Simple synthesis of endophenazine G and other phenazines and their evaluation as anti-methicillin-resistant Staphylococcus aureus agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.079

Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has become a severe health concern because of its treatment difficulties. Herein, we report the synthesis and biological evaluation of two phenazine natural products and a series o

Full text of DOI:10.1016/j.ejmech.2016.09.079

Accepted Manuscript
Simple synthesis of endophenazine G and other phenazines and their evaluation as
anti-methicillin-resistant Staphylococcus aureus agents
Martin Conda-Sheridan, Venkatareddy Udumula, Jennifer L. Endres, Caleb N.
Harper, Lee Jaramillo, Haizhen A. Zhong, Kenneth W. Bayles
PII:
S0223-5234(16)30815-7
10.1016/j.ejmech.2016.09.079
EJMECH 8945
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 August 2016
Revised Date: 23 September 2016
Accepted Date: 24 September 2016
Please cite this article as: M. Conda-Sheridan, V. Udumula, J.L. Endres, C.N. Harper, L. Jaramillo, H.A.
Zhong, K.W. Bayles, Simple synthesis of endophenazine G and other phenazines and their evaluation
as anti-methicillin-resistant Staphylococcus aureus agents, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.09.079.
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ACCEPTED MANUSCRIPT

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Simple Synthesis of Endophenazine G and Other Phenazines and Their Evaluation as Anti-
Methicillin-Resistant Staphylococcus Aureus Agents
Martin Conda-Sheridan,^, * ,§ Venkatareddy Udumula,^,§ Jennifer L. Endres,Ŧ Caleb N. Harper,^
Lee Jaramillo, ^ Haizhen A. Zhong,+ Kenneth W. Bayles,Ŧ
^Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical
Center, Omaha, Nebraska 68198, United States of America
ŦCenter for Staphylococcal Research, Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska 68198, United States of America
+ Department of Chemistry, University of Nebraska at Omaha, Omaha, NE 68182, United States
of America

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1. Introduction
The number of bacterial infections continues to increase annually, and the emergence of
resistance in most pathogens has rendered many clinically used antibiotics ineffective, causing a
major health concern in humans.[1, 2] One such pathogen is methicillin-resistant Staphylococcus
aureus (MRSA), which is listed as one of the ESKAPE pathogens. S. aureus infections used to
occur more frequently in immunocompromised patients however, in recent years MRSA has
become a serious threat to otherwise healthy individuals.[3, 4] It is estimated that half million
Americans are hospitalized every year due to MRSA infections [5] accounting for thousands of
deaths annually. The past decade has also seen the rise of vancomycin-resistant S. aureus strains
(VRSA), one of the last resort antibiotics used for the treatment of many S. aureus infections. [6-
8] It is clear that there is a critical need to find new antibiotics against this pathogen; however,
the discovery of antibacterial drugs has seen a marked decline over the last 50 years.[9] Indeed,
only seven new antibiotics have been approved by the FDA in the last decade.[10]
For centuries, scientists have looked to nature in order to identify molecules with
antibacterial activity. An intriguing family of natural products known for its broad biological
activities are the phenazines, secondary metabolites isolated mainly from Pseudomonas and
Streptomyces.[11] Arguably, the most interesting characteristic of the phenazines are their
antibacterial properties. Since the discovery of the phenazine antibiotic pyocyanin (1, Figure 1),
many phenazines have been discovered and evaluated as antimicrobial agents.[11, 12] In recent
years there has been a revival in the interest of phenazine antibiotics. Recent reports have
introduced molecules such as D-Alanylgriseoluteic Acid [13] (2, Figure 1) and 2,4-dibromo-1-
hydroxiphenazine[14] (3) with activities in the low µM range against bacteria such as S. aureus,
E. faecalis, E. coli, and Salmonella.[13, 15-19] In particular, the work by the Huigens group has

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yielded promising phenazines with intriguing activities against MRSA and other pathogens in
vitro and in vivo.[15, 16, 20]
These reports prompted us to look further into phenazine natural products as anti-MRSA
drugs. Our search led us to two natural products: endophenazine A1 (4) and endophenazine G
(5), which were isolated from Kitasatospora (sp. HKI 714).[21] These phenazines are active
against various pathogens including P. aeruginosa, vancomycin-resistant Enterococcus faecalis
(VRE), and MRSA.[22] In this study, we report the first synthesis of endophenazine G (5) using
an efficient route and a series of phenazine analogues. In addition, we describe the anti-MRSA
activity of the synthesized compounds, basic tests to understand their mechanism of action, and
computational studies to establish initial structure-activity relationships (SAR).
2. Results and Discussion
2.1.Chemistry. The synthesis of the natural product 5 is shown on Scheme 1. Briefly, we
coupled 2-bromo-3-nitrobenzoic acid (6) with 2-bromoaniline (7) using Ullmann conditions.[15]
The obtained intermediate 8 was cyclized under reductive conditions in basic media to afford
phenazine 9. Esterification of the compound provided molecule 10, which was coupled with 1,1-
dimethylallyl alcohol (11) using a Mizoroki−Heck reaction.[23] Hydrolysis of compound 12
provided the natural product endophenazine G in 88% yield. The double bond of 5 was further
reduced in the presence of hydrogen using Pd-C as a catalyst to get analogue 13.[24]
During preliminary antibacterial assays, it was found that precursor 9 was as active as the
natural product 5 with minimum inhibitory concentrations (MIC) against MRSA of 32 µg/mL
(Table 1). Therefore, we decided to explore the result of substitutions in both molecules, 5 and 9,
to develop structure activity relationships (SAR). Using the same chemistry, the bromine atom of

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9 was placed at different positions within the ring (18 and 19, Scheme 2), or replaced with a
methyl group (20 [25]), or chlorine atom (21)[15] to understand how electronic properties affect
activity. Next, some of the compounds were esterified with methyl iodine under basic conditions
(22,[26] 23 and 24). Molecule 22 was reacted with the isoprenoid 11, to provide an ester with a
tail opposite to the ester group (25). Basic hydrolysis of the methyl ester provided the natural
product 26, which had been previously isolated from Streptomyces cinnamonensis.[27] The
carboxylic acid was converted into a dimethylamide derivative, via an acyl chloride intermediate,
and subjected to Heck coupling conditions to yield 27.
To further understand the role of electronegativity and hydrogen bonding on antibacterial
activity, the carboxylic acid was replaced by an N-(methylsulfonyl)amide bioisoster (28) using
CDI and methane sulfonamide [28] or the electron withdrawing nitrile group (29 and 31), which
was prepared by dehydration [29] of an amide [30] intermediate. Also, two secondary amine
derivatives 30 and 32, which do not have the ability to ionize or act as hydrogen bond donors,
were prepared via an acyl chloride intermediate as shown in Scheme 3.[31] During preliminary
tests, it was found the N-(methylsulfonyl)amide 28 presented promising activity (MIC 4 µg/mL,
Table 1). Thus, analogue 33 (Scheme 3) was prepared from molecule 21.[15] Our library was
completed by substituting the bromide atom of molecule 9 with a fluorine atom (Scheme 4)
providing analogue 35 [25] that was further functionalized to 37.
2.2 Biological Evaluation. The biological activity of the synthesized compounds against a
Community-Associated MRSA strain, LAC JE2, was evaluated by performing broth
microdilution minimum inhibitory concentrations (MIC) assays and the results are shown in
Table 1. Initially, we tried to understand the role of the alkyl moiety but found that 5 is more

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active than analogue 26 (MIC of 32 µg/mL vs 128 µg/mL), which has an alkyl tail opposite to
the carboxylic acid. Removing the unsaturation slightly decreases activity to 64 µg/mL (5 vs
13). Given that molecule 9 (bromine substitution) presented activity similar to the natural
product; we decided to explore analogues of this molecule do to its ease of synthesis. It was
found that moving the position of the Br increases MIC from 32 µg/mL (9) to 128 µg/mL (18)
and 64 µg/mL (19). The Br was also exchanged by CH3 and Cl giving analogues with MICs of
32 µg/mL (20), and 16 µg/mL (21), which validates our observation the alkyl chain is not needed
for activity. The smaller substituents H (35) and F (36) both provide MICs of 64 µg/mL. The
data suggest that an electron-withdrawing group at position 1 is not enough for activity as seen
with primary amides (36 and 38), substituted amides (27 and 32), nitriles (29 and 31) and esters
(i.e. 10, 12, and 37). The best activity was observed with the N-(methylsulfonyl)amide
derivatives 28 and 33. Both exhibited low MIC values of 4 µg/mL and 2 µg/mL, respectively,
comparable to that of vancomycin (2 µg/mL under our testing conditions).
The optimal anti-MRSA activity is obtained when the substituents are on the same side of
the phenazine core. Placing substituents at other positions decreased activity. The alkyl tail is not
key for activity although some bulkiness at position 9 is needed for good biological action. The
results indicate that size is more important than electronics in this position as seen with 9, 20, 21,
35, 36. This may indicate the activity is not linked to the ability of the phenazines to accept
electrons. However, a larger library and computational studies are needed to confirm this
observation. The result support the idea that electronegativity alone does not induce activity as
seen with the esters and nitrile examples. It seems that electronegative-ionizable groups are key
for the antibacterial activity of these molecules. It is worth to mention that the carboxylic acid
and the N-(methylsulfonyl)amide are expected to have similar pKa values (around 4.5 in water).

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2.3 Mechanism of action studies. Next, we decided to study the mechanism of action of the
most active compound, 33, and the natural product 5. Although the phenazines can exert their
biological effects by various mechanisms [13, 32-34] we focused in investigating membrane
disruption or interference with redox cascades. We decided to study their ability to disrupt
bacterial membranes. [18] because bacteria cannot (easily) alter their membrane composition.
Thus, if the phenazine damage the membrane, they could be antibiotics that can overcome
resistance. The other selection is based on the fact that interference with electron-flow and
production of reactive oxygen species (ROS) [35] is the most common mechanism of action of
the phenazines.
To assess whether our phenazines were disrupting the membrane, we monitored propidium
iodide (PI) uptake of 5 and 33. PI is a DNA intercalating agent that enters bacteria and binds to
the chromosomal DNA only when its membrane is damaged. As shown in figure 2, MRSA
treated with these compounds did not exhibit increased PI staining suggesting that the
compounds were not disrupting the membrane.[36] In agreement with these data, confocal
microscopy showed that compounds 5, and 33 did not induce PI uptake on the MRSA strain (see
SI Figure 1).
The next experiments sought to understand if interference with redox processes or generation
of ROS were responsible for the antibacterial action. [13] Thus, we studied the interference with
cellular redox cycling processes using an electron donor (NADPH) and an electron acceptor
(cytochrome c, cyt-c).[13] Our experiments indicated a concentration-dependence on the rate of
cyt-c reduction, a result that has been observed by the Bean and Bunz groups.[13, 18] However,
compound 5, which is less active than 33, shows a higher rate of reduction. A similar result is
observed with molecule 27 (see SI). It is assumed that cyt-c reduction occurs through a one-

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electron transfer process.[13] Therefore, we tested the generation of ROS using the radical
scavenger 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA).[13, 37] If radicals are
present, the absorbance of the ABDA molecule would shift from the visible to the UV spectrum.
The ABDA experiment did not indicate that radicals were generated. A single flow cytometry
experiment using the ROS indicator hydroxyphenyl fluorescein (HPF)[38] also suggested that
radicals were not generated (not shown). Thus, we concluded that even when the phenazines
interfere with the redox processes, this mechanism does not (exclusively) lead to bacterial killing
as observed by others.[13]
Given these results, we wondered whether our compounds had a static or cidal affect.
Although pyocyanin (1) and other phenazine compounds have been reported to be bactericidal
agents [11, 39], there have also been a few reports that indicate they could be bacteriostatic. [40,
41] Our results show that compounds 5 and 33 had a bacteriostatic effect on MRSA at 10X MIC
concentrations (Figure 4), whereas the vancomycin control reduced the number of viable bacteria
after 24 hrs (bactericidal compound). Thus, these phenazines are bacteriostatic in nature, in
agreement with the lack of PI staining and ROS generation.
Finally, we studied the toxicity of some of the active phenazines against HaCat cells
(immortal keratinocytes) to assess their potential as antibacterial drugs. The in vitro IC50 values
for the compounds were 306 mM (molecule 5), 345 mM (molecule 9), 118 mM (28) and 193
mM (33). The obtained IC50 values coupled with the MIC results indicate that phenazines 5, 9,
and 33 are promising leads for the development of MRSA drugs.
2.4 QSAR models of synthesized compounds. In order to develop a predictive model that can
be used to guide future phenazine optimization we calculated physicochemical parameters, and

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the linear analyses of the structures and their antimicrobial activities (Table 2). Three
physicochemical parameters were included in each QSAR model. For the quantum chemical
descriptors, such as the EHOMO and ELUMO energies, ꢀE (the difference between ELUMO and
EHOMO orbital energy), the dipole moments were first evaluated individually against ln (MIC).
The results showed that only the EHOMO energies were linearly correlated to the ln (MIC). Thus
only the EHOMO energies (eV) in the QM parameter set were included in the further QSAR
studies.
The first QSAR model was based on the physicochemical parameters SMR (molecular
refractivity, a description of the volume, or size of a compound), log S (solubility), and EHOMO
orbital energies (eV) following equation 1.
ln (MIC) = 30.80 - 0.37 * SMR + 4.09 * EHOMO (ev) - 0.35 * logS
(Equation 1)
N = 16, R2 = 0.63, Pearson’s Correlation R = 0.79, MAE= 0.65, RMSE = 0.71
Equation 1 suggested that the activities (ln (MIC)) was positively correlated with the
increase of EHOMO energies and negatively correlated with the increase of the logS and SMR,
indicating that decreasing solubility and decreasing the size of molecules while increasing the
EHOMO energies would likely enhance the activities. The R2 of 0.63 from the training set and the
Q2 of 0.40 from the test set, along with high Pearson’s correlation (0.79), and small mean
absolute error (0.65) and small root-mean-square (RMSE) error between the predicted and the
observed activities suggested that predictability of model 1 (Figure 5).

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We also used logP (a measurement of hydrophobicity), TPSA (topological polar surface
area, a measurement of polarity), and the EHOMO energies (eV) to develop a second QSAR model
(model 2, Fig. 1, right).
ln (MIC) = 25.97 + 3.31 * EHOMO (ev) - 0.03 * TPSA + 0.17 * logP
(Equation 2)
N = 16, R2 = 0.68, Pearson’s Correlation R = 0.82, MAE= 0.60, RMSE = 0.67
Equation 2 suggested that the activities (ln (MIC)) were positively correlated with the
increase of EHOMO energies and the hydrophobicity (logP), but negatively correlated with the
increase of the TPSA, indicating that decreasing hydrophilicity of molecules while increasing the
HOMO energies and increasing the hydrophobicity of the molecules would likely lead to more
potent compounds. The R2 of 0.68 from the training set and the Q2 of 0.73 from the test set,
along with high Pearson’s correlation (0.82), and small mean absolute error and small root-
mean-square error (Table 3) between the predicted and the observed.
Though eq. 1 and eq. 2 used different sets of physicochemical parameters, the models
supported each other. Table 4 showed that in both model 1 and 2, the Pearson’s correlation
(Pearson R) is very high for both training set, even for the test set, the Pearson R were pretty
high, suggested strong linear relationship between the predicted activities and the observed
activities. The mean absolute error and the root-mean-square errors in both models, both in
training sets and in test sets were small, less than one unit of ln (MIC). Therefore, both models
are predictive and thus can be used for ligand optimization. The QSAR study predicts that
compounds with decreased hydrophobicity and EHOMO energies, and increased water solubility
may show better antibacterial activity.

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3.Conclusions and perspectives
We are presenting, to the best of our knowledge, the first synthesis of a phenazine natural
product endophenzine G (5) using a simple route that can be applied to the preparation of other
endophenazines.[11] We have also identified two promising lead compounds containing an N-
(methylsulfonyl)amide substituent with MICs comparable to vancomycin. Even when 7
phenazine-containing sulfonylamides are listed in Scifinder,[42] their preparation and biological
activity has not yet been reported. Thus, this paper indicates the promising potential of
phenazines containing this group. No membrane disruption was observed. Based in our
experiments, it seems the phenazines interfere with redox cascades but those results do not
correlate with the antibacterial activity. Our studies indicate the active phenazines are
bacteriostatic. We also propose two simple QSAR models that can be used for the development
of new phenazine antibacterials. Further directions will seek to increase the library for in-depth
structure-activity-relationships and validation of the QSAR models, and further in-depth
mechanistic studies.
4.Experimental Section
4.1. Chemistry
4.1.1. General Methods.
All reagents and solvents were obtained from commercial sources (Sigma-Aldrich, VWR or
1
Fisher) and used without further purification. H NMR spectra were recorded on a Bruker 500
MHz spectrometer. 1H NMR chemical shifts are reported in ppm with the solvent as the internal
13
standard (CHCl3, 7.26 ppm). C NMR chemical shifts are reported in ppm with the solvent as

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the internal standard (DMSO-d6, 77 ppm). Compounds were analyzed for purity by HPLC.
Purities of synthesized compounds were all found to be >95% by the following HPLC method:
Agilent 1100 HPLC with VWD detectors, C18 column; Eluent: 20% water in methanol over 20
min; Flow rate 1.0 mL/min; Column temp. 25 °C; λ 254 nm. The Mass spectrometry (MS) was
carried out on a Micromass Q-Tof Mass Spectrometer with an electrospray ionization source.
Microscopy and flow cytometry were performed on a CARLZEISS LSM 710, Jena (Germany)
and a BD LSR II. The melting points were measured with a EZ-Melt automated melting point
apparatus from Stanford Research Systems.
4.1.2. Synthetic procedures:
4.1.3. (E)-9-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carboxylic acid (5)
To a solution of compound 12 (50 mg, 0.16 mmol) in THF-methanol (1:1, 10 mL) was
added sodium hydroxide (25 mg, 0.62 mmol) dissolved in water (5 mL). The reaction mixture
was heated to 40o C for 2 hr, the solvent was concentrated to half its volume, and acidified to pH
2 with hydrochloric acid (1 N), the precipitate was filtered, and dried under vacuum to afford
o
compound 5 as yellow solid (43 mg, 88%); mp: 151-153 C. IR (KBr) 3448, 2953, 2917, 1695,
1155, 751 cm-1; 1H NMR (500 MHz, CDCl3): δ 15.75 (s, 1 H), 8.98 (d, J = 6.5 Hz, 1 H), 8.54 (d,
J = 7.5 Hz, 1 H), 8.26 (d, J = 8.5 Hz, 1 H), 8.09-8.04 (m, 2 H), 7.96 (t, J = 8 Hz, 1 H), 7.58 (d, J
= 16 Hz, 1 H), 6.66 (d, J = 16 Hz, 1H), 1.58 (s, 6 H); 13C NMR (125 MHz, CDCl3): 165.9, 144.1,
144.0, 143.1, 139.0, 138.0, 137.3, 135.6, 134.9, 131.7, 130.4, 129.1, 128.8, 124.9, 120.4, 71.3,
29.7; HRESIMS calcd for C18H16N2O3 309.1234 (MH+), found 309.0418 (MH+). Anal. calcd for
C18H16N2O3. 0.5 HCl: C, 66.20; H, 5.09; N, 8.58. Found: C, 66.15; H, 5.24; N, 8.31.
4.1.4. 2-((2-Bromophenyl)amino)-3-nitrobenzoic acid (8)

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To a solution of 2-bromo-3-nitrobenzoic acid (6, 2.0 g, 8.1 mmol) and 2-bromoaniline (7,
2.1g, 12 mmol) in ethanol (100 mL) was added copper powder (26 mg, 0.4 mmol), copper (I)
bromide (117 mg, 0.81 mmol) and N-ethyl morpholine (2.0 mL, 16 mmol). The reaction mixture
was heated to reflux for 16 hr, diluted with aqueous ammonium hydroxide solution (0.1 N, 25
mL) and filtered over celite. The filtrate was acidified to pH 2 using hydrochloric acid (1 N), and
the solvent removed by filtration. The obtained yellow precipitate was dried under vacuum to
afford 2-((2-bromophenyl)amino)-3-nitrobenzoic acid 8 as yellow solid (1.6 g, 60 %, yield).
Which was used for the next step without further purification.
4.1.5. 9-Bromophenazine-1-carboxylic acid (9)
To a solution of 2-((2-bromophenyl)amino)-3-nitrobenzoic acid 8 (1.6 g, 4.7 mmol) in
aqueous sodium hydroxide (2 N, 100 mL) was added sodium borohydride (718 mmol, 19.0
o
mmol). The reaction mixture was heated to 100 C for 16 hr, cooled to room temperature, and
acidified to pH 2 using hydrochloric acid (1 N), the solid precipitate was filtered, washed with
water (2 X 20 mL), and dried the under vacuum to afford 9-bromophenazine-1-carboxylic acid 9
o
as yellow solid (800 mg, 56%); mp: 314-316 C. IR (KBr) 3034, 2905, 1716, 1503, 1428, 1245,
1206, 927 cm-1; 1H NMR (500 MHz, CDCl3): δ 9.08 (d, J = 7,1 Hz, 1 H), 8.60 (dd, J1 = 9.0 Hz
J2 = 1.5 Hz, 1 H), 8.37 (dd, J1 = 8.5, J2 = 7.5 Hz, 2H), 8.13 (t, J = 7.5 Hz, 1 H), 7.88 (t, J = 7.5
13
Hz, 1 H); C NMR (125 MHz, CDCl3): 165.5, 144.4, 143.8, 140.1, 138.2, 137.8, 135.7, 134.7,
131.8, 131.2, 129.8, 125.2, 122.6; HRESIMS calcd for C13H7N2O2Br 302.9764 (MH+), found
302.9136 (MH+).
4.1.6. Methyl 9-Bromophenazine-1-carboxylate (10)
To a solution of compound 9 (800 mg, 2.62 mmol) in dimethylformamide (5 mL) was
added potassium carbonate (1.8 g, 13 mmol) followed by methyl iodide (822 µL, 13.2 mmol).

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The reaction mixture was stirred under nitrogen atmosphere at room temperature for 24 hr. The
solvent was removed under vacuum, the obtained residue was diluted with dichloromethane (20
mL), the solid filtered off, the liquid filtrate collected. The solvent was removed under vacuum
and the obtained residue purified by silica gel column chromatography using ethyl acetate-
hexane (1:10 to 10:1) to afford methyl 9-bromophenazine-1-carboxylate 10 as yellow solid (590
o
mg, 1.83 mmol, 71 %); mp: 119-121 C. IR (KBr) 2929, 2831, 1716, 1507, 1417, 1372, 1262,
1
1209, 1017 cm-1; H NMR (500 MHz, CDCl3): 8.40 (d, J = 7.5 Hz, 1 H), 8.33 (d, J = 7.0 Hz, 1
H), 8.25 (t, J = 9.5 Hz, 2 H), 7.94 (t, J = 7.0 Hz, 1 H), 7.75 (t, J = 7.0 Hz, 1 H), 4.17 (s, 3H); 13C
NMR (125 MHz, CDCl3): 167.2, 143.7, 143.0, 141.0, 140.9, 134.0, 133.0, 132.8, 131.9, 131.2,
130.0, 129.4, 125.3, 52.8; HRESIMS calcd for C14H9N2O2Br 316.9120 (MH+), found 316.9113
(MH+).
4.1.7. Methyl (E)-9-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carboxylate (12)
A solution compound 10 (100 mg, 0.315 mmol) in dimethylformamide (15 mL) was
added to a thick-wall tube and the solution purge with nitrogen gas for 5 min. Then, in a
sequential order were added palladium acetate (3.54 mg, 15,7 µmol), triphenyl phosphine (8.31
mg, 31,5 µmol), potassium carbonate (87.2 mg, 0.63 mmol) and a catalytic amount of
tetrabutylammonium bromide. Nitrogen gas was bubbled into the solution for 5 minutes,
followed by addition of 3-methyl-2-buten-2-ol (11, 66 µL, 0.63 mmol). The tube was sealed with
o
a teflon cap and the reaction mixture heated to 110 C for 16 hr. The solvent was removed, the
residue was diluted with dichloromethane (20 mL), filtered through a celite pad, the liquid
filtrated collected, the solvent removed under vacuum, and the crude product was purified by
silica gel column chromatography (ethyl actetate1hexane, 1:1) to afford compound 12 as yellow
o
solid (81 mg, 78%); mp: 142-144 C. IR (KBr) 3476, 2949, 1658, 1523, 1417, 1278, 1119, 980,

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751 cm-1; 1H NMR (500 MHz, CDCl3): 8.38 (d, J = 8.5 Hz, 1 H), 8.28 (d, J = 7.2 Hz, 1 H), 8.13
(d, J = 8.5 Hz, 1 H), 7.97 (d, J = 6.5 Hz, 1 H), 7.89-7.83 (m, 3 H), 7.10 (d, J = 16.5 Hz, 1 H),
4.14 (s, 3H), 1.59 (s, 6H); 13C NMR (125 MHz, CDCl3): 167.5, 143.4, 142.4, 141.7, 141.7,
139.9, 136.3, 133.2, 132.2, 131.7, 131.3, 129.3, 128.3, 127.0, 121.7, 71.5, 52.7, 29.8; HRESIMS
calcd for C19H18N2O3 323.3715 (MH+), found 323.3656 (MH+).
4.1.8. 9-(3-Hydroxy-3-methylbutyl)phenazine-1-carboxylic acid (13)
Compound 5 (45 mg, 0.14 mmol) was dissolved in ethanol (5 mL) and reduced by
catalytic hydrogenation (1 atm H2 prssure) using 10% palladium on carbon to afford the title
compound. The catalyst was filtered off over a celite pad, the celite was washed with
dichloromethane, and the solvent was removed under vacuum. Compound 13 was isolated as a
o
yellow solid (30 mg, 70%), mp: 258-260 C. IR (KBr) 3469, 2953, 2921, 1699, 1458, 1254,
1193, 1127, 874, 759 cm-1; 1H NMR (500 MHz, CDCl3): δ 9.01 (d, J = 7.0 Hz, 1 H), 8.57 (d, J =
8.5 Hz, 1 H), 8.24 (d, J = 8.5 Hz, 1 H), 8.07 (t, J = 7.0 Hz, 1 H), 7.95-7.90 (m, 2 H), 3.52-3.49
13
(m, 2 H), 2.05-2.02 (m, 2 H), 1.45 (s, 6 H); C NMR (125 MHz, CDCl3): δ 166.0, 144.6, 143.0,
140.4, 139.1, 139.0, 137.2, 135.0, 132.2, 131.9, 130.2, 128.2, 124.8, 70.8, 44.5, 29.5, 26.8;
HRESIMS calcd for C18H18N2O3 311.1390 (MH+), found 311.0725 (MH+).
4.1.9. 6-Bromophenazine-1-carboxylic acid (18)
To a solution of 2-bromo-3-nitrobenzoic acid (6, 1.01 g, 4.06 mmol) and 3-bromoaniline
(14, 1.05 g, 6.10 mmol) in ethanol (50 mL) was added copper powder (13.0 mg, 0.20 mmol),
copper (I) bromide (58.3 mg, 0.41 mmol) and N-ethyl morpholine (1.00 mL, 8.12 mmol). The
reaction mixture was heated at reflux for 16 hr, diluted with aqueous ammonium hydroxide (0.1
N, 12 mL) and filtered over celite. The filtrate was acidified to pH 2 using hydrochloric acid (1
N) and the solvent concentrated to half. The formed yellow precipitate was filtered, and dried

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under vacuum to afford 2-((3-bromophenyl)amino)-3-nitrobenzoic acid as yellow solid (800 mg,
60%), which was used without further purification.. To a solution of 2-((3-bromophenyl)amino)-
3-nitrobenzoic acid (800 mg, 2.37 mmol) in aqueous sodium hydroxide (2 N, 50 mL) was added
sodium borohydride (359 mg, 9.50 mmol), and the reaction mixture was heated to 100 o C for 12
hr. The reaction mixture was cooled to room temperature, and acidified to pH 2 using
hydrochloric acid (1 N). The formed precipitated was filtered, washed with water (2 X 20 mL)
and dried under vacuum to afford 6-bromophenazine-1-carboxylic acid, 18, as yellow solid (450
mg, 62%); mp: 288-290 oC. IR (KBr) 1711, 1544, 1462, 1254, 996, 987, 751 cm-1; 1H NMR (500
MHz, DMSO-d6): 14.18 (s, 1 H), 8.56 (d, J = 8.5 Hz, 1 H), 8.52 (d, J = 7.0 Hz, 1 H), 8.46 (d, J =
13
7.0 Hz, 1 H), 8.41 (d, J = 8.5 Hz, 1 H), 8.14 (t, J = 8.5 Hz, 1 H), 7.97 (t, J = 8.5 Hz, 1 H); C
NMR (125 MHz, DMSO-d6): 166, 148, 146, 143, 142, 141, 137, 136, 135, 134, 132, 130, 123;
HRESIMS calcd for C13H7BrN2O2 304.9743 (MH+), found 304.9072 (MH+).
Compounds 19,[15] 20,[25] and 21[15] were prepared following a similar procedure to the one
used for 17. Their spectroscopic data matched the literature reported values.
4.1.10. Methyl 6-Bromophenazine-1-carboxylate (22)
To a solution of compound 18 (400 mg, 1.3 mmol) in dimethylformamide (5 mL) was
added potassium carbonate (915 mg, 6.61 mmol) followed by methyl iodide (411 µL, 6.63
mmol) and the reaction mixture was stirred under a nitrogen atmosphere for 24 hr at room
temperature. The solvent was removed under vacuum, the residue diluted with dichloromethane,
the solid filtered off, and the filtrate collected. The solvent was removed under vacuum and the
compound purified the product by silica gel column chromatography (dichloromethane) to afford
o
methyl 6-bromophenazine-1-carboxylate 22 as a yellow solid (310 mg, 74%), mp: 314-315 C.
IR (KBr) 1714, 1258, 1213, 1123, 1024, 927, 845, 743 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.55

ACCEPTED MANUSCRIPT
(d, J = 9.0 Hz, 1 H), 8.34 (d, J = 9.0 Hz, 1 H) 8.33 (d, J =3.0 Hz, 1 H), 8.24 (d, J = 7 Hz, 1 H),
13
7.93 (t, J = 8.5 Hz, 1 H), 7.75 (t, J = 8.0 Hz, 1H ), 4.14 (s, 3 H); C NMR (125 MHz, CDCl3):
165.4, 144.4, 143.7, 140.1, 138.1, 137.7, 135.7, 134.7, 131.7, 131.2, 129.7, 125.2, 122.6;
HRESIMS calcd for C14H9BrN2O2 318.9900 (MH+), found 318.9417 (MH+).
4.1.11. Methyl 9-Methylphenazine-1-carboxylate (24):
To a solution of compound 20 (70 mg, 0.29 mmol) in DMF (10 mL) was added methyl
iodide (93 µL, 1.5 mmol), followed by K2CO3 (285 mg, 2.05 mmol), and the reaction mixture
was stirred at room temperature for 24 hr under nitrogen. After that, the solvent was removed
under vacuum, the residue was dissolved in dichloromethane (25 mL), the solids were filtered
off, the filtrate was collected, the solvent removed under vacuum, and the crude product was
purified by silica gel column chromatography (dichlorometane) to obtain compound 24 as yellow
solid (62 mg, 84%); mp: 214-216 oC. The spectroscopic data of compound 24 matched with the
literature reported values.[43, 44] IR (KBr) 2909, 1699, 1520, 1421, 1270, 1184, 1066, 1013,743
cm-1; 1H NMR (500 MHz, CDCl3): δ 8.38 (dd, J 1= 8.5 Hz, J 2 = 1.0 Hz, 1 H), 8.20 (dd, J 1 = 7.0
Hz, J 2 = 1.0 Hz, 1 H), 8.06 (d, J = 8.5 Hz, 1 H), 7.85 (t, J = 7.0 Hz, 1 H), 7.76
(t, J = 6.5 Hz,
1 H), 7.69 (bd, 1 H), 4.11 (s, 3 H), 2.92 (s, 3 H).
4.1.12. Methyl (E)-6-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carboxylate (25)
Compound 22 (100 mg, 0.32 mmol) was dissolved dimethylformamide (10 mL) and
added to a high-pressure (thick wall) tube. The solution was purged with nitrogen for 5 min, and
then were sequentially added: palladium acetate (3.54 mg, 0.016 mmol), triphenyl phosphine
(8.33 mg, 0.032 mmol), potassium carbonate (87.2 mg, 0.630 mmol) and a catalytic amount of
tetrabutylammonium bromide. Nitrogen gas was bubbled into the solution for another 5 minutes,
followed by the addition of 3-methyl-2-buten-2-ol (11, 66 µL, 0.63 mmol). The test tube was

ACCEPTED MANUSCRIPT
o
sealed with a teflon cap and the reaction mixture heated at 110 C for 16 hr. The solvent was
removed under vacuum, the residue diluted with dichloromethane (10 mL) and filtered through
celite pad, the filtrated collected, the celite was washed with dichloromethane (10 mL), the
solvent removed under vacuum, and the crude product purified by silica gel column
chromatography (ethyl acetate-hexane, 1-3) to afford compound 25 as yellow solid (81 mg,
o
1
78%); mp: 117-119 C. IR (KBr) 3346, 2958, 1711, 1523, 1442, 1262, 1041, 804, 755 cm-1; H
NMR (500 MHz, CDCl3): δ 8.44 (d, J = 8.5 Hz, 1 H), 8.24 (dd, J1 = 7.0 Hz, J2 = 4.5 Hz, 2 H),
8.00 (d, J = 8.0 Hz, 1 H), 7.96 (d, J = 17 Hz, 1 H), 7.84 (t, J = 7 Hz, 2 H), 6.81 (d, J = 16.5 Hz, 1
H), 4.13 (s, 3 H), 1.58 (s, 6 H); 13C NMR (125 MHz, CDCl3): δ 167.1, 143.7, 142.0, 141.5,
140.8, 140.8, 136.0, 134.0, 132.2, 131.2, 130.9, 129.4, 128.7, 126.2, 121.3, 71.5, 52.7, 29.9;
HRESIMS calcd for C19H18N2O2 323.1390 (MH+), found 323.0830 (MH+).
4.1.13. (E)-6-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carboxylic acid (26)
To a solution of compound 25 (50 mg, 0.155 mmol) in THF-methanol (1:1, 10 mL) was
added a solution of sodium hydroxide (25 mg, 0.62 mmol) in water (5 mL) and the reaction
o
mixture was heated at 40 C for 2 hr. The solvent was concentrated to half its volume and the
solution acidified with hydrochloric acid (1 N) to pH 2. The obtained precipitate was filtered and
o
dried under vacuum to afford compound 26 as a yellow solid (40 mg, 83%); mp: 146-148 C. IR
1
(KBr) 3484, 2986, 1695, 1401, 1270, 1148, 972, 902,759 cm-1; H NMR (500 MHz, CDCl3): δ
8.98 (d, J = 6.0 Hz, 1 H), 8.58 (d, J = 9.0 Hz, 1 H), 8.15 (d, J = 8.5 Hz, 1 H), 8.12 (d, J = 7.0 Hz,
1 H), 8.05-7.98 (m, 3 H), 6.86 (d, J = 16.5 Hz, 1 H), 1.60 (s, 6H); 13C NMR (125 MHz, CDCl3):
δ 166.0, 142.5, 142.1, 142.0, 139.9, 139.8, 137.4, 136.9, 135.5, 133.2, 130.0, 126.7, 126.6, 124.8,
120.6, 71.6, 30.0; HRESIMS calcd for C18H16N2O3 309.1194 (MH+), found 308.9907 (MH+).
4.1.14. 6-Bromo-N,N-Dimethylphenazine-1-carboxamide

ACCEPTED MANUSCRIPT
To a solution of compound 18 (100 mg, 0.32 mmol) in dichloromethane (10 mL) was
added a catalytic amount of dimethylformamide (2 drops), the reaction mixture was cooled to 0 o
o
C, and oxalyl chloride (110 µL, 1.29 mmol) was added dropwise at 0 C under nitrogen
atmosphere. After completion of the addition, the reaction mixture was stirred at room
temperature for 16 hr, the volatiles were removed under vacuum, the acid chloride intermediate
was dissolved in dry THF (5 mL), the reaction mixture cooled to 0 o C, and diethyl amine in THF
(2 M, 1 mL) was added. The reaction mixture was stirred at room temperature for 6 hr, the
volatiles were removed under vacuum, and the crude product was used for next step without
further purification to prepare compound 27.
4.1.15.(E)-6-(3-Hydroxy-3-methylbut-1-en-1-yl)-N,N-dimethylphenazine-1-carboxamide
(27)
6-Bromo-N,N-Dimethylphenazine-1-carboxamide (45 mg, 0.136 mmol) was dissolved in
dimethylformamide (2 mL) and the solution transferred to a thick wall tube. To this solution
were sequentially added; palladium acetate (1.5 mg, 6.8 µmol), triphenyl phosphine (3.6 mg, 14
µmol), potassium carbonate (37.6 mg, 0.272 mmol) and a catalytic amount of
tetrabutylammonium bromide. Nitrogen gas was bubbled into the solution for 5 minutes, then 3-
methyl-2-buten-2-ol 6 (28 µL, 0.272 mmol) was added and the tube sealed with a teflon cap.
o
The reaction mixture was heated to 110 C for 16 hr, the solvent removed, the residue diluted
with dichloromethane (10 mL), filtered through celite pad, and the celite was washed with
dichloromethae (10 mL). The filtrated was collected, the solvent removed under vacuum, and the
crude product was purified by preparative TLC (dichloromethane) to afford compound 27 as
o
1
yellow solid (32 mg, 70%); mp: 199-201 C. IR (KBr) 1703, 1352, 1206, 519 cm-1; H NMR
(500 MHz, CDCl3): δ 8.31 (t, J = 5.0 Hz, 1 H), 8.14 (d, J = 8.5 Hz, 1 H), 7.96 (d, J = 16.5 Hz, 1

ACCEPTED MANUSCRIPT
H), 7.93 (d, J = 7 Hz, 1 H), 7.83 (d, J = 5.0 Hz, 2H ), 7.78 (t, J = 7.5 Hz, 1 H), 6.78 (d, J = 16.5
Hz, 1 H), 3.35 (s, 3 H), 2.81 (s, 3 H), 1.57 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 169.4, 143.6,
142.0, 141.7, 140.8, 140.1, 137.2, 136.1, 131.1, 130.5, 129.6, 129.2, 128.3, 126.0, 121.3, 71.5,
38.8, 35.0, 30.0; HRESIMS calcd for C20H21N3O2 366.1707 (MH+) 358.1526 (MNa+), found
336.0933 (MH+) 358.0727 (MNa+).
4.1.16. General Procedure for the preparation Methanesulfonamide phenazines 28 and 33.
To a solution of the desired acid (0.13 mmol) in dimethylformamide (3 mL) was added carbonyl
o
diimidazole (CDI, 26 mg, 0.16 mmol) and the reaction mixture was heated at 60 C for 2 hr
under nitrogen atmosphere. The reaction mixture was cooled to room temperature, methane
sulfonamide (126 mg, 1.32 mmol) was added, followed by the addition of triethylamine (220 µL,
1.58 mmol) and a catalytic amount of dimethylaminopyridine (DMAP). After completion of the
addition, the reaction mixture was stirred overnight at room temperature under nitrogen
atmosphere. The solvent was removed under vacuum, and the residue was acidified using
hydrochloric acid (1 N). The yellow precipitated was filtered and dried under vacuum. The crude
product was purified by preparative TLC, using dichloromethane as mobile phase, to obtain the
products as yellow solids.
4.1.16.A. 9-Bromo-N-(methylsulfonyl)phenazine-1-carboxamide (28). Yellow solid (21.1 mg,
o
43% yield); mp: 165-167 C. IR (KBr) 2917, 1676, 1429, 1332, 1258, 1160, 751 cm-1; 1H NMR
(500 MHz, CDCl3): δ 13.97 (s, 1 H), 9.10 (q, J1 = 7.5 Hz, J2 =1.5 Hz,, 1 H), 8.59 (q, J1 = 9.0 Hz,
J2 =1.5 Hz, 1 H) 8.33 (m, 2 H), 8.11 (dd, J1 = 9.0 Hz, J1 =1.5 Hz, 1 H), 7.85 (dd, J = 7.5 Hz, 1
H), 3.56 (s, 3 H); 13C NMR (125 MHz, CDCl3): δ 163.3, 144.1, 143.7, 139.7, 138.8, 137.6,
135.6, 135.4, 131.8, 130.7, 129.5, 126.7, 123.7; HRESIMS calcd for C14H10BrN3O3S 379.9699
(MH+), found 379.8847 (MH+).

ACCEPTED MANUSCRIPT
4.1.16.B 9-Chloro-N-(methylsulfonyl)phenazine-1-carboxamide (33). See general procedure
for the synthesis of 28 and 33. Yellow crystals (20 mg, 46% yield); mp: 193-195 oC. IR (KBr)
2923, 2840, 1675, 1428, 1110, 760 cm-1; 1H NMR (500 MHz, CDCl3): δ = 14.12 (s, 1 H), 9.05
(d, J = 6.5 Hz, 1 H), 8.54 (d, J = 7.5 Hz, 1 H), 8.24 (d, J = 9.0 Hz, 1 H), 8.06 – 8.09 (m, 2 H),
7.87-7.90 (dd, J1 = 7.5 Hz, J2 =1.5 Hz, 1 H), 3.52 (s, 1 H); 13C NMR (125 MHz, CDCl3): δ
163.2, 144.1, 143.5, 139.3, 137.7, 137.5, 135.6, 132.8, 131.7, 131.3, 130.8, 128.7, 126.6;
HRESIMS calcd for C14H10ClN3O3S 334.7545 (MH-), found 334.7024 (MH-).
4.1.17. 9-Bromophenazine-1-carbonitrile (29)
To a solution of compound 9 (100 mg, 0.321 mmol) in dichloromethane (10 mL) was
added a catalytic amount of dimethylformamide (2 drops). The reaction mixture was cooled to 0
o C, and oxalyl chloride (110 µL, 1.29 mmol) was added dropwise under nitrogen atmosphere.
After completion of the addition, the reaction mixture was stirred at room temperature for 16 hr,
and the volatiles were removed under vacuum. The obtained acid chloride was dissolved in dry
o
dichloromethane (5 mL), the reaction mixture was cooled to 0 C, aqueous ammonia (30%, 0.5
mL) was added dropwise, and the reaction mixture stirred for 4 hr. The solvent was removed
under vacuum, the residue diluted with water (10 mL), the solid filtered, and dried under vacuum
to afford 9-bromophenazine-1-carboxamide as brownish yellow solid. Due to the difficulty of
purification of the compound, we proceed to the next step. To the crude compound from the
previous step (80 mg), was added thionyl chloride (10 mL), and the reaction mixture was heated
to reflux for 12 hr. The thionyl chloride was removed under vacuum, and the crude nitrile
compound was purified by silica gel column chromatography (ethyl acetate-hexane, 1:10 to 1:1)
to afford the title compound as a yellow solid (41 mg, 53% yield over 2 steps); mp: < 400 oC. IR
(KBr) 2222, 1508, 1487, 1413, 1277, 1119, 759 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.52 (d, J

ACCEPTED MANUSCRIPT
= 8.5 Hz, 1 H), 8.37 (d, J = 7 Hz, 1 H), 8.30 (d, J = 7 Hz, 1 H), 8.26 (d, J = 8.5 Hz, 1 H), 7.97 (t,
13
J = 7.0 Hz, 1 H), 7.81 (t, J = 8.5 Hz, 1 H); C NMR (125 MHz, CDCl3): 144.7, 142.6, 141.9,
141.3, 137.7, 135.1, 134.6, 131.9, 129.9, 129.5, 125.0, 115.8, 114.0; HRESIMS calcd for
C13H6BrN3 284.9725 (MH+), found 285.0769 (MH+).
4.1.18. 9-Bromo-N,N-dimethylphenazine-1-carboxamide (30)
To a solution of compound 9 (100 mg, 0.32 mmol) in dichloromethane (10 mL) was
added a catalytic amount of dimethylformamide (2 drops), the reaction mixture was cooled to 0 o
o
C, and oxalyl chloride (110 µL, 1.29 mmol) was added dropwise at 0 C under nitrogen
atmosphere. After completion of the addition, the reaction mixture was stirred at room
temperature for 16 hr, the volatiles were removed under vacuum, the acid chloride intermediate
o
was dissolved in dry THF (5 mL). The reaction mixture was cooled to 0 C, and diethyl amine
in THF (2 M, 1 mL) was slowly added. The reaction mixture was stirred at room temperature for
6 hr, the volatiles were removed under vacuum, and the crude product was purified by silica gel
column chromatography (dichloromethane) to afford compound 30 as yellow solid (81 mg,
o
74%); mp: 148-150 C. IR (KBr) 1617, 1274, 755 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.31 (d,
J = 8.5 Hz, 1 H), 8.23 (d, J = 9.0 Hz, 1 H), 8.19 (d, J = 7 Hz, 1 H), 7.98 (d, J = 6.5 Hz, 1 H), 7.94
(t, J = 7.5 Hz, 1 H), 7.72 (t, J = 7.5 Hz, 1 H), 3.38 (s, 3 H), 2.90 (s, 3 H); 13C NMR (125 MHz,
CDCl3): δ 168.8, 143.9, 142.9, 140.6, 140.0, 137.8, 133.8, 131.1, 131.0, 130.3, 129.6, 129.5,
125.0, 39.2, 35.2; HRESIMS calcd for C15H12BrN3O 332.0216 (MH+), found 331.9575 (MH+).
4.1.19. (E)-9-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carbonitrile (31)
To a solution compound 29 (45 mg, 0.16 mmol) in dimethylformamide (5 mL) placed in
a test tube were sequentially added: palladium acetate (1.78 mg, 8 µmol), triphenyl phosphine
(4.2 mg, 16 µmol), potassium carbonate (44 mg, 32 µmol) and a catalytic amount of

ACCEPTED MANUSCRIPT
tetrabutylammonium bromide. Nitrogen gas was bubbled into the solution for 5 minutes,
compound 29 was added, the test tube sealed with a teflon cap, and the reaction mixture heated
o
to 110 C for 16 hr. The solvent was removed, the residue diluted with dichloromethane (20
mL), the mixture filtered through celite pad, the pat was washed with dichloromethane (10 mL),
the filtrated collected, and the solvent removed under vacuum. The crude product was purified
by silica gel column chromatography to afford compound 31 as yellow solid (19 mg, 42%); mp:
152-154 oC. IR (KBr) 2970, 2905, 2210, 1515, 759 cm-1; 1H NMR (500 MHz, CDCl3): δ 8.51 (d,
J = 8.5 Hz, 1 H), 8.32 (d, J = 7.0 Hz, 1 H), 8.18 (d, J = 8.5 Hz, 1 H), 8.04 (d, J = 6.5 Hz, 1 H),
7.95-7.91 (m, 2 H), 7.79 (d, J = 16.5 Hz, 1 H), 7.35 (d, J = 16 Hz, 1 H), 1.60 (s, 6 H); 13C NMR
(125 MHz, CDCl3): 144.5, 143.3, 142.1, 141.9, 141.1, 136.7, 136.3, 134.7, 132.0, 129.2, 128.6,
128.3, 121.5, 116.3, 113.9, 71.6, 29.8; HRESIMS calcd for C18H15N3O 312.1107 (MNa+), found
312.0390 (MNa+).
4.1.20.(E)-9-(3-Hydroxy-3-methylbut-1-en-1-yl)-N,N-dimethylphenazine-1-carboxamide
(32)
To a solution compound 31 (55 mg, 0.166 mmol) in dimethylformamide taken in a test
tube were sequentially added palladium acetate (1.86 mg, 0.0083 mmol), triphenyl phosphine
(4.4 mg, 17 µmol), potassium carbonate (46 mg, 33 µmol) and a catalytic amount of
tetrabutylammonium bromide. Nitrogen gas was bubbled into the solution for 5 minutes, then
compound 6 (53 µL, 0.5 mmol) was added, the test tube sealed with a teflon cap, and the
o
reaction mixture heated to 110 C for 16 hr. The solvent was removed under vacuum, the
residue diluted with dichloromethane (10 mL), filtered through celite pad, the filtrated collected,
the solvent was removed under vacuum, and the crude product was purified by preparative TLC
o
(dichloromethane) to afford compound 32 as yellow solid (33 mg, 59%); mp: 149-151 C. IR

ACCEPTED MANUSCRIPT
1
(KBr) 3344, 2917, 1609, 1409, 1257, 1151, 910, 766 cm-1; H NMR (500 MHz, CDCl3): δ 8.23
(d, J = 8 Hz, 1 H), 8.11 (d, J = 9.0 Hz, 1 H), 7.92-7.88 (m, 4H), 7.80 (d, J = 15.5 Hz, 1 H), 6.80
13
(d, J = 16 Hz, 1H ), 3.36 (s, 3 H), 2.83 (s, 3 H), 1.54 (s, 6 H); C NMR (125 MHz, CDCl3):
169.3, 143.6, 142.5, 141.4, 141.0, 139.1, 137.5, 136.2, 131.0, 130.4, 130.2, 128.6, 128.4, 126.0,
121.4, 71.3, 39.0, 35.1, 30.0, 29.7; HRESIMS calcd for C20H21N3O2 336.4145 (MH+) 358.1526
(MNa+), found 336.0933 (MH+) 358.0635 (MNa+).
4.1.21. 9-Fluorophenazine-1-carboxylic acid (35) [25]
To a solution of 2-bromo-3-nitrobenzoic acid (6, 1.01 g, 4.06 mmol) and 2,6-
difluoroaniline (34, 1.05 g, 6.10 mmol) in methanol (50 mL) was added copper powder (13.0
mg, 0.20 mmol), copper (I) bromide (58.3 mg, 0.41 mmol) and N-ethyl morpholine (1.00 mL,
8.12 mmol). The reaction mixture was heated at reflux for 16 hr, diluted with aqueous
ammonium hydroxide (0.1 N, 12 mL) and filtered over celite. The solvent concentrated to half its
volume under vacuum and then acidified to pH 2 using hydrochloric acid (1 N). The formed
yellow precipitate was filtered, and dried under vacuum to afford 2-((2,6-difluorophenyl)amino)-
3-nitrobenzoic acid as yellow solid (800 mg, 60%). To a solution of 2-((2,6-
difluorophenyl)amino)-3-nitrobenzoic acid (650 mg, 2.21 mmol) in aqueous sodium hydroxide
(2 N, 50 mL) was added sodium borohydride (336 mg, 8.84 mmol), and the reaction mixture was
heated to reflux for 1 hr. The reaction mixture was cooled to room temperature, and acidified to
pH 2 using hydrochloric acid (1 N). The formed precipitated was filtered, washed with water (2
X 20 mL) and dried under vacuum to afford 9-fluorophenazine-1-carboxylic acid 35, as yellow
o
solid (224 mg, 31%); mp: 233-235 C. IR (KBr) 3047, 1728, 1515, 1458, 1246, 894, 763 cm-1;
1H NMR (500 MHz, CDCl3): δ 15.17 (s, 1 H), 9.02 (d, J = 7.0 Hz, 1 H), 8.54 (d, J = 9.0 Hz, 1
H), 8.16 (d, J = 9.0 Hz, 1 H), 8.09 (t, J = 9 Hz, 1 H), 7.95-7.91 (m, 1 H), 7.69 (t, J = 8.5 Hz, 1 H);

ACCEPTED MANUSCRIPT
13C NMR (125 MHz, CDCl3): 165.4, 144.4, 143.8, 138.1, 135.0, 131.2, 130.8, 130.8, 125.9,
125.8, 125.3, 115.5, 115.3; 19F NMR (470 MHz, CDCl3): δ -123.7; HRESIMS calcd for
C13H7N2O2F 242.0492 (MH+), found 242.9999 (MH+)
4.1.22. 9-Fluorophenazine-1-carboxamide (36)
Compound 35 (121 mg, 0.50 mmol) was dissolved in dichloromethane (10 mL), the
o
reaction mixture was cooled to 0 C, and oxalyl chloride (110 µL, 1.29 mmol) was added
dropwise under nitrogen atmosphere. Then, a catalytic amount of dimethylformamide (2 drops)
was added. After completion of the addition, the reaction mixture was stirred at room
temperature for 16 hr, the volatiles were removed under vacuum. The obtained acid chloride
o
was dissolved in dichloromethane (5 mL), the reaction mixture cooled to 0 C, aqueous
ammonia (30%, 0.5 mL) was added dropwise, and the reaction mixture was stirred for 4 hr. The
solvent was removed under vacuum, and the compound purified by silica gel column
chromatography. The title compound was obtained as an orange solid (87 mg, 0.36 mmol, 72%
o
yield); mp: 270-272 C. IR (KBr) 3354, 1668, 1580, 1410, 1067, 755 cm-1; 1H NMR (500 MHz,
DMSO-d6): δ 9.68 (brs, 1 H), 8.73 (d, J = 7Hz, 1 H), 8.45 (d, J = 9Hz, 1 H), 8.18 (brs, 1 H), 8.13
13
(t, J = 7 Hz, 2 H), 8.02 (q, J = 7.5 Hz, 1 H), 7.89 (t, J = 9.5 Hz, 1 H); C NMR (125 MHz,
DMSO-d6): δ 165.8, 157.4, 155.3, 143.7, 143.6, 140.1, 135.3, 133.6, 133.1, 133.0, 131.7, 131.4,
131.3, 126.0, 115.1, 115.0; 19F NMR (470 MHz, CDCl3): δ -125.6 HRESIMS calcd for
C13H8FN3O 242.0724 (MH+) 264.0544 (MNa+), found 242.0189 (MH+) 263.9966 (MNa+).
Compounds 37- 39[15, 26] were prepared according to literature procedures and their
data was identical to published reports.
4.2. Biology
4.2.1. Minimum Inhibitory Concentration Assays

ACCEPTED MANUSCRIPT
Broth microdilution assays were performed as described in the third edition of the ASM
Clinical Microbiology Procedures Handbook. Briefly, a stock solution of the compound at pH 7
was prepared and then serial 2-fold dilutions were made in Muller Hinton II broth, cation-
adjusted, CAMHB, (Becton, Dickinson and Company, Sparks, MD), containing 5% DMSO in
Costar 96-well microtiter plates (Corning, Kennebunk, ME). Bacterial cultures were prepared
using the direct colony suspension method to 0.5 McFarland units and each well was inoculated
with 10 ul. Plates were incubated statically at 37˚C for 24 h. MIC values are reported as the
lowest concentration of antibiotic at which no growth was observed. Vancomycin was used as
positive control and media was used as negative control.
4.2.2. Killing Assays
Killing assays were performed by diluting an overnight culture 1:1000 into fresh
CAMHB. After two hours of growth at 37˚C with agitation, 1-ml aliquots were removed and
treated with 10X MIC of the compounds. Samples were removed every two hours and bacterial
viability was quantified by serial dilution and plating on Tryptic Soy Agar (TSA).
4.2.3. Membrane disruption experiments
Staphylococcus aureus JE2 was resuspended in PBS to an OD600 of 1.0 and then treated
with 1X, 2X or 10X MIC of compounds 5 and 33. 200 ul of each sample was placed into wells of
a black 96-well microtiter plate (Corning Inc, Corning, NY), and the relative fluorescent units
(RFU) was measured in 15 min intervals for 4 hr in a Tecan Infinite 200 PRO plate reader
(Tecan Trading AG, Switzerland).
4.2.4. Rate of reduction of cytochrome-c
This procedure was based on previous literature precedents[18] and modified accordingly
by addition of DMSO and 0.1 M NaOH to increase solubility of the tested compounds. A

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solution of DMSO and NaOH was prepared with 95% DMSO and 5% NaOH (0.1 M) by volume.
Each compound was then dissolved in the DMSO/NaOH solution to make a 10 mM stock
solution. A 200 µM solution of each compound was prepared by diluting the aforementioned
solution with Dulbecco's Phosphate-Buffered Saline (DPBS, 1 X). In order to maintain an overall
5% DMSO/NaOH to 95% DPBS ratio, the needed volume of DMSO/NaOH solution was added.
The solutions were then serially diluted (100 µM, 50 µM, 25 µM). To the dilutions containing,
which were placed in a 96-well plate, was added NADPH (100 µM) followed by with
cytochrome-c derived from equine heart (20 µM). The extinction of cyt-c (peak at 550nm) was
monitored over time using a photospectrometer for a period of 900 s at 30 s intervals. As a
reference for baseline correction, the extinction coefficient at 700 nm (a wavelength were neither
the phenazine or cyt-c absorbs) was also monitored in order to account for lap-signal
fluctuations.[18] Solutions containing only cytochrome-c (20 µM) in 95% DPBS-5%
DMSO/NaOH buffer, cyt-c (20 µM) and NAPDH (100 µM) in PBS/DMSO/NaOH buffer, and
95% DPBS-5% DMSO/NaOH were used as controls.
4.2.5. Superoxide scavenging activity
The generation of superoxides was monitored by using (9,10- Anthracenediyl-
bis(methylene)dimalonic acid, ABDA). ABDA has an absorbance shift upon reaction with
superoxides in solution. Therefore, the decay of the ABDA was measured at 479 nm and
monitored over the period of 500 s at 30 s intervals using a photospectrometer. We followed the
exact same protocol of Hayden and co-workers, “The extinction at 600 nm was also plotted and
used as a reference baseline to account for any fluctuations in the lamp signal, as neither the
phenazine compounds nor ABDA has any extinction at this wavelength. These measurements
were performed at 3 concentrations for each compound (0.11, 0.22, 0.33, 0.44, and 0.55 mM)

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with three repeats per concentration. ABDA (5 ꢁM) and NADPH (0.01 mM) concentrations were
maintained constant throughout”[18]. The only difference was in the sample preparation. A stock
solution of DMSO and NaOH was prepared with 95% DMSO and 5% NaOH (0.1 M) by volume.
Each compound was then dissolved in the DMSO/NaOH solution to make a 10 mM solution.
The solutions were serially diluted while re-adding the DMSO/NaOH solution after each serial
dilution to maintain a 5% DMSO/NaOH concentration. In a 96-well plate, solutions containing
the experimental compounds (0 mM, 0.11 mM, 0.22 mM, 0.33 mM, 0.44 mM, 0.55 mM) and
NADPH (100 µM) were injected with ABDA (5 µM, final concentration) and monitored for
ADBA signal extinction. Solutions containing ABDA (5 µM) in 95% DPBS-5% DMSO/NaOH
buffer and 95% DPBS-5% DMSO/NaOH were used as controls.
4.2.6. Cytotoxicity experiments
Selected phenazine samples (~1.3 mg) were dissolved in 100 µL of a 2:3 mixture of
NaOH (0.01 M) and DMSO. The samples were diluted with Dulbecco's Modified Eagle Medium
(DMEM) containing 10% FBS and 1% penicillin streptomycin to a final concentration of 4 mM
(3% final DMSO concentration). Then, an aliquot of this stock solution was taken and serial
dilutions performed using DMEM buffer. Cytotoxicity was then performed using the celltiter
blue assay[45] and following the providers’ protocols (promega).[46] Cell death was measured
by observing the change if absorbance of resazurin using a spectrophotometer. The results were
plotted as Absorbance vs phenazine concentration and the IC50 (concentration that kills 50% of
the cell population) calculated.
4.3. Computational Methods
To generate the physicochemical parameters, we built the 3D models of all compounds in
MOE. The physicochemical parameters such as logP, TPSA, SMR, and logS were calculated in

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MOE. The EHOMO and ELUMO energies (in eV) were calculated using Gaussian09 after each
compound was energetically minimized using the density functional theory (DFT) method
B3LYP at the 6-31G(d) level of theory. All minimized structures were further subject to
frequency calculations which showed no negative frequencies, an indication of a true minimum
being found. After all physicochemical parameters being assigned, all compounds were
randomly divided into a training set and a test set. The activity data were converted to ln (MIC).
Correlations between selected physicochemical parameters and the ln (MIC) were established
using the Partial Least Square Fit (PLS) method. To evaluate the QSAR models, the resulting
QSAR models were used to predict the activity data of the compounds in the test set.
ACKNOWLEDGEMENTS
This work was support by the University of Nebraska Medical Center (Start-up funds, MC-S)
and by NIH grant number PO1-AI083211 (KB). We thank Dr. David Oupicky for providing
resources and space to conduct the cell studies. We thank Janice A. Taylor and James R. Talaska
of the Advanced Microscopy Core Facility at the University of Nebraska Medical Center for
providing assistance with confocal microscopy. Data reported in this manuscript was supported
by an Institutional Development Award (IDeA) from the National Institute of General Medical
Sciences of the National Institutes of Health under grant number P30 GM106397. The
CARLZEISS LSM is supported by the National Institutes of Health Shared Instrument Grant
program: NIH S10-RR-027301. The NMR Research Facility at UNMC (part of the Fred &
Pamela Buffett Cancer Center) is supported by NIH grant number P30-CA036727.
ABBREVIATIONS