Synthesis, antibacterial and antitumor activity of methylpyridinium salts of pyridoxine functionalized 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles

Article abstract of DOI:10.1080/00397911.2018.1501487

A library of 29 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles functionalized with a pyridoxine moiety was synthesized using a three-component one-pot reaction of aldehyde derivative of pyridoxine, malononitrile, and thiophenol. The obtained bipyridine structures were converted into methylpyridinium salts. Several compounds demonstrated expressed antibacterial activity with MICs (minimum inhibitory concentrations) in the range of 0.5–4 μg/mL against the three studied Gram-positive strains and 8–64 μg/mL against the Gram-negative E. coli strain, which was comparable or better than the activity of the reference antimicrobial agents. At the same time, all the synthesized compounds were inactive against the Gram-negative P. aeruginosa. Several compounds also demonstrated high cytotoxic activity against the studied tumor cells, but without selectivity for the normal HSF (human foreskin fibroblast) cells. Despite the preliminary character of the performed biological studies, the obtained results make the obtained structural chemotype a promising starting point for the design of physiologically active compounds.

Full text of DOI:10.1080/00397911.2018.1501487

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis, antibacterial and antitumor activity
of methylpyridinium salts of pyridoxine
functionalized 2-amino-6-sulfanylpyridine-3,5-
dicarbonitriles
Arthur A. Grigor’ev, Nikita V. Shtyrlin, Raylya R. Gabbasova, Marina I. Zeldi,
Denis Yu. Grishaev, Oleg I. Gnezdilov, Konstantin V. Balakin, Oleg E. Nasakin
& Yurii G. Shtyrlin
To cite this article: Arthur A. Grigor’ev, Nikita V. Shtyrlin, Raylya R. Gabbasova, Marina I. Zeldi,
Denis Yu. Grishaev, Oleg I. Gnezdilov, Konstantin V. Balakin, Oleg E. Nasakin & Yurii G. Shtyrlin
(2018): Synthesis, antibacterial and antitumor activity of methylpyridinium salts of pyridoxine
functionalized 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles, Synthetic Communications, DOI:
10.1080/00397911.2018.1501487
To link to this article: https://doi.org/10.1080/00397911.2018.1501487
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https://doi.org/10.1080/00397911.2018.1501487
Synthesis, antibacterial and antitumor activity of
methylpyridinium salts of pyridoxine functionalized
2-amino-6-sulfanylpyridine-3,5-dicarbonitriles
Arthur A. Grigor’eva, Nikita V. Shtyrlinb, Raylya R. Gabbasovab, Marina I. Zeldib,
Denis Yu. Grishaevb, Oleg I. Gnezdilovc, Konstantin V. Balakinb,d
Oleg E. Nasakina, and Yurii G. Shtyrlinb
,
b
aI. N. Ul’yanov Chuvash State University, Cheboksary, Russia; Scientific and Educational Center of
c
Pharmaceutics, Kazan (Volga region) Federal University, Kazan, Russia; Kazan Institute of Biochemistry
d
and Biophysics, FRC Kazan Scientific Center of RAS, Kazan, Russia; I.M. Sechenov First Moscow State
Medical University, Moscow, Russia
ABSTRACT
ARTICLE HISTORY
Received 27 April 2018
A library of 29 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles function-
alized with a pyridoxine moiety was synthesized using a three-
component one-pot reaction of aldehyde derivative of pyridoxine,
malononitrile, and thiophenol. The obtained bipyridine structures
were converted into methylpyridinium salts. Several compounds
demonstrated expressed antibacterial activity with MICs (minimum
inhibitory concentrations) in the range of 0.5–4 mg/mL against the
three studied Gram-positive strains and 8–64 mg/mL against the
Gram-negative E. coli strain, which was comparable or better than
the activity of the reference antimicrobial agents. At the same time,
all the synthesized compounds were inactive against the Gram-
negative P. aeruginosa. Several compounds also demonstrated
high cytotoxic activity against the studied tumor cells, but without
selectivity for the normal HSF (human foreskin fibroblast) cells. Despite
the preliminary character of the performed biological studies, the
obtained results make the obtained structural chemotype a promising
starting point for the design of physiologically active compounds.
KEYWORDS
2-Amino-6-sulfanylpyridine-
3,5-dicarbonitriles;
antibacterial activity;
antitumor activity;
pyridoxine; quaternary
ammonium salts
GRAPHICAL ABSTRACT
Introduction
Variously substituted 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles (general structure I,
Fig. 1) represent an interesting structural chemotype with promising biological activities.
CONTACT Yurii G. Shtyrlin
yurii.shtyrlin@gmail.com
Scientific and Educational Center of Pharmaceutics, Kazan
(Volga region) Federal University, Kremlyovskaya St. 18, Kazan 420008, Russia.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2018 Taylor & Francis

2
A. A. GRIGOR’EV ET AL.
They have been described as potent antimicrobial[1] and antitumor[2] agents, inhibitors
of tyrosine kinases,[3] modulators of androgen receptor function,[4] inhibitors of DNA
methyltransferase 1[5], agents for the treatment of prion diseases,[6] and agonists of
adenosine receptors.[7,8] One of the most known agents is Capadenoson (II, Fig. 1), a
highly potent selective partial adenosine A1 receptor agonist (EC50 ¼ 0.1 nM), which has
completed a phase-II clinical trial at Bayer for use in patients with atrial fibrillation.[8a]
Its close structural analog, Neladenoson bialanate, an oral partial A1 receptor agonist,
is in phase-II clinical development for the treatment of patients with chronic
heart failure.[8b]
The structural diversity of the 2-amino-6-sulfanylpyridine-3,5-dicarbonitrile pharma-
cophore can be achieved by chemical modification of positions 2, 4 and 6 of the studied
molecular scaffold I. Of particular interest is position 4 which can be occupied by vari-
ous R3-substituents for modification of biological activity profile. The most actively
investigated type of R3-substituents are variously substituted phenyl rings (for example,
Capadenoson II). At the same time, different aryl, heteroaryl and alkyl substituted
derivatives can also possess interesting biological activities. Thus, 4-(2-naphtyl)-
substituted compounds have been reported as potent antitumor agents.[2] 4-(2-Furyl)-
and 4-(2-thienyl)-substituted derivatives demonstrate expressed activity against
replication of the infectious prion isoform (PrPSc).[6b,6c] Bioisosteric 4-(3-pyridyl)-[7d]
and 4-(1H-pyrazol-3-yl)-[7e] substituted analogs of Capadenoson have been described as
highly potent adenosine A1 agonists selective over A2A and A2B receptors. 4-Ethyl-
substituted agents are potent inhibitors of DNA methyltransferase 1, potential antitumor
agents.[5] To complete the picture, 4-unsubstituted compounds (R3 ¼ H) are able
to selectively suppress recombinant human Plk-1 tyrosine kinase activity and display
antiproliferative activity against non-small lung cancer A549 cells.[9]
These promising pharmacological results encouraged us to develop novel synthetic
routes to various derivatives of 2-amino-6-sulfanylpyridine-3,5-dicarbonitrile, including
their heterocyclic analogs.[10] As a continuation of this work, in the present paper, we
report a novel structural chemotype III (Fig. 1) in which the substituent at position 4
of the pyridine-3,5-dicarbonitrile scaffold represents a methylpyridinium derivative
of pyridoxine.
Figure 1. General formula of 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles (I); example of a selective
partial adenosine A1 receptor agonist Capadenoson which has completed a phase II clinical trial (II);
compounds synthesized in this work (III).

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From the “pyridoxine-centric” point of view, the obtained compounds III represent
5-heteroaryl substituted derivatives of pyridoxine (vitamin B6), a key cofactor of many
important enzymes. In our group, we have systematically studied chemistry and biology
of the physiologically active pyridoxine derivatives. In particular, we have described a
wide series of 5-substituted pyridoxine derivatives, including 5-alkenyl-substituted com-
pounds[11], as well as ammonium and phosphonium salts[12], which possess promising
pharmacological potential as antibacterial and antitumor drugs. We have also reported
N-methylpyridoxinium salts with potent anticholinesterase activity.[13]
The mentioned examples demonstrate the excellent potential of 2-amino-6-
sulfanylpyridine-3,5-dicarbonitriles and 5-substituted pyridoxines (including their
ammonium salts) in the design of novel pharmacological agents. Molecules III synthe-
sized in this work contain both pharmacophore motifs that make the designed hybrid
structures a promising object for biological and pharmacological studies. The obtained
compounds have been tested in vitro for their ability to inhibit growth of Gram-positive
and Gram-negative bacterial pathogens, as well as tumor cells.
Results and discussion
Chemistry
A good synthetic accessibility of various 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles
favors to their active synthetic and biological studies. The target compounds can be
obtained using a three-component one-pot reaction of aldehyde, malononitrile, and
thiol actively studied by Evdokimov et al.[14] The reaction is typically catalyzed by
bases[15], but examples of successful use of Lewis acids[16], boronic acid[17], ionic
liquids[18], nanoparticles of various composition[19] and organometallic compounds[20]
as catalysts have been reported. The reaction can also be promoted by microwaves[21]
and ultrasound.[22]
As the key aldehyde reagents for the multicomponent reaction, we used aldehyde
derivatives of pyridoxine with various substituents at the acetal carbon atom, that were
prepared from pyridoxine hydrochloride 1 in two synthetic stages (Scheme 1). At the
first step, the cyclic ketal 2a and acetals 2 b and 2d were obtained according to a
literature procedure by reaction of 1 with the corresponding carbonyl reagents in the
presence of acid.[12a,23] Acetal 2c was obtained by reaction of 1 with n-hexanal in the
presence of para-toluenesulphonic acid under reflux. The variation of substituents at
the acetal carbon atom makes it possible to vary compound’s lipophilicity and thus
investigate the lipophilicity-activity relationships. Compounds 2a-d were then treated
with MnO2 in chloroform to obtain aldehydes 3a-d in good yields (72–86%).
Scheme 1. Synthesis of aldehydes 3a-d.

4
A. A. GRIGOR’EV ET AL.
At the next stage, aldehydes 3a-d were introduced into a multicomponent reaction
with 2 molar equivalents of malononitrile and 1 molar equivalent of three commercially
available thiophenols (thiophenol, p-thiocresol, 4-chlorothiophenol). It should be noted
that in this type of multicomponent reactions, only aromatic thiols can be used. The
use of triethylamine, in accordance with the method developed by Evdokimov et al.[14]
,
led to the target compounds in low yields (5-10%). The use of 10% (mol.) KOH
reported by Khan et al.[24] was more successful and led to the target pyridine-3,5-
dicarbonitriles 4a-m in 25–40% yield (Scheme 2). The reaction also resulted in a
number of side products, which were problematic to separate and identify. All further
attempts to increase the reaction yields by varying the experimental conditions (time,
temperature, base nature) were unsuccessful.
Compounds 4a-m appeared to be completely insoluble in water, and therefore it was
problematic to perform their biological studies in aqueous media. In an attempt to
improve solubility, ketals 4a and 4j with different R3-substituents were treated with
aqueous HCl to deprotect the hydroxyl groups of the pyridoxine part (Scheme 3). The
resulting diols 5a,b were obtained in 85% and 77% yields, respectively, as stable
crystalline substances. Unfortunately, the deprotection did not significantly improve the
solubility in water, and compounds 5a,b was still problematic to evaluate in biological
tests. In view of very poor solubility of compounds 5a,b with R3 ¼ H and Cl, we did
not synthesize the third possible diol with a more lipophilic R3-substituent (R3 ¼ CH3).
It is widely known that many quaternary ammonium compounds (QACs) possess
expressed antibacterial properties.[25] In our previous works, we have reported several
chemotypes of pyridoxine-based QACs with potent antimicrobial activity.[12] To
increase solubility and enhance the antimicrobial potential of the obtained structures,
we converted compounds 4a-m into the corresponding pyridinium salts (Scheme 4).
The reaction of 4a-m with methyl bromide led to selective methylation of the pyridox-
ine nitrogen atom to give compounds 6a-m in 50–70% yields. The chemoselective
1
character of this reaction can be unequivocally confirmed by H NMR spectral data
which demonstrate an expressed downfield shift of signals from protons belonging to 2-
and 6-substituents of the pyridoxine ring adjacent to the endocyclic N1 atom. Thus, as
a result of the conversion of compounds 4a-m to 6a-m, we observed a shift from
Scheme 2. Synthesis of compounds 4a-m.

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5
Scheme 3. Synthesis of compounds 5a,b with deprotected hydroxyl groups.
Scheme 4. Synthesis of methylpyridinium salts 6a-m.
2.40–2.42 ppm to 2.68–2.71 ppm for the three protons of 2-methyl group, and a shift
from 7.96–8.04 ppm to 8.96–9.02 ppm for the aromatic proton at position 6. Similar
observations were reported in our recent work in which we obtained N-methylated
pyridoxine derivatives.[13] At the same time, we did not observe any changes in
13C NMR signals from carbon atoms of the pyridine-3,5-dicarbonitrile ring, and this
fact indicated that the endocyclic nitrogen atom of this fragment remained intact during
the methylation reaction. Interestingly, even a prolonged treatment with an excess
of ethyl bromide or decyl bromide under reflux did not result in the corresponding
pyridinium slats, probably, due to steric hindrance.
At the final synthetic step, three compounds 6a, 6e and 6j with different R3-
substituents were treated with aqueous HBr to deprotect the hydroxyl groups of the
pyridoxine part (Scheme 5). The resulting diols 7a-c were obtained in good yields
(70–75%) as stable crystalline substances.
The developed synthetic route to compounds 4-7 is straightforward and well
reproducible. The obtained pyridinium salts 6a-m and 7a-c were soluble enough in the
water that allowed us to evaluate their antibacterial and antitumor activity.

6
A. A. GRIGOR’EV ET AL.
Scheme 5. Synthesis of compounds 7a–c with deprotected hydroxyl groups.
Table 1. In vitro antimicrobial activity of compounds 6a-m and 7a-c.
MICs (mg/mL)
Gram-positive bacteria
Gram-negative bacteria
S. aureus
S. epidermidis
E. coli
P. aeruginosa
Compounds
ATCC 29213
(clinical isolate) B. subtilis 168
???? 25922
ATCC 27853
AlogPS
6a
6b
6c
6d
6e
6f
6g
6h
6j
6k
6l
6m
7a
7b
7c
>64
64
4
>64
32
4
>64
64
4
>64
>64
32
>64
>64
64
>64
8
>64
64
32
32
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
0.34 1.5
0.35 1.5
0.98 1.5
1.18 1.5
0.51 1.5
0.53 1.5
1.2 1.5
1.6 1.5
0.69 1.5
0.7 1.5
1.4 1.5
1.6 1.5
ꢀ0.32 1.5
ꢀ0.19 1.5
ꢀ0.02 1.5
4.5 1.5
–
2
1
1
>64
32
4
>64
16
4
0.5
>64
32
2
2
>64
>64
>64
2
>64
16
4
2
1
>64
32
4
>64
32
4
2
2
>64
>64
>64
4
>64
>64
>64
2
Miramistin
Benzalkonium
chloride
2
2
32
4
64
In vitro antibacterial activity
The antibacterial activity of 15 new synthesized compounds was evaluated against three
Gram-positive and two Gram-negative bacterial strains. Table 1 shows the MIC values
of the tested compounds 6a-m and 7a-c. The lipophilicity of the synthesized
ammonium salts was expressed in terms of their partition coefficient values (logP)
calculated using AlogPS algorithm.[26]
As reference drugs, two widely used antiseptic quaternary ammonium compounds,
miramistin and benzalkonium chloride, were used. Due to broad-spectrum efficacy,
substantivity for the skin, and low irritation, benzalkonium chloride belongs to the
most widely used biocides in antiseptic products, in particular in handwashing and oral
products but also as disinfectants and preservatives.[27] Miramistin, which has been
developed in the 1980s in the former USSR as an antiseptic for the treatment of skin of
cosmonauts at orbital stations[28], is one of the most popular antimicrobial agents on

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7
Figure 2. Log10MIC (S. aureus) versus calculated AlogPS relationship for compounds 6a-m. A
polynomial trend line is shown. For three inactive compounds 6a, 6e and 6j (MICs > 64 mg/mL), MICs
were accepted equal to 128 mg/mL.
the pharmaceutical market of the Russian Federation. Five bacterial test organisms, S.
aureus, S. epidermidis, B, subtilis, E. coli and P. aeruginosa were selected as the most
common Gram-positive and Gram-negative hospital pathogens.[27]
At least five compounds demonstrated expressed antibacterial activity with MICs
in the range of 0.5–4 mg/mL against the three studied Gram-positive strains and
8–64 mg/mL against the Gram-negative E. coli strain, which was comparable or better
than the activity of the reference agents. However, all the obtained compounds were
inactive against the Gram-negative P. aeruginosa. The decreased activity of against
Gram-negative pathogens is in agreement with literature data on cationic disinfectants
(for example,[25]).
The antibacterial activity was positively correlated with the length of the substituents
at the acetal carbon atom (octyl > pentyl > ethyl), reaching a maximum for octyl
substituent (MIC 0.5–4 mg/mL for compounds 6d, 6h, and 6m). In agreement with this
observation, a strong correlation between the lipophilicity and antimicrobial activity of
quaternary ammonium pyridoxine derivatives was found. Figure 2 demonstrates the
relationships between the experimental MICs (expressed in a decimal logarithm scale)
for S. aureus and calculated AlogPS values for the studied quaternary ammonium pyri-
doxine derivatives 6a-m. Similar relationships were found for the other studied bacterial
strains. It can be suggested that the found relationships reflect the important features of
the active compounds essential for their effective interaction with the hydrophobic
membrane core of bacterial cells.
Compounds 7a-c with the deprotected hydroxyl groups were inactive in this
experiment. This result might be explained by their hydrophilic nature (AlogPS in the
range of ꢀ0.02 ꢁ ꢀ0.32).
In vitro cytotoxicity
The synthesized compounds were then studied for antitumor activity and cytotoxicity
in vitro (Table 2). The study of antitumor activity was carried out on breast

8
A. A. GRIGOR’EV ET AL.
Table 2. Antitumor activity and cytotoxicity in vitro of
compounds 6a-m and 7a-c.
IC50 (mM)
Compounds
MCF-7
SNB-19
HCT-116
HSF
6a
6b
6c
6d
141.5
27.7
6.9
106.4
35.5
13.9
5.1
91.3
8.3
8
272.4
11.2
7
2.8
2.8
2.8
6e
6f
6g
6h
6j
6k
6l
6m
99.2
17.0
3.8
14.4
138.8
8.4
5.6
2.3
119.4
130.2
15.7
0.2
99.7
22.2
13.2
9.4
101.5
13.8
13.6
10.3
96.2
91.8
37.6
0.1
66.1
4.1
6.6
6.1
161.4
8.1
4.2
3.4
121.6
94.4
15.7
0.1
264.1
14.6
3.4
15.7
241.9
7.5
4.4
3.6
266
268.1
16.2
0.9
7a
7b
7c
Doxorubicin
adenocarcinoma MCF-7, glioblastoma SNB-19, and human colon colorectal carcinoma
HCT-116 cells. Cytotoxicity was studied on primary human foreskin fibroblast (HSF)
cells. Doxorubicin, one of the most widely used cytostatic agents, was used as a
reference drug.
Several compounds (for example, 6d, 6 g, 6m) demonstrated a relatively high
antitumor activity against the studied tumor cells in a low-micromolar concentration
range. However, their activity was at least one order of magnitude lower than that
of Doxorubicin. In addition, their cytotoxicity against the normal HSF cells was
comparable to antitumor activity thus suggesting poor selectivity of their action and
high in vivo toxicity.
In general, the obtained results demonstrate the interesting pharmacological potential
of the described novel chemotype. Several compounds can be considered as antimicro-
bial agents active against Gram-positive bacterial pathogens.
Conclusions
In conclusion, we have synthesized a diverse library of 29 new pyridoxine functionalized
2-amino-6-sulfanylpyridine-3,5-dicarbonitriles and studied their antibacterial activity
and cytotoxicity in vitro. The synthetic route is based on a three-component one-pot
reaction of the corresponding aldehyde, malononitrile and thiol reactants, that leads to
the target bipyridine scaffold in moderate-to-good yields. To increase solubility and to
enhance the antimicrobial potential of the obtained structures, they were regioselectively
converted into methylpyridinium salts. Mild acidic hydrolysis led to the corresponding
compounds with deprotected hydroxyl groups. To the best of our knowledge, the
obtained structures represent a first reported example of 5-aryl-substituted pyridoxine
derivatives. In the primary biological experiments, several compounds demonstrated a
potent antibacterial activity, which was more expressed with respect to the studied
Gram-positive strains compared to Gram-negative pathogens. A strong correlation
between the lipophilicity and antimicrobial activity of the obtained compounds was

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9
observed. Several compounds also demonstrated high antitumor activity against the
studied tumor cells, though without any selectivity against the normal HSF cells.
Despite the preliminary character of the performed biological studies, the described
active compounds can be considered as a starting point for the development of new
antibacterial therapies. In a more general sense, the presence of two strong pharmaco-
phore motifs in the designed hybrid structures makes them a highly interesting object
for further biological and pharmacological studies. From one hand, they belong to
4-heteroaryl substituted analogs of 2-amino-6-sulfanylpyridine-3,5-dicarbonitriles that
have been reported as potent antitumor agents,[2] inhibitors of replication of the
infectious prion isoform (PrPSc),[6b,6c] and adenosine A1 agonists.[7d,7e] From the other
hand, they represent 5-substituted derivatives of pyridoxine that possess promising
pharmacological potential as antibacterial and antitumor drugs.[11,12]
Experimental
All reagents were obtained from commercial sources and were used without further
purification unless otherwise stated.
Acetals and ketals 2a, 2 b and 2d were synthesized according to literature procedur-
es.[12a,23] Compound 3a was synthesized according to a literature procedure.[29] 1H and
13C NMR spectra were recorded on a “Bruker AVANCE 400” instrument at operating
frequencies 400.17 and 100.62 MHz, respectively. Chemical shifts were measured with
reference to the residual protons of the solvent (DMSO-d6, 1H, 2.50 ppm, 13C,
1
39.52 ppm; CDCl3, H, 7.26 ppm, 13C, 77.16 ppm). Coupling constants (J) are given in
Hertz (Hz). The following abbreviations are used to describe coupling: s - singlet; d -
doublet; t - triplet; m - multiplet; q - quartet, br s - broad singlet, AB - AB system.
Melting points were determined using a Stanford Research Systems MPA-100 OptiMelt
melting point apparatus and are uncorrected. For TLC analysis, silica gel plates from
Sorbfil (Krasnodar, Russia) were used with UV light (t, 254 nm/365 nm) or iron (III)
chloride as developing agent. Column chromatography was performed on silica gel
(60–200 mesh) from Acros. All reactions were carried out in flasks.
High-resolution mass spectroscopy mass spectra were obtained on a quadrupole
time-offlight (t, qTOF) AB Sciex Triple TOF 5600 mass spectrometer using turbo-ion
spray source (nebulizer gas nitrogen, a positive ionization polarity, needle voltage
5500 V). Recording of the spectra was performed in “TOF MS” mode with collision
energy 10 eV, declustering potential 100 eV and with resolution more than 30,000 full-
width half-maximum. Samples with the analyte concentration 5 lmol/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.
Synthesis of 5-hydroxymethyl-8-methyl-2-pentyl-4H-[1,
3]dioxino[4,5-c]pyridine (2c)
n-Hexanal (0.78 ml, 9.6 mmol) and p-toluenesulfonic acid monohydrate (0.93 g,
4.9 mmol) were added to a suspension of compound 1 (1.00 g, 4.9 mmol) in 120 mL of
benzene. The reaction mixture was refluxed with a Dean-Stark trap for 7 h. Then the

10
A. A. GRIGOR’EV ET AL.
solvent was evaporated under reduced pressure, and the residue was neutralized with
aqueous solution of NaHCO3. The precipitate was collected by filtration, washed with
1
benzene and dried. Yield 75% (0.91 g), white solid, mp 60-64 ꢂC. H NMR (CDCl3) d:
3
0.88 (t, 3 H, JHH ¼ 7.2 Hz, CH3CH2CH2CH2CH2), 1.31–1.34 (m, 4 H, CH3CH2CH2
CH2CH2), 1.47–1.51 (m, 2 H, CH3CH2CH2CH2CH2), 1.81–1.85 (m, 2 H, CH3CH2CH2
CH2CH2), 2.35 (s, 3 H, CH3), 4.47 (s, 2 H, CH2OH), 4.95 (s, 2 H, CH2O), 4.96 (t, 1 H,
3JHH ¼ 5.1 Hz, CHC5H11), 7.82 (s, 1 H, CHPyr.). 13 C NMR (CDCl3) d: 14.04, 17.89,
22.59, 23.29, 31.59, 34.28, 59.82, 64.25, 100.15, 128.08, 130.39, 138.67, 146.85, 148.00.
ESI-HRMS m/z: 252.1589 [MþH]þ (calculated for [C14H22NO3]þ - 252.1594).
General procedure for the synthesis of compounds 3b-d
Activated MnO2 (6.52 g, 75.0 mmol) was added to a solution of the corresponding
pyridoxine acetal 2b-d (5.00 mmol) in 100 ml of dichloromethane, and the reaction
mixture was stirred at 20ꢂC for 1 h. The formed precipitate was filtered off, and the
solvent was evaporated in vacuo. The product was purified by column chromatography
on silica gel (eluent ethyl acetate). Analytical characteristics of compounds 3c,d are
described in the Electronic Supplementary Material.
2 -Ethyl-8-methyl-4H-[1, 3]dioxino[4,5-c]pyridine-5-carbaldehyde (3b)
The general procedure was followed using compound 2 b (1.05 g, 5.0 mmol). Yield 72%
1
(0.75 g), yellow oil. H NMR (CDCl3) d: 1.07 (t, 3H,3JHH ¼ 7.5 Hz, CHCH2CH3), 1.85 –
3
1.94 (m, 2 H, CHCH2CH3), 2.51 (s, 3H, CH3), 4.95 (t, 1H, JHH ¼ 5.1 Hz, CHCH2CH3),
2
5.14, 5.28 (AB, 2 H, JHH ¼ 18.0 Hz, CH2O), 8.46 (s, 1H, CH), 10.00 (s, 1H, C(O)H).
13 C NMR (CDCl3) d: 7.86, 19.52, 27.53, 65.87, 100.79, 126.41, 128.53, 147.08, 148.45,
154.10, 191.93. HRMS-ESI: 208.0968 [MþH]þ (calculated for [C11H14NO3]þ -208.0968).
General procedure for the synthesis of compounds 4a-m
A mixture of the corresponding aldehyde 3a-d (1 mol. equiv.), malononitrile (2 mol.
equiv.), KOH (0.1 mol. equiv.), thiol (1 mol. equiv.) and ethanol (10 ml) was placed into
a 25 mL round bottom flask fitted with reflux condenser. The reaction mixture was
refluxed for approximately 1 h until a clear solution was obtained. Then, the reaction
mixture was slowly cooled to 2 ꢂC. The solid product was collected by filtration, washed
with ethanol and dried. Analytical characteristics of compounds 4c-m are described in
the Electronic Supplementary Material.
2 -Amino-6-(phenylthio)-4-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-
yl)pyridine-3,5-dicarbonitrile (4a)
The general procedure was followed using compound 3a (638 mg, 3.0 mmol), malononi-
trile (407 mg, 6.0 mmol), KOH (17 mg, 0.3 mmol) and benzenethiol (339 mg, 3.0 mmol).
Yield 30% (400 mg), yellow solid, mp 126-128 ꢂC (decomp.). 1H NMR (DMSO-d6)
d: 1.55 (s, 6 H, 2CH3), 2.40 (s, 3 H, CH3Pyr), 4,72 (s, 2 H, CH2O), 7.50–7,62 (m, 5 H,

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SYNTHETIC COMMUNICATIONSV
11
C6H5Ar), 7.97 (br.s, 2 H, NH2), 8.03 (s, 1 H, CHpyr). 13 C NMR (DMSO-d6) d: 18.58,
24.17, 24.44, 58.15, 87.82, 93.73, 100.56, 114.64 (CN), 114.99 (CN), 124.53, 124.75,
127.00, 129.55, 129.83, 134.84, 138.51, 145.34, 148.90, 153.28, 159.48, 166.23. ESI-HRMS
m/z: 430.1338 [MþH]þ (calculated for [C23H20N5O2S]þ - 430.1332).
2 -Amino-4-(2-ethyl-8-methyl-4H-[1, 3]dioxino[4,5-c]pyridin-5-yl)-6-
(phenylthio)pyridine-3,5-dicarbonitrile (4b)
The general procedure was followed using compound 3 b (697 mg, 3.4 mmol), malono-
nitrile (444 mg, 6.8 mmol), KOH (19 mg, 0.3 mmol) and benzenethiol (370 mg,
1
3.4 mmol). Yield 30% (430 mg), pale yellow solid, mp 112–114 ꢂC (decomp.). H NMR
3
(DMSO-d6) d: 1.03 (t, 3 H, JHH=6.88 Hz, CH3CH2), 1.82–1.87 (m, 2 H, CH3CH2), 2.42
2
(s, 3 H, CH3Pyr), 4.70, 4.84 (AB, 2 H, JHH ¼ 16.26 Hz, CH2O), 5.16 (t, 1 H,
3JHH=5.00 Hz, CH), 7.48–7.63 (m, 5 H, C6H5Ar), 7.97 (br.s, 2 H, NH2), 8.04 (s, 1 H,
CHpyr). 13 C NMR (DMSO-d6) d: 7.64, 18.40, 26.90, 63.47, 87.82, 93.82, 100.92, 114.50
(CN), 114.90 (CN), 124.90, 125.97, 126.99, 129.52, 129.81, 134.81, 139.07, 146.88,
148.44, 153.13, 159.58, 166.12. ESI-HRMS m/z: 430.1338 [MþH]þ (calculated for
[C23H20N5O2S]þ - 430.1332).
General procedure for the synthesis of compounds 5a,b
A mixture of the corresponding compound 4 (0.5 mmol) and 0.4 ml of concentrated
HCl in 10 ml of water was refluxed for 30 min. Then the reaction mixture was slowly
cooled to 2 ꢂC. The solid product was collected by filtration, washed with water and
dried. Analytical characteristics of compound 5 b are described in the Electronic
Supplementary Material.
2 ’-Amino-5-hydroxy-4-(hydroxymethyl)-6-methyl-6’-(phenylthio)-[3,4’-bipyridine]-
3’,5’-dicarbonitrile (5a)
1
Yield 85% (180 mg), pale yellow solid, mp 198–202 ꢂC (decomp.). H NMR (DMSO-d6)
d: 2.64 (s, 3 H, CH3Pyr), 4.68 (s, 2 H, CH2O), 7.49–7.62 (m, 5 H, C6H5Ar), 7.93 (br.s, 2 H,
NH2), 8.35 (s, 1 H, CHpyr). 13 C NMR (DMSO-d6) d: 16.83, 56.48, 88.30, 94.34, 114.65
(CN), 115.01 (CN), 126.97, 129.58, 129.86, 133.82, 134.84, 136.71, 146.01, 150.91,
152.05, 159.14, 165.45. ESI-HRMS m/z: 390.1025 [M-Cl]þ (calculated for [C20H16N5
O2S]þ - 390.1019).
Preparation of methyl bromide solution in acetonitrile
Potassium bromide (12.39 g, 104.1 mmol) and water (0.34 ml, 18.9 mmol) were added to
dimethyl sulfate (11.88 ml, 125.4 mmol), and the reaction mixture was heated at 160 ꢂC
for 4 h. Evolved methyl bromide was absorbed by 200 ml of cold acetonitrile.
Methyl bromide concentration was determined by an increase in mass of the acetonitrile
solution.

12
A. A. GRIGOR’EV ET AL.
General procedure for the synthesis of compounds 6a-m
The corresponding compound 4a-m (0.1–0.7 mmol) was added to a solution of methyl
bromide (5.0 mmol) in acetonitrile (10 mL). and the reaction mixture was heated at
70ꢂC for 24 h. Then, the solvent was evaporated under reduced pressure, and the
residue was washed with diethyl ether. The precipitate was collected by filtration,
washed with diethyl ether and dried. Analytical characteristics of compound 6c-m are
described in the Electronic Supplementary Material.
5 -(2-Amino-3,5-dicyano-6-(phenylthio)pyridin-4-yl)-2,2,7,8-tetramethyl-4H-[1,
3]dioxino[4,5-c]pyridin-7-ium bromide (6a)
The general procedure was followed using compound 4a (270 mg, 0.6 mmol). Yield 64%
1
(210 mg), gray solid, mp 162–167 ꢂC (decomp.). H NMR (DMSO-d6) d: 1.63 (s, 6 H,
2CH3), 2.69 (s, 3 H, CH3Pyr), 4.30 (s, 3 H, CH3-N), 5,03 (s, 2 H, CH2O), 7.46–7,63 (m,
5 H, C6H5Ar), 8.22 (br.s, 2 H, NH2), 8.98 (s, 1 H, CHpyr). 13 C NMR (DMSO-d6) d: 16.71,
24.45, 24.65, 47.03, 58.97, 88.08, 93.63, 103.70, 114.56 (CN), 114.97 (CN), 126.98,
127.64, 130.11, 130.52, 130.52, 133.62, 135.33, 136.36, 149.05, 149.19, 149.27, 159.76,
167.31. ESI-HRMS m/z: 444.1494 [M-Br]þ (calculated for [C24H22N5O2S]þ - 444.1489).
5 -(2-Amino-3,5-dicyano-6-(phenylthio)pyridin-4-yl)-2-ethyl-7,8-dimethyl-4H-[1,
3]dioxino[4,5-c]pyridin-7-ium bromide (6b)
The general procedure was followed using compound 4 b (200 mg, 0.4 mmol), Yield
1
61% (149 mg), light green solid, mp 200-205 ꢂC (decomp.). H NMR (DMSO-d6) d: 1.05
3
(t, 3 H, JHH ¼ 7.50, CH3CH2), 1.89–1.96 (m, 2 H, CH3CH2), 2.71 (s, 3 H, CH3Pyr), 4.31
3
(s, 3 H, CH3-N), 5,09 (s, 2 H, CH2O), 5.42 (t, 1 H, JHH ¼ 5.00, CH), 7.51–7.63 (m, 5 H,
C6H5Ar), 8.20 (br.s, 2 H, NH2), 8.99 (s, 1 H, CHpyr). 13 C NMR (DMSO-d6) d: 7.34,
13.19, 26.52, 46.56, 63.67, 87.53, 93.09, 102.56, 113.96 (CN), 114.33 (CN), 126.48,
127.31, 130.03, 134.70, 134.88, 136.41, 138.78, 148.13, 148.70, 150.04, 159.25, 166.73.
ESI-HRMS m/z: 444.1494 [M-Br]þ (calculated for [C24H22N5O2S]þ - 444.1489).
General procedure for the synthesis of compounds 7a-c
A mixture of the corresponding compound 6 (0.3 mmol) and 0.4 ml of concentrated
HBr in 10 ml of water was stirred at 25ꢂC for 10 h. Then, the solvent was evaporated
under reduced pressure, and the residue was washed with chloroform and dried.
Analytical characteristics of compounds 7b,c are described in the Electronic
Supplementary Material.
2 ’-Amino-3’,5’-dicyano-5-hydroxy-4-(hydroxymethyl)-1,6-dimethyl-6’-(phenylthio)-
[3,4’-bipyridin]-1-ium bromide (7a)
1
Yield 75% (109 mg), yellow solid, mp 161–164 ꢂC (decomp.). H NMR (DMSO-d6) d:
2.73 (s, 3 H, CH3Pyr), 4.29 (s, 3 H, CH3-N), 4.76 (s, 2 H, CH2O), 7.51 – 7,61 (m, 5 H,
C6H5Ar), 8.03 (br.s, 2 H, NH2), 8.86 (s, 1 H, CHpyr). 13 C NMR (DMSO-d6) d: 14.10,

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SYNTHETIC COMMUNICATIONSV
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46.52, 56.96, 88.04, 93.95, 114.40 (CN), 114.79 (CN), 126.71, 129.64, 129.98, 134.72,
136.72, 143.47, 147.75, 152.02, 152.91, 159.07, 165.79. ESI-HRMS m/z: 404.1181 [M-Br]þ
(calculated for [C21H18N5O2S]þ - 404.1176).
Antibacterial activity
The antibacterial activity of the obtained compounds was evaluated on three Gram-
R
positive (Staphylococcus aureus ATCCV 29213TM, Staphylococcus epidermidis (clinical
R
isolate), Bacillus subtilis 168) and two Gram-negative bacteria (Escherichia coli ATCCV
R
25922TM, Pseudomonas aeruginosa ATCCV 27853TM). MICs were determined by using
the broth microdilution method in 96-well plates. Two-fold serial dilutions of the test
samples in 10% dimethyl sulfoxide (DMSO) were prepared to obtain a final test range
of 64–0.5 lg/mL. Two microliters of each dilution and 200 lL of test organism
(s, 2–9 ꢃ 104 cfu mLꢀ1) in the LB-broth medium were then dispensed into each well.
The MIC was defined as the lowest concentration of compound at which no visible
growth could be seen after 24 h of cultivation at 37 ꢂC. All experiments were carried out
in triplicate.
In vitro cytotoxicity studies
Breast adenocarcinoma MCF-7 (ATCC HTB-22), glioblastoma SNB-19 (ATCC
CRL-2219), colorectal carcinoma HCT-116 (GSM136288) and primary human foreskin
fibroblast cells (HSF) isolated from the skin explant according to conventional protocol
were cultured in a-MEM (PanEko, Russia) supplemented with 10% fetal bovine serum
(PAA, Australia), L-glutamine, and 1% penicillin-streptomycin at 37 C in a 5% humidi-
fied CO2 atmosphere in air. Cells were removed from the culture substrate by treatment
with trypsin-EDTA with subsequent inactivation of trypsin by adding a-MEM-contain-
ing serum. Cell suspensions were precipitated by centrifugation at 500 g and pellet was
resuspended in phosphate-buffered saline (PBS). Cell viability and density were meas-
ured in a Neubauer chamber by using 0.4% solution of trypan blue. Suspensions with
the amount of viable cells no less than 90% were used in the experiments. The effect of
synthesized compounds on the proliferation of the cells was measured by MTT-assay
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Promega). The cells
were seeded in a 96-well plate at a concentration of 2000 cells per well and were cul-
tured in supplemented a-MeM (180 lL) according to the standard culturing conditions
for 1 day. The cell viability was measured at 72 h after treatment with the test agent (20
lL). Then, the medium was exchanged for the supplemented medium (80 lL). MTT
solution (5 mg mLꢀ1) was added to a volume of 20 lL in each well and incubated for
3.5 h. Then the solution was removed and DMSO (100 lL) was added to solubilize the
formazan crystals. After 10 min, absorbance was measured at 555 nm (the reference
wavelength 650 nm) by a TECAN plate reader (Switzerland). The results were presented
as the percent ratio to the control sample treated with PBS. For each compound tested,
the IC50 values (l < 0.05) were generated from the dose-response curves for each
cell line.

14
A. A. GRIGOR’EV ET AL.
Funding
The work has been supported by the Russian Foundation for Basic Research (RFBR) (project No.
17-33-50020 “mol_nr”).
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