Discovery of novel N4-alkylcytidines as promising antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2021.113212

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. In order to find new compounds that effectively inhibit the growth of pathogenic bacteria and fungi, we synthesized a set of N⁴-derivat

Full text of DOI:10.1016/j.ejmech.2021.113212

European Journal of Medicinal Chemistry 215 (2021) 113212
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel N4-alkylcytidines as promising antimicrobial
agents
,
*
Liudmila A. Alexandrova a, Maxim V. Jasko a, Sergey D. Negrya a, Pavel N. Solyev a
,
Oleg V. Shevchenko a, Andrei P. Solodinin a, Daria P. Kolonitskaya a, Inna L. Karpenko a,
Olga V. Efremenkova b, Alla A. Glukhova b, Yuliya V. Boykova b, Tatiana A. Efimenko b,
Natalya V. Kost c, Darya A. Avdanina d, Gulgina K. Nuraeva e, Ivan A. Volkov e,
Sergey N. Kochetkov a, Alexander A. Zhgun d
a Engelhardt Institute of Molecular Biology RAS, 32 Vavilova St., 119991, Moscow, Russia
b Gause Institute of New Antibiotics, 11 Bol’shaya Pirogovskaya St., 119021, Moscow, Russia
c Federal State Budgetary Scientific Institution “Mental Health Research Centre”, 34 Kashirskoe Highway, 115522, Moscow, Russia
d Research Center of Biotechnology RAS, 33 Leninsky Ave, 119071, Moscow, Russia
e Moscow Institute of Physics and Technology (National Research University), 9 Institutsky Alley, 141700, Dolgoprudny, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new
drugs. In order to find new compounds that effectively inhibit the growth of pathogenic bacteria and
fungi, we synthesized a set of N4-derivatives of cytidine, 20-deoxycytidine and 5-metyl-20-deoxycytidine
bearing extended N4-alkyl and N4-phenylalkyl groups. The derivatives demonstrate activity against a
Received 9 October 2020
Received in revised form
18 November 2020
Accepted 12 January 2021
Available online 1 February 2021
number of Gram-positive bacteria, including Mycobacterium smegmatis (MIC ¼ 24e200
Staphylococcus aureus (MIC ¼ 50e200 M), comparable with the activities of some antibiotics in medical
use. The most promising compound appeared to be N4-dodecyl-5-metyl-20-deoxycytidine 4h with ac-
tivities of 24 and 48 M against M. smegmatis and S. aureus, respectively, and high inhibitory activity of
mM) and
m
Keywords:
Nucleoside
Cytidine
Antifungal
Antibacterial
Preservation
m
0.5 mM against filamentous fungi that can, among other things, damage works of art, such as tempera
painting. Noteworthy, some of other synthesized compounds are active against fungal growth with the
inhibitory concentration in the range of 0.5e3 mM.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
ever-increasing proportion of resistant strains in relation to total
pathogen populations, gradually rendering antibiotic therapy
Microorganisms are closely tied to all aspects of human life,
bringing both benefit and harm. A fundamentally new level of
healthcare has been reached with the introduction of antibiotics
into medical practice. As a result, the mortality rate from infectious
diseases has decreased drastically. Unfortunately, microbial resis-
tance to most classes of antibiotics has been an emerging challenge
ever since. Every new antibiotic introduced to therapeutic use so far
has eventually promoted a rapid spread of drug resistance in mi-
croorganisms towards the respective antibiotic, a direct conse-
quence of antibiotic intake by the pathogens [1]. This leads to an
ineffective [2]. According to the World Health Organization (WHO),
current situation with multiple drug resistance poses a serious
threat to human society and continues to worsen each year [3]. It is
believed that humanity is entering a post-antibiotic era, when even
common infections or minor injuries can be life-threatening [4,5].
In this light, one of the most important tasks of modern medicinal
chemistry is to find new types of compounds that would inhibit
pathogen growth and become effective antibacterial, antiviral and
antifungal drugs [6].
The idea of using analogues and derivatives of nucleic acid
components (i.e. nucleic bases, nucleosides and nucleotides) as
medicine was proposed in the middle of the XX century. At present,
analogues and derivatives of nucleic acid components are impor-
tant elements of anticancer, antiviral [4,7e9] and antifungal ther-
apy [9e11]. Many of the antiviral and antifungal nucleoside drugs
* Corresponding author.
E-mail addresses: ala2004_07@mail.ru (L.A. Alexandrova), solyev@gmail.com
(P.N. Solyev).
https://doi.org/10.1016/j.ejmech.2021.113212
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
that have been in common practice for many decades also appear to
be effective antagonists of bacterial growth [4].
the prodrugs strategy, which is often used to optimize the phar-
macological and physicochemical properties of drugs, and thus
improve their solubility and pharmacokinetic features and decrease
their toxicity [29].
Systematic studies of antibacterial (primarily antimycobacterial)
as well as antifungal activity of nucleoside derivatives have been
conducted over the past two decades; several reviews have been
published on modified nucleosides that suppress the growth of
bacteria [9,12e15]. 5-Modified pyrimidine nucleosides with
extended substituents introduced through alkynyl, hydroxymethyl
or triazolyl moiety were shown to exhibit good inhibitory activity
in vitro against Mycobacterium tuberculosis, M. avium and M. bovis
[9,14e18]. Even today, the exact mechanism of action of these
compounds remains unclear. However, it was demonstrated that
the cell wall of M. tuberculosis is lysed in the presence of 5-modified
20-deoxyuridines or 5-modified 50-norcarbocyclic pyrimidine nu-
cleosides with extended substituents [19,20].
Previously, we synthesized a number of water-soluble forms of
modified pyrimidine 20-deoxynucleosides [30,31]. They turned out
to be at least two orders of magnitude more soluble than the
starting compounds. They also had lower cytotoxicity and exhibited
activity against a number of Gram-positive bacteria. Similarly, in
the current work, we’ve introduced triethylene glycol residues at
the 30 position of the carbohydrate fragment of the most active
compounds 4g and 4h to obtain the 30-O-[(8-hydroxy-3,6-
dioxaoctyloxy)carbonyl]-N4-alkyl-5-methyl-20-deoxycytidines 4n
and 4o using the method described in Refs. [30,32] as a basis.
Namely, we first protected the 50 hydroxyl of thymidine 5 with the
In addition to medical use, antimicrobial compounds (natural
antibiotics and xenobiotics) have found application in different
areas of human activity, such as food industry, agriculture, and so
on [21]. Nowadays, an alternative application of nucleoside de-
rivatives is being thoroughly researched: their use as antifungal
agents also has a good potential [9e11].
TBDMS-group,
then
synthesized
30-O-(8-hydroxy-3,6-
dioxaoctyloxycarbonyl)-50-О-(tert-butyldimethylsilyl)thymidine 7
by sequential treatment of 6 with N,N0-carbonylditriazole and tri-
ethylene glycol followed by protection of the
the glycol fragment with the TBDMS-group and then used the
u
ꢀhydroxyl group of
synthon
8
(30-O-(8-tert-butyldimethylsilyloxy-3,6-
We suggest that N4-modified cytidine derivatives bearing
extended lipophilic alkyl or phenylalkyl substituents could exhibit
antibacterial and/or antifungal activity in the same way it was
observed for 5-modified uridine derivatives [14e20,22].
dioxaoctyloxycarbonyl)-50-О-(tert-butyl)dimethylsilylthymidine)
to obtain N4-modified 5-methyl-20-deoxycytidines 4n and 4o
(Scheme 2).
As a result, we obtained a representative library (Table 1) of N4-
substituted cytidine derivatives.
The goal of this work was to synthetize a set of new N4-modified
cytidine derivatives bearing extended N4-alkyl and N4-phenylalkyl
groups as potential microorganism growth inhibitors as well as to
evaluate their toxicity to biological objects along with their anti-
bacterial and antifungal activity towards a wide range of microor-
ganisms. As test objects, we used various clinical isolates and
bacteria strains provided by the Gause Institute of New Antibiotics
and the collection of degrader filamentous fungi isolated from
ancient Russian tempera painting from the State Tretyakov Gallery
collection.
2.2. Chemical and enzymatic stability
The chemical and enzymatic stability of 30-O-carbonyl tri-
ethylene glycol derivatives of N4-alkyl-5-methyl-20-deoxycytidines
4n and 4o was assessed using the method described in Refs. [30,31],
thus yielding estimates of their half-life (t1/2). The enzymatic hy-
drolysis of compounds 4n and 4o was performed in human blood
serum. The compounds were hydrolyzed with t1/2 ꢁ 24 h, slightly
exceeding the half-life optimal for prodrug forms [33]. The hydro-
lysis yields the only parent nucleosides 4j and 4h. On the other
hand, these compounds were stable in buffer solutions at two pH
values (2.2 and 7.5) for more than a week, and only slightly hy-
drolyzed at pH 9.0. This indicates that the studied compounds were
chemically stable and their hydrolysis in human blood serum was
almost exclusively enzymatic.
2. Results and discussion
2.1. Chemistry
Synthesis of some N4-modified pyrimidine nucleosides was re-
ported in a number of previous works [23e26]. Some of them
demonstrated antiviral activity against the human immunodefi-
ciency viruses (HIV-1, HIV-2), the herpes virus (HSV-1) [23,24] and
the hepatitis B virus (HBV) [25,26], but their antibacterial and/or
antifungal activities have not been reported. In our study, we pre-
sent evidence of antimicrobial activities of N4-modified derivatives
of cytidine and 20-deoxycytidine.
2.3. Cytotoxicity
The cytotoxicity of the synthesized compounds (CD50) was
estimated by MTT assay [34] in A549 cell line (human pulmonary
adenocarcinoma) (Table 3). The compounds demonstrated cyto-
The synthesis of N4-modified cytidine derivatives bearing
extended N4-alkyl and N4-phenylalkyl groups was performed using
the method of Divakar and Reese [27] in its later modification [28],
by coupling 30,50-diacetyl-20-deoxyuridines (1a), 30,50-diacetylth-
ymidines, (1b) or 20,30,50-triacetyluridines (1c) with 1,2,4-triazole
and 2-chlorophenyl dichlorophosphate in pyridine to obtain C4-
triazolyl derivatives followed by their condensation with alkyl- or
phenylalkyl amines (Scheme 1). The reaction of C4-triazolyl de-
rivatives with alkylamines was accompanied by the partial removal
of O-acetyl protecting groups. Therefore, in order to completely
remove the protective groups, the cleavage was carried out using
ammonia solution (controlled by TLC). The isolation of N4-alkyl-
cytidines proved difficult due to their chromatographic mobilities
being close to those of the starting amines.
toxic activity at concentrations of 80e200
the glycol derivatives 4n and 4o (90 M) was 2 times higher than
that of the parent nucleosides 4g and 4h (about 160 M). This is
probably due to the better solubility of compounds 4n and 4o in
aqueous media.
mM. The cytotoxicity of
m
m
2.4. Bacterial growth inhibition
Antibacterial effect of the obtained compounds was studied by
their ability to inhibit the growth of a number of microorganisms
in vitro using the same method as in our previous works [30,35].
The antibacterial activities were tested against such Gram-positive
bacteria as: Bacillus subtilis, meticillin-resistant Staphylococcus
aureus strain MRSA (MRSA strains are wide-spread and cause
nosocomial infections that resist the modern antibiotic therapy);
streptococcus-like bacteria Leuconostoc mesenteroides (strain
distinguished by a high native resistance to glycopeptide antibiotics
N4-Modified cytidines with extended N4-alkyl and N4-phenyl-
alkyl substituents are poorly soluble in water, the fact that signifi-
cantly complicates their study. To solve this problem, we applied
2

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
Scheme: 1. Synthesis of compounds 4a-m. Reagents and conditions: i) 1H-1,2,4-triazole, 2-chlorophenyl dichlorophosphate, Py, r.t., 16 h; ii) ReNH2, dioxane, r.t., 16 h; iii) NH3/H2O,
dioxane, r.t., 16 h.
Scheme: 2. Reagents and conditions: i: TBDMSCl, Py, r.t.; ii: CDT, DMF, (37 ꢂC); iii: triethylene glycol, dioxane, (37 ꢂC), 16 h; iv: 1H-1,2,4-triazole, 2-chlorophenyl dichlorophosphate,
Py, r.t., 16 h; v: ReNH2, dioxane, r.t., 16 h; vi: Bu4NF$3H2O, dioxane, r.t.
Table 1
against Gram-positive bacteria as well as mycobacteria (Table 3),
and there is no activity towards Gram-negative microorganisms.
20-Deoxynucleosides had a higher activity than the respective
ribo derivatives with the same alkyl substituent. Moreover, in most
cases, N4-modified 5-methyl derivatives of 20-deoxycytidine had a
slightly greater inhibitory effect than the compounds without such
methyl group at the C-5 position of the cytosine residue. The data
obtained are comparable with the activities of a number of anti-
biotics in medical use. For example, MIC of widely used first line
Obtained N4-alkylcytidines.
antibiotics against M. tuberculosis are: 800
(one of the first line antituberculosis drugs) [36] and 50 mM of
mM of pyrazinamide
ꢂ
ꢂ
N
R
R1
R2 R3
N
R
R1
R2 R3
amikacin (Table 3).
4a С8H17
H
H
H
H
H
H
H
H
H
H
H
H
OH 4i
OH 4j
OH 4k C8H17
OH 4l C10H21
C2H4eC6H5
C4H8eC6H5
CH3
H
H
H
H
OH
OH
Substituting linear alkyl residues for phenylalkyl residues at the
N4 position of the nucleobase did not lead to an increase in anti-
microbial activity. For instance, compounds 4i and 4j did not
demonstrate any inhibitory activity. This might be explained by the
low solubility of such nucleosides in aqueous solutions. E.g., a
compound is expected to be poorly soluble if it features an
extended linear substituent at N4 (4d). On the other hand, the
introduction of the hydrophylic triethylene glycol fragment at N4 of
5-methyl-20-deoxycytidines (4k) resulted in high water solubility
of the compound, but this also led to a complete loss of the anti-
bacterial action.
4b
4c
4d
C10H21
C12H25
C14H29
OH OH
OH OH
H
H
H
H
4e C6H13 CH3
4f C8H17 CH3
4g
4h
OH 4m (C2H4O)3CH3 CH3
OH
OH 4n C10H21
OH 4o C12H25
OH
CH3
CH3
OC(O)(OC2H4)3OH
OC(O)(OC2H4)3OH
C
C
10H21 CH3
12H25 CH3
of the vancomycin group, which often appear effective towards
pathogenic bacteria with multidrug resistance) and Micrococcus
luteus NCTC 8340; mycobacteria: two strains of Mycobacterium
smegmatis (which are used for the preliminary assessment of the
activity followed by the analysis of promising compounds against
the strains of the causative agent of tuberculosis, i.e. Mycobacterium
tuberculosis); Gram-negative bacteria: Escherichia coli and Pseudo-
monas aeruginosa (opportunistic human pathogen causing diffi-
culty in the treatment of nosocomial infection).
The 30-O-carbonyl triethylene glycol derivatives of N4-alkyl-5-
methyl-20-deoxycytidines 4n and 4o demonstrated lower inhibi-
tory effect than the parent nucleosides 4g and 4h; this difference is
especially noticeable for the 4h/4o pair. Since they also turned out
to be twice as toxic, this modification did not lead to an improve-
ment in the selectivity index.
Thus, among the studied compounds, nucleosides 4g and, above
all, 4h are the most promising candidates for antibacterial agents.
The compounds under investigation demonstrate activity
3

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
Table 3
Minimal bacterial growth inhibitory concentration (
m
M) and cytotoxicity (
mM).
Bacteria
Bacillus
Leuconostoc Mycobacterium Mycobacterium Staphylococcus Staphylococcus Micrococcus Cytotoxicity in
Compounds
subtilis
АТСС 6633
mesenteroides
VKPM B-4177
smegmatis
smegmatis
VKPM Ac-1339 FDA 209 P
aureus
aureus
INA 00761
luteus
NCTC 8340
A549 human
cells
mc2155
4 a, d-f, i-m
4b
4c
4g
4h
4n
4o
>200
140
70
50
24
>200
140
70
50
24
>200
140
70
50
24
>200
140
70
50
24
>200
>140
70
50
48
>200
>140
70
50
48
>200
140
70
50
24
90e200
200
100
156
158
90
90
68
90
68
54
68
54
68
54
50
54
50
>45
50
90
Antibiotic
AN 7
VA>275
AN 50
CIP 15
INZ 29
RFP 5
AN 29
CIP 12
INZ 2
RFP 10
AN 3,4
CIP 3
OX 2.5
AN 5
CIP 12
OX 80
AN 1
sensitivitya
a
Amikacin (AN), Ciprofloxacin (CIP), Isoniazid (INZ), Rifampicin (RFP), Oxacillin (OX), Vancomycin (VA).
Recently we demonstrated the destruction of the bacterial cell
wall of M. tuberculosis H37Rv strain treated with 5-modified py-
rimidine nucleosides with extended substituents using trans-
mission electron microscopy (TEM), suggesting that the
mechanism of action of these compounds may be related to their
interactions with bacterial cell walls [28,29]. It is likely that N4-
modified cytidines bearing extended N4-alkyl groups have the
same mode of antibacterial action.
(Cladosporium halotolerans STG-52B, C. parahalotolerans STG-93B)
and one representative for each of the families of Pleosporaceae
(Ulocladium chartarum STG-36), Cordycipitaceae (Simplicilium
lamellicola STG-96) and Microascaceae (Microascus paisii STG-103).
These strains were dominant among all microbiome from STG,
characterized by metagenomic profiling (SRA records Accession:
PRJNA606688, https://www.ncbi.nlm.nih.gov/sra/PRJNA606688).
In this regard, an important task for the preventive treatment of
icons from STG is to seek out an inhibitor with a broad spectrum
against these 10 fungi biodestructors. The toxicity effect was esti-
mated quantitatively as the percentage of colony growth inhibition
on CDA medium with an addition of N4-modified cytidines or 5FC
according to the method described in Ref. [45] (the control medium
contained no additions) (Fig. 2).
2.5. Acute and chronic toxicity
Acute and chronic toxicity of the most promising nucleoside 4h
were estimated by administering intraperitoneal injections of the
compound in 30% Tween-80 in mice.
The sublethal dose of inoculum was evaluated by drop and
dilution assay (Fig. 3A), as in Refs. [46,47]. The fungal growth in-
hibition (FGI) increases in series 4fe4ge4h (Fig. 3B). The modifi-
cation of 4g to 4n leads to a significant loss of inhibitory ability
(Fig. 3). The strongest inhibition effect was observed for nucleoside
4h (Figs. 2 and 3). The preliminary screening of antifungal activity
was assessed in the range of 0.1 and 3.5 mM of compounds. To
analyze the inhibition timescale of N4-modified cytidines or 5FC we
used concentrations of 0.5 and 3 mM; measurements were taken
every 3 days.
The compound was found to exhibit low toxicity: the LD50 of 4h
was found to be 270 mg/kg for acute administration, 215 mg/kg for
subchronic administration and 200 mg/kg for the delayed effect of
subchronic administration (Fig. 1). A decrease in subchronic LD50 of
the compound 4h compared to the respective value of acute
toxicity by 26% indicates a cumulative toxic effect of the drug,
without tolerance development.
2.6. Fungi growth inhibition
Fig. 4 shows the measurement results for the time range of 6e48
days, taken every 6 days. The strongest inhibition of fungi growth
was demonstrated for 3 mM solution of 4h. However, after 24e30
days, the majority of strains from the Aspergillaceae family over-
came the inhibition effected by the addition of 3 mM solution of 4h.
The genera most sensitive to any of the tested N4-modified cyti-
dines were Cladosporium and Ulocladium. The dynamic of growth
inhibition follows the same trend as FGI, with an increase in the
series 4fe4ge4h. 5FC inhibited Cladosporiacea (STG-52B, STG-93B)
and A. amoenus STG-106 at the same level as 4h. It also inhibited all
other representatives of the Aspergillaceae family (STG-25G, STG-
57, STG-86 and STG-93W) at the level between those of 4h and
4g, and was practically inactive against Pleosporaceae (STG-36) and
Sordariomycetas (STG-96 and STG-103). N4-Modified cytidines also
inhibited the growth of Aspergillus, slightly weaker than Cladospo-
rium and Ulacladium; 5FC inhibited Aspergillaceae (STG-25G, STG-
57, STG-86 and STG-93W) at the level slightly lower than that of
4g, and inhibited STG-106 at the level slightly higher than that of
4h. The most resistant filamentous fungi among the tested species
was Simplicillium STG-96, both to N4-modified cytidines and 5FC.
The antifungal activity of N4-modified 20-deoxycytidines 4n and
4f demonstrated close inhibitory activity. Four of the 10 strains
(STG-93W, STG-36, STG-96 and STG-106) demonstrated resistance
Microbiological community plays a crucial role in the destruc-
tion of cultural heritage [37,38]. Microorganisms from various
systematic groups (filamentous fungi especially) capable of
damaging works of art, e.g., tempera painting, oil painting on
canvas, have been extensively studied in recent years [39]. Tradi-
tional antiseptics used for tempera paintings have their limitations
[40,41] and there is an urgent need for the development of novel
modern antiseptics with broad-spectrum antimicrobial activity and
high efficacy [41,42] which at the same time would be safe for
paintings as well as the museum staff [38,43]. In this regard, the
challenge is to develop a new generation of biocides, with a wide
spectrum of action, inhibiting not only bacteria, but fungi as well,
while not damaging the materials of the cultural heritage [38,44].
Recently, we isolated a set of filamentous fungi from Paintings of
the Ancient Rus Halls, the State Tretyakov Gallery (STG), Russia,
Moscow [38]. The antifungal activities of N4-modified cytidines and
5-fluorocytosine (5FC) as a positive control were evaluated against
10 representatives of filamentous fungi known to cause biodeteri-
oration activity against organic materials used in tempera painting
of 15e16th centuries and in restoration [38]. Five species were from
the Aspergillaceae family (Aspergillus versicolor STG-25G, A. creber
STG-57, A. versicolor STG-86, Aspergillus sp. STG-93W, A. amoenus
STG-106); two species were from the Cladosporiacea family
4

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
Fig. 1. Toxicity of the nucleoside 4h. A. Acute toxicity. B. Subchronic and long-term toxicity. Each solution contained 30% Tween-80 and a respective does of 4h. No toxicity was
detected for negative control containing Tween-80 only (data not shown). Data are means SD.
Fig. 2. Growth of fungi strains on CDA medium with the addition of 0.5 mM nucleoside 4h or without addition (control). 24 days after inoculation.
to 3 mM 5FC at early stages (6e18 days) (Fig. 4). During this period
3 mM 4h completely inhibited growth of all the 10 strains. The
inoculum dose used in determination of growth inhibition against
control was 5 ꢃ 105 Colony Forming Units (CFU). The majority of
Aspergillaceae overcame the inhibitory effect of 3 mM 4h at day 48.
Using drop and dilution assay we demonstrated that an inoculum
dose equal to or lower than 5 ꢃ 104 CFU was completely inhibited
by 3 mM 4h at any given point. In this regard, the nucleoside 4h can
be perceived as a possible candidate for the preventive processing
of icons at the early stages of filamentous fungi infection (a test on
mock-layers is required).
Our experiments on 10 filamentous fungi from various sys-
tematic groups demonstrated that the inhibitory effect of the
studied N4-modified cytidines depends mainly on the nature and
concentration of the inhibitor itself (Fig. 4). Among representatives
of the 3 classes of Ascomycota (Eurotiomycetes, Dothideomycetes and
Sordariomycetes), no species were identified that would exhibit
specific resistance. In addition,
4 strains showed increased
5

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
Fig. 3. Growth of fungi strains on CDA medium with the addition of 0.5 mM 5FC, 0.5 mM nucleoside 4f-4h and 4n, or without addition (control). 6 days after inoculation. A. Drop
and dilution assay. B. Growth inhibition (%). Data are means SD, n ¼ 3. ND, not detected.
6

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
Fig. 4. The dynamic of growth inhibition (%) for STG strains on CDA medium with 5FC, 4f-4h and 4n (all tested at concentrations of 0.5 mM and 3 mM). Data on 6, 12, 18, 24, 30, 36,
42 and 48 days after inoculation.
resistance to 5FC (STG-93W, STG-36, STG-96, STG-103), and 3
strains showed increased sensitivity to the same compound (STG-
106, STG-52B, STG-93B). On the other hand, earlier for these 10
strains of filamentous fungi, we obtained a differential effect when
studying the fungicidal properties of chitosan [48]. The highest
sensitivity to chitosan was shown by representatives of the Clado-
sporiaceae family (STG-52B and STG-93B), the highest resistance
was shown by a representative of the Pleosporaceae family (STG-36)
and some representatives of Aspergillaceae (STG-93W and STG-106)
[48]. These results correlate with the mechanism of action of chi-
tosan on fungal cell membranes. Membranes of fungi with a high
content of polyunsaturated fatty acids (such as linoleic acid, char-
acteristic of Cladosporiaceae) are highly sensitive to chitosan,
membranes with low fluidity, enriched with saturated fatty acids
(such as stearic or palmitic, characteristic of Aspergillaceae), are
resistant to chitosan. The demonstrated broad spectrum inhibition
of filamentous fungi by N4-modified cytidines may be associated
with some universal mechanism of action on fungi cell, which can
be used for the development of a broad-spectrum antiseptic. We
plan to investigate this in the future.
Filamentous fungi (or molds) are taxonomically diverse organ-
isms from phyla Zygomycota and Ascomycota characterized by
filamentous hyphae and their ability to produce airborne spores or
conidia. Currently, more than 70 000 molds are known, some of
them contain unique and unusual biochemical pathways. A number
of products from those pathways (especially the secondary
metabolite pathways) are used as important pharmaceuticals,
including antibiotics, statins and immunosuppressors [38,49e52].
Furthermore, filamentous fungi have a variety of enzymatic activ-
ities leading to biodegradation of various materials, e.g., materials
used in tempera painting [38]. Finally, the third group of enzymatic
activities is associated with a variety of resistance mechanisms that
are also unique to different groups [38,46]. In this regard, the new
compounds, N4-modified cytidines, demonstrated a rather inter-
esting effect against the studied fungi responsible for destroying
tempera painting, exhibiting a broad-spectrum inhibitory activity.
The toxic effect increased proportionally with the extension of the
alkyl radical (from C8 to C12 length) in the range of the tested
concentrations (0.5 mM and 3 mM).
3. Conclusions
In summary, we have employed a simple method for prepara-
tion of N4-modified cytidines bearing extended N4-alkyl or N4-
phenylalkyl groups. The compounds demonstrated activity against
M. smegmatis, S. aureus and some other Gram-positive bacteria. The
range of activity is comparable with that of some antibiotics in
medical practice, compound 4h with activities 24 and 48 mM to-
wards M. smegmatis and S. aureus, respectively, being the best in the
synthesized series. None of the obtained compounds are active
against Gram-negative bacteria. Of most interest is the finding that
N4-modified cytidines exhibit high inhibitory activity against fila-
mentous fungi that can damage works of art, such as tempera
painting. Sublethal doses of this type of antifungal inhibitory ac-
tivity are in the range of 0.1e0.5 mM, and lethal doses are in the
range of 0.5e3 mM; the most promising nucleosides are 4g and 4h.
The latter completely blocks the growth of filamentous fungi at a
concentration of 0.5 mM. The convenience of the method used to
synthesize these N4-modified cytidines derivatives is that the re-
actions are carried out "in one pot" using inexpensive and readily
available thymidine. This simplicity will make it possible to obtain
novel series of effective inhibitors of bacterial and fungal
replication.
7

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
4. Experimental section
362.2050, found: 362.2063.
4.2.2. N4-Decyl-20-deoxycytidine (4b). Prepared according to the
general procedure from 1a (156 mg) and decylamine (157 mg,
4.1. Reagents and equipment
200
DMSO‑d6)
m
l). Yield 141 mg (77%). UV: lmax 273 nm. 1H NMR (300 MHz,
Commercial reagents were purchased from Acros, Aldrich and
Fluka. Column chromatography was performed on silica gel 60
0.040e0.063 mm (Merck, Germany). Thin layer chromatography
was performed on silica gel 60 F254 plates (Merck, Germany). NMR
spectra were registered on an AMX III-400 spectrometer (Bruker
BioSpin GmbH, Germany) with the working frequency of 400 MHz
for 1H NMR (Me4Si as an internal standard for organic solvents) and
101 MHz for 13C NMR (with the carboneproton interaction
decoupling). The spectra of compounds 4c, 4e, 4h, 4k, 4m, 4o were
registered on an AM-300 spectrometer (Bruker BioSpin GmbH,
Germany) with the working frequency of 300 MHz for 1H NMR and
75 MHz for 13C NMR (with the carboneproton interaction decou-
pling). UV spectra were recorded on a Perkin Elmer lambda 25
(Perkin Elmer, USA) in ethanol. High-resolution mass spectra were
recorded on a Bruker Daltonics micrOTOF-Q II device by electro-
spray ionization mass spectrometry (ESI-MS). Measurements were
carried out in positive ion mode in accordance with the previously
applied conditions [29,35].
d
0.85 (t, J ¼ 6.7 Hz, 3H, eCH2-CH3), 1.20e1.31 (m, 14H,
eNHe(CH2)2-(CH2)7-), 1.41e1.53 (m, 2H, eNHeCH2-CH2-), 1.93
(ddd, J ¼ 13.2, 7.5, 6.0 Hz, 1H, 20-Ha), 2.10 (ddd, J ¼ 13.2, 6.0, 3.1 Hz,
1H, 20-Hb), 3.17e3.28 (m, 2H, eNHeCH2-), 3.53 (ddd, J ¼ 11.9, 5.3,
4.1 Hz, 1H, 50-Hb), 3.57 (ddd, J ¼ 11.9, 5.3, 3.8 Hz, 1H, 50-Ha), 3.77
(ddd, J ¼ 4.2, 3.8, 3.0 Hz, 1H, 40-H), 4.20 (ddt, J ¼ 6.0, 4.2, 3.1 Hz, 1H,
30-H), 4.94 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.17 (d, J ¼ 4.2 Hz, 1H, 30-OH),
5.74 (d, J ¼ 7.5 Hz,1H, 5-H), 6.16 (dd, J ¼ 7.5, 5.9 Hz,1H, 10-H), 7.64 (t,
J ¼ 5.5 Hz, 1H, N4eH), 7.72 (d, J ¼ 7.5 Hz, 1H, 6-H). 13C NMR
(101 MHz, DMSO‑d6) d 13.87 (-CH2-CH3), 22.03, 26.44, 28.52, 28.64,
28.74, 28.91, 28.97, 31.23 (eNHeCH2-(CH2)8-), 39.71 (20-C), 40.25
(eNHeCH2-), 61.39 (50-C), 70.43 (30-C), 84.79 (10-C), 87.11 (40-C),
94.63 (5-C), 139.56 (6-C), 155.11 (2-C), 163.24 (4-C). HRMS (ESI) of
C
19H33N3O4, m/z: calcd [MþH]þ 368.2544, found: 368.2540; calcd
[MþNa]þ 390.2363, found: 390.2363.
4.2.3. N4-Dodecyl-20-deoxycytidine (4c). Prepared according to
the general procedure from 1a (156 mg) and dodecylamine
(185 mg). Yield 149 mg (75%). UV: lmax 274 nm. 1H NMR (300 MHz,
All research work with laboratory animals was carried out in
accordance with generally accepted ethical standards for the
treatment of animals, which comply with the rules adopted by the
European Convention [53].
DMSO‑d6)
d
0.86 (t, J ¼ 6.7 Hz, 3H, eCH2-CH3), 1.21e1.31 (m, 18H,
eNHe(CH2)2-(CH2)9-), 1.42e1.52 (m, 2H, eNHeCH2-CH2-), 1.93
(ddd, J ¼ 13.2, 7.5, 6.1 Hz, 1H, 20-Ha), 2.10 (ddd, J ¼ 13.2, 6.0, 3.1 Hz,
1H, 20-Hb), 3.17e3.27 (m, 2H, eNHeCH2-), 3.52 (ddd, J ¼ 11.7, 5.3,
4.1 Hz, 1H, 50-Hb), 3.57 (ddd, J ¼ 11.7, 5.3, 3.8 Hz, 1H, 50-Ha), 3.76
(ddd, J ¼ 4.2, 3.8, 3.0 Hz, 1H, 40-H), 4.20 (ddt, J ¼ 6.1, 4.2, 3.1 Hz, 1H,
30-H), 4.93 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.16 (d, J ¼ 4.2 Hz, 1H, 30-OH),
5.73 (d, J ¼ 7.5 Hz, 1H, 5-H), 6.16 (dd, J ¼ 7.5, 5.9 Hz,1H,10-H), 7.63 (t,
J ¼ 5.4 Hz,1H, N4eH), 7.71 (d, J ¼ 7.5 Hz,1H, 6-H). 13C NMR (75 MHz,
4.2. General method for the synthesis of compounds 4a-4m
2-Chlorophenyl dichlorophosphate (188 mg, 126 ml, 0.77 mmol)
was added to a solution of acetyl protected 20-deoxyuridine (1a), 5-
methyl-20-deoxyuridine (1b) or uridine (1c) (0.5 mmol) and 1,2,4-
triazole (200 mg, 3 mmol) in anhydrous pyridine, cooled to 0 ꢂC.
The mixture was stirred for 20 h at room temperature, then evap-
orated. The residue was partitioned between chloroform and 0.5 M
sodium bicarbonate, the chloroform layer was washed with water,
dried over Na2SO4, evaporated and dissolved in anhydrous dioxane
(3 ml). The corresponding 1-alkylamine (1 mmol) was added to a
solution, cooled to 0 ꢂC. The mixture was stirred for 20 h at room
temperature, then 3 ml of conc. aq. ammonia solution were added
and the mixture was stirred for 40 h at room temperature, then
evaporated, the compounds were purified on a column of silica gel
(2 ꢃ 15 cm) in chloroform or ethyl acetate were isolated by column
chromatography in CHCl3 or ethyl acetate eluted with a gradient of
ethanol in chloroform (0e15%) or in or ethyl acetate (0e10%),
respectively. The target fractions were evaporated in a vacuum to
give the expected compounds yields as colorless amorphous mass
with the 60e79% yields.
DMSO‑d6)
d 13.90 (-CH2-CH3), 22.05, 26.46, 28.54, 28.67, 28.76,
28.97, 29.01, 31.26 (eNHeCH2-(CH2)10-), 39.71 (20-C), 40.24
(eNHeCH2-), 61.40 (50-C), 70.44 (30-C), 84.78 (10-C), 87.11 (40-C),
94.61 (5-C), 139.60 (6-C), 155.07 (2-C), 163.23 (4-C). HRMS (ESI) of
C
21H37N3O4, m/z: calcd [MþH]þ 396.2857: found: 396.2854; calcd
[MþNa]þ 418.2676, found: 418.2674.
4.2.4. N4-Tetradecyl-20-deoxycytidine (4d). Prepared according to
the general procedure from 1a (156 mg) and tetradecylamine
(213 mg). Yield 176 mg (79%). UV: lmax 273 nm. 1H NMR (400 MHz,
DMSO‑d6)
d
0.85 (t, J ¼ 6.6 Hz, 3H, eCH2-CH3), 1.20e1.30 (m, 22H,
eNHe(CH2)2-(CH2)11-), 1.41e1.50 (m, 2H, eNHeCH2-CH2-), 1.92
(ddd, J ¼ 13.3, 7.2, 6.0 Hz, 1H, 20-Ha), 2.08 (ddd, J ¼ 13.2, 6.0, 3.2 Hz,
1H, 20-Hb), 3.16e3.24 (m, 2H, eNHeCH2-), 3.51 (ddd, J ¼ 11.7, 5.3,
4.1 Hz, 1H, 50-Hb), 3.56 (ddd, J ¼ 11.7, 5.3, 3.8 Hz, 1H, 50-Ha), 3.75
(ddd, J ¼ 4.1, 3.6, 3.0 Hz, 1H, 40-H), 4.19 (ddt, J ¼ 6.0, 4.2, 3.2 Hz, 1H,
30-H), 4.94 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.17 (d, J ¼ 4.2 Hz, 1H, 30-OH),
5.73 (d, J ¼ 7.5 Hz,1H, 5-H), 6.15 (dd, J ¼ 7.2, 6.2 Hz,1H, 10-H), 7.67 (t,
J ¼ 5.8 Hz, 1H, N4eH), 7.71 (d, J ¼ 7.4 Hz, 1H, 6-H). 13C NMR
4.2.1. N4-Octyl-20-deoxycytidine (4a). Prepared according to the
general procedure from 1a (156 mg) and octylamine (129 mg,
(101 MHz, DMSO‑d6)
d 13.94 (-CH2-CH3), 22.09, 26.50, 28.56, 28.71,
164
m
l). Yield 132 mg (78%). UV: lmax 272 nm1H NMR (300 MHz,
28.80, 29.02, 29.05, 31.30 (eNHeCH2-(CH2)12-), 39.74 (20-C), 40.27
(eNHeCH2-), 61.41 (50-C), 70.46 (30-C), 84.81 (10-C), 87.15 (40-C),
94.66 (5-C), 139.64 (6-C), 155.03 (2-C), 163.17 (4-C). HRMS (ESI) of
DMSO‑d6)
d
0.86 (t, J ¼ 6.7 Hz, 3H, eCH2-CH3), 1.21e1.34 (m, 10H,
eNHe(CH2)2-(CH2)5-), 1.42e1.53 (m, 2H, eNHeCH2-CH2-), 1.93
(ddd, J ¼ 13.2, 7.5, 6.0 Hz, 1H, 20-Ha), 2.10 (ddd, J ¼ 13.2, 6.0, 3.2 Hz,
1H, 20-Hb), 3.17e3.27 (m, 2H, eNHeCH2-), 3.52 (ddd, J ¼ 11.8, 5.3,
4.1 Hz, 1H, 50-Hb), 3.57 (ddd, J ¼ 11.8, 5.3, 3.8 Hz, 1H, 50-Ha), 3.76
(ddd, J ¼ 4.2, 3.7, 3.3 Hz, 1H, 40-H), 4.20 (ddt, J ¼ 6.0, 4.2, 3.4 Hz, 1H,
30-H), 4.93 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.16 (d, J ¼ 4.2 Hz, 1H, 30-OH),
5.73 (d, J ¼ 7.5 Hz,1H, 5-H), 6.16 (dd, J ¼ 7.5, 6.0 Hz,1H, 10-H), 7.63 (t,
J ¼ 5.5 Hz, 1H, N4eH), 7.71 (d, J ¼ 7.5 Hz, 1H, 6-H). 13C NMR
C
23H41N3O4, m/z: calcd [MþNa]þ 446.2989, found 446.2979.
4.2.5. N4-Hexyl-5-methyl-20-deoxycytidine (4e). Prepared ac-
cording to the general procedure from 1b (163 mg) and hexylamine
(101 mg, 138
l). Yield 119 mg (73%). UV: lmax 272 nm. 1H NMR
(300 MHz, DMSO‑d6)
0.81 (t, J ¼ 6.6 Hz, 3H, eCH2-CH3), 1.18e1.26
m
d
(m, 6H, eNHe(CH2)2-(CH2)3-), 1.40e1.52 (m, 2H, eNHeCH2-CH2-),
1.80 (d, J ¼ 1.0 Hz, 3H, 5-CH3), 1.91 (ddd, J ¼ 13.3, 7.7, 6.0 Hz, 1H, 20-
Ha), 2.02 (ddd, J ¼ 13.2, 6.0, 3.2 Hz, 1H, 20-Hb), 3.19e3.28 (m, 2H,
eNHeCH2-), 3.49 (ddd, J ¼ 11.8, 5.3, 3.9 Hz, 1H, 50-Hb), 3.55 (ddd,
J ¼ 11.8, 5.3, 3.9 Hz,1H, 50-Ha), 3.71 (td, J ¼ 3.9, 3.1 Hz,1H, 40-H), 4.17
(ddt, J ¼ 6.0, 4.2, 3.2 Hz,1H, 30-H), 4.93 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.12
(d, J ¼ 4.2 Hz, 1H, 30-OH), 6.14 (dd, J ¼ 7.6, 5.9 Hz, 1H, 10-H), 7.06 (t,
(101 MHz, DMSO‑d6) d 13.89 (-CH2-CH3), 22.05, 26.48, 28.55, 28.64,
28.73, 31.22 (eNHeCH2-(CH2)6-), 39.73 (20-C), 40.28 (eNHeCH2-),
61.42 (50-C), 70.46 (30-C), 84.83 (10-C), 87.15 (40-C), 94.70 (5-C),
139.60 (6-C), 155.18 (2-C), 163.29 (4-C). HRMS (ESI) of C17H29N3O4,
m/z: calcd [MþH]þ 340.2231, found: 340.2246; calcd [MþNa]þ
8

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
J ¼ 5.7 Hz,1H, N4eH), 7.52 (d, J ¼ 1.2 Hz,1H, 6-H). 13C NMR (75 MHz,
J ¼ 13.2, 5.9, 3.3 Hz, 1H, 20-Hb), 2.84 (dd, J ¼ 8.6, 6.5 Hz, 2H,
eNHeCH2-CH2-), 3.51e3.56 (m, 2H, eNHeCH2-), 3.56e3.57 (m, 1H,
50-Ha), 3.60 (ddd, J ¼ 11.8, 5.3, 4.0 Hz, 1H, 50-Hb), 3.76 (td, J ¼ 3.9,
3.0 Hz, 1H, 40-H), 4.22 (dddd, J ¼ 6.1, 4.3, 3.4, 3.0 Hz, 1H, 30-H), 4.97
(t, J ¼ 5.3 Hz, 1H, 50-OH), 5.17 (d, J ¼ 4.2 Hz, 1H, 30-OH), 6.20 (dd,
J ¼ 7.6, 5.9 Hz, 1H, 10-H), 7.16e7.20 (m, 1H, p-Ph), 7.20e7.22 (m, 2H,
o-Ph), 7.22e7.26 (m, 1H, N4eH), 7.26e7.32 (m, 2H, m-Ph), 7.60 (d,
DMSO‑d6)
d 13.11 (5-CH3), 13.87 (-CH2-CH3), 22.04, 26.13, 28.56,
31.06 (eNHe(CH2)4-), 40.12 (20-C), 40.21 (eNHeCH2-), 61.42 (50-C),
70.46 (30-C), 84.58 (10-C), 87.06 (40-C), 101.68 (5-C), 137.15 (6-C),
155.13 (2-C), 162.65 (4-C). HRMS (ESI) of C16H27N3O4, m/z: calcd
[MþH]þ 326.2074, found 326.2063.
4.2.6. N4-Octyl-5-methyl-20-deoxycytidine (4f). Prepared accord-
ing to the general procedure from 1b (163 mg) and octylamine
J ¼ 1.2 Hz, 1H, 6-H). 13C NMR (101 MHz, DMSO‑d6)
d 13.04 (5-CH3),
(129 mg, 164
m
l). Yield 138 mg (78%). UV: lmax 273 nm. 1H NMR
34.53 (eNHeCH2-CH2-), 40.15 (20-C), 41.73 (eNHeCH2-), 61.42 (50-
C), 70.45 (30-C), 84.64 (10-C), 87.10 (40-C),101.65 (5-C),126.04 (p-Ph),
128.30 (o-Ph), 128.58 (m-Ph), 137.37 (6-C), 139.53 (i-Ph), 155.07 (2-
C), 162.66 (4-C). HRMS (ESI) of C18H23N3O4, m/z: calcd [MþH]þ
346.1761, found 346.1763; calcd [MþNa]þ 368.1581, found:
368.1581.
(400 MHz, DMSO‑d6)
d
0.84 (t, J ¼ 6.8 Hz, 3H, eCH2-CH3), 1.21e1.30
(m, 10H, eNHe(CH2)2-(CH2)5-), 1.46e1.55 (m, 2H, eNHeCH2-CH2-),
1.84 (s, 3H, 5-CH3), 1.95 (ddd, J ¼ 13.3, 7.7, 6.0 Hz, 1H, 20-Ha), 2.06
(ddd, J ¼ 13.1, 6.0, 3.2 Hz,1H, 20-Hb), 3.25e3.31 (m, 2H, eNHeCH2-),
3.54 (ddd, J ¼ 12.0, 5.3, 3.9 Hz, 1H, 50-Ha), 3.59 (ddd, J ¼ 11.8, 5.5,
3.8 Hz, 1H, 50-Hb), 3.75 (td, J ¼ 3.9, 3.3 Hz, 1H, 40-H), 4.21 (ddt,
J ¼ 6.0, 4.3, 3.3 Hz, 1H, 30-H), 4.96 (t, J ¼ 5.3 Hz, 1H, 50-OH), 5.15 (d,
J ¼ 4.3 Hz, 1H, 30-OH), 6.17 (dd, J ¼ 7.6, 6.0 Hz, 1H, 10-H), 7.10 (t,
J ¼ 5.7 Hz, 1H, N4eH), 7.56 (s, 1H, 6-H). 13C NMR (101 MHz,
4.2.10. N4-(4-Phenylbutyl)-20-deoxycytidine (4j). Prepared ac-
cording to the general procedure from 1a (156 mg) and 4-
phenylbutylamine (149 mg, 158
m
l). Yield 127 mg (71%). UV: lmax
272 nm. 1H NMR (400 MHz, DMSO‑d6)
d
1.45e1.54 (m, 2H, -CH2-
DMSO‑d6)
d
13.05 (5-CH3), 13.84 (-CH2-CH3), 22.01, 26.43, 28.54,
CH2-C6H5), 1.54e1.64 (m, 2H, eNHeCH2-CH2-), 1.93 (ddd, J ¼ 13.3,
7.6, 6.0 Hz,1H, 20-Ha), 2.10 (ddd, J ¼ 13.2, 6.0, 3.2 Hz,1H, 20-Hb), 2.59
(t, J ¼ 7.5 Hz, 2H, -CH2-C6H5), 3.20e3.30 (m, 2H, eNHeCH2-), 3.53
(ddd, J ¼ 11.8, 5.2, 4.0 Hz, 1H, 50-Hb), 3.57 (ddd, J ¼ 12.0, 5.2, 4.0 Hz,
1H, 50-Ha), 3.77 (td, J ¼ 4.0, 3.0 Hz, 1H, 40-H), 4.20 (ddt, J ¼ 6.0, 4.3,
3.2 Hz, 1H, 30-H), 4.93 (t, J ¼ 5.2 Hz, 1H, 50-OH), 5.17 (d, J ¼ 4.3 Hz,
1H, 30-OH), 5.73 (d, J ¼ 7.5 Hz, 1H, 5-H), 6.17 (t, J ¼ 7.3, 6.2 Hz, 1H, 10-
H), 7.10e7.18 (m, 1H, p-Ph), 7.18e7.22 (m, 2H, o-Ph), 7.22e7.34 (m,
2H, m-Ph), 7.66 (t, J ¼ 5.6 Hz, 1H, N4eH), 7.72 (d, J ¼ 7.5 Hz, 1H, 6-H).
28.61, 28.76, 31.19 (eNHeCH2-(CH2)6-), 40.05 (20-C), 40.17
(eNHeCH2-), 61.39 (50-C), 70.42 (30-C), 84.56 (10-C), 87.03 (40-C),
101.63 (5-C), 137.09 (6-C), 155.09 (2-C), 162.61 (4-C). HRMS (ESI) of
C
18H31N3O4, m/z: calcd [MþH]þ 354.2387, found 354.2383; calcd
[MþNa]þ 376.2207, found: 376.2204.
4.2.7. N4-Decyl-5-methyl-20-deoxycytidine (4g). Prepared ac-
cording to the general procedure from 1b (163 mg) and decylamine
(157 mg, 200
(400 MHz, DMSO‑d6)
m
l). Yield 147 mg (77%). UV: lmax 273 nm. 1H NMR
d
0.84 (t, J ¼ 6.8 Hz, 3H, eCH2-CH3), 1.21e1.28
13C NMR (101 MHz, methanol-d4)
d 29.62 (-CH2-CH2-C6H5), 29.90
(m, 14H, eNHe(CH2)2-(CH2)7-), 1.45e1.55 (m, 2H, eNHeCH2-CH2-),
1.84 (d, J ¼ 1.0 Hz, 3H, 5-CH3), 1.95 (ddd, J ¼ 13.3, 7.8, 6.1 Hz, 1H, 20-
Ha), 2.06 (ddd, J ¼ 13.1, 6.0, 3.2 Hz, 1H, 20-Hb), 3.25e3.31 (m, 2H,
eNHeCH2-), 3.54 (ddd, J ¼ 11.7, 5.3, 4.0 Hz, 1H, 50-Ha), 3.59 (ddd,
J ¼ 11.7, 5.3, 3.8 Hz, 1H, 50-Hb), 3.75 (ddd, J ¼ 4.2, 3.7, 3.1 Hz, 1H, 40-
H), 4.21 (ddt, J ¼ 6.0, 4.2, 3.2 Hz, 1H, 30-H), 4.96 (t, J ¼ 5.3 Hz, 1H, 50-
OH), 5.16 (d, J ¼ 4.2 Hz, 1H, 30-OH), 6.17 (dd, J ¼ 7.6, 5.9 Hz, 1H, 10-H),
7.09 (t, J ¼ 5.7 Hz, 1H, N4eH), 7.56 (d, J ¼ 1.2 Hz, 1H, 6-H). 13C NMR
(eNHeCH2-CH2-), 36.47 (-CH2-C6H5), 41.39 (20-C), 41.89
(eNHeCH2-), 62.86 (50-C), 72.09 (30-C), 87.41 (10-C), 88.73 (40-C),
97.06 (5-C), 126.74 (p-Ph), 129.30 (o-Ph), 129.42 (m-Ph), 140.90 (6-
C), 143.54 (i-Ph), 158.62 (2-C), 165.40 (4-C). HRMS (ESI) of
C
19H25N3O4, m/z: calcd [MþH]þ 360.1918, found 360.1929.
4.2.11. N4-Octylcytidine (4k). Prepared according to the general
procedure from 1c (185 mg) and octylamine (129 mg, 164
139 mg (78%). UV: lmax 272 nm. 1H NMR (300 MHz, DMSO‑d6)
0.86 (t, 6.7 Hz, 3H, eCH2-CH3), 1.25e1.28 (m, 10H,
ml). Yield
(101 MHz, DMSO‑d6)
d
13.08 (5-CH3), 13.87 (-CH2-CH3), 22.05,
d
J
¼
26.45, 28.57, 28.67, 28.83, 28.94, 28.99, 31.26 (eNHeCH2-(CH2)8-),
40.14 (20-C), 40.20 (eNHeCH2-), 61.42 (50-C), 70.45 (30-C), 84.59 (10-
C), 87.05 (40-C), 101.66 (5-C), 137.11 (6-C), 155.12 (2-C), 162.64 (4-C).
HRMS (ESI) of C20H35N3O4, m/z: calcd [MþH]þ 382.2700, found
382.2707; calcd [MþNa]þ 404.2520, found 404.2527.
eNHe(CH2)2-(CH2)5-), 1.44e1.53 (m, 2H, eNHeCH2-CH2-),
3.18e3.27 (m, 2H, eNHeCH2-), 3.54 (ddd, J ¼ 12.1, 5.3, 3.5 Hz, 1H,
50-Ha), 3.66 (ddd, J ¼ 12.0, 5.1, 3.1 Hz, 1H, 50-Hb), 3.82 (ddd, J ¼ 4.3,
3.5, 3.0 Hz, 1H, 40-H), 3.89e3.99 (m, 2H, 20-H þ 30-H), 4.94 (d,
J ¼ 4.8 Hz, 1H, 30-OH), 5.01 (t, J ¼ 5.2 Hz, 1H, 50-OH), 5.24 (d,
J ¼ 4.7 Hz,1H, 20-OH), 5.72 (d, J ¼ 7.5 Hz,1H, 5-H), 5.77 (d, J ¼ 3.5 Hz,
1H, 10-H), 7.65 (t, J ¼ 5.5 Hz, 1H, N4eH), 7.77 (d, J ¼ 7.5 Hz, 1H, 6-H).
4.2.8. N4-Dodecyl-5-methyl-20-deoxycytidine (4h). Prepared ac-
cording to the general procedure from 1b (163 mg) and dodecyl-
amine (185 mg). Yield 147 mg (77%). UV: lmax 273 nm. 1H NMR
13C NMR (101 MHz, DMSO‑d6)
d 13.87 (-CH2-CH3), 22.01, 26.43,
(300 MHz, DMSO‑d6)
d
0.86 (t, J ¼ 6.8 Hz, 3H, eCH2-CH3), 1.23e1.28
28.47, 28.60, 28.68, 31.18 (eNHeCH2-(CH2)6-), 39.83 (eNHeCH2-),
60.60 (50-C), 69.41 (30-C), 73.93 (20-C), 84.07 (40-C), 89.12 (10-C),
94.61 (5-C), 140.14 (6-C), 155.05 (2-C), 162.91 (4-C). HRMS (ESI) of
(m, 18H, eNHe(CH2)2-(CH2)9-), 1.47e1.56 (m, 2H, eNHeCH2-CH2-),
1.84 (d, J ¼ 1.0 Hz, 3H, 5-CH3), 1.95 (ddd, J ¼ 13.2, 7.8, 6.0 Hz, 1H, 20-
Ha), 2.06 (ddd, J ¼ 13.1, 6.0, 3.2 Hz, 1H, 20-Hb), 3.19e3.32 (m, 2H,
eNHeCH2-), 3.53 (ddd, J ¼ 11.8, 5.3, 4.2 Hz, 1H, 50-Hb), 3.59 (ddd,
J ¼ 11.7, 5.3, 3.9 Hz, 1H, 50-Ha), 3.75 (ddd, J ¼ 4.1, 3.6, 3.0 Hz, 1H, 40-
H), 4.21 (dddd, J ¼ 6.0, 4.2, 3.2, 2.8 Hz, 1H, 30-H), 4.97 (t, J ¼ 5.3 Hz,
1H, 50-OH), 5.16 (d, J ¼ 4.2 Hz, 1H, 30-OH), 6.18 (dd, J ¼ 7.6, 6.0 Hz,
1H, 10-H), 7.10 (t, J ¼ 5.7 Hz, 1H, N4eH), 7.56 (d, J ¼ 1.2 Hz, 1H, 6-H).
C
17H29N3O5, m/z: calcd [MþH]þ 356.2180, found 356.2174; calcd
[MþH]þ 378.1999, found 378.1999.
4.2.12. N4-Decylcytidine (4l). Prepared according to the general
procedure from 1c (185 mg) and decylamine (157 mg, 200
132 mg (78%). (4l). UV: lmax 272 nm. 1H NMR (400 MHz, DMSO‑d6)
0.85 (t, 6.8 Hz, 3H, eCH2-CH3), 1.23e1.28 (m, 14H,
ml). Yield
d
J
¼
13C NMR (75 MHz, DMSO‑d6)
d
13.11 (5-CH3), 13.90 (-CH2-CH3),
eNHe(CH2)2-(CH2)7-), 1.44e1.50 (m, 2H, eNHeCH2-CH2-),
3.18e3.24 (m, 2H, eNHeCH2-), 3.53 (ddd, J ¼ 12.1, 5.4, 3.5 Hz, 1H,
50-Ha), 3.64 (ddd, J ¼ 12.1, 5.2, 3.1 Hz, 1H, 50-Hb), 3.80 (ddd, J ¼ 4.1,
3.5, 3.0 Hz, 1H, 40-H), 3.89e3.96 (m, 2H, 20-H þ 30-H), 4.92 (d,
J ¼ 5.2 Hz, 1H, 30-OH), 4.99 (t, J ¼ 5.2 Hz, 1H, 50-OH), 5.23 (d,
J ¼ 5.1 Hz,1H, 20-OH), 5.71 (d, J ¼ 7.5 Hz, 1H, 5-H), 5.75 (d, J ¼ 3.7 Hz,
1H, 10-H), 7.63 (t, J ¼ 5.5 Hz, 1H, N4eH), 7.75 (d, J ¼ 7.4 Hz, 1H, 6-H).
22.06, 26.45, 28.57, 28.68, 28.84, 29.00, 29.03, 31.27 (eNHeCH2-
(CH2)10-), 40.11 (20-C), 40.24 (eNHeCH2-), 61.40 (50-C), 70.43 (30-C),
84.58 (10-C), 87.07 (40-C), 101.65 (5-C), 137.18 (6-C), 154.93 (2-C),
162.50 (4-C). HRMS (ESI) of C21H37N3O4, m/z: calcd [MþH]þ
410.3013, found 410.3016.
4.2.9. N4-(2-Phenylethyl)-5-methyl-20-deoxycytidine (4i). Pre-
pared according to the general procedure from 1b (163 mg) and 2-
13C NMR (101 MHz, DMSO‑d6)
d 13.87 (-CH2-CH3), 22.03, 26.45,
phenylethylamine (121 mg, 125
m
l). Yield 147 mg (89%). UV: lmax
28.53, 28.64, 28.75, 28.92, 28.97, 31.23 (eNHeCH2-(CH2)8-), 39.75
(eNHeCH2-), 60.64 (50-C), 69.43 (30-C), 73.93 (20-C), 84.03 (40-C),
89.19 (10-C), 94.54 (5-C), 140.12 (6-C), 155.32 (2-C), 163.18 (4-C).
276 nm. 1H NMR (400 MHz, DMSO‑d6)
d
1.84 (d, J ¼ 1.1 Hz, 3H, 5-
CH3), 1.97 (ddd, J ¼ 13.2, 7.6, 6.1 Hz, 1H, 20-Ha), 2.08 (ddd,
9

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
HRMS (ESI) of C19H33N3O5, m/z: calcd [MþH]þ 384.2493, found
384.2492; calcd [MþNa]þ 406.2312, found 406.2311.
dissolved in anhydrous dioxane (3 ml). A corresponding 1-
alkylamine (1 mmol) was added to the dioxane solution, cooled
to 0 ꢂC. The mixture was stirred for 20 h at room temperature, then
3 ml of conc. aq. ammonia solution were added and the mixture
was stirred for 40 h at room temperature. The solvents were
evaporated; the compounds were purified on a silica gel column
(2 ꢃ 15 cm) in a gradient of concentrations of ethanol in chloroform
(0e15%) or of ethanol in ethyl acetate (0e10%). The target fractions
were evaporated in vacuum to give the expected compounds as an
amorphous mass in each case. 30-O-(8-tert-Butyldimethylsilyloxy-
3,6-dioxaoctyloxycarbonyl)-50-О-(tert-butyldimethylsilyl)-N4-
alkyl-5-methyl-20-deoxycytidines were dissolved in dioxane (3 ml),
then tetrabutylammonium fluoride trihydrate (380 mg, 1.2 mmol)
was added. The mixture was stirred at 25 ꢂC for 8 h and then the
solvents were evaporated. The products (4n-4o) were isolated by
column chromatography in a chloroform:ethanol (9:1 v/v) eluting
system.
4.2.13. N4-(3,6,9-Trioxadecyl)-5-methyl-20-deoxycytidine (4 m).
Prepared according to the general procedure from 1b (163 mg,
0.5 mmol) and 1-amino-3,6,9-trioxadecane (106 mg, 0.65 mmol).
Yield 132 mg (68%). UV: lmax 272 nm. 1H NMR (300 MHz, DMSO‑d6)
d
1.86 (d, J ¼ 1.0 Hz, 3H, 5-CH3),1.97 (ddd, J ¼ 13.3, 7.6, 6.0 Hz,1H, 20-
Ha), 2.09 (ddd, J ¼ 13.1, 6.0, 3.3 Hz, 1H, 20-Hb), 3.24 (s, 3H, -O-CH3),
3.39e3.44 (m, 2H, eNHeCH2-), 3.44e3.56 (m, 10H,
NHeCH2eCH2eOeCH2eCH2eOeCH2eCH2eO-), 3.56e3.59 (m, 1H,
50-Hb), 3.61 (ddd, J ¼ 11.8, 5.3, 3.8 Hz, 1H, 50-Ha), 3.77 (ddd, J ¼ 4.2,
3.7, 3.0 Hz, 1H, 40-H), 4.23 (ddt, J ¼ 6.0, 4.3, 3.3 Hz, 1H, 30-H), 4.97 (t,
J ¼ 5.3 Hz, 1H, 50-OH), 5.16 (d, J ¼ 4.3 Hz, 1H, 30-OH), 6.19 (dd, J ¼ 7.5,
6.0 Hz, 1H, 10-H), 7.11 (t, J ¼ 5.3 Hz, 1H, N4eH), 7.61 (d, J ¼ 1.1 Hz, 1H,
6-H). 13C NMR (75 MHz, Acetonitrile-d3)
d
12.37 (5-CH3), 40.35 (20-
C), 40.40 (eNHeCH2-), 57.97 (-O-CH3), 61.83 (50-C), 68.67, 69.98,
70.02, 70.10, 71.63 (NHeCH2eCH2eOeCH2eCH2eOeCH2eCH2eO-
), 70.88 (30-C), 86.25 (10-C), 87.42 (40-C), 102.55 (5-C), 138.11 (6-C),
156.25 (2-C), 163.48 (4-C). HRMS (ESI) of C17H29N3O7, m/z: calcd
[MþH]þ 388.2078, found 388.2080; calcd [MþNa]þ 410.1898, found
410.1900.
4.3.2.1. 30-O-(8-Hydroxy-3,6-dioxaoctyloxycarbonyl)-N4-decyl-20-
deoxycytidines (4n). Prepared according to the general procedure
from 8 and decylamine (1 mmol, 199
ml). Yield 175 mg (63%). UV:
lmax 273 nm. 1H NMR (400 MHz, DMSO‑d6)
d
0.85 (t, J ¼ 6.8 Hz, 3H,
eCH2-CH3), 1.23e1.29 (m, 14H, eNHe(CH2)2-(CH2)7-), 1.46e1.56
(m, 2H, eNHeCH2-CH2-), 1.84 (d, J ¼ 1.1 Hz, 3H, 5-CH3), 2.19 (ddd,
J ¼ 14.2, 8.8, 5.9 Hz, 1H, 20-Ha), 2.28 (ddd, J ¼ 14.0, 5.7, 1.8 Hz, 1H, 20-
Hb), 3.25e3.29 (m, 2H, eNHeCH2-), 3.40e3.65 (m, 12H, 50-СН2 þ -
CH2OC2H4OC2H4OH), 4.02 (td, J ¼ 3.6, 1.8 Hz, 1H, 40-H), 4.20e4.25
(m, 2H, eCO3-CH2-), 4.54 (t, J ¼ 5.5 Hz, 1H, eCH2-CH2-OH), 5.13 (dt,
J ¼ 5.9, 1.8 Hz, 1H, 30-H), 5.15 (t, J ¼ 5.3 Hz, 1H, 50-OH), 6.17 (dd,
J ¼ 8.8, 5.6 Hz, 1H, 10-H), 7.14 (t, J ¼ 5.7 Hz, 1H, N4eH), 7.54e7.58 (m,
4.3. 30-O-(8-Hydroxy-3,6-dioxaoctyloxycarbonyl)-N4-alkyl-20-
deoxycytidines (4n-4o)
4.3.1. 30-O-(8-tert-Butyldimethylsilyloxy-3,6-
dioxaoctyloxycarbonyl)-50-О-(tert-butyldimethylsilyl)thymidine (8)
50-O-(tert-Butyldimethylsilyl)thymidine (6, 820 mg, 2.3 mmol)
was dissolved in dry dimethylformamide (3 ml) and N,N0-carbon-
ylditriazole was added (1.5 g, 0.84 mmol). The mixture was heated
at 37 ꢂC for 24 h. Then anhydrous triethylene glycol (1.65 g, 1.5 ml,
9.2 mmol) and dioxane (3 ml) were added. The mixture was heated
at 37 ꢂC for 24 h, and then solvents were evaporated. The product
was extracted in (chloroform þ hexane, 2:1 v/v) - water system, the
1H, 6-H). 13C NMR (101 MHz, DMSO‑d6)
d 13.09 (5-CH3), 13.88
(-CH2-CH3), 22.02, 26.41, 28.51, 28.63, 28.78, 28.90, 28.95, 31.23
(eNHeCH2-(CH2)8-), 36.96 (20-C), 40.18 (eNHeCH2-), 60.18 (-CH2-
CH2-OH), 61.37 (50-C), 67.01, 68.06, 69.70, 69.74, 72.31 (-CO3-
C2H4OC2H4OCH2eCH2eOH), 78.62 (30-C), 84.04 (10-C), 84.60 (40-C),
102.02 (5-C), 136.91 (6-C), 153.84 (-CO3-),154.90 (2-C),162.65 (4-C).
HRMS (ESI) of C27H47N3O9, m/z: calcd [MþH]þ 558.3385, found
558.3400.
organic
layer
was
evaporated.
30-O-(8-Hydroxy-3,6-
dioxaoctyloxycarbonyl)-50-О-(tert-butyldimethylsilyl)thymidine
(7) was isolated by column chromatography in chloroform:ethyl
acetate:ethanol (5:5:0.1 v/v) eluting system to give 840 mg (73%).
The product 7 (820 mg, 1.7 mmol) was dissolved in dry pyridine
(5 ml) and tert-butyldimethylchlorosilane (453 mg, 3 mmol) was
added. The mixture was stirred for 16 h at 4ꢂС, then solvents were
evaporated. The product 8 was isolated by column chromatography
in chloroform:ethanol (20:1 v/v) eluting system to give 680 mg
4.3.2.2.
30-O-(8-Hydroxy-3,6-dioxaoctyloxycarbonyl)-N4-
dodecyl-20-deoxycytidines (4o). Prepared according to the general
procedure from 8 and dodecylamine (1 mmol, 185 mg). Yield
191 mg (65%). UV: lmax 273 nm. 1H NMR (300 MHz, DMSO‑d6)
d
0.85 (t,
J
¼
6.6 Hz, 3H, eCH2-CH3), 1.21e1.30 (m, 18H,
eNHe(CH2)2-(CH2)9-), 1.46e1.57 (m, 2H, eNHeCH2-CH2-), 1.86 (s,
3H, 5-CH3), 2.20 (ddd, J ¼ 14.1, 8.8, 5.8 Hz, 1H, 20-Ha), 2.30 (ddd,
J ¼ 14.1, 5.8, 1.8 Hz, 1H, 20-Hb), 3.26e3.33 (m, 2H, eNHeCH2-),
3.40e3.66 (m, 12H, 50-СН2 þ -CH2OC2H4OC2H4OH), 4.04 (td, J ¼ 3.6,
1.8 Hz, 1H, 40-H), 4.18e4.27 (m, 2H, eCO3-CH2-), 4.55 (t, J ¼ 5.4 Hz,
1H, eCH2-CH2-OH), 5.15 (dt, J ¼ 5.8, 1.8 Hz, 1H, 30-H), 5.19 (t,
J ¼ 5.4 Hz, 1H, 50-OH), 6.19 (dd, J ¼ 8.8, 5.6 Hz, 1H, 10-H), 7.19 (t,
J ¼ 5.6 Hz,1H, N4eH), 7.59 (s, 1H, 6-H). 13C NMR (75 MHz, DMSO‑d6)
(49%). 1H NMR (400 MHz, CDCl3)
SieOeCH2eCH2-), 0.12 (s, 6H, 50-OSi(C(CH3)3)(CH3)2), 0.88 (s, 9H,
(CH3)2((CH3)3C)SieOeCH2eCH2-), 0.92 (s, 9H,
50-
d 0.05 (s, 6H, (CH3)2((CH3)3C)
OSi(C(CH3)3)(CH3)2), 1.91 (s, 3H, 5-CH3), 2.10 (ddd, J ¼ 14.0, 8.8,
6.3 Hz, 1H, 20-Ha), 2.49 (ddd, J ¼ 13.9, 5.4, 1.2 Hz, 1H, 20-Hb), 3.54 (t,
J ¼ 5.4 Hz, 2H, (CH3)2((CH3)3C)SieOeCH2eCH2-), 3.65 (s, 4H, eCH2-
O-CH2-CH2-O-CH2-),
3.74
(m,
4H,
eCO3-CH2-CH2-
þ(CH3)2((CH3)3C)SieOeCH2eCH2-), 3.89 (m, 1H, 50-Hb), 3.92 (m,
1H, 50-Ha), 4.18 (m, 1H, 40-H), 4.29 (m, 2H, eCO3-CH2-), 5.15 (m, 1H,
30-H), 6.34 (dd, J ¼ 9.2, 5.3 Hz, 1H, 10-H), 7.50 (s, 1H, 6-H), 10.79 (q,
J ¼ 1.3, 0.9 Hz, 1H, 3-NH).
d 13.10 (5-CH3), 13.86 (-CH2-CH3), 22.09, 26.51, 28.59, 28.73, 28.90,
29.05, 29.08, 31.31 (eNHeCH2-(CH2)10-), 37.12 (20-C), 40.27
(eNHeCH2-), 60.25 (-CH2-CH2-OH), 61.43 (50-C), 67.02, 68.13,
69.76, 69.81, 72.38 (-CO3-C2H4OC2H4OCH2eCH2eOH), 78.72 (30-C),
84.18 (10-C), 84.74 (40-C), 102.15 (5-C), 136.90 (6-C), 153.90 (-CO3-),
155.08 (2-C), 162.72 (4-C). HRMS (ESI) of C29H51N3O9, m/z: calcd
[MþH]þ 586.3698, found 586.3717.
4.3.2. General method for the synthesis of compounds 4n-4o
2-Chlorophenyl dichlorophosphate (188 mg, 126 ml, 0.77 mmol)
was added to a solution of 30-O-(8-tert-butyldimethylsilyloxy-3,6-
dioxaoctyloxycarbonyl)-50-О-(tert-butyldimethylsilyl)thymidine
(8, 324 mg, 0.5 mmol) and 1,2,4-triazole (200 mg, 3 mmol) in
anhydrous pyridine, cooled to 0 ꢂC. The mixture was stirred for
20 h at room temperature, with solvents evaporated afterwards.
The residue was partitioned between chloroform and 0.5 M sodium
bicarbonate, the chloroform layer was washed with water, dried
over Na2SO4, the solvent was evaporated and the residue was
4.4. Chemical hydrolysis of compounds 4n and 4o
Compounds 4n and 4o (5 mg) were dissolved in dioxane
(0.25 ml) and these aliquots were subjected to chemical hydrolysis.
Each sample was added and incubated for 24 h at 37 ꢂC in the three
following media: 0.2 M glycine þ0.2 M HCl buffer (pH 2.2); 0.1 M
potassium phosphate buffer (pH 7.5, 0.25 ml); 0.2 M glycine þ 0.2 M
10

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
NaOH buffer (pH 9.0). Aliquots were taken out and analyzed by TLC
4.5.5. Fungal growth inhibition
(in chloroform:ethanol 9:1 v/v).
4.5.5.1. Fungi strains. The following fungi strains were used in the
study: Aspergillus versicolor STG-25G (SRX7729174; MK260015.1),
Ulocladium chartarum STG-36 (SRX7729176), Cladosporium hal-
otolerans STG-52B (SRX7729178; MK258720.1), Aspergillus creber
STG-57 (SRX7729180; MK266993.1), Aspergillus versicolor STG-86
4.5. Biological evaluation
4.5.1. Cytotoxic activity
The line of lung carcinoma A549 (ATCC N CCL-185) was ob-
tained from the collection of the Engelhardt Institute of Molecular
Biology, Russian Academy of Sciences.
Adherent A549 cells being passaged were cultured in DMEM
medium containing 10% fetal calf serum, 2 mM glutamine, at 37ꢂС
in an atmosphere of 5% СО2 at a humidity of 90%. A suspension
culture of Jurkat cells was cultured in medium RPMI containing 10%
fetal calf serum, 2 mM glutamine, under the same conditions.
Сytotoxicity of the compounds was determined by the MTT test
[34]. The CC50 value is the concentration of a compound required to
inhibit 50% cell culture growth.
ꢂ
(SRX7729182;
(SRX7729186),
MK262781.1),
Cladosporium
Aspergillus
parahalotolerans
sp.
STG-93W
STG-93B
(SRX7729188; MK262909.1), Simplicilium lamellicola STG-96
(SRX7729190; MK262921.1), Microascus paisii STG-103
(SRX7729192), and Aspergillus amoenus STG-106 (SRX7729194;
MK268342.1).
4.5.5.2. Fungal growth inhibition. The filamentous fungi cultivated
on Czapek-Dox agar (CDA) medium (30 g/l sucrose, 2 g/l NaNO3,1 g/
l K2HPO4, 0.5 g/l MgSO4 ꢃ 7H2O, 0.5 g/l KCl, 0.01 g/l FeSO4 ꢃ 7H2O,
20 g/l agar, рН 7.0e7.4). To determine the toxicity effect of N4-
modified cytidines on the growth of fungal cells the drop-dilution
method was used with some modifications as described earlier
[45e47]. Cells were collected from agar slants and diluted with 0.9%
NaCl solution up to OD600 ¼ 0.3 (basic concentration), followed by
4.5.2. Acute and chronic toxicity of nucleoside 4h
Acute and chronic toxicity of nucleoside 4h were estimated by
intraperitoneally injections of compound into white mice (average
weight of the mouse 30e35 g, 6 mice for each of the 8 doses of 4h, 3
males and 3 females). To determine acute toxicity, nucleoside 4h
was dissolved in 30% aqueous solution of Tween-80 and injected
into mice 0.2 ml intraperitoneally at doses of 80e400 mg/kg (0.2 ml
of 30% Tween-80 used as a positive control) and the animals were
observed for a week.
To determine the toxicity of nucleoside 4h with subchronic
administration, the drug was administered daily one time per day
for 5 consecutive days at doses less than LD50 (<270 mg/kg) and the
animals were observed for four weeks.
serial tenfold dilutions with the same solvent. Then, 1.5 ml of cell
suspension was inoculated onto Petri dishes with CDA prepared
with or without the addition of compounds, N4-modified cytidines
or 5-fluorocytosine (5FC) for positive control in the concentration
0.5e3 mM. The inoculated plates were incubated for 48 days at
26ꢂС. The inhibitory effects of the compounds were measured
every three days after inoculation and evaluated by ratio of colony
growth on CDA medium supplemented with N4-modified cytidines
or 5FC to the control growth (CDA medium without any additions).
To determine the percent of fungal growth inhibition (FGI) used the
formula: FGI % ¼ [(DceDt)/Dc] ꢃ 100, where Dc indicates the colony
diameter in control set, and Dt indicates the colony diameter in
treatment set. The data recorded were measured in triplicate and
repeated at least twice.
Statistical analysis was performed using the Statistics sub-
package from SciPy.
4.5.3. Hydrolysis of compounds 4n and 4o in human blood serum
Human blood serum (100%, 99
tion of compounds 4n and 4o in DMSO (0.5
incubated at 37 ꢂC. Aliquots were taken out after certain intervals,
m
l) was added to 100 mM solu-
m
l), and the mixture was
Declaration of competing interest
ethanol (40
ml) was added, the mixture was kept for
20 min at ꢀ20 ꢂC, and centrifuged. The supernatants were evapo-
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
rated, the residues were dissolved in ethanol (50
by HPLC or TLC.
ml) and analyzed
4.5.4. In vitro antibacterial assay
Acknowledgments
4.5.4.1. Bacterial strains. The following test strains were used:
Gram-positive bacteria Bacillus subtilis АТСС 6633, Staphylococcus
aureus INA 00761 (MRSA), Leuconostoc mesenteroides VKPM B-4177;
mycobacteria Mycobacterium smegmatis mc2155, and M. smegmatis
VKPM Ac-1339; Gram-negative bacteria Pseudomonas aeruginosa
ATCC 27853 and fungi Aspergillus niger and Saccharomyces cer-
evisiae INA 01129 from the collection of the Gause Institute of New
Antibiotics.
The synthesis, the cytotoxicity and antibacterial studies were
supported by the Russian Foundation for Basic Research (RFBR,
projects 20-04-00536 and 20-34-90056. The study of the anti-
fungal effects was supported by the RFBR (project 17-29-04349).
G.K. Nuraeva and I.A. Volkov are grateful to the Ministry of Science
and Higher Education of the Russian Federation for support of the
work on cultivation of filamentous fungi within the framework of
the state contract no. 075-00337-20-03 (project identifier FSMG-
2020-0007). Mass-spectrometry studies were supported by the
Program of fundamental research for state academies for
2013e2020 (project identifier 01201363818).
4.5.4.2. In vitro study of the antibacterial effect. Test strains were
incubated in modified Gause’s nutrient medium N 2. The level of
ꢂ
infection with test cultures was 106 cells/ml. A compound being
tested was dissolved in 50% aq. methanol. Ten volume percent of
tested compound was added to the nutrient medium. Samples
without the addition of substances, antibiotics in medical use
(amikacin, ciprofloxacin, isoniazidi, rifampicin, oxacillin and van-
comycin) and samples of medium supplemented with a mixture of
solvents served as controls of the test culture growth. Fungal test
cultures and L. mesenteroides were incubated at 28 ꢂС, and all other
strains were incubated at 37 ꢂС.
The authors are grateful to Dr. R.A. Novikov (Engelhardt Institute
of Molecular Biology RAS) for NMR investigations.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113212.
11

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
I.G. Sumarukova, I.L. Karpenko, S.N. Kochetkov, L.A. Alexandrova, Novel 5-
substituted derivatives of 2’-deoxy-6-azauridine with antibacterial activity,
J. Antibiot. (Tokyo) 72 (2019) 535e544, https://doi.org/10.1038/s41429-019-
0158-z.
References
[1] E.D. Brown, G.D. Wright, Antibacterial drug discovery in the resistance era,
Nature 529 (2016) 336e343, https://doi.org/10.1038/nature17042.
[2] S.A. Kraemer, A. Ramachandran, G.G. Perron, Antibiotic pollution in the
environment: from microbial ecology to public policy, Microorganisms 7
(2019), https://doi.org/10.3390/microorganisms7060180.
[3] Antibacterial Agents in Preclinical Development: an Open Access Database,
World Health Organization, 2019, pp. 1e20. https://apps.who.int/iris/
bitstream/handle/10665/330290/WHO-EMP-IAU-2019.12-eng.pdf?ua¼1.
[4] A.E.J. Yssel, J. Vanderleyden, H.P. Steenackers, Repurposing of nucleoside- and
nucleobase-derivative drugs as antibiotics and biofilm inhibitors,
J. Antimicrob. Chemother. 72 (2017) 2326e2333, https://doi.org/10.1093/jac/
dkx151.
[5] H.D. Marston, D.M. Dixon, J.M. Knisely, T.N. Palmore, A.S. Fauci, Antimicrobial
resistance, JAMA, J. Am. Med. Assoc. 316 (2016) 1193e1204, https://doi.org/
10.1001/jama.2016.11764.
[6] D.M. Livermore, Discovery research: the scientific challenge of finding new
antibiotics, J. Antimicrob. Chemother. 66 (2011) 1941e1944, https://doi.org/
10.1093/jac/dkr262.
[23] H.K. Akula, H. Kokatla, G. Andrei, R. Snoeck, D. Schols, J. Balzarini, L. Yang,
M.K. Lakshman, Facile functionalization at the C4 position of pyrimidine nu-
cleosides via amide group activation with (benzotriazol-1-yloxy)tris(dime-
thylamino)phosphonium hexafluorophosphate (BOP) and biological
evaluations of the products, Org. Biomol. Chem. 15 (2017) 1130e1139,
https://doi.org/10.1039/C6OB02334G.
[24] D. Loakes, D.M. Brown, N. Mahmood, J. Balzarini, E. De Clercq, Antiviral ac-
tivity of N4-aminocytidine derivatives related to AZT, Antivir. Chem. Chemo-
ther. 6 (1995) 9e16, https://doi.org/10.1177/095632029500600102.
[25] Zhigang Lv, W. He, X. Tian, J. Kang, Y. Liu, L. Peng, Q. Zheng, W. Yu Wang,
J. Chang, Design, synthesis, and biological evaluation of new N 4-Substituted
20-deoxy-20-fluoro-40-azido cytidine derivatives as potent anti-HBV agents,
Eur. J. Med. Chem. 101 (2015) 103e110, https://doi.org/10.1016/
j.ejmech.2015.06.030.
[26] Q. Yang, J. Kang, L. Zheng, X.J. Wang, N. Wan, J. Wu, Y. Qiao, P. Niu, S.Q. Wang,
Y. Peng, Q. Wang, W. Yu, J. Chang, Synthesis and biological evaluation of 4-
substituted fluoronucleoside analogs for the treatment of hepatitis B virus
infection, J. Med. Chem. 58 (2015) 3693e3703, https://doi.org/10.1021/
jm5012963.
[7] L.P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Advances in the develop-
ment of nucleoside and nucleotide analogues for cancer and viral diseases,
Nat. Rev. Drug Discov. 12 (2013) 447e464, https://doi.org/10.1038/nrd4010.
[8] E. De Clercq, G. Li, Approved antiviral drugs over the past 50 years, Clin.
Microbiol. Rev. 29 (2016) 695e747, https://doi.org/10.1128/CMR.00102-15.
[9] M. Serpi, V. Ferrari, F. Pertusati, Nucleoside derived antibiotics to fight mi-
crobial drug resistance: new utilities for an established class of drugs? J. Med.
[27] K.J. Divakar, C.B. Reese, 4-(1,2,4-Triazol-1-yl)- and 4-(3-nitro-1,2,4-triazol-1-
yl)-1-(
b-D-2,3,5-tri-O-acetylarabinofuranosyl)pyrimidin-2(1H)-ones.
Valu-
able intermediates in the synthesis of derivatives of 1-(
b
-D-arabinofuranosyl)
cytosine (ara-C), J. Chem. Soc. Perkin Trans. 1 (1982) 1171e1176, https://
doi.org/10.1039/P19820001171.
Chem.
acs.jmedchem.6b00325.
59
(2016)
10343e10382,
https://doi.org/10.1021/
[28] T.S. Lin, Y.S. Gao, W.R. Mancini, Synthesis and biological activity of various 30-
azido and 30-amino analogues of 5-substituted pyrimidine deoxyribonucleo-
sides, J. Med. Chem. 26 (1983) 1691e1696, https://doi.org/10.1021/
jm00366a006.
[10] G. Niu, J. Zheng, H. Tan, Biosynthesis and combinatorial biosynthesis of anti-
fungal nucleoside antibiotics, Sci. China Life Sci. 60 (2017) 939e947, https://
doi.org/10.1007/s11427-017-9116-0.
[11] T.D.H. Bugg, R.V. Kerr, Mechanism of action of nucleoside antibacterial natural
product antibiotics, J. Antibiot. (Tokyo) 72 (2019) 865e876, https://doi.org/
10.1038/s41429-019-0227-3.
[12] B.P. Duckworth, D.J. Wilson, K.M. Nelson, H.I. Boshoff, C.E. Barry, C.C. Aldrich,
Development of a selective activity-based probe for adenylating enzymes:
profiling MbtA involved in siderophore biosynthesis from mycobacterium
tuberculosis, ACS Chem. Biol. 7 (2012) 1653e1658, https://doi.org/10.1021/
cb300112x.
[13] S. Van Calenbergh, S. Pochet, H. Munier-Lehmann, Drug design and identifi-
cation of potent leads against Mycobacterium tuberculosis thymidine
monophosphate kinase, Curr. Top. Med. Chem. 12 (2012) 694e705, https://
doi.org/10.2174/156802612799984580.
[29] D.H. Jornada, G.F. Dos Santos Fernandes, D.E. Chiba, T.R.F. De Melo, J.L. Dos
Santos, M.C. Chung, The prodrug approach: a successful tool for improving
drug
solubility,
Molecules
21
(2016),
https://doi.org/10.3390/
molecules21010042.
[30] S.D. Negrya, O.E. Efremenkova, V.O. Chekhov, M.A. Ivanov, P.N. Solyev,
I.G. Sumarukova, I.L. Karpenko, S.N. Kochetkov, L.A. Alexandrova Synthesis of
water-soluble prodrugs of 5-modified 2ʹ-deoxyuridines and their antibacterial
activity, J. Antibiot. (Tokyo) 73 (2020) 236e246, https://doi.org/10.1038/
s41429-019-0273-x.
[31] S.D. Negrya, M.V. Jasko, D.A. Makarov, P.N. Solyev, I.L. Karpenko,
O.V. Shevchenko, O.V. Chekhov, A.A. Glukhova, B.F. Vasilyeva, T.A. Efimenko,
I.G. Sumarukova, O.V. Efremenkova, S.N. Kochetkov, L.A. Alexandrova glycol
and phosphate depot-forms of 4- and/or 5-modified nucleosides exhibiting
antibacterial activity, Mol. Biol. Rus 54 (2021) N1.
[32] L. Alexandrova, S. Zicari, E. Matyugina, A. Khandazhinskaya, T. Smirnova,
S. Andreevskaya, L. Chernousova, C. Vanpouille, S. Kochetkov, L. Margolis,
Dual-targeted anti-TB/anti-HIV heterodimers, Antivir. Res. 145 (2017)
175e183, https://doi.org/10.1016/j.antiviral.2017.07.011.
[33] E. De Clercq, Antiviral drugs: current state of the art, J. Clin. Virol. 22 (2001)
73e89, https://doi.org/10.1016/S1386-6532(01)00167-6.
[14] N. Shakya, G. Garg, B. Agrawal, R. Kumar, Chemotherapeutic interventions
against tuberculosis, Pharmaceuticals
10.3390/ph5070690.
[15] E.R. Shmalenyuk, S.N. Kochetkov, L.A. Alexandrova, Novel inhibitors of
Mycobacterium tuberculosis growth based on modified pyrimidine nucleo-
sides and their analogues, Russ. Chem. Rev. 82 (2013) 896e915, https://
doi.org/10.1070/rc2013v082n09abeh004404.
[16] E.R. Shmalenyuk, L.N. Chernousova, I.L. Karpenko, S.N. Kochetkov,
T.G. Smirnova, S.N. Andreevskaya, A.O. Chizhov, O.V. Efremenkova,
L.A. Alexandrova, Inhibition of Mycobacterium tuberculosis strains H37Rv and
MDR MS-115 by a new set of C5 modified pyrimidine nucleosides, Bioorg.
Med.
j.bmc.2013.07.003.
[17] L.A. Alexandrova, E.R. Shmalenyuk, S.N. Kochetkov, V.
5 (2012) 690e718, https://doi.org/
[34] M. Niks, M. Otto, Towards an optimized MTT assay, J. Immunol. Methods 130
(1990) 149e151, https://doi.org/10.1016/0022-1759(90)90309-J.
[35] L.A. Aleksandrova, O.V. Efremenkova, V.L. Andronova, G.A. Galegov,
P.N. Solyev, I.L. Karpenko, S.N. Kochetkov, 5-(4-alkyl-1,2,3-triazol-1-yl)methyl
derivatives of 20-deoxyuridine as inhibitors of viral and bacterial growth, Russ.
Chem.
21
(2013)
4874e4884,
https://doi.org/10.1016/
J.
Bioorg.
Chem.
42
(2016)
677e684,
https://doi.org/10.1134/
V
Erokhin,
S1068162016050022.
T.G. Smirnova, S.N. Andreevskaia, L.N. Chernousova, New 5-modified pyrim-
idine nucleoside inhibitors of mycobacterial growth, Acta Naturae 2 (2010)
108e110, https://doi.org/10.32607/actanaturae.10780.
[36] Y. Zhang, K. Post-Martens, S. Denkin, New drug candidates and therapeutic
targets for tuberculosis therapy, Drug Discov. Today 11 (2006) 21e27, https://
doi.org/10.1016/S1359-6446(05)03626-3.
[18] E. Matyugina, A. Khandazhinskaya, L. Chernousova, S. Andreevskaya,
T. Smirnova, A. Chizhov, I. Karpenko, S. Kochetkov, L. Alexandrova, The syn-
thesis and antituberculosis activity of 5’-nor carbocyclic uracil derivatives,
Bioorg. Med. Chem. 20 (2012) 6680e6686, https://doi.org/10.1016/
j.bmc.2012.09.019.
[19] A.L. Khandazhinskaya, L.A. Alexandrova, E.S. Matyugina, P.N. Solyev,
O.V. Efremenkova, K.W. Buckheit, M. Wilkinson, R.W. Buckheit,
L.N. Chernousova, T.G. Smirnova, S.N. Andreevskaya, O.G. Leonova,
V.I. Popenko, S.N. Kochetkov, K.L. Seley-Radtke, Novel 50-norcarbocyclic py-
rimidine derivatives as antibacterial agents, Molecules 23 (2018), https://
doi.org/10.3390/molecules23123069.
ꢀ
ꢀ
ꢀ
[37] M. del M. Lopez-Miras, I. Martín-Sanchez, A. Yebra-Rodríguez, J. Romero-
~
Noguera, F. Bolívar-Galiano, J. Ettenauer, K. Sterflinger, G. Pinar, Contribution
of the microbial communities detected on an oil painting on canvas to its
biodeterioration, PloS One
journal.pone.0080198.
8 (2013), e80198, https://doi.org/10.1371/
[38] A. Zhgun, D. Avdanina, K. Shumikhin, N. Simonenko, E. Lyubavskaya, I. Volkov,
V. Ivanov, Detection of potential biodeterioration risks for tempera painting in
16th century exhibits from State Tretyakov Gallery, PloS One 15 (2020),
e0230591, https://doi.org/10.1371/journal.pone.0230591.
[39] Q. Li, B. Zhang, Z. He, X. Yang, Distribution and diversity of bacteria and fungi
colonization in stone monuments analyzed by high-throughput sequencing,
PloS One 11 (2016), e0163287, https://doi.org/10.1371/journal.pone.0163287.
[40] F. Poyatos, F. Morales, A.W. Nicholson, A. Giordano, Physiology of biodeteri-
oration on canvas paintings, J. Cell. Physiol. 233 (2018) 2741e2751, https://
doi.org/10.1002/jcp.26088.
[20] A.L. Khandazhinskaya, E.S. Matyugina, L.A. Alexandrova, V.A. Kezin,
L.N. Chernousova, T.G. Smirnova, S.N. Andreevskaya, V.I. Popenko,
O.G. Leonova, S.N. Kochetkov, Interaction of 5-substituted pyrimidine nucle-
oside analogues and M.Tuberculosis: a view through an electron microscope,
Biochimie
j.biochi.2020.03.004.
171e172
(2020)
170e177,
https://doi.org/10.1016/
[41] K. Sterflinger, Fungi: their role in deterioration of cultural heritage, Fungal
Biol. Rev. 24 (2010) 47e55, https://doi.org/10.1016/J.FBR.2010.03.003.
[21] P.J.G. Henriksson, A. Rico, M. Troell, D.H. Klinger, A.H. Buschmann, S. Saksida,
M.V. Chadag, W. Zhang, Unpacking factors influencing antimicrobial use in
global aquaculture and their implication for management: a review from a
systems perspective, Sustain. Sci. 13 (2018) 1105e1120, https://doi.org/
10.1007/s11625-017-0511-8.
~
[42] K. Sterflinger, G. Pinar, Microbial deterioration of cultural heritage and works
of art–tilting at windmills? Appl. Microbiol. Biotechnol. 97 (2013) 9637e9646,
https://doi.org/10.1007/s00253-013-5283-1.
[43] A. Unger, A.P. Schniewind, W. Unger, Conservation of Wood Artifacts : a
Handbook, Springer Berlin Heidelberg, 2001.
[22] S.D. Negrya, O.V. Efremenkova, P.N. Solyev, V.O. Chekhov, M.A. Ivanov,
[44] A.G. Domratcheva, A.A. Zhgun, N.V. Novak, V.V. Dzhavakhiya, The influence of
12

L.A. Alexandrova, M.V. Jasko, S.D. Negrya et al.
European Journal of Medicinal Chemistry 215 (2021) 113212
750e755, https://doi.org/10.1134/S0026261720060193.
[49] A.A. Zhgun, M.A. Ivanova, A.G. Domracheva, M.I. Novak, M.A. Elidarov,
K.G. Skryabin, Y.E. Bartoshevich, Genetic transformation of the mycelium
fungi Acremonium chrysogenum, Appl. Biochem. Microbiol. 44 (2008)
600e607, https://doi.org/10.1134/S0003683808060070.
chemical mutagenesis on the properties of the cyclosporine a high-producer
strain tolypocladium inflatum VKM F-3630D, Appl. Biochem. Microbiol. 54
(2018) 53e57, https://doi.org/10.1134/S0003683818010027.
€
€
[45] M.T. Hyvonen, T.A. Keinanen, G.K. Nuraeva, D.V. Yanvarev, M. Khomutov,
€ €
E.N. Khurs, S.N. Kochetkov, J. Vepsalainen, A.A. Zhgun, A.R. Khomutov, Hy-
droxylamine analogue of agmatine: magic bullet for arginine decarboxylase,
Biomolecules 10 (2020) 1e16, https://doi.org/10.3390/biom10030406.
[46] M.V. Dumina, A.A. Zhgun, I.V. Kerpichnikov, A.G. Domracheva, M.I. Novak,
A.Y. Valiachmetov, D.A. Knorre, F.F. Severin, M.A. Eldarov, Y.E. Bartoshevich,
Functional analysis of MFS protein CefT involved in the transport of beta-
lactam antibiotics in Acremonium chrysogenum and Saccharomyces cer-
evisiae, Appl. Biochem. Microbiol. 49 (2013) 368e377, https://doi.org/
10.1134/S0003683813040042.
[47] A.A. Zhgun, G.K. Nuraeva, M.V. Dumina, T.M. Voinova, V.V. Dzhavakhiya,
M.A. Eldarov, 1,3-Diaminopropane and spermidine upregulate lovastatin
production and expression of lovastatin biosynthetic genes in Aspergillus
terreus via LaeA regulation, Appl. Biochem. Microbiol. 55 (2019) 244e255,
https://doi.org/10.1134/S0003683819020170.
[50] M.V. Dumina, A.A. Zhgun, A.G. Domracheva, M.I. Novak, M.A. El’darov, Chro-
mosomal polymorphism of Acremonium chrysogenum strains producing
cephalosporin C, Russ. J. Genet. 48 (2012) 778e784, https://doi.org/10.1134/
S1022795412050067.
[51] M. V Dumina, A.A. Zhgun, M.I. Novak, A.G. Domratcheva, D. V Petukhov, V. V
Dzhavakhiya, M.A. Eldarov, I.E. Bartoshevitch, Comparative gene expression
profiling reveals key changes in expression levels of cephalosporin
C
biosynthesis and transport genes between low and high-producing strains of
Acremonium chrysogenum, World J. Microbiol. Biotechnol. 30 (2014)
2933e2941, https://doi.org/10.1007/s11274-014-1721-1.
[52] A.A. Zhgun, M.V. Dumina, T.M. Voinova, V.V. Dzhavakhiya, M.A. Eldarov, Role
of acetyl-CoA synthetase and LovE regulator protein of polyketide biosyn-
thesis in lovastatin production by wild-type and overproducing Aspergillus
terreus strains, Appl. Biochem. Microbiol. 54 (2018) 188e197, https://doi.org/
10.1134/S0003683818020138.
[48] A.A. Zhgun, D.A. Avdanina, B.T. Shagdarova, E.V. Troyan, G.K. Nuraeva,
M.P. Potapov, A.V. Il’ina, M.V. Shitov, V.P. Varlamov, Search for efficient
chitosan-based fungicides to protect the 15th‒16th centuries tempera
painting in exhibits from the state Tretyakov Gallery, Microbiology 89 (2020)
[53] European Convention for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes (ETS 123). Strasbourg, 1986.
13