An Efficient Buchwald-Hartwig/Reductive Cyclization for the Scaffold Diversification of Halogenated Phenazines: Potent Antibacterial Targeting, Biofilm Eradication, and Prodrug Exploration

Article abstract of DOI:10.1021/acs.jmedchem.7b01903

Bacterial biofilms are surface-attached communities comprised of nonreplicating persister cells housed within a protective extracellular matrix. Biofilms display tolerance toward conventional antibiotics, occur in ～80% of infections, and lead to >500000 deaths annually. We recently identified halogenated phenazine (HP) analogues which demonstrate biofilm-eradicating activities against priority pathogens; however, the synthesis of phenazines presents limitations. Herein, we report a refined HP synthesis which expedited the identification of improved biofilm-eradicating agents. 1-Methoxyphenazine scaffolds were generated through a Buchwald-Hartwig cross-coupling (70% average yield) and subsequent reductive cyclization (68% average yield), expediting the discovery of potent biofilm-eradicating HPs (e.g., 61: MRSA BAA-1707 MBEC = 4.69 μM). We also developed bacterial-selective prodrugs (reductively activated quinone-alkyloxycarbonyloxymethyl moiety) to afford HP 87, which demonstrated excellent antibacterial and biofilm eradication activities against MRSA BAA-1707 (MIC = 0.15 μM, MBEC = 12.5 μM). Furthermore, active HPs herein exhibit negligible cytotoxic or hemolytic effects, highlighting their potential to target biofilms.

Full text of DOI:10.1021/acs.jmedchem.7b01903

Subscriber access provided by Kaohsiung Medical University
An Efficient Buchwald-Hartwig/Reductive Cyclization for the
Scaffold Diversification of Halogenated Phenazines: Potent
Antibacterial Targeting, Biofilm Eradication and Prodrug Exploration
Aaron T. Garrison, Yasmeen Abouelhassan, Dimitris Kallifidas, Hao Tan, Young
Sik Kim, Shouguang Jin, Hendrik Luesch, and Robert William Huigens III
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01903 • Publication Date (Web): 11 Apr 2018
Downloaded from http://pubs.acs.org on April 11, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 62
Journal of Medicinal Chemistry
An Efficient Buchwald-Hartwig/Reductive Cyclization for the Scaffold Diversification of
Halogenated Phenazines: Potent Antibacterial Targeting, Biofilm Eradication and Prodrug
Exploration
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
†
†
†
‡
‡
‡
Aaron T. Garrison , Yasmeen Abouelhassan , Dimitris Kallifidas , Hao Tan , Young S. Kim , Shouguang Jin ,
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
†
†*
Hendrik Luesch , 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
‡
Department of Molecular Genetics & Microbiology, College of Medicine, University of Florida, Gainesville,
Florida 32610, United States
*
Corresponding author Eꢀmail: rhuigens@cop.ufl.edu
Abstract:
Bacterial biofilms are surfaceꢀattached communities comprised of nonꢀreplicating persister cells housed within
a protective extracellular matrix. Biofilms display tolerance towards conventional antibiotics, occur in ~80% of
infections and lead to >500,000 deaths annually. We recently identified halogenated phenazine (HP)
analogues which demonstrate biofilmꢀeradicating activities against priority pathogens; however, the synthesis
of phenazines presents limitations. Herein we report of a refined HP synthesis which expedited the
identification of improved biofilmꢀeradicating agents. 1ꢀMethoxyphenazine scaffolds were generated through a
BuchwaldꢀHartwig crossꢀcoupling (70% average yield) and subsequent reductive cyclization (68% average
yield), expediting the discovery of potent biofilmꢀeradicating HPs (e.g. 61; MRSA BAAꢀ1707 MBEC = 4.69 ꢁM).
We also developed bacterialꢀselective prodrugs (reductivelyꢀactivated quinoneꢀalkyloxycarbonyloxymethyl
moiety) to afford HP 87, which demonstrated excellent antibacterial and biofilm eradication activities against
MRSA BAAꢀ1707 (MIC = 0.15 ꢁM, MBEC = 12.5 ꢁM). Furthermore, active HPs herein exhibit negligible
cytotoxic or hemolytic effects, highlighting their potential to target biofilms.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 62
Introduction:
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
New antibacterial agents that operate through unique mechanisms of action are of significant importance to
combat the emergence of antibioticꢀresistant and tolerant bacteria. Bacteria utilize multiple mechanisms (e.g.,
target modification, drug inactivation, overexpression of efflux pumps) to acquire, or gain, resistance to
1
antibiotics following drug exposure during treatment. In contrast to acquired antibiotic resistance, freeꢀfloating
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
bacteria can collectively form surfaceꢀattached biofilm communities resulting in an innate tolerance towards
2–4
antibiotic therapies due to metabolically dormant persister cells (nonꢀreplicating bacteria).
Persistent
biofilms are credited as the underlying cause of chronic and recurring bacterial infections, which is recognized
as a major clinical problem resulting in an estimated 17 million new biofilm infections and >500,000 deaths
5,6
each year.
Each of our conventional antibiotic classes was initially discovered as growthꢀinhibiting agents that targeted
rapidlyꢀdividing planktonic bacteria in phenotypic screens/assays. In addition, the large majority of our
antibiotic classes were discovered preꢀ1970, which was 25 years before bacterial biofilms were recognized as
7
medically relevant. As such, it should be no surprise that our clinically used antibiotic growth inhibitors have
essentially no effect on nonꢀreplicating biofilm bacteria. Unfortunately, biofilmꢀassociated infections remain an
unanswered clinical problem as there are currently no biofilmꢀeradicating agents available to treat persistent
7
and chronic biofilmꢀassociated infections. Biofilmꢀassociated infections are highly prevalent, occurring in
~
80% of all bacterial infections, and play a critical role in numerous infections and diseases, including: hospitalꢀ
acquired infections, implanted medical device infections (e.g., prosthetic joint replacement), catheterꢀbased
infections, skin/burn wounds, endocarditis, periodontitis, caries (tooth decay) and lung infections in Cystic
7
Fibrosis patients.
Innovative strategies are needed to address the many concerns that drugꢀresistance and antibioticꢀtolerance
(
biofilm infections) have placed on mankind. With the large majority of clinically used antibiotics being products
of microbial warfare (e.g., penicillin, streptomycin, vancomycin), we reasoned that biofilmꢀeradicating microbial
compounds exist and had likely not been identified or harnessed therapeutically. It is known that young Cystic
8
Fibrosis (CF) patients endure persistent Staphylococcus aureus lung infections. As these CF patients age,
Pseudomonas aeruginosa coꢀinfects the lungs of these patients and eradicates S. aureus. It is known that P.
ACS Paragon Plus Environment

Page 3 of 62
Journal of Medicinal Chemistry
aeruginosa is able to kill S. aureus through the use of phenazine antibiotics, which established the framework
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
8–10
for our biofilm eradication strategy design as the initial S. aureus infections were likely biofilmꢀassociated.
We discovered that halogenated phenazine (HP) analogues of the marine phenazine 2ꢀbromoꢀ1ꢀ
hydroxyphenazine 1 demonstrate potent antibacterial and biofilmꢀkilling activities (Figure 1) against drugꢀ
resistant, Gramꢀpositive pathogens (e.g. methicillinꢀresistant Staphylococcus aureus isolates) and
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
11–14
Mycobacterium tuberculosis, which is responsible for 1.5 million deaths globally each year.
With the dual
planktonic and biofilmꢀkilling activity demonstrated by select halogenated phenazines, we find this class of
natural product derived compounds to be exciting and unique. The majority of biofilmꢀeradicating agents
operate through the destruction of bacterial membranes; however, halogenated phenazines operate through a
12,15
metal(II)ꢀdependent mechanism that enables a high degree of bacterial targeting.
Through the course of
our investigations, we have found the chemical synthesis of halogenated phenazines, and highly substituted
phenazines in general, to be quite challenging. Synthetic challenges related to scaffold diversification of
biologically active heterocycles remain, yet motivated these studies and enabled us to generate novel
halogenated phenazines that displayed an array of antibacterial and biofilmꢀeradicating activities. Here we
report an efficient BuchwaldꢀHartwig/Reductive Cyclization (BHꢀRC) route enabling extensive biological
investigations of novel HP analogues not readily accessed using previous routes (Figure 2). In addition, we
report our investigations regarding the exploration of several phenolicꢀbased prodrug strategies in an attempt
to improve water solubility and target bacteria to avoid offꢀtarget metalꢀcation binding of HP analogues.
Together, these investigations demonstrate the potential for halogenated phenazine analogues to be
developed using multiple medicinal chemistry strategies.
Results and Discussion:
Construction of the Halogenated Phenazine (HP) Scaffold:
Although our previous HP analogues show great promise as antibacterial and biofilmꢀeradicating agents, a
major hindrance in the rapid assembly of HP libraries was the inherently poor means of synthesizing the
phenazine heterocycle. The reported phenazine syntheses by which the aforementioned halogenated
phenazines were generated are not without shortcomings (Figure 2A). The phenylenediamineꢀquinone
condensation used previously by our group suffers from a lack of regioselectivity with regard to substituents at
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 62
11,12
the 6ꢀ, 7ꢀ, 8ꢀ, and 9ꢀpositions of the phenazine heterocycle.
This leads to complications when structural
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
fineꢀtuning is necessary for optimization of structure activity relationship (SAR) profiles. The regioisomers
obtained from the condensation of an orthoꢀquinone and a monoꢀsubstituted phenylenediamine yields a
mixture of products that are often not easily separable. Furthermore, structural characterization of the two
resulting methoxyphenazine isomers is no trivial task. The WohlꢀAue reaction, at present, also leaves much to
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
15
be desired in terms of synthetic efficiency to afford halogenated phenazine compounds. The yields for
phenazines synthesized from the WohlꢀAue reaction are often low, owing to the formation of diphenylamine
16–18
and nitroso compounds as the major products.
Additionally, the conditions by which cyclization occurs in
the WohlꢀAue reaction are very harsh (e.g. refluxing in a highly alkaline chemical environment).
There are a handful of remaining phenazine syntheses reported in literature; however, most either utilize
19–21
homocoupling or offer little to no regioselectivity for asymmetric phenazines.
Perhaps the most elegant
phenazine synthesis, reported by the Ellman group, incorporated a rhodium(III)ꢀcatalyzed [3 + 3] annulation by
22
C−H amination with aromatic azides and diazobenzenes.
Using this route, phenazines with asymmetric
substitution can be obtained in good yield but for our purposes, the synthesis would necessitate the
preparation of either a diazobenzene or aryl azide diversityꢀincorporating coupling partner for each analogue.
Consequently, improved methods by which halogenated phenazines can be rapidly synthesized needed to be
developed. A primary goal of this work was to develop a modular methodology such that phenazine scaffolds
could be generated using readily available materials (e.g. anilines, aryl halides), permitting a high degree of
diversity with no additional synthetic steps.
One of the few synthetic methods known to regioselectively afford phenazine compounds is the reductive
23–25
cyclization of 4ꢀaminoꢀ2ꢀnitrodiphenylamines under basic conditions using sodium borohydride.
This
protocol has the potential to incorporate a multitude of commercially available anilines, nitrobenzenes, and aryl
halides into the synthesis of the diarylamine intermediate via one of several wellꢀdescribed methods (e.g.
26–32
BuchwaldꢀHartwig amination, JourdanꢀUllman coupling, nucleophilic aromatic substitution).
Thus, reductive
cyclization was selected for the synthesis of halogenated phenazines to accommodate diverse substitution at
the 6ꢀ, 7ꢀ, 8ꢀ, and 9ꢀpositions. We envisioned 1ꢀmethoxyphenazine intermediates could be obtained via
reductive cyclization through two orientations of diarylamine intermediates: 2 and 3 (Figure 2B). In addition to
expanding the scope of this synthesis, the use of two orientations of diarylamine intermediates can ostensibly
ACS Paragon Plus Environment

Page 5 of 62
Journal of Medicinal Chemistry
1
3
remedy one caveat with the synthesis: C2 rotation of the phenyl moiety housing the R through R substituents
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
of intermediate 2 inverts the orientation of the R and R substituents such that two regioisomers can form in
the reductive cyclization. In the case of singly orthoꢀsubstituted anilines, cyclization can only occur through one
position (the remaining ortho position), which makes these substrates a nonꢀissue for the proposed reactions.
However, in the case where a metaꢀsubstituted aniline is used as the diversityꢀhousing coupling partner, two
regioisomers can form following reductive cyclization of the diarylamine intermediate (6ꢀ and 8ꢀsubstituted
phenazines). Fortunately, the use of both “standard” and “inverted” diarylamines as shown in Figure 2B paired
with careful selection of coupling partners can, in theory, provide complete regioselectivity at the 6ꢀ through 9ꢀ
positions.
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
Our synthesis commenced with investigation into coupling conditions for the generation of diarylamine
intermediates. A copperꢀcatalyzed Ullmann coupling, although frequently used to couple anilines to aryl
halides, yielded no desired product in the reaction between anilines and 2ꢀbromoꢀ1ꢀmethoxyꢀ3ꢀnitrobenzene
28–30
(
4, Figure 3A).
With this shortcoming in mind, we then turned our attention toward a palladiumꢀcatalyzed
BuchwaldꢀHartwig (BH) amination reaction as this chemistry has been very useful due to typical high yields
26,27
and a broad substrate scope of coupling partners.
Initially, we attempted a BH crossꢀcoupling between
iodobenzene and 2ꢀaminoꢀ1ꢀmethoxyꢀ3ꢀnitrobenzene (5), but observed no product formation (Figure 3B). We
suspect this result is due to the electronꢀpoor nature of the aniline coupling partner when positioned ortho to
the nitro group on substrate 5, although others have reported success with similar BH couplings using electronꢀ
33,34
poor anilines.
We also sought to design the synthesis such that a TBSꢀprotected hydroxyphenazine could
be generated from the reductive cyclization. Since we anticipated the necessity for demethylation of every
successfully cyclized phenazine, this strategy would allow for far simpler deprotections en route to the
corresponding HP target structures. However, the attempt to couple anilines to (2ꢀbromoꢀ3ꢀnitrophenoxy)(tertꢀ
butyl)dimethylsilane (8) were also met with failure (Figure 3C).
To our delight, simple inversion of the anilinic and aryl halide coupling partners from the example shown in
Figure 3B (e.g., from 5 and aryl halides to 4 and anilines) cleanly afforded both orientations of desired
diarylamine intermediates in good yield (2 and 3, Figures 3D and 3E). Anilines were coupled to 2ꢀbromoꢀ1ꢀ
methoxyꢀ3ꢀnitrobenzene (4) using 6 mol % tris(dibenzylideneacetone)dipalladium(0) with 18 mol % (±)ꢀ2,2′ꢀ
bis(diphenylphosphino)ꢀ1,1′ꢀbinaphthalene (racꢀBINAP) as a ligand to afford 2ꢀseries diarylamines in 71%
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 62
average yield and inverted 3ꢀseries diarylamine intermediates in 25% average yield. With diarylamine
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
intermediates in hand, the reductive cyclization (RC) reactions were conducted using a procedure adapted
24
from previously reported syntheses. Using six equivalents of sodium borohydride in ethanolic solutions of 2 N
sodium ethoxide, the reductive cyclizations proceeded smoothly for the 2ꢀseries diarylamines, affording 1ꢀ
methoxyphenazines in 70% average yield (22 examples; Figure 4). Interestingly, the 3ꢀseries diarylamines
yielded no desired 1ꢀmethoxyphenazine products from reductive cyclization. This is likely due to electron
donation from the orthoꢀpositioned anisole oxygen, rendering the nitro group nitrogen atom much less
electrophilic.
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
Nonetheless, the successful formation of 17 1ꢀmethoxyphenazine scaffolds was achieved as planned (Figure
4). In several cases, unexpected 1ꢀmethoxyphenazine products (31, 41, 44, 45) were obtained following
reductive cyclization of the corresponding diarylamines (19, 22, 23, 24). From these examples, it is apparent
that select halogenated anilines are subject to substitution or overꢀreduction during cyclization and, as such,
may not be ideal substrates for BHꢀRC. This result was initially surprising as analogous reductive ring closures
24,25
have been previously reported to yield halogenꢀbearing phenazine products.
However, there is also
literature precedence for the selective displacement of fluorine atoms during reductive ring closures en route to
35
phenazine scaffolds. Although initially discouraging, it is likely that this fluorine displacement could be utilized
in future studies for regioselective cyclization from asymmetrical anilines or the introduction of nucleophilic
groups during cyclization, as observed in the formation of 7ꢀethoxyꢀ1ꢀmethoxyphenazine (41). In the case of
the trifluoromethylated diarylamine 23, formation of an orthoester was observed during reductive cyclization,
which could be isolated under basic workup conditions. Although acidꢀpromoted hydrolysis of this orthoester
afforded the corresponding ethyl 6ꢀmethoxyphenazineꢀ2ꢀcarboxylate (44) in quantitative yield, we found that
direct conversion from diarylamine 23 to this ethyl ester analogue via standard reductive cyclization conditions
followed by acidic workup afforded 44 in an improved 84% yield following column chromatography. However,
despite these synthetic challenges related to halogenated aniline substrates, we were able to synthesize 7ꢀ
chloroꢀ1ꢀmethoxyphenazine 42 and 7,9ꢀdichloroꢀ1ꢀmethoxyphenazine 43 using this BHꢀRC route.
It was also noted, however, that silyl ethers were unstable toward these reductive cyclization conditions (Figure
4). Ring closure of diarylamine intermediate 24 afforded 1ꢀmethoxyphenazine 45 with loss of the silyl protecting
group as the sole product in 64% yield. Anticipating the difficulty in selectively brominating the demethylated
ACS Paragon Plus Environment

Page 7 of 62
Journal of Medicinal Chemistry
1
,7ꢀdiol counterpart of this analogue, we discontinued its advancement. Although desired TBSꢀprotected
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
products could be obtained in low yield (e.g. 49 was obtained from 28 in 31% yield), it was found that removal
of the TBS group prior to reductive cyclization allowed for substantial improvements in 1ꢀmethoxyphenazine
yields (e.g. a 31% yield for TBSꢀcontaining 49 compared to a 64% yield for the corresponding alcohol 50).
Shortcomings notwithstanding, this twoꢀstep BuchwaldꢀHartwig/reductive cyclization (BHꢀRC) protocol yielded
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
21 discrete 1ꢀmethoxyphenazines with an average coupling yield of 70% and an average reductive cyclization
yield of 68% (Figure 4). With this collection of 1ꢀmethoxyphenazines in hand, we advanced analogues toward
HP final products for biological evaluation. Of the 21 novel 1ꢀmethoxyphenazines generated, 13 were
demethylated using boron tribromide to afford 1ꢀhydroxyphenazines in 89% average yield (Figure 5). Several
1ꢀmethoxyphenazines presented difficulty with this demethylation reaction, resulting in decomposition (40, 51;
structures found in Figure 4) or formation of unexpected products. Surprisingly, demethylation of 1ꢀ
methoxyphenazines 48 and 50 resulted in concomitant bromide displacement of the primary alcohol or silyl
ether to afford alkyl bromides 70 and 71 in 87% and 53% yield, respectively (Figure 6). Initially, this result was
discouraging as the intention was to utilize the desired primary alcohol products for lateꢀstage derivatization.
These transformations, however, were perhaps fortuitous as reactions of the primary bromides with
nucleophiles are likely preferable to attempting to selectively react the primary alcohols without undesired side
reactions at the 1ꢀposition phenol.
Dibromination reactions of 1ꢀhydroxyphenazines were conducted using 2.2 equivalents of Nꢀbromosuccinimide
in dichloromethane to afford HP target structures in 69% average yield (Figure 5A; analogues 52ꢀ65). It should
be noted that attempts to dibrominate the 1ꢀhydroxyphenazine obtained from 47 at only the 2ꢀ and 4ꢀpositions
were unsuccessful, as reactions using NBS or bromine in acetic acid both yielded 2,4,6ꢀtribrominated HP 64
(and to a lesser extent, dealkylated side products). Despite this complication, the tribrominated analogue 64
was advanced to biological studies.
In our previous work, we disclosed 4ꢀposition halogenated HPs (i.e. without 2ꢀposition halogenation) to
11,12
demonstrate excellent antibacterial activities against Mycobacterium tuberculosis.
These findings motivated
the synthesis of a small subꢀset of HPs for investigations into novel antiꢀMtB agents (66 – 69, Figure 5B).
These four target analogues were chosen on the basis of the highly potent MIC activity (versus S. aureus) of
the corresponding 2,4ꢀdibrominated counterparts 52, 53, 56, and 61 (vide infra). The chosen 1ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 62
methoxyphenazines were selectively brominated at the 4ꢀposition using Nꢀbromosuccinimide in refluxing
dichloromethane to afford 4ꢀbromoꢀ1ꢀmethoxyphenazines which subsequently underwent boron tribromide
demethylation to afford 4ꢀbromoꢀ1ꢀhydroxyphenazine antiꢀMtB designed analogues 66 – 69 in 72% average
yield over two steps.
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
Initially, the structureꢀactivity relationships of this new BHꢀRC HP series (antibacterial activity discussed in the
next section) suggested that structural modifications could be made to select positions of the phenazine
scaffold (particularly the 7ꢀposition) that would result in activity gains against bacterial pathogens. This BHꢀRC
synthetic route grants the opportunity to introduce functional groups to the phenazine scaffold, which can be
used as handles for lateꢀstage derivatization and development (e.g. 70 and 71, Figure 6). To this end, we
sought to utilize alkyl bromide compounds 70 and 71 for analogue synthesis with the goal of using
functionalized 2,4ꢀdibrominated HPs as starting materials. A lastꢀstep copperꢀcatalyzed click reaction was
chosen as the derivatization step due to generally high reaction yields and broad range of viable alkyne
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
36,37
substrates.
Although 7ꢀ(2ꢀbromoethyl)phenazinꢀ1ꢀol (71) was prone to elimination of HBr (likely owing to favorable
conjugation of the resulting olefin with the phenazine heterocycle), 70 proved to be an robust analogue to
functionalize. A reaction of sodium azide with 70 in N,Nꢀdimethylformamide afforded a primary azide in
quantitative yield. However, subsequent 2,4ꢀdibromination of this product proved troublesome, yielding the
desired product in only 31% yield (best of three attempts). To circumvent this issue, 70 was first brominated at
the 2ꢀ and 4ꢀpositions, generating HP 65 in 73% yield (Figure 6). This dibrominated product was then
subjected to azide displacement, yielding the crude azidophenazine 72. To avoid complications arising from
undesired hydroxyphenazineꢀcopper chelation during the lastꢀstep click reaction, the phenol of crude 72 was
protected using acetyl chloride and a catalytic amount of DMAP in dichloromethane to afford the acylated
azidophenazine 73 in 59% yield over two steps. The final click reaction proceeded smoothly, reacting
azidophenazine
73
with
1ꢀpentyne,
copper(II)
sulfate,
and
sodium
ascorbate
in
tertꢀ
butanol:water:dichloromethane (1:2:1) to afford triazoleꢀHP 74 in 96% yield. For a full library of HP analogues
synthesized in this study, see Supporting Information Figure 1.
Antibacterial Investigations:
ACS Paragon Plus Environment

Page 9 of 62
Journal of Medicinal Chemistry
Following the chemical synthesis of our new series of halogenated phenazine small molecules, we began
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
11,12,15
antibacterial investigations using minimum inhibitory concentration (MIC) assays
to determine the growthꢀ
inhibitory activities against planktonic bacteria. MIC assays are operationally simple and provide a rapid
approach to generating antibacterial profiles of HPs that allow for the identification of compounds to select for
further investigation. We have found this to be a useful approach as HPs that demonstrate potent antibacterial
activities in MIC assays typically perform well at eradicating biofilms using Calgary Biofilm Device (CBD)
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
11,12,15
assays (discussed in later section).
The 17 novel HP analogues obtained from these BHꢀRC studies were first evaluated for antibacterial activities
against several drugꢀresistant strains of major bacterial pathogens (Table 1). This library of HPs hosts several
11,12
analogues which demonstrate improved MIC activity from that of previous lead HPs.
Halogenated
phenazines 61 and 63, particularly, report MIC activity of <0.1 ꢁM against methicillinꢀresistant Staphylococcus
aureus (0.038 ꢁM and 0.075 ꢁM, respectively against MRSA BAAꢀ1707) while also demonstrating potent
activity against methicillinꢀresistant S. epidermidis (MRSE 35984; MIC 0.1 ꢁM for 61 and 63) and vancomycinꢀ
resistant Enterococcus faecium (VRE 700221; MIC 0.39 ꢁM and 0.2 ꢁM for 61 and 63, respectively).
12,15
From mechanistic investigations regarding HP analogues, which are able to chelate select metal(II) cations
,
we suspected that substitution of the 9ꢀposition of the HP scaffold would not be well tolerated in terms of
antibacterial activities due to steric interference with chelating metal(II)ꢀcations. Expectedly, 9ꢀalkylated HP
analogues 57 and 58 were completely inactive against MRSA, MRSE, and VRE (MICs >100 ꢁM). To further
evaluate the nature of metal interaction observed from these inactive HPs, UVꢀvis determination of chelation
kinetics was conducted. Therein, we observed little to no loss of absorbance at the λmax wavelength for HPs 57
and 58 following addition of ammonium iron(II) sulfate hexahydrate (see Supporting Information). In contrast,
addition of iron(II) to active HP 61 presents a distinct loss of absorbance at the λmax. To supplement kinetic
chelation experiments, UVꢀvis evaluation of HP:metal(II) stoichiometry was conducted, wherein it was found
that inactive HP 58 exhibited no chelation to copper(II) in contrast to active HP 61, which chelates copper(II) in
a manner representative of expected 2:1 HP:metal(II) stoichiometry (see Supporting Information). Thus, it was
concluded that inactive HP analogues 57 and 58 were unable to chelate metal(II) cations due to 9ꢀposition
steric bulk perturbing the metalꢀbinding site of the HP scaffold, whereas potent antibacterial agent HP 61
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 62
efficiently chelated iron(II) and copper(II) at rates analogous to those observed with previously reported active
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
12,15
HPs.
We were surprised to learn that monoꢀhalogenated HPs 66 – 69 reported only good to moderate activity (MICs
of 6.25 – 50 ꢁM; Figure 5B) against MtB, suggesting this new series did not conform to our previously
established predictive method for MtB activities (i.e. active dibrominated HPs against MRSA would correlate to
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
11,12,15
active monoꢀhalogenated counterparts against MtB).
Fortunately, we were pleased to learn that
dihalogenated HP 61 (lead agent versus MRSA in MIC assays) reported excellent MIC activity against MtB
11,12,15
(
3.13 ꢁM), proving to be equipotent to our previously reported antiꢀMtB lead.
It was also discovered that triazoleꢀHP 74 showed very poor activity against all bacterial pathogens tested
against during these studies, with the most potent MIC being 18.8 ꢁM against MRSE (Table 1). The analogue
was tested as the acetylated HP as our previous reports have shown good activity from acetylated HP 1 as
24,38,39
well as structurallyꢀrelated halogenated quinolines.
These MIC results precluded further analogue
synthesis from the HP click chemistry protocol.
HeLa Cytotoxicity and Hemolysis Assessment:
Several HPs that demonstrated potent antibacterial activities against MRSA BAAꢀ1707, or proved to have antiꢀ
MtB activities, were evaluated for mammalian cytotoxicity against HeLa cells in LDHꢀrelease assays (Table 1).
Cytotoxicity from HeLa cells generated from these assays were used to provide selectivity indexes (SI) for
these HPs to quantify the targeting of bacterial cells. This series of HPs demonstrated excellent cytotoxicity
profiles using this approach. Despite HPs 53 and 54 reporting cytotoxicity against HeLa cells at 100 ꢁM
(highest concentration tested; IC >50 ꢁM), the remaining 11 HP analogues tested (52, 55, 56, 59, 61, 63, 64,
50
66, 67-69) reported minimal, if any, cytotoxicity against HeLa cells (IC50 >100 ꢁM). Using these results,
selectivity indices were generated (HeLa cell IC divided by MRSA BAAꢀ1707 MIC) for these HPs to quantify
50
their high degree of selectivity and bacterial targeting relative to HeLa cells (SI >169 to >2,632 for HPs active
against MRSA BAAꢀ1707; Table 1).
In addition to mammalian cytotoxicity assessment, we conducted hemolysis assays with human red blood cells
(
RBCs). No HP analogue exceeded 3% hemolysis at 200 ꢁM (Table 2). Most importantly, this finding (in
addition to demonstrating a favorable safety profile) suggests that our new HP analogues act through a
ACS Paragon Plus Environment

Page 11 of 62
Journal of Medicinal Chemistry
mechanism that does not involve membrane lysis. QACꢀ10 is a known membraneꢀlysing agent that
demonstrates antibacterial activities in addition to biofilm eradication. When tested alongside with our HP
small molecules, QACꢀ10 served as a positiveꢀcontrol and demonstrated >99% hemolysis of RBCs at 200 ꢁM.
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
Biofilm Eradication Activities:
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
Nine potent HPs (52 – 56, 59, 61, 63, 64; Table 2) were identified during initial MIC studies and advanced to
biofilm eradication assays against MRSA, MRSE and VRE using Calgary Biofilm Devices (CBD; several potent
HPs presented in Figure 7 to illustrate SAR and antibacterial profiles). CBDs are specialized 96ꢀwell plates that
have lids containing pegs designed to sit down in microtiter wells (one peg per microtiter well) and provide
40,41
bacteria with a surface for biofilm formation.
Biofilm eradication assays have three distinct phases, which
include: 1) biofilm establishment (media and bacteria only, biofilms are established on CBD pegs), 2)
compound treatment (compounds in media; test compounds have the chance to eradicate biofilms during this
phase), and 3) growth and dispersion of viable biofilms (media only). Each of these phases involves static
incubation (24 hours at 37 °C), a wash of the CBD pegs containing biofilms with subsequent lid transfer to new
96ꢀwell plate at the end of phases one and two; however, at the end of the final phase, the microtiter wells are
evaluated for bacterial growth (turbidity). Upon completion of the assay, microtiter wells that are turbid from
bacterial growth correspond to wells that contained viable biofilms that dispersed bacteria into the media
followed by bacterial growth during phase 3. Microtiter wells without bacterial growth, or turbidity, at the end of
the CBD assay corresponds to microtiter wells that contained eradicated biofilms (≥99.9% of biofilm cells
12,15
eradicated based on previous studies
) that were unable to grow and disperse viable bacteria during phase
3
(see Figure 8A and supporting information for CBD plate images). HPs and control compounds were tested
in 2ꢀfold serial dilution and the lowest test concentration of a compound that is required to eradicate biofilms
(from nonꢀturbid microtiter wells) is known as the minimum biofilm eradication concentration (MBEC) value.
The Calgary Biofilm Device also allows for planktonic toxicity (determination of minimum bactericidal
concentration or MBC values) to be evaluated alongside the biofilm eradication activity, providing ideal
information regarding planktonic and biofilm killing dynamics (MBC:MBEC ratios) from a single bacterial culture
11,12,15
under the same experimental conditions.
During these investigations, we identified several new HPs with potent biofilm eradication activities against
MRSA, MRSE and VRE biofilms (Table 2). Against MRSAꢀ1707 biofilms, 7ꢀsubstituted HPs 55 (MBEC = 4.69
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 62
ꢁ
M; 7ꢀtertꢀbutylꢀHP), 61 (MBEC = 4.69 ꢁM; 7ꢀchloroꢀHP), 63 (MBEC = 2.35 ꢁM; 7ꢀphenoxyꢀHP) and 64 (MBEC
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
=
4.69 ꢁM; 7ꢀdiethylamineꢀ6ꢀbromoꢀHP) demonstrated potent killing activities (Figure 7). In addition, 6,8ꢀ
dimethylꢀHP 56 demonstrated potent MRSA BAAꢀ1707 biofilm eradication (MBEC = 4.69 ꢁM). Several HPs
demonstrated excellent biofilm eradication activities against MRSAꢀ2 (e.g., HP 55; MBEC = 3.13 ꢁM) and
MRSA BAAꢀ44 (e.g., HP 55; MBEC = 9.38 ꢁM). Multiple frontꢀrunning MRSA antibiotics, including vancomycin,
daptomycin and linezolid, demonstrated no biofilm eradication activities against MRSA biofilms at 2,000 ꢁM
(highest concentration tested) when tested alongside this series of halogenated phenazine small molecules.
Against MRSE 35984 biofilms, HPs 55 (MBEC = 3.13 ꢁM), 56 (MBEC = 4.69 ꢁM) and 63 (MBEC = 4.69 ꢁM)
demonstrated highly potent eradication activities; however, several analogues reported MBEC values ≤ 25 ꢁM
against MRSE 35984 biofilms. In addition, several HPs from this series demonstrated potent, subꢀmicromolar
MBEC activities against VRE 700221 biofilms with 55, 61 (MBEC = 0.59 ꢁM), 56, 63 and 64 (MBEC = 0.78
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
ꢁ
M) proving to be the most active agents against this pathogen. Viable biofilm cell counts were carried out
using CBD pegs from select experiments with HP biofilmꢀeradicating agents and demonstrated ≥ 3 log
reduction of viable biofilm cells at their corresponding MBEC values (≥99.9% biofilm cell killing), similar to
12,15
previous findings (see supporting information).
11,12,15
In addition to select antibiotic comparators, various biofilm eradication controls (e.g. QACꢀ10, CCCP)
12,15
along with metalꢀchelating agents (e.g. EDTA, TPEN)
were assayed alongside new halogenated
phenazines. These comparator agents proved to be significantly less active, or inactive, in their ability to
eradicate established biofilms in CBD assays when compared to these HPs. These data collectively point to
the unique mechanism and biofilmꢀkilling potency displayed by these HP small molecules. Similar to previous
studies, the planktonic killing (MBC) to biofilm killing (MBEC) ratios generated from these Calgary Biofilm
Device assays demonstrated that HPs have near equipotent killing activities against planktonic and biofilm
bacteria (MBC:MBEC ratios 1ꢀ3; see Table 2), a profile that conventional antibiotic therapies do not possess.
Following biofilm eradication investigations in CBD assays, lead HP 61 was tested against biofilms of MRSE
35984 in live/dead staining experiments (Figure 8B). After a 24ꢀhour biofilm establishment, HP 61 was added
at 1, 5, and 10 ꢁM then left to incubate against MRSE 35984 biofilms for an additional 24 hours at 37 °C.
Images were then taken of treated and untreated biofilms using fluorescence microscopy to further
demonstrate the highly potent eradication and clearance of MRSE 35984 biofilms with HP 61.
ACS Paragon Plus Environment

Page 13 of 62
Journal of Medicinal Chemistry
HPꢀProdrug Strategy and Synthesis:
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
Due to the inherently high CLogP values and potential for offꢀtarget metalꢀchelating events of lead HP
analogues, we sought to preemptively address these concerns through the development of phenolicꢀbased
prodrug strategies (Figure 9). Although we were initially excited about the incorporation of waterꢀsoluble
moieties at the 6ꢀ through 8ꢀpositions attainable by way of BHꢀRC, the lack of activity observed with triazoleꢀ
HP 74 discouraged us from this endeavor. In lieu of 6ꢀ through 8ꢀposition modifications, prodrug strategies
were sought wherein conjugation of waterꢀsoluble groups to the phenol group would afford HP analogues with
reduced CLogP values. Although we initially devised our phenolic prodrug approach to attain improved water
solubility and suppress offꢀtarget metal chelation, we realized the proposed functionalization strategy could
ostensibly be used to impart bacterial selectivity onto our HP series. To this end, a library of bacterialꢀselective
prodrugs (Figure 9FꢀI; HPꢀprodrugs synthesized using various alkylation reactions) was assembled (see
Supporting Information for further descriptions of the employed prodrug strategies and proposed mechanisms
of activation/HP release). Thus, a library of diverse, CLogPꢀguided prodrugs was synthesized from active HP
scaffolds (Figure 9AꢀE; HPꢀprodrugs synthesized using acylation, alkylation and sugarꢀbased syntheses).
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
Biological Evaluation of HPꢀProdrugs:
We initiated the biological evaluation of this HPꢀprodrug library with MIC and MBEC assays against MRSA,
MRSE, and VRE (Table 3). Among the CLogPꢀguided analogues, HP prodrugs 75, 76, 77, and 78 were found
to be active in MIC assays, reporting MICs between 0.0005 to 3.13 ꢁM and MBECs of 0.78 to 75 ꢁM against
all strains (Table 3). Surprisingly, carbonate prodrugs 77 and 78 reported MICs of 0.0005 ꢁM and 0.1 ꢁM
respectively against MRSA BAAꢀ1707 (up to ~76ꢀfold increase in potency relative to the corresponding HPs).
As prodrug activity is not expected to exceed that of the active agent to which it is metabolized, we suspect the
12
carbonate prodrugs temporarily protect HPs from ionization (phenolic pKa = ~6.7 ), allowing for improved
membrane permeability prior to intracellular activation of the prodrug (see Supporting Figures 4 and 5).
Although these intriguing activities may warrant further investigation, the apparent susceptibility of prodrugs 75
– 78 to rapidly undergo enzymatic or chemical hydrolysis (implied by their highly potent antibacterial activities)
may limit their utility in vivo in future endeavors.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 62
Interestingly, the HPꢀglycoconjugate (79, Figure 9D) and HPꢀalkyloxycarbonyloxymethyl (AOCOM) prodrugs
0 and 82 (Figure 9E) demonstrated no activity in MIC assays. We suspect that differences in carbonate
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
8
stability of the AOCOM series relative to HPꢀcarbonates 77 and 78 are likely responsible for this activity
disparity. Fortunately, human serum stability assays for HPs 80 and 82 reveal favorable serum halfꢀlives (t )
1/2
of 15.8 minutes and 7.3 minutes, respectively, which are in line with reported halfꢀlives for structurally related
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
42
prodrugs. These data suggest the HPꢀAOCOM prodrugs could have potential for further in vivo experiments
wherein hostꢀdependent prodrug cleavage could occur.
In an effort to develop more stable HP prodrugs, we investigated etherꢀbased prodrug moieties. We targeted
the synthesis of boronꢀbased ether 83 and betaꢀlactam 84. Each of these HPꢀprodrugs were designed to target
different host response/bacterial features relevant to infection. Pinacol boronꢀHP 83 was designed to undergo
a host inflammationꢀinduced oxidation of boron, followed by the liberation of an active HP compound at the site
43,44
of bacterial infection.
Betaꢀlactam 84 was designed to target penicillinꢀbinding proteins/betaꢀlactamases for
45,46
an initial betaꢀlactam cleavage, followed by the liberation of the active HP compound.
Although we were
able to isolate etherꢀlinked prodrugs 83 and 84 (Figure 9F, 9G), we found them to be unstable and, thus,
discontinued further investigations of these analogues.
47,48
We then moved to exploiting the bacterial cytoplasm for the intracellular release of active HPs.
The
bacterial cytoplasm is a reductive environment and we drew inspiration from the natural product mitomycin C, a
potent antibacterial agent and cytotoxin, that requires bioreductive activation of its quinone moiety before it can
47
carry out its mode of action (DNA crosslinking). Interestingly, reductively activated HPꢀdisulfide prodrug 85
(
Figure 9H) reported no antibacterial activity against any pathogen tested. We then designed two quinoneꢀ
AOCOM (QuAOCOM) prodrugs 86 and 87 (Figure 9I) and found these agents to demonstrate antibacterial
activities which were near equipotent to the corresponding HP counterparts with MICs = 2.35 ꢁM and 0.15 ꢁM,
respectively, versus MRSA BAAꢀ1707. The exceptional antibacterial activity of QuAOCOM 87 was confirmed
via agar diffusion assay, wherein this prodrug reported activity near that of the parent HP 61 (Figure 8D).
Additionally, the QuAOCOM prodrugs exhibited good to potent biofilm eradication activities against MRSA
BAAꢀ1707 (MBECs = 75 ꢁM and 12.5 ꢁM for 86 and 87, respectively). To ensure this activity was not a result
of prodrug activation in the assay medium, stability experiments were conducted in lysogeny broth (LB),
wherein no loss of the prodrug moiety was observed at up to 16 hours at 37 °C (Figure 8C). Additionally,
ACS Paragon Plus Environment

Page 15 of 62
Journal of Medicinal Chemistry
serum stability assays for QuAOCOM prodrugs revealed halfꢀlives of 6.3 ± 3.3 minutes and 11.4 ± 2.8 minutes
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
for 86 and 87, respectively (Table 3). Finally, QuAOCOM 87 proved to have an outstanding cytotoxicity (IC50
>
100 ꢁM against HeLa cells; SI > 667) and hemolysis (2.7% hemolysis of RBCs at 200 ꢁM) profile, similar to
parent HPs identified during these studies. Based on our stability assays for this prodrug class along with the
wellꢀunderstood reductive conditions of the bacterial cytoplasm, we can conclude that the QuAOCOM prodrugs
exhibit activities due to a bacterialꢀselective release mechanism. We are very encouraged by the initial activity
profiles of these HPꢀQuAOCOM prodrugs and believe the reductive trigger of the quinone moiety in
conjunction with the AOCOM linker to be an ideal platform for future developments regarding HPꢀbased biofilm
therapies.
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
Conclusions:
The design, optimization, and utilization of the BHꢀRC protocol reported herein has not only broadened the
substrate scope for phenazine synthesis, but has also offered substantial improvement of regioselective control
for modification at the 6ꢀ through 9ꢀpositions of the phenazine heterocycle. We have shown that intermediate
1ꢀmethoxyphenazines can be efficiently generated through a BuchwaldꢀHartwig crossꢀcoupling (71% average
yield) and a subsequent, relatively mild reductive cyclization (68% average yield) en route to final HP products.
These disclosed syntheses have permitted the identification of novel HP agents which efficaciously eradicate
biofilms of harmful, drugꢀresistant pathogens. We also now have an improved understanding of the importance
of metal chelation in the antibacterial mode of action as demonstrated by the apparent liability of 9ꢀposition
functionalization in HP analogues (HPs 57 and 58). Novel lead HP 61 demonstrates the remarkable ability to
eradicate biofilms of domesticallyꢀthreatening MRSA (MBEC = 4.69 ꢁM) while also reporting good activity in
antibacterial susceptibility assays against the worldwide bacterial threat Mycobacterium tuberculosis (MIC =
3.13 ꢁM).
Furthermore, prodrug investigations afforded the discovery of analogues with potential therapeutic utility.
AOCOM prodrugs 80 and 82 are endowed with improved CLogP values and were shown to be activated in
human serum. In addition, the bacterialꢀselective QuAOCOM prodrugs 86 and 87 were designed to target the
reductive cytoplasm of bacteria for requisite bioreduction of their quinone moiety, which triggers HP release.
Development of HP prodrugs herein has strengthened our understanding of what the synthetic capabilities are
for phenolic functionalization of this compound class. Naturally, all prodrug avenues have not yet been
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 62
exhausted and further development of HP prodrugs will be forthcoming. The potent biofilm eradication activities
of new halogenated phenazines in conjunction with negligible hemolytic toxicity or mammalian cytotoxicity
represent a unique profile compared to our current antibiotic arsenal. Thus, halogenated phenazines and
prodrugs thereof may prove to be invaluable agents for combatting the growing threat of biofilmꢀassociated
bacterial infections worldwide.
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
Experimental:
I) General Information. All synthetic reactions were carried out under an inert atmosphere of argon unless
otherwise specified. All reagents for chemical synthesis were purchased from commercial sources and used
without further purification. Reagents were purchased at ≥ 95% purity and commercially available controls
were used in our biological investigations without further purification. All microwave reactions were carried out
in sealed tubes in an Anton Paar Monowave 300 Microwave Synthesis Reactor. A constant power was applied
to ensure reproducibility. Temperature control was automated via IR sensor and all indicated temperatures
correspond to the maximal temperature reached during each experiment. Analytical thin layer chromatography
(TLC) was performed using 250 ꢂm 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
1
13
1
VNMRꢀJ software (400 MHz for H and 100 MHz for C) and a Bruker Avance II (600 MHz for H NMR; 150
13
MHz for C NMR). All spectra are presented using MestReNova 11.0 (Mnova) software and are displayed
without the use of the signal suppression function. Spectra were obtained in the following solvents (reference
1
13
1
13
peaks also included for H and C NMRs): CDCl ( H NMR: 7.26 ppm; C NMR: 77.23 ppm) and d ꢀDMSO
3
6
1
13
( H NMR: 2.50 ppm; C NMR: 39.52 ppm). All NMR experiments were performed at room temperature.
1
13
1
Chemical shift values (δ) are reported in parts per million (ppm) for all H NMR and C NMR spectra. 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.
ACS Paragon Plus Environment

Page 17 of 62
Journal of Medicinal Chemistry
All compounds evaluated in biological assays were determined to be ≥ 95% pure via LCꢀMS using a Shimadzu
Prominence HPLC system, AB Sciex 3200 QTRAP spectrometer and a Kinetex C18 column (50 mm × 2.1 mm
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
×
2.6 ꢁm) with a 35ꢀminute linear gradient from 10ꢀ65% acetonitrile in 0.5% 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)
(
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
methicillinꢀresistant Staphylococcus epidermidis (MRSE strain ATCC 35984), and vancomycinꢀresistant
Enterococcus faecium (VRE strain ATCC 700221). 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.
II) Chemistry. This chemistry section includes the following items, in numerical order: (a) synthetic procedures,
(
b) compound characterization data for analogues synthesized via general procedures, (c) UVꢀvis experiments
for HP complex formation with iron(II) and (d) spectroscopic determination of prodrug stability in LB media.
II.a) Synthetic procedures
1) General Procedure for the Buchwald-Hartwig Amination (9-26, 28, 30). To a stirring solution of 4 (436.2
mg, 1.88 mmol) and desired aniline (2.26 mmol) in toluene was added trisꢀ
(dibenzylideneacetone)dipalladium(0) (103.5 mg, 0.11 mmol), (±)ꢀ2,2′ꢀBis(diphenylphosphino)ꢀ1,1′ꢀ
binaphthalene (210.5 mg, 0.34 mmol), and sodium tertꢀbutoxide (234.6 mg, 2.44 mmol). The reaction was
allowed to stir for 16 hours at 100 °C. The mixture was then allowed to cool to room temperature and
transferred to a separatory funnel containing ethyl acetate and saturated sodium bicarbonate. The organic
layer was sequentially washed with sodium bicarbonate and brine before the organic layer was collected. The
organic layer was then dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude
solid was purified via flash column chromatography using hexanes:ethyl acetate (95:5 to 80:20) to elute pure
diarylamines.
2) General Procedure for the Removal of TBS Ethers (27, 29). To a stirring solution of tertꢀbutylꢀdiꢀmethyl
silyl ethers (561.7 mg, 1.45 mmol) in anhydrous tetrahydrofuran (15 mL) was added 2.17 mL (2.17 mmol) of a
1
M solution of tetrabutyl ammonium fluoride in tetrahydrofuran. The reaction mixture was allowed to stir for 2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 62
hours at room temperature. The reaction was then quenched with water and transferred to a separatory funnel
containing ethyl acetate and brine. The organic layer was sequentially washed with brine before the organic
layer was collected. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The crude solid was purified via flash column chromatography using hexanes:ethyl acetate (99:1 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
85:15) to elute pure alcohols as red oily residues.
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) General Procedure for Reductive Cyclization with Sodium Borohydride and Sodium Ethoxide (31-51).
To a stirring solution of diarylamine (1.27 mmol) in 2 N sodium ethoxide in ethanol (35 mL) was added sodium
borohydride (290 mg, 7.66 mmol). The reaction was allowed to stir for 2 hours at 60 °C. The reaction was
cooled to room temperature, quenched with water and then transferred to a separatory funnel containing ethyl
acetate and brine. The organic layer was sequentially washed with brine before the organic layer was
collected. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The
crude solid was purified via flash column chromatography using hexanes:ethyl acetate (99:1 to 85:15) to elute
pure 1ꢀmethoxyphenazine analogues.
4) General Procedure for Dibromination of 1-Hydroxyphenazines (52-65): Desired 1ꢀhydroxyphenazines
(
0.24 mmol) and Nꢀbromosuccinimide, (86.2 mg, 0.48 mmol) were dissolved in dichloromethane (15 mL) and
allowed to stir at room temperature for 2 hours. The reaction was diluted with dichloromethane and quenched
with brine (3 x 20 mL). The organic layer was dried with sodium sulfate, filtered and concentrated. The
reaction contents were then concentrated, adsorbed onto silica gel and purified via column chromatography
using dichloromethane to elute pure 2,4ꢀdibromohydroxyphenazine analogues as yellow solids.
5) General Procedure for Boron Tribromide Demethylation (66-71; 97-109 see ESI). To a round bottom
flask was added the desired 1ꢀmethoxyphenazine (1.07 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 (6.4 mL, 6.41 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
ACS Paragon Plus Environment

Page 19 of 62
Journal of Medicinal Chemistry
column chromatography using dichloromethane to elute pure 1ꢀhydroxyphenazines as yellow solids. Note:
Analogous procedures were used for all demethylation reactions using boron tribromide (BBr3).
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) Synthesis of 7-(azidomethyl)-2,4-dibromophenazin-1-yl acetate (73): HP 65 (33.6 mg, 0.08mmol) was
added to a roundꢀbottom flask and dissolved in N,Nꢀdimethylformamide (4 mL). Sodium azide (12.2 mg, 0.188
mmol) was added and the reaction was stirred at room temperature for 2 hours. Following completion by TLC,
the reaction was diluted with ethyl acetate and quenched with brine (3 x 50 mL). The organic layer was dried
with sodium sulfate, filtered and concentrated. The crude solid was then dissolved in dichloromethane (10
mL). Triethylamine (6 ꢁL, 0.04 mmol), a catalytic amount of 4ꢀdimethlyaminopyridine, then acetyl chloride (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
ꢁ
L, 0.03 mmol) were added at room temperature. The reaction was allowed to stir for one hour before being
quenched with an aqueous solution of saturated sodium bicarbonate. The resulting mixture was then
transferred to a separatory funnel and extracted with dichloromethane. The organic layer was then dried with
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude solid was purified via flash column
chromatography using dichloromethane as the eluent to afford 73 as a yellow oily residue (20.1 mg, 59% over
1
two steps). H NMR (400 MHz, CDCl ): δ 8.35 (s, 1H), 8.30 (m, 1H), 8.25 (d, J = 9.0 Hz, 1H), 7.83 (dd, J = 9.0,
3
13
1
1
4
.9 Hz, 1H), 4.66 (s, 2H), 2.61 (s, 3H). C NMR (100 MHz, CDCl ): δ 168.3, 145.2, 143.4, 143.1, 140.6, 139.9,
3
+
37.9, 136.1, 132.1, 130.7, 128.4, 122.3, 117.5, 54.7, 20.9. HRMS (ESI): calc. for C H Br N O [M+H] :
15
10
2
5
2
51.9176, found: 451.9176.
) Synthesis of 2,4-dibromo-7-((4-propyl-1H-1,2,3-triazol-1-yl)methyl)phenazin-1-yl acetate (74):
7
Anhydrous copper sulfate (2.2 mg, 0.01 mmol) and sodium ascorbate (8.0 mg, 0.04 mmol) were dissolved in a
solution of tertꢀbutanol:H O (1:2, 300 ꢁL) and was added to a roundꢀbottom flask containing 73 (12.2 mg, 0.06
2
mmol). 1ꢀPentyne (16.0 ꢁL, 0.16 mmol) was added, followed by dichloromethane (3.0 mL). The biphasic
mixture was vigorously stirred at room temperature for 16 hours until starting material was fully consumed as
determined by TLC analysis. The mixture was quenched with brine (2 x 50 mL) and the product was extracted
with dichloromethane. The organics were collected, dried with sodium sulfate, filtered and concentrated to
1
afford pure 74 (96%, 13.5 mg) as a yellow solid. H NMR (600 MHz, DMSOꢀd ): δ 8.68 (s, 1H), 8.28 (d, J = 9.0
6
Hz, 1H), 8.08 – 8.06 (m, 2H), 7.93 (dd, J = 9.0, 1.9 Hz, 1H), 5.93 (s, 2H), 2.62 (t, J = 7.5 Hz, 2H), 2.57 (s, 3H),
13
1
.62 (sextet, J = 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). C NMR (150 MHz, DMSOꢀd ): δ 168.0, 147.3, 144.5,
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 62
1
42.5, 142.1, 141.2, 139.7, 137.1, 135.8, 132.6, 129.9, 127.3, 122.7, 121.6, 117.2, 52.2, 27.1, 22.2, 20.4, 13.6.
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
+
HRMS (ESI): calc. for C H Br N O [M+H] : 519.9803, found: 519.9793. MP: 203 – 205 °C.
20
18
2
5
2
8) General Procedure for the Synthesis of HP Esters/Carbamate Prodrugs (75, 76). To a stirring solution
HP 1 (62.0 mg, 0.18 mmol), triethylamine (48 ꢁL, 0.35 mmol), and a catalytic amount of 4ꢀ
dimethlyaminopyridine in dichloromethane (20 mL) was added the acid chloride or carbamoyl chloride reagent
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
(
0.35 mmol) at room temperature. The reaction was allowed to stir for two hours before being quenched with
an aqueous solution of saturated sodium bicarbonate. The resulting mixture was then transferred to a
separatory funnel and ethyl acetate was added to extract the product. The organic layer was sequentially
washed with water and brine before being collected. The organic layer was then dried with anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The respective ester or carbamate derivative was purified via flash
column chromatography using hexanes:ethyl acetate (99:1 to 80:20) to elute, yielding pure products as yellow
solids.
9) General Procedure for the Synthesis of HP-Carbonates (77, 78): Tetraethyleneglycol monomethyl ether
(101.4 ꢁL, 0.48 mmol) was placed in an ovenꢀdried roundꢀbottomed flask and dissolved in anhydrous
dichloromethane (2 mL) and cooled to 0 °C. Pyridine (46.4 ꢁL, 0.58 mmol) and triethylamine (16.8 ꢁL 0.12
mmol) was then added via syringe, followed by triphosgene (71.2 mg, 0.24 mmol) dissolved in
dichloromethane (2 mL). The resulting mixture was stirred from 0 °C to room temperature and continued to stir
at room temperature for 5 hours. The reaction was then cooled to 0 °C before the addition of solution of 61
(
118.2 mg, 0.30 mmol) and triethylamine (63 ꢁL 0.45 mmol) in anhydrous dichloromethane was added to the
reaction dropwise. The reaction was allowed to reach ambient temperature for reaction overnight. After the
reaction was complete by TLC, the reaction mixture was poured into a separatory funnel containing brine (20
mL). The organic layer was drawn and the extracts were collected, dried over sodium sulfate, filtered, and
concentrated under vacuum. The resulting crude material was purified using flash column chromatography with
3:1 hexanes:ethyl acetate to 100% ethyl acetate as eluent to yield 77 (183.1 mg, 99% yield) as a yellow solid.
10) Synthesis of HP-glucoconjugate prodrug 79 ((2S,3R,4S,5S,6R)-2-((2,4-dibromophenazin-1-yl)oxy)-6-
(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol): To a sealed microwave vial was added HP 1 (50.0 mg,
0.14 mmol), potassium carbonate (39.0 mg, 0.28 mmol) in methanol (5 mL). The resulting mixture was heated
to 80 °C in the microwave reactor for a single 5 minute cycle. After that time, acetobromoꢀαꢀDꢀglucose (145
ACS Paragon Plus Environment

Page 21 of 62
Journal of Medicinal Chemistry
mg, 0.35 mmol) was added to the reaction vial. The reaction was cooled to room temperature and the solvent
was removed in vacuo. The crude residue was taken up in ethyl acetate, transferred to a separatory funnel
and then washed with water and extracted with ethyl acetate three times. The organic layers were combined,
dried with anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude solid was dissolved in
methanol (6 mL) and sodium ethoxide (40.8 mg, 0.60 mmol) was added. The reaction was allowed to stir 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
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 hours, then was diluted with dichloromethane and transferred to a separatory funnel. The organic layer was
drawn and the extracts were collected, dried over sodium sulfate, filtered, and concentrated under vacuum.
The crude solid was rinsed with iceꢀcold water and methanol, then dried in vacuo to afford pure desired
1
product as a pale yellow solid (17.7 mg, 24% over 2 steps). H NMR (600 MHz, DMSOꢀd ): δ 8.56 (s, 1H), 8.38
6
–
4
8.26 (m, 2H), 8.14 – 8.02 (m, 2H), 6.03 (d, J = 7.7 Hz, 1H), 5.57 (d, J = 4.9 Hz, 1H), 5.11 (d, J = 5.2 Hz, 1H),
.98 (d, J = 5.2 Hz, 1H), 4.20 (t, J = 5.7 Hz, 1H), 3.59 – 3.40 (m, 2H), 3.38 – 3.25 (m, 2H, partially buried under
13
water signal), 3.14 (m, 2H). C NMR (150 MHz, DMSOꢀd ): δ 148.9, 142.3, 141.4, 139.8, 137.8, 136.4, 132.6,
6
1
32.5, 129.3, 129.3, 118.0, 115.8, 104.0, 77.8, 76.9, 74.8, 69.9, 60.8. HRMS (DART): calc. for C H Br N O
18 17 2 2 6
+
[
M+H] : 516.9429, found: 516.9435. MP: 199 – 201 °C.
11) Synthesis of alkyloxycarbonyloxymethyl (“AOCOM”) carbonate prodrugs (80-81, 85-87): To a stirring
solution of the desired alkyloxy chloromethyl carbonate (0.08 mmol) in acetone (2 mL) was added sodium
iodide (10.5 mg, 0.07 mmol). The reaction was allowed to stir for two hours at room temperature. Then, this
mixture was added to a stirring solution of 1 (23.5 mg, 0.07 mmol) and potassium carbonate (11.0 mg, 0.08
mmol) in acetone (2 mL). After stirring for 14 additional hours, the reaction was quenched by addition of
deionized water. The resulting mixture was then transferred to a separatory funnel and ethyl acetate was
added to extract the product. The organic layer was sequentially washed with sodium bicarbonate and brine
before being collected. The organic layer was then dried with anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The resulting crude material was purified using flash column chromatography with 3:1
hexanes:ethyl acetate to 100% ethyl acetate as eluent to yield AOCOM prodrugs as yellow solids.
12) Synthesis of ((2,4-dibromophenazin-1-yl)oxy)methyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)
carbonate (82): To a stirring solution of 81 (25.8 mg, 0.03 mmol) at 0 °C in 6 mL of anhydrous tetrahydrofuran
was added 34 ꢁL of a 1 M solution of tetrabutylammonium fluoride (0.03 mmol). The reaction mixture was
allowed to stir for 6 hours, slowly reaching ambient temperature. The reaction was then quenched with water
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 62
and transferred to a separatory funnel containing ethyl acetate and brine. The organic layer was sequentially
washed with brine before the organic layer was collected. The organic layer was dried with anhydrous sodium
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
sulfate, filtered, and concentrated in vacuo. The crude solid was purified via flash column chromatography
1
using ethyl acetate to elute pure 82 as a yellow oily residue (60% yield, 11.0 mg). H NMR (400 MHz, CDCl ):
3
δ 8.37 (m, 1H), 8.32 (s, 1H), 8.28 (m, 1H), 7.97 – 7.89 (m, 2H), 6.26 (s, 2H), 4.39 – 4.23 (m, 2H), 3.73 – 3.68
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
(
m, 4H), 3.68 – 3.54 (m, 10H). C NMR (100 MHz, CDCl ): δ 154.7, 149.7, 143.5, 143.0, 140.7, 138.4, 136.5,
3
132.3, 132.0, 130.2, 129.8, 120.5, 116.7, 92.4, 72.7, 70.9, 70.9, 70.7, 70.5, 69.0, 67.7, 62.0. HRMS (ESI): calc.
+
for C H Br N O [M+H] : 604.9954, found: 604.9951.
22
25
2
2
8
II.b) Compound characterization data for analogues synthesized via general procedures, in numerical
order. Additional information can be found in the Supporting Information document.
1
2
-Methoxy-6-nitro-N-phenylaniline (9). Yield: 81%; 206.0 mg was isolated as a red oily residue. H NMR
(
400 MHz, CDCl ): δ 8.18 (br. s, 1H), 7.71 (dd, J = 8.5, 1.4 Hz, 1H), 7.28 – 7.17 (m, 2H), 7.09 (dd, J = 8.0, 1.4
3
13
Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.98 (tt, J = 7.2, 1.0 Hz, 1H), 6.85 – 6.79 (m, 2H), 3.75 (s, 3H). C NMR (100
MHz, CDCl ): δ 152.7, 142.3, 140.5, 129.6, 128.7, 122.4, 120.7, 118.6, 118.1, 116.3, 56.3. MP: 55 – 57 °C.
3
+
HRMS (ESI): calc. for C H N O [M+H] : 245.0921, found: 245.0930. Note: Compound has been previously
13
13
2
3
reported (CAS: 7575ꢀ27ꢀ1), but no spectra were found for comparison.
2-Methoxy-6-nitro-N-(p-tolyl)aniline (10). Yield: 94%; 460.8 mg was isolated as a red oily residue. H NMR
1
(
400 MHz, CDCl ): δ 8.18 (br. s, 1H), 7.71 (dd, J = 8.5, 1.4 Hz, 1H), 7.08 (dd, J = 8.1, 1.4 Hz, 1H), 7.04 (d, J =
3
13
8
.3 Hz, 2H), 6.97 (dd, J = 8.5, 8.0 Hz, 1H), 6.75 (d, J = 8.3 Hz, 2H), 3.75 (s, 3H), 2.31 (s, 3H). C NMR (100
MHz, CDCl ): δ 152.4, 139.9, 139.8, 132.1, 130.4, 129.3, 120.1, 118.9, 118.2, 116.3, 56.4, 20.9. HRMS (ESI):
3
+
calc. for C H N O [M+H] : 259.1077, found: 259.1085. MP: 65 – 67 °C.
14
15
2
3
1
N-(4-Ethylphenyl)-2-methoxy-6-nitroaniline (11). Yield: 71%; 83.3 mg was isolated as a red oily residue. H
NMR (400 MHz, CDCl ): δ 8.21 (br. s, 1H), 7.72 (dd, J = 8.5, 1.4 Hz, 1H), 7.12 – 7.05 (m, 3H), 6.98 (dd, J =
3
13
8
.5, 8.0 Hz, 1H), 6.85 – 6.68 (m, 2H), 3.76 (s, 3H), 2.62 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). C NMR
(
100 MHz, CDCl ): δ 152.4, 139.9, 138.5, 130.3, 128.1, 120.1, 118.9, 118.1, 116.3, 56.3, 28.3, 15.8. Note:
3
13
One C signal missing in the aromatic region, likely due to signal overlap. HRMS (ESI): calc. for
+
C H N O [M+H] : 273.1234, found: 273.1243.
15
7
2
3
ACS Paragon Plus Environment

Page 23 of 62
Journal of Medicinal Chemistry
2
-Methoxy-6-nitro-N-(4-propylphenyl)aniline (12). Yield: 71%; 453.3 mg was isolated as a red oily residue.
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
H NMR (400 MHz, CDCl ): δ 8.24 (br. s, 1H), 7.72 (dd, J = 8.5, 1.4 Hz, 1H), 7.14 – 7.03 (m, 3H), 6.98 (dd, J =
3
8.5, 8.0 Hz, 1H), 6.84 – 6.73 (m, 2H), 3.75 (s, 3H), 2.57 (dd, J = 8.5, 6.7 Hz, 2H), 1.76 – 1.44 (m, 2H), 0.97 (t, J
13
=
7.3 Hz, 3H). C NMR (100 MHz, CDCl ): δ 152.3, 139.9, 139.7, 136.8, 130.2, 128.6, 120.0, 118.7, 118.0,
3
+
116.2, 56.2, 37.4, 24.7, 13.9. HRMS (ESI): calc. for C H N O [M+H] : 287.1390, found: 287.1398.
16
19
2
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
N-(4-(tert-Butyl)phenyl)-2-methoxy-6-nitroaniline (13). Yield: 63%; 477.0 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.22 (br. s, 1H), 7.73 (dd, J = 8.5, 1.4 Hz, 1H), 7.32 – 7.22 (m, 2H), 7.11
3
13
(
dd, J = 8.1, 1.4 Hz, 1H), 6.99 (dd, J = 8.5, 8.0 Hz, 1H), 6.85 – 6.72 (m, 2H), 3.77 (s, 3H), 1.33 (s, 9H). C
NMR (100 MHz, CDCl ): δ 152.5, 145.3, 140.0, 139.7, 130.1, 125.4, 120.2, 118.3, 118.1, 116.3, 56.3, 34.3,
3
+
31.6. HRMS (ESI): calc. for C H N O [M+H] : 301.1547, found: 301.1547.
17
21
2
3
N-(3,5-Dimethylphenyl)-2-methoxy-6-nitroaniline (14). Yield: 67%; 366.1 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.02 (br. s, 1H), 7.70 (dd, J = 8.5, 1.5 Hz, 1H), 7.11 (dd, J = 8.1, 1.4 Hz,
3
13
1H), 7.01 (dd, J = 8.5, 8.1 Hz, 1H), 6.65 (m, 1H), 6.48 (m, 2H), 3.79 (s, 3H), 2.27 (q, J = 0.7 Hz, 6H). C NMR
(100 MHz, CDCl ): δ 152.7, 142.1, 140.4, 138.4, 129.6, 124.2, 120.4, 118.0, 116.3, 116.1, 56.3, 21.5. HRMS
3
+
(
ESI): calc. for C H N O [M+H] : 273.1234, found: 273.1237.
15
17
2
3
N-(2-Methoxy-6-nitrophenyl)-2,3-dimethylaniline (15). Yield: 65%; 315.8 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.10 (br. s, 1H), 7.74 (dd, J = 8.6, 1.4 Hz, 1H), 7.10 (dd, J = 8.0, 1.4 Hz,
3
13
1H), 7.00 – 6.94 (m, 2H), 6.91 (m, 1H), 6.56 (m, 1H), 3.74 (s, 3H), 2.38 (s, 3H), 2.35 (s, 3H). C NMR (100
MHz, CDCl ): δ 152.1, 140.7, 139.3, 137.2, 131.4, 127.2, 125.0, 124.8, 119.5, 118.0, 116.7, 116.2, 56.3, 20.7,
3
+
13.7. HRMS (ESI): calc. for C H N O [M+H] : 273.1234, found: 273.1233.
15
17
2
3
1
N-(2-Ethylphenyl)-2-methoxy-6-nitroaniline (16). Yield: 60%; 260.2 mg was isolated as a red oily residue. H
NMR (400 MHz, CDCl ): δ 8.17 (br. s, 1H), 7.68 (dd, J = 8.5, 1.5 Hz, 1H), 7.19 (dd, J = 7.4, 1.8 Hz, 1H), 7.06 –
3
6
2
1
.94 (m, 3H), 6.91 (dd, J = 8.5, 7.9 Hz, 1H), 6.60 (dd, J = 7.9, 1.5 Hz, 1H), 3.65 (s, 3H), 2.75 (q, J = 7.6 Hz,
13
H), 1.31 (t, J = 7.6 Hz, 3H). C NMR (100 MHz, CDCl ): δ 152.2, 140.1, 139.5, 134.0, 131.1, 128.5, 125.8,
3
+
23.0, 119.8, 118.7, 118.0, 116.3, 56.2, 24.7, 13.8. HRMS (ESI): calc. for C H N O [M+H] : 273.1234,
15
17
2
3
1
found: 273.1233. Note: TMS used for reference of H NMR spectrum (0.00 ppm).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 62
N-(2-Methoxy-6-nitrophenyl)-3,4,5-trimethylaniline (17). Yield: 61%; 427.6 mg was isolated as a red oily
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
residue. H NMR (400 MHz, CDCl ): δ 8.00 (br. s, 1H), 7.69 (dd, J = 8.5, 1.4 Hz, 1H), 7.09 (dd, J = 8.0, 1.4 Hz,
3
1H), 6.97 (dd, J = 8.6, 8.0 Hz, 1H), 6.52 (pentet, J = 0.6 Hz, 2H), 3.78 (s, 3H), 2.22 (s, 3H), 2.22 (s, 3H), 2.12
13
(
s, 3H). C NMR (100 MHz, CDCl ): δ 152.5, 139.9, 139.2, 136.9, 130.3, 129.4, 119.9, 118.2, 118.0, 116.1,
3
+
5
6.4, 20.9, 15.0. HRMS (ESI): calc. for C H N O [M+H] : 287.1390, found: 287.1399.
16
19
2
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
N-(2-Methoxy-6-nitrophenyl)pyridin-3-amine (18). Yield: 76%; 249.1 mg was isolated as a yellow oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.25 (br. s, 1H), 8.18 – 8.10 (m, 2H), 7.67 (dd, J = 8.4, 1.5 Hz, 1H), 7.15
3
13
–
7.05 (m, 2H), 7.05 – 6.94 (m, 2H), 3.70 (s, 3H). C NMR (100 MHz, CDCl ): δ 152.5, 142.9, 140.8, 140.7,
3
+
1
38.7, 128.2, 124.9, 123.0, 121.6, 117.9, 116.5, 56.2. HRMS (ESI): calc. for C H N O [M+H] : 246.0873,
12 12
3
3
found: 246.0884.
N-(4-Fluorophenyl)-2-methoxy-6-nitroaniline (19). Yield: 81%; 401.8 mg was isolated as a red oily residue.
1
H NMR (400 MHz, CDCl ): δ 8.22 (br. s, 1H), 7.71 (dd, J = 8.5, 1.4 Hz, 1H), 7.09 (dd, J = 8.1, 1.4 Hz, 1H),
3
13
6.99 (d, J = 8.3 Hz, 1H), 6.96 – 6.89 (m, 2H), 6.83 – 6.78 (m, 2H), 3.73 (s, 3H). C NMR (100 MHz, CDCl ): δ
3
158.6 (d, J = 240.8 Hz), 152.2, 139.8, 138.4 (d, J = 2.7 Hz), 130.2, 120.5 (d, J = 8.0 Hz), 120.3, 118.0, 116.4,
+
1
15.2 (d, J = 22.7 Hz), 56.2. HRMS (ESI): calc. for C H FN O [M+H] : 263.0826, found: 263.0823.
13
12
2
3
N-(4-Chlorophenyl)-2-methoxy-6-nitroaniline (20). Yield: 83%; 515.0 mg was isolated as a red oily residue.
1
H NMR (400 MHz, CDCl ): δ 8.14 (br. s, 1H), 7.70 (dd, J = 8.3, 1.5 Hz, 1H), 7.21 – 7.13 (m, 2H), 7.11 (dd, J =
3
13
8
.3, 1.5 Hz, 1H), 7.03 (dd, J = 8.3, 8.3 Hz, 1H), 6.79 – 6.67 (m, 2H), 3.76 (s, 3H). C NMR (100 MHz, CDCl ):
3
δ 152.5, 141.0, 140.5, 129.0, 128.6, 127.0, 121.2, 119.7, 118.0, 116.4, 56.2. HRMS (ESI): calc. for
+
C H ClN O [M+H] : 279.0531, found: 279.0538.
13
12
2
3
2,4-Dichloro-N-(2-methoxy-6-nitrophenyl)aniline (21). Yield: 60%; 354.7 mg was isolated as an orange
1
solid. H NMR (400 MHz, CDCl ): δ 8.12 (br. s, 1H), 7.71 (dd, J = 7.6, 2.3 Hz, 1H), 7.36 (d, J = 2.3 Hz, 1H),
3
13
7
.18 – 7.10 (m, 2H), 7.03 (ddd, J = 8.7, 2.3, 0.5 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H), 3.79 (s, 3H). C NMR (100
MHz, CDCl ): δ 153.0, 141.9, 137.9, 129.0, 127.2, 126.8, 126.2, 123.5, 122.6, 118.4, 118.0, 116.6, 56.4.
3
+
HRMS (ESI): calc. for C H Cl N O [M+H] : 313.0141, found: 313.0146. MP: 120 – 122 °C.
13
11
2
2
3
N-(4-Bromophenyl)-2-methoxy-6-nitroaniline (22). Yield: 52%; 289.5 mg was isolated as a red oily residue.
1
H NMR (400 MHz, CDCl ): δ 8.11 (br. s, 1H), 7.70 (dd, J = 8.2, 1.5 Hz, 1H), 7.34 – 7.27 (m, 2H), 7.11 (dd, J =
3
ACS Paragon Plus Environment

Page 25 of 62
Journal of Medicinal Chemistry
13
8
.2, 1.5 Hz, 1H), 7.04 (dd, J = 8.2, 8.2 Hz, 1H), 6.72 – 6.59 (m, 2H), 3.76 (s, 3H). C NMR (100 MHz, CDCl ):
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
δ 152.6, 141.5, 140.7, 131.5, 128.8, 121.3, 120.1, 118.0, 116.5, 114.4, 56.3. HRMS (ESI): calc. for
+
C H BrN O [M+H] : 323.0026, found: 323.0037.
13
12
2
3
2-Methoxy-6-nitro-N-(4-(trifluoromethyl)phenyl)aniline (23). Yield: 21%; 124.5 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.10 (br. s, 1H), 7.73 (dd, J = 7.7, 2.1 Hz, 1H), 7.52 – 7.40 (m, 2H), 7.21
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
–
1
7.06 (m, 2H), 6.83 – 6.74 (m, 2H), 3.81 (s, 3H). C NMR (100 MHz, CDCl ): δ 153.3, 145.5 (d, J = 1.4 Hz),
3
42.0, 127.4, 126.1 (q, J = 3.8 Hz), 123.5 (q, J = 123.2 Hz), 123.3, 122.6, 118.0, 117.4, 116.6, 56.4. HRMS
+
(
ESI): calc. for C H F N O [M+H] : 313.0795, found: 313.0788.
14
12
3
2
3
N-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-2-methoxy-6-nitroaniline (24). Yield: 53%; 474.2 mg was
1
isolated as a red oily residue. H NMR (400 MHz, CDCl ): δ 8.36 (br. s, 1H), 7.71 (dd, J = 8.6, 1.4 Hz, 1H), 7.04
3
(
dd, J = 8.0, 1.4 Hz, 1H), 6.91 (dd, J = 8.6, 7.9 Hz, 1H), 6.79 – 6.68 (m, 4H), 3.69 (s, 3H), 0.99 (s, 9H), 0.19 (s,
13
6
H). C NMR (100 MHz, CDCl ): δ 152.0, 151.5, 139.1, 136.2, 131.6, 121.1, 120.1, 119.3, 118.2, 116.4, 56.2,
3
+
25.9, 18.4, ꢀ4.3. HRMS (ESI): calc. for C H N O Si [M+H] : 375.1735, found: 375.1744.
19
27
2
4
2-Methoxy-6-nitro-N-(4-phenoxyphenyl)aniline (25). Yield: 76%; 646.9 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.29 (br. s, 1H), 7.73 (dd, J = 8.5, 1.4 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.14
3
13
– 7.05 (m, 2H), 7.03 – 6.98 (m, 3H), 6.97 – 6.91 (m, 2H), 6.89 – 6.83 (m, 2H), 3.76 (s, 3H). C NMR (100
MHz, CDCl ): δ 158.1, 152.2, 152.0, 139.7, 138.1, 130.3, 129.8, 122.8, 120.4, 120.2, 119.7, 118.1, 116.4, 56.2.
3
13
Note: One C signal missing from aromatic region, likely due to signal overlap. HRMS (ESI): calc. for
+
C H N O [M+H] : 337.1183, found: 337.1193.
19
17
2
4
N ,N -Diethyl-N -(2-methoxy-6-nitrophenyl)benzene-1,4-diamine (26). Yield: 50%; 298.7 mg was isolated
1
1
4
1
as a dark purple oily residue. H NMR (400 MHz, CDCl ): δ 8.38 (br. s, 1H), 7.71 (dd, J = 8.7, 1.4 Hz, 1H), 7.02
3
(
(
dd, J = 8.0, 1.4 Hz, 1H), 6.89 – 6.75 (m, 3H), 6.67 – 6.54 (m, 2H), 3.71 (s, 3H), 3.32 (q, J = 7.1 Hz, 4H), 1.15
13
t, J = 7.1 Hz, 6H). C NMR (100 MHz, CDCl ): δ 151.8, 144.7, 138.2, 132.4, 131.5, 121.6, 118.3, 118.3,
3
+
1
16.2, 112.7, 56.4, 44.8, 12.7. HRMS (ESI): calc. for C H N O [M+H] : 316.1656, found: 316.1641.
17
22
3
3
(
4-((2-Methoxy-6-nitrophenyl)amino)phenyl)methanol (27). Yield: 99%; 422.1 mg was isolated as a red oily
1
residue. H NMR (400 MHz, CDCl ): δ 8.12 (br. s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.10
3
(
d, J = 8.2 Hz, 1H), 7.01 (dd, J = 8.2, 8.2 Hz, 1H), 6.79 (d, J = 8.3 Hz, 2H), 4.59 (s, 2H), 3.75 (s, 3H), 1.97 (br.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 62
13
s, 1H). C NMR (100 MHz, CDCl ): δ 152.7, 141.8, 140.5, 134.8, 129.4, 127.8, 120.8, 118.5, 118.1, 116.3,
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
+
6
5.2, 56.3. HRMS (ESI): calc. for C H N O [M+H] : 275.1026, found: 275.1024.
14
15
2
4
N-(4-(2-((tert-Butyldimethylsilyl)oxy)ethyl)phenyl)-2-methoxy-6-nitroaniline (28). Yield: 83%; 695.1 mg
1
was isolated as a red oily residue. H NMR (400 MHz, CDCl ): δ 8.21 (br. s, 1H), 7.70 (dd, J = 8.5, 1.4 Hz, 1H),
3
7
.13 – 7.05 (m, 3H), 6.97 (m, 1H), 6.78 (d, J = 8.5 Hz, 2H), 3.81 (t, J = 7.0 Hz, 2H), 3.74 (s, 3H), 2.79 (t, J = 7.0
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
Hz, 2H), 0.91 (s, 9H), 0.02 (s, 6H). C NMR (100 MHz, CDCl ): δ 152.4, 140.4, 139.9, 133.2, 129.9, 129.3,
3
+
1
20.2, 118.6, 117.9, 116.2, 64.7, 56.2, 39.0, 26.0, 18.4, ꢀ5.3. HRMS (ESI): calc. for C H N O Si [M+H] :
2
1
31
2
4
403.2048, found: 403.2061.
2
-(4-((2-Methoxy-6-nitrophenyl)amino)phenyl)ethan-1-ol (29). Yield: 97%; 297.4 mg was isolated as a red
1
oily residue. H NMR (400 MHz, CDCl ): δ 8.10 (br. s, 1H), 7.68 (dd, J = 8.5, 1.4 Hz, 1H), 7.13 – 7.03 (m, 3H),
3
6
.98 (t, J = 8.3 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 3.79 (t, J = 6.6 Hz, 2H), 3.75 (s, 3H), 2.79 (t, J = 6.6 Hz, 2H),
13
1
.94 (br. s, 1H). C NMR (100 MHz, CDCl ): δ 152.5, 140.7, 140.1, 132.4, 129.7, 129.3, 120.5, 118.7, 118.0,
3
ꢀ
116.2, 63.8, 56.3, 38.6. HRMS (ESI): calc. for C H N O [MꢀH] : 287.1037, found: 287.1024.
15
15
2
4
1
-(4-((2-Methoxy-6-nitrophenyl)amino)phenyl)-N-methylmethanesulfonamide (30). Yield: 46%; 228.1 mg
1
was isolated as a red solid. H NMR (400 MHz, DMSOꢀd ): δ 8.08 (s, 1H), 7.52 (dd, J = 8.3, 1.3 Hz, 1H), 7.38
6
(dd, J = 8.3, 1.4 Hz, 1H), 7.24 (dd, J = 8.3, 8.3 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 6.84 (q, J = 4.9 Hz, 1H), 6.63
13
(d, J = 8.5 Hz, 2H), 4.16 (s, 2H), 3.80 (s, 3H), 2.51 (d, J = 4.9 Hz, 3H). C NMR (100 MHz, DMSOꢀd ): δ 154.2,
6
1
44.0, 143.8, 131.1, 125.3, 123.5, 120.9, 116.5, 116.1, 114.9, 56.4, 55.5, 28.9. HRMS (ESI): calc. for
+
C H N O SNa [M+Na] : 374.0781, found: 374.0791. MP: 156 – 158 °C.
15
17
3
5
1
1-Methoxy-7-methylphenazine (32). Yield: 83%; 265.9 mg was isolated as a yellow solid. H NMR (400 MHz,
CDCl ): δ 8.15 (m, 1H), 7.82 (m, 1H), 7.66 (dd, J = 8.9, 1.1 Hz, 1H), 7.56 (dd, J = 8.9, 7.5 Hz, 1H), 7.51 (dd, J
3
13
=
8.9, 1.9 Hz, 1H), 6.86 (dd, J = 7.6, 1.1 Hz, 1H), 4.02 (s, 3H), 2.48 (d, J = 1.2 Hz, 3H). C NMR (100 MHz,
CDCl ): δ 155.1, 144.1, 143.5, 141.5, 140.9, 136.2, 133.1, 130.2, 129.6, 127.3, 121.3, 105.9, 56.4, 22.2.
3
+
HRMS (ESI): calc. for C H N O [M+H] : 225.1022, found: 225.1026. MP: 124 – 126 °C. Note: Compound has
14
13
2
been previously reported (CAS: 13860ꢀ49ꢀ6), but no spectra were found for comparison.
-Ethyl-1-methoxyphenazine (33). Yield: 97%; 294.4 mg was isolated as a yellow solid. H NMR (400 MHz,
1
7
CDCl ): δ 8.05 (d, J = 8.9 Hz, 1H), 7.70 (m, 1H), 7.52 (dd, J = 8.9, 1.1 Hz, 1H), 7.41 – 7.33 (m, 2H), 6.68 (dd, J
3
ACS Paragon Plus Environment

Page 27 of 62
Journal of Medicinal Chemistry
13
=
7.6, 1.1 Hz, 1H), 3.86 (s, 3H), 2.64 (qd, J = 7.5, 1.1 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H). C NMR (100 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
CDCl ): δ 155.3, 147.7, 144.3, 143.9, 141.4, 136.4, 132.5, 130.5, 129.9, 126.0, 121.4, 106.2, 56.6, 29.4, 14.6.
3
+
HRMS (ESI): calc. for C H N O [M+H] : 239.1179, found: 239.1182. MP: 106 – 108 °C.
15
15
2
1
1
-Methoxy-7-propylphenazine (34). Yield: 70%; 277.3 mg was isolated as a yellow solid. H NMR (400 MHz,
CDCl ): δ 8.24 (dd, J = 9.0, 0.6 Hz, 1H), 7.91 (dq, J = 1.8, 0.9 Hz, 1H), 7.74 (dd, J = 9.0, 1.1 Hz, 1H), 7.67 –
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
7
0
1
2
.56 (m, 2H), 6.96 (dd, J = 7.8, 1.1 Hz, 1H), 4.10 (s, 3H), 2.81 (td, J = 7.5, 0.9 Hz, 2H), 1.92 – 1.52 (m, 2H),
13
.96 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, CDCl ): δ 155.2, 146.1, 144.3, 143.7, 141.3, 136.4, 132.6, 130.4,
3
+
29.8, 126.8, 121.4, 106.1, 56.5, 38.4, 23.6, 13.9. HRMS (ESI): calc. for C H N O [M+H] : 253.1335, found:
16
17
2
53.1335. MP: 70 – 72 °C.
-(tert-Butyl)-1-methoxyphenazine (35). Yield: 94%; 363.8 mg was isolated as a yellow oily residue. H NMR
1
7
(
400 MHz, CDCl ): 8.10 (dd, J = 9.2, 0.6 Hz, 1H), 7.89 (dd, J = 2.2, 0.6 Hz, 1H), 7.69 (dd, J = 9.2, 2.1 Hz, 1H),
3
13
7
.53 (dd, J = 8.8, 1.1 Hz, 1H), 7.43 – 7.32 (m, 1H), 6.68 (dd, J = 7.6, 1.1 Hz, 1H), 3.86 (s, 3H), 1.22 (s, 9H). C
NMR (100 MHz, CDCl ): δ 154.8, 153.8, 143.9, 143.3, 140.7, 136.1, 129.9, 129.6, 129.2, 123.4, 120.9, 105.7,
3
+
56.1, 35.2, 30.5. HRMS (ESI): calc. for C H N O [M+H] : 267.1492, found: 267.1502.
17
19
2
1
6
-Methoxy-1,3-dimethylphenazine (36). Yield: 86%; 233.1 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl ): δ 7.91 (m, 1H), 7.75 (dd, J = 8.8, 1.1 Hz, 1H), 7.57 (dd, J = 8.8, 7.6 Hz, 1H), 7.37 (m, 1H), 6.93
3
13
(dd, J = 7.6, 1.1 Hz, 1H), 4.08 (s, 3H), 2.78 (dd, J = 1.0, 1.0 Hz, 3H), 2.49 (d, J = 1.0 Hz, 3H). C NMR (100
MHz, CDCl ): δ 154.9, 143.0, 142.5, 142.0, 140.6, 136.8, 136.3, 132.9, 129.2, 126.2, 121.9, 106.2, 56.4, 22.3,
3
+
1
7.6. HRMS (ESI): calc. for C H N O [M+H] : 239.1179, found: 239.1177. MP: 117 – 119 °C.
15
15
2
1
9-Methoxy-1,2-dimethylphenazine (37). Yield: 44%; 121.3 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl ): δ 7.92 (d, J = 8.9 Hz, 1H), 7.75 (dd, J = 8.8, 1.1 Hz, 1H), 7.63 (dd, J = 8.8, 7.5 Hz, 1H), 7.58 (d, J
3
13
=
8.9 Hz, 1H), 6.95 (dd, J = 7.5, 1.1 Hz, 1H), 4.10 (s, 3H), 2.86 (s, 3H), 2.49 (s, 3H). C NMR (100 MHz,
CDCl ): δ 155.4, 143.2, 142.4, 141.7, 137.6, 136.0, 134.8, 134.7, 129.9, 126.1, 121.2, 106.3, 56.5, 20.8, 13.4.
3
+
HRMS (ESI): calc. for C H N O [M+H] : 239.1179, found: 239.1179. MP: 157 – 159 °C, lit. 165 – 168 °C.
15
15
2
1
49
Note: H NMR tabulation and melting point match previously reported values.
-Ethyl-9-methoxyphenazine (38). Yield: 46%; 104.5 mg was isolated as a yellow solid. H NMR (400 MHz,
1
1
CDCl ): δ 8.03 (m, 1H), 7.78 (dd, J = 8.9, 1.2 Hz, 1H), 7.74 (dd, J = 8.7, 6.9 Hz, 1H), 7.68 (dd, J = 8.8, 7.5 Hz,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 62
1
H), 7.61 (dq, J = 6.9, 1.2 Hz, 1H), 6.99 (dd, J = 7.6, 1.1 Hz, 1H), 4.11 (s, 3H), 3.49 (q, J = 7.5 Hz, 2H), 1.45 (t,
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
13
J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl ): δ 155.6, 144.1, 143.9, 143.7, 141.4, 136.2, 131.0, 130.4, 127.5,
3
+
127.2, 121.3, 106.6, 56.7, 23.8, 14.5. HRMS (ESI): calc. for C H N O [M+H] : 239.1179, found: 239.1184.
15
15
2
MP: 124 – 126 °C.
1
6-Methoxy-1,2,3-trimethylphenazine (39). Yield: 60%; 213.2 mg was isolated as a yellow solid. H NMR (400
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
MHz, CDCl ): δ 7.93 (s, 1H), 7.74 (dd, J = 8.8, 1.1 Hz, 1H), 7.55 (dd, J = 8.8, 7.6 Hz, 1H), 6.89 (dd, J = 7.6, 1.1
3
13
Hz, 1H), 4.07 (s, 3H), 2.77 (s, 3H), 2.44 (d, J = 1.1 Hz, 3H), 2.32 (s, 3H). C NMR (100 MHz, CDCl ): δ 154.9,
3
143.1, 142.2, 141.3, 141.2, 138.5, 135.7, 133.4, 129.0, 126.3, 121.9, 105.8, 56.3, 21.9, 16.8, 13.4. HRMS
+
(
ESI): calc. for C H N O [M+H] : 253.1335, found: 253.1339. MP: 196 – 198 °C.
16 17
2
1
6-Methoxypyrido[2,3-b]quinoxaline (40). Yield: 42%; 49.4 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl ): δ 9.37 (dd, J = 3.9, 2.0 Hz, 1H), 8.76 (dd, J = 8.7, 2.0 Hz, 1H), 7.97 (dd, J = 8.9, 1.1 Hz, 1H),
3
13
7.84 (dd, J = 8.9, 7.6 Hz, 1H), 7.78 (dd, J = 8.7, 3.9 Hz, 1H), 7.14 (dd, J = 7.6, 1.1 Hz, 1H), 4.20 (s, 3H). C
NMR (100 MHz, CDCl ): δ 156.9, 155.1, 149.8, 145.9, 139.1, 137.9, 137.7, 132.2, 125.4, 122.1, 107.3, 56.7.
3
+
HRMS (ESI): calc. for C H N O [M+H] : 212.0818, found: 212.0822. MP: 141 – 143 °C. Note: Compound has
12
10
3
been previously published (CAS: 54696ꢀ72ꢀ9), but no characterization data was reported for the isolated
50
product.
1
7-Ethoxy-1-methoxyphenazine (41). Yield: 79%; 110.0 mg was isolated as a yellow solid. H NMR (400 MHz,
CDCl ): δ 8.12 (d, J = 9.5 Hz, 1H), 7.61 (dd, J = 8.8, 1.4 Hz, 1H), 7.56 (dd, J = 8.8, 7.3 Hz, 1H), 7.35 (dd, J =
3
9
1
1
.5, 2.7 Hz, 1H), 7.21 (d, J = 2.7 Hz, 1H), 6.84 (dd, J = 7.4, 1.4 Hz, 1H), 4.10 (q, J = 7.0 Hz, 2H), 4.02 (s, 3H),
13
.40 (t, J = 7.0 Hz, 3H). C NMR (100 MHz, CDCl ): δ 161.0, 155.5, 145.4, 144.2, 139.5, 135.1, 131.3, 130.6,
3
51
26.4, 120.9, 105.6, 104.9, 64.4, 56.6, 14.7. MP: 149 – 151 °C, lit. 148 – 149 °C. Note: Compound has been
previously reported (CAS: 58476ꢀ65ꢀ6), but only melting point was found for comparison.
-Chloro-1-methoxyphenazine (42). Yield: 71%; 380.0 mg was isolated as a yellow solid. H NMR (400 MHz,
CDCl ): δ 8.14 (dd, J = 9.2, 0.6 Hz, 1H), 8.00 (dd, J = 2.4, 0.6 Hz, 1H), 7.64 – 7.47 (m, 3H), 6.87 (dd, J = 6.2,
1
7
3
13
2.4 Hz, 1H), 4.02 (s, 3H). C NMR (100 MHz, CDCl ): δ 155.0, 144.3, 143.2, 140.3, 136.7, 136.5, 131.4,
3
+
131.3, 131.1, 127.6, 121.2, 106.6, 56.4. HRMS (ESI): calc. for C H ClN O [M+H] : 245.0476, found:
13
10
2
ACS Paragon Plus Environment

Page 29 of 62
Journal of Medicinal Chemistry
52
2
45.0479. MP: 162 – 164 °C, lit. 164 – 165 °C. Note: Compound has been previously reported (CAS: 13554ꢀ
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
2ꢀ4), but only melting point was found for comparison.
1
1,3-Dichloro-9-methoxyphenazine (43). Yield: 15%; 10.2 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl ): δ 8.17 (d, J = 2.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.86 – 7.75 (m, 2H), 7.11 (dd, J = 6.5, 2.3 Hz,
3
13
1
1
1
H), 4.18 (s, 3H). C NMR (100 MHz, CDCl ): δ 155.6, 145.1, 143.7, 137.6, 136.8, 136.1, 135.0, 132.5, 130.9,
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
+
27.3, 121.1, 107.6, 56.9. HRMS (ESI): calc. for C H Cl N O [M+H] : 279.0086, found: 279.0082. MP: 186 –
13
9
2
2
88 °C.
1
Ethyl 6-methoxyphenazine-2-carboxylate (44). Yield: 84%; 77.5 mg was isolated as a yellow solid. H NMR
(
400 MHz, CDCl ): δ 8.97 (dd, J = 1.8, 0.8 Hz, 1H), 8.43 (dd, J = 9.1, 0.7 Hz, 1H), 8.37 (dd, J = 9.1, 1.8 Hz,
3
1
7
1
H), 7.84 (dd, J = 8.9, 1.2 Hz, 1H), 7.77 (dd, J = 8.9, 7.4 Hz, 1H), 7.10 (dd, J = 7.4, 1.2 Hz, 1H), 4.48 (q, J =
13
.1 Hz, 2H), 4.18 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, CDCl ): δ 165.9, 155.3, 145.0, 143.6,
3
42.8, 137.9, 132.7, 132.5, 131.3, 130.6, 129.3, 121.9, 107.6, 61.9, 56.8, 14.5. HRMS (ESI): calc. for
+
C H N O [M+H] : 305.0897, found: 305.0908. MP: 139 – 141 °C. Note: Product obtained from an acidic
16
15
2
3
workup following reductive cyclization of diarylamine intermediate 23.
-Methoxyphenazin-2-ol (45). Yield: 64%; 183.0 mg was isolated as a yellow solid. H NMR (400 MHz,
DMSOꢀd ): δ 10.86 (s, 1H), 8.13 (d, J = 9.4 Hz, 1H), 7.77 (dd, J = 8.8, 7.6 Hz, 1H), 7.64 (dd, J = 8.8, 1.1 Hz,
1
6
6
13
1H), 7.55 (dd, J = 9.4, 2.6 Hz, 1H), 7.31 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 7.6, 1.1 Hz, 1H), 4.04 (s, 3H). C
NMR (100 MHz, DMSOꢀd ): δ 159.8, 155.2, 144.6, 143.7, 138.1, 134.1, 131.1, 130.9, 125.8, 119.9, 106.9,
6
+
105.8, 55.9. HRMS (ESI): calc. for C H N O [M+H] : 249.0634, found: 249.0636. MP: 210 °C (decomp), lit.
13
11
2
2
53
200 °C (decomp).
1
1-Methoxy-7-phenoxyphenazine (46). Yield: 71%; 388.2 mg was isolated as a yellow solid. H NMR (400
MHz, CDCl ): δ 8.35 (dd, J = 9.5, 0.5 Hz, 1H), 7.74 – 7.61 (m, 3H), 7.49 – 7.39 (m, 2H), 7.34 (dd, J = 2.7, 0.5
3
13
Hz, 1H), 7.29 – 7.16 (m, 3H), 6.96 (dd, J = 6.2, 2.5 Hz, 1H), 4.13 (s, 3H). C NMR (100 MHz, CDCl ): δ 160.5,
3
155.2, 154.7, 144.5, 144.2, 139.5, 135.5, 131.8, 130.7, 130.3, 125.4, 125.3, 121.0, 120.8, 109.9, 105.8, 56.4.
+
HRMS (ESI): calc. for C H N O [M+H] : 303.1128, found: 303.1140. MP: 163 – 165 °C. Note: TMS used for
19
15
2
2
1
reference of H NMR spectrum (0.00 ppm).
ACS Paragon Plus Environment