Synthesis of triazolylmethyl-linked nucleoside analogs via combination of azidofuranoses with propargylated nucleobases and study on their cytotoxicity

Article abstract of DOI:10.1007/s10593-018-2248-4

[Figure not available: see fulltext.] Copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition reactions (CuAAC) between azidofuranoses and propargyl-nucleobases were carried out in the presence of CuSO₄·5H₂O and sodium ascorbate as catalytic system to provide the corresponding 1,4-disubstituted-1,2,3-triazole-bridged nucleoside analogs in good yields. Twelve new sugar-based triazolylmethyl-linked nucleoside analogs were synthesized and screened for their cytotoxic activity against MDA-MB-231, Hep3B, PC-3, SH-SY5Y, and HCT-116 cancer cell lines and control cell line (L929). Most of the compounds were moderately effective against all the cancer cell lines assayed. Particularly, among the tested compounds, 1,2,3-triazole-linked 5-fluorouracil–mannofuranose hybrid was found to be the most potent cytotoxic agent against HCT-116, Hep3B, SH-SY5Y cells with IC₅₀ values of 35.6, 71.1, and 75.6 μM, respectively. None of the triazolylmethyl-linked nucleoside analogs exhibited cytotoxic effect against the control cells L929.

Full text of DOI:10.1007/s10593-018-2248-4

DOI 10.1007/s10593-018-2248-4
Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
We dedicated this article to Prof. Dr. Hüseyin Anıl for his 70th birthday.
He influenced our lives and our thinking in a very positive way.
Synthesis of triazolylmethyl-linked nucleoside analogs
via combination of azidofuranoses with propargylated nucleobases
and study on their cytotoxicity
Erkan Halay1, Emriye Ay2, Emine Şalva3, Kadir Ay4*, Tamer Karayıldırım5
1 Scientific Analysis Technological Application and Research Center, Uşak University,
1 Eylul Campus, Uşak 64500, Turkey; e-mail: erkan.halay@usak.edu.tr
2 Department of Food Technology, Şebinkarahisar School of Applied Sciences, Giresun University,
Yayci Village, Giresun 28800, Turkey; e-mail: emriye.ay@giresun.edu.tr
3 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, İnönü University,
Elazığ Highway, Malatya 44280, Turkey; e-mail: emine.salva@inonu.edu.tr
4 Department of Chemistry, Faculty of Arts and Sciences, Manisa Celal Bayar University,
Şehit Prof. Dr. İlhan Varank Campus, Manisa 45030, Turkey; e-mail: kadir.ay@cbu.edu.tr
5 Department of Chemistry, Faculty of Science, Ege University,
Erzene St., İzmir 35100, Turkey; e-mail: tamer.karayildirim@ege.edu.tr
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2018, 54(2), 158–166
Submitted May 5, 2017
Accepted after revision November 9, 2017
Copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition reactions (CuAAC) between azidofuranoses and propargyl-nucleobases were
carried out in the presence of CuSO4·5H2O and sodium ascorbate as catalytic system to provide the corresponding 1,4-disubstituted-1,2,3-
triazole-bridged nucleoside analogs in good yields. Twelve new sugar-based triazolylmethyl-linked nucleoside analogs were synthesized
and screened for their cytotoxic activity against MDA-MB-231, Hep3B, PC-3, SH-SY5Y, and HCT-116 cancer cell lines and control cell
line (L929). Most of the compounds were moderately effective against all the cancer cell lines assayed. Particularly, among the tested
compounds, 1,2,3-triazole-linked 5-fluorouracil–mannofuranose hybrid was found to be the most potent cytotoxic agent against
HCT-116, Hep3B, SH-SY5Y cells with IC50 values of 35.6, 71.1, and 75.6 μM, respectively. None of the triazolylmethyl-linked
nucleoside analogs exhibited cytotoxic effect against the control cells L929.
Keywords: azido sugars, nucleobases, nucleosides, triazoles, anticancer activity, click chemistry.
1,2,3-Triazoles, heterocyclic moieties readily synthe-
sized from azide and alkyne derivatives of many com-
pounds thanks to the copper-catalyzed 1,3-dipolar cyclo-
addition reaction (CuAAC, a type of click reactions), have
found widespread usage in a broad range of disciplines
such as medicinal chemistry, materials chemistry, and
combinatorial chemistry.1–5 Despite the fact that these
synthetically produced triazole compounds do not exist in
nature, they are commercially employed as agrochemicals,
anticorrosive agents, dyes,6,7 and biologically important
molecules possessing activities such as antibacterial,8–10
antifungal,11–13 anticancer,14–18 antiviral,19–21 antitubercu-
lar,22–24 antiproliferative,25–27 and various enzyme inhi-
bitory.28–30 Besides all these, 1,2,3-triazoles are used as
linkers to connect two biologically beneficial units into one
molecule to generate novel hybrids. In this regard, the
significance of related compounds has increased
considerably in the field of medicinal chemistry. Thus,
0009-3122/18/54(2)-0158©2018 Springer Science+Business Media, LLC
158

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
Figure 1. Examples of carbohydrate-triazole conjugates and ribofuranosyl pyrimidine nucleoside analogs with enzyme inhibitory activity.
1,2,3-triazole moieties have been emerging as powerful
pharmacophoric unit in their own right. As a result of this,
many reports have appeared dealing with the synthesis of a
variety of triazole derivatives including carbohydrate-
triazole conjugates as shown in Figure 1.31–34
Azido sugars represent important and versatile products
of the carbohydrate chemistry.35 In the past twenty years, there
have been reports on preparative approaches which provide
many synthetic options for the use of such compounds.36
Among these, the CuAAC reaction is one of the best, finding
extensive application in the preparation of carbohydrates
with attached triazole moiety from monosubstituted
aliphatic acetylenes or compounds containing the
acetylenic function linked to sugars (Fig. 1).37–39
toward the synthesis of nucleobase derivatives (especially
nucleosides) have been reported in the literature.41,42 In a
similar article that is connected with our work, a series of ribo-
furanosyl pyrimidine nucleosides and their corresponding
1,2,3-triazole derivatives were synthesized and tested for
their inhibitory action on ribonuclease A enzyme (Fig. 1).30
In addition to our ongoing research on creating a 1,2,3-tri-
azole library by using pyranosyl azido sugars and in
continuation of our drug delivery/discovery program, herein
we report the synthesis of twelve novel 1,4-disubstituted-
1,2,3-triazole nucleoside analogs from azidofuranoses and
propargylated nucleobases via CuAAC reaction.
The selected monosaccharides were firstly protected
with isopropylidene (for mannose and glucose) or trichloro-
ethylidene (for ribose) groups. Afterwards, the introduction
of a leaving group followed by its exchange with sodium
azide afforded azidofuranoses 1–3 in moderate to good
yields over two steps (Scheme 1).43–46
Likewise, nucleobases or their modified analogs have been
found to be excellent structural motifs in medicinal chemistry,
and a number of nucleoside- and nucleotide-type drugs are
currently on the market.40 Therefore, many new approaches
Scheme 1
Me
Me
Me
Me
Me
Me
O
O
O
O
O
O
Me2CO
SOCl2
O
O
O
NaN3
N3
D-mannose
O
O
O
O
O
O
H2SO4, CuSO4
Py, CHCl3
DMF
67%
OH
Cl
Me Me
Me Me
Me Me
1
Me
Me
Me
Me
Me
O
O
O
O
OTf
O
Me
Tf2O
O
O
Me2CO
NaN3
O
O
D-glucose
D-ribose
OH
Py, CH2Cl2
H2SO4, CuSO4
DMF
50%
O
O
O
Me
Me
Me
N3
O
O
O
Me
Me
2
Me
TsO
HO
N3
O
O
O
Cl3CCHO
H2SO4
NaN3
TsCl
Py
O
O
O
CCl3
CCl3
CCl3
DMF
77%
OH
OH
O
OH
O
O
H
H
H
3
159

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
Scheme 2
On the other hand, the commercially available nucleo-
bases (uracil, thymine, 5-fluorouracil, and adenine) were
alkylated with propargyl bromide (80% solution in toluene)
and K2CO3 in DMF at 50°C for 8–12 h and gave required
monopropargylated nucleobases 4–7 with moderate yields
that were almost the same as those reported earlier in the
literature (Scheme 2).47
These two steps were followed by Cu(I)-catalyzed
CuAAC reaction to obtain a library of 1,4-disubstituted
1,2,3-triazole nucleoside analogs as illustrated in Scheme 3.
Compounds 8–19 were prepared in good to excellent yields
by performing the CuAAC reactions between azido sugars
1–3 and N-propargylated nucleobases 4–7 at a molar ratio
of azide:alkyne = 1.1:1 in solvent system THF–t-BuOH–
H2O, 3:1:1, using CuSO4·5H2O (0.1 equiv) and sodium
ascorbate (0.5 equiv) at 50°C for 3–6 h along with TLC
monitoring.
The exact structure and regiochemistry of these new
5-fluorouracil) nucleoside analogs 8–16 were observed at
4.87–5.11 ppm, while they were at 5.43–5.47 ppm for
purine (adenine) nucleoside analogs 17–19 as another proof
for triazole formation. Moreover, to establish the linking
1
nucleoside analogs was established by IR, MS, H, 13C,
1
1
19F NMR, H–13C HSQC, and H–13C HMBC data. In
1H NMR spectra, 1,2,3-triazole protons gave the characte-
ristic resonance at about 8.00 ppm. The methylene linker
protons (CH2N) for pyrimidine (uracil, thymine, and
1
points and full structural confirmation, H–13C HSQC and
1H–13C HMBC spectra of compounds 13, 14, and 15 were
Scheme 3
CuSO4·5H2O
Na ascorbate
R1
N
N
HC
N
R1 N3
1–3
+
R2
THF–t-BuOH–H2O, 3:1:1
4–7
R2
50°C, 3–6 h
8–19
6''
N
Me
Me
Me
Me
Me
Me
6''
O
O
O
O
O
N
N
5'' O
N
N
5''
5'' O
O
1''
N
N
O
O
O1''
N
N
1''
4'
5' 4''
4''
4''
O
O
O
O
O
CCl3
5'
4'
3''
OH2''
O
N
Me
Me
N
3''
2''
3''
2''
O
N 2
O
N
N
O
6
N
O
HN
Me Me
H
Me
Me
NH
5'
5
4'
NH
4
O
O
Me
O
10 (80%)
11 (87%)
O
N
O
8 (88%)
O
HN
Me
O
O
Me
Me
Me
Me
O
O
O
9 (90%)
Me
N
O
O
N
O
N
N
N
N
O
O
O
O
N
Me
Me
O
N
N
N
N
N
O
O
N
O
O
O
Me
Me
O
Me
N
O
Me
CCl3
NH
OH
O
O
N
F
HN
N
O
O
H
Me
14 (92%)
HN
HN
Me
F
13 (75%)
O
Me
Me
O
12 (85%)
15 (85%)
O
O
Me
Me
N
O
O
N
N
N
Me
N
N
N
O
O
N
N
O
N
N
N
N
O
O
O
O
O
Me
CCl3
O
O
OH
O
CCl3
N
N
N
N
N
N
N
HN
H
Me
OH
Me
O
F
N
N
H
N
N
N
O
16 (72%)
NH2
NH2
NH2
17 (89%)
19 (70%)
18 (90%)
160

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
examined. For instance, for compound 14, specific HSQC
cytotoxic effect against HCT-116 and MDA-MB-231 cells,
respectively. Compounds 10–12, 14, and 19 were the most
potent against all cancer cell lines. Specifically, compound
14 showed higher cytotoxic activities against HCT-116 and
SH-SY5Y cells at 35.6 and 75.6 μM, respectively, whereas
compound 11 showed higher activity (66.1 μM) against
Hep3B, compound 10 showed higher activity (76.9 μM)
against PC-3, and compound 16 showed higher activity
(126.0 μM) against MDA-MB-231 cells than other nucleo-
side analogs. Moreover, all tested compounds showed no
cytotoxic activity against L929 cells that were used as non-
tumor cells. The IC50 values for the tested compounds are
shown in Table 1.
Generally, with respect to the results, Hep3B was
confirmed as the most susceptible cell line. Apart from
some exceptions, higher concentrations of the tested
compounds were required for the inhibition of other four
cell lines. Regarding the activity of the tested compounds
against Hep3B cell line, compounds 8, 11, 12, 14, 16, 17,
and 19 exhibited more potent cytotoxic effects (66.1–92.8 μM)
than other synthesized compounds, while compounds 8–11
and 19 were more active than their nucleobase
intermediates 4, 5, and 7, respectively. The highest activity
against HCT-116 cells was observed for compound 14
(IC50 35.6 μM), which was nearly four times more potent
than 5-fluoro-1-(prop-2-yn-1-yl)uracil (6) (IC50 134.2 μM).
Among other nucleoside analogs, compound 14 was also
more active than others against Hep3B (71.1 μM) and
SH-SY5Y (75.6 μM) cells, while it showed less activity
than nucleobase intermediate 6.
correlations at 8.17 ppm for C-6 (130 ppm), 8.05 ppm for
C-5' (124.2 ppm), 6.24 ppm for C-1'' (88.5 ppm), and
4.91 ppm for CH2–N (42.9 ppm) were established. In
HMBC spectrum of compound 14, the correlations of
carbon nuclei C-2 (149.8 ppm), C-6 (130.0 ppm), and
C-5' (124.2 ppm) to the methylene linker protons CH2N
(4.91 ppm) and the correlation of triazole C-5' (124.2 ppm)
atom to the anomeric 1''-CH proton of mannose moiety
(6.24 ppm) strongly established the formation of 1,4-di-
substituted 1,2,3-triazole nucleoside analogs. The proposed
structure 14 was confirmed by positive ion mode TOF-MS
(ESI) spectra with molecular ion peak [M+H]+ (see
Supplementary information file). For the remaining eleven
triazole nucleoside analogs, similar HSQC and HMBC
correlations were observed and their structures were
characterized on the basis of these data.
Analysis of literature data has revealed that protected
sugars with increased molecular volume may show high
permeation across cell membrane contrary to more polar
sugars. In other words, in some occasions a correlation was
made between increased hydrophobicity and increased
cytotoxic activity.34,48–50 This prompted us to test the cyto-
toxic activity of nucleoside analogs 8–19 in their O-pro-
tected form. The newly synthesized purine/pyrimidine
nucleoside analogs 8–19 were evaluated for their in vitro
cytotoxic activity against MDA-MB-231, Hep3B, PC-3,
SH-SY5Y, and HCT-116 cancer cell lines using 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The cytotoxic effects of compounds were studied at
five different concentrations (200, 100, 50, 25, and 10 μM).
Among the N-propargylated nucleobases 4–7, 5-fluoro-
uracil derivative 6 was more potent than the others against
Hep3B, PC-3, and SH-SY5Y cells. On the other hand,
thymine and adenine derivatives 5 and 7 showed strong
Based on the cytotoxic activity results, it was
determined that integration of nucleobase units into the
monosaccharide platform via 1,2,3-triazole linkage actually
enhanced biological activities for some pairs of compo-
nents and that was a good way to generate novel triazoles.
Table 1. IC50 value of 1,2,3-triazole-bridged nucleoside analogs 4–19, µM*
Compound
MDA-MB-231
Hep3B
PC-3
SH-SY5Y
HCT-116
>200
177.9 0.13
73.58 0.17
70.42 0.15
81.95 0.17
92.82 0.15
105.3 0.06
112.0 0.14
66.1 0.16
>200
120.8 0.14
67.87 0.16
53.77 0.15
91.73 0.15
127.9 0.09
>200
177.5 0.12
122.4 0.10
134.2 0.09
179.0 0.08
158.0 0.07
166.1 0.08
105.5 0.07
124.9 0.12
74.68 0.05
110.8 0.07
35.62 0.10
151.5 0.06
157.8 0.11
175.3 0.06
156.8 0.08
109.1 0.08
4
195.0 0.15
125.4 0.18
108.4 0.08
>200
118.5 0.14
92.74 0.13
167.5 0.11
162.8 0.15
144.2 0.07
76.91 0.17
119.5 0.12
96.63 0.15
151.5 0.11
125.2 0.15
132.7 0.11
132.2 0.12
178.8 0.07
>200
5
6
7
8
192.3 0.11
150.9 0.16
>200
9
>200
10
11
12
13
14
15
16
17
18
19
135.0 0.11
185.7 0.35
126.8 0.10
75.64 0.24
167.1 0.12
175.0 0.016
84.25 0.08
>200
193.0 0.14
131.3 0.13
144.5 0.12
187.4 0.01
126.0 0.09
178.6 0.09
143.6 0.13
172.0 0.19
87.25 0.11
133.8 0.18
71.13 0.12
127.3 0.23
80.08 0.18
84.42 0.13
154.0 0.13
72.26 0.18
122.6 0.12
>200
* Data presented the mean standard deviation of each compound from three independent experiments.
161

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
Besides, the results suggested that some features such as
open column chromatographic studies, flash chromato-
graphy was performed using 230–400 mesh silica gel
(Merck 9385) with the indicated solvent mixture. All
starting materials, solvents, and other chemicals were
purchased from Merck, Fluka, Carlo Erba, Alfa Aesar, or
Sigma-Aldrich and were used without purification.
Synthesis of starting materials, namely, protected azido
sugar compounds 1–343–46 and propargylated nucleobases
4–747 were prepared according to the previously reported
procedures.
1-Azido-1-deoxy-2,3:5,6-di-O-propan-2-ylidene-β-D-
mannofuranose (1) was prepared from D-mannose.43,44
Yield over two steps 3.7 g (67%), white solid, mp 74–75°C.
IR spectrum, ν, cm–1: 1068 (C–O–C), 2122 (–N3), 2987 and
2937 (CH).
different substituents (sugar and/or nucleobase), position of
linkage, and steric hindrance markedly influence the
cytotoxic efficacy of compounds. In this regard, these
findings clearly indicate that the glycosidic linkage to the
triazole ring had a remarkable effect on the cytotoxic
activity. Accordingly, as said above, compound 14 exhi-
bited the strongest activity with IC50 value of 35.6 μM. On
the other hand, ribose-derived nucleoside analogs with the
triazole linkage attached to the C-6' atom displayed less
activity (72 ˃ IC50 > 200 μM) than the nucleoside analogs
derived from mannose. Additionally, allose-derived
nucleoside analogs containing the triazole linker at the C-3'
position were defined to be the least potent among all
newly synthesized nucleoside analogs.
In conclusion, we reported the synthesis, characteri-
zation, and biological evaluation of a novel series of
triazole-linked nucleoside analogs derived from azido
furanoses and propargylated nucleobases. All synthesized
compounds were assayed for cytotoxic activity against five
different human cancer cell lines. The nucleoside analog
derived from mannose and 5-fluorouracil was identified as
the most potent cytotoxic agent with promising potential
against colon cancer HCT-116 cells with IC50 value of
35.6 μM. At the same time, thymine–mannofuranose
hybrid showed reasonable activity against liver cancer
Hep3B cell as well as most compounds. In this regard, it
was determined that all nucleoside analogs were suitable
for inhibiting Hep3B cell line and showed good activity
against other tested cell lines. Consequently, with the
guidance of these in vitro assays, we aimed to provide a
nucleoside analog library to compare advantage/disadvantage
of the carbohydrate and nucleobase units. As expected,
neither triazole nor sugar moiety is responsible for the
activity, rather it is the whole structural motif including
nucleobases. We hope that these results will help the
scientists in future studies and, with higher number of
available triazole-linked nucleoside analogs, a more clear
structure–activity relationship will emerge.
3-Azido-3-deoxy-1,2:5,6-di-O-propan-2-ylidene-α-D-
allofuranose (2) was prepared from D-glucose.45 Yield
over two steps 3.0 g (50%), colorless syrup. IR spectrum,
ν, cm–1: 1065 (C–O–C), 2111 (–N3), 2988 (CH).
5-Azido-5-deoxy-1,2-O-(S)-(2,2,2-trichloroethylidene)-
α-D-ribofuranose (3) was prepared from D-ribose.46 Yield
over two steps 2.6 g (77%), white solid, mp 129–131°C.
IR spectrum (KBr), ν, cm–1: 1120 (C–O–C), 2102 (–N3),
3006, 2976, 2944 and 2908 (CH), 3512 (–OH).
1-(Prop-2-yn-1-yl)uracil (4) was synthesized from
uracil and propargyl bromide.47 Yield 1.9 g (50%), white
solid, mp 168–169°C (mp 154–166°C47). IR spectrum,
ν, cm–1: 1620 (C=C), 1715 and 1688 (C=O), 2120 (C≡C),
2923 and 2851 (CH), 3114 (C≡CH), 3243 (–NH).
1-(Prop-2-yn-1-yl)thymine (5) was synthesized from
thymine and propargyl bromide.47 Yield 2.1 g (65%), white
solid, mp 158–160°C (mp 154–156°C47). IR spectrum,
ν, cm–1: 1680 and 1704 (C=O), 2124 (C≡C), 2833, 2893
and 2932 (CH), 3160 (C≡CH), 3254 (–NH).
5-Fluoro-1-(prop-2-yn-1-yl)uracil (6) was synthesized
from 5-fluorouracil and propargyl bromide.47 Yield 1.2 g
(40%), white solid, mp 147–148°C (mp 146–148°C47).
IR spectrum, ν, cm–1: 1651 (C=C), 1705 (C=O), 2132
(C≡C), 2825 (CH), 3030 (C≡CH), 3225 (–NH).
9-(Prop-2-yn-1-yl)adenine (7) was synthesized from
adenine and propargyl bromide.47 Yield 1.5 g (69%), white
solid, mp 214–215°C (mp 213–214°C47). IR spectrum,
ν, cm–1: 1686 (C=N), 2110 (C≡C), 2863 and 2910 (CH),
3139 (C≡CH), 3245 and 3372 (–NH2).
1,2,3-Triazole syntheses by the CuAAC reaction
(General method). To a well stirred solution of azido sugar
1–3 (1.1 equiv) and propargylated nucleobase 4–7 (1.0 equiv)
in solvent system THF–t-BuOH–H2O, 3:1:1, CuSO4·5H2O
(0.11 equiv) and sodium ascorbate (0.55 equiv) were
added.51 The mixture was then stirred at 50°C for 3–6 h
along with TLC monitoring. After completion of the
reaction, the mixture was diluted with water and extracted
with CHCl3 (3×30 ml). The combined organic fractions
were dried over anhydrous Na2SO4, and the solvent was
evaporated under reduced pressure. The crude residue was
subjected to flash column chromatography using CH2Cl2–
MeOH system and afforded the pure products 8–19.
1-{[1-(2,3:5,6-Di-O-propan-2-ylidene-β-D-manno-
furanosyl)-1H-1,2,3-triazol-4-yl]methyl}uracil (8) was
Experimental
IR spectroscopy was performed on a PerkinElmer
Spectrum 100 FTIR spectrophotometer equipped with ATR
apparatus. Optical rotations were measured on a Rudolph
Research Analytical Autopol II automatic polarimeter. All
1D and 2D NMR spectra (1H, 13C, 19F, HSQC and HMBC)
were recorded on a Varian AS 400 MHz spectrometer in
CDCl3 or DMSO-d6 using TMS as internal standard. High-
resolution mass spectra analyses were performed on an
Agilent 6230 TOF-MS equipped with Jet Stream ESI
source operated in positive ion mode. Melting points were
determined using a Gallenkamp Electrothermal WA11373
melting point apparatus in open glass capillaries. All
reactions were monitored by thin-layer chromatography
using precoated silica gel G-60F254 (Merck 5554)
aluminum plates with detection under UV light (254 and
366 nm) or sprayed with 20% H2SO4 solution in water
followed by heating at 120°C or basic KMnO4 solution.
While 70–230 mesh silica gel (Merck 7734) was used for
162

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
obtained by the reaction of azido sugar 1 (600 mg,
br. s, NH). 13C NMR spectrum (DMSO-d6), δ, ppm: 42.7
(CH2N); 50.9 (C-5''); 72.7 (C-3''); 78.7 (C-4''); 81.7 (C-2'');
100.1 (CCl3); 101.6 (C-5); 105.4 (C-1''), 109.1 (CHCCl3),
124.8 (C-5'); 142.7 (C-4'); 145.9 (C-6); 151.1 (C-2); 164.1
(C-4). Found, m/z: 454.0058 [M+H]+. C14H15Cl3N5O6.
Calculated, m/z: 454.0082.
2.1 mmol) and propargyluracil 4 (287 mg, 1.9 mmol) and
purified by flash chromatography using CH2Cl2–MeOH,
100:0→100:2. Yield 728 mg (88%), white foam, mp 126–
20
128°C. [α]D +57 (c 2.5 mg/ml, DMSO). IR spectrum,
ν, cm–1: 1077 (C–O–C), 1373 (C–N), 1601 (C=C), 2989,
2946, 2893 and 2870 (–CH), 3418 (N–H). 1H NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 1.22 (3H, s, CH3);
1.26 (3H, s, CH3); 1.31 (3H, s, CH3); 1.39 (3H, s, CH3); 3.90–
4.03 (3H, m, 4''-CH, 6''-CH2); 4.28–4.40 (1H, m, 5''-CH);
4.84–4.88 (2H, m, 2'',3''-CH); 4.95 (2H, s, CH2N); 5.57
(1H, d, J5,6 = 8.0, 5-CH); 6.24 (1H, d, J1'',2'' = 2.8, 1''-CH); 7.74
(1H, d, J5,6 = 8.0, 6-CH); 8.00 (1H, s, 5'-CH); 11.27 (1H,
br. s, NH). 13C NMR spectrum (DMSO-d6), δ, ppm: 24.5,
25.7, 25.8, 27.1 (4CH3); 42.9 (CH2N); 66.4 (C-6''); 73.0
(C-5''); 79.0 (C-4''); 79.4 (C-2''); 80.2 (C-3''); 88.8 (C-1'');
101.9 (C-5); 113.2, 108.8 (2(CH3)2C); 124.3 (C-5'); 142.9
(C-4'); 146.1 (C-6); 151.4 (C-2); 164.3 (C-4). Found, m/z:
436.1958 [M+H]+. C19H26N5O7. Calculated, m/z: 436.1827.
1-{[1-(3-Deoxy-1,2:5,6-di-O-propan-2-ylidene-α-D-
allofuranos-3-yl)-1H-1,2,3-triazol-4-yl]methyl}uracil (9)
was obtained by the reaction of azido sugar 2 (600 mg,
2.1 mmol), and propargyluracil 4 (287 mg, 1.9 mmol) and
purified by flash chromatography using CH2Cl2–MeOH,
100:0→100:2. Yield 744 mg (90%), white foam, mp 110–
1-{[1-(2,3:5,6-Di-O-propan-2-ylidene-β-D-manno-
furanosyl)-1H-1,2,3-triazol-4-yl]methyl}thymine
(11)
was obtained by the reaction of azido sugar 1 (600 mg,
2.1 mmol) and propargylthymine 5 (313 mg, 1.9 mmol)
and purified by flash chromatography using CH2Cl2–
MeOH, 100:0→100:2. Yield 742 mg (87%), white foam,
20
mp 246–248°C. [α]D +56 (c 2.5 mg/ml, CHCl3).
IR spectrum, ν, cm–1: 1047 (C–O–C), 1368 (C–N), 1667
(C=C), 1697 (C=O), 2825 and 2990 (–CH), 3170 (C=CH),
1
3390 (N–H). H NMR spectrum (CDCl3), δ, ppm (J, Hz):
1.32 (3H, s, CH3); 1.39 (3H, s, CH3); 1.45 (3H, s, CH3); 1.52
(3H, s, CH3); 1.89 (3H, s, 5-CH3); 3.78 (1H, dd, J4'',5'' = 7.8,
J3'',4'' = 4.0, 4''-CH); 4.05 (1H, dd, J6''a,6''b = 8.8, J5'',6''b = 4.0,
6''-CHb); 4.13 (1H, dd, J6''a,6''b = 9.0, J5'',6''a = 6.2, 6''-CHa);
4.51 (1H, ddd, J4'',5'' = 7.8, J5'',6''a = 6.4, J5'',6''b = 4.0, 5''-CH);
4.88 (1H, dd, J2'',3'' = 5.8, J3'',4'' = 4.0, 3''-CH); 4.95 (1H, dd,
J2'',3'' = 5.8, J1'',2'' = 3.6, 2''-CH); 4.96 (1H, d, J = 15.2) and
5.05 (1H, d, J = 15.2, CH2N); 6.11 (1H, d, J1'',2'' = 3.6,
1''-CH); 7.37 (1H, d, J6,5-CH3 = 0.8, 6-CH); 7.96 (1H, s,
5'-CH); 9.66 (1H, br. s, NH). 13C NMR spectrum (CDCl3),
δ, ppm: 12.4 (5-CH3); 24.2, 25.2, 25.5, 27.1 (4CH3); 42.7
(CH2N); 67.0 (C-6''); 72.7 (C-5''); 79.4 (C-4''); 79.6 (C-2'');
79.8 (C-3''); 89.1 (C-1''); 111.3 (C-5); 109.8, 114.1
(2(CH3)2C); 124.4 (C-5'); 140.3 (C-6); 141.8 (C-4'); 151.1
(C-2); 164.6 (C-4). Found, m/z: 450.2073 [M+H]+.
C20H28N5O7. Calculated, m/z: 450.1983.
20
112°C. [α]D +56 (c 2.5 mg/ml, CHCl3). IR spectrum,
ν, cm–1: 1046 (C–O–C), 1373 (C–N), 1678 (C=O), 2987
1
(–CH), 3410 (N–H). H NMR spectrum (CDCl3), δ, ppm
(J, Hz): 1.12 (3H, s, CH3); 1.20 (3H, s, CH3); 1.34 (3H, s,
CH3); 1.64 (3H, s, CH3); 3.69 (1H, dd, J6''a,6''b = 8.8, J5'',6''b = 4.8,
6''-CHb); 4.02 (1H, dd, J6''a,6''b = 8.8, J5'',6''a = 6.8, 6''-CHa);
4.16–4.22 (1H, m, 5''-CH); 4.41 (1H, dd, J3'',4'' = 9.2,
J4'',5'' = 6.2, 4''-CH); 4.84 (1H, t, J2'',3'' = 4.4, 2''-CH); 4.94
(1H, d, J = 15.2) and 5.11 (1H, d, J = 15.2, CH2N); 5.13–
5.17 (1H, m, 3''-CH); 5.71 (1H, d, J5,6 = 8.0, 5-CH); 5.97
(1H, d, J1'',2'' = 3.2, 1''-CH); 7.57 (1H, d, J5,6 = 8.0, 6-CH);
7.95 (1H, s, 5'-CH); 10.01 (1H, br. s, NH). 13C NMR
spectrum (CDCl3), δ, ppm: 24.9, 26.1, 26.5, 26.8 (4CH3);
42.9 (CH2N); 63.8 (C-3''); 66.5 (C-6''); 76.0 (C-5''); 78.4
(C-4''); 79.3 (C-2''); 102.8 (C-5); 104.5 (C-1''); 110.1, 114.1
(2(CH3)2C); 124.5 (C-5'); 141.8 (C-4'); 144.5 (C-6); 151.2
(C-2); 164.1 (C-4). Found, m/z: 436.1822 [M+H]+.
C19H26N5O7. Calculated m/z: 436.1827.
1-{[1-(3-Deoxy-1,2:5,6-di-O-propan-2-ylidene-α-D-
allofuranos-3-yl)-1H-1,2,3-triazol-4-yl]methyl}thymine (12)
was obtained by the reaction of azido sugar 2 (600 mg,
2.1 mmol) and propargylthymine 5 (313 mg, 1.9 mmol)
and purified by flash chromatography using CH2Cl2–
MeOH, 100:0→100:2. Yield 725 mg (85%), white foam,
20
mp 201–203°C. [α]D +48 (c 2.5 mg/ml, CHCl3).
IR spectrum, ν, cm–1: 1023 (C–O–C), 1374 (C–N), 1678
(C=O), 2931 and 2986 (–CH), 3044 (C=CH), 3139 (N–H).
1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 1.12 (3H, s,
CH3); 1.20 (3H, s, CH3); 1.33 (3H, s, CH3); 1.64 (3H, s,
4CH3); 1.89 (3H, s, 5-CH3); 3.66 (1H, dd, J6''a,6''b = 8.0,
1-{[1-(5-Deoxy-1,2-O-(S)-(2,2,2-trichloroethylidene)-α-D-
ribofuranos-5-yl)-1H-1,2,3-triazol-4-yl]methyl}uracil (10)
was obtained by the reaction of azido sugar 3 (600 mg,
2.0 mmol) and propargyluracil 4 (268 mg, 1.8 mmol) and
purified by flash chromatography using CH2Cl2–MeOH,
100:0→100:2. Yield 652 mg (80%), white foam, mp 134–
J
5'',6''b = 6.0, 6''-CHb); 4.01 (1H, dd, J6''a,6''b = 8.0, J5'',6''a = 7.6,
6''-CHa); 4.16–4.22 (1H, m, 5''-CH); 4.41 (1H, dd,
J4'',3'' = 9.5, J4'',5'' = 6.0, 4''-CH); 4.83 (1H, t, J2'',3'' = 4.0,
2''-CH); 4.89 (1H, d, J = 15.6) and 5.08 (1H, d, J = 15.6,
CH2N); 5.12 (1H, dd, J3'',4'' = 9.5, J2'',3'' = 4.4, 3''-CH); 5.96
(1H, d, J1'',2'' = 3.2, 1''-CH); 7.38 (1H, s, 6-CH); 7.93 (1H, s,
5'-CH); 9.71 (1H, br. s, NH). 13C NMR spectrum (CDCl3),
δ, ppm: 12.4 (5-CH3); 24.9, 26.1, 26.5, 26.8 (4CH3); 42.7
(CH2N); 63.7 (C-3''); 66.5 (C-6''); 76.0 (C-5''); 78.4 (C-4'');
79.3 (C-2''); 104.5 (C-1''); 111.3 (C-5); 110.1, 114.1
(2(CH3)2C); 124.4 (C-5'); 140.3 (C-6); 142.0 (C-4'); 151.2
(C-2); 164.5 (C-4). Found, m/z: 450.1980 [M+H]+.
C20H28N5O7. Calculated, m/z: 450.1983.
20
136°C. [α]D +52 (c 2.5 mg/ml, DMSO). IR spectrum,
ν, cm–1: 1089 (C–O–C), 1359 (C–N), 1661 and 1708
1
(C=O), 3203 (C=CH), 3388 (N–H). H NMR spectrum
(DMSO-d6), δ, ppm (J, Hz): 3.62–3.74 (1H, m, 3''-CH);
4.09 (1H, br. t, J3'',4'' = 7.2, 4''-CH); 4.46 (1H, dd,
J5''a,5''b = 14.0, J4'',5''b = 7.2, 5''-CHb); 4.68 (1H, d,
J
5''a,5''b = 14.0, 5''-CHa); 4.76–4.83 (1H, m, 2''-CH); 4.92 (2H,
s, CH2N); 5.57 (1H, d, J5,6 = 7.6, 5-CH); 5.75 (1H, br. s, OH);
5.86 (1H, s, CHCCl3); 6.04 (1H, d, J1'',2'' = 2.8, 1''-CH); 7.72
(1H, d, J5,6 = 7.6, 6-CH); 8.00 (1H, s, 5'-CH); 11.24 (1H,
1-{[1-(5-Deoxy-1,2-O-(S)-(2,2,2-trichloroethylidene)-α-D-
ribofuranos-5-yl)-1H-1,2,3-triazol-4-yl]methyl}thymine (13)
163

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
was obtained by the reaction of azido sugar 3 (600 mg,
(2H, s, CH2N); 5.17 (1H, dd, J3'',4'' = 9.6, J2'',3'' = 4.8, 3''-CH);
5.90 (1H, d, J1'',2'' = 3.6, 1''-CH); 8.11 (1H, d, J6,F = 6.8, 6-CH);
8.15 (1H, s, 5'-CH); 11.80 (1H, br. s, NH). 13C NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 25.2; 26.2; 26.7 and
26.7 (4CH3); 42.9 (CH2–N); 62.8 (C-3''); 65.9 (C-6''); 75.8
(C-5''); 77.7 (C-4''); 79.4 (C-2''); 104.6 (C-1''); 109.3 and
113.0 (2(CH3)2C); 124.6 (C-5'); 130.1 (d, J = 33.3, C-6);
140.1 (d, J = 228.6, C-5); 142.2 (C-4'); 149.8 (C-2); 157.8
(d, J = 25.6, C-4). 19F NMR spectrum (376 MHz, DMSO-d6),
δ, ppm (J, Hz): –169.2 (1F, d, JF,H6 = 7.2). Found, m/z:
454.1798 [M+H]+. C19H25FN5O7. Calculated, m/z: 454.1733.
1-{[1-(5-Deoxy-1,2-O-(S)-(2,2,2-trichloroethylidene)-α-D-
ribofuranos-5-yl)-1H-1,2,3-triazol-4-yl]methyl}-5-fluoro-
uracil (16) was obtained by the reaction of azido sugar 3
(600 mg, 2.0 mmol) and 5-fluoropropargyluracil 6 (301 mg,
1.8 mmol) and purified by flash chromatography using
CH2Cl2–MeOH, 100:0→100:1. Yield 610 mg (72%), white
2.0 mmol) and propargylthymine 5 (294 mg, 1.8 mmol)
and purified by flash chromatography using CH2Cl2–
MeOH, 100:0→100:2. Yield 631 mg (75%), white foam,
20
mp 119–120°C. [α]D +76 (c 2.5 mg/ml, DMSO).
IR spectrum, ν, cm–1: 1094 (C–O–C), 1214 (C–N), 1625
(C=C), 1676 (C=O), 2927 (–CH), 3205 (N–H), 3505
1
(–OH). H NMR spectrum (DMSO-d6), δ, ppm (J, Hz):
1.73 (3H, s, 5-CH3); 3.66–3.70 (1H, m, 3''-CH); 4.07–4.11
(1H, m, 4''-CH); 4.46 (1H, dd, J5''a,5''b = 14.4, J4'',5''b = 7.6,
5''-CHb); 4.67 (1H, br. d, J5''a,5''b = 14.4, 5''-CHa); 4.75–4.82
(1H, m, 2''-CH); 4.88 (2H, s, CH2N); 5.71–5.75 (1H, m,
OH); 5.86 (1H, s, CHCCl3); 6.04 (1H, d, J1'',2'' = 4.0,
1''-CH); 7.59 (1H, s, 6-CH); 7.99 (1H, s, 5'-CH); 11.27
(1H, s, NH). 13C NMR spectrum (DMSO-d6), δ, ppm: 12.3
(5-CH3); 42.5 (CH2–N); 50.9 (C-5''); 72.7 (C-3''); 78.7
(C-4''); 81.7 (C-2''); 100.1 (CCl3); 105.4 (C-1''); 109.1
(CHCCl3); 109.3 (C-5); 124.8 (C-5'); 141.6 (C-6); 142.9
(C-4'); 151.1 (C-2); 164.7 (C-4). Found, m/z: 468.0217
[M+H]+. C15H17Cl3N5O6. Calculated, m/z: 468.0239.
20
foam, mp 185–186°C. [α]D +52 (c 2.5 mg/ml, DMSO).
IR spectrum, ν, cm–1: 1089 (C–O–C), 1241 (NH), 1383 (C–N),
1608 (C=C), 1672 and 1688 (C=O), 2855 and 2924 (CH),
3058 and 3133 (C=CH), 3338 (N–H), 3432 (OH). 1H NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 3.70 (1H, dd, J3'',4'' = 9.2,
1-{[1-(2,3:5,6-Di-O-propan-2-ylidene-β-D-manno-
furanosyl)-1H-1,2,3-triazol-4-yl]methyl}-5-fluorouracil (14)
was obtained by the reaction of azido sugar 1 (600 mg,
2.1 mmol) and 5-fluoropropargyluracil 6 (321 mg,
1.9 mmol) and purified by flash chromatography using
CH2Cl2–MeOH, 100:0→100:1. Yield 792 mg (92%), white
J3'',2'' = 4.8, 3''-CH); 4.09 (1H, td, J3'',4'',5b'' = 8.4, J4'',5a'' = 2.4,
4''-CH); 4.45 (1H, dd, J5''a,5''b = 14.4, J4'',5''b = 7.6, 5''-CHb);
4.69 (1H, dd, J5''a,5''b = 14.4, J4'',5''a = 2.4, 5''-CHa); 4.79 (1H,
t, J1'',2'',3'' = 4.8, 2''-CH); 4.87 (2H, s, CH2N); 5.85 (1H, s,
CHCCl3); 6.03 (1H, d, J1'',2'' = 3.6, 1''-CH); 8.03 (1H, s, 5'-CH);
8.08 (1H, d, J6,F = 6.4, 6-CH). 13C NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 43.0 (CH2N); 51.0 (C-5''); 72.7 (C-3''); 78.7
(C-4''); 81.7 (C-2''); 100.1 (CCl3); 105.4 (C-1''); 109.1
(CHCCl3); 124.8 (C-5'); 129.9 (d, J = 33.4, C-6); 140.3 (d,
J = 229.4, C-5); 142.7 (C-4'); 150.6 (C-2); 158.8 (d, J = 24.5,
C-4). 19F NMR spectrum (376 MHz, DMSO-d6), δ, ppm
(J, Hz): –168.9 (1F, d, JF,H-6 = 6.8). Found, m/z: 493.9858
[M+Na]+. C14H13Cl3FN5NaO6. Calculated, m/z: 493.9808.
9-{[1-(2,3:5,6-Di-O-propan-2-ylidene-β-D-manno-
furanosyl)-1H-1,2,3-triazol-4-yl]methyl}adenine (17) was
obtained by the reaction of azido sugar 1 (600 mg,
2.1 mmol) and propargyladenine 7 (331 mg, 1.9 mmol) and
purified by flash chromatography using CH2Cl2–MeOH,
100:0→100:4. Yield 774 mg (89%), white foam, mp 111–
20
foam, mp 120–122°C. [α]D +40 (c 2.5 mg/ml, DMSO).
IR spectrum, ν, cm–1: 1043 (C–O–C), 1209 (NH), 1374 (C–N),
1664 (C=C), 1693 (C=O), 2827, 2987 (–CH), 3082, 3179
(C=CH), 3530 (N–H). 1H NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 1.23 (3H, s, CH3); 1.27 (3H, s, CH3); 1.32
(3H, s, CH3); 1.40 (3H, s, CH3); 3.90‒4.03 (3H, m, 4''-CH,
6''-CH2); 4.32–4.38 (1H, m, 5''-CH); 4.84‒4.88 (2H, m,
2'',3''-CH); 4.91 (2H, s, CH2N); 6.24 (1H, d, J1'',2'' = 3.2,
1''-CH); 8.05 (1H, s, 5'-CH); 8.17 (1H, d, J6,F = 6.4, 6-CH),
11.80 (1H, br. s, NH). 13C NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 24.3, 25.4, 25.5, 26.9 (4CH3); 42.9 (CH2N);
66.2 (C-6''); 72.7 (C-5''); 78.8 (C-4''); 79.1 (C-2''); 79.9
(C-3''); 88.5 (C-1''); 108.6, 112.9 (2(CH3)2C); 124.2 (C-5');
130.2 (d, J = 33.3, C-6), 140.1 (d, J = 228.6, C-5); 142.3
(C-4'); 149.8 (C-2); 157.8 (d, J = 25.6, C-4). 19F NMR
spectrum (376 MHz, DMSO-d6), δ, ppm (J, Hz): –169.2
(1F, d, JF,H-6 = 7.2). Found, m/z: 454.1723 [M+H]+.
C19H25FN5O7. Calculated, m/z: 454.1733.
20
113°C. [α]D +62 (c 2.5 mg/ml, DMSO). IR spectrum,
ν, cm–1: 1042 (C–O–C), 1208 (C–N), 1574 (C–NH2), 1596
(C=C), 1645 (C=N), 2985 (–CH), 3158 and 3331 (–NH2).
1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 1.18 (3H,
s, CH3); 1.25 (3H, s, CH3); 1.30 (3H, s, CH3); 1.30 (3H, s,
CH3); 3.94–3.88 (2H, m, 6''-CH2); 3.99 (1H, t, J4'',5'' = 6.0,
4''-CH); 4.28–4.38 (1H, m, 5''-CH); 4.83 (2H, br. s, 2''-CH,
3''-CH); 5.47 (2H, s, CH2N); 6.22 (1H, br. d, J1'',2'' = 1.6,
1''-CH); 7.24 (2H, s, NH2); 8.09 (1H, s, 5'-CH); 8.13 (1H, s,
2-CH); 8.21 (1H, s, 8-CH). 13C NMR spectrum (DMSO-d6),
δ, ppm: 24.2, 25.3, 25.5, 26.9 (4CH3); 38.4 (CH2N); 66.1
(C-6''); 72.7 (C-5''); 78.7 (C-4''); 79.1 (C-2''); 79.9 (C-3'');
88.5 (C-1''); 108.5, 112.9 (2(CH3)2C); 119.0 (C-5); 124.2
(C-5'); 141.0 (C-8); 143.0 (C-4'); 149.7 (C-4); 153.0 (C-2);
156.4 (C-6). Found, m/z: 459.2111 [M+H]+. C20H27N8O5.
Calculated, m/z: 459.2099.
1-{[1-(3-Deoxy-1,2:5,6-di-O-propan-2-ylidene-α-D-allo-
furanos-3-yl)-1H-1,2,3-triazol-4-yl]methyl}-5-fluoro-
uracil (15) was obtained by the reaction of azido sugar 2
(600 mg, 2.1 mmol) and 5-fluoropropargyluracil 6
(321 mg, 1.9 mmol) and purified by flash chromatography
using CH2Cl2–MeOH, 100:0→100:1. Yield 732 mg (85%),
white foam, mp 245–247°C. [α]D20 +56 (c 2.5 mg/ml, DMSO).
IR spectrum, ν, cm–1: 1068 (C–O–C), 1207 (NH), 1380 (C–N),
1691 (C=O), 2979 (–CH), 3078 and 3146 (C=CH), 3380
1
(N–H). H NMR spectrum (DMSO-d6), δ, ppm (J, Hz):
1.07 (3H, s, CH3); 1.12 (3H, s, CH3); 1.23 (3H, s, CH3);
1.51 (3H, s, CH3); 3.55 (1H, dd, J6''a,6''b = 8.4, J5'',6''b = 5.2,
6''-CHb); 3.95 (1H, dd, J6''a,6''b = 8.4, J5'',6''a = 7.2, 6''-CHa);
4.12–4.18 (1H, m, 5''-CH); 4.52 (1H, dd, J4'',3'' = 9.7,
J4'',5'' = 5.6, 4''-CH); 4.83 (1H, t, J1'',2'' = 4.4, 2''-CH); 4.90
9-{[1-(3-Deoxy-1,2:5,6-di-O-propan-2-ylidene-α-D-allo-
furanos-3-yl)-1H-1,2,3-triazol-4-yl]methyl}adenine (24)
164

Chemistry of Heterocyclic Compounds 2018, 54(2), 158–166
was obtained by the reaction of azido sugar 2 (600 mg,
ATCC CCL-247) cell lines were used. L929 (control cell
line, ATCC CRL-6364) was used to test cell viability and
cytotoxicity. All cell lines were obtained from the
American Type Culture Collection (USA). The cells were
maintained in Dulbecco's Modified Eagles's Medium
supplemented with 10% (v/v) of fetal bovine serum
obtained from Gibco, Carlsbad, CA, USA. Cells were
routinely grown in 25 and 75 cm2 flasks in an environment
containing 5% CO2 and passed every 3 days. Cells were
plated in 96 multiwell plate (5 × 103 cells/well) for 24 h
before treatment with the test compounds to allow
attachment of the cells. Compounds 4–19 were dissolved in
dimethyl sulfoxide (DMSO, Sigma D2650). The final
concentration of DMSO was 0.1%. After 24 h of growth,
the medium was replaced with fresh medium containing
compounds 4–19 at the different concentrations (200, 100,
50, 25, and 10 μM). Triplicate wells were prepared for each
individual concentration. Cells were incubated with the
compounds for 48 h at 37°C and in atmosphere of 5% CO2.
After the incubation period, 10 µl MTT was added to each
well and incubated overnight. The MTT-containing
medium was removed and 100 µl solubilization buffer
(10% sodium dodecyl sulfate in 0.01 M HCl) was added to
the formazan crystal formed by live cells. Plates were
incubated for overnight and absorbances were measured at
550 and 600 nm using a multimode microplate reader
(Biotek, USA).
2.1 mmol) and propargyladenine 7 (331 mg, 1.9 mmol) and
purified by flash chromatography using CH2Cl2–MeOH,
100:0→100:4. Yield 783 mg (90%), white foam, mp 198–
20
199°C. [α]D +56 (c 2.5 mg/ml, DMSO). IR spectrum,
ν, cm–1: 1044 (C–O–C), 1212 (C–N), 1579 (C–NH2), 1596
(C=C), 1662 (C=N), 2986 (–CH), 3127 (C=CH), 3333 and
3397 (–NH2). 1H NMR spectrum (DMSO-d6), δ, ppm
(J, Hz): 0.93 (3H, s, CH3); 1.07 (3H, s, CH3); 1.20 (3H, s,
CH3); 1.48 (3H, s, CH3); 3.50 (1H, dd, J6''a,6''b = 8.4,
J5'',6''b = 5.6, 6''-CHb); 3.91 (1H, dd, J6''a,6''b = 8.4, J5'',6''a = 7.2,
6''-CHa); 4.09–4.16 (1H, m, 5''-CH); 4.48 (1H, dd,
J4'',3'' = 9.7, J4'',5'' = 5.6, 4''-CH); 4.82 (1H, t, J1'',2'' = 4.1,
2''-CH); 5.16 (1H, dd, J3'',4'' = 10.2, J2'',3'' = 4.8, 3''-CH); 5.44
(2H, s, CH2N); 5.90 (1H, d, J1'',2'' = 3.6, 1''-CH); 7.23 (2H,
s, NH2); 8.13 (1H, s, 5'-CH); 8.15 (1H, s, 2-CH); 8.18 (1H,
s, 8-CH). 13C NMR spectrum (DMSO-d6), δ, ppm: 25.2,
26.1, 26.7, 26.7 (4CH3); 38.3 (CH2N); 62.7 (C-3''); 65.9
(C-6''); 75.7 (C-5''); 77.6 (C-4''); 79.3 (C-2''); 104.6 (C-1'');
109.2, 113.0 (2(CH3)2C); 119.0 (C-5); 124.7 (C-5'); 140.9
(C-8); 142.6 (C-4'); 149.7 (C-4); 152.9 (C-2); 156.4 (C-6).
Found, m/z: 459.2096 [M+H]+. C20H27N8O5. Calculated, m/z:
459.2099.
1-{[1-(5-Deoxy-1,2-O-(S)-(2,2,2-trichloroethylidene)-
α-D-ribofuranos-5-yl)-1H-1,2,3-triazol-4-yl]methyl}ade-
nine (19) was obtained by the reaction of azido sugar 3
(600 mg, 2.0 mmol) and propargyladenine 7 (310 mg,
1.8 mmol) and purified by flash chromatography using
CH2Cl2–MeOH, 100:0→100:4. Yield 600 mg (70%), white
The percentage cell viability was calculated by
expressing the absorbance of treated cells as a percentage
of untreated control cells. Three separate experiments with
triplicate data were performed to obtain mean cell viability.
The IC50 value is the concentration (μM) of a compound
which was able to cause 50% cell death with respect to the
control culture. IC50 values were calculated according to
the inhibition ratios using GraphPad Prism 7.02.
20
foam, mp 198–199°C. [α]D +56 (c 2.5 mg/ml, DMSO).
IR spectrum, ν, cm–1: 1054 (C–O–C), 1241 (C–N), 1579
(C–NH2), 1597 (C=C), 1637 (C=N), 2919 (–CH), 3146
1
(C=CH), 3261 and 3325 (–NH2), 3480 (OH). H NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 3.64–3.69 (1H, m,
3''-CH); 4.09 (1H, br. t, J3'',4'',5''b = 8.1, 4''-CH); 4.45 (1H,
dd, J5''a,5''b = 13.2, J4'',5''b = 7.6, 5''-CHb); 4.67 (1H, dd,
J5''a,5''b = 13.2, J4'',5''a = 1.6, 5''-CHa); 4.79 (1H, t, J1'',2'',3'' = 4.0,
2''-CH); 5.43 (2H, s, CH2N); 5.71–5.75 (1H, m, OH); 5.84
(1H, s, CHCCl3); 6.02 (1H, d, J1'',2'' = 3.6, 1''-CH); 7.19
(2H, s, NH2); 8.04 (1H, s, 5'-CH); 8.14 (1H, d, J8,2 = 0.8,
2-CH); 8.18 (1H, d, J8,2 = 0.8, 8-CH). 13C NMR spectrum
(DMSO-d6), δ, ppm: 38.4 (CH2N); 50.9 (C-5''); 72.7
(C-3''); 78.7 (C-4''); 81.7 (C-2''); 100.1 (CCl3); 105.4
(C-1''); 109.1 (CHCCl3); 118.9 (C-5); 124.9 (C-5'); 141.1
(C-8); 142.9 (C-4'); 149.8 (C-4); 153.0 (C-2); 156.4 (C-6).
Found, m/z: 477.0416 [M+H]+. C15H16Cl3N8O4. Calculated,
m/z: 477.0355.
Supplementary information file containing spectral data
of the synthesized compounds is available at the journal
website at http://link.springer.com/journal/10593.
This work was supported by TUBITAK-Research
Council of Turkey with the project number 114Z757.
Author E. Halay also offers his profound thanks to
TUBITAK-BIDEB 2211A for their bursary support.
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