Synthesis and anticancer structure activity relationship investigation of cationic anthraquinone analogs

Article abstract of DOI:10.1016/j.ejmech.2014.02.060

We have synthesized a series of novel 4,9-dioxo-4,9-dihydro-1H-naphtho[2,3- d][1,2,3]triazol-3-ium salts, which can be viewed as analogs of cationic anthraquinones. Unlike the similar analogs that we have reported previously, these compounds show relative

Full text of DOI:10.1016/j.ejmech.2014.02.060

European Journal of Medicinal Chemistry 77 (2014) 96e102
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer structure activity relationship investigation
of cationic anthraquinone analogs
*
Jaya P. Shrestha, Marina Y. Fosso, Jeremiah Bearss, Cheng-Wei Tom Chang
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a series of novel 4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-3-ium
salts, which can be viewed as analogs of cationic anthraquinones. Unlike the similar analogs that we
have reported previously, these compounds show relatively weak antibacterial activities but exert strong
Received 20 September 2013
Received in revised form
11 February 2014
Accepted 27 February 2014
Available online 28 February 2014
anticancer activities (low mM to nM GI50), in particular, against melanoma, colon cancer, non-small cell
lung cancer and central nervous system (CNS) cancer. These compounds are structurally different from
their predecessors by having the aromatic group, instead of alkyl chains, directly attached to the cationic
anthraquinone scaffold. Further investigation in the structureeactivity relationship (SAR) reveals the
significant role of electron donating substituents on the aromatic ring in enhancing the anticancer ac-
tivities via resonance effect. Steric hindrance of these groups is disadvantageous but is less influential
than the resonance effect. The difference in the attached groups at N-1 position of the cationic
anthraquinone analog is the main structural factor for the switching of biological activity from anti-
bacterial to anticancer. The discovery of these compounds may lead to the development of novel cancer
chemotherapeutics.
Keywords:
Cancers
Chemotherapeutics
Cationic anthraquinones
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
designed compound 3 to serve as an analog of compound 2. Our
initial goal is to investigate the importance of methylene group
The development of novel antitumor agents has long been the
focused area in cancer chemotherapy [1,2]. Molecules bearing an
anthraquinone motif have attracted great interest due to their po-
tential uses as cancer chemotherapeutics [3,4]. We have previously
reported the synthesis of a library of cationic anthraquinone ana-
logs and their antibacterial activities [5,6] and mode of action study
[7]. Most of these compounds exhibit strong antibacterial activities
against Gram positive (Gþ) bacteria with minimum inhibitory
between the cationic anthraquinone scaffold and the benzene ring.
2. Design, synthesis and evaluation of anticancer activities
Using commercially available 1,4-naphthoquinone (4) and
phenyl azide (5), compound 3 can be synthesized following the
protocol reported previously (Scheme 1) [5]. The first step is a
cycloaddition using excess naphthoquinone followed by an
oxidation to yield compound 6. Methylation of compound 6 pro-
vided compound 3 as a triflate salt, which can be converted into
the corresponding chloride salt after ion-exchange column chro-
matography. To our delight, compound 3 displays anticancer ac-
tivities comparable to 1 which proves that the methylene bridge
between the cationic anthraquinone scaffold and aromatic ring
can be omitted. Without the presence of this methylene group, it
is expected that substituents at the ortho or para positions on the
benzene ring of compound 3 will have higher electronic influence
due to the resonance effect. Since ortho-substituents may cause
higher steric hindrance toward the naphthoquinone core, we used
compound 3 as the new lead and synthesized a library of de-
rivatives of compound 3 with para-monosubstituted benzene
rings.
concentrations (MIC’s) lower than 1 mg/mL against Staphylococcus
aureus (ATCC 25923). By comparison, two analogs, compounds 1
and 2, exert only modest antibacterial activity with MIC’s of 2 and
4e8
manifest significant anticancer activities with GI50 values ranging
from low M to nM in the five-dose 60 cell lines assay performed by
mg/mL, respectively (Fig. 1). However, these two compounds
m
National Cancer Institute (NCI). Further derivatization of Com-
pound 2 will be relatively difficult. In addition, derivatives of
compound 1 having substituents, such as a p-methoxy group on the
benzene ring have been found to be unstable. Therefore, we
* Corresponding author.
E-mail address: tom.chang@usu.edu (C.-W.T. Chang).
http://dx.doi.org/10.1016/j.ejmech.2014.02.060
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
97
Fig. 1. Cationic anthraquinone analogs.
The synthesis started with the preparation of the corresponding
diazonium salts from para-substituted anilines, 7aee using NaNO2
and HCl (Scheme 2), followed by a nucleophilic aromatic substi-
tution using NaN3 to obtain the desired para-substituted phenyl-
azides 8aee. Due to their relatively low boiling points, these aryl
azides are used as concentrated solutions in ether without further
purification or complete removal of solvents, which contribute to
the relatively lower yields for the cycloaddition/oxidation step for
the formation of compounds 6aee. Meanwhile, we have also
discovered that the counter ion of the cationic anthraquinone an-
alogs has no effect on the anticancer activities. Thus, these new
members were prepared and tested as trifluoromethanetriflate
(OTfꢀ) salts following the N-methylation step.
Compounds 3aee were tested in the single dose 60 cell lines
assay conducted through the Developmental Therapeutic Program
(DTP) of NCI (Table 1). The results of mean cell growth percentages
strongly indicate that the strong electron-donating methoxy group,
as in compound 3a, significantly increases the anticancer activities.
Compound 3b, with a moderate electron-donating methyl group
that can provide only inductive effect to increase the electron
density on the benzene ring, is less active than compound 3a.
Electron-withdrawing groups, such as trifluoromethyl and chloro
groups, further decrease the activities. Interestingly, the fluoro
derivative 3e bearing the most strongly electron-withdrawing
group that we have introduced, does not follow this trend. It is
possible that smaller steric hindrance of the fluoro group attributes
to the partial restoration of the anticancer activities.
importance of resonance effect from the electron-donating group,
methoxy group. For example, the methoxy groups attached at the
meta positions of benzene ring on compound 3f can only increase
the electron density of benzene ring and the cationic anthraqui-
none scaffold through the inductive effect. However, the 3,5-
dimethoxy groups increase the steric hindrance. As a result, the
activities of 3f are even lower than 3a. The methoxy group attached
to the para position can increase the electron density of benzene
ring and the cationic anthraquinone scaffold through the resonance
effect. As predicted, both 3g and 3h show better activities than 3f
and even 3a. With less steric hindrance, compound 3h is slightly
more active than compound 3g.
Compounds 3, 3aec and 3eeh were selected to five-dose cell
lines assay also conducted through the DTP of NCI. Overall, these
compounds displayed higher anticancer activities (low mM to nM
GI50) against melanoma, colon cancers, non-small cell lung can-
cers and central nervous system (CNS) cancers [8]. The GI50 (50%
growth inhibition) from the test results are summarized in
Tables 3e6. In general, the activities are consistent with the re-
sults from single dose assay with 3, 3a and 3h as the most active
analogs. However, slight differences have been noted. For
example, based on the average GI50, 3h is most active against
melanoma, non-small cell lung cancer and CNS cancer followed
by either 3a or 3 whereas 3 and 3a are more active against colon
cancer than 3h.
We have also noticed that certain cell lines appear to be less
susceptible to these cationic anthraquinone analogs. For example,
among the cell lines of melanoma, these compounds are more
active against MDA-MB-435 than LOX IMVI. A similar tendency
has also been found in the anticancer studies of spongistatin 1
[9]. Nevertheless, even with the available information on the
mutations of cell lines, it is difficult to provide satisfactory
explanation on the difference. For instance, SK-MEL-5 that ex-
presses mutant B-Raf (V600E) and wildtype N-Ras [10] is sensi-
tive to the anticancer action of cationic anthraquinone analogs. A
similar cell line, SK-MEL-28 that also expresses mutant B-Raf
(V600E) and wildtype N-Ras [10] is much less susceptible to
these compounds.
After confirming the positive role of strong electron-donating
group, we decided to further explore the effect of having multiple
methoxy groups. The designs and syntheses of these new members
are shown in Scheme 3. Although all the desired aryl azides can be
prepared accordingly, the cycloaddition/oxidation step using aryl
azides 8i and 8j offered products mixed with inseparable by-
products. Therefore, only compounds 7f, 7g and 7h were con-
verted to the corresponding cationic anthraquinone analogs 3f, 3g
and 3h, respectively.
The anticancer activities from single dose 60 cell lines assay of
compounds 3feh are shown in Table 2. The results confirm the
Scheme 1. Synthesis of cationic anthraquinone analogs.

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J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
Scheme 2. Synthesis of cationic anthraquinone analogs with substituted aryl groups.
Table 1
Table 2
Single dose 60 cell lines assay of compounds 3 and 3aee.
Single dose 60 cell lines assay of compounds 3feh.
Compound
Mean cell growth percentage (%)
N-1 substituent
Compound
Mean cell growth percentage (%)
N-1 substituent
3
ꢀ10.18
ꢀ24.70
11.53
58.69
101.70
43.38
Phenyl
3f
3g
3h
23.23
ꢀ27.06
ꢀ31.51
3,5-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
3,4-Dimethylphenyl
3a
3b
3c
3d
3e
4-Methoxyphenyl
4-Methylphenyl
4-Trifluoromethylphenyl
4-Chlorophenyl
4-Fluorophenyl
density via the resonance effect and steric hindrance of the N-1 aryl
groups play important roles in the anticancer activities. From the
mean cell growth percentage and average GI50 values, a general
tendency that indicates the presence of electron-donating sub-
stituents at the N-1 aryl group enhance the anticancer activity when
the tested compounds are arranged in order of decreasing electron
density (Figs. 2 and 3). Further analysis shows that only the groups
that can increase the electron density via the resonance effect can
enhance the anticancer activity. As mentioned previously, both
3. Discussion of anticancer and antibacterial SAR
We are currently in the process of investigating the possible
anticancer mode of action and the potential cellular targets of these
cationic anthraquinone analogs. Based on the structureeactivity
relationship (SAR) that has been revealed, we believe that electron
Scheme 3. Synthesis of cationic anthraquinone analogs with substituted aryl groups.

J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
99
Table 3
Table 5
Anticancer activities (GI50) against melanoma.a
Anticancer activities against non-small cell lung cancers.a
Cell line
Compound
3a 3b 3c
Cell line
Compound
3a 3b 3c
A549/ATCC (Adenocarcinoma) 0.35 0.33 1.11 0.86 0.67 0.78 1.68 0.40
3
3e 3f
3g 3h
3
3e
3f
3g 3h
LOX IMVI (Malignant)
MALME-3M (Malignant)
M14
1.16 1.24 1.80 2.68 1.35 1.71 1.19 1.14
0.21 0.24 0.32 1.84 0.35 0.41 0.30 0.21
0.20 0.21 0.26 0.54 0.34 0.50 0.48 0.33
EKVX (Adenocarcinoma)
HOP-62 (Adenocarcinoma)
HOP-92 (Large cell)
NCI-H226 (Squamous)
NCI-H23 (Adenocarcinoma)
NCI-H322M (Carcinoma)
NCI-H460 (Large cell
carcinoma)
1.89 2.42 4.16
0.76 0.98 2.16
23.4
3.28 NDb ND ND
2.12 2.40 1.51 1.31 1.23
MDA-MB-435 (Adenocarcinoma) 0.20 0.19 0.17 1.09 0.26 0.31 0.19 0.19
4.17 3.47 0.42 >100
10.7 7.07 1.92 2.27
2.26 1.74 1.80 1.30
SK-MEL-2 (Malignant)
SK-MEL-28 (Malignant)
SK-MEL-5 (Malignant)
UACC-257
UACC-62
Average GI50
2.18 2.05 2.17 12.5 2.27 2.08 1.75 1.40
1.19 1.13 2.65 12.8 4.27 3.20 1.37 1.34
0.18 0.16 NDb 0.16 0.17 0.18 0.24 0.16
1.70 1.45 1.85 3.24 0.60 1.20 1.37 1.49
1.09 1.01 1.65 1.67 1.55 1.41 1.23 1.22
0.90 0.85 1.36 4.06 1.24 1.22 0.90 0.83
ND ND 2.93
0.98 1.05 0.65
ND ND 0.88
0.30 0.30 0.33
10.1
0.36 0.82 1.26 1.94 1.06
0.83 0.45 0.36 0.40 0.30
1.40 0.63 0.56 0.79 0.36
NCI-H522 (Adenocarcinoma) 11.5 2.05 8.14
14.3 12.1 2.54 1.71 2.00
Average GI50
2.85 1.51 2.31
17.04 3.70 1.98 1.44 1.12
a
Unit:
m
M.
b
a
ND: Not determined.
Unit:
m
M.
b
ND: Not determined.
compounds 3f and 3b, which contain electron-donating groups but
cannot increase the electron density via the resonance effect, are less
active than 3, which has no substituent attached. This observation
suggests the adverse steric effect in anticancer activity.
We have reported the synthesis and antibacterial activity of a
library of cationic anthraquinone analogs bearing two alkyl groups
at the N-1 and N-3 positions (Fig. 4) [5,6]. One of the lead com-
pounds, 9 [5] which is similar to compound 2 but has an octyl group
instead of methyl group at the N-1 position, exerts only poor
Table 6
Anticancer activities (GI50) against CNS cancer.a
Cell line
Compound
3a
1.64 1.72 3.14 18.7
3
3b
3c
3e
3f
3g
3h
SF-268 (Astrocytoma)
3.67 3.68 0.87 1.23
SF-295 (Glioblastoma- 0.54 0.63 1.04
Multiforme)
0.54 0.58 0.18 0.54 0.20
SF-539
1.09 1.28 1.97
4.68 1.83 1.64 0.40 0.50
1.45 1.37 1.25 1.40 1.14
1.07 1.94 1.63 1.43 0.82
SNB-19 (Glioblastoma) 1.07 1.01 1.39
SNB-75 (Astrocytoma) 0.64 1.25 2.09
U251 (Glioblastoma)
anticancer activities (mean cell growth percentage
Table 7). These results are consistent with the negative effect of
steric hindrance in the anticancer activities.
¼
44.8%,
1.26 1.16 2.43 14.0
7.86 1.53 1.54 1.34
Average GI50
1.04 1.18 2.01
6.74 2.88 1.65 1.03 0.87
a
When comparing the SAR of anticancer and antibacterial ac-
tivities (Table 7), we conclude that a small alkyl chain (CH3, entry
11, compound 2) or aromatic ring at the N-1 position increases the
anticancer activities. A longer alkyl chain is crucial for antibacterial
activities but not the anticancer activities (octyl, entry 12, com-
pound 9) [5,6]. Overall, reduced steric hindrance and increased
electron-density via resonance are crucial for tuning the biological
activity of cationic anthraquinone analogs from being antibacterial
to anticancer and vice versa. Although we are investigating the
possible modes of anticancer activity and targets, we have noted
that compound 2 also has a similar profile of anticancer activities:
particularly active against melanoma, colon cancer, non-small cell
lung cancer and CNS cancer [11]. This interesting finding implies
that the cationic anthraquinone is the structural motif responsible
for the anticancer activity and that the N-1 substituent plays an
auxiliary role. It also points to two possible mode of actions:
intercalation of nucleic acids and disruption of cellular redox pro-
cesses. The former is favored by small steric hindrance as in the case
of compound 2, and the latter can be affected by the electronic
property (redox potential) of the compounds.
Unit: mM.
120
100
80
60
40
20
0
3g
3h
3f
3a
3b
3
3c
3d
3e
-20
-40
Fig. 2. Comparison of mean cell growth percentage.
18
16
14
12
10
8
Finally, for compounds with substituted benzene rings at N-1
position, we have summarized the SAR as following: 1) a methylene
group between the benzene and triazole moieties is not needed; 2)
Table 4
melonama
colon cancer
lung cancer
CNS cancer
Anticancer activities (GI50) against colon cancers.a
Cell line
Compound
3a
COLO 205 (Adenocarcinoma) 0.19 0.23 0.43 1.32 0.35 0.40 0.39 0.31
3
3b
3c
3e
3f
3g
3h
6
HCC-2998 (Carcinoma)
HCT-116 (Carcinoma)
HCT-15 (Adenocarcinoma)
HT29 (Adenocarcinoma)
KM12 (Adenocarcinoma)
SW-620 (Adenocarcinoma)
Average GI50
0.34 0.45 1.13 1.52 1.40 1.50 1.63 1.65
0.32 0.37 1.55 3.42 1.50 2.66 0.46 1.16
1.29 1.23 2.44 3.48 3.72 1.61 1.35 1.11
0.40 0.38 2.10 4.10 2.51 1.10 0.34 0.55
0.82 0.97 1.38 8.17 2.40 3.11 2.11 1.38
0.55 0.38 1.76 14.0 6.12 3.00 1.99 1.66
0.56 0.57 1.54 5.14 2.57 1.91 1.18 1.12
4
2
0
3g 3h 3f 3a 3b
3
3c 3e
a
Unit:
m
M.
Fig. 3. Comparison of GI50 (nM).

100
J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
resulting solution was heated at 60 ꢁC for 2 days. After removal of
the solvent, diethyl ether (25 mL) was added and the reaction
mixture was stirred until the products precipitated. The precipi-
tated products were collected with a Hirsh funnel, washed with
more diethyl ether (25 mL), and dried under vacuum. The yields
were calculated based on the estimated amount of aryl azides used.
5.2.1. 1-Phenyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (6)
This is a known compound [12] and was synthesized according
to our general procedures for the preparation of aryl azides and
[3 þ 2] cycloaddition/oxidation with 27% yield. 1H NMR (300 MHz,
Fig. 4. Dialkyled cationic anthraquinone analogs.
CDCl3)
d 8.4 (m, 1H), 8.2 (m, 1H), 7.8e7.9 (m, 2H), 7.7e7.8 (m, 2H),
electron-donating substituents at the para position of the benzene
ring increase the anticancer activities; and 3) reduced steric hin-
drance is beneficial for anticancer activities.
7.6e7.7 (m, 3H). 13C NMR (100 MHz, CDCl3)
d 177.2, 174.5, 146.2,
135.5, 135.4, 134.7, 133.6, 133.4, 133.3, 131.1, 129.5(2C), 128.0, 127.9,
125.3(2C).
4. Conclusion
5.2.2. 1-(4-Methoxyphenyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-
dione (6a)
This is a known compound [13] and was synthesized according
to our general procedures for the preparation of aryl azides and
[3 þ 2] cycloaddition/oxidation with 33% yield.
In conclusion, we have developed a concise synthesis of cationic
anthraquinone analogs bearing aryl substituents. The simplicity in
the synthesis can enable a scale-up production when needed and
greatly increase the chance of developing practical therapeutics.
Investigation of the SAR also reveals the structural factors for
tuning the biological activities (anticancer vs. antibacterial) of these
compounds. These discoveries may lead to the development of
novel anticancer therapeutics. It will also help to the development
of antibacterial agents with less cytotoxicity.
5.2.3. 1-p-Tolyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (6b)
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
oxidation with 45% yield. 1H NMR (300 MHz, CDCl3)
d 8.38
(dd, J ¼ 7.5, 1.4 Hz, 1H), 8.21 (dd, J ¼ 7.4, 1.4 Hz, 1H), 7.84 (m, 2H),
7.61 (d, J ¼ 8.4 Hz, 2H), 7.41 (d, J ¼ 8.2 Hz, 2H), 2.50 (s, 3H); 13C NMR
5. Experimental section
(75 MHz, CDCl3) d 177.1, 174.4, 145.6, 141.4, 135.2, 134.6, 133.4, 133.3,
133.2, 132.9, 129.9 (2C), 127.8, 127.7, 124.9 (2C), 21.5; ESI/APCI calcd.
5.1. General procedure for the preparation of aryl azides
for C17H11N3Oþ2 ([MH]þ) m/z 290.0924; measured m/z 290.0931.
To a solution of substituted anilines (46.7 mmol) in water (50 mL),
conc. HCl (11.2 mL, 37% v/v) was added. The reaction mixture was
cooled to 0 ꢁC and NaNO2 (46.7 mol) dissolved into 10 mL water was
added. After being stirred for 10 min, NaN3 (60.0 mmol) was added
portion wise. The mixture was stirred for 1.5 h at r.t. Formation of an
oily compound was observed. The mixture was extracted with ether
(3 ꢂ 25 mL), and then the ether extracts were washed with water
(2 ꢂ 50 mL) and once with brine (50 mL). The organic layer was dried
over anhydrous MgSO4 or Na2SO4, filtered and concentrated to about
1/10 of the original volume. The resulting aryl azides were used
without further purification.
5.2.4. 1-(4-Trifluoromethylphenyl)-1H-naphtho[2,3-d][1,2,3]
triazole-4,9-dione (6c)
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
oxidation with 30% yield. 1H NMR (300 MHz, CDCl3)
d
8.4 (m, 1H),
176.5,
8.2 (m, 1H), 7.8e8.0 (m, 6H); 13C NMR (100 MHz, CDCl3)
d
174.5, 146.0, 133.8, 133.0,133.7, 135.6, 134.9,133.2, 128.1, 127.9, 126.7
(2C), 125.7 (2C); ESI/APCI calcd. for C17H9F3N3Oþ2 ([MH]þ) m/z
344.0641; measured m/z 344.0643.
5.2.5. 1-(4-Chlorophenyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-
dione (6d)
5.2. General procedure for [3 þ 2] cycloaddition/oxidation:
synthesis of compounds 6 and 6aej
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
oxidation with 32% yield. 1H NMR (300 MHz, CDCl3)
d 8.37
To a solution of aryl azides (estimated 3.2 mmol) in DMF
(10 mL), 1,4-naphthoquinone (1.0 g, 6.3 mmol) was added, and the
(dd, J ¼ 7.6, 1.7 Hz, 1H), 8.21 (dd, J ¼ 7.6, 1.7 Hz, 1H), 7.83 (dq, J ¼ 7.6,
1.7 Hz, 2H), 7.6e7.7 (m,2H), 7.5e7.6 (m, 2H); 13C NMR (75 MHz,
Table 7
CDCl3)
d 176.9, 174.9, 146.1, 137.1, 135.4, 134.8, 134.7, 133.8, 133.4,
Comparison of antibacterial and anticancer activities.
133.1, 133.1, 129.6 (2C), 127.9, 127.7, 126.4 (2C); ESI/APCI calcd. for
16H9ClN3Oþ2 ([MH]þ) m/z 310.0378; measured m/z 310.0381.
Entry
Compound
MIC against
Mean cell growth
percentage (%)
C
S. aureusa
(mg/mL)
1
2
3
4
5
6
7
8
3
3a
3b
3c
3d
3e
3f
3g
3h
1
8
4e8
4
ND
ND
ND
16
32
4
4e8
2
0.032
ꢀ10.18
ꢀ24.70
11.53
58.69
101.70
43.38
5.2.6. 1-(4-Fluorophenyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-
dione (6e)
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
oxidation with 30% yield. 1H NMR (300 MHz, CDCl3)
d 8.38
23.23
(dd, J ¼ 7.6, 1.7 Hz, 1H), 8.21 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.8e7.9 (m, 2H),
ꢀ27.06
ꢀ31.51
ꢀ37.9
ꢀ46.0
44.8
7.7e7.8 (m, 2H), 7.3 (m, 2H); 13C NMR (100 MHz, CDCl3)
d
177.1,
9
174.5, 163.9 (d, JCeF ¼ 251 Hz), 146.1, 135.5, 134.8, 133.8, 133.5, 133.2
10
11
12
2
9
(2C), 131.5, 128.0, 127.9, 127.4 (d, JCeF ¼ 9 Hz, 2C), 116.6 (d, JCe
¼ 23.5 Hz, 2C). ESI/APCI calcd. for C16H9FN3Oþ2 ([MH]þ) m/z
F
a
Staphylococcus aureus (ATCC 25923).
394.0673; measured m/z 294.0678.

J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
101
5.2.7. 1-(3,5-Dimethoxyphenyl)-1H-naphtho[2,3-d][1,2,3]triazole-
4,9-dione (6f)
5.3.3. 4,9-Dioxo-3-methyl-1-p-tolyl-naphtho[2,3-d][1,2,3]triazol-
3-ium trifluoromethanesulfonate (3b)
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 79% yield. 1H NMR (300 MHz,
oxidation with 41% yield. 1H NMR (3200 MHz, CDCl3)
d
8.37
CD3OD)
d
8.4 (m, 1H), 8.3 (m, 1H), 8.0 (m, 21H), 7.76 (d, J ¼ 8.5 Hz,
(d, J ¼ 7.3 Hz, 1H), 8.22 (d, J ¼ 8.2 Hz, 1H), 7.8e7.9 (m, 2H), 6.88
2H), 7.57 (d, J ¼ 8.2 Hz, 2H), 4.81 (s, 3H), 2.54 (s, 3H); 13C NMR
(d, J ¼ 1.9 Hz, 2H), 6.6e6.7 (m, 1H), 3.87 (s, 6H); 13C NMR (100 MHz,
(75 MHz, CD3OD) d 172.8,171.0,144.1,136.1,135.8,133.6,131.9,130.6
CDCl3)
d
177.2, 174.3, 161.2, 146.1, 136.8, 135.3, 134.7, 133.5 (3C),
(2C), 130.1, 127.9, 127.6, 125.2, 121.2, 65.6, 40.0, 20.1, 14.1; ESI/APCI
127.9, 103.7, 103.1, 56.0 (2C); ESI/APCI calcd. for C18H14N3O4þ
calcd. for C18H14N3Oþ2 ([M]þ) m/z 304.1081; measured m/z 304.1081.
([MH]þ) m/z 336.0979; measured m/z 336.0971.
5.3.4. 4,9-Dioxo-3-methyl-1-(4-trifluoromethylphenyl)naphtho
[2,3-d][1,2,3]triazol-3-ium trifluoromethanesulfonate (3c)
5.2.8. 1-(3,4,5-Trimethoxyphenyl)-1H-naphtho[2,3-d][1,2,3]
triazole-4,9-dione (6g)
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 90% yield. 1H NMR (300 MHz,
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
CD3OD)
13C NMR (100 MHz, CD3OD)
132.3, 128.2, 127.9, 127.1 (2C), 126.9, 40.5; ESI/APCI calcd. for
18H11F3N3Oþ2 ([M]þ) m/z 358.0798; measured m/z 358.0788.
d
8.3 (m, 1H), 8.1e8.2 (m, 1H), 7.8e8.0 (m, 6H), 4.67 (s, 3H);
oxidation with 43% yield. 1H NMR (300 MHz, CDCl3)
d 8.7
d
172.6, 171.1, 136.6, 136.3, 136.2, 132.9,
(dd, J ¼ 1.7, 7.2 Hz,1H), 8.04 (dd, J ¼ 1.7, 6.8,1H), 7.8e7.9 (m, 2H), 7.01
(s, 2H), 3.97 (s, 3H), 3.95 (s, 6H); 13C NMR (75 MHz, CDCl3)
d
177.1,
C
174.3, 153.4 (2C), 145.9, 139.9, 135.3, 134.6, 133.3, 133.0, 130.7, 127.8
(2C), 102.8 (2C), 61.2, 56.6; ESI/APCI calcd. for C19H16N3Oþ5 ([MH]þ)
m/z 366.1084; measured m/z 366.1084.
5.3.5. 1-(4-Chlorophenyl)-4,9-dioxo-3-methylnaphtho[2,3-d][1,2,3]
triazol-3-ium trifluoromethanesulfonate (3d)
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 99% yield. 1H NMR (300 MHz,
5.2.9. 1-(3,4-Dimethoxyphenyl)-1H-naphtho[2,3-d][1,2,3]triazole-
4,9-dione (6h)
CD3OD)
d
8.1e8.2 (m, 1H), 7.9 (m, 2H), 7.7e7.9 (m, 3H), 7.7 (m, 2H),
172.5, 170.9, 139.2, 136.2,
4.76 (s, 3H). 13C NMR (75 MHz, CH3OD)
d
This compound was synthesized according to the general pro-
cedures for the preparation of aryl azides and [3 þ 2] cycloaddition/
135.9, 135.8, 132.9, 132.2 (2C), 129.9 (2C), 128.0, 127.7, 127.2 (2C),
40.3; ESI/APCI calcd. for C17H11ClN3Oþ2 ([M]þ) m/z 324.0534;
measured m/z 324.0544.
oxidation with 45% yield. 1H NMR (300 MHz, CDCl3)
d 8.6
(d, J ¼ 7.2 Hz, 1H), 8.21 (d, J ¼ 7.6 Hz, 1H), 7.8 (m, 2H), 7.32
(d, J ¼ 8.6 Hz, 2H), 7.03 (d, J ¼ 8.6 Hz, 1H), 3.99 (s, 3H), 3.96 (s, 3H);
5.3.6. 1-(4-Fluorophenyl)-4,9-dioxo-3-methylnaphtho[2,3-d][1,2,3]
triazol-3-ium trifluoromethanesulfonate (3e)
13C NMR (75 MHz, CDCl3)
d 177.1, 174.4, 150.9, 149.2, 145.9, 135.2,
134.5, 133.3, 133.1, 128.2, 127.8 (3C), 117.7, 110.7, 108.6, 56.4, 56.3;
ESI/APCI calcd. for C18H14N3Oþ4 ([MH]þ) m/z 336.0979; measured m/
z 336.098.
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 69% yield. 1H NMR (300 MHz,
CD3OD)
7.9e8.0 (m, 2H), 7.5 (m, 2H), 4.80 (s, 3H); 13C NMR (100 MHz,
CD3OD)
d 8.4 (m, 1H), 8.3 (m, 1H), 8.0e8.1 (m, 2H), 7.9e8.0 (m, 2H).
5.3. General procedure for N-3 alkylation: synthesis of compounds
3 and 3aeh
d
171.2, 169.7, 163.7 (d, JCeF ¼ 252 Hz), 134.7(2C), 134.5 (2C),
131.5, 128.4, 127.0 (d, JCeF ¼ 9 Hz, 2C), 126.6, 126.3, 115.3 (d, JCe
¼ 24.1 Hz, 2C), 38.8. ESI/APCI calcd. for C17H11FN3Oþ2 ([M]þ) m/z
F
To a solution of compounds 6aej (0.35 mmol) in anhydrous
toluene (10 mL), methyl trifluoromethanesulfonate (0.15 mL,
1.38 mmol) was added, and the resulting solution was heated at
100 ꢁC overnight. After removal of the solvent, diethyl ether (25 mL)
was added and the reaction mixture was stirred for 30 min. The
precipitated products were collected with a Hirsh funnel, washed
with more diethyl ether (25 mL), and dried under the vacuum.
308.0832; measured m/z 308.0838.
5.3.7. 1-(3,5-Dimethoxyphenyl)-4,9-dioxo-3-methylnaphtho[2,3-d]
[1,2,3]triazol-3-ium trifluoromethanesulfonate (3f)
This compound was synthesized according to the general pro-
cedures for N-3 alkylation with 95% yield. 1H NMR (300 MHz,
CD3OD)
(d, J ¼ 2.4 Hz, 2H), 6.91 (t, J ¼ 2.0 Hz, 1H), 4.81 (s, 3H), 3.88 (s, 6H);
13C NMR (75 MHz, DMSO-d6)
173.3, 171.4, 161.4 (2C), 137.1, 136.7,
d 8.3e8.4 (m, 1H), 8.04 (m, 1H), 8.0 (m, 2H), 7.04
5.3.1. 4,9-Dioxo-3-methyl-1-phenylnaphtho[2,3-d][1,2,3]triazol-3-
ium chloride (3)
d
135.9, 135.0, 133.4, 132.1, 128.5, 128.2, 104.7 (2C), 56.7 (2C), 40.3;
ESI/APCI calcd. for C19H16N3Oþ4 ([M]þ) m/z 350.1135; measured m/z
350.1137.
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 74% yield. 1H NMR (300 MHz,
CD3OD)
d
8.4 (m, 1H), 8.3 (m, 1H), 7.9e8.1 (m, 4H), 7.8e7.9 (m, 1H),
172.7,
7.7e7.8 (m, 2H), 4.83 (s, 3H); 13C NMR (75 MHz, CD3OD)
d
5.3.8. 4,9-Dioxo-3-methyl-1-(3,4,5-trimethoxyphenyl)naphtho[2,3-
d][1,2,3]triazol-3-ium trifluoromethanesulfonate (3g)
171.0, 136.2, 136.1, 135.9, 138.8, 133.9, 133.1, 132.8, 132.3, 129.6(2C),
128.0, 127.7, 125.8(2C), 40.3; ESI/APCI calcd. for C17H12N3Oþ2 ([M]þ)
m/z 290.0924; measured m/z 290.0924.
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 99% yield. 1H NMR (300 MHz, DMSO-
d6)
d
8.4 (m, 1H), 8.3 (m, 1H), 8.0 (m, 2H), 7.19 (s, 2H), 4.7e4.8
173.4, 171.5,
5.3.2. 4,9-Dioxo-1-(4-methoxyphenyl)-3-methylnaphtho[2,3-d]
[1,2,3]triazol-3-ium trifluoromethanesulfonate (3b)
(m, 3H), 3.88 (s, 9H); 13C NMR (75 MHz, DMSO-d6)
d
153.7, 148.4 (2C), 139.9, 137.0, 136.6, 135.9, 133.4, 132.2, 128.6, 128.1,
124.2, 104.1 (2C), 60.9, 57.1 (2C), 40.3. ESI/APCI calcd. for
C
This compound was synthesized according to the general pro-
cedure for N-3 alkylation with 80% yield. 1H NMR (300 MHz,
20H18N3Oþ5 ([M]þ) m/z 380.1241; measured m/z 380.1231.
CD3OD)
7.2e7.3 (m, 2H), 4.81 (s, 3H), 3.98 (s, 3H); 13C NMR (100 MHz,
CD3OD) 172.9, 171.1, 163.4, 136.2 (2C), 135.9, 135.6, 133.2, 132.3,
d 8.4 (m, 1H), 8.3 (m, 1H), 8.0e8.1 (m, 2H), 7.8e7.9 (m, 2H),
5.3.9. 1-(3,4-Dimethoxyphenyl)-4,9-dioxo-3-methylnaphtho[2,3-d]
[1,2,3]triazol-3-ium trifluoromethanesulfonate (3h)
d
128.1, 127.7, 127.2 (2C), 126.3, 114.7 (2C), 55.4, 40.2; ESI/APCI calcd.
This compound was synthesized according to the general pro-
for C18H14N3Oþ3 ([M]þ) m/z 320.1030; measured m/z 320.1038.
cedure for N-3 alkylation with 94% yield. 1H NMR (300 MHz, DMSO-

102
J.P. Shrestha et al. / European Journal of Medicinal Chemistry 77 (2014) 96e102
d6)
d
8.3 (m, 1H), 8.2 (m, 1H), 8.0e8.1 (m, 2H), 7.56 (s, 1H), 7.50
Appendix A. Supplementary data
(dd, J ¼ 2.0, 8.6 Hz, 1H), 7.33 (d, J ¼ 8.6 Hz, 1H), 4.75 (s, 3H), 3.90
(s, 3H), 3.80 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d
173.4, 171.5,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.060.
152.5, 149.4, 137.0, 136.6, 136.1, 136.0, 133.4, 132.2, 128.5, 128.2,
123.2, 118.9, 112.1, 109.5, 56.7, 56.6, 41.7. ESI/APCI calcd. for
C
19H16N3Oþ4 ([M]þ) m/350.1135; measured m/z 350.1129.
References
5.4. Procedure for MIC determination
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A solution of selected bacteria (S. aureus) was inoculated in the
Trypticase Soy broth at 35 ꢁC for 1e2 h, after which the bacterial
concentration was found and diluted with broth, if necessary, to an
absorption value of 0.08e0.1 at 625 nm. The adjusted inoculated
medium (100
to a 96-well microliter plate (50
m
L) was diluted with 10 mL of broth, and then applied
L). A series of solutions (50 mL
m
each in 2-fold dilution) of the tested compounds was added to the
testing wells. The 96-well plate was incubated at 35 ꢁC for 12e18 h.
The minimum inhibitory concentration (MIC) is defined as the
minimum concentration of compound needed to inhibit the
growth of bacteria. The MIC results are repeated at least three
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We thank the NCI for conducting the single- and five-dose
assays. We thank the support from Utah State University and
Department of Chemistry and Biochemistry, Utah State
University.