5-Ethynyl-1-β-d-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide (ETCAR) and its analogues: Synthesis and cytotoxic properties

Article abstract of DOI:10.1016/j.bmc.2011.05.050

Efficient Pd(0)-catalysed synthesis of 5-alkynyl-1-β-d-ribofuranosyl- 1H-[1,2,3]triazole-4-carboxylic acid amide depends on the presence of different protecting groups of the ribose moiety. Peracetylated 5-iodo substrate (15) couples with terminal alkynes or trimethyl-[(tributylstannyl)ethynyl]silane in 50-71% and 72% yield (ETCAR), respectively, although its hydrodehalogenation to 19 is noticeable. On the other hand, hydrodehalogenation of acetonide (16) predominates over coupling with terminal alkyne and slightly decreases a yield of cross-coupling reaction with trimethyl[(tributylstannyl)ethynyl]silane. Alternative conditions of reaction with terminal alkynes, to exclude so far identified hydride sources to produce hydridopalladium species, have been established for acetonide 16 and allowed to achieve 72% of coupling. Fluoromethyl derivative (42) was prepared from its 5-hydroxymethyl precursor by fluorination with DAST. Additionally, X-ray structural analysis of 42 was performed. All 1,2,3-triazolonucleosides and two synthesized cycloSal-pronucleotides were evaluated for cytotoxic activity against K562, HeLa and HUVEC cells.

Full text of DOI:10.1016/j.bmc.2011.05.050

Bioorganic & Medicinal Chemistry 19 (2011) 4386–4398
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Ethynyl-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide
(ETCAR) and its analogues: Synthesis and cytotoxic properties
Tomasz Ostrowski a, Piotr Januszczyk a, Marcin Cieslak b, Julia Kazmierczak-Baranska b, Barbara Nawrot b,
Elzbieta Bartoszak-Adamska c, Joanna Zeidler a,
⇑
a Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
b Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland
c Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2011
Revised 5 May 2011
Accepted 9 May 2011
Available online 30 May 2011
Efficient Pd(0)-catalysed synthesis of 5-alkynyl-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid
amide depends on the presence of different protecting groups of the ribose moiety. Peracetylated 5-iodo
substrate (15) couples with terminal alkynes or trimethyl-[(tributylstannyl)ethynyl]silane in 50–71% and
72% yield (ETCAR), respectively, although its hydrodehalogenation to 19 is noticeable. On the other hand,
hydrodehalogenation of acetonide (16) predominates over coupling with terminal alkyne and slightly
decreases a yield of cross-coupling reaction with trimethyl[(tributylstannyl)ethynyl]silane. Alternative
conditions of reaction with terminal alkynes, to exclude so far identified hydride sources to produce
hydridopalladium species, have been established for acetonide 16 and allowed to achieve 72% of cou-
pling. Fluoromethyl derivative (42) was prepared from its 5-hydroxymethyl precursor by fluorination
with DAST. Additionally, X-ray structural analysis of 42 was performed. All 1,2,3-triazolonucleosides
and two synthesized cycloSal-pronucleotides were evaluated for cytotoxic activity against K562, HeLa
and HUVEC cells.
Keywords:
1,2,3-Triazole nucleosides
Ribavirin
Pd-catalysed coupling
Hydrodehalogenation
Cytotoxic activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
1-b-
D
-ribofuranosylimidazolium-5-olate-4-carboxylic acid amide,
-ribofuranosylimidazole-4-carbox-
5) and EICAR (5-ethynyl-1-b-
D
Nucleoside analogues possessing five-membered heterocyclic
bases have become interesting objects to design and synthesize
since the discovery of antiviral potency of ribavirin
ylic acid amide, 6).5,6 While the immunosuppressant 5 has been li-
censed as a drug in Japan, 6 has been known for antileukemic
activity in vivo and antiviral potency against a broad spectrum of
DNA and RNA viruses. It is worthy to note that EICAR exceeds 1
10- to 100-fold in respect of the level of antiviral activity.7 The com-
pounds 1, 5 and 6 are metabolized to 50-monophosphates (RMP,
mizoribine-MP, EICAR-MP) and in this form act as powerful inhibi-
tors of inosine 50-monophosphate dehydrogenase (IMPDH) of differ-
ent organisms, occupying a substrate binding site. However, each of
the inhibitors binds to the enzyme in its specific way: competitively
(RMP), or as an analogue of transient-state (mizoribine-MP), or by
forming a covalent bond (EICAR-MP).2,8,9 Interestingly, C-nucleo-
sides 2 and 3, after being metabolized to the enzyme cofactor
NAD+ analogues – tiazofurin adenine dinucleotide (TAD) and sele-
nazofurin adenine dinucleotide (SAD), respectively, inhibit IMPDH
binding into the cofactor domain of the enzyme.4b,3 Inhibition of
IMPDH leads to a depletion of guanine nucleotides pools and in con-
sequence is responsible for antiviral or antileukemic, or immuno-
suppressant activities demonstrated by AICAR analogues. Another
(1-b-
1, Fig. 1).1 Ribavirin has been licensed for treatment of RSV infec-
tion in children and, in combination with interferon , in therapy
of HCV infections.2 Significant antileukemic activity in vivo of tia-
zofurin (2-b- -ribofuranosyl-thiazole-4-carboxylic acid amide, 2)
D-ribofuranosyl-1H-[1,2,4]triazole-3-carboxylic acid amide,
a
D
guaranteed its further evaluation and finally the C-nucleoside 2
has been granted an orphan-drug status for treatment of chronic
myelogenous leukemia in accelerated phase of blast crisis.3 Antivi-
ral potency of 2 and its close congener selenazofurin (3) have also
been documented.4 The presence of carbamoyl group attached to
heterocycles of 1–3 is indispensible for biological activity of the
compounds. It assures they structurally resemble AICA riboside
(5-aminoimidazo-4-carboxamide riboside, 4), whose 50-mono-
phosphate is a key intermediate in de novo biosynthesis of purine
nucleotides.
Additionally, 4,5-disubstituted imidazole ribosides have
drawn much attention – naturally occurring mizoribine (bredinin,
synthesized triazolonucleoside – the isomer of 1 (1-b-D-ribofurano-
syl-1H-[1,2,3]triazole-4-carboxylic acid amide, 7) has been gener-
ally inactive, though its 50-monophosphate matched ribavirin
monophosphate in potency to inhibit IMPDH. 10
⇑
Corresponding author.
E-mail address: joandler@ibch.poznan.pl (J. Zeidler).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.050

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
CONH2
4387
CONH2
CONH2
Se
N
N
S
N
N
N
HO
HO
HO
O
O
O
OH
OH
HO
HO
OH
selenazofurin, 3
HO
tiazofurin, 2
ribavirin, 1
CONH2
CONH2
CONH2
O
+
N
N
HN
NH2
N
N
O
N
O
HO
HO
HO
O
OH
HO
OH
OH
HO
HO
EICAR, 6
AICAR, 4
bredinin, 5
CONH2
CONH2
CONH2
O
+
N
N
N
HN
N
N
N
N
N
HO
HO
HO
R
O
O
O
OH
HO
OH
HO OH
HO
9
R = H
8
7
10 R = CH3
11 R = C6H5
12 R = C6H4CH3
Figure 1.
Not many 4,5-disubstituted-1,2,3-triazole nucleosides analo-
gous to AICA riboside have been synthesized so far and, in partic-
ular, evaluated for their biological activity. For example, in the
of coupling reactions of 5-iodo-1,2,3-triazole could be observed in
comparison with 5-iodoimidazole, considering that both heterocy-
cles possessed the same substituent (carbamoyl) in position 4.6
However, it did not pose a difficulty when a carbocyclic moiety
was attached to 1,2,3-triazole and a coupling reaction could be car-
ried out at 155 °C.12
Although a multicomponent one-pot method to efficiently syn-
thesize 5-iodo-1,4-disubstituted triazole has been newly estab-
lished on a small scale,13 we exploited the diazotization-iodination
reaction to transform 5-aminotriazolonucleoside. It had been pre-
pared earlier by 1,3-dipolar cycloaddition of cyanoacetamide and
case of 1-b-D-ribofuranosyl-1H-[1,2,3]triazole-5-olate-4-carboxylic
acid amide (8), isosteric to 5, accessible data are scarce and relate
to the antiviral activity of 8 against measles and herpes simplex
viruses.10 It seems, that 5-substituted 4-carbamoyl-1,2,3-triazole
ribosides constitute the next subgroup of AICA riboside analogues,
biological activity of which should be thoroughly evaluated. It
makes them also interesting objects for synthesis, especially the
ones with attached alkynyl groups in position 5.
Mechanism-based inhibition of IMPDH by EICAR-MP is a conse-
quence of irreversible addition of Sꢀ nucleophile of cysteine residue
(e.g., Cys-331 in human IMPDH) to the ethynyl group of nucleotide.
1-b-D-ribofuranosyl azide, carrying 2,3-O-isopropylidene blockade
to avoid any side reaction in basic conditions.14 Appropriately pro-
tected 5-aminonucleosides 1315 and 14 upon diazotization with or-
ganic nitrite in diiodomethane6 were iodinated to the pivotal
compounds 15 and 16 in 50–60% yield (Scheme 1), similarly to the
respective 1,2,3-triazolo-carbanucleoside.12
We designed 5-ethynyl-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-
carboxylic acid amide (ETCAR, 9), which after being transformed
into 50-monophosphate, should form readily a covalent adduct with
IMPDH.
We investigated coupling reactions of 5-iodo-1-b-D-ribofurano-
syl-1,2,3-triazole-4-carboxylic acid amide in triacetate form (15)
with phenylacetylene and tolueneacetylene according to copper-
free Sonogashira protocol, using bis(benzonitrile)palladium dichlo-
ride as a catalyst, triethylamine as a base and dimethylformamide
(Scheme 2). The reactions had to be performed at 100 °C and for
20 h to achieve couplings, the 5-alkynyl products 17 and 18 were
isolated in a good yield (70% and 71%, respectively), regardless of
a minor reaction course – hydrodehalogenation of the substrate
15 to the known triazole derivative 19. The conditions were subse-
2. Chemistry
Organopalladium chemistry was exploited for the synthesis of 9
and its congeners such as 5-propynyl (10), 5-phenylethynyl (11)
and toluenethynyl (12), but we encountered a serious problem –
thermal instability of a 5-iodo precursor, while performing the
introductory experiments.11 It narrowed the scope of reaction con-
ditions we intended to investigate, since the significant retardation

4388
T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
Y
Y
N
N
i
N
N
I
N
NH2
N
R1O
R1O
O
O
3
2O
R
O
OR3
R2O
R
13 R1 = R2 = R3 = Ac, Y = CONH2
14 R1 = Ac R2, R3 = CMe2, Y = CONH2 iii
21 R1 = R2 = R3 = H, Y = CONH2
26 R1 = R2 = R3 = Ac, Y = CN
15 R1 = R2 = R3 = Ac, Y = CONH2
16 R1 = Ac R2, R3 = CMe2, Y = CONH2
22 R1 = R2 = R3 = H, Y = CONH2
25 R1 = R2 = R3 = Ac, Y = CN
ii
ii
ii
31 R1 = H R2, R3 = CMe2, Y = CONH2
Scheme 1. Reagents and conditions: (i) isoamyl nitrite, CH2I2, 100 °C; (ii) MeONa/MeOH, Dowex H+; (iii) Py, TsCl, rt.
CONH2
CONH2
N
N
N
N
N
N
+
R1O
1O
i
R
R
O
O
15, 16
OR3
3
2O
O
R
R
2O
R
17 R = C6H5, R1 = R2 = R3 = Ac
18 R = (C6H4)CH3, R1 = R2 = R3 = Ac
20 R = CH3, R1 = R2 = R3 = Ac
19 R1 = R2 = R3 = Ac
iii
10, 11, 12
28 R1 = Ac, R2 = R3 = CMe2
iii
30 R1 = H, R2 = R3 = CMe2
27 R = CH3, R1 = Ac R2 = R3 = CMe2
29 R = C6H5, R1 = H, R2 = R3 = CMe2
35 R = C6H5, R1 = Ac, R2 = R3 = CMe2
CONH2
N
CONH2
N
N
N
O
N
N
O
ii
+
1O
R
R1O
Si(CH3)3
H
OR3
R2
OR3
24 R1 = R2 = R3 = Ac
O
2O
R
iii
iii
32 R1 = Ac, R2 = R2 = CMe2
23 R1 = R2 = R3 = Ac
9
9
iii
33 R1 = Ac, R2 = R2 = CMe2
34 R1 = H, R2 = R3 = CMe2
34
iii
Scheme 2. Reagents and conditions: (i) alkyne, Et3N, (PhCN)2PdCl2, DMF, 100 °C or phenylacetylene, K2CO3, (PhCN)2PdCl2, 1,4-dioxane, 100 °C; (ii) TMSC„CSnBu3,
(PhCN)2PdCl2, CH3CN, 90 °C; (iii) MeONa/MeOH, Dowex H+, 0 °C.
quently applied to a more demanding reaction with propyne, it
was necessary to maintain the temperature at 100 °C and to use
approximately 60 equiv of alkyne to achieve efficient coupling.
Standard purification followed and we obtained 5-propynyl deriv-
ative 20 in 51% yield and the deiodinated 19 (15%) as well. Triazol-
onucleosides 13, 15, 17, 18, and 20 were then subjected to
methanolysis to afford 21,15 22, 10–12, respectively. Regarding
the synthesis of ETCAR we could easily presume the formation of
dimeric product upon coupling of 15 and TMS–acetylene in the
presence of a base, due to an anticipated lability of TMS group
attached to a preformed product. Afterwards, we turned to the
methods offering neutral conditions and attempted a Kosugi–Migi-
ta–Stille reaction, bearing in mind that the coupling with tri-
methyl[(tributylstannyl)ethynyl]silane (TMSE-n-Bu3Sn) had been
used previously for the synthesis of EICAR (77% of the protected
product).6 First, we carried out coupling experiments for the iodo-
nucleoside 15 with TMSE-n-Bu3Sn in DMF at elevated temperature
using either conventional heating or a microwave oven. The results
were generally unsatisfactory and we replaced DMF by acetoni-
trile. Despite the fact that iodonucleoside 15 was more prone to
hydrodehalogenation than expected and, that we could not elabo-
rate conditions to prevent this side reaction (22% loss of 15), we
achieved the coupling conventionally, maintaining the tempera-
ture at 90 °C for several hours. Purification of fully protected 23
by chromatography could not be omitted and resulted in a partial
loss of TMS group – the oily products 23 and 24 were isolated in
the total 72% yield, comparable to that one of EICAR. Methanolysis
of both products afforded ETCAR (9), in a high yield. Coupling
experiments performed next were aimed to minimize the loss of
substrate 15 being dehalogenated to 19. The synthesis of ETCAR

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
4389
could be attempted with 4-cyano-5-iodo-precursor 25, to reduce
steric congestion near the reaction centre, accordingly to the suc-
cessful alternative approach to EICAR synthesis (76% yield).6 For
this purpose we transformed the 5-amino derivative 13 into the ni-
trile 26, which was further iodinated to 25. Contrary to our expec-
tations, we did not succeed in replacing the carboxamide 15 by the
carbonitrile 25, due to an instability of the latter under conditions
required to achieve the coupling.
of nucleoside drugs by nucleoside kinases, which does not proceed
efficiently in some cases and in consequence, it lowers the efficacy
of a drug. CycloSal-pronucleotide concept introduced originally by
Meier20 has been an example of the kinase by-pass approach. This
interesting proposition has been based upon nonenzymatic, step-
wise pH driven hydrolysis of cyclic saligenyl esters of nucleoside
50-monophosphates within the cell. It has been particularly advan-
tageous, when applied to anti-HIV drugs and anticancer FdUMP.21
However, as ribonucleosides are concerned, only two applications
of the discussed pronucleotide approach have been reported so
far.22 The first one deals with the above-mentioned tiazofurin 2
and has been realized to prevent toxic effects of nonphosphory-
lated 2 on the central nervous system. The second has been carried
out for cytostatic 6-(het)aryl-7-deazapurine ribosides to improve
their pharmacological properties. Exactly for that reason we
decided to prepare cycloSal-pronucleotides of inactive 5-unsubsti-
tuted derivative 7 and ETCAR 9 (Scheme 3). The latter, according to
our assumption, could be the most active compound of 1,2,3-tria-
zole series. Phosphitylation of 30 and 34 was performed with sal-
icylchlorophosphane in the presence of triethylamine and followed
by in situ oxidation to phosphotriesters 36 and 37, which we iso-
lated as diastereomeric mixtures in 45% and 48% yield, respec-
tively. Subsequently, we applied the standard method (80% aq
TFA) to remove isopropylidene groups and the final cycloSal-pro-
nucleotides 38 and 39 were purified by chromatography and char-
acterized as mixtures (1:1) of diastereoisomers (RP, SP).
Commonly applied in coupling reactions soluble palladium(0)
complexes – dba2Pd(0) and [PPh3]4Pd(0) catalysts appeared to be
useless in our case, as expected. The presence of phosphine was
a driving force of hydrodehalogenation of 15 (63% of the only prod-
uct 19). Our further experiments performed with the more rigid
substrate 16, revealed substantially different reactivity of this com-
pound while coupled with terminal alkynes or trimethyl-[(tribu-
tylstannyl)ethynyl]silane (TMSE-n-Bu3Sn). Coupling of 16 with
terminal alkynes generally gave unsatisfactory results, for exam-
ple, 32% yield of the propynylated product 27, and proceeded in fa-
vor of hydrodehalogenation to the compound 28 (50% yield). In
turn we found almost equal amounts of both anticipated products
29 and 30 (ca. 30% each) resulting from a reaction of acetonide 31
with phenylacetylene. Fortunately, the cross-coupling of 16 with
TMSE-n-Bu3Sn could be achieved in 62% total yield (7% of fully pro-
tected 32, 55% of the desilylated product 33), but the minor course
of reaction should not be disregarded (18% of the byproduct 28).
Nevertheless, after conventional deprotection of 50-hydroxyl group
acetonides 30 and 34 could be transformed further into masked
nucleoside 50-monophosphates.
Prior to any biological assays we conducted a model addition of
thiolate nucleophile to ETCAR with protected
L-cysteine
Recently, many accounts have presented studies on hydrido
complexes of palladium and their significance as intermediates in
reactions performed with, for example, aryl halides.16–18 For exam-
ple, Zawisza and Muzart investigated a particular role of a com-
monly used solvent for Pd-catalysed coupling reactions (DMF), as
a hydride source, and proposed a mechanism of this reaction. In
accordance with the literature we assumed that Pd-catalysed hyd-
rodehalogenation of iodosubstrates 15, 16 or 31 was initiated by
oxidative addition of a Pd(0) catalyst to produce an organopalla-
dium(II) complex, which instead of reacting with a terminal al-
kyne, could coordinate to an amine present in the reaction
milieu. A further hydride transfer from the already bound amine
to the palladium atom would lead to the formation of hydridoor-
ganopalladium complex. This unstable intermediate could undergo
easily reductive elimination to yield triazolonucleosides 19, 28 or
30 and a regenerated Pd(0) catalyst. Regarding possible hydride
sources present in this case, both dimethylformamide (precursor
of dimethylamine)17 and triethylamine18 should be taken into con-
sideration. Although others have proved that H-transfer from a ter-
tiary amine occurs even in a presence of DMF,19 we decided to
circumvent the problem by elaborating alternative reaction condi-
tions: Et3N and DMF were changed for potassium carbonate and
1,4-dioxane, respectively. According to this modified protocol, we
achieved a successful coupling of the acetonide 16 with phenyl-
acetylene – 72% of the desired product 35, while hydrodehalogen-
ation produced only 6% of the triazolonucleoside 28. However, as
far as cross-coupling reactions of 15 and 16 with organostannane
are concerned, a source of the hydridopalladium complex remains
unknown. In the course of our investigations we demonstrated that
hydrodehalogenation could be a drawback in the synthesis of
1,2,3-triazolonucleosides via organopalladium chemistry. Particu-
larly it should not be disregarded for substrates of increased steric
congestion, which slows down dramatically a reaction of a pre-
formed organopalladium(II) complex with a terminal alkyne.
Nucleoside 50-monophosphates (NMPs) can be converted into
nonpolar derivatives (pronucleotides), usually phosphotriesters,
which after entering cells, can be enzymatically and/or chemically
converted into NMPs. It allows to avoid enzymatic phosphorylation
(N-acetyl- -cysteine methyl ester) in the presence of catalytic
L
amount of triethylamine in 0–5 °C (Scheme 4). The reaction pro-
ceeded readily and we observed the formation of both isomeric
products Z (40) and E (41), isolated in 44% and 12% yield. On the
other hand, the reaction of triacetate 24 with sodium methanethi-
olate, conducted in aqueous methanol at ambient temperature,
afforded products of intra and intermolecular addition, respec-
tively. Occurrence of the former reaction has been a measure of
enhanced acidity of 20-hydroxyl group of ETCAR (9), if compared
to that of EICAR (6). It should be mentioned here, that the
formation of an analogous product, namely 1-b-D-ribofuranosyl-
5,20-O-cycloethenoimidazole-4-carbonitrile has been reported to
proceed under drastic conditions – with methanolic ammonia at
120 °C.23
Additionally, we aimed to synthesize the triazole derivative car-
rying 5-fluoromethyl substituent (42) bearing in mind that some
inosine or AICA riboside analogues possessing an electron-with-
drawing group proximal to C-2 or C-5, respectively, exhibited
noticeable IMPDH inhibitory activity, provided that they had been
earlier efficiently 50-phosphorylated.24 The synthesis could be
accomplished by a Heck reaction of 15 to introduce a 5-alkenyl
substituent to be further transformed into the 5-hydroxymethyl
product (43) via a multistep procedure. We abandoned this ap-
proach due to a discouraging yield of coupling with methyl acry-
late. The alternative method has been already described25 and
according to it we synthesized compound 43 in Huisgen reaction
of 1-b-D-ribofuranosyl azide triacetate with methyl 4-hydroxy-2-
butynoate, followed by deblocking of the triazole 5-hydroxymethyl
group. Subsequently, we approached fluorination of 43 with DAST
(Scheme 5), and settled the conditions required to obtain the ester
(44) in a satisfactory yield (62%). Upon methanolysis, compound 44
was transformed into the desired amide (42), which was then puri-
fied by crystallization to afford material suitable for X-ray analysis.
Thus, we envisioned a possibility to compare compound 42 and
antiviral nucleoside 1 in respect of hydrogen bonding properties,
which for the latter compound have been of great importance for
its biological activity. The antiviral potency of ribavirin (1) on
RNA viruses may be explained by several mechanisms.2 One of

4390
T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
CONH2
R
CONH2
N
N
O
P
N
O
P
N
R
N
N
ii
i
30, 34
O
O
O
O
O
O
O
O
OH
HO
O
O
36 R = ethynyl
37 R = H
38 R = ethynyl
39 R = H
Scheme 3. Reagents and conditions: (i) salicylchlorophosphane, Et3N, CH3CN, 0 °C, then t-BuOOH, 0 °C to rt; (ii) 80% aq CF3CO2H, 0 °C.
O
CONH2
S
CONH2
N
N
CO2Me
O
i
N
N
NAc
9
+
S
N
N
CO2Me
HO
HO
O
O
NAc
OH
OH
OH
40
OH
41
Scheme 4. Reagents and conditions: (i) N-acetyl-
L
-cysteine methyl ester, Et3N, CH3CN, 0–5 °C.
CO2CH3
CONH2
CO2CH3
N
N
N
i
ii
N
N
N
CH2OH
CH2F
N
CH2F
N
N
AcO
HO
AcO
O
O
O
OAc
OH
OH
AcO
43
OAc
AcO
42
44
Scheme 5. Reagents and conditions: (i) DAST, CH2Cl2, ꢀ35 °C; (ii) MeOH sat NH3, rt, 48 h.
them is based on mutagenic effect of the compound on viral geno-
mic RNA that leads to depletion of the virus. The phenomenon has
been known as lethal mutagenesis or ‘error catastrophe’.26 It is
considered to be a consequence of base-pairing of ribavirin 50-tri-
phosphate with both cytosine (C) and uracil (U) due to rotation
of the carboxamide group.27
3. Crystal and molecular structure of 42
The investigated compound (42) crystallizes in tetragonal
P43212 space group with one molecule of 5-fluoromethyl-1-b-D-
ribofuranosyl)-1H-[1,2,3]triazole-4-carboxylic acid amide in the
asymmetric unit. The molecular structure and atom numbering of
42 is presented in Figure 2. The geometry of the ribose fragment
generally agrees with the data found by Prusiner & Sundaralingam
in two polymorphic forms, V1 and V2, of ribavirin (1).28 In the pres-
ent structure the conformation of the ribose ring is an envelope
C30-endo (3E). In terms of pseudorotation the ribose conformation
Figure 2. An ORTEP plot of the asymmetric unit contents of the crystal 42. The
atomic displacement ellipsoids have been drawn at 40% probability level. H atoms
are represented as spheres of arbitrary radius.
is characterized by P = 20.2(1)° and sm = 40.9(1)°. The glycosidic
torsion angle, O(40)–C(10)–N(1)–C(5), which describes the relative
orientation of the base with respect to the sugar is in the high anti
region (
c
v
= ꢀ87.2(2)°). The torsion angle O(50)–C(50)–C(40)–C(30),
pseudorotation phase angle P and
anti (
= 10.4°), 3T2 (P = 11.7°) and gauche+ while in V2 they are high
anti (
= 119.0°), 2T1 (P = 335.8°) and trans.28 In the present struc-
c torsion angle are, respectively,
= 46.5(2)°, indicates that the side chain has the preferred gauche+
v
v
conformation. For comparison, in V1 the glycosidic torsion angle,

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
4391
ture the five-membered ring of the base is flat. The angle between
the planes through the 1,2,3-triazole ring and the amide group is
2.7(2)°. The bond lengths of the 1,2,3-triazole ring show a delocal-
ization of electrons and a prevalent double character of the
N(2)–N(3) and C(4)–C(5) bonds. The heterocyclic N(3) and amide
N(6) atom are on the same side of the triazole moiety (torsion angle
N(3)–C(4)–C(6)–N(3) = 2.8(2)°). The geometry of the CH2F group is
perturbed due to disorder.
The crystal and molecular structure of 1,2,3-triazolonucleoside
42 is stabilized by classical hydrogen bonds, C–HꢁꢁꢁO and C–HꢁꢁꢁF
weak interactions and stacking forces between aromatic moieties
of nitrogen bases (Fig. 3). One structural motif, a pair of hydro-
gen-bonded bases, is particularly interesting for us to observe
(Fig. 4). The N(6) atom of the carboxamide group of 42 donates
hydrogen to the O(6) oxygen atom of a neighboring molecule
and a short intermolecular N–H. . .O hydrogen bond is formed with
the length of 2.911(2) Å. Two hydrogen-bonded bases are related
by a two-fold axis along the diagonal [1 1 0] direction and they
form an eight-membered ring with two donors and two acceptors,
R22(8). A second motif, R22(16) ring, is a result of sugar. . .base inter-
actions via two O(30)–HꢁꢁꢁN(3) hydrogen bonds, related by another
two-fold axis ( Fig. 4). These two C2-symmetric motifs form a
ribbon-like chain of rings running along the c axis. The O(50) and
O(30) atoms act as donors and acceptors in a system of homodro-
mic cooperative O(20)–HꢁꢁꢁO(50)–HꢁꢁꢁO(30)–HꢁꢁꢁN(3) hydrogen
Figure 4. Base pairing in the crystal of 42. Hydrogen bonds are indicated by the
dotted lines.
4. Cytotoxicity assays
At the beginning of systematic screening of their biological
activity, several 1,2,3-triazolonucleosides (7, 9–12, 21, 22, 42)
and two pronucleotides (38, 39) were evaluated for their cytotoxic
properties against K562 (leukemia), HeLa (cervix carcinoma) and
HUVEC (normal) cells. As the control (100% viability in the MTT as-
say) cells treated with DMSO (1%) were used. The viability of cells
was determined at four different drug concentrations: 1, 1 ꢂ 10ꢀ2
,
1 ꢂ 10ꢀ4 and 1 ꢂ 10ꢀ6 mM.
bonds. A co-operative effect combined with p–p stacking interac-
Cytotoxic activities of EICAR and its congeners have been dem-
onstrated toward a broad panel of human cancer cell lines.6 In that
study EICAR has been selected as the most promising anticancer
agent comparable with 5-fluorouracil. Contrary to our expectations,
1,2,3-triazolonucleosides exhibited marginal to low levels of cyto-
toxic activity against the used cell lines, or were inactive (Table 1).
Results of the assay in human chronic myelogenous leukemia cells
tions stabilize additionally the structure of a ‘double-ribbon’. The
‘double-ribbon’ propagated by a left-handed four-fold screw axis
is a building block of the crystal architecture (Fig. 3).
Comparison of hydrogen bonding systems in 42, and 1 (V1,V2)
revealed differences between these structures, with two excep-
tions: (i) only one of the amino hydrogen atoms is engaged in
classical hydrogen bonding in all compared crystals and (ii) the
O(30)–HꢁꢁꢁN(3) hydrogen bond found in 42 is present in V1.
Although, in the crystals of ribavirin (V1 and V2) base pairing does
not occur at all, in the crystal structure of the investigated 1,2,3-tri-
azole-4-carboxamide derivative 42 only one rotamer is present
with a syn conformation which is assumed to base-pair with uracil.
(K 562): IC50 90, 400 and 300
led us to general conclusion that an ethynyl group was the optimal
substituent in position 5 of triazole (IC50 0.64
M for EICAR6). When
lM for ETCAR, 10 and 12, respectively,
l
it was replaced by a larger alkynyl group we observed decrease of
activity. Surprisingly, pronucleotide 38 only matched parent nucle-
oside 9 in cytotoxicity against K 562 cells, and its final toxicity
toward HUVEC cells was four times higher than that of 9. Transfor-
mation of inactive nucleoside 7 (IC50 >1 mM), into the form of cyclo-
Sal-pronucleotide (39) assured its cytotoxicity, albeit at a low level,
against HeLa cells. Cytotoxic effect of hydrophobic compound 22
seemed to be time-dependent, but the above-mentioned com-
pounds did not display any selectivity of their cytotoxic effect
against cancer versus normal cells. Compounds 21 and 42 were
inactive toward cells used in the study (IC50 >1 mM). Interestingly,
a series of 4-substituted 1,2,3-triazolonucleosides, and compound
Table 1
Inhibitory effects of 1,2,3-triazolonucleosides 7, 9–12, 21, 22, 42 and pronucleotides
38, 39 on K562, HeLa and HUVEC cells
a
Compd
K562 IC50
24 h
(
l
M)
HeLa IC50
24 h
(
l
M)
HUVEC IC50
24 h
(lM)
48 h
48 h
48 h
7
9
>1000
90
>1000
90
>1000
200
>1000
150
>1000
70
>1000
200
10
11
12
21
22
38
39
42
>1000
400
nd
300
>1000
80
90
200
>1000
>1000
>1000
>1000
>1000
500
1000
100
>1000
>1000
>1000
700
>1000
250
100
90
>1000
>1000
>1000
>1000
>1000
700
40
700
>1000
>1000
>1000
>1000
>1000
70
50
200
>1000
ndb
800
>1000
600
100
200
>1000
a
The concentration of test compound required to reduce the cell survival fraction
to 50% of the control.
Figure 3. Crystal packing of 42. Four unit-cells are viewed along c axis. Hydrogen
atoms are omitted for clarity.
b
Not determined.

4392
T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
7 as well, were evaluated by others, for cytostatic activity in vitro
(DMSO-d6) d 7.48 and 7.13 (2ꢂ s, 2H, CO–NH2), 6.65 (s, 2H, NH2),
6.26 (s, 1H, 10-H), 5.46 (d, 1H, 20-H), 4.97 (dd, 1H, 30-H), 4.35 (dt,
1H, 40-H), 4.08 and 3.85 (2ꢂ dd, 2H, 50-H), 1.98 (s, 3H, Ac), 1.51
and 1.35 (2ꢂ s, 6H, 2ꢂ CH3).
against leukemia cell lines (L1210, Molt4/C8, CEM).29 The best
antiproliferative effect (IC50 36–152 lM) was established for the
4-(p-methoxyphenyl) derivative.
5.3. 5-Iodo-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide (15)
5. Experimental
5.1. General methods
To a suspension of 1315 (1.255 g, 3.26 mmol, dried in vacuo over
P2O5, at 90 °C, for 4 h) in diiodomethane (52.2 g, 195 mmol) was
added isoamyl nitrite (1.335 g, 11.4 mmol). The mixture was stir-
red at 110 °C for 1.5 h, then it was applied onto a silica gel column.
The column was eluted with CH2Cl2 (in order to collect diiodo-
methane) followed by CH2Cl2/MeOH (95:5) to give crude product.
It was next rechromatographed using toluene/MeOH (95:5 ? 9:1)
to afford homogenous 15 as a solid (960 mg, 59% yield). 1H NMR
(DMSO-d6) d 7.95 and 7.60 (2ꢂ s, 2H, CO–NH2), 6.22 (d, 1H, 10-
H), 6.05 (dd, 1H, 20-H), 5.67 (dd, 1H, 30-H), 4.50 (m, 1H, 40-H),
4.32 and 4.02 (2ꢂ dd, 2H, 50-H), 2.13, 2.10 and 1.88 (3ꢂ s, 9H,
3ꢂ Ac). 13C NMR (DMSO-d6) d 169.87, 169.56 and 169.39 (3ꢂ
CO–CH3), 160.98 (CO–NH2), 143.72 (C-4), 89.58 (C-10), 87.45 (C-
5), 80.23 (C-40), 73.23 (C-20), 70.20 (C-30), 62.04 (C-50), 20.37 and
20.30 (3ꢂ CO–CH3).
Melting points were determined on MEL-TEMP II capillary melt-
ing point apparatus and are uncorrected. Elemental analyses were
performed with a microanalyser Elemental Vario ELIII by Microan-
alytical Laboratories of the A. Mickiewicz University, Poznan. 1H
and 13C NMR spectra were recorded on a Bruker 400 spectrometer
operating at 400 MHz and 100.6 MHz, respectively, or on Unity 300
Varian spectrometer operating at 300 and 75.4 MHz, respectively
(tetramethylsilane or DMSO as internal standard). 31P NMR and
19F spectra were recorded on a Bruker 400 spectrometer at 162
and 376 MHz, respectively (H3PO4 or trifluoroacetic acid as exter-
nal standard). The chemical shifts are reported in ppm (d scale),
spectra are described. Mass spectra were recorded using Bruker
microTOF-q mass spectrometer in positive (ESI+) or negative (ESI
ꢀ) mode. UV spectra were taken with Perkin Elmer Lambda
EZ201 spectrophotometer. LC/UV analyses were performed on
Merck Hitachi HPLC system: pump model L-7100, diode array
detector model L-7450, autosampler model L-7200 and Superspher
100 (TM) RP-18 column (250 ꢂ 2 mm; Merck), gradient% MeOH in
H2O: 10% MeOH (0–2 min), 10–30% MeOH (2.1–8 min), 30–80%
MeOH (8.1–12 min), 80% MeOH (12.1–18 min), 80–100% MeOH
(18.1–20 min), the flow rate was 0.2 ml minꢀ1. Thin-layer chroma-
tography (TLC) was performed on Merck precoated 60 F254 silica
gel plates. Column chromatography was carried out on Merck silica
5.4. 1-(5-O-Acetyl-2,3-O-isopropylidene-b-D-ribofuranosyl)-5-
iodo-1H-[1,2,3]triazole-4-carboxylic acid amide (16)
Prepared from 14 by the reaction procedure described for 15.
For chromatography was used CH2Cl2 followed by CH2Cl2/MeOH
(97:3 ? 95:5) to give the product which was next rechromato-
graphed with EtOAc/hexane (1.5:1 ? 2:1) to afford 16 as solid
foam (53% yield). 1H NMR (DMSO-d6) d 7.98 and 7.64 (2ꢂ s, 2H,
CO–NH2), 6.29 (s, 1H, 10-H), 5.67 (d, 1H, 20-H), 5.12 (dd, 1H, 30-H),
4.52 (dt, 1H, 40-H), 4.07 and 3.87 (2ꢂ dd, 2H, 50-H), 2.00 (s, 3H,
Ac), 1.60 and 1.43 (2ꢂ s, 6H, 2ꢂ CH3). HRMS [M+Na]+ calcd for
gel 60H (40–63 lm). Anhydrous dimethylformamide (DMF), tetra-
hydrofuran (THF) and diethyl ether were supplied in Sure/Seal bot-
tles (Aldrich). Methanol HPLC grade was purchased from J.T. Baker,
acetone and alkynes were supplied from Aldrich. Other anhydrous
solvents were prepared as follows: CH3CN and pyridine were dis-
tilled with P2O5 and stored over 3 and 4 Å molecular sieves, respec-
tively, MeOH and EtOH by treatment with magnesium turnings/
iodine. Triethylamine was freshly distilled with calcium hydride.
Anhydrous 1,4-dioxane was prepared by distillation with calcium
hydride first, followed by distillation with sodium/benzophenone.
Trimethyl[(tributylstannyl)ethynyl]silane (bp 96 °C, 0.01 Torr)
was prepared as described.30
C13H17IN4O6Na: 475.0085; found: 475.0095.
5.5. General procedure for the preparation of compounds 17, 18
and 20
A solution of 15 (dried in vacuo over P2O5, at 60 °C, for 3 h) and
bis(benzonitrile)palladium dichloride (0.1 equiv) in anhyd DMF
(12 ml/1 mmol of 15) was vigorously deoxygenated with argon.
Anhyd triethylamine (6 equiv) was added, followed by phenylacet-
ylene (5 equiv), 4-methylphenylacetylene (3 equiv) or a solution of
propyne (60 equiv) in anhyd DMF, respectively. The reaction mix-
ture was heated at 100 °C overnight in a Parr reactor, then evapo-
rated. The residue was chromatographed on silica gel column using
EtOAc/hexane (1:1 ? 2:1) for 17 and 18 or (2:1 ? 5:1) for 20. Fur-
ther elution with EtOAc/hexane (6:1) led to the isolation of deiodi-
nated product 19.
5.2. 1-(5-O-Acetyl-2,3-O-isopropylidene-b-D-ribofuranosyl)-5-
amino-1H-[1,2,3]triazole-4-carboxylic acid amide (14)
To a cooled (0 °C) solution of KOH (1.753 g, 31.25 mmol) in
DMF–H2O (7:1; 50 ml) was added cyanoacetamide (2.628 g,
31.25 mmol). The mixture was stirred at 0 °C until all of the solid
material had dissolved. Next, a solution of 2,3-O-isopropylidene-
b-D
-ribofuranosyl azide14 (5.38 g, 25 mmol) in DMF (24 ml) was
5.6. 5-Phenylethynyl-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-
1H-[1,2,3]triazole-4-carboxylic acid amide (17)
added and the whole was stirred at room temperature for 5 h.
The cooled (0 °C) mixture was neutralized with 80% aq AcOH and
the volatiles were evaporated. The residue was co-evaporated with
anhyd pyridine, then treated with anhyd pyridine (70 ml) and ace-
tic anhydride (7.657 g, 75 mmol). The resulting mixture was stirred
at room temperature overnight, then the reaction was quenched by
addition of anhyd MeOH (1 ml). After evaporation the residue was
taken up into CH2Cl2 and extracted with H2O. The organic layer
was dried over Na2SO4 and evaporated. The material obtained
was treated with EtOAc (40 ml), the insoluble crystalline product
was separated by filtration, washed with CH2Cl2 and dried under
vacuum to give 7.68 g of 14 (mp 153 °C, 90% yield). 1H NMR
Oily product, 70% yield. 1H NMR (CDCl3) d 7.64–7.67 and
7.37–7.48 (2ꢂ m, 5H, Ph), 6.98 and 5.65 (2ꢂ s, 2H, CO–NH2),
6.29 (d, 1H, 10-H), 6.07 (dd, 1H, 20-H), 5.80 (t, 1H, 30-H), 4.51 (m,
1H, 40-H), 4.44 and 4.18 (2ꢂ dd, 2H, 50-H), 2.15, 2.14 and 2.05
(3ꢂ s, 9H, 3ꢂ Ac). 13C NMR (CDCl3) d 170.51, 169.41 and 169.20
(3ꢂ CO–CH3), 160.58 (CO–NH2), 142.21 (C-4), 132.24, 130.28,
128.56 and 120.77 (Ph), 123.82 (C-5), 104.73 (C„C–Ph), 88.58
(C-10), 81.12 (C-40), 74.00 (C-20), 72.45 (C„C–Ph), 70.86 (C-30),
62.75 (C-50), 20.61, 20.47 and 20.43 (3ꢂ CO–CH3). HRMS [M+Na]+
calcd for C22H22N4O8Na: 493.1330; found 493.1340.

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
4393
5.7. 5-(4-Methylphenylethynyl)-1-(2,3,5-tri-O-acetyl-b-
D
-
5.12. 5-Phenylethynyl-1-b-
D
-ribofuranosyl-1H-[1,2,3]triazole-4-
ribofuranosyl)-1H-[1,2,3]triazole-4-carboxylic acid amide (18)
carboxylic acid amide (11)
Oily material, 71% yield. 1H NMR (CDCl3) d 7.54 and 7.21 (2ꢂ
d, 4H, Ph), 6.98 and 5.69 (2ꢂ s, 2H, CO–NH2), 6.29 (d, 1H, 10-H),
6.06 (dd, 1H, 20-H), 5.80 (t, 1H, 30-H), 4.51 (m, 1H, 40-H), 4.44
and 4.18 (2ꢂ dd, 2H, 50-H), 2.40 (s, 3H, CH3), 2.15, 2.14 and
2.05 (3ꢂ s, 9H, 3ꢂ Ac). 13C NMR (CDCl3) d 170.50, 169.39 and
169.17 (3ꢂ CO–CH3), 160.70 (CO–NH2), 142.00 (C-4), 140.81,
132.14, 129.31 and 117.66 (Ph), 123.97 (C-5), 105.18 (C„C–Ph),
88.51 (C-10), 81.06 (C-40), 73.98 (C-20), 71.97 (C„C–Ph), 70.85
(C-30), 62.76 (C-50), 21.69 (Ph–CH3), 20.63, 20.48 and 20.43 (3ꢂ
CO–CH3). HRMS [M+Na]+ calcd for C23H24N4O8Na: 507.1486;
found 507.1487.
Chromatographically purified solid product (68% yield) was
next crystallized from iPrOH to give white needles: mp 166–
169 °C. 1H NMR (DMSO-d6) d 7.98 and 7.67 (2ꢂ s, 2H, CO–
NH2), 7.62–7.66 and 7.48–7.55 (2ꢂ m, 5H, Ph), 6.03 (d, 1H, 10-
H), 5.71 (d, 1H, 20-OH), 5.34 (d, 1H, 30-OH), 4.82 (t, 1H, 50-OH),
4.72 (dd, 1H, 20-H), 4.27 (dd, 1H, 30-H), 4.01 (dd, 1H, 40-H),
3.40–3.61 (m, 2H, 50-H). 13C NMR (DMSO-d6) d 160.45 (CO–
NH2), 143.25 (C-4), 131.70, 130.40, 129.03 and 120.36 (Ph),
122.16 (C-5), 102.25 (C„C–Ph), 90.77 (C-10), 86.31 (C-40), 73.78
(C-20), 73.70 (C„C–Ph), 70.54 (C-30), 61.66 (C-50). Anal. Calcd
for C16H16N4O5: C, 55.81; H, 4.68; N, 16.27. Found: C, 55.73; H,
4.77; N, 16.17.
5.8. 5-Propynyl-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide (20)
5.13. 5-(4-Methylphenylethynyl)-1-b-D-ribofuranosyl-1H-
[1,2,3]triazole-4-carboxylic acid amide (12)
Oily product, 51% yield. 1H NMR (CDCl3) d 6.96 and 5.73 (2ꢂ s,
2H, CO–NH2), 6.21 (d, 1H, 10-H), 6.03 (dd, 1H, 20-H), 5.78 (t, 1H, 30-
H), 4.49 (m, 1H, 40-H), 4.42 and 4.15 (2ꢂ dd, 2H, 50-H), 2.25 („C–
CH3), 2.14, 2.13 and 2.05 (3ꢂ s, 9H, 3ꢂ Ac). 13C NMR (CDCl3) d
170.49, 169.39 and 169.17 (3ꢂ CO–CH3), 160.85 (CO–NH2),
141.77 (C-4), 124.08 (C-5), 103.53 (C„C–CH3), 88.24 (C-10), 80.97
(C-40), 73.85 (C-20), 70.85 (C-30), 63.68 (C„C–Ph), 62.72 (C-50),
20.62, 20.48 and 20.44 (3ꢂ CO–CH3), 5.42 („C–CH3). HRMS
[M+Na]+ calcd for C17H20N4O8Na: 431.1173; found 431.1175.
Crude solid product (83% yield) was subsequently crystallized
from iPrOH to result in white crystalline material: mp 195–
196 °C. 1H NMR (DMSO-d6) d 7.96 and 7.65 (2ꢂ s, 2H, CO–NH2),
7.53 and 7.33 (2ꢂ d, 4H, Ph), 6.02 (d, 1H, 10-H), 5.70 (d, 1H, 20-
OH), 5.33 (d, 1H, 30-OH), 4.82 (t, 1H, 50-OH), 4.71 (dd, 1H, 20-H),
4.27 (dd, 1H, 30-H), 4.01 (dd, 1H, 40-H), 3.40–3.61 (m, 2H, 50-H),
2.37 (s, 3H, CH3). 13C NMR (DMSO-d6) d 160.50 (CO–NH2), 143.07
(C-4), 140.52, 131.65, 129.66 and 117.36 (Ph), 122.34 (C-5),
102.61 (C„C–Ph), 90.71 (C-10), 86.28 (C-40), 73.78 (C-20), 73.22
(C„C–Ph), 70.56 (C-30), 61.69 (C-50), 21.18 (Ph–CH3). Anal. Calcd
for C17H18N4O5: C, 56.98; H, 5.06; N, 15.63. Found: C, 56.90; H,
5.34; N, 15.34.
5.9. 1-(2,3,5-Tri-O-acetyl-b-D-ribofuranosyl)-1H-[1,2,3]triazole-
4-carboxylic acid amide (19)
White solid, 5–15% yield. 1H NMR (CDCl3) d 8.39 (s, 1H, 5-H),
7.07 and 5.89 (2ꢂ s, 2H, CO–NH2), 6.20 (d, 1H, 10-H), 5.84 (dd,
1H, 20-H), 5.56 (t, 1H, 30-H), 4.51 (m, 1H, 40-H), 4.41 and 4.25 (2ꢂ
dd, 2H, 50-H), 2.13, 2.12 and 2.10 (3ꢂ s, 9H, 3ꢂ Ac). 13C NMR
(CDCl3) d 170.34, 169.42 and 169.16 (3ꢂ CO–CH3), 161.38 (CO–
NH2), 142.93 (C-4), 125.27 (C-5), 90.45 (C-10), 81.05 (C-40), 74.38
(C-20), 70.30 (C-30), 62.51 (C-50), 20.65, 20.42 and 20.34 (3ꢂ CO–
CH3).
5.14. 5-Iodo-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic
acid amide (22)
Crude product (74% yield) was crystallized from MeOH to give
crystalline material: mp >132 °C (dec). 1H NMR (DMSO-d6) d 7.89
and 7.55 (2ꢂ s, 2H, CO–NH2), 5.91 (d, 1H, 10-H), 5.69 (d, 1H, 20-
OH), 5.37 (d, 1H, 30-OH), 4.80 (t, 1H, 50-OH), 4.75 (dd, 1H, 20-H),
4.26 (dd, 1H, 30-H), 3.98 (dd, 1H, 40-H), 3.40 and 3.54 (2ꢂ m, 2H,
50-H). 13C NMR (DMSO-d6) d 161.09 (CO–NH2), 143.15 (C-4),
91.66 (C-10), 87.54 (C-5), 86.20 (C-40), 73.73 (C-20), 70.58 (C-30),
61.67 (C-50). Anal. Calcd for C8H11IN4O5: C, 25.96; H, 3.00; N,
15.14. Found: C, 25.74; H, 3.14; N, 15.13.
5.10. General procedure for the preparation of compounds
10–12 and 22
A solution of compound 17, 18, 20 or 15 in anhyd MeOH
(10 ml/1 mmol of substrate) was cooled to 0 °C and 0.2 M MeONa
in anhyd MeOH (0.2–0.5 equiv) was added. The mixture was al-
lowed to react at +5 °C overnight. It was cooled again to 0 °C
and neutralized with Dowex 50WX8-100 ion exchange resin,
which was then separated by filtration. The solution was evapo-
rated and the resulting oil was chromatographed on silica gel
column with CH2Cl2/MeOH (9:1 ? 6:1) for 10 and 22 or
(95:5 ? 9:1) for 11 and 12.
5.15. 1-(2,3,5-Tri-O-acetyl-b-
thynyl-1H-[1,2,3]triazole-4-carboxylic acid amide (23) and 5-
ethynyl-1-(2,3,5-tri-O-acetyl-b- -ribofuranosyl)-1H-[1,2,3]
D-ribofuranosyl)-5-trimethylsilyle
D
triazole-4-carboxylic acid amide (24)
A solution of 15 (685 mg, 1.38 mmol, dried in vacuo over P2O5,
at 60 °C, for 3h) and bis(benzonitrile)palladium dichloride (53 mg,
0.14 mmol) in anhyd CH3CN (18 ml) was vigorously deoxygenated
with argon. Trimethylsilylethynyltributylstannane (802 mg, 2.07
mmol) was added and the mixture was heated at 90 °C for 18 h
in a glass pressure tube. It was next diluted with CH3CN (30 ml),
extracted with hexane (3 ꢂ 50 ml) and evaporated. The residue
was chromatographed on silica gel column with EtOAc/hexane
(1.5:1 ? 3:1) as an eluent. Product 23 was isolated as an oil
(325 mg, 51% yield). 1H NMR (CDCl3) d 6.93 and 5.72 (2ꢂ s, 2H,
CO–NH2), 6.21 (d, 1H, 10-H), 5.94 (dd, 1H, 20-H), 5.77 (t, 1H, 30-H),
4.41–4.50 (m, 2H, 40,50-H), 4.18 (dd, 1H, 50-H), 2.14, 2.13 and 2.06
(3ꢂ s, 9H, 3ꢂ Ac), 0.31 (s, 9H, TMS). HRMS [M+Na]+ calcd for
5.11. 5-Propynyl-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-
carboxylic acid amide (10)
Crude solid product (90% yield) was subjected to crystallization
from iPrOH to give white crystalline material: mp 180 °C. 1H NMR
(DMSO-d6) d 7.83 and 7.57 (2ꢂ s, 2H, CO–NH2), 5.91 (d, 1H, 10-H),
5.65 (d, 1H, 20-OH), 5.29 (d, 1H, 30-OH), 4.79 (t, 1H, 50-OH), 4.67 (dd,
1H, 20-H), 4.24 (dd, 1H, 30-H), 3.98 (dd, 1H, 40-H), 3.38–3.57 (m, 2H,
50-H). 13C NMR (DMSO-d6) d 160.54 (CO–NH2), 142.43 (C-4), 122.89
(C-5), 102.07 (C„C–Ph), 90.18 (C-10), 86.16 (C-40), 73.68 (C-20),
70.59 (C-30), 64.31 (C„C–Ph), 61.72 (C-50), 4.60 („C–CH3). Anal.
Calcd for C11H14N4O5: C, 46.81; H, 5.00; N, 19.85. Found: C,
46.72; H, 5.10; N, 19.68.
C
19H26N4O8SiNa: 489.1412; found: 489.1434. Desilylated product
24 was obtained as an oil (114 mg, 21% yield). 1H NMR (CDCl3) d
6.98 and 5.84 (2ꢂ s, 2H, CO–NH2), 6.23 (d, 1H, 10-H), 6.04 (dd,

4394
T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
1H, 20-H), 5.76 (t, 1H, 30-H), 4.41–4.50 (m, 2H, 40,50-H), 4.18 (dd, 1H,
50-H), 4.00 (s, 1H, C„CH), 2.14, 2.13 and 2.06 (3ꢂ s, 9H, 3ꢂ Ac).
HRMS [M+Na]+ calcd for C16H18N4O8Na: 417.1017; found:
417.1033. Further elution with EtOAc/hexane (6:1) gave 19
(113 mg, 22% yield).
20.39 (3ꢂ CO–CH3). HRMS [M+Na]+ calcd for C14H15IN4O7Na:
500.9883; found: 500.9925.
5.19. 1-(5-O-Acetyl-2,3-O-isopropylidene-b-
propynyl-1H-[1,2,3]triazole-4-carboxylic acid amide (27) and 1-
(5-O-acetyl-2,3-O-isopropylidene-b- -ribofuranosyl)-1H-
D-ribofuranosyl)-5-
D
5.16. 5-Ethynyl-1-b-
D
-ribofuranosyl-1H-[1,2,3]triazole-4-
[1,2,3]triazole-4-carboxylic acid amide (28)
carboxylic acid amide (ETCAR, 9)
Prepared from 16 by the procedure described for 20. After col-
umn chromatography with EtOAc/hexane (2:1 ? 3:1) as an eluent,
the oily product 27 was isolated in 32% yield. 1H NMR (DMSO-d6) d
7.88 and 7.62 (2ꢂ s, 2H, CO–NH2), 6.24 (s, 1H, 10-H), 5.50 (d, 1H, 20-
H), 5.03 (dd, 1H, 30-H), 4.45 (dt, 1H, 40-H), 4.08 and 3.87 (2ꢂ dd, 2H,
50-H), 2.23 (s, 3H, „C–CH3), 1.96 (s, 3H, Ac), 1.53 and 1.35 (2ꢂ s,
6H, 2ꢂ CH3). HRMS [M+Na]+ calcd for C16H20N4O6Na: 387.1275;
found 387.1284. Further elution with EtOAc/hexane (6:1) gave
deiodinated product 28 in 50% yield. 1H NMR (DMSO-d6) d 8.68
(s, 1H, 5-H), 7.93 and 7.53 (2ꢂ s, 2H, CO–NH2), 6.38 (s, 1H, 10-H),
5.43 (d, 1H, 20-H), 5.00 (dd, 1H, 30-H), 4.50 (dt, 1H, 40-H), 4.10
and 3.97 (2ꢂ dd, 2H, 50-H), 1.91 (s, 3H, Ac), 1.52 and 1.34 (2ꢂ s,
6H, 2ꢂ CH3). HRMS [M+Na]+ calcd for C13H19N4O6Na: 349.1119;
found 349.1132.
A solution of 23 or 24 in anhyd MeOH (8 ml/1 mmol of sub-
strate) was cooled to 0 °C and 0.2 M MeONa in anhyd MeOH
(0.5 equiv) was added under argon. The mixture was allowed to re-
act at +5 °C under an argon atmosphere overnight. It was cooled
again to 0 °C and neutralized with Dowex 50WX8-100 ion ex-
change resin, which was then separated by filtration. The solution
was evaporated and the resulting oil was chromatographed on sil-
ica gel column with CH2Cl2/MeOH (6:1) to afford 9 as solid foam in
57–80% yield. It was dissolved in EtOAc/MeOH, concentrated and
kept at +5 °C to give white crystalline material: mp 106–108 °C.
1H NMR (DMSO-d6) d 7.93 and 7.66 (2ꢂ s, 2H, CO–NH2), 5.92 (d,
1H, 10-H), 5.67 (d, 1H, 20-OH), 5.33 (s, 1H, C„CH), 5.31 (d, 1H, 30-
OH), 4.79 (t, 1H, 50-OH), 4.69 (dd, 1H, 20-H), 4.24 (dd, 1H, 30-H),
3.98 (dd, 1H, 40-H), 3.39–3.58 (m, 2H, 50-H). 13C NMR (DMSO-d6)
d 160.24 (CO–NH2), 143.62 (C-4), 121.84 (C-5), 95.66 (C„CH),
90.40 (C-10), 86.33 (C-40), 73.64 (C-20), 70.54 (C-30), 68.04 (C„CH),
61.59 (C-50). Anal. Calcd for C10H12N4O5ꢁ1H2O: C, 41.96; H, 4.93; N,
19.57. Found: C, 42.01; H, 5.05; N, 19.31.
5.20. 1-(2,3-O-Isopropylidene-b-D-ribofuranosyl)-5-iodo-1H-
[1,2,3]triazole-4-carboxylic acid amide (31)
Prepared by methanolysis of 16 according to the standard pro-
cedure. 1H NMR (DMSO-d6) d 7.89 and 7.57 (2ꢂ s, 2H, CO–NH2),
6.17 (s, 1H, 10-H), 5.59 (d, 1H, 20-H), 4.98 (dd, 1H, 30-H), 4.90 (t,
1H, 50-OH), 4.20 (m, 1H, 40-H), 3.18 (m, 2H, 50-H), 1.52 and 1.37
(2ꢂ s, 6H, 2ꢂ CH3). HRMS [M+Na]+ calcd for C11H15IN4O5Na:
432.9979; found 432.9986.
5.17. 5-Amino-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-1H-
[1,2,3]triazole-4-carbonitrile (26)
4-Toluenesulfonyl chloride (534 mg, 2.8 mmol) was added to a
solution of 13 (771 mg, 2.0 mmol) in dry pyridine (20 ml). After
being stirred at room temperature for 24 h, the reaction was
quenched by addition of anhyd EtOH (2 ml) at 0 °C and the volatiles
were removed by evaporation. The residue was treated with EtOAc/
H2O (1:1), the separated organic layer was washed with brine, dried
with Na2SO4 and evaporated. The residue was chromatographed on
a silica gel column with CH2Cl2/MeOH (99:1 ? 97:3) as an eluent to
afford the product which was purified on a next silica gel column
using toluene-EtOH (9:1). Isolated oily 26 turned into solid foam
on drying in vacuo, 437 mg (59% yield). 1H NMR (CDCl3) d 5.98 (d,
1H, 10-H), 5.95 (dd, 1H, 20-H), 5.50 (t, 1H, 30-H), 5.12 (s, 2H, NH2),
4.49 (m, 1H, 40-H), 4.34 (m, 2H, 50-H), 2.14 and 2.04 (2ꢂ s, 9H, 3ꢂ
Ac). 13C NMR (CDCl3) d 170.32, 169.56 and 169.50 (3ꢂ CO–CH3),
147.44 (C-5), 111.88 (CN), 105.22 (C-4), 88.42 (C-10), 81.64 (C-40),
72.42 (C-20), 70.22 (C-30), 62.46 (C-50), 20.52, 20.43 and 20.39 (3ꢂ
CO–CH3). HRMS [M+Na]+ calcd for C14H17N5O7Na: 390.1026; found:
390.1076.
5.21. 1-(2,3-O-Isopropylidene-b-D-ribofuranosyl)-5-phenyl-
ethynyl-1H-[1,2,3]triazole-4-carboxylic acid amide (29)
Prepared from 31 by the procedure described for 17. After col-
umn chromatography with ethanol in toluene (5–10%) as an elu-
ent, the oily product 29 was isolated in 27% yield. 1H NMR
(DMSO-d6) d 7.98 and 7.68 (2ꢂ s, 2H, CO–NH2), 7.66–7.63 and
7.55–7.49 (2ꢂ m, 5H, Ph), 6.33 (s, 1H, 10-H), 5.58 (d, 1H, 20-H),
4.99–4.93 (dd overlap. t, 2H, 30-H, 50-OH), 4.24 (m, 1H, 40-H), 3.27
(m, 2H, 50-H) 1.52 and 1.36 (2ꢂ s, 6H, 2ꢂ CH3). HRMS [M+Na]+
calcd for C19H20N4O5Na: 407.1326; found 407.1332. Further elu-
tion with ethanol in toluene (20%) afforded 30 as a solid in 33%
yield.
5.22. 1-(5-O-Acetyl-2,3-isopropylidene-b-
methylsilylethynyl-1H-[1,2,3]triazole-4-carboxylic acid amide
(32) and 1-(5-O-acetyl-2,3-isopropylidene-b- -ribofuranosyl)-
D-ribofuranosyl)-5-tri
D
5.18. 5-Iodo-1-(2,3,5-tri-O-acetyl-b-
D
-ribofuranosyl)-1H-
5-ethynyl-1H-[1,2,3]triazole-4-carboxylic acid amide (33)
[1,2,3]triazole-4-carbonitrile (25)
Prepared from 16 by the procedure described for 23 and 24.
After column chromatography with EtOAc/hexane (1:1 ? 1.5:1)
as an eluent, oily products 32 and 33 were isolated in 7% and
55% yield, respectively. 32: 1H NMR (CDCl3) d 6.89 and 5.57 (2ꢂ
s, 2H, CO–NH2), 6.32 (s, 1H, 10-H), 5.45 (d, 1H, 20-H), 5.00 (dd, 1H,
30-H), 4.55 (dt, 1H, 40-H), 4.01–4.15 (m, 2H, 50-H), 2.03 (s, 3H, Ac),
1.61 and 1.41 (2ꢂ s, 6H, 2ꢂ CH3), 0.31 (s, 9H, TMS). HRMS
[M+Na]+ calcd for C18H26N4O6SiNa: 445.1514; found: 445.1521.
33: 1H NMR (CDCl3) d 6.94 and 5.68 (2ꢂ s, 2H, CO–NH2), 6.34 (s,
1H, 10-H), 5.52 (d, 1H, 20-H), 5.02 (dd, 1H, 30-H), 4.55 (dt, 1H, 40-
H), 4.01–4.15 (m, 2H, 50-H), 4.00 (s, 1H, C„CH), 2.03 (s, 3H, Ac),
1.61 and 1.41 (2ꢂ s, 6H, 2ꢂ CH3). HRMS [M+Na]+ calcd for
To a suspension of 26 (423 mg, 1.15 mmol) in diiodomethane
(18.48 g, 69 mmol) isoamyl nitrite (472 mg, 4.03 mmol) was
added. The resulting mixture was stirred at 100 °C for 1 h, then it
was applied onto a silica gel column. The column was eluted with
CH2Cl2 (in order to collect diiodomethane) followed by CH2Cl2/
MeOH (99:1 ? 98:2) to give the title product which was rechro-
matographed using toluene-EtOH (98:2 ? 96:4) for elution to iso-
late 25 as an oil (254 mg, 46% yield). 1H NMR (CDCl3) d 6.06 (d, 1H,
10-H), 6.08 (dd, 1H, 20-H), 5.74 (dd, 1H, 30-H), 4.52 (m, 1H, 40-H),
4.39 and 4.16 (2ꢂ dd, 2H, 50-H), 2.16, 2.14 and 2.03 (3ꢂ s, 9H,
3ꢂ Ac). 13C NMR (CDCl3) d 170.37, 169.37 and 169.26 (3ꢂ CO–
CH3), 129.95 (C-4), 110.74 (CN), 90.80 (C-10), 88.01 (C-5), 81.81
(C-40), 74.04 (C-20), 70.62 (C-30), 62.25 (C-50), 20.54, 20.41 and
C
15H18N4O6Na: 373.1119; found 373.1122. Further elution with
EtOAc/hexane (6:1) gave 28 in 18% yield.

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
4395
5.23. 1-(5-O-Acetyl-2,3-O-isopropylidene-b-
phenylethynyl-1H-[1,2,3]triazole-4-carboxylic acid amide (35)
D
-ribofuranosyl)-5-
5.26. cycloSaligenyl-5-ethynyl-(20,30-O-isopropylidene-b-
D-
ribofuranosyl)-1H-[1,2,3]triazole-4-carboxamide-50-phosphate
(36)
A solution of 16 (90 mg, 0.2 mmol, dried in vacuo over P2O5, at
60 °C, for 3 h) and bis(benzonitrile)palladium dichloride (8 mg,
0.02 mmol) in anhyd 1,4-dioxane (3 ml) was vigorously deoxy-
genated with argon. To the solution was added K2CO3 (28 mg,
0.2 mmol, dried as described for 16), followed by phenylacetylene
(102 mg, 1.0 mmol). The reaction mixture was heated at 100 °C
overnight, then evaporated. The residue was chromatographed
on silica gel column using EtOAc/hexane (1.5:1 ? 2:1) to give
35 (61 mg, 72% yield). Further elution with EtOAc/hexane (8:1)
led to the isolation of deiodinated product 28 (4 mg, 6% yield).
35: 1H NMR (DMSO-d6) d 7.99 and 7.68 (2ꢂ s, 2H, CO–NH2),
7.64–7.68 and 7.49–7.55 (2ꢂ m, 5H, Ph), 6.39 (s, 1H, 10-H), 5.59
(d, 1H, 20-H), 5.04 (dd, 1H, 30-H), 4.50 (m, 1H, 40-H), 3.88–4.10
(m, 2H, 50-H), 1.94 (s, 3H, Ac), 1.53 and 1.37 (2ꢂ s, 6H, 2ꢂ
CH3). HRMS [M+Na]+ calcd for C21H22N4O6Na: 449.1432; found
449.1450.
0.135 g of colorless oil, yield 45%. 1H NMR (DMSO-d6) d 7.97,
7.70 (2ꢂ s, 2ꢂ 2H, CO–NH2), 7.37 (t, 1H, H-4aryl), 7.33 (t, 1H, H-
4
aryl), 7.26–7.16 (m, 4H, H-5aryl, H-6aryl) 7.11 (d, 1H, H-3aryl), 7.02
(d, 1H, H-3aryl), 6.24 (s, 1H, 10-H), 6.22 (s, 1H, 10-H), 5.48–5.27 (m,
8H, CH2OP, 20H, C„CH), 4.99 (ddd, 2H, 30-H), 4.46 (m, 2H, 40-H),
4.18 (m, 2H, H-5’’) 4.00 (m, 2H, H-50), 1.50, 1.33 (2ꢂ s, 2ꢂ 6H,
(CH3)2C). 13C NMR (DMSO-d6) d 160.06 (CO–NH2), 149.32 (d, C-
2aryl), 143.76 (C-4), 129.75, 129.67 (C-6aryl), 125.99 (C-4aryl),
124.46, 124.39 (C-5aryl), 121.80 (C-5), 120.83, 120.70 (2ꢂ d, C-
1aryl), 118.25, 118.13 (2ꢂ d, C-3aryl), 113.12 (CH3)2C, 96.12
(C„CH), 91.26 (C-1), 86.51, 86.41 (C-40), 83.57 (C-20), 80.83 (C-
30), 68.50, 68.35 (2ꢂ d, Cbenzyl), 67.78 (s, C„CH), 66.70 (m, C-
50), 26.59, 26.54, 24.91, 24.88 (CH3)2C. 31P NMR (DMSO-d6) d -
10.21. HRMS [M+Na]+ calcd for C20H21N4O8PNa: 499.0989; found:
499.0990.
5.27. cycloSaligenyl-(20,30-O-isopropylidene-b-
D-ribofuranosyl)-
5.24. 1-(2,3-O-Isopropylidene-b-
zole-4-carboxylic acid amide (30) and 5-ethynyl-1-(2,3-O-isop
ropylidene-b- -ribofuranosyl)-1H-[1,2,3]triazole-4-carboxylic
D-ribofuranosyl)-1H-[1,2,3]tria
1H-[1,2,3]triazole-4-carboxamide-50-phosphate (37)
D
0.136 g of colorless oil, yield 48%. 1H NMR (DMSO-d6) d 8.69,
8.66 (2ꢂ s, 2ꢂ 1H, 5-H), 7.94, 7.56 (2ꢂ s, 2ꢂ 2H, CO–NH2), 7.38–
7.32 (m, 2H, H-4aryl), 7.26–7.17 (m, 4H, H-5aryl, H-6aryl), 7,12 (d,
1H, H-3aryl), 7.05 (d, 1H, H-3aryl), 6.42 (d, 1H, 10-H), 6.39 (d, 1H,
10-H), 5.50–5.27 (m, 6H, CH2OP, 20-H), 4.96 (dd, 1H, 30-H), 4.93
(dd, 1H, 30-H), 4.45 (m, 2H, 40-H), 4.25 (m 2H, H-5’’), 4.12 (m, 2H,
H-50), 1.50 and 1.31 (2ꢂ s, 6H, 2ꢂ CH3), 1.31 (d, 6H, (CH3)2C). 13C
NMR (DMSO-d6) d 161.09 (CO–NH2), 149.30, 149.24 (dd, C-2aryl),
143.19, 143,13 (C-4), 129.73 (d, C-6aryl), 126.54, 126.46 (C-5),
126.01 (C-4aryl), 124.44 (C-5aryl), 120.90, 120.80 (2ꢂ d, C-1aryl),
118.24, 118,12 (2ꢂ d, C-3aryl), 113,16 (CH3)2C); 92.64, 92.58 (C-
10), 85.80, 85.69 (2ꢂ d, C-40), 83.92 (C-20), 80.83 (C-20), 68.52,
68.34 (2ꢂ d, Cbenzyl), 67.10, 66.86 (C-50), 26.59, 24.92 (CH3)2C).
31P NMR (DMSO-d6) d ꢀ9.72, ꢀ9.69. HRMS [M+Na]+ calcd for
acid amide (34)
Methanolysis of compounds 28, 32 and 33 was carried out
according to general procedure described for preparation of 10–
12 and 22. The resulting crude products were chromatographed
on silica gel with EtOAc/hexane (3:1) to afford 30 and 34 as solid
foam (90–95% yield). 30: 1H NMR (DMSO-d6) d 8.70 (s, 1H, 5-H),
7.93, 7.54 (2ꢂ s, 2H, CO–NH2) 6.31 (d, 1H, 10-H), 5.31 (dd, 1H, 20-
H), 5.06 (b s, 1H, 50-OH), 4.93 (dd, 1H, 30-H), 4.28 (dt, 1H, 40-H),
3.39 (d, 2H, 50-H), 1.52, 1.34 (2ꢂ s, 6H, 2ꢂ CH3); 13C NMR
(DMSO-d6) d 161.35 (CO–NH2), 143.20 (C-4), 125.87 (C-5), 112.85
(CH3)2C, 93.68 (C-10), 88.14 (C-40), 84.28 (C-20), 81.67 (C-30), 61.22
(C-50), 26.78, 25.02 (CH3)2C. HRMS [M+Na]+ calcd for C11H16N4O5-
Na: 207.1013; found: 307.1003. 34: 1H NMR (DMSO-d6) d 7.95,
7.68 (2ꢂ s, 2H, CO–NH2), 6.19 (s, 1H, 10-H), 5.54 (d, 1H, 20-H),
5.33 (s, 1H, C„CH), 4.97 (dd, 1H, 30-H), 4.92 (t, 1H, 50-OH), 4.21
(td, 1H, 40-H), 3.25 (m, 2H, 50-H), 1.51, 1.35 (2ꢂ s, 6H, 2ꢂ CH3);
13C NMR (DMSO-d6) d 160.13 (CO–NH2), 143.78 (C-4), 121.74 (C-
5), 112.73 (CH3)2C, 95.97 (C„CH), 91.52 (C-10), 88.98 (C-40),
83.28 (C-20), 81.75 (C-30), 67.95 (C„CH), 60.88 (C-50), 26.65,
24.93 (CH3)2C. HRMS [M+Na]+ calcd for C13H16N4O5Na: 331.1013;
found: 331.1021.
C18H21N4O8PNa: 475.0989; found: 475.1006.
5.28. cycloSaligenyl-5-ethynyl-b-D-ribofuranosyl-1H-
[1,2,3]triazole-4-carboxamide-50-O-phosphate (38)
A solution of 36 (0.135 g, 0.28 mmol) in 80% aq TFA (5 ml) was
stirred at 0 °C for 1 h. The volatiles were removed under reduced
pressure and the residue coevaporated with anh ethanol
(3 ꢂ 10 ml). The product was purified by chromatography on a sil-
ica gel short column with 10% MeOH in CH2Cl2 as an eluent. It
afforded 0.109 g of 38 as glassy oil (88% yield). An analytical sam-
ple was precipitated with methanol-hexane (mp 75 °C). 1H NMR
(DMSO-d6) d 7.92, 7.68 (2ꢂ s, 2ꢂ 2H, CO–NH2), 7.38–7.33 (m, 2H,
H-4aryl), 7.25–7.13 (m, 4H, H-5aryl, H-6aryl), 7.08 (d, 1H, H-3aryl),
7.03(d, 1H, H-3aryl), 5.92 (d, 2H, 10-H), 5.83 (m, 2H, 20-OH), 5.54–
5.30 (m, 8H, CH2OP, 30-OH, C„CH), 4.59 (m, 2H, 20-H), 4.34–4.28
(m, 4H, 30-H, 40-H), 4.18–4.13 (m, 4H, 50-H). 13C NMR (DMSO-d6)
d 160.17 (CO–NH2), 149.34, 149.25(2ꢂ d, C-2aryl), 143.53 (C-4),
129.66 (C-6aryl), 125.98, 125.94 (C-4aryl), 124.34, 124.31(C-5aryl),
121.73, 121.69 (C-5), 120.80, 120.68 (2ꢂ d, C-1aryl), 118.17,
118.05 (2ꢂ d, C-3aryl), 95.76, 95.70 (C„CH), 90.50 (C-10), 82.83,
82.74 (C-40), 73.74, 73.67 (C-20), 70.02 (C-30), 68.42, 68.33 (2ꢂ d,
Cbenzyl); 67.91, 67.87 (C„CH), 67.46, 67.30 (2ꢂ d, C-50). 31P
5.25. General procedure for the preparation of compounds 36
and 37
The reactions were carried out in an argon atmosphere under
strictly anhydrous conditions.
To a cooled (0 °C) and stirred solution of nucleoside 30 or 34
(0.63 mmol) in CH3CN (20 ml) was injected triethylamine
(2.0 equiv, 1.26 mmol).). Next, a solution of salicylchlorophos-
phane (2.0 equiv, 1.26 mmol) in CH3CN (2 ml) was added dropwise
within 5 min. The reaction was complete after stirring for further
15 min, as determined by TLC (CH2Cl2/MeOH 9:1). Oxidation of
intermediate phosphites was performed immediately by adding
tert-butylhydroperoxide (2 equiv of 5.5 M solution in decane) to
the reaction mixture at 0 °C. It was stirred for 30 min and allowed
to warm up to room temperature. The solvent was removed under
reduced pressure and the residue was chromatographed on a silica
gel short column with 5% MeOH in CH2Cl2 to yield the pronucleo-
tides 36 and 37.
NMR (DMSO-d6)
d -10.13, -9.97. UV (MeOH) kmax (e) = 244
nm (15100). HRMS (ESIꢀ) (M-1) calcd for: C17H16N4O8P
435.0711, found 435.0687. HRMS (ESI+) [M+Na]+ calcd for
C17H17N4O8PNa: 459.0676; found: 459.0687. Analytical HPLC tR:
22.50 min.

4396
T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
5.29. cycloSaligenyl-b-
D
-ribofuranosyl-1H-[1,2,3]triazole-4-
(dd, 1H, 30-H), 4.93, 4.87 (2 overlap. dd, 2H, CH2OH), 4.47 (m, 1H,
carboxamide-50-O-phosphate (39)
40-H), 4.35 and 4.07 (2ꢂ dd, 2H, 50-H), 3.87 (s, 3H, OCH3), 2.11,
2.07, 1.92 (3ꢂ s, 9H, 3ꢂ Ac). 1-(2,3,5-Tri-O-acetyl-b-
D-ribofurano-
Compound 37 (0.13 g, 0.29 mmol) was deprotected with 80% aq
TFA (5 ml) according to the procedure described for 36. Pronucle-
otide 39 was purified analogously to 38. It afforded 0.1 g of 39 as
glassy oil (85% yield). 1H NMR (DMSO-d6) d 8.69, 8.68 (2ꢂ s, 2ꢂ
1H, 5-H), 7.89, 7.53 (2ꢂ s, 2ꢂ 2H, CO–NH2), 7.35–7.32 (m, 2H, H-
syl)-4-hydroxymethyl-1H-[1,2,3]triazole-5-carboxylic acid methyl
1
ester: H NMR (DMSO-d6) d 6.70 (s, 1H, 10-H), 5.96 (d, 1H, 20-H),
5.67 (t, 1H, CH2OH), 5.29 (t, 1H, 30-H), 4.69 (d, 2H, CH2OH), 4.46
(m, 1H, 40-H), 4.30 and 4.02 (2ꢂ m, 2H, 50-H), 3.90 (s, 3H, OCH3),
2.13, 2.08, 1.89 (3ꢂ s, 9H, 3ꢂ Ac).
4aryl), 7.25–7.16 (m, 4H, H-5aryl, H-6aryl), 7.12 (d, 1H, H-3aryl), 7.07
(d, 1H, H-3aryl), 6.00 (d, 2H, 10-H), 5.76 (m, 2H, 20-OH), 5.50–5.35
(m, 6H, CH2OP, 30-OH), 4.40 (m, 2H, 20-H), 4.37–4.16 (m, 8H, 30-
H, 40-H, 50-H). 13C NMR (DMSO-d6) d 161.24 (CO–NH2), 149.40,
149.30 (2ꢂ d, C-2aryl), 143.17 (C-4), 129.69 (C-6aryl), 126.01 (C-5),
125.73, 125.67 (C-4aryl), 124.37 (C-5aryl), 120.86, 120.75 (2ꢂ d, C-
5.32. 5-Fluoromethyl-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-
1H-[1,2,3]triazole-4-carboxylic acid methyl ester (44)
A reaction was carried out in argon atmosphere under anhy-
drous conditions at –35 °C. To a cooled flask containing CH2Cl2
(7 ml) was injected DAST (0.280 g, 2.16 mmol). Likewise, a solution
of substrate 43 (0.298 g, 0.72 mmol) in CH2Cl2 (7 ml) was added in
5 min. and a reaction mixture was stirred for 3 h and 30 min.
According to TLC only trace amount of substrate remained and a
reaction mixture was neutralized with cold 0.5 M NaHCO3 adjust-
ing temperature to ꢀ5 °C. After CO2 evolution ceased, EtOAc was
added (100 ml) to the mixture and organic layer was separated,
carefully washed with water, brine and finally with water. Next,
the organic solution was dried with Na2SO4, filtered and reduced
in vacuo to yellow oil (0.330 g). Fluorinated product was purified
on a silica gel short column with EtOAc/hexane (1:1). It afforded
44 as a white solid: 0.187 g (62% yield). 1H NMR (DMSO-d6) d
6.47 (d, 1H, 10-H), 6.00 (dd, 1H, 20-H), 5.91, 5.78 (d, 2ꢂ 1H,
2JH,F = 48 Hz, CH2F), 5.64 (m, 1H, 30-H), 4.51 (m, 1H, 40-H), 4.31
and 4.06 (2ꢂ dd, 2H, 50-H), 3.90 (s, 3H, OCH3), 2.12, 2.09, 1.88
(3ꢂ s, 9H, 3ꢂ Ac). 13C NMR (DMSO-d6) d 169.82, 169.48, 169.22
(3ꢂ COCH3), 160.40 (4-CO2CH3), 137.59 (C-4), 136.30 (d,
JC,F = 19 Hz, C-5), 88.33 C-10), 73.28 (C-20), 70.77 (d, JC,F = 164 Hz,
CH2F), 70.03 (C-30), 52.31 (OCH3), 20.26 (3ꢂ COCH3). HRMS
[M+Na]+ calcd for C16H20FN3O9Na: 440.1076; found: 440.1087.
1
aryl), 118.20, 118.10 (2ꢂ d, C-3aryl), 92.08 (C-10), 82.59, 82.50 (C-
40), 74.38, 74.32 (C-20), 69.89, 69.86 (C-30), 68.44, 68.35 (2ꢂ d, Cben-
zyl), 67.66, 67.52 (2ꢂ d, C-50); 31P NMR (DMSO-d6) d -9.95, -9.84. UV
(MeOH) kmax (e
) = 266 nm (2300). HRMS (ESIꢀ) (M-1) calcd for:
C15H16N4O8P 411.0711, found 411.0690. HRMS (ESI+) [M+Na]+
calcd for C15H17N4O8PNa: 435.0676; found: 435.0686. Analytical
HPLC tR: 22.44 min.
5.30. (Z)- and (E)-5-{2-[2-(Acetylamino)-2-(methoxycarbonyl)
ethylthio]vinyl}-1-b-D-ribofuranosyl-1H-[1,2,3]triazole-4-carbo
xylic acid amide (40 and 41)
To a cooled (0 °C) solution of 9 (188 mg, 0.7 mmol) in anhyd
CH3CN (14 ml) was added N-acetyl-L-cysteine methyl ester
(124 mg, 0.7 mmol) and anhyd triethylamine (21 mg, 0.21 mmol).
The mixture was allowed to react at 5 °C overnight, next it was
neutralized at 0 °C with AcOH and evaporated. The residue was
chromatographed on a silica gel column with CH2Cl2/MeOH (9:1)
to isolate oily products 41 (isomer E, 20 mg, 6% yield) and 40 (iso-
mer Z, 139 mg, 45% yield). 41: 1H NMR (DMSO-d6) d 8.51 (d, 1H,
NH), 7.95 (d, 1H, 5-CH@CH, J = 15.6 Hz), 7.88 and 7.52 (2ꢂ s, 2H,
CO–NH2), 6.66 (d, 1H, CH@CH, J = 15.6 Hz), 5.83 (d, 1H, 10-H),
5.56 (d, 1H, 20-OH), 5.24 (d, 1H, 30-OH), 4.79 (m, 2H, 50-OH and
CH), 4.55 (m, 1H, 20-H), 4.25 (dd, 1H, 30-H), 3.99 (dd, 1H, 40-H),
3.66 (s, 3H, OCH3), 3.35–3.58 (m, 2H, 50-H), 3.04–3.19 (m, 2H,
CH2), 1.86 (s, 3H, N-Ac). HRMS [M+Na]+ calcd for C16H23N5O8SNa:
468.1165; found: 468.1151. 40: 1H NMR (DMSO-d6) d 8.36 (d,
1H, NH), 7.79 and 7.45 (2ꢂ s, 2H, CO–NH2), 6.90 (d, 1H, 5-CH@CH,
J = 10.4 Hz), 6.43 (d, 1H, CH@CH, J = 10.4 Hz), 5.71 (d, 1H, 10-H),
5.55 (d, 1H, 20-OH), 5.25 (d, 1H, 30-OH), 4.76 (m, 1H, 50-OH), 4.55
(m, 1H, 20-H), 4.47 (m, 1H, CH), 4.25 (dd, 1H, 30-H), 3.95 (dd, 1H,
40-H), 3.63 (s, 3H, OCH3), 3.35–3.58 (m, 2H, 50-H), 3.04–3.19 (m,
2H, CH2), 1.85 (s, 3H, N-Ac). 13C NMR (DMSO-d6) d 170.72 (CO-
OCH3), 169.59 (CO–CH3), 161.69 (CO–NH2), 139.49 (C-4), 137.77
(5-CH@CH), 134.69 (C-5), 110.48 (5-CH@CH), 90.35 (C-10), 85.71
(C-40), 74.04 (C-20), 70.55 (C-30), 61.84 (C-50), 52.48 (NH-CH),
52.16 (O–CH3), 34.77 (S-CH2), 22.22 (CO–CH3). HRMS [M+Na]+
calcd for C16H23N5O8SNa: 468.1165; found: 468.1160.
5.33. 5-Fluoromethyl-1-b-D-ribofuranosyl)-1H-[1,2,3]triazole-4-
carboxylic acid amide (42)
To a carefully dried compound 44 (0.155 g, 0.38 mmol) was
added methanol saturated with ammonia (30 ml, 10 M solution)
and resulted solution was stirred in a pressure tube at 20 °C for
2 days. After this time according to TLC a single product was
formed and no starting material remained in the reaction mixture.
The volatiles were removed under reduced pressure, and the resi-
due was co-evaporated with ethanol to ease evacuation of residual
acetamide to afford carboxamide 42: 0.096 g as colorless oil (89%).
Analytical sample (68 mg) was crystallized from MeOH/EtOH (1:3)
to give 43 mg of crystals (mp 190 °C), solvated with alcohols
(according to 1H NMR: 8% and 6%, respectively). The filtrate affor-
ded next portion of crystals on standing at 8 °C for several days,
this material was suitable for X-ray analysis. 42: 1H NMR
(DMSO-d6) d 8.08, 7.52 (2ꢂ s, 2H, CO–NH2), 5.99 (2ꢂ d, 1H,,
JH,H = 16 Hz, JH,F = 48 Hz CH2F), 5.96 (d, 1H, 10-H), 5.83 (2ꢂ d, 1H,
JH,H = 16 Hz, JH,F = 48 Hz CH2F), 5.63 (b s, 1H, 20-OH), 5.29 (b s, 1H,
30-OH), 4.79 (t, 1H, 50-OH), 4.66 (b s, 1H, 20_H), 4.25 (d, 1H, 30-H),
3.99 (dd, 1H, 40-H), 3.56 and 3.42 (2ꢂ m, 2H, 50-H). 13C NMR
(DMSO-d6) d 161.69 (CO–NH2), 140.24 (d, JC,F = 4 Hz, C-4), 133.93
(d, JC,F = 18 Hz, C-5), 90.78 (C-10), 86.06 (C-40), 74.06 (C-20), 70.85
(d, JC,F = 162 Hz, CH2F), 70.36 (C-30), 61.47 (C-50). 19F NMR
(DMSO-d6) d -135.91 (t). Anal. Calcd for C9H13 FN4O5: C, 39.13; H,
4.74; N, 20.28. Found: C, 39.09; H, 4.94; N, 20.33.
5.31. 5-Hydroxymethyl-1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-
1H-[1,2,3]triazole-4-carboxylic acid methyl ester (43)
Compound 43 has been prepared according to a procedure of
Earl and Townsend25 by 1,3-cycloaddition of 1-b-
D-ribofuranosyl
azide triacetate31 (1.77 g, 5.9 mmol) with methyl 4-hydroxy-2-
butynoate32 (0.875 g, 7.67 mmol). The reaction was conducted in
toluene at 100 °C for 24 h and resulted in the formation of two
regioisomers, separated by chromatography on silica gel to afford
faster moving product, 5-hydroxymethyl-isomer 43: 1.47 g as a
colorless solid (62% yield) and a slower moving 4-hydroxy-
methyl-isomer 0.128 g as a solid (6%). 43: 1H NMR (DMSO-d6) d
6.47 (d, 1H, 10-H), 5.91 (dd, 1H, 20-H), 5.77 (t, 1H, CH2OH), 5.64
5.34. Crystallographic procedure
X-ray diffraction measurements of a single crystal of 42 were
carried out at room temperature using graphite-monochromated

T. Ostrowski et al. / Bioorg. Med. Chem. 19 (2011) 4386–4398
4397
Table 2
Table 3
Crystal data and structure refinement for 5-fluoromethyl-1-b-D-ribofuranosyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide (42)
Geometry of the hydrogen bonds (Å, o) for 5-fluoromethyl-1-b-D-ribofuranosyl)-1H-
[1,2,3]triazole-4-carboxylic acid amide, 42
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C9H13FN4O5
276.25
294 K
1.54184 Å
tetragonal
P 43212
D–HꢁꢁꢁA
d(D–H)
d(HꢁꢁꢁA)
d(DꢁꢁꢁA)
<(DHA)
O(20)–H(20)ꢁꢁꢁO(50)i
O(30)–H(30)ꢁꢁꢁN(3)ii
O(50)–H(50)ꢁꢁꢁO(30)iii
N(6)–H(61)ꢁꢁꢁO(6)iv
C(7)–H(7B)ꢁꢁꢁO(6)
C(30)–H(310)ꢁꢁꢁO(50)
C(7)–H(71)ꢁꢁꢁO(6)v
C(7)–H(72)ꢁꢁꢁO(40)iii
C(40)–H(40)ꢁꢁꢁF(7A)v
C(10)–H(H10)ꢁꢁꢁF(7)vi
0.87(2)
0.89(2)
0.90(3)
0.81(2)
0.96
0.93(2)
0.96(5)
0.94(3)
0.95(2)
0.97(2)
2.00(3)
1.91(2)
1.94(3)
2.10(2)
2.39
2.61(2)
2.50(4)
2.70(3)
2.62(2)
2.60(2)
2.830(2)
2.776(2)
2.801(2)
2.911(2)
3.057(2)
2.981(2)
3.422(2)
3.403(2)
3.098(5)
3.138(3)
160(2)
165(2)
160(2)
173(2)
126
104(1)
161(4)
132(2)
112(1)
115(1)
Unit cell dimensions
a = 12.55938(3) Å
c = 14.62874(5) Å
2307.51(1) Å3
8
Volume
Z
Calculated density
Absorption coefficient
F(0 0 0)
Crystal size
h range for data collection
Limiting indices
1.590 g/cm3
1.223 mmꢀ1
1152
Symmetry codes: (i) x + 0.5, ꢀy + 0.5, ꢀz + 0.25; (ii) ꢀy, ꢀx, ꢀz + 0.5; (iii) y ꢀ 0.5,
ꢀx + 0.5, z + 0.25; (iv) ꢀy, ꢀx, ꢀz + 0.75; (v) ꢀy + 0.5, x + 0.5, z ꢀ 0.25; (vi) y, x,
ꢀz + 1.
0.48 ꢂ 0.38 ꢂ 0.22 mm
9.31–74.39o
ꢀ15 6 h 6 15
ꢀ15 6 k 6 15
ꢀ18 6 l 6 18
94350/2350 [Rint = 0.0389]
99.2%
0.7747 and 0.5913
Full-matrix least-squares on F2
2350/0/234
air atmosphere. 7 ꢂ 103 cells were seeded on each well on 96-well
plate (Nunc). 24 h later cells were exposed to the test compounds
for another 24 or 48 h. Stock solutions (100 mM) of test com-
pounds were freshly prepared in DMSO. The final concentrations
Reflections collected/unique
Completeness to = 26.37°
Max. and min. transmission
Refinement method
Data/restraints/parameters
of the compounds tested in the cell cultures were: 1, 1 ꢂ 10ꢀ2
,
Goodness-of-fit on F2
1.083
1 ꢂ 10ꢀ4 and 1 ꢂ 10ꢀ6 mM. The concentration of DMSO in the cell
Final R indices [I >2(I)]
R indices (all data)
Extinction coefficient
R1 = 0.0280, wR2 = 0.0781
R1 = 0.0281, wR2 = 0.0782
0.0021(4)
culture medium was 1%.
The values of IC50 (the concentration of test compound required
to reduce the cell survival fraction to 50% of the control) were
calculated from dose-response curves and used as a measure of
cellular sensitivity to a given treatment. The cytotoxicity of all
compounds was determined by the MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] assay, as described previ-
ously.39 Briefly, after 24 or 48 h of incubation with drugs, the cells
were treated with the MTT reagent and incubation was continued
for 2 h. MTT-formazan crystals were dissolved in 20% SDS and 50%
DMF at pH 4.7 and absorbance was measured at 562 and 630 nm
on an ELISA-PLATE READER (FLUOstar Omega, BMG Labtech, Ger-
many). As a control (100% viability), we used cells grown only in
the presence of vehicle (1% DMSO).
Absolute structure parameter
Largest diff. peak and hole
0.03(18)
0.154 and ꢀ0.139 e Åꢀ3
Cu Ka
radiation and a KM4-CCD diffractometer.33 Integrated inten-
sities were obtained using the CrysAlisPro program.33 The struc-
ture was solved by direct methods with SHELXS-97 and refined
by full-matrix least squares minimization of
R
w(F2o ꢀ F2c )2 using
SHELXL-97.34 All hydrogen atoms were derived from a difference
Fourier map and included in the refinement with isotropic B fac-
tors. The final model contains two CH2F units with a common
C(7) centre and two sets of fluorine atoms populated with 0.71/
0.29 occupancy. Two hydrogen atoms of the less populated CH2F
group were generated geometrically and during the refinement
process their positions and Biso were fixed. The crystal data and de-
tails of data processing are given in Table 2. Table 3 contains the
hydrogen bonds geometry. Molecular illustrations were prepared
using the ORTEPII35 and the Mercury programs,36 and the XP pack-
age.37 Ring puckering analysis was done using WinGX (version
1.64.05) system of programs.38 Atomic parameters in CIF format
are available as Electronic Supplementary Publication from Cam-
Acknowledgments
This work was supported by the Polish Ministry of Science and
Higher Education grants no. N N405 2516 33 and PBZ-MNiSW-07/
1/2007. We wish to thank Dr. Lukasz Marczak for MS spectra mea-
surements and Stefan Donevski and Karolina Krolewska for their
assistance in performing cytotoxicity assays.
bridge Crystallographic Data Centre (CCDC 823702)
.
Supplementary data
5.35. Cells and cytotoxicity assay
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.050.
Human umbilical vein endothelial cells (HUVEC) were isolated
from freshly collected umbilical cords and cultured in plastic
dishes coated with gelatin, in RPMI 1640 medium supplemented
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