.S. and W.R.W. conducted the in-life
portion of the animal studies. S.M. and M.C. directed the all studies. The manuscript was
written and proofread by all authors.
Notes
The authors declare the following financial interest: US patent 9,776,975 has been issued for
the quinazolinones.
ACKNOWLEDGMENTS
Methicillin-resistant S. aureus (MRSA) strains NRS70, NRS119, and USA300 were provided
by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for
distribution by BEI Resources, NIAID, NIH. This project is supported by grants AI116548
(to M.C.) and AI104987 (to S.M.) from the National Institutes of Health. Y.Q. is a Ruth L.
Kirschtein National Research Service Award Fellow of the Chemistry-Biochemistry-Biology
Interface Program at the University of Notre Dame, supported by Training Grant
T32GM075762 from the National Institute of General Medical Sciences.
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ABBREVIATIONS USED
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AcOH, acetic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt,
hydroxybenzotriazole; HATU, 1 N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; EtOAc, ethyl acetate
AUC, area-underthecurve; CL, clearance; CFU, colony forming unit; iv, intravenous; MIC,
minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; PBP,
penicillin-binding protein; PK, pharmacokinetics; po, oral; PPB, plasma protein binding;
SEM, standard error of the mean; TZP, piperacillin-tazobactam; UPLC, ultraperformance
liquid chromatography; UV, ultraviolet; Vd, volume of distribution; XTT, 2,3-bis(2-methoxy-
4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
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REFERENCES
1. Centers for Disease Control and Prevention (CDC) Antibiotic Resistance Threats in the
United States, 2019, 1–50.
2. Pourmand, A.; Mazer-Amirshahi, M.; Jasani, G.; May, L. Emerging Trends in Antibiotic
Resistance: Implications for Emergency Medicine. Am. J. Emerg. Med. 2017, 35, 1172–1176.
3. Hofer, U. The Cost of Antimicrobial Resistance. Nat. Rev. Microbiol. 2019, 17, 3.
4. Rice, L. B. Federal Funding for the Study of Antimicrobial Resistance in Nosocomial
Pathogens: No ESKAPE. J. Infect. Dis. 2008, 197, 1079–1081.
5. Bush, K. Synergistic Antibiotic Combinations. Top. Med. Chem. 2018, 25, 69–88.
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