Synthesis and structure-activity relationship of novel bisindole amidines active against MDR Gram-positive and Gram-negative bacteria

Article abstract of DOI:10.1016/j.ejmech.2018.03.031

A series of novel diamidines with N-substituents on an amidine N-atom were synthesized and evaluated for their cytotoxicity and in vitro antibacterial activity against a range of Gram-positive and Gram-negative bacterial strains. Based on structure-activity relationship, N-substituents with a branched chain and a shorter carbon chain on the amidine N-atom exhibited more promising activity against Gram-negative and MDR-Gram-positive bacteria; compounds 5c and 5i were the most powerful candidate compounds. Compound 5c showed greater efficacy than levofloxacin against most drug-resistant Gram-positive bacteria and exhibited broad-spectrum antibacterial activity against Gram-negative bacteria, with MIC values in the range of 2–16 μg/mL. Slightly more potent antibacterial activity against Klebsiella pneumoniae, Acinetobacter calcoaceticus, Enterobacter cloacae, and Proteus mirabilis was observed for 5i in comparison with 5c. Compound 5i also showed remarkable antibacterial activity against NDM-1-producing Gram-negative bacteria, with MIC values in the range of 2–4 μg/mL, and was superior to the reference drugs meropenem and levofloxacin. Effective antibacterial activity of 5i was also shown in vivo in a mouse model of Staphylococcus aureus MRSA strain, with an ED₅₀values of 2.62 mg/kg.

Full text of DOI:10.1016/j.ejmech.2018.03.031

Accepted Manuscript
Synthesis and structure-activity relationship of novel bisindole amidines active against
MDR Gram-positive and Gram-negative bacteria
Yonghua Liu, Xinxin Hu, Yanbin Wu, Weixing Zhang, Xiaofang Chen, Xuefu You,
Laixing Hu
PII:
S0223-5234(18)30274-5
10.1016/j.ejmech.2018.03.031
EJMECH 10296
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 September 2017
Revised Date: 8 March 2018
Accepted Date: 9 March 2018
Please cite this article as: Y. Liu, X. Hu, Y. Wu, W. Zhang, X. Chen, X. You, L. Hu, Synthesis and
structure-activity relationship of novel bisindole amidines active against MDR Gram-positive and Gram-
negative bacteria, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and structure-activity relationship of novel bisindole amidines active against
MDR Gram-positive and Gram-negative bacteria
Yonghua Liu, Xinxin Hu, Yanbin Wu, Weixing Zhang, xiaofang Chen, Xuefu You*, Laixing Hu*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences
Tian Tan Xi Li 1 , Beijing 100050, China
& Peking Union Medical Collage,
#
* Corresponding author:
Yonghua liu, Tel.: +86 10 63170561; E-mail address: lyh75112@163.com
Laixing Hu, Tel.: +86 10 63132113; fax: +86 10 63017302; E-mail address: hulaixing@hotmail.com
Xuefu You, Tel.: +86 10 63165290; fax: +86 10 63017302; E-mail address: xuefuyou@hotmail.com
Abstract
A series of novel diamidines with N-substituents on an amidine N-atom were synthesized and
evaluated for their cytotoxicity and in vitro antibacterial activity against a range of Gram-positive
and Gram-negative bacterial strains. Based on structure-activity relationship, N-substituents with a
branched chain and a shorter carbon chain on the amidine N-atom exhibited more promising
activity against Gram-negative and MDR-Gram-positive bacteria; compounds 5c and 5i were the
most powerful candidate compounds. Compound 5c showed greater efficacy than levofloxacin
against most drug-resistant Gram-positive bacteria and exhibited broad-spectrum antibacterial
activity against Gram-negative bacteria, with MIC values in the range of 2-16 µg/mL. Slightly
more potent antibacterial activity against Klebsiella pneumoniae, Acinetobacter calcoaceticus,
Enterobacter cloacae, and Proteus mirabilis was observed for 5i in comparison with 5c.
Compound 5i also showed remarkable antibacterial activity against NDM-1-producing
Gram-negative bacteria, with MIC values in the range of 2-4 µg/mL, and was superior to the
reference drugs meropenem and levofloxacin. Effective antibacterial activity of 5i was also shown
in vivo in a mouse model of Staphylococcus aureus MRSA strain, with an ED50values of 2.62
mg/kg.
Keywords: Antibacterial; Diamidine; Indole; Broad-spectrum; MRSA; Gram-negative bacteria;
NDM-1
1. Introduction

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The rapid emergence and dissemination of drug-resistant bacteria has become a global threat to
human health. The Gram-negative Klebsiella pneumonia (K. pneumoniae), Acinetobacter
baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Enterobacter species,
in addition to the Gram-positive Enterococcus faecium (E. faecium) and Staphylococcus aureus (S.
aureus), collectively referred to as "ESKAPE" pathogens, have become a prominent cause of
morbility and mortality in the last few decades [1]. The extended-spectrum β-lactamase
(ESBL)-producing Enterobacteriaceae and carbapenemase-producing K. pneumoniae (KPC)
represent the most severe antibiotic resistance crisis, and are associated with high mortality rates
[2]. There are only a limited number of antibacterial agents available to treat these highly resistant
organisms, such as tobramycin, amikacin, tigecycline, minocycline, and colistin, most of which
have significant toxicity [3].
NDM-1 (New Delhi Metallo-beta-lactamase-1), a newly emerged carbapenemase, has generated
worldwide public concern because of its resistance to almost all beta-lactam antibiotics [4].
Additionally, owing to the rapid spread of the plasmid-encoded NDM-1 gene, NDM-1 has been
found mostly among Escherichia coli and K. pneumoniae [5, 6]. Given the increasing number of
reports of their occurrence and detection, multidrug-resistant bacteria (MDR) harbouring the
beta-NDM-1 gene have become a formidable threat to human health, which prompted the World
Health Organization to issue a global warning [7]. At present, the most effective option to treat
MDR NDM-1-producing bacteria is colistin, which has significant side effects, including
nephrotoxicity. Colistin has become a "last resort" antibiotic against MDR Gram-negative bacteria.
However, the isolation of colistin-resistant strains of bacteria has become more frequent, a
colistin-resistance gene was found on a plasmid that could easily move between bacterial species
[8, 9]. The rapid spread of these drug-resistant genes would make the thread of "superbugs" a
genuine possibility.
Despite the approval of 58 new antibiotics by Food and Drug Administration (FDA) or entry
into advanced clinical development (Phase II or III) since 2000, most have mainly targeted
Gram-positive bacteria and only two new compounds have been used specifically against
Gram-negative bacteria. Furthermore, these Gram-negative antibiotics candidates are not capable
of providing coverage against all four serious pathogens described above [10, 11]. Therefore, there
is an urgent need of new classes and broad-spectrum Gram-negative antibiotics.

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Fig. 1 Chemical structures of diamidine compounds
Although diamidine is known to show various biological activities, such as antibacterial,
antiparasitic, anti-inflammatory, and antimalarial effects [12-15], its representative activity is its
antibacterial and antifungal effects. Maciejewska et al. designed and synthesized pentamidine
analogues with unsubstituted or N-alkyl substituted amidines that had promising antibacterial and
antifungal activities (1, Fig. 1) [16]. Williams et al. discovered a series of triaryl amidine
compounds with bis-5 or 6-cyclo amidino structures with potent antibacterial against various
bacterial strains (2, Fig. 1) [17, 18]. Recently, they also found that these compounds were potent
antifungal agents [19]. However, they did not conduct systematic research into the effect of the
N-substituent of an amidine N-atom on the antibacterial activity. Previous mechanisms studies of
action have shown that these compounds target DNA synthesis by binding to the minor groove of
DNA [20-22]. Oldfield et al. recently found that some diamidine derivatives with bis-5 or 6-cyclo
amidino structures with best antibacterial activities bind to AT-rich DNA in addition to inhibiting
undecaprenyl diphosphate synthase (UPPS), that is involved in bacterial cell wall biosynthesis and
humans do not possess this kind of synthase [23-24]. The find of the novel antibacterial drug
target inspired the researchers to synthesize more diamidine compounds in order to improve
antibacterial activities, overcome bacterial resistance and improve selectivity [24].
To continue our efforts into the development of novel antibacterial agents, we conducted a
systematic study on the antibacterial effects of N-substituted amidines and discovered a series of
bisamidines with N-substituted groups that have potent activity against antibiotic-resistant bacteria
including MRSA, MRSE, and VRE (3 and 4, Fig. 1) [25-27]. The diphenyl ether bisamidines with
a N,N-dimethylamino substituent (4) not only showed significant activity against anti-Gram
positive drug-resistant bacterial strains (MRSA, MRSE, and VRE: 0.25-2 µg/mL), but also

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displayed promising activity against Gram-negative bacteria, especially for the NDM-1-producing
K. pneumoniae strain (MIC = 8 µg/mL) [27]. This pharmaceutical salience of N-substituted
amidine was the impetus for our study of novel N-substituted amidines.
In order to find more potent antibiotics against Gram-negative strains and further extend the
structure-activity relationship (SAR) analysis for this class of compounds, we replaced the core
structure of lead compound 4 with phenyl, naphthyl, or other heterocyclic aryl groups to design
and synthesize 25 new diamidine compounds, and evaluated their antibacterial effect against
Gram-positive and Gram-negative bacterial strains. We identified phenyl bisindole derivatives
with N-substituted amidines that showed high antibacterial activities against MDR Gram-positive
bacteria and some Gram-negative pathogens in the panel; specifically, compounds with
N,N-dimethylamino substituent groups possessed remarkable activity against NDM-1-producing
Gram-negative pathogens.
2. Chemistry
In order to explore the effect of different N-substituent groups of amidine in phenyl-linked
bisindole compounds on antibacterial activity, we prepared compounds 5a-s. The synthesis route
of compound 5a-s is depicted in Scheme 1. Compound 7 was prepared from the
piperidine-catalyzed condensation of 4-methyl-3-nitrobenzonitrile and terephthaldehyde in
sulfolane and subsequent heating with neat triethylphosphite. Treatment of the dinitrile in the
presence of dry HCl in anhydrous ethanol gave an imidate ester 8, which was then reacted with
various amines to produce the desired target compounds 5a-r, from which 5c, 5f, and 5g were
known and identified with potential antifungal activity [19]. Known compound 5s, a reference
standard, was synthesized by the treatment of the dinitrile 7 with alkyldiamine in the presence of
catalytic phosphorous pentasulfide in heat-sealed tubes, as previously reported [17].

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Scheme 1. Reagents and conditions: (a) piperidine, sulfolane, 150 °C, 45 min; (b) P(OEt)3, 165 °C, 2 days; (c) HCl
(gas), anhydrous EtOH, 35 °C, 5 days; (d) the corresponding amines, anhydrous MeOH, rt, 48 h.
From the SAR studies into amidine functionality, it was found that compound 5i, with
dimethylamino functionality, showed better antibacterial activities against a range of bacterial
strains. In order to determine the limits of linker structure changes, we synthesized analogues of
compound 5i with five-membered, six-membered, or naphthyl-linker moieties, such as
thiazolyl-linked 18, pyrazinyl-linked 19, pyridinyl-linked 20 and 22, and naphthyl-linked 21 and
24 (Scheme 2). The diintriles 11-17 were prepared by double Stille coupling reaction of the
commercially available dibromo compounds with
N-Boc-2-(trimethylstannyl)-1H-indole-6-carbonitrile (10), which was obtained from the
reaction of N-Boc-6-nitrile-indole (9) with lithium diisopropylamide and trimethyltin chloride [28].
The nitrile compounds were then converted to the corresponding diamidine compounds 18-24 by
using the same process described above.

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Scheme 2. Reagents and conditions: (a) (Boc)2O, DMAP, CH3CN, rt; (b) LDA, (CH3)3SnCl, THF, -20 °C - rt; (c),
2,5-dibromothiazole (for 18), Pd(PPh3)4, DMF, 110 °C; (2,5-dibromopyrazine for 19, 2,5-dibromopyridine for 20,
2,6-dibromonaphthalene for 21, 3,5-dibromopyridine for 22, 1,3-dibromobenzene for 23, and
2,7-dibromonaphthanlene for 24); (d) (1) HCl (gas), anhydrous EtOH, 35 °C, 3-5 days, (2) N,N-dimethylamine,
anhydrous MeOH, rt, 48 h.
The 3,5-pyridinyl-linked diamidine analogue 22 which had a clearly different shape from the
linear molecule 5i, displayed similarly potent activity against most strains. In order to explore the
SAR, further experiments focused on 3,5-pyridinyl-linked diamidine analogues. We selected
several carbon chains with good activity in phenyl-linked derivatives to design the
3,5-pyridinyl-linked diamidine analogues 25-28. By following approaches similar to those
outlined in Scheme 1, compounds 25-28 were synthesized by the treatment of various alkyl
amines with the imidate ester, which was obtained by treatment of the nitrile compound 15 with
HCl (gas) in anhydrous EtOH for 3 days.
Scheme 3. reagents and conditions: (a) (1) HCl (gas), anhydrous EtOH, 35 °C, 3 days; (2) the
corresponding amines, anhydrous MeOH, rt, 48 h.
3. Results and discussion
3.1 In vitro structure -activity relationships and cytotoxicity
All the synthesized diamidines were evalued for their potential antibacterial activity in vitro
against 15 Gram-positive and 21 Gram-negative bacterial strains from 16 different species. The
minimum inhibitory concentration (MIC) values for all strains are presented in the supplementary

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data. The MIC values for the phenyl diamidine compounds against representative common human
pathogens including MRSA/MRSE, VRE, drug-resistant Enterobacteriaceae, P. aeruginosa and A.
calcoaceticus strains are shown in Table 1 and below all the results showing antibacterial activities
of synthesized compounds are referred to tested strains present in Table 1. The lead compounds 4
and 5s, and clinic drug levofloxacin were used as the reference standards. The cytotoxicity (CC50)
of each analogue against HEK 293T cells was determined and is shown in Table 1.
The data generated from this study showed that all tested phenyl diamidine compounds
displayed good activity against the selected Gram-positive strains, including MRSA, MRSE, and
VRE, with MIC values in the range of 0.125−16 µg/mL. Of the 19 compounds tested, the
anti-MRSE activity of all compounds, except for 5q, were better than or comparable with that of
levofloxacin, the anti-MRSA activity was 4- to 128-fold greater than that of levofloxacin, and the
anti-VRE activity was 4- to 512-fold more potent than that of levofloxacin. Most compounds
showed improved activity against the Gram-negative strains E. coli and K. pneumoniae than that
of corresponding diphenyl ether diamidine compounds [27], with MIC values in the range of 1−32
µg/mL. Some compounds exhibited broad-spectrum antibacterial activity against both
Gram-positive and Gram-negative bacterial strains.
First, we investigated the effect of the N-substituent group of amidine in phenyl-linked
bisindole compounds on the antibacterial activity. We selected some amidino groups with good
activity in diphenyl ether diamidine compounds to replace the 6-cyclo amidino structure of
compound 5s and prepared compounds 5a−5e, 5i-5l, and 5o-5r. The comparison of the
antibacterial activity of the thirteen phenyl-linked diamidine compounds to the corresponding
diphenyl ether-linked compounds revealed that there was a general improvement in the
antibacterial potency. Of the phenyl-linked diamidine compounds, 5a, 5c, 5d, and 5i displayed
1−8-fold greater activity against MDR Gram-positive bacteria than the corresponding diphenyl
ether-linked compounds, an inhibitory profile similar to that of the known compound 5s, with
MIC values in the range of 0.125−2 µg/mL, and 16−512-fold superiority to that of the clinic drug,
levofloxacin. It is noteworthy that the phenyl-linked compounds showed superior coverage of
Gram-negative bacteria compared with diphenyl ether-linked compounds. All the
phenyl-substituted compounds displayed potent antibacterial activity against E. coli and K.
pneumoniae strains, with MIC values in the range of 2−64 µg/mL. Compounds 5c−5d and 5i−5j

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displayed greater to comparable activity against E. coli and K. pneumoniae strains than that of the
lead compound 4, with MIC values in the range of 1−8 µg/mL. Compounds 5a−5k and 5p−5r
showed more potent activity against P. aeruginosa than compound 4, with MIC values in the
range of 2−64 µg/mL. Compounds 5c−5f and 5i−5o exhibited more potent inhibitory activity
against A. calcoaceticus than compound 4 (MIC > 128 µg/mL) by 4−32-fold and compound 5s
(MIC = 64 µg/mL) by 2−16-fold, with MIC values in the range of 4−32 µg/mL. Compounds
5a−5q showed 4−64-fold greater inhibitory activity against E. cloacae than compound 4, with
MIC values in the range of 1−16 µg/mL. Compounds 5c−5e and 5i−5l displayed 2−4-fold greater
inhibitory activity against P. mirabilis than compound 4 and 8−16-fold activity than compound 5s,
with MIC values in the range of 8−16 µg/mL. All compounds displayed more potent activity
against the NDM-1-producing K. pneumoniae ATCC BAA-2146 than levofloxacin. Of these
compounds, 5i showed the most powerful antibacterial activity (MIC = 2 µg/mL). Based on the
SAR of compounds 5a−e and 5i−k, we found that a branched chain and a shorter carbon chain on
the amidine N-atom resulted in more powerful activity. The result was similar to that of the
diphenyl ether diamidines. In the comparisons of 5b with 5a, 5d with 5c, 5j with 5i, a longer
carbon chain resulted in weaker antibacterial activity. In the comparisons of 5a with 5i, 5b with 5c,
and 5b with 5j, the introduction of methyl-branched substitution resulted in a great improvement
of inhibitory activity against Gram-negative bacteria such as K. pneumoniae, A. calcoaceticus, E.
cloacae and P. mirabilis strains, with a 4−32-fold increase in antibacterial activity. More
broad-spectrum antibacterial activity was observed in compound 5c, with MIC values in the range
of 0.125−0.25 µg/mL against Gram-positive strains and 2−16 µg/mL against the 10 Gram-negative
strains (see Supplementary data). The activity against P. aeruginosa of compound 5c was the best,
with MIC values in the range of 2−4 µg/mL. Slightly more potent antibacterial activity against A.
calcoaceticus, E. cloacae, and P. mirabilis strains was observed for compounds 5i and 5j in
comparison with compound 5c. The toxicity towards mammalian cells was evaluated for these
synthesized compounds in HEK 293T cells: compounds 5c−5d and 5i−5j, which had excellent
antibacterial activity against Gram-negative bacteria, displayed lower or equivalent cytotoxicity
against HEK 293T cells than the reference compound 5s.
Table 1. Antibacterial activity (MIC µg/mL) and cytotoxicity (CC50) of compounds 5a−5s against

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Gram-positive and Gram-negative strains
Based on the study by O’Shea and Moser, which reported that anti-Gram-negative agents are
generally more polarity and more positively charged than anti-Gram-positive agents [29], we
synthesized compounds 5f−5h and 5o−5r with an oxygen- or tertiary amino-substituent group.
Unexpectedly, the introduction of the tertiary amino-substitution in these compounds led to a
complete loss of antibacterial activity against A. calcoaceticus and P. mirabilis strains. Compound
5g used an N,N-dimethylamino group to replace the methoxyl group of compound 5f and
compound 5p used an N-methyl to substitute for the oxygen atom of compound 5o, which had
moderate potency against K. pneumoniae, P. aeruginosa, and E. cloacae, but were devoid of
activity against A. calcoaceticus and P. mirabilis. Similar trends were observed in compounds 5h,
5q, and 5r. The compounds with lipophilic amino substituents, such as isopropylamino (5c),
isobutylamino (5d), 3-pentylamino (5e), N,N-dimethylamino (5i), N-methyl-N-ethylamino (5j),
pyrrolidine (5l), and 3,6-dihydro-2H-pyridine (5n), showed valuable antibacterial activity against
A. calcoaceticus, with MIC values in the range of 4–16 µg/mL. The introduction of polarity and
positively charged displayed no obvious effect on the antibacterial activity against P. aeruginosa,
but it was notable that compounds 5a–j (MIC: 2–16 µg/mL), with alkyl chain amino substitution,
showed more potent activity against P. aeruginosa strains than compounds 5k–r with cyclic
amino substitution (MIC: 16–128 µg/mL) and superiority to the lead compound 4 (MIC: 128
µg/mL). The polarity and positively charged groups also exerted no effect on the activity against K.
pneumoniae strains. Compounds 5f–h showed similar activity against K. pneumoniae strains, with
MIC values in the range of 8–16 µg/mL, and the MIC values of 5o–r were in the range of 8–64
µg/mL. Compounds 5g–h and 5o–r also showed relatively low cytotoxicity against 293T cells,
with CC50 ≥ 16 µg/mL values.
In order to further understand the SAR of the amidine position, we prepared compound 5m,
with a 2,5-dihydro-1H-pyrrol ring, and compound 5n, with a 3,6-dihydro-2H-pyridine ring, to
investigate the effect of alkene-containing N-heterocycle substitutions on antibacterial activity.
Compound 5n, with an alkene-containing six-membered ring had a similar potency to 5l with a
five membered ring, but much lower cytotoxicity. Compound 5m showed similar cytotoxicity to
5n, but two-fold lower antibacterial activity.

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Table 2. Antibiotic activity (MIC µg/mL) and cytotoxicity (CC50) of compounds 18–28 against
Gram-positive and Gram-negative strains.
Next, we explored the effect of different central linkers on the antibacterial properties of the
diamidines with a dimethylamino group (Table 2). The replacement of the phenyl group with the
2,5-thiazolyl (18) and 3,5-pyridinyl (22) groups resulted in similar potency against most of the
bacteria strains to that of compound 5i, but higher cytotoxicity. Suboptimal activity resulted from
the replacement of phenyl with the 2,5-pyrazine (19) and 2,5-pyridinyl (20) groups. The
introduction of 1,3-phenyl (23) and naphthyl (21 and 24) groups resulted in a loss of activity
against Gram-negative strains.
Although compound 22 has a clearly different shape from the linear molecule 5i, it displayed
similar antibacterial activity against most strains in the panel. This result inspired us to explore
some new structural compounds. A series of new 3,5-pyridinyl-linked analogues (25–28) with
different N-substituted amidines were constructed and their antibacterial activity was evaluated
(Table 2). Unexpectedly, compounds 25–27 showed only weak activity against Gram-positive
bacteria and no antibacterial activity against Gram-negative bacteria. Compound 28 had no
activity against all strains. This result confirmed the importance of the dimethylamino-substituted
amidine moiety in the antibacterial activity against Gram-negative bacteria.
3.2 In vitro antibacterial activity against NDM-1-producing and carbapenem-resistant bacteria
strains
Because of the unexpected antibacterial activity against NDM-1-producing K. pneumoniae of
compound 5i, we further measured the activity against different NDM-1-producing Gram-negative
strains, including E. coli, K. pneumoniae, A. calcoaceticus, and E. cloacae. Compound 5i
exhibited broad-spectrum antibacterial activity against the seven different NDM-1-producing
Gram-negative strains with MIC values in the range of 2–4 µg/mL. The activity was superior to
the reference drugs meropenem, aztreonam, and levofloxacin, comparable with tigecycline, and
slightly weaker than colistin. In addition, compound 5i exhibited potent antibacterial activity
against carbapenem-resistant K. pneumoniae, A. calcoaceticus, and E. aerogenes with MICs
values in the range of 8–16 µg/mL, which were superior to meropenem and aztreonam, and
comparable with levofloxacin.

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Table 3. Antibiotic activity (MIC µg/mL) of compound 5i against NDM-1-producing and
carbapenem-resistant Gram-negative strains
3.3 In vivo efficacy of compound 5i
Owing to the excellent in vitro efficacy of compound 5i against S. aureus MRSA (MIC
0.125–0.5 µg/mL), it was selected for the evaluation of in vivo efficacy in a murine systemic
infection model of the S. aureus MRSA (ATCC 33591) strain. Compound 5i displayed potent in
vivo efficacy (ED50 = 2.62 mg/kg and ED95 = 4.62 mg/kg) (Table 4).Further evaluation of the in
vivo efficacy of compound 5i against NDM-1-producing Gram-negative bacteria is in progress.
Table 4. In vivo efficacy of compound 5i in a murine S. aureus infection model
4. Conclusion
Herein, we have described the design and synthesis of a series of diamidines with
N-substituents on an amidine N-atom that show remarkable broad-spectrum antibacterial
properties, including against MRSA, MRSE, VRE, ESBLs-producing, and NDM-1-producing
pathogens. SAR studies confirmed that a branched chain and a shorter carbon chain on the
amidine N-atom exhibited more powerful activity against Gram-negative and MDR Gram-positive
pathogens. Compound 5c with isopropylamino, 5d with isobutylamino, 5i with
N,N-dimethylamino, and 5j with N-methyl-N-ethylamidino groups are highly promising candidate
compounds with MIC values below 16 µg/mL. Compound 5c showed broad-spectrum
antibacterial activity against most Gram-positive bacteria and Gram-negative bacteria and much
greater efficacy against Gram-positive pathogens than the standard drug levofloxacin.
Furthermore, owing to its greater potency against P. aeruginosa (MIC values = 2–8 µg/mL),
compound 5c could act as a lead compound for specific antibacterial activity against P.
aeruginosa. Slightly more potent antibacterial activity against K. pneumoniae, A. calcoaceticus, E.
cloacae, and P. mirabilis was observed for compound 5i in comparison with compound 5c.
Compound 5i also displayed broad-spectrum antibacterial activity against NDM-1-producing and
carbapenem-resistant Gram-negative strains. Further investigation of the in vivo efficacy in a
mouse model of S. aureus MRSA demonstrated the effective antibacterial activity of 5i. The
amidines with the N,N-dimethylamino substituent group illustrated their advantage in different

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centrally linked compounds. Further investigation of this class of compounds with N-substituted
amidines, including in vivo efficacy against NDM-1-producinggram-negative pathogens, is in
progress.
5. Experimental Section
5.1 Chemistry
General: Experiments were carried out under an inert atmosphere unless otherwise stated.
Solvents were purified by standard procedures. All starting materials were obtained from
1
13
commercial suppliers and used as received. H and C NMR spectra were recorded at 400 MHz
and 100 MHz or 500 MHz and 125 MHz by using a Bruker DRX 400 or 500 instrument
1
respectively. H spectra were recorded in deuterated solvents as detailed and at ambient probe
temperature (300 K). Chemical shifts are reported in parts per million (ppm) and are referenced to
the residual solvent peak. The following notations are used: singlet (s); doublet (d); triplet (t);
quartet (q); multiplet (m); broad (br). Data are reported in the following manner: chemical shift
(multiplicity, coupling constant if appropriate, integration). Signals are quoted as δ values in ppm
and coupling constants (J) are reported in Hertz. Using residual protonated solvent signals as
internal standard (1H: δ (CDCl3) = 7.26 ppm, δ ((CD3)2SO-d6) = 2.50 ppm, δ (CD3OD) = 3.31 ppm,
δ (H2O) = 4.67 ppm; and 13C: δ (CDCl3) = 77.1, δ ((CD3)2SO-d6) = 39.5 ppm, δ (CD3OD) = 49.0
ppm. HRESIMS data were measured using a Thermo LTQ Orbitrap XL mass spectrometer. Thin
layer chromatography (TLC) was performed on Merck Kieselgel 60 F254 glass plates and
visualised under UV light. Column chromatography was performed by using Sephadex LH-20
with distilled solvents.
5.1.1 1,4-Bis(6-cyanoindol-2-yl)benzene (7)
To a solution of 4-cyano-2-nitrotoluene (4.0 g, 24.8 mmol) in sulfolane (10 mL) was added
terephthaldehyde (1.66 g, 12.4 mmol) and piperidine (1.0 g, 12.6 mmol). The resulting mixture
was heated at 150 °C for 45 min, forming an orange precipitate. The reaction was cooled to 25 °C,
and diluted with CHCl3 (20 mL). The mixture was stirred for 30 min at 25 °C, then the precipitate
was filtered and rinsed with additional CHCl3 (12 mL). The resulting solids were dried to yield
compound 6 (4.1 g) as an orange powder. The orange powder was added in triethyl phosphite
(50mL). The mixture was heated to reflux for 2 days and then cooled to 25 °C, and
dichloromethane (50 mL) was added. The suspension was stirred for 30 min at 25 °C, then the

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solids were filtered and rinsed with additional CH2Cl2 (20 mL). The resulting solids were dried to
yield compound 7 (2.1 g, 64%) as gold-yellow powder; 1H NMR (400 Hz, DMSO-d6): δ 12.21 (s,
2H), 8.07 (s, 4H), 7.86 (s, 2H), 7.73 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.19 (s, 2H).
5.1.2 1,4-Bis{6-[ethoxy(imino)methyl]indol-2-yl}benzene (8; as hydrochloric acid salt)
1,4-Bis(6-cyanoindole-2-yl)benzene (7, 210 mg, 0.58 mmol) was suspended in anhydrous
ethanol (200 mL) and treated with HCl gas at -5 °C for 45 min. The resulting mixture was heated
to 35 °C (oil bath) in a sealed tube for 5 days. The ethanol and HCl were then concentrated under
reduced pressure until the volume of residuum was about 15 mL, adding anhydrous ether (about
150 mL) to precipitate dark green solid, which was filtrated and dried to provide product as a
1
green solid (220 mg, 84%) H NMR (400 Hz, DMSO-d6) δ 11.83 (s, 2H), 8.72 (s, 2H), 8.00 (s,
4H), 7.87 (s, 2H), 7.56(m, 4H), 7.05 (s, 2H), 4.29 (q, 4H), 1.37 (t, 6H).
5.1.3 1,4-Bis[6-(N-methylamidino)indol-2-yl]benzene (5a; as hydrochloric acid salt)
To a suspension of 1,4-bis{6-[ethoxy(imino)methyl]indol-2-yl}benzene (8, 135 mg, 0.30 mmol)
in anhydrous MeOH (20 mL) was added a solution of methylamine 30% in methanol (4.5 mL).
The reaction mixture was stirred at room temperature for 48 h and concentrated under reduced
pressure to provide a dark green solid which was chromatographed on Sephadex LH-20 column
(gradient: 95% ethanol /0.5% HCl) to provide product as a brown powder (85 mg, 56%); 1H NMR
(400 MHz, DMSO-d6) δ 12.39 (s, 2H), 9.74 (s, br, 2H), 9.42 (s, br, 2H), 8.82 (s, br, 2H), 8.13 (s,
4H), 7.87 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.39 (dd, J = 8.0, 1.6 Hz, 2H), 7.20 (d, J = 1.6 Hz, 2H),
3.17 (d, J = 4.8 Hz, 3H), 3.05 (d, J = 4.8 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.3, 141.1,
136.3, 132.2, 131.0, 126.1, 121.4, 121.2, 118.8, 111.9, 99.6, 29.6; HRMS (ESI+) m/z calcd for
C26H25N6 [M+H]+ 421.2135, found 421.2140.
5.1.4 1,4-Bis[6-(N-ethylamidino)indol-2-yl]benzene (5b; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and ethylamine (108 µL, 1.8 mmol), compound 5b was obtained as
green solid (78 mg, 50% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 2H), 9.14 (br s, 6H),
8.11 (s, 4H), 7.84 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.18 (s, 2H), 3.46 (q,
4H), 1.28 (t, 6H); 13C NMR (100 MHz, CD3OD): δ 166.5, 143.1, 138.1, 134.7, 132.9, 127.3, 123.0,
122.0, 119.6, 112.5, 100.8, 39.1, 13.3; HRMS (ESI+) calcd for C28H29N6 [M+H]+ 449.2448, found
449.2460.

ACCEPTED MANUSCRIPT
5.1.5 1,4-Bis[6-(N-isopropylamidino)indol-2-yl]benzene (5c; as hydrochloric acid salt)
Following the procedure for preparation of compound 4a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 2-propylamine (154 µL, 1.8 mmol), compound 5c was obtained as
yellow solid (105 mg, 64% yield); 1H NMR (400 MHz, CD3OD): δ 8.00 (s, 4H), 7.84 (s, 2H), 7.76
(d, J = 8.0 Hz, 2H), 7.37 (dd, J = 8.0, 2.0 Hz, 2H), 7.07 (s, 2H), 4.05 (m, 2H), 1.42 (d, J = 6.4 Hz,
12H); 13C NMR (125 MHz, CD3OD): δ 165.7, 143.1, 138.1, 134.6, 132.9, 127.3, 123.3, 122.0,
119.7, 112.6, 100.8, 46.8, 21.7; HRMS (ESI+) calcd for C30H33N6 [M+H]+ 477.2761, found
477.2777.
5.1.6 1,4-Bis[6-(N-isobutylamidino)indol-2-yl]benzene (5d; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 2-butylamine (182 µL, 1.8 mmol), compound 5d was obtained as
1
green solid (79 mg, 46% yield); H NMR (500 MHz, CD3OD): δ 8.01 (s, 4H), 7.89 (s, 2H), 7.76
(d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.06 (s, 2H), 3.87 (m, 2H), 1.77 (m, 4H), 1.39 (d, J =
6.0 Hz, 6H), 1.08 (t, J = 7.0 Hz, 6H); 13C NMR (100 MHz, CD3OD): δ 166.2, 143.1, 138.0, 134.6,
132.9, 127.3, 123.3, 122.0, 119.7, 112.6, 100.8, 52.5, 29.8, 19.7, 10.9; HRMS (ESI+) calcd for
C32H37N6 [M+H]+ 505.3074, found 505.3090.
5.1.7 1,4-Bis{6-[N-(3-pentyl)-amidino]indol-2-yl}benzene (5e; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 3-pentylamine (209 µL, 1.8 mmol), compound 5e was obtained as
green solid (76 mg, 42% yield); 1H NMR (500MHz, CD3OD): δ 8.01 (s, 4H), 7.89 (s, 2H), 7.77 (d,
J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.07 (s, 2H), 3.74 (s, 2H), 1.82 (m, 4H), 1.72 (m, 4H),
1.08 (t, J = 7.0 Hz, 6H); 13C NMR (100 MHz, CD3OD): δ 167.0, 143.1, 138.0, 134.6, 132.9, 127.3,
123.4, 122.0, 119.6, 112.5, 100.8, 58.4, 28.0, 10.7; HRMS (ESI+) calcd for C34H41N6 [M+H]+
533.3387, found 533.3402.
5.1.8 1,4-Bis[6-(N-(2-methoxyethyl)-amidino)indol-2-yl]benzene (5f; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 2-methoxyethylamine (115 µL, 1.8 mmol), compound 5f was
1
obtained as green solid (117 mg, 67% yield); H NMR (400 MHz, DMSO-d6): δ 12.48 (s, 2H),
9.73 (br s, 2H), 9.43 (br s, 2H), 8.97 (br s, 2H), 8.14 (s, 4H), 7.87 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H),
7.36 (dd, J = 8.0, 1.6 Hz, 2H), 7.18 (s, 2H), 3.64 (m, 4H), 3.35 (s, 6H), 3.29 (s, 4H); HRMS (ESI+)

ACCEPTED MANUSCRIPT
calcd for C30H33N6O2 [M+H]+ 509.2660, found 509.2670.
5.1.9 1,4-Bis{6-[N-(2-N,N-dimethylaminoethyl)-amidino]indol-2-yl}benzene (5g; as hydrochloric
acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), 20 mL of methanol and N,N-dimethylaminoethylamine (195 µL, 1.8 mmol), compound 5g
was obtained as green solid (127 mg, 70% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.35 (s, 2H),
9.34 (br s, 6H), 8.11 (s, 4H), 7.84 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.37 (dd, J = 8.0, 1.6 Hz, 2H),
7.19 (s, 2H), 3.55 (t, J = 6.0 Hz, 4H), 2.60 (t, J = 6.0 Hz, 4H), 2.66 (s, 6H); 13C NMR (100 MHz,
DMSO-d6): δ 164.2, 141.1, 136.2, 132.3, 131.0, 126.1, 121.6, 120.3, 119.0, 112.1, 99.7, 56.5, 44.9;
HRMS (ESI+) calcd for C32H39N8 [M+H]+ 535.3292, found 535.3307.
5.1.10
1,4-Bis[6-(N-(3-N,N-dimethylaminopropyl)-amidino)indol-2-yl]benzene
(5h;
as
hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and N,N-dimethylaminopropylamine (229 µL, 1.8 mmol), compound 5h
was obtained as green solid (144 mg, 76% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.50 (s, 2H),
9.34 (br s, 6H), 8.14 (s, 4H), 7.88 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.38 (dd, J = 8.0, 1.6 Hz, 2H),
7.18 (s, 2H), 3.46 (t, J = 6.4 Hz, 4H), 2.36 (t, J = 6.4 Hz, 4H), 2.20 (s, 6H), 1.84 (m, 4H); 13C
NMR (125 MHz, CD3OD): δ 166.8, 143.2, 138.1, 134.7, 132.8, 127.3, 122.7, 122.0, 119.5, 112.5,
100.8, 57.1, 45.2, 42.4, 26.6; HRMS (ESI+) calcd for C34H43N8 [M+H]+ 563.3605, found
563.3619.
5.1.11 1,4-Bis[6-(N, N-dimethylamidino)indol-2-yl]benzene (5i; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and N,N-dimethylamine (1.5 mL, 2M in THF, 3.0 mmol), compound 5i
1
was obtained as green solid (89 mg, 57% yield); H NMR (400 Hz, DMSO-d6): δ 12.36 (s, 2H),
9.26 (br s, 2H), 8.89 (br s, 2H), 8.10 (s, 4H), 7.75 (d, J = 8.4 Hz, 2H), 7.66 (s, 2H), 7.19 (d, J = 8.4,
2H), 7.17 (s, 2H), 3.26 (s, 6H), 3.08 (s, 6H); 13C NMR (125 MHz, CD3OD): δ 168.8, 142.6, 138.0,
133.6, 132.9, 127.2, 122.9, 122.0, 120.0, 112.9, 100.7, 42.9, 39.4; HRMS (ESI+) calcd for
C28H29N6 [M+H]+ 449.2448, found 449.2455.
5.1.12 1,4-Bis[6-(N-methyl-N-ethylamidino)indol-2-yl]benzene (5j; as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30

ACCEPTED MANUSCRIPT
mmol), methanol (20 mL) and N-methyl-N-ethylamine (0.26 mL, 3.0 mmol), compound 5j was
1
obtained as green-yellow solid (69 mg, 42% yield); H NMR (400MHz, DMSO-d6): δ 12.36 (s,
2H), 9.03 (br s, 2H), 8.08 (s, 4H), 7.73 (d, J = 8.4 Hz, 2H), 7.61 (s, 2H), 7.14 (s, 2H), 7.13 (d, J =
8.4 Hz, 2H), 3.42 (s, 4H), 3.12 (s, 6H), 1.19 (t, J = 6.5 Hz, 6H); 13C NMR (100 MHz, DMSO-d6):
δ 166.2, 140.1, 136.3, 131.0, 130.7, 125.9, 120.5, 120.4, 118.6, 112.5, 101.5, 29.1, 28.6, 12.3;
HRMS (ESI+) calcd for C30H33N6 [M+H]+ 477.2761, found 477.2768.
5.1.13 1,4-Bis{6-[(trimethyleneimino-1-yl)(imino)methyl]indol-2-yl}benzene (5k; as hydrochloric
acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and azetidine (202 µL, 3.0 mmol), compound 5k was obtained as green
solid (110 mg, 67% yield); 1H NMR (500 MHz, CD3OD): δ 8.00 (s, 4H), 7.79 (s, 2H), 7.75 (d, J =
6.4 Hz, 2H), 7.28 (d, J = 6.4, 2H), 7.05 (s, 2H), 4.51 (t, J = 5.6 Hz, 4H), 4.47 (t, J = 5.6 Hz, 4H),
2.55 (m, 4H); 13C NMR (100 MHz, CD3OD): δ 166.2, 142.9, 138.0, 134.1, 132.8, 127.3, 122.1,
120.3, 119.7, 112.8, 100.8, 56.2, 53.4, 16.6; HRMS (ESI+) calcd for C30H29N6 [M+H]+ 473.2448,
found 473.2443.
5.1.14 1,4-Bis{6-[(pyrrolidin-1-yl)(imino)methyl]indol-2-yl}benzene (5l; as hydrochloric acid
salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and pyrrolidene (296 µL, 3.0 mmol), compound 5l was obtained as
green-yellow solid (130 mg, 76% yield); 1H NMR (500 MHz, CD3OD): δ 7.99 (s, 4H), 7.77 (d, J
= 6.4 Hz, 2H), 7.73 (s, 2H), 7.25 (d, J = 6.4, 2H), 7.05 (s, 2H), 3.65 (m, 8H), 2.23(m, 4H), 2.00 (s,
4H); 13C NMR (100 MHz, CD3OD): δ 165.7, 142.5, 137.9, 133.5, 132.9, 127.3, 127.2, 122.0,
119.6, 112.4, 100.7, 53.5, 46.6, 26.0, 25.0; HRMS (ESI+) calcd for C32H33N6 [M+H]+ 501.2761,
found 501.2773.
5.1.15
1,4-Bis{6-[(2,5-dihydro-1H-pyrrol-1-yl)(imino)methyl]indol-2-yl}benzene
(5m;
as
hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL), Et3N (0.43 mL, 3.0 mmoL) and 2,5-dihydro-1H-pyrrole hydrochloride
1
(127 mg, 1.2 mmol), compound 5m was obtained as green solid (130 mg, 76% yield); H NMR
(400 MHz, DMSO-d6): δ 12.37 (s, 2H), 9.47 (br s, 2H), 8.94 (br s, 2H), 8.11 (s, 4H), 7.75 (d, J =

ACCEPTED MANUSCRIPT
8.0 Hz, 2H), 7.72 (s, 2H), 7.30 (dd, J = 8.0, 1.6, 2H), 7.17 (d, J = 1.2 Hz, 2H), 6.07 (dt, J = 6.8,
2.0 Hz, 2H), 5.93 (dt, J = 6.8, 2.0 Hz, 2H), 4.44 (s, 4H), 4.29 (s, 4H); HRMS (ESI+) calcd for
C32H29N6 [M+H]+ 497.2448, found 497.2459.
5.1.16 1,4-Bis{6-[(3,6-dihydro-2H-pyridine-1-yl)(imino)methyl]indol-2-yl}benzene (5n; as
hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 1,2,3,6-tetrahydropyridine (109 µL, 1.4 mmol), compound 5n was
1
obtained as green-yellow solid (75 mg, 42% yield); H NMR (400 MHz, DMSO-d6): δ 12.40 (s,
2H), 9.50 (m, 2H), 9.20 (m, 2H), 8.13 (s, 4H), 7.81 (d, J = 8.0 Hz, 2H), 7.71 (s, 2H), 7.23 (s, 4H),
6.06 (d, 2:1, 2H), 5.90 (d, 2:1, 2H), 4.23 (s, 3H), 4.00 (d, 1:1, 3H), 3.54 (s, 3H), 2.32 (s, 3H); 13C
NMR (100 MHz, CD3OD): δ 168.4, 142.7, 138.1, 133.9, 132.9, 127.2, 122.6, 122.3, 119.8, 112.8,
100.8, 51.9, 47.5, 45.8, 26.4, 25.6; HRMS (ESI+) calcd for C34H33N6 [M+H]+ 525.2761, found
525.2778.
5.1.17 1,4-Bis{6-[(1,4-oxazine-4-yl)(imino)methyl]indol-2-yl}benzene(5o; as hydrochloric acid
salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and morpholine (122 µL, 1.4 mmol), compound 5o was obtained as
green-yellow solid (118 mg, 65% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.47 (s, 2H), 9.45 (m,
6H), 8.11 (s, 4H), 7.76 (d, J = 8.4 Hz, 2H), 7.71 (s, 2H), 7.21 (dd, J = 8.4, 1.6 Hz, 2H), 7.18 (s,
2H), 3.75 (m, 12H), 3.03 (d, 4H); HRMS (ESI+) calcd for C32H33N6O2 [M+H]+ 533.2660, found
533.2676.
5.1.18 1,4-Bis{6-[(4-methylpiperazin-1-yl)(imino)methyl]indol-2-yl}benzene (5p; as hydrochloric
acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and methylpiperazine (155 µL, 1.4 mmol), compound 5p was obtained
1
as yellow solid (142 mg, 67% yield); H NMR (400 MHz, DMSO-d6): δ 12.37 (s, 2H), 9.40 (m,
6H), 8.10 (s, 4H), 7.77 (d, J = 8.4 Hz, 2H), 7.68 (s, 4H), 7.18 (m, 4H), 3.66 (m, 8H), 2.50 (m, 8H),
2.25 (s, 6H); 13C NMR (100 MHz, CD3OD): δ 168.3, 142.8, 138.1, 134.0, 132.8, 127.3, 122.3,
122.2, 120.3, 113.4, 100.8, 55.5, 54.5, 51.1,47.5, 45.6; HRMS (ESI+) calcd for C34H39N8 [M+H]+
559.3292, found 559.3313.

ACCEPTED MANUSCRIPT
5.1.19 1,4-Bis{6-[(4-(1-pyrrolidinyl)piperidin-1-yl)(imino)methyl]indol-2-yl}benzene (5q; as
hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 4-(1-pyrrolidinyl)piperidine (216 mg, 1.4 mmol), compound 5q
1
was obtained as yellow solid (173 mg, 71% yield); H NMR (400 MHz, DMSO-d6): δ 12.35 (s,
2H), 9.37 (br s, 3H), 9.15 (br s, 3H), 8.10 (s, 4H), 7.77 (d, J = 8.4 Hz, 2H), 7.68 (s, 2H), 7.20 (d, J
= 8.4 Hz, 2H), 7.19 (s, 2H), 4.14 (m, 2H), 3.64 (m, 2H), 3.45 (m, 2H), 2.50 (m, 8H), 2.09 (m, 4H),
1.91 (m, 3H), 1.74 (m, 13H); HRMS (ESI+) calcd for C42H51N8 [M+H]+ 667.4231, found
667.4220.
5.1.20 1,4-Bis{6-[(8-methyl-3,8-diazaspiro[4,5]decen-1-yl)(imino)methyl]indol-2-yl}benzene (5r;
as hydrochloric acid salt)
Following the procedure for preparation of compound 5a, from compound 8 (135 mg, 0.30
mmol), methanol (20 mL) and 8-methyl-3,8-diazaspiro[4,5]decene (231 mg, 1.5 mmol),
compound 5r was obtained as yellow solid (105 mg, 43% yield); 1H NMR (400 MHz, DMSO-d6):
δ 12.31 (s, 2H), 8.87 (br s, 4H), 8.09 (s, 4H), 7.74 (d, J = 8.4 Hz, 2H), 7.68 (s, 2H), 7.24 (d, J =
8.4 Hz, 2H), 7.16 (s, 2H), 4.35 (s, 2H), 3.62 (s, 4H), 3.49 (s, 2H), 2.33 (m, 4H), 2.13 (m, 10H),
13
1.87 (m, 4H), 1.57 (m, 8H); C NMR (100 MHz, CD3OD): δ 167.5, 142.0, 141.9, 138.1, 132.9,
132.8, 127.1, 121.8, 119.5, 111.9, 100.6, 77.6, 64.3, 53.6, 46.2, 41.1, 40.2; HRMS (ESI+) calcd
for C42H51N8 [M+H]+ 667.4231, found 667.4260.
5.1.21 1,4-Bis[6-(3,4,5,6-tetrahydropyrimidin-2-yl)indol-2-yl]benzene (5s; as trifluoroacetic acid
salt)
To a suspension of 1,4-bis(6-cyanoindole-2-yl)benzene (340 mg, 0.94 mmol) in propane-1,3-
diamine (5 mL) was added phosphorous pentasulfide (218 mg, 0.66 mmol). The suspension was
heated at 120 °C in a sealed vessel for 2.0 h, then cooled to room temperature and poured into
water (70 mL). The resulting yellow suspension was stirred for 30 min, then filtered. The solids
were rinsed with water (50 mL) and then dried under vacuum to yield a yellow powder. The
powder was dissolved in trifluoroacetic acid (5 mL) and MeOH (15 mL) with heating at 60 °C.
The yellow solution was cooled to room temperature and diluted with Et2O (120 mL). The
resulting solids were filtered, rinsed with Et2O (50 mL), and dried to provide product 5s (460 mg ,
70%) as a yellow powder; 1H NMR (400 MHz, DMSO-d6) δ 11.80 (br s, 6H), 8.01 (s, 4H), 7.81 (s,

ACCEPTED MANUSCRIPT
2H), 7.56 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.04 (s, 2H), 3.43 (s, 8H), 1.80 (s, 4H);
HRMS (ESI+) alcd for C30H29N6 [M+H]+ 473.2448, found 473.2466.
5.1.22 N-Boc-2-(trimethylstannyl)-1H-indole-6-carbonitrile (10)
To a solution of N-Boc-1H-indole-6-carbonitrile [23] (3.6 g, 15 mmol) and Me3SnCl (3.39 g, 17
mmol) in THF (50 mL) was slowly added 2.0 M LDA in THF (9 mL, 18 mmol) while keeping the
temperature below –20 °C. The reaction mixture was warmed to r.t. and left to stir for 6 h.
Another portion of Me3SnCl (1.01 g, 5.1 mmol) and 2.0 M LDA in THF (2.7 mL, 5.4 mmol) were
added at –20 °C to the reaction mixture, which was stirred at r.t. for 24 h. TLC analysis showed
only traces of starting material. After quenching the reaction with H2O (3 mL), the solvent was
removed under reduced pressure and the residue was extracted with Et2O (80 mL). The organic
phase was washed with 10% NaF solution (10 mL) and H2O (40 mL), dried (anhyd MgSO4) and
concentrated to provide a residue, which was subjected to flash chromatography on silica gel with
1
5% EtOAc/hexane to provide product (4.8 g, 80%) as a white solid; H NMR (400 MHz,
DMSO-d6): δ 8.21 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0, 1.2 Hz, 1H), 6.94 (s, 1H),
1.69 (s, 9H), 0.32 (s, 9H).
5.1.23 General procedure of Stille coupling reaction
The reactions were carried out in an Ar atmosphere using flame-dried apparatus. To a stirred
solution of N-Boc-2-(trimethylstannyl)-1H-indole-6-carbonitrile (10, 483 mg, 1.2 mmol) and
2,5-dibromothiazole (146 mg, 0.60 mmol) in DMF (4.0 mL) at r.t. was added Pd(PPh3)4 (34 mg,
0.06 mmol). The resulting solution was heated to 110 °C for about 12 h. When the reaction was
determined by TLC analysis to have reached completion, the mixture was cooled to r.t. and
concentrated. The residual oil was subjected to flash chromatography (silica gel, EtOAc/petroleum
ether: 0-75%) to provide 2,5-Bis(6-cyanoindol-2-yl)thiazole (11) as yellow solid (170 mg, 78%);
1H NMR (400 MHz, DMSO-d6): δ 12.61 (s, 1H), 12.44 (s, 1H), 8.50 (s, 1H), 7.90 (s, 1H), 7.87 (s,
1H), 7.81 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.40 (m, 2H), 7.28 (s, 1H), 7.04 (s, 1H) ;
13C NMR (100 MHz, DMSO-d6): δ 158.4, 141.5, 136.5, 136.4, 135.0, 133.0, 131.9, 131.5, 130.7,
122.7, 121.8, 120.9, 120.7, 117.2, 116.5, 104.9, 140.0, 103.7, 102.6.
5.1.24 2,5-Bis(6-cyanoindol-2-yl)pyrazine (12)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (725 mg, 1.8 mmol), DMF (4 mL) and 2,5-dibromopyrazine (142 mg, 0.6

ACCEPTED MANUSCRIPT
1
mmol). compound 12 was obtained as yellow solid (117 mg, 54% yield); H NMR (400 MHz,
DMSO-d6): δ 12.46 (s, 2H), 9.44 (s, 2H), 7.92 (s, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.58 (s, 2H), 7.40
(d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6): δ 144.0, 142.1, 138.5, 136.8, 131.9, 122.8,
122.6, 120.8, 118.2, 117.3, 104.6, 103.2.
5.1.25 2,5-Bis(6-cyanoindol-2-yl)pyridine (13)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (725 mg, 1.8 mmol), DMF (4 mL) and 2,5-dibromopyridine (141 mg, 0.6
1
mmol), 13 was obtained as yellow-green solid (144 mg, 67% yield); H NMR (400 MHz,
DMSO-d6): δ 12.37 (s, 1H), 12.32 (s, 1H), 9.27 (d, J = 1.6 Hz, 1H), 8.44 (dd, J = 8.4, 2.4 Hz, 1H),
8.24 (d, J = 8.4 Hz, 1H), 7.90 (s, 1H), 7.89 (s, 1H), 7.77 (t, J = 8.0 Hz, 2H), 7.40-7.37 (m, 3H),
13
7.33 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 149.0, 147.0, 141.0, 139.0, 136.6, 136.5, 134.2,
132.1, 132.0, 126.7, 122.4, 121.9, 121.3, 121.0, 117.2, 116.5, 104.0, 103.7, 102.2, 101.5.
5.1.26 2,6-Bis(6-cyanoindol-2-yl)naphthalene (14)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (483 mg, 1.2 mmol), DMF (4 mL) and 2,6-dibromonaphthalene (172 mg, 0.6
1
mmol), compound 14 was obtained as green solid (217 mg, 89% yield); H NMR (400 MHz,
DMSO-d6): δ 12.35 (s, 2H), 8.52 (s, 2H), 8.10 (m, 4H), 7.90 (s, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.38
(dd, J = 8.0, 1.6 Hz, 2H), 7.27 (d, J = 1.6 Hz, 2H).
5.1.27 3,5-Bis(6-cyanoindol-2-yl)pyridine (15)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (483 mg, 1.2 mmol), DMF (4 mL) and 3,5-dibromopyridine (142 mg, 0.6
1
mmol), compound 15 was obtained as gray solid (120 mg, 56% yield); H NMR (400 MHz,
DMSO-d6): δ 12.39 (s, 2H), 9.15 (d, J = 1.6 Hz, 2H). 8.83 (t, J = 2.4 Hz, 1H). 7.95 (s, 2H), 7.82 (d,
J = 8.4 Hz, 2H), 7.42 (dd, J = 8.4, 1.6 Hz, 2H), 7.35 (d, J = 1.6 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 146.4, 138.9, 136.5, 132.0, 130.1, 127.8, 122.9, 122.0, 120.9, 116.7, 103.8, 101.7.
5.1.28 1,3-Bis(6-cyanoindol-2-yl)phene (16)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (483 mg, 1.2 mmol), DMF (4 mL) and 1,3-dibromophene (142 mg, 0.6 mmol),
compound 16 was obtained as gray solid (86 mg, 40 % yield); 1H NMR (400 MHz, DMSO-d6): δ
12.25 (s, 2H), 8.52 (s, 1H). 7.96 (dd, J = 8.0, 1.5 Hz, 2H). 7.90 (s, 2H), 7.78 (d, J = 8.0 Hz, 2H),

ACCEPTED MANUSCRIPT
7.67 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.0, 1.0 Hz, 2H), 7.25 (s, 2H).
5.1.29 2,7-Bis(6-cyanoindol-2-yl)naphthalene (17)
Following the general procedure of Stille coupling reaction for preparation of compound 11,
from compound 10 (483 mg, 1.2 mmol), DMF (4 mL) and 2,7-dibromonaphthalene (171 mg, 0.6
1
mmol), compound 17 was obtained as gray solid (110 mg, 45% yield); H NMR (400 MHz,
DMSO-d6): δ 12.41 (s, 2H), 8.52 (s, 2H). 8.11 (d, J = 2.5 Hz, 4H). 7.92 (s, 2H), 7.78 (d, J = 8.0 Hz,
2H), 7.39 (dd, J = 8.0, 1.5 Hz, 2H), 7.28 (s, 2H).
5.1.30 2,5-Bis[6-(N, N-dimethylamidino)indol-2-yl]thiazole (18; as hydrochloric acid salt)
2,5-Bis(6-cyanoindole-2-yl)thiazole (11, 110 mg, 0.30 mmol) was suspended in anhydrous
ethanol (100 mL) and treated with HCl gas at -5 °C for 45 min. The resulting mixture was heated
at 35 °C (oil bath) in a sealed tube for 5 days. The reaction solution was then evaporated in vacuo
to provide dark green solid, which was added 20 mL of absolute methanol and N,N-dimethylamine
(1.5 mL, 2M in THF, 3.0 mmol) and stirred for 48 h at r.t.. The mixture solution was evaporated
and chromatographed on Sephadex LH-20 (gradient: 95% ethanol / 0.5% HCl) to provide
1
2,5-Bis[6-(N, N-dimethylamidino)indol-2-yl]thiazole as a yellow powder (90 mg, 66%); H
NMR(400 MHz, DMSO-d6) δ 12.64 (s, 1H), 12.61 (s, 1H), 9.30 (br s, 2H), 8.94 (br s, 2H), 8.55 (s,
1H), 7.83 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.68 (s, 1H), 7.67 (s, 1H), 7.28 (d, J = 1.2
Hz, 1H), 7.23 (dt, J = 8.0, 1.6 Hz, 2H), 7.01(d, J = 1.6 Hz, 1H), 3.26 (s, 6H), 3.05 (s, 6H); 13C
NMR (100 MHz, DMSO-d6): δ 165.7, 165.5, 157.8, 140.9, 136.2, 136.1, 133.8, 130.1, 123.6,
122.5, 121.4, 120.5, 119.4, 119.3, 112.8, 112.2, 102.8, 101.2, 41.9, 33.9; HRMS (ESI+) calcd for
C25H26N7S [M+H]+ 456.1965, found 456.1964.
5.1.31 2,5-Bis[6-(N, N-dimethylamidino)indol-2-yl]pyrazine (19; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 12 (120 mg, 0.33
mmol), compound 19 was obtained as green-yellow solid (35 mg, 24% yield); 1H NMR (400 MHz,
DMSO-d6): δ 12.50 (s, 1H), 12.49 (s, 1H), 9.45 (s, 2H), 9.29 (br s, 2H), 8.89 (br s, 2H), 7.85 (d, J
= 8.4 Hz, 2H), 7.72 (s, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.24 (dd, J = 8.4, 1.6 Hz, 2H), 3.26 (s, 6H),
3.06 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ 165.6, 143.7, 141.5, 137.3, 136.5, 130.5, 123.2,
121.3, 119.2, 112.8, 102.3, 41.9, 28.9; HRMS (ESI+) calcd for C26H27N8 [M+H]+ 451.2353, found
451.2352.
5.1.32 2,5-Bis[6-(N, N-dimethylamidino)indol-2-yl]pyridine (20; as hydrochloric acid salt)

ACCEPTED MANUSCRIPT
Following the procedure for preparation of compound 18, from compound 13 (100 mg, 0.29
1
mmol), compound 20 was obtained as brown solid (96 mg, 74% yield); H NMR (400 MHz,
DMSO-d6): δ 12.70 (s, 1H), 12.39 (s, 1H), 9.34 (s, 1H), 9.30 (d, J = 5.4 Hz, 2H), 8.94 (d, J = 54
Hz, 2H), 8.50 (dd, J = 8.4, 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.80 (t, J = 8.4 Hz, 2H), 7.71 (s,
2H), 7.40 (d, J = 1.2 Hz, 1H), 7.31 (d, J = 1.2 Hz, 1H), 7.20 (m, 2H), 3.28 (s, 3H), 3.27 (s, 3H),
3.08 (s, 3H), 3.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 165.8, 136.5, 130.7, 130.5, 126.7,
121.3, 120.7, 119.2, 112.9, 112.5, 102.3, 100.7, 42.0, 34.0; HRMS (ESI+) calcd for C27H28N7
[M+H]+ 450.2401, found 450.2401.
5.1.33 2,6-Bis[6-(N,N-dimethylamidino)indol-2-yl]naphthalene (21; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 14 (150 mg, 0.37
1
mmol), compound 21 was obtained as brown solid (110 mg, 60% yield); H NMR (400 MHz,
DMSO-d6): δ 12.59 (s, 2H), 9.30 (s, 2H), 8.97 (s, 2H), 8.58 (s, 2H), 8.19 (d, J = 8.8 Hz, 1H), 8.08
(d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (s, 2H), 7.25 (s, 2H), 7.21 (dd, J = 8.4, 1.6 Hz,
2H), 3.29 (s, 6H), 3.09 (s, 6H); 13C NMR (125 MHz, DMSO-d6): δ 165.9,140.79, 136.4, 131.1,
128.8, 124.7, 123.9, 121.8, 120.4, 119.0, 112.3, 99.8, 42.0, 33.9; HRMS (ESI+) calcd for
C32H31N6 [M+H]+ 499.2605, found 499.2604.
5.1.34 3,5-Bis[6-(N, N-dimethylamidino)indol-2-yl]pyridine (22; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 15 (100 mg, 0.28
1
mmol), compound 22 was obtained as yellow solid (45 mg, 36% yield); H NMR (400 MHz,
DMSO-d6): δ 12.55 (s, 2H), 9.28 (br s, 2H), 8.95 (br s, 2H), 7.77 (s, 2H), 7.74 (d, J = 8.0 Hz, 2H),
7.64 (s, 2H), 7.20 (dd, J = 8.4, 1.2 Hz, 2H), 6.90 (s, 2H), 3.27 (s, 6H), 3.07 (s, 6H); 13C NMR (125
MHz, DMSO-d6): δ 165.8, 136.0, 135.0, 134.2, 130.9, 126.2, 121.9, 120.2, 119.2, 112.1, 99.3,
42.0, 33.9; HRMS (ESI+) calcd for C27H28N7 [M+H]+ 450.2401, found 450.2400.
5.1.35 1,3-Bis[6-(N, N-dimethylamidino)indol-2-yl]phene (23; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 16 (100 mg, 0.28
1
mmol), Compound 23 was obtained as brown solid (45 mg, 37% yield); H NMR (400 MHz,
DMSO-d6): δ 12.67 (s, 2H), 9.31 (br s, 2H), 8.99 (br s, 2H), 8.85 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H),
7.77 (d, J = 8.4 Hz, 2H), 7.71 (s, 2H), 7.62 (t, J = 8.0 Hz, 1H), 7.28 (s, 2H), 7.20 (d, J = 8.4 Hz,
2H), 3.28 (s, 6H), 3.09 (s, 6H); 13C NMR (125 MHz, DMSO-d6): δ 165.9, 140.6, 136.1, 132.1,
130.9, 129.6, 124.9, 121.7, 120.3, 118.9, 112.3, 99.4, 42.0, 33.9; HRMS (ESI+) calcd for

ACCEPTED MANUSCRIPT
C28H29N6 [M+H]+ 448.2375, found 449.2447.
5.1.36 2,7-Bis[6-(N, N-dimethylamidino)indol-2-yl]naphthalene (24; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 17 (160 mg, 0.27
1
mmol), compound 24 was obtained as brown solid (102 mg, 76% yield); H NMR (400 MHz,
DMSO-d6): δ 12.62 (s, 2H), 9.30 (br s, 2H), 8.95 (br s, 2H), 8.56 (s, 2H), 8.12 (m, 4H), 7.80 (d, J
= 8.4 Hz, 2H), 7.73 (s, 2H), 7.25 (s, 2H), 7.22 (d, J = 8.4 Hz, 2H), 3.28 (s, 6H), 3.10 (s, 6H); 13C
NMR (125 MHz, DMSO-d6): δ 165.9, 140.7, 136.4, 129.7, 128.5, 124.4, 124.1, 121.7, 120.3,
119.0, 112.4, 99.8, 42.0, 33.9; HRMS (ESI+) calcd for C32H31N6 [M+H]+ 499.2605, found
499.2603.
5.1.37 3,5-Bis[6-(N-methylamidino)indol-2-yl]pyridine (25; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 15 (110 mg, 0.31
mmol) and a solution of methylamine 30% in methanol (4.5 mL), compound 25 was obtained as
yellow solid (52 mg, 42% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.67 (s, 2H), 9.77 (br s, 2H),
9.46 (br s, 2H), 9.20 (s, 2H), 9.14 (s, 1H), 8.84 (br s, 2H), 7.92 (s, 2H), 7.83 (d, J = 8.0 Hz, 2H),
7.43 (dd, J = 8.4, 1.6 Hz, 2H), 7.40 (s, 2H), 3.05 (s, 6H); 13C NMR (125 MHz, DMSO-d6): δ 164.2,
138.3, 136.3, 131.8, 127.4, 121.7, 120.5, 118.9, 112.1, 100.6, 29.7; HRMS (ESI+) calcd for
C25H24N7 [M+H]+ 422.2088, found 422.2086.
5.1.38 3,5-Bis[6-( N-isopropylamidino)indol-2-yl]pyridine (26; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 15 (110 mg, 0.31
mmol) and 2-propylamine (154 µL, 1.8 mmol), compound 26 was obtained as gray solid (46 mg,
32% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.63 (s, 2H), 9.53 (br s, 2H), 9.37 (br s, 2H), 9.12
(s, 1H), 8.91 (br s, 2H), 7.87 (s, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.38 (m, 4H), 4.09 (m, 2H), 2.50 (s,
13
12H); C NMR (100 MHz, DMSO-d6): δ 162.6, 136.3, 131.5, 122.6, 120.7, 119.3, 112.3, 101.7,
42.8, 39.7, 21.3, 20.3; HRMS (ESI+) calcd for C29H32N7 [M+H]+ 478.2714, found 478.2712.
5.1.39 3,5-Bis[6-(N-isobutylamidino)indol-2-yl]pyridine (27; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 15 (110 mg, 0.31
mmol) and 2-butylamine (182 µL, 1.8 mmol), compound 27 was obtained as green-yellow solid
1
(65 mg, 40% yield); H NMR (400 MHz, DMSO-d6): δ 12.73 (s, 2H), 9.48 (d, J = 8.4 Hz, 2H),
9.37 (s, 2H), 9.21 (br s, 3H), 8.94 (s, 2H), 7.88 (s, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.42 (s, 2H), 7.40
(dd, J = 8.4, 1.2 Hz, 2H), 3.91(m, 2H), 1.66 (m, 4H), 1.29 (d, J = 6.4 Hz, 6H), 0.97 (t, J = 6.0 Hz,

ACCEPTED MANUSCRIPT
6H); 13C NMR (125 MHz, DMSO-d6): δ 163.2, 137.2, 136.3, 131.5, 122.8, 120.7, 119.3, 112.3,
101.5, 50.4, 28.1, 19.2, 10.4; HRMS (ESI+) calcd for C31H36N7 [M+H]+ 506.3027, found
506.3025.
5.1.40 3,5-Bis[6-(N-(2-methoxyethyl)-amidino)indol-2-yl]pyridine (28; as hydrochloric acid salt)
Following the procedure for preparation of compound 18, from compound 15 (110 mg, 0.31
mmol) and 2-methoxyethylamine (115 µL, 1.8 mmol), compound 28 was obtained as yellow solid
1
(35 mg, 22% yield); H NMR (400 MHz, DMSO-d6): δ12.68 (s, 2H), 9.77 (s, 2H), 9.46 (s, 2H),
9.22 (br s, 1H), 9.15 (br s, 1H), 8.96 (s, 2H), 7.90 (s, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.42 (dd, J =
8.4, 1.2 Hz, 2H), 7.40 (s, 2H), 3.65 (s, 6H), 3.36 (s, 8H); 13C NMR (125 MHz, DMSO-d6): δ 164.2,
138.4, 136.2, 131.8, 122.0, 120.5, 120.4, 119.1, 112.4, 112.2, 69.0, 58.2, 42.6; HRMS (ESI+)
calcd for C29H32N7O2 [M+H]+ 510.2612, found 510.2610.
5.2 Minimum Inhibitory Concentration Testing
Minimum inhibitory concentrations (MICs) of the target compounds were determined using the
broth microdilution protocol according to the methods of the Clinical and Laboratory Standards
Institute (CLSI) [30]. Organisms used in this study included strains from the ATCC collection and
clinical isolates. The test medium was Mueller-Hinton broth, and the inoculum was 10 000 colony
forming units (cfu)/spot. Levofloxacin was used as positive control. All of the test compounds
were dissolved in DMSO and then serially diluted in growth medium. The final concentrations of
compounds ranged from 0.03 to 128 µg/mL. Culture plates were incubated at 35-37 °C for 18 h,
and MICs were then recorded. The MIC was defined as the lowest concentration that prevented
visible growth of the bacteria. The microdilution broth susceptibility testing was performed in
triplicate.
5.3 Determination of mammalian cytotoxcity.
Human embryonic kidney 293t cells were seeded onto a 96-well plate at a concentration of 1
x105 Vero cells per mL and a volume of 200 µL per well. Following 24 h incubation at 37 °C, a
confluent cell monolayer was confirmed. Test compounds were diluted with DMSO reach the
different concentrations. Negative control dilution of DMSO at 1% was also included. 2 µL /well
of each diluted compound or DMSO was added to the plates in triplicate. After incubation at 37
°C with 5% CO2 for 48 h, cell supernatants were replaced with fresh medium containing MTS

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reagent (110 µL medium containing 10 µL MTS reagent per well). The trays were further
incubated for 2 h to allow MTS production. The absorbance was determined with an ELISA
reader at a test wavelength of 490 nm. Data were calculated as the percentage of inhibition using
the following formula: inhibition % = [100 – (At/As) x 100] %; At and As refer to the absorbance
of the test substances and the solvent control, respectively. CC50 values, defined as the
concentration of 50% cellular cytotoxicity (CC50) of test compounds.
5.4 Murine Systemic Infection Model
S. aureus MRSA (ATCC 33591) strain: ICR mice (half male and half female) weighing between
18 and 22 g were used in this study, with ten mice in each group. A lethal systemic infection was
given to the mice by intraperitoneal injection of 0.5 mL of inoculum of S. aureus MRSA ATCC
33591 strain, 5 × 105 CFU/mL. Compounds were administered intravenously at 1.0 and 5.0 h
postinfection at doses of 2.0, 2.5, 3.0, 3.5, and 4.0 mg/kg for 5i. The ED50 was calculated 7 day
after treatment by the method of Bliss.
Acknowledgments
The authors thank the National Natural Science Foundation of China (Grants 81573298, 81321004,
81361138020) and the National High-tech R&D Program (863 Program) (Young Scientist Grants
2015AA020911) for financial support.
Supplementary material
Supplementary data (the activity data of all compounds for 41 strains) associated with this article
can be found in the online version.
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