Tuning the biological activity of cationic anthraquinone analogues specifically toward Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2018.08.018

Development of new antibacterial agents against drug resistant bacteria is an imminent task, especially against methicillin-resistant Staphylococcus aureus (MRSA). While MRSA can still be treated with broad spectrum antibiotics, the use of which often leads to the disruption of normal microbial flora leading to Clostridium difficile infection (CDI). Herein, a new class of antibacterial agent, cationic anthraquinone analogues specifically against MRSA, has been developed. Through the variation and optimization of substituents, these agents are selective toward MRSA, and not Gram negative bacteria which may avoid the problem of CDI. In addition, newly discovered lead compounds also show significantly reduced cytotoxicity against normal mammalian cells than cancerous cells. This interesting finding can alleviate the toxicity and side effect problems often associate with the use of antibiotics.

Full text of DOI:10.1016/j.ejmech.2018.08.018

Accepted Manuscript
Tuning the biological activity of cationic anthraquinone analogues specifically toward
Staphylococcus aureus
Yagya Prasad Subedi, Madher N. Alfindee, Jaya P. Shrestha, Cheng-Wei Tom Chang
PII:
S0223-5234(18)30676-7
10.1016/j.ejmech.2018.08.018
EJMECH 10629
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 April 2018
Revised Date: 27 May 2018
Accepted Date: 4 August 2018
Please cite this article as: Y.P. Subedi, M.N. Alfindee, J.P. Shrestha, C.-W. Tom Chang, Tuning the
biological activity of cationic anthraquinone analogues specifically toward Staphylococcus aureus,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.018.
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60
50
40
30
20
10
0
MIC MRSA (ATCC 33591)
MIC MRSA (ATCC 43300)
IC A549 (Lungs cancer cells)
50
IC Colo205 (Colon cancer cells)
50
IC BEAS-2B (Lungs normal cells)
50
IC CCD-841-CoN (Colon normal cells)
50

ACCEPTED MANUSCRIPT
Tuning the Biological Activity of Cationic Anthraquinone Analogues
Specifically toward Staphylococcus aureus
Yagya Prasad Subedi, Madher N. Alfindee, Jaya P. Shrestha, Cheng-Wei Tom Chang*
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan,
UT 84322-0300, USA
Email: tom.chang@usu.edu
Abstracts
Development of new antibacterial agents against drug resistant bacteria is an imminent task,
especially against methicillin-resistant Staphylococcus aureus (MRSA). While MRSA can still
be treated with broad spectrum antibiotics, the use of which often leads to the disruption of
normal microbial flora leading to Clostridium difficile infection (CDI). Herein, a new class of
antibacterial agent, cationic anthraquinone analogues specifically against MRSA, has been
developed. Through the variation and optimization of substituents, these agents are selective
toward MRSA, and not Gram negative bacteria which may avoid the problem of CDI. In
addition, newly discovered lead compounds also show significantly reduced cytotoxicity against
normal mammalian cells than cancerous cells. This interesting finding can alleviate the toxicity
and side effect problems often associate with the use of antibiotics.
Key words
cationic anthraquinone analogues, antibiotic, MRSA, vancomycin-resistant Staphylococcus
aureus (VRSA), Clostridium difficile infection (CDI), cytotoxicity
Introduction
Infectious diseases caused by pathogenic and opportunistic Gram positive (G+) bacteria remain a
severe public health problem. Among these bacteria, methicillin-resistant Staphylococcus aureus
(MRSA)[1-3] and vancomycin-resistant Staphylococcus aureus (VRSA)[4] are of particular
interest due to their prevalence in community, multi-drug resistance to many clinically used
antibiotics, and the severity of illness caused by these bacteria. The burden of MRSA is
significant. In 2009, there were an estimated 463,017 MRSA-related hospitalizations in the
United States [5]. Although VRSA infection is still rare, a more serious concern is the transfer of

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vancomycin resistance from common bacteria, such as Enterococci, to MRSA, leading to the
increase in the VRSA infection [6, 7].
While MRSA can still be successfully treated with vancomycin, linezolid, daptomycin,
teicoplanin, quinupristine-dalfopristine, and tigecycline, resistance against these antibacterial
agents has been noted or reported. Approved by the FDA in 2003, daptomycin (Cubicin ®) is the
latest novel antibiotic[8], however, drug-resistance has been observed in MRSA in 2005 [9], and
reduced susceptibility has been reported in VRSA in 2006 [10]. Approved in 2001 by FDA,
linezolid (Zyvox®) is one of the few clinically used antibiotics for treating enterococcus
infection, however, linezolid drug-resistance by enterococci was noted in 2007 [11].
On the other hand, a common practice for treatment of bacterial infection is to apply broad-
spectrum antibiotics, such as ciprofloxacin, imipenem and cefepime. Nevertheless, the use of the
broad-spectrum antibiotics often eradicates beneficial bacteria, disrupts the normal microbial
flora, and leads to the over-growth of Clostridium difficile and C. difficile infection (CDI) [12,
1
3]. CDI can range in severity from asymptomatic to severe and life threatening. In the United
States alone, CDI increases the cost of healthcare by over 1 billion each year [14]. In short, these
reports exemplify the need for the development of new antibiotics selectively against G+
bacterial pathogens, like MRSA. Finally, it is also important that the new antibiotics manifest
low cytotoxicity toward normal mammalian cells to alleviate the problem of side effects.
Rationale and Design
In an effort to develop countermeasure against MRSA, our group has been studying the use of
cationic anthraquinone analogues (CAAs) as antibacterial agents (Table 1) [15-17]. These
antibacterial CAAs have two mode of actions (MOAs): (1) by disrupting the redox processes of
bacteria or (2) by acting as membrane disrupting agents. The redox processes of bacteria can
occur at the bacteria membrane, a site that can be accessible easier by CAAs. The latter MOA
requires the attachment of relatively long linear alkyl chains (C8-C16) [18]. In general, these
CAAs regardless of the type of structural modifications (N-1 and N-3 positions) all exert
significant antibacterial activity against drug-susceptible S. aureus and MRSA with minimum
inhibitory concentrations (MICs) less than 2 µg/mL. The activity against Gram negative (G-)
bacteria, such as Escherichia coli, varies drastically upon structural modifications of CAAs. For
example, most of the CAAs bearing alkyl groups at N-1 and N-3 positions (1a, 1b and 1c) are
more active than those with aryl groups at N-1 positions (2b, 2c and 2d). This finding prompts
our interest in the development of selective antibiotic against MRSA.
a
Table 1. Antibacterial activity (MIC) of CAAs

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Entry Compound
R/R′
S. aureus
ATCC25923)
S. aureus
MRSA
ATCC 33591)
E. coli
ATCC25922)
(
(
(
b
1
1a
1b
Me/Me
32-64
8
4-8
4-8
b
2
3
4
5
6
7
8
C H /Me
0.032-0.064
1
0.25 – 0.5
-
0.5 - 1
0.5
8
17
b
1c
C
16
H
33/Me
0.5-1
>128
8-16
>128
128
0.125-0.25
c
1d
2a
2b
C8H17/Et
Bn/Me
0.25 – 0.5
b
2
1-2
1
d
4-MeOPh/Me
Ph/Me
4-ClPh/Me
d
2c
0.5
0.5
d
2d
>128
1
a
b
c
d
Unit: µg/mL; ref:[17]; ref:[15]; ref:[19].
At the meantime, several members of antibacterial CAAs also show significant anticancer
activity (IC in low µg/mL), which raises the concern that the antibacterial CAAs might be too
5
0
toxic to mammalian cells (Table 2) [19-21]. Therefore, we further investigated the cytotoxicity
of CAAs bearing alkyl groups at N-1 and N-3 positions and those with aryl groups at N-1
positions. The results show that these CAAs manifest various degree of cytotoxicity upon
changing the substituents at the N-1 and N-3 positions. Furthermore, the structure of substituents
appears to have profound effect on the cytotoxicity of normal and cancerous mammalian cells.
For example, CAAs with only alkyl substituents (entries 1-3) show relatively low selectivity
between normal (BEAS-2B) and cancerous (A549) mammalian cells. However, with the
introduction of aromatic scaffold (entries 4-8), increasing toxicity toward cancerous cells was
observed (indicated as increased selectivity). Additionally, the electronic effect of the
substituents of the aromatic scaffold (entries 5-7) and the alkyl groups at N-3 (entries 7 vs. 8)
seems to perturb the selectivity. CAA with electron-donating group, 2b (OMe, entry 5) has
similar toxicity as CAA with electron-withdrawing group, 2d (Cl, entry 7) toward normal
mammalian cell. However, 2b is more toxic than 2d toward cancerous cell.
a
Table 2. Cytotoxicity (IC50) of CAAs

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b
Entry
Compound
R/R′
BEAS-2B (CCL-
22) (lungs
A549 (CCL-
185) (lungs
cancerous cell)
0.49±0.05
Selectivity
2
normal cell
0.62±0.05
0.60±0.02
0.22±0.05
2.32±1.20
5.80 ± 2.03
5.76 ± 0.40
6.43±2.44
1
1a
1b
1c
2a
2b
2c
Me/Me
1.27
1.40
0.36
5.16
7.44
5.77
2.18
2
3
4
5
6
7
C H /Me
0.43±0.03
8
17
C
16
H33/Me
0.61±0.01
Bn/Me
4-MeOPh/Me
Ph/Me
0.45±0.06
0.78 ± 0.16
1.00±0.15
2d
b
4-ClPh/Me
2.95±1.07
a
Unit: µg/mL; selectivity is calculated as IC (normal cell)/IC (cancer cell).
5
0
50
Combining the results from the antibacterial and cytotoxicity studies, we decided to investigate
the electronic effect from the substituents of the aromatic scaffold at N-1 and the alkyl groups at
N-3. Our goal is to identify new CAAs that are active specifically or selectively toward
pathogenic S. aureus, not G- E. coli while maintaining low toxicity toward normal mammalian
cells. To accomplish this goal, we opted to synthesize a library of new CAAs with variation on
the substituents of the aromatic scaffold at N-1 first. Following the identification of the lead(s),
more variation on the chain length of the alkyl group at N-3 will be commenced.
Synthesis, Result and discussion
Six additional CAAs were selected or synthesized for further examination (Figure 1).
Compounds 2f, 2g and 2h have moderate to strong electron-withdrawing groups on the aromatic
scaffold. Compound 2e and 2i have ethyl group at N-3 and compound 2j has an extra methoxy
group. Minimum inhibitory concentration (MIC) of these compounds towards MRSA can be
compared with 2c. The synthesis of compounds 2f and 2j has been reported previously [19].
Compounds 2e, 2g, 2h and 2i were synthesized following the reported protocol (Scheme 1).
Figure 1. CAAs for examining the effect of substituents of the aromatic scaffold

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Scheme 1.
The antibacterial activity of the newly selected/synthesized CAAs summarized in Table 3.
Additional strain of MRSA (ATCC43300) was also tested. All of these new members show
comparable activity as the parent compounds, 2b and 2c, and are inactive (MICs ≥ 128 µg/mL)
against E. coli. It is worth of mentioning that vancomycin is much less active against MRSA
(ATCC43300) while all of the tested CAAs still maintain the same level of antibacterial activity.
The high MIC of vancomycin against MRSA (ATCC43300) makes this strain of S. aureus
actually falls into the category of VRSA or vancomycin intermediate (resistant) S. aureus
(VISA).
a
Table 3. Antibacterial activity (MIC) of CAAs

ACCEPTED MANUSCRIPT
O
O
R'
OTf
N
N
N
R
Compound
R/R′
S. aureus
ATCC25923)
MRSA
(ATCC
MRSA
(ATCC
43300)
E. coli
ATCC25922)
(
(
3
3591)
0.5
0.5
1
2
b
4-OMe/Me
H/H
4 ClPh/Et
4-F/Me
>128
128
1-2
1
2-4
1
2
2
2
1
2
2
2
2
2
c
e
>128
>128
>128
>128
128
2
f
0.5
1
2
g
4-CN/Me
2
2
h
4-NO
2
/Me
2
1
2
i
4-OMe/Et
4-8
2
2
j
3,4-
dimethoxy
>128
2
> 256
4
2 - 4
2
2
256
2
4
Neomycin
Vancomycin
-
-
> 256
32
a
Unit: µg/mL
In addition of the BEAS-2B (CCL-222) (lungs normal cell)/A549 (CCL-185) (lungs cancer cell)
pair, CCD-841-CoN (CRL-1790) (colon normal cells)/Colo205 (CCL-222) (colon cancer cells)
pair of mammalian cells were also used for the cytotoxicity evaluation. Compound 2i that has
ethyl group at N-3 displays similar toxicity toward normal cells and toward cancerous cells
resulting in similar selectivity but the antibacterial activity dropped. Despite similar activity of
2
2
e, ethyl at N-3, towards MRSA it is more toxic towards mammalian cells than methyl analog,
d. CAA with additional methoxy group exert increased toxicity toward normal and reduced
selectivity. In contrast, CAAs with electron-withdrawing group(s) on the aromatic scaffold
become less toxic toward normal cells. However, the decrease in toxicity toward cancerous cells
was not as significant resulting in the increase in selectivity. Considering the selectivity and the
cytotoxicity toward normal cells, compound 2h was chose as the lead for investigating the effect
of the alkyl chain length at N-3.
a
Table 4. Cytotoxicity (IC50) of CAAs
b
b
Compound
BEAS-2B
CCL-222)
lungs
normal cell
A549
(Lungs
Cancer
Cell)
Selectivity CCD-841- Colo205
Selectivity
(
CoN
(CRL-
1790)
(CCL-
222)
(colon
(

ACCEPTED MANUSCRIPT
(
colon
cancer
cells)
normal
cells)
2
2
b
c
5.80 ± 2.03
5.76 ± 0.40
0.78 ± 0.16
1.00±0.15
7.44
5.77
5.89±0.81 0.70±0.02
8.10 ±
0.90 ±
.05
8.41
9.00
0
0
.80
6.65±1.342 0.78 ± 0.06
ND ND
2
e
4.22 ± 0.94
3.76±0.29
14.23±1.86
30.15±4.55
52.86±6.82
0.85 ± 0.06
1.60±0.55
2.22±1.10
5.20±0.46
3.50±1.07
4.96
2.35
6.44
8.53
2
2
2
j
f
g
-
8.16 ± 0.23 2.86±0.38
2.85
5.52
11.99
2.37
32.65±0.35 5.91± 0.28
2h
15.09
55.75±5.12 4.64± 0.25
3
2
i
5.00 ± 0.22
0.73 ± 0.08
6.85
7.85± 1.04
0.82±
.06
9.57
0
a
b
Unit: µg/mL; selectivity is calculated as IC50(normal cell)/IC50(cancer cell).
Using 2h as the lead, the ethyl, butyl, hexyl, octyl and decyl groups were incorporated at the N-3
position (Scheme 2). These compounds were tested again for their activity against S. aureus and
toxicity (Tables 5 and 6). Overall antibacterial activity of these analogs with extended alkyl
chain were mostly inferior to the parent compound, 2h. In general, the antibacterial activity
decreases with decreasing chain length. There is an interesting structure-activity relationship
(SAR) between the cytotoxicity and chain length. Except 2h, the cytotoxicity decreased
(
increased IC ) initially as the chain length increased, reached minimum with butyl or hexyl
5
0
groups and increased (decreased IC ) as the chain length further increased. Same SARs were
5
0
observed for all of the examined cell lines (normal and cancerous). Overall, the parent 2h still
has the lowest toxicity toward normal cells and best antibacterial activity against S. aureus
Scheme 2.
a
Table 5. Antibacterial activity (MIC) of CAAs
Compound
R
S. aureus
MRSA
MRSA

ACCEPTED MANUSCRIPT
(ATCC25923)
(ATCC 33591)
(ATCC 43300)
2
k
ethyl
butyl
hexyl
octyl
decyl
8-16
4
1-2
2
4-8
8-16
128
32
2
l
2
m
2-4
8
2-4
1
2
n
16
8
2
o
8-16
a
Unit: µg/mL
a
Table 6. Cytotoxicity (IC ) of CAAs
5
0
b
b
Compound BEAS-2B A549
Selectivity CCD-841-
Colo205 Selectivity
(
CCL-222) (Lungs
CoN (CRL- (CCL-
(
lungs
Cancer
Cell)
1790)
(colon
normal
cells)
222)
(colon
cancer
cells)
normal
cell
2
k
43.56±2.67 7.40±0.28 5.89
49.75±5.73 44.89±5.17 1.11
51.26±5.86
7.72±0.40 6.64
2
l
42.79 ± 1.48 43.17 ±
.53
0.99
4
2
m
38.58±0.81 48.88±2.66 0.79
40.19 ± 7.18 21.25 ±
.24
30.98 ± 3.57 8.53 ±
0.71
1.89
3.63
4.20
3
2
n
20.98 ±
.37
15.59 ±
4.411
4.76
2
2
o
7.39 ± 0.65 7.02 ± 2.16 1.05
30.64 ± 5.30 7.29 ±
0
.24
a
b
Unit: µg/mL; selectivity is calculated as IC (normal cell)/IC (cancer cell).
5
0
50
Following these SAR investigation, the electronic effect from the substituents of the aromatic
scaffold at N-1 and the effect of alkyl groups at N-3 on the antibacterial activity and cytotoxicity
are illustrated in Figures 2 and 3. It is clear that having electron-withdrawing groups (ex. NO
2
and CN) on the aromatic scaffold at N-1produces CAAs with superior antibacterial activity and
selective cytotoxicity toward cancerous cells (Figure 2). Such selectivity between antibacterial
and anticancer, and normal and cancerous mammalian cells diminishes as weaker electron-
withdrawing group (Cl) or electron-donating group (OMe) are attached. With few exceptions, the
general trend regarding the SAR of varying chain-length at N-3 is that short alkyl chain (Me) is
better than longer alkyl chains (Et, butyl, hexyl and octyl) (Figure 3).
Figure 2. Electronic effect from the substituents of the aromatic scaffold

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6
5
4
3
2
1
0
0
0
0
0
0
0
IC50 BEAS-2B
IC50 CCD-841-CoN
IC50 A549
IC50 Colo205
MIC MRSA (ATCC 33591)
MIC MRSA (ATCC 43300)
-
10
2
b
2c
2d
2f
2g
2h
Figure 3. Effect of variation of alkyl groups at N-3
IC50 BEAS-2B
IC50 CCD-841-CoN
IC50 A549
1
1
1
40
20
00
IC50 Colo205
MIC MRSA (ATCC 33591)
MIC MRSA (ATCC 43300)
8
6
4
2
0
0
0
0
0
2
h
2k
2l
2m
2n
2o
In general, most of the reported CAAs possess significant antibacterial activity against S. aureus.
These CAAs are more active than previously reported quinone based antibacterial compounds
like phaeosphenone, Lawson and Lawson derivatives [22-24] and have similar activity like
naphthazarins [25]. We have previously reported that the anticancer CAAs exert the activity by
inducing the production of reactive oxygen species (ROSs) leading to the damage of
mitochondria membrane, a site that is not readily accessible as bacteria membrane [21]. In
addition, we have also noticed that the attachment of electron-withdrawing group on the aryl
group of CAAs can lower the cytotoxicity due to the perturbation of redox potential. We have
discovered that cancerous cells are more prone toward the oxidative stress (high ROS level)
induced by CAAs as compared to normal cells. Combining these finding, it provides the

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rationale for supporting the reported observation. First, the redox active CAAs exert antibacterial
activity by disrupting the redox process of bacteria via, likely, the production of ROSs: a
common MOA that has been noticed in other quinone-based antibacterial compounds[26].
Second, since cancerous cells are prone to oxidative stress, the presence of nitro group makes
compound 2h less toxic toward normal mammalian cells than cancerous cells. Third, the sites
targeted by CAAs for the antibacterial and anticancer MOAs are different (bacterial vs.
mitochondrial membranes). Therefore, it is possible that the easy accessibility of bacterial
membrane leads to the observation that the electronic effect of the substituents on CAAs does
not influence their antibacterial activity as much as their anticancer activity.
To augment the application of the lead compound, 2h, the antibacterial activity using
combination of 2h with commercially used antibacterial compounds fosfomycin and
ciprofloxacin, and previously reported membrane disrupting antibacterial compounds 7 (Figure
4
) [27] were studied. Result showed that 2h does not have any synergistic effect with the tested
compounds.
Figure 4. Structure of 7
Conclusion-
CAAs are generally highly active against various strains of G+ bacterial pathogens, including
MRSA. Most of the CAAs are much less active against G- bacteria, an advantage that can be
utilized for the development of selective antibiotic and avoid CDI. However, several members of
CAAs also exert moderate to significant cytotoxicity toward mammalian cells. It has been noted
that the substituents at the N-1 and N-3 positions of CAAs play significant role in the observed
biological activity. In order to tune the activity of CAAs toward MRSA while maintaining low
cytotoxicity toward mammalian cells, a library of CAAs was synthesized and assayed to reveal
the SAR. Results from cytotoxicity antibacterial assay showed that N-1 and N-3 alkyled CAAs
have either no selectivity or very poor selectivity between G+ bacteria and mammalian cells
(normal and cancerous). Nevertheless, when attaching aryl motif with electron-withdrawing
groups at N-1 position, CAAs become much less toxic toward mammalian cells while still
maintaining the antibacterial activity. Further screening the CAAs with various electron-

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withdrawing groups against normal and cancerous cells allows the identification of 2h equipped
with NO as the lead compound. SAR based on the variation of alky chain length revealed that
2
methyl group is the optimal structural motif. Compound 2h stands out with the best selectivity
between bacteria vs. mammalian cells, and normal mammalian cells vs. cancerous cells. The
discovery of 2h may pave the way toward the development of new antibacterial agent against
MRSA and VRSA.
Experimental section
General procedure A for the synthesis of 2e, 2i, 2k: Ethyl triflate used on the synthesis of
these compounds was synthesized from the reaction of ethanol with triflic anhydride using
pyridine as the base. 8 equivalents of triflic anhydride was added dropwise to the stirred solution
of 1 equivalent of ethanol and 8 equivalents of the pyridine in the anhydrous toluene maintaining
o
the temperature below 0 C. Reaction mixture was stirred for 2 hours and filtered through the
anhydrous sodium sulfate. Ethyl triflate in filtrate was added to the 1 equivalent of the 6a, 6b, or
o
6
d and refluxed at 100 C overnight. Toluene from reaction mixture was remove under reduced
pressure and the residue washed with the diethyl ether to obtain pure products.
General procedure B for the synthesis of 2l–2o: When pyridine was used as the base to
synthesize these compounds as the general procedure A using alcohols with different chain
length, less than 5% of the expected products were obtained. The side products of those reaction
were the alkyl substituted pyridine. To avoid the alkylation of the pyridine, less nucleophilic base
triethylamine was used. 8 equivalents of triflic anhydride was added dropwise to the stirred
solution of 1 equivalent of alkyl alcohol and 8 equivalents of triethylamine in anhydrous toluene
o
over the thirty minutes keeping the temperature below 0 C. Then the reaction keeps stirred for
o
another 2 hours. To the reaction flasks 6d was added and refluxed at 100 C overnight. Next day
reaction was cooled to room temperature and toluene removed under reduced pressure. Thus,
obtained residue washed with water and diethyl ether to obtain final products.
General procedure for the antibacterial activity- Minimum inhibitory concentration of the
compounds were tested against E. coli (ATCC25922), S. aureus (ATCC25923), S. aureus
MRSA (ATCC 33591), and S. aureus MRSA (ATCC 43300) as the reported procedure[28].
General procedure for the cytotoxicity- Cytotoxicity of the compounds was evaluated by
colorimetric assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
dye. A549 (CCL-185), BEAS-2B (CCL-222), Colo205 (CCL-222), and CCD-841-CoN (CRL-
1
790) cells were used to assess the cytotoxicity of the compounds. A549 and BEAS-2B were
grown in the Dulbecco's modification of eagle's medium (DMEM), (Corning) supplemented with
0 percent fetal bovine serum (FBS) (Hyclone, SH3007103). Similarly, Colo205 cells were
1
grown in RPMI1640 (ATCC 302001) and CCD-841-CoN cells in Eagle's minimum essential
medium (EMEM) (ATCC 302003) supplemented with 10% FBS. 100 U/mL of streptomycin and
1
00 U/mL of penicillin were treated to all these culture media.

ACCEPTED MANUSCRIPT
2
00 uL of the growing medium containing enough cells to get sub confluent culture and good
absorption value of at the end of the assay, 5000 cells for A549 and BEAS-2B cells and 15,000
cells for colo205 and CCD-841-CoN, were added to the 96 wells cell culture plate (Corning).
After 24 hours media were removed and the different concentration of drugs (0.1, 1, 10, 100
µg/mL) diluted in culture medium were added and incubated for 48 hours in the incubator at 37
o
C with 5 % CO2 environment. Then 20 uL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) dye (5 mg/mL) was added and incubated in the same
condition. After 5 hours, media was removed, and the reduced formazan dye was dissolved in
DMSO. Absorption of the dissolved formazan was taken at 570 nm with 650 nm as background
in micro titer plate reader (BioTek). Relative viability of the cells was calculated by comparing
the absorption of the compound treated cells to control cells, which is considered to have 100%
cell viability.
Material and Methods
Solvents and reagents were purchased from the commercial source. Alcohols used in the
synthesis of the alkyltriflate was dried over molecular sieve for 48 and toluene was dried using
1
13
calcium hydride. H NMR and C NMR spectra were taken in Bruker Ascend 500 under 500
MHZ and 75 MHZ respectively. Melting point of the compounds were recorded using DigiMelt
MPA160.
4
-(4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-1-yl)benzonitrile
(6c)-
This
compound is synthesized as discribed[19] by the cyclization of naphthoquinone with aryl azide
o
1
with 60 % yield. 6c is yellowish white solid with melting point more than 260 C. H NMR (500
MHz, DMSO-d ) δ 8.26 (dd, J = 9.0 Hz, J = 1.5 Hz, 1H), 8.21 (d, J = 8.5 Hz, 2H) 8.15 (dd, J =
.5 Hz, J = 1.5 Hz, 1H) 8.07 (d, J = 8.5 Hz, 2H) 7.9 – 8.0 (m, 2H)); C NMR (125 MHz,
DMSO-d6) δ 177.46, 174.39, 145.47, 139.00, 135.62, 135.49, 135.31, 134.01(2C), 133.73,
6
1
3
7
+
1
3
33.12, 127.58, 127.42, 126.90 (2C), 118.42, 113.81. ESI/APCI calculated for [C H N O ]:
01.0726; Measured m/e: 301.0725.
1
7
9
4
2
1
-(4-cyanophenyl)-3-methyl-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2g) – 2g is synthesized by the reaction of compound 6c with
methyltriflate[19] with 98 % yield. This compound is light grey solid with melting point 240-241
o
1
C. H NMR (500 MHz, DMSO- d ) δ 8.3 – 8.4 (m, 3H), 8.2 – 8.3 (m, 3H), 8.1 – 8.2 (m, 2H),
6
1
3
4
1
4
.84 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 173.02, 171.47, 137.15, 136.89, 136.80, 136.66,
35.99, 134.71 (2C), 133.08, 132.17, 128.51, 128.31, 127.40 (2C), 117.89, 116.22,
1.69.ESI/APCI calculated for[C H N O ]: 315.0877; measured m/e: 315.0866.
+
2
1
8
11
4
3
-methyl-1-(4-nitrophenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2h)- This is synthesized by following the same procedure as
o
1
compound 2g with 96 % yield. 2h is grey solid with melting point 248-249 C. H NMR (500
MHz, Methanol- d ) δ 8.65 (d, J = 9.0 Hz, 2H), 8.4 – 8.5 (m, 1H), 8.3 – 8.4 (m, 1H), 8.24 (d, J =
3
1
3
9
1
.0 Hz, 2H) 8.1 – 8.2 (m, 2H), 4.89 (s, 3H); C NMR (125 MHz, Methanol-D3) δ 172.34,
70.91, 150.44, 137.62, 136.18, 136.14 (2C), 136.02, 132.73, 132.14, 127.98, 127.74, 127.40

ACCEPTED MANUSCRIPT
+
4 +
(
2C), 124.81 (2C), 40.42. ESI/APCI calculated for [C H N O
MeOH]: 367.1037;
1
7
11
4
Measured m/e: 367.1113.
3
-ethyl-1-(4-methoxyphenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2i)- Compound is synthesized by following the general procedure
o
1
A with 95 % yield. 2i is yellow solid with melting point 155-156 C. H NMR (500 MHz,
Methanol- d ) δ 8.4 -8.5 (m, 1H), 8.3 – 8.4 (m, 1H), 8.0 – 8.1 (m, 2H), 7.8-7.9 (m, 2H) 7.2 – 7.3
3
1
3
(
m, 2H), 5.25 (q, J = 7.0 Hz, 2H), 1.83 (t, J = 7.0 Hz, 3H). C NMR (125 MHz, Methanol-D3) δ
1
72.71, 171.14, 163.17, 135.92, 135.69, 132.82, 132.26, 127.82, 127.57, 127.06 (2C), 126.27,
+
1
14.48 (2C), 55.16, 50.43, 12.77. ESI/APCI calculated for[C H N O ]: 334.1186;
1
9
16
3
3
Measured m/e:- 334.1205.
1
-(4-chlorophenyl)-3-ethyl-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2e)- This compound is synthesized by following the general
o
1
procedure A with 92 %Yield. 2e is greenish white solid with melting point 197-198 C. H NMR
500 MHz, Methanol- d ) δ 8.4 – 8.5 (m, 2H), 8.3 – 8.54 (m, 1H), 8.0 – 8.1 (m, 2H), 7.95 (dt, J =
(
3
9
7
1
.0 Hz, J = 3.0 Hz, 2H) 7.83 (dt, J = 9.0 Hz, J = 3.0 Hz, 2H) 5.28 (q, J = 7.0 Hz 2H), 1.84 (t, J =
.0 Hz 3H); C NMR (125 MHz, Methanol-D3) δ 172.48, 171.07, 139.05, 136.04, 135.89,
35.76, 132.25, 130.22, 129.79 (3C), 127.90, 127.70, 127.24 (2C), 123.13, 50.76, 12.75.
1
3
ESI/APCI calculated for338.0691
; Measured m/e: 338.0687.
3
-ethyl-1-(4-nitrophenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2k): Compound 2k is synthesized by following the general
procedure A with the 0.331 gm 99 % yield. This compound is grey in color with melting point
o
1
2
03-205 C. H NMR (500 MHz, Methanol- d ) δ 8.6 – 8.7 (m, 2H), 8.4 – 8.5 (m, 1H), 8.3-8.4
3
(
m, 1H) 8.2 – 8.3 (m, 2H), 8.0 – 8.1 (m, 2H), 5.33 (q, J = 7.5 Hz, 2H), 1.86 (t, J = 7.5 Hz, 3H).
C NMR (125 MHz, Methanol-D3) δ 172.31, 171.04, 150.44, 137.73, 136.37, 136.13, 136.06,
1
3
1
35.85, 132.59, 132.24, 127.97, 127.80, 127.42 (2C), 124.80 (2C), 51.07, 12.74. ESI/APCI
+
calculated for [C H N O ]
:
334.1186; Measured m/e: 334.1205.
1
9
16
3
3
3
-butyl-1-(4-nitrophenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2l)- Compounds is synthesized by following the general procedure
B with the 0.504 gm (95.81 %) yield. This compound is grey in color with melting point 199-200
o
1
C. H NMR (500 MHz, Methanol- d ) δ 8.6 -8.7 (m, 2H), 8.4 – 8.5 (m, 1H), 8.3-8.4 (m, 1H) 8.2
3
–
1
1
8.3 (m, 2H), 8.0 – 8.1 (m, 2H), 5.29 (t, J = 7.5 Hz, 2H), 2.2 – 2.3 (m, 2H), 1.6 – 1.7 (m, 2H),
.10 (t, J = 7.0 Hz, 3H). C NMR (125 MHz, Methanol-D3) δ 172.33, 170.99, 150.46, 137.66,
1
3
36.41, 136.18, 136.09, 135.85, 132.58, 132.24, 127.99, 127.83, 127.40 (2C), 124.82 (2C),
+
5
4.85, 30.58, 19.12, 12.27. ESI/APCI calculated for [C H N O ]
:
377.1244; Measured
2
0
17
4
4
m/e: 377.1214.
3
-hexyl-1-(4-nitrophenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2m)- Compounds is synthesized by following the general
procedure B with the 0.509 gm (91.87 %) yield. This compound is grey in color with melting
o
1
point 195-196 C. H NMR (500 MHz, Methanol- d ) δ 8.67 (d, J = 9.0 Hz, 2H), 8.4 – 8.5 (m,
3
1
H), 8.3-8.4 (m, 1H) 8.25 (d, J = 9.0 Hz, 2H), 8.0 – 8.1 (m, 2H), 5.28 (t, J = 7.5 Hz, 2H), 2.2 –

ACCEPTED MANUSCRIPT
1
3
2
.3 (m, 2H), 1.5 – 1.6 (m, 2H), 1.3 - 1.5 (m, 4H), 0.97 (t, J = 7.0 Hz, 3H). C NMR (125 MHz,
Methanol-D3) δ 172.33, 171.02, 150.44, 137.68, 136.41, 136.41, 136.15, 136.09, 135.85, 132.59,
32.25, 127.97, 127.82, 127.42 (2C), 124.81 (2C), 55.07, 30.79, 28.62, 25.55, 22.03, 12.27.
1
+
ESI/APCI calculated for [C22
21 4 4
H N O ]: 405.1557; Measured m/e: 405.1583.
1
-(4-nitrophenyl)-3-octyl-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2n)- Compounds is synthesized by following the general procedure
B with the 0.546 gm (93.97 %) yield. This compound is grey in color with melting point 92-93
o
1
C. H NMR (500 MHz, CDCl
3
) δ 8.32 (d, J = 9.0 Hz, 2H), 8.2 – 8.3 (m, 1H), 8.1-8.2 (m, 1H)
7
1
1
5
.8 - 8.0 (m, 4H), 8.0 – 8.1 (m, 2H), 4.85 (t, J = 7.5 Hz, 2H), 2.0 - 2.1 (m, 2H), 1.2 – 1.5 (m,
1
3
0H), 0.90 (t, J = 7.5 Hz, 3H). C NMR (125 MHz, CDCl ) δ 172.27, 171.16, 149.78, 137.92,
3
36.25, 135.69, 135.62(2C), 132.42, 132.19, 128.17, 127.99, 127.91, 124.55 (2C), 123.74 (2C),
4.96, 31.67, 28.91, 28.72, 28.29, 26.29, 25.57, 22.57, 14.05. ESI/APCI calculated for
+
[C H N O ]: 433.1870; Measured m/e: 433.1900.
2
4
25
4
4
3
-decyl-1-(4-nitrophenyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
trifluoromethanesulfonate (2o)- Compound is synthesized by following the general procedure
o
1
B with the 96 % yield. 2o is brown solid with melting point 82-83 C. H NMR (500 MHz,
CDCl ) δ 8.3 – 8.4 (m, 2H), 8.2 – 8.3 (m, 1H), 8.1-8.2 (m, 1H) 7.8 - 8.0 (m, 4H), 4.85 (t, J = 7.5
3
1
3
Hz, 2H), 2.0 - 2.1 (m, 2H), 1.2 – 1.5 (m, 14H), 0.89 (t, J = 7.5 Hz, 3H). C NMR (125 MHz,
CDCl ) δ 172.26, 171.16, 149.79, 137.92, 136.25, 135.69, 135.63 (2C), 132.43, 132.19, 128.19,
3
1
1
28.00, 127.91 (2C), 124.56 (2C), 54.97, 31.84, 29.45, 29.27, 29.23, 28.91, 28.77, 26.30, 22.66,
+
4.10. ESI/APCI calculated for [C H N O ]: 461.2183; Measured m/e: 461.2158.
2
6
29
4
4
Acknowledgement
We acknowledge the support from NSF Award CHE-1429195 for 500 MHz Bruker NMR. We thank the
support from Department of Chemistry and Biochemistry, Utah State University.
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Highlights
1
2
3
. Substituent at N-3 can tune the selectively of compound toward G+ bacteria.
. Compound 2h have antibacterial activity against MRSA but not G- bacteria.
. Compound 2h is less toxic towards normal mammalian cells compared to cancerous cells.