Triphenylphosphonium conjugates of 1,2,3-triazolyl nucleoside analogues. Synthesis and cytotoxicity evaluation

Article abstract of DOI:10.1007/s00044-020-02629-x

A series of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl analogues of several pyrimidine nucleosides was synthesized and evaluated for the in vitro cytotoxicity against human cancer cell lines M-HeLa, MCF-7, PANC-1, PC-3, DU145, SKOV-3, A275,

Full text of DOI:10.1007/s00044-020-02629-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02629-x
ORIGINAL RESEARCH
Triphenylphosphonium conjugates of 1,2,3-triazolyl nucleoside
analogues. Synthesis and cytotoxicity evaluation
Irina Yu. Strobykina1 Olga V. Andreeva1 Mayya G. Belenok1 Marina N. Semenova2 Victor V. Semenov3
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Roman N. Chuprov-Netochin4 Anastasiya S. Sapunova1 Alexandra D. Voloshina1 Alexey B. Dobrynin1
Vyacheslav E. Semenov
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Vladimir E. Kataev1
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Received: 18 July 2020 / Accepted: 3 September 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl analogues of several pyrimidine nucleosides was
synthesized and evaluated for the in vitro cytotoxicity against human cancer cell lines M-HeLa, MCF-7, PANC-1, PC-3,
DU145, SKOV-3, A275, and normal human cell line WI-38. In these TPP-conjugates triphenylphosphonium cation was
attached via a tetramethylene chain to the N-3 atom of the heterocycle moiety (uracil, thymine, quinazoline-2,4-dione),
which was coupled with the D-ribofuranosyl-1,2,3-triazol-4-yl fragment via methylene or tetramethylene linker. It was
shown for the first time that the conjugation of 1,2,3-triazolyl derivatives of uridine, its analogues featuring quinazoline-2,4-
dione fragment as well as uracil and thymine derivatives, having propargyl or a 1,2,3-triazolyl substituent at the N-1 atom,
with a TPP-butyl cation endowed some of them with cytotoxic activity against human cancer cells. Among all human cancer
cell lines used, DU-145 and A375 cells were the most sensitive to these TPP conjugates. At the same time, all tested
compounds did not inhibit growth of normal cells WI-38. Propargyl containig TPP-conjugates of uracil 4f, 4j, and thymine
5f showed the highest cytotoxicity with IC50 values in the low micromolar concentration range. The present findings suggest
that TPP-conjugates of uracil and thymine derivatives would be promising for further development as an anticancer agent.
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Keywords Nucleoside analogues 1,2,3-Triazole Triphenylphosphonium TPP conjugates Cytotoxicity
Introduction
The mitochondrion is a vital organelle in most eukaryotic
cells. One of the main functions of mitochondria is the
synthesis of adenosine triphosphate (ATP), an universal
form of chemical energy in any living cell, via oxidative
phosphorylation. Moreover, mitochondria play a key role
in many other metabolic processes in a cell, such as
modulating calcium homeostasis, the tricarboxylic acid
and urea cycles, fatty acid oxidation, iron sulfur center
and heme biosynthesis, amino acid metabolism, and redox
signaling [1–4] Furthermore, mitochondria are critically
involved in the regulation of programmed cell death, the
intrinsic pathway of apoptosis [1–4]. Mitochondrial
damage that disrupts ATP synthesis and calcium home-
ostasis is a key component of necrotic cell death [5]. For
all these reasons, mitochondrial malfunction disrupts the
function of cells, tissues, and organs and contributes to a
remarkably wide range of diseases [2, 5]. Mitochondrial
dysfunction has been implicated in neurodegenerative and
neuromuscular disorders, ischemia-reperfusion injury,
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02629-x) contains supplementary
material, which is available to authorized users.
* Vyacheslav E. Semenov
vesemenov@mail.ru
1
Arbuzov Institute of Organic and Physical Chemistry, Federal
Research Center “Kazan Scientific Center of the Russian Academy
of Sciences”, Arbuzov Str., 8, Kazan 420088, Russian Federation
2
N. K. Kol’tsov Institute of Developmental Biology, Russian
Academy of Sciences, Vavilov Street, 26, Moscow 119334,
Russian Federation
3
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy
of Sciences, Leninsky Prospect, 47, Moscow 119991, Russian
Federation
4
Moscow Institute of Physics and Technology (State University),
Institutskiy per. 9, Dolgoprudny, Moscow Region 141701,
Russian Federation

Medicinal Chemistry Research
diabetes, obesity, inherited mitochondrial diseases and
most importantly, cancer [2, 6]. In nearly all cases where
mitochondrial dysfunction contributes to disease, a major
cause of damage is reactive oxygen species (ROS) pro-
duced by mitochondria, either directly or as a secondary
consequence of other malfunctions [5]. One approach to
addressing these challenges is to target antioxidants to
mitochondria by conjugation to a lipophilic cation, such
as the triphenylphosphonium (TPP) cation [5]. The large
membrane potential of 150–180 mV (negative inside)
across the mitochondrial inner membrane is used to
deliver lipophilic cations or molecules to mitochondria.
Lipophilic TPP cation passes easily through lipid bilayers
because its charge is dispersed over a large surface area
and the potential gradient drives the accumulation of TPP
cation into the mitochondrial matrix [7]. A wide range of
antioxidants were targeted to mitochondria by conjuga-
tion to the TPP moiety, and those that have been
employed to date include TPP-conjugated derivatives of
ubiquinone, tocopherol, lipoic acid, spin traps, and the
peroxidase mimetic ebselen [7–10]. Compared with nor-
mal cells, cancer cells exhibit a markedly higher mito-
chondrial transmembrane potential that provides the
selective accumulation of the TPP cation inside cancer
cell mitochondria. Hence, the bulky lipophilic TPP
cations are assumed to be promising carriers for a drug
accumulation in mitochondria [11, 12]. Notably, a con-
jugation of well-known anticancer agents (doxorubicin,
salicylaldazine, chlorambucil, glycyrrhetinic acid, curcu-
min, etc) with TPP has resulted in both the targeting of
mitochondria in cancer cells and the increase of cyto-
toxicity [8,13–15].
The literature has provided many examples of nucleo-
side analogues, which also displayed anticancer activity.
Among them 2′-deoxy-2′,2′-difluorocytidine (Gemcita-
bine) was approved for the treatment of breast cancer,
ovarian cancer, non-small cell lung cancer, pancreatic
cancer, bladder cancer and 2′-deoxy-5-fluorouridine
(Floxuridine) is an oncology drug to treat colorectal cancer
[16]. 3′-(4-Chlorophenyl-1,2,3-triazolyl)thymidine exhib-
ited high potential in bringing down tumor cell prolifera-
tion and induced their apoptosis [17]. Several nucleoside
analogues containing substituted 1,2,3-triazolyl moieties
at the C-5′ position of ribofuranose residue demonstrated
significant cytotoxicity against cancer cell lines A549, HT-
29, MCF-7, A-375 [18]. A 2′-deoxyuridine derivarive
featuring perfluorodecyl substituted 1,2,3-triazole moiety
attached to the C-5 position of the uracil ring exhibited
significant growth inhibition of PC-3, MDA-MB-231, and
ACHN cancer cells [19]. Surprisingly, there is only one
publication reporting the synthesis of the TPP conjugate of
a 1,2,3-triazolyl pyrimidine analog, that penetrated selec-
tively the mitochondria of cancer cells [20].
Apparent scarcity of literature data regarding TPP-
conjugates of 1,2,3-triazolyl nucleoside analogues promp-
ted us to synthesize a series of 1,2,3-triazolyl pyrimidine
analogues conjugated with a TPP cation and to study their
cytotoxicity against a panel of human cancer cell lines. In
these TPP-conjugates TPP cation was attached via a tetra-
methylene chain to the N-3 atom of the heterocycle moiety
(uracil, thymine, quinazoline-2,4-dione), which was cou-
pled with the D-ribofuranosyl-1,2,3-triazol-4-yl fragment
via methylene or tetramethylene linker. In addition, a series
of model compounds, namely derivatives of uracil and
thymine containing ω-alkyne or a 1,2,3-triazolyl substituent
at the N-1 atom as well as the TPP-butyl moiety at the N-3
atom of nucleobase was synthesized and evaluated for
cytotoxicity against a panel of human cancer cells.
Material and methods
General chemistry
The 1H NMR spectra were recorded on 400 MHz and
600 MHz Brucker Advance. 13C NMR spectra were
obtained in the above instruments operating at 100.6 MHz,
31P NMR spectra at 161 MHz. Melting points were obtained
on an Electrothermal IA 9000 instrument (Electrothermal,
Great Britain). Mass spectra (MALDI) were recorded in a
positive ion mode on a Bruker Ultraflex III TOF/TOF mass
spectrometer for 10−3 mg/mL solutions. The ESI MS mea-
surements were performed using an AmazonX ion trap
mass spectrometer (Bruker Daltonic GmbH, Germany) in
positive mode in the mass range of 70–3000. The capillary
voltage was 3500 V, nitrogen drying gas 10 L/min, deso-
lvation temperature 250 °C. A methanol/water solution
(70:30) was used as a mobile phase at a flow rate of 0.2 mL/
min by binary pump (Agilent 1260 chromatograph, USA).
The sample was dissolved in methanol to a concentration of
10−6 g/L. Flash chromatography was performed on silica
gel 60 (40–63 μm, Buchi, Sepacore). Thin-layer chromato-
graphy was carried out on plates with silica gel (Sorbfil,
Russia). Spots of compounds were visualized by using
ultraviolent fluorescence under a short wavelength (254 nm)
followed by heating the plates (at ca. 150 °C) after
immersion in a solution of 5% H2SO4 and 95% H2O. All
reactions sensitive to air and/or moisture were carried out
under an argon atmosphere with anhydrous solvents, which
were purified and dried (where appropriate) according to
standard procedures.
1,2,3-Triazolyl pyrimidine analogues 1a, 1d, 1e, 1h, 2a,
2d, 3a, 3d and pyrimidines 4b, 5b, 4i having ω-alkyne
substituent at the N-1 atom were synthesized according a
procedure earlier described [21]. Uracil and thymine deri-
vatives 4g, 5c, 5g were prepared by analogy with the

Medicinal Chemistry Research
methods previously described [22–24]. 1-Azidopentane and
(4-bromobutyl)TPP bromide were synthesized by known
methods [25–27]. Uracil, thymine, quinazoline-2,4(1H,3H)-
dione and propargyl bromide were purchased from Sigma-
Aldrich.
1-{[1-(2′′,3′′,5′′-Tri-O-acetyl-β-D-ribofuranosyl)-1′H-
1′,2′,3′-triazol-4-yl]butyl}-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (1f)
1
Foam; yield: 0.20 g (65%); H NMR (CD3OD, 400 MHz):
General procedure for synthesis of
triphenylphosphonium salts
δ = 7.90–7.71 (16H, m, H-5′, 6H-12, 6H-13, 3H-14), 7.53
(1H, d, J = 7.8 Hz, H-6), 6.21 (1H, d, J = 3.4 Hz, H-1′′),
5.87–5.84 (1H, m, H-3′′), 5.67–5.60 (2H, m, H-5, H-2′′),
4.49–4.45 (1H, m, H-4′′), 4.36 (1H, dd, J = 12.3, 3.4 Hz, H-
5′′a), 4.17 (1H, dd, J = 12.2, 4.4 Hz, H-5′′b), 3.95 (2H, t,
J = 6.6 Hz, H-7), 3.79–3.72 (2H, m, H-15), 3.51–3.43 (2H,
m, H-10), 2.77–2.70 (2H, m, H-18), 2.10, 2.09, 1.98 (3H, s,
OAc), 1.89–1.82 (2H, m, H-8), 1.73–1.60 (6H, m, H-9, H-
16, H-17); 13C NMR (CDCl3, 100 MHz): δ = 170.31,
169.40, 169.26 (C, CH3CO), 163.29 (C, C-4), 151.25 (C, C-
2), 147.45 (C, C-4′), 143.41 (CH, C-6), 135.09 (CH, d, J =
2.9 Hz, C-14), 133.57 (CH, d, J = 9.9 Hz, C-12), 130.44
(CH, d, J = 12.5 Hz, C-13), 121.18 (CH, C-5′), 117.97 (C,
d, J = 85.8 Hz, C-11), 100.92 (CH, C-5), 89.65 (CH, C-1′′),
80.51 (CH, C-4′′), 74.10 (CH, C-3′′), 70.78 (CH, C-2′′),
62.98 (CH2, C-5′′), 49.23 (CH2, C-15), 39.10 (CH2, C-7),
28.18 (CH2, C-18), 27.82 (CH2, d, J = 16.5 Hz, C-8), 25.84
(CH2, C-16), 24.78 (CH2, C-17), 21.91 (CH2, d, J =
51.0 Hz, C-10), 20.62, 20.41, 20.38 (CH3, CH3CO), 19.42
(CH2, d, J = 3.7, C-9); 31P NMR (CDCl3, 161 MHz): δ =
24.66; ESI MS m/z: 810.3 [M-Br]+ (calcd. 810.3); Anal.
Calcd. for C43H49BrN5O9P: C, 57.98; H, 5.54; Br, 8.97; N,
7.86; P, 3.48. Found: C, 57.90; H, 5.48; Br, 9.04; N, 7.78;
P, 3.57.
A mixture of an uracil derivative (compounds 1a, 1e, 4b,
4d, 4g, 4i) or a thymine derivative (compounds 2a, 5b, 5d,
5g) or quinazoline-2,4-dione derivative 3a (1 mmol), (4-
bromobutyl)triphenylphosphonium bromide (1 mmol) and
K2CO3 (3 mmol) in dimethylformamide (DMF) (3–5 ml)
was stirred for 24 h in an argon atmosphere at the tem-
perature 40 °C. The reaction mixture was filtered, and DMF
was removed under reduced pressure at the temperature
40–50 °C. The residue was dissolved in CH2Cl2, washed
with a saturated solution of NaCl and H2O, dried with
Na2SO4. The resulting mixture was concentrated under a
vacuum to 3–5 mL, and 5 mL Et2O was added. The product,
after decanting the solvent, was dried in a high vacuum at
40–50 °C.
1-{[1-(2′′,3′′,5′′-Tri-O-acetyl-β-D-ribofuranosyl)-1′H-
1′,2′,3′-triazol-4-yl]methyl}-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (1b)
Amorphous powder; yield: 0.17 g (57%); 1H NMR (CDCl3,
400 MHz): δ = 8.32 (1H, s, H-5′), 7.83–7.65 (15H, m, 6H-
12, 6H-13, 3H-14), 7.59 (1H, d, J = 7.9 Hz, H-6), 6.25 (1H,
d, J = 3.4 Hz, H-1′′), 5.84–5.81 (1H, m, H-3′′), 5.63 (1H, t,
J = 5.5 Hz, H-2′′), 5.57 (1H, d, J = 7.9 Hz, H-5), 5.10–5.00
(2H, m, H-15), 4.46–4.41 (1H, m, H-4′′), 4.37 (1H, dd, J =
12.2, 3.3 Hz, H-5′′a), 4.17 (1H, dd, J = 12.2, 4.8 Hz, H-5′′
b), 3.93 (2H, t, J = 6.3 Hz, H-7), 3.88–3.77 (2H, m, H-10),
2.09, 2.09, 2.04 (3H, s, OAc), 2.03–1.97 (2H, m, H-8),
1.69–1.53 (2H, m, H-9); 13C NMR (CDCl3, 100 MHz): δ =
170.33, 169.37, 169.19 (C, CH3CO), 163.12 (C, C-4),
151.22 (C, C-2), 142.87 (C, C-4′), 142.20 (CH, C-6),
135.04 (CH, d, J = 2.9 Hz, C-14), 133.59 (CH, d, J =
10.1 Hz, C-12), 130.43 (CH, d, J = 12.6 Hz, C-13), 124.52
(C, C-5′), 118.04 (C, d, J = 86.0 Hz, C-11), 101.43 (CH, C-
5), 89.82 (CH, C-1′′), 80.59 (CH, C-4′′), 74.13 (CH, C-3′′),
70.74 (CH, C-2′′), 62.94 (CH2, C-5′′), 43.45, 39.28 (CH2,
C-7, C-15), 27.86 (CH2, d, J = 16.7 Hz, C-8), 21.93 (CH2,
d, J = 50.6 Hz, C-10), 20.61, 20.38, 20.35 (CH3, CH3CO),
19.52 (CH2, d, J = 3.8 Hz, C-9); 31P NMR (CDCl3,
161 MHz): δ = 25.11; MALDI MS m/z: 768.5 [M-Br]+
(calcd. 768.3); Anal. Calcd. for C40H43BrN5O9P: C, 56.61;
H, 5.11; Br, 9.42; N, 8.25; P, 3.65. Found: C, 56.72; H,
5.08; Br, 9.52; N, 8.15; P, 3.78%.
5-Methyl-1-{[1-(2′′,3′′,5′′-tri-O-acetyl-β-D-
ribofuranosyl)-1′H-1′,2′,3′-triazol-4-yl]methyl}-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (2b)
Amorphous powder; yield: 0.19 g (68%); 1H NMR
(CD3OD, 400 MHz): δ = 8.13 (1H, s, H-5′), 7.90–7.70
(15H, m, 6H-12, 6H-13, 3H-14), 7.54 (1H, br. s, H-6), 6.26
(1H, d, J = 3.3 Hz, H-1′′), 5.86–5.82 (1H, m, H-3′′), 5.62
(1H, t, J = 5.6 Hz, H-2′′), 5.00 (2H, s, H-15), 4.51–4.46
(1H, m, H-4′′), 4.35 (1H, dd, J = 12.4, 3.2 Hz, H-5′′a), 4.17
(1H, dd, J = 12.3, 4.1 Hz, H-5′′b), 3.97 (2H, t, J = 6.6 Hz,
H-7), 3.50–3.41 (2H, m, H-10), 2.09, 2.08, 1.95 (3H, s,
OAc), 1.90–1.80 (5H, m, CH3-5, H-8), 1.70–1.60 (2H, m,
H-9); 13C NMR (CDCl3, 100 MHz): δ = 170.34, 169.39,
169.22 (C, CH3CO), 163.84 (C, C-4), 151.17 (C, C-2),
142.34 (C, C-4′), 138.67 (CH, C-6), 135.01 (CH, d, J =
2.6 Hz, C-14), 133.60 (CH, d, J = 9.9 Hz, C-12), 130.41
(CH, d, J = 12.8 Hz, C-13), 124.27 (CH, C-5′), 118.07 (C,
d, J = 85.8 Hz, C-11), 109.76 (C, C-5), 89.86 (CH, C-1′′),
80.58 (CH, C-4′′), 74.15 (CH, C-3′′), 70.70 (CH, C-2′′),
62.93 (CH2, C-5′′), 43.33, 39.58 (CH2, C-7, C-15), 28.05

Medicinal Chemistry Research
(CH2, d, J = 16.6 Hz, C-8), 21.96 (CH2, d, J = 50.6 Hz, C-
10), 20.60, 20.38, 20.37 (CH3, CH3CO), 19.65 (CH2, d, J =
4.0 Hz, C-9), 12.86 (CH3, CH3-C-5); 31P NMR (CDCl3,
161 MHz): δ = 25.57; MALDI MS m/z: 782.5 [M-Br]+
(calcd. 782.3); Anal. Calcd. for C41H45BrN5O9P: C, 57.08;
H, 5.26; Br, 9.26; N, 8.12; P, 3.59. Found: C, 56.92; H,
5.18; Br, 9.35; N, 8.20; P, 3.67.
100 MHz): δ = 165.31 (C, C-4), 152.78 (C, C-2), 145.16 (C,
C-4′), 143.57 (CH, C-6), 136.33 (CH, d, J = 2.6 Hz, C-14),
134.88 (CH, d, J = 9.9 Hz, C-12), 131.57 (CH, d, J =
12.5 Hz, C-13), 125.10 (CH, C-5′), 119.83 (C, d, J =
86.2 Hz, C-11), 102.04 (CH, C-5), 51.46 (CH2, C-16), 44.99
(CH2, C-15), 40.76 (CH2, C-7), 30.95 (CH2, C-17), 29.64
(CH2, C-18) 29.34 (CH2, d, J = 17.3 Hz, C-8), 23.09 (CH2,
C-19), 22.49 (CH2, d, J = 51.4 Hz, C-10), 20.83 (CH2, d,
J = 3.7 Hz, C-9), 14.23 (CH3, C-20); 31P NMR (CD3OD,
161 MHz): δ = 23.98; ESI MS m/z: 580.4 [M-Br]+ (calcd.
580.3); Anal. Calcd. for C34H39BrN5O2P: C, 61.82; H, 5.95;
Br, 12.10; N, 10.60; P, 4.69. Found: C, 61.85; H, 5.81; Br,
12.24; N, 10.51; P, 4.77.
1-{[1-(2′′,3′′,5′′-Tri-O-acetyl-β-D-ribofuranosyl)-1′H-
1′,2′,3′-triazol-4-yl]methyl}-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
quinazolinedione bromide (3b)
The product was purified by dry column chromatography
with CHCl3/MeOH from 100:0.1 to 100:2 solvent system to
afford pure compound in 21% yield (0.08 g); 1H NMR
(CDCl3, 400 MHz): δ = 8.16 (1H, s, H-5′), 8.01 (1H, d, J =
7.7 Hz, H-5), 7.81–7.60 (17H, m, H-7, H-8, 6H-16, 6H-17,
3H-18), 7.20–7.15 (1H, m, H-6), 6.25 (1H, d, J = 3.0 Hz,
H-1′′), 5.78–5.74 (1H, m, H-3′′), 5.60 (1H, t, J = 5.6 Hz, H-
2′′), 5.38–5.27 (2H, m, H-19), 4.42–4.37 (1H, m, H-4′′),
4.32 (1H, dd, J = 12.2, 3.0 Hz, H-5′′a), 4.14–4.06 (3H, m,
H-11, H-5′′b), 3.89–3.80 (2H, m, H-14), 2.13–2.02 (8H, m,
H-12, 2OAc), 1.96 (3H, s, OAc), 1.70–1.60 (2H, m, H-13);
13C NMR (CDCl3, 100 MHz): δ = 170.27, 169.33, 169.19
(C, CH3CO), 161.80 (C, C-4), 150.63 (C, C-2), 142.66 (C,
C-4′), 139.42 (C, C-9), 135.41, 128.55, 124.02 (CH, C-6,
C-7, C-8), 134.90 (CH, d, J = 2.7 Hz, C-18), 133.59 (CH, d,
J = 10.0 Hz, C-16), 130.33 (CH, d, J = 12.7 Hz, C-17),
123.04 (CH, C-5′), 118.06 (C, d, J = 85.8 Hz, C-15),
115.11 (C, C-10), 114.59 (CH, C-5), 89.80 (CH, C-1′′),
80.48 (CH, C-4′′), 74.14 (CH, C-3′′), 70.63 (CH, C-2′′),
62.88 (CH2, C-5′′), 40.10, 39.06 (CH2, C-19, C-11), 28.19
(CH2, d, J = 17.0 Hz, C-12), 21.99 (CH2, d, J = 50.9 Hz, C-
14), 20.51, 20.36, 20.34 (CH3, CH3CO), 19.67 (CH2, d, J =
3.9 Hz, C-13); 31P NMR (CDCl3, 161 MHz): δ = 24.73;
MALDI MS m/z: 818.5 [M-Br]+ (calcd. 818.3); Anal.
Calcd. for C44H45BrN5O9P: C, 58.80; H, 5.05; Br, 8.89; N,
7.79; P, 3.45. Found: C, 58.95; H, 5.10; Br, 8.77; N, 7.68;
P, 3.51.
1-Propargyl-3-(n-butyltriphenylphosphonium)-2,4
(1H,3H)-pyrimidinedione bromide (4f)
1
Foam; yield: 0.15 g (27%); H NMR (CDCl3, 400 MHz):
δ = 7.86–7.65 (15H, m, 6H-12, 6H-13, 3H-14), 7.58 (1H,
d, J = 7.9 Hz, H-6), 5.64 (1H, d, J = 7.9 Hz, H-5), 4.57
(2H, d, J = 2.5 Hz, H-15), 3.95 (2H, t, J = 6.3 Hz, H-7),
3.88–3.78 (2H, m, H-10), 2.45 (1H, t, J = 1.9 Hz, H-17),
2.07–1.96 (2H, m, H-8), 1.66–1.55 (2H, m, H-9); 13C
NMR (CDCl3, 100 MHz): δ = 162.95 (C, C-4), 150.90 (C,
C-2), 141.41 (CH, C-6), 135.03 (CH, d, J = 2.9 Hz, C-14),
133.71 (CH, d, J = 9.9 Hz, C-12), 130.46 (CH, d, J =
12.6 Hz, C-13), 118.15 (C, d, J = 86.0 Hz, C-11), 101.74
(CH, C-5), 76.48 (C, C-16), 75.18 (CH, C-17), 39.47,
38.06 (CH2, C-7, C-15), 27.84 (CH2, d, J = 16.5 Hz, C-8),
22.01 (CH2, d, J = 50.7 Hz, C-10), 19.56 (CH2, d, J =
3.8 Hz, C-9); 31P NMR (CDCl3, 161 MHz): δ = 24.60;
MALDI MS, m/z: 467.5 [M-Br]+ (calcd. 467.2); Anal.
Calcd. for C29H28BrN2O2P: C, 63.63; H, 5.16; Br, 14.60;
N, 5.12; P, 5.66. Found: C, 63.75; H, 5.09; Br, 14.72; N,
5.05; P, 5.58.
1-Methyl-3-(n-butyltriphenylphosphonium)-2,4
(1H,3H)-pyrimidinedione bromide (4h)
1
Foam; yield: 0.20 g (26%); H NMR (CD3OD, 400 MHz):
δ = 7.90–7.72 (15H, m, 6H-12, 6H-13, 3H-14), 7.50 (1H, d,
J = 7.8 Hz, H-6), 5.65 (1H, d, J = 7.9 Hz, H-5), 3.96 (2H, t,
J = 6.7 Hz, H-7), 3.51–3.43 (2H, m, H-10), 3.31 (3H, s,
CH3-1), 1.91–1.83 (2H, m, H-8), 1.71–1.61 (2H, m, H-9);
13C NMR (CDCl3, 100 MHz): δ = 163.46 (C, C-4), 151.66
(C, C-2), 144.13 (CH, C-6), 135.10 (CH, C-14), 133.61
(CH, d, J = 10.0 Hz, C-12), 130.47 (CH, d, J = 12.4 Hz, C-
13), 118.01 (C, d, J = 75.3 Hz, C-11), 100.84 (CH, C-5),
39.13 (CH2, C-7), 36.97 (CH3, CH3-N-1), 27.81 (CH2, d,
J = 16.6 Hz, C-8), 22.01 (CH2, d, J = 50.9 Hz, C-10), 19.43
(CH2, C-9); 31P NMR (CDCl3, 161 MHz): δ = 24.64;
MALDI MS m/z: 443.4 [M-Br]+ (calcd. 443.2); Anal.
Calcd. for C27H28BrN2O2P: C, 61.96; H, 5.39; Br, 15.27;
1-[(n-Pentyl-1′H-1′,2′,3′-triazol-4′-yl)methyl]-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (4e)
Amorphous powder; yield: 0.19 g (59%); 1H NMR
(CD3OD, 400 MHz): δ = 7.96 (1H, s, H-5′), 7.90–7.71
(15H, m, 6H-12, 6H-13, 3H-14), 7.65 (1H, d, J = 7.9 Hz, H-
6), 5.69 (1H, d, J = 7.8 Hz, H-5), 5.00 (2H, s, H-15), 4.36
(2H, t, J = 7.2 Hz, H-16), 3.95 (2H, t, J = 6.7 Hz, H-7),
3.51–3.42 (2H, m, H-10), 1.91–1.81 (4H, m, H-8, H-17),
1.72–1.60 (2H, m, H-9), 1.39–1.22 (4H, m, H-18, H-19),
0.88 (3H, t, J = 7.1 Hz, H-20); 13C NMR (CD3OD,

Medicinal Chemistry Research
N, 5.35; P, 5.92. Found: C, 62.10; H, 5.32; Br, 15.34; N,
5.28; P 5.86.
1-Propargyl-3-(n-butyltriphenylphosphonium)-5-
methyl-2,4(1H,3H)-pyrimidinedione bromide (5f)
1-(Hex-5′-yn-1′-yl)-3-(n-
butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (4j)
Amorphous powder; yield: 0.15 g (44%); 1H NMR (CDCl3,
400 MHz): δ = 7.86–7.66 (15H, m, 6H-12, 6H-13, 3H-14),
7.30 (1H, d, J = 0.9 Hz,.H-6), 4.52 (2H, d, J = 2.5 Hz, H-
15), 3.97 (2H, t, J = 6.4 Hz, H-7), 3.95–3.89 (2H, m, H-10),
2.44 (1H, t, J = 3.0 Hz, H-17), 2.06–1.99 (2H, m, H-8),
1.86 (3H, d, J = 1.0 Hz, CH3-5), 1.66–1.57 (2H, m, H-9);
13C NMR (CDCl3, 100 MHz): δ = 163.66 (C, C-4), 150.89
(C, C-2), 137.25 (CH, C-6), 134.98 (CH, d, J = 3.1 Hz, C-
14), 133.66 (CH, d, J = 9.9 Hz, C-12), 130.41 (CH, d, J =
12.5 Hz, C-13), 118.17 (C, d, J = 86.0 Hz, C-11), 110.06
(C, C-5), 76.75 (C, C-16), 74.81 (CH, C-17), 39.67, 37.63
(CH2, C-7, C-15), 27.98 (CH2, d, J = 16.7 Hz, C-8), 21.87
(CH2, d, J = 50.5 Hz, C-10), 19.59 (CH2, d, J = 3.8 Hz, C-
9), 12.95 (CH3, CH3-C-5); 31P NMR (CDCl3, 161 MHz):
δ = 24.69; MALDI MS m/z: 481.1 [M-Br]+ (calcd. 481.2);
Anal. Calcd. for C30H30BrN2O2P: C, 64.18; H, 5.39; Br,
14.23; N, 4.99; P, 5.52. Found: C, 64.02; H, 5.40; Br,
14.38; N, 5.00; P, 5.44.
1
Foam; yield: 0.19 g (41%); H NMR (CD3OD, 400 MHz):
δ = 7.92–7.72 (15H, m, 6H-12, 6H-13, 3H-14), 7.55 (1H, d,
J = 7.9 Hz, H-6), 5.67 (1H, d, J = 7.9 Hz, H-5), 3.96 (2H, t,
J = 6.7 Hz, H-7), 3.75 (2H, t, J = 7.3 Hz, H-15), 3.52–3.43
(2H, m, H-10), 2.25–2.17 (3H, m, H-18, H-20), 1.91–1.83
(2H, m, H-8), 1.79–1.61 (4H, m, H-9, H-17), 1.53–1.44
(2H, m, H-16); 13C NMR (CDCl3, 100 MHz): δ = 163.23
(C, C-4), 151.24 (C, C-2), 143.13 (CH, C-6), 135.02 (CH,
d, J = 2.9 Hz, C-14), 133.60 (CH, d, J = 9.9 Hz, C-12),
130.43 (CH, d, J = 12.5 Hz, C-13), 118.05 (C, d, J =
85.8 Hz, C-11), 101.04 (CH, C-5), 83.52 (C, C-19), 68.97
(CH, C-20), 49.05 (CH2, C-15), 39.20 (CH2, C-7), 27.96
(CH2, C-18), 27.79 (CH2, d, J = 16.9 Hz, C-8), 25.06 (CH2,
C-16), 21.86 (CH2, d, J = 42.6 Hz, C-10), 19.47 (CH2, d,
J = 3.7 Hz, C-9), 17.94 (CH2, C-17); 31P NMR (CDCl3,
161 MHz): δ = 24.68; ESI MS m/z: 509.4 [M-Br]+ (calcd.
509.2); Anal. Calcd. for C32H34BrN2O2P: C, 65.20; H, 5.81;
Br, 13.55; N, 4.75; P, 5.25. Found: C, 65.12; H, 5.8; Br,
13.63; N, 4.68; P, 5.19.
1,5-Dimethyl-3-(n-butyltriphenylphosphonium)-2,4
(1H,3H)-pyrimidinedione bromide (5h)
1
Foam; yield: 0.22 g (31%); H NMR (CD3OD, 400 MHz):
δ = 7.92–7.72 (15H, m, 6H-12, 6H-13, 3H-14), 7.38 (1H, d,
J = 1.2 Hz, H-6), 3.98 (2H, t, J = 6.7 Hz, H-7), 3.53–3.45
(2H, m, H-10), 3.29 (3H, s, CH3-1), 1.90–1.84 (2H, m, H-
8), 1.84 (3H, d, J = 1.0 Hz, CH3-5), 1.70–1.60 (2H, m, H-
9); 13C NMR (CDCl3, 100 MHz): δ = 164.09 (C, C-4),
151.60 (C, C-2), 140.06 (CH, C-6), 135.06 (CH, C-14),
133.62 (CH, d, J = 9.9 Hz, C-12), 130.43 (CH, d, J =
12.4 Hz, C-13), 118.05 (C, d, J = 86.0 Hz, C-11), 109.07
(C, C-5), 39.37 (CH2, C-7), 36.62 (CH3, CH3-N-1), 27.97
(CH2, d, J = 16.6 Hz, C-8), 22.05 (CH2, d, J = 50.9 Hz, C-
10), 19.51 (CH2, d, J = 2.2 Hz, C-9), 12.75 (CH3, CH3-C-
5); 31P NMR (CDCl3, 161 MHz): δ = 24.60; MALDI MS
m/z: 457.5 [M-Br]+ (calcd. 457.2); Anal. Calcd. for
C28H30BrN2O2P: C, 62.58; H, 5.63; Br, 14.87; N, 5.21; P,
5.76. Found: C, 62.62; H, 5.57; Br, 15.05; N, 5.12; P, 5.83.
1-[(n-Pentyl-1′H-1′,2′,3′-triazol-4′-yl)methyl]-3-(n-
butyltriphenylphosphonium)-5-methyl- 2,4(1H,3H)-
pyrimidinedione bromide (5e)
Amorphous powder; yield: 0.30 g (63%); 1H NMR
(CD3OD, 400 MHz): δ = 7.99 (1H, s, H-5′), 7.90–7.71
(15H, m, 6H-12, 6H-13, 3H-14), 7.53 (1H, s, H-6), 4.98
(2H, s, H-15), 4.36 (2H, t, J = 7.2 Hz, H-16), 3.97 (2H, t,
J = 6.7 Hz, H-7), 3.55–3.44 (2H, m, H-10), 1.92–1.85
(4H, m, H-8, H-17), 1.84 (3H, d, J = 1.0 Hz, CH3-5),
1.71–1.60 (2H, m, H-9), 1.37–1.22 (4H, m, H-18, H-19),
0.88 (3H, t, J = 7.2 Hz, H-20); 13C NMR (CD3OD,
100 MHz): δ = 165.75 (C, C-4), 152.79 (C, C-2), 143.80
(C, C-4′), 141.15 (CH, C-6), 136.36 (CH, d, J = 2.9 Hz, C-
14), 134.88 (CH, d, J = 10.3 Hz, C-12), 131.57 (CH, d,
J = 12.5 Hz, C-13), 125.02 (CH, C-5′), 119.86 (C, d, J =
86.2 Hz, C-11), 110.72 (C, C-5), 51.47 (CH2, C-16), 44.75
(CH2, C-15), 40.99 (CH2, C-7), 30.95 (CH2, C-17), 29.65
(CH2, C-18), 29.46 (CH2, d, J = 16.9 Hz, C-8), 23.10
(CH2, C-19), 22.53 (CH2, d, J = 52.7 Hz, C-10), 20.88
(CH2, d, J = 3.7 Hz, C-9), 14.24 (CH3, C-20), 13.01 (CH3,
CH3-C-5); 31P NMR (CD3OD, 161 MHz): δ = 24.51; ESI
MS m/z: 594.4 [M-Br]+ (calcd. 594.3); Anal. Calcd. for
C35H41BrN5O2P: C, 62.31; H, 6.13; Br, 11.84; N, 10.38; P,
4.59. Found: C, 62.50; H, 6.08; Br, 12.05; N, 10.29;
P, 4.71.
General procedure for synthesis of unprotected
triphenylphosphonium salts of 1,2,3-triazolyl
nucleoside analogues 1b, 1f, 2b, 3b
The protected TPP salts of 1,2,3-triazolyl nucleoside
analogues 1b, 1f, 2b, 3b were dissolved in anhydrous
MeOH at room temperature and the pH was adjusted to 9.0
using a solution of 0.1 N MeONa/MeOH. The deacetyla-
tion procedure was monitored by TLC and upon its
completion the pH adjusted to 7.0 with acidic ion-
exchange resin Amberlyst 15. After filtration, the filtrate

Medicinal Chemistry Research
was concentrated under reduced pressure to afford the
corresponding target compounds.
δ = 24.58; MALDI MS m/z: 684.6 [M-Br]+ (calcd. 684.3);
Anal. Calcd. for C37H43BrN5O6P: C, 58.12; H, 5.67; Br,
10.45; N, 9.16; P, 4.05. Found: C, 58.31; H, 5.58; Br, 10.54;
N, 9.08; P, 3.98.
1-{[1-(β-D-ribofuranosyl)-1′H-1′,2′,3′-triazol-4-yl]
methyl}-3-(n-butyltriphenylphosphonium)-2,4
(1H,3H)-pyrimidinedione bromide (1c)
1-{[1-(β-D-ribofuranosyl)-1′H-1′,2′,3′-triazol-4-yl]
methyl}-3-(n-butyltriphenylphosphonium)-5-methyl-
2,4(1H,3H)-pyrimidinedione bromide (2c)
1
Foam; yield: 0.05 g (68%); H NMR (CD3OD, 400 MHz):
δ = 8.27 (1H, s, H-5′), 7.90–7.70 (15H, m, 6H-12, 6H-13,
3H-14), 7.67 (1H, d, J = 7.8 Hz, H-6), 6.01 (1H, d, J =
4.2 Hz, H-1′′), 5.68 (1H, d, J = 7.9 Hz, H-5), 5.02 (2H, s,
H-15), 4.48 (1H, t, J = 4.6 Hz, H-3′′), 4.29 (1H, t, J =
5.0 Hz, H-2′′), 4.13–4.09 (1H, m, H-4′′), 3.94 (2H, t, J =
6.8 Hz, H-7), 3.77 (1H, dd, J = 12.2, 3.2 Hz, H-5′′a), 3.67
(1H, dd, J = 12.2, 4.1 Hz, H-5′′b), 3.52–3.42 (2H, m, H-
10), 1.92–1.82 (2H, m, H-8), 1.72–1.59 (2H, m, H-9); δ 13C
NMR (CD3OD, 100 MHz): δ = 165.32 (C, C-4), 152.79 (C,
C-2), 145.17 (CH, C-6), 143.87 (C, C-4′), 136.34 (CH, s, C-
14), 134.86 (CH, d, J = 10.0 Hz, C-12), 131.58 (CH, d, J =
12.7 Hz, C-13), 123.96 (C, C-5′), 119.79 (C, d, J = 86.3 Hz,
C-11), 102.10 (CH, C-5), 94.48 (CH, C-1′′), 87.37 (CH,
C-4′′), 77.11 (CH, C-3′′), 71.92 (CH, C-2′′), 62.80 (CH2,
C-5′′), 44.97, 40.81 (CH2, C-7, C-15), 29.34 (CH2, d, J =
16.5 Hz, C-8), 22.54 (CH2, d, J = 51.7 Hz, C-10), 20.84
(CH2, s, C-9); 31P NMR (CD3OD, 161 MHz): δ = 25.15;
MALDI MS m/z: 642.5 [M-Br]+ (calcd. 642.2); Anal.
Calcd. for C34H37BrN5O6P: C, 56.52; H, 5.16; Br, 11.06; N,
9.69; P, 4.29. Found: C, 56.68; H, 5.08; Br, 11.15; N, 9.58;
P, 4.34%.
1
Foam; yield: 0.06 g (75%); H NMR (CD3OD, 400 MHz):
δ = 8.26 (1H, s, H-5′), 7.90–7.70 (15H, m, 6H-12, 6H-13,
3H-14), 7.54 (1H, s, H-6), 6.00 (1H, d, J = 4.0 Hz, H-1′′),
4.99 (2H, s, H-15), 4.47 (1H, t, J = 4.6 Hz, H-3′′), 4.29 (1H,
t, J = 5.0 Hz, H-2′′), 4.13–4.08 (1H, m, H-4′′), 3.96 (2H, t,
J = 6.6 Hz, H-7), 3.77 (1H, dd, J = 12.1, 3.2 Hz, H-5′′a),
3.66 (1H, dd, J = 12.3, 4.2 Hz, H-5′′b), 3.52–3.43 (2H, m,
H-10), 1.91–1.79 (5H, m, CH3-5, H-8), 1.70–1.59 (2H, m,
H-9); 13C NMR (CD3OD, 100 MHz): δ = 165.74 (C, C-4),
152.79 (C, C-2), 144.37 (C, C-4′), 141.15 (CH, C-6),
136.34 (CH, d, J = 3.3 Hz, C-14), 134.86 (CH, d, J =
9.9 Hz, C-12), 131.57 (CH, d, J = 12.5 Hz, C-13), 123.88
(C, C-5′), 119.82 (C, d, J = 86.5 Hz, C-11), 110.76 (C, C-
5), 94.45 (CH, C-1′′), 87.34 (CH, C-4′′), 77.10 (CH, C-3′′),
71.92 (CH, C-2′′), 62.80 (CH2, C-5′′), 44.75 (CH2, C-15),
41.01 (CH2, C-7), 29.46 (CH2, d, J = 16.9 Hz, C-8), 22.55
(CH2, d, J = 51.7 Hz, C-10), 20.87 (CH2, d, J = 4.0 Hz, C-
9), 13.00 (CH3, CH3-C-5); 31P NMR (CD3OD, 161 MHz):
δ = 25.45; MALDI MS m/z: 656.5 [M-Br]+ (calcd. 656.3);
Anal. Calcd. for C35H39BrN5O6P: C, 57.07; H, 5.34; Br,
10.85; N, 9.51; P, 4.21. Found: C, 57.20; H, 5.27; Br,
10.98; N, 9.46; P, 4.14.
1-{[1-(β-D-ribofuranosyl)-1′H-1′,2′,3′-triazol-4-yl]
butyl}-3-(n-butyltriphenylphosphonium)-2,4(1H,3H)-
pyrimidinedione bromide (1g)
1-{[1-(β-D-ribofuranosyl)-1′H-1′,2′,3′-triazol-4-yl]
methyl}-3-(n-butyltriphenylphosphonium)-2,4
(1H,3H)-quinazolinedione bromide (3c)
A foam; yield: 0.05 g (63%); 1H NMR (CD3OD, 400 MHz):
δ = 8.34 (1H, s, H-5′), 7.95–7.71 (15H, m, 6H-12, 6H-13,
3H-14), 7.58 (1H, d, J = 7.7 Hz, H-6), 6.06 (1H, d, J =
4.0 Hz, H-1′′), 5.66 (1H, d, J = 7.7 Hz, H-5), 4.51 (1H, t,
J = 4.4 Hz, H-3′′), 4.30 (1H, t, J = 5.0 Hz, H-2′′), 4.18–4.13
(1H, m, H-4′′), 3.95 (2H, t, J = 6.8 Hz, H-7), 3.84-3.75 (3H,
m, H-15 and H-5′′a), 3.70 (1H, dd, J = 12.1, 3.9 Hz, H-5′′b),
3.54–3.44 (2H, m, H-10), 2.86–2.78 (2H, m, H-18),
1.92–1.82 (2H, m, H-8), 1.75–1.60 (6H, m, H-9, H-16, H-
17); 13C NMR (CD3OD, 100 MHz): δ = 165.46 (C, C-4),
152.94 (C, C-2), 147.46 (C, C-4′), 145.58 (CH, C-6), 136.36
(CH, C-14), 134.88 (CH, d, J = 10.2 Hz, C-12), 131.59 (CH,
d, J = 12.7 Hz, C-13), 123.64 (C, C-5′), 119.83 (C, d, J =
86.6 Hz, C-11), 101.68 (CH, C-5), 95.56 (CH, C-1′′), 87.79
(CH, C-4′′), 77.31 (CH, C-3′′), 71.78 (CH, C-2′′), 62.55
(CH2, C-5′′), 50.22 (CH2, C-15), 40.74 (CH2, C-7), 29.40
(CH2, d, J = 16.6 Hz, C-8), 29.13 (CH2, C-18), 26.70 (CH2,
C-16), 25.00 (CH2, C-17), 22.50 (CH2, d, J = 51.9 Hz,
C-10), 20.89 (CH2, s, C-9); 31P NMR (CD3OD, 161 MHz):
1
Foam; yield: 0.06 g (74%); H NMR (CDCl3, 400 MHz):
δ = 8.26 (1H, s, H-5′), 8.03 (1H, d, J = 7.7 Hz, H-5),
7.85–7.65 (16H, m, H-7, 6H-16, 6H-17, 3H-18), 7.53 (1H,
d, J = 8.4 Hz, H-8), 7.26 (1H, t, J = 7.5 Hz, H-6), 5.98 (1H,
d, J = 4.0 Hz, C-1′′), 5.40 (2H, s, C-19), 4.45 (1H, t, J =
4.4 Hz, C-3′′), 4.27 (1H, t, J = 5.0 Hz, C-2′′), 4.13 (2H, t,
J = 7.5 Hz, C-11), 4.10–4.06 (1H, m, C-4′′), 3.73 (1H, dd,
J = 12.5, 3.3 Hz, H-5′′a), 3.63 (1H, dd, J = 12.3, 4.2 Hz, H-
5′′b), 3.57–3.47 (2H, m, H-14), 2.02–1.92 (2H, m, H-12),
1.74–1.63 (2H, m, H-13); 13C NMR (CD3OD, 100 MHz):
δ = 163.54 (C, C-4), 152.36 (C, C-2), 144.23 (C, C-4′),
140.99 (C, C-9), 136.68, 129.59, 124.41 (CH, C-6, C-7, C-
8), 136.27 (CH, d, J = 2.9 Hz, C-18), 134.85 (CH, d, J =
10.2 Hz, C-16), 131.52 (CH, d, J = 12.5 Hz, C-17), 123.67
(CH, C-5′), 119.79 (C, d, J = 85.8 Hz, C-15), 116.54 (C, C-
10), 115.88 (CH, C-5), 94.43 (CH, C-1′′), 87.28 (CH, C-4′′),
77.05 (CH, C-3′′), 71.85 (CH, C-2′′), 62.74 (CH2, C-5′′),

Medicinal Chemistry Research
41.38, 40.08 (CH2, C-11, C-19), 29.61 (CH2, d, J = 16.1 Hz,
C-12), 22.60 (CH2, d, J = 51.3 Hz, C-14), 20.83 (CH2, d,
J = 4.4 Hz, C-13); 31P NMR (CD3OD, 161 MHz): δ =
24.65; MALDI MS m/z: 692.6 [M-Br]+ (calcd. 692.3); Anal.
Calcd. for C38H39BrN5O6P: C, 59.07; H, 5.09; Br, 10.34; N,
9.06; P, 4.01. Found: C, 59.18; H, 5.00; Br, 10.26; N, 9.12;
P, 4.13.
(CH2, C-7), 29.76, 28.48 (CH2, C-9, C-10), 21.93 (C-11),
13.70 (CH3, C-12); ESI MS m/z: 264.1 [M + H]+ (calcd.
264.1); Anal. Calcd. for C12H17N5O2: C, 54.74; H, 6.51; N,
26.60. Found: C, 54.91; H, 6.40; N, 26.48.
1-[(n-Pentyl-1′H-1′,2′,3′-triazol-4′-yl)methyl]-5-
methyl-2,4(1H,3H)-pyrimidinedione (5d)
1
General procedure for synthesis of compounds 4c,
4d, 5d
Amorphous powder; yield: 0.28 g (80%); H NMR (CDCl3,
400 MHz): δ = 9.70 (1H, s, H-3), 7.70 (1H, s, H-5′), 7.34
(1H, d, J = 1.1 Hz, H-6), 4.95 (2H, s, H-7), 4.30 (2H, t, J =
7.3 Hz, H-8), 1.93–1.83 (5H, m, H-9, CH3-5), 1.37–1.23
(4H, m, H-10, H-11), 0.87 (3H, t, J = 7.0 Hz, H-12); 13C
NMR (CDCl3, 100 MHz): δ = 164.28 (C, C-4), 151.16 (C,
C-2), 142.01 (C, C-4′), 140.18 (CH, C-6), 123.47 (CH, C-5′),
111.22 (C, C-5), 50.48 (CH2, C-8), 42.96 (CH2, C-7), 29.80,
28.53, 21.98 (CH2, C-9, C-10, C-11), 13.73 (CH3, C-12),
12.20 (CH3, CH3 - C-5); ESI MS m/z: 278.1 [M + H]+
(calcd. 278.2); Anal. Calcd. for C13H19N5O2: C, 56.30; H,
6.91; N, 25.25. Found: C, 56.41; H, 6.80; N, 25.19.
To a solution of equimolar amounts of N-1-propargyl pyr-
imidine-2,4-dione 4b (or 5b) and trimethylsilyl azide (or 1-
azidopentane) in a mixture of 1:1 tert-butanol/water was added
freshly prepared solution of equimolar amounts of sodium
ascorbate in 2 mL of water and CuSO4 × 5 H2O in 2 mL of
water. The reaction mixture was stirred at 40 °C for 48 h, then
was concentrated under reduced pressure. The residue was
taken up in methylene chloride, washed successively with
water, dried over anhydrous sodium sulfate, and concentrated
under a vacuum to provide the desired compounds.
X-ray analysis
1-[(2′H-1′,2′,3′-Triazol-4′-yl)methyl]-2,4(1H,3H)-
pyrimidinedione (4c)
The X-ray diffraction experiments for 4c was carried out on
a Bruker KAPPA APEX II CCD diffractometer (graphite-
monochromated Mo Kα (0.71073 Å) radiation). Data col-
lection: images were indexed, integrated, and scaled using
the APEX2 data reduction package and corrected for
absorption using SADABS program for empirical X-ray
absorption correction. The structure of 4c was solved by the
direct methods and refined by anisotropic (isotropic for all
H atoms) full-matrix least-squares method against F2 of all
reflections using SHELX programs. The positions of the
hydrogen were calculated geometrically and refined in
riding model.
Trimethylsilyl azide (1.5 ml, 11.3 mmol) was added to a
solution of N-1-propargyl uracil 4b (0.2 g, 1.33 mmol) in
anhydrous toluene (5 mL). The mixture was heated in an oil
bath (120 °C) for 35 h under argon and volatiles were dis-
tilled off. The residue was dissolved in dichloromethane and
was purified by flash chromatography using petroleum
ether–ethyl acetate 10:1–1:1 as eluent. White crystal; yield:
0.06 g (20%); mp 237.2–237.8 °C; 1H NMR (DMSO,
400 MHz): δ = 11.29 (1H, s, NH), 7.80 (1H, s, H-5′), 7.67
(1H, d, J = 7.7 Hz, H-6), 5.59 (1H, d, J = 7.7 Hz, H-5), 4.94
(2H, s, H-7); 13C NMR (DMSO-d6, 100 MHz): δ = 164.56
(C, C-4), 151.21 (C, C-2), 146.13 (CH, C-6), 142.07 (C, C-
4′), 128.81 (CH, C-5′), 101.71 (CH, C-5), 42.66 (CH2, C-7);
ESI MS m/z: 194.2 [M + H]+ (calcd. 194.1); Anal. Calcd.
for C7H7N5O2: C, 43.53; H, 3.65; N, 36.26. Found: C,
43.45, H, 3.79, N, 36.31%.
Crystallographic data for 4c: C7H7N5O2, M 193.18,
monoclinic, P21/n, a 4.1712(3), b 17.0328(13), c 11.4114
(9) Å,
β 90.091(4)°, V Z 4, Dcalcd
810.75(11) Å3,
1.583 g·cm–3, μ(Mo-Kα) 0.122 mm–1, F(000) 400, (θ
3.0–28.0°, completeness 99.3%), T 296(2) К, colorless
prism, (0.05 × 0.05 × 0.2) mm3, 10349 measured reflections
in index range −5 < = h < = 5, −22 < = k < = 22, −14 < =
l < = 15, 1948 independent (Rint 0.068), 127 parameters,
R1 = 0.0567 (for 1491 observed I > 2σ(I)), wR2 = 0.2047
(all data), GOOF 0.82, largest diff. peak and hole 0.46 and
−0.32 e.A3.
1-[(n-Pentyl-1′H-1′,2′,3′-triazol-4′-yl)methyl]-2,4
(1H,3H)-pyrimidinedione (4d)
Amorphous powder; yield: 0.28 g (72%); 1H NMR (CDCl3,
400 MHz): δ = 10.05 (1H, s, H-3), 7.70 (1H, s, H-5′), 7.53
(1H, d, J = 7.9 Hz, H-6), 5.68 (1H, d, J = 7.9 Hz, H-5), 4.98
(2H, s, H-7), 4.30 (2H, t, J = 7.3 Hz, H-8), 1.92–1.83 (2H,
m, H-9), 1.36–1.22 (4H, m, H-10, H-11), 0.86 (3H, t, J =
7.0 Hz, H-12); 13C NMR (CDCl3, 100 MHz): δ = 163.89 (C,
C-4), 151.13 (C, C-2), 144.35 (CH, C-6), 141.68 (C, C-4′),
123.51 (CH, C-5′), 102.62 (C, C-5), 50.45 (CH2, C-8), 43.13
Crystallographic data for 4c have been deposited at the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 2033250. Copy of the data can be
obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB21EZ, UK (fax: +44 122 3336033 or
e-mail: deposit@ccdc.cam.ac.uk; internet: www.ccdc.cam.
ac.uk).

Medicinal Chemistry Research
Biological assays
were washed with filtered seawater and fertilized by adding
drops of diluted sperm. Embryos were cultured at room
temperature under gentle agitation with a motor-driven
plastic paddle (60 rpm) in filtered seawater. The embryos
were observed with a Biolam light microscope (LOMO, St.
Petersburg, Russia). For treatment with the test compounds,
5 mL aliquots of embryo suspension were transferred to six-
well plates and incubated as a monolayer at a concentration
up to 2000 embryos/mL. Stock solutions of compounds
were prepared in DMSO at 10–20 mM concentration fol-
lowed by a tenfold dilution with 96% EtOH. This procedure
enhanced the solubility of the test compounds in the salt-
containing medium (seawater), as evidenced by micro-
scopic examination of the samples. The maximal tolerated
concentrations of DMSO and EtOH in the in vivo assay
were determined to be 0.05% and 1%, respectively. Higher
concentrations of either DMSO (~0.1%) or EtOH (>1%)
caused nonspecific alteration and retardation of the sea
urchin embryo development independent of the treatment
stage. The antimitotic activity was assessed by exposing
fertilized eggs (8–15 min after fertilization, 45–55 min
before the first mitotic cycle completion) to twofold
decreasing concentrations of the compound. Normally at the
minimal effective (threshold) concentration a tested com-
pound caused 100% cleavage alteration and embryo death
before hatching, whereas at twofold lower concentration the
compound failed to produce any effect. Cleavage alteration
and arrest were observed at 2.5–5.5 h after fertilization,
when control embryos reached 8-cell and early blastula
stages, respectively. Experiments with the sea urchin
embryos fulfill the requirements of biological ethics. The
artificial spawning does not cause animal death, embryos
develop outside the female organism, and both post
spawned adult sea urchins and the excess of intact embryos
are returned to the sea, their natural habitat.
Human cell culture
M-Hela human cervix epitheloid carcinoma cells, human
breast adenocarcinoma cells (MCF-7), PANC-1 human
pancreatic cancer cells, and Wi-38 human fibroblast-like fetal
lung cells were presented by the Institute of Cytology
(Russian Academy of Sciences) and cultured in a standard
Eagle’s nutrient medium manufactured at the Chumakov
Institute of Poliomyelitis and Virus Encephalitis (PanEco
company). DU145 human prostate epithelial carcinoma cells
derived from brain metastatic site (HTB-81™) were cultured
with Eagle’s Minimum Essential Medium. PC-3 human
prostate epithelial adenocarcinoma grade IVcells, derived
from bone metastatic site (CRL-1435™) were cultured with
Ham’s F-12K (Kaighn’s) Medium. SKOV-3 human ovary:
ascites epithelial adenocarcinoma cells (HTB-77™) were
cultured with McCoy’s 5a Modified Medium. All cancer cell
cultures were performed in media contained 10% fetal bovine
serum, penicillin (100 units/mL) and streptomycin (100 µg/mL)
at 37 °C under a 5% CO2 humidified atmosphere.
Cell toxicity MTT assay
The cells were seeded in 96-well plates (Eppendorf, Cat. №
0030730119), 200 μL of cell suspension in culture media
(Gibco) with 10% fetal bovine serum (FBS, Gibco) per well
at a density of 5 × 103 cells per well. Cells were incubated in
a humidified atmosphere with 5% CO2. Stock solutions of
test compounds were prepared in dimethylsulfoxide
(DMSO, Sigma). Next day (overnight incubation) cells
were treated for 48 h with test compounds titrated in Dul-
becco’s modified eagle medium (DMEM) media. Doxor-
ubicin (Sigma-Aldrich) and tubercidin (Sigma-Aldrich)
served as a positive control. The cytotoxicity was deter-
mined by the colorimetric cell proliferation assay MTT
(Thiazolyl Blue Tetrazolium Bromide, Sigma). For that
20 μL of MTT-reagent in Dulbecco’s phosphate-buffered
saline was added per each well (final concentration of
0.5 mg/mL). The plates were incubated at 37 °C for 2–3 h
under a 5% CO2 humidified atmosphere. The absorbance
was recorded at 570 nm using a CLARIO star microplate
reader (BMG LABTECH). Experiments for all compounds
were repeated three times. IC50 values were determined
using GraphPad Prism 6 software.
Results and discussion
Chemistry
The synthetic route of TPP-conjugates of 1,2,3-triazolyl
nucleoside analogues 1b, c, f, g; 2b, c; 3b, c is outlined in
Scheme 1. Starting material (1a, e; 2a; 3a) was synthesized
as has been reported [21]. (4-Bromobutyl)triphenylpho-
sphonium bromide (TPP-butyl bromide) was prepared by
the reaction of triphenylphosphine with fivefold excess of
1,4-dibromobutane under heating in an oil bath at 90 °C
without any solvent similarly to a procedure described [26].
Then it was reacted with equimolar amounts of starting
compounds 1a, e; 2a; 3a in the presence of K2CO3 in
according to the method previously described [29] to afford
the target TPP-conjugates 1b, f; 2b; 3b in 57%, 65%, 68%,
Phenotypic sea urchin embryo assay [27, 28]
Adult sea urchins, Paracentrotus lividus L. (Echinidae),
were collected from the Mediterranean Sea on the Cyprus
coast and kept in an aerated seawater tank. Gametes were
obtained by intracoelomic injection of 0.5 M KCl. Eggs

Medicinal Chemistry Research
Scheme 1 Synthesis of triphenylphosphonium conjugates of 1,2,3-triazolyl nucleoside analogues. The numbering of atoms of target compounds
is shown
21% yields, respectively. The attachment of the TPP-butyl
3.0–3.4 Hz, respectively, that confirmed a β-orientation of
the glycosidic bonds [21]. Finally, the removal of the O-
acetyl protective groups of 1b, f; 2b; 3b with 0.1 N MeONa/
MeOH solution furnished TPP-conjugates 1c, g; 2c; 3c
bearing unprotected OH groups in yields of 76–68%.
Similarly, the removal of the O-acetyl protection of the
starting 1,2,3-triazolyl nucleoside analogues 1a, e; 2a; 3a
afforded their derivatives 1d, h; 2d; 3d possessing unpro-
tected OH groups with good yields (89–98%).
In addition, a series of model compounds, namely deri-
vatives of uracil and thymine containing ω-alkyne or a 1,2,3-
triazolyl substituent at the N-1 atom as well as the TPP-butyl
moiety at the N-3 atom was synthesized (Scheme 2). N-1-
Propargyl derivatives of uracil 4b and thymine 5b were
synthesized in 48% and 41% yields, respectively, by an
alkylation of monosodium salts of uracil 4a and thymine 5a
prepared by their reaction with NaH according to known
procedure [21]. The N-1-ω-alkyne derivative of uracil 4i was
prepared in two steps similarly to a method previously
described [21]. At first, uracil 4a was reacted with an excess
of hexamethyldisilazane (HMDS) in toluene in the presence
of H2SO4 to afford a bissilylated derivative followed by
alkylation with 1-iodo-hex-5-yne to obtain 4i in 45% yield.
Then the Cu alkyne-azide cycloaddition reaction of the alkyne
derivatives 4b and 5b with trimethylsilylazide (TMSN3)
afforded N-1-(1,2,3-triazol-4-yl) derivatives 4c and 5c and
this reaction with n-pentyl azide furnished N-1-(pentyl-1H-
1,2,3-triazol-4-yl) derivatives 4d and 5d (Scheme 2).
moiety to starting compounds 1a, e; 2a; 3a was indicated by
1
the 31P, H, and 13C NMR spectra of compounds 1b, f; 2b;
3b. In the 31P NMR spectra of these compounds the signal
for the P+ atom appeared as a singlet within the range
23.98–25.57 ppm. The protons of the P+-CH2 moiety in the
1H NMR spectra of compounds 1b, f; 2b; 3b resonated as
multiplets within the range 3.40–3.95 ppm. The carbon of
the P+-CH2 bond in the 13C NMR spectra of TPP-
conjugates 1b, f; 2b; 3b resonated as a doublet at 22 ppm
with a coupling constant within the range 50.6–52.7 Hz.
The phenyl rings of these compounds were observed in
the 1H NMR spectra as multiplets within the range
7.65–7.95 ppm and as four doublets within the range
120–135 ppm in the 13C NMR spectra. The CH2 unit at the
N-1 atom of the pyrimidine rings of TPP-conjugates 1b, f;
1
2b occurred in their H NMR spectra as a triplet within the
range 3.93–3.97 ppm (3J = 6.3–6.6 Hz) and a singlet within
the range 39–40 ppm in their 13C NMR spectra. The same
1
unit of the quinazoline’s conjugate 3b resonated in its H
NMR spectrum as a triplet at 4.13 with a vicinal coupling
constant of 7.5 Hz and a singlet at 39 ppm in its 13C NMR
spectrum. All spectral data were in full accordance with the
spectral features of TPP-conjugates of nitrogen containing
1
heterocyclic compounds [29–32]. The H NMR spectra of
TPP-conjugates of 1,2,3-triazolyl nucleoside analogues 1b,
1f; 2b; 3b displayed the anomeric protons as doublets at
6.25, 6.21, 6.26, 6.25 ppm with a vicinal constant of

Medicinal Chemistry Research
O
N
R
R
NH
TMSN3
4с; R = H (20%)
5c; R = Me (48%)
O
N
1. HMDS,
PhCH3,
H2SO4
NH
14
13
N
13
7-10
O
N
O
12
Br
2.
12
11
O
N
4
R
3
NH
N
R
5
PPh2Br
4
Br
NH
2O
PPh3Br
C5H11N3
4
6
O
N1
15
O
N3'
N
DMF
CuSO4,
K2CO3, DMF,
40 oC, 24 h
4'
5'
N
2'
N
Na L-ascorbate
N1'
N
tBuOH / H2O
16-19
4b; R = H (48%)
5b; R = Me (41%)
CH3
CH3
20
4
4
4d; R = H (72%)
5d; R = Me (80%)
4e; R = H (59%)
5e; R = Me
(63%)
O
O
Br
R
PPh3BrH
4
N
PPh3Br
R
4
NH
O
N
K2CO3, DMF,
40 oC, 24 h
O
N
H
4f; R = H (27%)
5f; R = Me (44%)
4a; R = H
5a; R = Me
1. HMDS,
PhCH3,
H2SO4
O
O
R
R
Br
PPh3BrH
4
2. CH3I
N
NH
PPh3Br
4
O
O
K2CO3, DMF,
40 oC, 24 h
N
CH3
N
CH3
4h; R = H (26%)
5h; R = Me (31%)
4g; R = H (51%)
5g; R = Me (85%)
1. HMDS,
PhCH3,
H2SO4
I
2.
O
R
O
N
NH
Br
O
R
4
PPh3BrH
4
PPh3Br
4
N
O
K2CO3, DMF,
40 oC, 24 h
N
DMF
4
4
4i; R = H (45%)
4j; R = H (41%)
Scheme 2 Synthesis of triphenylphosphonium conjugates of uracil and thymine. The numbering of atoms of target compounds is shown
The structure of 4c was confirmed by single crystal X-ray
diffraction analysis (Fig. 1). Attaching the TPP-butyl moi-
ety to 4d and 5d in the presence of K2CO3 in DMF by
analogy with a procedure described [29] afforded TPP-
conjugates 4e and 5e. Similarly, TPP-butyl moiety has been
attached to the N-3 atom of the N-1-propargyl derivatives of
uracil 4b, thymine 5b and N-1-ω-alkyne derivative of uracil
4i to obtain TPP-conjugates 4f, 5f, 4j (Scheme 2). Besides,
uracil 4a and thymine 5a were preliminary alkylated at the
N-1 position in two steps in order to provide the attachment
of the TPP-butyl moiety to the N-3 atom. At first, treatment
of 4a and 5a with hexamethyldisilazane (HMDS) in toluene
in the presence of H2SO4 afforded bissilylated derivatives
which then were alkylated at the N-1 position with CH3I to
obtain pyrimidines 4g and 5g (Scheme 2). Then, 4g and 5g
were alkylated with TPP-butyl bromide to obtain TPP-
conjugates 4h and 5h.
Fig. 1 X-ray crystal structure of compound 4c

Medicinal Chemistry Research
Table 1 Cytotoxic activity of several 1,2,3-triazolyl nucleoside analogues against human cancer and human normal cell lines (IC50 values in μM
with standard errors)
IC50 (µM)
Normal
Compound
Structure
Cancer cell lines
cell line
aM-HeLa
>100
bMCF-7
>100
cPANC-1
>100
dPC-3
eDU145
fA375
>50
gSKOV-3
>50
hWI38
>100
>250
16.4 1.10
>250
>100
>100
>100
1b
1c
1d
>100
>100
>100
>100
>100
>100
>100
>100
>250
>50
>250
>50
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
68.7 3.87
44.6 2.52
nd
>100
>250
nd
>100
>250
nd
>100
>100
nd
1f
1g
1h
>100
92.5 7.3
81 6.2
>100
>100
>100
>100
>100
>100
>250
>250
>250
>250
>250
>250
>250
>250
2a
2b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
nd
>250
>50
nd
>250
>50
nd
>250
>100
>100
2c
2d
3a
nd
>100
>100
>100
>100
49.8 2.36 20.5 1.08 62.6 2.76
>100
3b
>100
>100
>100
>100
>100
>100
>100
>100
nd
nd
nd
nd
nd
nd
>100
>100
3c
3d
Doxoru-
bicin
Tuber-
cidin
3.0 0.2 3.0 0.1 0.09 0.01 1.09 0.01 0.07 0.01 0.02 0.01 0.03 0.01
0.64 0.02 0.82 0.01 nd 0.77 0.01 0.89 0.01 0.59 0.01 0.8 0.01
nd
nd
nd not determined
aM-Hela is a human cervix epitheloid carcinoma
bMCF-7 is a human breast adenocarcinoma (pleural fluid)
cPANC-1 is a human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin
dPC-3 is a human prostate cancer cell line

Medicinal Chemistry Research
eDU-145 is a human prostate cancer cell line
fA375 is a human malignant melanoma
gSKOV-3 is a human ovarian adenocarcinoma
hWi-38 is a diploid human cell strain composed of fibroblasts derived from lung tissue of a 3-months gestation aborted female fetus
Biological activity
at the N-1 atom showed weak cytotoxicity against cell lines
DU145 and M-HeLa with IC50 values of 56.8 μM and
75.8 μM, respectively. The attachment of a pentyl sub-
stituent to the N-1′ atom of the 1,2,3-triazole moiety of
compounds 4c and 5c (i.e., the transition to compounds 4d
and 5d) has caused activity loss (Table 2). Similarly to
1,2,3-triazolyl nucleoside analogues, attachment of the
TPP-butyl moiety to the N-3 atom of the pyrimidine frag-
ment of inactive compounds 4d and 5d (i.e., the transition
to compounds 4e and 5e) resulted in the appearance of the
ability to inhibit the growth of cancer cell lines DU145,
A375, and SKOV-3, with the TPP conjugate 5e being the
most potent. This compound inhibited the in vitro growth of
cancer cell lines DU145, A375, and SKOV-3 with IC50
values of 14.5 μM, 13.2 μM, and 16.1 μM, respectively.
Therefore, the coupling of uracil and thymine with the TPP
moiety afforded cytotoxic molecules exemplified by uracil
derivative 4h and thymine derivative 5h (Table 2). More-
over, the TPP conjugates of N-1-ω-alkyne derivatives of
uracil 4f and thymine 5f showed considerable cytotoxicity
against DU145 with IC50 values of 6.1 μM and 9.3 μM,
respectively. In addition, 4j inhibited the growth of A375
cancer cells with the IC50 value of 5.1 μM.
In vitro cytotoxicity of the TPP-conugates of 1,2,3-triazolyl
nucleoside analogues as well as alkyne and 1,2,3-triazolyl
derivatives of uracil and thymine
1,2,3-Triazolyl pyrimidine analogues 1d, h; 2a, d; 3a, d as
well as their conjugates with n-butyltriphenylphosphonium
bromide (TPP-conjugates) 1b, c, f, g; 2b,c; 3b,c were
evaluated for in vitro cytotoxicity against seven human
cancer cell lines: M-HeLa cervical epitheloid carcinoma,
MCF-7 breast adenocarcinoma, PANC-1 pancreatic carci-
noma, PC-3 and DU145 prostate carcinoma, SKOV-3
ovarian adenocarcinoma, A375 malignant melanoma, as
well as a diploid human cell strain WI-38 composed of
fibroblasts. The results are presented in Table 1. As shown
in the table, all tested 1,2,3-triazolyl nucleoside analogues
were inactive against all human cancer cell lines, whereas
some of their TPP-conjugates exhibited cytotoxicity against
human cancer cell lines M-HeLa, DU145, A375, and
SKOV-3. Thus, a weak inhibition of cell growth was
demonstrated by TPP-conjugates 1f, 1g, and 3b in DU145
(IC50 values of 68.7 μM, 44.6 μM, and 49.8 μM, respec-
tively), TPP-conjugate 2b in M-HeLa (IC50 = 81 μM), and
TPP-conjugate 3b in SKOV-3 (IC50 = 62.6 μM). The most
potent compounds, TPP-conjugates 1c and 3b, exhibited
Note, the growth of MCF-7, PANC-1, and PC-3 cells
was not suppressed by the tested molecules at 100 μM
concentration. It should be emphasized that all tested
compounds failed to induce cytotoxic effects on normal WI-
38 fibroblasts.
cytotoxicity against DU145 and A375 cells with IC50
=
16.4 μM and 20.5 μM, respectively (Table 1). These results
suggested that attachment of the TPP moiety to the 1,2,3-
triazole pyrimidine analogues endowed some of them with
cytotoxic activity.
In vivo sea urchin embryo tests
To identify the pharmacophore essential for cytotoxicity,
a series of model compounds (4b–j, 5b–h) was synthesized
(Scheme 2), and their ability to inhibit the in vitro growth of
human cancer cell lines M-HeLa, MCF-7, PANC-1, PC-3,
DU145, A375, and SKOV-3 as well as normal human cell
line WI-38 was evaluated. The results are presented in
Table 2. As anticipated, uracil 4a and thymine 5a them-
selves were inactive. The addition of a propargyl fragment
to the N-1 atom of these nucleobases resulted in moderate
cytotoxicity against M-HeLa. Namely, compounds 4b and
5b inhibited the growth of the cancer cell line M-HeLa with
IC50 values of 26 μM and 32.4 μM, respectively (Table 2).
The derivatives of uracil (compound 4c) and thymine
(compound 5c) with the 1,2,3-triazol-4-yl-methyl fragment
All compounds synthesized were evaluated for antimitotic
activity using in vivo phenotypic screening on a sea urchin
embryo model [27, 28]. This simple organism provides
rapid and highly reproducible assessment of both anti-
proliferative/antimitotic effect and/or systemic toxicity
together with information about mode of action, in parti-
cular, targeting tubulin and microtubules. Multiple advan-
tages of the sea urchin embryo allows for its consideration
as a relevant model organism for biomedical research
[33, 34]. It was found that all tested molecules failed to
cause developmental abnormalities of sea urchin embryos
when applied to fertilized eggs. The results suggest that
uracil and thymine derivatives including their TPP con-
jugates were nontoxic toward this animal model.

Medicinal Chemistry Research
Table 2 Cytotoxic activity of several N-1-alkyne and N-1-1,2,3-triazolyl derivatives of uracil and thymine as well as their TPP-conjugates (IC50
values in μM with standard errors)
IC50 (µM)
Normal cell
Compound
Structure
Cancer cell lines
lines
aM-Hela
>100
bMCF-7
>100
cPANC-1
>100
dPC-3
eDU145
fA375
>250
gSKOV-3
>250
hWI38
>100
>250
nd
>100
>100
4a
4b
26 2.0
>100
>100
>100
>100
>100
>100
>100
>50
>50
58.6 5.8
>250
>250
>250
>100
>100
>100
>100
>100
>100
>100
4c
4d
4e
4f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
>250
24.8 0.87 60.2 3.65 54.6 4.98
6.1 0.09
>50
>50
53.6 4.0 84.0 7.17 75.7 3.34
4h
4j
62.8 5.1 13.4 0.22 5.1 0.19 20.9 0.38
>100
>100
>100
>250
nd
>250
>50
>250
>50
5a
32.4 2.6
75.8 6.2
>100
>100
>100
>100
>100
>100
5b
5c
>250
>250
>250
100
>100
>100
>100
>250
>250
>250
>100
5d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.5 0.28 13.2 0.40 16.1 1.13
9.3 0.21 17.8 0.12 23.4 0.14
29.6 1.14 21.2 2.09 62.3 2.83
>100
>100
>100
5e
5f
87.7 7.2
5h
Doxoru-
bicin
Tuber-
cidin
3.0 0.2 3.0 0.1 0.09 0.01 1.09 0.01 0.07 0.01 0.02 0.01 0.03 0.01
0.64 0.02 0.82 0.01 nd 0.77 0.01 0.89 0.01 0.59 0.01 0.8 0.01
nd
nd
nd not determined
aM-Hela is human cervix epitheloid carcinoma
bMCF-7 is human breast adenocarcinoma (pleural fluid)
cPANC-1 is human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin

Medicinal Chemistry Research
dPC-3 is human prostate cancer cell line
eDU-145 is human prostate cancer cell line
fA375 is human malignant melanoma
gSKOV-3 is human ovarian adenocarcinoma
hWi-38 is diploid human cell strain composed of fibroblasts derived from lung tissue of a 3-months gestation aborted female fetus
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Conclusion
A series of TPP conjugates of 1,2,3-triazolyl analogues of
several pyrimidine nucleosides was synthesized and eval-
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DU145 cell line with IC50 values of 6.1 μM, 13.4 μM, and
9.3 μM, respectively. In addition, 4j was the most potent
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Acknowledgements The authors from Arbuzov Institute of Organic
and Physical Chemistry, FRC Kazan Scientific Center, RAS are
grateful to the Assigned Spectral-Analytical Center of FRC Kazan
Scientific Center of RAS for technical assistance in research.
Funding Synthesis and cytotoxicity evaluation were supported by the
Russian Science Foundation (grant no. 19-13-00003). Phenotypic sea
urchin embryo assay was performed by MNS under the IDB RAS
Government basic research program in 2020 no. 0088-2019-0005. IYS
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
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