Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium compounds based on pyridoxine and fatty acids

Article abstract of DOI:10.1016/j.ejmech.2020.113100

A diverse series of 43 novel “soft antimicrobials” based on quaternary ammonium pyridoxine derivatives which include six-membered acetals and ketals of pyridoxine bound via cleavable linker moieties (amide, ester) with a fragment of fatty carboxylic acid was designed. Nine compounds exhibited in vitro promising antibacterial activity against Gram-positive and Gram-negative bacterial strains with MIC values comparable with reference antiseptics miramistin, benzalkonium chloride and chlorohexidine. On various clinical isolates, the lead compounds 6i and 12a exhibited antibacterial activity comparable with that of benzalkonium chloride while higher than that of miramistin. Moreover, 6i and 12a were able to kill bacteria embedded into the matrix of mono- and dual species biofilms. The treatment of bacterial cells by either 6i and 12a lead to fast depolarization of the membrane suggesting that the membrane is an apparent molecular target of compounds. 6i and 12a were non mutagenic neither in SOS-chromotest nor in Ames test and non-toxic in vivo at acute oral (LD₅₀ > 2000 mg/kg) and cutaneous administration (LD₅₀ > 2500 mg/kg) on mice. Taken together, our data allow suggesting described active compounds as promising starting point for the new antibacterial agents development.

Full text of DOI:10.1016/j.ejmech.2020.113100

Journal Pre-proof
Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium
compounds based on pyridoxine and fatty acids
Sergey V. Sapozhnikov, Alina E. Sabirova, Nikita V. Shtyrlin, Anastasia Y. Druk,
Mariya N. Agafonova, Milana N. Chirkova, Renata R. Kazakova, Denis Y. Grishaev,
Tatyana V. Nikishova, Elena S. Krylova, Elena V. Nikitina, Airat R. Kayumov, Yurii G.
Shtyrlin
PII:
S0223-5234(20)31072-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.113100
EJMECH 113100
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 October 2020
Revised Date: 7 December 2020
Accepted Date: 7 December 2020
Please cite this article as: S.V. Sapozhnikov, A.E. Sabirova, N.V. Shtyrlin, A.Y. Druk, M.N. Agafonova,
M.N. Chirkova, R.R. Kazakova, D.Y. Grishaev, T.V. Nikishova, E.S. Krylova, E.V. Nikitina, A.R.
Kayumov, Y.G. Shtyrlin, Design, synthesis, antibacterial activity and toxicity of novel quaternary
ammonium compounds based on pyridoxine and fatty acids, European Journal of Medicinal Chemistry,
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Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium
compounds based on pyridoxine and fatty acids
Sergey V. Sapozhnikov, Alina E. Sabirova, Nikita V. Shtyrlin, Anastasia Y. Druk, Mariya N.
Agafonova, Milana N. Chirkova, Renata R. Kazakova, Denis Y. Grishaev, Tatyana V.
Nikishova, Elena S. Krylova, Elena V. Nikitina, Airat R. Kayumov, Yurii G. Shtyrlin

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Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium compounds
based on pyridoxine and fatty acids
Sergey V. Sapozhnikov, Alina E. Sabirova, Nikita V. Shtyrlin, Anastasia Y. Druk, Mariya N.
Agafonova, Milana N. Chirkova, Renata R. Kazakova, Denis Y. Grishaev, Tatyana V. Nikishova,
Elena S. Krylova, Elena V. Nikitina, Airat R. Kayumov, Yurii G. Shtyrlin

Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage:www.elsevier.com/locate/ejmec
Design, synthesis, antibacterial activity and toxicity of novel
quaternary ammonium compounds based on pyridoxine and fatty
acids
Sergey V. Sapozhnikov†, Alina E. Sabirova†, Nikita V. Shtyrlin†, Anastasia Y. Druk, Mariya N.
Agafonova, Milana N. Chirkova, Renata R. Kazakova, Denis Y. Grishaev, Tatyana V.
Nikishova, Elena S. Krylova, Elena V. Nikitina, Airat R. Kayumov, Yurii G. Shtyrlin
Kazan (Volga region) Federal University, Kremlyovskaya St. 18, Kazan, 420008, Russian Federation
† Authors contributed equally to this manuscript.
ARTICLE INFO
ABSTRACT
Keywords:
Pyridoxine
Antibacterial activity
Quaternary ammonium compounds
Soft drugs
A diverse series of 43 novel “soft antimicrobials” based on quaternary
ammonium pyridoxine derivatives which include six-membered acetals
and ketals of pyridoxine bound via cleavable linker moieties (amide, ester)
with a fragment of fatty carboxylic acid was designed. Nine compounds
exhibited in vitro promising antibacterial activity against Gram-positive
and Gram-negative bacterial strains with MIC values comparable with
reference antiseptics miramistin, benzalkonium chloride and
chlorohexidine. On various clinical isolates, the lead compounds 6i and
12a exhibited antibacterial activity comparable with that of benzalkonium
chloride while higher than that of miramistin. Moreover, 6i and 12a were
able to kill bacteria embedded into the matrix of mono- and dual species
biofilms. The treatment of bacterial cells by either 6i and 12a lead to fast
depolarization of the membrane suggesting that the membrane is an
apparent molecular target of compounds. 6i and 12a were non mutagenic
neither in SOS-chromotest nor in Ames test and non-toxic in vivo at
acute oral (LD50 > 2000 mg/kg) and cutaneous administration (LD50
>2500 mg/kg) on mice. Taken together, our data allow suggesting the
described active compounds as promising starting point for the new
antibacterial agents development.
Cytotoxicity
2020 Elsevier Ltd. All rights reserved.
1.
Introduction
The bacterial resistance to antimicrobials is one of serious challenges of healthcare
worldwide. The multicenter monitoring notices the continuous spread of bacterial strains
resistant to many external factors, and pathogenic bacteria quickly become resistant to existing
commercially available antibiotics and antiseptics [1, 2]. A similar increase is observed for both
nosocomial hospital infections arising due to the direct spread of bacteria from patient to patient
and for community-acquired infections [3, 4].
Since the 1930s, quaternary ammonium compounds (QACs) are widely used as
antiseptics and disinfectants [5]. A number of QACs such as the benzalkonium chloride [5],
dequalinium chloride [6], cetylpyridinium chloride [7] have been widely used for a variety of
Corresponding author. Tel.: +7-843-233-7363; fax: +7-843-233-7531. E-mail: yurii.shtyrlin@kpfu.ru

clinical purposes (e.g., preoperative disinfection of intact skin, application to mucous
membranes, disinfection of noncritical surfaces etc.). According to literature [7], the general
mechanism of the antibacterial activity of QACs is the damage of the cytoplasmic and outer
membrane lipid bilayers via association of the positively charged quaternary nitrogen with the
anionic head groups of acidic phospholipids and interaction of the lipophilic tail with the
hydrophobic membrane core. Because of universal basic architecture of cellular membranes,
these properties of QAСs have disadvantage such as high toxicity [8]. Moreover, many classical
representatives of these compounds has a long half-life in environment, increasing the frequency
of bacterial resistance developing to QACs [9, 10]. One of possible ways to solve this problem
could be the strategy of soft drugs, i.e. substances that are readily degraded into nontoxic and
biologically inactive products both in vivo and in the environment. The inclusion of a
metabolically sensitive fragment into the structure of designed products allows predicting the
main metabolic pathway for degradation and avoiding the toxic products formation. The most
suitable for that are ester and amide bonds, which do not affect significantly the hydrophilic-
lipophilic balance of molecules while keeping their antibacterial activity [11]. The concept of
soft antibacterial agents was proposed by Bodor in 1980 [8] who synthesized isosteric analogues
of cetylpyridinium chloride with comparable antimicrobial activity, significantly less toxic and
undergo facile hydrolytic cleavage, leading to their deactivation (Fig. 1). Since then, a series of
soft QAC antimicrobials have been synthesized [12‒15]. Among them, the QAC Miramistin
occupies leading position in Russian antiseptics market structurally related to soft drugs.
Soft antimicrobials reported by Bodor [8]
R1
R1
N+
Cl-
O
O
N+
N
O
O
Cl-
H2n+1Cn
CnH2n+1
n=7, 11, 13, 15 R1=H, CH3
C13H27
N+
N+
NH
O
CnH2n+1
Cl-
Cl-
n=8,10,12,14,16,18
Structure of Benzalkonium chloride
Structure of Miramistin
Cl
NH
Cl
NH
NH
NH
N
N
HN
H
H
NH
Structure of Chlorhexidine
Fig.1. Structures of quaternary ammonium compounds.
N
N
H
H
In our group, we have systematically studied chemistry and biological activity of
pyridoxine (vitamin B6) derivatives including a series of QAC based on pyridoxine derivatives
[16‒20] (Fig.2). Some of them possess high antibacterial activity against various clinical Gram-
positive and Gram-negative pathogenic bacteria (minimal inhibitory concentration (MIC) 0.5-16
µg/ml) and low toxicity in vitro.

Fig.2. Structures of the QAC based on pyridoxine derivatives obtained previously [17‒20].
In this work, we present a series of novel “soft antimicrobials” based on quaternary
ammonium pyridoxine derivatives which include cleavable moieties (amide, ester) and a six-
membered acetals and ketals of pyridoxine bound via linker moiety with a fragment of fatty
carboxylic acid (Fig. 3). The relationship between the structure of synthesized compounds and
their antibacterial activity in vitro were investigated by variations of the substitutents at the
acetal carbon atom, the length of the linker, the nature of the bond between linker and carboxylic
acid fragment, the length of the carboxylic acid and the position of the quaternary ammonium
fragment in the pyridine ring of pyridoxine. Identified lead compounds inhibited growth of
Gram-positive and Gram-negative pathogens, including biofilm-embedded bacteria, while
exhibiting low toxicity and resistance development risk.
Fig.3. Design of the soft antimicrobials based on quaternary ammonium pyridoxine derivatives.
2.
Results and discussion
2.1.
Chemistry and primary screening of antibacterial activity in vitro
At first, a series of amides 3a–i with different lipophilicity were obtained by reaction of
different fatty carboxylic acids (caproic acid, lauric acid, myristic acid, palmitic acid and stearic
acid) with N,N-dimethylethylenediamine or N,N-dimethylpropylene-1,3-diamine in toluene in
the presence of p-toluenesulfonic acid [21]. Then, in the reaction of the corresponding amides
3a-i with chlorine derivatives of pyridoxine 5a-d containing various substitutents at the acetal
carbon atom [19, 22] in ethanol at 70 °C the ammonium salts 6a-u were obtained (Scheme 1).

Sheme 1. Synthesis of QACs 6a-u. (a) toluene, reflux (Dean-Stark trap), 48h, 40-74%; (b) NaHCO3, C2H5OH, 70
oC, 8h, 40-76%.
The antibacterial activity of compounds 6a-u was evaluated on various strains of Gram-
positive and Gram-negative bacteria. Table 1 shows the MIC of compounds 6a-u in comparison
with benzalkonium chloride, miramistin, chlorhexidine and their structural analog 6v without
amide moiety described in our recent paper [19]. The lipophilicity of the synthesized ammonium
salts was expressed in terms of their partition coefficient values (logP) calculated using online
platform Chemicalize (ChemAxon) [23]. For derivatives containing fragments of stearic and
caproic acids a sharp decrease in antibacterial activity was observed. For compounds containing
identical fragments of carboxylic acid and linker, the antibacterial activity were decreased with
the increasing of alkyl chain length at acetal carbon atom (2CH3~C3H7 > C5H11 > C8H17). The
number of methylene fragments in the linker between the quaternary and amide nitrogen atoms
did not affect the antibacterial activity. The most active compounds 6b, 6f, 6i and 6j contain the
myristic and lauric acids residues in the amide fragment. Their activity against Gram-positive
and Gram-negative bacteria, in general, was comparable with the reference drugs and more than
previously reported ammonium salt 6v without amide moiety.
Table 1
Antibacterial activity of quaternary ammonium salts 6a-u
Compound
MICs (µg/ml)
Gram-negative bacteria
E. сoli P. aeruginosa
MG1655 АТСС 27853
Gram-positive bacteria
S. aureus B. subtilis M. luteus
АТСС
168
LogP
29213
1
2
1
4
8
4
4
8
4
4
1
2
32
8
32
>64
32
2
2
0.52
1.41
2.93
3.19
6a
6b
6c
6d
8

8
0.5
4
4
1
1
8
>64
1
2
2
>64
2
>64
>64
>64
>64
4
8
2
2
8
2
16
1
2
4
2
64
8
>64
>64
4
64
8
8
8
32
0.58
1.47
2.36
3.25
1.35
2.24
3.13
4.02
1.41
2.30
3.19
4.08
3.19
4.53
-2.09
-1.25
2.34
4.35
-
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
2
1
32
8
16
>64
4
2
4
16
>64
4
2
2
>64
>64
8
2
2
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
>64
1
>64
2
>64
>64
>64
>64
>64
4
>64
>64
>64
>64
16
0.5
6v
1
Benzalkonium
chloride
Miramistin
Chlorhexidine
2
2
4
1
2
0.5
8
1
1
4
2.43
4.51
In the second experimental series, we have obtained bioisostere analogues of compounds
6a-u where the amide fragment was replaced by ester. The synthesis of derivatives based on
stearic and caproic acids isn’t performed, because these compouds contain an amide fragment
(6d, 6h, 6l, 6p) exhibiting low antibacterial activity. Esters 8a-f were prepared by Steglich
esterification with amine 7a-b and fatty carbonic acids [24]. Then, in the reaction of the
corresponding esters 8a-f with chlorine derivatives of pyridoxine 5a-d in ethanol at 70 °C the
ammonium salts 9a-n were performed (Scheme 2).
Sheme 2. Synthesis of QACs 9a-n. (a) DCC, DMAP, CH2Cl2, rt, 4h, 47-61%; (b) NaHCO3, C2H5OH, 70 °C, 8h, 39-
73%.

The antibacterial activity of compounds 9a-n is shown in Table 2. In general, the effect of
obtained bioisostere esters was comparable with a series of amides 6a-u and references drugs.
The highest influence was observed for compounds 9b, 9h and 9j containing fragments of
myristic and lauric acids.
Table 2
Antibacterial activity of quaternary ammonium salts 9a-n
MICs (µg/ml)
Compound
Gram-positive bacteria
Gram-negative bacteria
S. aureus B. subtilis M. luteus
E. сoli
P. aeruginosa LogP
АТСС
168
MG1655 АТСС 27853
29213
4
4
1
1
1
2
8
2
4
4
4
0.5
0.5
2
2
2
2
0.5
1
1
1
8
8
>64
16
0.5
16
8
>64
8
16
>64
4
16
>64
4
>64
>64
>64
>64
>64
8
64
1
4
32
16
32
32
1
8
32
4
32
>64
>64
>64
1
1.25
2.14
3.03
1.31
2.20
3.09
2.08
2.97
3.86
2.14
3.03
3.92
3.92
5.25
4.35
-
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
0.5
0.5
2
2
16
1
0.5
2
2
2
32
8
2
2
32
4
>64
>64
4
>64
4
1
9n
6v
Benzalkonium
chloride
Miramistin
Chlorhexidine
2
2
4
1
2
0.5
8
1
1
4
2.43
4.51
Synthesis of compounds 12a-d and 15a-d in which quaternary ammonium fragment is
located in the sixth position of the pyridine ring was carried according to the Scheme 3.
Qaternary ammonium salts 12a-d and 15a-d were obtained from mono-chlorine derivates 11 and
14 in the reaction with myristic acid amides 3b,f or esters 8b,e. Chlorides 11 and 14 were
obtained from acetal 10 [25] and ketal 13 [26] by selective chlorination by equimolar quantities
of N-chlorocuccinimide in dichloromethane. The regioselective chlorination proceeded similarly
to the previously described regioselective bromination of diol 10 under the action of N-
13
bromosuccinimide [26] and also confirmed by C chemical shift of CH2Cl group in the ortho-
position of pyridine ring (~45 ppm). The minor products (~15-20 %) of the reactions were
dichlorides. The traces of the second regio-isomers were not observed.

Sheme 3. Synthesis of QACs 12a-d, 15a-d (a) NCS (1 eqviv.), Ph3P, CH2Cl2, rt, 0.5h, 44-54%; (b) C2H5OH, 70 °C,
8h, 44-83%.
Table 3
Antibacterial activity of quaternary ammonium salts 12a-d, 15a-d
MICs (µg/ml)
Compound
Gram-positive bacteria
S. aureus B. subtilis M. luteus
Gram-negative bacteria
E. сoli P. aeruginosa
MG1655 АТСС 27853
LogP
АТСС
168
29213
1
1
2
1
2
2
2
2
64
1
1
0.5
2
0.5
2
2
1
2
64
4
2
1
1
2
4
2
1
2
64
0.5
8
>64
64
0.73
1.46
0.79
1.52
1.56
2.29
1.62
2.35
-0.78
-
12a
12b
12c
12d
15a
15b
15c
15d
15e
32
16
64
64
64
64
64
64
8
>64
>64
>64
>64
>64
>64
>64
1
Benzalkonium
chloride
Miramistin
Chlorhexidine
2
2
4
1
2
0.5
8
1
1
4
2.43
4.51
The antibacterial activity of esters and amides of pyridoxine derivatives 12a-d and 15a-d
on Gram-positive bacteria was comparable with derivatives containing ammonium fragments in
the fifth position of pyridoxine and reference drugs. However, their effect on Gram-negative
bacteria P. aeruginosa and E. coli was significantly lower. It should be noted that the previously
obtained compound 15e [18] without ether or amide fragments was practically inactive.
Thus compounds 6b, 6f, 6i, 6g, 9b, 9h, 9j, 12a and 12b with promising activities in the
primary assay were selected for further in-depth investigation in vitro.
2.2. Structure-Activity Relationship (SAR)

To identify quantitative relationships “structure–antibacterial activity” in a series of
synthesized QACs based on pyridoxine and fatty acids derivatives, the dependences of
antibacterial activity on calculated in ChemAxon lipophilicity [23] were evaluated (Figure 4, for
S. aureus). MIC values are shown in µg/ml for practical reasons. A strong correlation between
the lipophilicity and antimicrobial activity of quaternary ammonium pyridoxine derivatives on S.
aureus was observed. Thus, all the most active compounds had logP values in the range of 1–3,
while compounds with logP > 5 and logP < 0 were almost inactive. Similar relationships were
found for the other studied bacteria. Apparently, relationships reflect the important features of
the active compounds essential for their effective interaction with the hydrophobic membrane
core of bacterial cells.
S. aureus
0.5
0
-0.5
-1
-1.5
-2
-2.5
-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0
LogP
Fig 4. Lg(1/MIC) versus calculated logP relationship for QAS based on pyridoxine and fatty acids. The
parabolic line shows the polynomial approximation by using a fifth order model.
2.3. Cytotoxicity
Cytotoxicity of compounds 6b, 6f, 6i, 6j, 9b, 9h, 9j, 12a and 12b was evaluated on
human embryonic kidney cells (HEK-293), human mesenchymal stem cells (MSK) and primary
human skin fibroblasts (HSF) in comparison with benzalkonium chloride, miramistin and
chlorhexidine (Table 4). Compounds 6b, 6f and 9j demonstrated the lowest cytotoxicity on all
cell lines among the studied molecules. However, their toxicity was greater than that of
chlorhexidine and miramistin. The most toxic were compounds 6i and 9b whose toxicity
exceeded benzalkonium chloride on HEK-293 cells. All other compounds were less toxic than
benzalkonium chloride.
Table 4
Cytotoxicity (CC50, µg/mL mean ± SD) of novel pyridoxine-based QACs
Compound
СС50, µg/mL
HEK-293
MSK
HSF
6b
6f
6i
6j
9b
9h
1.18 ± 0.33
1.61 ± 0.40
0.15 ± 0.01
1.16 ± 0.92
0.17 ± 0.04
1.00 ± 0.13
2.16 ± 0.80
2.21 ± 0.71
1.15 ± 0.52
1.38 ± 0.74
1.08 ± 0.54
1.28 ± 0.52
3.03 ± 0.91
3.14 ± 0.80
1.79 ± 0.80
2.15 ± 0.42
1.94 ± 0.42
2.18 ± 0.50

9j
12а
12b
2.15 ± 0.67
1.32 ± 0.75
0.88 ± 0.14
6.08 ± 2.14
0.59 ± 0.21
7.28 ± 1.50
2.42 ± 0.74
1.74 ± 0.44
1.02 ± 0.17
4.16 ± 0.86
0.82 ± 0.17
5.12 ± 1.20
4.32 ± 0.74
3.21 ± 0.70
2.24 ± 0.52
7.21 ± 1.92
1.14 ± 0.40
6.32 ± 1.24
Miramistin
Benzalkonium chloride
Chlorhexidine
2.4.Genotoxicity
Genotoxicity of compounds 6b, 6f, 6i, 6j, 9b, 9h, 9j, 12a, 12b and benzalkonium
chloride was evaluated in SOS-chromotest and Ames test. In SOS-chromotest, the β-
galactosidase activity was normalized to the amount of cells estimated from the OD600 values
and SOS induction factor was calculated as a ratio of β-galactosidase activity in the presence of
compounds and the solvent control. Mitomycin C (1 µg/ml) was used as positive control. No
significant dose-dependent increase more than 2-fold was observed in SOS-chromotest (Table 5)
thus indicating the lack of DNA-damage action for all compounds under concentrations tested.
Table 5
DNA-damage activity of novel pyridoxine-based QACs (ratio, fold increase over the solvent control), mean ± SD
Compound
QACs concentration, µg/mL
750
150
75
15
7.5
0.4±0.03
1.0±0.17
0.3±0.02
0.3±0.03
0.4±0.03
0.3±0.02
0.5±0.04
0.4±0.04
0.3±0.02
0.8±0.17
0.5±0.06
0.5±0.04
0.5±0.02
0.5±0.04
0.6±0.10
1.3±0.51
1.1±0.15
0.7±0.10
0.9±0.07
0.7±0.04
0.7±0.08
0.7±0.18
0.7±0.13
0.9±0.11
1.2±0.11
1.5±0.06
1.5±0.13
0.7±0.15
1.2±0.33
1.1±0.11
1.5±0.10
1.5±0.20
1.6±0.10
1.6±0.04
1.6±0.14
1.5±0.17
1.4±0.14
1.8±0.19
1.6±0.13
1.5±0.10
1.5±0.06
1.5±0.16
1.6±0.04
1.5±0.09
1.6±0.27
1.4±0.10
1.4±0.19
1.6±0.30
1.8±0.14
1.5±0.25
6b
6f
6i
6j
9b
9h
9j
12a
12b
Benzalkonium
chloride
12.9±2.41 (1 µg/mL)
Mitomicin C
Since compounds demonstrated antibacterial acitivity on S. typhymuriym strains (data not
shown), the spot-test modification of the Ames test has been used instead of classic technique.
For that, 50 µg of compound in water was dropped onto 5-mm whatmann disk placed onto agar
surface (see Fig. S1). While almost all compounds led to increase of revertants amount on at
least one strain in the Ames test. Only compounds 6b, 6i and 12a could be considered as non-
mutagenic in Ames test (Table 6).
Table 6
Mutagenicity of novel pyridoxine-based QACs (ratio, fold increase over the solvent control in Ames spot-test)
Compound
S. typhimurium strain
TA1535
TA1537
TA98
TA100
TA102
0.8
0.4
0.5
0.5
0.9
0.4
0.06
0.03
0.03
0.03
0.02
0.04
1.7
2.6
1.9
4.4
4.6
4.7
1.3
2.5
1.1
0.7
1.4
1.2
0.1
0.2
0.1
0.7
4.8
0.2
6b
6f
6i
6j
9b
9h

0.5
1.1
0.4
0.03
0.01
0.05
1.8
1.9
2.5
2.4
1.7
2.1
0.1
0.4
0.4
9j
12a
12b
Methyl
4-Nitro-o-
4-Nitro-o-
Methyl
methanesu phenylenediam phenylenediam methanesulfo Mitomycin C
Positive
control
lfonate
ine
ine
nate
30
3.7
4.2
2.7
1.9
2.5. In vitro antibacterial activity on clinical strains
The antibacterial activity of 6b, 6f, 6i, 6j, 9b, 9h, 9j, 12a and 12b was further studied on
various Gram-positive and Gram-negative clinical isolates (see Table S2 for their antibiotic
resistance profile), miramistin and benzalkonium chloride were used as reference drugs (Table
7).
The most active compounds 6i and 12a exhibited antibacterial activity comparable with
benzalkonium chloride on Gram-positive bacteria, while being less active on Gram-negative
ones. At the same time, 6i and 12a were more active in comparison with miramistin on both
Gram-positive and Gram-negative bacteria.

Table 7
In vitro antimicrobial activity of 6b, 6f, 6i, 6j, 9b, 9h, 9j, 12a, 12b on an extended panel of clinical bacterial pathogens
MICs (µg/ml)
6b
6f
6i
6j
9b
9h 9j
12a 12b Benzalkonium Miramistin
chloride
Strain
Gram-positive bacteria
S. aureus 713 MRSA
S. aureus 983 MRSA
S. aureus MRSA 1053
S. intermedius MRSI
1061
4
64
4
16
64
8
4
16
2
2
16
1
8
64
8
2
64
2
16
32
8
4
32
4
2
32
2
8
16
4
64
>64
32
4
8
4
2
8
2
16
4
4
4
64
S. aureus MRSA 1065
S. aureus MRSA 1131
S. intermedius MRSI
1143
8
16
4
8
16
16
2
4
4
2
2
2
8
16
8
2
4
4
16
16
16
2
4
4
4
4
4
4
4
4
32
32
64
S. aureus 25
E. faecalis 23
E. faecalis 3063
4
2
2
8
4
4
2
2
2
2
1
1
8
4
4
2
4
4
8
4
4
2
1
1
2
2
2
2
1
2
16
4
16
Gram-negative bacteria
Acinetobacter spp. 3
Pseudomonas spp. 5
Klebsiella spp. 11
Proteus spp. 17
64
64
64
64
>64
>64
>64
>64
>64
>64
>64
32
32
16
>64
8
16
2
16
64
64
32
32
32
64
64
64
64
64
32
64
32
32
32
32
32
64
64
64
16
16
16
64
8
16
1
32
64
>64
>64
>64
>64
>64
64
>64
16
64
64
64
>64
32
64
4
64
64
64
>64 >64 >64 >64 >64 >64
E. coli 718
8
16
1
8
64
32
64
4
64
64
32
64
1
8
64
8
16
64
2
32
64
2
Moraxella sp. 765
K. pneumoniae 1813
P. aeruginosa 1945
S. marcescens 1966
S. ureilytica 1972
64
>64
32
64
>64 >64 >64 >64
>64
>64
>64 >64 >64 >64 >64 >64

Thus based on in vitro data of antibacterial activity and toxicity of 43 synthesized
quaternary ammonium pyridoxine derivatives compounds 6i and 12a were selected as lead
compounds for further investigations.
2.6. Anti-biofilm activity
The anti-biofilm activity of compounds 6i and 12a has been tested on mono- and dual
species biofilms. Firstly, 48h-old biofilms of S. aureus, M. luteus, E. coli or P. aeruginosa were
prepared in 24-well plates in basal medium (BM) broth, washed with sterile phosphate-buffered
saline (PBS) and filled with 1 mL of fresh BM broth containing 6i, 12a or benzalkonium
chloride in concentrations of their respective 1-32 × minimum bactericidal concentrations
(MBCs) (see Table S1). After 24 hours of incubation, the viability of biofilm-embedded cells
was evaluated by colony-forming units (CFUs) counting. No statistically significant difference in
the activity of 6i, 12a and benzalkonium chloride has been observed. All studied antimicrobials
provided a 3-log drop of CFUs of all biofilm-embedded bacteria at nearly the same relative
concentration (expressed as fold excess of respective MBC), which varied by only one dilution
step (Fig. 5). Thus, both 6i and 12a demonstrated anti-biofilm activity comparable with that of
the benzalkonium chloride.
Next, the action of 6i and 12a against bacterial dual-species biofilms was studied in a
similar experimental design. A 48-h old biofilms formed by S. aureus and E. coli or S. aureus
and P. aeruginosa were incubated with different concentrations of antimicrobials for 24 h and
then quantified by differential CFUs counting. In these experiments, effects of both 6i and 12a
against both S. aureus and E. coli in dual-species culture remained more or less similar to
benzalkonium chloride (Fig. 6). By contrast, the influence of both compounds worsened against
S. aureus - P. aeruginosa dual-species biofilms, apparently because of changed metabolism of
these bacteria in their consortium.
These data allow suggesting 6i and 12a as a promising topical antimicrobials with
action comparable with benzalkonium chloride.
Fig. 5. Antimicrobial effect of novel pyridoxine-based QACs 6i and 12a against biofilm-embedded bacteria.
Benzalkonium chloride served as reference. Antimicrobials were added to 48 hours-old biofilms. After 24 h

incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended
and CFUs were counted. The median values with (IQRs) from six independent measurements are shown.
Fig. 6. Antimicrobial effect of novel pyridoxine-based QAСs 6i and 12a against dual-species biofilms.
Benzalkonium chloride served as reference. Antimicrobials were added to 48 hours-old biofilms. After 24 h
incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended
and CFUs were differentially counted on Endo-agar, Cetrimide agar and Salt-mannitol agar. The median values with
IQRs from six independent measurements are shown.
2.7. In vitro resistance development
Spontaneous development of resistance to 6i and 12a in comparison with miramistine and
benzalkonium chloride was studied on S. aureus and E. coli strains. MICs of all antimicrobials
increased significantly (up to 32-fold) on the 8th passage on S. aureus, and 4-fold on E. coli (Fig.
7). After removal of antimicrobials, the MIC values decreased significantly suggesting that
observed resistance is rather phenotypic than genetically determined.

Fig. 7. The development of S.aureus (A) and E.coli (B) resistance to the novel pyridoxine-based QACs 6i and 12a.
A series of 2-fold serial dilutions of the test compounds were inoculated with 107 CFUs of S. aureus or E. coli. After
24 h incubation, cultures from the wells with the highest antibiotic concentration permitting growth to the equivalent
of a 0.5 McFarland standard were used as inoculum for the next round of MIC determination. This procedure was
repeated for 14 passages, and MICs were recorded at the end of every passage. Next 7 passages were performed in
absence of antimicrobials and MIC on 21st passage was determined. Benzalkonium chloride and miramistin were
served as references.
2.8. In vivo toxicity
For lead compounds 6i and 12a in vivo acute oral and dermal toxicity on mice were
perfomed as described in section 4.5.
In acute oral toxicity study after administration of 6i and 12a in dose 2000 mg/kg the
death of animals occurred within the first 7 days (Table 8). Necropsy of compound 6i treated
group revealed one case of enlarged spleen, pale liver and kidneys. Autopsy of mice received
12a revealed a pale liver, pale gastric mucosa in 3 cases (one of them had black contents in the
stomach, presumably blood). Despite this, compounds 6i and 12a were considerably less toxic
(LD50 > 2000 mg/kg) than benzalkonium chloride (LD50 180 mg/kg [11]), miramistine (LD50
1000 mg/kg [27]), and chlorhexidine (LD50 1260 mg/kg) [28]).
During 14 days after the single cutaneous administration of compounds 6i and 12a at
dose 2500 mg/kg no toxicity-related clinical symptoms or mortalities were revealed (Table 8).
For compound 12a a weak skin irritation was observed, which completely disappeared on day 5.
At necropsy, no remarkable findings were noted in the control and tested compound treatment
groups. Thus, we can conclude that compounds 6i and 12a are non-toxic at cutaneous
administration.
Based on results of acute oral and dermal in vivo studies compounds 6i and 12a belong
to category 5 of globally harmonized system of classification and labeling of chemicals [29]. It
should be noted that the in vivo toxicity data did not correlate with the in vitro data, according to
which compounds 6i and 12a were more toxic than chlorhexidine and miramistin.
Table 8
Acute oral and dermal toxicity data of compounds 6i and 12a on mice
Acute oral toxicity
Acute dermal toxicity
Compound
Dead/total
animals
Dead/total
animals
0/12
LD50,
mg/kg
LD50, mg/kg
2/12
5/12
6i
12a
˃ 2000
> 2500
0/12
2.9. Unraveling the mechanism of antibacterial activity of 6i and 12a
The cell membrane damage causing changes in the membrane potential is a well-known
mechanism of antimicrobial activity exhibited by various quaternary ammonium salts [7]. To test
whether 6i and 12a can damage the cell membrane thereby leading to the cells death, the
membrane potential of bacterial cells was measured by detection of DioC2(3) fluorescence.
S. aureus and E. coli were grown for 18 h, harvested and washed with PBS. Cells were re-

suspended until reaching the final density of 105 CFU/mL in PBS supplemented with DioC2(3)
(10 µM). After 30 min preincubation at 25 °C, antimicrobials have been added at their respective
1×MIC and 1-4×MBCs (see Tables 1,3, S1) and the fluorescence was measured for 35 min with
5-minute intervals. As expected, a significant dose-dependent decrease of fluorescence has been
observed in all cells treated with either 6i or 12a at their respective 1×MIC and higher, similarly
to benzalkonium chloride and miramistine regardless of the cell wall traits, suggesting that the
membrane potential decreased due to the membrane damage (Fig. 8). As could be seen from the
figure, the absolute drop in RFUs was 1.5-fold higher for Gram positive than on Gram negative
strains and was characterized by the time-dependent drop of membrane potential apparently
assuming their continuous damage. By contrast, for the Gram negative strains fast drop with no
further changes in relative membrane potential was detected allowing speculating that probably
the outer membrane becomes broken while the inner membrane remains intact.
Fig. 8. Relative membrane potential of S.aureus (A) and E.coli (B) treated with QAСs 6i and 12a (1×MIC, 1×, 2× or
4× of corresponding MBC, for values see Tables 1, 3 and S1).Benzalkonium chloride and miramistin served as
references drugs. Bacteria in the late exponential growth phase were harvested, washed by PBS and resuspended in
it, then supplemented by 10 µM of DioC2(3). After 30 min preincubation, compounds were added as indicated and
the fluorescence was measured for 35 min with 5-minute interval. Lines represent the median values with IQRs from
5 independent measurements.
3.
Conclusion
In conclusion, a diverse library of 43 novel “soft antimicrobials” based on quaternary
ammonium pyridoxine derivatives was designed and their antibacterial activity against six Gram-
positive and Gram-negative bacterial strains was evaluated in vitro. The structure of obtained
compounds include six-membered acetals and ketals of pyridoxine bound via cleavable linker
moieties (amide, ester) with a fragment of fatty carboxylic acid. Nine compounds exhibit
promising antibacterial activity with MIC values comparable with that of miramistin,
benzalkonium chloride and chlorohexidine. SOS-chromotest in S. typhimurium showed the lack
of DNA-damage activity for all active compounds and they were non-mutagenic in the Ames
test. Cytotoxicity studies on HEK-293, MSK and HSF cells demonstrated that some of the active

compounds were less toxic than the benzalkonium chloride, but more toxic than chlorhexidine
and miramistin. The investigation of antibacterial activity on various Gram-positive and Gram-
negative clinical isolates showed that the most active compounds 6i and 12a exhibited
antibacterial activity comparable with benzalkonium chloride and higher than miramistin. The
further in-depth investigations showed that compounds 6i and 12a have comparable with
benzalkonium chloride activity against mono- and dual species biofilms. Compounds 6i and 12a
are less toxic in vivo (LD50 > 2000 mg/kg) than benzalkonium chloride, miramistine and
chlorhexidine at oral administration and non-toxic at dermal administration (LD50 > 2500 mg/kg)
on mice. The mechanism of antibacterial activity of 6i and 12a includes membrane integrity
damage. The obtained results make the described active compounds a promising starting point
for new antibacterial agents development.
4.
Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a “Bruker AVANCE 400” at operating
frequency 400 and 101.56ꢀMHz, respectively. Chemical shifts were measured with reference to
1
1
the residual protons of the solvent (DMSO-d6, H, 2.50ꢀppm, 13C, 39.52ꢀppm; CDCl3, H,
7.26ꢀppm, 13C, 77.16ꢀppm). Coupling constants (J) are given in Hertz (Hz). The following
abbreviations are used to describe coupling: sꢀ=ꢀsinglet; dꢀ=ꢀdoublet; tꢀ=ꢀtriplet; mꢀ=ꢀmultiplet; br
sꢀ=ꢀbroad singlet, ABꢀ=ꢀAB system. Melting points were determined using a Stanford Research
Systems MPA-100 OptiMelt melting point apparatus and are uncorrected. For thin layer
chromatography analysis, silica gel plates from Sorbfil (Krasnodar, Russia) were used with UV
light (254ꢀnm/365ꢀnm) or iron (III) chloride as developing agent. Column chromatography was
performed on silica gel (60-200ꢀmesh) from Acros or reversed-phase chromatography on PF-
15C18HP column from Interchim.
High-resolution (HRMS) mass spectra were obtained on a quadrupole time-of-flight
(qTOF) AB Sciex Triple TOF 5600 mass spectrometer using turbo-ion spray source (nebulizer
gas nitrogen, a positive ionization polarity, needle voltage 5500ꢀV). Recording of the spectra was
performed in “TOF MS” mode with collision energy 10ꢀeV, declustering potential 100ꢀeV and
with resolution more than 30 000 full-width half-maximum. Samples with the analyte
concentration 5ꢀμmol/l were prepared by dissolving the test compounds in a mixture of methanol
(HPLC-UV Grade, LabScan) and water (LC-MS Grade, Panreac) in 1:1 ratio.
Analytical reversed-phase HPLC was used for determination of uncalibrated purity of the
compounds and conducted using a Zorbax RX-SIL column (5µm, 250*4.6 mm); eluent A, 0.77
% solution of ammonium acetate in water; eluent B CH3CN; isocratic elution A:B = 1:9; flow
rate was 1.6 mL/min. HPLC analysis was performed at 40 oC during 17 min at 285 nm.
4.1.1. General procedure for preparation of сompounds 3a-h
The amine 2a-b (1.4 equiv) was added to carboxylic acid 1a-d (1 equiv) in 50 ml of
toluene. The reaction mixture was refluxed with a Dean-Stark trap for 48 h. The progress of
reaction was monitored for water formed in the Dean-Stark trap. Then the solvent was
evaporated under reduced pressure. The product was recrystallized from acetone.
4.1.1.1. N-(2-(Dimethylamino)ethyl)laurylamide (3a)
The reaction was carried out following the general procedure with compound 1a (5.00 g,
25.0 mmol) and compound 2a (3.82 ml, 35.0 mmol). Yield 74% (5.00 g); white solid; mp 48 °C.
3
1H NMR (CDCl3) δ: 0.86 (t, 3Н, JHH = 6.8 Hz, CH3C10H22), 1.23-1.29 (m, 16Н, 8СН2), 1.56-

3
1.64 (m, 2Н, СН2), 2.15 (t, 2Н, JHH = 7.6 Hz, СН2С(О)), 2.23 (s, 6Н, (СН3)2N), 2.40 (t, 2Н,
13
3JHH = 5.8 Hz, СН2N), 3.29-3.33 (m, 2Н, СН2NH), 6.07 (br s, 1Н, NH). C NMR (CDCl3) δ:
14.22 (СН3), 22.79 (СН2), 25.91 (СН2), 29.44 (СН2), 29.48 (СН2), 29.62 (СН2), 29.72 (СН2),
32.02 (СН2), 36.71 (СН2), 36.88 (СН2NH), 45.21 ((СН3)2N), 58.03 (СН2N), 173.42 (С=О). ESI-
HRMS m/z: 271.2749 [М+H]+ (calculated for [C16H35N2O]+ - 271.2744).
4.1.1.2. N-(2-(Dimethylamino)ethyl)myristylamide (3b)
The reaction was carried out following the general procedure with compound 1b (5.00 g,
21.9 mmol) and compound 2a (3.35 ml, 30.7 mmol). Yield 69% (4.51 g); white solid; mp 52 °C.
3
1H NMR (CDCl3) δ: 0.87 (t, 3Н, JHH = 6.7 Hz, CH3C12H24), 1.24-1.30 (m, 20Н, 10СН2), 1.57-
3
1.65 (m, 2Н, СН2), 2.17 (t, 2Н, JHH = 7.6 Hz, СН2С(О)), 2.27 (s, 6Н, (СН3)2N), 2.46 (t, 2Н,
13
3JHH = 5.9 Hz, СН2N), 3.32-3.36 (m, 2Н, СН2NH), 6.18 (br s, 1Н, NH). C NMR (CDCl3) δ:
14.25 (СН3), 22.82 (СН2), 25.92 (СН2), 29.46 (СН2), 29.48 (СН2), 29.50 (СН2), 29.65 (СН2),
29.75 (СН2), 29.78 (СН2), 29.80 (СН2), 32.05 (СН2), 36.56 (СН2), 36.88 (СН2), 45.12
((СН3)2N), 58.08 (СН2N), 173.51 (С=О). ESI-HRMS m/z: 299.3062 [М+H]+ (calculated for
[C18H39N2O]+ - 299.3057).
4.1.1.3 N-(2-(Dimethylamino)ethyl)palmitamide (3c)
The reaction was carried out following the general procedure with compound 1c (5.00 g,
19.5 mmol) and compound 2a (2.98 ml, 27.3 mmol). Yield 64% (4.08 g); white solid; mp 64 °C.
3
1H NMR (CDCl3) δ: 0.87 (t, 3Н, JHH = 6.8 Hz, CH3C14H28), 1.24-1.32 (m, 24H, 12СН2), 1.57-
3
1.64 (m, 2Н, СН2), 2.17 (t, 2Н, JHH = 7.9 Hz, СН2С(О)), 2.31 (s, 6Н, (СН3)2N), 2.52 (t, 2Н,
13
3JHH = 5.7 Hz, СН2N), 3.37-3.39 (m, 2Н, СН2NH), 6.45 (br s, 1Н, NН). C NMR (CDCl3) δ:
14.28 (CH3), 22.83 (СН2), 25.91 (СН2), 29.49 (СН2), 29.52 (СН2), 29.66 (СН2), 29.77 (СН2),
29.80 (СН2), 29.83 (СН2), 32.06 (СН2), 36.33 (СН2), 36.84 (СН2), 44.88 ((СН3)2N), 58.01
(СН2N), 173.63 (С=О). ESI-HRMS m/z: 327.3375 [М+H]+ (calculated for [C20H43N2O]+ -
327.3370).
4.1.1.4. N-(2-(Dimethylamino)ethyl)stearamide (3d)
The reaction was carried out following the general procedure with compound 1d (7.14 g,
25.1 mmol) and compound 2a (3.83 ml, 35.1 mmol). Yield 73% (6.49 g); white solid; mp 72 °C.
3
1H NMR (CDCl3) δ: 0.87 (t, 3H, JHH = 6.8 Hz, CH3C16H32), 1.24-1.32 (m, 28Н, 14СН2), 1.57-
1.64 (m, 2Н, СН2), 2.17 (t, 2Н, 3JHH = 7.9 Hz, СН2С(О)), 2.32 (s, 6Н, (СН3)2N), 2.52 (t, 2Н, 3JHH
13
= 5.8 Hz, СН2N), 3.35-3.39 (m, 2Н, СН2NH), 6.44 (br s, 1Н, NH). C NMR (CDCl3) δ: 14.28
(СН3), 22.84 (СН2), 25.91 (СН2), 29.49 (СН2), 29.52 (СН2), 29.67 (СН2), 29.77 (СН2), 29.80
(СН2), 29.84 (СН2), 32.07 (СН2), 36.33 (СН2), 36.85 (СН2), 44.89 ((СН3)2N), 58.02 (СН2N),
173.63 (С=О). ESI-HRMS m/z: 355.3683 [М+H]+ (calculated for [C22H47N2O]+ - 355.3683).
4.1.1.5. N-(3-(Dimethylamino)propyl)laurylamide (3e)
The reaction was carried out following the general procedure with compound 1a (5.00 g,
25.0 mmol) and compound 2b (4.37 ml, 35.0 mmol). Yield 70% (4.97 g); white solid; mp 34 °C.
3
1H NMR (CDCl3) δ: 0.84 (t, 3Н, JHH = 6.0 Hz, CH3C10H22), 1.22-1.28 (m, 16Н, 8СН2), 1.53-
3
1.59 (m, 2Н, СН2), 1.62-1.68 (m, 2Н, СН2), 2.11 (t, 2Н, JHH = 7.6 Hz, СН2C(O)), 2.24 (s, 6Н,
3
(СН3)2N), 2.40 (t, 2Н, JHH = 6.4 Hz, СН2N), 3.26-3.31 (m, 2Н, СН2NH), 6.97 (br s, 1Н, NH).
13C NMR (CDCl3) δ: 14.17 (СН3), 22.74 (СН2), 25.86 (СН2), 26.08 (СН2СH2CH2NH), 29.41
(СН2), 29.47 (СН2), 29.58 (СН2), 29.64 (СН2), 29.69 (СН2), 31.97 (СН2), 37.01 (СН2), 38.99
(СН2NH), 45.19 ((СН3)2N), 58.34 (СН2N), 173.28 (С=О). ESI-HRMS m/z: 285.2906 [М+H]+
(calculated for [C17H37N2O]+ - 285.2900).
4.1.1.6. N-(3-(Dimethylamino)propyl)myristylamide (3f)
The reaction was carried out following the general procedure with compound 1a (10.00 g,
43.8 mmol) and compound 2b (7.66 ml, 61.3 mmol). Yield 65% (8.89 g); white solid; mp 51 °C.

3
1H NMR (CDCl3) δ: 0.87 (t, 3H, JHH = 6.7 Hz, CH3C12H24), 1.24-1.31 (m, 20Н, 10СН2), 1.55-
3
1.62 (m, 2Н, СН2), 1.64-1.70 (m, 2Н, СН2), 2.14 (t, 2Н, JHH = 7.7 Hz, СН2С(О)), 2.27 (s, 6Н,
3
(СН3)2N), 2.42 (t, 2H, JHH = 6.4 Hz, СН2N), 3.33-3.35 (m, 2Н, СН2NH), 6.95 (br s, 1Н, NH).
13C NMR (CDCl3) δ: 14.26 (СН3), 22.83 (СН2), 25.90 (СН2), 26.09 (СН2СH2CH2NH), 29.48
(СН2), 29.53 (СН2), 29.64 (СН2), 29.78 (СН2), 32.05 (СН2), 37.11 (СН2), 39.21 (СН2), 45.37
((СН3)2N), 58.63 (СН2N), 173.27 (С=О). ESI-HRMS m/z: 313.3219 [М+H]+ (calculated for
[C19H41N2O]+ - 313.3213).
4.1.1.7. N-(3-(Dimethylamino)propyl)palmitamide (3g)
The reaction was carried out following the general procedure with compound 1b (4.00 g,
15.6 mmol) and compound 2b (2.73 ml, 21.8 mmol). Yield 59% (5.31 g); white solid; mp 57 °C.
3
1H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 6.7 Hz, CH3C14H28), 1.24-1.32 (m, 24Н, 12СН2), 1.55-
3
1.62 (m, 2Н, СН2), 1.66-1.73 (m, 2Н, СН2), 2.14 (t, 2Н, JHH = 7.8 Hz, СН2С(О)), 2.30 (s, 6Н,
3
(СН3)2N), 2.47 (t, 2H, JHH = 6.5 Hz, СН2N), 3.30-3.35 (m, 2Н, СН2NH), 6.98 (br s, 1Н, NH).
13C NMR (CDCl3) δ: 14.27 (СН3), 22.83 (СН2), 25.91 (СН2), 25.94 (СН2СH2CH2NH), 29.47
(СН2), 29.50 (СН2), 29.54 (СН2), 29.66 (СН2), 29.77 (СН2), 29.80 (СН2), 29.83 (СН2), 32.06
(СН2), 37.08 (СН2), 38.87 (СН2), 45.08 ((СН3)2N), 58.17 (СН2N), 173.39 (С=O). ESI-HRMS
m/z: 341.3532 [М+H]+ (calculated for [C21H45N2O]+ - 341.3526).
4.1.1.8. N-(3-(Dimethylamino)propyl)stearamide (3h)
The reaction was carried out following the general procedure with compound 1c (10.00 g,
35.2 mmol) and compound 2b (6.15 ml, 49.2 mmol). Yield 61% (7.91 g); white solid; mp 64 °C.
3
1H NMR (CDCl3) δ: 0.87 (t, 3H, JHH = 6.8 Hz, CH3C16H32), 1.24-1.32 (m, 28Н, 14СН2), 1.56-
3
1.63 (m, 2Н, СН2), 1.69-1.75 (m, 2Н, СН2), 2.15 (t, 2Н, JHH = 7.8 Hz, СН2С(О)), 2.33 (s, 6Н,
3
(СН3)2N), 2.51 (t, 2H, JHH = 6.5 Hz, СН2N), 3.30-3.35 (m, 2Н, СН2NH), 6.95 (br s, 1Н, NH).
13C NMR (CDCl3) δ: 14.26 (СН3), 22.83 (СН2), 25.86 (СН2), 25.92 (СН2СH2CH2NH), 29.49
(СН2), 29.54 (СН2), 29.66 (СН2), 29.70 (СН2), 29.79 (СН2), 29.84 (СН2), 32.06 (СН2), 37.05
(СН2), 38.57 (СН2), 44.84 ((СН3)2N), 57.79 (СН2N), 173.48 (С=О). ESI-HRMS m/z: 369.3845
[М+H]+ (calculated for [C23H49N2O]+ - 369.3839).
4.1.1.9. N-(3-(Dimethylamino)propyl)hexanamide (3i)
The reaction was carried out following the general procedure with compound 1e (4.50 g,
38.7 mmol) and compound 2b (5.80 ml, 46.5 mmol). Then the solvent was evaporated under
reduced pressure. Oily residue then was refluxed in petroleum ether during 1h and organic layer
1
3
was decanted. Yield 40% (3.16 g); yellow oil. H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.4 Hz,
CH3C4H8), 1.24-1.31 (m, 4Н, 2CH2), 1.53-1.60 (m, 2Н, СН2), 1.64-1.71 (m, 2Н,
СН2СН2СН2NH), 2.12 (t, 2Н, 3JHH = 7.6 Hz, СН2С(О)), 2.28 (s, 6Н, (СН3)2N), 2.45 (t, 2H, 3JHH
13
= 6.5 Hz, СН2N), 3.27-3.31 (m, 2Н, СН2NH), 7.05 (br s, 1Н, NH). C NMR (CDCl3) δ: 14.00
(СН3), 22.47 (СН2), 25.49 (СН2), 25.91 (СН2СH2CH2NH), 31.51 (СН2), 36.88 (СН2), 38.75
(СН2), 45.05 ((СН3)2N), 58.14 (СН2N), 173.40 (С=О). ESI-HRMS m/z: 201.1967 [М-Cl]+
(calculated for [C11H25N2O]+ - 201.1961).
4.1.2. General procedure for preparation of compound 8a-f
The amine 7a-b (1 equiv), carboxylic acid 1a-c (1 equiv) and DMAP (0.05 equiv) was
dissolved in 30 ml of methylene chloride then DCC was added slowly during 15 min. The
reaction mixture was stirred for 4 h at 25 °C. Then the formed precipitate was filtered. Filtrate
was concentrated and purified by column chromatography (eluent methylene chloride). Then the
eluate was evaporated under reduced pressure. The dry residue was emulsified in water and
neutralized with hydrochloric acid (pH=7). Then the water was evaporated under reduced
pressure. Then dry residue was refluxed in 30 ml of petroleum ether for 1 h and the precipitate
was filtered. The precipitate was dissolved in water and NaHCO3 was added (1 equiv). Then the

solvent was evaporated under reduced pressure. The dry residue was dissolved in methylene
chloride and NaCl was filtered. Then the solvent was evaporated under reduced pressure.
4.1.2.1. 2-(Dimethylamino)ethyl laurylate (8a)
The reaction was carried out following the general procedure with compound 1a (5.00 g,
25.0 mmol), compound 7a (2.51 ml, 25.0 mmol), DCC (5.15 g, 25.0 mmol), DMAP (0.15 g, 1.3
1
3
mmol). Yield 61% (4.15 g); colorless oil. H NMR (CDCl3) δ: 0.84 (t, 3Н, JHH = 6.8 Hz,
CH3C10H22), 1.21-1.28 (m, 16Н, 8СН2), 1.54 -1.61 (m, 2Н, СН2), 2.24 (s, 6Н, (СН3)2N), 2.28 (t,
3
3
3
2Н, JHH = 7.6 Hz, СН2С(О)), 2.52 (t, 2Н, JHH = 5.8 Hz, СН2N), 4.13 (t, 2Н, JHH = 5.8 Hz,
СН2O). 13C NMR (CDCl3) δ: 14.19 (СН3), 22.75 (СН2), 25.00 (СН2), 29.20 (СН2), 29.34 (СН2),
29.41 (СН2), 29.53 (СН2), 29.67 (СН2), 31.98 (СН2), 34.32 (СН2), 45.77 ((СН3)2N), 57.92
(СН2N), 62.05 (СН2O), 174.03 (С=О). ESI-HRMS m/z: 272.2590 [М+H]+ (calculated for
[C16H34N2O]+ - 272.2584).
4.1.2.2. 2-(Dimethylamino)ethyl myristate (8b)
The reaction was carried out following the general procedure with compound 1b (5.00 g,
21.9 mmol), compound 7a (2.20 ml, 21.9 mmol), DCC (4.52 g, 21.9 mmol), DMAP (0.13 g, 1.1
1
3
mmol). Yield 52% (3.42 g); colorless oil. H NMR (CDCl3) δ: 0.81 (t, 3H, JHH = 6.7 Hz,
CH3C12H24), 1.19-1.26 (m, 20Н, 10СН2), 1.51-1.59 (m, 2Н, СН2), 2.22 (s, 6Н, (СН3)2N), 2.26 (t,
3
3
3
2Н, JHH = 7.3 Hz, СН2СО), 2.50 (t, 2H, JHH = 5.7 Hz, СН2N), 4.11 (t, 2H, JHH = 5.7 Hz,
СН2O). 13C NMR (CDCl3) δ: 14.15 (СН3), 22.72 (СН2), 24.96 (СН2), 29.17 (СН2), 29.31 (СН2),
29.39 (СН2), 29.50 (СН2), 29.63 (СН2), 29.68 (СН2), 29.71 (СН2), 31.96 (СН2), 34.27 (СН2),
45.68 ((СН3)2N), 57.85 (СН2N), 61.95 (СН2O), 173.90 (С=О). ESI-HRMS m/z: 300.2903
[М+H]+ (calculated for [C18H38NO2]+ - 300.2897).
4.1.2.3. 2-(Dimethylamino)ethyl palmitate (8c)
The reaction was carried out following the general procedure with compound 1c (5.00 g,
19.5 mmol), compound 7a (1.96 ml, 19.5 mmol), DCC (4.02 g, 19.5 mmol), DMAP (0.12 g, 0.9
1
3
mmol). Yield 52% (3.32 g); colorless oil. H NMR (CDCl3) δ: 0.87 (t, 3H, JHH = 6.8 Hz,
CH3C12H24), 1.24-1.30 (m, 24Н, 12СН2), 1.57-1.64 (m, 2Н, СН2), 2.31 (s, 6Н, (СН3)2N), 2.32 (t,
3
3
3
2Н, JHH = 7.7 Hz, СН2С(О)), 2.59 (t, 2Н, JHH = 5.7 Hz, СН2N), 4.18 (t, 2Н, JHH = 5.7 Hz,
СН2O). 13C NMR (CDCl3) δ: 14.27 (СН3), 22.83 (СН2), 25.07 (СН2), 29.27 (СН2), 29.41 (СН2),
29.50 (СН2), 29.60 (СН2), 29.74 (СН2), 29.79 (СН2), 29.83 (СН2), 32.06 (СН2), 34.40 (СН2),
45.72 ((СН3)2N), 57.89 (СН2N), 61.90 (СН2O), 174.06 (С=О). ESI-HRMS m/z: 328.3216
[М+H]+ (calculated for [C20H42NO2]+ - 328.3210).
4.1.2.4. 3-(Dimethylamino)propyl laurylate (8d)
The reaction was carried out following the general procedure with compound 1a (5.00 g,
25.0 mmol), compound 7b (2.95 ml, 25.0 mmol), DCC (5.15 g, 25.0 mmol), DMAP (0.15 g, 1.3
1
3
mmol). Yield 51% (3.64 g); colorless oil. H NMR (CDCl3) δ: 0.85 (t, 3Н, JHH = 6.7 Hz,
CH3C10H22), 1.23-1.31 (m, 16Н, 8СН2), 1.55-1.62 (m, 2Н, СН2), 1.73-1.80 (m, 2Н,
CH2СН2CH2O), 2.20 (s, 6Н, (СН3)2N), 2.26 (t, 2Н, 3JHH = 7.5 Hz, СН2С(О)), 2.30 (t, 2Н, 3JHH
=
3
13
7.0 Hz, СН2N), 4.09 (t, 2Н, JHH = 6.1 Hz, СН2O). C NMR (CDCl3) δ: 14.23 (СН3), 22.79
(СН2), 25.09 (СН2), 27.12 (c, CH2CH2CH2O), 29.26 (СН2), 29.38 (СН2), 29.44 (СН2), 29.57
(СН2), 29.71 (СН2), 32.01 (СН2), 34.45 (СН2), 45.59 ((СН3)2N), 56.37 (СН2N), 62.68 (СН2O),
174.02 (С=О). ESI-HRMS m/z: 286.2746 [М+H]+ (calculated for [C17H36N2O]+ - 286.2741).
4.1.2.5. 3-(Dimethylamino)propyl myristate (8e)
The reaction was carried out following the general procedure with compound 1b (5.00 g,
21.9 mmol), compound 7b (2.59 ml, 21.9 mmol), DCC (4.52 g, 21.9 mmol), DMAP (0.13 g, 1.1
1
3
mmol). Yield 47% (3.28 g); colorless oil. H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 6.8 Hz,
CH3C12H24), 1.24-1.32 (m, 20Н, 10СН2), 1.57-1.63 (m, 2Н, СН2), 1.77-1.84 (m, 2Н, СН2), 2.24

(s, 6Н, (СН3)2N), 2.28 (t, 6H, 3JHH = 7.6 Hz, СН2С(О)), 2.35 (t, 2Н, 3JHH = 7.5 Hz, СН2N), 4.10
3
13
(t, 2H, JHH = 6.5 Hz, СН2O). C NMR (CDCl3) δ: 14.26 (СН3), 22.82 (СН2), 25.12 (СН2),
27.00 (CH2CH2CH2O), 29.29 (СН2), 29.41 (СН2), 29.49 (СН2), 29.60 (СН2), 29.77 (СН2), 32.05
(СН2), 34.47 (СН2), 45.48 ((СН3)2N), 56.35 (СН2N), 62.63 (СН2O), 174.03 (С=О). ESI-HRMS
m/z: 314.3059 [М+H]+ (calculated for [C19H40NO2]+ - 314.3054).
4.1.2.6. 3-(Dimethylamino)propyl palmitate (8f)
The reaction was carried out following the general procedure with compound 1c (5.00 g,
19.5 mmol), compound 7b (2.31 ml, 19.5 mmol), DCC (4.02 g, 19.5 mmol), DMAP (0.12 g, 0.9
1
3
mmol). Yield 51% (3.42 g); colorless oil. H NMR (CDCl3) δ: 0.87 (t, 3H, JHH = 6.7 Hz,
CH3C14H28), 1.24-1.35 (m, 24Н, 12СН2), 1.56-1.64 (m, 2Н, СН2), 1.78-1.85 (m, 2Н, СН2), 2.26
(s, 6Н, (СН3)2N), 2.28 (t, 2Н, 3JHH = 7.6 Hz, СН2С(О)), 2.37 (t, 2Н, 3JHH = 7.3 Hz, СН2N), 4.11
3
13
(t, 2H, JHH = 6.5 Hz, СН2O). C NMR (CDCl3) δ: 14.27 (СН3), 22.83 (СН2), 25.12 (СН2),
26.94 (CH2CH2CH2O), 29.30 (СН2), 29.41 (СН2), 29.50 (СН2), 29.61 (СН2), 29.75 (СН2), 29.79
(СН2), 29.83 (СН2), 32.06 (СН2), 34.47 (СН2), 45.44 ((СН3)2N), 56.34 (СН2N), 62.59 (СН2O),
174.02 (С=О). ESI-HRMS m/z: 342.3372 [М+H]+ (calculated for [C21H44NO2]+ - 342.3367).
4.1.3. General procedure for preparation of quaternary ammonium salts 6a-u, 9a-n
A solution of the corresponding chloride 5a–e (1 equiv) in 20 ml of ethanol was
neutralized with aqueous solution of NaHCO3 (1 equiv). The solvent was evaporated under
reduced pressure and the residue was dissolved in ethanol (20 ml), and the formed NaCl was
filtered off. Compounds 3a-i, 8a-f (1 equiv) were added to the filtrate. The reaction mixture was
heated at 70 °C for 8 h, then the solvent was evaporated under reduced pressure. The product
was recrystallized from acetone (compounds 6a-u, 9b-g, 9i, 9k-n) or purified by column
chromatography (compound 9a, 9h, 9j) on reversed-fase column (eluent isopropanol/water =
from 0:100 to 100:0).
4.1.3.1.
5-(Methylene(N,N-dimethyl-N-(2-lauroylaminoethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6a)
The reaction was carried out following the general procedure with compound 5a (0.66 g,
2.5 mmol), NaHCO3 (0.21 g, 2.5 mmol) and compound 3a (0.68 g, 2.5 mmol). Yield 71% (0.89
1
3
g); white solid; mp 140-142 °C (dec). H NMR (CDCl3) δ: 0.84 (t, 3Н, JHH = 6.8 Hz,
СН3С10Н20), 1.18-1.28 (m, 16H, 8CH2), 1.51 (s, 6Н, (СН3)2С), 1.52-1.58 (m, 2H, CH2), 2.23 (t,
3
4Н, JHH = 7.6 Hz, СН2С(О)), 2.42 (s, 3Н, CH3Pyr), 3.34 (s, 6Н, (СН3)2N+), 3.80-3.83 (m, 2Н,
СН2), 3.99-4.02 (m, 2Н, СН2), 4.95 (s, 2Н, СН2), 5.10 (s, 2Н, СН2), 8.26 (s, 1Н, СНPyr), 8.71 (t,
3
13
1Н, JHH = 5.4 Hz, NH). C NMR (CDCl3) δ: 14.23 (СН3), 18.60 (СH3Pyr), 22.78 (СH2), 24.95
(C(CH3)2), 25.61 (СH2), 29.44 (СH2), 29.50 (СH2), 29.55 (СH2), 29.65 (СH2), 29.72 (СH2),
29.75 (СH2), 32.00 (СH2), 34.34 (СH2), 36.39 (СH2), 49.99 (СH3N+), 59.43 (СH2), 62.82 (СH2),
64.30 (СH2), 100.78 ((СН3)2С), 117.27 (СPyr), 129.16 (СPyr), 143.53 (СPyr), 147.06 (СPyr), 150.88
(СPyr), 174.98 (С=О). ESI-HRMS m/z: 462.3696 [М-Cl]+ (calculated for [C27H48N3O3]+ -
462.3690).
4.1.3.2. 5-(Methylene(N,N-dimethyl-N-(2-myristoylaminoethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6b)
The reaction was carried out following the general procedure with compound 5a (0.37 g,
1.4 mmol), NaHCO3 (0.12 g, 1.4 mmol) and compound 3b (0.41 g, 1.4 mmol). Yield 53% (0.59
o
1
3
g); beige solid; mp 132-135 C (dec). H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 7.0 Hz,
CH3C12H24), 1.21-1.28 (m, 20H, 10CH2), 1.52 (s, 6H, (CH3)2C), 1.55-1.59 (m, 2H, CH2), 2.25 (t,
3
2H, JHH = 7.5 Hz, СH2C(O)), 2.41 (s, 3H, CH3Pyr), 3.29 (s, 6H, (CH3)2N+), 3.82-3.86 (m, 2H,
CH2), 4.01 (br s, 2H, CH2), 4.84 (s, 2H, CH2), 5.07 (s, 2H, CH2), 8.18 (s, 1H, CНPyr), 8.63 (t, 1H,
13
3JHH = 5.0 Hz, NH). C NMR (CDCl3) δ: 14.22 (CH3), 18.75 (СН3Pyr), 22.78 (СН2), 24.94
((СH3)2С), 25.61 (СH2), 29.45 (CH2), 29.50 (CH2), 29.56 (CH2), 29.65 (CH2), 29.77 (CH2),

32.01 (CH2), 34.32 (CH2), 36.38 (CH2), 49.97 (CH3N+), 59.40 (CH2), 62.90 (CH2), 64.30 (CH2),
100.69 ((СН3)2С), 117.08 (CPyr), 128.84 (CPyr), 143.82 (CPyr), 146.95 (CPyr), 151.07 (CPyr),
174.98 (C=O). ESI-HRMS m/z: 490.4003 [М-Cl]+ (calculated for [C29H52N3O3]+ - 490.4003).
4.1.3.3. 5-(Methylene(N,N-dimethyl-N-(2-palmitoylaminoethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6c)
The reaction was carried out following the general procedure with compound 5a (0.34 g,
1.3 mmol), NaHCO3 (0.11 g, 1.3 mmol) and compound 3с (0.43 g, 1.3 mmol). Yield 36% (0.26
1
3
g); beige solid; mp 135-136 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.6 Hz,
CH3C14H28), 1.20-1.27 (m, 24H, 12CH2), 1.50 (s, 6H, (СН3)2С), 1.55 (br s, 2H, CH2), 2.24 (t,
2H, 3JHH = 7.3 Hz, СH2C(O)), 2.40 (s, 3H, CH3Pyr), 3.32 (s, 6H, (CH3)2N+), 3.81 (br s, 2H, CH2),
4.00 (br s, 2H, CH2), 4.89 (s, 2H, CH2), 5.08 (s, 2H, CH2), 8.19 (s, 1H, CНPyr), 8.72 (t, 1H, 3JHH
=
13
5.4 Hz, NH). C NMR (CDCl3) δ: 14.18 (СH3), 18.94 (СH3Pyr), 22.73 (СH2), 24.89 ((СH3)2С),
25.57 (СH2), 29.41 (СH2), 29.47 (СH2), 29.53 (СH2), 29.62 (СH2), 29.75 (СH2), 31.97 (CH2),
34.26 (CH2), 36.33 (CH2), 49.88 (CH3N+), 59.32 (CH2), 62.94 (CH2), 64.22 (CH2), 100.47
((СН3)2С), 116.80 (CPyr), 128.25 (CPyr), 144.35 (CPyr), 146.69 (CPyr), 151.28 (CPyr), 174.91
(C=O). ESI-HRMS m/z: 518.4316 [М-Cl]+ (calculated for [C31H56N3O3]+ - 518.4316).
4.1.3.4. 5-(Methylene(N,N-dimethyl-N-(2-stearoylaminoethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6d)
The reaction was carried out following the general procedure with compound 5a (0.58 g,
2.2 mmol), NaHCO3 (0.18 g, 2.2 mmol) and compound 3d (0.78 g, 2.2 mmol). Yield 44% (0.56
1
3
g); beige solid; mp 143 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.3 Hz, CH3C16H32),
3
1.20-1.30 (m, 28H, 14CH2), 1.51 (s, 6H, (СН3)2С), 1.54 (br s, 2H, CH2), 2.22 (t, 2H, JHH = 7.4
Hz, СH2C(O)), 2.41 (s, 3H, CH3Pyr), 3.30 (s, 6H, (CH3)2N+), 3.82 (br s, 2H, CH2), 3.98 (br s, 2H,
13
CH2), 4.89 (s, 2H, CH2), 5.08 (s, 2H, CH2), 8.24 (s, 1H, CНPyr), 8.62 (br s, 1H, NH). C NMR
(CDCl3) δ: 14.25 (СH3), 18.67 (СH3Pyr), 22.80 (CH2), 24.94 ((СH3)2С), 25.62 (CH2), 29.48
(CH2), 29.53 (CH2), 29.60 (CH2), 29.69 (CH2), 29.78 (CH2), 29.83 (CH2), 32.04 (CH2), 34.20
(CH2), 36.38 (CH2), 49.94 (CH3N+), 59.37 (CH2), 62.86 (CH2), 64.31 (CH2), 100.71 ((СН3)2С),
117.21 (CPyr), 129.00 (CPyr), 143.70 (CPyr), 146.96 (CPyr), 150.91 (CPyr), 175.02 (C=O). ESI-
HRMS m/z: 546.4629 [М-Cl]+ (calculated for [C33H60N3O3]+ - 546.4629).
4.1.3.5. 5-(Methylene(N,N-dimethyl-N-(3-lauroylaminopropyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6e)
The reaction was carried out following the general procedure with compound 5a (0.29 g,
1.1 mmol), NaHCO3 (0.09 g, 1.1 mmol) and compound 3e (0.31 g, 1.1 mmol). Yield 45% (0.25
o
1
3
g); white solid; mp 109-111 C (dec). H NMR (CDCl3) δ: 0.85 (t, 3Н, JHH = 6.7 Hz,
СН3С10Н20), 1.17-1.29 (m, 16H, 8CH2), 1.52 (s, 6Н, (СН3)2С), 1.52-1.58 (m, 2H, CH2), 2.11-
2.18 (m, 2H, CH2CH2CH2NH), 2.24 (t, 4Н, 3JHH = 7.6 Hz, СН2С(О)), 2.43 (s, 3Н, CH3Pyr), 3.22
(s, 6Н, (СН3)2N+), 3.32-3.35 (m, 2Н, СН2), 3.97-4.00 (m, 2Н, СН2), 4.90 (s, 2Н, СН2), 5.11 (s,
13
2Н, СН2), 8.12 (br s, 1Н, NH), 8.23 (s, 1Н, СНPyr). C NMR (CDCl3) δ: 14.23 (СН3), 18.53
(СH3Pyr), 22.78 (СH2), 23.11 (CH2СH2CH2NH), 24.97 (C(CH3)2), 25.93 (СH2), 29.46 (СH2),
29.56 (СH2), 29.66 (СH2), 29.71 (СH2), 29.74 (СH2), 29.79 (СH2), 32.01 (СH2), 36.36 (СH2),
36.46 (СH2), 49.39 (СH3N+), 59.41 (СH2), 62.39 (СH2), 63.39 (СH2), 100.85 ((СН3)2С), 117.45
(СPyr), 129.40 (СPyr), 143.21 (СPyr), 147.15 (СPyr), 150.72 (СPyr), 174.81 (С=О). ESI-HRMS m/z:
476.3853 [М-Cl]+ (calculated for [C28H50N3O3]+ - 476.3847).
4.1.3.6.
5-(Methylene(N,N-dimethyl-N-(3-myristoylaminopropyl)ammonium))-2,2,8-trimethyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6f)
The reaction was carried out following the general procedure with compound 5a (1.00 g,
3.8 mmol), NaHCO3 (0.32 g, 3.8 mmol) and compound 3f (1.18 g, 3.8 mmol). Yield 40% (0.82
o
1
3
g); beige solid; mp 123-125 C (dec). H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 6.8 Hz,

CH3C12H24), 1.21-1.29 (m, 20H, 10CH2), 1.52 (s, 6H, (СН3)2С), 1.56 (br s, 2H, CH2), 2.13-2.19
3
(m, 2H, CH2CH2CH2NH), 2.27 (t, 2H, JHH = 7.7 Hz, СH2C(O)), 2.41 (s, 3H, CH3Pyr), 3.20 (s,
6H, (СН3)2N+), 3.34-3.38 (m, 2H, CH2), 4.03-4.06 (m, 2H, CH2), 4.84 (s, 2H, CH2), 5.09 (s, 2H,
3
13
CH2), 8.11 (t, 1H, JHH = 5.4 Hz, NH), 8.16 (s, 1H, CНPyr). C NMR (CDCl3) δ: 14.24 (СH3),
18.89 (СH3Pyr), 22.80 (CH2), 23.10 (CH2СH2CH2NH), 24.96 ((СH3)2С), 25.93 (CH2), 29.35
(CH2), 29.48 (CH2), 29.57 (CH2), 29.66 (CH2), 29.74 (CH2), 29.78 (CH2), 29.82 (CH2), 32.04
(CH2), 34.40 (CH2), 36.48 (CH2), 49.39 (CH3N+), 59.35 (CH2), 62.56 (CH2), 63.58 (CH2),
100.63 ((СН3)2С), 116.92 (CPyr), 128.56 (CPyr), 143.93 (CPyr), 146.87 (CPyr), 151.31 (CPyr),
174.99 (C=O). ESI-HRMS m/z: 504.4160 [М-Cl]+ (calculated for [C30H54N3O3]+ - 504.4160).
4.1.3.7.
5-(Methylene(N,N-dimethyl-N-(3-palmitoylaminopropyl)ammonium))-2,2,8-trimethyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6g)
The reaction was carried out following the general procedure with compound 5a (0.34 g,
1.3 mmol), NaHCO3 (0.11 g, 1.3 mmol) and compound 3g (0.45 g, 1.3 mmol). Yield 43% (0.32
1
3
g); beige solid; mp 114-115 °C (dec). H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 6.8 Hz,
CH3C14H28), 1.21-1.29 (m, 24H, 12CH2), 1.51 (s, 6H, (СН3)2С), 1.56 (br s, 2H, CH2), 2.13-2.20
3
(m, 2H, CH2CH2CH2NH), 2.26 (t, 2H, JHH = 7.6 Hz, СH2C(O)), 2.41 (s, 3H, CH3Pyr), 3.19 (s,
6H, (СН3)2N+), 3.34-3.38 (m, 2H, CH2), 4.00-4.04 (m, 2H, CH2), 4.82 (s, 2H, CH2), 5.08 (s, 2H,
3
13
CH2), 8.11 (t, 1H, JHH = 5.5 Hz, NH), 8.15 (s, 1H, CНPyr). C NMR (CDCl3) δ: 14.19 (СH3),
18.81 (СH3Pyr), 22.76 (СH2), 23.10 (CH2СH2CH2NH), 24.91 ((СH3)2С), 25.89 (СH2), 29.35
(CH2), 29.44 (CH2), 29.55 (CH2), 29.63 (CH2), 29.67 (CH2), 29.70 (CH2), 29.74 (CH2), 29.79
(CH2), 31.99 (CH2), 34.47 (CH2), 36.42 (CH2), 49.39 (CH3N+), 59.29 (CH2), 62.46 (CH2), 63.36
(CH2), 100.55 ((СН3)2С), 117.03 (CPyr), 128.52 (CPyr), 144.00 (CPyr), 146.77 (CPyr), 151.11
(CPyr), 174.89 (C=O). ESI-HRMS m/z: 532.4474 [М-Cl]+ (calculated for [C32H58N3O3]+ -
532.4473).
4.1.3.8. 5-(Methylene(N,N-dimethyl-N-(3-stearoylaminopropyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (6h)
The reaction was carried out following the general procedure with compound 5a (0.34 g,
1.3 mmol), NaHCO3 (0.11 g, 1.3 mmol) and compound 3h (0.49 g, 1.3 mmol). Yield 49% (0.39
1
3
g); beige solid; mp 134-136 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.7 Hz,
CH3C16H32), 1.21-1.23 (m, 28H, 14CH2), 1.51 (s, 6H, (CH3)2C), 1,55 (br s, 2H, CH2), 2.17 (br s,
3
2H, CH2CH2CH2NH), 2.25 (t, 2H, JHH = 7.5 Hz, СH2C(O)), 2.40 (s, 3H, CH3Pyr), 3.19 (s, 6H,
(CH3)2N+), 3.33-3.37 (m, 2H, CH2), 3.97-4.01 (m, 2H, CH2), 4.82 (s, 2H, CH2), 5.08 (s, 2H,
3
13
CH2), 8.11 (t, 1H, JHH = 5.4 Hz, NH), 8.16 (s, 1H, CНPyr). C NMR (CDCl3) δ: 14.18 (CH3),
18.79 (CH3Pyr), 22.74 (CH2), 23.09 (CH2CH2CH2NH), 24.90 ((СH3)2С), 25.88 (CH2), 29.34
(CH2), 29.42 (CH2), 29.54 (CH2), 29.63 (CH2), 29.72 (CH2), 29.79 (CH2), 31.98 (CH2), 34.45
(CH2), 36.41 (CH2), 49.37 (CH3N+), 59.29 (CH2), 62.42 (CH2), 63.31 (CH2), 100.53 ((СН3)2С),
117.03 (CPyr), 128.52 (CPyr), 143.98 (CPyr), 146.76 (CPyr), 151.08 (CPyr), 174.86 (C=O). ESI-
HRMS m/z: 560.4786 [М-Cl]+ (calculated for [C34H62N3O3]+ - 560.4786).
4.1.3.9.
5-(Methylene(N,N-dimethyl-N-(2-lauroylaminoethyl)ammonium))-2-propyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6i)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3a (0.34 g, 1.2 mmol). Yield 45% (0.29
1
3
g); white solid; mp 125 °C (dec). H NMR (CDCl3) δ: 0.84 (t, 3Н, JHH = 6.8 Hz, СН3С10Н20),
3
0.96 (t, 3Н, JHH = 7.4 Hz, СН3СН2СН2СН), 1.16-1.25 (m, 16Н, 8СН2), 1.45-1.54 (m, 4Н,
2СН2), 1.73-1.85 (m, 2Н, СН2), 2.21 (t, 2Н, 3JHH = 7.6 Hz, СН2С(О)), 2.42 (s, 3Н, CH3Pyr), 3.30
(s, 3Н, CH3N+), 3.31 (s, 3Н, CH3N+), 3.79 (m, 2Н, СН2), 3.95 (br s, 2Н, СН2), 4.89, 4.92 (AB,
2JHH = -13.6 Hz, 2H, CH2), 5.04, 5.34 (AB, 2JHH = -16.4 Hz, 2H, CH2), 5.09 (t, 1H, 3JHH = 5.1 Hz,
3
13
СН3СН2СН2СН), 8.25 (s, 1Н, СНPyr), 8.63 (t, 1H, JHH = 4.9 Hz, NH). C NMR (CDCl3) δ:
13.99 (СН3CH2CH2), 14.21 (СН3), 16.90 (СН3CH2CH2), 18.52 (СН3Pyr), 22.76 (СН2), 25.59

(СН2), 29.43 (СН2), 29.49 (СН2), 29.55 (СН2), 29.64 (СН2), 29.71 (СН2), 29.74 (СН2), 31.99
(СН2), 34.23 (СН2), 36.25 (CH2), 36.36 (СН2), 49.94 (СН3N+), 50.09 (СН3N+), 62.87 (СН2),
64.23 (СН2), 65.28 (СН2), 100.58 (СН3СН2СН2CH), 117.53 (СPyr), 130.66 (СPyr), 144.24 (СPyr),
148.46 (СPyr), 150.46 (СPyr), 174.93 (С=О). ESI-HRMS m/z: 476.3852 [М-Cl]+ (calculated for
[C28H50N3O3]+ - 476.3847). HPLC analysis: retention time 8.8 min; purity 99.6 %.
4.1.3.10 5-(Methylene(N,N-dimethyl-N-(2-myristoylaminoethyl)ammonium))-2-propyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6j)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3b (0.36 g, 1.2 mmol). Yield 58% (0.38
1
3
g); beige solid; mp 136-139 °C (dec). H NMR (CDCl3) δ: 0.86 (t, 3H, JHH = 6.7 Hz,
CH3C12H24), 0.98 (t, 3H, 3JHH = 7.4 Hz, СН3СН2СН2СН), 1.21-1.28 (m, 20H, 10CH2), 1.48-1.59
3
(m, 4H, 2CH2), 1.74-1.86 (m, 2H, CH2), 2.24 (t, 3H, JHH = 7.7 Hz, СH2C(O)), 2.43 (s, 3H,
CH3Pyr), 3.29 (s, 3H, CH3N+), 3.30 (s, 3H, CH3N+), 3.81-3.84 (m, 2H, CH2), 3.97-4.00 (m, 2H,
2
2
CH2), 4.82, 4.88. (AB, 2H, JHH = -13.6 Hz, CH2), 5.04, 5.32 (AB, 2H, JHH = -16.4 Hz, CH2),
5.07 (t, 3H, 3JHH = 5.1 Hz, СН3СН2СН2CH), 8.19 (s, 1H, CHPyr), 8.62 (t, 1H, 3JHH = 5.2 Hz, NH).
13C NMR (CDCl3) δ: 13.96 (СН3CH2CH2), 14.17 (СH3), 16.89 (СН3CH2CH2), 18.84 (СН3Pyr),
22.73 (СH2), 25.56 (СH2), 29.41 (СH2), 29.47 (СH2), 29.53 (СH2), 29.62 (СH2), 29.74 (СH2),
31.97 (СH2), 34.20 (СH2), 36.26 (CH2), 36.32 (СH2), 49.88 (CH3N+), 50.01 (CH3N+), 62.97
(CH2), 64.19 (CH2), 65.21 (CH2), 100.43 (СН3СН2СН2CH), 117.11 (CPyr), 129.81 (CPyr), 144.95
(CPyr), 148.18 (CPyr), 150.86 (CPyr), 174.89 (C=O). ESI-HRMS m/z: 504.4160 [М-Cl]+
(calculated for [C30H54N3O3]+ - 504.4160).
4.1.3.11. 5-(Methylene(N,N-dimethyl-N-(2-palmitoylaminoethyl)ammonium))-2-propyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6k)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3c (0.41 g, 1.2 mmol). Yield 46% (0.33
1
3
g); beige solid; mp 141-142 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.5 Hz,
CH3C14H28), 0.96 (t, 3H, 3JHH = 7.3 Hz, СН3СН2СН2СН), 1.19-1.28 (m, 24H, 12CH2), 1.47-1.56
3
(m, 4H, 2CH2), 1.73-1.83 (m, 2H, CH2), 2.21 (t, 3H, JHH = 7.6 Hz, СH2C(O)), 2.39 (s, 3H,
CH3Pyr), 3.27 (s, 3H, CH3N+), 3.29 (s, 3H, CH3N+), 3.80 (br s, 2H, CH2), 3.95 (br s, 2H, CH2),
2
2
4.82, 4.87 (AB, 2H, JHH = -14.0 Hz, CH2), 5.01, 5.30 (AB, 2H, JHH = -16.1 Hz, CH2), 5.05 (t,
3
3
13
3H, JHH = 4.8 Hz, СН3СН2СН2CH), 8.19 (s, 1H, CНPyr), 8.63 (t, 1H, JHH = 5.0 Hz, NH). C
NMR (CDCl3) δ: 13.97 (СН3CH2CH2), 14.18 (СH3), 16.91 (СН3CH2CH2), 18.82 (СH3Pyr), 22.75
(CH2), 25.58 (CH2), 29.43 (CH2), 29.46 (CH2), 29.49 (CH2), 29.56 (CH2), 29.65 (CH2), 29.73
(CH2), 29.78 (CH2), 31.98 (CH2), 34.15 (CH2), 36.27 (CH2), 36.32 (CH2), 49.86 (CH3N+), 50.00
(CH3N+), 62.96 (CH2), 64.21 (CH2), 65.18 (CH2), 100.43 (СН3СН2СН2CH), 117.18 (CPyr),
129.86 (CPyr), 144.94 (CPyr), 148.19 (CPyr), 150.80 (CPyr), 174.94 (C=O). ESI-HRMS m/z:
532.4437 [М-Cl]+ (calculated for [C32H58N3O3]+ - 532.4437).
4.1.3.12. 5-(Methylene(N,N-dimethyl-N-(2-stearoylaminoethyl)ammonium))-2-propyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6l)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3d (0.44 g, 1.2 mmol). Yield 46% (0.34
1
3
g); beige solid; mp 143 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.7 Hz, CH3C16H32),
3
0.97 (t, 3H, JHH = 7.4 Hz, СН3СН2СН2СН), 1.19-1.27 (m, 28H, 14CH2), 1.46-1.59 (m, 4H,
2CH2), 1.76-1.83 (m, 2H, CH2), 2.22 (t, 2H, 3JHH = 7.4 Hz, CH2С(O)), 2.41 (s, 3H, CH3Pyr), 3.30
(s, 3H, CH3N+), 3.31 (s, 3H, CH3N+), 3.79-3.82 (m, 2H, CH2), 3.95-3.99 (m, 2H, CH2), 4.84,
4.90 (AB, 2H, 2JHH = 13.6 Hz, CH2), 5.03, 5.32 (AB, 2H, 2JHH = 16.4 Hz, CH2), 5.06 (t, 1H, 3JHH
= 5.1 Hz, CH), 8.20 (s, 1H, CHPyr), 8.64 (br s, 1H, NH). 13C NMR (CDCl3) δ: 13.99
(СН3CH2CH2), 14.21 (СH3), 16.92 (СН3CH2CH2), 18.84 (СH3Рyr), 22.77 (СH2), 25.59 (СH2),
29.44 (СH2), 29.51 (СH2), 29.57 (СH2), 29.67 (СH2), 29.71 (СH2), 29.75 (СH2), 29.80 (СH2),

32.00 (СH2), 34.20 (СH2), 36.29 (СH2), 36.36 (СH2), 49.88 (CH3N+), 50.04 (CH3N+), 63.02
(CH2), 64.24 (CH2), 65.22 (CH2), 100.47 (СН3СН2СН2CH), 117.14 (CPyr), 129.89 (CPyr), 144.91
(CPyr), 148.24 (CPyr), 150.90 (CPyr), 174.97 (C=O). ESI-HRMS m/z: 560.4786 [М-Cl]+
(calculated for [C34H62N3O3]+ - 560.4786).
4.1.3.13. 5-(Methylene(N,N-dimethyl-N-(3-lauroylaminopropyl)ammonium))-2-propyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6m)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3e (0.35 g, 1.2 mmol). Yield 44% (0.29
1
3
g); white solid; mp 130-131 °C (dec). H NMR (CDCl3) δ: 0.84 (t, 3Н, JHH = 6.8 Hz,
3
СН3С14Н28), 0.97 (t, 3Н, JHH = 7.4 Hz, СН3СН2СН2СН), 1.20-1.27 (m, 16Н, 8СН2), 1.46-1.55
(m, 4Н, 2СН2), 1.73-1.86 (m, 2Н, СН2), 2.11-2.14 (m, 2H, CH2CH2CH2NH), 2.23 (t, 2Н, 3JHH
=
7.3 Hz, СН2С(О)), 2.43 (s, 3Н, CH3Pyr), 3.20 (s, 3Н, CH3N+), 3.25 (s, 3Н, CH3N+), 3.30-3.35 (m,
2Н, СН2), 3.87-3.99 (m, 2Н, СН2), 4.90 (s, 2H, CH2), 5.07, 5.34 (AB, 2H, 2JHH = -16.4 Hz, CH2),
5.14 (t, 1H, 3JHH = 5.1 Hz, СН3СН2СН2СН), 5.02 (s, 2Н, СН2), 8.08 (t, 1H, 3JHH = 5.6 Hz, NH),
8.22 (s, 1Н, СНPyr). 13C NMR (CDCl3) δ: 14.02 (СН3CH2CH2), 14.23 (СН3), 16.92
(СН3CH2CH2), 18.53 (СН3Pyr), 22.78 (СН2), 23.21 (CH2CH2CH2NH), 25.90 (СН2), 29.46 (СН2),
29.55 (СН2), 29.64 (СН2), 29.71 (СН2), 29.74 (СН2), 29.79 (СН2), 32.01 (СН2), 36.28 (CH2),
36.46 (СН2), 49.22 (СН3N+), 49.64 (СН3N+), 62.72 (СН2), 63.11 (СН2), 65.30 (СН2), 100.62
(СН3СН2СН2СН), 117.63 (СPyr), 130.90 (СPyr), 143.90 (СPyr), 148.56 (СPyr), 150.43 (СPyr),
174.78 (С=О). ESI-HRMS m/z: 490.4009 [М-Cl]+ (calculated for [C29H52N3O3]+ - 490.4003).
4.1.3.14.
5-(Methylene(N,N-dimethyl-N-(3-myristoylaminopropyl)ammonium))-2-propyl-8-
methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6n)
The reaction was carried out following the general procedure with compound 5b (0.36 g,
1.3 mmol), NaHCO3 (0.11 g, 1.3 mmol) and compound 3f (0.41 g, 1.3 mmol). Yield 58% (0.42
1
3
g); beige solid; mp 130-132 °C (dec). H NMR (CDCl3) δ: 0.84 (t, 3H, JHH = 6.8 Hz,
CH3C12H24), 0.95 (t, 3H, 3JHH = 7.4 Hz, СН3СН2СН2СН), 1.19-1.27 (m, 20H, 10CH2), 1.47-1.54
(m, 4H, 2CH2), 1.75-1.81 (m, 2H, CH2), 2.07-2.15 (m, 2H, CH2СH2CH2NH), 2.21 (t, 2H, 3JHH
=
7.6 Hz, CH2С(O)), 2.38 (s, 3H, CH3Pyr), 3.18 (s, 3H, CH3N+), 3.22 (s, 3H, CH3N+), 3.30-3.33 (m,
2
2H, CH2), 3.83-3.98 (m, 2H, CH2), 4.82, 4.86 (AB, 2H, JHH = 14.8 Hz, CH2), 5.02, 5.30 (AB,
2
3
3
2H, JHH = 16.2 Hz, CH2), 5.09 (t, 3H, JHH = 5.1 Hz, СН3СН2СН2СН), 8.08 (t, 1H, JHH = 5.4
Hz, CH), 8.16 (s, 1H, NH). 13C NMR (CDCl3) δ: 14.02 (СН3CH2CH2), 14.22 (СH3), 16.96
(СН3CH2CH2), 18.79 (СH3Pyr), 22.78 (СH2), 23.34 (СH2CH2CH2NH), 25.89 (CH2), 29.47 (CH2),
29.59 (CH2), 29.67 (CH2), 29.77 (CH2), 29.82 (CH2), 32.02 (CH2), 36.32 (СН3CH2CH2), 36.48
(CH2NH), 49.54 (CH3N+), 49.94 (CH3N+), 63.19 (CH2), 65.31 (CH2), 100.47 (СН3СН2СН2СН),
117.42 (CPyr), 130.13 (CPyr), 144.76 (CPyr), 148.27 (CPyr), 150.69 (CPyr), 174.88 (C=O). ESI-
HRMS m/z: 518.4316 [М-Cl]+ (calculated for [C31H56N3O3]+ - 518.4316).
4.1.3.15.
5-(Methylene(N,N-dimethyl-N-(3-palmitoylaminopropyl)ammonium))-2-propyl-8-
methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6o)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3g (0.42 g, 1.2 mmol). Yield 57% (0.41
1
3
g); beige solid; mp 130-132 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.7 Hz,
CH3C14H28), 0.96 (t, 3H, 3JHH = 7.3 Hz, СН3СН2СН2СН), 1.20-1.27 (m, 24H, 12CH2), 1.48-1.54
(m, 4H, 2CH2), 1.75-1.81 (m, 2H, CH2), 2.12 (br s, 2H, CH2CH2CH2NH), 2.21 (t, 3H, 3JHH = 7.4
Hz, CH2С(O)), 2.39 (s, 3H, CH3Pyr), 3.17 (s, 3H, CH3N+), 3.22 (s, 3H, CH3N+), 3.33 (br s, 2H,
2
CH2), 3.74-3.93 (m, 2H, CH2), 4,82 (s, 2H, CH2), 5,01, 5.28 (AB, 2H, JHH = - 16.9 Hz, CH2),
5.09 (t, 1H, 3JHH = 5.3 Hz, СН3СН2СН2СН), 8.07 (t, 1H, 3JHH = 5.3 Hz, NH), 8.17 (s, 1H, CНPyr).
13C NMR (CDCl3) δ: 13.99 (СН3CH2CH2), 14.19 (СH3), 16.92 (СН3CH2CH2), 18.79 (СH3Pyr),
22.75 (CH2), 23.26 (СH2CH2CH2NH2), 25.86 (CH2), 29.43 (CH2), 29.56 (CH2), 29.66 (CH2),
29.73 (CH2), 29.80 (CH2), 31.99 (CH2), 36.29 (CH2), 36.41 (CH2), 49.28 (CH3N+), 49.73

(CH3N+), 62.82 (CH2), 62.96 (CH2), 65.13 (CH2), 100.40 (СН3СН2СН2СН), 117.42 (CPyr),
130.05 (CPyr), 144.71 (CPyr), 148.19 (CPyr), 150.60 (CPyr), 174.74 (C=O). ESI-HRMS m/z:
546.4630 [М-Cl]+ (calculated for [C33H64N3O3]+ - 546.4629).
4.1.3.16.
5-(Methylene(N,N-dimethyl-N-(3-stearoylaminopropyl)ammonium))-2-propyl-8-
methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6p)
The reaction was carried out following the general procedure with compound 5b (0.33 g,
1.2 mmol), NaHCO3 (0.11 g, 1.2 mmol) and compound 3g (0.46 g, 1.2 mmol). Yield 46% (0.35
1
3
g); beige solid; mp 143 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3H, JHH = 6.8 Hz, CH3C16H32),
3
0.97 (t, 3H, JHH = 7.4 Hz, СН3СН2СН2СН), 1.22-1.28 (m, 28H, 14CH2), 1.47-1.56 (m, 4H,
3
2CH2), 1.75-1.85 (m, 2H, CH2), 2.13 (br s, 2H, CH2CH2CH2NH2), 2.24 (t, 2H, JHH = 7.9 Hz,
CH2С(O)), 2.41 (s, 3H, CH3), 3.18 (s, 3H, CH3N+), 3.22 (s, 3H, CH3N+), 3.32-3.36 (m, 2H,
CH2N+), 3.84-3.98 (m, 2H, CH2), 4.82, 4.86 (AB, 2H, JHH = -14.4 Hz, CH2), 5.04, 5.31 (AB,
2
2
3
3
2H, JHH = -16.4 Hz, CH2), 5.11 (t, 1H, JHH = 5.1 Hz, СН3СН2СН2СН), 8.05 (t, 1H, JHH = 5.4
Hz, NH), 8.17 (s, 1H, CHPyr). C NMR (CDCl3) δ: 13.98 (СН3CH2CH2), 14.17 (СH3), 16.91
13
(СН3CH2CH2), 18.74 (СH3Рyr), 22.74 (СH2), 23.20 (СH2СH2CH2NH2), 25.85 (СH2), 29.42
(СH2), 29.54 (СH2), 29.63 (СH2), 29.72 (СH2), 29.79 (СH2), 31.97 (СH2), 36.28 (CH2), 36.40
(СН2), 49.26 (CH3N+), 49.67 (CH3N+), 62.77 (СH2), 62.99 (СH2), 65.15 (СH2), 100.41
(СН3СН2СН2СН), 117.39 (CPyr), 130.11 (CPyr), 144.61 (CPyr), 148.22 (CPyr), 150.61 (CPyr),
174.76 (C=O). ESI-HRMS m/z: 574.4943 [М-Cl]+ (calculated for [C35H64N3O3]+ - 574.4942).
4.1.3.17.
5-(Methylene(N,N-dimethyl-N-(3-myristoylaminopropyl)ammonium))-2-pentyl-8-
methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6q)
The reaction was carried out following the general procedure with compound 5c (0.80 g,
2.6 mmol), NaHCO3 (0.22 g, 2.6 mmol) and compound 3f (0.82 g, 2.6 mmol). Yield 57% (0.90
1
3
g); white solid; mp 122 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3Н, JHH = 6.9 Hz, СН3С14Н28),
3
0.89 (t, 3Н, JHH = 6.9 Hz, СН3(CН2)4СН), 1.20-1.27 (m, 20Н, 10СН2), 1.30-1.34 (m, 4Н,
2СН2), 1.44-1.56 (m, 4Н, 2СН2), 1.75-1.85 (m, 2Н, СН2), 2.12 (br s, 2H, CH2CH2CH2NH), 2.23
(t, 2Н, 3JHH = 7.6 Hz, СН2С(О)), 2.40 (s, 3Н, CH3Pyr), 3.18 (s, 3Н, CH3N+), 3.22 (s, 3Н, CH3N+),
2
3.31-3.34 (m, 2Н, СН2), 3.84-4.00 (m, 2Н, СН2), 4.82, 4.86 (AB, 2H, JHH = -14.0 Hz, CH2),
2
3
5.02, 5.30 (AB, 2H, JHH = -16.4 Hz, CH2), 5.08 (t, 1H, JHH = 5.2 Hz, СН3(CН2)4СН), 8.10 (t,
3
13
1H, JHH = 5.1 Hz, NH), 8.16 (s, 1Н, СНPyr). C NMR (CDCl3) δ: 14.08 (СН3), 14.23 (СН3),
18.92 (СН3Pyr), 22.61 (СН2), 22.78 (CH2CH2CH2NH), 23.22 (СН2), 25.90 (СН2), 29.47 (СН2),
29.57 (СН2), 29.66 (СН2), 29.73 (СН2), 29.76 (СН2) 29.81 (СН2), 31.61 (СН2), 32.01 (СН2),
34.26 (СН2), 36.27 (СН2), 36.46 (СН2), 49.21 (СН3N+), 49.57 (СН3N+), 62.84 (СН2), 63.15
(СН2), 65.24 (СН2), 100.68 (СН3(CН2)4СН), 117.14 (СPyr), 129.98 (СPyr), 144.65 (СPyr), 148.29
(СPyr), 150.94 (СPyr), 174.79 (С=О). ESI-HRMS m/z: 546.4635 [М-Cl]+ (calculated for
[C33H60N3O3]+ - 546.4629).
4.1.3.18. 5-(Methylene(N,N-dimethyl-N-(3-myristoylaminopropyl)ammonium))-2-octyl-8-methyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6r)
The reaction was carried out following the general procedure with compound 5d (0.56 g,
1.6 mmol), NaHCO3 (0.13 g, 1.6 mmol) and compound 3f (0.50 g, 1.6 mmol). Yield 76% (0.76
g); beige solid; mp 162-165 °C (dec). 1H NMR (CDCl3) δ: 0.85 (t, 3Н, 3JHH = 6.9 Hz, СН3), 0.86
(t, 3Н, 3JHH = 5.9 Hz, СН3), 1.20-1.34 (m, 30Н, 15СН2), 1.43-1.52 (m, 4Н, 2СН2), 1.73-1.87 (m,
3
2Н, СН2), 2.12 (br s, 2H, CH2CH2CH2NH), 2.23 (t, 2Н, JHH = 7.6 Hz, СН2С(О)), 2.40 (s, 3Н,
CH3Pyr), 3.18 (s, 3Н, CH3N+), 3.22 (s, 3Н, CH3N+), 3.31-3.34 (m, 2Н, СН2), 3.84-4.00 (m, 2Н,
2
СН2), 4.84 (c, 2H, CH2), 5.02, 5.30 (AB, 2H, JHH = -16.4 Hz, CH2), 5.08 (t, 1H, 3JHH = 5.1 Hz,
СН3(CН2)7СН), 8.09 (t, 1H, 3JHH = 5.4 Hz, NH), 8.16 (s, 1Н, СНPyr). 13C NMR (CDCl3) δ: 14.22
(СН3), 18.91 (СН3Pyr), 22.77 (СН2), 23.19 (CH2CH2CH2NH), 23.56 (СН2), 25.90 (СН2), 29.34
(СН2), 29.47 (СН2), 29.50 (СН2), 29.57 (СН2), 29.67 (СН2) 29.74 (СН2), 29.77 (СН2), 29.82
(СН2), 31.96 (СН2), 32.02 (СН2), 34.32 (СН2), 36.26 (СН2), 36.45 (СН2), 49.20 (СН3N+), 49.57

(СН3N+), 62.83 (СН2), 63.15 (СН2), 65.23 (СН2), 100.68 (СН3(CН2)7СН), 117.15 (СPyr), 129.97
(СPyr), 144.65 (СPyr), 148.29 (СPyr), 150.93 (СPyr), 174.79 (С=О). ESI-HRMS m/z: 588.5104 [М-
Cl]+ (calculated for [C36H66N3O3]+ - 588.5099).
4.1.3.19. 5-(Methylene(N,N-dimethyl-N-(3-hexanoylaminopropyl)ammonium))-2,2,8-trimethyl-
4H-[1,3]dioxino[4,5-c]pyridin chloride (6s)
The reaction was carried out following the general procedure with compound 5a (0.37 g,
1.4 mmol), NaHCO3 (0.12 g, 1.4 mmol) and compound 3i (0.28 g, 1.4 mmol). Yield 63% (0.38
g); white solid; mp 124-125 °C (dec). 1H NMR (CDCl3) δ: 0.79 (t, 3Н, 3JHH = 6.9 Hz, СН3С4Н8),
1.18-1.25 (m, 4H, 2CH2), 1.48 (s, 6Н, (СН3)2С), 1.50-1.56 (m, 2H, CH2), 2.09-2.17 (m, 2H,
3
CH2CH2CH2NH), 2.22 (t, 4Н, JHH = 6.8 Hz, СН2С(О)), 2.36 (s, 3Н, CH3Pyr), 3.20 (s, 6Н,
(СН3)2N+), 3.29-3.33 (m, 2Н, СН2), 3.95-3.98 (m, 2Н, СН2), 4.85 (s, 2Н, СН2), 5.06 (s, 2Н,
3
13
СН2), 8.16 (s, 1Н, СНPyr), 8.20 (t, 1Н, JHH = 5.3 Hz, NH). C NMR (CDCl3) δ: 14.06 (СН3),
18.96 (СH3Pyr), 22.52 (СH2), 23.10 (CH2СH2CH2NH), 24.90 (СH2), 25.51 (СH2), 31.55 (СH2),
36.31 (СH2), 49.28 (СH3N+), 59.31 (СH2), 62.47 (СH2), 63.23 (СH2), 100.51 ((СН3)2С), 116.87
(СPyr), 128.32 (СPyr), 144.11 (СPyr), 146.71 (СPyr), 151.24 (СPyr), 174.71 (С=О). ESI-HRMS m/z:
392.2913 [М-Cl]+ (calculated for [C22H38N3O3]+ - 392.2908).
4.1.3.20.
5-(Methylene(N,N-dimethyl-N-(3-hexanoylaminopropyl)ammonium))-2-propyl-8-
methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6t)
The reaction was carried out following the general procedure with compound 5b (0.39 g,
1.4 mmol), NaHCO3 (0.12 g, 1.4 mmol) and compound 3i (0.27 g, 1.4 mmol). Yield 40% (0.24
g); white solid; mp 99-100 °C (dec). 1H NMR (CDCl3) δ: 0.80 (t, 3Н, 3JHH = 6.7 Hz, СН3С4Н8),
0.96 (t, 3Н, 3JHH = 7.3 Hz, СН3СH2CH2СH), 1.16-1.25 (m, 4H, 2CH2), 1.44-1.54 (m, 2H, CH2),
1.72-1.80 (m, 2H, CH2CH2CH2CH), 2.11 (br s, 2H, CH2CH2CH2NH), 2.17 (t, 4Н, 3JHH = 7.6 Hz,
СН2С(О)), 2.36 (s, 3Н, CH3Pyr), 3.17 (s, 3Н, СН3N+), 3.22 (s, 3Н, СН3N+), 3.30-3.32 (m, 2Н,
2
СН2), 3.74-3.78 (m, 2Н, СН2), 4.76, 4.82 (AB, 2H, JHH = -14.1 Hz, CH2), 5.00, 5.24 (AB, 2H,
2JHH = -16.2 Hz, CH2), 5.09 (t, 3H, 3JHH = 4.9 Hz, CH), 8.08 (br s,1H, NH), 8.17 (s, 1H, CHPyr).
13C NMR (CDCl3) δ: 14.03 (СН3CH2CH2), 14.10 (СH3), 16.95 (СН3CH2CH2NH), 18.85
(СН3Pyr), 22.55 (СH2), 23.26 (СH2СH2СH2), 25.51 (СH2), 31.60 (СH2), 36.22 (СH2), 36.31
(CH2), 36.36 (CH2), 49.29 (CH3N+), 49.72 (CH3N+), 62.90 (CH2), 63.09 (CH2), 65.16 (CH2),
100.45 (CHCH2CH2CH3), 117.38 (CPyr), 130.05 (CPyr), 144.73 (CPyr), 148.24 (CPyr), 150.74
(CPyr), 174.82 (C=O). ESI-HRMS m/z: 406.3070 [М-Cl]+ (calculated for [C23H40N3O3]+ -
406.3064).
4.1.3.21. 5-(Methylene(N,N-dimethyl-N-(3-hexanoylaminopropyl)ammonium))-2-(undecan-2-yl)-
8-methyl-4H-[1,3]dioxino[4,5-c]pyridin chloride (6u)
The reaction was carried out following the general procedure with compound 5e (0.35 g,
0.9 mmol), NaHCO3 (0.08 g, 0.9 mmol) and compound 3i (0.18 g, 0.9 mmol). Yield 40% (0.2 g);
1
white solid; mp 137-140 °C (dec). H NMR (CDCl3) δ (mixture of diastereomers): 0.83 (t, 3Н,
3JHH = 7.4 Hz, СН3), 0.86 (t, 3Н, 3JHH = 6.7 Hz, СН3), 0.96 (t, 3Н, 3JHH = 7.3 Hz, СН3), 0.98-1.01
(m., 3Н, СН3(СH2)8CH(CH3)), 1.21-1.32 (m, 17H, 9CH2), 1.35-1.44 (m, 2H, CH2), 1.51-1.62
(m, 4H, 2CH2), 1.87 (br s, 1H, СН3(СH2)8CH(CH3)), 2.09-2.19 (m, 2H, CH2CH2CH2NH), 2.25
(t, 4Н, 3JHH = 7.6 Hz, СН2С(О)), 2.41 (s, 3Н, CH3Pyr), 3.18 (s, 3Н, СН3N+), 3.22 (s, 3Н, СН3N+),
2
3.31-3.36 (m, 2Н, СН2), 3.87-4.04 (m, 2Н, СН2), 4.84, 4.87 (AB, 2H, JHH = -14.2 Hz, CH2),
4.93 (t, 3H, 3JHH = 4.9 Hz, CH), 5.03, 5.35 (AB, 2H, 2JHH = 16.2 Hz, CH2), 8.15 (br s, 1H, NH),
13
8.18 (s, 1H, CHPyr). C NMR (CDCl3) δ (mixture of diastereomers): 13.49 (СН3), 13.70 (СН3),
14.11 (СН3), 14.23 (СH3), 18.85 (СН3Pyr), 18.90 (СН3Pyr), 22.57 (СH2), 22.79 (СH2), 23.20
(СH2), 25.55 (СH2), 27.00 (СH2), 27.08 (СH2), 29.45 (СH2), 29.74 (СH2), 29.94 (СH2), 30.92
(СH2), 31.04 (СH2), 31.61 (СH2), 32.02 (СH2), 36.25 (СH2), 36.40 (СH2), 37.34 (СH2), 49.24
(CH3N+), 49.58 (CH3N+), 62.91 (CH2), 63.31 (CH2), 65.34 (CH2), 103.24
(СН3(СH2)8CH(CH3)СH), 103.39 (СН3(СH2)8CH(CH3)), 117.14 (CPyr), 130.03 (CPyr), 144.59

(CPyr), 148.49 (CPyr), 150.99 (CPyr), 174.85 (C=O). ESI-HRMS m/z: 518.4323 [М-Cl]+
(calculated for [C31H56N3O3]+ - 518.4316).
4.1.3.22.
5-(Methylene(N,N-dimethyl-N-(2-lauroyloxyethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (9a)
The reaction was carried out following the general procedure with compound 5a (0.40 g,
1.5 mmol), NaHCO3 (0.13 g, 1.5 mmol) and compound 8a (0.42 g, 1.5 mmol). Yield 47% (0.36
g); colorless oil. 1H NMR (CDCl3) δ: 0.85 (t, 3Н, 3JHH = 6.9 Hz, СН3С10Н20), 1.18-1.28 (m, 16H,
3
8CH2), 1.50 (s, 6Н, (СН3)2С), 1.52-1.60 (m, 2H, CH2), 2.30 (t, 4Н, JHH = 6.5 Hz, СН2С(О)),
2.40 (s, 3Н, CH3Pyr), 3.39 (s, 6Н, (СН3)2N+), 4.29 (br s, 2Н, СН2), 4.61 (br s, 2Н, СН2), 5.16 (s,
4Н, 2СН2), 8.21 (s, 1Н, СНPyr). 13C NMR (CDCl3) δ: 14.20 (СН3), 18.92 (СH3Pyr), 22.76 (СH2),
24.73 ((СН3)2С), 25.00 (СH2), 29.19 (СH2), 29.33 (СH2), 29.41 (СH2), 29.53 (СH2), 29.68
(СH2), 31.98 (СH2), 34.12 (СH2), 50.12 (СH3N+), 57.76 (СH2), 59.76 (СH2), 62.72 (СH2), 63.48
(СH2), 100.62 ((СН3)2С), 117.10 (СPyr), 129.02 (СPyr), 144.32 (СPyr), 146.89 (СPyr), 151.12
(СPyr), 172.89 (С=О). ESI-HRMS m/z: 463.3538 [М-Cl]+ (calculated for [C27H47N2O4]+ -
463.3530).
4.1.3.23. 5-(Methylene(N,N-dimethyl-N-(2-myristoyloxyethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (9b)
The reaction was carried out following the general procedure with compound 5a (0.34 g,
1.3 mmol) NaHCO3 (0.11 g, 1.3 mmol) and compound 8c (0.38 g, 1.3 mmol). Yield 49% (0.33
1
3
g); beige solid; mp 126-129 °C (dec). H NMR (CDCl3) δ: 0.85 (t, 3Н, JHH = 6.4 Hz,
СН3С12Н24), 1.22-1.28 (m, 20Н, 10СН2), 1.50 (s, 6Н, (СН3)2С), 1.52-1.56 (m, 2H, CH2), 2.30 (t,
2Н, 3JHH = 7.6 Hz, СН2С(О)), 2.41 (s, 3Н, CH3Pyr), 3.39 (s, 6Н, (СН3)2N+), 4.28 (br s, 2Н, СН2),
4.61 (br s, 2Н, СН2), 5.17 (s, 4Н, 2СН2), 8.23 (s, 1Н, СНPyr). 13C NMR (CDCl3) δ: 14.22 (СН3),
19.00 (СH3Pyr), 22.77 (СН2), 24.72 ((СH3)2С), 24.97 (СН2), 29.19 (СН2), 29.34 (СН2), 29.44
(СН2), 29.55 (СН2), 29.69 (СН2), 29.73 (СН2), 29.76 (СН2), 32.00 (СН2), 34.10 (СН2), 50.07
(СН3N+), 57.75 (СН2), 59.67 (СН2), 62.67 (СН2), 63.45 (СН2), 100.54 ((СН3)2С), 117.00 (СPyr),
128.80 (СPyr), 144.43 (СPyr), 146.80 (СPyr), 151.18 (СPyr), 172.90 (С=О). ESI-HRMS m/z:
491.3849 [М-Cl]+ (calculated for [C29H51N2O4]+ - 491.3843).
4.1.3.24. 5-(Methylene(N,N-dimethyl-N-(2-palmitoyloxyethyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (9c)
The reaction was carried out following the general procedure with compound 5a (0.32 g,
1.2 mmol), NaHCO3 (0.10 g, 1.4 mmol) and compound 8e (0.40 g, 1.2 mmol). Yield 41% (0.28
1
3
g); white solid; mp 125-128 °C (dec). H NMR (CDCl3) δ: 0.87 (t, 3Н, JHH = 6.6 Hz,
СН3С14Н28), 1.24-1.29 (m, 24Н, 12CH2), 1.56 (s, 6Н, (СН3)2С), 1.56-1.60 (m, 2Н, СН2), 2.33 (t,
2Н, 3JHH = 7.3 Hz, СН2С(О)), 2.52 (s, 3Н, СH3Pyr), 3.42 (s, 6Н, (СН3)2N+), 4.32 (br s, 2Н, СН2),
4.63 (br s, 2Н, СН2), 5.27 (s, 2Н, СН2), 5.28 (s, 2Н, СН2), 8.39 (s, 1Н, СНPyr). 13C NMR
(CDCl3) δ: 14.27 (СН3), 17.70 (СH3Рyr), 22.83 (СН2), 24.79 ((СH3)2С), 25.10 (СН2), 29.26
(СН2), 29.40 (СН2), 29.50 (СН2), 29.62 (СН2), 29.76 (СН2), 29.80 (СН2), 29.84 (СН2), 32.06
(СН2), 34.19 (СН2), 50.38 (СН3N+), 57.77 (СН2), 59.96 (СН2), 63.11 (СН2), 63.17 (СН2),
101.47 ((СН3)2С), 118.55 (СPyr), 131.88 (СPyr), 141.74 (СPyr), 147.90 (СPyr), 149.68 (СPyr),
172.98 (С=О). ESI-HRMS m/z: 519.4162 [М-Cl]+ (calculated for [C31H55N2O4]+ - 519.4156).
4.1.3.25. 5-(Methylene(N,N-dimethyl-N-(3-lauroyloxypropyl)ammonium))-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridin chloride (9d)
The reaction was carried out following the general procedure with compound 5a (0.42 g,
1.6 mmol), NaHCO3 (0.13 g, 1.6 mmol) and compound 8b (0.46 g, 1.6 mmol). Yield 46% (0.38
1
3
g); beige solid; mp 159-160 °C (dec). H NMR (DMSO-d6) δ: 0.85 (t, 3Н, JHH = 6.7 Hz,
СН3С10Н20), 1.23-1.28 (m, 16H, 8CH2), 1.50-1.56 (m, 2H, CH2), 1.58 (s, 6Н, (СН3)2С), 2.10-
2.17 (m, 2H, CH2CH2CH2O), 2.32 (t, 4Н, 3JHH = 7.5 Hz, СН2С(О)), 2.55 (s, 3Н, CH3Pyr), 3.09 (s,