Synthesis and evaluation of 1,2,4-triazolo[1,5- a ]pyrimidines as antibacterial agents against Enterococcus faecium

Article abstract of DOI:10.1021/jm501831g

Rapid emergence of antibiotic resistance is one of the most challenging global public health concerns. In particular, vancomycin-resistant Enterococcus faecium infections have been increasing in frequency, representing 25% of enterococci infections in intensive care units. A novel class of 1,2,4-triazolo[1,5-a]pyrimidines active against E. faecium is reported herein. We used a three-component Biginelli-like heterocyclization reaction for the synthesis of a series of these derivatives based on reactions of aldehydes, β-dicarbonyl compounds, and 3-alkylthio-5-amino-1,2,4-triazoles. The resulting compounds were assayed for antimicrobial activity against the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These analyses identified a subset of 1,2,4-triazolo[1,5-a]pyrimidines that had good narrow-spectrum antibacterial activity against E. faecium and exhibited metabolic stability with low intrinsic clearance. Macromolecular synthesis assays revealed cell-wall biosynthesis as the target of these antibiotics.

Full text of DOI:10.1021/jm501831g

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Article
Synthesis and Evaluation of 1,2,4-Triazolo[1,5-a]pyrimidines
as Antibacterial Agents Against Enterococcus faecium
Huan Wang, Mijoon Lee, Zhihong Peng, Blas Blázquez, Elena Lastochkin,
Malika Kumarasiri, Renee Bouley, Mayland Chang, and Shahriar Mobashery
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501831g • Publication Date (Web): 29 Apr 2015
Downloaded from http://pubs.acs.org on May 6, 2015
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Synthesis and Evaluation of 1,2,4ꢀTriazolo[1,5ꢀ
a]pyrimidines as Antibacterial Agents Against
Enterococcus faecium
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Huan Wang, Mijoon Lee, Zhihong Peng, Blas Blázquez, Elena Lastochkin, Malika Kumarasiri,
Renee Bouley, Mayland Chang*and Shahriar Mobashery*
Department of Chemistry and Biochemistry and Eck Institute for Global Health, University of
Notre Dame, Notre Dame, IN 46556, USA
KEYWORDS: triazolopyrimidines, Enterococcus faecium, antibacterial activity
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ABSTRACT: Rapid emergence of antibiotic resistance is one of the most challenging global
public health concerns. In particular, vancomycinꢀresistant Enterococcus faecium infections have
been increasing in frequency, representing 25% of enterococci infections in intensive care units.
A novel class of 1,2,4ꢀtriazolo[1,5ꢀa]pyrimidines active against E. faecium is reported herein.
We used a threeꢀcomponent Biginelliꢀlike heterocyclization reaction for the synthesis of a series
of these derivatives based on reactions of aldehydes, βꢀdicarbonyl compounds and 3ꢀalkylthioꢀ5ꢀ
aminoꢀ1,2,4ꢀtriazoles. The resulting compounds were assayed for antimicrobial activity against
the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These
analyses identified a subset of 1,2,4ꢀTriazolo[1,5ꢀa]pyrimidines that had good narrowꢀspectrum
antibacterial activity against E. faecium, and exhibited metabolic stability with low intrinsic
clearance. Macromolecular synthesis assays revealed cellꢀwall biosynthesis as the target of these
antibiotics.
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INTRODUCTION
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Antibacterial resistance is on the increase, becoming one of the most serious public health
challenges.1,2,3 The ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are
important pathogens, not only because they account for nearly one third of all nosocomial
infections in the United States,4,5 but also because they are broadly resistant to existing
antibiotics.4,6 Enterococci have been important clinically for some time. They are either
intrinsically resistant to antibiotics or have acquired resistant mechanisms to many commonly
used antibiotics.7,8,9 This is especially true for bloodstream infections, for which they are the
third most common bacterial pathogen.10 E. faecalis and E. faecium are the major enterococcal
species.11 Although E. faecium is known to be a harmless commensal inhabitant of the
gastrointestinal tract, it causes invasive diseases such as neonatal meningitis,12 endocarditis,13
urinary tract infections,14 surgical wound infections15 and bloodstream infections,16 accounting
for 20% of enterococcal infections in the United States.11 It shows high resistance to many
commonly used antibacterial reagents.17 Vancomycinꢀresistant E. faecium (VRE) emerged in the
1980s in the United Kingdom and France.18,19 The VRE strains disseminated globally rapidly.19
Infections by VRE represent 25% of enterococci infections in intensiveꢀcare units,20 and in some
medical centers more than 70% of E. faecium isolates are vancomycinꢀresistant.11,21 There is a
genuine need for new antibacterial agents against this organism and agents selective for this
organism are especially sought. The draw of selective agents is that they would be useful in
treatment of infections by E. faecium, yet they would spare other organisms in the microbiota,
mitigating the opportunity for rapid emergence of resistance clinically.
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We report herein the discovery of a novel class of antibacterial agents, the triazolopyrimidines,
which emerged from an in silico screening against a penicillinꢀbinding protein (PBP), which is
an essential enzyme for peptidoglycan biosynthesis.22 The in silico screening was performed
against 1.2 million compounds. Of the top 2,500 hits, we purchased 118 compounds, which were
then evaluated against the living bacteria of the ESKAPE panel. The triazolopyrimidine emerged
as a template with antibacterial activity, of which we synthesized 68 analogs. These derivatives,
along with 18 additional compounds that could be purchased commercially, were evaluated for
antibacterial activity. We disclose herein the structural template and describe the structureꢀ
activity relationship (SAR) for antibacterial activity for this set of molecules.
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RESULTS AND DISCUSSION
Discovery of the lead template. We used in silico docking and scoring of 1.2 million drugꢀ
like compounds from the ChemDiv subset of the ZINC database23 against the Xꢀray structure of
PBP2a (PDB ID: 1VQQ),24 in discovery of antibiotics. We reasoned that identification of the
leads from the in silico screening should result in discovery of compounds that would have the
ability to crossꢀinhibit more than one PBP, as the active sites of these enzymes are very similar.
Whereas these in silico methodologies have come a long way in sophistication in the past few
years, there still exists a degree of uncertainty in identifying molecules that indeed bind to the
target site for which they were screened. As such, as will be described, experimental verification
is critical. We analyzed the top 2,500 best scoring compounds and clustered them in groups
according to structural similarity using hierarchical clustering. From these, we selected 118
compounds for purchase from ChemDiv and screened them for antibacterial activity against the
ESKAPE panel of organisms. Therefore, the bar was set high from the outset, as demonstration
of antibacterial activity with living bacteria was selected as the minimal criterion for a molecule
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to move forward. This is in contrast to the possibility that the compound inhibiting avidly the
recombinant target in vitro, which might turn out to be devoid of any activity with living bacteria.
Compound 1 (Scheme 1) emerged from this screening, with an MIC of 8 ꢀg/mL against E.
faecium. No antibacterial activity was observed against the other bacteria in the ESKAPE panel.
Hence, this compound fits the narrowꢀspectrum antibiotic definition for enterococci, which is
desirable, as described earlier. However, as will be elaborated below—and as a cautionary note
on in silico drug discovery—compound 1 did not inhibit PBP2a, which was the basis of the
screening. Hence, notwithstanding the prediction by computation that this compound should
inhibit PBP2a, it does not. Yet, it possesses antibacterial activity, as activity against living
bacteria was set as the minimal criterion for moving forward. Optimization of the antibacterial
activity became the goal of the diversification of structure that we undertook.
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Chemistry. The general procedure for the syntheses of triazolopyrimidine derivatives is
outlined in Scheme 1. We made 68 analogs of compound 1 by this method. Compounds III, as
key intermediates in these syntheses, were prepared by the reaction of 3ꢀaminoꢀ5ꢀthiolꢀ1,2,4ꢀ
triazole with alkyl bromides in the presence of triethyl amine as the base. Subsequently, equal
amounts of derivatives I, II, and III were heated in DMF for roughly 15ꢀ20 min (with a
temperature of 130ꢀ160 °C), which yielded the desired compounds after the work up. This threeꢀ
component Biginelliꢀlike heterocyclization method25,26 afforded the rapid and efficient synthesis
of a focused library of these compounds, using commercially available reagents. In light of the
essentially singleꢀpot reactions that led to these molecules, only the final compounds have been
characterized in this report.
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Scheme 1. Syntheses of the 1,2,4ꢀtriazolo[1,5ꢀa]pyrimidine derivatives.
Antibacterial activity and SAR analysis. The SAR of the compound class was investigated
by synthesizing structural variations at each of the three sites designated as SAR1, SAR2, and
SAR3 (Figure 1) and by determination of their minimalꢀinhibitory concentration (MIC) values
against E. faecium. Initial studies of the scaffold focused on modifications of the aromatic ring in
the pyrimidine segment (SAR1). The antibacterial activities (as MICs) for these compounds are
shown in Table 1. In the SAR1 study, the synthesis was driven by preserving the original
functionalities for the SAR2 and SAR3 regions in the lead compound, while the SAR1 position
was varied. Various functional groups, such as substituted phenyl and heterocycles, were
introduced in SAR1. Substitution of the 4ꢀiꢀpropyl group with a hydrogen atom resulted in loss
of antibacterial activity (2), which indicates addition of a substituent on the phenyl ring is
necessary. An evaluation of derivatives with lipophilic steric bulk showed that the antiꢀE.
faecium activity is linked with the size of the lipophilic group (ꢀPhꢀ4ꢀiꢀBu (5)/ꢀPhꢀ4ꢀiꢀPr (1)/ ꢀPhꢀ
4ꢀEt (4) > ꢀPhꢀ4ꢀMe (3)/ ꢀPhꢀ4ꢀH (2)). Assessment of compounds with bulky oxygen and sulfur
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substituents at the paraꢀposition also indicates a similar trend. Introduction of 4ꢀOꢀiꢀPr (10) and
4ꢀSEt (13) into the scaffold showed 32ꢀfold improvement of the antiꢀE. faecium activity,
compared to the 4ꢀOCH3 (7) and 4ꢀSMe (12) derivatives, which were devoid of activity. A series
of analogs with nitrogen (or alkylated nitrogen) at this position, ꢀPhꢀ4ꢀNH2 (15), ꢀPhꢀ4ꢀNMe2
(16), ꢀPhꢀ4ꢀNEt2 (17), ꢀPhꢀ4ꢀ(1ꢀpyrrolidinyl) (18) and ꢀPhꢀ4ꢀNHCOCH3 (19), were prepared.
Compound 16 with ꢀPhꢀ4ꢀNMe2 was the best in this series. It showed 32ꢀfold improved potency
over compounds 15 (ꢀPhꢀ4ꢀNH2) and 19 (ꢀPhꢀ4ꢀNHCOCH3), 4ꢀfold better antimicrobial activity
than compound 18 (ꢀPhꢀ4ꢀ(1ꢀpyrrolidinyl)), and slightly better activity than compound 17 (ꢀPhꢀ4ꢀ
NEt2); also 2ꢀfold improved antibacterial activity over the lead compound 1. Introduction of
small electronꢀdonating groups at the paraꢀposition of the phenyl ring resulted in loss of
antimicrobial activity, as observed for compounds 6, 7, 8, and 9. On the other hand, introduction
of electronꢀwithdrawing groups at the paraꢀposition of the phenyl ring retained activity (11, 21
and 24), but not in all cases (14, 25 and 26), as compared to compound 1. Analogs with halogen
substituents did not show any activity (22 and 23). The variant 20 had no activity. From all the
above findings, compounds with ꢀPhꢀ4ꢀiꢀPr (1), ꢀPhꢀ4ꢀEt (4), ꢀPhꢀ4ꢀOꢀiꢀPr (10), ꢀPhꢀ4ꢀOCF3 (11),
ꢀPhꢀ4ꢀNMe2 (16), ꢀPhꢀ4ꢀNEt2 (17) and ꢀPhꢀ4ꢀCF3 (21) groups showed good activity (MIC of ≤ 8
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ꢀg/mL).
In general, the substituents that gave good activity at the paraꢀposition of the ring, gave poorer
or no activity when placed at the metaꢀ or orthoꢀpositions. Compounds with ꢀPhꢀ3ꢀCF3, ꢀPhꢀ3ꢀiꢀ
Pr, ꢀPhꢀ3ꢀEt, ꢀPhꢀ3ꢀOꢀiꢀPr, and ꢀPhꢀ3ꢀOCF3 substitutions (28, 29, 30, 31, and 32) were examples
of these analogs. Also, compound 27 with CF3 at the orthoꢀposition of the phenyl group was
inactive. Substitutions of the phenyl group with heterocyclic rings were also tried, including
thiophene, furan, pyrrole, imidazole, indole, pyridine rings (compounds 33 to 41); however,
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these changes were generally not tolerated, resulting in loss of activity. The nonꢀaromatic
cyclohexyl moiety in the SAR1 position (42) was devoid of activity. Based on the SAR1 findings,
the phenyl ring was necessary for activity. Furthermore, substituents at the paraꢀposition
improved the activity. Introduction of bulky electronꢀdonating groups proved useful in
improving activity against E. faecium. We selected ꢀPhꢀ4ꢀNMe2 (16) from SAR1 for its
favorable logP value and for its MIC of 4 ꢀg/mL.
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Figure 1. SAR of 1,2,4ꢀtriazolo[1,5ꢀa]pyrimidine derivatives.
Next, our exploration shifted to modifications at the amide position within the bicyclic system
SAR2 (Figure 1 and Table 1). Considering that the antibacterial activity favors the presence of
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the ꢀPhꢀ4ꢀNMe2 (of the SAR1), the subsequent analogs were investigated with this moiety
retained. Replacing the carboxamide with an ester, Nꢀmethylcarboxamide, and Nꢀ
phenylcarboxamide generated analogs 43, 45, and 51, respectively. While the substituted amides
(45 and 51) led to slight reduction of activity compared to compound 16, the ester derivative (43)
lost antimicrobial activity. Introduction of cyclohexenone next to the 1,6ꢀdihydropyridine (46)
abolished activity. This series of molecules shows that replacement of the carboxamide with an
ester or cyclic ketone led to loss of activity. We also evaluated the commercially available
derivatives 44, 47ꢀ50, and 52ꢀ63. Unfortunately, all were devoid of antibacterial activity. These
analyses indicated that the carboxamide segment of the molecular scaffolding was desirable for
activity.
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Our attention turned to the SAR3 series bearing the ꢀPhꢀ4ꢀNMe2 (SAR1) and ꢀCONH2 (SAR2)
moieties on the structural template (Figure 1 and Table 1). Replacement of the Sꢀbenzyl moiety
with smaller groups (64ꢀ71) resulted in loss of activity. Introduction of longer R3 functionalities
(than Sꢀbenzyl) in compounds 72, 73 and 74 were not tolerated. Further exploration focused on
the necessity of the phenyl ring and methylene of the Sꢀbenzyl group on the SAR3 region.
Compound 75 was generated by replacing the phenyl ring with a pyridine ring; compound 76
was generated by removing the methylene from 75. These changes resulted in loss of activity,
which indicated the need for the phenyl ring and the methylene group. These results indicate the
requirement for the Sꢀbenzyl group, which was retained in the next group of compounds that was
prepared. A subset of analogs was made to investigate whether the antibacterial activity could be
improved via substituents on the Sꢀbenzyl ring at the SAR3 position. A total of 10 paraꢀ
substituted analogues (77ꢀ86) were made. While the ones with electronꢀdonating groups at the
paraꢀposition did not show good activity (77ꢀ79), compounds with electronꢀwithdrawing groups
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at the paraꢀposition, such as the trifluoromethylmercaptan (82) and the cyano (84) were as active
as compound 1 and slightly less active than compound 16. The nitro (83) analog was as good as
compound 16. Compounds 80 and 81 had no activity. The SAR3 revealed that the Sꢀbenzyl
group and electronꢀwithdrawing groups at the paraꢀposition are favored.
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Table 1. Minimalꢀinhibitory concentrations (MICs, ꢁg/mL) of SAR1, SAR2, and SAR3
compounds against E. faecium NCTC 7171a
MICs of SAR1 compounds against E. faecium NCTC 7171
Compd
R1
MIC Compd
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MIC
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ꢀPh
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ꢀPhꢀ4ꢀCl
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>128
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>128
>128
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ꢀPhꢀ4ꢀMe
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ꢀPhꢀ4ꢀCOOH
ꢀPhꢀ2ꢀCF3
ꢀPhꢀ3ꢀCF3
ꢀPhꢀ3ꢀiꢀPr
ꢀPhꢀ3ꢀEt
3
ꢀPhꢀ4ꢀEt
64
4
ꢀPhꢀ4ꢀiꢀBu
ꢀPhꢀ4ꢀOH
8
>128
>128
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>128
>128
>128
>128
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ꢀPhꢀ4ꢀOCH3
ꢀPhꢀ3ꢀOHꢀ4ꢀOCH3
ꢀPhꢀ3,4ꢀ(OCH3)2
ꢀPhꢀ4ꢀOꢀiꢀPr
ꢀPhꢀ4ꢀOCF3
ꢀPhꢀ4ꢀSMe
7
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ꢀPhꢀ3ꢀOꢀiꢀPr
ꢀPhꢀ3ꢀOCF3
ꢀ2ꢀthienyl
>128
16
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>128
>128
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ꢀPhꢀ4ꢀSEt
ꢀPhꢀ4ꢀSCF3
4
>128
>128
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ꢀ3ꢀthienyl
ꢀ2ꢀfuryl
>128
>128
>128
32
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ꢀPhꢀ4ꢀNH2
ꢀ2ꢀpyrrolyl
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ꢀ2ꢀimidazolyl
ꢀ3ꢀindolyl
ꢀPhꢀ4ꢀNEt2
8
>128
>128
128
ꢀPhꢀ4ꢀ(1ꢀpyrrolidinyl)
ꢀPhꢀ4ꢀNHCOCH3
ꢀPhꢀ4ꢀOCH2CH2NEt2
ꢀPhꢀ4ꢀCF3
16
ꢀ3ꢀpyridyl
128
>128
8
ꢀ3ꢀpyridylꢀ(2ꢀOCH3)
ꢀ4ꢀpyridyl
>128
>128
ꢀcyclohexyl
MICs of SAR2 compounds against E. faecium NCTC 7171
Compd
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R1
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R3
MIC
>128
>128
8
ꢀPhꢀ4ꢀNMe2
ꢀ2ꢀthienyl
ꢀPhꢀ4ꢀNMe2
ꢀOC2H5
ꢀOC2H5
ꢀNHCH3
ꢀSCH2Ph
ꢀSCH2Phꢀ4ꢀCO2H
ꢀSCH2Ph
44
45
Compd
R1
R3
MIC
Compd
R1
R3
MIC
>128
ꢀ
ꢀPhꢀ4ꢀ
NMe2
ꢀPhꢀ3,4ꢀ
(OMe)2
ꢀSCH2Ph
>128
SCH2Phꢀ
4ꢀF
46
49
ꢀSCH2ꢀ
ꢀSCH2Phꢀ4ꢀ
CO2H
ꢀPhꢀ4ꢀOMe
ꢀPhꢀ2ꢀOEt
>128
>128
ꢀPhꢀ4ꢀCl
naphthale >128
nyl
47
48
50
ꢀSCH2Phꢀ4ꢀ
CO2H
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R3
Compd
51
R1
ꢀPhꢀ4ꢀNMe2
ꢀPhꢀ4ꢀMe
ꢀPhꢀ4ꢀMe
ꢀPhꢀ4ꢀMe
ꢀPhꢀ4ꢀEt
R2
ꢀH
MIC
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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59
60
ꢀSCH2Ph
ꢀSCH2Phꢀ2ꢀMe
ꢀSCH2Phꢀ3ꢀMe
ꢀSCH2Phꢀ3ꢀMe
ꢀSCH2Phꢀ3ꢀOMe
ꢀSCH2Phꢀ3ꢀOMe
ꢀSCH2Ph
>128
>128
>128
>128
>128
>128
>128
>128
16
ꢀH
52
ꢀ2ꢀMe
ꢀ2,4ꢀMe2
ꢀH
53
54
55
ꢀPhꢀ4ꢀEt
ꢀ2ꢀMe
ꢀ2ꢀOMe
56
ꢀPhꢀ4ꢀEt
57
ꢀSCH2Phꢀ2,5ꢀMe2
ꢀSCH2Phꢀ3ꢀMe
ꢀSCH2Ph
ꢀPhꢀ4ꢀOMe
ꢀPhꢀ4ꢀOMe
ꢀPhꢀ3ꢀOMeꢀ4ꢀOH
ꢀPhꢀ4ꢀF
ꢀ2ꢀMe
58
ꢀ2,4ꢀMe2
ꢀ2,4ꢀMe2
ꢀ2,4ꢀMe2
ꢀ2,4ꢀMe2
ꢀ2,4ꢀMe2
59
60
ꢀSCH2Phꢀ3ꢀMe
ꢀCO2ꢀiꢀPr
>128
16
61
ꢀPhꢀ4ꢀCl
62
ꢀSCH2Phꢀ3ꢀCl
ꢀ2ꢀthienyl
>128
63
MICs of SAR3 compounds against E. faecium NCTC 7171
Compd
64
R3
ꢀH
MIC
>128
>128
>128
Compd
77
R3
MIC
>128
>128
>128
ꢀSCH2Phꢀ4ꢀCH3
ꢀSCH2Phꢀ4ꢀOCH3
ꢀSCH2Phꢀ4ꢀSCH3
ꢀNH2
66
78
ꢀCO2CH3
67
79
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ꢀCO2CH2CH3
ꢀSCH3
>128
>128
>128
>128
ꢀSCH2Phꢀ4ꢀCF3
ꢀSCH2Phꢀ4ꢀOCF3
ꢀSCH2Phꢀ4ꢀSCF3
ꢀSCH2Phꢀ4ꢀNO2
ꢀSCH2Phꢀ4ꢀCN
>128
>128
8
4
5
6
7
8
9
68
69
70
71
72
75
76
80
81
82
83
84
85
86
ꢀSCH2CONH2
ꢀSCH2CO2Et
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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52
53
54
55
56
57
58
59
60
4
ꢀSCH2CH2NHCO2CH2Ph >128
8
ꢀSCH2ꢀ(4ꢀpyridyl)
ꢀSꢀ2ꢀpyridyl
128
ꢀSCH2Phꢀ4ꢀCOOH
ꢀSCH2Phꢀ4ꢀCOOCH3
>128
>128
>128
Compd
65
R1
R3
H
MIC
ꢀ2ꢀthienyl
ꢀPhꢀ4ꢀiꢀPr
ꢀPhꢀ4ꢀCO2H
>128
>128
>128
ꢀSCH2CH2NHCO2CH2Ph
73
ꢀSCH2CH2NHCO2CH2 Ph
74
aCompounds with MIC ≤ 16 ꢀg/mL are highlighted in gray background.
The most active compounds (1, 10, 13, 16, 83 and 84) were further evaluated for their MICs
with several additional Enterococci strains, both from commercial as well as the clinical sources
(Table 2). These included strains that were resistant to erythromycin, tetracycline, vancomycin
and teicoplanin.
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Table 2. Minimalꢀinhibitory concentrations (MICs, µg/mL) of compounds 1, 10, 13, 16, 83, 84
and Vancomycin with additional E. faecium strains
9
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
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40
41
42
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44
45
46
47
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50
51
52
53
54
55
56
57
58
59
60
MIC (ꢀg/mL)
1
10 13 16 83 84 Vancomycin
E. faecium ATCC
BAA 2127a
4
16 16
4
4
32
16
8
8
1
E. faecium 119ꢀ
39A (Van S)b
4
4
8
0.5
16
E. faecium ATCC
700221(Van R)c
4
8
4
8
8
4
4
8
8
8
4
E. faecium C9d
16
2
E. faecium C38d 16 32 16 16 32 16
E. faecium C68d,e 16 16 16
64
4
32
4
64
aResistant to copper, erythromycin, and tetracycline, and sensitive to vancomycin and
teicoplanin. bVancomycinꢀsusceptible clinical isolate. cIsolated from human feces, vanA positive,
d
resistant to vancomycin and teicoplanin. Clinical strains isolated in Cleveland hospitals, gifts
from Dr. Robert A. Bonomo. eMost prevalent VRE clinical strain from Cleveland hospitals.
In vitro metabolic stability, plasmaꢀprotein binding, and cytotoxicity. The most active
compounds (1, 10, 13, 16, 83, and 84) were evaluated for metabolic stability using human liver
S9, which contains microsomal and cytosolic fractions capable of phase I and II metabolism. In
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general, the compounds were metabolically stable, with halfꢀlives of around 1 h or longer (Table
3). Replacement of the ꢀPhꢀ4ꢀiꢀPr (1) with ꢀPhꢀ4ꢀOꢀiꢀPr (10) did not change the halfꢀlife,
however ꢀPhꢀ4ꢀSEt (13) and ꢀPhꢀ4ꢀNMe2 (16) resulted in increased metabolic stability and lower
intrinsic clearance. Retention of ꢀPhꢀ4ꢀNMe2 at R1 and ꢀSCH2Phꢀ4ꢀNO2 at R3 (83) and ꢀSCH2Phꢀ
4ꢀCN at R3 (84) also increased the halfꢀlives, and consequently resulted in the lowest intrinsic
clearances. We also ascertained plasmaꢀprotein binding for these compounds in human plasma
using equilibrium dialysis (Table 3). Compound 1 showed 98.7 ± 0.7% plasmaꢀprotein binding.
Plasmaꢀprotein binding of compounds 10, 13, and 83 was high. Compound 16 had 97.6 ± 0.1%
plasmaꢀprotein binding, while compound 84 had the lowest protein binding at 96.8 ± 0.4%. Forty
three percent of the 1,500 frequently prescribed drugs on the market show protein binding
greater than 90%, and 27% of antiꢀinflammatory drugs have protein binding above 99%.27
Moreover, many marketed antibiotics, including daptomycin,28 oxacillin,29 teicoplanin,30
rifampicin,31 and clindamycin31 have plasma protein binding of >91%. Compounds 1, 10, 13, 16,
83 and 84 were evaluated for in vitro toxicity in the hemolysis assay (Table 3). The most active
of these compounds (e.g., compounds 16 and 84) showed 1ꢀ2% hemolysis of red blood cells at a
fixed concentration of 64 µg/mL for the antibiotic.
10
11
12
13
14
15
16
17
18
19
20
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23
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27
28
29
30
31
32
33
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39
40
41
42
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44
45
46
47
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50
51
52
53
54
55
56
57
58
59
60
The minimalꢀbactericidal concentrations (MBCs) of compounds 1, 10, 13, 16, 83 and 84 were
determined using E. faecium NCTC7171. The MBC values were 32, 16, 16, 32, 16, and 16
µg/mL for the given antibiotics, respectively. This reveals that cidal effects are seen at 2ꢀ to 4ꢀ
fold above the MIC values.
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Table 3. Metabolic stability, apparent intrinsic clearance, plasmaꢀprotein binding and hemolysis
7
8
9
of red blood cells of selected antibiotics.
10
11
12
13
14
15
16
17
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57
58
59
60
MIC of E.
Apparent
Halfꢀlife
Plasmaꢀ
protein
binding (%)
Hemolysis at
64 µg/mL
(%)
faecium
C68
intrinsic
in human
liver S9
(min)
clearance
(ꢀg/mL)
(mLminꢀ1kgꢀ1)
Compd
1
R1
R3
ꢀPhꢀ4ꢀi-Pr
ꢀPhꢀ4ꢀOꢀi-Pr
ꢀPhꢀ4ꢀSEt
ꢀPhꢀ4ꢀNMe2
ꢀSCH2Ph
ꢀSCH2Ph
ꢀSCH2Ph
ꢀSCH2Ph
16
16
16
4
56 ± 1
57 ± 8
>60
11.1
10.9
98.7 ± 0.7
99.9 ± 0.2
99.9 ± 0.4
97.6 ± 0.1
99.9 ± 0.5
96.8 ± 0.4
22%
<1%
46%
2%
10
<10.4
<10.4
<10.4
<10.4
13
>60
16
ꢀPhꢀ4ꢀNMe2 ꢀSCH2Phꢀ4ꢀNO2
ꢀPhꢀ4ꢀNMe2 ꢀSCH2Phꢀ4ꢀCN
32
4
>60
23%
1%
83
>60
84
Mechanism of Action. Compound 16 was selected to investigate the mode of action by using
macromolecular synthesis assays with E. faecium NCTC7171. Incorporation of radiolabeled
precusors [methylꢀ3H]ꢀthymidine, [5,6ꢀ3H]ꢀuridine, ꢀ[4,5ꢀ3H]ꢀleucine, or ꢀ[2,3ꢀ3H]ꢀalanine into
L D
DNA, RNA, protein, or peptidoglycan, respectively, was monitored in the logarithmic phase of
growth of this bacterium. Ciprofloxacin, rifampicin, tetracycline, and meropenem, which are
known inhibitors of each corresponding pathway, were used as positive controls. Compound 16
showed notable inhibition of peptidoglycan biosynthesis. It did not inhibit replication,
transcription, or translation appreciably by these assays (Figure 2). These experiments with
living bacteria were followed up by additional in vitro transcription and translation assays. A
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transcriptAid T7 highꢀyield transcription kit was used for in vitro transcription assay; an E. coli
S30 extract for circular DNA kit plus a βꢀgalactosidase assay were used for a tandem in vitro
transcription and translation assay. Compound 16 did not show any effect on transcription and/or
translation (Figure 3) in these assays.
10
11
12
13
14
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16
17
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59
60
100
Compound 16 Positive control
80
60
40
20
0
DNA
RNA
Protein
Peptidoglycan
ꢀ20
Figure 2. Macromolecular synthesis assays. Compound 16 at 2 × MIC. Positive controls for
DNA, RNA, protein, and peptidoglycan syntheses are ciprofloxacin (4 ꢀg/mL), rifampicin (4
ꢀg/mL), tetracycline (248 ng/mL) and meropenem (4 ꢀg/mL), respectively. The inhibition
reported for compound 16 is at 120 min of the incubation; other time points of incubation are
provided in the Supporting Information.
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B
A
1.2
1
1.2
1
0.8
0.6
0.4
0.2
0
7
8
9
0.8
0.6
0.4
0.2
0
10
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0 0.5 1
2
4
8 16 32
0
0.5
1
2
4
8
16 32
Compound 16 (µg/mL)
Compound 16 (µg/mL)
Figure 3. (A) In vitro transcription assay of compound 16. (B) In vitro transcription and
translation assay of compound 16 to evaluate βꢀgalactosidase expression levels.
Binding assays for PBP2a of S. aureus and PBP5fm of E. faecium. Compound 1 was
conceived from in silico screening of PBP2a of S. aureus. We explored if recombinant purified
PBP2a was actually inhibited by this compound. At concentrations of up to 100 ꢀg/mL we
observed no inhibition. We then turned to PBP5fm, an important PBP of E. faecium, as these
compounds had activity against this organism. Similarly, at up to 100 ꢀg/mL we saw no
inhibition of this enzyme. We also investigated compound 84, an active compound that emerged
from the SAR analyses. This compound also lacked the ability to inhibit both the recombinant
proteins. As a last experiment, we also investigated if other PBPs in the E. faecium membrane
fractions were inhibited in the presence of these compounds. The data from the membrane
fractions were less clear due to the low copy numbers of PBPs in this organism. Regardless, no
inhibition was apparent in membranes exposed to these antibiotics. It would appear that
triazolopyrimidines are not inhibitors of PBPs.
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CONCLUSION
5
6
7
8
In summary, we synthesized 68 compounds and investigated the SAR of these and 18
additional compounds that were purchased. The most active compounds were evaluated in
several E. faecium strains, including clinical VRE isolates, as well as assessed for metabolic
stability, plasmaꢀprotein binding, and cytotoxicity. Compound 16 has promising activity against
E. faecium, it is metabolically stable, has low intrinsic clearance, and the lower plasmaꢀprotein
binding.
9
10
11
12
13
14
15
16
17
18
19
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24
25
26
27
28
29
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Notwithstanding the fact that the in silico segment of screening was done against the PBP2a
structure, compounds 1 and 84 did not inhibit this enzyme in vitro, nor the PBP5fm of E. faecium
or likely the PBPs in the E. faecium membranes. This exercise reveals the limitations of in silico
search. Experimental verification is critical. We state that by setting the bar high at the outset for
the experimental verification, we went directly for documentation of the antibacterial activity
with living bacteria, rather than the in vitro exploration of inhibition of recombinant PBP2a. If
the bar had not been set high, by mere inhibition assays for PBPs the triazolopyrimidine class of
antibacterials would not have been discovered.
An important aspect of the work with the triazolopyrimidine template is that a large set of
molecules of this class can be prepared essentially in singleꢀpot threeꢀcomponent reactions, using
the Biginelliꢀlike heterocyclization reaction. This ease in preparation should lend itself to further
exploration of the SAR. Notwithstanding our observation that cellꢀwall biosynthesis is inhibited
by this class of antibiotics, the identification of the actual target in the pathway is open for
elucidation.
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EXPERIMENTAL SECTION
General chemistry. All chemicals, reagents, and solvents were used directly as purchased
7
8
9
1
13
without further purification. Chemical shifts in H NMR and C NMR spectra are reported in
10
11
12
13
14
15
16
17
18
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22
23
24
25
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28
29
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60
1
parts per million (ppm) relative to tetramethylsilane (TMS). H and 13C NMR spectra were
recorded on Bruker AVANCE III HD 400 or Bruker AVANCE III HD 500 spectrometers
1
(Bruker Daltonik, Bremen, Germany) equipped with BBFO probes and operating at H
resonance frequencies of 400.13 and 500.13 MHz, respectively, or using Varian UnityPlus 300
spectrometer (Varian Inc., Palo Alto, CA, USA). Highꢀresolution mass spectra were measured
using a Bruker micrOTOF/Q2 mass spectrometer in ESI ionization. Compounds 1, 44, 47ꢀ50,
52ꢀ63 were purchased from ChemDiv with >95% purity. All synthetic compounds had >95%
purity as determined by highꢀperformance liquid chromatography.
General procedure for syntheses of the 1,2,4ꢀtriazolo[1,5ꢀa]pyrimidine analogs.
The 1,2,4ꢀtriazolo[1,5ꢀa]pyrimidine analogs were synthesized by using a modification of a
known method.26 Equimolar mixtures of compounds I, II, and III (0.80ꢀ2.0 mmol), were heated
in 0.30ꢀ0.50 mL of DMF within the temperature range of 130ꢀ160 °C for 15ꢀ20 min. After
cooling, acetone (10ꢀ25 mL) was added to the solution. The mixture was allowed to stand for a
minimum of 2 h or overnight (depending on the nature of the derivative), at which time the
product precipitates. The precipitate was filtered to obtain the desired product in the range of 6.0ꢀ
82% of yield. Spectral data for the six active compounds (1, 10, 13, 16, 83, and 84) are given
below. The spectral data of the remaining 63 compounds are given in Supporting Information.
Compounds III were synthesized by a modification of a known method.32 3ꢀAminoꢀ5ꢀthiolꢀ
1,2,4ꢀtriazole (6.0 mmol) was stirred in anhydrous DMF (10 mL) for 5 min. Triethylamine (7.0
mmol) was added dropwise to this solution, and the mixture was stirred under a nitrogen
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atmosphere at room temperature for 15 min. Alkyl bromide (7.0 mmol) was added to the
solution, followed by stirring under a nitrogen atmosphere at room temperature for 3 h. The
solvent was evaporated to dryness, and the resulting residue was partitioned between ethyl
acetate (30 mL) and water (20 mL). The organic layer was washed with brine (10 mL), dried
over anhydrous MgSO4, and it was concentrated under reduced pressure. The crude product was
typically purified by column chromatography with hexane/ethyl acetate.
10
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2ꢀ(Benzylthio)ꢀ7ꢀ(4ꢀisopropylphenyl)ꢀ5ꢀmethylꢀ4,7ꢀdihydro[1,2,4]triazolo[1,5ꢀ
a]pyrimidineꢀ6ꢀcarboxamide (1). Compound 1 was prepared from 4ꢀisopropylꢀbenzaldehyde
(0.30 g, 2.0 mmol), acetoacetamide (0.20 g, 2.0 mmol) and 3ꢀaminoꢀ5ꢀ(benzylthio)ꢀ1,2,4ꢀtriazole
1
(0.41 g, 2.0 mmol) in 44% yield as a white solid (0.37 g). H NMR (DMSOꢀd6, 400 MHz): δ
10.09 (s, 1H, NH), 7.29−7.12 (m, 10H, 9ArH + NH), 6.96 (s, 1H, NH), 6.31 (s, 1H, CHN), 4.24
(A of AB, 1H, J = 13.4 Hz, one of SCH2), 4.16 (B of AB, 1H, J = 13.4 Hz, one of SCH2), 2.86
(m, 1H, CH(CH3)2), 2.17 (s, 1H, =C−CH3), 1.18 (d, J = 7.1 Hz, 6H, CH(CH3)2). 13C NMR
(DMSOꢀd6, 75 MHz): δ 167.8, 157.2, 148.5, 148.1, 138.2, 138.1, 136.0, 128.8, 128.3, 127.2,
127.0, 126.4, 103.4, 59.7, 34.8, 33.2, 23.9, 17.4. HRMS (ESI) m/z: 442.1672 calcd for
C23H25N5NaOS [M+Na]+; 442.1697 obsd
2ꢀ(Benzylthio)ꢀ7ꢀ(4ꢀisopropoxyphenyl)ꢀ5ꢀmethylꢀ4,7ꢀdihydroꢀ[1,2,4]triazolo[1,5ꢀ
a]pyrimidineꢀ6ꢀcarboxamide (10). Compound 10 was prepared from 4ꢀisopropoxyꢀ
benzaldehyde (0.16 g, 1.0 mmol), acetoacetamide (0.10 g, 1.0 mmol) and 3ꢀaminoꢀ5ꢀ
1
(benzylthio)ꢀ1,2,4ꢀtriazole (0.21 g, 1.0 mmol) in 80% yield as a white solid (0.35 g). H NMR
(DMSOꢀd6, 400 MHz): δ 10.08 (s, 1H, NH), 7.41−7.18 (m, 6H, 5ArH + NH), 7.13 (d, 2H, J =
8.7 Hz, ArH), 6.97 (s, 1H, NH), 6.85 (d, 2H, J = 8.7 Hz, ArH), 6.28 (s, 1H, CHN), 4.56 (m, 1H,
OCH(CH3)2), 4.23 (A of AB, 1H, J = 13.2 Hz, one of SCH2), 4.15 (B of AB, 1H, J = 13.2 Hz,
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one of SCH2), 2.17 (s, 3H, =C−CH3), 1.25 (d, 6H, J = 5.9 Hz, OCH(CH3)2). 13C NMR (DMSOꢀ
5
6
7
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d6, 125 MHz): δ 167.7, 157.3, 157.0, 148.3, 138.1, 135.8, 132.5, 128.7, 128.5, 128.2, 127.0,
115.1, 103.4, 69.1, 59.4, 34.8, 21.8, 17.3. HRMS (ESI) m/z: 458.1621 calcd for C23H25N5NaO2S
[M+Na]+; 458.1644 obsd.
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2ꢀ(Benzylthio)ꢀ7ꢀ(4ꢀethylthiophenyl]ꢀ5ꢀmethylꢀ4,7ꢀdihydro[1,2,4]triazolo[1,5ꢀ
a]pyrimidineꢀ6ꢀcarboxamide (13). Compound 13 was prepared from 4ꢀmethylthioꢀ
benzaldehyde (0.20 g, 1.2 mmol), acetoacetamide (0.12 g, 1.2 mmol) and 3ꢀaminoꢀ5ꢀ
1
(benzylthio)ꢀ1,2,4ꢀtriazole (0.25 g, 1.2 mmol) in 79% yield as a lightꢀyellow solid (0.42 g). H
NMR (DMSOꢀd6, 400 MHz): δ 10.13 (s, 1H, NH), 7.27−7.15 (m, 10H, 9ArH + NH), 6.99 (s, 1H,
NH), 6.31 (s, 1H, CHN), 4.24 (A of AB, 1H, J = 13.4 Hz, one of SCH2Ph), 4.16 (B of AB, 1H, J
= 13.4 Hz, one of SCH2Ph), 2.97 (q, 2H, J = 7.3 Hz, SCH2CH3), 2.17 (s, 3H, =C−CH3), 1.23 (t,
3H, J = 7.3 Hz, SCH2CH3). 13C NMR (DMSOꢀd6, 100 MHz): δ 167.5, 157.2, 148.4, 138.0, 137.8,
136.3, 136.0, 128.6, 128.1, 127.8, 127.4, 126.9, 103.1, 59.5, 34.7, 25.8, 17.3, 14.1. HRMS (ESI)
m/z: 460.1236 calcd for C22H23N5NaOS2 [M+Na]+; 460.1260 obsd.
2ꢀ(Benzylthio)ꢀ7ꢀ(4ꢀdimethylaminophenyl)ꢀ5ꢀmethylꢀ4,7ꢀdihydro[1,2,4]triazolo[1,5ꢀ
a]pyrimidineꢀ6ꢀcarboxamide (16). Compound 16 was prepared from 4ꢀdimethylaminoꢀ
benzaldehyde (0.22 g, 1.5 mmol), acetoacetamide (0.15 g, 1.5 mmol) and 3ꢀaminoꢀ5ꢀ
1
(benzylthio)ꢀ1,2,4ꢀtriazole (0.31 g, 1.5 mmol) in 33% yield as an offꢀwhite solid (0.21 g) H
NMR (DMSOꢀd6, 300 MHz): δ 10.02 (s, 1H, NH), 7.30−7.17 (m, 6H, 5ArH + NH), 7.05 (d, 2H,
J = 8.6 Hz, ArH), 6.94 (s, 1H, NH), 6.65 (d, 2H, J = 8.6 Hz, ArH), 6.21 (s, 1H, CHN), 4.23 (A of
AB, 1H, J = 13.2 Hz, one of SCH2), 4.15 (B of AB, 1H, J = 13.2 Hz, one of SCH2), 2.87 (s, 6H,
13
N(CH3)2), 2.16 (s, 3H, =C−CH3). C NMR (DMSOꢀd6, 75 MHz): δ 167.8, 156.8, 150.2, 148.3,
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138.1, 135.6, 128.7, 128.2, 128.0, 127.0, 112.0, 103.6, 59.5, 40.1, 34.8, 17.3. HRMS (ESI) m/z:
421.1805 calcd for C22H25N6OS [M+H]+; 421.1808 obsd.
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2ꢀ(4ꢀNitrobenzylthio)ꢀ7ꢀ(4ꢀdimethylaminophenyl)ꢀ5ꢀmethylꢀ4,7ꢀ
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dihydro[1,2,4]triazolo[1,5ꢀa]pyrimidineꢀ6ꢀcarboxamide (83). Compound 83 was prepared
from 4ꢀdimethylaminoꢀbenzaldehyde (0.22 g, 1.5 mmol), acetoacetamide (0.15 g, 1.5 mmol) and
3ꢀaminoꢀ5ꢀ[4ꢀnitro(benzylthio)]ꢀ1,2,4ꢀtriazole (0.38 g, 1.5 mmol) in 20% yield as a lightꢀyellow
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solid (0.14 g). H NMR (DMSOꢀd6, 400 MHz): δ 10.00, (s, 1H, NH), 7.99(d, 2H, J = 8.7 Hz,
ArH), 7.47 (d, 2H, J = 8.7 Hz, ArH), 7.16 (s, 1H, NH), 7.04 (d, 2H, J = 8.7 Hz, ArH), 6.93 (s, 1H,
NH), 6.64 (d, 2H, J = 8.7 Hz, ArH), 6.20 (s, 1H, CHN), 4.34 (A of AB, 1H, J = 14.0 Hz, one of
SCH2), 4.23 (B of AB, 1H, J = 14.0 Hz, one of SCH2), 2.89 (s, 6H, N(CH3)2), 2.16 (s, 3H,
13
=C−CH3). C NMR (DMSOꢀd6, 100 MHz): δ 167.7, 155.9, 150.1, 148.4, 146.7, 146.2, 135.5,
129.9, 128.0, 123.1, 111.8, 103.5, 59.5, 39.9, 33.7, 17.2. HRMS (ESI) m/z: 466.1656 calcd for
C22H24N7O3S [M+H]+; 466.1631 obsd.
2ꢀ(4ꢀCyanobenzylthio)ꢀ7ꢀ(4ꢀdimethylaminophenyl)ꢀ5ꢀmethylꢀ4,7ꢀdihydroꢀ
[1,2,4]triazolo[1,5ꢀa]pyrimidineꢀ6ꢀcarboxamide (84). Compound 84 was prepared from 4ꢀ
dimethylaminoꢀbenzaldehyde (0.12 g, 0.80 mmol), acetoacetamide (0.08 g, 0.80 mmol) and 3ꢀ
aminoꢀ5ꢀ(4ꢀcyanobenzylthio)ꢀ1,2,4ꢀtriazole (0.19 g, 0.80 mmol) in 15% yield as a yellow solid
1
(53 mg). H NMR (DMSOꢀd6, 400 MHz): δ 9.99 (s, 1H, NH), 7.62 (d, 2H, J = 8.7 Hz, ArH),
7.43 (d, 2H, J = 8.7 Hz, ArH), 7.15 (s, 1H, J = 8.7 Hz, NH), 7.03 (d, 2H, J = 8.7 Hz, ArH), 6.92
(s, 1H, NH), 6.65 (d, 2H, J = 8.7 Hz, ArH), 6.19 (s, 1H, CHN), 4.28 (A of AB, 1H, J = 13.8 Hz,
one of SCH2), 4.20 (B of AB, 1H, J = 13.8 Hz, one of SCH2), 2.89 (s, 6H, N(CH3)2), 2.16 (s, 3H,
=C−CH3). 13C NMR (DMSOꢀd6, 75 MHz): δ 167.8, 156.1, 150.2, 148.4, 144.5, 135.6, 132.0,
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129.8, 128.14, 128.07, 118.8, 112.0, 109.6, 103.6, 59.6, 40.1, 34.2, 17.3. HRMS (ESI) m/z:
446.1758 calcd for C23H24N7OS [M+H]+; 446.1752 obsd.
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Bacterial strains. S. aureus ATCC 29213 , K. pneumonia ATCC 700603, A. baumanii ATCC
17961, P. aeruginosa ATCC 27853, E. aerogenes ATCC 35029, E. coli ATCC 25922, E.
faecium NCTC 7171 (ATCC 19734), E. faecium ATCC 700221, and E. faecium ATCC BAA
2127 were used for the initial screen and were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, U.S.A.). Strain E. faecium 119ꢀ39A was collected from
Wayne State University School of Medicine. The strains E. faecium C9, C38, and C68 were
generously supplied by Dr. Robert A. Bonomo.
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Determination of Minimalꢀinhibitory concentrations (MICs) and MinimalꢀBactericidal
Concentraions (MBCs). The MIC assays of each compound were conducted in triplicate in 96ꢀ
well plates with cationꢀadjusted Mueller Hinton II Broth (MH) according to broth microdilution
method described by the Clinical and Laboratory Standards Institute.33 Bacteria were grown to
OD600 of approximately 0.60ꢀ0.80, then the suspension was diluted 100ꢀfold with fresh MHꢀ
medium. The final bacterial innoculum was 5 × 105 CFU/mL. Each compound was prepared
from a 5 mg/mL DMSO stock solution. The stock solution was diluted with MHꢀmedium to give
various concentrations of antibiotics in a manner that the final concentration of DMSO was kept
to 2% (v/v) with a supplement of 0.25% (v/v) glacial acetic acid. After the plates were incubated
at 35 °C for 16ꢀ20 h, MICs were read. All MIC determinations were performed in triplicate.
Determination of MBCs was performed by a reported method.34 E. faecium NCTC 7171 was
used for the determination of MBCs.
In vitro metabolic stability. Pooled human liver S9 (1 mg/mL, BD Biosciences, Woburn,
MA), NADPH (1 mM), and a given synthetic compound (10 ꢀM) in sodium phosphate buffer
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(PBS, 0.20 M sodium phosphate, 0.15 M NaCl, pH 7.4) were incubated at 37 °C for 60 min.
Aliquots were taken at each of six time points, and the reaction was terminated by addition of
two volumes of acetonitrile containing the internal standard (5 ꢀM of 24). The mixture was
centrifuged (20 min, 20,000 g) and the supernatant was analyzed by ultraꢀperformance liquid
chromatography (UPLC) with multiple reaction monitoring (MRM). Halfꢀlives of the
compounds in human liver S9 were calculated based on the firstꢀorder rate constants that were
measured. Intrinsic clearance was calculated by the following equation:35
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CLint = (0.693/in vitro t1/2) × (mL incubation /mg microsomal protein) × (45 mg microsomal
protein/gram liver) × (20 g liver/kg body weight)
The metabolic stability for each compound was determined in duplicates.
Determination of plasmaꢀprotein binding. The plasmaꢀprotein binding of compounds 1, 10,
13, 16, 83, and 84 was determined in a rapid equilibrium dialysis device (Pierce Biotechnology,
ThermoFisher Scientific, Waltham, MA). Human plasma was thawed and centrifuged at 1,200 g
for 10 min to remove any particulates. The compounds were dissolved in DMSO and incubated
in human plasma at a final concentration of 10 ꢀM and 0.1% DMSO. A 200ꢀꢀL aliquot was
added into the sample chamber and 350 ꢀL of 0.1 M sodium phosphate buffer, pH 7.4,
containing 0.15 mM NaCl was added into the adjacent chamber. The compounds were dialyzed
at 37 °C in an orbital shaker overnight. A 150ꢀꢀLꢀaliquot from the buffer side was mixed with
300 ꢀL internal standard solution (24 with final concentration of 1 ꢁM in acetonitrile), and an
equivalent sample volume from the plasma side was also mixed with 300 ꢀL internal standard
solution. The solutions were vortexed and centrifuged (20 min, 20,000 g). A 400ꢀꢀL aliquot of
the supernatant was concentrated to dryness, and then dissolved in 120 ꢀL of acetonitrile,
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followed by centrifugation. A 100ꢀꢀL aliquot of the supernatant was analyzed by UPLCꢀMRM.
The plasmaꢀprotein binding (B%) was calculated according to the following equation:
B% = (CpꢀCf)/Cp × 100, where Cp and Cf are the total plasma concentration and the free
concentration of the compound.
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Macromolecular Synthesis Assays. Macromolecular synthesis assays were performed
according to a previously reported method.36 E. faecium NCTC 7171 was grown in brainꢀheart
infusion culture at 37 °C until earlyꢀlog phase was reached. Radiolabeled precursor (15 ꢀL, 1
ꢀCi/mL of [methylꢀ3H]ꢀthymidine, [5,6ꢀ3H]ꢀuridine, ꢀ[4,5ꢀ3H]ꢀleucine, or ꢀ[2,3ꢀ3H]ꢀalanine)
L D
was added into each Falcon tube of cell culture, followed by incubation at 37 °C for 5 min. The
radiolabeled precursors were purchased from Perkin Elmer (Waltham, MA, USA). Negative
control (DMSO) and positive controls (ciprofloxacin, rifampicin, tetracycline, and meropenem),
as well as compound 16 (8 ꢀg/mL) were added into each tube and allowed to growth for an
additional 2 h. The positive controls were purchased from Sigma Aldrich (SigmaꢀAldrich, MO,
USA). Samples (650 ꢀL) were collected every 20 min and were mixed with 650 ꢀL of 20% iceꢀ
cold trichoroacetic acid (TCA), and allowed to sit on ice for 1 h. The sample (1.3 mL) in each
case was transferred to a glass microfiber filter (GF/A grade, whatman), which was washed with
5% TCA (2×) and with 95% ethanol (2×), before letting it dry. The microfiber filter was placed
in 20ꢀmL scintillation vials with 5 mL of scintillation cocktail for radioactive measurement.
Scintillation vials were counted using a Beckman LSꢀ100 liquid scintillation counter (Beckman
Coulter, Inc., Brea, CA, USA). Duplicates aliquots were taken at each time point for
measurements.
In Vitro Transcription Assay. In vitro transcription and the tandem transcription/translation
assays were performed using a previous reported method.36
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High performance liquid chromatography (HPLC). The HPLC instrument consisted of a
Perkin Elmer series 200 autosampler, series 200 UV/VIS detector, series 200 pump, using
TotalChrom Navigator software (PerkinElmer, Shelton, CT USA). The HPLC conditions
consisted of isocratic elution on a ZORBAX RxꢀC8 5 µm, 4.6×250 mm column (Agilent
Technologies , Santa Clara, CA, USA) at a flow rate of 1 mL/min with 50% A/50% B or 60%
A/40% B for 15 min (A = 0.1% trifluoroacetic acid/water; B = 0.1% trifluoroacetic
acid/acetonitrile). Effluent was detected at 254 nm. All synthetic compounds were ≥95% pure.
Mass spectrometry and sample analysis. Mass spectra were acquired on a Waters Acquity
UPLC Binary Solvent Manager (Waters Corporation, Milford, Massachusetts, USA), using
MASSLYNX V4.1 software. The instrument consists of a Sample Manager, PDA Detector, and
Triple Quadrupole Detector. The experiments were performed in the ESI positive mode with
MRM. The parameters of capillary voltage, cone voltage, extractor voltage, RF lens voltage were
set at 3.6 kV, 30 V, 3 V, 0.3 V, respectively. The source temperature was 150 °C, desolvation
temperature was 350 °C. Desolvation gas flow rate (N2) was 650 L/hour and the cone gas (Xe)
flow rate was 10 L/hour. The dwell time was 0.1 s and collision energy was 20 V. Samples were
analyzed by UPLCꢀMRM on a Waters Acquity UltraPerformance Thermo Scientific AcclaimTM
RSLC 120 C18 2.2 ꢁm, 120 Å, 2.1×100 mm column at 40 °C. Compounds 1, 10, and 13 were
eluted at 0.4 mL/min with 80% A/20% B for 2 min, followed by a 8ꢀmin linear gradient to 10%
A/90% B.( A = 0.1% formic acid/water; B = 0.1% formic acid/acetonitrile). For compounds 16,
83, 84 the flow was changed to 0.5 mL/min; the other conditions were same. The transitions
monitored for each compound were: 420 →403 for 1, 437 →378 for 10, 439 →422 for 13, 421
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→404 for 16, 466 →122 for 83, 446 →122 for 84, and 403 →91 for the internal standard 24.
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Purification of PBP5fm. The pbp5 gene corresponding to amino acids 34 to 678 for PBP5
from E. faecium D63r was custom synthesized (GenScript) for optimal expression in E. coli.
This synthetic gene was cloned into plasmid pETꢀ28a(+) with a Hisꢀtag in the Nꢀterminal
position to give the recombinant plasmid pET28ꢀPBP5fm. This construct lacks the signal peptide
for delivery to the periplasm, hence the recombinant protein is expresses in the cytoplasm. For
the enzyme purification, E. coli BL21 (DE3) harboring the pET28ꢀPBP5fm plasmid was grown
at 37 °C in LuriaꢀBertani (LB) supplemented with kanamycin (50 ꢁg/mL) to OD600 of 0.6.
Protein expression was then induced using 0.4 mM IPTG and the culture incubated at 25 °C for
an additional 6 h. The protein was purified using a MacroꢀPrep High Q anion exchange column
(BioRad) equilibrated with 50 mM Tris pH 7.5 and eluted using a gradient from 0ꢀ0.5 M NaCl in
50 mM Tris pH 7.5. The fractions containing PBP5fm were loaded onto a HiTrap Chelating
column (GE Healthcare) and eluted in a 0.5 M imidazole gradient. After removing the imidazole,
protein concentration was determined spectrophotometrically using the predicted extinction
coefficient (ꢂε280 = 57760 Mꢀ1 cmꢀ1) and purity was assessed by resolution on 12% SDSꢀPAGE.
The enzyme was stored at 4 °C in 50 mM Tris pH 7.5, 0.5 M NaCl.
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Binding Assays for PBP2a and PBP5fm. Purification of PBP2a was reported earlier and was
followed in the present study.37 The assay is identical for PBP5fm, so we only explain the
procedure for PBP2a here.
BOCILLIN FL, a fluorescent penicillin (Molecular Probes, Inc., CA), is used in this assay as a
labeling reagent for the active sites of PBP2a.38 A 19ꢀꢁL reaction solution was prepared on ice
by adding 1 ꢁL PBP2a (final concentration 2 ꢀM) into 18 ꢁL of the inhibitor (1 and 83) in 25
mM Hepes, 1 M NaCl (pH 7.0). The mixture was incubated at 37 °C for 10 min. A 1ꢀꢁL
BOCILLIN FL solution was added into the reaction mixture to give the final concentration of 20
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ꢀM, and the mixture was allowed to incubate for an additional 10 min at 37 °C. A total of 15 ꢀL
of a SDS sample buffer was added to the reaction mixture, and it was boiled for 5 min. The
samples at different antibiotic concentrations were loaded onto 12% SDSꢀPAGE and the gel was
developed at 125 V for 120 min. The gels were scanned using Storm840 Fluorimager and the
bands were quantified with IMAGEQUANT 5.2 software. A control sample without inhibitor
was also prepared at the same time.
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The assay for PBP5fm is similar to that for PBP2a. The differences were the final
concentration of PBP5fm (40 ꢀΜ) and BOCILLIN FL (30 ꢀM), and the incubation time was
extended from 10 min to 20 min.
Supporting Information.
Additional experimental information and 1H and 13C NMR spectra of all the other compounds.
This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
mchang@nd.edu or mobashery@nd.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by grant AI90818 from the NIH. H.W. was supported by a
Predoctoral Fellowship from the Eck Institute for Global Health, University of Notre Dame. R.B.
was supported by training grant T32GM075762 and by an individual Ruth L. Kirschstein
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