Synthesis and antibacterial activity study of a novel class of cationic anthraquinone analogs

Article abstract of DOI:10.1016/j.bmc.2010.11.001

Reported previously by our group, one-pot cycloaddition using naphthoquinone, sodium azide and alkyl halides can lead to the formation of both 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones. Herein, the effect of leaving group and additive i

Full text of DOI:10.1016/j.bmc.2010.11.001

Bioorganic & Medicinal Chemistry 19 (2011) 498–503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antibacterial activity study of a novel class of cationic
anthraquinone analogs
Jianjun Zhang a, Nathan Redman a, Anthony Phillip Litke a, Jia Zeng b, Jixun Zhan b, Ka Yee Chan a,
Cheng-Wei Tom Chang a,
⇑
a Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300, USA
b Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Reported previously by our group, one-pot cycloaddition using naphthoquinone, sodium azide and alkyl
halides can lead to the formation of both 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones.
Herein, the effect of leaving group and additive in dictating the selectivity between the formation of
1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones has been further investigated. In the pro-
cess of investigating the factors that control the selectivity and the biological activity associated with
these two compounds, a novel class of antibacterial cationic anthraquinone analogs has been developed.
Although these compounds are structurally similar, different antibacterial profiles are noted. One lead
Received 14 September 2010
Revised 28 October 2010
Accepted 1 November 2010
Available online 4 November 2010
Keywords:
Antibacterials
Antibiotics
compound, 4e manifests high potency (MIC < 1 lg/mL) and selectivity against Gram positive (G+) patho-
gens including methicillin-resistant Staphylococcus aureus (MRSA) while exerting only modest activity
against Gram negative (Gꢀ) bacteria. Other lead compounds (4f and 4g) exhibit broad antibacterial activ-
ity including MRSA and vancomycin-resistant Enterococcus faecalis (VRE) that is comparable to other
commercially available cationic antiseptic chemicals. This unique difference in antibacterial profile
may pave the way for the development of new therapeutic agents.
Anthraquinone derivatives
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
and alkyl halides, we have recently reported a concise one-pot
divergent synthesis of 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-
Molecules containing naphthoquinone and anthraquinone scaf-
folds have long attracted great interest due to their important
biological and pharmaceutical applications leading to the develop-
ment and discovery of numerous therapeutics (Fig. 1).1,2 For exam-
ple, 1,4-naphthoquinone derivatives have been studied for their
diverse biological activities including uses as antibacterial,3,4 anti-
fungal,5 antimalarial,6 and antitumor agents,7,8 or being employed
as inhibitors against vitamin K dependent carboxylase,9 protein
kinase,10 coenzyme Q,11 and even as growth stimulator for bif-
idobacteria.12 Anthraquinone, which bears the structural core of
anthracycline, has also attracted great interest due to its applica-
tions as antibiotics or anticancer agents.13 Finally, naphthoquinone
and anthraquinone are known to uncouple mitochondria oxidative
phosphorylation leading to mechanistic investigations in their
redox chemistry and related applications, such as the use as new
material for photocell.14
d]triazole-4,9-diones (2 and 3), both of which can be viewed as
analogs of anthraquinone or naphthoquinone fused with 1,2,3-tri-
azole (Fig. 2).16 Since a wide range of biological activities and
O
OH
OH
O
O
O
OH
OH
Cl
Cl
O
Me
OH
OH
O
O
pleosporone
antibacterial
Phygon
antifungal
psychorubrin
antitumor
O
OH
OH
O
OH
O
OH
OH
CH3
The syntheses of naphthoquinone and anthraquinone deriva-
tives, in general, require multi-step processes and begin from var-
ious starting materials.15 Using naphthoquinone, 1, sodium azide,
OCH3
O
O
Daunorubicin
O
naphthazarin
anticancer
antibiotic
O
antibacterial
H3C
HO
NH2
⇑
Corresponding author. Tel.: +1 435 797 3545.
E-mail addresses: tom.chang@usu.edu, chang@cc.usu.edu
Figure 1. Examples of molecules containing naphthoquinone and anthraquinone
(Cheng-Wei Tom Chang).
scaffolds.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.001

J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 498–503
499
O
O
O
and 3 vs 4). The poorest leaving group, CF3CO2, gave the lowest ra-
tio of 2d/3d, or better relative yield for the formation of 2-alkyl-
2H-naphtho[2,3-d]triazole-4,9-dione (entry 7). Nevertheless, the
overall yields when poor leaving groups were employed decreased
dramatically probably due to the degradation of products or
intermediates.
O
O
NaN3
N
N
N
R
Br
N
+
N
R
solvent
120oC
N
R
1
O
2-alkyl-2H-naphthotriazole-
4,9-dione, 3
1-alkyl-1H-naphthotriazole-
4,9-dione, 2
2.2. Synthesis of cationic anthraquinone analogues
Figure 2. One-pot divergent synthesis of 1-alkyl-1H- and 2-alkyl-2H-naphtho[2,3-
d]triazole-4,9-diones.
In our initial studies on the synthesis of 1-alkyl-1H-naph-
tho[2,3-d]triazole-4,9-diones, carbohydrates were incorporated as
the alkyl group (R) group.19 Unfortunately, the carbohydrate moi-
eties were unsuitable for the synthesis of 2-alkyl-2H-naph-
tho[2,3-d]triazole-4,9-diones following our one-pot protocol.16
Only alkyl groups are applicable in the synthesis of both 1-alkyl-
1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones. However,
both 1-alkyl-1H- and 2-alkyl-2H-alkylated naphtho[2,3-d]tria-
zole-4,9-diones were completely insoluble in aqueous media mak-
ing these two classes of compounds not suitable for biological
evaluation. Thus, our effort was directed to improve the solubility
for these anthraquinone analogs. One of the feasible approaches is
to convert these molecules into cationic compounds via methyla-
tion at the N-3 of the triazole motif. After several attempts, meth-
ylation using MeOTf proves to be effective in methylating the
1-alkyl-1H-alkylated naphtho[2,3-d]triazole-4,9-diones but not
the 2-alkyl-2H-alkylated naphtho[2,3-d]triazole-4,9-diones (Scheme 1).
The latter appears to yield intermediate or product that is too
unstable to be isolated upon methylation. Similar results were
noted with carbohydrates or adamantane attached at N-1 of
1-alkyl-1H-alkylated naphtho[2,3-d]triazole-4,9-diones. After meth-
ylation, the TfOꢀ anion was exchanged with Clꢀ using ion-
exchange resin. This protocol enables the synthesis of compounds
4a–h. The synthesis of compound 4i began with a Boc-protected 1-
(4-(N-tert-butoxycarbonylpiperidinyl))-1H-naphtho[2,3-d]triazole-
4,9-dione (2i)19 (Scheme 2). During the methylation step, the
by-product TfOH also triggered the deprotection of Boc group
and provided the desired product after ion-exchange. The synthe-
sis of 4j started with the synthesis of 2-picolyl azide followed by
the cycloaddition protocol as described previously (Scheme 2).19
The resulting cycloaddition product 2j can be converted to the
practical applications can be associated with these compounds, we
began to investigate the factors that can govern the selectivity be-
tween these two classes of compound.
2. Results and discussion
2.1. Investigation of the effect in controlling selectivity
In our one-pot divergent synthesis of 1-alkyl-1H- and 2-alkyl-
2H-naphtho[2,3-d]triazole-4,9-diones, we have investigated the ef-
fect of solvents on the product ratio by using DMF, and toluene as
the solvents. We have noted that less polar solvents, such as tolu-
ene, favor slightly the formation of 2-alkyl-2H-naphtho[2,3-d]tria-
zole-4,9-diones.16 However, the cycloaddition reactions run in
toluene offer less satisfactory yields, probably, due to the degrada-
tion of the products or intermediates. Therefore, we started to look
for other factors, such as leaving group and additive that may offer
the selectivity without scarifying the overall yields of both 1-alkyl-
1H- and 2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones.
We employed pentyl group as the alkyl group to be incorpo-
rated. Various leaving groups including bromide, chloride, tosylate
(TsO), mesylate (MsO), and trifluoroacetate (CF3CO2), were investi-
gated in the optimal condition established previously (Table 1).
The pKa of the conjugated acids of leaving groups has been used
as the measurement of leaving group capability.17 Based on the
documented pKa’s of HI (ꢀ10), HBr (ꢀ9), HCl (ꢀ8), TsOH (ꢀ2.8),
MsOH (ꢀ2.6), and trifluoroacetic acid (0.23),18 we estimate that io-
dide is the best leaving group and trifluoroacetate is the poorest.
From the ratio of 2d/3d determined by 1H NMR, we observed an
approximate trend that better leaving groups (Br) favor the forma-
tion of 1-alkyl-1H-naphtho[2,3-d]triazole-4,9-diones while poor
leaving groups (MsO and CF3CO2) prefer the formation of 2-alkyl-
2H-naphtho[2,3-d]triazole-4,9-diones. Bromide is a good leaving
group for the formation of 1-alkyl-1H-naphtho[2,3-d]triazole-4,9-
diones offering a higher ratio of 2d/3d. Iodide is even a better leav-
ing group as compared to chloride or bromide. The results from
addition of TBAI, which can generate iodide as the best leaving
group in situ, further corroborate this observation (entries 1 vs 2
O
O
O
O
O
O
Me
Me
MeOTf
toluene
100oC
Dowex 1X-8
(Cl-form)
N
N
N
N
N
N
N
N
N
R
Cl-
R
TfO-
R
4a-h
2a-h
Compound
2a16
R
Product
4a
Yield (%)
81%
CH3
Table 1
The ratio of 2d/3d
2b16
CH2CH3
4b
82%
O
O
O
O
2c16
CH2(CH2)2CH3
CH2(CH2)3CH3
CH2(CH2)6CH3
CH2(CH2)10CH3
CH2(CH2)14CH3
Bn
4c
84%
O
3
NaN3
C5H
X: leaving groups
DMF, 120oC
N
N
N
N2
+
11 X
N
C5H11
2d16
4d
84%
N1
C5H11
2e16
4e
99%
3d
2d
O
1
Entry Leaving group (X)/additive 1H:2H ratio (2d/3d) Overall yield (%)
2f16
4f
88%
1
2
3
4
5
6
7
Bromide/TBAI
Bromide
Chloride/TBAI
Chloride
Tosylate (TsO)
Mesylate (MsO)
Trifluoroacetate (CF3CO2)
7.12:1.00
5.00:1.00
1.27:1.00
1.02:1.00
1.58:1.00
1.17:1.00
1.00:1.06
98
75
76
99
75
29
38
2g16
4g
43%
2h16
4h
85%
Scheme 1.

500
J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 498–503
1) MeOTf, toluene
creases as the number of carbon of the alkyl group increases. How-
O
O
O
O
100oC
Me
Cl-
ever, significant antibacterial activity emerges as the chain length
reaches eight carbons (octyl group, 4e), and such high activity re-
mains even with sixteen carbons (hexadodecyl group, 4g). Never-
theless, compound 4e (C8) has different antibacterial profile as
compared to compounds 4f (C12) and 4g (C16) particularly against
Gꢀ bacteria and E. faecalis. The former compound, 4e shows high
antibacterial activity selectively toward G+ bacteria except
E. faecalis. Compounds with shorter linear alkyl chain than 4e also
display similar antibacterial profile as 4e. The latter two com-
pounds, 4f and 4g manifest rather broad antibacterial activity
against Gꢀ and G+ strains comparable to the commonly used cat-
ionic control, HTB.
2) Dowex 1X-8
(Cl-form)
N
N
N
N
N
N
4i
2i
99% (2 steps)
N
N
H
Boc
1) MeOTf, toluene
100oC
O
1) NaN3, nBu4N-HSO4
CH2Cl2, NaHCO3(aq)
2) Dowex 1X-8
(Cl-form)
N
N
N
Cl
2) DMF, 120oC, 2 days
O
N
HCl
O
Enterococci are facultative anaerobic organisms that can thrive
in both oxygen-rich and oxygen-deficient environments.20 The lack
of activity of 4e against E. faecalis is specially interesting since it
implies that 4e and compounds with shorter alkyl chains (4a–d)
have a different antibacterial mode of action from 4f and 4g. Com-
mercially available cationic antibacterial agents, such as HTB, carry
a lipophilic alkyl chain with length around twelve to eighteen car-
bons, which often lowers the solubility of these agents in aqueous
media. The shorter chain length of 4e is potentially advantageous
since it can be quite soluble in aqueous media unlike HTB. Finally,
all the cationic compounds including HTB are not very activity
against P. aeruginosa, which is known to exert drug resistance via
lowering its membrane permeability.21
Two structural motifs are expected to be responsible for the ob-
served antibacterial activity: the cationic anthraquinone analog
and the alky groups at the N-1 position. As mentioned previously,
the neutral 1-alkyl-1H-naphtho[2,3-d]triazole-4,9-diones, com-
pound 2a–j are insoluble in aqueous media making it difficult to
evaluate the antibacterial activity of these compounds. We have
previously prepared 1-alkyl-1H-naphtho[2,3-d]triazole-4,9-diones
with carbohydrates attached to the N-1 position which enables
these compounds to have moderate to excellent solubility in aque-
ous media (Fig. 3).19 However, these compounds were found to be
inactive against E. coli (ATCC 25922) and S. aureus (ATCC 25923) in
the diffusion assay with no observable zone of inhibition. There-
fore, the cationic form of the anthraquinone analog is crucial for
the antibacterial activity.
The chain length of the linear alkyl groups also plays important
role with the optimal number of carbon ranging from C8 to C16.
Commonly used antiseptic quaternary ammonium compounds of-
ten contain linear lipophilic alkyl chains (C12–C18). The amphi-
philic property of these cationic agents allows the molecules to
exert their antibacterial activity by disrupting the bacterial mem-
brane.22 Since compound 4e (C8) also exert excellent antibacterial
activity as compared to 4f and 4g, membrane disruption that is re-
lated to the lipophilicity of the commercially used cationic antisep-
tic agents may not offer sole explanation for the observed activity
of 4e. We speculate that it is a combination of the cationic nature
and the octyl (C8) group that contributes to the exceptionally high
antibacterial activity of 4e via an unknown mode of action, which
seems to be specific toward G+ bacteria. Noticeable antibacterial
activities, which are also specific toward G+ bacteria for com-
pounds with shorter alkyl chains (4a–d, C2–C5) implies that the
cationic anthraquinone moiety plays an important role in the anti-
bacterial activity of these compounds. As the chain length ex-
tended to C12 and C16, the increased lipophilicity of 4f and 4g
begins to exert broad antibacterial activity like other cationic anti-
septic agents. The antibacterial profile of 4f and 4g falls in-line
with other cationic compounds suggests that the dominant anti-
bacterial mode of action is still on the perturbation of bacterial
membrane for these two compounds. Thus, the lead compound
with octyl group, 4e insinuates an optimal combination of cationic
anthraquinone moiety and the lipophilicity of octyl group to
2j
N
60%
O
O
Me
Cl-
N
N
N
O
4j
N
85% (2 steps)
Scheme 2.
corresponding cationic adduct 4j via the same procedure. Overall,
above investigations on the factors for selective synthesis of 1-al-
kyl-1H-naphtho[2,3-d]triazole-4,9-dione is helpful in optimizing
the synthesis of the novel cationic analogs (4a–j).
2.3. Antibacterial study and discussion
With the structural character of both cation and naphthoqui-
none, the resulting molecules may have duel properties found in
cationic antibiotics and naphthoquinone-based therapeutics. A ser-
ies of these cationic 1-alkyl-3-methyl-1H-naphtho[2,3-d]triazole-
4,9-diones were tested against various Gram positive (G+) and
Gram negative (Gꢀ) bacteria including Escherichia coli (Gꢀ, ATCC
25922), Staphylococcus aureus (G+, ATCC 25923), Klebsiella pneumo-
niae (Gꢀ, ATCC 13883), Pseudomonas aeruginosa (Gꢀ, ATCC 27853),
Mycobacterium smegmatis (G+, ATCC 14468), methicillin-resistant
S. aureus (G+, ATCC 33591) (MRSA), vancomycin-resistant Entero-
coccus faecalis (G+, ATCC 51299) (VRE), and E. faecalis (G+, ATCC
29212). Similar cationic compounds, such as benzyldimethyl-
hexadecylammonium chloride (BDC), hexadecylpyridinium bro-
mide (HPB), hexadecyltrimethylammonium bromide (HTB), and
clinical used antibiotics were employed as the controls. Due to
the relatively modest solubility of BDC and HPB in aqueous media,
most of the assay employed only HTB as the control.
In general, these compounds are much more active against G+
bacteria than Gꢀ bacteria. From the minimum inhibition concen-
tration (MIC) generated from compounds with linear alkyl group,
we were impressed with the high potency of some of the com-
pounds against, in particular, the Gram positive bacteria (Table
2). For example, against S. aureus and MRSA, the MIC’s for 4e, 4f,
and 4g are lower than all the controls employed in this study.
The MIC’s of 4e and 4f are even in mid-nanomolar range. Com-
pounds with non-linear alkyl groups, 4h (Bn), 4i (4-piperidinyl),
and 4j (2-picolyl) are less active than those with linear alkyl
groups. Although compound 4h seems to be the most active one
among these three, which could imply the positive role of lipophil-
icity of the Bn group, the pool of this type of compounds is too
small to make affirmative conclusion. Rather clearer structure–
activity relationship (SAR) can be deduced from the compounds
with linear alkyl groups. The antibacterial activity slightly in-

J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 498–503
501
Table 2
Minimum inhibitory concentration (MIC)a
Entry
Compounds
E. colib
S. aureusc
K. pneumoniaed
S. aureuse
P. aeruginosaf
M. smegmatisg
E. faecalish
E. faecalisi
j
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Neomycin
Kanamycin
Vancomycin
Amikacin
BDC
HPB
HTB
4a
4b
4c
4d
4e
4f
4g
4h
4i
4
2–4
P250
1
ND
1
1–2
1
0.5–1
2–4
2–4
0.5–1
4–8
1–2
2–4
2
0.032–0.064
0.032
0.125–0.25
2
2
125
64
ND
1–2
ND
16–32
ND
1–2
32–64
ND
ND
125–250
ND
125
P250
ND
ND
0.5–1
P250
1
ND
ND
125–250
0.5–1
16
1
4
2
4–8
1–2
1–2
125–250
P250
0.25
ND
ND
0.25
ND
ND
1
ND
1
1
64
16–32
125
2–4
4–8
32–64
16–32
16–32
8–16
8
32–64
16–32
16
8–16
1
1–2
2–4
8–16
16
P250
P250
P250
P250
125–250
4–8
125–250
P250
P250
125–250
125–250
4–8
32–64
32–64
32
8–16
2–4
2–4
8–16
64
P250
P250
P250
P250
16
P250
125–250
64
1–2
0.5–1
0.25–0.5
0.25–0.5
0.5–1
2–4
2–4
0.5–1
8–16
32–64
16–32
2–4
4–8
P250
P250
P250
P250
P250
P250
2–4
2–4
4j
16–32
2–4
P250
16
a
b
c
d
e
f
Unit: lg/mL.
Escherichia coli (ATCC 25922).
Staphylococcus aureus (ATCC 25923).
Klebsiella pneumoniae (ATCC 13883).
Staphylococcus aureus (ATCC 33591) (MRSA).
Pseudomonas aeruginosa (ATCC 27853).
Mycobacterium smegmatis (ATCC 14468).
Enterococcus faecalis (ATCC 29212).
E. faecalis (ATCC51299) (VRE).
g
h
i
j
ND: not determined.
O
N
N
N
naphtho[2,3-d]triazole-4,9-diones, which results the novel cationic
anthraquinone analogs with impressive antibacterial activity. With
the growing interest in using triazoles and ‘Click’ chemistry as the
tools for constructing bioactive molecules, this methylation proto-
col may broaden the potential applications. The prominent activity
and selectivity of 4e may lead to the development of antibacterial
agent and avoid CDI. The broad spectrum activity of 4f and 4g
could have significance of being used as antiseptic agents. These
cationic compounds that contain a combination of alkyl groups
and the cationic anthraquinone or naphthoquinone moieties could
pave the way for the development of novel antibiotics. Ongoing ef-
fort has been directed to the investigation of the possible antibac-
terial modes of action.
O
O
5
(OH)n
Figure 3. 1H-Naphtho[2,3-d]triazole-4,9-diones with N-1 carbohydrates.
achieve the observed significant and selective antibacterial activ-
ity. The interesting selectivity of these compounds against G+ bac-
teria may find additional applications. For example, it may be
possible to employ compound 4e as antibacterial agent and avoid
Clostridium difficile infection (CDI).23 C. difficile is a G+ anaerobic
bacterium and is the most significant cause of pseudomembranous
colitis, a severe infection of the colon, often appears after normal
gut flora is eradicated by the use of antibiotics following surgery.
Many associated deaths have been reported, especially among
the elderly. The presence of beneficial bacteria within our intes-
tines is necessary to help the human body develop properly and
to remain healthy. Since compound 4e is less active against
Gꢀ bacteria, it may be possible to selectively ‘kill’ pathogenic G+
bacteria and avoid CDI. Finally, quaternary ammonium com-
pounds, such as HTB, BDC or HPB are often considered too toxic,
and are limited to topical uses as antiseptics, disinfectants, or pre-
servatives.24 The reduced lipophilicity of compound 4e with short-
er alkyl chains could have potential of being employed as effective
antibiotics. Further investigations on the possible modes of anti-
bacterial action are currently undertaken.
4. Experimental
4.1. General procedure of methylation
To a solution of starting material (ca. 0.05 g) in toluene (10 mL),
MeOTf (4 equiv) was added. The reaction mixture was stirred at
100 °C for 24 h. After completion of the reaction, the solvent was
removed and the crude product was loaded with a short column
packed with Dowex 1X-8 resin (Clꢀ form). The column was eluted
with MeOH (ca. 20 mL). After removal of the solvent, the product
was obtained as brownish solid.
4.2. 1-(2-Picolyl)-1H-naphtho[2,3-d]triazole-4,9-dione (2j)
This compound is synthesized using procedure described in Ref.
19. 1H NMR (CDCl3, 300 MHz) d 8.51 (d, J = 4.8 Hz, 1H), 8.34 (dd,
J = 7.6, 1.4 Hz, 1H), 8.20 (dd, J = 7.5, 1.7 Hz, 1H), 7.85 (td, J = 7.2,
1.4 Hz, 1H), 7.79 (td, J = 7.6, 1.7 Hz, 1H), 7.70 (td, J = 7.6, 1.7 Hz,
1H), 7.32 (d, J = 7.9 Hz, 1H), 7.2 (m, 1H), 6.18 (s, 2H); 13C (CDCl3,
100 MHz) d 177.0, 175.6, 153.4, 150.2, 145.7, 137.4, 135.4, 134.5,
134.1, 133.7, 133.1, 128.1, 127.6, 123.7, 122.3, 55.0; ESI/APCI calcd
3. Conclusion
In conclusion, we have further investigated the effect of leaving
groups in the chemoselectivity for the synthesis of 1-alkyl-1H- and
2-alkyl-2H-naphtho[2,3-d]triazole-4,9-diones. In an effort of
improving the bioavailability of the triazole constructs, the meth-
ylation approach proved be an effective method for 1-alkyl-1H-
for C16H11N14O2 ([M+H]+) m/z 291.0877; measure m/z 291.0879.
þ

502
J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 498–503
4.3. Compound 4a
100 MHz) d 172.6, 172.5, 136.4, 136.2, 136.1, 135.3, 132.9, 132.7,
131.6, 129.8, 129.5 (2 carbons), 129.2 (2 carbons), 128.0, 127.9,
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 4.70
(s, 6H); 13C NMR (CD3OD, 75 MHz) d 172.4 (2 carbons), 136.0 (2
carbons), 135.8 (2 carbons), 132.6 (2 carbons), 127.8 (2 carbons),
57.2, 40.1; ESI/APCI calcd for C18H14N3O2 ([M]+) m/z 304.1081;
þ
measure m/z 304.1077.
47.1 (2 carbons); ESI/APCI calcd for C12H10N3O2 ([M]+) m/z
þ
4.11. Compound 4i
228.0768; measure m/z 228.0772.
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 5.90 (tt,
J = 8.2, 4.1 Hz, 1H), 4.75 (s, 3H), 3.7 (m, 2H), 3.55 (td, J = 14.4,
3.1 Hz, 2H), 2.6 (m, 4H); 13C NMR (CD3OD, 100 MHz) d 172.6,
172.5, 136.22, 136.16 (2 carbons), 136.0, 133.0, 132.5, 128.1,
128.0, 59.7, 42.2 (2 carbons), 40.4, 27.6 (2 carbons); ESI/APCI calcd
4.4. Compound 4b
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 5.14 (q,
J = 7.2 Hz, 2H), 4.71 (s, 3H), 1.74 (t, J = 7.2 Hz, 3H); 13C NMR
(CD3OD, 75 MHz) d 172.6, 172.4, 136.1, 135.9, 135.88, 135.5,
for C16H17N4O2 ([M]+) m/z 297.1346; measure m/z 297.1346.
þ
132.8, 132.6, 127.8, 127.7, 50.0, 39.8, 13.0; ESI/APCI calcd for
C
13H12N3O2 ([M]+) m/z 242.0924; measure m/z 242.0929.
þ
4.12. Compound 4j
1H NMR (CD3OD, 300 MHz) d 8.99 (d, J = 5.5 Hz, 1H), 8.68 (td,
J = 1.7, 0.5 Hz, 1H), 8.4 (m, 3H), 8.2 (t, J = 6.0 Hz, 1H), 8.0 (m, 2H),
6.80 (s, 2H), 4.77 (s, 3H); 13C NMR (CD3OD, 100 MHz) d 172.5,
172.2, 147.3, 145.2, 143.6, 136.5, 136.4, 136.3, 136.1, 132.8,
132.7, 128.6, 128.1, 128.0 (2 carbons), 53.4, 40.7; ESI/APCI calcd
4.5. Compound 4c
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.0 (m, 2H), 5.09 (t,
J = 7.6 Hz, 2H), 4.71 (s, 3H), 2.1 (m, 2H), 1.51 (m, 2H), 1.03 (t,
J = 7.6 Hz, 3H); 13C NMR (CD3OD, 75 MHz) d 172.5, 172.4, 136.1,
136.0, 135.9, 135.5, 132.8, 132.5, 127.8, 127.7, 54.0, 39.8, 30.7,
for C17H13N4O2 ([M]+) m/z 305.1033; measure m/z 305.1034.
þ
19.1, 12.3; ESI/APCI calcd for C15H16N3O2 ([M]+) m/z 270.1237;
þ
4.13. Procedure for MIC determination
measure m/z 270.1242.
A solution of selected bacteria was inoculated in the Trypticase
Soy broth at 35 °C for 1–2 h. After which, the bacteria concentra-
tion was found, and diluted with broth, if necessary, to an absorp-
tion value of 0.08–0.1 at 625 nm. The adjusted inoculated medium
4.6. Compound 4d
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.0 (m, 2H), 5.08 (t,
J = 7.2 Hz, 2H), 4.72 (s, 3H), 2.1 (m, 2H), 1.5 (m, 4H), 0.96 (t,
J = 6.9 Hz, 3H); 13C NMR (CD3OD, 100 MHz) d 172.7, 172.6, 136.3,
(100
lL) was diluted with 10 mL broth, and then applied to a 96-
well microtiter plate (50
l
L). A series of solutions (50 L each in
l
136.1, 136.0, 135.7, 132.9, 132.7, 127.9, 127.8, 54.4, 40.0, 28.6,
28.1, 21.9, 12.9; ESI/APCI calcd for
C
16H18N3O2 ([M]+) m/z
þ
twofold dilution) of the tested compounds was added to the testing
wells. The 96-well plate was incubated at 35 °C for 12–18 h. The
minimum inhibitory concentration (MIC) is defined as the mini-
mum concentration of compound needed to inhibit the growth of
bacteria. The MIC results are repeated at least three times.
284.1394; measure m/z 284.1399.
4.7. Compound 4e
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 5.08 (t,
J = 7.6 Hz, 2H), 4.71 (s, 3H), 2.1 (m, 2H), 1.5 (m, 2H), 1.3 (m, 8H),
0.90 (t, J = 6.9 Hz, 3H); 13C NMR (CD3OD, 75 MHz) d 172.6, 172.5,
136.2, 135.9, 135.87, 135.6, 132.9, 132.6, 127.8, 127.7, 54.2, 39.9,
Acknowledgment
We acknowledge the support from Department of Chemistry
and Biochemistry, Utah State University.
31.6, 28.8, 28.7 (2 carbons), 25.9, 22.4, 13.2; ESI/APCI calcd for
þ
C
19H24N3O2 ([M]+) m/z 326.1863; measure m/z 326.1866.
References and notes
4.8. Compound 4f
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Sons, Inc.: New York, 1998.
2. Gringauz, A. Introduction to Medicinal Chemistry, How Drugs Act and Why?;
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3. Zhang, C.; Ondeyka, J. G.; Zink, D. L.; Basilio, A.; Vicente, F.; Collado, J.; Platas, G.;
Bills, G.; Huber, J.; Dorso, K.; Motyl, M.; Byrne, K.; Singh, S. B. J. Nat. Prod. 2008,
71, 1304.
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1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 5.08 (t,
J = 7.6 Hz, 2H), 4.71 (s, 3H), 2.1 (m, 2H), 1.4 (m, 2H), 1.3 (m,
16H), 0.88 (t, J = 6.5 Hz, 3H); 13C NMR (CD3OD, 100 MHz) d 172.5,
172.4, 136.1, 135.9 (2 carbons), 135.5, 132.8, 132.5, 127.8, 127.7,
54.2, 39.8, 31.7, 29.4 (2 carbons), 29.3, 29.1 (2 carbons), 28.8,
28.7, 25.8, 22.4, 13.1; ESI/APCI calcd for C23H32N3O2 ([M]+) m/z
þ
5. Meazza, G.; Dayan, F. E.; Wedge, D. E. J. Agric. Food Chem. 2003, 51, 3824.
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382.2489; measure m/z 382.2492.
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504.
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13. For example: (a) Fan, E.; Shi, W.; Lowary, T. L. J. Org. Chem. 2007, 72, 2917; (b)
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4.9. Compound 4g
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.1 (m, 2H), 5.08 (t,
J = 7.6 Hz, 2H), 4.71 (s, 3H), 2.1 (m, 2H), 1.5 (m, 2H), 1.3 (m,
24H), 0.88 (t, J = 6.5 Hz, 3H); 13C NMR (CD3OD, 100 MHz) d 172.6,
172.5, 136.2, 136.1 (2 carbons), 135.6, 132.9, 132.6, 127.9, 127.8,
54.3, 39.9, 31.9, 29.6 (5 carbons), 29.4 (2 carbons), 29.2 (2 carbons),
28.9, 28.8, 26.0, 22.2, 13.2; ESI/APCI calcd for C27H40N3O2 ([M]+)
þ
m/z 438.3115; measure m/z 438.3120.
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4.10. Compound 4h
1H NMR (CD3OD, 300 MHz) d 8.4 (m, 2H), 8.0 (m, 2H), 7.6 (m,
2H), 7.4 (m, 3H), 6.30 (s, 2H), 4.94 (s, 3H); 13C NMR (CD3OD,
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