Novel fluorinated pyrrolomycins as potent anti-staphylococcal biofilm agents: Design, synthesis, pharmacokinetics and antibacterial activities

Article abstract of DOI:10.1016/j.ejmech.2016.08.017

Staphylococcus aureus (SA) is a major cause of hospital- and community-associated bacterial infections in the U.S. and around the world. These infections have become increasingly difficult to treat due to the propensity to develop antibiotic resistance and form biofilm. To date, no antibiofilm agents are available for clinical use. To add to the repertoire of antibiotics for clinical use and to provide novel agents for combating both SA and biofilm infections, we previously reported marinopyrroles as potent anti-SA agents. In this study, we used fragment-based and bioisostere approaches to design and synthesize a series of novel fluorinated pyrrolomycins for the first time, performed analyses of their physicochemical and drug-like properties, and investigated structure activity relationships and pharmacokinetics. These promising fluorinated pyrrolomycins demonstrate potent antibacterial activity against SA with favorable drug-like properties and pharmacokinetic profiles. Importantly, these compounds kill staphylococcal biofilm-associated cells with a lack of mammalian cell cytotoxicity and no occurrence of bacterial resistance. Our novel fluorinated pyrrolomycin 4 has a clogP value of 4.1, an MIC of 73 ng/mL, MBC of 4 μg/mL, kill staphylococcal-associated biofilm at 8 μg/mL, bioavailability of 35%, and the elimination half-life of 6.04 h and 6.75 h by intravenous and oral administration, respectively. This is the first report of comprehensive drug discovery studies on pyrrolomycin-based antibiotics.

Full text of DOI:10.1016/j.ejmech.2016.08.017

Accepted Manuscript
Novel fluorinated pyrrolomycins as potent anti-staphylococcal biofilm agents: Design,
synthesis, pharmacokinetics and antibacterial activities
Zunhua Yang, Yan Liu, Jongsam Ahn, Zhen Qiao, Jennifer L. Endres, Nagsen
Gautam, Yunlong Huang, Jerry Li, Jialin Zheng, Yazen Alnouti, Kenneth W. Bayles,
Rongshi Li
PII:
S0223-5234(16)30660-2
10.1016/j.ejmech.2016.08.017
EJMECH 8813
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 June 2016
Revised Date: 20 July 2016
Accepted Date: 9 August 2016
Please cite this article as: Z. Yang, Y. Liu, J. Ahn, Z. Qiao, J.L. Endres, N. Gautam, Y. Huang, J. Li, J.
Zheng, Y. Alnouti, K.W. Bayles, R. Li, Novel fluorinated pyrrolomycins as potent anti-staphylococcal
biofilm agents: Design, synthesis, pharmacokinetics and antibacterial activities, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.017.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel fluorinated pyrrolomycins as potent anti-staphylococcal biofilm agents with high
ligand efficiency, a lack of mammalian cell cytotoxicity, no occurrence of bacterial
resistance, favorable physicochemical and drug-like properties, and pharmacokinetic
profiles.

ACCEPTED MANUSCRIPT
Novel Fluorinated Pyrrolomycins as Potent Anti-Staphylococcal Biofilm Agents: Design,
Synthesis, Pharmacokinetics and Antibacterial Activities
a,1
a
b
a
b
a
Zunhua Yang, Yan Liu, Jongsam Ahn, Zhen Qiao, Jennifer L. Endres, Nagsen Gautam,
c
a,2
b,c
a
b,d
Yunlong Huang, Jerry Li, Jialin Zheng, Yazen Alnouti, Kenneth, W. Bayles, Rongshi
a,d,e,
Li
*
a
UNMC Center for Drug Discovery and Department of Pharmaceutical Sciences
b
Department of Pathology and Microbiology
c
Department of Pharmacology and Experimental Neuroscience
d
Center for Staphylococcal Research
e
Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 986125
Nebraska Medical Center, Omaha, NE 68198, United States
ABSTRACT
Staphylococcus aureus (SA) is a major cause of hospital- and community-associated bacterial
infections in the U.S. and around the world. These infections have become increasingly difficult
to treat due to the propensity to develop antibiotic resistance and form biofilm. To date, no
antibiofilm agents are available for clinical use. To add to the repertoire of antibiotics for clinical
use and to provide novel agents for combating both SA and biofilm infections, we previously
reported marinopyrroles as potent anti-SA agents. In this study, we used fragment-based and
bioisostere approaches to design and synthesize a series of novel fluorinated pyrrolomycins for
the first time, performed analyses of their physicochemical and drug-like properties, and
investigated structure activity relationships and pharmacokinetics. These promising fluorinated
1

ACCEPTED MANUSCRIPT
pyrrolomycins demonstrate potent antibacterial activity against SA with favorable drug-like
properties and pharmacokinetic profiles. Importantly, these compounds kill staphylococcal
biofilm-associated cells with a lack of mammalian cell cytotoxicity and no occurrence of
bacterial resistance. Our novel fluorinated pyrrolomycin 4 has a clogP value of 4.1, an MIC of
7
3 ng/mL, MBC of 4 µg/mL, kill staphylococcal-associated biofilm at 8 µg/mL, bioavailability
of 35%, and the elimination half-life of 6.04 hours and 6.75 hours by intravenous and oral
administration, respectively. This is the first report of comprehensive drug discovery studies on
pyrrolomycin-based antibiotics.
Keywords
Antibiofilm, Antibiotic Drug Discovery, Pyrrolomycin, SAR Optimization, Staphylococcus
aureus
1
. INTRODUCTION
Staphylococcus aureus (SA), including methicillin-resistant SA (MRSA), is one of the
most common causes of infection in the U.S., attributable in large part to its propensity to
become multidrug-resistant [1, 2]. The Infectious Disease Society of America has classified it as
an “ESKAPE” pathogen that can readily develop resistance to the biocidal action of antibiotics
[3, 4]. Currently, glycopeptides, such as vancomycin [5], are the most common treatment [6].
However, its overuse has resulted in an increase in vancomycin-resistant SA strains [1, 7-9].
Further, except for the addition of linezolid [10] in 2000, daptomycin [11] in 2003, and the
FDA’s recent approval of ceftaroline [12], tedizolid [13], and dalbavancin [14], options to treat
SA are limited. Additionally, although these recently approved and late-stage antibiotics in
2

ACCEPTED MANUSCRIPT
development may prove invaluable in combating SA resistance, these bacteria will almost
inevitably develop resistance to new antibacterial agents introduced to the clinic [15]. For
instance, reports of resistance to linezolid [16, 17] and daptomycin [18, 19] have quickly
emerged shortly after their introduction. In essence, antibiotic resistance is occurring faster than
new compounds can be introduced into clinical practice [20, 21]. Compounding this problem is
the propensity of SA to form biofilms [22], which are a leading cause of chronic and medical
device associated infections [23] and are tolerant to most currently available antibiotics [24]. To
date, no antibiofilm agents are available for clinical use [25]. Thus, new drug leads to combat
biofilm-related infections are urgently needed.
2
. RESULTS AND DISCUSSION
2
.1. Design of novel fluorinated pyrrolomycins that have favorable physicochemical and drug-
like properties
We previously designed, synthesized and reported “nonsymmetrical” marinopyrrole
derivatives 1 and 2 as potent anti-MRSA agents [26-28]. However, physicochemical properties
of these marinopyrroles are not optimal [28] because their clogP values are beyond the desired
range of therapeutic drugs formulated by Lipinski’s rule of five (RO5) [29]. For example, clogP
values of 1 and 2 are 6.5 and 6.1, respectively (Table 1). In order to improve their
physicochemical and drug-like properties, we first dissected the bis-pyrrole marinopyrroles into
their truncated monomer (fragment derivatives of marinopyrroles by breaking the 1', 3- N-C
bond as shown in Table 1). Then we introduced fluorine atoms to both aromatic rings of
pyrrolomycin (Li and Liu, U.S. Patent Application 62/192,192; Li, U.S. Patent Applications
6
2/219,289 and 62/299,588; Li, Liu and Bayles, U.S. Patent Application 62/309,685).
3

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Investigation of both natural and synthetic pyrrolomycins has revealed that some of
pyrrolomycin derivatives like 3 (Table 1) have anti-staphylococcal biofilm activities [30, 31].
Like marrinopyrroles 1 and 2, pyrrolomycin 3 reported in the literature [31] also has a clogP
value of 6.0. After analysis of the pyrrolomycin structures in the literature, we were surprised to
find that there were no fluorine analogues of pyrrolomycins either in the ortho- and para-
positions of the phenol or in the pyrrole ring ever reported, and the efforts toward optimization of
structure-activity relationships (SARs) were limited to substitutions with only chlorine and
bromine atoms in both pyrrole and benzene rings [32].
Table 1. Design of marinopyrroles and novel pyrrolomycins.
Cl
HN
Z
O
Cl
O
W
O
HO
N
3
1
X1
Fragment & Bioisostere
Approaches
O
Y1
Y2
W
'
O
N
X1
X2
X3
Y1
Y3
X2
X3
Y2
Marinopyrroles Y3
Pyrrolomycins
a
b
ID
1
2
3
4
5
6
7
8
X
1
X
2
X
H
H
Br
H
H
Br
H
H
3
Y
H
H
Br
Cl
Cl
Cl
Cl
F
F
F
F
F
1
Y
2
Y
H
F
Br
Cl
Cl
Cl
Cl
F
F
F
F
F
3
W
H
H
H
H
H
H
H
Me
H
Me
Me
H
Z
-
-
clogP
Cl
Cl
Br
F
Br
F
H
H
H
Br
Br
Br
Br
H
Cl
Cl
Br
Br
Br
Br
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
6.5
6.1
6.0
4.1
4.7
4.9
3.4
2.1
2.7
4.1
4.3
5.0
5.5
4.2
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
9
H
1
0
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
1
1
1
1
2
3
4
F
Cl
F
Cl
H
H
1
a
b
Z group for marrinopyrroles 1 and 2 is a second pyrrolomycin core; Calculated using
ChemAxon MarvinSketch version 15.7.6.0, Hungary.
4

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To test the fluorine effects on both clogP and anti-SA activity with substitutions in the 2-
position (X ) of the pyrrole ring and in the ortho- and para-positions (Y and Y ) of the phenol
1
1
3
ring, we designed a series of novel pyrrolomycins except for a known compound 7 [33] as shown
in Table 1. We then investigated the role of the fluorine atoms on the physicochemical and drug-
like properties of these derivatives, specifically by lowering the clogP values to the desired
range. Indeed, our introduction of fluorine atoms to both aromatic rings in pyrrolomycins lowers
the clogP values from 6.0 (3) to the range 2.1–5.5 (4, 6, 8–13, Table 1). For example, compound
4
has a clogP value of 4.1, 0.6 log-unit lower than 5 after replacing a bromine with a fluorine
atom at the X position of the pyrrole. Similar results were observed with compounds 7 and 9
0.7 log-unit reduction by replacing chlorine with fluorine atoms in the ortho- and para-position
of the phenol ring, Y and Y ), and compounds 6 and 13 (0.6 log-unit reduction by replacing a
1
(
1
3
bromine with a fluorine atom in the X position). Compounds 4, 5 and 14 were designed
1
specifically to study the fluorine effects on anti-staphylococcal activity in the 2-position of the
pyrrole ring (see Table 2 in section 2.3 for details). Compounds 4 and 6 were designed to see if a
3
bromine substitution at X on the pyrrole ring is necessary. Compound 7 was designed to
determine if substitutions with fluorine or bromine atoms on the pyrrole ring are essential for
activity by comparing its anti-staphylococcal activity with those of compounds 4–6. Also,
compounds 7 and 9 were designed to see if the fluorine atoms have any advantages to their
chlorine counterpart in the benzene ring (Y and Y ) without substitutions in the pyrrole ring.
1
3
Finally, compounds 10–13 were designed to investigate the importance of substitutions W and Z.
.2. Chemistry
,5-Difluoro-2-hydroxybenzoic acid (15) was synthesized in 95% yield from 2,3,5-
2
3
trifluorobenzoic acid (Scheme 1) using a published protocol [34]. Acylation of pyrrole with the
5

ACCEPTED MANUSCRIPT
ester 16, obtained from methylation of 15, furnished the fluoro-pyrrolomycin cores 8 and 9 for
the first time with 58% and 11% yields, respectively. Compound 9 was formed as a byproduct,
due to demethylation of 8 by MeMgI present in the reaction mixture. Bromination of 8 using N-
bromosuccinimide (NBS) yielded 10 in 70% yield. N-methylation on pyrrole NH furnished 11 in
8
4% yield. O-demethylation of 10 with boron tribromide (BBr ) and 11 with aluminum chloride
3
(
AlCl ) furnished 13 and 12 with 85% and 80% yields, respectively.
3
Scheme 1. Synthesis of novel pyrrolomycin (I).
Similarly, the ester 18 was obtained from dichlorosalicylic acid 17 in 95% yield (Scheme
2
). Acylation of pyrrole with the ester 18 furnished 7 and 19 with 22% and 25% yields,
respectively. Similar to 8 and 9 in Scheme 1, demethylation of 19 by MeMgI yielded 7 as a
byproduct. Bromination of 7 using three-equivalent or one-equivalent of NBS furnished 5 or 14
with 29% and 40% yields, respectively. Further, fluorination of pyrrole 19 yielded a complex
mixture that was used as crude intermediates without purification. Fluorination with xenon
2 3
difluoride (XeF ), bromination with NBS, demethylation by AlCl followed by HPLC
purification furnished two novel pyrrolomycins 6 and 4 for the first time with unoptimized 7%
and 1% yields, respectively, in three steps from 19. Reaction optimization is ongoing and the
results will be reported in due course.
6

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Scheme 2. Synthesis of novel pyrrolomycin (II).
.3. Antibacterial activity, ligand efficiency, mammalian cell cytotoxicity and bacterial
resistance
As shown in Table 2, all pyrrolomycins designed in Table 1 were evaluated for their
ability to inhibit growth of a clinical MRSA isolate, USA300 JE2, with MIC values ranged from
.073 to 4.0 µg/mL (highest concentration tested). The ligand efficiency (LE) was calculated to
measure the binding energy per non-hydrogen atom of a ligand [35, 36]. The LE values of 4 and
are 0.57 and 0.54, respectively. These LE values double that of their dimer derivative
2
0
5
counterpart marinopyrroles 1 and 2 (LE = 0.29 and 0.28, respectively in Table 1), suggesting that
the non-hydrogen atoms of these pyrrolomycins are much more efficient binders than those of
marinopyrroles. Mammalian cell cytotoxicity was determined for all active pyrrolomycins (MIC
values < 0.20 µg/mL) and marinopyrroles 1 and 2 using human Hela cells. Selectivity Index (SI)
was defined as a ratio of IC of human Hela cell cytotoxicity/MIC against SA (in µg/mL).
5
0
Mammalian cell cytotoxicity shows that all active compounds have SI > 229, demonstrating that
they are selective antibacterial agents without toxicity at concentrations several hundred-fold
higher than their MICs. The SI of our novel pyrrolomycins is remarkable because a SI > 200 was
considered a good safety margin for clinical development of the fluoroquinolone antibiotics [37].
7

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An important consideration in the assessment of novel antibiotic compounds is the ease
in which bacteria can develop resistance to the compounds as this could have a dramatic impact
on their therapeutic potential. In addition, a determination of the chromosomal mutations that
accumulate in resistant organisms can provide key insight into the mechanism of action of the
compounds. To date, we have been unable to isolate mutant strains that exhibit high-level
resistance to these pyrrolomycin compounds. We have attempted to identify resistant mutants
using several strategies. First, we have looked for resistant mutants within zones of clearing
within a lawn of S. aureus cells grown on an agar surface on which a pyrrolomycin-soaked disk
had been placed. The zones of clearing examined lacked any resistant mutants. Second, we
attempted to “train” S. aureus cells to become resistant to these compounds by growing them in
liquid culture in the presence of 1x MIC levels of antibiotic. Although some growth was seen in
the presence of these low levels of antibiotic, consistent growth under these conditions was not
observed.
Table 2. Ligand efficiency, antibacterial activity and mammalian cell cytotoxicity.
a
b
c
d
ID
1
MIC
LE
IC50
SI
0.078
0.19
0.29
0.28
0.57
0.54
0.51
>40.9
>52.8
>35.3
>41.4
>43.2
ND
>524
>280
>484
>567
>229
ND
2
4
0.073
0.073
0.19
5
6
e
f
7
>4.0
ND
8
>4.0
>4.0
3.0
ND
ND
ND
ND
ND
0.54
ND
ND
ND
9
ND
ND
1
0
ND
ND
11
>4.0
>4.0
0.17
>4.0
ND
ND
1
1
1
2
3
4
ND
ND
>46.0
ND
>275
ND
8

ACCEPTED MANUSCRIPT
a
MIC: Minimum Inhibitory Concentration of pyrrolomycin in µg/mL against Staphyloccocus
b
aureus USA300 (n ≥ 3) with vancomycin as a positive control (MIC = 2.0 µg/mL); LE: Ligand
Efficiency ≈ –∆G/HAC, defined as the free energy of binding divided by the number of non-
hydrogen atoms (Heavy Atom Count) [35, 36]; IC : Concentration of the pyrrolomycin in
c
50
µg/mL at which the human Hela cell proliferation was inhibited by 50% with respect to the
d
untreated growth control, the highest concentration tested was 100 µM; SI: Selectivity Index
e
was defined as a ratio of IC of human Hela cell cytotoxicity to MIC; Highest concentration
tested was 4 µg/mL; ND: not determined.
5
0
f
2
.4. Structure activity relationship (SAR)
Our novel pyrrolomycins 4 and 5 are equally potent against SA, with MIC values of
.073 µg/mL similar to that of marinopyrrole 1 although the LE values of the former almost
0
twice that of the latter (see Table 2). Compounds 4 and 5 have the same anti-staphylococcal
activity while their structures differ by only one atom (fluorine vs. bromine) at X position.
1
1
Compound 14 (X = H) is at least 54-fold (MIC > 4.0 µg/mL) less active than its fluorine or
bromine congeners 4 and 5, suggesting that X must be either fluoro- or bromo-substituted to
1
maintain antibacterial activity (Table 1 and Table 2). Although there is no obvious fluorine effect
on anti-staphylococcal activity (MIC) observed between compound 4 and 5, they do have
different bactericidal activities as detailed in the bactericidal and anti-staphylococcal biofilm
section 2.5. Compounds 7 and 9 are 21–57-fold less potent than compounds 4–6, further
reinforcing the fact that X
1
and X
2
must be substituted by either fluorine or bromine atom in the
position is
pyrrole ring. Compound 6 is less active than 4, suggesting that substitution at the X
3
not necessary. Compound 10 with methyl substituted at W is 18-fold less potent than 13. Both 11
and 12 are 24-fold less active than 13. These results suggest that neither the pyrrole nitrogen nor
the hydroxyl group in the benzene ring can be substituted to achieve potent anti-SA activity.
2
.5. Bactericidal and anti-staphylococcal biofilm activity
9

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Our minimum bactericidal concentration (MBC) assays, using vancomycin as a positive
control according to a published protocol [38], have confirmed that these pyrrolomycins are
bactericidal. The MBC of pyrrolomycin 4 with a fluorine substitution at the X position is 4.0
1
µg/mL whereas its bromine counterpart 5 kills only 92.5% of bacteria at a concentration of 8.0
µg/mL (Figure 1, MBC of vancomycin control = 8.0 µg/mL, n ≥ 3). This finding supports our
design and rationale for fluorine substitution to improve both the physicochemical properties and
antibacterial activities of these pyrrolomycins. Furthermore, our introduction of fluorine atoms to
these pyrrolomycin compounds not only reduced the clogP values from 4.7 to 4.1 (5 to 4 in the
desired range of RO5), but the fluorine atoms can also serve as electron-withdrawing substituents
that stabilize the pyrroles to avoid potential toxicity [39, 40], and also potentially improve their
pharmacokinetic and pharmacodynamic (PK/PD) profiles [41-43]. More significantly,
fluorinated pyrrolomycin 4 disrupts pre-formed staphylococcal biofilms completely at the
concentration of 8.0 µg/mL in five days as shown in Figure 2. Compound 4 kills 84% biofilms in
2
9
4 hours while vancomycin kills only 11% at 32 µg/mL. Pyrrolomycin 4 continues to achieve
7% biofilm killing at the concentration of 8.0 µg/mL, while vancomycin kills only 39% at 32
µg/mL in 48 hours.
100
Vancomycin
8 µg/mL)
(
4
(4 µg/mL)
50
5
(8 µg/mL)
0
Figure 1. Bactericidal activity of novel pyrrolomycins 4 and 5.
1
0

ACCEPTED MANUSCRIPT
100
Vancomycin
(32 µg/mL)
4
(8 µg/mL)
50
0
Figure 2. Anti-staphylococcal biofilm killing by pyrrolomycin 4.
2
.6. Pharmacokinetics of pyrrolomycin 4
A sensitive and specific LC-MS/MS method has been developed for determination of 4 in
mouse plasma [44-47]. Following intravenous (IV) and oral dosing in mice, the oral
bioavailability of 4 was found to be 35%. The plasma concentrations vs. time profiles after IV
and oral administration of 4 in mice are shown in Figure 3. The compound was rapidly absorbed
with maximum plasma concentration (Cmax) observed after 0.25 h of oral administration.
Maximum plasma concentrations were declined in a biphasic manner with rapid decline showing
distribution phase for up to 4 h, and elimination phase for up to 72 h. Similarly, a biphasic
decline in plasma concentration was observed after intravenous administration. Further, these
plasma concentrations vs. time profiles were used to calculate the following pharmacokinetic
parameters. The elimination half-life was 6.04 hours and 6.75 hours following IV and oral
administration, respectively. The area under the curve (AUC) for the IV dose at 1 mg/kg was 3.8
µg*h/mL and 13.2 µg*h/mL for the oral dose at 10 mg/kg, respectively. After both IV and oral
administration, parent compound 4 was detectable in plasma up to 72 hours with the detection
limit of 0.8 ng/mL.
1
1

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1
0000
000
00
Oral dose 10mg/kg
IV dose 1mg/kg
1
1
10
1
0.1
0
20
40
60
80
Time (h)
Figure 3. Plasma concentration vs. time profile of pyrrolomycin 4.
3
. CONCLUSIONS
We designed, synthesized and investigated a series of novel fluorinated pyrrolomycins as
potent anti-staphylococcal biofilm agents for the first time. Our fragment-based and bioisostere
approaches yielded a series of novel pyrrolomycins that possess high ligand efficiencies, desired
physicochemical and drug-like properties, a lack of mammalian cell cytotoxicity, no occurrence
of bacterial resistance, excellent bactericidal activity, and favorable pharmacokinetic profiles.
The most active fluorinated pyrrolomycin 4 exhibited potent antibacterial activity against SA
with MIC values of 0.073 µg/mL and MBC of 4.0 µg/mL. Significantly, this compound also
exhibited 100% staphylococcal biofilm killing at the concentration of 8.0 µg/mL. Bioavailability
of compound 4 is 35% with the elimination half-life of 6.04 hours and 6.75 hours by intravenous
and oral administration, respectively. These compounds provide promising scaffolds for the
development of new antibiotics useful for the treatment of S. aureus infections, including those
are biofilm associated.
1
2

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4
. EXPERIMENTAL SECTION
4
.1. Synthesis of novel pyrrolomycins
All chemicals and solvents were purchased from commercial suppliers and used without
further purification. Melting points were determined on a Stanford Research Systems EZ-Melt
apparatus and are uncorrected. Flash column chromatography was performed on silica gel 60,
1
13
0
.040–0.063 µm (EMD chemicals, Billerica, MA, USA). H NMR (500 MHz) and C NMR
(125 MHz) spectra were recorded on a Bruker Avance III at ambient temperature. High-
resolution mass spectra were obtained on a Micromass GC-T by electron impact or on a
Micromass Q-TOF Ultima with electrospray ionization. Analytical HPLC was performed on an
Agilent 1260 Infinity with diode array detectors and auto samplers. Preparative HPLC was
performed on a Shimadzu LC-20A series. All target compounds after HPLC purification
(preparative C18 column, 5 µm, 150 x 21.2 mm and analytical C18 column, 5 µm, 150 x 4.6
mm, UV 214 nm, 254 and 280 nm) possessed purities of ≥95%.
4
.1.1. 3,5-Difluoro-2-hydroxybenzoic acid (15)
After a mixture of 2,3,5-trifluorobenzoic acid (5 g, 28.4 mmol), NaOH (4.5 g, 112.5
mmol) and anhydrous DMSO (60 mL) was stirred at 130 ºC for 3 h, the reaction mixture was
poured into ice water (200 mL), acidified to ~pH 1, and extracted with ether (3 x 100 mL). The
organic layer was washed with brine, dried over anhydrous MgSO
4
, filtered and evaporated to
1
give a crude product, 3,5-difluorosalicylic acid 15 as a white solid (4.7 g, 95%). H NMR (500
MHz, CDCl3)
Hz, 1H).
δ 10.22 (s, 1H), 7.41 (ddd, J = 8.3, 3.0, 2.0 Hz, 1H), 7.15 (ddd, J = 10.9, 8.0, 3.0
4
.1.2. Methyl 3,5-difluoro-2-methoxybenzoate (16)
1
3

ACCEPTED MANUSCRIPT
2 3
To a solution of 15 (6.6 g, 37.9 mmol) in acetone (80 mL) were added K CO (15 g,
1
1
08.7 mmol) and dimethyl sulfate (10 mL, 105.6 mmol). The resulting mixture was refluxed for
8 h, cooled to room temperature, and solvent was evaporated. The residue was partitioned
between ether and water. The organic layer was washed with 1N NaOH solution and brine, dried
over anhydrous MgSO , filtered and evaporated to dryness. The crude product was purified by
4
silica gel column chromatography (4% ethyl acetate in hexane) to afford the desired product as a
1
white solid 16 (5.3 g, 69%). H NMR (500 MHz, CDCl
3
)
δ
7.28 (ddd, J = 8.5, 3.0, 2.0 Hz, 1H),
7
.02 (ddd, J = 10.8, 7.9, 3.1 Hz, 1H), 3.94 (d, J = 0.9 Hz, 3H), 3.92 (s, 3H).
4
.1.3. (3,5-Difluoro-2-methoxyphenyl)(1H-pyrrol-2-yl)methanone (8)
A solution of methyl iodide (CH I, 1.24 mL, 19.9 mmol) in ether (10 mL) was added
3
dropwise to a stirred magnesium turning (0.45 g, 18.8 mmol) in ether (5 mL) to maintain a gentle
reflux. After the mixture was stirred for 0.5 h at room temperature, pyrrole (1.50 mL, 20.0 mmol)
was added dropwise to maintain a gentle reflux. The resulting mixture was refluxed for 0.5 h and
cooled to room temperature. To the pyrrylmagnesium iodide was added dropwise a solution of
1
6 (2.0 g, 9.9 mmol) in ether (40 mL). After stirred at room temperature for 2 h, the reaction
mixture was quenched with saturated NH Cl solution and extracted with ether (3 x 100 mL). The
4
organic layer was washed with brine, dried over anhydrous MgSO , filtered and evaporated to
4
dryness. The residue was purified by silica gel column chromatography (9% ethyl acetate in
1
hexane) to afford the desired product 8 as a brown solid (1.36 g, 58%). mp 85.7 – 87.1 ºC. H
NMR (500 MHz, CDCl3) δ 10.65 (s, 1H), 7.22–7.16 (m, 1H), 7.02–6.91 (m, 2H), 6.71–6.66 (m,
1
3
1
3
3
H), 6.32–6.25 (m, 1H), 3.87 (d, J = 1.3 Hz, 3H); C NMR (125 MHz, CDCl ) δ 182.1 (dd, J =
3
.2, 2.0 Hz), 157.3 (dd, J = 245.6, 11.0 Hz), 155.7 (dd, J = 250.8, 11.9 Hz), 142.4 (dd, J = 12.0,
.9 Hz), 135.4 (dd, J = 7.8, 2.5 Hz), 131.7 (s), 127.5 (s), 121.6 (s), 111.6 (s), 111.3 (dd, J = 23.8,
1
4

ACCEPTED MANUSCRIPT
+
3
.7 Hz), 107.0 (dd, J = 26.6, 23.2 Hz), 62.9 (dd, J = 4.6, 0.6 Hz); HRMS (EI-TOF) M calcd for
C H F NO 237.0601, found 237.0603; HPLC purity, 95.6%.
12
9
2
2
4
.1.4. (3,5-difluoro-2-hydroxyphenyl)(1H-pyrrol-2-yl)methanone (9)
After silica gel column purification of the reaction mixture above (4.1.3) and purification
by preparative HPLC, a demethylated byproduct 9 was obtained as a yellow solid (0.25 g, 11%).
1
mp 161.7 – 162.5 ºC. H NMR (500 MHz, CDCl
3
)
δ
11.50 (s, 1H), 9.56 (s, 1H), 7.59–7.51 (m,
1
3
1
1
H), 7.25–7.18 (m, 1H), 7.14–7.06 (m, 2H), 6.47–6.41 (m, 1H); C NMR (125 MHz, CDCl ) δ
3
84.9 (t, J = 2.9 Hz), 153.9 (dd, J = 241.3, 10.4 Hz), 151.8 (dd, J = 251.4, 11.6 Hz), 147.5 (dd, J
=
=
12.6, 3.1 Hz), 129.7 (s), 126.9 (s), 120.6 (dd, J = 7.8, 4.0 Hz), 120.4 (s), 112.5 (s), 111.5 (dd, J
+
23.7, 4.0 Hz), 110.0 (dd, J = 27.1, 21.3 Hz); HRMS (EI-TOF) M calcd for C H F NO
1
1
7
2
2
2
23.0445, found 223.0444; HPLC purity, 100%.
4
.1.5. (3,5-Difluoro-2-methoxyphenyl)(3,4,5-tribromo-1H-pyrrol-2-yl)methanone (10)
To a solution of 8 (100 mg, 0.42 mmol) in acetonitrile (2 mL) was added N-
bromosuccinimide (NBS, 225 mg, 1.27 mmol) at 0 ºC. The resulting mixture was stirred at room
temperature for 20 h and evaporated to dryness. The residue was partitioned between ether and
water. The organic layer was washed with brine, dried over anhydrous MgSO , filtered and
4
evaporated. The residue was purified by silica gel column chromatography (5% ethyl acetate in
1
hexane) to afford 10 as a white solid (140 mg, 70%). mp 178.9 – 179.7 ºC. H NMR (500 MHz,
6
Acetone-d ) δ 12.37 (s, 1H), 7.26 (ddd, J = 11.6, 8.5, 3.0 Hz, 1H), 7.06 (ddd, J = 7.9, 2.8, 1.8 Hz,
1
3
1
1
1
H), 3.83 (d, J = 1.5 Hz, 3H). C NMR (125 MHz, Acetone-d ) δ 180.5 (dd, J = 3.5, 2.1 Hz),
6
58.5 (dd, J = 244.8, 11.1 Hz), 156.0 (dd, J = 250.7, 12.2 Hz), 142.7 (dd, J = 12.0, 3.9 Hz),
35.5 (dd, J = 8.4, 2.9 Hz), 131.2 (s), 111.4 (s), 111.2 (dd, J = 24.3, 3.7 Hz), 108.2 (s), 107.7 (dd,
1
5

ACCEPTED MANUSCRIPT
+
6 3 2 2
J = 27.0, 23.4 Hz), 106.2 (s), 62.6 (d, J = 5.1 Hz); HRMS (EI-TOF) M calcd for C12H Br F NO
4
70.7917, found 470.7919; HPLC purity, 100%.
.1.6. (3,5-Difluoro-2-hydroxyphenyl)(3,4,5-tribromo-1H-pyrrol-2-yl)methanone (13)
To a solution of 10 (60 mg, 0.13 mmol) in CH Cl (3 mL) was added a solution of boron
4
2
2
tribromide (BBr , 0.38 mL, 1M solution in CH Cl ) dropwise at -78 ºC. The resulting mixture
3
2
2
was stirred at this temperature for 2 h, quenched with water and extracted with CH Cl . The
2
2
4
organic layer was washed with brine, dried over anhydrous MgSO , filtered and evaporated to
dryness. The residue was purified by silica gel column chromatography (6% ethyl acetate in
hexane) to afford the desired product as a yellow solid. This compound was purified by
1
preparative HPLC to yield 50 mg (85%): mp 151.8 – 152.7 ºC. H NMR (500 MHz, Acetone-d )
6
1
3
δ
10.98 (br s, 2H, O-H and N-H), 7.38–7.23 (m, 1H, C6-H), 7.19–7.06 (m, 1H, C4-H); C NMR
125 MHz, Acetone-d 182.5 (dd, J = 3.3, 2.3 Hz, C-7), 155.6 (dd, J = 240.1, 10.5 Hz, C-5 or
C3), 152.2 (dd, J = 245.2, 11.8 Hz, C-3 or C5), 142.4 (dd, J = 14.9, 3.1 Hz, C-2), 131.2 (s), 127.5
dd, J = 8.1, 3.0 Hz, C-1), 112.2 (dd, J = 24.2, 3.9 Hz, C-6), 110.5 (s), 108.3 (dd, J = 27.3, 22.5
(
6
) δ
(
1
9
Hz, C-4), 107.6 (s), 106.1 (s); F NMR (470 MHz, Acetone-d ) δ -122.7 (t, J = 8.2 Hz, C5-F), -
6
+
1
4
4
32.8 (d, J = 10.8 Hz, C3-F); HRMS [M+H] calcd for C11
H Br
5 3
F
2
NO
2
457.7838, found
57.7852; HPLC purity, 98.6%.
.1.7. (3,5-Difluoro-2-methoxyphenyl)(3,4,5-tribromo-1-methyl-1H-pyrrol-2-yl)methanone (11)
To a solution of 10 (63 mg, 0.133 mmol) in DMF (1.0 mL) at room temperature were
added K CO (55 mg, 0.40 mmol) and iodomethane (0.083 mL, 1.33 mol). The resulting mixture
2
3
was stirred at room temperature for 3 h, and evaporated to dryness. The residue was partitioned
between ether and water. The organic layer was washed with brine, dried over anhydrous
MgSO , filtered and evaporated to afford 11 as a white solid (55 mg, 84%). This compound was
4
1
6

ACCEPTED MANUSCRIPT
1
purified by preparative HPLC. mp 107.4 – 108.1 ºC. H NMR (500 MHz, CDCl )
δ 6.99 (ddd, J
3
=
11.2, 8.1, 3.0 Hz, 1H), 6.83 (ddd, J = 7.6, 2.9, 1.7 Hz, 1H), 4.01 (s, 3H), 3.85 (d, J = 1.8 Hz,
1
3
3
1
H). C NMR (125 MHz, CDCl ) δ 182.1 (dd, J = 3.5, 2.2 Hz), 157.7 (dd, J = 246.7, 10.9 Hz),
3
55.2 (dd, J = 252.2, 11.7 Hz), 142.4 (dd, J = 11.8, 3.9 Hz), 135.2 (dd, J = 8.0, 3.1 Hz), 130.6
(s), 117.0 (s), 111.0 (dd, J = 23.9, 3.7 Hz), 109.32 (s), 107.7 (dd, J = 26.5, 23.1 Hz), 104.9 (s),
+
6
4
4
2.2 (d, J = 5.7 Hz), 37.6 (s); HRMS (EI-TOF) M calcd for C13
H
8
Br
3
F
2
NO
2
484.8073, found
84.8072; HPLC purity, 100%.
.1.8. (3,5-Difluoro-2-hydroxyphenyl)(3,4,5-tribromo-1-methyl-1H-pyrrol-2-yl)methanone (12)
A mixture of 11 (45 mg, 0.092 mmol) and AlCl (74 mg, 0.55 mmol) in CH Cl (2 mL)
3
2
2
was stirred at room temperature for 2 h to give compound 12 as a yellow solid (35 mg, 80%).
1
This compound was purified by preparative HPLC. mp 156.2 – 157.2 ºC. H NMR (500 MHz,
3
CDCl ) δ 11.01 (s, 1H), 7.25 – 7.21 (m, 1H), 7.16 (ddd, J = 10.6, 8.0, 2.9 Hz, 1H), 3.79 (s, 3H);
1
3
C NMR (125 MHz, CDCl ) δ 188.5 (t, J = 3.0 Hz), 153.6 (dd, J = 241.7, 10.1 Hz), 151.3 (dd, J
3
=
251.4, 11.2 Hz), 147.5 (dd, J = 12.7, 2.9 Hz), 129.6 (s), 120.2 (dd, J = 7.8, 3.8 Hz), 114.5 (s),
1
14.2 (dd, J = 23.8, 4.1 Hz), 111.6 (dd, J = 27.2, 21.2 Hz), 106.1 (s), 104.3 (s), 36.5 (s); HRMS
+
(
EI-TOF) M calcd for C12
H
6
Br
3
F
2
NO
2
470.7917, found 470.7914; HPLC purity, 100%.
4
.1.9. Methyl 3,5-dichloro-2-methoxybenzoate (18)
To a solution of 3,5-dichlorosalicylic acid (17, 20.0 g, 0.097 mol) in DMF (150 mL) on
an ice water bath were added K CO (32.0 g, 0.232 mol) and iodomethane (24.0 mL, 0.384 mol).
2
3
The resulting mixture was stirred at room temperature for 24 h, and evaporated to dryness. The
residue was partitioned between ether and water. The organic layer was washed with 1N NaOH
solution and brine, dried over anhydrous MgSO , filtered and evaporated to afford the desired
4
1
7

ACCEPTED MANUSCRIPT
1
product 18 as a white solid (21.6 g, 95%). H NMR (500 MHz, CDCl )
δ
7.68 (d, J = 2.7 Hz,
3
1
H), 7.54 (d, J = 2.7 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H).
4
.1.10. (3,5-dichloro-2-methoxyphenyl)(1H-pyrrol-2-yl)methanone (19)
3
A solution of CH I (1.33 mL, 21.3 mmol) in ether (10 mL) was added dropwise to a
stirred magnesium turning (0.51 g, 21.2 mmol) in ether (5 mL) to maintain a gentle reflux. After
stirred for 0.5 h at room temperature, pyrrole (1.50 mL, 21.6 mmol) was added dropwise to
maintain a gentle reflux. The resulting mixture was refluxed for 0.5 h and cooled to room
temperature. To the pyrrylmagnesium iodide solution was added dropwise the solution of 18 (2
g, 8.5 mmol) in ether (40 mL). After stirred at room temperature for 2 h, the reaction mixture
was quenched with saturated NH Cl solution and extracted with ether. The organic layer was
4
washed with brine, dried over anhydrous MgSO , filtered and evaporated. The residue was
4
purified by silica gel column chromatography (9% ethyl acetate in hexane) to afford the desired
1
product 19 as a brown solid (0.58 g, 25%). H NMR (500 MHz, CDCl )
δ
9.45 (br s, 1H), 7.51
3
(
d, J = 2.6 Hz, 1H), 7.33 (d, J = 2.5 Hz, 1H), 7.19–7.15 (m, 1H), 6.71–6.67 (m, 1H), 6.37–6.29
m, 1H), 3.85 (s, 3H).
(
4
.1.11. (3,5-Dichloro-2-hydroxyphenyl)(1H-pyrrol-2-yl)methanone (7)
After silica gel column purification of the reaction mixture above (4.1.10), a
1
demethylated byproduct 7 was obtained as a yellow solid (0.5 g, 22%). mp 158.0 – 159.0 ºC. H
NMR (500 MHz, CDCl3)
δ
12.27 (s, 1H), 9.56 (br s, 1H), 7.96 (d, J = 2.5 Hz, 1H), 7.58 (d, J =
+
2
.4 Hz, 1H), 7.26–7.21 (m, 1H), 7.13–7.08 (m, 1H), 6.48–6.42 (m, 1H); HRMS (EI-TOF) M
calcd for C11 Cl NO 254.9854, found 254.9862; HPLC purity, 98.5%.
.1.12. (4,5-Dibromo-1H-pyrrol-2-yl)(3,5-dichloro-2-hydroxyphenyl)methanone (5)
H
7
2
2
4
1
8

ACCEPTED MANUSCRIPT
3
To a stirred solution of 7 (30 mg, 0.12 mmol) in CH CN (1.0 mL) was added a solution
of NBS (63 mg, 0.35 mmol) in CH CN (0.5 mL) dropwise on an ice water bath under argon
3
atmosphere. The resulting mixture was stirred at room temperature for 40 h. The precipitate was
collected by filtration and washed with ice-cold CH CN to give 5 as a yellow solid (15 mg,
3
1
2
1
9%). This compound was purified by preparative HPLC. H NMR (500 MHz, Acetone-d ) δ
6
1
3
2.35 (s, 1H), 11.61 (s, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 7.28 (s, 1H). C
NMR (125 MHz, Acetone-d
6
) δ 184.1 (s), 156.6 (s), 135.3 (s), 132.0 (s), 129.9 (s), 124.6 (s),
+
1
24.3 (s), 123.1 (s), 122.7 (s), 113.4 (s), 102.3 (s); HRMS (EI-TOF) M calcd for
11 5 2 2 2
C H Br Cl NO 410.8064, found 410.8065; HPLC purity, 100%.
4
.1.13. (4-Bromo-1H-pyrrol-2-yl)(3,5-dichloro-2-hydroxyphenyl)methanone (14)
To a stirred solution of 7 (100 mg, 0.4 mmol) in CH CN (5.0 mL) was added a solution
3
of NBS (71.2 mg, 0.4 mmol) in CH CN (1 mL) dropwise on an ice water bath under argon
3
atmosphere. The resulting mixture was stirred at room temperature for 4 h. The precipitate was
collected by filtration and washed with ice-cold CH CN to give 14 as a yellow solid (52.8mg,
3
1
4
0%). This compound was purified by preparative HPLC. mp 222.2–223.1 ºC. H NMR (500
MHz, Acetone-d6) δ 11.89 (s, 1H), 11.70 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.75 (d, J = 2.5 Hz,
1
3
1
1
H), 7.49 (dd, J = 3.1, 1.3 Hz, 1H), 7.25 (m, 1H). C NMR (125 MHz, Acetone-d ) δ 184.9 (s),
6
56.8 (s), 135.1 (s), 130.5 (s), 129.8 (s), 128.3 (s), 124.3 (s), 124.1 (s), 122.5 (s), 122.1 (s), 99.2
+
(
s). HRMS (EI-TOF) M calcd for C11
H
6
BrCl
2
NO
2
332.8959, found 332.8961; HPLC purity:
1
4
00%.
.1.14. (3,4-Dibromo-5-fluoro-1H-pyrrol-2-yl)(3,5-dichloro-2-hydroxyphenyl)methanone (6)
To a stirred solution of 19 (1.0 g, 3.7 mmol) in CH CN (20 mL) on an ice water bath
3
were added XeF (1.0 g, 5.9 mmol) under argon atmosphere. The resulting mixture was heated to
2
1
9

ACCEPTED MANUSCRIPT
3
0 ºC for 48 h, and then evaporated to dryness. The residue was treated with NBS (1.3 g, 7.4
mmol) followed by AlCl (1.98 g, 14.8 mmol). After flash chromatography (silica gel/ 2%–9%
3
ethyl acetate in hexane and purification by preparative HPLC, 111 mg of 6 (7%) was obtained.
1
mp 171.9–172.7 ºC. H NMR (500 MHz, CDCl )
δ
10.51 (s, 1H, O-H), 9.31 (s, 1H, N-H), 7.73
3
1
3
(
d, J = 2.5 Hz, 1H, C4-H), 7.59 (d, J = 2.4 Hz, 1H, C6-H); C NMR (125 MHz, CDCl
3
) δ 184.8
(s, C-7), 154.8 (s, C-2), 148.2 (d, J = 274.6 Hz, C-5’), 135.3 (s, C-6), 130.9 (s, C-4), 124.1 (s),
1
9
1
23.5 (s), 120.6 (s), 119.8 (s), 109.0 (s), 84.6 (d, J = 14.0 Hz, C-4’); F NMR (470 MHz,
+
Acetone-d ) δ -128.94 (s, C5’-F); HRMS (EI-TOF) M calcd for C H Br Cl FNO 428.7970,
6
11
4
2
2
2
found 428.7962; HPLC purity, 97.8%.
4
.1.15. (4-bromo-5-fluoro-1H-pyrrol-2-yl)(3,5-dichloro-2-hydroxyphenyl)methanone (4)
Same as above (4.1.14), after flash chromatography (silica gel/ 2%–9% ethyl acetate in
hexane and purification by preparative HPLC, 13 mg of 4 (1%) was obtained. mp 207.7 – 208.9
1
ºC. H NMR (500 MHz, Acetone-d )
δ
12.23 (s, 1H, O-H), 11.69 (s, 1H, N-H), 7.92 (d, J = 2.5
6
1
3
Hz, 1H, C4-H), 7.71 (d, J = 2.5 Hz, 1H, C6-H), 7.23 (d, J = 4.3 Hz, 1H, C3’-H); C NMR (125
MHz, Acetone-d ) δ 184.2 (d, J = 3.0 Hz, C-7), 156.4 (s, C-2), 150.9 (d, J = 269.4 Hz, C-5’),
6
1
1
35.1 (s, C-6), 129.8 (s, C-4), 124. (s), 124.2 (s), 122.7 (s, C-3’), 122.6 (s), 122.1 (s), 78.5 (d, J =
1
9
6
4.9 Hz, C-4’); F NMR (470 MHz, Acetone-d ) δ -130.40 (d, J = 4.0 Hz, C5’-F); HRMS (EI-
+
TOF) M calcd for C11
5
H BrCl
2
FNO
2
350.8865, found 350.8853; HPLC purity, 100%.
4
.2. Mammalian cell cytotoxicity
HeLa (ATCC, Manassas, VA, USA) cells were cultured in 96-well plates at a density of 2
10 cells/well in Dulbecco’s modified Eagles medium (DMEM, GIBCO Invitrogen Corp,
4
×
Carlsbad, CA) with 10% heat inactivated fetal bovine serum (FBS) (GIBCO) and an antibiotic
mixture containing penicillin and streptomycin (Life Technologies, Grand Island, NY). Cells
2
0

ACCEPTED MANUSCRIPT
were treated with various concentrations of the PL compounds for 24 h in growth medium. Cell
®
viability was determined by a colorimetric CellTiter 96 AQ
One Solution Cell Proliferation
ueous
Assay (Promega, Madison, WI) based on the manufacture’s instruction. Data were recorded at
the absorbance of 490 nm. Each condition was tested in triplicate.
4
.3. Minimum Inhibitory Concentration
Broth microdilution assays were performed as described in the third edition of the ASM
Clinical Microbiology Procedures Handbook. Briefly, serial 2-fold dilutions of pyrrolomycins
were made in Muller Hinton II broth (cation-adjusted)(CAMHB) (Becton, Dickinson and
Company, Sparks, MD), containing 1%–5% DMSO in Costar 96-well microtiter plates (Corning,
Kennebunk, ME). Bacterial cultures were prepared using the direct colony suspension method to
0
.5 McFarland units and each well was inoculated with 10 µL of this suspension. Plates were
incubated statically at 37 ºC for 20–24 h. MIC values were reported as the lowest concentration
of antibiotic in which no growth was seen.
4
.4. Minimum Bactericidal Concentration
MBC assays were performed as previously described [38]. Overnight cultures grown in
CAMHB were adjusted to an OD600 of 0.05 in 25 mL of CAMHB in a 250-mL flask. They were
then allowed to grow at 37 ºC with agitation at 250 rpm to exponential phase (OD =0.5) at
6
00
6
which point the cultures were diluted to achieve a concentration of 5x10 colony-forming units
(cfu)/mL. One hundred microliters of the diluted, exponential phase bacteria was added to 1 mL
of the pyrrolomycin and control antibiotic vancomycin in a 10-mL tube. Bacteria were harvested
at 0, 1, 3 and 24 h and plated for viable cell counts and reported as cfu/mL.
4
.5. Anti-staphylococcal biofilm activity
2
1

ACCEPTED MANUSCRIPT
4
3
Static biofilms were generated as previously described. Briefly, overnight cultures of S.
aureus were diluted to an OD600 of 0.05 in TSB-NaCl/Glc, and 200 µL was inoculated into wells
of a Costar 3596 plate (Corning life Sciences, Acton, MA), that had been coated overnight at 4
ºC with 20% human plasma (Sigma). After 24 h of static growth, at 37 ºC the biofilms were
washed two times with PBS and the remaining biofilm was treated with pyrrolomycin
derivatives in Muller Hinton II broth (cation-adjusted) (Becton, Dickinson and Company,
Sparks, MD) supplemented with 5% DMSO (Sigma). After 24, 48 or 120 h of treatment, the
remaining biofilm was washed two times with PBS and then re-suspended in 100 µL PBS, serial
diluted and plated for cfu.
4
.6. Pharmacokinetics of pyrrolomycin 4
A Waters ACQUITY ultra-performance liquid chromatography (UPLC) system (Waters,
®
Milford, MA) coupled to a 4000 Q TRAP quadrupole linear ion trap hybrid mass spectrometer
with an electrospray ionization (ESI) source (Applied Biosystems, MDS Sciex, Foster City, CA)
was used throughout. All chromatographic separations were performed with an ACQUITY
®
UPLC BEH Shield RP18 column (2.1×100mm, 1.7µm; Waters) equipped with an ACQUITY
UPLC C guard column (Waters, Milford, MA). Mobile phase A consisted of 0.1% acetic acid
1
8
and mobile phase B was methanol (MeOH). The initial mobile phase composition was 80% B for
the first 3 min and was gradually increased to 95%B in 4.25 min and held constant for 1.25 min.
Mobile phase B was then reset to 80% in 0.25 min and the column was equilibrated for 1 min
before the next injection. A flow rate of 0.25 mL/min was used and the injection volume of all
samples was 10 µL. MS/MS analyses were performed with negative ESI mode and using the
following parameters: ion spray voltage, -4500 V; source temperature, 500 °C; curtain gas
(nitrogen), 10 arbitrary units; and collision gas (nitrogen), “High”. Specific detection was
2
2

ACCEPTED MANUSCRIPT
performed by monitoring the transition 349.67→160.8 m/z for 4. In addition, for the detection
of potential 4 metabolites, multiple reaction monitoring–information-dependent acquisition-
enhanced product ion, neutral loss, precursor ion and enhanced MS scans were used as described
previously [44-47].
Plasma samples were prepared by protein precipitation using ice-cold methanol. Sixty-
five µL of methanol was added to 25 µL plasma samples pre-spiked with 10 µL of internal
standard (500 ng/ml). Samples were then vortexed, and centrifuged at 16,000 × g for 10 min.
After centrifugation at 16,000 × g for 10 min, 80 µL supernatant were mixed with 40 µL of 10%
2
methanol in H O. Further, 10 µL of each sample was used for LC-MS/MS analysis.
Eight-week-old healthy male Balc/c mice were purchased from Charles River
Laboratories (Wilmington, MA). Sterilized 7012 Teklad diets (Harlan) were used for mice, and
water was provided ad libitum. A 10 mg/kg oral dose was administered as 250 µL of solution (4,
1
mg/mL) in a mixture of DMSO-PEG400-PG-EtOH-Cremophore-PBS (2/20/10/10/5/53 % v/v)
to a 25 g mouse. A 1.0 mg/kg intravenous dose was administered as a 100 µL of solution (4, 0.25
mg/mL) in the same DMSO-PEG400-PG-EtOH-Cremophore-PBS mixture to a 25 g mouse.
Oral doses were administered via oral gavage, whereas intravenous doses were administered via
the tail vein. Blood samples were collected at 0, 5, 15, 30, 45 min, and 1, 2, 4, 8, 24, 48, and 72 h
post oral dose, and at 0, 5, 30 min, and 1, 3, 8, 24, 48, and 72 h post intravenous dose. Plasma
was separated by centrifugation of blood samples at 2000 × g for 5 min at 4 °C within 1 h of
sample collection. The pharmacokinetic parameters were determined using the
noncompartmental analysis module of WinNonlin (version 5.1, Pharsight, Mountain View, CA).
The absolute bioavailability (F) was calculated as the ratio between the AUC0-infinity from oral
and intravenous routes, after dose normalization using the following equation:
2
3

ACCEPTED MANUSCRIPT
AUC xDose
Oral
i.v.
%
FAbsolute
=
x100
AUC xDose
i.v.
oral
AUTHOR INFORMATION
Corresponding Author. Department of Pharmaceutical Sciences, UNMC Center for Drug
*
Discovery, College of Pharmacy; Cancer Genes and Molecular Regulation Program, Fred and
Pamela Buffett Cancer Center, University of Nebraska Medical Center, 986125 Nebraska
Medical Center, Omaha, NE 68198, United States. Phone: +1-402-559-6609. Fax: +1-402-559-
5
418. E-mail address: Rongshi.li@unmc.edu
Present Addresses
1
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, 18 Yunwan Road,
Wanli, Nanchang, China 330004.
2
College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109.
Author Contributions
All authors have contributed to the manuscript and given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work is partially supported by the Nebraska Research Initiative grant (University of
Nebraska) and startup funds (University of Nebraska Medical Center) to R. L., and by PO1-
2
4

ACCEPTED MANUSCRIPT
AI083211 (National Institutes of Health) to K.W.B. The NMR Core Facility is partially
supported by the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727).
ABBREVIATIONS USED
AUC, the area under the curve; cfu, colony-forming units; Cmax, maximum plasma concentration;
ESKAPE pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia,
Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp; IV, intravenous; LC-
MS, Liquid chromatography-mass spectrometry; LE, Ligand Efficiency; MBC, minimum
bactericidal concentration; MIC, minimum inhibitory concentration; RO5, rule of five; SA,
Staphylococcus aureus; SAR, structure activity relationship; SI, selectivity index.
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