A Modular Synthetic Route Involving N-Aryl-2-nitrosoaniline Intermediates Leads to a New Series of 3-Substituted Halogenated Phenazine Antibacterial Agents

Article abstract of DOI:10.1021/acs.jmedchem.1c00168

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

Full text of DOI:10.1021/acs.jmedchem.1c00168

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A Modular Synthetic Route Involving N‑Aryl-2-nitrosoaniline
Intermediates Leads to a New Series of 3‑Substituted Halogenated
Phenazine Antibacterial Agents
Hongfen Yang, Shivani Kundra, Michaelle Chojnacki, Ke Liu, Marisa A. Fuse, Yasmeen Abouelhassan,
Dimitris Kallifidas, Peilan Zhang, Guangtao Huang, Shouguang Jin, Yousong Ding, Hendrik Luesch,
Kyle H. Rohde, Paul M. Dunman, José A. Lemos, and Robert W. Huigens, III*
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ABSTRACT: Pathogenic bacteria demonstrate incredible abilities
to evade conventional antibiotics through the development of
resistance and formation of dormant, surface-attached biofilms.
Therefore, agents that target and eradicate planktonic and biofilm
bacteria are of significant interest. We explored a new series of
halogenated phenazines (HP) through the use of N-aryl-2-
nitrosoaniline synthetic intermediates that enabled functionaliza-
tion of the 3-position of this scaffold. Several HPs demonstrated
potent antibacterial and biofilm-killing activities (e.g., HP 29,
against methicillin-resistant Staphylococcus aureus: MIC = 0.075
μM; MBEC = 2.35 μM), and transcriptional analysis revealed that
HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against
Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29
demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to
significant advances in the treatment of challenging infections.
impede protein synthesis (e.g., macrolides, tetracyclines, and
aminoglycosides), while other classes inhibit cell wall synthesis
(e.g., beta-lactams and glycopeptides).1,5,6 In addition, select
antibiotic therapies inhibit DNA synthesis (e.g., quinolones),
RNA polymerase (e.g., rifamycin), and folate synthesis (e.g.,
sulfonamides), while polymyxins target and disrupt bacterial
membranes.1,5,6
Significant efforts to identify novel antibiotics that operate
through unique modes of action have been made to overcome
resistant and tolerant bacterial infections. A few recent
discoveries in the antibiotic arena include the identification
of teixobactin (targeting lipid II),24 darobactin (inhibits BamA,
an essential chaperone and translocator that folds outer
membrane proteins),25 and G0775 (synthetic arylomycin
that covalently modifies and inhibits LepB, a membrane-
bound protease that cleaves signal sequences from prepro-
teins),26 the development of eNTRy rules to guide synthetic
1. INTRODUCTION
Novel agents that target multidrug-resistant bacteria are of
critical importance due to significant clinical challenges posed
by these important human pathogens.1−5 During infection,
bacteria can rapidly develop resistance to conventional
antibiotic therapies using a multitude of mechanisms, which
include (1) target mutation to impede antibiotic binding, (2)
alterations in membrane chemistry to reduce drug penetration,
(3) increased efflux pump activity to reduce intracellular
concentrations of the antibiotic, (4) overproduction of the
antibacterial target, and (5) enzymatic deactivation of
antibiotics.1,5−10 In contrast to resistance, free-floating
planktonic bacteria communicate through quorum sensing to
coordinate virulent behaviors,11−13 including the formation of
surface-attached biofilm communities composed of enriched
populations of dormant persistent cells innately tolerant to
antibiotics.14−20 Bacterial biofilms are credited as the primary
cause of chronic and recurring infections.1,20−23 As such, new
agents capable of targeting antibiotic-resistant bacteria through
mechanisms that eradicate surface-attached biofilms are of
considerable interest to human health.
Received: January 29, 2021
Despite extraordinary chemical diversity, conventional
antibiotics operate through relatively few modes of action.5,6
Multiple classes of antibiotics inhibit bacterial ribosomes to
https://doi.org/10.1021/acs.jmedchem.1c00168
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conversion of Gram-positive antibacterials into broad-spec-
trum agents,27−29 and new group A streptogramin antibiotic
analogues that overcome virginiamycin acetyltransferase (Vat)
resistance.30 In addition, recent progress has been made to
identify agents that can effectively treat persister/biofilm
infections in mouse models, including the ClpP protease-
activating agent ADEP-4 (synthetic acyldepsipeptide)31 and
membrane-disrupting retinoid CD437.32
Our group has identified a series of halogenated phenazines
(HP) that demonstrate antibacterial and biofilm eradication
activities through a unique mechanism (Figure 1).33−39 We
Figure 2. Select halogenated phenazine analogues that demonstrate
potent antibacterial and biofilm eradication activities.
which aligned with our previous findings that HPs bind
metal(II) cations, including iron(II).36−38 Overall, we have
identified a synthetically tunable series of HPs that
demonstrates potent antibacterial activity against planktonic
cells and eradicates biofilms through a rapid iron starvation
mode of action.
Our efforts toward exploring the HP scaffold began with the
condensation of 4,5-disubstutied o-phenylenediamines 8 and
quinone 9 to yield 1-methoxyphenazines, which were then
subjected to (1) boron tribromide (BBr3) demethylation and
(2) N-bromosuccinimide (NBS)-mediated bromination to
final HPs 7 (Figure 3).35,36 In later work, we fused a series
of diverse aniline building blocks 10 with 2-nitroanisole 11
through a Wohl−Aue reaction to access new 1-methoxyphe-
nazine compounds, which were transformed to target HPs for
biological investigation.37 Most recently, our lab reported the
use of 2-bromo-3-nitroanisole 12 in a modular Buchwald−
Hartwig cross-coupling reaction with diverse anilines 10
followed by a reductive cyclization under basic conditions to
generate new 1-methoxyphenazines that were advanced to new
HPs.38 Here, we report the utilization of N-aryl-2-nitrosoani-
line intermediates to enable rapid access to HPs functionalized
at the 3-position from anilines 10 and 2-nitro-5-chloroanisole
13 to further explore structure−activity relationships of this
potent antibacterial scaffold.
Figure 1. Marine phenazine 2-bromo-1-hydroxyphenazine 1 demon-
strates antibacterial activities, and synthetic analogues 2 can eradicate
both planktonic cells and established biofilms.
were initially interested in phenazine antibiotics (e.g.,
pyocyanin and phenazine-1-carboxylic acid) utilized by
Pseudomonas aeruginosa to eradicate established Staphylococcus
aureus infections during Cystic Fibrosis (CF) disease
progression.40−43 Inspired by the interspecies competition
between P. aeruginosa and S. aureus,40,42 we synthesized a series
of phenazine antibiotics and non-natural phenazines, which
were tested for antibacterial activities against S. aureus and
Staphylococcus epidermidis.33 We discovered that 2-bromo-1-
hydroxyphenazine 1, initially isolated from a marine
Streptomyces strain, displayed the most potent antibacterial
activities against S. aureus among the naturally occurring
phenazines in our collection (1, MIC = 6.25 μM against S.
aureus and S. epidermidis). Since our initially discovery that 1
exhibits good antibacterial activities against Gram-positive
pathogens, we have synthesized a diversity of HPs 2 that have
shown incredible potency and utility as probes to better
understand biofilm viability.
1.1. Design and Synthesis of New Halogenated
Phenazines Using a Modular Route. Our previous efforts
to develop HP agents have not included synthetic chemistry
that enables exploration at the 3-position of this scaffold. We
́
Our previous studies have focused on the (1) synthesis of
diverse HPs to explore structure−activity relationships (SAR,
see HPs 3−6; Figure 2)36−38 and (2) understanding HP-14’s
mode of action by transcript profiling using RNA-seq
technology.39 Through the synthesis and microbiological
testing of >100 HP analogues, we have shown that several
substituents in the 6-, 7-, and 8-positions of the HP scaffold
significantly improve antibacterial/biofilm eradication potency,
while substitution in the 9-position abolishes antibacterial
activity. Transcript profiling of established MRSA BAA-1707
biofilms demonstrated that HP-14 rapidly induces the
transcription of several gene clusters involved in iron
acquisition (isd, iron-regulated surface determinant; sbn,
staphyloferrin B, siderophore; sfa, staphyloferrin A, side-
rophore; and MW0695, ferrichrome ABC transporter),39
came across an interesting method reported by Wrobel and co-
workers utilizing aniline starting materials to access N-aryl-2-
nitrosoaniline intermediates (e.g., 14, Scheme 1) which were
then cyclized to phenazines upon treatment with the silylating
agent N,O-bis(trimethylsilyl)acetamide (BSA) in N,N-dime-
thylformamide.44,45 In Wrobel’s approach, 3-chloro-1-methox-
́
yphenazines (15, R = H) were accessed and we envisioned this
as an entry point to investigate novel 3-subsituted HPs (e.g.,
16).
During the course of these investigations, 10 diverse,
commercially available anilines 10 were reacted with potassium
tert-butoxide (tBuOK) in the presence of 2-nitro-5-chloroani-
sole 13 to yield N-aryl-2-nitrosoaniline intermediates (Scheme
2). We had concerns regarding the stability of the
corresponding N-aryl-2-nitrosoaniline intermediates, so they
B
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Figure 3. Overview of synthesis strategies utilized to access new HPs for microbiological investigations.
Scheme 1. Utilization of N-Aryl-2-nitrosoaniline Intermediates for the Synthesis of New HPs
were each taken on crude and immediately treated with BSA to
yield diverse 3-chloro-1-methoxyphenazines 15. This pathway
proved to be fruitful as all 10 aniline starting materials were
transformed to target phenazines in 34−82% yield (average
yield = 66%) using this synthetic approach. Select N-aryl-2-
nitrosoanilines were confirmed through MS analysis during
these investigations; however, they were not fully characterized
(see the Supporting Information). Following synthesis of the
phenazine nucleus (e.g., 16), each 1-methoxyphenazine was
subjected to (1) boron tribromide (BBr3) demethylation to the
corresponding 1-hydroxyphenazine (18−27, average yield =
98%) and (2) bromination using N-bromosuccinimide (NBS)
to generate target HP analogues (28−37, average yield =
59%).
reactions with three thiol nucleophiles to generate 3-thiolated
HP analogues 45-47. During these studies, thiols were reacted
with 3-chlorophenazine compounds 19 and 38 to yield the
corresponding 3-thiolated phenazine 39−44 (Scheme 3).
Despite this reaction working well with potassium carbonate
(K2CO3) in N,N-dimethylformamide (DMF) under oil bath
heating, this reaction required 7 days to complete. Encourag-
ingly, we found that microwave conditions allowed the desired
SNAr reaction to occur in 1−3.5 min with good yields (47−
100%, Scheme 3A). Interestingly, the 3-methoxy group of
phenazine 38 was labile in the presence of the thiol nucleophile
under these reaction conditions, yielding a mixture of 1-
methoxy and 1-hydroxyphenazine (from demethylation)
products that were readily separated via column chromatog-
raphy. To circumvent problems with product mixtures, we
performed the SNAr reaction on 1-hydroxy-3-chloro-6-
In addition to accessing 3-chloro-HP analogues 18-37, we
utilized the chlorine atom as a synthetic handle in SNAr
C
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Scheme 2. Modular Synthesis of 3-Substituted HPs from Diverse Anilines 10 and 2-Nitro-5-chloroanisole 13
D
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a
Scheme 3. Chemical Synthesis of 3-Thiolated HPs Synthesized via Nucleophilic Aromatic Substitution
a
Method A: NBS, CH2Cl2, rt. Method B: NBS, PhMe, 50 °C.
methylphenazine 19 to yield the desired 3-thiolated products
in 75−89% yields (Scheme 3A, entry 2 & 5). Each of the 3-
thiol-1-hydroxyphenazines (42−44) was then subjected to
NBS bromination to yield target 3-thiolated HP analogues 45−
47 in 27−73% yield (Scheme 3B).
atoms at the 2- and 4-position of the HP scaffold, suggesting
that the 3-chlorine atom dramatically impacts the SAR profile
of these agents. Analogue 21 (subseries A) demonstrated
potent activities against Staphylococcus strains MRSA-1707,
MRSA-44, and MRSE 35984 (MIC = 0.30−0.78 μM, Table 1),
while demonstrating good antibacterial potency against the
other pathogens in the panel (VRE 700221, MIC = 1.17 μM;
E. faecalis OG1RF, MIC = 2.35 μM; Mtb H37Ra, MIC = 6.25
μM; Mtb CDC1551, MIC = 3.27 μM). HP analogue 24 also
demonstrated a similar antibacterial profile with some reduced
potency against the Enterococcus strains when compared to 21.
The remaining 3-chloro-1-hydroxyphenazines 18−20, 22, 23,
and 25−27 demonstrated a range of reduced antibacterial
activities; however, we found these profiles to be incredibly
insightful to our SAR investigations.
The 2,4-dibromo-3-chloro-1-hydroxyphenazine subseries (B,
10 analogues; Table 1) demonstrated incredible potency
against the panel of pathogenic bacteria. As an overview of
subseries B activities, seven HPs demonstrated MIC = 0.08−
0.30 μM against MRSA-1707 (vancomycin, MIC = 0.39 μM;
comparator), six HPs reported MIC = 0.08−0.20 μM against
MRSA-44 (vancomycin, MIC = 0.39 μM), eight HPs gave
MIC = 0.05−0.30 μM against MRSE 35984 (vancomycin,
MIC = 0.78 μM), seven HPs showed MIC = 0.05−0.30 μM
against VRE 700221 (vancomycin, MIC >100 μM), and five
HPs proved MIC = 0.08−0.39 μM against E. faecalis OG1RF
1.2. In Vitro Antibacterial Studies. We evaluated the new
series of 23 HP analogues functionalized at the 3-position in
antibacterial assays against a panel of pathogens, including
several antibiotic-resistant strains (methicillin-resistant S.
aureus strains MRSA-1707 and MRSA-44; methicillin-resistant
S. epidermidis MRSE 35984; vancomycin-resistant Enterococcus
faecium VRE 700221 and Enterococcus faecalis OG1RF; and
Mycobacterium tuberculosis, Mtb strains H37Ra and
CDC1551). These new HPs were categorized into three
subseries to explore and define new structure−activity
relationships regarding the HP scaffold, including subseries:
(A) 3-chloro-1-hydroxyphenazines (10 nonbrominated HP
analogues, 18−27), (B) 2,4-dibromo-3-chloro-1-hydroxyphe-
nazines (10 dibrominated HPs, 28−37), and (C) 2,4-dibromo-
3-thio-1-hydroxyphenazines (3 thiolated HPs, 45−47). Each of
the three HP subseries produced active, submicromolar potent
antibacterial agents against MRSA strains, while demonstrating
outstanding activity profiles against all bacterial pathogens
investigated (Table 1).
This was the first time that we observed potent antibacterial
activities regarding HP analogues not containing both bromine
E
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Table 1. Summary of MIC Values Obtained during Antibacterial Assessment for 3-Functionalized HP Analogues and
a
Comparator Compounds, Including Several Antibiotics
compound
MRSA-1707
3.13
1.56
25
MRSA-44
3.13
MRSE 35984
3.13
VRE 700221
6.25
4.69
E. faecalis OG1RF
S. pneumoniae 6303
Mtb H37Ra
25
Mtb CDC1551
13.7
b
1
3
18.8
c
c
b
b
c
1.56
2.35
12.5
b
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
45
46
47
18.8
>50
3.13
3.13
1.17
4.69
18.8
2.35
18.8
b
1.56
2.35
0.30
1.56
2.35
b
b
b
2.35
2.35
12.5
2.35
b
b
b
b
0.30
0.78
3.13
6.25
3.13
3.27
2.31
b
3.13
6.25
0.30
4.69
9.38
3.13
b
b
18.8
b
b
b
b
b
0.30
1.17
9.38
b
b
b
b
9.38
18.8
b
>50
3.13
0.59
>50
3.13
1.17
0.78
0.15
0.08
0.08
0.59
0.15
0.15
0.59
0.30
0.59
0.15
0.30
1.56
b
b
b
b
0.59
3.13
0.30
6.25
6.25
4.37
1.59
3.62
0.88
3.01
1.71
0.80
3.91
b
b
b
b
0.08
0.08
0.08
0.10
0.08
0.50
b
b
d
b
≤0.05
0.20
0.08
b
b
d
b
1.17
0.10
0.20
0.78
≤0.05
0.15
b
b
0.10
0.20
0.15
0.78
3.13
6.25
b
b
0.78
0.08
0.15
0.30
1.17
b
b
d
b
b
≤0.05
0.30
b
b
0.20
1.56
0.78
0.10
0.39
0.78
1.56
0.78
b
b
0.10
b
b
b
0.30
0.59
b
b
b
0.30
0.30
0.39
12.5
3.13
>50
1.69
b
6.25
2.35
6.25
4.69
4.69
2.35
b
b
b
b
48 (1-OHP)
49 (QAC-10)
EDTA
TPEN
50
4.69
50
2.35
37.5
>50
3.13
500
b
b
b
b
2.35
2.35
25
46.9
125
46.9
b
b
b
46.9
vancomycin
daptomycin
linezolid
streptomycin
0.39
3.13
12.5
0.78
>100
0.78
1.32
a
b
c
All MIC values are reported in micromolar (μM) concentrations. Midpoint of a twofold range in observed MIC values. Midpoint of a fourfold
d
range in observed MIC values. Lowest test concentration. The test range was 0.05−50 μM for all HPs and higher (e.g., 100 μM) for comparators.
EDTA and TPEN are metal-binding agent comparators. Each MIC was determined from three to nine independent experiments.
Figure 4. MIC assay results focused on selected 3-chloro HP analogues against MRSA-1707 and E. faecalis OG1RF. Note: “Van.” is for
vancomycin.
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(vancomycin, MIC = 0.78 μM). HPs 29, 30, 32, 34, and 35
each recorded submicromolar MICs against all Gram-positive
strains in the panel (see representative MIC assays, Figure 4).
Select analogues from subseries B were also evaluated against
M. tuberculosis strains and found to have good to excellent anti-
TB activities, including HPs 29 (H37Ra, MIC = 6.25 μM;
CDC1551, MIC = 1.59 μM), 31 (CDC1551, MIC = 0.88
μM), 32 (H37Ra, MIC = 3.13 μM; CDC1551, MIC = 3.01
μM), and 34 (H37Ra, MIC = 6.25 μM; CDC1551, MIC =
0.80 μM). In addition, HP 29 displayed excellent antibacterial
activity against Streptococcus pneumoniae 6303 (MIC = 0.50
μM), which is the first time we have shown HP activity against
this pathogen.
In addition to HPs bearing a chlorine atom in the 3-position
(subseries A and B), three 3-thiol HP analogues were tested for
antibacterial activities. We also wanted to utilize the SNAr
chemistry to explore water-solubilizing side chains (e.g., PEG in
HP 47). We found HP 45 bearing an ethyl thiol moiety to
have potent antibacterial activities against all Gram-positive
strains tested (MRSA, MIC = 0.30 μM; MRSE 35984, MIC =
0.30 μM; VRE 700221, MIC = 0.39 μM); however, this was a
reduction in activity compared to HP 29, which has a single
group change from 3-ethyl thiol to 3-chlorine (29 and 45
possess a 6-methyl group on the HP scaffold). The two 3-thiol
HP analogues aimed at improving water solubility (HP 46
bearing a 2-hydroxylethyl thiol moiety at the 3-position; HP 47
containing a four PEG unit thiol at the 3-position)
demonstrated significant reductions in antibacterial activities
(MICs = 2.35−12.5 μM against MRSA, MRSE, and VRE;
Table 1).
HP analogues 3, 29, 34, and 45 were evaluated in MIC
assays against six MRSA clinical isolates (Supporting
Information, Table S2) obtained from patients treated at
Shands Health Hospital (Gainesville, FL). Investigating new
agents against clinical isolates is an important early step when
working to translate interesting leads. MIC results against
MRSA clinical isolates aligned with findings in lab strains as
new HPs (29, 34, and 45) were significantly more active than
parent HP 3 (MIC = 1.25−2.5 μM). HP 29 demonstrated
outstanding antibacterial potency with MIC values between
0.06 and 0.12 μM against the MRSA isolates, which was
considerably more active than vancomycin (MIC = 0.39−0.59
μM). In addition, HP 45 (MIC = 0.31−0.63 μM) and 34
(MIC = 0.47−1.25 μM) were found to have good to excellent
activity profiles. Overall, HP analogues 29, 34, and 45 showed
remarkable activities as three of six clinical isolates were
multidrug resistant (methicillin, MIC ≥37.5 μM; ciprofloxacin,
MIC >100 μM for MDR strains).
1.3. Cytotoxicity Assessment. Following antibacterial
investigations, we evaluated a select panel of active HP
analogues against multiple mammalian cell types to determine
cytotoxicity and bacterial targeting. From this series, 12 new
HPs and parent HP 3 were evaluated against mammalian cell
lines, including (1) three nonbrominated 3-chloro HPs
(subseries A; 21, 24, and 27), (2) eight 2,4-dibromo-3-chloro
HPs (subseries B; 28−35), and (3) one 2,4-dibromo-3-thiol
HP (subseries C; 45). The cell lines used to determine
cytotoxicity of HPs included HeLa (cervical cancer cell line; 24
h LDH release assay,46 HPs were evaluated at 25, 50, and 100
μM), J774 MΦ (macrophages; 24 h Alamar Blue assay,47 HPs
tested in twofold dilutions up to 200 μM), HepG2
(hepatocellular carcinoma; 24 h Alamar Blue assay, HPs tested
in twofold dilutions up to 200 μM), and HEK-293 (human
embryonic kidney cells; 72 h MTT assay48 only HP 29 was
evaluated in twofold dilutions up to 200 μM).
Table 2. Summary of HP Cytotoxicity against HeLa, J774
a b
,
MΦ, Hep G2, and HEK-293 Cell Lines
HeLa
J774 MΦ Hep G2
HEK-293
cytotox.
(IC50)
MRSA-1707 cytotox.
cytotox.
(IC50)
cytotox.
(IC50)
compound
MIC
(IC50)
3
1.56
0.30
0.30
0.78
0.15
0.08
0.08
0.59
0.15
0.15
0.59
0.30
0.30
>100
>100
>100
100
>100
>100
>100
>200
>200
>100
>200
>200
21
24
27
28
29
30
31
32
33
34
35
45
>200
>200
>200
>200
>200
>200
>200
>200
>100
>200
>200
>200
>200
>200
>200
>200
>200
>200
18.3 5.8
>100
>25
>100
100
a
b
All results are reported in micromolar (μM) concentrations. Assay
information: HeLa cytotoxicity (24 h, LDH release assay), J774 MΦ
cytotoxicity (24 h, Alamar Blue assay), HepG2 cytotoxicity (24 h,
Alamar Blue assay), HEK-293 cytotoxicity (72 h, MTT assay). Note:
All experimental results are reported from a minimum of three
independent cytotoxicity experiments.
Results from the cytotoxicity assessment of new HPs were
very encouraging (Table 2). The majority of new HPs reported
IC50 > 100 μM against HeLa cells (six of nine new HPs
evaluated) with HP 33 reporting the most cytotoxicity against
HeLa cells with an IC50 > 25 μM. When tested against J774
MΦ and HepG2 cells, nearly all new HPs reported IC50 > 200
μM, resulting in a selectivity index (SI) of >2000 when
considering the MIC values of HP analogues 29 and 30 against
MRSA, MRSE, and VRE strains (MIC = 0.05−0.10 μM).
HEK-293 cells demonstrated increased sensitivity toward HP
29 (IC50 = 18.3
5.8 μM), however, gave a very good
selectivity index (SI = 244) when comparing its IC50 against
HEK-293 to MIC values against MRSA/MRSE strains (MIC =
0.075 μM). Overall, this new series of HPs demonstrates very
good to outstanding bacterial targeting results based on relative
cytotoxicity profiles to antibacterial activities.
1.4. Biofilm Eradication and Iron Starvation. Initial
MIC assays allowed us to determine planktonic growth
inhibition activities of new HP analogues; however, HPs
eradicate surface-attached bacterial biofilms with excellent
potency, which require different microbiological assays. To
investigate the biofilm-eradication activities of select com-
pounds, our group has utilized calgary biofilm device (CBD)
assays to determine minimum biofilm eradication concen-
tration (MBEC) values.35−38 The CBD is a 96-well assay that
has a specialized lid with anchored pegs that are submerged
into microtiter wells, allowing biofilm formation and
subsequent transfer to fresh 96-well plates as the assay
progresses.49,50 Biofilm eradication assays have three phases,
including the (1) biofilm establishment phase (inoculated
media in a 96-well plate with CBD pegs submerged into
microtiter wells providing a surface for biofilms to establish; 24
h incubation under static conditions), (2) compound treat-
ment phase (pegs with established biofilms are gently rinsed to
remove planktonic cells and transferred to a new 96-well plate
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a
Table 3. Summary of Biofilm Eradication Studies against MRSA, MRSE, VRE, and E. faecalis Biofilms
MRSA-1707
MBC/MBEC
MRSA 44
MBC/MBEC
MRSE 35984
MBC/MBEC
VRE 700221
MBC/MBEC
E. faecalis OG1RF
MBC/MBEC
% hemolysis at
compound
200 μM
c
c
b
b
c
c
b
c
c
d
3
21
24
27
28
29
30
31
32
33
34
35
36
37
45
50 /100
37.5 /75
25 /50
18.8 /12.5
25 />200
≤1
≤1
≤1
1.9
≤1
≤1
≤1
27
2.6
≤1
≤1
≤1
5.1
3.0
5.7
>99
≤1
≤1
b
b
18.8 /37.5
18.8 /75
37.5 /37.5
50/50
1.56 /2.35
b
b
b
b
b
b
c
c
b
b
b
b
d
37.5 /75
12.5 /25
1.17 /0.59
3.13 /2.35
4.69 /1.17
1.56 /1.56
12.5/12.5
0.59 /0.30
2.35 /2.35
6.25/6.25
6.25/6.25
12.5 /4.69
3.13/3.13
250/>2000
4.69 /2.35
75 />200
c
b
b
c
c
b
b
b
b
b
4.69 /6.25
0.59 /0.59
6.25/18.8
b
b
c
b
c
b
18.8 /4.69
0.78 /0.30
b
b
b
b
c
c
75 /75
2.35 /0.20
1.56 /0.78
18.8 /75
b
b
b
c
c
c
b
b
9.38 /2.35
12.5 /18.8
2.35 /3.13
4.69 /4.69
4.69 /6.25
0.78 /0.39
c
b
c
b
3.13 /1.17
b
c
c
c
b
b
b
c
c
b
6.25 /6.25
0.39 /0.20
3.13 /1.17
6.25/6.25
b
b
c
b
b
b
b
b
c
6.25 /18.8
0.59 /0.30
b
12.5/12.5
4.69 /0.59
b
b
12.5/9.38
4.69 /3.13
c
c
b
b
c
50 /200
9.38 /3.13
b
b
b
b
49 (QAC-10)
TPEN
EDTA
Vancomycin
Daptomycin
Linezolid
93.8 /93.8
3.0 /3.0
b
c
375 />2000
>2000/>2000
7.8/>2000
125/>2000
31.3/>2000
188 />2000
500/>2000
>2000/>2000
11.7 />2000
1000/>2000
c
b
b
7.8/>2000
3.0 />2000
750 />2000
b
375/93.8
b
4.69 /1.56
a
b
c
All biological results are reported in micromolar (μM) concentrations. Midpoint of a twofold range in observed values. Midpoint of a fourfold
d
range in values. Partial turbidity observed at the highest test concentration. All values in this table resulted from a minimum of three independent
experiments. Halogenated phenazines and QAC-10 were tested at concentrations of up to 200 μM. Conventional antibiotics (e.g., vancomycin),
EDTA, and TPEN (metal-binding agents) were tested at concentrations of up to 2000 μM in CBD assays.
Figure 5. (A) CBD assay of a panel of HPs, vancomycin and TPEN against MRSA-1707. (B) MRSA-1707 biofilm cell killing (CFU/peg) for HPs
29 and 34 obtained from CBD pegs.
containing a twofold serially diluted test compound in fresh
media; 24 h incubation), and (3) recovery phase (lid bearing
compound-treated biofilms on CBD pegs is transferred to a
final 96-well plate with fresh media only; 24 h incubation).
Upon completion of CBD assays, biofilm eradication is
determined by a turbidity readout. Microtiter wells that are
turbid result from viable biofilms on CBD peg surfaces (live
biofilms will disperse planktonic bacteria into fresh media and
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Figure 6. (A) Treating established MRSA-1707 biofilms with HPs 3, 28, and 29 leads to a rapid induction of iron starvation. RT-qPCR results of
HPs 3, 28, and 29 upregulating iron uptake genes in MRSA-1707 biofilms following 4 h treatment at (B) 1 μM or 1/10 x MBEC; *p value ≤ 0.05,
**p ≤ 0.01 (Student’s T-test). (C) UV−vis spectroscopy of HPs 18, 28, and 29 binding iron(II). Note: In previous work, we show that EDTA and
TPEN do not upregulate this panel of iron uptake genes at 5 μM.39
undergo replication/bacterial growth), whereas nonturbid
microtiter wells result from completely eradicated biofilms.
The lowest concentration at which biofilm eradication occurs
is referred to as the minimum biofilm eradication concen-
tration (MBEC) value.
The CBD allows one to determine planktonic versus biofilm
cell killing dynamics of small molecules as these assays allow
for the determination of minimum bactericidal concentration
(MBC) and MBEC values simultaneously. This is a significant
advantage over comparing MBEC to MIC values obtained
from significantly different microbiological assays. In the past,
we have found HPs to report MBC/MBEC ratios of 1:1 to 1:3
from CBD assays, demonstrating near equipotent planktonic-
and biofilm-killing properties.35−38
Since biofilm eradication assays are more sophisticated than
MIC assays and HP’s antibacterial potency typically correlates
to biofilm-killing activities, we advanced a subset of 14 new
HPs to MBEC assays against MRSA-1707 (Table 3). We
investigated three nonbrominated HPs from subseries A (21,
24, and 27) and found these analogues to have good activities
with HPs 21 and 24 demonstrating the highest levels of biofilm
killing (MBEC = 37.5 μM, Table 3). We evaluated all subseries
B (dibrominated HP) analogues bearing a 3-chloro substituent
in MBEC assays since these compounds demonstrated MIC
values ≤0.59 μM against MRSA-1707. Each of the 10
dibrominated HP analogues (subseries B) reported MBEC
values ≤75 μM with five analogues demonstrating outstanding
biofilm killing with MBECs at 2.35−4.69 μM (HPs 29, 30, 32,
34, and 35, see Figure 5A). Finally, 3-thiolated HP 45
demonstrated relatively weak biofilm-killing activities against
MRSA-1707 (MBEC = 200 μM).
In addition, dose−response of biofilm killing was deter-
mined from CFU counts from CBD pegs against MRSA-1707
treated with HPs 29 (2.55
0.39 log10 reduction of viable
biofilm cells at the MBEC value, Figure 5B) and 34 (3.03
0.21 log10 reduction of viable MRSA-1707 biofilm cells).
Together, HPs 29 and 34 demonstrated ∼3 log10 reduction or
∼99.9% kill of MRSA-1707 biofilm cells at the corresponding
MBEC value, similar to previous findings.35−38 Subseries B
analogues 29, 32, 34, and 35 were also evaluated for biofilm
eradication activities against MRSA BAA-44 and demonstrated
excellent biofilm-killing potencies with MBEC values between
6.25 and 18.8 μM (Table 3).
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Figure 7. MRSE 35984 biofilm eradication results with HPs 29 and 34 from CBD assays. (A) Turbidity results to determine planktonic and biofilm
eradication (MBC/MBEC values) and (B) biofilm killing from viable cell counts on CBD pegs.
In addition to MBEC assays, we showed that HPs 3, 28, and
29 rapidly induce iron starvation in MRSA-1707 biofilms using
RT-qPCR experiments (Figure 6), which is in line with
previous findings regarding 4 (HP-14).39 Briefly, established
MRSA-1707 biofilms were treated for 4 h with 3, 28, and 29
with 1/10 × MBEC and 1 μM of each compound before RNA
was extracted from HP- and vehicle-treated biofilm samples.
RT-qPCR was then performed to investigate transcript levels
from four different MRSA genes involved in iron uptake: isdB
(iron regulated surface determinant; heme iron acquisition),
sfaA (staphyloferrin A; siderophore), sbnC (staphyloferrin B;
siderophore), and MW0695 (ferrichrome ABC transporter).
Results from these experiments show that HPs induce a rapid
upregulation of gene clusters involved in iron uptake (Figure
6B), and we conclude that biofilm eradication occurs due to
iron starvation. HPs 18, 28, 29, and 34 were confirmed to
directly bind iron(II) via UV−vis spectroscopy (see Figure 6C
and Supporting Information), aligning with our RT-qPCR
results related to the iron starvation of MRSA biofilms.
Following initial biofilm-killing studies in MRSA, select HPs
were evaluated against other Gram-positive pathogens in CBD
assays (Table 3). Several HPs demonstrated good (MBEC =
12.5−25 μM) to outstanding (MBEC = 0.30−6.25 μM)
biofilm-killing potencies against MRSE 35984 with 29 (MBEC
= 0.59 μM), 31 (MBEC = 1.17 μM), 32 (MBEC = 1.56 μM),
and 34 (MBEC = 0.30 μM) proving to be the most active.
Dose-dependent killing of MRSE 35984 biofilm bacteria was
determined by CFU counts from CBD pegs treated with HPs
29 (3.65 1.33 log10 reduction of viable MRSE biofilm cells at
In addition, HP analogues from subseries B demonstrated
good to excellent biofilm eradicating activities against enter-
ococci strains (Table 3). Seven HPs showed remarkable
biofilm-killing activity against E. faecium 700221 with MBEC
values 0.20−0.59 μM (HP 31 and 34, MBEC = 0.20 μM).
Select HPs were evaluated against E. faecalis OG1RF biofilms
with 31 (MBEC = 0.78 μM), 34 (MBEC = 1.17 μM), and 35
(MBEC = 6.25 μM) proving to have the highest killing
activities.
Vancomycin, daptomycin, and linezolid are frontline anti-
biotics used to treat MRSA infections and, in our CBD assays,
failed to eradicate MRSA-1707 biofilms despite effective
planktonic killing (e.g., vancomycin, MBC = 7.8 μM against
planktonic cells, MBEC >2000 μM against MRSA biofilms;
Table 3). In addition, QAC-10 is a membrane-lysing
quaternary ammonium cation that displays good biofilm-
eradicating activities51 and serves as a valuable comparator in
these investigations. QAC-10 eradicated MRSA-1707 biofilms
with an MBEC of 93.8 μM in CBD assays, which is 20-fold less
potent than our most active HPs in subseries B. In addition,
EDTA and TPEN (membrane-permeable agent) are general
metal-chelating agents and are unable to eradicate biofilms at
the highest test concentration (MBECs >2000 μM; Table 3).
We then assessed the ability of our most active HP
analogues to lyse red blood cells (RBCs). Hemolysis assays
are used to determine the membrane-lysis activity of a
compound, which is of particular interest to this work as
membrane-lysing agents (e.g., QACs) can eradicate biofilms.
During these investigations, we observed minimal hemolytic
activity for nearly all new HP compounds at 200 μM (≤5%
hemolysis; Table 3), with the exception of 31 which reported
27% hemolysis of RBCs. QAC-10 was tested alongside HP
analogues as an active membrane-lysing agent (comparator)
and caused >99% hemolysis of RBCs at 200 μM.
the MBEC value; Figure 7B) and 34 (3.18
0.40 log10
reduction of viable MRSE biofilm cells at the MBEC value). In
addition, these dose−response experiments show that HPs 29
and 34 eradicate ∼99.999% MRSE 35984 biofilm cells at 3.13
μM (∼5 log10 reduction of viable CFUs).
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Figure 8. In vivo assessment of HP 29 in dorsal wound infections of S. aureus and E. faecalis in mice. (A) HP 29 reduces S. aureus UAMS-1 bacterial
load in BALB/c mice (Student’s T-test: p value = 0.010; seven mice per group). (B) HP 29 reduces E. faecalis OG1RF bacterial load in C57BL/6J
mice (comparing mice treated with 29 and infection control; ANOVA: day 3, p value = 0.068).
Figure 9. Structure−activity relationship profiles for new analogues functionalized at the 3-position of the HP scaffold. Note: MRSA data refer to
MRSA-1707 findings in this SAR figure.
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1.5. In Vivo Assessment of HP 29 in Dorsal Wound
Infection Models. Based on its potent antibacterial activities,
rapid induction of iron starvation in MRSA biofilms, and
excellent cytotoxicity profile, HP 29 was evaluated for in vivo
efficacy against S. aureus and E. faecalis using dorsal wound
infection models in mice. S. aureus and E. faecalis are highly
prevalent in both hospital-acquired and wound infections.52,53
In addition, these pathogens are notorious for their antibiotic-
resistant phenotypes and propensity to form tolerant
biofilms.54,55
For the in vivo experiments, HP 29 was formulated in a
PEG-based ointment52 for topical application. In separate
experiments, wounds were created on the mouse’s dorsal mid-
section using a biopsy punch to remove the dermal layer. The
resulting wound was inoculated with either S. aureus UAMS-1
(1 × 107 CFU/mL) or E. faecalis OG1RF (6 × 1010 CFU/mL)
to establish infection. Following infection, PEG ointment
containing HP 29 or PEG ointment alone (vehicle control)
was directly applied to the infected wounds twice (S. aureus
infection) or once (E. faecalis infection) for three days before
mice were sacrificed, and the bacterial load in each wound was
determined as CFU per lesion (Figure 8).
Results from the dorsal wound infection experiments
demonstrated that HP 29 treatment led to a significant
decolonization of both S. aureus and E. faecalis. HP 29-treated
mice led to a 0.82-log10 reduction in CFU per lesion of S.
aureus UAMS-1 compared to vehicle-treated mice following 3
consecutive days of treatment (Figure 8A). Against E. faecalis
OG1RF, HP 29 showed more efficacious decolonization with
1.73-log10 reduction compared to vehicle control after
treatment (Figure 8B). Collectively, these results indicate
that HP agents are a useful alternative for the topical treatment
of wounds infected by Gram-positive pathogens.
1.6. Structure−Activity Relationship Analysis. This
new collection of HPs has significantly expanded structure−
activity relationship profiles for these antibacterial agents
(Figure 9). In addition to the synthesis and biological
assessment of 3-substituted HPs, we determined pKa and
CLogP values (ChemDraw) of select analogues for further
analysis.
New key insights into HP antibacterial agents from these
studies include the following: (1) a 3-chlorine atom on the HP
scaffold increases the antibacterial/biofilm eradication activ-
ities, acidity of the phenolic proton, and CLogP values (see
subseries B, Figure 9), (2) bromine/chlorine/iodine atoms at
the 2- and 4-positions of the HPs are not critical for
antibacterial activities, as previously determined,33,35−38 based
on new findings with subseries A analogues 21 and 24 that
demonstrate potent antibacterial activities and very good
biofilm eradication activities, and (3) thiol substituents at the
3-position diminished antibacterial activities compared to 3-
chlorinated HP analogues (comparing subseries C to subseries
B analogues) and reduced acidity of the phenolic proton.
As noted, the 3-chlorine atom has a significant impact on the
acidity of the phenolic proton of HP analogues, and we believe
that this primes the HP scaffold for iron binding as the alkoxy
anion. In addition, the 3-chlorine atom increases the CLogP
value of HP analogues (that are largely anionic in biological
assays performed at pH ∼ 7), which could lead to rapid
diffusion through bacterial membranes to bind intracellular
iron(II). For instance, parent HP 3 is without a 3-chlorine
atom and has an MIC = 1.56 μM (MRSA), MBEC = 100 μM
(MRSA), a pKa of 7.12, and a CLogP value of 4.68, whereas
HP 28 has the addition of a chlorine atom in the 3-position of
the HP and has an MIC = 0.15 μM (MRSA), MBEC = 50 μM
(MRSA), a pKa of 5.93, and a CLogP of 5.12 (Figure 9). A
similar profile can be observed when comparing HPs 6 (MIC =
0.30 μM, MRSA; MBEC = 6.25 μM, MRSA; CLogP = 5.18;
pKa not determined) and 29 (MIC = 0.08 μM, MRSA; MBEC
= 2.35 μM, MRSA; CLogP = 5.62; pKa = 6.79; Figure 9).
Several HPs from subseries B demonstrate high levels of
planktonic and biofilm-killing activities against Gram-positive
pathogens, including multidrug-resistant strains. HP 29
demonstrated outstanding in vitro activities in addition to
encouraging in vivo efficacy in dorsal wound infections against
S. aureus UAMS-1 and E. faecalis OGRF1. Multiple HPs
demonstrated rapid iron starvation in MRSA biofilms as a
result of their ability to bind iron between the hydroxyl oxygen
at the 1-position of the HP scaffold and nitrogen at the 10-
position. Active HPs identified during these studies are ideal
candidates for prodrug development through functionalization
of the 1-hydroxyl group aimed to enhance physicochemical
properties, prevent rapid metabolism (phenol conjugation),
mitigate off-target metal binding that could lead to toxicity, and
release via bacteria-specific mechanisms.37,38,56,57 Prodrug
efforts related to new HP scaffolds from this study are
underway, and findings will be reported in due course.
2. CONCLUSIONS
In conclusion, we utilized aniline building blocks to rapidly
access a diverse series of N-aryl-2-nitrosoaniline intermediates
en route to new halogenated phenazine antibacterial agents.
This chemistry enabled the first study of HPs functionalized at
the 3-position with chlorine or thiol substituents. This
collection of >20 HP analogues demonstrated highly potent
antibacterial and biofilm eradication activities against Gram-
positive pathogens (e.g., HP 29, MIC = 0.075 μM, MBEC =
2.35 μM against MRSA-1707), and multiple HPs were shown
to induce rapid iron starvation in MRSA biofilms. In addition,
several new HPs reported good to excellent activities against
M. tuberculosis (e.g., HP 34, MIC = 0.80 μM against M.
tuberculosis CDC1551). Three diverse subseries of HPs
provided significant SAR insights into this antibacterial
scaffold, and HP 29 demonstrated in vivo efficacy against S.
aureus and E. faecalis in wound infection models in mice. These
findings could lead to significantly improved treatment options
for antibiotic-resistant and -tolerant bacterial infections,
including wound and chronic biofilm-associated infections.
3. EXPERIMENTAL SECTION
3.1. General Information. All reagents for chemical synthesis
were purchased at ≥95% purity from commercial sources and used
without further purification. All microwave reactions were carried out
using an Anton Paar Monowave 300 microwave synthesis reactor. A
constant power was applied to ensure reproducibility regarding
microwave reactions as temperature control was automated via the IR
sensor and all indicated temperatures correspond to the maximal
temperature reached during each experiment. Analytical thin layer
chromatography (TLC) was performed using 250 μm silica gel 60
F254 precoated plates (EMD Chemicals Inc.) and used to monitor all
reactions. Flash column chromatography was performed using 230−
400 Mesh 60Å silica gel from Sorbent Technologies. Melting points
were obtained, uncorrected, using a Mel-Temp capillary melting point
apparatus from Laboratory Services, Inc.
NMR experiments were recorded using broadband probes on a
Varian Mercury-Plus-400 spectrometer via VNMR-J software (400
MHz for 1H and 101 MHz for 13C), Varian Mercury-Plus-500
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1
spectrometer via VNMR-J software (500 MHz for H and 126 MHz
for 13C), Bruker Avance III (500 MHz for 1H; 126 MHz for 13C), and
Bruker Avance II (600 MHz for 1H; 151 MHz for 13C). All spectra are
presented using MestReNova 11.0 (Mnova) software and are
displayed without the use of the signal suppression function. Spectra
silica gel chromatography using hexanes/ethyl acetate (99:1 to 85:15)
to afford a yellow solid as a second product batch, which was
combined with the solid obtained above to afford 57 (413 mg, 80%
yield).
3.2.1.2. 3-Chloro-1-methoxy-6-methylphenazine (38). Yield:
1
1
were obtained in the following solvents (reference peaks for H and
80%; 578 mg was isolated as a yellow solid. H NMR (400 MHz,
13C NMRs are included): CDCl3 (1H NMR, 7.26 ppm; 13C NMR,
CDCl3): δ 8.18 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.74−
7.62 (m, 2H), 6.98 (d, J = 2.0 Hz, 1H), 4.16 (s, 3H), 2.87 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 155.6, 143.8, 143.3, 142.4, 137.8, 136.3,
135.2, 130.7, 130.6, 128.2, 120.7, 108.7, 57.0, 17.8. HRMS (ESI):
calcd for C14H12ClN2O [M + H]+, 259.0633; found, 259.0642. mp
178−180 °C.
3.2.1.3. 3-Chloro-1-methoxyphenazine (50). Yield: 44%; 105 mg
was isolated as a yellow solid. Note: 1H NMR and 13C NMR spectral
data match those previously reported for this compound (CAS:
1346682-87-8).44
77.23 ppm) and DMSO-d6 (1H NMR, 2.50 ppm; 13C NMR, 39.52
ppm). All NMR experiments were performed at room temperature.
Chemical shift values (δ) are reported in parts per million (ppm) for
all 1H NMR and 13C NMR spectra. 1H NMR multiplicities are
reported as follows: s = singlet, br. s = broad singlet, d = doublet, t =
triplet, q = quartet, and m = multiplet. HSQC was used to identify a
few challenging 13C signals, and these spectra are reported in the
Supporting Information. High-resolution mass spectrometry (HRMS)
was obtained for all new compounds from the Chemistry Department
at the University of Florida.
3.2.1.4. 8-Chloro-6-methoxy-1,2-dimethylphenazine (51). Yield:
1
72%; 211 mg was isolated as a yellow solid. H NMR (400 MHz,
Multiple controls and comparator agents (e.g., antibiotics and
EDTA) were purchased and used in biological assays. All synthesized
compounds evaluated in biological assays were determined to be
≥95% pure using a Shimadzu Prominence HPLC system, an AB Sciex
3200 QTRAP spectrometer, and a Kinetex C18 column (50 mm ×
2.1 mm × 2.6 μm) with a 21 min linear gradient from 10 to 80%
acetonitrile in 0.1% formic acid at a flow rate of 0.25 mL/min (traces
and purity analysis can be viewed in the Supporting Information). All
compounds were stored as DMSO stocks at room temperature in the
absence of light for several months at a time without observing losses
in biological activity. To ensure the integrity of DMSO stock solutions
of test compounds, they were not subjected to freeze−thaw cycles.
Bacterial strains used during these investigations include S. aureus
BAA-1707 (“MRSA-1707”), BAA-44 and UAMS-1, S. epidermidis
ATCC 35984, E. faecium ATCC 700221, E. faecalis OG1RF (ATCC
47077), S. pneumoniae ATCC 6303, M. tuberculosis H37Ra (ATCC
25177), and CDC1551. All animal experiments performed were
conducted in compliance with institutional guidelines.
3.2. Chemistry. This chemistry section includes the following
items: (a) synthetic procedures and compound characterization
(ordered by the synthetic route; note: most 1-methoxyphenazines
synthesized during these studies are not presented in the manuscript;
however, their full structures can be viewed in the Supporting
Information Figure 2 and spectra section), (b) UV−vis for HPs
binding iron(II), and (c) pKa determination of select HP analogues.
3.2.1. Synthetic Procedures and Compound Characterization.
3.2.1.1. General Two-Step Procedure for the Synthesis of 3-Chloro-
1-methoxyphenazines (38, 50−58).44,45 Step 1. 4-Bromoaniline
(303 mg, 1.76 mmol) dissolved in 2 mL of N,N-dimethylformamide
was added dropwise to a stirring solution of potassium tert-butoxide
(416 mg, 4.80 mmol) in N,N-dimethylformamide (12 mL) at −60 °C.
Then, a solution of 5-choloro-2-nitroanisole 13 (300 mg, 1.60 mmol)
in N,N-dimethylformamide (2 mL) was added to the mixture which
continued to stir at −60 °C for 7 h. Upon completion of this reaction,
the resulting mixture was transferred to a separatory funnel containing
saturated aqueous ammonium chloride (80 mL) and the crude
product was extracted with ethyl acetate (3 × 30 mL). The organic
layers were then combined, washed with brine, and dried with sodium
sulfate. The resulting organic layer was then filtered and concentrated
in vacuo to obtain the crude nitroso intermediate that was used
directly in the next step. Step 2. The crude nitroso intermediate (oil)
was dissolved in N,N-dimethylformamide (10 mL) before N,O-
bis(trimethylsilyl)acetamide (1.97 mL, 8.00 mmol) was added to the
solution. The resulting mixture was allowed to stir at 50 °C for 16 h
until completion. After this time, 2 mL of water was added to the
mixture and stirring continued at room temperature for an additional
10 min before the precipitate was filtered and washed with cold ethyl
acetate (this solid was a batch of the desired product). The filtrate was
then transferred to a separatory funnel containing brine (50 mL) and
extracted with ethyl acetate (3 × 30 mL) to isolate additional product.
After extraction of the filtrate, the organic layers were combined and
washed with water (3 × 50 mL), dried with sodium sulfate, filtered,
and concentrated in vacuo. The resulting crude solid was purified via
CDCl3): δ 8.10 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 1.5, 1H), 7.64 (d, J =
9.0 Hz, 1H), 6.98 (d, J = 1.5 Hz, 1H), 4.17 (s, 3H), 2.82 (s, 3H), 2.57
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 155.7, 143.7, 143.3, 141.2,
139.0, 136.1, 134.6, 134.5, 134.2, 127.1, 120.7, 108.3, 57.0, 21.0, 13.3.
HRMS (ESI): calcd for C15H14ClN2O [M + H]+, 273.0789; found,
273.0799. mp 174−176 °C.
3.2.1.5. 2-Bromo-8-chloro-6-methoxy-1-methylphenazine (52).
1
Yield: 72%; 388 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl3): δ 8.04 (d, J = 9.3 Hz, 1H), 7.90 (d, J = 9.3 Hz, 1H),
7.84 (d, J = 2.0 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 4.17 (s, 3H), 2.95
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 155.6, 143.6, 143.4, 141.2,
137.5, 137.2, 135.1, 135.0, 128.7, 127.4, 120.6, 109.0, 57.1, 17.6.
HRMS (ESI): calcd for C14H11BrClN2O [M + H]+, 336.9738; found,
336.9734. mp 218−220 °C.
3.2.1.6. 3-Chloro-1-methoxy-6-phenoxyphenazine (53). Yield:
1
69%; 445 mg was isolated as a yellow solid. H NMR (600 MHz,
CDCl3): δ 8.08 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.68
(dd, J = 8.8, 7.6 Hz, 1H), 7.47−7.41 (m, 2H), 7.26−7.21 (m, 3H),
7.08 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 2.0 Hz, 1H), 4.20 (s, 3H). 13C
NMR (151 MHz, CDCl3): δ 156.4, 155.5, 154.1, 143.2, 143.0, 137.7,
137.0, 135.9, 130.2, 130.1, 124.8, 124.3, 120.8, 120.8, 114.7, 109.3,
57.0. HRMS (ESI): calcd for C19H14ClN2O2 [M + H]+, 337.0738;
found, 337.0752. mp 178−180 °C. Note: TMS was used as the
reference in this 1H NMR spectrum at 0.00 pm due to the chloroform
peak being buried in proton signals.
3.2.1.7. 3-Chloro-8-fluoro-1-methoxyphenazine (54). Yield: 82%;
344 mg was isolated as a yellow solid. 1H NMR (400 MHz, CDCl3): δ
8.21 (dd, J = 9.5, 5.9 Hz, 1H), 7.96 (dd, J = 9.4, 2.8 Hz, 1H), 7.82 (d,
J = 2.0 Hz, 1H), 7.68 (ddd, J = 9.5, 7.9, 2.8 Hz, 1H), 7.03 (d, J = 2.0
Hz, 1H), 4.18 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 163.2 (d, J =
255.7 Hz), 155.5, 143.6 (d, J = 2.8 Hz), 142.7 (d, J = 13.7 Hz), 141.7,
136.8 (d, J = 1.8 Hz), 136.0, 131.9 (d, J = 10.2 Hz), 123.6 (d, J = 28.3
Hz), 120.3 (d, J = 1.2 Hz), 112.6 (d, J = 21.5 Hz), 109.5, 57.1. HRMS
(ESI): calcd for C13H9ClFN2O [M + H]+, 263.0382; found, 263.0394.
mp 221−223 °C.
3.2.1.8. 1,3,8-Trichloro-6-methoxyphenazine (55). Yield: 34%;
175 mg was isolated as a yellow solid. 1H NMR (400 MHz, CDCl3): δ
8.26 (s, 1H), 7.98−7.86 (m, 2H), 7.03 (s, 1H), 4.17 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 155.5, 143.7, 142.2, 139.4, 138.2, 136.3,
135.7, 134.2, 132.0, 127.9, 120.6, 110.1, 57.2. HRMS (DART): calcd
for C13H8Cl3N2O [M + H]+, 312.9697; found, 312.9696. mp 221−
223 °C.
3.2.1.9. 6-Bromo-3-chloro-1-methoxyphenazine (56). Yield: 61%;
183 mg was isolated as a yellow solid. 1H NMR (500 MHz, CDCl3): δ
8.34 (dd, J = 8.8, 1.3 Hz, 1H), 8.20 (dd, J = 7.3, 1.3 Hz, 1H), 7.99 (d,
J = 2.0 Hz, 1H), 7.68 (dd, J = 8.8, 7.3 Hz, 1H), 7.05 (d, J = 2.0 Hz,
1H), 4.19 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 155.5, 144.2,
142.6, 141.5, 137.9, 136.0, 134.7, 130.5, 130.3, 124.1, 120.6, 109.6,
57.1. HRMS (ESI): calcd for C13H9BrClN2O [M + H]+, 322.9581;
found, 322.9578. mp 137−139 °C.
3.2.1.10. 8-Bromo-3-chloro-1-methoxyphenazine (57). Yield:
1
80%; 413 mg was isolated as a yellow solid. H NMR (400 MHz,
M
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Featured Article
CDCl3): δ 8.55 (d, J = 2.1 Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.89
(dd, J = 9.2, 2.1 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 2.0 Hz,
1H), 4.17 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 155.7, 144.1,
142.7, 142.3, 137.5, 135.9, 135.3, 132.3, 130.7, 125.1, 120.3, 109.5,
57.1. HRMS (ESI): calcd for C13H9BrClN2O [M + H]+, 322.9581;
found, 322.9580. mp 225−227 °C.
3.2.1.11. 3-Chloro-8-iodo-1-methoxyphenazine (58). Yield: 65%;
388 mg was isolated as a yellow solid. 1H NMR (400 MHz, CDCl3): δ
8.83 (d, J = 1.9 Hz, 1H), 8.06 (dd, J = 9.1, 1.9 Hz, 1H), 7.89 (d, J =
9.1 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 4.17
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 155.8, 144.1, 143.0, 142.5,
140.2, 139.3, 137.6, 135.7, 130.4, 120.2, 109.4, 97.2, 57.1. HRMS
(ESI): calcd for C13H9ClIN2O [M + H]+, 370.9443; found, 370.9448.
mp 224−226 °C.
3.2.1.12. Boron Tribromide Demethylation of 3-Chloro-1-
methoxyphenazines to 3-Chloro-1-hydroxyphenazines (18−27).
Compound 38 (200 mg, 0.77 mmol) was dissolved in anhydrous
dichloromethane (50 mL) in a round bottom flask. The solution was
then cooled to −78 °C before a 1 M solution of boron tribromide
(4.6 mL, 4.6 mmol in dichloromethane) was added dropwise. The
resulting reaction mixture was allowed to stir at −78 °C for 1 h before
being warmed to room temperature overnight. After this time, the
reaction was heated to reflux until completion (monitored by TLC).
Upon completion, brine (50 mL) was added to the mixture to quench
the reaction. The resulting mixture was then transferred to a
separatory funnel and extracted with dichloromethane. The combined
organic layers were dried with sodium sulfate, filtered, and
concentrated in vacuo. The resulting solid was purified via column
chromatography using dichloromethane to elute compound 19 as a
yellow solid (191 mg, 100%).
3.2.1.13. 3-Chlorophenazin-1-ol (18). Yield: 89%; 84.0 mg was
isolated as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.27 (br s,
1H), 8.26−8.18 (m, 2H), 7.92−7.83 (m, 2H), 7.79 (d, J = 2.0 Hz,
1H), 7.22 (d, J = 2.0 Hz, 1H). 13C NMR (101 MHz, CDCl3): δ 152.4,
145.0, 143.8, 141.2, 138.3, 133.5, 131.6, 131.0, 129.9, 129.4, 118.9,
111.0. HRMS (ESI): calcd for C12H8ClN2O [M + H]+, 231.0320;
found, 231.0329. mp 213−215 °C. HPLC purity: 98.7%.
3.2.1.18. 3-Chloro-8-fluorophenazin-1-ol (23). Yield: 100%; 95
mg was isolated as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ
11.32 (br s, 1H), 8.29 (ddd, J = 9.4, 6.1, 0.7 Hz, 1H), 8.01−7.91 (m,
2H), 7.73 (d, J = 2.2 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6): δ 162.5 (d, J = 253.0 Hz), 154.6, 143.0 (d, J =
2.5 Hz), 141.6 (d, J = 13.9 Hz), 141.0, 136.2 (d, J = 1.7 Hz), 135.0,
131.9 (d, J = 10.5 Hz), 123.6 (d, J = 28.4 Hz), 117.6, 112.0, 111.7 (d,
J = 21.1 Hz). HRMS (ESI): calcd C12H5FClN2O for [M − H]−,
247.0080; found, 247.0078. mp 214−216 °C. HPLC purity: >99.9%.
3.2.1.19. 3,6,8-Trichlorophenazin-1-ol (24). Yield: 96%; 116 mg
1
was isolated as a yellow solid. H NMR (600 MHz, CDCl3): δ 8.15
(d, J = 2.2 Hz, 1H), 8.06 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.92 (d, J =
2.0 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H). 13C NMR (101 MHz, CDCl3):
δ 152.1, 143.4, 141.2, 140.2, 139.5, 136.1, 134.7, 134.1, 132.1, 127.0,
119.4, 112.6. HRMS (ESI): calcd for C12H4Cl3N2O [M − H]−,
296.9395; found, 296.9403. mp 234−236 °C. HPLC purity: 97.6%.
3.2.1.20. 6-Bromo-3-chlorophenazin-1-ol (25). Yield: 100%; 141
1
mg was isolated as a yellow solid. H NMR (400 MHz, CDCl3): δ
8.26−8.18 (m, 2H), 8.15 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.71 (dd, J
= 8.7, 7.3 Hz, 1H), 7.26 (m, 1H, partially buried in the reference
signal). 13C NMR (151 MHz, CDCl3): δ 152.2, 143.9, 142.3, 141.7,
139.1, 134.8, 133.9, 130.8, 129.3, 124.7, 119.4, 111.9. HRMS (ESI):
calcd for C12H5BrClN2O [M − H]−, 306.9279; found, 306.9277. mp
223−225 °C. HPLC purity: 98.2%.
3.2.1.21. 8-Bromo-3-chlorophenazin-1-ol (26). Yield: 100%; 103
mg was isolated as a yellow solid. 1H NMR (600 MHz, DMSO-d6): δ
11.30 (br s, 1H), 8.45 (d, J = 2.1 Hz, 1H), 8.11 (d, J = 9.2 Hz, 1H),
8.05 (dd, J = 9.2, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 7.16 (d, J =
2.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 154.8, 143.5, 142.0,
141.4, 136.9, 135.0, 134.9, 131.2, 130.8, 124.1, 117.5, 111.9. HRMS
(ESI): calcd for C12H5BrClN2O [M − H]−, 306.9279; found,
306.9274. mp 251−253 °C. HPLC purity: 98.1%.
3.2.1.22. 3-Chloro-8-iodophenazin-1-ol (27). Yield: 100%; 110
mg was isolated as a yellow solid. 1H NMR (600 MHz, DMSO-d6): δ
11.28 (br s, 1H), 8.69 (d, J = 1.8 Hz, 1H), 8.19 (dd, J = 9.1, 1.8 Hz,
1H), 7.96 (d, J = 9.1 Hz, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.18 (d, J =
2.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 154.9, 143.6, 142.3,
141.8, 140.0, 137.9, 136.9, 134.8, 130.4, 117.5, 111.8, 98.3. HRMS
(ESI): calcd for C12H5ClIN2O [M − H]−, 354.9141; found,
354.9145. mp 233−235 °C. HPLC purity: 97.9%.
3.3. General Procedures for Nucleophilic Aromatic Sub-
stitution (NAS). 3.3.1. NAS Reaction Method A (39, 40, and 42). 3-
Chloro-1-methoxy-6-methylphenazine 38 (160 mg, 0.62 mmol) and
potassium carbonate were suspended in anhydrous N,N-dimethylfor-
mamide (6 mL) in a sealed tube. The resulting mixture was then
purged with argon for 30 min before 2-mercaptoethanol (350 μL,
4.94 mmol) was added. The sealed tube was then closed, and the
reaction was allowed to stir at 85 °C for 7 days. Upon completion, the
reaction mixture was transferred to a separatory funnel containing
brine (100 mL) and extracted with ethyl acetate (3 × 50 mL). The
resulting organic extracts were then combined, washed with water,
dried with anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The resulting crude solid was then purified via flash column
chromatography using hexanes/ethyl acetate (4:1 to 1:1) to afford 43
as a yellow solid (30 mg, 17%) and 40 as a yellow solid (96 mg, 52%).
3.3.2. NAS Reaction Method B (41, 43 and 44). 3-Chloro-1-
methoxy-6-methylphenazine 38 (147 mg, 0.57 mmol) was dissolved
in N,N-dimethylformamide (6 mL) in a microwave reaction vessel.
Then, potassium carbonate (392 mg, 2.84 mmol) and 2,5,8,11-
tetraoxatridecane-13-thiol 61 (956 mg, 4.54 mmol) were added to the
reaction vessel, which was then purged with argon for 3 min before
being sealed and heated to 200 °C for 152 s. Upon completion, the
reaction mixture was then transferred to a separatory funnel
containing ethyl acetate (50 mL) and brine (50 mL). The product
was extracted, and the organic layer was collected, washed with brine
(3 × 50 mL), dried with anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give a crude red oil. The crude products were
purified via column chromatography using hexane/ethyl acetate (1:1
to 1:99) to afford 44 (43 mg, 18%) as red oil and 41 as red oil (210
mg, 82%).
3.2.1.14. 3-Chloro-6-methylphenazin-1-ol (19). Yield: 100%; 191
mg was isolated as a yellow solid. 1H NMR (600 MHz, DMSO-d6): δ
11.16 (br s, 1H), 8.08 (dd, J = 8.5, 1.6 Hz, 1H), 7.86−7.76 (m, 2H),
7.72 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 2.2 Hz, 1H), 2.79 (s, 3H). 13C
NMR (101 MHz, DMSO-d6): δ 154.8, 142.8, 142.7, 141.4, 137.0,
136.2, 134.4, 130.9, 130.7, 127.4, 117.8, 111.3, 17.3. HRMS (ESI):
calcd for C13H10ClN2O [M + H]+, 245.0476; found, 245.0464. mp
185−187 °C. HPLC purity: 97.8%.
3.2.1.15. 3-Chloro-6,7-dimethylphenazin-1-ol (20). Yield: 93%;
123 mg was isolated as a yellow solid. 1H NMR (400 MHz, CDCl3): δ
8.19 (br s, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H),
7.60 (d, J = 8.9 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 2.79 (s, 3H), 2.56
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 152.2, 144.3, 142.8, 140.0,
138.9, 137.2, 134.8, 134.5, 132.2, 126.0, 119.2, 110.3, 21.0, 13.4.
HRMS (ESI): calcd for C14H12ClN2O [M + H]+, 259.0633; found,
259.0642. mp 183−185 °C. HPLC purity: 99.1%.
3.2.1.16. 7-Bromo-3-chloro-6-methylphenazin-1-ol (21). Yield:
1
100%; 122 mg was isolated as a yellow solid. H NMR (400 MHz,
CDCl3): δ 8.14 (s, 1H), 7.97−7.89 (m, 2H), 7.83 (d, J = 2.0 Hz, 1H),
7.22 (d, J = 2.1 Hz, 1H), 3.00 (s, 3H). 13C NMR (151 MHz, CDCl3):
δ 152.3, 144.4, 143.1, 140.2, 138.5, 138.0, 135.4, 132.8, 127.7, 127.4,
119.3, 111.3, 17.8. HRMS (ESI): calcd for C13H7BrClN2O [M − H]−,
320.9436; found, 320.9421. mp 210−212 °C. HPLC purity: 99.3%.
3.2.1.17. 3-Chloro-6-phenoxyphenazin-1-ol (22). Yield: 100%;
118 mg was isolated as a yellow solid. 1H NMR (500 MHz, CDCl3): δ
8.23 (s, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.92 (dd, J = 8.8, 1.1 Hz, 1H),
7.69 (dd, J = 8.8, 7.6 Hz, 1H), 7.48−7.41 (m, 2H), 7.28−7.20 (m,
4H), 7.08 (dd, J = 7.6, 1.1 Hz, 1H). 13C NMR (101 MHz, CDCl3): δ
156.3, 154.5, 152.2, 142.8, 141.9, 138.4, 138.2, 133.7, 130.5, 130.3,
125.1, 123.2, 120.9, 119.5, 114.5, 111.5. HRMS (ESI): calcd for
C18H12ClN2O2 [M + H]+, 323.0582; found, 323.0570. mp 168−170
°C. HPLC purity: 97.4%.
N
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3.3.2.1. 3-(Ethylthio)-1-methoxy-6-methylphenazine (39). Yield:
31%; 51 mg was isolated as a yellow solid (NAS method A). 1H NMR
(600 MHz, CDCl3): δ 8.14 (dd, J = 8.4, 1.8 Hz, 1H), 7.63−7.55 (m,
2H), 7.50 (d, J = 1.8 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 4.11 (s, 3H),
3.16 (q, J = 7.4 Hz, 2H), 2.84 (s, 3H), 1.46 (t, J = 7.4 Hz, 3H). 13C
NMR (151 MHz, CDCl3): δ 154.4, 143.8, 143.3, 142.1, 141.7, 137.3,
135.6, 130.3, 129.4, 128.2, 115.1, 107.0, 56.6, 26.3, 17.8, 13.8. HRMS
(ESI): calcd for C16H17N2OS [M + H]+, 285.1056; found, 285.1069.
mp 89−91 °C.
extracted with dichloromethane. The organic layer extracts were
collected, dried with sodium sulfate, filtered, and concentrated in
vacuo. The resulting solid was purified via column chromatography
using 100% dichloromethane to elute 37 (107 mg, 74%) as a yellow
solid.
3.4.2. Bromination Reaction Method B (Synthesis of 34, 36, 46,
and 47). Compound 24 (102 mg, 0.34 mmol) was dissolved in
toluene (7 mL) before N-bromosuccinimide (133 mg, 0.75 mmol)
was added to the solution. The resulting reaction mixture was then
heated to 50 °C for 6 h until completion. After this time, the reaction
mixture was cooled to room temperature and concentrated via
rotavap. The crude material was then absorbed onto silica gel (dry
loaded) and purified via column chromatography using 100%
dichloromethane to elute 34 (98 mg, 63%) as a yellow solid.
3.4.2.1. 2,4-Dibromo-3-chlorophenazin-1-ol (28). Yield: 46%; 31
3.3.2.2. 2-((4-Methoxy-9-methylphenazin-2-yl)thio)ethan-1-ol
(40). Yield: 52%; 96 mg was isolated as a yellow solid (NAS method
1
A). H NMR (500 MHz, CDCl3): δ 8.20 (m, 1H), 7.83 (s, 1H),
7.74−7.65 (m, 2H), 6.91 (d, J = 2.0 Hz, 1H), 4.16 (s, 3H), 4.01 (t, J =
6.2 Hz, 2H), 3.42 (t, J = 6.2 Hz, 2H), 2.92 (s, 3H). 13C NMR (126
MHz, CDCl3): δ 154.8, 143.6, 143.4, 142.0, 140.6, 137.4, 135.7,
130.6, 129.9, 128.3, 116.3, 107.4, 60.6, 56.8, 35.4, 17.9. HRMS (ESI):
calcd for C16H17N2O2S [M + H]+, 301.1005; found, 301.1016. mp
158−160 °C.
1
mg was isolated as a yellow solid (method A). H NMR (400 MHz,
DMSO-d6): δ 8.37 (m, 1H), 8.32 (m, 1H), 8.11−8.03 (m, 2H). 13C
NMR (101 MHz, DMSO-d6): δ 152.2, 143.4, 141.2, 139.7, 137.2,
133.9, 132.7, 132.3, 129.4, 128.9, 112.5, 106.1. HRMS (DART): calcd
for C12H6Br2ClN2O [M + H]+, 388.8509; found, 388.8525. mp 218−
220 °C. HPLC purity: 99.5%.
3.3.2.3. 3-((2,5,8,11-Tetraoxatridecan-13-yl)thio)-1-methoxy-6-
methylphenazine (41). Yield: 82%; 210 mg was isolated as a red
1
residue (NAS method B). H NMR (600 MHz, CDCl3): δ 8.18 (m,
1H), 7.72−7.63 (m, 3H), 6.89 (d, J = 1.7 Hz, 1H), 4.14 (s, 3H), 3.88
(t, J = 6.6 Hz, 2H), 3.72−3.66 (m, 6H), 3.66−3.60 (m, 4H), 3.54−
3.51 (m, 2H), 3.40 (t, J = 6.6 Hz, 2H), 3.36 (s, 3H), 2.90 (s, 3H).13C
NMR (151 MHz, CDCl3): δ 154.6, 143.3, 142.9, 142.2, 141.9, 137.1,
135.8, 130.8, 129.7, 128.3, 115.2, 107.3, 72.1, 70.9, 70.8, 70.8, 70.7,
69.4, 59.2, 56.8, 32.1, 18.0. Note: one carbon signal is missing in our
spectra due to overlap at 70.8 ppm (determined by HSQC; see
spectra for details). HRMS (ESI): calcd for C23H31N2O5S [M + H]+,
447.1948; found, 447.1940.
3.4.2.2. 2,4-Dibromo-3-chloro-6-methylphenazin-1-ol (29).
1
Yield: 75%; 434 mg was isolated as a yellow solid (method A). H
NMR (400 MHz, DMSO-d6): δ 11.91 (s, 1H), 8.16 (d, J = 8.4 Hz,
1H), 7.95−7.85 (m, 2H), 2.85 (s, 3H). 13C NMR (101 MHz, DMSO-
d6): δ 152.0, 142.7, 141.4, 138.6, 137.4, 136.7, 133.4, 132.1, 131.4,
126.6, 112.9, 106.1, 16.9. HRMS (DART): calcd for C13H8Br2ClN2O
[M + H]+, 402.8665; found, 402.8671. mp 216−218 °C. HPLC
purity: 98.8%.
3.4.2.3. 2,4-Dibromo-3-chloro-6,7-dimethylphenazin-1-ol (30).
3.3.2.4. 3-(Ethylthio)-6-methylphenazin-1-ol (42). Yield: 37%; 58
1
Yield: 72%; 112 mg was isolated as a yellow solid (method A). H
1
mg was isolated as a yellow solid (NAS method A). H NMR (500
NMR (400 MHz, DMSO-d6): δ 11.66 (br s, 1H), 8.06 (d, J = 8.9 Hz,
1H), 7.84 (d, J = 8.9 Hz, 1H), 2.80 (s, 3H), 2.57 (s, 3H). 13C NMR
(101 MHz, DMSO-d6): δ 151.8, 142.4, 140.0, 139.7, 138.3, 136.3,
135.6, 133.7, 132.4, 125.3, 112.8, 105.3, 20.1, 12.6. HRMS (DART):
calcd for C14H10Br2ClN2O [M + H]+, 416.8822; found, 416.8816. mp
223−225 °C. HPLC purity: 95.5%.
MHz, CDCl3): δ 8.13 (s, 1H), 7.93 (dd, J = 7.4, 2.5 Hz, 1H), 7.64−
7.58 (m, 2H), 7.46 (d, J = 1.9 Hz, 1H), 7.04 (d, J = 1.9 Hz, 1H), 3.16
(q, J = 7.4 Hz, 2H), 2.86 (s, 3H), 1.48 (t, J = 7.4 Hz, 3H). 13C NMR
(126 MHz, CDCl3): δ 151.1, 144.1, 143.6, 143.4, 140.6, 137.8, 133.5,
130.2, 129.7, 127.1, 114.0, 108.9, 26.3, 18.0, 13.9. HRMS (ESI): calcd
C15H15N2OS for [M + H]+, 271.0900; found, 271.0896. mp 119−121
°C.
3.3.2.5. 3-((2-Hydroxyethyl)thio)-6-methylphenazin-1-ol (43).
Yield: 75%; 44 mg was isolated as a yellow solid (NAS method B).
1H NMR (600 MHz, DMSO-d6): 10.72 (br s, 1H), 8.06 (m, 1H),
3.4.2.4. 2,4,7-Tribromo-3-chloro-6-methylphenazin-1-ol (31).
1
Yield: 33%; 51 mg was isolated as a yellow solid (method A). H
NMR (500 MHz, DMSO-d6): δ 8.12 (d, J = 9.3 Hz, 1H), 8.07 (d, J =
9.3 Hz, 1H), 2.92 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 151.9,
142.5, 140.2, 138.8, 137.5, 136.9, 135.7, 133.2, 127.6, 127.1, 112.7,
106.4, 16.9. HRMS (DART): calcd for C13H7Br3ClN2O [M + H]+,
478.7792; found, 478.7801. mp 243−245 °C. HPLC purity: >99.9%.
3.4.2.5. 2,4-Dibromo-3-chloro-6-phenoxyphenazin-1-ol (32).
7.79−7.73 (m, 2H), 7.46 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 2.0 Hz,
1H), 5.13 (t, J = 5.6 Hz, 1H), 3.73 (q, J = 5.7 Hz, 2H), 3.28 (t, J = 6.6
Hz, 2H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6): δ 153.0,
143.2, 142.8, 142.5, 140.7, 136.7, 134.6, 130.4, 129.6, 127.3, 112.6,
109.8, 59.5, 33.7, 17.4. HRMS (ESI): calcd for C15H15N2O2S [M +
H]+, 287.0849; found, 287.0855. mp 154−156 °C.
1
Yield: 38%; 47 mg was isolated as a yellow solid (method A). H
NMR (500 MHz, DMSO-d6): δ 8.11 (d, J = 8.7 Hz, 1H), 7.96 (t, J =
8.2 Hz, 1H), 7.44 (t, J = 7.8 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.26−
7.16 (m, 3H). 13C NMR (151 MHz, DMSO-d6): δ 156.8, 152.7,
152.1, 142.2, 138.9, 137.2, 133.9, 132.1, 130.1, 124.1, 123.6, 119.3,
117.5, 112.9, 106.6. Note: despite multiple experiments, one carbon
signal is missing likely due to overlap. HRMS (DART): calcd for
C18H10Br2ClN2O2 [M + H]+, 478.8792; found, 478.8813. mp 239−
241 °C. HPLC purity: 98.1%.
3.4.2.6. 2,4-Dibromo-3-chloro-8-fluorophenazin-1-ol (33). Yield:
73%; 89 mg was isolated as a yellow solid (method A). 1H NMR (500
MHz, DMSO-d6): δ 12.02 (br s, 1H), 8.39 (dd, J = 9.5, 5.9 Hz, 1H),
8.04 (ddd, J = 9.5, 8.2, 2.7 Hz, 1H), 8.00 (dd, J = 9.5, 2.7 Hz, 1H).
13C NMR (126 MHz, DMSO-d6): δ 163.2 (d, J = 255.6 Hz), 151.9,
141.7 (d, J = 14.2 Hz), 141.1, 139.2 (d, J = 1.8 Hz), 137.0, 134.1,
132.4 (d, J = 10.7 Hz), 124.3 (d, J = 28.4 Hz), 112.6, 111.1 (d, J =
21.5 Hz), 106.9. HRMS (ESI): calcd C12H3Br2ClFN2O for [M −
H]−, 402.8290; found, 402.8286. mp 236−238 °C. HPLC purity:
99.6%.
3.3.2.6. 3-((2,5,8,11-Tetraoxatridecan-13-yl)thio)-6-methylphe-
nazin-1-ol (44). Yield: 18%; 43 mg was isolated as a red residue
1
(NAS method B). H NMR (600 MHz, DMSO-d6): δ 10.73 (br s,
1H), 8.06 (m, 1H), 7.78−7.74 (m, 2H), 7.46 (d, J = 1.8 Hz, 1H), 7.03
(d, J = 1.8 Hz, 1H), 3.76 (t, J = 6.3 Hz, 2H), 3.59 (dd, J = 5.9, 3.5 Hz,
2H), 3.54 (dd, J = 5.9, 3.5 Hz, 2H), 3.51 (dd, J = 5.9, 3.5 Hz, 2H),
3.49−3.45 (m, 4H), 3.40−3.38 (m, 4H), 3.20 (s, 3H), 2.80 (s, 3H).
13C NMR (151 MHz, DMSO-d6): δ 153.1, 143.1, 142.5, 142.5, 140.7,
136.7, 134.6, 130.3, 129.6, 127.3, 112.9, 109.8, 71.3, 69.8, 69.8, 69.6,
68.6, 58.0, 30.9, 17.4. Note: two carbon signals from the PEG chain
could not be viewed in our 13C NMR spectra due to signal overlap at
70 ppm, which is supported by HSQC (see the spectra section of the
Supporting Information for details). HRMS (ESI): calcd for
C22H29N2O5S [M + H]+, 433.1792; found, 433.1800.
3.4. General Procedures for Bromination of 3-Chloro-1-
hydroxyphenazines to Target HPs. 3.4.1. Bromination Reaction
Method A (Synthesis of 28−33, 35, 37, and 45). Compound 27
(100 mg, 0.28 mmol) and N-bromosuccinimide (104 mg, 0.59 mmol)
were dissolved with dichloromethane (75 mL) in a round bottom
flask. The resulting reaction mixture was then allowed to stir at room
temperature for 3 h until completion. After this time, the contents of
the reaction were transferred to a separatory funnel with brine and
3.4.2.7. 2,4-Dibromo-3,6,8-trichlorophenazin-1-ol (34). Yield:
1
63%; 98 mg was isolated as a yellow solid (method B). H NMR
(500 MHz, DMSO-d6): δ 8.39 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 2.1
Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δ 152.2, 141.4, 139.5,
138.7, 138.4, 135.8, 134.7, 133.7, 132.1, 126.6, 112.6, 107.8. HRMS
O
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(DART): calcd for C12H4Br2Cl3N2O [M + H]+, 458.7703; found,
458.7696. mp 246−248 °C. HPLC purity: 99.9%.
halogenated phenazine−iron(II) complex formation [in a 2:1 HP/
iron(II) ratio] yielded a loss in absorbance due to precipitation.
3.4.4. pKa Determination of Select HP Analogues.36 Dissociation
constants (pKa) for select HP analogues were determined using UV−
vis spectroscopy and the Henderson−Hasselbalch equation. Buffers
were prepared using potassium phosphate monobasic (KH2PO4) and
sodium phosphate dibasic (Na2HPO4) in a 1:1 solution of water/
methanol to achieve a pH range of 4.07−9.66 (ammonium hydroxide
was added to prepare buffer pH over 9.80). Compounds were added
from 10 mM stock solutions in dimethyl sulfoxide (25 μL) to 1975
μL of each buffer to evaluate each analogue to yield a final compound
concentration of 125 μM. Full spectral scans were performed from
200 to 800 nm in 2 nm increments to determine λmax values for the
protonated phenol (HA) and the deprotonated phenolate species
(A−). The change in absorption at each determined λmax in relation to
pH was monitored in each buffer and plotted as absorbance versus pH
for each species. The pKa was first estimated by determining the pH of
the point of intersection of the two linear curves (see the Supporting
Information). The visual estimation was confirmed by plotting pH
versus log[A−/HA]. The resulting plot yielded a linear regression line
with a Y-intercept corresponding to a calculated pKa value. As a
method validation, the pKa of 4-nitrophenol (lit. pKa = 7.15) was
determined to be 7.52 under these experimental parameters.
3.4.2.8. 2,4,6-Tribromo-3-chlorophenazin-1-ol (35). Yield: 38%;
1
30 mg was isolated as an orange solid (method A). H NMR (500
MHz, DMSO-d6): δ 8.45 (dd, J = 7.3, 0.7 Hz, 1H), 8.35 (dd, J = 8.8,
0.7 Hz, 1H), 7.93 (dd, J = 8.6, 7.3 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6): δ 152.1, 141.9, 140.6, 139.8, 138.0, 135.5, 134.3, 132.4,
129.0, 123.5, 112.7, 107.1. HRMS (ESI): calcd for C12H3Br3ClN2O
[M − H]−, 462.7490; found, 462.7486. mp 193−195 °C. HPLC
purity: >99.9%.
3.4.2.9. 2,4,8-Tribromo-3-chlorophenazin-1-ol (36). Yield: 36%;
1
46 mg was isolated as a yellow solid (method A). H NMR (400
MHz, DMSO-d6): δ 8.55 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 9.3 Hz,
1H), 8.17 (dd, J = 9.3, 2.1 Hz, 1H). 13C NMR (101 MHz, DMSO-
d6): δ 151.9, 142.0, 141.3, 139.7, 137.5, 135.5, 134.1, 131.1, 130.4,
125.5, 112.5, 106.8. HRMS (ESI): calcd for C12H3Br3ClN2O [M −
H]−, 462.7490; found, 462.7469. mp 250 °C (decomp). HPLC
purity: 98.9%.
3.4.2.10. 2,4-Dibromo-3-chloro-8-iodophenazin-1-ol (37). Yield:
1
74%; 107 mg was isolated as an orange solid (method A). H NMR
(600 MHz, DMSO-d6): δ 8.76 (d, J = 1.8 Hz, 1H), 8.28 (dd, J = 9.1,
1.8 Hz, 1H), 8.07 (d, J = 9.1 Hz, 1H). 13C NMR (101 MHz, DMSO-
d6): δ 152.2, 142.4, 141.7, 140.7, 139.8, 137.6, 137.2, 133.9, 130.7,
112.7, 106.8, 100.4. HRMS (ESI): calcd for C12H3Br2ClIN2O [M −
H]−, 510.7351; found, 510.7351. mp 258−260 °C. HPLC purity:
99.7%.
3.5. Biological Studies. This section includes (a) in vitro testing
of HP analogues and (b) in vivo testing of HP 29.
3.5.1. In Vitro Testing of HP Analogues. 3.5.1.1. Minimum
Inhibitory Concentration Susceptibility Assay for MRSA-1707,
MRSA-44, MRSE 35984, VRE 700221, and E. faecalis OG1RF.33−38
The minimum inhibitory concentration (MIC) for each test
compound was determined by the broth microdilution method as
recommended by the Clinical and Laboratory Standards Institute
(CLSI).58 In a 96-well plate, 11 twofold serial dilutions of each
compound were made in a final volume of 100 μL of lysogeny broth
(LB, MRSA-1707, MRSA-44, and MRSE 35984; brain heart infusion,
BHI, VRE 700221, and E. faecalis OG1RF). Each well was inoculated
with ∼105 bacterial cells at the initial time of incubation, prepared
from a fresh log phase culture (OD600 of 0.5−1.0 depending on
bacterial strain). The MIC was defined as the lowest concentration of
the compound that prevented bacterial growth after incubating for 16
h at 37 °C (MIC values were further supported by spectrophoto-
metric readings at OD600). The concentration range tested for each
HP compound during this study was 0.05−50 μM. DMSO served as
our vehicle and negative control in each microdilution MIC assay.
DMSO was serially diluted with a top concentration of 0.5% v/v. All
compounds were tested in a minimum of three independent
experiments.
3.4.2.11. 2,4-Dibromo-3-(ethylthio)-6-methylphenazin-1-ol (45).
1
Yield: 73%; 35 mg was isolated as a yellow solid (method A). H
NMR (500 MHz, CDCl3): δ 8.62 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H),
7.81 (dd, J = 8.5, 7.0 Hz, 1H), 7.76 (d, J = 7.0 Hz, 1H), 3.17 (q, J =
7.4 Hz, 2H), 2.99 (s, 3H), 1.32 (t, J = 7.3 Hz, 3H). 13C NMR (126
MHz, CDCl3): δ 149.1, 144.5, 141.9, 140.2, 139.3, 139.3, 133.2,
132.5, 131.1, 126.6, 124.2, 111.5, 31.4, 17.7, 14.9. HRMS (DART):
calcd for C15H13Br2N2OS [M + H]+, 428.9090; found, 428.9094. mp
163−165 °C. HPLC purity: 97.8%.
3.4.2.12. 2,4-Dibromo-3-((2-hydroxyethyl)thio)-6-methylphena-
zin-1-ol (46). Yield: 28%; 25 mg was isolated as a yellow solid
1
(method B). H NMR (600 MHz, DMSO-d6): δ 11.52 (br s, 1H),
8.16 (d, J = 8.6 Hz, 1H), 7.91 (dd, J = 8.6, 6.8 Hz, 1H), 7.87 (d, J =
6.8 Hz, 1H), 4.88 (br s, 1H), 3.59 (t, J = 7.1 Hz, 2H), 3.17 (t, J = 7.1
Hz, 2H), 2.87 (s, 3H). 13C NMR (151 MHz, DMSO-d6): δ 150.8,
142.8, 141.5, 139.7, 138.6, 137.6, 134.2, 132.3, 131.1, 126.5, 122.0,
112.3, 60.5, 38.9, 17.0. The 13C signal at 38.9 ppm was determined by
HSQC and can be viewed in the spectra section. HRMS (ESI): calcd
for C15H11Br2N2O2S [M − H]−, 442.8893; found, 442.8905. mp
178−180 °C. HPLC purity: 95.7%.
3.5.1.2. MIC Assay for Mycobacterium tuberculosis (Mtb) H37Ra
(ATCC 25177).35−38 M. tuberculosis H37Ra (ATCC 25177) was
inoculated in 10 mL of Middlebrook 7H9 medium and allowed to
grow for 2 weeks. The culture was then diluted with fresh medium to
an OD600 = 0.01. Aliquots of 200 μL were then added to each well of
a 96-well plate starting from the second column. Test compounds
were dissolved in DMSO at a final concentration of 10 mM. Each
compound (7.5 μL) along with DMSO (negative control) and
streptomycin (positive control-40 mg/mL stock solution) was added
to 1.5 mL of the diluted cultures, resulting in 50 μM final
concentration of each halogenated phenazine analogues and 340
μM for streptomycin. The final DMSO concentration was maintained
at 0.5%. Aliquots of 400 μL were added to wells of the first column of
the 96-well plate and serially diluted twofold (200 μL) per well across
the plate to obtain final concentrations that range from 0.024 to 50
μM for the test compounds and 0.16−340 μM for streptomycin. The
plates were then incubated at 37 °C for 7 days. Minimum inhibitory
concentrations are reported as the lowest concentration at which no
bacterial growth was observed. OD600 absorbance was recorded using
SpectraMax M5 (Molecular Devices). All compounds were tested in a
minimum of three independent experiments.
3.4.2.13. 3-((2,5,8,11-Tetraoxatridecan-13-yl)thio)-2,4-dibromo-
6-methylphenazin-1-ol (47). Yield: 27%; 16 mg was isolated as a
yellow residue (method B). 1H NMR (600 MHz, CDCl3): δ 8.66 (br
s, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.82 (dd, J = 8.6, 6.7 Hz, 1H), 7.76
(d, J = 6.7 Hz, 1H), 3.71 (t, J = 6.8 Hz, 2H), 3.62−3.54 (m, 10H),
3.51 (dd, J = 5.8, 3.6 Hz, 2H), 3.36 (s, 3H), 3.32 (t, J = 6.8 Hz, 2H),
2.98 (s, 3H). 13C NMR (151 MHz, CDCl3): δ 149.2, 144.5, 141.9,
140.2, 139.3, 139.3, 133.2, 132.6, 131.1, 126.6, 124.2, 111.3, 72.1,
70.8, 70.8, 70.7, 70.7, 70.6, 70.5, 59.2, 36.6, 17.7. HRMS (ESI): calcd
for C22H27Br2N2O5S [M + H]+, 590.9983; found, 590.9992. HPLC
purity: >99.9%.
3.4.3. UV−Vis for HPs Binding Iron(II).36−38 Halogenated
phenazine−iron(II) complex formation was determined using UV−
vis spectrometry. Ammonium iron(II) sulfate hexahydrate (0.5 equiv)
was added to a stirring solution of an HP analogue (10 mM for HPs
18, 28, and 29; 5 mM for HP 34) in dimethyl sulfoxide. Aliquots of
50 μL (HPs 18, 28, and 29) or 100 μL (HP 34) were then removed
from the resulting mixture and added to 1 mL of dimethyl sulfoxide in
a cuvette. Spectral scanning was performed from 200 to 800 nm in 2
nm increments and a loss of absorbance at λmax (free HP) in the UV−
vis spectrum, and apparent formation of a halogenated phenazine−
iron(II) complex was observed over time. The disappearance of HPs
28, 29, and 34 was observed over the indicated time points, and the
3.5.1.3. MIC Assay for Mtb CDC1551. A bioluminescent M.
tuberculosis CDC1551 reporter strain (Mtb-lux) was grown in
Middlebrook 7H9 media supplemented with 0.05% Tween 80 and
P
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10% oleic acid-albumin-dextrose-catalase (OADC) under kanamycin
(50 μg/mL) selection until an optical density (OD600) of 0.4−0.8. A
twofold serial drug dilution series (0.003−200 μM) was prepared at
2× in media with 2% dimethyl sulfoxide (DMSO) in 15 μL in white
384-well plates (white with solid bottom, Corning #3570). Mtb-lux
was diluted to OD600 0.02 and added to the drug dilution plate (15
μL/well). Luminescence was measured with a Synergy H4 plate
reader after 5 days of treatment (37 °C, 5% CO2) and compared to
the negative control (1% DMSO) and positive control (10 μM
rifampicin) used to calculate % inhibition of growth. Dose−response
curves were generated using GraphPad Prism, and MIC values were
determined using a modified Gompertz model.
3.5.1.4. MIC Assay for S. pneumoniae (ATCC 6303). The MIC of
29 against S. pneumoniae ATCC 6303 was determined according to
the following procedure. Fresh overnight cultures were inoculated at
1% in microtiter wells containing tryptic soy broth with 5% sheep
blood in the presence of 29 ranging from 0.5 to 1000 nM. The plate
was incubated at 37 °C with 5% CO2 for 16−20 h. After this time, the
lowest test concentration of 29 that resulted in a complete lack of
turbidity (bacterial growth) was determined to be the MIC. All tests
were performed in at least three independent experiments.
extracted using the RiboPure RNA purification kit, bacteria
(Invitrogen, cat# AM1925) according to the manufacturer’s protocols.
Genomic DNA was digested using the materials supplied by the kit.
Each experiment was performed in three replicates. RNA quality
control information: RNA concentration was determined on a Qubit
2.0 fluorometer (ThermoFisher/Invitrogen, Grand Island, NY), and
RNA quality was assessed using the Agilent 2100 bioanalyzer (Agilent
Technologies, Inc.). Total RNA with RNA integrity numbers (RIN)
≥ 7 were used for RT-qPCR validation. Quantitative real-time PCR
(qPCR) for select gene transcripts (validation): Total RNA was
isolated from MRSA BAA-1707 biofilms treated and untreated with
HPs (see the Supporting Information for RNA quality control data).
Real-time PCR reactions were performed using the Power SYBR
Green RNA-to-CT 1-Step kit (Applied Biosystems 4389986) using the
manufacturer’s guidelines. SYBR, primers Rt enzyme RNA, and water
were added to a 1.5 mL Eppendorf tube on ice. After all contents were
added to Eppendorf tubes, they were mixed by centrifugation for 1
min at 10,000g. 20 μL was then removed from the reaction tubes and
was added to each well of a MicroAmp optical 96-well reaction plate
with barcode (Applied Biosystems 4306737) on ice. The plate was
then sealed with the MicoAmp optical adhesive film (Applied
Biosystems 4311971). The plate was centrifuged for 2 min at 1200g.
qPCR was carried out on an ABI 7300 sequence detection system
using the thermocycler program: 30 min at 50 °C, 10 min at 95 °C,
15 s at 95 °C (40 cycles), and 1 min at 60 °C. Relative gene
expression changes were calculated using the ΔΔCT method. For
each experiment, the CT values of each gene tested were normalized
to the CT values of the housekeeping gene ptaA. Graphs were plotted
and data analysis was performed using GraphPad Prism 6. All primers
used during these studies are listed in the Supporting Information. All
qPCR data were generated from three independent experiments.
3.5.1.7. Lactate Dehydrogenate (LDH) Release Assay for HeLa
Cytotoxicity Assessment.46 HeLa cytotoxicity was assessed using the
LDH release assay described by CytoTox96 (Promega G1780). HeLa
cells were grown in DMEM (Gibco) supplemented with 10% FBS at
37 °C with 5% CO2. When the HeLa cultures exhibited 70−80%
confluence, halogenated phenazines were then diluted by DMEM
(10% FBS) at concentrations of 25, 50, and 100 μM and added to
HeLa cells. Triton X-100 (at 2% v/v) was used as the positive control
for maximum LDH activity in this assay (i.e., complete cell death),
while “medium only” lanes served as negative control lanes (i.e., no
cell death). DMSO was used as our vehicle control. HeLa cells were
treated with compounds for 24 h, and then 50 μL of the supernatant
was transferred into a fresh 96-well plate where 50 μL of the reaction
mixture was added to the 96-well plate and incubated at room
temperature for 30 min. Finally, stop solution (50 μL) was added to
the incubating plates, and the absorbance was measured at 490 nm.
Results are from three independent experiments.
3.5.1.8. Cytotoxicity Assay against J774 Macrophages and
HepG2 Cells.47 J774 macrophages and HepG2 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented
with 10% heat-inactivated fetal calf serum (Atlanta Biologicals), 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100
mg/mL streptomycin in T75 flasks until confluent. J774 macrophages
were scraped in PBS, pelleted at 1000 rpm for 10 min, and
resuspended in macrophage infection media (DMEM + 10% fetal
bovine serum, FBS, 1% L-glutamine, and 1% sodium pyruvate, no
phenol red). HepG2 cells were lifted with 0.25% trypsin, pelleted at
1000 rpm for 10 min, and resuspended in macrophage infection
media. Both cell types were seeded into 384-well plates (black with
clear bottom, Corning #3712) at 25,000 cells/well (in 24 μL total
volume). After 4 h of attachment (37 °C, 5% CO2), 6 μL of 5× drugs
(twofold dilution series spanning 0.003−200 μM test concentrations,
prepared in water) was added to each well. Cells were treated
overnight (16−18 h), followed by the addition of 6 μL/well Alamar
Blue (0.02% resazurin in water). After 4 h, fluorescence was measured
using a Synergy H4 plate reader (λex = 530 nm/λem = 590 nm).
Viability was calculated as a percent of DMSO control. 2% Triton X-
100 was used as a positive control.
3.5.1.5. CBD Experiments.36−38 Biofilm eradication experiments
were performed using the CBD to determine MBC/MBEC values for
various compounds of interest (Innovotech, product code: 19111).
The CBD (96-well plate with a lid containing pegs to establish
biofilms) was inoculated with 125 μL of a mid-log phase culture
diluted 1000-fold in tryptic soy broth with 0.5% glucose (TSBG; BHI
for E. faecalis OG1RF) to establish bacterial biofilms after incubation
at 37 °C for 24 h. The lid was then removed, washed, and transferred
to another 96-well plate containing twofold serial dilutions of the test
compounds (the “challenge plate”). The total volume of media with
the compound in each well in the challenge plate is 150 μL. The CBD
device was incubated at 37 °C for 24 h, the lid was then removed
from the challenge plate, and MBC/MBEC values were determined
using different experimental pathways. To determine MBC values, 20
μL of the challenge plate was transferred into a fresh 96-well plate
containing 180 μL of TSBG (BHI for E. faecalis OG1RF) and
incubated overnight at 37 °C. The MBC values were determined as
the concentration, giving a lack of visible bacterial growth (i.e.,
turbidity). For determination of MBEC values, the CBD lid (with
attached pegs/treated biofilms) was transferred to a new 96-well plate
containing 150 μL of fresh TSBG (BHI for E. faecalis OG1RF) media
in each well and incubated for 24 h at 37 °C to allow viable biofilms
to disperse and grow, resulting in turbidity after the incubation period.
MBEC values were determined as the lowest test concentration that
resulted in eradicated biofilms (i.e., wells that had no turbidity after
the final incubation period). In selected experiments, both treated and
untreated CBD pegs were removed from the lid after final incubation,
sonicated for 30 min in phosphate-buffered saline (PBS), and plated
out to determine biofilm cell killing in colony-forming units per peg
(CFU/peg). All data were obtained from a minimum of three
independent experiments.
3.5.1.6. RT-qPCR Protocol to Determine Iron Starvation in MRSA
Biofilms.39 Biofilm formation: MRSA BAA-1707 was grown in TSBG
to an OD600 ∼ 0.8−1.0. Then, 1 mL of this culture was added to a 24-
well plate coated with 0.1% gelatin. The plate was then incubated for
20 h at 37 °C under static conditions to form biofilms. Following
biofilm formation, the contents of the well were discarded, leaving
only the biofilm. Treating the established biofilms with compounds:
HPs 3, 28, and 29 were added to the established MRSA BAA-1707
biofilms in TSBG at the desired concentration (1 μM or 1/10 MBEC
value). In addition, the same volume of DMSO (vehicle) was added
as a negative control. The plate was then incubated under static
conditions for 4 h at 37 °C. After the incubation period, the liquid
culture was discarded, leaving only the surface-attached biofilm.
Extraction of total RNA from MRSA BAA-1707 biofilms: 0.5 mL of
the RNAprotect bacteria reagent (Qiagen) was added for 5 min to the
plate, and the biofilm suspension was scraped and transferred into 2
mL tubes. The bacterial cells were then centrifuged for 1 min at
15,000g; then, the supernatant was removed. Total RNA was
Q
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3.5.1.9. Cytotoxicity Assay against HEK-293 Cells.48 The viability
of HEK-293 cells was assessed by the MTT assay. The cells were
cultured in DMEM medium containing 10% fetal bovine serum and
100 U/mL penicillin and streptomycin. The cells (104 cells/well)
were seeded onto a 96-well plate and maintained at 37 °C in a
humidified incubator under 5% CO2 overnight. Serial concentrations
of 29 (final concentrations at 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and
200 μM) were then added to the wells (n = 6). After incubation at 37
°C for 72 h, 10 μL of MTT (5 mg/mL) in PBS was added to each
well and incubated for 4 h, followed by the aspiration of the medium.
Dimethyl sulfoxide (DMSO, 100 μL) was then added to each well to
dissolve the MTT in the wells, and the plate was agitated for 1 h. The
optical density (OD) was measured at 570 nm using a UV/vis
microplate spectrophotometer (BioTek). The percent inhibition was
calculated as follows: inhibition (%) = (1 − test OD570/nontreated
OD570) × 100%. The data were analyzed using Origin.
3.5.1.10. Hemolysis Assay with Red Blood Cells. Freshly drawn
human red blood cells [hRBC with ethylenediaminetetraacetic acid
(EDTA) as an anticoagulant] were washed with Tris-buffered saline
[0.01 M Tris-base, 0.155 M sodium chloride (NaCl), pH 7.2] and
centrifuged for 5 min at 3500 rpm. The washing was repeated three
times with the buffer. In a 96-well plate, test compounds were added
to the buffer from DMSO stocks. Then, 2% hRBCs (50 μL) in buffer
were added to test compounds to give a final concentration of 200
μM. The plate was then incubated for 1 h at 37 °C. After incubation,
the plate was centrifuged for 5 min at 3500 rpm before 80 μL of the
supernatant was transferred to another 96-well plate to obtain an
optical density (OD) read at 405 nm. DMSO served as the negative
control (0% hemolysis), while Triton X served as the positive control
(100% hemolysis) in these experiments. The percent hemolysis was
calculated as (OD405 of the compound treated RBCs − OD405 DMSO
control)/(OD405 Triton X treated RBCs − OD405 buffer) from three
independent experiments.
3.5.2. In Vivo Testing of HP 29 for Efficacy. 3.5.2.1. Preparation
of Polyethylene Glycol (PEG) Ointment. PEG ointments were
prepared based on reported procedures with minor modifications.52
For these investigations, the PEG ointment base was prepared by
mixing PEG 400 (70%, wt/vol) with PEG 3350 (30%, wt/vol) as
described by the U.S. Pharmacopeia and The National Formulary
(USP 24-NF 19). PEG 400 (7 mL) was added to a flask containing
PEG 3550 (3 g), and the resulting mixture was heated at 60 °C until
the mixture liquified. Then, 1 mL of the resulting PEG liquid was
transferred to a vial containing HP 29 (20 mg) to create a 2%
suspension. The mixture was then allowed to stir at 60 °C for 30 min
before being cooled to room temperature where the suspension
solidified. A similar procedure was used to create vehicle control
(without 29).
Shenandoah IA) in PBS. Meloxicam (2 mg kg−1, Henry Schein
Animal Health, Portland, ME) was administered prior to dermal
wounding. The dorsal mid-section of the mouse was shaved and
cleaned with a series of betadine scrub (Fisher Scientific), povidone-
iodine pads (Professional Disposables International Inc; Orangeburg,
NY), and isopropyl alcohol pads (Fisher Scientific) for a total contact
time of 2 min. A wound was created in this sterile field on the mouse
with a 4.5 mm biopsy punch (Fisher Scientific) to remove only the
dermal layer and not disrupt the underlying musculature. The wounds
of the mice were inoculated with 1 × 107 S. aureus strain UAMS-1 by
pipetting 10 μL of culture directly onto the wound. Mice were then
treated with ointment formulations (50 μL) containing either vehicle
alone or test compound 45 min post inoculation; treatments were
repeated every 12 h for three consecutive days Mice were then
euthanized via CO2 asphyxiation and cervical dislocation. The wound
and underlying muscle were excised with a 7 mm biopsy punch and
placed in microcentrifuge tubes containing 1.4 mm ceramic beads
(Fisher Scientific) and 1 mL of freshly made PBS. Samples were
homogenized for 1 min using the Fisherbrand bead mill homogenizer,
serially diluted, and plated on ChromoAgar plates. Plates were
incubated for 16 h at 37 °C, and the number of S. aureus was
enumerated.
3.5.2.4. E. faecalis Dorsal Wound Infection Model. To test the
efficacy of HP 29 for the treatment of wounds infected with E. faecalis,
a 3 mm incision dorsal wound was created using a biopsy punch on 7
week-old female C57BL/6J mice and the wound was infected with 20
μL of 6 × 1010 CFU/mL of E. faecalis OG1RF, and the infected
wound was covered with Tegaderm (Tegaderm, 3 M, St Paul
Minnesota). 24 h after infection, the dressing was removed and the
wounds were treated topically with HP 29 ointment or with the PEG
vehicle control once a day. On day 3 (2 days of treatment)
postinfection, animals were euthanized, the wounds were aseptically
excised using a surgical blade, and the tissues were homogenized in 1
mL of sterile PBS. The homogenates were serially diluted and plated
on trypticase soy (TSA) agar containing rifampicin and fusidic acid
for bacterial enumeration.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00168.
Supporting figures, 1H/13C NMR spectra, dose−
response curves, UV−vis results for iron(II) binding,
pKa determination, and purity analysis (HPLC traces)
for HPs evaluated in biological assays (PDF)
3.5.2.2. Bacterial Strains Used in Animal Models of Infection. S.
aureus strain UAMS-1, a well-characterized antibiotic-susceptible
clinical isolate commonly used to study the organism’s biofilm
formation and colonization properties, was used in animal experi-
ments during these studies.59 Female BALB/c mice 4−6 weeks of age
were obtained from Charles River Laboratories International, Inc.
(Wilmington, MA) and housed individually according to approved
University of Rochester Medical Center Council on Animal Research
(UCAR) protocol UCAR-101864/2017-022. The UAMS-1 wound
infection experiments were performed by the Dunman lab at the
University of Rochester Medical Center.
E. faecalis strain OG1RF was used in animal experiments during
these investigations. Seven week-old female C57BL/6J mice were
purchased from Jackson Laboratories Ltd. and housed according to
the University of Florida’s approved IACUC protocol 201709769.
The OG1RF wound infection experiments were performed by the
Lemos lab at the University of Florida.
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Robert W. Huigens, III − Department of Medicinal
Chemistry, Center for Natural Products, Drug Discovery and
Development (CNPD3), College of Pharmacy, University of
Florida, Gainesville, Florida 32610, United States;
orcid.org/0000-0003-3811-2721; Phone: (352) 273-
7718; Email: rhuigens@cop.ufl.edu
Authors
Hongfen Yang − Department of Medicinal Chemistry, Center
for Natural Products, Drug Discovery and Development
(CNPD3), College of Pharmacy, University of Florida,
Gainesville, Florida 32610, United States; orcid.org/
0000-0001-8367-4104
Shivani Kundra − Department of Oral Biology, College of
Dentistry, University of Florida, Gainesville, Florida 32610,
United States; orcid.org/0000-0003-0711-1646
3.5.2.3. S. aureus Dermal Wound Infection Model. The effects of
ointment compilations were evaluated for in vivo antimicrobial activity
using a dermal wound infection mouse model, as previously
described.52 Briefly, mice were anesthetized by intraperitoneal
injection with a mixture of 100 mg kg−1 ketamine (Hospira Inc.,
Lake Forest IL) and 10 mg kg−1 xylazine (Lloyd Laboratories,
R
https://doi.org/10.1021/acs.jmedchem.1c00168
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Featured Article
Michaelle Chojnacki − Department of Microbiology and
Immunology, University of Rochester, Rochester, New York
14642, United States; orcid.org/0000-0003-0245-9918
Ke Liu − Department of Medicinal Chemistry, Center for
Natural Products, Drug Discovery and Development
(CNPD3), College of Pharmacy, University of Florida,
Gainesville, Florida 32610, United States; orcid.org/
0000-0001-6827-6706
R35GM128742 to Y.D.; R21AI37446 to J.A.L.). All high-
resolution mass spectra were obtained for new compounds
from the University of Florida’s Mass Spectrometry Research
and Education Center and supported by NIH S10 OD021758-
01A1.
ABBREVIATIONS
■
1-OHP, 1-hydroxyphenazine; BHI, brain heart infusion; BSA,
bis(trimethylsilyl)acetamide; CBD, calgary biofilm device;
CFU, colony-forming unit; CLogP, calculated logarithm of
partition coefficient between n-octanol and water; CO2, carbon
dioxide; d, day; DMEM, Dulbecco’s modified Eagle’s medium;
DMF, N,N-dimethylformamide; EDTA, ethylenediaminetetra-
acetic acid; FBS, fetal bovine serum; h, hour; HP, halogenated
phenazine; hRBCs, human red blood cells; LB, lysogeny broth;
LDH, lactate dehydrogenase; MBC, minimum bactericidal
concentration; MBEC, minimum biofilm eradication concen-
tration; MDR, multidrug resistant; MIC, minimum inhibitory
concentration; μM, micromolar; min, minute; MRSA,
methicillin-resistant Staphylococcus aureus; MRSE, methicillin-
resistant Staphylococcus epidermidis; Mtb, Mycobacterium tuber-
culosis; NAS, nucleophilic aromatic substitution; NBS, N-
bromosuccinimide; OD, optical density; PBS, phosphate-
buffered saline; PEG, polyethylene glycol; pKa, dissociation
constant; QAC-10, quaternary ammonium cation-10; rt, room
temperature; RT-qPCR, real-time quantitative polymerase
chain reaction; SI, selectivity index; TPEN, N,N,N′,N′-
tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; TSA, trypticase
soy agar; TSB, tryptic soy broth; TSBG, tryptic soy broth
supplemented with 0.5% glucose; VRE, vancomycin-resistant
Enterococcus faecium
Marisa A. Fuse − Burnett School of Biomedical Sciences,
College of Medicine, University of Central Florida, Orlando,
Florida 32827, United States
Yasmeen Abouelhassan − Department of Medicinal
Chemistry, Center for Natural Products, Drug Discovery and
Development (CNPD3), College of Pharmacy, University of
Florida, Gainesville, Florida 32610, United States
Dimitris Kallifidas − Department of Medicinal Chemistry,
Center for Natural Products, Drug Discovery and
Development (CNPD3), College of Pharmacy, University of
Florida, Gainesville, Florida 32610, United States
Peilan Zhang − Department of Medicinal Chemistry, Center
for Natural Products, Drug Discovery and Development
(CNPD3), College of Pharmacy, University of Florida,
Gainesville, Florida 32610, United States; orcid.org/
0000-0001-6417-6417
Guangtao Huang − Department of Molecular Genetics &
Microbiology, College of Medicine, University of Florida,
Gainesville, Florida 32610, United States
Shouguang Jin − Department of Molecular Genetics &
Microbiology, College of Medicine, University of Florida,
Gainesville, Florida 32610, United States; orcid.org/
0000-0002-6447-6978
Yousong Ding − Department of Medicinal Chemistry, Center
for Natural Products, Drug Discovery and Development
(CNPD3), College of Pharmacy, University of Florida,
Gainesville, Florida 32610, United States; orcid.org/
0000-0001-8610-0659
Hendrik Luesch − Department of Medicinal Chemistry,
Center for Natural Products, Drug Discovery and
Development (CNPD3), College of Pharmacy, University of
Florida, Gainesville, Florida 32610, United States;
orcid.org/0000-0002-4091-7492
Kyle H. Rohde − Burnett School of Biomedical Sciences,
College of Medicine, University of Central Florida, Orlando,
Florida 32827, United States; orcid.org/0000-0001-
9838-3238
Paul M. Dunman − Department of Microbiology and
Immunology, University of Rochester, Rochester, New York
14642, United States
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ACKNOWLEDGMENTS
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We acknowledge the National Institute of General Medical
Sciences and the National Institute of Allergy and Infectious
Diseases of the National Institutes of Health for providing
support for this work (R35GM128621 to R.W.H.;
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