Biological evaluation of tetracationic compounds based on two 1,4-diazabicyclo[2.2.2]octane moieties connected by different linkers

Article abstract of DOI:10.1016/j.bmc.2016.09.064

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2?h. Furthermore, compounds 1a–c with aromatic linkers and compound 1e showed the highest antiviral activity.

Full text of DOI:10.1016/j.bmc.2016.09.064

Accepted Manuscript
Biological evaluation of tetracationic compounds based on two 1,4-diazabicy-
clo[2.2.2]octane moieties connected by different linkers
Ekaterina A. Burakova, Irina V. Saranina, Nina V. Tikunova, Zhanna K.
Nazarkina, Pavel P. Laktionov, Lubov’ A. Karpinskaya, Vadim B. Anikin,
Vladimir V. Zarubaev, Vladimir N. Silnikov
PII:
S0968-0896(16)30817-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.064
BMC 13317
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2016
20 September 2016
24 September 2016
Please cite this article as: Burakova, E.A., Saranina, I.V., Tikunova, N.V., Nazarkina, Z.K., Laktionov, P.P.,
Karpinskaya, L.A., Anikin, V.B., Zarubaev, V.V., Silnikov, V.N., Biological evaluation of tetracationic compounds
based on two 1,4-diazabicyclo[2.2.2]octane moieties connected by different linkers, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.064
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Biological evaluation of tetracationic compounds based on two 1,4-
diazabicyclo[2.2.2]octane moieties connected by different linkers
Ekaterina A. Burakovaa*, Irina V. Saraninaa, Nina V. Tikunovaa, Zhanna K. Nazarkinaa, Pavel P.
Laktionova, Lubov’ A. Karpinskayab, Vadim B. Anikinb, Vladimir V. Zarubaevb, Vladimir N.
Silnikova
a
Institute of Chemical Biology and Fundamental Medicine, Lavrent’ev Ave. 8, Novosibirsk, 630090, Russia
b
Influenza Research Institute, Prof. Popova Str. 15/17, St. Petersburg, 197376, Russia
* Corresponding author. Tel.: +7-383-363-5183; Fax: +7-383-363-5182.
E-mail: kat7@ngs.ru
Abstract
A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated
for activity against several strains of both Gram-positive and Gram-negative bacteria including
drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All
compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The
minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl
linker and compound 1e with a propylene aliphatic linker were found to be low and were
comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and
Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the
bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and
Ps. aeruginosa after 2 h. Furthermore, compounds 1a–c with aromatic linkers and compound
1e showed the highest antiviral activity.
Keywords: Antimicrobial activity; antiviral activity; cytotoxicity; 1,4-diazabicyclo[2.2.2]octane
(DABCO); quaternary ammonium compounds (QACs)

1. Introduction
The wide application of antibiotics has led to the emergence of resistant strains of
microorganisms at the beginning of the 21st century [1–7]. The discovery and development of
new antimicrobials is one of the main challenges of modern medicinal chemistry. Among the
potential candidates in the search for new drugs, attention may be drawn to quaternary
ammonium compounds (QACs), which are used as antimicrobial, disinfectant and sterilizing
agents [8–10]. These compounds have been found to exert antimicrobial activity against both
Gram-positive and Gram-negative bacteria as well as against some pathogenic species of fungi
and protozoa, and may be active against enveloped viruses. QACs are cationic amphiphilic
organic molecules with at least one major hydrophobic long alkyl chain associated to one
quaternary nitrogen atom. Cationic amphiphiles have the ability to perturb the lipid membranes
of microbes through electrostatic and hydrophobic interactions [8]. At first, the positively
charged sites of cationic molecules provide strong interaction with the predominantly anionic
components of the microorganism’s surface (lipopolysaccharides for Gram-negative bacteria and
teichoic acid for Gram-positive bacteria, as well as phospholipids of both cells) by electrostatic
interactions. Then, the hydrophobic groups of the cationic amphiphiles provide compatibility
with the hydrophobic end of the phospholipid of the cytoplasmic membrane and allow the
molecules to be inserted into the cell membrane, resulting in disruption of the cytoplasmic
membrane. Damage of the membrane results in the release of potassium ions and other
cytoplasmic constituents, finally leading to the death of the cell [11].
Conventional QAC applications are limited, due to reduced susceptibility to QACs as well as
cross- and co-resistance to antibiotics [12]. Thus, new types of QACs as antimicrobial agents are
needed. Among the different classes of QACs, bis-type QACs, called gemini amphiphiles, have
shown a comparatively higher antimicrobial activity than their mono-ammonium analogues
[11,13,14]. Bis-QACs contain two quaternized nitrogen atoms, each bearing a hydrophobic tail,
covalently connected by a linker. Because of the linker in bis-QACs, the two hydrophilic polar
head groups are so close that the charge densities of these surfactants are higher than those of
conventional QACs. Therefore, it is reasonable to assume that bis-QACs would be able to more
rapidly adsorb to the bacteria surface than conventional QACs and result in bacteria death [15].
The antimicrobial activity of QACs strongly depends on the alkyl chain length, the structure of
the cationic group and the linker [10,15–17].
It is known from the literature that QACs based on 1,4-diazabicyclo[2.2.2]octane (DABCO)
such as mono-, di- and polycationic DABCO derivatives exhibit antibacterial and antifungal
activity [18–21]. The DABCO moiety, with its ability to have two positive charges, has higher
fixed charge density than other commonly used cationic antimicrobial agents [22]. In our recent

work we reported synthesis of a series of tetracationic QACs, each with two DABCO moieties
substituted at the bridge nitrogen atom with alkyl chains of length C2–C18 connected by a 1,5-
pentenyl linker [23]. We studied the effect of alkyl chain length on microbial activity and
showed that antibacterial activity was enhanced with an increase in chain length, exhibiting a
cut-off effect at C12 chain length. Further increase of the alkyl chain length leads to the reduction
of antibacterial activity. The influence of the length of the alkyl tails in the compounds on their
antimicrobial activity may be explained by the balance of hydrophobicity/hydrophilicity and
solubility. The investigation of dodecyl tails containing tetracationic DABCO-based derivatives
is the aim of the present study.
Recently, we studied ribonuclease activity of the DABCO derivatives [24]. Some compounds
capable of phosphodiester bond cleavage displayed antiviral activity [25,26].
Hence, we evaluated the antimicrobial activity of a series of compounds on the basis of
quaternary salts of DABCO with dodecyl tails connected by different linkers (Fig. 1, Scheme 1,
1a–n) against a panel of bacteria and a fungus, as well as antiviral activity against influenza virus
A/Puerto Rico/8/34 (H1N1). We also carried out cytotoxicity assays of the compounds and
calculated selectivity indexes. Minimum inhibitory concentration (MIC), minimum bactericidal
concentration (MBC) as well as 50% effective concentration (EC50) and 50% cytotoxic
concentration (CC50) were tested. The most active compounds were evaluated in a time-kill
kinetic studies assay to determine effective time of incubation and the influence of these
compounds on Gram-positive and Gram-negative bacteria growth. Structure–activity relationship
(SAR) was delineated by varying the structure of linkers: long or short, hydrophilic or
hydrophobic, flexible or rigid.
2. Experimental protocols
2.1. Chemistry (see Scheme 1)
See the Supporting Information for full experimental data.
2.2. Biology
2.2.1 Antimicrobial assays
Bacterial strains Ps. aeruginosa (ATCC 9027), Pr. vulgaris (ATCC 6380), C. freundii (ATCC
8090), S. enterica (ATCC 14028), E. coli (ATCC 25922), St. aureus (ATCC 25923, CEMTC 675,
CEMTC 707, CEMTC 1695, CEMTC 1732, CEMTC 1733, CEMTC 1850), E. faecalis (ATCC
51299) and one yeast C. albicans (CEMTC 34) were obtained from the collection of
extremophilic microorganisms and type cultures of the Siberian branch of the Russian Academy
of Science (Novosibirsk, Russia). All tested strains were routinely cultivated in Luria broth (LB)

at 37 °C with shaking overnight. The drugs ciprofloxacin and gentamicin, and clotrimazole
were used as positive controls for antibacterial and antifungal activity, respectively. Sterile water
was used as a negative control.
2.2.1.1 MIC and MBC (MFC) determination
Screening of the susceptibility of different bacterial strains and fungus to the active
compounds that was revealed from a modified method was performed by quantitative assay of
MIC (μg/mL) based on liquid medium serial microdilutions [27]. All studied compounds except
1l–n were water-soluble at room temperature. Compounds 1l–n were solubilized in water by
heating. Stock solutions were prepared by serial dilution of the compounds using sterilized Milli-
Q water. Two-fold serial dilutions of each compound solution along with control antibiotics were
prepared in polypropylene 96-well microliter plates with Mueller–Hinton broth (MHB)
(Sabouraud Dextrose broth (SDB) for C. albicans) followed by the addition of organism
suspension. The final concentration of organism inoculum was approximately 5 × 105
CFU/mL in the final volume of 200 μL. The plates were then incubated at 37 °C with
continuous orbital shaking (580 rpm) in a shaker incubator. Aliquots were withdrawn to
perform colony counts after 2, 4, 6, 8 and 22 h. MICs were determined by means of
spectrophotometric readings at 595 nm (Uniplan, PICON, Russia) and were defined as the
lowest concentration of the compounds found to prevent growth of the tested bacterium. MBC or
MFC was defined as lowest concentration at which no visible growth was observed. All
experiments were carried out in duplicate in two separate experiments. MIC and MBC values of
the compounds 1a–n are presented in Table 1.
2.2.1.2 Time-kill assay
Overnight cultures of St. aureus and Ps. aeruginosa were adjusted in MHB to contain initial
inoculum ~ 8 × 105 and ~ 2.5 × 105 CFU/mL, respectively. Compound 1c or 1e was added
to the St. aureus bacterial solution at concentrations of 1.25, 2.5 and 5 μg/mL and to the Ps.
aeruginosa bacterial solution at concentrations of 2.5, 5 and 10 μg/mL. The mixtures were then
incubated at 37 °C with shaking (580 rpm) for 22 h. Aliquots were withdrawn to perform
colony counts after 2, 4, 6, 8 and 22 h. The number of viable bacteria was determined by
counting the colonies that grew on the plates. The limit of quantification by these methods is
100 CFU/mL. All experiments were carried out in triplicate. Time-kill profiles of compound 1c
or 1e against both bacterial strains are shown in Table 3.
2.2.2 Toxicity study
HEK293T human embryonic kidney cell line and MCF-7 human breast cancer cell line were
used for cytotoxicity assays.

Cells were grown as a monolayer in Iscove’s Modified Dulbecco’s Medium (IMDM),
supplemented with 10% fetal bovine serum (FBS; Sigma), 100 mg/mL streptomycin and
100 units/mL penicillin, at 37 °C in an atmosphere of 5% CO2. HEK293T or MCF-7 cells
were seeded at 20,000 cells into each well of a 96-well plate in 100 µL of culture medium to
grow overnight. The next day, the medium was replaced with fresh medium supplemented with
the compounds at 10–250 µg/mL doses. Compounds at each concentration were tested in
duplicate in a single experiment and each experiment was repeated at least two times
independently. The stock solutions of the compounds were prepared in water and further
dilutions were made with fresh medium. The plates were incubated at 37 °C in an atmosphere
of 5% CO2 for 24 h. After 24 h the medium was replaced with fresh medium, 10 µL 12 mM
MTT reagent (Molecular Probes) was added to each well, and then plates were incubated for
another 4 h in CO2 at 37 °C. SDS-HCl solution (100 µL) was added to each well. The plates
were incubated at 37 °C in an atmosphere of 5% CO2 overnight to dissolve the precipitate. The
next day, the absorbance was read at 570 nm. Cell viability was expressed as percentage
compared to untreated control cells (100% viability). The percentage of cell viability was
calculated based on the following formula:
% cell viability = (A/Acontrol) × 100
where A represents the sample absorbance at a given concentration of compound and Acontrol
represents untreated cells.
Results are presented as mean CC50 (cytotoxic concentration of the tested compound allowing
survival of 50% of the cells) and summarized in Table 4.
2.2.3 Antiviral assay
The influenza virus A/Puerto Rico/8/34 (H1N1) from the virus collection of the Influenza
Research Institute (St. Petersburg, Russia) was used. The virus was cultivated in 10–12 day-old
chicken embryos at 37 °C for 48 h. MDCK cells (ATCC CCL 34) or U-87 MG cells in
minimal essential medium (MEM; PAA, Austria, Cat.# E15-825) were seeded onto 96-well
plates (Orange Scientific No. 5530100) and incubated at 37 °C in 5% CO2 until a confluent
monolayer formed. To cultivate the virus, 5% albumin, trypsin (1 µg/mL), and 16 mM HEPES
(pH 7.6) were added. Oseltamivir was used as the reference drug. The cytotoxicity of compounds
1a–c and e–i against the MDCK cells was evaluated in parallel with antiviral activity as
described [28].
The compounds at appropriate concentrations were incubated with MDCK cells at 37 °C for
1 h. The cell culture was then infected with 10-fold dilutions of the virus (10−1–10−6). Plates

were incubated at 37 °C in the presence of 5% CO2 for 48 h. The infection activity of the virus
was evaluated by the hemagglutination reaction with chicken erythrocytes. A virus-containing
solution (100 µL) was placed into wells of a round-bottom plate. An equal amount of a 1%
suspension of chicken erythrocytes in saline was then added. The reaction was evaluated after
incubation at room temperature for 60 min. Each compound concentration was tested in
triplicate. A virus titer was considered as reciprocal to the decimal logarithm of the maximum
dilution that caused complete agglutination of erythrocytes and was expressed as logarithms of
the 50% experimental infection dose (log10 EID50). The antiviral activity of the compounds was
estimated by a decrease in the virus titer compared to the control. The EC50 of a drug, i.e. the
concentration at which the virus production is decreased two-fold (virus titer per 0.3 log10
EID50), and the selectivity index (CC50 to EC50 ratio) were calculated from the data obtained and
are presented in Table 5.
3. Results
3.1 Chemistry
For better SAR, DABCO-based compounds were studied such as: relatively rigid linkers
containing an aromatic core (phenyl (1a–c) and biphenyl (1d)), olefinic (1f) and acetylenic
fragments (1g), a triazole ring (1m–n), flexible aliphatic linkers (1e, 1h–j) and flexible linkers
with a nitrogen atom (1k–l).
The tetracationic DABCO derivatives can be readily prepared by sequential alkylation of
DABCO in the appropriate solvent. Previously, we have published the synthesis of compounds
1a–j from mono-salt 7 with the appropriate dihalide in acetonitrile or dimethylformamide (DMF)
as solvent [24,29] (Fig. 1). In our work [23] we replaced the solvent with methanol to enable us
to prepare compounds on a large scale. Compound 1k with a linker containing a nitrogen atom
was synthesized by the reaction of mono-salt 7 with dichloride 3 in methanol with a high yield
(Scheme 1).

N+ C12H25
N+
N+ C12H25
N+
N+
C12H25 N+
N+
C12H25 N+
1e
1a
N+
N+ C12H25
4X-
C12H25 N+
N+
1f
N+ C12H25
N+
N+
N+ C12H25
4X-
N+
C12H25 N+
1b
C12H25 N+
N+
1g
N+ C12H25
N+
4X-
C12H25 N+
C12H25 N+
C12H25 N+
N+
N+
N+ C12H25
N+
C12H25 N+
1h
1c
N+ C12H25
N+
N+
1i
N+
C12H25 N+
4X-
N+ C12H25
N+
N+
N+ C12H25
N+
1d
1j
X = Cl, Br
Figure 1. Structures of compounds 1a-j.
New compounds 1l–n with linkers containing functional groups were designed to produce a
new hybrid antibiotic with improved antibacterial properties. Development of bifunctional
antibacterials with distinct modes of action is a radical therapeutic approach for the production of
compounds displaying an extended spectrum of activity, including also drug-resistant pathogens
[30,31]. For it to be successful the hybrid agents must be more effective than the sum of their
parts and so we evaluated antimicrobial activity of the compounds 1l–n.
For preparation of these compounds we began with the synthesis of the linker groups 6, 13
and 14 (Scheme 1). We chose a number of functional groups such as carbonyl, azide and alkyne.
These functionalities provide a platform for further modifications. The reaction between a
carbonyl and an amine (hydrazine) is highly chemoselective and is compatible with other
functional groups present in molecules. The resulting imine (hydrazone) bond is covalent but
reversible, and both its stability and dynamics strongly depend on the nature of the amine and the
carbonyl. Introduction of the alkyne or azide group into the compound linker permits facile
chemical modification of such compounds using an azide–alkyne 1,3-dipolar cycloaddition
reaction. This reaction between an azide and a terminal alkyne is being widely explored because
of some interesting properties like quantitative yield, high tolerance of functional groups [32],

simple reaction conditions, high chemoselectivity with no side products, chemical inertness of
the 1,2,3-triazole ring and ease of product isolation. Moreover, the 1,2,3-triazoles formed are N-
heterocyclic compounds which display high biological activity including antimicrobial and
antiviral activity [33,34]. Compounds 6, 13 and 14 were prepared in two steps from diols 5, 11
and 12, respectively. The final products 1l–n were synthesized by the reaction of mono-salt 7
with the corresponding compounds 6, 13 and 14 in methanol (Scheme 1) with high yields. All
1
new compounds were characterized by H NMR, 13C NMR, and ESI-MS as given in the
Supporting Information.
Scheme 1. Synthesis of compounds 1k-n.
3.2 Biological studies
3.2.1 MIC and MBC (MFC) assays
The effect of linker structure on antimicrobial activity was studied on five Gram-negative
bacteria (Pseudomonas aeruginosa ATCC 9027, Proteus vulgaris ATCC 6380, Citrobacter
freundii ATCC 8090, Salmonella enterica ATCC 14028, Escherichia coli ATCC 25922), eight
Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC

51299 and methicillin-resistant St. aureus (MRSA) strains CEMTC 675, 707, 1695, 1732, 1733
and 1850) and one fungus – the yeast Candida albicans CEMTC 34. The common antibacterial
drugs ciprofloxacin and gentamicin, and antimycotic drug clotrimazole were used as standards
for antibacterial and antifungal activity, respectively. The in vitro antimicrobial activities of the
compounds and standard drugs were tested by serial dilutions in Mueller-Hinton broth (MHB).
The dependencies between changes of optical density and concentration of the compounds
against some tested strains can be found in the Supporting Information.
As illustrated in Table 1, compounds based on the quaternary salts of DABCO exhibited
inhibitory activity against both Gram-positive and Gram-negative bacteria with MIC values from
0.25 to 100 μg/mL. All compounds showed high antifungal activity toward C. albicans with
MIC values from 1.56 to 25.0 μg/mL, that is 16 to 250-fold better values than clotrimazole
(MIC 400.0). The MBC value was one or two times the MIC in all strains of bacteria and the
minimum fungicidal concentration (MFC) value was one or two times the MIC in the case of C.
albicans for the compounds studied, typical for bactericidal (fungicidal) compounds. The
bactericidal/fungicidal activity is defined as a ratio of MBC/MFC to MIC of ≤ 4 [35].
The Gram-positive strains St. aureus and E. faecalis were the most sensitive to the action of
the compounds. Their growth was completely inhibited with all the compounds studied at a
concentration of 0.3–3.1 µg/mL. All compounds exhibited better anti-St. aureus activity than
ciprofloxacin (MIC 6.3) and gentamicin (MIC 50.0) which were used as positive controls. Most
of the compounds tested were found to have moderate to high antibacterial activities against
Gram-negative bacteria (E. coli, C. freundii, S. enterica and Ps. aeruginosa) except Pr. vulgaris.
E. coli and Ps. aeruginosa from the Gram-negative strains tested were the most sensitive to the
action of these compounds. In this study, most compounds exhibited activity against these
bacteria at a concentration of 1.6–6.3 µg/mL. In the case of Ps. aeruginosa the MIC and MBC
values of compounds 1b, c, e–f and h are equal to the values of the standard drug ciprofloxacin
and better than the values of the drug gentamicin. The discovery of high anti-Ps. aeruginosa
activity of these compounds has essential interest; most of the known QACs exhibit low or no
activity against this bacterium. Among Gram-negative strains, Ps. aeruginosa is mostly a
nosocomial pathogen. This bacterium is a highly adaptable organism [36] and has developed
resistance to multiple antimicrobial agents with the greatest frequency, in some cases expressing
resistance to all clinically available compounds [37].

Table 1. Antimicrobial activity of compounds 1а-n (MICa and MBCa (MFCa) in µg/mL).
№
Compound
test strains
Gram negative bacteria
S. enterica C. freundi
Gram positive bacteria
St. aureus E. faecalis
yeast
C. albicans
Ps.
E. coli
Pr. vulgaris
aeruginosa
MBC
1.2
1.6
1.2
1.2
1.2
1.6
3.1
1.2
1.2
1.6
1.2
3.1
3.1
3.1
12.0
100.0
nd
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MFC MIC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
0.6
0.6
0.6
0.6
0.3
1.2
1.6
0.6
0.6
0.6
0.6
1.6
1.6
1.6
6.3
1.6
1.6
1.6
1.6
3.1
1.6
3.1
1.6
1.6
1.6
1.6
3.1
3.1
1.6
0.8
1.6
1.6
0.8
0.8
1.6
1.6
1.6
1.6
1.6
1.6
0.8
1.6
1.6
1.6
0.4
12.5
3.1
3.1
6.3
3.1
3.1
12.5
3.1
6.3
6.3
6.3
1.6
1.6
3.1
1.6
1.6
3.1
1.6
3.1
3.1
1.6
6.3
6.3
3.1
1.6
3.1
3.1
3.1
3.1
3.1
3.1
12.5
3.1
3.1
3.1
3.1
3.1
3.1
3.1
0.8
1.6
1.6
1.6
1.6
1.6
3.1
6.3
1.6
1.6
1.6
1.6
3.1
3.1
1.6
0.4
3.1
3.1
3.1
3.1
3.1
3.1
12.5
3.1
3.1
6.3
3.1
3.1
3.1
3.1
1.6
1.6
1.6
1.6
1.6
1.6
6.3
1.6
1.6
3.1
1.6
3.1
3.1
1.6
3.1
3.1
1.6
1.6
3.1
3.1
12.0
1.6
3.1
6.3
3.1
3.1
3.1
3.1
1.6
1.6
1.6
1.6
1.6
1.6
6.3
1.6
1.6
3.1
3.1
3.1
1.6
1.6
<0.2
0.4
nd
25.0
25.0
>100 100.0
100.0 50.0
>100 100.0
>100 100.0 12.5
>100 100.0 12.5
>100 25.0
>100 >100 12.5
>100 100.0 12.5
50.0
>100 100.0 25.0
>100
12.5
25.0
12.5
12.5
6.3
6.3
6.3
12.5
12.5
3.1
1.6
3.1
12.5
12.5
6.3
6.3
6.3
12.5
25.0
6.3
12.5
nd
6.3
6.3
50.0
12.5
12.5
12.5
6.3
100
12.5
12.5
nd
100.0 50.0
15 ciprofloxacin
16
17
3.1
<0.2 <0.2 <0.2
<0.2
1.6
nd
<0.2
0.8
nd
gentamicin
clotrimazole
50.0 100.0 50.0 >400 >400 100.0 50.0
1.6
0.8
0.8
nd
nd
nd nd nd nd nd nd nd
nd
nd
nd
400
400
aValues are the average of two independent experiments run in duplicate. The best values for each strain are given in bold.
The grey highlighting indicates the most active compounds.

For compounds 1e and h–j with flexible aliphatic linkers we observed an optimal distance
between DABCO moieties of three bonds (compound 1e) for high antibacterial activity against
all microorganisms. Among compounds 1a–d containing aromatic linkers (o-, m-, p-
phenylenebismethyl, biphenyl) compounds 1c and 1d displayed the highest inhibitory effect
against all strains tested, near to that of compound 1e. The structure of linker groups containing
the same amount of carbon atoms (C4, compounds 1c, f–h, m and n) insignificantly influences
the antibacterial activity of the compounds. Nevertheless, for some strains, MICs differed two to
four-fold. We supposed that compounds 1m and n would be more active than analogues 1c or 1f
because the 1,2,3-triazole ring in the linkers of these compounds may contribute to the
antibacterial activity against the tested bacteria since triazoles are effective antibacterial
functional groups. However, the presence of the triazole ring increased MIC values, and
compounds 1m and n exhibited equal or comparatively lower (two or four-fold) antimicrobial
activity than that of analogues 1c and 1f.
Drug resistance is one of the major problems at the present time. The emergence of MRSA
strains has complicated the treatment of St. aureus infections [38–40]. This type of
staphylococcal strain was first discovered in 1961. At present, MRSA are resistant to all β-
lactams and to some other widely used agents and belong to the so-called multidrug-resistant
strains. One of the few drugs effective in combating infections caused by MRSA is ciprofloxacin
[41]. Excellent antibacterial activity defined for a drug-susceptible strain of reference St. aureus
made us check that the most active compounds 1c and 1e were also effective against MRSA
strains. As shown in Table 2, compounds 1c and 1e exhibited inhibitory activity against all
MRSA strains with MIC values from 0.5 to 1.0 μg/mL. Reference drugs at the same
concentrations did not exhibit any bactericidal properties (ciprofloxacin), or acted selectively
only on some of the tested strains (gentamicin).
Table 2. Activity of compounds 1c, 1e against hospital strains MRSA (MIC and MBC in
μg/mL).
№
Compound
Staphylococcus aureus strains
1695 1732
675
707
1733
1850
MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC
1
2
3
4
1c
1e
1.0
1.0
>8
0.5
0.5
>8
1.0
1.0
`>8
1
0.5
0.5
>8
1
1.0
1.0
>8
1.0
1.0
8
1.0
1.0
2
0.5
0.5
1
1.0
1.0
>8
1
1.0
1.0
8
1.0
1.0
>8
0.5
1.0
8
ciprofloxacin
gentamicin
>100 >100
>100 >100 >100 >100
1
>100
10
3.2.2 Time-kill assays
In order to obtain some insight and information on the mode of action of these compounds,
derivatives 1c and 1e, which showed the lowest MIC values against almost all species tested,

were selected to conduct time-kill assays. The MBC assay may give a general indication of
bactericidal activity, while time-kill curves give a more meaningful measurement [42]. In
previous work it was revealed that the compound bearing dodecyl tails (1i) showed clear
bactericidal activity against both low (104) and high (107) inoculum of St. aureus and Ps.
aeruginosa at concentrations of 10 μg/mL. In this work, compounds 1c and 1e, bearing dodecyl
tails but other linkers, were tested in time-kill studies against Gram-positive St. aureus and
Gram-negative Ps. aeruginosa (inoculum ~ 105). The compounds were compared at 1.25, 2.5
and 5 μg/mL against St. aureus and 2.5, 5 and 10 μg/mL against Ps. aeruginosa.
Dependencies between changing microorganism populations and interaction times for
compounds 1c and 1e are presented in Table 3. The bacterial growth curves under the influence
of compounds 1c and 1e are shown in Figure 2.
Table 3. Bactericidial effect of the compounds 1c, 1e against Ps. aeruginosa and St. aureus
Strain Compound
Population of microorganisms, CFU/ml (reduction, %)
С
0 h
2 h
4 h
6 h
8 h
22 h
MBC/MIC
1c
3.1/1.6
(μg/ml)
10
<102 (≥99,9) <102 (≥99,9) <102
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
2,8x105
2,8x105
2,8x105
2,8x105
2,8x105
2,8x105
(≥99,9)
<102 (≥99,9) <102
5
3,3x102
(99,0)
(≥99,9)
(≥99,9)
102
(≥99,9)
2.5
10
5
6,8x103
(90,0)
1,3x102
102
3,0x102
(≥99,9)
(≥99,9)
(≥99,9)
1e
3.1/1.6
<102 (≥99,9) <102 (≥99,9) <102
<102 (≥99,9) <102
(≥99,9)
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
1,7x109
<102 (≥99,9) <102 (≥99,9) <102
(≥99,9)
<102
2.5
3,3x102
(99,0)
102 (≥99,9)
(≥99,9)
control
0
5
3,6x105
1,2x107
108
2,7x108
2,8x105
8,0x105
1c
1.2/0.6
<102 (≥99,9) <102 (≥99,9) <102
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
2,9x109
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
2.5
1.25
5
103 (99,0)
8,0x105
8,0x105
8,0x105
8,0x105
8,0x105
8,0x105
6,7x102
(≥99,9)
1e
1.2/0.3
<102 (≥99,9) <102 (≥99,9) <102
(≥99,9)
<102 (≥99,9) <102
(≥99,9)
2.5
1.25
0
3,3x102
(≥99,9)
<102 (≥99,9) <102 (≥99,9) <102
(≥99,9)
control
106
7,8x106
1,2x107
2,7x108
Bactericidal activity was defined as a reduction of 99.9% (≥ 3log10) of the total count of
colony forming units (CFU) in the starting inoculum. Both compounds at the lowest
concentration of 1.25 μg/mL (~ 2 × MIC and ~ 4 × MIC, respectively) were rapidly
bactericidal against St. aureus, causing a 1000-fold reduction in bacteria count in 2 h, while
their bactericidal activity against Ps. aeruginosa was observed at the lowest concentration after

4 h of incubation. In the case of Ps. aeruginosa, compounds displayed a concentration-
dependent bactericidal effect, with faster killing kinetics at higher concentrations. After 2 h of
incubation compounds 1e and 1c inhibited bacterial growth at concentrations of 5.0 μg/mL
(~ 3 × MIC) and 10.0 μg/mL (~ 6 × MIC), respectively. The ratio of MBC to MIC and
time-kill results were consistent for both compounds.
Figure 2. Time-kill kinetics of compounds 1c and 1e against (a) Ps. aeruginosa (at 2.5
μg/mL) and (b) S. aureus (at 1.25 μg/mL).
3.2.3 Cytotoxicity
To investigate whether the antimicrobial activities of the compounds related specifically to
their selective toxicity toward the tested strains, we carried out cytotoxicity assays of our
compounds. Compounds 1a–c and e–i were tested on normal (HEK293T) and cancer (MCF-7)
cell lines. The results showed that the majority of the compounds had similar effects in the two

cell lines (HEK293T and MCF-7). Compounds showed cytotoxicity within CC50 values ranging
from 4 to 23 μg/mL (Table 4). The selectivity index (SI), an important pharmaceutical
parameter, was determined as the ratio of the CC50 value for the normal cell line (HEK293T) to
the MIC values of the microbial strains (SI = CC50/MIC). According to literature reports, SI
values higher than 8 are considered indicative of a potential therapeutic agent that would merit
further pharmaceutical and pre-clinical studies [42-48]. The compounds exhibited high in vitro
antimicrobial activities at nontoxic concentrations within SI values ranging from 2.5 to 26.7 for
all bacterial strains except Pr. vulgaris and from < 1 to 2 for C. albicans. Although compound
1e was one of the most active because of its high toxicity, it showed high selectivity only for St.
aureus (SI = 22). In the case of compounds containing aromatic linkers, derivatives possessing
para- and meta- substitution patterns, 1a and 1b showed high selectivity against all bacterial
strains (SI ≥ 10). At the same time, the ortho-isomer 1c showed low therapeutic indexes
(SI ≤ 7).
Table 4. Cytotoxicity and the calculated SIs.
№
Compound
a
CC50
16.0
17.0
4.0
5.0
8.0
23.0
4.0
7.0
60
CC50
16.0
16.0
4.0
SIb
27
27
7
22
8
14
7
13
>10
NDc
SI
10
10
5
4
6
14
3
5
>150
ND
SI
3
10
3
4
6
7
3
2
>38
SI
10
10
3
4
3
4
3
5
SI
10
10
3
4
6
4
3
5
SI
10
10
3
4
6
4
3
5
SI
1
SI
1
1
1
2
<1
2
<1
1
ND
«1
1
2
1a
1b
<1
<1
<1
<1
<1
<1
<1
3
1c
4
1e
6.5
5
1f
10.0
22.0
4.0
7.5
>60
6
7
1g
1h
8
1i
9
ciprofloxacin
>150 >300 >300 >300
ND ND ND ND
10
clotrimazole
5
7
ND
aValues are the average of two independent experiments run in duplicate.
bSI is the ratio of CC50 on normal HEK293T cells to MIC for bacterial cells. The best values of
SI are given in bold.
cND= Not determined.
3.2.4 Antiviral activity
To evaluate more widely the biological properties of DABCO derivatives and on the basis of
reported antiviral activities of some compounds against a number of viruses [25, 26], we tested
compounds 1a–c and e–n in a cell-based assay against influenza virus A/Puerto Rico/8/34
(H1N1). Influenza still remains a serious public health problem because of the appearance of
resistant strains.

The cytotoxicity of compounds 1a–c and e–n against MDCK cells was evaluated in parallel
with antiviral activity. Most compounds showed different toxic effects against MDCK cells than
in the HEK293T and MCF-7 cell lines. It is noteworthy that for compound 1g, a significantly
higher toxicity was observed in MDCK cells than in the two other cell lines. In contrast,
compound 1e showed lower toxicity in MDCK cells. The fact that the toxicity of compounds
was dependent on the type of cell used to assay toxicity was observed both in our recent work
[23] and earlier [49].
The antiviral potency and selectivity of these compounds were lower than that of the
reference drug oseltamivir (EC50 0.1, SI 1000) as illustrated in Table 5. A relationship between
compound structure and antiviral activity may be analyzed. Among compounds 1e and h–j with
flexible aliphatic linkers, antiviral activity decreased with the increase of linker length from
propylene to octamethylene. Compound 1e was the most potent and showed the highest
selectivity (SI = 7). In compounds containing aromatic linkers (o-, m-, p-phenylenebismethyl),
1a–c displayed a slight increase in potency over 1e, but derivatives possessing para- and ortho-
substitution patterns, 1a and 1c exhibited potent viral inhibitory activity together with high
toxicity, which indicated a low therapeutic index (SI = 2). At the same time, the most active
and least toxic compound was the meta-isomer 1b with a therapeutic index of 6. Replacement of
the CH2 group in 1i (2.4 μg/mL, SI = 4) by NH as in 1k (2.2 μg/mL, SI = 4) did not
result in a decrease in potency, while introduction of a substituent into a linker resulted in a
significant loss of activity as seen in compounds 1l–n. Although these compounds were less
toxic, they showed low selectivity (SI ≤ 3).
Table 5. Antiviral activity of compounds 1a–c and e-n against the influenza virus A/Puerto
Rico/8/34 (H1N1).
a
SIb
a
Compounds
CC50
EC50
1
2
3
4
5
6
7
8
9
10
11
12
13
1a
1b
1c
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
2.9 0.4
8.4 0.8
1.9 0.3
12.8 0.9
6.8 0.1
4.0 0.0
6.8 0.4
9.7 0.4
4.5 0.3
7.7 0.2
14.9 0.8
20.6 1.1
12.9 0.6
300 21
1.0 0.2
1.3 0.2
1.0 0.3
1.9 0.4
2.6 0.4
>3
3
6
2
7
3
<1
<2
4
2
4
>3
2.4 0.3
2.2 0.2
2.2 0.3
10.0 1.2
>11
1
<2
3
4.2 0.5
0.3 0.1
14 oseltamivir
1000
a Values of EC50, CC50 are given inμg/mL. The best values of EC50 are given in bold.
bSI is the CC50/EC50 ratio.
The grey highlighting indicates the most effective compounds.

As in the case of antimicrobial activity, the highest antiviral activity was exhibited by
compound 1e with a propylene aliphatic linker and compounds 1a–c with aromatic linkers, with
the highest SIs found in compounds 1e and 1b.
4. Discussion
In the case of bis-QACs, alkyl hydrophobic tails and linkers influence biological activity. In
previous work [23] we studied the effect of the alkyl chain length of tetracationic QACs
consisting of two DABCO head groups connected through a 1,5-pentenyl linker and bearing two
hydrophobic tails on antimicrobial activity. It was shown that tetracationic molecules with decyl
or dodecyl tails exhibited high activity, while mono- and bis-derivatives with the same (decyl or
dodecyl) alkyl length exhibited low activity. The required parameters for high activity were
revealed: two hydrophobic tails (decyl or dodecyl) and two DABCO cationic fragments.
Furthermore, compound with dodecyl tails had strong bactericidal activity and could rapidly kill
Gram-positive St. aureus as well as Gram-negative Ps. aeruginosa. This study is intended to
develop novel DABCO derivatives with different linkers to investigate the role of linker
fragments in microbial activity, keeping the dodecyl chain length.
Although in most cases the biological activity showed little difference according to the
variation of linker moieties, on the basis of the MIC data we can identify a number of the most
active compounds (Table 1 and Figure 3). Thus, among the screened compounds (1a–n)
compounds 1c (o-phenylenebismethyl), 1d (biphenyl) and 1e (propylene linker) showed the most
potent activity against all tested strains. The high antibacterial activity of these compounds may
be explained by linker length. It was seen that the distance between the two DABCO moieties of
the most active compounds is close and shorter in comparison to other derivatives, so the ionic
radius is smaller. The smaller ionic radius of the cation causes stronger electrostatic interactions
with the negatively charged cell surface of microorganisms and, as a consequence, higher
biocidal activity.
Figure 3. Comparison of antibacterial activity of compounds 1a-n (MIC in μg/mL).

In most cases the MIC values of DABCO compounds defined for Gram-positive bacteria were
slightly lower than for Gram-negative bacteria, in accordance with the literature about QAC
activity. In general, QACs usually exhibit greater activity against Gram-positive bacteria than
against Gram-negative bacteria, which is due to the structural differences of the two cell types.
Gram-positive and negative organisms have a similar cytoplasmic membrane inside the outer
wall, containing phospholipids and membrane proteins; the outer walls are very different. Gram-
positive bacteria have a very simple cell wall, consisting mainly of a mesh-like structure, while
Gram-negative bacterial cell walls contain a layer of peptidoglycan between an outer membrane
consisting of lipopolysaccharides (LPS) and the cytoplasmic membrane. Although the influence
of the composition and domain structure of the lipid membranes of strains on antibacterial
activity is less well studied and understood [50], it is possible that these parameters are important
in directing selectivity toward specific microorganisms. For instance, in this work Pr. vulgaris
was resistant to the action of all compounds, and their MICs were found to be much higher in
comparison to the reference drugs. The loss of activity against the Pr. vulgaris strain could be
explained by features of the membrane composition of Pr. vulgaris.
Thus, previously and in this study, we have shown that activity is controlled by several
parameters. The structural features of both the compounds and the microbes play significant
roles in determining the antimicrobial activity of the compounds. Investigation of cell wall
structure and membrane composition of microorganisms, positive charge density, hydrophilicity,
lipophilicity and geometry of the compounds is required.
The major problem in using QACs is their degree of selectivity between microbial and
mammalian cells. Cell selectivity of QACs can be attributed to the differences of the lipid
components of the cell membranes of bacterial and mammalian cells. While bacterial membrane

is largely composed of the negatively charged lipids phosphatidylglycerol, cardiolipin and LPS,
the mammalian cell membrane is mainly composed of zwitterionic lipids such as
phosphatidylcholine and sphingomyelin [51]. In addition, eukaryotic cell membranes are rich in
cholesterol, while bacterial cell membranes are devoid of it. The positively charged QACs
preferably bind to the more negatively charged bacterial membrane. The tetracationic DABCO-
based derivatives were more toxic to bacteria than mammalian cells. Nevertheless, compounds
showed high selectivity only for St. aureus (SI≥ 7). The most active compounds 1c and 1e were
found to be nontoxic against HEK293T cell line until concentrations of 4 and 6.5 μg/mL,
respectively. The high selectivity against all bacterial strains was showed only compounds 1a
and 1b. These compounds were not toxic against HEK293T cell line until concentration of 16
μg/mL.
According to the MBC and time-kill assays, activity was bactericidal and the compounds
discussed exhibited rapid bactericidal effect on Gram-positive and Gram-negative bacteria. Such
properties of the compounds suggest that they might act as amphiphilic membrane-active agents
interacting with the cytoplasmic membrane of bacteria. While disruption of cytoplasmic
membranes as the main mechanism of action of QACs has been well studied, their interaction
with other targets of microorganisms is little known, although they may interact with several
cytoplasmic components and lock their activity. In particular, cationic QACs may be expected to
interact with negatively charged polymers such as DNA and RNA, for which they are known to
have very high affinity [52]. The bis-quaternary DABCO salts possess high affinity for
phosphate anions due to steric factors [53] and can induce conformational transitions of DNA
[54,55]. Previously, it was shown that the DABCO-based compounds studied here act as
chemical ribonucleases, cleaving phosphodiester bonds of different RNAs [24]. Therefore, we
have supposed that the mechanism of antimicrobial activity of DABCO-based QACs can include
disruption of the cytoplasmic membrane of bacteria, followed by interaction with negatively
charged DNA or RNA. Antibacterials with distinct modes of action and rapid killing can display
broad-spectrum activity, including against drug-resistant pathogens, and can also counter the
development of bacterial resistance. Thus, our compounds represent a promising class of
antimicrobial agents for combating increasing bacterial resistance. The subject of our further
research is to study the mechanism of action and to optimize the structure to achieve high
selectivity.
5. Conclusion
In conclusion, the experiments in vitro showed selective toxicity of the compounds studied
toward bacterial cells over mammalian cells. The cytotoxic doses of all compounds were higher

than their effective doses against bacterial strains. Compounds exhibited high antibacterial
activity, in some cases equal or higher to that of standards ciprofloxacin and gentamicin. Indeed,
most of the tested compounds have an interesting biological activity, especially against Gram-
negative bacteria like E. coli or Ps. aeruginosa and a Gram-positive bacterium like St. aureus.
The most active compounds (1c, 1e) inhibited growth of laboratory strain St. aureus and a series
of clinical isolates of methicillin-resistant Staphylococcus with MIC values in the 0.3–
1.6 μg/mL range. They were found to be 10 to 50-fold more potent than the reference drugs.
Additionally, time-kill assays revealed the rapid bactericidal effect on Gram-positive and Gram-
negative bacteria. Furthermore, compound 1e with a propylene aliphatic linker and compounds
1a–c with aromatic linkers displayed the highest antiviral activity. A number compounds could
be used as lead compounds for further development of new antimicrobial drugs readily
obtainable from inexpensive commercial products.
Appendix A. Supplementary data

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Graphical abstract
Highlights
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A series of DABCO derivatives were evaluated for antimicrobial activity
Some compounds were comparable or better to the reference drug ciprofloxacin
1e and 1c exhibited the most potent activity against Ps. aeruginosa and St. aureus
Bactericidal activity was showed against Gram-positive and Gram-negative bacteria