In Vitro and in Vivo Evaluation of Fully Substituted (5-(3-Ethoxy-3-oxopropynyl)-4-(ethoxycarbonyl)-1,2,3-triazolyl-glycosides as Original Nucleoside Analogues to Circumvent Resistance in Myeloid Malignancies

Article abstract of DOI:10.1021/acs.jmedchem.6b01803

A series of nucleoside analogues bearing a 1,4,5-trisubstituted-1,2,3-triazole aglycone was synthesized using a straightforward click/electrophilic addition or click/oxidative coupling tandem procedures. SAR analysis, using cell culture assays, led to the

Full text of DOI:10.1021/acs.jmedchem.6b01803

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Article
In vitro and in vivo evaluation of fully substituted (5–(3-ethoxy-3-
oxopropynyl)-4-(ethoxycarbonyl)-1,2,3-triazolyl-glycosides as original
nucleoside analogs to circumvent resistance in myeloid malignancies
Hella Amdouni, Guillaume Robert, Mohsine Driowya, Nathan Furstoss, Camille Métier,
Alix Dubois, Maeva Dufies, Marwa Zerhouni, François Orange, Sandra Lacas-
Gervais, Khalid Bougrin, Anthony R. Martin, Patrick AUBERGER, and Rachid Benhida
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01803 • Publication Date (Web): 17 Jan 2017
Downloaded from http://pubs.acs.org on January 18, 2017
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In vitro and in vivo evaluation of fully substituted
(5–(3ꢀethoxyꢀ3ꢀoxopropynyl)ꢀ4ꢀ(ethoxycarbonyl)ꢀ
1,2,3ꢀtriazolylꢀglycosides as original nucleoside
analogs to circumvent resistance in myeloid
malignancies
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Hella Amdouni,a‡ Guillaume Robert,b‡ Mohsine Driowya,a‡ Nathan Furstoss,b Camille Métier,a
Alix Dubois,b Maeva Dufies,b Marwa Zerhouni,b François Orange,c Sandra Lacas-Gervais,c
Khalid Bougrin,d Anthony R. Martin,a Patrick Aubergerb†* and Rachid Benhida a†*
aUniversité Côte d’Azur, CNRS, Institut de Chimie de Nice UMR7272 – 06108 Nice, France;
bCentre Méditerranéen de Médecine Moléculaire UMR INSERM U1065 – 06204 Nice, France;
d
cCentre Commun de Microscopie Appliquée – 06108 Nice, France; Laboratoire de Chimie des
Plantes et de Synthèse Organique et Bioorganique, URAC23, Université Mohammed V, Faculté
des Sciences, B.P. 1014 Rabat, Morocco.
KEYWORDS: triꢀsubstituted 1,2,3ꢀtriazoles, leukemia, antiꢀleukemic effects, myelodysplasic
syndromes, autophagy.
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ABSTRACT A series of nucleoside analogs bearing a 1,4,5ꢀtrisubstitutedꢀ1,2,3ꢀtriazole aglycone
was synthesized using a straightforward click/electrophilic addition or click/oxidative coupling
tandem procedures. SAR analysis, using cell culture assays, led to the discovery of a series of
compounds belonging to the 5ꢀalkynylꢀ1,2,3ꢀtriazole family that exhibits potent antileukemic
effects on several hematologic malignancies including chronic myeloid leukemia (CML) and
myelodysplastic syndromes (MDS) either sensitive or resistant to their respective therapy.
Compound 4a also proved efficient in vivo on mice xenografted with SKM1ꢀR MDS cell line.
Additionally, some insights in its mode of action revealed that this compound induced cell death
by caspase and autophagy induction.
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Introduction
Nucleoside analogs (NAs) constitute since decades a therapeutic armamentarium of choice in the
treatment of malignant hemopathies. The anticancer nucleosides include several analogs of
pyrimidine and purine derivatives.1,2 The currently available analogues of purine are cladribine,
clofarabine and fludarabine, and those of pyrimidine are cytarabine, gemcitabine and azacitidine.
NAs mimic natural nucleosides by using their physiological nucleosides transporters to enter into
the cells before being phosphorylated to their active triphosphate form inside the cell by specific
kinases. These compounds are classified as antiꢀmetabolites drugs that inhibit directly or
indirectly the DNA and/or RNA synthesis thus inducing apoptotic cell death.3,4
Recently, we reported that acadesine (5ꢀaminoimidazoleꢀ4ꢀcarboxamideꢀ1ꢀβꢀDꢀriboside, Aca)
acts as a peculiar antineoplastic agent that inhibits CML progression of naïve and resistant cells.5
Despite good efficiency in sensitive and resistant CML cells, the amount of acadesine needed to
kill the cells was excessive (IC50 on K562 = 0.8 mM). However, its mode of action resulting in
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an autophagic cell death remains of great interest.5 Thus, the production of more potent analogs
of acadesine that retain its original mode of action could be highly desirable. To ensure a rapid
and straightforward access to a wide variety of acadesine analogs we replaced its original
imidazole core by a more convenient 1,2,3ꢀtriazole structure (Figure 1).
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Figure 1. From acadesine to potent analogs.
Herein, we report our efforts towards the discovery of potent analogs of acadesine and their
biological evaluation against various hematological malignancies, including CML and
myelodysplastic syndromes, either sensitive or resistant to their reference treatment. We also
disclose some insights in the mode of action of our compounds and the in vivo evaluation of a
representative structure on aggressive and resistant MDS mouse model is presented.
Results and discussion
Chemistry
We intended to develop potent antiproliferative compounds and at the same time envisioned to
grant access to them through very short reaction sequences. For this purpose, we limited our
investigations to single or twoꢀstep synthetic pathways. We based our syntheses on the copper
catalyzed azideꢀalkyne cycloaddition reaction (CuAAC),6ꢀ8 which is known for its robustness and
versatility, and included it in modified procedures. Indeed, instead of using regular CuAAC
which only yields 1,4ꢀdisubstituted 1,2,3ꢀtriazoles and narrows the accessible structural variety,
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we employed modified CuAAC procedures that allow for the synthesis of 1,4,5ꢀtrisubstituted
1,2,3ꢀtriazoles, and thus expand the structural modularity. Moreover, the use of azidoꢀsugars as
1,3ꢀdipole would allow a direct access to fully functionalized triazolylꢀnucleosides. It should be
noted that in practice syntheses of such analogs have been limited to disubstituted 1,4ꢀ
derivatives.6ꢀ8 Fully decorated 1,2,3ꢀtriazoles can be obtained through several synthetic
pathways;9ꢀ11 herein, we report the use of two strategies, Scheme 1; both of them relying on the
trapping of the 5ꢀcupratedꢀ1,2,3ꢀtriazole intermediate (A) generated during the CuAAC reaction.
On the one hand, A can be trapped by various electrophiles.12,13 (path a). When the electrophile
is a halogen, a subsequent Pdꢀcatalyzed coupling could be applied to increase the structural
diversity. On the other hand, under oxidative conditions, the CuAAC reaction can yield, in a
single step, 5ꢀalkynylꢀ1,2,3ꢀtriazoles (path b).14ꢀ16 Thus, we will resort to these two routes
leading to fully decorated 1,2,3ꢀtriazoles to build up the library surveyed in the biology section.
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Scheme 1. Synthetic access to the 1,2,3ꢀtrisubstituted triazolylꢀnucleosides.
Path a, CuAAC / Electrophilic trapping. A first series of 1,4,5ꢀtrisubstituted 1,2,3ꢀtriazoles
has been synthesized following a CuAAC/ electrophilic trapping sequence (Scheme 2). This
methodology, previously reported by Wu12 and Benhida,13 takes advantage on the nucleophilicity
of the 5ꢀtriazolyl copper intermediate of the CuAAC (A) to introduce, in a multicomponent
fashion, an electrophile at the 5ꢀposition. In the present study, we used as electrophilic sources:
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iodine (1a-1c) and acyl chlorides (1d-1f). Thus, starting from the conveniently protected 1ꢀ
deoxyꢀ1ꢀazidoꢀβꢀDꢀribofuranose,17,18 through this singleꢀstep method, we prepared 6 compounds
(1a-1f) with nonꢀoptimized yields ranging from 32 to 77%.
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a
Reaction conditions: azide (1.0 mmol), alkyne (2.0 mmol), CuI (1.2 mmol), I2 (2.0 mmol),
b
CAN (1.0 mmol) and DIPEA (5.0 mmol) in THF. azide (1.0 mmol), alkyne (2.0 mmol), CuI
(1.2 mmol), acyl chloride (3.0 mmol) and DIEA (1.5 mmol), in THF.
Scheme 2. Synthesis of triazoles 1a-1f through a CuAAC/Electrophilic trapping sequence
To broaden the structural variety of our acadesine analogs, 1a was subjected to Sonogashira
couplings using either pꢀfluorophenylacetylene or nonꢀ1yne as the alkyne (Scheme 3).
i) 1a (1.0 mmol), alkyne (5.0 mmol), triethylamine (1.0 mmol), CuI (0.05 mmol) and
PdCl2(PPh3)2 (0.05 mmol), in anhydrous toluene at 60 °C.
Scheme 3. Functionalization of 1a through Sonogashira coupling.
Finally, a Stille coupling (Scheme 4) was performed on 1a and 1c to afford the compounds 3a
and 3b in 70% and 76% yield, respectively.
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i) 1a or 1c (1.0 mmol), 2ꢀ(tributylstannyl)thiophene (2.0 mmol), CuI (0.05 mmol) and
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PdCl2(PPh3)2 (0.1 mmol), in anhydrous toluene at 80 °C.
Scheme 4. Functionalization of 1a and 1c through a Stille coupling.
Path b, CuAAC / oxidative coupling. During his research on the cycloaddition of
bromomagnesium acetylides, Krasiński evidenced 4ꢀalkynylꢀ1,2,3ꢀtriazoles as trace
byproducts.19 In 2006, Porco Jr. developed a new methodology to gain access to this class of
compound in decent yields (31ꢀ68 %).14 The reported protocol relies on a formal CuAAC /
oxidative coupling cascade (Scheme 5). This sequence was further explored by others and
adapted to a wider range of substrates.15,16
Scheme 5. CuAAC / Oxidative coupling developed by Porco Jr.
In the present study, we first tried to apply the reported conditions to the synthesis of
compounds 4aꢀ4o. However, we eventually faced reactivity issues and very poor yields (<5%)
were obtained when somewhat acidic alkynes, such as ethyl propiolate, were used. This
prompted us to adapt the reaction conditions to our specific substrates. A rapid screening of the
reaction parameters of this CuAAC / oxidative coupling revealed that copper cyanide was best
suited as the Cu(I) source, N,Nꢀdiisopropylethylamine as the base/ligand, hydrogen peroxide as
terminal oxidant20 and tetrahydrofuran as the solvent (THF). Using our adapted protocol for the
CuAAC / oxidative coupling, we next undertook the preparation of compounds 4a-4o, dedicated
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to biological evaluations. Thus a collection of 17 compounds was synthesized in poor to good
yields (21ꢀ72%, Scheme 6).
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i): azide (1.0 mmol), alkyne (4.0 mmol), CuCN (1.2 mmol), H2O2 (5.0 mmol) and DIEA (2.0
mmol), in THF at 0 °C for 10 min. then r.t.
Scheme 6. Synthesis of triazoles 4aꢀ4q through a CuAAC/oxidative coupling reaction.
Biology
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In vitro anti-leukemic effect of triazolyl nucleoside analogs in the chronic myelogenous
leukemia K562 cell line. The first step in the investigation of the therapeutic potential of all the
reported compounds was devoted to determine their activity on the viability of the K562 CML
cell line. Cell viability assays were performed using the XTT assay. According to this first screen
(Table 1) a robust structureꢀactivity relationship was established. Indeed, the first 3 series of
compounds (1a-1f, 2a-2b and 3a-3b) did not exhibit inhibitory activities on K562 cell
metabolism at 48 h (up to 10 ꢀM). Remarkably, most of the compounds belonging to the fourth
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series (4a-4q) were highly efficient and displayed IC50 ranging from 0.15 to 2.50 ꢀM. It is worth
noting that these series of compounds are 320ꢀ to >5000ꢀfold more active than the parent
acadesine, used as a control (Table 1). Hence, concerning the aglycone ring, the structureꢀ
activity relationship revealed that substitution on the 5ꢀposition of the 1,2,3ꢀtriazole
tremendously modulated the activity. The presence of halide, heteroaryl or acyl groups on this
position resulted in no antiꢀproliferative efficiency (series 1ꢀ3), while alkynyl substituents
terminated by an ester function led to high activity (series 4). Moreover, the nature of the ester
(Me, Et, tBu – 4aꢀ4c) did not influence significantly the IC50 values, albeit the tBu substituent
may be slightly less favorable (4c and 4j vs. 4a). Finally, it is worth noting that the 5ꢀalkynyl
substituent must be terminated by an ester function as its replacement by a non electronꢀ
withdrawing moiety induces a loss of activity (series 2). Conversely to the aglycone moiety, the
saccharide motif tolerates a broader variety of substituents without dramatically affecting the
activity.21 Indeed, various combinations of 2’,3’ꢀOꢀalkylidene, acetyl or methyl Oꢀsubstitutions
(4aꢀ4k) did not significantly affect the IC50 although the best efficacies were observed with
larger 2’,3’ꢀOꢀalkylidene (4g, 4h, 4m). This may underpin the need for some hydrophobic bulk
on this part of the scaffold. Regarding the nature of the saccharide, the replacement of the
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ribofuranose structure (4i) by a glucopyranose (4l), a 2’ꢀdeoxyribose (4n), a xylose (4o) or even
a Lꢀribose moiety (4q and 4p) resulted in no notable variation of the IC50 value.
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Table 1. Evaluation of the compounds efficiency against K562 cell lines.
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IC50 (ꢀM)a
>10
IC50 (ꢀM)a
0.50 (0.01)
0.15 (0.01)
0.45 (0.01)
0.15 (0.01)
0.70 (0.04)
2.50 (0.15)
0.80 (0.06)
0.70 (0.03)
0.50 (0.02)
0.20 (0.01)
0.20 (0.02)
0.20 (0.01)
0.20 (0.01)
800
Cpd
Cpd
1a
1b
1c
1d
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1f
4e
4f
>10
>10
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4h
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>10
>10
4j
>10
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2b
3a
3b
4a
4b
4c
4k
4l
>10
>10
4m
4n
4o
4p
4q
Aca
>10
0.37 (0.01)
1.45 (0.01)
1.15 (0.04)
0.55 (0.02)
4d
a
IC50 determined by XTT assay (mitochondrial metabolism measurement) after 48 h
incubation time. Standard deviation is given in parenthesis.
Following the first screen of all the compounds on the leukemic K562 cell line, we selected 4a as
a representative compound of the 4th series and get it screened on a panel of 60 cancer cell lines
by the National Cancer Institute.22 In line with the effect of 4a on K562 cancer cells, it exhibited
a strong activity, reported as the 50% growth inhibition, in most of the screened cancer cell lines
and in particular in hematopoietic cell lines (see ESI for the full NCI60 results).
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Table 2. GI50 of 4a against selected cancer cell lines from NCI60 assay.
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a
a
Cell lines
GI50 Cell lines
GI50
3.11
CCRFꢀCEMb
HLꢀ60b
1.33 M14c
2.07 MDAꢀMB435c 2.03
K562b
1.28 SKꢀMELꢀ2c
3.07 SRc
12.20
1.22
1.77
3.96
MOLTꢀ4b
RPMIꢀ8226b
LOX IMVIc
MALMEꢀ3Mc
2.23 MCF7d
1.71 BTꢀ549d
2.39 MDAꢀMB468d 1.66
a Values in ꢀM. b leukemia cell lines. c melanoma cell lines. d breast cancer cell lines.
Resistance to targeted therapies occurs in a significant percentage of CML patients. To complete
these observations, we evaluated the capacity of our lead compound to eradicate imatinib
sensitive and resistant CML cells. Thus, 4a was assessed against the ImaꢀR cell line which
derives from parental K562 after chronic exposition to imatinib for 6 months.23,24 Importantly,
compound 4a was able to eliminate resistant cells as efficiently as sensitive cells (Figure 2) with
an IC50 of 0.4 µM.
120
100
K562
80
60
40
20
0
Ima-R
0
2
4
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10
4a [µM]
Figure 2. Comparison of the effect of 4a on K562 and ImaꢀR cell lines.
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We further extend our study to other hematopoietic cell lines. The SKM1–S and –R cells
constitute a valuable model to study the resistance of myelodysplastic syndromes (MDS) cell
lines to azacitidine (Aza), a gold standard drug used in the treatment for elderly patients, and
classified as hypomethylating agent.25,26 Hence, we assessed the effect of 4a on the
mitochondrial metabolism (IC50 – Table 2) and the cell viability (EC50 – Table 2) of both cell
types, and the mitochondrial metabolism. We observed that the EC50 of 4a were 0.24 and 0.40
µM for the SKM1ꢀS and the SKM1ꢀR, respectively. Its IC50 fall in the same range with values of
0.34 and 0.50 µM on the sensitive and resistant cell lines, respectively. We also confirm that
azacitidine failed to kill resistant cells contrariwise to sensitive ones. Therefore, compound 4a is
highly efficient at least against two leukemic cell lines resistant to two different chemotherapies,
imatinib and azacitidine.
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Table 2. Evaluation of 4a versus azacitidine in SKM1ꢀS and SKM1ꢀR cell lines.
SKM1ꢀS
SKM1ꢀR
IC50 (ꢀM)a EC50 (ꢀM)b
0.50 (0.04) 0.40 (0.03)
IC50 (ꢀM)a EC50 (ꢀM)b
0.34 (0.03) 0.24 (0.01)
4a
Aza
0.50 (0.07) 0.51 (0.03)
>10
>10
a
IC50 of the mitochondrial metabolism determined by XTT assay after 48 h incubation time.
bDL50 determined by flow cytometry (DAPI incorporation) method after 48 h incubation time.
To gain insights into the mechanism of action of 4a, we next analyzed the morphology of cells
incubated for 24 h in the presence of 4a. Electron microscopy images of SKM1 revealed the
presence of autophagic vacuoles after 24 h treatment (Figure 3). Moreover, 4a induced a striking
disorganization of mitochondrial structure with an important swelling of mitochondrial crest,
more particularly in sensitive cells. These observations suggest autophagy and mitochondrial
impairment in the antiꢀleukemic effects of 4a.
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Figure 3. 4a induces the formation of autolysosomal vacuoles and disorganization of
intramitochondrial structures.
We next compared the effect of azacitidine and 4a, both at 1 µM, on the signaling pathways
involved in cell death. We first confirmed that azacitidine is able to induce apoptotic cell death in
sensitive SKM1ꢀS cells with a robust cleavage of caspase 3. As expected, SKM1ꢀR cells fail to
respond to azacitidine treatment (Figure 4) even at 10 µM (Table 2). Meanwhile, compound 4a
induced both caspase activation and an increase in LC3 lipidation, a hallmark of autophagy
induction (Figure 4). Resistant cells, SKM1ꢀR, exhibit a low but detectable activation of caspase
3 under 4a treatment.
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Figure 4. 4a induces both apoptosis and autophagic cell deaths.
The results presented above show that resistant MDS cell lines harbor defect in apoptosis and are
thus less sensitive to apoptosisꢀinducing agents as azacitidine. In this context, the original mode
of cell death induced by 4a could be used to circumvent resistance to apoptosis in cancer cells.
To validate the effect of 4a in vivo, we implanted highly aggressive human SKM1ꢀR cells
subcutaneously on the flank of athymic mice. These were separated in two groups, one receiving
a vehicle and the other one receiving 5 mg/kg of 4a five days a week. 4a treatment was found to
reduce the tumor volume by 50% as compared to untreated mice, after 35 days (Figure 5, left
panel). Moreover, tumor free weight was comparable in both groups of mice indicating that
compound 4a did not induce acute toxicity in treated mice (Figure 5, right panel).
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Figure 5. 4a inhibits tumor growth in nude mice without any apparent acute toxicity.
Finally, the effect of compound 4a was assessed in bone marrow samples from azacitidine
resistant MDS and acute myeloid leukemia (AML) patients. Conversely to azacitidine which
fails to induce cell death in these resistant cells as expected, compound 4a killed these cells in a
dose dependent manner (Figure 6A). Importantly, Zvad, a panꢀcaspase inhibitor was found to
partially counteract the effect of 4a, hence confirming that it induced cell death by both caspaseꢀ
dependent and independent mechanisms.
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Figure 6. Effect of 4a on primary cancer cells extracted from azacitidineꢀresistant patients.
CONCLUSION
In conclusion, we herein reported a series of nucleoside analogs bearing a 1,4,5ꢀtrisubstitutedꢀ
1,2,3ꢀtriazole aglycone synthetized using a straightforward click/electrophilic addition or
click/oxidative coupling tandem procedures. These original compounds acted as potent antiꢀ
leukemic agent in several hematopoietic cell lines. They exhibited strong activities compared to
acadesine (>5000ꢀfold) and were highly efficient on imatinibꢀ and azacitidineꢀresistant myeloid
cell lines. Moreover, we observed that one of our lead compound (4a), also induced tumor
regression in nude mice xenotransplanted with azacitidineꢀresistant MDS cells, without
significant toxicity. Finally, we established that the antiꢀleukemic effect of compound 4a was
related to its ability to induce both caspase activation and autophagy.
EXPERIMENTAL SECTION
Cell cultures. The human CML K562 cell line was provided by ATCC and was grown in RPMI
1630 medium (Lonza, Walkersville, MD, USA) in the presence of 5% FCS. The human MDS
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SKM1, cell line was provided by DSMZ and was grown in RPMI 1630 medium (Lonza,
Walkersville, MD, USA) in the presence of 10% FCS. All cell cultures were grown at 37°C
under 5% CO2, 50 units/ml penicillin and 50 mg/ml streptomycin to minimize contamination.
Isolation of bone marrow patient primary. Bone marrow samples were collected from patients
suffering MDS or AML in the course of azacitidine (vidaza®) treatment as part of an
institutionally approved cellular sample collection protocol. Informed consent has been obtained
according to institutional guidelines. Mononuclear cells were isolated from bone marrow
samples by density centrifugation (FicollꢀPaqueTM Plus), washed with PBS, 5% SVF, 2 mM
EDTA and resuspended in cell culture medium (IMDM, 10% fetal bovine serum) and incubated
overnight at 37°C in a 5% CO2 incubator before CD34+ cells isolation. MDS or AML cells were
labelled with CD34 microbeads isolated by magnetic positive selection (StemSepTM Human
CD34 Selection Kit; StemCell, Vancouver, BC, Canada). Purity was estimated to at least 90% by
FACS analysis. Experiments were performed using a StemSpanR SFEM medium (StemCell,
Vancouver, BC, Canada) supplemented with 100 ng/ml human recombinant SCF, FLT3ꢀL and
20 ng/ml human recombinant ILꢀ3, ILꢀ6 and GꢀCSF (Peprotech, Rocky Hill, NJ, USA).
Reagents and antibodies. Sodium fluoride and orthovanadate, phenylmethylsulfonyl fluoride,
aprotinin and leupeptin were purchased from Sigma (SaintꢀLouis, MO, USA). AntiꢀantiꢀLC3ꢀb,
antiꢀcaspase 3, and HRP conjugated antiꢀrabbit antibodies were from Cell Signaling Technology
(Beverly, MA, USA). AntiꢀHsp60 and antiꢀactin antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). HRPꢀconjugated antiꢀmouse and antiꢀgoat antibodies
were from Dakopatts (Glostrup, Denmark).
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Measurement of cell metabolism (XTT). K562 cells (20.103 cells/100ml) were incubated in a
96ꢀwell plate with indicated concentration of STS for 6 h. About 50 ꢀl of XTT reagent (sodium
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3’ꢀ[1ꢀ(phenylaminocarbonyl)ꢀ3,4ꢀtetrazolium]ꢀbis (4–methoxy–6–nitro) benzene sulfonic acid
hydrate) (MPP) was added to each well as described previously. The absorbance of the formazan
product, reflecting cell viability, was measured at 490 nm. Each assay was performed in
quadruplicate.
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Measurement of cell death. After 4a stimulation, cells were stained with propidium iodide.
Then, stained cells were analyzed by flow cytometry.
Electronic microscopy. K562 cell pellets were collected, fixed with 1.6% glutaraldehyde, postꢀ
fixed in 1% OsO4, dehydrated in alcohol series, and embedded in epoxy resin. Thin sections
were contrasted with uranyl acetate and lead citrate. Preparations were observed either with a
Philips CM12 electron microscope operating at 80 kV (FEI, Eindhoven, The Netherlands) or
with a Jeol 1400 (Tokyo, Japan) mounted with CCD cameras (Morada, Olympus SIS, Germany).
Samples were analysed with Jeol 1200 XII Philipps electron microscope.
Western blot. After stimulation, cells were lysed at 4°C in lysis buffer. Lysates were centrifuged
at 10 000g for 10 min at 4°C and supernatants were supplemented with concentrated SDS sample
buffer. A total of 30 mg of protein were separated on 12% polyacrylamide gel and transferred
onto polyvinylidene difluoride (PVDF) membrane (ImmobilonꢀP, Millipore, Bedford, MA,
USA). After blocking nonꢀspecific binding sites, the membranes were incubated with specific
antibodies, washed three times and finally incubated with HRPꢀconjugated antibody for 1 h at
room temperature. Immunoblots were revealed using the enhanced chemiluminescence detection
kit (Amersham Biosciences, Uppsala, Sweden).
Tumor regression experiments in nude mice. Female Nude NMRI Mice (Janvier, Le Genest
Saint Ile, France) were randomized into two experimental groups, each containing 7 animals.
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Animals in both groups received a 200 µl injection of 1.106 SKM1ꢀR leukemia cells on the left
flank. When tumors reached 100 mm3, animals were injected intraperitoneally with vehicle, or
4a at a dose level of 5 mg/kg of body weight, 5 days a week. The growths of leukemic cells
composing the tumor were visualized and quantified every 2 days in the animal with an
electronic calliper.
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General procedures. Reagents were obtained from commercial suppliers (SigmaꢀAldrich or
Alfa Aesar) and used as received. Anhydrous solvents were obtained according to standard
procedures. The reactions were monitored by thinꢀlayer chromatography (TLC, Merck silica gel
60 F254 plates) and revealed by visualization under UV light (254, 315 & 365 nm), then by
spraying ethanolic solution of either vanillin/H2SO4 or pꢀanisaldehyde/H2SO4. Column
chromatography purifications were performed on 40–63 ꢀm silica gel. All NMR spectra
(1H, 13C{1H}) were recorded on a 200 MHz Bruker Avance Spectrometer. Mass spectra (MS)
were recorded on an Esquire 3000 Plus apparatus with ESI in both positive and negative mode.
High resolution mass spectra (HRMS) were recorded on ESIꢀLTQOrbitrapU3000 RSLC
spectrometer. HPLC analysis was performed on a Jasco LCꢀNet II /ADC apparatus using
phenomenex columns (conditions: unless otherwise stated: 1.0 mL/min, gradient 75% A / 25% B
(1 min.) then increasing to 0% A / 100% B in 6 min. and a plateau of 3 min. before returning to
75% A / 25% B in 1 min. A is water and B is CH3CN, both containing 1‰ HCOOH). Purity of
all compounds was found to be ≥95% as determined by HPLC using UV detection at 260 nm.
General procedure A: preparation of 5-iodo-1,2,3-triazoles (1a-1c). To a solution of azide
(1.0 mmol) in THF or DCM (10.0 mL), were added successively the corresponding alkyne (2.0
mmol), CuI (1.2 mmol), I2 (2.0 mmol), cerium ammonium nitrate (CAN) (1.0 mmol) and DIPEA
(5.0 mmol). The reaction mixture was allowed to react at r.t. until complete consumption of the
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starting material (0.5ꢀ1 h). Then, it was filtered on calcite and the solvent was removed under
vacuum. The crude material was purified over a silica gel column (cyclohexane/ethyl acetate: 9/1
to 8/2) to yield the desired product.
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General procedure B: preparation of 5-acylated triazoles (1d-1e). To a solution of azide (1.0
mmol) in THF or DCM (10.0 mL), were added successively the corresponding alkyne (2.0
mmol), CuI (1.2 mmol), acyl chloride (3.0 mmol) and DIPEA (1.5 mmol). The reaction mixture
was allowed to react at r.t. until complete consumption of the starting material (3ꢀ5 h). Then, it
was filtered on calcite and the solvent was removed under vacuum. The crude material was
purified over a silica gel column eluted with cyclohexane/ethyl acetate: 9/1 to 8/2 to yield the
desired product.
General procedure C: Sonogashira coupling (2a-2d). To a solution of 5ꢀiodoꢀtriazole (1.0
mmol) in anhydrous toluene at 60 °C were added the corresponding alkyne (5.0 mmol),
triethylamine (1.0 mmol), CuI (0.05 mmol) and PdCl2(PPh3)2 (0.05 mmol). The reaction mixture
was allowed to react until complete consumption of the starting material (overnight). The
reaction mixture was then filtered on calcite and the solvent was removed under vacuum. The
crude material was purified over a silica gel column (cyclohexane/ethyl acetate: 9/1 to 7/3) to
yield the desired product.
General procedure D: Stille coupling (3a, 3b)
To a solution of 5ꢀiodoꢀtriazole (1.0 mmol) in anhydrous toluene at 80 °C were added 2ꢀ
(tributylstannyl)thiophene (2.0 mmol), CuI (0.05 mmol) and PdCl2(PPh3)2 (0.1 mmol). The
reaction mixture was allowed to react until complete consumption of the starting material (16 h).
The reaction mixture was then filtered on calcite and washed with ethyl acetate. The filtrate was
concentrated under reduced pressure and the crude residue purified on silica gel column
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chromatography (cyclohexane/ethyl acetate: 8/2 to 3/7) followed by a recrystallization in
diethylether to yield the desired product.
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General procedure E: preparation of 5-alkynl-triazoles (4a-4q). To a solution of azide (1.0
mmol) in THF (10.0 mL) at 0 °C 10 min, were added successively the corresponding alkyne (4.0
mmol), CuCN (1.2 mmol), H2O2 (5.0 mmol) and DIPEA (2.0 mmol). The reaction mixture was
allowed to react at r.t until complete consumption of the starting material (3ꢀ7h). The reaction
mixture was then filtered on calcite and the solvent was removed under vacuum. The crude
material was purified over a silica gel column eluted with cyclohexane/ethyl acetate: 9/1 to 7/3 to
yield the desired product.
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1-Deoxy-1-(5-iodo-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-O-isopropylidene-β-D-
ribofuranose (1a). The title compound was obtained, according to the general procedure A and
starting from the corresponding azide (6.0 g, 28.0 mmol), as a brown powder (4.0 g) in 32%
1
yield. H NMR (CDCl3, 200 MHz) δ 1.38ꢀ1.46 (m, 6H), 1.62 (s, 3H), 3.04 (br, 1H), 3.58ꢀ3.64
(m, 1H), 3.73ꢀ3.81 (m, 1H), 4.45 (q, J 7.1 Hz, 2H), 4.52ꢀ4.57 (m, 1H), 5.06 (dd, J 5.8 and 1.7
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Hz, 1H), 5.38 (dd, J 5.8 and 1.9 Hz, 1H), 6.27 (d, J 1.9 Hz, 1H); C{1H} NMR (CDCl3, 50
MHz) δ 14.2, 25.1, 27.0, 61.8, 63.2, 82.0, 85.3, 89.6, 95.2, 113.9, 142.0, 156.7, 159.9; HRMS
(ESI+): calcd for [M+H]+ C13H19O6N3I, 440.0313, found 440.0314; HPLC (λ260) 97% purity.
1-Deoxy-1-(5-iodo-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-O-isopropylidene-β-D-
ribofuranose (1b). The title compound was obtained, according to the general procedure A and
starting from the corresponding azide (1.0 g, 4.6 mmol), as a pinkish powder (691 mg) in 35%
yield. 1H NMR (CDCl3, 200 MHz) δ 1.39 (s, 3H), 1.62 (s, 3H), 2.98 (m, 1H), 3.55ꢀ3.67 (m, 1H),
3.72ꢀ3.80 (m, 1H), 3.98 (s, 3H), 4.54 (t, J 3.2 Hz, 1H), 5.06 (dd, J 5.8 and 1.7 Hz, 1H), 5.39 (dd,
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J 5.8 and 1.9 Hz, 1H), 6.27 (d, J 1.8 Hz, 1H); C{1H} NMR (CDCl3, 50 MHz) δ 25.1, 27.0,
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52.5, 63.1, 81.9, 85.3, 89.6, 95.2, 113.9, 141.9, 156.7, 160.3; HRMS (ESI+): calcd for [M+H]+
C12H17O6N3I, 426.0156, found 426.0157; HPLC (λ260) 99% purity.
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1-Deoxy-1-(5-iodo-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3,5-tri-O-acetyl-β-D-
ribofuranose (1c). The title compound was obtained, according to the general procedure A and
starting from the corresponding azide (1.1 g, 3.7 mmol), as a brown powder (780 mg) in 40%
yield. 1H NMR (CDCl3, 200 MHz) δ 1.39 (t, J 7.1 Hz, 3H), 2.00 (s, 3H), 2.10 (s, 6H), 4.09 (dd, J
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12.3 and 4.0 Hz, 1H), 4.33ꢀ4.46 (m, 4H), 5.74 (t, J 5.4 Hz, 1H), 6.07ꢀ6.15 (m, 2H); C{1H}
NMR (CDCl3, 50 MHz) δ 14.1, 20.3, 20.4, 20.5, 61.6, 62.4, 70.8, 73.9, 81.4, 85.7, 90.2, 142.1,
159.8, 169.1, 169.3, 170.4; HRMS (ESI+): calcd for [M+H]+ C16H21O9N3I, 526.0317, found
526.0319; HPLC (λ260) 97% purity.
1-Deoxy-1-(5-(furan-2-carbonyl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3,5-tri-O-
acetyl-β-D-ribofuranose (1d). The title compound was obtained, according to the general
procedure B and starting from the corresponding azide (1.0 g, 3.3 mmol), as a white powder
(1.13 g) in 65% yield. 1H NMR (CDCl3, 200 MHz) δ 1.13 (t, J 7.1 Hz, 3H), 2.04 (s, 3H), 2.09 (s,
6H), 4.06 (dd, J 12.4 and 4.2 Hz, 1H), 4.09ꢀ4.28 (m, 3H), 4.38ꢀ4.40 (m, 1H), 5.68 (t, J 5.3 Hz,
13
1H), 6.12ꢀ6.20 (m, 2H), 6.64ꢀ6.65 (m, 1H), 7.28 (d, J 4.1 Hz, 1H), 7.66 (s, 1H); C{1H} NMR
(CDCl3, 50 MHz) δ 13.8, 20.3, 20.4, 20.6, 61.7, 62.4, 70.7, 73.7, 81.6, 89.4, 113.5, 121.6, 135.9,
139.4, 148.7, 152.0, 159.5, 169.1, 169.4, 170.5, 171.8; HRMS (ESI+): calcd for [M+H]+
C21H24O11N3, 494.1405, found 494.1407; HPLC (λ260) 99% purity.
1-Deoxy-1-(5-(thiophen-2-carbonyl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3,5-tri-O-
acetyl-β-D-ribofuranose (1e). The title compound was obtained, according to the general
procedure B and starting from the corresponding azide (553 mg, 1.83 mmol), as a brown powder
(717 mg) in 77% yield. 1H NMR (CDCl3, 200 MHz) δ 1.03 (t, J 7.1 Hz, 3H), 2.01ꢀ2.06 (m, 9H),
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4.00ꢀ4.27 (m, 4H), 4.32ꢀ4.36 (m, 1H), 5.62 (t, J 4.4 Hz, 1H), 6.07ꢀ6.10 (m, 2H), 7.10ꢀ7.15 (m,
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1H), 7.42 (d, J 3.8 Hz, 1H), 7.85 (d, J 4.1 Hz, 1H); C{1H} NMR (CDCl3, 50 MHz) δ 13.5,
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20.2, 20.3, 20.5, 61.6, 62.4, 70.7, 73.5, 81.7, 89.2, 128.7, 136.3, 136.9, 137.3, 138.6, 142.7,
156.6, 159.2, 169.0, 169.3, 170.3, 177.0; HRMS (ESI+): calcd for [M+H]+ C21H24O10N3S,
510.1177, found 510.1178; HPLC (λ260) 96% purity.
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1-Deoxy-1-(5-(p-methylbenzoyl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3,5-tri-O-
acetyl-β-D-ribofuranose (1f) The title compound was obtained, according to the general
procedure B and starting from the corresponding azide (650 mg, 1.83 mmol), as a brown powder
(552 mg) in 72% yield. 1H NMR (CDCl3, 200 MHz) δ 0.98 (t, J 7.2 Hz, 3H), 2.01 (s, 3H), 2.07
(s, 6H), 2.42 (s, 3H), 3.99ꢀ4.26 (m, 4H), 4.31ꢀ4.35 (m, 1H), 5.64 (t, J 4.7 Hz, 1H), 6.11ꢀ6.15 (m,
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2H), 7.27 (d, J 8.5 Hz, 2H), 7.64 (d, J 8.4 Hz, 2H); C{1H} NMR (CDCl3, 50 MHz) δ 13.1,
19.8, 19.9, 20.1, 21.4, 26.4, 61.1, 62.1, 70.3, 73.2, 81.2, 89.0, 129.2, 129,3, 133.3, 137.2, 138.3,
146.0, 168.7, 169.0, 169.8, 185.0; HRMS (ESI+): calcd for [M+H]+ C24H18O10N3, 518.1769,
found 518.1772; HPLC (λ260) >99% purity.
Ethyl
5-((4-fluorophenyl)ethynyl)-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-1H-1,2,3-
triazole-4-carboxylate (2a). The title compound was obtained, according to the general
procedure C starting from compound 1a (200 mg, 0.46 mmol), as a brown oil (22 mg) in 11%
1
yield. H NMR (CDCl3, 200 MHz) δ 1.38 ꢀ1.46 (m, 6H), 1.61 (s, 3H), 3.25 (br, 1H), 3.64ꢀ3.72
(m, 1H), 3.81ꢀ3.87 (m, 1H), 4.45 (q, J 7.1 Hz, 2H), 4.52ꢀ4.61 (m, 1H), 5.06 (dd, J 5.8 and 1.36
Hz, 1H), 5.32 (dd, J 5.8 and 1.8 Hz, 1H), 6.35 (d, J 1.8 Hz, 1H), 7.06ꢀ7.15 (m, 2H), 7.57ꢀ7.10
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(m, 2H); C{1H} NMR (CDCl3, 50 MHz) δ 14.3, 25.1, 27.0, 61.6, 63.3, 82.0, 85.5, 89.6, 93.6,
104.1, 113.8, 116.2 (d, J 22.4 Hz), 116.7 (d, J 3.6 Hz), 124.9, 134.2 (d, J 8.8 Hz), 134.3, 140.2,
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159.7, 163.7 (d, J 253.3 Hz); HRMS (ESI+): calcd for [M+H]+ C21H23O6N3F, 432.1565, found
432.1566. HPLC (λ260) 94% purity.
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Ethyl
1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(non-1-yn-1-yl)-1H-1,2,3-triazole-4-
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carboxylate (2b). The title compound was obtained, according to the general procedure C
starting from compound 1a (200 mg, 0.46 mmol), as a brown oil (26 mg) in 13% yield. 1H NMR
(CDCl3, 200 MHz) δ 0.89 (t, J 6.5 Hz, 3H), 1.25ꢀ1.45 (m, 14H), 1.61ꢀ1.75 (m, 5H), 2.57 (t, J 7.0
Hz, 2H), 3.76ꢀ3.60 (m, 1H), 3.84 (dd, J 12.6 and 3.1 Hz, 1H), 4.44 (q, J 7.1 Hz, 2H), 4.51ꢀ4.59
(m, 1H), 5.06 (dd, J 5.8 and 1.5 Hz, 1H), 5.27 (dd, J 5.8 and 2.0 Hz, 1H), 6.28 (d, J 2.0 Hz, 1H);
13C{1H} NMR (CDCl3, 50 MHz) δ 14.1, 14.2, 19.9, 22.6, 25.1, 27.0, 27.9, 28.7, 28.8, 31.6, 61.5,
63.4, 64.4, 82.0, 85.6, 89.5, 93.4, 108.4, 113.7, 125.6, 139.8, 159.9; HRMS (ESI+): calcd for
[M+H]+ C22H34O6N3, 436.2442, found 436.2444. HPLC (λ260) 96% purity.
Ethyl
1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(thiophen-2-yl)-1H-1,2,3-triazole-4-
carboxylate (3a). The title compound was obtained, according to the general procedure D and
starting from 1a (1.0 g, 2.2 mmol), as a white powder (630 mg) in 70% yield. 1H NMR (CDCl3,
200 MHz) δ 1.31ꢀ1.38 (m, 6H), 1.52 (s, 3H), 3.36 (dd, J 8.4 and 4.8 Hz, 1H), 3.63ꢀ3.76 (m, 1H),
3.80ꢀ3.90 (m, 1H), 4.37 (q, J 7.1 Hz, 2H), 4.52 (br, 1H), 5.10 (dd, J 5.7 and 1.7 Hz, 1H), 5.42
(dd, J 5.8 and 2.2 Hz, 1H), 6.00 (d, J 2.2 Hz, 1H), 7.20 (dd, J 5.1 and 3.6 Hz, 1H), 7.43 (d, J 3.7
and 1.3 Hz, 1H), 7.66 (dd, J 5.1 and 1.2 Hz, 1H); 13C{1H} NMR (CDCl3, 50 MHz) δ 14.1, 25.1,
26.9, 61.5, 63.5, 82.2, 85.4, 89.2, 92.6, 113.7, 123.1, 127.4, 130.7, 132.5, 135.8, 137.1, 160.4;
HRMS (ESI+): calcd for [M+H]+ C17H22O6N3S, 396.1224, found 396.1226; HPLC (λ260) >99%
purity.
Ethyl
1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-5-(thiophen-2-yl)-1H-1,2,3-triazole-4-
carboxylate (3b) The title compound was obtained, according to the general procedure D and
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starting from 1c (1.0 g, 1.9 mmol), as a white powder (701 mg) in 76% yield. 1H NMR (CDCl3,
200 MHz) δ 1.31 (t, J 7.1 Hz, 3H), 2.06 (s, 6H), 2.09 (s, 3H), 4.14 (dd, J 13.0 and 5.0 Hz, 1H),
4.30ꢀ4.46 (m, 4H), 5.79ꢀ5.86 (m, 2H), 6.15 (dd, J 5.3 and 3.2 Hz, 1H), 7.22 (dd, J 5.1 and 3.7
Hz, 1H), 7.43 (d,d J 3.7 and 1.2 Hz, 1H), 7.65 (dd, J 5.1 and 1.2 Hz, 1H); 13C{1H} NMR (CDCl3,
50 MHz) δ 14.1, 20.4, 20.4, 20.6, 61.3, 62.5, 71.0, 74.0, 81.3, 87.9, 123.3, 127.4, 130.5, 132.2,
135.8, 137.3, 156.6, 160.4, 169.2, 169.3, 170.5; HRMS (ESI+): calcd for [M+H]+ C20H24O9N3S,
482.1228, found 482.1230; HPLC (λ260) >99% purity.
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1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-
O-isopropylidene-β-D-ribofuranose (4a). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (1.0 g, 4.6 mmol), as a brown
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powder (1.0 g) in 64% yield. H NMR (CDCl3, 200 MHz) δ 1.32ꢀ1.46 (m, 9H), 1.61 (s, 3H),
2.86 (br, 1H), 3.66 (dd, J 12.4 and 4.7 Hz, 1H), 3.80 (dd, J 12.4 and 3.6 Hz, 1H), 4.34 (q, J 7.1
Hz, 2H), 4.45 (q, J 7.1 Hz, 2H), 4.52ꢀ4.57 (m, 1H), 5.04 (dd, J 5.9 and 1.8 Hz, 1H), 5.34 (dd, J
5.8 and 2.2 Hz, 1H), 6.29 (d, J 2.2 Hz, 1H); 13C{1H} NMR (CDCl3, 50 MHz) δ 13.9, 14.0, 25.1,
26.9, 62.1, 63.0, 67.5, 81.8, 85.1, 89.4, 93.8, 94.0, 114.1, 122.0, 142.7, 152.2, 156.7, 158.9;
HRMS (ESI+): calcd for [M+H]+ C18H24O8N3, 410.1558, found 410.1558; HPLC (λ260) >99%
purity.
1-Deoxy-1-(5-(3-methoxy-3-oxoprop-1-yn-1-yl)-4-(methoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
2,3-O-isopropylidene-β-D-ribofuranose (4b). The title compound was obtained, according to
the general procedure E and starting from the corresponding azide (231 mg, 1.1 mmol) as a
brown powder (127 mg) in 31% yield. 1H NMR (CDCl3, 200 MHz) δ 1.35 (s, 3H), 1.58 (s, 3H),
2.87 (br, 1H), 3.61 (dd, J 12.3 and 4.8 Hz, 1H), 3.74 (dd, J 12.3 and 3.9 Hz, 1H), 3.86 (s, 3H),
3.96 (s, 3H), 4.50 (br, 3H), 5.01 (d, J 5.9 Hz, 1H), 5.36 (d, J 5.9 Hz, 1H), 6.25 (br, 1H); 13C{1H}
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NMR (CDCl3, 50 MHz) δ 25.0, 26.8, 52.7, 53.4, 62.7, 67.8, 81.7, 84.8, 89.4, 93.7, 114.0, 122.0,
142.4, 152.5, 159.2; HRMS (ESI+): calcd for [M+H]+ C16H20O8N3, 382.1245, found 382.1245;
HPLC (λ260) 96% purity.
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1-Deoxy-1-(5-(3-tert-butoxy-3-oxoprop-1-yn-1-yl)-4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-
1-yl)-2,3-O-isopropylidene-β-D-ribofuranose (4c). The title compound was obtained,
according to the general procedure E and starting from the corresponding azide (500 mg, 2.3
1
mmol) as a violet powder (600 mg) in 60% yield. H NMR (CDCl3, 200 MHz) δ 1.39 (s, 3H),
1.54 (s, 9H), 1.62 (s, 12H), 2.87 (br, 1H), 3.67ꢀ3.85 (m, 2H), 4.52ꢀ4.57 (m, 1H), 5.04 (dd, J 5.8
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and 1.8 Hz, 1H), 5.28ꢀ5.32 (m, 1H), 6.29 (d, J 2.2 Hz, 1H); C{1H} NMR (CDCl3, 50 MHz) δ
25.2, 27.0, 27.9, 28.0, 63.1, 65.9, 81.8, 83.6, 85.2, 85.3, 89.3, 93.7, 94.9, 114.0, 121.7, 143.6,
151.2, 157.9; HRMS (ESI+): calcd for [M+H]+ C22H32O8N3, 466.2184, found 466.2186; HPLC
(λ260) >99% purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-
O-isopropylidene--5-O-methyl-β-D-ribofuranose (4d). The title compound was obtained,
according to the general procedure E and starting from the corresponding azide (456 mg, 1.9
1
mmol) as a brown powder (396 mg) in 49% yield. H NMR (CDCl3, 200 MHz) δ 1.33ꢀ1.46 (m,
9H), 1.60 (s, 3H), 3.22 (s, 3H), 3.39ꢀ3.33 (m, 2H), 4.34 (q, J 7.1 Hz, 2H), 4.40ꢀ4.53 (m, 3H),
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4.98 (dd, J 6.0 and 2.4 Hz, 1H), 5.55 (dd, J 6.0 and 0.9 Hz, 1H), 6.27ꢀ6.34 (m, 1H); C{1H}
NMR (CDCl3, 50 MHz) δ 13.9, 14.0, 25.1, 59.2, 63.0, 68.0, 72.2, 82.3, 84.2, 87.9, 92.9, 93.9,
114.1, 122.0, 143.0, 152.3, 159.1; HRMS (ESI+): calcd for [M+H]+ C19H26O8N3, 424.1714,
found 424.1718; HPLC (λ260) >99% purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-
O-isopropylidene-5-O-acetyl-β-D-ribofuranose (4e). The title compound was obtained,
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according to the general procedure E and starting from the corresponding azide (3.0 g, 11.7
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mmol) as a yellow oil (3.3 g) in 63% yield. H NMR (CDCl3, 200 MHz) δ 1.26ꢀ1.39 (m, 9H),
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1.53 (s, 3H), 1.93 (s, 3H), 3.91ꢀ4.07 (m, 2H), 4.22ꢀ4.51 (m, 5H), 4.96 (dd, J 5.89 and 2.4 Hz,
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1H), 5.57ꢀ5.45 (m, 1H), 6.25 (s, 1H); C{1H} NMR (CDCl3, 50 MHz) δ 13.8, 13.8, 20.4, 26.6,
61.8, 62.8, 63.2, 67.6, 81.7, 83.9, 86.7, 92.2, 93.7, 114.1, 122.0, 142.8, 152.0, 158.8, 170.1;
HRMS (ESI+): calcd for [M+H]+ C20H26O9N3, 452.1664, found 452.1666; HPLC (λ260) 99%
purity.
1-Deoxy-1-(5-(3-methoxy-3-oxoprop-1-yn-1-yl)-4-(methoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
2,3-O-isopropylidene-5-O-acetyl-β-D-ribofuranose (4f). The title compound was obtained,
according to the general procedure E and starting from the corresponding azide (3.0 g, 11.7
mmol) as an orange oil (2.2 g) in 45% yield. 1H NMR (CDCl3, 200 MHz) δ 1.31 (s, 3H), 1.50 (s,
3H), 1.91 (s, 3H), 3.80 (s, 3H), 3.90 (s, 3H), 3.95ꢀ3.98 (m, 2H), 4.46 (dt, J 5.8, 5.7 and 2.4 Hz,
1H), 4.93 (dd, J 5.9 and 2.4 Hz, 1H), 5.50ꢀ5.53 (m, 1H), 6.22 (s, 1H); 13C{1H} NMR (CDCl3, 50
MHz) δ 20.4, 24.9, 26.8, 52.5, 63.1, 67.9, 81.6, 83.8, 86.7, 92.3, 93.4, 114.0, 122.0, 142.5, 152.4,
159.2, 170.0; HRMS (ESI+): calcd for [M+H]+ C18H22O9N3, 424.1351, found 424.1352; HPLC
(λ260) >99% purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-
O-cyclohexylidene-β-D-ribofuranose (4g). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (301 mg, 1.2 mmol) as a brown
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powder (610 mg) in 42% yield. H NMR (CDCl3, 200 MHz) δ 1.33ꢀ1.47 (m, 6H), 1.91ꢀ1.53ꢀ
1.91 (m, 10H), 3.68 (dd, J 12.6 and 4.7 Hz, 1H), 3.83 (dd, J 12.4 and 3.3 Hz, 1H), 4.35 (q, J 7.1
Hz, 2H), 4.47 (q, J 7.2 Hz, 2H), 4.57 (s, 1H), 5.03 (dd, J 5.8 and 1.6 Hz, 1H), 5.32 (dd, J 5.8 and
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2.2 Hz, 1H), 6.31 (d, J 2.1 Hz, 1H); C{1H} NMR (CDCl3, 50 MHz) δ 13.9, 14.0, 23.6, 23.9,
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24.8, 34.6, 36.8, 62.1, 63.1, 63.2, 67.6, 81.4, 84.9, 89.6, 94.1, 115.0, 122.0, 142.8, 152.2, 158.9;
HRMS (ESI+): calcd for [M+H]+ C21H27O8N3, 450.1871, found 450.1872; HPLC (λ260) >99%
purity.
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1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-2,3-
O-(hept-4-ylidene)- β-D-ribofuranose (4h). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (366 mg, 1.3 mmol) as a white
powder (620 mg) in 48% yield. 1H NMR (CDCl3, 200 MHz) δ 0.87ꢀ1.01 (m, 6H), 1.32ꢀ1.81 (m,
14H), 2.87 (m, 1H), 3.60ꢀ3.84 (m, 2H), 4.29ꢀ4.51 (m, 4H), 4.56 (br, 1H), 5.04 (d, J 6.0 Hz, 1H),
5.31ꢀ5.33 (m, 1H), 6.31 (br, 1H); 13C{1H} NMR (CDCl3, 50 MHz) δ 14.0, 14.0, 14.3, 16.7, 17.5,
39.0, 39.1, 62.1, 63.1, 63.2, 67.5, 81.9, 85.5, 89.7, 94.0, 117.9, 122.1, 142.7, 152.2, 158.9;
HRMS (ESI+): calcd for [M+H]+ C22H32O8N3, 466.2184, found 466.2185; HPLC (λ260) >99%
purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
2,3,5-tri-O-acetyl-β-D-ribofuranose (4i). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (3.0 g, 9.8 mmol), as a white
powder (3.1 g) in 63% yield. 1H NMR (CDCl3, 200 MHz) δ 1.36 (t, J 7.1 Hz, 3H), 1.42 (t, J 7.1
Hz, 3H), 2.04 (s, 3H), 2.13 (s, 6H), 4.14 (dd, J 12.3 and 4.2 Hz, 1H), 4.29ꢀ4.50 (m, 6H), 5.72 (t,
J 5.5 Hz, 1H), 6.05 (dd, J 5.5 and 3.5 Hz, 1H), 6.21 (d, J 3.45 Hz, 1H); 13C{1H} NMR (CDCl3,
50 MHz) δ 3.9, 14.0, 20.4, 20.5, 20.6, 62.0, 62.4, 63.1, 67.6, 70.7, 73.7, 81.6, 89.1, 94.0, 122.2,
143.0, 152.3, 158.9, 169.1, 169.4, 170.5; HRMS (ESI+): calcd for [M+H]+ C21H26O11N3,
496.1562, found 496.1564; HPLC (λ260) >99% purity.
1-Deoxy-1-(5-(3-tert-butoxy-3-oxoprop-1-yn-1-yl)-4-(tert-butoxycarbonyl)-1H-1,2,3-triazol-
1-yl)-2,3,5-O-tri-acetyl-β-D-ribofuranose (4j). The title compound was obtained, according to
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the general procedure E and starting from the corresponding azide (679 mg, 13.3 mmol) as a pale
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violet powder (264 mg) in 21% yield. H NMR (CDCl3, 200 MHz) δ 1.51 (s, 9H), 1.59 (s, 9H),
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2.02 (s, 3H), 2.11 (s, 6H), 4.06ꢀ4.16 (m, 1H), 4.35ꢀ4.46 (m, 2H), 5.70 (t, J 5.5 Hz, 1H), 5.99ꢀ6.03
(m, 1H), 6.18 (d, J 3.0 Hz, 1H); 13C{1H} NMR (CDCl3, 50 MHz) δ 20.3, 20.4, 20.6, 27.8, 28.0,
62.4, 66.0, 70.6, 73.7, 81.4, 83.4, 85.2, 89.0, 94.7, 121.7, 143.7, 151.2, 157.8, 169.1, 169.3,
170.4; HRMS (ESI+): calcd for [M+H]+ C25H34O11N3, 552.2188, found 552.2189; HPLC (λ260)
98% purity.
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Ethyl 1-(2,3,5-tri-O-methyl-β-D-ribofuranosyl)-5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-1H-1,2,3-
triazole-4-carboxylate (4k). The title compound was obtained, according to the general
procedure E and starting from the corresponding azide (217 mg, 1.0 mmol) as a brown powder
1
(160 mg) in 40% yield. H NMR (CDCl3, 200 MHz) δ 1.35 (t, J 7.1 Hz, 3H), 1.41 (t, J 7.1 Hz,
3H), 3.31 (s, 3H), 3.49 (s, 3H), 3.50 (s, 3H), 3.53 (d, J 2.2 Hz, 1H), 3.55 (d, J 1.2 Hz, 1H), 4.21
(t, J 5.2 Hz, 1H), 4.27ꢀ4.50 (m, 5H), 4.63 (dd, J 4.9 and 3.6 Hz, 1H), 6.17 (d, J 3.6 Hz, 1H);
13C{1H} NMR (CDCl3, 50 MHz) δ 13.9, 14.0, 58.4, 58.9, 59.4, 61.9, 63.0, 68.0, 72.5, 79.4, 82.1,
82.7, 89.6, 93.8, 122.2, 142.8, 152.2, 159.1; HRMS (ESI+): calcd for [M+H]+ C18H26O8N3,
412.1714, found 412.1716; HPLC (λ260) 99% purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (4l). The title compound was obtained, according to
the general procedure E and starting from the corresponding azide (300 mg, 0.8 mmol) as a
brown powder (326 mg) in 72% yield. 1H NMR (CDCl3, 200 MHz) δ 1.33ꢀ1.44 (m, 6H), 1.85 (s,
3H), 2.01ꢀ2.04 (m, 9H), 4.01 (ddd, J 9.7, 4.7 and 2.4 Hz, 1H) 4.13ꢀ4.27 (m, 3H), 4.30ꢀ4.48 (m,
4H), 5.27 (t, J 9.7 Hz, 1H), 5.41 (t, J 9.2 Hz, 1H), 5.78ꢀ5.94 (m, 2H); 13C{1H} NMR (CDCl3, 50
MHz) δ 13.9, 13.9, 20.0, 20.4, 26.8, 30.0, 61.4, 62.0, 63.0, 67.3, 67.8, 69.2, 72.5, 75.2, 85.8,
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94.3, 121.8, 143.2, 152.1, 158.8, 168.5, 169.0, 169.9, 170.4; HRMS (ESI+): calcd for [M+H]+
C24H30O13N3, 568.1773, found 568.1775; HPLC (λ260) 99% purity.
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Ethyl
5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-1-((2R,3aR,4R,6R,6aR)-6-(hydroxymethyl)-2-
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methyl-2-phenyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1H-1,2,3-triazole-4-carboxylate
(4m). The title compound was obtained, according to the general procedure E and starting from
the corresponding azide (529 mg, 1.8 mmol) as a white powder (402 mg) in 47% yield. 1H NMR
(CDCl3, 200 MHz) δ 1.35ꢀ1.46 (m, 6H), 1.68 (s, 3H), 2.95ꢀ3.02 (m, 1H), 3.62ꢀ3.85 (m, 2H),
4.31ꢀ4.50 (m, 5H), 5.22 (dd, J 6.0 and 2.0 Hz, 1H), 5.44 (dd, J 6.0 and 2.4 Hz, 1H), 6.15 (d, J 2.4
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Hz, 1H), 7.34ꢀ7.45 (m, 3H), 7.57ꢀ7.62 (m, 2H); C{1H} NMR (CDCl3, 50 MHz) δ 13.9, 14.0,
28.2, 62.1, 63.0, 63.2, 67.4, 82.3, 86.1, 88.8, 93.0, 94.0, 114.2, 121.9, 124.9, 128.4, 128.5, 142.5,
152.1, 158.9; HRMS (ESI+): calcd for [M+H]+ C23H26O8N3, 472.1714, found 472.1717; HPLC
(λ260) 98% purity.
1,2-Dideoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
3,5-di-O-acetyl-β-D-ribofuranose (4n). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (630 mg, 1.8 mmol) as a brown
1
oil (561 mg) in 48% yield. H NMR (CDCl3, 200 MHz) δ 1.31ꢀ1.44 (m, 6H), 2.03 (s, 3H), 2.12
(s, 3H), 2.57ꢀ2.69 (m, 1H), 3.38ꢀ3.51 (m, 1H), 4.07ꢀ4.14 (m, 1H), 4.28ꢀ4.49 (m, 6H), 5.53 (s,
1H), 6.45 (t, J 6.2 Hz, 1H); 13C{1H} NMR (CDCl3, 50 MHz) δ 13.9, 14.0, 20.6, 20.8, 36.5, 61.9,
63.0, 63.1, 68.1, 74.2, 84.0, 87.6, 93.7, 122.0, 143.1, 152.3, 159.1, 170.2, 170.4; HRMS (ESI+):
calcd for [M+H]+ C19H24O9N3, 438.1507, found 438.1508; HPLC (λ260) >99% purity.
1-Deoxy-1-(5-(3-ethoxy-3-oxoprop-1-yn-1-yl)-4-(ethoxycarbonyl)-1H-1,2,3-triazol-1-yl)-
2,3,5-tri-O-acetyl-β-D-xylofuranose (4o). The title compound was obtained, according to the
general procedure E and starting from the corresponding azide (222 mg, 0.7 mmol) as a white
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