Marinopyrrole derivatives as potential antibiotic agents against methicillin-resistant Staphylococcus aureus (III)

Article abstract of DOI:10.3390/md12052458

The marine natural product, marinopyrrole A (1), was previously shown to have significant antibiotic activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Although compound (1) exhibits a significant reduction in MRSA activity in the presence of human serum, we have identified key modifications that partially restore activity. We previously reported our discovery of a chloro-derivative of marinopyrrole A (1a) featuring a 2-4 fold improved minimum inhibitory concentration (MIC) against MRSA, significantly less susceptibility to serum inhibition and rapid and concentration-dependent killing of MRSA. Here, we report a novel fluoro-derivative of marinopyrrole A (1e) showing an improved profile of potency, less susceptibility to serum inhibition, as well as rapid and concentration-dependent killing of MRSA.

Full text of DOI:10.3390/md12052458

Mar. Drugs 2014, 12, 2458-2470; doi:10.3390/md12052458
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Marinopyrrole Derivatives as Potential Antibiotic Agents
against Methicillin-Resistant Staphylococcus aureus (III)
1
,2,3,†
4,5,†
5
3
4,5
Yan Liu
, Nina M. Haste
, Wdee Thienphrapa , Jerry Li , Victor Nizet ,
5
1,2,3,6,
Mary Hensler and Rongshi Li
*
1
Department of Pharmaceutical Sciences, Center for Drug Discovery, College of Pharmacy,
University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198,
USA; E-Mail: Yan.Liu@unmc.edu
2
3
Cancer Genes and Molecular Regulation Program, Buffett Cancer Center, University of Nebraska
Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA
Department of Drug Discovery, Chemical Biology & Molecular Medicine Program, H. Lee Moffitt
Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA;
E-Mail: Jerry.Li@ucsf.edu
4
5
6
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego,
La Jolla, CA 92093, USA; E-Mails: nhaste@ucsd.edu (N.M.H.); vnizet@ucsd.edu (V.N.)
Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA;
E-Mails: wdee.ucsd@gmail.com (W.T.); mhensler@ucsd.edu (M.H.)
Department of Oncologic Sciences, Morsani College of Medicine, University of South Florida,
1
2901 Bruce B. Downs, Tampa, FL 33612, USA
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: Rongshi.Li@unmc.edu;
Tel.: +1-402-559-5965; Fax: +1-402-559-8270.
Received: 10 February 2014; in revised form: 21 March 2014 / Accepted: 24 March 2014 /
Published: 30 April 2014
Abstract: The marine natural product, marinopyrrole A (1), was previously shown to have
significant antibiotic activity against Gram-positive pathogens, including methicillin-resistant
Staphylococcus aureus (MRSA). Although compound (1) exhibits a significant reduction
in MRSA activity in the presence of human serum, we have identified key modifications
that partially restore activity. We previously reported our discovery of a chloro-derivative
of marinopyrrole A (1a) featuring a 2–4 fold improved minimum inhibitory concentration
(MIC) against MRSA, significantly less susceptibility to serum inhibition and rapid and
concentration-dependent killing of MRSA. Here, we report a novel fluoro-derivative of

Mar. Drugs 2014, 12
marinopyrrole A (1e) showing an improved profile of potency, less susceptibility to serum
2459
inhibition, as well as rapid and concentration-dependent killing of MRSA.
Keywords: antibiotics; non-symmetrical marinopyrroles; MRSA; SAR
1
. Introduction
Since we reported the synthesis of novel non-symmetrical marinopyrrole derivatives retaining their
potent activity against methicillin-resistant Staphylococcus aureus (MRSA), yet less susceptible to
human serum inhibition [1], several research publications on the topic of marinopyrroles have
appeared [2–7]. Biosynthesis of marinopyrrole A via an N,C-bipyrrole homocoupling catalyzed by two
flavin-dependent halogenases was reported by the Moore group [2], and racemic marinopyrrole B by
total synthesis and a review of the marinopyrroles were reported by the Clive group [3,4]. After
optimization of the key step to avoid the formation of an oxazepine byproduct [5] that was reported in
our first total synthesis of marinopyrroles [8], we published the most potent symmetrical
marinopyrrole derivative against MRSA and methicillin-resistant Staphylococcus epidermidis
(
MRSE) [6]. Recently we reported a series of cyclic marinopyrroles as disruptors of Mcl-1 and Bcl-x
L
binding to Bim [7] and a series of novel marinopyrroles with potential as anticancer agents [9].
The World Health Organization recognizes antibiotic resistance as a serious threat to human
health [10]. The global crisis of antibiotic resistance has spread rapidly over the last several decades,
with multidrug-resistant MRSA as a major cause of serious infections in the United States [11–15].
From 1999 to 2005, estimated MRSA hospitalizations in the U.S. more than doubled, increasing from
1
27,000 to 280,000, and accounted for roughly 94,000 infections and close to 19,000 deaths in
005 [16]. That same year, more people in the U.S. died from MRSA infections than HIV/AIDS
2
(16,000 people). MRSA infections cost U.S. hospitals between $3.2 and $4.2 billion annually [17].
Recent survey documents have shown that MRSA remains one of the most prevalent
multidrug-resistant organisms causing healthcare-associated infections, and the MRSA prevalence in
2
010 is higher than that reported in 2006 [18]. The introduction of new MRSA antibiotics to clinical
practice has been limited primarily to the oxazolidinone, linezolid [19], in 2000, the lipopeptide,
daptomycin [20], in 2003, and ceftaroline [21]. Vancomycin remains the most commonly used first
line treatment against MRSA. However, overreliance on this drug has resulted in an increase in MRSA
with reduced susceptibility to vancomycin [22,23]. The minimum inhibitory concentration (MIC) shift
(“the MIC creep”) for vancomycin has been especially noticeable in MRSA compared to other
S. aureus [24]. In fact, vancomycin efficacy continues to decline, due to pathogen-developed
resistance [22,24]. Instances of daptomycin [25] and linezolid [26,27] resistance have also surfaced.
There are now several late-stage products in development, including tedizolid, dalbavancin,
oritavancin and ceptobiprole. Although these antibiotics may add to the arsenal for combating MRSA
resistance, bacteria inevitably develop resistance to all antibacterial agents that are introduced to the
clinic [28]. Novel antibiotic agents of new structural classes and further advances in discovery research
are urgently needed to overcome the problem of MRSA resistance.

Mar. Drugs 2014, 12
2460
The relative abandonment of the discovery and development of antibiotics by the pharmaceutical
industry has opened opportunities for academic researchers to discover new antibiotics that treat these
increasingly problematic infections. Here, we report our design and synthesis of novel marinopyrrole
derivatives with excellent antibiotic activity against MRSA, but only limited serum inactivation.
2
. Results and Discussion
2
.1. Synthesis and Structural Activity Relationships of Non-Symmetrical Marinopyrrole Derivatives
We classified marinopyrroles as “symmetrical” and “asymmetrical/non-symmetrical” in our
previous publication to facilitate structure-activity relationship (SAR) discussions [1]. “Symmetrical”
derivatives bear the same substituents and patterns on both phenyl Rings A and B attached to the
carbonyl groups, while “non-symmetrical” marinopyrroles are those with different substituents for
Rings A and B (Chart 1). As we envisaged that the “non-symmetrical” marinopyrrole derivatives
should have different and possibly more favorable biological activity than their symmetrical
counterparts, in particular, the molecules with diverse functional groups decorated on this unique
1
,3-bispyrrole system may adopt specific conformations, due to the restricted free rotation of the chiral
axis. Indeed, a series of novel non-symmetrical marinopyrrole derivatives that we designed and
synthesized showed potent anti-MRSA activity with a superior antibiotic profile to the parent
marinopyrrole A (1) [1].
Chart 1. Marinopyrroles.
To continue our efforts of structure-activity relationship (SAR) optimization, we designed and
synthesized a series of novel non-symmetrical marinopyrroles and evaluated their anti-MRSA activity.
As shown in Chart 1 and Scheme 1, while Ring A was kept constant, substitutions with different
halogen (F, Cl and Br) in the different position of Ring B were examined. Indeed, the effects of
different halogen (F, Cl and Br) in Ring B on SARs, physicochemical properties and pH-dependent
microspecies are observed, as detailed in Figures 1 and 2.
Chemistries to access both symmetrical and non-symmetrical marinopyrrole derivatives have been
reported [1,6,8,29]. Briefly, Friedel–Crafts arylation of mono acylated bispyrrole 2 [1,29] with the acid
chlorides, 4, generated in situ from the corresponding carboxylic acids, 3, with thionyl chloride,
afforded a series of marinopyrrole precursors, 5c–5f, in a 48%–71% yield. A novel series of
non-symmetrical marinopyrrole derivatives, 6c–6f, were obtained in a 21%–60% yield by
tetrachlorination of the corresponding 5c–5f using 4.1 equivalents of sulfuryl chloride (SO
2
2
Cl ) in
DCM at 0 °C . Demethylation of 6c–6f using BBr
3
in DCM at −78 °C furnished 1c–1f in a 31%–72%
yield (Scheme 1).

Mar. Drugs 2014, 12
Scheme 1. Synthesis of novel non-symmetrical marinopyrrole derivatives.
2461
Figure 1. Physicochemical properties and structure-activity relationship (SAR) of
marinopyrrole derivatives against methicillin-resistant Staphylococcus aureus (MRSA).
f
a
a
a
c
c
Compound
(parent)
MW
pK
a
1
pK
a
2
Clog p
THB
THB + 20% Serum
b
b
b
d
d
1
510.15
544.60
528.14
528.14
528.14
589.05
1485.72
7.8
8.4
5.6
0.75
94–188
12.5–25
d
d
d
1
a
7.3
7.2
7.0
7.6
7.5
8.2
8.2
8.1
8.3
8.2
6.1
5.7
5.7
5.7
6.4
0.19–0.39
3.13
e
1
c
ND
1
d
0.78
ND
25–50
ND
1
e
0.19–0.78
1.56
1
f
g
Vanco
0.85–1.7
0.85–1.7
a
b
Calculated using ChemAxon Software Version 5.12.3 (ChemAxon, Budapest, Hungary); reported in our
c
previous paper [6]; MIC, minimum inhibitory concentration in μM in Todd-Hewitt broth (THB) in the
absence or presence of 20% human serum; all data were generated from experiments repeated four times;
d
results, except for the calculated pK
a
and log p-values, from our previous paper [1] for SAR discussions;
e
f
g
not determined; MW, molecular weight; Vanco, vancomycin.

Mar. Drugs 2014, 12
2462
Figure 2. pH-dependent microspecies of marinopyrroles.
Microspecies
1
1a
1c
1d
1e
1f
a
I
85.3/68.5/29.9
7.1/14.3/24.8
7.1/14.3/24.8
0.6/3.0/20.5
0.0/0.0/0.01
63.2/38.4/9.7
61.2/36.3/9.0
50.3/26.7/5.9 79.8/59.4/21.3 75.3/52.7/16.7
II
29.2/44.4/45.0 31.2/46.5/45.7 42.0/56.1/48.8 6.6/12.4/17.6 17.1/30.0/38.0
III
IV
5.2/8.0/8.1
2.4/8.0/37.2
0.0/0.0/0.01
5.1/7.6/7.4
2.6/9.7/37.9
0.0/0.0/0.01
4.2/5.6/4.8
3.5/11.7/40.4
0.0/0.0/0.01
12.5/23.4/33.4 6.2/11.0/13.9
1.0/4.9/27.6
0.0/0.0/0.02
1.4/6.3/31.4
0.0/0.0/0.01
V
a
Percentage (%) of microspecies distributions at pH 7.0/7.4/8.0, respectively, calculated using ChemAxon Software
Version 5.12.3 (ChemAxon, Budapest, Hungary); numbers in italics are the percent of microspecies distributions at
pH 7.0.
The anti-MRSA activity of the novel non-symmetrical marinopyrroles was evaluated against a
USA300 strain of community-associated MRSA using marinopyrrole A (1) as a control. The MIC values
of 1a [(4-chloro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrole-2-yl)
methanone], which we reported previously [1], were used to facilitate the SAR discussions. Similar to
the effects from chlorine substituted marinopyrroles [1], fluorine substitutions in Ring B have
significant contributions to the antibacterial activity. Although non-symmetrical marinopyrrole with
ortho-substitution of the carbonyl group (1c) is four times less potent than 1, those marinopyrroles
with para-(1d) and meta-fluoro (1e) substitutions display similar or better activities than 1. One of the
derivatives, (5-fluoro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)
methanone (1e), exhibited potent antibacterial activity similar to that of 1a [1]. The 1–4 fold
improvement in antibacterial activity from the parent compound, 1, was observed for this novel
fluoro-substituted marinopyrrole derivative, 1e, as shown in Figure 1. Compound 1f with meta-bromo
substitution also exhibited antibacterial activity, although the MIC value is two-fold less potent than 1.
Most significantly, not only did Compound 1e show increased antibacterial activity compared to the
parent compound, marinopyrrole A (1), but its activity was also less inhibited upon the addition of
2
0% human serum (MIC 25–50 μM vs. 94–188 μM). In comparison to contemporary MRSA agents,
Compound 1e is more potent than vancomycin against USA300 MRSA strain TCH1516 [30].
To understand the significant effects of the physicochemical properties on antibacterial activity, we
calculated the pK
marinopyrroles (1c–1e) have lower Clog p-values than 1, while their chloro-(1a) or bromo-(1f)
counterparts are up to half a log unit higher. Although pK 2 values do not vary much (8.1–8.4), the
pK 1 values change from 7.0 to 7.8, due to the substitution of halogen atoms in different positions of
Ring B. Careful analysis of pK data reveals that there are five microspecies, I–V, present, and their
a a
1, pK 2 and log p of all marinopyrrole derivatives (Figure 2). All fluoro-substituted
a
a
a
distributions depend on the pH, as shown in Figure 2. Although our MIC assays were performed at
pH 7.0, microspecies distributions at pH 7.4 and 8.0 are also tabulated, as the latter conditions are

Mar. Drugs 2014, 12
2463
usually used for other assays [7,9]. At pH 7.0, 50%–85% of all marinopyrroles are in the form of
Microspecies I, with the parent marinopyrrole, 1, being the most predominant (85%) Microspecies I
(Figure 2); marinopyrroles 1 and 1e have similar distributions of Microspecies II, 7.1% and 6.6%,
respectively; the rest are increasing from 17% (1f) to 42% (1d) Microspecies II; the variation of
Microspecies III distributions is small from 4.2% (1d) to 12.5% (1e); both Microspecies IV and V are
from 0.0% to 3.5% at pH 7 and may be considered negligible. Microspecies I–IV distributions of
marinopyrroles vary significantly at pH 7.4 and 8.0 (Figure 2). Microspecies I is found in the free
hydroxyl form for both phenol groups, which can serve as both hydrogen bond donors and acceptors.
Microspecies II and III can provide one free hydroxyl and one phenoxide group, as shown in Figure 2.
The former can provide both a hydrogen bond donor and acceptor in Ring A and only a hydrogen bond
acceptor or phenoxide for ionic interactions in Ring B, and vice versa for the latter. Microspecies IV
has both phenol groups in phenoxide form, which are only available as hydrogen bond acceptors or for
ionic interactions. The microspecies and their distributions at different pH should have a significant
impact on their antibacterial activity.
Figure 3. In vitro time-kill analysis for marinopyrrole derivatives 1a (A) and 1e (B)
against the USA300 community-associated MRSA strain TCH1516. MRSA was subjected
to increasing concentrations of 1× (0.39 μM), 10× (3.9 μM) and 20× (7.8 μM) the MIC of
the assayed derivatives or the vehicle control (none). Both derivatives showed potent
concentration-dependent killing kinetics. At 20× (7.8 μM), Derivative 1e demonstrated
more rapid bacterial killing at 3 h.
2
.2. In Vitro Time-Kill of Marinopyrrole Derivative 1e
Our previous data showed that the marinopyrrole derivative, 1a, exhibited rapid killing kinetics, and
we investigated whether 1e might also show similar kinetics compared to the parent molecule.
Derivative 1e displayed strong concentration-dependent MRSA killing similar in profile to the parent
compound [30]. The potency of Derivative 1e was especially evident at 20× the MIC (7.8 μM),
yielding greater than a 4-log kill of MRSA at 4 h (Figure 3B). These killing kinetics parallel the effects
previously seen with the natural product parent compound, (−)-1 [30]. Importantly, the tested
concentration of 1e (MIC 0.39 μM) was half of the concentration of the parent natural product tested in
time-kill analyses (MIC 0.75 μM or 0.375 μg/mL) [30]. Secondly at 3.9 μM (10× MIC), bacterial
counts were reduced by nearly two log units at three hours incubation and on average by three log

Mar. Drugs 2014, 12
2464
units at six hours. In comparison, at six hours of incubation, the parent natural product only yielded a
two log decrease in bacterial counts at concentrations two fold higher (7.5 μM) than that tested for
Derivative 1e (3.9 μM) [30]. Furthermore, the actual tested compound concentration of (−)-1 was
7
.5 μM, two fold higher than that of Derivative 1e (3.9 μM) (Figure 3). In summary, we have
discovered a second novel derivative of a natural product with favorable bactericidal activity against
MRSA, even in the presence of human serum. These results provide additional data showing that the
marinopyrrole A scaffold is amenable to modifications to increase its antibacterial activity.
3
. Experimental Section
3
.1. Synthesis of Compounds 5c–6f
All chemicals and solvents were purchased from commercial suppliers and used without further
purification. Preparative flash column chromatography was performed on silica gel 60, 0.040–0.063 mm
1
(
EMD Chemicals, Billerica, MA, USA). H NMR (400 MHz) spectra were recorded on a Varian
AS400 with a 60-place automated sample changer (Thermo, Madison, WI, USA). High resolution
ESI-MS spectra were recorded on an Agilent ESI-TOF LC-MS 6200 system (Agilent Technologies,
Santa Clara, CA, USA). Preparative HPLC was performed on a Gilson HPLC system with UV
detectors and a Gilson 215 liquid handler for auto injection and fraction collections (customized by HT
Labs, San Diego, CA, USA). Analytical HPLC was performed on an Agilent 1100 series with diode
array detectors and auto samplers (Agilent Technologies, Santa Clara, CA, USA). The specifications of
HPLC analysis are as follows: flow rate, 1 mL/min; column, Intertsil, 2.5 μm, 4.6 × 150 mm;
wavelength, 254 and 280 nm; mobile phase, A: H
B in 25 min. All tested compounds possessed a purity of not less than 95%.
2-(6-Fluoro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (5c). Into a
solution of 6-fluoro-2-methoxybenzoic acid (116 mg, 0.67 mmol, 1.2 equivalent) in benzene (1.0 mL),
SOCl (1.0 mL) was added at room temperature, and the resulting solution was refluxed for 2 h. The
reaction mixture was concentrated under vacuum to generate 6-fluoro-2-methoxybenzoyl chloride 4a,
which was used directly in the next step without purification. A solution of 4a in CH Cl (DCM, 2 mL)
was added to a slurry of AlCl (97 mg, 1.3 equivalent) in DCM (2.5 mL) at 0 °C, and then, a solution
2 2
O with 0.1% HCO H, B: MeOH, gradient of 50%–95%
(
2
2
2
3
of 1′H-1,3′-bipyrrol-2′-yl(2-methoxyphenyl)methanone (2) [1] (150 mg, 0.56 mmol, 1.0 equivalent) in
DCM (1.5 mL) was added dropwise. The resulting solution was allowed to warm to room temperature
and stirred overnight. A saturated solution of NaHCO
3
(10 mL) and DCM (10 mL) was then added,
®
and the resulting mixture was stirred for 1 h and then filtered through Celite (Sigma-Aldrich,
St. Louis, MO, USA). The mixture was extracted with DCM (3 × 10 mL). The organic layer was
dried over anhydrous Na
2
SO
4
, and purified by flash column chromatography (silica gel,
1
hexanes:DCM:EtOAc 4:4:1) to afford 168 mg of 5c as a white solid, 71% yield. H NMR (400 MHz,
CDCl
3
) δ 9.79 (br s, 1H), 7.31–7.27 (m, 1H), 7.22–7.14 (m, 2H), 7.07 (t, J = 4.0 Hz, 1H), 6.75–6.68 (m,
H), 6.54–6.53 (m, 1H), 6.41–6.40 (m, 1H), 6.34 (t, J = 4.0 Hz, 1H), 5.70 (dd, J = 4.0, 2.6 Hz, 1H),
.80 (s, 3H), 3.76 (s, 3H). The same procedure as described above was followed to obtain 5d–5f.
2-(4-Fluoro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (5d). After
flash column chromatography (silica gel, DCM:EtOAc 9:1), 113 mg of 5d was obtained as a yellowish
4
3
(

Mar. Drugs 2014, 12
2465
1
solid, 48% yield. H NMR (400 MHz, CDCl
3
) δ 9.71 (br s, 1H), 7.22–7.14 (m, 3H), 7.07–7.03
(
m, 1H), 6.71–6.60 (m, 5H), 6.32 (dd, J = 4.0, 1.7 Hz, 1H), 6.29 (t, J = 2.8 Hz, 1H), 5.83 (dd, J = 4.0,
.6 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 3H).
2-(5-Fluoro-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (5e). After
2
(
flash column chromatography (silica gel, DCM:EtOAc 9:1), 118 mg of 5e was obtained as an
1
off-white solid, 50% yield. H NMR (400 MHz, CDCl
3
) δ 9.45 (br s, 1H), 7.24–7.19 (m, 2H), 7.08–7.04
(
m, 2H), 6.89–6.86 (m, 2H), 6.73–6.68 (m, 3H), 6.35–6.32 (m, 2H), 5.85 (dd, J = 4.0, 2.6 Hz, 1H), 3.76
s, 3H), 3.70 (s, 3H).
2-(5-Bromo-2-methoxybenzoyl)-1′H-1,3′-bipyrrol-2′-yl)(2-methoxyphenyl)methanone (5f). After
(
(
flash column chromatography (silica gel, DCM:EtOAc 9:1), 112 mg of 5f was obtained as a yellowish
1
solid, 48% yield. H NMR (400 MHz, CDCl
3
) δ 9.71 (br s, 1H), 7.45 (dd, J = 8.8, 2.5 Hz, 1H),
7
.26–7.21 (m, 1H), 7.20–7.15 (m, 2H), 7.07 (t, J = 3.0 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.73
dt, J = 11.3, 8.4 Hz, 3H), 6.34–6.29 (m, 2H), 5.89 (dd, J = 4.0, 2.6 Hz, 1H), 3.75 (s, 3H), 3.69 (s, 3H).
6-Fluoro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-[1,3′-bipyrrole]-2-yl)
methanone (6c). To a solution of Compound 5c (150 mg, 0.36 mmol, 1 equivalent) in DCM (4 mL) at
(
(
0
0
°C, SO
2
Cl
2
(119 μL, 1.48 mmol, 4.1 equivalent) was added dropwise, and the solution was stirred at
solution (2 mL) was added, and the resulting mixture was
°C for 1 h. Saturated aqueous NaHCO
3
4
extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO ,
filtered and concentrated. The residue was purified by flash column chromatography (silica gel,
1
DCM:hexane:EtOAc 1:1:0.1) to afford 6c (118 mg, 59% yield) as an off-white solid. H NMR
(
400 MHz, CDCl
d, J = 8.4 Hz, 1H), 6.77–6.72 (m, 2H), 6.67 (t, J = 7.5 Hz, 1H), 6.37 (s, 1H), 3.80 (s, 3H),
.77 (s, 3H).
4-Fluoro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-[1,3′-bipyrrole]-2-yl)
methanone (6d). To a solution of compound 5d (125 mg, 0.30 mmol, 1 equivalent) in DCM (4 mL) at
3
) δ 10.48 (br s, 1H), 7.34 (td, J = 8.4, 6.7 Hz, 1H), 7.25–7.18 (m, 2H), 6.80
(
3
(
0
0
°C, SO
2
Cl
2
(99 μL, 1.23 mmol, 4.1 equivalent) was added dropwise, and the solution was stirred at
solution (2 mL) was added, and the resulting mixture was
°C for 1 h. Saturated aqueous NaHCO
3
4
extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO ,
filtered and concentrated. The residue was purified by flash column chromatography (silica gel,
1
DCM:hexane:EtOAc 1:1:0.1) to afford 6d (100 mg, 60% yield) as an off-white solid. H NMR
(
400 MHz, CDCl
ddd, J = 8.1, 6.2, 3.8 Hz, 3H), 6.32 (s, 1H), 3.80 (s, 3H), 3.72 (s, 3H).
5-Fluoro-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-[1,3′-bipyrrole]-2-yl)
methanone (6e). To a solution of Compound 5e (270 mg, 0.64 mmol, 1 equivalent) in DCM (7 mL) at
3
) δ 10.60 (br s, 1H), 7.27–7.18 (m, 3H), 6.76 (d, J = 8.6 Hz, 1H), 6.67
(
(
0
0
°C, SO
2
Cl
2
(210 μL, 2.63 mmol, 4.1 equivalent) was added dropwise, and the solution was stirred at
solution (4 mL) was added, and the resulting mixture was
°C for 1 h. Saturated aqueous NaHCO
3
4
extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO ,
filtered and concentrated. The residue was purified by flash column chromatography (silica gel,
1
DCM:hexane:EtOAc 4:4:0.5) to afford 6e (110 mg, 31% yield) as an off-white solid. H NMR
(
(
(
3
400 MHz, CDCl ) δ 9.81 (br s, 1H), 7.28 (dt, J = 2.7, 1.8 Hz, 1H), 7.17 (dd, J = 7.5, 1.7 Hz, 1H), 7.11
ddd, J = 9.1, 7.9, 3.1 Hz, 1H), 6.88 (ddd, J = 8.0, 6.8, 3.6 Hz, 2H), 6.79 (d, J = 8.3 Hz, 1H), 6.71
td, J = 7.5, 0.8 Hz, 1H), 6.34 (s, 1H), 3.78 (s, 3H), 3.74 (s, 3H).

Mar. Drugs 2014, 12
2466
(5-Bromo-2-methoxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-methoxybenzoyl)-1′H-[1,3′-bipyrrole]-2-yl)
methanone (6f). To a solution of Compound 5f (110 mg, 0.23 mmol, 1 equivalent) in DCM (3 mL) at
0
0
°C, SO
2
Cl
2
(75 μL, 0.92 mmol, 4.1 equivalent) was added dropwise, and the solution was stirred at
solution (2 mL) was added, and the resulting mixture was
°C for 1 h. Saturated aqueous NaHCO
3
4
extracted with DCM (3 × 4 mL). The combined organic layers were dried with anhydrous MgSO ,
filtered and concentrated. The residue was purified by flash column chromatography (silica gel,
1
DCM:hexane:EtOAc 4:4:0.5) to afford 6f (30 mg, 21% yield) as an off-white solid. H NMR
(400 MHz, CDCl
3
) δ 10.53 (br s, 1H), 7.50 (dd, J = 8.8, 2.5 Hz, 1H), 7.32–7.27 (m, 1H), 7.20
(dd, J = 8.6, 2.1 Hz, 2H), 6.81 (dd, J = 14.3, 8.6 Hz, 2H), 6.74 (td, J = 7.5, 0.7 Hz, 1H), 6.33 (s, 1H),
3
.77 (s, 3H), 3.72 (s, 3H).
3
.2. Synthesis of Compounds 1c–1f
(
6-Fluoro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)
methanone (1c). To a solution of 6c (118 mg, 0.21 mmol) in anhydrous DCM (2 mL) was slowly
added 1.0 M solution of BBr in DCM (848 μL, 0.84 mmol, 4 equivalent) via a syringe under N
at −78 °C. After being stirred for 0.5 h, the mixture was quenched by the addition of MeOH (0.5 mL)
and then H O (3 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried
over anhydrous MgSO , filtered and concentrated in vacuum. The residue was purified by column
3
2
2
4
chromatography (silica gel, hexanes:EtOAc 10:1) to give 1c (40 mg, 36% yield) as a yellow solid.
1
3
H NMR (400 MHz, CDCl ) δ 10.47 (s, 1H), 10.01 (s, 1H), 9.80 (br s, 1H), 7.43–7.36 (m, 2H),
7
6
5
.35–7.29 (m, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 5.2 Hz, 1H),
+
.68–6.62 (m, 1H), 6.49 (t, J = 7.6 Hz, 1H). HRMS (ESI-TOF) [M + H] calcd. for C22
H12Cl
4
FN
2
O
4
26.9530, found 526.9521; HPLC purity, 95.1%.
(
4-Fluoro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)
methanone (1d). To a solution of 6d (100 mg, 0.18 mmol) in anhydrous DCM (2 mL) was slowly
added a 1.0 M solution of BBr in DCM (720 μL, 0.72 mmol, 4 equivalent) via a syringe under N
at –78 °C. After being stirred for 0.5 h, the mixture was quenched by the addition of MeOH (0.5 mL)
and then H O (4 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried
over anhydrous MgSO
3
2
2
4
, filtered and concentrated in vacuum. The residue was purified by column
1
chromatography (silica gel, hexanes:EtOAc 10:1) to give 1d (29 mg, 31% yield) as a yellow solid. H
NMR (400 MHz, CDCl
3
) δ 11.60 (s, 1H), 10.41 (s, 1H), 9.84 (br s, 1H), 7.57 (dd, J = 8.9, 6.5 Hz, 1H),
7
1
.45 (dd, J = 8.0, 1.6 Hz, 1H), 7.39–7.34 (m, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.71 (s, J = 10.3, 2.5 Hz,
+
H), 6.67 (d, J = 1.7 Hz, 1H), 6.65–6.58 (m, 1H), 6.56–6.50 (m, 1H). HRMS (ESI-TOF) [M + H]
calcd. for C22
5-Fluoro-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)
methanone (1e). To a solution of 6e (160 mg, 0.29 mmol) in anhydrous DCM (4 mL) was slowly
added a 1.0 M solution of BBr in DCM (1160 μL, 1.16 mmol, 4 equivalent) via a syringe under N
at –78 °C. After being stirred for 0.5 h, the mixture was quenched by the addition of MeOH (0.5 mL)
and then H O (5 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were dried
over anhydrous MgSO , filtered and concentrated in vacuum. The residue was purified by column
chromatography (silica gel, hexanes:EtOAc 10:1) to give 1e (110 mg, 72% yield) as a yellow solid.
4 2 4
H12Cl FN O 526.9530, found 526.9533; HPLC purity, 95.2%.
(
3
2
2
4

Mar. Drugs 2014, 12
2467
1
H NMR (400 MHz, CDCl
3
) δ 10.91 (s, 1H), 10.36 (s, 1H), 9.71 (br s, 1H), 7.41–7.36 (m, 2H),
7
1
5
.27–7.22 (m, 1H), 7.14 (dd, J = 8.8, 3.0 Hz, 1H), 6.99 (dd, J = 9.1, 4.5 Hz, 1H), 6.94 (d, J = 8.4 Hz,
+
4 2 4
H), 6.72 (s, 1H), 6.54 (t, J = 7.6 Hz, 1H). HRMS (ESI-TOF) [M + H] calcd. for C22H12Cl FN O
26.9530, found 526.9531; HPLC purity, 98.4%.
(
5-Bromo-2-hydroxyphenyl)(4,4′,5,5′-tetrachloro-2′-(2-hydroxybenzoyl)-1′H-1,3′-bipyrrol-2-yl)
methanone (1f). To a solution of 6f (30 mg, 0.05 mmol) in anhydrous DCM (2 mL) was slowly added
a 1.0 M solution of BBr in DCM (196 μL, 0.196 mmol, 4 equivalent) via a syringe under N at –78 °C.
After being stirred for 0.5 h, the mixture was quenched by addition of MeOH (0.5 mL) and then H
3
2
2
O
(2 mL) and extracted with DCM (3 × 5 mL). The combined organic layers were dried over anhydrous
MgSO
4
, filtered and concentrated in vacuum. The residue was purified by column chromatography
1
(silica gel, hexanes:EtOAc 10:1) to give 1f (12 mg, 42% yield) as a yellow solid. H NMR (400 MHz,
1
3 3
CDCl ) δ H NMR (400 MHz, CDCl ) δ 11.12 (s, 1H), 10.35 (s, 1H), 9.82 (s, 1H), 8.10–8.04 (m, 2H),
7
1
5
.83 (d, J = 8.2 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.58 (dd, J = 8.8, 2.3 Hz, 1H), 7.44 (t, J = 7.6 Hz,
+
H), 6.92 (d, J = 8.9 Hz, 1H), 6.86 (s, 1H). HRMS (ESI-TOF) [M + H] calcd. for C22
86.8729, found 586.8731; HPLC purity, 95.6%.
4 2 4
H12BrCl N O
3
.3. In Vitro Antibacterial Assays
TCH1516, a USA300 strain of community-associated MRSA, was obtained from the American
Type Culture Collection (Manassas, VA, USA) and used for biological assays. MICs were determined
by broth microdilution in 96-well tissue-culture treated plates (Falcon Becton Dickson, Franklin Lakes,
NJ, USA) according to Clinical and Laboratory Standards Institute guidelines, except that Todd-Hewitt
broth (THB) was used in place of Mueller-Hinton broth. Vancomycin (NOVAPLUS Hospira, Inc.
Lake Forest, IL, USA) served as a control antibiotic. MIC assays in 20% human serum were assessed
by bacterial metabolic activity in resazurin, as described [30].
3
.4. In Vitro Time-Kill Analysis
The bactericidal activity of Derivative 1e against the MRSA isolate TCH1516 was assessed by
time-kill analysis, as described previously [30–33]. Briefly, MRSA was grown overnight in
Todd-Hewitt broth (THB) at 37 °C with shaking. Following overnight growth, MRSA was inoculated
in fresh media for growth to the mid-logarithmic phase. At the start of the time-kill assay, bacteria
5
(
starting inoculum ~5 × 10 colony forming units (CFU/mL)) were added to duplicate 5-mL polystyrene
round-bottom tubes (Falcon, Bedford, MA, USA) containing 20×, 10× or 1× of the MIC of
Derivative 1e (0.39 μM) or an equivalent amount of dimethyl sulfoxide (DMSO, Sigma-Aldrich,
St. Louis, MO, USA) vehicle control (Figure 3, none). These cultures were incubated in a shaking
3
7 °C incubator for 24 h. To determine the rate of antibiotic killing, small aliquots were removed from
tubes at 0, 3, 6 and 24 h and serially diluted for CFU enumeration on Todd-Hewitt agar plates
Hardy Diagnostics, Santa Maria, CA, USA). The limit of detection for the time-kill assay was
.6 (log10 CFU/mL).
(
1

Mar. Drugs 2014, 12
. Conclusions
In our continuation of studies of novel non-symmetrical derivatives of the marine natural product,
2468
4
marinopyrrole A, we identified a derivative, designated as 1e, with favorable bactericidal activity
against MRSA (MIC = 0.19–0.78 μM). Furthermore, our time-kill studies indicate potent
concentration-dependent killing with 1e that is at least comparable or slightly better than the parent
natural product in parallel studies. One of the main drawbacks of the natural product has been its
significant reduction in anti-MRSA activity (a 128 to 256 fold increase in MIC) in the presence of
human serum. Importantly, 1e is clearly less serum-inhibited with only a 32–64 fold increase in MIC
in 20% human serum (Figure 3). Future derivatization and SAR optimization will continue to identify
more potent analogs with activity in human serum.
Acknowledgments
This work was partially supported by start-up funds from Moffitt Cancer Center and University of
Nebraska Medical Center (R.L.) and the National Institutes of Health (NIH) grant HD071600 (V.N.).
Work by N.M.H. was supported by the Ruth L. Kirschstein National Research Service Award (NRSA)
from the NIH (5 F31 GM090658-02). We are grateful to ChemAxon Software (ChemAxon, Budapest,
a
Hungary) for pK and log p calculations.
Author Contributions
Conceived and designed the experiments: MH, NMH, RL, VN. Performed the experiments: JL,
NMH, WT, YL. Analyzed the data: MH, NMH, RL, VN. Wrote the first draft of the manuscript: RL.
Contributed to the writing of the manuscript: JL, MH, NMH, RL, VN.
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2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(