Structure-Activity Relationships of a Diverse Class of Halogenated Phenazines That Targets Persistent, Antibiotic-Tolerant Bacterial Biofilms and Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.5b02004

Persistent bacteria, including persister cells within surface-attached biofilms and slow-growing pathogens lead to chronic infections that are tolerant to antibiotics. Here, we describe the structure-activity relationships of a series of halogenated phena

Full text of DOI:10.1021/acs.jmedchem.5b02004

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Article
Structure-Activity Relationships of a Diverse Class of
Halogenated Phenazines that Targets Persistent, Antibiotic-
Tolerant Bacterial Biofilms and Mycobacterium tuberculosis
Aaron T. Garrison, Yasmeen Abouelhassan, Verrill M Norwood, Dimitris Kallifidas, Fang Bai, Minh Thu
Nguyen, Melanie Rolfe, Gena M. Burch, Shouguang Jin, Hendrik Luesch, and Robert William Huigens III
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b02004 • Publication Date (Web): 28 Mar 2016
Downloaded from http://pubs.acs.org on March 31, 2016
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StructureꢀActivity Relationships of a Diverse Class of Halogenated Phenazines
that Targets Persistent, AntibioticꢀTolerant Bacterial Biofilms and
Mycobacterium tuberculosis
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Aaron T. Garrison , Yasmeen Abouelhassan , Verrill M. Norwood IV , Dimitris Kallifidas , Fang Bai ,
†
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†
‡
†
⊥
⊥
⊥
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Minh Thu Nguyen , Melanie Rolfe , Gena M. Burch , Shouguang Jin , Hendrik Luesch , Robert W.
†
⊥
Huigens III*
†
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610;
‡
⊥
Department of Molecular Genetics & Microbiology, College of Medicine, University of Florida; Center for
Natural Products, Drug Discovery and Development (CNPD3), University of Florida
Abstract. Persistent bacteria, including persister cells within surfaceꢀattached biofilms and slowꢀgrowing
pathogens lead to chronic infections that are tolerant to antibiotics. Here, we describe the structureꢀactivity
relationships of a series of halogenated phenazines (HP) inspired by 2ꢀbromoꢀ1ꢀhydroxyphenazine 1. Using
multiple synthetic pathways, we probed diverse substitutions of the HP scaffold in the 2ꢀ, 4ꢀ, 7ꢀ and 8ꢀpositions
providing critical information regarding their antibacterial and bacterial eradication profiles. Halogenated
phenazine 14 proved to be the most potent biofilmꢀeradicating agent (≥99.9% persister cell killing) against
MRSA (MBEC < 10 µM), MRSE (MBEC = 2.35 µM) and VRE (MBEC = 0.20 µM) biofilms while 11 and 12
demonstrated excellent antibacterial activity against M. tuberculosis (MIC = 3.13 µM). Unlike antimicrobial
peptide mimics that eradicate biofilms through the general lysing of membranes, HPs do not lyse red blood
cells. HPs are promising agents that effectively target persistent bacteria while demonstrating negligible
toxicity against mammalian cells.
INTRODUCTION
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Current antibiotics operate primarily through growthꢀdependent mechanisms and suffer from an inability to
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effectively treat persistent and chronic infections involving bacterial biofilms (surfaceꢀattached communities)
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and Mycobacterium tuberculosis (MtB). Bacteria prefer to exist in biofilms that are composed of specialized,
nonꢀreplicating persister cells encased within an extracellular matrix of biomolecules and demonstrate tolerance
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,6,7
toward every class of antibiotic therapy.
Antibiotic tolerance is an innate bacterial phenotype attributed to
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metabolically dormant persister cells and distinct from acquired antibiotic resistance gained by rapidlyꢀdividing,
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freeꢀfloating (planktonic) bacteria induced by antibiotic treatment (Figure 1).
Chronic and recurring
bacterial infections are attributed to antibiotic tolerance which results in an estimated 17 million new biofilm
10,11
infections and >500,000 annual deaths in the United States.
An unproductive antibiotic pipeline over the
last 45 years has led to many BigPharma companies eliminating or significantly downsizing antibacterial
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2
discovery programs. In addition, we have only begun to understand the importance of biofilms in human
health over the last 20 years and currently there are no biofilmꢀ or persisterꢀeradicating therapeutics available to
effectively treat chronic infections. In order to address persistent and biofilmꢀassociated bacterial infections,
innovative strategies are required to identify novel small molecules capable of targeting and killing nonꢀ
replicating persister cells through unique, growthꢀindependent mechanisms.
Many efforts have been made to modulate bacterial biofilms through the perturbation of bacterial signaling
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(quorum sensing) processes or the identification of nonꢀgrowth altering biofilm inhibitors and dispersal
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14ꢀ18
agents ; however, very few classes of biofilmꢀeradicating agents have been reported.
Unlike biofilm
inhibitors and dispersal agents, biofilmꢀeradicating agents kill persister cells within surfaceꢀattached biofilms
and have the potential to be standꢀalone therapeutics for the treatment of biofilmꢀassociated infections. The
most prevalent class of biofilmꢀeradicating agents are the antimicrobial peptides (AMPs) and mimics thereof,
1
4ꢀ18
which operate through bacterial membrane disruption and lysis.
The development of AMPꢀbased
antibacterial therapeutics is indeed promising; however, effective AMPs must target bacterial membranes over
mammalian cell membranes to reduce or eliminate potential human toxicity concerns. New biofilmꢀeradicating
agents that operate through complimentary mechanisms are critical to address biofilmꢀassociated bacterial
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infections and have the potential to address other problems associated with chronic bacterial infections, such as
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targeting slowꢀgrowing Mycobacterium infections.
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The marine environment is an extensive source of microbial diversity and new antibacterial agents, thus it is
fertile ground for the discovery of antibacterial agents that operate through new modes of action and could lead
to effective biofilmꢀeradicating agents. Marine sources have provided diverse classes of natural products able
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to modulate quorum sensing,
inhibit biofilm formation and/or disperse established biofilms.
Recently,
25
our group found that 2ꢀbromoꢀ1ꢀhydroxyphenazine 1 (Figure 2), a phenazine originally isolated by Cushman
and coꢀworkers from a marine Streptomyces species, demonstrates potent antibacterial activity against
Staphylococcus aureus and S. epidermidis (minimum inhibitory concentration or MIC of 6.25 µM; 1.7
26
µg/mL). The antibacterial potency of this marine phenazine antibiotic is enhanced with a second bromine
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atom installed at the 4ꢀposition of the phenazine heterocycle (2
; MIC 0.78ꢀ1.56 µM, or 0.27ꢀ0.55 µg/mL
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against S. aureus and S. epidermidis). Halogenated phenazine (HP) 2 displayed effective eradication activity
against methicillinꢀresistant Staphylococcus aureus (MRSA) biofilms (minimum biofilm eradication
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concentration or MBEC ~ 150 µM). In addition, we recently reported the identification of a small series of HP
analogues that demonstrated potent biofilm eradication activities (MBEC = 0.2ꢀ12.5 µM) against several major
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human pathogens, including: MRSA, MRSE and VRE. Here, we report the full account of our investigations
detailing the chemical synthesis, biological evaluation and structureꢀactivity relationships of a series of 29 HP
analogues.
RESULTS AND DISCUSSION
Chemical Synthesis of Marine Halogenated Phenazine Analogues.
Our chemical synthesis efforts were motivated by our preliminary success in identifying potent biofilmꢀ
eradicating agents, and thus, newly synthesized HP analogues would be evaluated in an array of biological
assays. Our initial goals were to synthesize a focused, yet diverse set of marine phenazine analogues with a
primary emphasis on functionalizing the 7ꢀ and 8ꢀpositions (Figure 2) of the phenazine heterocycle. These
synthetic goals can be achieved through the following transformations: (1) condensation between a quinone and
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2
5,28
29
phenylenediamine
and (2) WohlꢀAue condensation between anilines and nitroarenes. In addition, we were
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interested in probing alternative substitution patterns with various combinations of halogens and alkyl
substituents of the phenazine scaffold which required multiple synthetic pathways (Scheme 1).
We began our chemistry efforts by synthesizing dichlorinated and diiodinated versions of HP 2 (Scheme 1.A).
Although the dibromination reaction of 1ꢀhydroxyphenazine was highꢀyielding when using Nꢀ
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bromosuccinimide (NBS; 99% yield), the synthesis of 2,4ꢀdichloroꢀ1ꢀhydroxyphenazine 3 and 2,4ꢀdiiodoꢀ1ꢀ
hydroxyphenazine 4 from 1ꢀhydroxyphenazine 42 gave considerably lower yields. Chlorination at the 2ꢀ and 4ꢀ
positions of 1ꢀhydroxyphenazine using Nꢀchlorosuccinimide (NCS) proceeded in a 47% yield to give 2 while
iodination using Nꢀiodosuccinimide (NIS) yielded 3 in only 19%. We also synthesized a series of mixed
halogenated analogues 5ꢀ10 via a regioselective monohalogenation reaction at the 4ꢀposition of 1ꢀ
methoxyphenazine 32 to give phenazines 33ꢀ35 (68ꢀ76% yield), followed by demethylation using boron
tribromide (BBr ) to give phenazine intermediates 39ꢀ41 (86ꢀ97% yield). A final halogenation reaction at the 2ꢀ
3
position of 1ꢀhydroxyphenazines 39ꢀ41 yielded mixed halogenated analogues 5ꢀ10 (34ꢀ91% yield).
1
ꢀMethoxyphenazines 20, 30, and 31 (synthesized via quinoneꢀphenylenediamine condensation; see Supporting
Information) were demethylated using boron tribromide to afford 1ꢀhydroxyphenazines 21, 43, and 44 in 78ꢀ
9% yield (Scheme 1.A). A dibromination reaction at the 2ꢀ and 4ꢀpositions of the phenazine heterocycle was
carried out using two equivalents of Nꢀbromosuccinimide (NBS) to afford HP analogues 16, 17 and 19 in 38ꢀ
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4% yield. This synthetic approach enabled us to access key HP analogues with diversity at the 7ꢀ and 8ꢀ
positions of the phenazine ring; however, the dibromination reaction with NBS proved to be troublesome and
gave inconsistent results (i.e., low yields, impurities difficult to remove) during these studies.
To circumvent the problems encountered during the dihalogenation reaction, we used 1ꢀmethoxyphenazines 20,
3
0 and 31 to sequentially install a single halogen atom at the 4ꢀposition of the phenazine ring upon treatment
with one equivalent of halogenating agent (NCS, NBS, NIS or KI/NaIO ) to give phenazines 36ꢀ38 in 59ꢀ96%
4
yield (Scheme 1.A). Following this monohalogenation reaction at the 4ꢀposition, 1ꢀmethoxyphenazines 36ꢀ38
were demethylated using boron tribromide to yield 1ꢀhydroxyphenazines 11ꢀ13 in 91ꢀ97% yield. Final
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halogenation at the 2ꢀposition of the phenazine heterocycle cleanly afforded HP analogues 14 and 15 in 52%
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and 65% yields, respectively.
To gain further insight into the structureꢀactivity relationships of HP antibacterial agents, we employed multiple
routes to generate new analogues for our biological studies. In the first route, we utilized a WohlꢀAue reaction
between 4ꢀbromoaniline 45 and 2ꢀnitroanisole 46 which afforded 8ꢀbromoꢀ1ꢀhydroxyphenazine 57 (Supporting
Information) in 2% yield after refluxing with potassium hydroxide (KOH) in toluene. Despite this low yield,
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we were able to advance 57 through the BBr demethylation/dibromination route to afford HP 18 in 45% yield
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over the final 2 steps (Scheme 1.B). For the second synthetic pathway, we carried out a Suzuki coupling
between 35 and nꢀbutylboronic acid pinacol ester using 20 mol% tetrakis(triphenylphosphine)palladium(0),
followed by subsequent BBr demethylation and NBS bromination reactions to afford lone HP analogue 22 in
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17% yield over 3 steps (Scheme 1.C). For the third synthetic route, we employed an Oꢀallylation/Claisen
rearrangement sequence leading to 2ꢀallylated HP analogues 23 (85% over 3 steps) and 24 (34% over 3 steps)
(Scheme 1.D). Finally, we synthesized a small series of halogenated quinoxalines (25ꢀ28, see Supporting
Information for details). Although these diverse synthetic routes have not been exhausted, we were able to
make several HP analogues that were critical in probing various positions of the HP scaffold.
Antibacterial Evaluation and HeLa Cytotoxicity Studies.
Our biological investigations were initiated with the screening of our HPꢀinspired library (including
halogenated quinoline 29) in MIC assays against a panel of pathogenic bacteria, including: S. aureus, S.
epidermidis, Enterococcus faecium, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella
pneumoniae and Escherichia coli. Lead antibacterial agents from these initial studies were then evaluated
against M. tuberculosis (H37Ra). Several bacterial strains used in this panel were drugꢀresistant (i.e., MRSAꢀ2,
MRSA BAAꢀ44, MRSA BAAꢀ1707, S. epidermidis 35984 are methicillinꢀresistant; E. faecium 700221 is
vancomycinꢀresistant; Table 1).
During initial MIC assays, 4,7,8ꢀtrihalogenated phenazine 12 proved to be the most potent analogue against
methicillinꢀresistant S. aureus (MRSA) planktonic cells by reporting MIC values of 0.15ꢀ0.59 µM (Table 1 and
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Supporting Table 2 for a panel of five additional MRSA isolates). HP 12 also demonstrated potent antibacterial
activity against methicillinꢀsensitive S. epidermidis (MIC = 0.39 µM; ATCC 12228) and methicillinꢀresistant S.
epidermidis (MIC = 0.78 µM; MRSE ATCC 35984). This was our first encounter of an active HP analogue that
did not possess a bromine atom in the 2ꢀposition of the phenazine. Based on this finding, the two chlorine
atoms in the 7ꢀ and 8ꢀpositions of 12 override the previously established structureꢀactivity relationship (SAR)
requirements of having a bromine in the 2ꢀposition to be an active antibacterial agent. In addition, HP
analogues 7, 8, 11, 14 and 18 demonstrated subꢀmicromolar growth inhibition activities against at least one
MRSA strain while 12 and 18 demonstrated this level of antibacterial potency against all three MRSA strains
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(MIC 0.15ꢀ0.78 µM; Table 1). Quinoxaline analogues 25ꢀ28 demonstrated a range of antibacterial activities
against MRSA strains (MIC 0.78ꢀ25 µM, Table 1), with 28 proving to be the most potent quinoxaline analogue.
Select HP analogues (11, 12, 14, 22, 26, 28; Supporting Table 2) were evaluated against a panel of five
additional MRSA isolates and reported potent antiꢀMRSA activities
HP analogues that had potent antibacterial agents against MRSA strains demonstrated similar antibacterial
activities against S. epidermidis. Against methicillinꢀsensitive S. epidermidis 12228, we observed a 12ꢀ to 15ꢀ
fold increase in antibacterial potency for halogenated phenazines 14ꢀ17 (MIC 0.08ꢀ0.10 µM; Table 1) compared
to parent HP 2 (MIC 1.17 µM). HP analogues 15, 16 and 22 (MIC 0.30ꢀ0.59 µM) proved to be the most potent
antibacterial agents against methicillinꢀresistant S. epidermidis (MRSE 35984).
When evaluated for antibacterial activity against VRE, HPs 14ꢀ18 and 22 demonstrated an 8ꢀ to 64ꢀfold
increase in antibacterial activities (MIC 0.10ꢀ0.39 µM) compared to parent HP 2 (MIC = 6.25 µM; 2.2 µg/mL)
with 14 proving to be the most potent analogue (MIC = 0.10 µM; 0.05 µg/mL). HP analogues 11 and 12 (MIC
3
.13ꢀ6.25 µM) demonstrated near equipotent antibacterial activities compared to 2 against VRE, despite
demonstrating improvements in antibacterial activities against staphylococcal pathogens. The remaining HP
analogues demonstrated antibacterial activities against VRE comparable to parent HP 2 (Table 1).
There is a critical need to identify new antibacterial agents that target the slowꢀgrowing pathogen
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Mycobacterium tuberculosis (MtB), which results in 1.5 million deaths each year worldwide.
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Mycobacterium pathogens are difficult to treat as these persistent bacteria are involved in many chronic and
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recurring infections, similar to bacterial biofilms. We tested a small panel of our most potent HP analogues
against M. tuberculosis (MtB) H37Ra (ATCC 25177) in MIC assays (Table 1). HP 2 demonstrated moderate
antibacterial activity against M. tuberculosis with an MIC value of 25 µM. Four halogenated phenazine
analogues were identified to be more potent than 2, including 14 (MIC 12.5 µM), 16 (MIC 6.25 µM) with the
most potent analogues 11 and 12 reporting MIC values of 3.13 µM against M. tuberculosis. HP analogues 17
and 28 were found to be inactive (MIC > 50 µM) against M. tuberculosis in these assays. HPs 11 and 12
demonstrated antibacterial potency against M. tuberculosis near that of streptomycin (MIC 1.32 µM; positive
control). Select HPs possess impressive antibacterial activities that could be useful in treating lifeꢀthreatening
MtB infections.
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Following our investigations with gramꢀpositive pathogens and M. tuberculosis, we evaluated 12 HP analogues
against gramꢀnegative pathogens, including: A. baumannii (ATCC 19606), P. aeruginosa (PAO1), K.
pneumoniae (ATCC 13883) and E. coli (UAECꢀ1, clinical isolate from a bloodstream infection at the
University of Arkansas Med. School). Quinoxaline analogue 25 was the only HP analogue to demonstrate
good antibacterial activities against the gramꢀnegative pathogens A. baumannii 19606 (MIC 6.25 µM) and E.
coli UAECꢀ1 (MIC 12.5 µM) with moderate antibacterial activity against K. pneumoniae 13883 (MIC 37.5 µM)
(Supporting Information). All 12 HP analogues evaluated in this panel, including quinoxaline 25, proved to be
inactive against P. aeruginosa (PAO1; MIC > 100 µM).
Select HP analogues were then evaluated for mammalian cytotoxicity in lactate dehydrogenate (LDH) release
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2
assays against HeLa cells to determine if HP analogues target bacteria and not mammalian cells, a critical
aspect of developing new antibacterial agents (Table 1). In LDH release assays, all 15 HP analogues that were
evaluated against HeLa cells demonstrated excellent cytotoxicity profiles with several HP analogues not
showing any observable cytotoxicity at 100 µM. Only HP 14 demonstrated cytotoxicity at 100 µM; however,
no cytotoxicity was observed for this analogue at 50 µM. Considering the subꢀmicromolar antibacterial activity
of many HP analogues against MRSA, MRSE and VRE, our HeLa cell data demonstrates that HP analogues
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demonstrate a very high degree of specificity targeting bacterial cells over mammalian cells, which is promising
for this new class of antibacterial agents.
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Bacterial Biofilm Eradication and Hemolysis Studies.
Following the initial antibacterial assessment, 24 HP analogues were advanced to biofilm eradication studies
against clinical isolate MRSAꢀ2 alongside conventional antibiotics and other controls agents. For biofilm
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9
0
1
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7
8
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0
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7
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0
3
3ꢀ35
eradication assays, we used the Calgary Biofilm Device (CBD)
established on pegs that are anchored to the lid of a 96ꢀwell plate and submerged in inoculated media. Pegs
i.e., CBD lid) with established bacterial biofilms are then transferred to a second 96ꢀwell plate containing serial
which allows bacterial biofilms to be
(
dilutions of test compound (i.e., halogenated phenazine, antibiotic) for biofilm eradication. After compound
treatment, the CBD pegs are then transferred to a third 96ꢀwell plate containing only fresh media to allow any
viable biofilms to recover and disperse planktonic cells from the viable biofilm, resulting in turbid wells.
Microtiter wells that are not turbid result from eradicated biofilms (the lowest test concentration that results in a
lack of turbidity is considered to be a compound’s minimal biofilm eradication concentration or MBEC).
Using the CBD assay, we were able to determine the relative killing dynamics for a compound against both
planktonic and biofilm cells in a single assay as the CBD assay can also be used to determine minimum
bactericidal concentrations from the media in the test plate (MBC; planktonic killing; Table 2). In addition to
MRSAꢀ2, we evaluated panels of 8, 7, 18 and 14 HP analogues and various control compounds (i.e.,
conventional antibiotics) against MRSA BAAꢀ1707, MRSA BAAꢀ44, MRSE 35984 and VRE 700221 biofilms
using the CBD assay, respectively (Table 2). We found the CBD assay to be highly robust for these phenotypic
2
8,36
screening purposes
in addition to being useful in quantifying biofilm (persister) cell killing with select
compounds from treated and untreated CBD pegs.
Of the 24 HP analogues that were evaluated against MRSAꢀ2, eight analogues demonstrated more potent
biofilmꢀeradicating activity than HP 2 (MBEC = 93.8 µM; Table 2), including: 8, 11, 12, 14, 15, 16, 17 and 18.
Seven of the eight more potent HP analogues contain additional halogen atom substitutions in the 7ꢀ and/or 8ꢀ
positions of the phenazine heterocycle. Several of these HP analogues were then evaluated against BAAꢀ1707
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Journal of Medicinal Chemistry
(
Figure 3) and BAAꢀ44, all proving to be potent eradicating agents against these MRSA biofilms. HP analogue
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
2
2
2
3
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5
5
5
6
1
4 (2ꢀbromoꢀ7,8ꢀdichloroꢀ4ꢀiodoꢀ1ꢀhydroxyphenazine) reported the most potent activity against MRSA
biofilms with MBEC values of 6.25ꢀ9.38 µM (i.e., 10ꢀ to 60ꢀfold more potent than parent HP 2) while HP
analogues 16ꢀ18 also demonstrated potent biofilmꢀeradicating activities against all MRSA strains (MBEC =
9
.38ꢀ50 µM). At the MBEC value against MRSAꢀ2, HP 14 eradicates >3ꢀlog (>99.9%) of persister cells within
0
1
2
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4
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9
0
the corresponding biofilms (viable biofilm cell count determined from CBD peg; Figure 5).
Frontꢀrunning antibiotics used to treat MRSA infections (i.e., vancomycin, daptomycin and linezolid) were
tested alongside our HP analogues. Vancomycin, daptomycin and linezolid were unable to eradicate MRSA
biofilms at the highest test concentrations (MBEC >2,000 µM; Figure 3; Table 2) despite demonstrating
moderate to potent planktonic killing in the same experiment (MBC 3.0ꢀ125 µM). These results are illustrative
of the innate ability of biofilms to tolerate high levels of antibiotics despite having susceptible planktonic
counterparts. During these investigations, doxycycline and rifampin were both found to eradicate MRSAꢀ2
biofilms in CBD assays (MBEC = 46.9 µM); however, the MBEC:MBC ratio for these conventional antibiotics
is >23 compared to MBEC:MBC ratios of 1ꢀ6 for most active HP analogues in the same assays (Table 2).
In addition to conventional antibiotics, select biofilmꢀeradicating agents and persister cell killing agents were
also evaluated in our CBD assays as positive controls to determine the effectiveness of our HP analogues on a
headꢀtoꢀhead basis. This panel of known biofilmꢀ or persisterꢀeradicating agents includes: 64 (quaternary
1
7
ammonium cationꢀ10 or QACꢀ10; an AMP mimic and membraneꢀlysing agent), carbonyl cyanide mꢀ
3
7
38
chlorophenyl hydrazine (CCCP; membraneꢀactive ionophore) , Nꢀacetyl cysteine (NAC; antioxidant) and
4
,39,40
pyrazinamide (persister killer).
From this panel of control agents, we found 64 to have the most potent
biofilm eradication activities (MBEC = 125 µM) against MRSAꢀ2. CCCP reported weak biofilm eradication
activities against MRSAꢀ2 (MBEC = 1,000 µM) while NAC and pyrazinamide did not show any biofilm
eradication activities (MBEC > 2,000 µM) using the CBD assay. Compared to our lead HP 14, this small panel
of known biofilmꢀeradicating agents and persister cell killers were found to be 12ꢀ to >200ꢀfold less potent at
eradicating MRSAꢀ2 biofilms (Table 2).
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From the panel of 18 HP analogues that were evaluated against MRSE biofilms, 13 demonstrated more potent
biofilm eradication activities than HP 2 (MBEC = 250 µM; Table 2). As with our MRSA biofilm eradication
studies, we found HP 14 to demonstrate unparalleled biofilmꢀeradicating activities against MRSE (MBEC =
1
2
3
4
5
6
7
8
9
1
1
1
1
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1
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5
6
2
.35 µM; >100ꢀfold more potent than 2; Figure 3). At the MBEC values against MRSE, HPs 14 and 16
eradicated >4ꢀlog (>99.99%) of persister cells within the corresponding biofilms while demonstrating 1ꢀ2 log
reduction in viable biofilm cells at subꢀMBEC values (viable biofilm cells determined from CBD peg; Figure 4;
see 16 in Supporting Information). Several HP analogues (15, 16, 17, 18) that possess halogen atoms in the 7ꢀ
and/or 8ꢀposition of the phenazine heterocycle proved to eradicate MRSE biofilms with a high level of potency
0
1
2
3
4
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6
7
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8
9
0
(MBEC = 3.13ꢀ25 µM). In addition, HPs 4 and 22 (nonꢀsubstituted analogues in the 7ꢀ and 8ꢀposition) reported
MBEC values of 23.5 µM against MRSE biofilms. In these investigations, MRSE biofilms did show some
susceptibility to various control compounds (64, MBEC = 31.3 µM; CCCP, MBEC = 93.8 µM; rifampin,
MBEC = 15.6 µM); however, vancomycin was unable to eradicate MRSE biofilms at the highest concentration
tested (MBEC >2,000 µM; Table 2).
VRE biofilms were found to be highly sensitive to several of our HP analogues, including parent HP 2 (MBEC
=
4
9.38 µM, Figure 3). Nine of the 14 HP analogues we tested demonstrated more potency compared to 2, with
HP analogues (14, 16ꢀ18) reporting subꢀmicromolar MBEC values against VRE biofilms (Table 2). HP 14
demonstrated the most potent biofilm eradication activity against VRE biofilms and gave an MBEC value of
.20 µM (47ꢀfold more potent than 2). 64 potently eradicates VRE biofilms (MBEC = 3.0 µM) similar to
0
1
7
previous reports; however, NAC demonstrates no biofilm eradication activity in our CBD assays, despite a
3
8
previous report of E. faecium biofilm eradication.
In addition to Calgary Biofilm Device experiments, we evaluated select HPs against staphylococcal biofilms in
live/dead staining experiments. HP 14 was added to 24 hour old MRSE biofilms at 0.1, 1 and 10 µM and
allowed to incubate at 37 °C for an additional 24 hours. Following this, images of the treated and untreated S.
epidermidis biofilm were taken using fluorescence microscopy (Figure 5). Halogenated phenazine 14 removed
all detectable MRSE biofilm cells from the glass surface at 10 µM and a significant amount of the biofilm cells
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Journal of Medicinal Chemistry
at 1 µM. Interestingly, at 0.1 µM of 14 a significant amount of MRSE biofilm cells were eradicated, however,
not cleared from within the biofilm. We previously observed HPs 12 and 14 to clear MRSAꢀ2 biofilms at subꢀ
1
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2
8
micromolar concentrations, well below their corresponding MBEC values in CBD experiments. It appears
that halogenated phenazine analogues are able to eradicate bacterial biofilms, in part, due to an effective
clearance mechanism.
0
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0
HP analogues were also evaluated for hemolysis activity against human red blood cells (RBCs; Table 2) at 200
µM to probe if HP analogues operated through a membraneꢀlysing mechanism, typical of antimicrobial
1
7
peptides.
We found HP analogues do not generally lyse RBCs as only analogues 6 (5%) and 22 (12%)
demonstrated low levels of hemolytic activity at 200 µM while the remainder of the HP analogues demonstrated
insignificant hemolysis (i.e., < 3%). We found AMPꢀmimic and known membraneꢀlysing agent 64 to cause
>99% hemolysis of RBCs in these assays alongside our HP analogues. This hemolysis data provides further
support that HP analogues preferentially target bacterial cells over mammalian cell types, similar to our
observations with HeLa cells.
StructureꢀActivity Relationships (SAR) and Profiles of Halogenated Phenazine Analogues.
Our focused, yet structurally diverse subꢀclasses of halogenated phenazines (2ꢀ24), quinoxalines (25ꢀ28) and
quinoline (29) small molecules (Figure 6) provided significant structureꢀactivity relationship details for this
subset of potent biofilmꢀeradicating agents while illuminating interesting biological profiles (Figure 7). Active
HP analogues demonstrated good to outstanding gramꢀpositive antibacterial and biofilm eradication activities.
In addition, HP analogues demonstrated potent antibacterial activities for select HPs against M. tuberculosis
(Table 1, Figure 7). Interestingly, all HP analogues were found to be completely inactive against our panel of
gramꢀnegative bacteria.
Chlorine atom substitution at the 2ꢀ and/or 4ꢀposition of 2 resulted in a loss of MRSAꢀ2 biofilm eradication
activities (see HP 3, 5, 7, 9; Figure 6) with the only exception to this trend being HP 6 which demonstrated
essentially equipotent biofilm eradication activities compared to 2. 2,4ꢀDiiodoꢀ1ꢀhydroxyphenazine 4
maintained biofilm eradication activities compared to 2, while 2ꢀbromoꢀ4ꢀiodoꢀ1ꢀhydroxyphenazine 8 gave a
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slight (<2ꢀfold) increase in biofilm eradication activities against MRSAꢀ2. Reversing the mixed halogenated
pattern of 8 to afford HP 10 (4ꢀbromoꢀ2ꢀiodoꢀ1ꢀhydroxyphenazine) resulted in a 4ꢀfold reduction in biofilm
eradication activity between these two mixed HP analogues.
1
2
3
4
5
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7
8
9
1
1
1
1
1
1
1
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2
2
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5
5
5
5
5
6
The 7ꢀ and 8ꢀpositions of the HP scaffold (i.e., structure 2) proved to be critical for potent biofilm eradication
activities as the introduction of a single bromine atom at the 8ꢀposition of HP 18 resulted in a 4ꢀfold increase in
biofilm eradication potency against MRSAꢀ2 compared to 2 (Figures 6 & 7). HP 18 also proved to be 10ꢀ to
0
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8
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0
3
0ꢀfold more potent at eradicating biofilms against our panel of MRSA (BAAꢀ1707 and BAAꢀ44), MRSE and
VRE strains compared to 2 (Table 2). Introducing a fourth bromine atom at the 7ꢀposition (from HP 18)
corresponds to HP 17, which resulted in a 2ꢀfold loss of biofilm eradication activity against MRSAꢀ2 compared
to 18. This loss in activity was also observed in the other two MRSA strains (Table 2); however, HP 17 proved
to be more potent than 18 against MRSE and VRE biofilms (Figure 7). 7,8ꢀDichlorohalogenated phenazine
analogues 15 and 16 demonstrated equipotent biofilm eradication potencies to HP 17 and 18 against MRSAꢀ2.
HP 16 also demonstrated potent biofilm eradication activities against MRSE and VRE that were on pace with
17. HP analogue 14 is very similar to 16 except the 2ꢀbromine atom is exchanged with a 2ꢀiodide atom, which
resulted in a significant increase in biofilmꢀeradicating potencies across all MRSA, MRSE and VRE strains
tested. This single halogen atom difference resulted in a 5ꢀfold increase in potency for HP 14 against MRSAꢀ2
compared to HP 16, and a 10ꢀfold increase in biofilm eradication activity compared to parent HP 2. HP 14
demonstrated the most potent biofilm eradication across the entire panel of gramꢀpositive pathogens,
corresponding to a 60ꢀfold increase against MRSA BAAꢀ1707, 100ꢀfold increase against MRSE and 47ꢀfold
increase in VRE biofilm eradication activities compared to parent HP 2 (Figure 6). Interestingly, dibrominated
benzophenazine analogue 19 (containing a fused aromatic ring at the 7ꢀ and 8ꢀposition of the HP scaffold) did
not show any biofilm eradication activities at the highest concentration tested, resulting in a >2ꢀ to >20ꢀfold loss
in activity compared to active HPs 2 and 14, respectively (Table 2).
26
From our previous studies, we determined that the bromine atom in the 2ꢀposition of the HP scaffold is crucial
for antibacterial activities; however, when the 7ꢀ and 8ꢀpositions of the HP scaffold are chlorinated, HP
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Journal of Medicinal Chemistry
analogues bearing a bromine (11) or iodide (12) atom in the 4ꢀposition without a halogen atom in the 2ꢀposition
demonstrate potent biofilm eradication activities overriding our previous SAR knowledge of this class of
antibacterial agents (Figure 7). Despite HP 11 and 12 being 3ꢀfold more potent than parent HP 2, these
analogues were less active than lead HP 14 against MRSAꢀ2 biofilms (Figure 6). HP 11 and 12 demonstrated
moderate eradication activities compared to other HP analogues against MRSE and VRE biofilms. Although
HPs 11 and 12 demonstrated a moderate level of biofilm eradication, these analogues were found to possess the
most potent antibacterial activities for any HPs evaluated against M. tuberculosis. Alternatively, when the two
chlorine atoms in the 7ꢀ and 8ꢀposition of 11 and 12 were replaced with bromine atoms (giving HP 13), there
was a significant loss in activity as no eradication was observed against MRSA, MRSE and VRE biofilms.
Removal of the 4ꢀhalogen atom of 7,8ꢀdichlorinated analogues 11 and 12 resulted in inactive HP 21,
demonstrating the necessity for the 4ꢀposition to be halogenated in this HP subꢀseries (Figure 7).
1
2
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7
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9
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0
We expanded our library to include alkylated HP analogues and we were delighted to discover that 4ꢀbutyl HP
analogue 22 (monoꢀbrominated only at 2ꢀposition) demonstrated potent biofilm eradication activities during
these investigations (Figures 6 & 7). Against MRSAꢀ2, HP 22 demonstrated a 2ꢀfold reduction in biofilm
eradication activities compared to 2; however, this trend was reversed as 22 was found to be more potent than 2
against MRSAꢀ1707 (6ꢀfold increase in potency), VRE (5ꢀfold increase in potency) and MRSE (10ꢀfold
increase in potency) biofilms. This was the first time that a monohalogenated HP analogue demonstrated
biofilm eradication activities and sets the stage for future analogue synthesis. Interestingly, switching the
positions of the alkyl and bromine groups results in a complete loss of antibacterial activity (i.e., HPs 23 and
2
4).
Simplified HP scaffolds, including quinoxalines 25ꢀ28 and quinoline 29, led to analogues with reduced or no
biofilm eradication activities against MRSAꢀ2 (Figures 6 & 7). From the quinoxaline series, unsubstituted
halogenated quinoxalines 25 and 26 were inactive as biofilm eradicating agents at the highest concentrations
tested, which are >20ꢀfold higher than active concentrations for HP 2. 2,3ꢀDimethylquinoxaline analogues gave
interesting results with dibrominated analogue 27 demonstrating weak biofilm eradication activities while the
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diiodinated analogue 28 was equipotent with HP 2 as a biofilm eradicator against MRSAꢀ2. Halogenated
quinoline 29 demonstrated a slight loss in biofilm eradication activities against MRSAꢀ2 compared to 2;
however, 29 is more potent than 2 against MRSE and VRE biofilms. Our group is also developing halogenated
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
36,41,42
quinoline antibacterial and antibiofilm agents as an elaboration of our halogenated phenazine program.
0
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8
9
0
Preliminary Mechanistic Insights.
Phenazine antibiotics, which include 1, are class of redoxꢀactive metabolites produced by Pseudomonas and
4
3,44
Streptomyces bacteria.
It has been proposed that the central ring of the phenazine heterocycle undergoes a
4
4
redox reaction that generates superoxide anions which, in turn, kills various bacteria and fungi. Several active
HP analogues were assayed in MIC experiments against MRSAꢀ2 in combination with tiron (1 mM, which is
45
nonꢀtoxic to MRSAꢀ2; Table 3), a superoxide quenching agent (S. epidermidis 12228 and VRE were also
evaluated, see Supporting Information). In these experiments, tiron did not suppress the antibacterial activity of
2
8
HP analogues despite eliminating the antibacterial activity of 8ꢀhydroxyquinoline (8ꢀHQ; positive control). In
analogous experiments with other free radical scavengers, we found that thiourea (500 µM), manganese (III)
tetrakis (4ꢀbenzoic acid)porphyrin (MnTBAP; 1 mM) or ascorbic acid (1 mM) did not suppress the antibacterial
activities of HP 2. Based on these findings, we conclude that HP analogues do not operate primarily through a
redoxꢀbased mechanism.
Halogenated quinolines (i.e., 29) and halogenated phenazines (2ꢀ22) share several structural features, including
a dihalogenated phenolic ring adjacent to the nitrogen atom of a heterocycle. Halogenated quinolines (i.e., 5,7ꢀ
dihaloꢀ8ꢀhydroxyquinolines) and 8ꢀhydroxyquinolines complex metal(II)ꢀcations allowing an array of diverse
3
6,42
46ꢀ49
bioactivity profiles which have been studied by our group
and others.
Biofilmꢀeradicating HP analogues
have a metalꢀbinding moiety that is analogous to 8ꢀhydroxyquinolines where the 1ꢀhydroxyl group and the
adjacent phenazine nitrogen atom would form a 5ꢀmembered ring upon metal complexation. Recently,
Beauvais and coꢀworkers reported that 1ꢀhydroxyphenazine chelates metal ions resulting antifungal activities
50
against Aspergillus fumigatus through ironꢀstarvation.
Although 1ꢀhydroxyphenazine is inactive against
2
6
staphylococcal pathogens, this metalꢀbinding moiety is critical to the potent activities of halogenated
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Journal of Medicinal Chemistry
phenazines as methylation of the phenolic hydroxyl group in 2 abolishes the antibacterial activity of the HP
1
2
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5
6
2
6
scaffold.
We coꢀtreated select HP analogues with 200 µM of copper(II), iron(II) and magnesium(II) in MIC assays
against MRSAꢀ2 (Table 3) to determine if certain metal(II) cations would complex to HP analogues and
eliminate their antibacterial activities. Upon coꢀtreatment with copper(II), HP analogues demonstrated a
significant decrease in antibacterial activities (up to 48ꢀfold loss determined by elevated MIC values by
copper(II) coꢀtreatment), while iron(II) slightly decreases antibacterial activity (up to 5ꢀfold elevated MIC
values) and magnesium(II) had no effect against MRSAꢀ2 (data not shown). We have observed similar
0
1
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5
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7
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6,42
activities with halogenated quinoline small molecules.
Using UVꢀVis spectroscopy, we demonstrated that HP 2 and halogenated quinoline 29 directly bind copper(II)
and iron(II) (Figure 8). When treating HP 2 (λmax = 440 nm; Figure 8A) with copper(II), an insoluble complex
forms which precipitates out of solution resulting in the disappearance of UVꢀVis absorbance. For halogenated
quinoline analogue 29 (λmax = 314 nm; Figure 8B) we observed a soluble copper(II)ꢀcomplex resulting in a shift
in absorbance to λmax = 394 nm. We also demonstrate that HP 2 (ligand) binds copper(II) in a 2:1
ligand:copper(II) ratio (see titration curve in Supporting Information), analogous to previous findings with 1ꢀ
50
46ꢀ49
hydroxyphenazine and halogenated quinolines.
resulting in a soluble complex and a concomitant shift in absorbance
metal binding could be influenced by the acidity of the phenolic proton, which we measured experimentally for
In addition, we demonstrated that HP 2 binds iron(II)
(
λmax 550 nm; Figure 8C). We felt that
=
2
+
HPs 2 (pKa = 6.74; most acidic, largest shift in MIC with Cu ), 11, 12 (pKa = 7.70; least acidic, smallest shift
2
+
in MIC with Cu ) and 14 (Table 3). Although pKa values correlated to changes in MIC with copper(II) coꢀ
treatment, changes in MIC values with iron(II) coꢀtreatment had less of a correlation to phenolic acidity of these
HP analogues. We then evaluated the general metalꢀchelating agent ethylenediaminetetraacetic acid (EDTA) in
biofilm eradication assays against MRSAꢀ2 to determine if metal sequestration was critical to the observed
biofilm eradication activities observed by our HP analogues; however, EDTA demonstrated no biofilm
eradication at the highest concentrations tested (MBEC > 2,000 µM).
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Interestingly, in kill curve experiments against bacteria in exponentialꢀgrowth phase, our HP analogues
demonstrated bacteriostatic activity as the minimum bactericidal concentration (MBC) is >4ꢀfold higher than
corresponding MIC values (see Supporting Information). At higher concentrations, we previously demonstrated
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
28
that 14 slowly kills stationary MRSAꢀ2 cells unlike 64, which is a rapid killer of stationary MRSA cells.
From these results, we conclude that HP analogues operate primarily through a unique metal(II)ꢀdependent
mechanism that is critical to persister cells in biofilms; however, detailed mechanistic investigations are
required to fully understand our preliminary findings.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
Here, we described the chemical synthesis, biological investigations and detailed structureꢀactivity relationships
of a new series of HP analogues, inspired by marine phenazine antibiotic 1. Halogenated phenazines
demonstrate potent biofilm eradication activities against multiple MRSA, MRSE and VRE biofilms and operate
through a nonꢀmembrane lysing mechanism, similar to antimicrobial peptide mimics. HP 14 proved to be the
most potent biofilmꢀeradicating agent against every drugꢀresistant isolate tested for biofilm eradication and
killed >99.9% of MRSA and MRSE persister cells at the corresponding MBEC value. In addition, HPs 11 and
1
2 demonstrated potent antibacterial activity against the slowꢀgrowing human pathogen M. tuberculosis (MIC =
3
.13 µM; ~1 µg/mL). We demonstrated that HP 2 directly binds copper(II) and iron(II), which likely plays a
role in the mode of action; however, detailed mechanistic studies are required. Halogenated phenazines are a
promising class of biofilmꢀeradicating agents that effectively target multiple persistent bacterial phenotypes
(i.e., biofilms, slowꢀgrowing MtB) and could lead to novel therapeutics to treat a spectrum of chronic and
recurring bacterial infections.
EXPERIMENTAL SECTION
General Information. All synthetic reactions were carried out under an inert atmosphere of argon unless
otherwise specified. Reagents for chemical synthesis were purchased from commercial sources and used
without further purification. All microwave reactions were carried out in an Anton Paar Monowave 300
Microwave Synthesis Reactor. Analytical thin layer chromatography (TLC) was performed using 250 ꢁm
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Journal of Medicinal Chemistry
Silica Gel 60 F254 preꢀcoated plates (EMD Chemicals Inc.). Flash column chromatography was performed
using 230ꢀ400 Mesh 60Å Silica Gel from Sorbent Technologies. All melting points were obtained, uncorrected,
using a MelꢀTemp capillary melting point apparatus from Laboratory Services, Inc. NMR experiments were
recorded using broadband probes on a Varian MercuryꢀPlusꢀ400 spectrometer via VNMRꢀJ software (400 MHz
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
13
for H and 100 MHz for C). Spectra for all new compounds were obtained in the following solvents
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
1
13
(
reference peaks also included for H and C NMRs): CDCl ( H NMR: 7.26 ppm; C NMR: 77.23 ppm), d -
3
6
1
13
1
13
DMSO ( H NMR: 2.50 ppm; C NMR: 39.52 ppm) and d ꢀMeOD ( H NMR: 3.31 ppm; C NMR: 49.00
4
1
ppm). H NMR multiplicities are reported as: s = singlet, br. s = broad singlet, d = doublet, t = triplet, q =
quartet, m = multiplet. HighꢀResolution Mass Spectrometry (HRMS) were obtained for all new compounds
from the Chemistry Department at the University of Florida. The purities of all compounds evaluated in
biological assays were confirmed to be ≥95% by LCꢀMS using a Shimadzu Prominence HPLC system, AB
Sciex 3200 QTRAP spectrometer, and a Kinetex C18 column (50 mm × 2.1 mm × 2.6 µm) with a 13 minute
linear gradient from 10ꢀ80% acetonitrile in 0.1% formic acid at a flow rate of 0.25 mL/min.
Bacterial strains used during these investigations include: methicillinꢀresistant Staphylococcus aureus (Clinical
Isolate from Shands Hospital in Gainesville, FL: MRSAꢀ2; ATCC strains: BAAꢀ1707, BAAꢀ44) methicillinꢀ
resistant Staphylococcus epidermidis (MRSE strain ATCC 35984; methicillinꢀsensitive strain ATCC 12228),
vancomycinꢀresistant Enterococcus faecium (VRE strain ATCC 700221), Acinetobacter baumannii (ATCC
1
9606), Pseudomonas aeruginosa (PAO1), Klebsiella pneumoniae (ATCC 13883) and Escherichia coli clinical
isolate (UAECꢀ1). All compounds were stored as DMSO stocks at room temperature in the absence of light for
several months at a time without observing any loss in biological activity. To ensure compound integrity of our
DMSO stock solutions, we did not subject DMSO stocks of our test compounds to freezeꢀthaw cycles.
Chemistry.
General dihalogenation procedure at the 2ꢀ and 4ꢀposition of the phenazine ring (3, 18): 8ꢀBromoꢀ1ꢀ
hydroxyphenazine 57 (43.2 mg, 0.16 mmol) and Nꢀbromosuccinimide, (57.5 mg, 0.32 mmol) were dissolved in
dichloromethane (10 mL) and allowed to stir at room temperature for 2 hours. The reaction contents were then
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Journal of Medicinal Chemistry
Page 18 of 47
concentrated, adsorbed onto silica gel and purified via column chromatography using dichloromethane to elute
8 as a dark orange solid (46%, 31.3 mg). Note: Analogous reaction conditions were used to synthesize
analogue 3.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
2
,4-Dichloro-1-hydroxyphenazine (3): 47% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.40 (m, 1H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
8
.36 (br. s, 1H), 8.26 (m, 1H), 7.96ꢀ7.89 (m, 2H), 7.91 (s, 1H). C NMR (100 MHz, CDCl ): δ 147.0, 143.9,
3
141.7, 139.0, 134.6, 132.3, 132.0, 131.9, 130.4, 129.1, 123.2, 114.6. HRMS (DART): calc. for C H N Cl O
12
7
2
2
+
[
M+H] : 264.9930, found: 264.9932. MP: 205 ꢀ 207 °C.
1
2
,4,8-Tribromo-1-hydroxyphenazine (18): 46% yield; orange solid. H NMR (400 MHz, d ꢀDMSO): δ 8.50 (d,
6
13
J = 2.2 Hz, 1H), 8.45 (s, 1H), 8.26 (d, J = 9.3 Hz, 1H), 8.15 (dd, J = 9.3, 2.2 Hz, 1H). C NMR (100 MHz, d6ꢀ
DMSO): δ 150.9, 141.5, 141.5, 139.6, 137.3, 135.8, 135.3, 131.3, 130.6, 125.7, 111.6, 105.3. HRMS (ESI):
+
calc. for C H Br N O [M+H] : 430.8025, found: 430.8010. MP: 239 ꢀ 241 °C.
1
2
6
3
2
Synthesis of 2,4,ꢀDiiodoꢀ1ꢀhydroxyphenazine (4): To a mixture of 1ꢀhydroxyphenazine 42 (68.1 mg, 0.35
mmol) in a 9:1 solution of glacial acetic acid:water (15 mL) was added sodium chloride (244 mg, 4.17 mmol),
sodium periodate (446 mg, 2.08 mmol), then potassium iodide (241 mg, 2.08 mmol). The reaction was then
allowed to stir at room temperature for 8 hours. Upon completion of this reaction (as determined by TLC
analysis using dichloromethane), the reaction contents were transferred to a separatory funnel containing a
solution of saturated sodium bicarbonate, and the aqueous layer was extracted with dichloromethane. The
organic layer was collected, dried with anhydrous sodium sulfate and concentrated in vacuo. The resulting solid
was adsorbed onto silica gel (dry loading with dichloromethane) and purified via column chromatography using
first hexanes to elute an undesired purple product, followed by dichloromethane to elute pure 4 (19%, 79.2 mg),
1
which was isolated as a yellow solid. H NMR (400 MHz, CDCl ): δ 8.78 (br. s, 1H), 8.65 (s, 1H), 8.40 (m,
3
1
3
1
H), 8.27 (m, 1H), 7.97 – 7.88 (m, 2H). C NMR (100 MHz, CDCl ): δ 153.9, 148.1, 144.9, 142.3, 141.7,
3
+
1
33.1, 132.1, 131.8, 130.3, 128.9, 89.6, 76.5. HRMS (DART): calc. for C H I N O [M+H] : 448.8642, found:
1
2
7 2
2
448.8623. MP: 193 ꢀ 195 °C.
General procedure for the monohalogenation at the 2ꢀposition of the phenazine ring (5ꢀ10, 15 and 22): 4ꢀ
Chloroꢀ1ꢀhydroxyphenazine 40 (75.4 mg, 0.33 mmol) and Nꢀbromosuccinimide (58.2 mg, 0.33 mmol) were
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Journal of Medicinal Chemistry
dissolved in 15 mL dichloromethane and allowed to stir at room temperature for 1 hour. The reaction contents
were then concentrated, adsorbed onto silica gel and purified via column chromatography using
dichloromethane to elute 2ꢀbromoꢀ4ꢀchloroꢀ1ꢀhydroxyphenazine 7, which was isolated as a yellow solid (91%,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
1.9 mg). Note: Analogous procedures were used for chlorination and iodination using N-chlorosuccinimide
and Nꢀiodosuccinimide, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4-Bromo-2-chloro-1-hydroxyphenazine (5): 34% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.44 –
3
13
8
1
.35 (m, 2H), 8.26 (m, 1H), 8.13 (s, 1H), 7.96 – 7.89 (m, 2H). C NMR (100 MHz, CDCl ): δ 147.6, 144.2,
3
41.6, 139.7, 135.4, 134.6, 132.3, 131.8, 130.4, 128.9, 115.2, 112.9. HRMS (DART): calc. for C H BrClN O
12
7
2
+
[
M+H] : 308.9425, found: 308.9437. MP: 229 ꢀ 231 °C.
1
2
-Chloro-4-iodo-1-hydroxyphenazine (6): 42% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.43 (br. s,
3
1
3
1
H), 8.39 (m, 1H), 8.38 (s, 1H), 8.26 (m, 1H), 7.96 – 7.89 (m, 2H). C NMR (100 MHz, CDCl ): δ 148.5,
3
144.6, 141.9, 141.7, 141.3, 134.0, 132.2, 131.7, 130.3, 128.8, 116.0, 88.9. HRMS (DART): calc. for
+
C H ClIN O [M+H] : 356.9286, found: 356.9281. MP: 183 ꢀ 185 °C.
12
7
2
1
2
-Bromo-4-chloro-1-hydroxyphenazine (7): 91% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.47 (br.
3
1
3
s, 1H), 8.39 (m, 1H), 8.25 (m, 1H), 8.03 (s, 1H), 7.99 ꢀ 7.86 (m, 2H). C NMR (100 MHz, CDCl ): δ 148.6,
3
1
44.0, 141.6, 139.4, 134.4, 134.0, 132.3, 131.9, 130.5, 129.1, 123.4, 102.6. HRMS (DART): calc. for
+
C H BrClN O [M+H] : 308.9425, found: 308.9434. MP: 212 ꢀ 214 °C.
12
7
2
1
2-Bromo-4-iodo-1-hydroxyphenazine (8): 65% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.57 (br. s,
3
13
1
H), 8.51 (s, 1H), 8.40 (m, 1H), 8.27 (m, 1H), 7.97 ꢀ 7.89 (m, 2H). C NMR (100 MHz, CDCl ): δ 150.2,
3
1
44.7, 143.8, 141.7, 141.7, 134.0, 132.2, 131.8, 130.3, 128.8, 104.2, 89.2. HRMS (DART): calc. for
+
C H BrIN O [M+H] : 400.8781, found: 400.8785. MP: 186 ꢀ 188 °C.
12
7
2
1
4
-Chloro-2-iodo-1-hydroxyphenazine (9): 82% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.67 (br. s,
3
1
3
1
H), 8.36 (m, 1H), 8.21 (m, 1H), 8.14 (s, 1H), 7.98 ꢀ 7.85 (m, 2H). C NMR (100 MHz, CDCl ): δ 152.2,
3
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1
44.1, 141.4, 139.8, 138.4, 133.3, 132.2, 131.9, 130.4, 129.1, 123.4, 74.4. HRMS (DART): calc. for
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
C H ClIN O [M+H] : 356.9286, found: 356.9299. MP: 175 ꢀ 177 °C.
12
7
2
1
4
-Bromo-2-iodo-1-hydroxyphenazine (10): 48% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.71 (br. s,
3
1
3
1
H), 8.36 (m, 1H), 8.35 (s, 1H), 8.22 (m, 1H), 7.95 ꢀ 7.87 (m, 2H). C NMR (100 MHz, CDCl ): δ 152.8,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
144.4, 141.7, 141.4, 140.6, 133.4, 132.2, 131.9, 130.3, 129.0, 113.3, 75.2. HRMS (DART): calc. for
+
C H BrIN O [M+H] : 400.8781, found: 400.8770. MP: 188 ꢀ 190 °C.
12
7
2
1
4,7,8-Tribromo-2-chloro-1-hydroxyphenazine (15): 65% yield; orange solid. H NMR (400 MHz, d ꢀDMSO): δ
6
13
8
.78 (s, 1H), 8.68 (s, 1H), 8.41 (s, 1H). C NMR (100 MHz, d ꢀDMSO): δ 149.5, 141.7, 140.4, 139.7, 136.3,
6
+
1
35.9, 133.3, 132.6, 128.2, 128.1, 116.5, 111.3. HRMS (DART): calc. for C H Br ClN O [M+H] : 464.7635,
12
5
3
2
found: 464.7614. MP: 242 ꢀ 244 °C.
1
2
1
1
-Bromo-4-butyl-1-hydroxyphenazine (22): 53% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.38 (br. s,
3
H), 8.28 (m, 1H), 8.21 (m, 1H), 7.90 – 7.80 (m, 2H), 7.67 (t, J = 0.8 Hz 1H), 3.26 (dt, J = 7.8, 0.9 Hz, 2H),
13
.83 – 1.75 (m, 2H), 1.47 (tq, J = 7.4, 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ): δ
3
147.2, 143.4, 142.0, 141.0, 134.7, 134.0, 132.9, 131.2, 130.6, 130.5, 129.0, 103.5, 32.9, 30.1, 22.9, 14.3.
+
HRMS (ESI): calc. for C H BrN O [M+H] : 331.0441, found: 331.0462. MP: 135 ꢀ 137 °C.
1
6
16
2
General procedure for boron tribromide demethylation (13, 40, 41, 43, 57, 58): To a round bottom flask
was added 7,8ꢀdichloroꢀ1ꢀmethoxyphenazine 20 (218 mg, 0.78 mmol) dissolved in anhydrous dichloromethane
(18 mL). The mixture was brought to ꢀ78 °C in a dry ice bath before dropwise addition of 1M boron tribromide
solution in dichloromethane (5.5 mL, 5.5 mmol). The reaction was left to stir at ꢀ78 °C for 1 hour, and then
allowed to reach ambient temperature for reaction overnight. The reaction was heated to reflux for 8 hours until
complete (monitored by TLC). The solution was transferred to a separatory funnel containing an aqueous
solution of saturated sodium bicarbonate, and then extracted with dichloromethane. Organic solvents were
dried with sodium sulfate, filtered through cotton, and removed in vacuo. The resulting solid was purified via
column chromatography using dichloromethane to elute compound 21 as an orange solid (>99%, 209 mg).
Note: Analogous procedures were used for all demethylation reactions using BBr3.
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Journal of Medicinal Chemistry
1
4
,7,8-Tribromo-1-hydroxyphenazine (13): 96% yield; orange solid. H NMR (400 MHz, CDCl ): δ 8.80 (s,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
1
H), 8.62 (s, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.04 (s, 1H), 7.18 (d, J = 8.1 Hz, 1H). C NMR (100 MHz,
CDCl ): δ 151.8, 143.2, 141.4, 140.2, 135.8, 135.6, 134.0, 132.7, 129.7, 129.1, 112.5, 110.7. HRMS (DART):
3
+
calc. for C H Br N O [M+H] : 430.8025, found: 430.8017. MP: >260 °C.
1
2
6
3
2
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-Chloro-1-hydroxyphenazine (40): 95% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.40 (m, 1H), 8.23
3
13
(
m, 1H), 8.17 (s, 1H), 7.97 – 7.86 (m, 2H), 7.87 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H). C NMR (100
13
MHz, CDCl ): δ 151.1, 144.2, 141.4, 140.3, 135.1, 131.6, 131.1, 130.4, 129.1, 122.5, 108.8. Note: One C
3
+
signal missing, likely due to overlap. HRMS (DART): calc. for C H ClN O [M+H] : 231.0320, found:
1
2
8
2
2
31.0330. MP: 196 ꢀ 198 °C.
1
4
-Iodo-1-hydroxyphenazine (41): 86% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.41 (m, 1H), 8.36 (d,
3
13
J = 8.0 Hz, 1H), 8.30 – 8.20 (m, 2H), 7.97 – 7.82 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H). C NMR (100 MHz,
CDCl ): δ 152.9, 144.9, 142.6, 141.6, 141.5, 134.9, 131.5, 131.5, 130.2, 128.9, 110.8, 88.4. HRMS (DART):
3
+
calc. for C H IN O [M+H] : 322.9676, found: 322.9689. MP: 193 ꢀ 195 °C.
1
2
8
2
1
8
-Bromo-1-hydroxyphenazine (57): >99% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.42 (dd, J = 2.2,
3
0
.5 Hz, 1H), 8.12 (dd, J = 9.2, 0.5 Hz, 1H), 8.09 (s, 1H), 7.90 (dd, J = 9.3, 2.2 Hz, 1H), 7.82 – 7.66 (m, 2H),
1
3
7
.26 (dd, J = 6.9, 1.7 Hz, 1H). C NMR (100 MHz, CDCl ): δ 151.8, 144.0, 142.9, 141.5, 135.0, 134.7, 132.5,
3
+
1
31.3, 131.1, 125.2, 120.2, 109.9. HRMS (ESI): calc. for C H BrN O [M+H] : 274.9815, found: 274.9826.
1
2
8
2
5
1
MP: 189 ꢀ 191 °C., lit. 176 ꢀ 177 °C.
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1
4
-Butyl-1-hydroxyphenazine (58): 90% yield; 33.2 mg of 58 was isolated as a yellow solid. H NMR (400
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MHz, CDCl ): δ 8.28 (m, 1H), 8.17 (m, 1H), 8.10 (br. s, 1H), 7.84 – 7.76 (m, 2H), 7.53 (dt, J = 7.5, 0.9 Hz,
3
1H), 7.15 (d, J = 7.5 Hz, 1H), 3.28 (td, J = 7.4 Hz, 0.9 Hz, 2H), 1.84 – 1.75 (m, 2H), 1.47 (tq, J = 7.4, 7.4 Hz,
13
2
H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ): δ 149.7, 143.5, 142.9, 140.8, 135.0, 132.6, 130.4,
3
+
1
30.4, 130.2, 129.6, 129.1, 108.7, 33.0, 30.3, 22.9, 14.3. HRMS (ESI): calc. for C H N O [M+H] : 253.1335,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16 17
2
found: 253.1325. MP: 138 ꢀ 140 °C.
General procedure for the halogenation of the 4ꢀposition to synthesize 23 and 24: 2ꢀAllylꢀ1ꢀ
hydroxyphenazine 62 (46.5 mg, 0.20 mmol) and Nꢀbromosuccinimide (35.1 mg, 0.20 mmol) were dissolved in
1
0 mL dichloromethane and allowed to stir at room temperature for 1 hour. The reaction contents were then
concentrated, adsorbed onto silica gel and purified via column chromatography using dichloromethane to elute
ꢀallylꢀ4ꢀbromoꢀ1ꢀhydroxyphenazine 23, which was isolated as a yellow solid (93%, 58.8 mg). Note: An
analogous procedure was used to synthesize 24.
2
1
2
-Allyl-4-bromo-1-hydroxyphenazine (23): 93%% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.35 (m,
3
1
H), 8.22 (s, 1H), 8.16 (m, 1H), 7.98 (s, 1H), 7.92 – 7.76 (m, 2H), 6.06 (ddt, J = 16.7, 10.1, 6.6 Hz, 1H), 5.21
13
(ddt, J = 16.7, 1.7, 1.6 Hz, 1H), 5.17 (ddt, J = 10.1, 1.7, 1.5 Hz, 1H) 3.66 (ddd, J = 6.6, 1.6, 1.5 Hz, 2H).
C
NMR (100 MHz, CDCl ): δ 148.2, 144.0, 141.4, 140.0, 136.9, 135.4, 134.8, 131.3, 131.0, 130.3, 128.9, 121.8,
3
+
1
17.1, 112.0, 33.8. HRMS (ESI): calc. for C H BrN O [M+H] : 315.0128, found: 315.0127. MP: 149 ꢀ 151
1
5
12
2
°
C.
1
2
-Allyl-4,7,8-tribromo-1-hydroxyphenazine (24): 39% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.74
3
(s, 1H), 8.57 (s, 1H), 8.07 (br. s, 1H), 8.03 (s, 1H), 6.04 (ddt, J = 16.8, 10.1, 6.6 Hz, 1H), 5.21 (dd, 16.8, 1.6 Hz,
1
3
1H), 5.19 (dd, 10.1, 1.6 Hz, 1H), 3.66 (d, J = 6.6 Hz, 2H). C NMR (100 MHz, CDCl ): δ 148.2, 142.7, 140.4,
3
1
40.2, 138.1, 135.3, 135.0, 134.0, 132.6, 128.9, 128.5, 123.1, 117.5, 112.2, 33.8. HRMS (ESI): calc. for
+
C H Br N O [M+H] : 472.8318, found: 472.8331. MP: 187 ꢀ 189 °C.
15 14
3
2
General procedure for monohalogenation at the 4ꢀposition of the phenazine (33, 36, 38): 7,8ꢀDichloroꢀ1ꢀ
methoxyphenazine 20 (151 mg, 0.54 mmol) was dissolved in dichloromethane (15 mL) before Nꢀ
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Journal of Medicinal Chemistry
bromosuccinimide (106 mg, 0.60 mmol) was added and the reaction was brought to reflux. The mixture was
left to stir overnight until complete (monitored by TLC with dichloromethane). At this time, the reaction was
concentrated and adsorbed onto silica gel (via dissolving the crude reaction contents and silica gel in
dichloromethane, then concentrating via rotavap) and purified via column chromatography using
dichloromethane to elute pure 4ꢀbromoꢀ7,8ꢀdichloroꢀ1ꢀmethoxyphenazine 36, which was isolated as a dark
yellow solid (96%, 185.2 mg). Notes: Analogous procedures were used for the chlorination and iodination of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ꢀmethoxyphenazines using N-chlorosuccinimide or Nꢀiodosuccinimide.
1
4
-Chloro-1-methoxyphenazine (33): 76% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.19 – 8.08 (m,
3
1
3
2
H), 7.71 – 7.61 (m, 2H), 7.53 (d, J = 8.2 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H). C NMR (100 MHz,
CDCl ): δ 154.0, 142.9, 141.8, 139.9, 136.6, 131.1, 130.8, 129.6, 129.5, 129.2, 123.5, 105.7, 56.3. HRMS
3
+
(DART): calc. for C H ClN O [M+H] : 245.0476, found: 245.0484. MP: 149 ꢀ 151 °C.
1
3
10
2
1
4
-Bromo-7,8-dichloro-1-methoxyphenazine (36): 96% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.56
3
1
3
(
s, 1H), 8.54 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 4.17 (s, 3H). C NMR (100 MHz,
CDCl ): δ 155.3, 142.2, 141.6, 140.9, 137.8, 136.8, 136.4, 134.4, 130.2, 130.2, 114.3, 107.9, 57.0. HRMS
3
+
(
DART): calc. for C H BrCl N O [M+H] : 356.9192, found: 356.9194. MP: 199 ꢀ 201 °C.
1
3
8
2
2
1
4
,7,8-Tribromo-1-methoxyphenazine (38): 86% yield; yellow solid. H NMR (400 MHz, CDCl ): δ 8.75 (s, 1H),
3
13
8
.72 (s, 1H), 8.10 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.16 (s, 3H). C NMR (100 MHz, CDCl ): δ
3
155.3, 142.5, 141.6, 141.2, 137.8, 134.5, 133.7, 133.6, 129.1, 128.6, 114.4, 107.9, 57.0. HRMS (DART): calc.
+
for C H Br N O [M+H] : 444.8181, found: 444.8181. MP: 241 ꢀ 243 °C.
1
3
8
3
2
Synthesis of 4ꢀiodoꢀ1ꢀmethoxyphenazine 35 (potassium iodide and sodium periodate): To a mixture of 1ꢀ
methoxyphenazine 32 (356 mg, 1.69 mmol) in 9:1 acetic acid:water (30 mL) was added sodium chloride (435
mg, 7.45 mmol), sodium periodate (869 mg, 4.06 mmol), then potassium iodide (675 mg, 4.06 mmol). The
reaction was heated to 65 °C and allowed to stir for 21 hours. After completion of this reaction as determined
by TLC analysis (with dichloromethane), the reaction mixture was washed with a saturated solution of sodium
bicarbonate and then partitioned with dichloromethane. The organic layers were then collected, dried with
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anhydrous sodium sulfate and concentrated in vacuo. The resulting solid was then dry loaded onto silica gel
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(using dichloromethane) and purified via column chromatography using first hexanes to elute purple side
1
product, followed by dichloromethane to elute 35 as a yellow solid (68%, 388.0 mg). H NMR (400 MHz,
CDCl ): δ 8.37 (m, 1H), 8.32 (m, 1H), 8.30 (d, J = 8.2 Hz, 1H), 7.89 – 7.79 (m, 2H), 6.81 (d, J = 8.2 Hz, 1H),
3
1
3
4
1
.12 (s, 3H). C NMR (100 MHz, CDCl ): δ 156.2, 144.1, 142.6, 140.2, 136.9, 131.4, 131.1, 129.8, 129.7,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
08.2, 91.0, 56.8. Note: One signal obscured likely due to overlap. HRMS (DART): calc. for C H IN O
1
3
10
2
+
[
M+H] : 336.9832, found: 336.9829. MP: 169 ꢀ 171 °C.
Synthesis of 7ꢀbromoꢀ1ꢀmethoxyphenazine (52): To a 100 mL roundꢀbottom flask was added 4ꢀbromoaniline
5 (1.74 mL, 14.2 mmol), 2ꢀnitroanisole 46 (2.45 g, 14.2 mmol), and potassium hydroxide (3.99 g, 71.1 mmol)
4
in toluene (20 mL). The reaction was then allowed to reflux for 24 hours. After the reaction was complete, the
reaction contents were allowed to cool to room temperature. The reaction mixture was transferred to a
separatory funnel, partitioned between ethyl acetate, and then washed with water and brine. The organic layers
were collected, dried with anhydrous sodium sulfate, filtered and then concentrated in vacuo. The resulting
crude solid was purified via column chromatography using 99:1 to 85:15 hexanes:ethyl acetate to afford 52,
1
which was isolated as a yellow solid (2%, 87.7 mg). H NMR (400 MHz, CDCl ): δ 8.58 (d, J = 2.2 Hz, 1H),
3
8.05 (d, J = 9.2 Hz, 1H), 7.86 (dd, J = 9.2, 2.2 Hz, 1H), 7.80 – 7.70 (m, 2H), 7.05 (dd, J = 7.1, 1.5 Hz, 1H), 4.15
1
3
(
s, 3H). C NMR (100 MHz, CDCl ): δ 155.2, 144.4, 142.5, 142.2, 137.2, 134.7, 132.3, 131.1, 130.8, 124.7,
3
+
1
21.6, 107.3, 56.7. HRMS (ESI): calc. for C H BrN O [M+H] : 288.9971, found: 288.9960. MP: 194 ꢀ 196
1
3
10
2
5
1
°C., lit. 187 ꢀ 188 °C.
OMe
N
N
56
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis of 4ꢀbutylꢀ1ꢀmethoxyphenazine (56):
To a 50 mL roundꢀbottom flask was added 4ꢀ
iodomethoxyphenazine 35 (146 mg, 0.43 mmol), tetrakis(triphenylphosphine)palladium(0) (100 mg, 0.1 mmol),
sodium hydroxide (347 mg, 8.67 mmol), and nꢀbutylboronic acid pinacol ester 64 (1.43 mL, 6.72 mmol) in a
2:1 solution of toluene:water (6 mL). The mixture was heated to 90 °C in an oil bath and left to stir for 48
hours. After cooling to room temperature, the reaction mixture was transferred to a separatory funnel,
partitioned between an aqueous solution of saturated sodium bicarbonate and ethyl acetate. The resulting
organic layer was washed with a 1 N solution of hydrochloric acid, and then brine. The organic layers were
combined and dried with anhydrous sodium sulfate and concentrated in vacuo. The resulting crude material
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was purified via column chromatography using 99:1 to 85:15 hexanes:ethyl acetate to afford 56 as a yellow
1
solid (34% yield, 38.9 mg). H NMR (400 MHz, CDCl ): δ 8.38 (m, 1H), 8.26 (m, 1H), 7.85 – 7.78 (m, 2H),
3
7
.54 (dt, J = 7.8, 0.9 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 4.15 (s, 3H), 3.30 (td, J = 7.6, 0.9 Hz, 2H), 1.87 – 1.72
13
(m, 2H), 1.47 (tq, J = 7.4, 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ): δ 153.4, 143.3,
3
1
43.0, 141.9, 137.2, 133.9, 130.4, 130.2, 130.1, 130.1, 128.4, 106.5, 56.5, 32.9, 30.6, 22.9, 14.3. HRMS (ESI):
+
calc. for C H N O [M+H] : 267.1492, found: 267.1497. MP: 77 ꢀ 79 °C.
17 19
2
General procedure for phenolic allylation (60, 61): Potassium carbonate (866 mg, 6.27 mmol) was added to
a stirring solution of 1ꢀhydroxyphenazine 42 (247 mg, 1.25 mmol) in 15 mL acetone. The resulting mixture was
allowed to stir at room temperature for 30 minutes before allyl bromide (0.13 mL, 1.51 mmol) was added to the
reaction. The resulting reaction mixture was allowed to stir for an additional 17 hours at reflux. The reaction
contents were then transferred to a separatory funnel and extracted with ethyl acetate. The organic contents
were then dried with anhydrous sodium sulfate and then removed in vacuo. The resulting crude solid was
purified via column chromatography using 99:1 to 90:10 hexanes:ethyl acetate to afford 1ꢀallyloxyphenazines
60, which was isolated as a yellow solid (85%, 251.0 mg). Note: An analogous procedure was used to
synthesize 61.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-(Allyloxy)phenazine (60): 85% yield; yellow solid. H NMR (100 MHz, CDCl ): δ 8.33 (m, 1H), 8.15 (m,
3
1
H), 7.79 – 7.70 (m, 3H), 7.63 (dd, J = 8.9, 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.17 (ddt, J = 17.3, 10.6, 5.5
Hz, 1H), 5.47 (ddt, J = 17.3, 1.6, 1.6 Hz, 1H), 5.32 (ddt, J = 10.6, 1.6, 1.6 Hz, 1H), 4.88 (ddd, J = 5.5, 1.6, 1.6
13
Hz, 2H). C NMR (100 MHz, CDCl ): δ 154.0, 144.3, 143.5, 142.3, 137.1, 132.7, 130.8, 130.4, 130.4, 130.1,
3
+
1
29.3, 121.6, 118.6, 108.2, 70.2. HRMS (ESI): calc. for C H N O [M+H] : 237.1022, found: 237.1026. MP:
15 13
2
1
17 ꢀ 119 °C.
1
7,8-Dibromo-1-allyloxyphenazine (61): 88% yield; orange solid. H NMR (400 MHz, CDCl ): δ 8.70 (s, 1H),
3
8
.51 (s, 1H), 7.76 – 7.69 (m, 2H), 7.07 (dd, J = 5.9, 2.8 Hz, 1H), 6.21 (ddt, J = 17.4, 10.6, 5.4 Hz, 1H), 5.53
1
3
(
ddt, J = 17.4, 1.5, 1.5 Hz, 1H), 5.39 (ddt, J = 10.6, 1.5, 1.3 Hz, 1H), 4.92 (ddd, J = 5.4, 1.5, 1.3 Hz, 2H).
C
NMR (100 MHz, CDCl ): δ 154.2, 144.8, 142.4, 141.2, 137.6, 134.1, 133.2, 132.6, 131.7, 128.0, 127.2, 121.7,
3
+
1
19.0, 109.1, 70.4. HRMS (ESI): calc. for C H Br N O [M+H] : 394.9213, found: 394.9211. MP: 170 ꢀ 172
1
5
10
2
2
°
C.
General procedure for Claisen rearrangement (62, 63): To an 8 mL sealed microwave vial was added 1ꢀ
allyloxy)phenazine 60 (19.4 mg, 0.08 mmol) in ethanol (4 mL). The resulting mixture was then heated at 150
C in the microwave reactor for 90 minutes. The solvent was removed in vacuo and the resulting solid was
(
°
purified via column chromatography using dichloromethane to elute, affording 2ꢀallylphenazine 62, which was
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Journal of Medicinal Chemistry
isolated as a yellow solid (>99%, 19.3 mg). Note: Analogous reaction conditions were used to synthesize 63
from 61.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
-Allyl-1-hydroxyphenazine (62): >99% yield; yellow solid. H NMR (100 MHz, CDCl ): δ 8.28 (br. s, 1H),
3
8
.21 (m, 1H), 8.13 (m, 1H), 7.81 – 7.74 (m, 2H), 7.72 (d, J = 9.1 Hz, 1H), 7.64 (d, J = 9.1 Hz, 1H), 6.09 (ddt, J
=
16.7, 10.1, 6.6 Hz, 1H), 5.18 (ddt, J = 16.7, 1.7, 1.7 Hz, 1H), 5.14 (ddt, J = 10.1, 1.7 Hz, 1.6 Hz, 1H) 3.68
1
3
(ddd, J = 6.7, 1.7, 1.6 Hz, 2H). C NMR (100 MHz, CDCl ): δ 148.2, 143.8, 143.0, 141.3, 136.0, 134.6, 134.5,
3
+
1
30.4, 130.4, 129.9, 129.2, 120.8, 119.6, 116.5, 34.0. HRMS (ESI): calc. for C H N O [M+H] : 237.1022,
1
5
13
2
found: 237.1017. MP: 112 ꢀ 114 °C.
1
2
-Allyl-7,8-dibromo-1-hydroxyphenazine (63): >99% yield; orange solid. H NMR (400 MHz, CDCl ): δ 8.59
3
(d, J = 0.4 Hz, 1H), 8.56 (d, J = 0.4 Hz, 1H), 8.07 (s, 1H), 7.74 – 7.69 (m, 2H), 6.07 (ddt, J = 17.1, 10.1, 6.6 Hz,
1
H), 5.19 (ddt, J = 17.1, 1.6, 1.6 Hz, 1H), 5.16 (ddt, J = 10.1, 1.6, 1.5 Hz, 1H) 3.69 (ddd, J = 6.6, 1.6, 1.5 Hz,
1
3
2
H). C NMR (100 MHz, CDCl ): δ 148.3, 143.5, 142.7, 140.2, 135.7, 135.6, 135.0, 133.7, 133.0, 127.7,
3
+
1
27.6, 122.2, 119.9, 116.8, 34.1. HRMS (ESI): calc. for C H Br N O [M+H] : 394.9213, found: 394.9216.
1
5
10
2
2
MP: 199 ꢀ 201 °C.
Biology.
Microdilution Minimum Inhibitory Concentration (MIC) Assay. The minimum inhibitory concentration
(MIC) for each compound was determined by the broth microdilution method as recommended by the Clinical
5
2
and Laboratory Standards Institute (CLSI).
In a 96ꢀwell plate, eleven twoꢀfold serial dilutions of each
5
compound were made in a final volume of 100 ꢁL Luria Broth. Each well was inoculated with ~10 bacterial
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cells at the initial time of incubation, prepared from a fresh log phase culture (OD600 of 0.5 to 1.0 depending on
bacterial strain). The MIC was defined as the lowest concentration of compound that prevented bacterial
growth after incubating 16 to 18 hours at 37 °C (MIC values were supported by spectrophotometric readings at
OD ). The concentration range tested for each phenazine analogue/antibacterial during this study was 0.10 to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
00
1
00 ꢁM. DMSO served as our vehicle and negative control in each microdilution MIC assay. DMSO was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
serially diluted with a top concentration of 1% v/v. All compounds were tested in two independent
experiments, active compounds were tested in a third independent experiment (lead compounds were tested in
more assays as positive controls during these studies).
Microdilution MIC Assay for Mycobacterium tuberculosis. M. tuberculosis H37Ra (ATCC 25177) was
inoculated in 10 mL Middlebrook 7H9 medium and allowed to grow for two weeks. The culture was then
diluted with fresh medium until an OD600 of 0.01 was reached. Aliquots of 200 µL were then added to each well
of a 96ꢀwell plate starting from the second column. Test compounds were dissolved in DMSO at final
concentration of 10 mM. 7.5 µL of each compound along with DMSO (negative control) and streptomycin
(positive controlꢀ40mg/ml stock solution) were added to 1.5 mL of the Mycobacterium 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 96ꢀwell plate and serially diluted twoꢀfold (200 µL) per well across the plate to obtain final
concentrations that ranges from 0.024 to 50 µM for the test compounds and 0.16 to 340 µM for streptomycin.
Three rows were reserved for each compound. The plates were then incubated at 37°C for seven 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). Data obtained from
three independent experiments were analyzed using Excel.
Calgary Biofilm Device (CBD) Experiments. Biofilm eradication experiments were performed using the
Calgary Biofilm Device to determine MBC/MBEC values for various compounds of interest (Innovotech,
product code: 19111). The Calgary device (96ꢀwell plate with lid containing pegs to establish biofilms on) was
inoculated with 125 µL of a midꢀlog phase culture diluted 1,000ꢀfold in tryptic soy broth with 0.5% glucose
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Page 29 of 47
Journal of Medicinal Chemistry
(
TSBG) to establish bacterial biofilms after incubation at 37 °C for 24 hours. The lid of the Calgary device was
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then removed, washed and transferred to another 96ꢀwell plate containing 2ꢀfold serial dilutions of the test
compounds (the “challenge plate”). The total volume of media with compound in each well in the challenge
plate is 150 µL. The Calgary device was then incubated at 37 °C for 24 hours. The lid was then removed from
the challenge plate and MBC/MBEC values were determined using different final assays. To determine MBC
values, 20 µL of the challenge plate was transferred into a fresh 96ꢀwell plate containing 180 µL TSBG 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 the determination of MBEC values, the Calgary device lid (with attached
pegs/treated biofilms) was transferred to a new 96ꢀwell plate containing 150 µL of fresh TSBG media in each
well and incubated for 24 hours at 37 °C to allow viable biofilms to grow and disperse resulting in turbidity
after the incubation period. MBEC values were determined as the lowest test concentration that resulted in
eradicated biofilm (i.e., wells that had no turbidity after final incubation period). In select experiments, pegs
from the Calgary device were removed from lead biofilm eradicators after final incubation, sonicated for 30
minutes in PBS and plated out to determine biofilm cell killing of lead biofilmꢀeradicating agents (i.e., colony
forming unit per milliliter, CFU/mL; Figure 4). All compounds were tested in two independent experiments,
active compounds were tested in a third independent experiment (lead compounds were tested in more assays as
positive controls during these studies). Antibiotic controls have been tested in a minimum of three independent
experiments. Note: MRSAꢀ2, S. aureus (ATCC strains: BAAꢀ1707, BAAꢀ44), S. epidermidis (ATCC 35984)
and VRE (ATCC 700221) were tested using these assay parameters.
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Live/Dead staining (Fluorescence Microscopy) of MRSE 35984 biofilms. A midꢀlog culture of MRSE
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5894 was diluted 1:1,000ꢀfold and 500 µL was transferred to each compartment of a 4 compartment
CELLview dish (Greiner BioꢀOne 627871). The dish was then incubated for 24 hours at 37 °C. After this time,
the cultures were removed and the plate was washed with 0.9% saline. The dish was then treated with the
compounds in fresh media at various concentrations. DMSO was used as our negative control in this assay. The
dish was incubated with the compound for 24 hours at 37 °C. After this time, the cultures were removed and the
dish was washed with 0.9% saline for 2 minutes. Saline was then removed and 500 µL of the stain (Live/Dead
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