Novel 50-norcarbocyclic pyrimidine derivatives as antibacterial agents

Article abstract of DOI:10.3390/molecules23123069

A series of novel 5⁰-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC₉₉ of 67 μg/mL (mc²155) and a MIC₉₉ of 6.7–67 μg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC₉₉ 28–61 μg/mL) and Mycobacterium bovis ATCC 35737 (MIC₉₉ 50–60 μg/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC₉₉ 20–50 μg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC₉₉ 20–50 μg/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Full text of DOI:10.3390/molecules23123069

molecules
Article
Novel 50-Norcarbocyclic Pyrimidine Derivatives as
Antibacterial Agents
Anastasia L. Khandazhinskaya 1,†, Liudmila A. Alexandrova 1,†, Elena S. Matyugina 1,
Pavel N. Solyev 1 , Olga V. Efremenkova 2 , Karen W. Buckheit 3, Maggie Wilkinson 3,
Robert W. Buckheit, Jr. 3, Larisa N. Chernousova 4, Tatiana G. Smirnova 4,
Sofya N. Andreevskaya 4, Olga G. Leonova 1, Vladimir I. Popenko 1, Sergey N. Kochetkov 1 and
Katherine L. Seley-Radtke 5,
*
1
Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, 32 Vavilov St.,
Moscow 119991, Russia; khandazhinskaya@bk.ru (A.L.K.); ala2004_07@mail.ru (L.A.A.);
matyugina@gmail.com (E.S.M.); solyev@gmail.com (P.N.S.); varjag@aport2000.ru (O.G.L.);
popenko@eimb.ru (V.I.P.); kochet@eimb.ru (S.N.K.)
Gause Institute of New Antibiotics, 11 Bol’shaya Pirogovskaya St., Moscow 119021, Russia; ovefr@yandex.ru
ImQuest BioSciences, 7340 Executive Way Suite R, Frederick, MD 21704, USA;
kbuckheit@imquestbio.com (K.W.B.); mwilkinson@imquestbio.com (M.W.);
rbuckheit@imquestbio.com (R.W.B.Jr.)
Central Tuberculosis Research Institute, 2 Yauzskaya Alley, Moscow 107564, Russia;
lchernousova@mail.ru (L.N.C.); s_tatka@mail.ru (T.G.S.); andsofia@mail.ru (S.N.A.)
Department of Chemistry & Biochemistry, University of Maryland, 1000 Hilltop Circle, Baltimore,
MD 21250, USA
2
3
4
5
*
Correspondence: kseley@umbc.edu; Tel.: +1-410-455-8684
†
A.L.K. and L.A.A. contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 October 2018; Accepted: 20 November 2018; Published: 23 November 2018
0
Abstract: A series of novel 5 -norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl
uracil were synthesized and the activity of the compounds evaluated against both Gram-positive
and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by
the most active compounds at a MIC99 of 67
Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains
of Mycobacterium tuberculosis ATCC 25177 (MIC99 28–61 g/mL) and Mycobacterium bovis ATCC
35737 (MIC99 50–60 g/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain
H37Rv (MIC99 20–50 g/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99
20–50 g/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial
µ µg/mL (VKPM
g/mL (mc2155) and a MIC99 of 6.7–67
µ
µ
µ
µ
cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting
that the mechanism of action for these compounds may be related to their interactions with bacteria
cell walls.
Keywords: 50-norcarbocyclic nucleosides; Mycobacterium tuberculosis; antibacterial activity; uracil
derivatives
1. Introduction
Antibiotic resistance has emerged for nearly all currently used therapeutics for many diseases,
including tuberculosis (TB) [1,2]. Despite this, the development of new classes of antibiotics has lagged
far behind the field’s growing need for drugs that can act on new targets and remain active against
resistant strains of pathogens. Nucleoside derivatives have historically served as potent antiviral and
anticancer agents [3], but it was not until 2000 that their anti-TB activity was reported [4]. Recently, several
Molecules 2018, 23, 3069; doi:10.3390/molecules23123069
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3069
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reports of modified nucleosides showing activity against Mycobacterium tuberculosis (M. tuberculosis),
Mycobacterium bovis (M. bovis) and Mycobacterium avium (M. avium) were published [4–18]. Among
them, the best activity was observed for several uridine analogues (MIC90 1 to 10 µg/mL) possessing
5-alkynyl (decynyl, dodecynyl, tetradecynyl, pyridylethynyl, etc.) substituents as well as modified
sugar moieties (Figure 1). In addition, several 20-deoxypyrimidine derivatives with extended
5-alkyloxymethyl or 5-alkyltriazolylmethyl substituents (5-dodecyloxy-methyl-20-deoxyuridine,
5-decyltriazolylmethyl-20-deoxyuridine, 5-dodecyltriazolylmethyl-20-deoxycytidine, etc.) were shown
to inhibit in vitro growth of both laboratory H37Rv and clinical multiple drug resistant (MDR) MS-115
M. tuberculosis strains with MIC99 values ~10 µg/mL (Figure 1) [5].
R
O
R
O
O
O
N
R
O
N
R
RO
HO
N
N
NH
O
NH
NH
NH
NH
N
N
N
O
N
O
O
O
H
HO
HO
HO
O
O
O
O
O
O
R2
R1
R = C8H17, C10H21
HO R3
HO
R = C10H21
HO
R = C8H17
HO
R = C8H17
,
,
,
,
,
R = C10H21
,
,
C11H23
C
C
10H21
12H25
,
C
12H25, 2-pyridyl
C
10H21
C11H23
C12H25
C12H25
R1 = OH, H, F
R2 = OH, R3 = H;
R2 = H, R3 = F
Figure 1. Previously reported anti-M. tuberculosis nucleosides.
In an effort to explore additional sugar modifications, a series of carbocyclic nucleoside analogues
with the same modifications were designed and synthesized. Because it had already been shown
that uracil analogues with short alkyne or halogen substituents at the C-5 position failed to inhibit
growth of M. tuberculosis [
exhibiting activity below 100
discussed below.
4
,
17
,
18], we chose to synthesize longer substituents. Several showed promise,
g/mL. Their synthesis and subsequent biological investigations are
µ
2. Results and Discussion
Carbocyclic nucleosides are analogues of natural nucleosides in which the oxygen atom of
the furanose ring is replaced by a methylene group. In addition, several carbocyclic nucleosides,
for example, aristeromycin and neplanocin A, have been found in Nature [19–22]. Nucleosides of this
type are recognized by many receptors and enzymes due to their structural similarity to the natural
nucleosides, however they display increased stability towards phosphorylase- and hydrolase-induced
cleavage of the pseudo-glycosidic bond. Moreover, they possess a wide spectrum of biological activity,
particularly, antiviral and anticancer properties [19,23–33]. Unfortunately, in some cases they have
exhibited significant toxicity as a result of their conversion to triphosphate forms, which closely
resemble natural nucleoside triphosphates (NTP), thus resulting in unwanted recognition by ATP
metabolizing enzymes [34–36].
To overcome the cellular cytotoxicity of carbocyclic nucleosides that inhibit their target
enzymes without preliminary phosphorylation (for example, aristeromycin and neplanocin A) [35],
the 50-norcarbocyclic nucleosides were designed. The switch of a secondary hydroxyl group for the
0
primary hydroxyl group at C-4 led to a lack of recognition by cellular kinases and as a result, they were
less toxic [36
uracil base (Figure 2) can act as promising anti-TB agents (MIC99 5–10
–
46]. Recently, we have shown that 50-norcarbocyclic nucleosides with a 5-substituted
µg/mL on both laboratory strain
M. tuberculosis H37Rv and clinical isolate M. tuberculosis MDR MS-115) [47
,
48]. Despite our best efforts,
however, their mechanism of action remains elusive.

Molecules 2018, 23, 3069
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O
N
R
O
N
H
R1
O
N
N
NH
O
R2
HO
HO
(±)
R = C10H21, C12H25
(±)
R1 = H, R2 = PhO;
R1 = 4-hydroxy-2-cyclopentenyl, R2 = H, Me, BuO
0
Figure 2. Examples of anti-Mtb 5 -norcarbocyclic nucleosides.
In that regard, the thymidylate kinases of M. tuberculosis were evaluated as
possible enzymatic targets of 1-(40-hydroxy-20-cyclopenten10-yl)-5-(phenylamino)uracils and
1,3-di-(40-hydroxy-20-cyclopenten-10-yl)-5-(phenylamino)uracils. No inhibition was found up to
200
µM [47]. Another possible mechanism of action that was considered was the inhibition of
M. tuberculosis thymidylate synthases, thus both the classical (ThyA) and flavin dependent thymidylate
synthase (ThyX)A were evaluated [46]. All of the derivatives lacked activity against the ThyA and
only one inhibited ThyX at a concentration of 8.32 µM. Thus it was assumed that the anti-TB activity
of 5-alkoxymethyl or 5-alkyltriazolylmethyl 20-deoxyuridine analogues could not be explained by
inhibition of thymidine kinases.
In an effort to further explore the mechanism of action for these compounds, we have now
designed a new hybrid scaffold possessing both modifications. Herein, we describe the synthesis
and preliminary antibacterial activity for a series of new 50-norcarbocyclic pyrimidine derivatives
containing extended 5-alkyloxymethyl or 5-alkyltriazolylmethyl residues (Figure 3).
O
O
O
O
R
R
N
N
HN
OR
HN
OR
HN
HN
N N
N N
N
N
O
O
N
N
O
O
AcO
HO
AcO
HO
(±)1: R = C10H21
(±)2: R = C11H23
(±)3: R = C12H25
(±)4: R = C10H21
(±)5: R = C11H23
(±)6: R = C12H25
(±)7: R = C8H17
(±)8: R = C10H21
(±)9: R = C12H25
(±)10: R = C8H17
(±)11: R = C10H21
(±)12: R = C12H25
Figure 3. Target compounds.
2.1. Chemistry
The key intermediate needed for the synthesis of both scaffolds for the new 5-modified
50-norcarbocyclic analogues is racemic 1-(40-acetoxycyclopent-10-yl)-5-(bromomethyl)-uracil
(
16).
1-(40-hydroxy-20-cyclopenten-10-yl)-thymine (13), which was obtained by the Trost procedure [49
using the coupling of epoxy-cyclopentene with thymine. Hydrogenation of 13 in the presence of
As shown in Scheme 1, 16 was synthesized in three steps starting from
]
0
0
palladium on carbon gave 1-(4 -hydroxycyclopent-1 -yl)-thymine (14) which was protected to provide
1-(40-acetoxycyclopent-10-yl)thymine (15). Following free radical bromination by the Barwolff and
Langen procedure [50], key intermediate 16 was obtained.

Molecules 2018, 23, 3069
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O
N
O
O
N
O
N
HN
HN
HN
HN
Br
O
O
N
O
O
HO
HO
AcO
AcO
(i)
(ii)
(iii)
(±)13
(±)14
Scheme 1. (i) Pd/C, H2, MeOH; (ii) Ac2O, Py; (iii) Br2, DCE, hv, 83 C.
The desired 5-alkyloxymethyl substituted analogues were then prepared by coupling with the
(±)15
(±)16
◦
1–3
corresponding 1-alcohol [51] and subsequent deprotection of the 4’-hydroxy group to provide targets
4–6 (Scheme 2).
O
N
O
N
O
N
HN
HN
HN
OR
Br
OR
O
O
O
AcO
AcO
HO
(ii)
(i)
(±)16
(iii)
(±)1: R = C10H21
(±)2: R = C11H23
(±)3: R = C12H25
(±)4: R = C10H21
(±)5: R = C11H23
(±)6: R = C12H25
R
R
N
N
N
N
O
N
N
O
N
O
N
HN
N3
HN
HN
O
N
O
O
AcO
AcO
HO
(iv)
(ii)
(±)17
(±)7: R = C8H17
(±)10: R = C8H17
(±)8: R = C10H21
(±)9: R = C12H25
(±)11: R = C10H21
(±)12: R = C12H25
Scheme 2. (i) ROH; (ii) K2CO3, MeOH; (iii) NaN3, DMF, 40 ◦C; (iv) RC
ascorbate, CH2Cl2/H2O (1:1).
≡
CH, CuSO4 5H2O, sodium
·
The 5-alkyltriazolylmethyl derivatives 7–12 were synthesized by the Lee method (Scheme 2) [52].
Azidation of 1-(40-acetoxycyclopent-10-yl)-5-(bromomethyl)uracil (16) to the corresponding
5-azidomethyl 17 was followed by 1,3-dipolarcycloaddition with various 1-acetylenes in a biphasic
system of dichloromethane-water under catalytic conditions using Cu(I) obtained in situ from copper
sulphate and sodium ascorbate to give
the desired 5-alkyltriazolylmethyl derivatives 10
be noted that the synthetic route from 16 to
centers, i.e., C-10 and C-40. Thus, because compound 16 is racemic all of the target compounds
12) were obtained as racemic mixtures. Moreover, following synthesis of the individual isomers
7–
9
. After deprotection of the 40-hydroxyl group of
12 were obtained in good yields (84–92%). It should
12 doesn’t change the configuration of the chiral
7–9,
–
1–
(1–
of the compound with the 5-dodecynyl substituent (Figure 2) no significant difference in the activity
was found (see [47]), thus we chose not to synthesize the individual enantiomers of the other
target compounds.

Molecules 2018, 23, 3069
5 of 18
2.2. Biological Evaluation and Structure-Activity Relationship Studies
In order to determine the lead compounds prior to evaluation against the virulent strains of
M. tuberculosis, the twelve compounds were initially evaluated for inhibition of an attenuated strain of
M. tuberculosis ATCC 25177 (H37Ra), which is closely related to a virulent laboratory strain H37Rv,
as well as the M. bovis ATCC strain 35737 in an in vitro assay (Table 1). When evaluated against
M. tuberculosis ATCC strain 25177, the 5-alkyloxymethyl substituted analogues
of 409, 423 and 54
g/mL, respectively. Deprotection of the 40-hydroxygroup resulted in targets
having MIC values of 183, 191 and 198 g/mL. The 5-alkyltriazolylmethyl derivatives 12 had
1–3 had MIC99 values
µ
4
–6
µ
7–
increased activities ranging from 29 µg/mL to 98 µg/mL.
Table 1. Preliminary evaluation of antimycobacterial activity of compounds 1–12.
M. tuberculosis
M. bovis ATCC
35737 MIC99
M. smegmatis
mc2155 MIC99
(µg/mL)
M. smegmatis
a
a
Compound
ATCC 25177
VKPM Ac 1339
a
a
MIC99 (µg/mL)
(µg/mL)
MIC99 (µg/mL)
(±) 1
(±) 2
409
423
54
183
191
198
54
29
61
98
53
409
423
54
366
381
>395
54
>100
>100
67
>100
>100
67
>100
67
>100
>100
67
>100
>100
-
-
1
>100
>100
67
>100
>100
67
>100
6.7
>100
>100
6.7
>100
10
-
-
2
(±) 3
(±) 4
(±) 5
(±) 6
(±) 7
(±) 8
57
(±) 9
488
195
105
446
<0.2
0.4
1.6
-
(±) 10
(±) 11
(±) 12
56
Rifampicin
Streptomycin
Kanamycin
Ciprofloxacin
Amikacin
Isoniazid
a
<0.2
0.4
1.6
-
-
-
-
-
<3.8
64
<3.8
1
The concentration of the drug at which the growth of the bacteria is inhibited by 99%.
Compounds
the same or within 2-fold of the MICs determined for M. tuberculosis. The exception was compounds
and 12, which exhibited an 8-fold decrease in activity against M. bovis relative to M. tuberculosis.
1–12 have similar activities against M. bovis ATCC strain 35737 with MICs either
9
Thus, the best antimycobacterial activity against both strains among the 5-alkyloxymethyl derivatives
1
–
6
was seen for compound
3
with the 40-acetyl group and the methyloxydodecyl substituent at the
0
5-position of the uracil moiety. In the series of 5-alkyltriazolylmethyl derivatives
analogues
7–
12, the 4 -acetylated
7
(5-octyltriazolylmethyl) and 8-(5-decyltriazolylmethyl) were observed to have a significant
inhibitory effect. DMSO (5%, equivalent to the same amount as would be present in the final solutions
of the tested compounds) was evaluated in parallel and was determined to be non-toxic to all of
the organisms.
The antibacterial activity of the synthesized compounds was also evaluated against Gram-negative
bacteria including Pseudomonas aeruginosa (P. aeruginosa) ATCC strain 27853 and Escherichia coli (E. coli)
ATCC strain 25922, Gram-positive bacteria including Bacillus subtilis (B. subtilis) ATCC strain 6633,
Staphylococcus aureus (S. aureus) INA 00761 (MRSA) and Leuconostoc mesenteroides (L. mesenteroides)
VKPM B-4177. Mycobacterium smegmatis (M. smegmatis) mc2155 and M. smegmatis VKPM Ac 1339,
were also used in the primary screening for potential antibiotic activity. Most of the bacteria were
not sensitive to the tested compounds. The only exception was against M. smegmatis, whose growth
was completely inhibited by compounds
3
,
6
,
8
and 11 at a concentration of 6.7–67
µg/mL (Table 1).
The MIC99 of the control antibiotics rifampicin and isoniazid were 10 and 1
µg/mL for M. smegmatis

Molecules 2018, 23, 3069
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VKPM Ac 1339, and > 100 and 64
µ
g/mL for M. smegmatis mc2155, respectively. Interestingly, the tested
compounds demonstrated selective activity only on mycobacteria, but not on other gram-positive
and gram-negative bacteria (data not shown), thus could represent a specific interaction that will
be pursued in subsequent studies. Three compounds (3, 7 and 8) were selected as leads against the
attenuated strain ATCC 25177 and subjected to additional antimycobacterial screening. The ability of
0
the new 5 -norcarbocyclic nucleosides to influence the growth of two virulent strains of M. tuberculosis,
including the laboratory strain M. tuberculosis H37Rv and the clinical strain M. tuberculosis MS-115
with multiple drug resistance were assessed in vitro. The antimycobacterial effect was studied by
measuring the growth dynamics of strains of M. tuberculosis in the enriched medium Middlebrook
7H9 in the automated growth registration system BACTEC MGIT 960. Different concentrations of the
compounds were compared to the growth of strains on media not containing preparations and media
containing control preparations at critical concentrations (rifampicin 1
µg/mL, isoniazid 0.1 µg/mL
and levofloxacin 1.5 g/mL). Each of the concentrations of the tested compounds as well as control
µ
samples were done in triplicate. In all positive samples, the culture of mycobacteria of the tuberculosis
complex was confirmed to grow and there was no contamination with nonspecific microflora. When
exposed to control preparations at critical concentrations, the growth of the sensitive culture was not
fixed, and the growth parameters of the MDR strain when incubated with isoniazid and rifampicin
did not differ from the control without the drug.
As shown in Table 2, the MIC99 against the laboratory sensitive strain M. tuberculosis H37Rv
were for compounds
3
(20
µg/mL),
7
(50
µg/mL) and
8
(50
µg/mL). Against M. tuberculosis
MS-115, the MIC99 values for compounds
3
,
7
, and were 20
8
µg/ mL, 20
µ
g/mL, and 50 g/mL,
µ
respectively. It should be noted that unlike the previously reported 5-alkynyl substituted compounds,
the 5-alkoxymethyl and 5-alkyl-triazolylmethyl derivatives proved to be quite toxic to the monocytic
cells. The cytotoxicity of compounds
was similar to or greater than the activity of the compounds against the mycobacteria with TC50 values
20.6 g/mL, 23.7 g/mL and 15.1 g/mL, respectively. Nonoxynol-9 was also evaluated in parallel as
3, 7 and 8 to the U-937 laboratory adapted monocytic cell line
µ
µ
µ
a positive control and was toxic at the expected concentration. So while it is clear these compounds are
too toxic to be regarded as drug candidates, they may be useful as a tool for better understanding the
mechanism of antimicobacterial action for this class of compounds.
Table 2. The antimycobacterial activity * of 50-norcarbocyclic derivatives of 5-substituted uracil and
cytotoxicity to U937 cells.
M. tuberculosis H37Rv
MIC99 (µg/mL)
M. tuberculosis MS-115
MIC99 (µg/mL)
Compound
U-937 TC50 (µg/mL)
(±) 3
(±) 7
20
50
50
1
0.1
1.5
-
20
20
50
>50
>100
1.5
-
20.6
23.7
15.1
>500
-
(±) 8
Rifampicin
Isoniazid
Levofloxacin
Nonoxynol-9
-
5.9
* For more details see the Table in Supplementary Material.
In contrast, the observed biological activity of these new derivatives of 5-substituted uracils
are in good correlation with previously reported data on the inhibition of M.tb growth by
nucleoside analogues containing 5-substituted pyrimidine bases and different sugars, as well as their
corresponding acyclic, or carbocyclic analogues [4,5,15–18,53] As a result, it appears these compounds
may have the same mechanism of antimicrobial action (likely cell wall disruption) and that it depends
primarily on the length and structure of the substituent at C-5 of the base moiety. Thus this new data
has added to the growing examples of similar compounds and their mechanism of action for inhibitors

Molecules 2018, 23, 3069
7 of 18
of this type. In an attempt to further support this hypothesis some additional experiments using
method of transmission electron microscopy were carried out.
Figure 4 shows electron micrographs of control M. tuberculosis H37Rv cells grown for 4 days in
enriched Dubois medium (A) and in the presence of DMSO/Twin-80/H2O buffer without compound
3
(B). The morphology of bacterial cells grown in the medium containing compound was markedly
3
different. As shown in Figure 4C,D, the bacterial wall was damaged (Figure 4C) or completely
destroyed (Figure 4D). This observation suggests that inactivation of M. tuberculosis may occur due
to the interaction of compound
difference of Mycobacteria from other bacteria is their thick cell wall containing unique lipids—mycolic
acids [54 55]. Thus speculatation that the synthesized compounds selectively act on mycolic acids of the
Mycobacteria cell wall or, like isoniazid, block the pathways involved in mycolic acids biosynthesis [56
3 with the bacterial wall and its subsequent destruction. The main
,
]
was logical. More testing however needs to be performed to confirm this observation, but if this
phenomenon is subsequently confirmed for the majority of species of gram-positive and gram-negative
bacteria, then these compounds may prove useful due to the lack of effect on the natural microflora of
the gut bacteria.
Figure 4. Transmission electron microscopy of M. tuberculosis H37Rv bacterial cells. Control bacterial
cells were grown for 4 days in enriched Dubois medium (A) or in enriched Dubois medium containing
DMSO/Twin-80/H2O solution without compound ). Bacterial cells after treatment with compound
3 (B
3 (50 µg/mL) for 4 days (C and D). Scale bar = 1 µm.
3. Materials and Methods
3.1. Chemistry
The reactions were performed with the use of commercial reagents acquired from Acros (Geel,
Belgium), Aldrich (St. Louis, MO, USA) and Fluka (Bucharest, Romania); anhydrous solvents were
purified according to the standard procedures. Column chromatography was performed on Silica Gel
60 0.040–0.063 mm (Merck, Darmstadt, Germany) columns. Thin layer chromatography (TLC) was
performed on Silica Gel 60 F254 aluminum-backed plates (Merck). Preparative layer chromatography
(PLC) was performed on Silica Gel 60 F254 glass-backed plates (Merck). NMR spectra were registered
on an AMX III-400 spectrometer (Bruker, Newark, Germany) with the working frequency of 400 MHz
1
for H-NMR (Me4Si as an internal standard for organic solvents) and 100.6 MHz for 13C-NMR
(with carbon-proton interaction decoupling). High resolution mass spectra (HRMS) were registered
on a Bruker Daltonics micrOTOF or a Bruker micrOTOF II instrument using electrospray ionization
(ESI HRMS) (Bruker Daltonics, Hamburg, Germany). The measurements were done in positive ion

Molecules 2018, 23, 3069
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mode (interface capillary voltage 4500 V) in a mass range from m/z 50 to m/z 3000 Da; external
or internal calibration was done with ESI Tuning Mix
USA). Nitrogen was applied as a dry gas (6 L/min); the interface temperature was set at 180 C,
™
(Agilent Technologies, Santa Clara, CA,
◦
nebulizer pressure: 0.4 Bar; flow rate: 3
chamber from the Agilent 1260 HPLC system equipped with an Agilent Poroshell 120 EC-C18 column
3.0 × 50 mm; 2.7 µm) and an identically packed security guard, using an autosampler. The samples
were injected from acetonitrile (LC-MS grade) solution. The column temperature was 30 ◦C and 5
of the sample solution was injected. The column was eluted in a gradient of concentrations of A
(acetonitrile) in B (water) with the flow rate of 400 L/min in the following gradient parameters: 0–15%
A for 6.0 min, 15%–85% A for 1.5 min, 85%–0% A for 0.1 min, 0% A for 2.4 min.
µL/min. Samples were injected into the mass spectrometer
(
µL
µ
(
)-1-(40-Hydroxy-20-cyclopenten-10-yl)-thymine (13). Epoxycyclopentene (0.8 g, 10 mmol) in freshly
±
distilled THF (15 mL) and tetrakis(triphenylphosphine)palladium (0) [Pd(PPh3)4] (308 mg; 0.26 mmol)
were added to a stirred solution of thymine (1 g, 8.0 mmol) in anhydrous DMF (40 mL). The reaction
mixture was stirred at room temperature for 18 h, then concentrated under vacuum, and the residue
dissolved in 98:2 CHCl3:MeOH (5 mL) and loaded onto a silica gel column. Elution with a 98:2 to 95:5
CHCl3:MeOH gave compound ( )-13 as a white powder (600 mg, 36%): Rf 0.32 (95:5 CHCl3:MeOH).
±
1H-NMR (DMSO-d6): 11.20 (1H, br. s, NH), 7.28 (1H, s, H-6), 6.14–6.11 (1H, m, H-20), 5.79–5.77 (1H,
m, H-30), 5.39–5.36 (1H, m, H-10), 5.20 (1H, d, J = 6.0 Hz, OH), 4.63–4.61 (1H, m, H-40), 2.74–2.70 (1H,
m, H-a50), 1.76 (3H, s, 5-CH3), 1.37–1.33 (1H, m, H-b50). 13C-NMR (DMSO-d6): 164.3, 151.3 (C-4, C-2),
140.4 (C-6), 137.7 (C-30), 131.4 (C-20), 109.9 (C-5), 73.8 (C-40), 58.3 (C-10), 39.9 (C-50), 12.6 (CH3).
(
±
)-1-(40-Hydroxycyclopent-10-yl)-thymine (14). To a solution of (
)-13 (0.6 g, 3 mmol) in anhydrous
±
MeOH (15 mL) 0.1 g 10% Pd/C was added under H2 atmosphere and the reaction mixture was stirred
at room temperature under H2 atmosphere for 18 h. The mixture was filtrated through a pad of Celite
and the filtrate was evaporated to dryness. The residue was dissolved in 98:2 CHCl3:MeOH (5 mL) and
loaded onto a silica gel column eluting with the same solvent system, followed by 95:5 CHCl3:MeOH
to give compound (
)-14 as an off white powder (510 mg, 81%): Rf 0.35 (95:5 CHCl3:MeOH). 1H-NMR
±
(CDCl3): 8.77 (1H, br. s, NH), 7.48 (1H, s, H-6), 5.03–4.95 (1H, m, H-10), 4.41 (1H, m, H-40), 2.39 (1H, d,
J = 8.0 Hz, OH), 2.36–2.30 (1H, m, H-a50), 2.20–2.13, 2.00–1.98 and 1.73–1.71 (4H, 3m, H-20and H-30),
1.93 (3H, s, 5-CH3), 1.70–1.67 (1H, m, H-b50). 13C-NMR (CDCl3): 164.2, 151.2 (C-4, C-2), 139.9 (C-6),
109.9 (C-5), 73.6 (C-40), 58.3 (C-10), 39.9 (C-50), 31.8 and 29.9(C-20 and C-30), 12.6 (CH3).
(
±
)-1-(40-Acetoxycyclopent-10-yl)-thymine (15). To a solution of (
)-14 (0.5 g, 2.4 mmol) in anhydrous
±
pyridine (15 mL), Ac2O (0.275 mL, 2.9 mmol) was added. The reaction mixture was stirred at room
temperature for 18 h, concentrated under vacuum, and the residue dissolved in CHCl3 (5 mL), loaded
onto a silica gel column. Elution with CHCl3 followed by 98:2 CHCl3:MeOH provided compound
(
±
)-15 as a white powder (0.5 g, 83%): Rf 0.36 (elution with 98:2 CHCl3:MeOH).01H-NMR (CDCl3):
0
9.13 (1H, br. s, NH), 7.21 (1H, s, H-6), 5.23–5.20 (1H, m, H-1 ), 5.14–5.10 (1H, m, H-4 ), 2.54–2.47 (1H, m,
H-a50), 2.20–2.16, 2.00–1.98 and 1.87–1.82 (4H, 3m, H-20and H-30), 2.08 (3H, s, Ac), 1.96 (3H, s, 5-CH3),
1.78–1.73 (1H, m, H-b50). 13C-NMR (CDCl3): 170.0 (C(O) acetyl), 163.7, 151.2 (C-4, C-2), 136.7 (C-6),
111.4 (C-5), 74.8 (C-4’), 53.9 (C-10), 37.8 (C-50), 31.8 and 29.9, (C-20 and C-30), 21.3 and 12.8 (2CH3).
0
0
(
±
)-1-(4 -acetoxycyclopent-1 -yl)-5-(bromomethyl)uracil (16). A solution of Br2 (0.2 mL, 2.4 mmol) in DCE
(5 mL) was added dropwise over a 3 h period with stirring to a boiling solution of ( )-15 (0.25 g,
1 mmol) in DCE (10 mL) with concomitant irradiation using an incandescent 300 W lamp under
an argon atmosphere. The progress of the reaction was monitored by TLC in EtOAc/hexane (1:1).
±
The mixture was evaporated under vacuum, coevaporated with toluene (3
dissolved in DMF (5 mL) and used without further purification in subsequent reactions.
×
10 mL), the crude 16

Molecules 2018, 23, 3069
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3.1.1. General Procedure for the Synthesis of
(±)-1-(40-Acetoxycyclopent-10-yl)-5-alkyloxy-methyluracils 1–3
The corresponding alcohol (1.5 mmol) was added to a solution of 1-(40-acetoxycyclopent-
10-yl)-5-(bromomethyl)uracil (13, 0.5 mmol) in dry DMF (10 mL). The mixture was stirred for 48 h at
◦
37 C under argon atmosphere, evaporated under vacuum, and the residue dissolved in CHCl3 (5 mL),
loaded onto a silica gel column, and eluted with CHCl3:MeOH (98:2) to provide the crude products
which were purified as described below.
(
±
)-1-(40-Acetoxycyclopent-10-yl)-5-decyloxymethyluracil (
1
). Crude
1 was purified by PLC eluting
with EtOAc/hexane (2:1) to give compound (
±
)-
1
as a white powder (92 mg, 45%): Rf 0.40 (98:2
CHCl3:MeOH). H-NMR (CDCl3): 8.76 (1H, br. s, NH), 7.49 (1H, s, H-6), 5.23–5.21 (1H, m, H-10),
5.15–5.11 (1H, m, H-40), 4.26 (2H, s, 5-CH2O), 3.51 (2H, t, J = 8.0 Hz, CH2CH2(CH2)7CH3), 2.55–2.47
(1H, m, H-a50), 2.22–2.18, 2.01–1.99 and 1.87–1.83 (4H, 3m, H-20 and H-30), 2.08 (3H, s, Ac), 1.78–1.72
1
(1H, m, H-b50), 1.61–1.56 (2H, m, CH2C
(3H, t, J = 8 Hz, CH2CH2(CH2)7C
138.0 (C-6), 112.9 (C-5), 74.8 (C-40), 71.6 (5-CH2O), 65.0 (
32.0, 31.9, 30.0, 29.8, 29.6
2, 29.5, 29.4, 26.2, 22.7, 21.3 (C-20, C-30, (CH2)8,
HRMS: found m/z 409.2698, calculated C22H36N2O5 [M + H]+ 409.26697.
H
2(CH2)7CH3), 1.29–1.25 (14H, m, CH2CH2(C
3). 13C-NMR (CDCl3): 170.3 (C(O) acetyl), 162.2, 150.9 (C-4, C-2),
H2CH2(CH2)7CH3), 54.4 (C-10), 38.0 (C-50),
H3C(O)), 14.2 (CH3).
H2)7CH3), 0.86
H
C
×
C
0
0
(
±
)-1-(4 -Acetoxycyclopent-1 -yl)-5-undecyloxymethyluracil (
2
). Crude
2 was purified by PLC eluting with
EtOAc/hexane (2:1) mixture to give compound (
±
)-2
as a white powder (99 mg, 47%): Rf 0.41 (98:2
CHCl3:MeOH). H-NMR (CDCl3): 8.58 (1H, br. s, NH), 7.49 (1H, s, H-6), 5.25–5.20 (1H, m, H-10),
5.17–5.11 (1H, m, H-40), 4.26 (2H, s, 5-CH2O), 3.52 (2H, t, J = 8.0 Hz, CH2CH2(CH2)8CH3), 2.58–2.47
(1H, m, H-a50), 2.22–2.18, 2.03–1.99 and 1.88–1.83 (4H, 3m, H-20and H-30), 2.09 (3H, s, Ac), 1.79–1.72
1
(1H, m, H-b50), 1.62–1.58 (2H, m, CH2C
H
2(CH2)8CH3), 1.30–1.26 (16H, m, CH2CH2(C
(3H, t, J = 8.0 Hz, CH2CH2(CH2)8CH3). 13C-NMR (CDCl3): 170.2 (C(O) acetyl), 162.1, 150.9 (C-4, C-2),
138.0 (C-6), 112.8 (C-5), 74.7 (C-4’), 71.5 (5-CH2O), 64.9 (
H2CH2(CH2)8CH3), 54.3 (C-10), 37.9 (C-50),
31.9, 31.8, 29.9, 29.6 H3C(O)), 14.1 (CH3).
3, 29.5, 29.3, 29.0, 26.1, 22.7, 21.2 (C-20, C-30, (CH2)9,
HRMS: found m/z 423.2853, calculated C23H38N2O5 [M + H]+ 423.2853.
H2)8CH3), 0.87
C
×
C
0
0
(
±
)-1-(4 -Acetoxycyclopent-1 -yl)-5-dodecyloxymethyluracil (
3
). Crude
3 was purified by PLC eluting with
EtOAc/hexane (2:1) to give compound ( )-
±
3 as a white powder (100 mg, 46%): Rf 0.42 (elution with
98:2 CHCl3:MeOH). 1H-NMR (CDCl3): 8.92 (1H, br. s, NH), 7.43 (1H, s, H-6), 5.18-5.14 (1H, m, H-10),
5.13-5.06 (1H, m, H-40), 4.20 (2H, s, 5-CH2O), 3.46 (2H, t, J=8 Hz, C0H2CH2(CH2)9CH3), 2.50–2.42 (1H,
0
0
m, H-a5 ), 2.16–2.12, 1.96–1.94 and 1.81–1.77 (4H, 3m, H-2 and H-3 ), 2.03 (3H, s, Ac), 1.72–1.67 (1H, m,
H-b50), 1.57–1.50 (2H, m, CH2CH2(CH2)9CH3), 1.25–1.19 (18H, m, CH2CH2(CH2)9CH3), 0.86 (3H, t,
J = 8.0 Hz, CH2CH2(CH2)9CH3). 13C-NMR (CDCl3): 170.3 (C(O) acetyl), 162.4, 151.1 (C-4, C-2), 138.1
(C-6), 113.0 (C-5), 74.9 (C-40), 71.7 (5-CH2O), 65.0 (
C
H2CH2(CH2)8CH3), 54.5 (C-10), 38.1 (C-50), 32.1,
0
0
32.0, 30.1, 29.8
×
5, 29.6, 29.5, 26.3, 22.8, 21.4 (C-2 , C-3 , (CH2)10,
C
H3C(O)), 14.2 (CH3). HRMS: found
m/z 437.3004, calculated C24H40N2O5 [M + NH]+ 437.3010.
0 0
( )-1-(4 -Acetoxycyclopent-1 -yl)-5-(azidomethyl)uracil (17). Sodium azide (160 mg, 2.5 mmol) was added
±
0
0
to a solution of 1-(4 -acetoxycyclopent-1 -yl)-5-(bromomethyl)uracil (13) (0.5 mmol) in dry DMF (10 mL).
◦
The mixture was stirred for 48 h at 37 C under argon atmosphere, evaporated under vacuum, and the
residue dissolved in CHCl3 (5 mL), applied onto a silica gel column, and eluted with CHCl3:MeOH
(98:2) to give crude product 17. It was then purified further by PLC eluted with EtOAc/hexane (1:1) to
1
provide the desired compound 17 as off white solid (143 mg, 97.5%). H-NMR (CDCl3): 8.43 (1H, br. s,
NH), 7.54 (1H, s, H-6), 5.31–5.27 (1H, m, H-10), 5.22–5.17 (1H, m, H-40), 4.23 (2H, s, 5-CH2), 2.60–2.53
(1H, m, H-a50), 2.31–2.27, 2.10–2.05 and 1.93–1.85 (4H, 3m, H-20and H-30), 2.07 (3H, s, Ac), 1.81–1.75
(1H, m, H-b50).

Molecules 2018, 23, 3069
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3.1.2. General Procedure for the Synthesis of
(±)-1-(40-Acetoxycyclopent-10-yl)-5-[(4-alkyl)-1,2,3-triazol-1-yl]methyluracils 7–9
To a solution of azide 17 (143 mg, 0.49 mmol) and corresponding 1-alkyne (0.75 mmol) in
dichloromethane (2 mL), copper sulphate (12.4 mg, 0.05 mmol), sodium ascorbate (30 mg, 0.15 mmol)
and H2O (2 mL) were added. The reaction mixture was stirred for 17 h at room temperature.
The solvents were evaporated under vacuum and the crude products were purified by PLC eluting
with EtOAc/hexane (2:1) to give compounds 7–9.
( 7). White powder (100 mg,
)-1-(40-Acetoxycyclopent-10-yl)-5-[(4-octyl)-1,2,3-triazol-1-yl]methyluracil (
±
47.5%). Rf 0.28 (EtOAc/hexane (2:1)).1H-NMR (CDCl3): 9.53 (1H, s, NH), 7.82 (1H, s, H-6), 7.60
(1H, s, Htriazol), 5.03–5.20 (3H, m, H-10and 5-CH2N), 5.16–5.08 (1H, m, H-40), 2.71 (2H, t, J = 8.0 Hz,
C
H2CH2(CH2)5CH3), 2.59–2.49 (1H, m, H-a050), 2.20–2.17, 2.03–1.99 and 1.90–1.79 (4H, 3m, H-20and
0
H-3 ), 2.18 (3H, s, Ac), 1.76–1.65 (1H, m, H-b5 ), 1.65–1.59 (2H, m, CH2C
H
2(CH2)5CH3), 1.29–1.24 (10H,
m, CH2CH2(CH2)5CH3), 0.88 (3H, t, J=8 Hz, CH2CH2(CH2)5CH3). 13C-NMR (CDCl3): 170.4 (C(O)
acetyl), 162.4, 150.5 (C-4, C-2), 148.7 (C-5triazole), 142.4 (C-6), 122.4 (C-4triazole), 109.0 (C-5), 74.4 (C-40),
55.2 (C-10), 46.6 (5-CH2N), 38.1 (C-5’), 31.9, 31.8, 30.2, 29.8, 29.3
×
3, 25.4, 22.7, 21.5 (C-20, C-30, (CH2)7,
CH3C(O)), 14.1 (CH3). HRMS: found m/z 432.2601, calculated C22H33N5O4 [M + H]+ 432.2605.
( 8), white powder (143 mg,
)-1-(40-Acetoxycyclopent-10-yl)-5-[(4-decyl)-1,2,3-triazol-1-yl]methyluracil (
±
63.5%). Rf 0.30 (EtOAc/hexane (2:1)).1H-NMR (CDCl3): 9.03 (1H, s, NH), 7.78 (1H, s, H-6), 7.55
(1H, s, Htriazol), 5.26-5.21 (3H, m, 5-CH2N and H-10), 5.09–5.05 (1H, m, H-40), 2.68 (2H, t, J = 8.0 Hz,
C
H2CH2(CH2)7CH3), 2.56–2.48 (1H, m, H-a050), 2.22–2.17, 2.01–1.99 and 1.89–1.80 (4H, 3m, H-20and
0
H-3 ), 2.15 (3H, s, Ac), 1.76–1.69 (1H, m, H-b5 ), 1.66–1.62 (2H, m, CH2C
H
2(CH2)7CH3), 1.30–1.24 (14H,
m, CH2CH2(CH2)7CH3), 0.86 (3H, t, J = 8.0 Hz, CH2CH2(CH2)7CH3). 13C-NMR (CDCl3): 170.4 (C(O)
acetyl), 162.5, 150.7 (C-4, C-2), 148.7 (C-5triazole), 142.2 (C-6), 122.1 (C-4triazole), 109.5 (C-5), 74.5 (C-40),
55.2 (C-10), 45.2 (5-CH2N), 38.2 (C-50), 32.0, 31.9, 30.3, 29.7
×
2, 29.4
×
4, 25.7, 22.8, 21.5 (C-20, C-30,
(CH2)9,
C
H3C(O)), 14.2 (CH3). HRMS: found m/z 460.2919, calculated C24H37N5O4 [M + H]+ 460.2918.
( 9), white powder (127 mg,
)-1-(40-Acetoxycyclopent-10-yl)-5-[(4-dodecyl)-1,2,3-triazol-1-yl]methyluracil (
±
53%). Rf 0.32 (EtOAc/hexane (2:1)).1H-NMR (CDCl3): 9.30 (1H, s, NH), 7.78 (1H, s, H-6), 7.55
(1H, s, Htriazol), 5.21 (3H, m, 5-CH2N and H-10), 5.09–5.05 (1H, m, H-40), 2.67 (2H, t, J = 8.0 Hz,
C
H2CH2(CH2)9CH3), 2.55–2.47 (1H, m, H-a50), 2.20–2.17, 2.02–1.99 and 1.93–1.83 (4H, 3m, H-20 and
H-30), 2.15 (3H, s, Ac), 1.76–1.70 (1H, m, H-b50), 1.65–1.61 (2H, m, CH2CH2(CH2)9CH3), 1.25–1.22
(18H, m, CH2CH2(CH2)9CH3), 0.86 (3H, t, J = 8.0 Hz, CH2CH2(CH2)9CH3). 13C-NMR (CDCl3): 170.4
(C(O) acetyl), 162.5, 150.7 (C-4, C-2), 148.7 (C-5triazole), 142.1 (C-6), 122.0 (C-4triazole), 109.5 (C-5), 74.4
(C-40), 55.1 (C-10), 46.1 (5-CH2N), 38.1 (C-50), 32.0, 31.8, 30.2, 29.7
(C-20, C-30, (CH2)11,
[M + H]+ 488.2232.
×
3, 29.6, 29.4
×
4, 25.6, 22.7, 21.4
CH3C(O)), 14.2 (CH3). HRMS: found m/z 488.2232, calculated C26H41N5O4
3.1.3. General Procedure for the Synthesis of
(±)-1-(40-Hydroxycyclopent-10-yl)-5-alkyloxy-methyluracils 4–6 and
(±)-1-(40-hydroxycyclopent-10-yl)-5-[(4-alkyl)-1,2,3-triazol-1-yl]methyl-uracils 10–12
To a stirred solution of the corresponding acetylated compound 1–3, 7–9 (0.15 mmol) in MeOH
(5 mL) K2CO3 (52 mg; 0.38 mmol) was added and refluxed during 2–4 h. The progress of the reaction
was monitored by TLC in 95:5 CHCl3:MeOH. The reaction mixture was concentrated in vacuum and
the residue purified by PLC eluting with 9:1 CHCl3-MeOH to give the desired compounds.
(
±
)-1-(40-Hydroxycyclopent-10-yl)-5-decyloxymethyluracil (
4
), white solid (47 mg, 85 %): Rf 0.47 (9:1
CHCl3:MeOH). H-NMR (CDCl3): 8.72 (1H, br. s, NH), 7.73 (1H, s, H-6), 5.00–4.92 (1H, m, H-10),
4.39 (1H, m, H-40), 4.24 (2H, s, 5-CH2O), 3.50 (2H, t, J = 8 Hz, CH2CH2(CH2)7CH3), 2.36–2.34
(1H, m, H-a50), 2.19–2.16, 1.99–1.89 (4H, 2m, H-20and H-30), 1.77–1.72 (1H, m, H-b50), 1.60–1.57
(2H, m, CH2CH2(CH2)7CH3), 1.28–1.25 (14H, m, CH2CH2(CH2)7CH3), 0.86 (3H, t, J = 8.0 Hz,
1

Molecules 2018, 23, 3069
11 of 18
CH2CH2(CH2)7CH3). 13C-NMR (CDCl3): 162.5, 151.0 (C-4, C-2), 140.8 (C-6), 112.6 (C-5), 72.2
(C-40), 71.5 (5-CH2O), 65.0 (
C
H2CH2(CH2)7CH3), 56.4 (C-10), 40.5 (C-50), 35.3, 32.0, 29.8, 29.7 × 3,
29.6, 29.4, 26.2, 22.8 (C-20, C-30, (CH2)8), 14.2 (CH3). HRMS: found m/z 367.2592, calculated
C20H34N2O4 [M + H]+ 367.2591.
(
±
)-1-(40-Hydroxycyclopent-10-yl)-5-undecyloxymethyluracil (
5
), white solid (51 mg, 88 %): Rf 0.49 (9:1
CHCl3:MeOH). H-NMR (CDCl3): 8.56 (1H, br. s, NH), 7.72 (1H, s, H-6), 4.98-4.94 (1H, m, H-10),
4.40 (1H, m, H-40), 4.24 (2H, s, 5-CH2O), 3.50 (2H, t, J = 8 Hz, CH2CH2(CH2)8CH3), 2.41–2.33 (2H, m,
H-a50, OH), 2.21–2.14, 2.05–1.96, 1.94–1.87 and 1.78–1.75 (4H, 4m, H-20and H-30), 1.74–1.69 (1H, m,
H-b50), 1.62-1.55 (2H, m, CH2CH2(CH2)8CH3), 1.29–1.25 (16H, m, CH2CH2(CH2)8CH3), 0.87 (3H, t,
J = 8.0 Hz, CH2CH2(CH2)8CH3). 13C-NMR (CDCl3): 162.4, 151.0 (C-4, C-2), 140.7 (C-6), 112.6 (C-5),
1
72.2 (C-40), 71.5 (5-CH2O), 65.0 (
4, 29.6, 29.4, 26.2, 22.8 (C-20, C-30, (CH2)9), 14.2 (CH3). HRMS: found m/z 381.2749, calculated
C21H36N2O4 [M + H]+ 381.2748.
C
H2CH2(CH2)8CH3), 56.4 (C-10), 40.5 (C-50), 35.3, 32.00, 29.8, 29.7
×
(
±
)-1-(40-Hydroxycyclopent-10-yl)-5-dodecyloxymethyluracil (
6
), white solid (51.5 mg, 87 %): Rf 0.50 (9:1
1
CHCl3:MeOH). H-NMR (CDCl3): 8.60 (1H, br. s, NH), 7.72 (1H, s, H-6), 4.97–4.94 (1H, m, H-10),
4.40 (1H, m, H-40), 4.24 (2H, s, 5-CH2O), 3.50 (2H, t, J = 8 Hz, CH2CH2(CH2)9CH3), 2.41–2.33 (1H,
m, H-a50), 2.21–2.14, 2.05–1.97, 1.94–1.87 and 1.75 (4H, 4m, H-20and H-30), 1.75–1.67 (1H, m, H-b50),
1.62–1.55 (2H, m, CH2C
H2(CH2)9CH3), 1.29–1.25 (18H, m, CH2CH2(CH2)9CH3), 0.86 (3H, t, J = 8.0 Hz,
CH2CH2(CH2)9CH3). 13C-NMR (CDCl3): 162.4, 151.0 (C-4, C-2), 140.7 (C-6), 112.6 (C-5), 72.2 (C-40),
71.5 (5-CH2O), 65.0 (
C
H2CH2(CH2)8CH3), 56.5 (C-10), 40.4 (C-50), 35.3, 32.0, 29.7
×
5, 29.6, 29.5, 29.4,
26.2, 22.8 (C-20, C-30, (CH2)10), 14.2 (CH3). HRMS: found m/z 395.2904, calculated C22H38N2O4
[M + H]+ 395.2904.
0
0
(
±
)-1-(4 -Hydroxycyclopent-1 -yl)-5-[(4-octyl)-1,2,3-triazol-1-yl]methyluracil (10), white solid (54 mg, 92%):
1
Rf 0.49 (9:1 CHCl3:MeOH). H-NMR (CDCl3): 9.41 (1H, s, NH), 8.22 (1H, s, H-6), 7.60 (1H, s,
Htriazole), 5.28–5.19 (2H, m, 5-CH2N), 5.13–5.07 (1H, m, H-10), 4.40 (1H, m, H-40), 2.67 (2H, t,
J = 8.0 Hz,
C
H2CH2(CH2)5CH3), 2.33–2.25 (1H, m, H-a50), 2.23–2.19, 1.98–1.84 and 1.67–1.64 (4H,
0
0
0
3m, H-2 and H-3 ), 1.74–1.70 (1H, m, H-b5 ), 1.62–1.57 (2H, m, CH2C
H
2(CH2)5CH3), 1.28–1.23 (10H, m,
CH2CH2(C
C-2), 148.3 (C-5triazole), 144.2 (C-6), 122.5 (C-4triazole), 109.1 (C-5), 72.0 (C-4 ), 55.7 (C-1 ), 46.5 (5-CH2N),
41.0 (C-5 ), 35.2, 31.9, 30.7, 29.8, 29.4
m/z 390.2501, calculated C20H31N5O3 [M + H]+ 390.2500.
H2)5CH3), 0.85 (3H, t, J = 8.0 Hz, CH2CH2(CH2)5CH
3). 13C-NMR (CDCl3): 162.8, 151.0 (C-4,
0
0
0
0 0
×
2, 29.3, 25.5, 22.7 (C-2 , C-3 , (CH2)7), 14.1 (CH3). HRMS: found
0
0
(
±
)-1-(4 -Hydroxycyclopent-1 -yl)-5-[(4-decyl)-1,2,3-triazol-1-yl]methyluracil (11), white solid (53 mg, 84%):
1
Rf 0.50 (9:1 CHCl3:MeOH). H-NMR (CDCl3): 9.71 (1H, s, NH), 8.17 (1H, s, H-6), 7.54 (1H, s,
Htriazole), 5.24-5.19 (2H, m, 5-CH2N), 5.16-5.06 (1H, m, H-1’), 4.41–4.40 (1H, m, H-40), 2.63 (2H, t,
J = 8.0 Hz, C
H-20and H-30), 1.77–1.73 (1H, m, H-b50), 1.64–1.57 (2H, m, CH2CH2(CH2)7CH3), 1.28–1.23 (14H, m,
CH2CH2(C 2)7CH3), 0.85 (3H, t, J = 8.0 Hz, CH2CH2(CH2)7C
3). 13C-NMR (CDCl3): 162.8, 150.2 (C-4,
C-2), 148.2 (C-5triazole), 144.2 (C-6), 122.6 (C-4triazole), 109.0 (C-5), 72.0 (C-4 ), 55.8 (C-1 ), 46.7 (5-CH2N),
H
2CH2(CH2)7CH3), 2.34–2.26 (1H, m, H-a50), 2.23–2.17, 1.91–1.84 and 1.74–1.65 (4H, 3m,
H
H
0
0
0
0 0
41.0 (C-5 ), 35.2, 32.0, 30.7, 29.7, 29.6, 29.4
m/z 418.2078, calculated C22H35N5O3 [M + H]+ 418.2813.
×
4, 25.5, 22.8 (C-2 , C-3 , (CH2)9), 14.2 (CH3). HRMS: found
(
±
)-1-(40-Hydroxycyclopent-10-yl)-5-[(4-dodecyl)-1,2,3-triazol-1-yl]methyluracil (12), white solid (519 mg,
1
88%): Rf 0.52 (9:1 CHCl3:MeOH). H-NMR (CDCl3): 9.02 (1H, s, NH), 8.11 (1H, s, H-6), 7.52 (1H,
s, Htriazole), 5.24–5.20 (2H, m, 5-CH2N), 5.16–5.04 (1H, m, H-10), 4.42–4.40 (1H, m, H-40), 2.65 (2H, t,
J = 8.0 Hz,
H-20and H-30), 1.73–1.70 (1H, m, H-b50), 1.64–1.59 (2H, m, CH2CH2(CH2)9CH3), 1.28–1.24 (18H, m,
CH2CH2(C 2)9CH3), 0.86 (3H, t, J = 8.0 Hz, CH2CH2(CH2)9C
3). 13C-NMR (CDCl3): 162.6, 150.9 (C-4,
C-2), 148.8 (C-5triazole), 143.8 (C-6), 122.0 (C-4triazole), 109.5 (C-5), 72.1 (C-4 ), 55.8 (C-1’), 46.1 (5-CH2N),
CH
2CH2(CH2)9CH3), 2.34–2.28 (1H, m, H-a50), 2.25–2.18, 1.94-1.83 and 1.69–1.66 (4H, 3m,
H
H
0

Molecules 2018, 23, 3069
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40.9 (C-5’), 35.3, 32.0, 30.6, 29.7
×
4, 29.5
×
2, 29.4
×
2, 25.8, 22.8 (C-20, C-30, (CH2)11), 14.2 (CH3).
HRMS: found m/z 446.3127, calculated C24H39N5O3 [M + H]+ 446.3127.
3.2. Antibacterial Experiments
3.2.1. Bacterial Strains
The M. tuberculosis ATCC 25177 and M. bovis ATCC 35737 strains used in these studies were
purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The laboratory
M.tuberculosis H37Rv strain and the MS-115 clinical strain resistant to five first line anti-TB drugs
(rifampicin, isoniazid, streptomycin, ethambutol, and pyrazinamide) were acquired from the Russian
Central Tuberculosis Research Institute Collection (Moscow, Russia). The bacteria were propagated
as recommended by the supplier. B. subtilis ATCC 6633, S. aureus INA 00761 (MRSA), M. smegmatis
mc2155, M. smegmatis VKPM Ac 1339, L. mesenteroides VKPM B-4177, P. aeruginosa ATCC 27853,
and E. coli ATCC 25922 were purchased from the collection of Gause Institute of New Antibiotics
(Moscow, Russia).
3.2.2. U-937 Cells
The U-937 cells used in the cytotoxicity evaluation were obtained from the AIDS Research and
Reference Program (Germantown, MD, USA). The cells were cultured as specified by the supplier in
RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin/streptomycin
solution and 1% L-glutamine.
3.2.3. Determination of Minimal Inhibitory Concentration (MIC) against M. tuberculosis ATCC 25177
The susceptibility of the M. tuberculosis ATCC 25177 to the test compounds was evaluated by
determining the MIC of each compound using a micro-broth dilution analysis [57–60]. A standardized
inoculum was prepared by direct suspension of bacteria from a Lowenstein-Jensen slant to Middlebrook
◦
7H9 broth with ADC enrichment. The broth culture was incubated for 26 days at 37 C. Following the
incubation, the culture was vortexed with glass beads resulting in an OD595 of 0.18 and then further
diluted 1:20 to yield 5
of a 96-well plate containing 100
This dilution scheme yielded final concentrations of the bacteria estimated to be 2.5
The plates were incubated for seven days at 37 C at which time 30
to each well and the cultures were incubated for an additional 24 h at 37 C. Following the incubation,
the plates were visually scored +/- for color change from blue (no growth) to pink (growth). The MIC
for each compound was determined as the lowest compound dilution that completely inhibited
microbial growth.
×
106 CFU/mL. One hundred microliters (100
µ
L) was added in triplicate wells
µL of test compound serially diluted 2-fold in the appropriate broth.
×
106 CFU/mL.
◦
µ
L of reazurin solution was added
◦
3.2.4. Determination of MIC against M. bovis ATCC 35737
The susceptibility of M. bovis ATCC 35737 to the test compounds was evaluated by determining
the MIC of each compound using a micro-broth dilution [57]. A standardized inoculum was prepared
by direct suspension of bacteria from a Lowenstein-Jensen slant to Middlebrook 7H9 broth with
ADC (albumin, dextrose, catalase) enrichment and 40 mM sodium pyruvate. The broth culture was
incubated for 23 days at 37 ◦C. Following the incubation, the culture was vortexed with glass beads
resulting in an OD595 = 0.203 which was further diluted 1:10 to yield 3
microliters (100 L) was added in triplicate wells of a 96-well plate containing 100
serially diluted 2-fold in the appropriate broth. This dilution scheme yielded final concentrations of
the bacteria estimated to be 1.5
107 CFU/mL. The plates were incubated for five days at 37 ◦C at
×
107 CFU/mL. One-hundred
L of test compound
µ
µ
×
which time 30
µ
L of reazurin solution was added to each well. The cultures were incubated for an
◦
additional 4 days at 37 C at which time the plates were visually scored +/- for color change from blue

Molecules 2018, 23, 3069
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(no growth) to pink (growth). The MIC for each compound was determined as the lowest compound
dilution that completely inhibited microbial growth.
3.2.5. Antituberculosis Tests with Virulent Strains of M. tuberculosis
The laboratory strain of M. tuberculosis H37Rv susceptible to anti-TB drugs and MDR strain
M. tuberculosis MS-115 were used. The MDR strain is resistant toward five first line anti-TB
drugs: rifampicin, isoniazid, streptomycin, ethambutol and pyrazinamide. The mycobacteria were
transformed into a suspension of single cells at the same growth phase and normalized by CFU [61
]
and studied using an automated BACTEC MGIT 960 system (BD, Westfield, MA, USA) [62]. Tested
compounds were dissolved in DMSO at a concentration of 40 mg/mL (stock solutions). Water and
Tween-80 were added to the stock solutions to provide DMSO/Tween-80/H2O (5:0.5:4.5, v/v/v).
The concentration of mycobacterial cells was 106 CFU/mL. Each sample, including the control
samples lacking the tested compound, were evaluated in triplicate as previously reported [28,29].
The antibacterial activity was analyzed with the TB Exist software [53]. The method is based on
comparison of the bacterial growth in the samples, containing the tested compounds, and the control
samples diluted 100 times if compared with the initially inoculated culture. The culture is considered
susceptible (i.e., the compound is active) if the optical density of the control sample is 400 GU (growth
units) and that of the tested sample is less than 100 GU. In addition, using the absolute concentrations
method, a delay in the bacterial growth of the tested sample was measured in comparison with the
control samples, which reflected a negative effect of the tested compounds at the concentrations lower
than MIC on the bacterial viability. The measurements were carried out automatically every hour and
registered with the Epicenter software (BD, Westfield, MA, USA).
3.2.6. Antibacterial Effects
The antibacterial effects for each of the compounds as well as rifampicin, isoniazid, ciprofloxacin
and amikacin were determined by the micro-broth delution method [63] against B. subtilis ATCC 6633,
S. aureus INA 00761 (MRSA), M. smegmatis mc2155, M. smegmatis VKPM Ac 1339, L. mesenteroides
VKPM B-4177, P. aeruginosa ATCC 27853, and E. coli ATCC 25922. These strains were purchased from
the collection of Gause Institute of New Antibiotics. Two-fold serial dilutions of the compounds
in nutrient medium No. 2 Gause (composition (%): glucose-1, peptone-0.5, tryptone-0.3, NaCl-0.5,
pH 7.2–7.4) were performed to include concentrations ranging from 300 µg/mL to 1 µg/mL, and the
test strains were cultured at 106 cells/mL. All cultures were incubated at 37 ◦C with the exception
of L. mesenteroides VKPM B-4177, which was incubated at 28 ◦C. The presence of growth and growth
intensity were determined after one day in all the strains with the exception of M. smegmatis, which
was evaluated after two days of growth.
3.2.7. Evaluation of U-937 Cytotoxicity
U-937 cells plated at 2.5
test compounds in a volume of 100
×
103 cells/well in a volume of 100
µ
L were incubated with serially diluted
◦
µL for 6 days at 37 C/5% CO2. Following the incubation, the cells
were stained with the tetrazolium dye XTT to determine cellular viability and the plates were read at
460/650 nm on a spectrophotometer. Toxicity values were calculated using linear regression analysis.
3.2.8. Electron Microscopy
For electron microscopy M. tuberculosis H37Rv cells (109 CFU/mL) were grown in enriched Dubois
medium with compound
concentration of compound
enriched Dubois medium or in the presence of DMSO/Twin-80/H2O solution without compound
3
dissolved in of DMSO/Twin-80/H2O solution as described above. The final
3
was 60 g/mL. In control experiments the cells were grown in pure
µ
3
.
After 4 days the cells were collected by centrifugation, washed in distilled water and fixed in 2.5%
glutaraldehyde. Aliquotes (80 mkL) of cell suspensions were centrifuged in the small centrifuge
chambers onto freshly glow-discharged carbon-collodion electron microscopic grids at 2500 g for

Molecules 2018, 23, 3069
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20 min [64,65]. Specimens were briefly rinsed in distilled water, stained for 1 min in 1% aqueous uranyl
acetate solution [66], air dried and studied on a JEM-100CX electron microscope (JEOL, Peabody,
MA, USA) in Core Facility “UNIQEM Collection” of Federal Research Centre “Fundamentals of
Biotechnology” of the Russian Academy of Sciences.
4. Conclusions
In summary, twelve new 50q5 ml-norcarbocyclic derivatives of 5-alkoxymethyl or
5-alkyl-triazolylmethyl uracil were synthesized. The antibacterial activity of the compounds was tested
against different classes of bacteria, including both Gram-positive and Gram-negative strains. It was
determined that this set of uracil derivatives possess selectivity towards Mycobacteria. Analogues
3
,
7
and
strain H37Rv and the MDR clinical strain MS-115, which is known to be clinically resistant to five
top antituberculosis drugs. Notably, analogue was 2.5 times more active against the MDR strain.
8 exhibited activities against two virulent strains of M. tuberculosis: laboratory sensitive
7
However, all three of the active compounds proved to be quite toxic to the monocytic cell line U937,
thus, additional work needs to be undertaken to attenuate this toxicity and improve the activity.
TEM studies of M. tuberculosis H37Rv bacterial cells treated with
3 as a representative of this set
demonstrated destruction of the bacterial cell wall. The results of these studies thus suggest that the
mechanism of action for these compounds is most likely related to interactions with the bacterial cell
wall. Ongoing studies currently underway will hopefully provide more insight on this interesting
finding and will be reported elsewhere as they become available.
Supplementary Materials: Copies of the NMR spectra are available online.
Author Contributions: A.A.K., E.S.M. and A.L.K. conceived, designed and performed the chemical synthesis;
K.W.B., M.W., E.B.L. and M.A.Z. designed and performed cell assays and evaluated biological properties of the
compounds; P.N.S. performed HRMS analysis and analyzed the relevant data. A.A.K., E.S.M., E.B.L., M.A.Z.,
P.N.S., S.N.K., K.L.S-R. and A.L.K. analyzed the data; R.W.B., S.N.K. contributed reagents/materials/analysis
tools; K.L.S.-R., A.A.K., E.S.M., E.B.L., P.N.S. and A.L.K. wrote the paper.
Funding: The work was supported by Russian Science Foundation, project No 14-50-00060.
Conflicts of Interest: The authors declare no conflict of interest.
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