2-Aryl-8-aza-3-deazaadenosine analogues of 5′-O-[N-(salicyl)sulfamoyl]adenosine: Nucleoside antibiotics that block siderophore biosynthesis in Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2016.05.037

A series of 5′-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deaz

Full text of DOI:10.1016/j.bmc.2016.05.037

Accepted Manuscript
2-Aryl-8-aza-3-deazaadenosine Analogues of 5’-O-[N-(Salicyl)sulfamoyl]ade-
nosine: Nucleoside Antibiotics that Block Siderophore Biosynthesis in Myco-
bacterium tuberculosis
Anna Krajczyk, Joanna Zeidler, Piotr Januszczyk, Surendra Dawadi, Helena I.
Boshoff, Clifton E. Barry III, Tomasz Ostrowski, Courtney C. Aldrich
PII:
S0968-0896(16)30368-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.037
BMC 13021
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2016
17 May 2016
19 May 2016
Please cite this article as: Krajczyk, A., Zeidler, J., Januszczyk, P., Dawadi, S., Boshoff, H.I., Barry, C.E. III,
Ostrowski, T., Aldrich, C.C., 2-Aryl-8-aza-3-deazaadenosine Analogues of 5’-O-[N-(Salicyl)sulfamoyl]adenosine:
Nucleoside Antibiotics that Block Siderophore Biosynthesis in Mycobacterium tuberculosis, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.037
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2-Aryl-8-aza-3-deazaadenosine Analogues of 5’-O-[N-
(Salicyl)sulfamoyl]adenosine: Nucleoside Antibiotics that Block
Siderophore Biosynthesis in Mycobacterium tuberculosis
Anna Krajczyk a, Joanna Zeidler a, Piotr Januszczyk a, Surendra Dawadi b,
Helena I. Boshoff c, Clifton E. Barry, III c, Tomasz Ostrowski a,*, Courtney C. Aldrich b,*
a Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704
Poznan, Poland
b
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455,
USA
c
Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases,
Bethesda, MD 20892, USA
* Corresponding authors.
Tel.: +1 612 625 7956; fax: +1 612 626 3114; E-mail address: aldri015@umn.edu
(C.C. Aldrich);
Tel.: +48 61 8528503; fax: +48 61 8520532; E-mail address: tostr@ibch.poznan.pl
(T. Ostrowski).
ABSTRACT
A series of 5’-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to
block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the
essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deazaadenosine
nucleosides featured sequential copper-free palladium-catalyzed Sonogashira coupling of a
precursor 4-cyano-5-iodo-1,2,3-triazolonucleoside with terminal alkynes and Minakawa-
Matsuda annulation reaction. These modified nucleosides were shown to inhibit MbtA with
apparent Ki values ranging from 6.1 to 25 nM and to inhibit Mtb growth under iron-deficient
conditions with minimum inhibitory concentrations ranging from 12.5 to >50 µM.
Keywords: 8-Aza-3-deazaadenosine; Modified Nucleoside; Tuberculosis; Siderophore
Biosynthesis, Adenylation Inhibitor.
1

1. Introduction
Tuberculosis (TB) is an infectious disease primarily caused by the bacterium
Mycobacterium tuberculosis (Mtb) that recently surpassed HIV as the leading cause of
infectious disease mortality [1]. Mtb is easily spread from an actively infected individual by
the aerosol route. Upon inhalation Mtb quickly replicates and becomes encased in
granulomatous lesions in the lungs. A majority of healthy individuals infected with Mtb are
asymptomatic and able to effectively contain, but not clear, the bacteria in these granulomas.
Latently infected individuals provide a vast reservoir of the disease and include nearly one-
third of the global population. However, individuals – who have an impaired immune system
from aging, malnutrition, or co-infection with HIV – are much more likely to develop active
TB or reactivate a latent infection. Active TB infections ensue when the granulomas rupture,
releasing the bacteria into the sputum leading to the clinical manifestations of hemoptysis and
cachexia. Treatment of simple drug-susceptible TB is very challenging, compared to most
bacterial infections, requiring 6–9 months of a four-drug regimen comprised of isoniazid,
rifampicin, ethambutol, and pyrazinamide [2]. The underlying cause of the persistence and
drug tolerance of Mtb in vivo that necessitates this long treatment course is still not fully
understood, but is likely multifactorial resulting from variability in the lesion environment and
phenotype of individual bacteria [3, 4]. Considering the unique challenges posed by drug
susceptible TB, the emergence and dissemination of multidrug resistance TB (MDR-TB) and
extensively drug resistant TB (XDR-TB), which are minimally resistant to the two most
effective antitubercular agents – isoniazid and rifampicin, is a global health crisis [5]. The
European region alone accounts for more than a quarter of the global MDR-TB burden [6]. In
order to bring TB back under control, a unified effort will be required to develop improved
diagnostics, effective vaccines, and new anti-tubercular agents with novel modes of action.
Mtb requires iron, a trace micronutrient that is highly restricted in a mammalian host, to
establish and maintain an infection. To circumvent the nutritional immunity imposed by the
host, Mtb synthesizes and secretes a family of small-molecule iron chelators or siderophores
known as the mycobactins that extract iron (Fe3+) from host proteins [7]. The Fe3+-mycobactin
complex is then imported back into the bacterium through a specialized transport system and
the iron is reductively released from the siderophore. The mycobactins share a common
biosynthetic pathway for construction of the conserved peptidic core using a mixed non-
ribosomal peptide synthetase–polyketide synthase (NRPS-PKS) assembly line of six proteins
designated MbtA through MbtF [8]. The nucleoside derivative 5’-O-[N-
2

(salicyl)sulfamoyl]adenosine (Sal-AMS, 2, Fig. 1) is a potent nanomolar inhibitor of MbtA,
which catalyzes the first committed step in mycobactin synthesis, through the activation of
salicylic acid to the mixed anhydride salicyl-AMP intermediate (1) and subsequent loading
onto MbtB [9-11]. Sal-AMS blocks synthesis of the mycobactins and correspondingly inhibits
growth of Mtb under iron-deficient conditions in vitro and in vivo [12]. Comprehensive SAR
studies of 2 demonstrated the N-(salicyl)sulfamoyl moiety is essential for activity and only
conservative substitutions to the salicyl ring were permitted [13]. By contrast, the nucleoside
is substantially more tolerant to modification with addition of nonpolar aromatic substituents
at C-2 exemplified by 2-phenyl-Sal-AMS (3) providing the most potent Sal-AMS derivatives
[14]. These studies also showed the N-3 nitrogen atom of adenosine is dispensable for activity
[14].
To extend the SAR investigation of base-modified Sal-AMS analogues we describe herein
the synthesis and evaluation of a series of compounds bearing the isosteric 8-aza-3-
deazaadenine nucleobase with various 2-aryl substituents (6a–f, Fig. 2) that uniquely
capitalize on the existing SAR. The 8-aza-3-deazaadenine nucleosides have several useful
attributes over adenosine and other modified purine nucleosides as elaborated below including
stability to cyclonucleoside degradation, lack of intrinsic biological activity, and improved
fluorescent properties. Purine nucleoside analogues containing an activated 5’-leaving groups
like a sulfamate as found in Sal-AMS are prone to cyclonucleoside formation through attack
of the N-3 purine atom onto the C-5’ ribose to afford a 3,5’-cyclonucleoside [15]. This
undesired degradation pathway is prevented in 8-aza-3-deazaadenines due to lack of a N-3
atom. As first reported by Franchetti, 8-aza-3-deazaadenosine is devoid of biological activity
suggesting it binds poorly to ATP-utilizing proteins, a feature that we exploit to minimize
potential off-target toxicity [16].
Figure 1. Structures of Sal-AMP (1), Sal-AMS (2) and 2-phenyl analog of Sal-AMS (3).
3

NH2
N
O
N
N
N
N
NH
NH2
N
N
HO
HO
O
O
OH OH
OH OH
4
5
NH2
N
N
OH
O
N
O
O
S
N
R
N
O
O
Et3NH
OH OH
6a, R = phenyl
6b, R = 4-methylphenyl
6c, R = 4-fluorophenyl
6d, R = 4-trifluoromethylphenyl
6e, R = naphthyl
6f, R = 6-methoxynaphthyl
Figure 2. Structures of 8-aza-3-deazaadenosine (4), 8-aza-3-deazaguanosine (5), and 2-aryl-
8-aza-3-deazaadenine analogs of Sal-AMS (6a–f).
2. Results and discussion
2.1 Chemistry
There are two general synthetic approaches for the preparation of 8-aza-3-deazaadenosine
(4) and the homologous guanosine derivative (5) that have been reported in the literature: (1)
glycosylation of a protected sugar with a triazolo[4,5-c]pyridine heterocycle and (2)
annulation onto a 1H-[1,2,3]triazole-1-β-D-ribofuranoside derivative generated from the
glycosylation of the corresponding aglycone, or the 1,3-dipolar cycloaddition of a
ribofuranosyl azide to an appropriate dipolarophile, or the Dimroth reaction. The
glycosylation strategy suffers from poor selectivity resulting in a mixture of N(1), N(2) and
N(3) regiosomeric products [16,17] while the latter procedure involving elaboration of a pre-
formed triazole does not allow facile incorporation of C-6 aryl substituents [18-22]. We
elected to adapt methodology first reported by Minakawa and Matsuda for the construction of
3-deazapurine nucleosides involving nucleophilic cyclization of 4-carbamoyl- or 4-cyano-
substitued derivatives of 5-ethynyl-1-β-D-ribofuranosylimidazole [23,24]. We were inspired
by the successful application of this method for the preparation of related carbocyclic 8-aza-3-
deazainosine derivatives reported by Agrofoglio and co-workers [25]. Herein we present the
synthesis of protected 5-alkynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carbonitriles and
their ring closure leading to the corresponding 2-aryl-8-aza-3-deazaadenosine analogues using
a modified Minakawa-Matsuda annulation strategy.
Synthesis of the triazole nucleoside building block 9 was accomplished as shown in
Scheme 1 starting from 5-amino-4-carboxamide-1,2,3-triazole nucleoside (7) prepared from
4

2,3-O-isopropylidene-β-D-ribofuranosyl azide as reported through Dimroth reaction with
cyanoacetamide [26-28]. Dehydration of the amide in 7 to the respective nitrile 8 was
accomplished by treatment with 4-toluenesulfonyl chloride [28]. Subsequent diazotization-
iodination of 8 with diiodomethane and isoamyl nitrite afforded 9 in 75% over two steps,
which was superior the inverse reaction sequence via 10 that provided a lower 41% overall
yield.
CN
N
N
N
NH2
O
AcO
O
CN
CN
I
N
N
N
N
N
N
NH2
NH2
i
ii
i
N
N
N
O
O
8
AcO
AcO
AcO
O
O
O
O
O
O
12
O
O
O
O
7
ii
9
N
N
NH2
N
I
AcO
O
OCH3
N
O
O
iii
N
10
N
N
Ph
HO
O
NH2
N
NH2
CN
C
iv
N
O
O
N
N
N
N
N
N
13
N
N
vi
v
N
AcO
C
O
R
R
AcO
HO
R
O
O
O
O
11a-f
a, R = phenyl
b, R = 4-methylphenyl
c, R = 4-fluorophenyl
15a-f
14a-f
O
O
O
O
d, R = 4-trifluoromethylphenyl
e, R = naphthyl
f, R = 6-methoxynaphthyl
vii
NH2
N
NH2
N
N
N
N
N
N
OH
O
O
O
S
O
O
N
S
viii
ix
R
N
H
O
R
H2N
O
6a-f
O
O
O
O
O
O
17a-f
16a-f
Scheme 1. Synthesis of compounds 6a–f. Reagents and conditions: (i) Pyr, TsCl, rt; (ii)
CH2I2, isoamyl nitrite, 100 ºC; (iii) alkyne, K2CO3, (PhCN)2PdCl2, 1,4-dioxane, H2O, 55–65
o
ºC; (iv) NH3–MeOH, 80 C; (v) NH3–DME, 85 ºC; (vi) MeONa–MeOH, 5 ºC, then Dowex
50WX8-100; (vii) NH2SO2Cl, MeCN, DMA, rt; (viii) salicylic acid, CDI, MeCN, 60 °C, then
substrate, DBU, rt; (ix) 4:1 TFA–H2O, 5 °C.
The Sonogashira coupling of 9 was first optimized with phenylacetylene. We explored a
wide variety of conditions, catalysts, and additives (Supplementary data, Table S1) [29].
Optimal conditions were found using bis(benzonitrile)palladium(II) chloride as reported by
Minakawa and co-workers [30], but employing K2CO3 as base in 1,4-dioxane with 10
equivalents of H2O at 60 ºC to afford 11a in 69% yield. These conditions minimized
formation of homocoupled products (not shown) as well as hydrodehalogenated 12 and were
used for the other terminal alkynes to afford products 11b–f in 43–77% yield. The requisite
5

terminal alkynes, except phenylacetylene and 1-ethynyl-4-methylbenzene, were prepared by
deprotection [31] of their trimethylsilyl precursors obtained using previously reported
procedures [31,32] (Supplementary data).
We undertook the key Minakawa–Matsuda annulation of 11a using MeOH saturated with
NH3, 80 °C [23,24]. However, we obtained the 4-methoxy-6-phenyl cyclized product 13 (68%
yield) due to competitive reaction with methanol. Thus, we next examined non-nucleophilic
solvents including 1,4-dioxane, THF, and 1,2-dimethoxyethane (DME). Using 1,4-dioxane
saturated with NH3, we obtained at 120 °C an encouraging 29% yield of the desired product
14a along with 61% unreacted 11a. The yield of 14a was further improved to 58% (along
with 32% recovered 11a) employing THF saturated with NH3. We attributed the improved
yield to the greater solubility of ammonia in THF. Based on this hypothesis, we saturated
DME with ammonia at -60 ºC then added 11a and heated at 85°C to obtain 14a in an
impressive 95% yield. This optimized method was then successfully used for the synthesis of
compounds 14b–f.
Methanolysis of compounds 14a–f afforded their corresponding 5’-deacetylated
congeners 15a–f, which were then converted to 5’-O-sulfamoyl derivatives 16a–f by
treatment with sulfamoyl chloride in MeCN–DMA [33]. These sulfamates 16a–f were
coupled to salicylic acid using CDI activation and DBU as a base in MeCN to provide 17a–f
[13]. Deprotection of the isopropylidene acetal was accomplished with 80% aqueous TFA and
the final products 6a–f were isolated as the triethylammonium salts following silica gel
chromatography with 0.5% triethylamine as eluent, which were all greater than 99% pure as
determined by HPLC.
We determined the fluorescence properties of compounds 6a–f in MeOH and in aqueous
solutions (Table 1). The emission maxima measured for all compounds in MeOH were in the
range λmax 409-420 nm (Stokes shifts 95.5-111.5 nm), and in H2O in the range λmax 420-442
nm (Stokes shifts 106.5-137.5 nm). These results are comparable with data given previously
for 8-aza-3-deazaadenosine (4) in H2O (λmax 430 nm, Stokes shift 140 nm) [17]. Compounds
6a and 6d–f in MeOH exhibited relatively high values of fluorescence quantum yield (ΦF =
0.32-0.5, compared with ΦF = 1 assumed for 2-aminopurine), while compounds 6b and 6c
were less fluorescent (ΦF = 0.06 and 0.11). In contrast, the quantum yield values of 6a–f in
H2O were decreased 30- to 250-fold.
6

Table 1. Fluorescence properties of compounds 6a–f.
Fluorescence emissiona
(MeOH)
Fluorescence emissiona
(H2O)
Compd
b
b
λmax (nm)
419
ΦF
λmax (nm)
433
ΦF
0.37
0.06
0.11
0.32
0.38
0.5
0.0015
0.0018
0.002
6a
6b
6c
6d
6e
6f
412
433
420
442
409
422
0.0019
0.0025
0.007
412
420
418
425
a Excitation at 305 nm. b Fluorescence quantum yields calculated relative to 2-aminopurine (ΦF = 1).
2.2. Enzyme inhibition and antitubercular activity
Inhibitors 6a–f were evaluated for enzyme inhibition against recombinant MbtA under
initial velocity conditions as previously described (see Experimental Section) [34]. The
apparent inhibition constants (appKi) were determined by fitting the concentration–response
plots to the Morrison equation (eq 1, see Experimental Section) since all compounds exhibited
tight-binding behavior. The appKi values ranged from 6.1 to 25 nM (Table 2). The first
analogue in the series 6a was 31-fold less potent than the isosteric analogue 2-phenyl-Sal-
AMS (3) demonstrating simultaneous deletion of the N-3 atom and introduction of an N-8
atom in 6a was not well tolerated. Nonetheless, 6a is still an exceptionally potent compound
and the appKi vastly underestimates the true potency since the assay was performed using
supersaturating concentrations of all substrates. Introduction of p-methyl, p-fluoro, and p-
trifluoromethyl in 6b, 6c, and 6d, respectively, had a relatively negligible impact on potency
and these analogues were only 2–3 fold less potent than 6a. Introduction of the 2-naphthyl
substituent in 6e provided the most potent 8-aza-3-deazaadenine analogue with an appKi of
6.1 nM commensurate with 2 while further modification of the naphthyl moiety with a 6-
methoxy substituent in 6f was not beneficial leading to a 2-fold loss of potency relative to 6e.
7

Table 2. Enzyme inhibition and antimycobacterial activity of 6a–f.
NH2
N
OH
O
N
O
O
S
N
N
R
N
O
O
Et3NH
OH OH
Inhibitor
R
appKi (nM)a
6.6 ± 1.5d
0.27 ± 0.07d
8.4 ± 1.0
MIC (µM)b
0.39d
0.049d
25
nac
nac
2
3
6a
6b
6c
6d
6e
6f
16.9 ± 1.9
13.7 ± 2.3
25.0 ± 1.9
6.1 ± 0.7
25
19
F
>50
CF3
12.5
19
10.5 ± 1.2
OMe
b
aAssay performed with 7 nM MbtA, 10 mM ATP, 250 µM salicylic acid, 1 mM PPi. M. tuberculosis H37Rv
c
grown in glycerol-alanine salts (GAS) medium without ferric ammonium citrate at pH 6.6. not applicable.
dreference 14.
Next, we evaluated these analogs against whole-cell M. tuberculosis H37Rv under iron-
deficient and iron-replete conditions as previously described [11]. These antibacterial agents
operate by a unique mechanism of action and target iron acquisition by inhibition of
siderophore biosynthesis. Under iron-replete conditions, none of the compounds displayed
any activity at the maximum concentration evaluated (50 µM) against whole-cell M.
tuberculosis H37Rv consistent with their designed mechanism of action. Activity was
revealed only under iron-deficient conditions. The minimum inhibitory concentrations (MIC)
that resulted in complete inhibition of observable growth under iron-deficient conditions are
shown in Table 1. The antitubercular activity of 6a–f excluding 6d ranged between 12.5 and
25 µM, consistent with their relatively flat biochemical SAR. Compared to the lead
compound 2, all of the compounds showed considerable loss in whole-cell activity despite
displaying similar biochemical potency. These results suggest the nucleoside modification
adversely impacted cellular accumulation.
3. Conclusion
8

The series of 2-aryl-8-aza-3-deazaadenine nucleobase analouges of 5’-O-[N-
(salicyl)sulfamoyl]adenosine (2) were synthesized as siderophore inhibitors based on prior
SAR studies and evaluated for biochemical activity against MbtA and whole-cell activity
under iron-deficient conditions with M. tuberculosis H37Rv. We developed an optimized
route to the 2-aryl-8-aza-3-deazaadenosine nucleosides using a modified Minakawa–Matsuda
annulation strategy that was highly efficient and enabled facile introduction of C-2 aryl
substituents. The SAR studies revealed concurrent introduction of an N-8 atom and deletion
of the N-3 atom in the purine base was detrimental to biochemical potency and whole-cell
activity. Despite these shortcomings, the compounds showed high chemical stability and
promising fluorescent properties that could be useful to study mycobacterial accumulation and
localization.
4. Experimental section
4.1. General chemistry methods
Melting points were determined on MEL-TEMP II capillary melting point apparatus and
1
are uncorrected. H and 13C NMR spectra were recorded on a Bruker 400 spectrometer
operating at 400.1 MHz and 100.6 MHz, respectively, or on Unity 300 Varian spectrometer
operating at 300 MHz and 75.4 MHz, respectively. 19F NMR spectra were recorded on a
Bruker 400 spectrometer at 376.4 MHz. The chemical shifts are reported in ppm (δ scale).
Mass spectra were recorded using ESI-MS Thermo Q Exactive and Bruker micrOTOF-Q
mass spectrometers. UV spectra were measured with Beckman Coulter DU 640
spectrophotometer. Fluorescence spectra were measured on a Shimadzu RF-5301 PC
fluorescence spectrophotometer (excitation at 305 nm); quantum yields were calculated
relative to 2-aminopurine (ΦF = 1) Microwave heating was performed with Ertec-Poland MW
reactor. Thin-layer chromatography (TLC) was carried out on Merck precoated 60 F254 silica
gel plates, while column chromatography on Merck silica gel 60H (40-63 μm). Anhydrous
1,4-dioxane, tetrahydrofuran (THF) and 1,2-dimethoxyethane (DME) were prepared by
stirring with iron(II) sulfate heptahydrate at room temperature, followed by drying with KOH,
distillation with calcium hydride (CaH2) and distillation with sodium/benzophenone. Other
anhydrous solvents were prepared as follows: MeOH by treatment with magnesium
turnings/iodine and distillation, MeCN and CH2Cl2 by distillation with P2O5, pyridine by
drying with KOH and distillation with P2O5, dimethylformamide (DMF) by drying with CaH2
9

and distillation, triethylamine by distillation with CaH2. Anhydrous dimethylacetamide
(DMA) was purchased from Acros. MeOH (Acros) and MeCN (J.T.Baker) used for UV and
fluorescence spectra were of HPLC grade.
4.2. Chemical synthesis
4.2.1. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-amino-1H-[1,2,3]triazole-4-
carbonitrile (8)
4-Toluenesulfonyl chloride (4.437 g, 22.81 mmol) was added to a solution of 7 [11]
(3.975 g, 11.64 mmol) in dry pyridine (55 ml). After being stirred at room temperature for 24
h, the reaction mixture was treated with 5% aq NaHCO3 (100 ml) at 0 ºC and with CH2Cl2
(400 ml). The organic layer was separated, while the aqueous one was extracted again with
CH2Cl2 (300 ml). Combined organic layers were dried with MgSO4 and evaporated. The
residue was chromatographed on a silica gel column with EtOAc-hexane (1:2→2:1) as an
1
eluent to afford solid 8 (3.398 g, 90% yield). UV (MeOH) λmax = 247.5 nm (5900). H NMR
(DMSO-d6) δ 7.42 (s, 2H, NH2), 6.22 (d, J1’,2’=0.8 Hz, 1H, 1’-H), 5.46 (dd, J2’,1’=0.8 Hz,
J2’,3’=5.8 Hz, 1H, 2’-H), 4.96 (dd, J3’,2’=5.8 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.37 (m, 1H, 4’-H),
4.02 (dd, J5’a,5’b=11.6 Hz, J5’a,4’=6.0 Hz, 1H, 5’a-H) 3.85 (dd, J5’b,5’a=11.6 Hz, J5’b,4’=7.2 Hz,
1H, 5’-H), 1.97 (s, 3H, OAc), 1.50 and 1.34 (2×s, 6H, 2×CH3). 13C NMR (DMSO-d6) δ
169.90 (CO), 148.51 (C-5), 113.25 ((CH3)2C), 112.81 (CN), 101.19 (C-4), 88.46 (C-1’), 85.28
(C-4’), 83.33 (C-2’), 81.48 (C-3’), 63.41 (C-5’), 26.61 and 24.94 ((CH3)2C), 20.42 (CO-CH3).
4.2.2. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-iodo-1H-[1,2,3]triazole-4-
carbonitrile (9)
To a suspension of 8 (628 mg, 1.94 mmol) in diiodomethane (31.18 g, 116.4 mmol)
isoamyl nitrite (797 mg, 6.8 mmol) was added. The resulting mixture was stirred at 100 ºC for
2.5 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 (9:1). Isolated crude product was
purified on a next silica gel column using EtOAc-hexane (1:2→2:1) to give 9 as a solid (704
mg, 83% yield). UV (MeOH) λmax 239 nm (5400). 1H NMR (DMSO-d6) δ 6.22 (s, 1H, 1’-H),
5.67 (d, J2’,3’=6.0 Hz, 1H, 2’-H), 5.04 (dd, J3’,2’=5.8 Hz, J3’,4’=1.8 Hz, 1H, 3’-H), 4.49 (m, 1H,
4’-H), 3.92 (dd, J5’a,5’b=12.0 Hz, J5’a,4’=5.2 Hz, 1H, 5’-H), 3.82 (dd, J5’b,5’a=11.8 Hz, J5’b, 4’=7.4
13
Hz, 1H, 5’-H), 1.91 (s, 3H, OAc), 1.53 and 1.36 (2×s, 6H, 2×CH3). C NMR (DMSO-d6) δ
169.72 (CO), 128.46 (C-4), 112.93 ((CH3)2C), 112.04 (CN), 95.35 (C-5), 93.75 (C-1’), 86.23
10

(C-4’), 83.51 (C-2’), 81.16 (C-3’), 62.86 (C-5’), 26.58 and 24.88 ((CH3)2C), 20.36 (CO-CH3).
HRMS [M+H]+ calcd for C13H16IN4O5: 435.0160; found: 435.0156.
4.2.3. General procedure for the coupling of 9 with terminal alkynes
A solution of 9 (dried in vacuo over P2O5 at 50 ºC) and bis(benzonitrile)palladium(II)
chloride (0.1 equiv) in anhyd 1,4-dioxane (20 ml/mmol) was vigorously deoxygenated with
argon for 15 min. To the solution was added K2CO3 (1.0-1.3 equiv), water (10 equiv) and
alkyne (3 equiv). The reaction mixture was heated at 55-65 ºC overnight, then evaporated.
The residue was chromatographed on silica gel column using toluene-EtOAc (98:2→95:5 or
95:5→9:1) to give a crude product. It was subsequently rechromatographed on next silica gel
column using hexane-EtOAc (9:1 or 9:1→4:1) to isolate pure 11a–f.
4.2.4.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-phenylethynyl-1H-
[1,2,3]triazole-4-carbonitrile (11a)
1
Solid foam, 69% yield. UV (MeCN): λmax 288.5 nm (14500), 306 nm (12600). H NMR
(DMSO-d6) δ 7.74 (m, 2H, Ph), 7.62-7.52 (m, 3H, Ph), 6.48 (s, 1H, 1’-H), 5.64 (d, J2’,3’=5.8
Hz, 1H, 2’-H), 5.04 (dd, J3’,2’=5.8 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.56 (m, 1H, 4’-H), 4.00 (m,
2H, 5’-H), 1.90 (s, 3H, OAc), 1.54 and 1.37 (2×s, 6H, 2×CH3). 13C NMR (DMSO-d6) δ
169.78 (CO), 132.12, 131.29 and 129.16 (Ph), 127.65 and 122.64 (C-4, C-5), 118.98 (Ph),
113.05 ((CH3)2C), 110.99 (CN), 105.54 (C≡CPh), 93.01 (C-1’), 86.30 (C-4’), 83.80 (C-2’),
81.14 (C-3’), 70.40 (C≡CPh), 63.13 (C-5’), 26.57 and 24.87 ((CH3)2C), 20.29 (CO-CH3).
HRMS [M+H]+ calcd for C21H21N4O5: 409.15120; found: 409.15039.
4.2.5. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(4-methylphenylethynyl)-1H-
[1,2,3]triazole-4-carbonitrile (11b)
1
Oily material, 77 % yield. UV (MeOH): λmax 296 nm (18500), 312 nm (16100). H NMR
(DMSO-d6) δ 7.62 (d, J=8.0 Hz, 2H, Ph), 7.36 (d, J=8.0 Hz, 2H, Ph), 6.45 (s, 1H, 1’-H), 5.63
(d, J2’,3’=6.0 Hz, 1H, 2’-H), 5.04 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.56 (m, 1H, 4’-
H), 4.02 (dd, J5’a,4’=5.0 Hz, J5’a,5’b=11.8 Hz, 1H, 5’a-H), 3.96 (dd, J5’b,4’=6.8 Hz, J5’b,5’a=12.0
Hz, 1H, 5’b), 2.38 (s, 3H, Ph-CH3), 1.90 (s, 3H, OAc), 1.53 and 1.37 (2×s, 6H, C(CH3)2). 13C
NMR (DMSO-d6) δ 169.77 (CO), 141.62 (C-4Ph), 132.06 (Ph) 129.76 (Ph), 127.81 and 122.45
(C-4, C-5), 115.95 (Ph), 113.04 ((CH3)2C), 111.03 (CN), 106.01 (C≡CPh), 92.89 (C-1’),
86.28 (C-4’), 83.80 (C-2’), 81.14 (C-3’), 69.99 (C≡CPh), 63.12 (C-5’), 26.56 and 24.86
11

((CH3)2C), 21.22 (Ph-CH3), 20.28 (CO-CH3). HRMS [M+H]+ calcd for C22H23N4O5:
423.16685; found: 423.16635.
4.2.6. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(4-fluorophenylethynyl)-1H-
[1,2,3]triazole-4-carbonitrile (11c)
Oily material, 43% yield. UV (MeOH): λmax 290 nm (16900), 306.5 nm (14200). 1H NMR
(DMSO-d6) δ 7.82 (m, 2H, Ph), 7.40 (m, 2H, Ph), 6.49 (s, 1H, 1’-H), 5.63 (d, J2’,3’=5.6 Hz,
1H, 2’-H), 5.04 (dd, J3’,2’=5.8 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.56 (m, 1H, 4’-H), 3.99 (m, 2H,
13
5’-H), 1.90 (s, 3H, OAc), 1.53 and 1.37 (2×s, 6H, C(CH3)2). C NMR (DMSO-d6) δ 169.79
(CO), 163.45 (d, JC,F=251.84 Hz, C-4Ph), 134.92 (d, JC,F=8.83 Hz, C-2Ph, C-6Ph), 127.56 and
122.63 (C-4, C-5), 116.61 (d, JC,F=22.74 Hz, C-3Ph, C-5Ph), 115.51 (C-1Ph), 113.07 ((CH3)2C),
110.97 (CN), 104.57 (C≡CPh), 92.99 (C-1’), 86.32 (C-4’), 83.83 (C-2’), 81.15 (C-3’), 70.26
(C≡CPh), 63.14 (C-5’), 26.57 and 24.88 ((CH3)2C), 20.29 (CO-CH3). 19F NMR (DMSO d6) δ
-30.69. HRMS [M+H]+ calcd for C21H20N4O5F: 427.14177; found: 427.14114.
4.2.7.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(4-
trifluoromethylphenylethynyl)-1H-[1,2,3]triazole-4-carbonitrile (11d)
1
Oily material, 69 % yield. UV (MeOH): λmax 287.5 nm (22600), 306 nm (17800). H
NMR (DMSO-d6) δ 7.97 (d, J=8.4 Hz, 2H, Ph) ,7.90 (d, J=8.4 Hz, 2H, Ph), 6.54 (s, 1H, 1’-H),
5.65 (d, J2’,3’=5.8 Hz, 1H, 2’-H), 5.05 (dd, J3’,2’=5.6 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.57 (m, 1H,
13
4’-H), 4.00 (m, 2H, 5’-H), 1.89 (s, 3H, OAc), 1.54 and 1.37 (2×s, 6H, C(CH3)2). C NMR
(DMSO-d6) δ 169.79 (CO), 133.02 (Ph), 130.73 (q, JC,F=32.4 Hz, C-4Ph), 127.09, 123.31 and
123.10 (C-4, C-5, Ph), 125.99 (q, JC,F=3.7 Hz, C-3Ph, C-5Ph), 123.66 (q, JC,F=272.8 Hz, CF3),
113.06 ((CH3)2C), 110.85 (CN), 103.52 (C≡CPh), 93.20 (C-1’), 86.39 (C-4’), 83.87 (C-2’),
19
81.15 (C-3’), 72.39 (C≡CPh), 63.15 (C-5’), 26.56 and 24.86 ((CH3)2C), 20.28 (CO-CH3). F
NMR (DMSO d6) δ 14.02. HRMS [M+H]+ calcd for C22H20N4O5F3: 477.13858; found:
477.13531.
4.2.8.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(naphth-2-ylethynyl)-1H-
[1,2,3]triazole-4-carbonitrile (11e)
From fractions containing homogenous 11e white crystalline material was separated,
while evaporation of the filtrate resulted in the oily residue (57% total yield). UV (MeCN):
1
λmax 266 nm (25900), 276.5 nm (35700), 312 nm (26700). H NMR (DMSO-d6) δ 8.44 (d,
J=0.4Hz, 1H, naphthyl), 8.07 (m, 2H, naphthyl), 8.02 (m, 1H, naphthyl), 7.73 (dd, J=1.4 Hz,
12

J=8.6 Hz, 1H, naphthyl), 7.66 (m, 2H, naphthyl), 6.54 (s, 1H, 1’-H), 5.66 (d, J2’,3’=6.0 Hz, 1H,
2’-H), 5.06 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.58 (m, 1H, 4’-H), 4.01 (m, 2H, 5’-H),
13
1.90 (s, 3H, OAc), 1.56 and 1.38 (2×s, 6H, 2×CH3). C NMR (DMSO-d6) δ 169.80 (CO),
133.46, 133.16, 132.28, 128.86, 128.40, 128.29, 127.88, 127.69, 127.47, 127.38, 122.66 and
116.23 (naphthyl, C-4, C-5), 113.06 ((CH3)2C), 111.05 (CN), 105.98 (C≡C-naphthyl), 92.99
(C-1’), 86.36 (C-4’), 83.86 (C-2’), 81.16 (C-3’), 70.65 (C≡C-naphthyl), 63.15 (C-5’), 26.58
and 24.87 ((CH3)2C), 20.30 (CO-CH3). HRMS [M+H]+ calcd for C25H23N4O5: 459.16685;
found: 459.16596.
4.2.9.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-(6-methoxynaphth-2-
ylethynyl)-1H-[1,2,3]triazole-4-carbonitrile (11f)
For rechromatography CH2Cl2 was used to give 11f as solid foam in 60% yield. UV
1
(MeCN): λmax 227 nm (21600), 281.5 nm (11800), 331.5 nm (11800). H NMR (DMSO-d6) δ
8.34 (d, J=0.8 Hz, 1H, naphthyl), 7.96 (m, 2H, naphthyl), 7.68 (dd, J=1.8 Hz, J=8.6 Hz, 1H,
naphthyl), 7.43 (d, J=2.4 Hz, 1H, naphthyl), 7.28 (dd, J=2.6 Hz, J=9.0 Hz, 1H, naphthyl), 6.51
(s, 1H, 1’-H), 5.65 (d, J2’,3’=6.8 Hz, 1H, 2’-H), 5.06 (m, 1H, 3’-H), 4.57 (m, 1H, 4’-H), 4.00
(m, 2H, 5’-H), 3.92 (s, 3H, OCH3), 1.90 (s, 3H, OAc), 1.55 and 1.38 (2×s, 6H, C(CH3)2). 13C
NMR (DMSO-d6) δ 169.80 (CO), 159.12 (C-6naphthyl), 135.27, 132.96, 129.96, 128.08, 127.88,
127.72, 127.64, 122.42 and 119.96 (naphthyl, C-4, C-5), 113.51 and 113.05 (naphthyl,
(CH3)2C), 111.10 (CN), 106.58 and 106.27 (naphthyl, C≡C-naphthyl), 92.88 (C-1’), 86.32 (C-
4’), 83.83 (C-2’), 81.16 (C-3’), 70.12 (C≡C-naphthyl), 63.15 (C-5’), 55.44 (OCH3), 26.59 and
24.87 ((CH3)2C), 20.30 (CO-CH3). HRMS [M+H]+ calcd for C26H25N4O6: 489.17741; found:
489.17714.
4.2.10 General procedure for cyclization reactions
To a solution of 1-(5-O-acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-5-alkynyl-1H-
[1,2,3]triazole-4-carbonitrile (11a–f; dried under vacuum at room temperature) in DME (10
ml/mmol) was added ca 10 M NH3/DME (30 ml/mmol) and the resulting mixture was heated
in a Parr reactor at 85 ºC for 40 h, then it was evaporated.
4.2.11.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-phenyl-1H-
[1,2,3]triazole[4,5-c]pyridine (14a)
Chromatography on silica gel column with hexane-EtOAc (2:1→1:1) resulted in 14a as
1
solid foam (95% yield). UV (MeOH): λmax 249 nm (22900), 315 nm (9900). H NMR
13

(DMSO-d6) δ 8.10 (m, 2H, Ph), 7.63 (s, 1H, 7-H), 7.47 (m, 2H, Ph), 7.40 (m, 1H, Ph), 7.30 (s,
2H, NH2), 6.70 (d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.72 (dd, J2’,1’=1.6 Hz, J2’,3’=6.0 Hz, 1H, 2’-H),
5.10 (dd, J3’,2’=6.0 Hz, J3’,4’=2.4 Hz, 1H, 3’-H), 4.48 (m, 1H, 4’-H), 4.06 (dd, J5’a,5’b=11.8 Hz,
J5’a,4’=5.4 Hz, 1H, 5’a-H), 3.87 (dd, J5’b,5’a=11.8 Hz, J5’b,4’=6.6 Hz, 1H, 5’b-H), 1.86 (s, 3H,
13
OAc), 1.58 and 1.39 (2×s, 6H, C(CH3)2). C NMR (DMSO-d6) δ 169.84 (CO), 152.65 and
151.41 (C-4, C-6), 139.49, 138.98, 130.71, 128.71, 128.39 and 126.87 (Ph, C-3a, C-7a),
113.05 ((CH3)2C), 90.85 and 90.48 (C-1’, C-7), 84.90 (C-4’), 83.44 (C-2’), 81.51 (C-3’),
63.41 (C-5’), 26.69 and 25.01 ((CH3)2C), 20.31 (CO-CH3). HRMS [M+H]+ calcd for
C21H24N5O5: 426.1772; found: 426.1787.
4.2.12. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-(4-methylphenyl)-
1H-[1,2,3]triazole[4,5-c]pyridine (14b)
1
Solid (95% yield). UV (MeOH): λmax 256.5 nm (31000), 316.5 nm (13500). H NMR
(DMSO-d6) δ 8.01 (d, J=8.0 Hz, 2H, Ph), 7.60 (s, 1H, 7-H), 7.27 (m, 4H, Ph, NH2), 6.69 (s,
1H, 1’-H), 5.72 (d, J2’,3’=5.6 Hz, 1H, 2’-H), 5.10 (dd, J3’,2’=5.8 Hz, J3’,4’=1.4 Hz, 1H, 3’-H),
4.48 (m, 1H, 4’-H), 4.06 (dd, J5’a,5’b=11.8 Hz, J5’a,4’=5.4 Hz, 1H, 5’a-H), 3.86 (dd, J5’b,5’a=11.8
Hz, J5’b,4’=6.6 Hz, 1H, 5’b-H), 2.35 (s, 3H, CH3), 1.87 (s, 3H, OAc), 1.57 and 1.39 (2×s, 6H,
C(CH3)2). 13C NMR (DMSO-d6) δ 169.84 (CO), 152.64 and 151.34 (C-4, C-6), 139.55,
138.18, 136.19, 130.61, 129.00 and 126.77 (Ph, C-3a, C-7a), 113.04 ((CH3)2C), 90.80 (C-1’),
89.92 (C-7), 84.89 (C-4’), 83.42 (C-2’), 81.52 (C-3’), 63.41 (C-5’), 26.69 and 25.01
((CH3)2C), 20.78 (Ph-CH3), 20.31 (CO-CH3). HRMS [M+H]+ calcd for C22H26N5O5:
440.1934; found: 440.1925.
4.2.13. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-(4-fluorophenyl)-
1H-[1,2,3]triazole[4,5-c]pyridine (14c)
Chromatography on silica gel column with hexane-EtOAc (2:1→1:2) resulted in 14c as
1
solid foam (80% yield). UV (MeOH): λmax 248.5 nm (37000), 318 nm (15500). H NMR
(DMSO-d6) δ 8.15 (m, 2H, Ph), 7.63 (s, 1H, 7-H), 7.30 (m, 4H, Ph, NH2), 6.69 (d, J1’,2’=1.2
Hz, 1H, 1’-H), 5.72 (dd, J2’,1’=1.2 Hz, J2’,3’=6.0 Hz, 1H, 2’-H), 5.10 (dd, J3’,2’=6.0 Hz,
J3’,4’=2.4 Hz, 1H, 3’-H), 4.48 (m, 1H, 4’-H), 4.07 (dd, J5’a,5’b=11.6 Hz, J5’a,4’=5.2 Hz, 1H, 5’a-
H), 3.87 (dd, J5’b,5’a=11.6 Hz, J5’b,4’=6.8 Hz, 1H, 5’b-H), 1.87 (s, 3H, OAc), 1.57 and 1.39
(2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 169.84 (CO), 162.62 (d, JC,F=245.88 Hz, C-4Ph),
151.58 and 151.41 (C-4, C-6), 139.54, 135.44 and 130.63 (C-1Ph, C-3a, C-7a), 128.93 (d,
JC,F=8.76 Hz, C-2Ph, C-6Ph), 115.22 (d, JC,F=21.22 Hz, C-3Ph, C-5Ph), 113.05 ((CH3)2C), 90.83
14

(C-1’), 90.28 (C-7), 84.94 (C-4’), 83.45 (C-2’), 81.53 (C-3’), 63.41 (C-5’), 26.68 and 25.00
((CH3)2C), 20.30 (CO-CH3). 19F NMR (DMSO d6) δ -37.96. HRMS [M+H]+ calcd for
C21H23N5O5F: 444.16832; found: 444.16778.
4.2.14.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-(4-
trifluoromethylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine (14d)
Chromatography on silica gel column with hexane-EtOAc (2:1) gave 14d as crystalline
material (80% yield), mp 154-157 °C. UV (MeOH): λmax 248 nm (25600), 322.5 nm (10900).
1H NMR (DMSO-d6) δ 8.32 (d, J=8.0 Hz, 2H, Ph), 7.84 (d, J=8.4 Hz, 2H, Ph), 7.79 (s, 1H, 7-
H), 7.42 (s, 2H, NH2), 6.72 (d, J1’,2’=0.8 Hz, 1H, 1’-H), 5.73 (dd, J2’,1’=1.2 Hz, J2’,3’=6.0 Hz,
1H, 2’-H), 5.11 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.49 (m, 1H, 4’-H), 4.06 (dd,
J5’a,5’b=11.8 Hz, J5’a,4’=5.4 Hz, 1H, 5’a-H), 3.87 (dd, J5’b,5’a=11.6 Hz, J5’b,4’=6.8 Hz, 1H, 5’b-
H), 1.86 (s, 3H, OAc), 1.58 and 1.39 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 169.83
(CO), 151.60 and 150.81 (C-4, C-6), 142.86 (Ph), 139.43 and 131.06 (C-3a, C-7a), 128.80 (q,
JC,F=31.70 Hz, C-4Ph), 127.47 (Ph), 125.35 (q, JC,F=3.51 Hz, C-3Ph, C-5Ph), 124.34 (q,
JC,F=272.20 Hz, CF3), 113.04 ((CH3)2C), 91.69 (C-7), 90.92 (C-1’), 85.05 (C-4’), 83.48 (C-
2’), 81.54 (C-3’), 63.39 (C-5’), 26.68 and 24.99 ((CH3)2C), 20.29 (CO-CH3). 19F NMR
(DMSO-d6) δ 14.75. HRMS [M+H]+ calcd for C22H23N5O5F3: 494.16513; found: 494.16504.
4.2.15. 1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-(naphth-2-yl)-1H-
[1,2,3]triazole[4,5-c]pyridine (14e)
Chromatography on silica gel column with hexane-EtOAc (2:1→1:1) afforded 14e as
1
solid (95% yield). UV (MeOH): λmax 252.5 nm (59200), 325 nm (18100). H NMR (DMSO-
d6) δ 8.67 (s, 1H, naphthyl), 8.27 (dd, J=1.6 Hz, J=8.8 Hz, 1H, naphthyl), 8.02-7.94 (m, 3H,
naphthyl), 7.82 (s, 1H, 7-H), 7.55 (m, 2H, naphthyl), 7.38 (s, 2H, NH2), 6.74 (s, 1H, 1‘-H),
5.76 (d, J2’,3’=6.0 Hz, 1H, 2’-H), 5.12 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.50 (m, 1H,
4’-H), 4.08 (dd, J5’a,5’b=11.6 Hz, J5’a,4’=5.6 Hz, 1H, 5’a-H), 3.88 (dd, J5’b,5’a=12.0 Hz,
13
J5’b,4’=6.8 Hz, 1H, 5’b-H), 1.87 (s, 3H, OAc), 1.59 and 1.40 (2×s, 6H, C(CH3)2). C NMR
(DMSO-d6) δ 169.85 (CO), 152.41 and 151.50 (C-4, C-6), 139.60, 136.40, 133.10, 132.95,
130.82, 128.41, 127.83, 127.52, 126.50, 126.44, 125.99 and 124.82 (naphthyl, C-3a, C-7a),
113.04 ((CH3)2C), 90.95 and 90.88 (C-1’, C-7), 84.97 (C-4’), 83.44 (C-2’), 81.55 (C-3’),
63.41 (C-5’), 26.71 and 25.03 ((CH3)2C), 20.32 (CO-CH3). HRMS [M+H]+ calcd for
C25H26N5O5: 476.19340; found: 476.19348.
15

4.2.16.
1-(5-O-Acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-4-amino-6-(6-
methoxynaphth-2-yl)-1H-[1,2,3]triazole[4,5-c]pyridine (14f)
Solid (88% yield). UV (MeOH): λmax 260 nm (39600), 297 nm (15200), 327.5 (18600). 1H
NMR (DMSO-d6) δ 8.59 (s, 1H, naphthyl), 8.22 (d, J=8.8 Hz, 1H, naphthyl), 7.90 (dd, J=3.4
Hz, J=8.6 Hz, 2H, naphthyl), 7.75 (s, 1H, 7-H), 7.34 (m, 3H, naphthyl, NH2), 7.19 (m, 1H,
naphthyl), 6.72 (s, 1H, 1’-H), 5.75 (d, J2’,3’=6.0 Hz, 1H, 2’-H), 5.11 (m, J3’,2’=6.0 Hz, J3’,4’=1.6
Hz., 1H, 3’-H), 4.50 (m, 1H, 4’-H), 4.07 (dd, J5’a,5’b=11.8 Hz, J5’a,4’=5.4 Hz, 1H, 5’a-H), 3.88
(m, 4H, 5’b-H, OCH3), 1.87 (s, 3H, OAc), 1.59 and 1.40 (2×s, 6H, C(CH3)2). 13C NMR
(DMSO-d6) δ 169.86 (CO), 157.78 (C-6naphthyl), 152.68 (C-6), 151.44 (C-4), 139.64, 134.51,
134.17, 130.70, 129.98, 128.34, 126.72, 125.90, 125.29 and 118.98 (naphthyl, C-3a, C-7a),
113.06 ((CH3)2C), 105.86 (naphthyl), 90.87 (C-1’), 90.36 (C-7), 84.94 (C-4’), 83.43 (C-2’),
81.56 (C-3’), 63.42 (C-5’), 55.23 (OCH3), 26.72 and 25.03 ((CH3)2C), 20.32 (CO-CH3).
HRMS [M+H]+ calcd for C26H28N5O6: 506.20396; found 506.20301.
4.2.17. General procedure for deacetylation reactions
To a cooled to 0 ºC solution of compound 14a–f in anhyd 1,4-dioxane/MeOH (1:1; 20
ml/mmol) or in anhyd CH2Cl2/MeOH (1:1; 20 ml/mmol) was added 0.25 M MeONa in
anhydrous MeOH (0.08 equiv). The mixture was allowed to react under argon at 5 ºC for 20
h. It was again cooled to 0 ºC and neutralized with Dowex 50WX8-100 ion exchange resin,
which was then separated by filtration. The filtrate was evaporated and the residue was
subjected to chromatography on silica gel column with CH2Cl2-MeOH (99:1→95:5) to afford
purified product 15a–f as solid or solid foam in 79-96% yield.
4.2.18.
4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-phenyl-1H-
[1,2,3]triazole[4,5-c]pyridine (15a)
Crude material after chromatography was crystallized from MeOH to give white crystals,
1
mp 105 ºC. H NMR (DMSO-d6) δ 8.10 (m, 2H, Ph), 7.68 (s, 1H, 7-H), 7.46 (m, 2H, Ph),
7.40 (m, 1H, Ph), 7.34 (s, 2H, NH2), 6.63 (d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.65 (dd, J2’,1’=1.8 Hz,
J2’,3’=6.2 Hz, 1H, 2’-H), 5.05 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 5.00 (t, J5’OH,5’H=5.6
Hz, 1H, 5’-OH), 4.23 (m, 1H, 4’-H), 3.30 (m, 2H, 5’-H), 1.56 and 1.38 (2×s, 6H, C(CH3)2).
13C NMR (DMSO-d6) δ 152.53 (C-6), 151.47 (C-4), 139.44, 139.05, 130.86, 128.77, 128.48
and 126.93 (Ph, C-3a, C-7a), 112.91 ((CH3)2C), 91.00 (C-1’), 90.73 (C-7), 87.86 (C-4’), 83.25
(C-2’), 81.72 (C-3’), 61.02 (C-5’), 26.80 and 25.05 ((CH3)2C). HRMS [M+H]+ calcd for
C19H22N5O4: 384.16718; found 384.16706.
16

4.2.19.
4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(4-methylphenyl)-1H-
[1,2,3]triazole[4,5-c]pyridine (15b)
1H NMR (DMSO-d6) δ 8.00 (d, J=8.0 Hz, 2H, Ph), 7.63 (s, 1H, 7-H), 7.32 (s, 2H, NH2),
7.27 (d, J=8.0 Hz, 2H, Ph), 6.61 (d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.64 (dd, J2’,1’=1.2 Hz, J2’,3’=6.0
Hz, 1H, 2’-H), 5.05 (dd, J3’,2’=6.0 Hz, J3’,4’=1.6 Hz, 1H, 3’-H), 4.96 (brs, 1H, 5’-OH), 4.24 (m,
1H, 4’-H), 3.31 (m, 2H, 5’-H), 2.35 (s, 3H, CH3), 1.57 and 1.38 (2×s, 6H, C(CH3)2). 13C NMR
(DMSO-d6) δ 152.22 (C-6), 151.25 (C-4), 139.43, 138.23, 135.98, 130.69, 129.01 and 126.79
(Ph, C-3a, C-7a), 112.86 ((CH3)2C), 90.99 (C-1’), 90.28 (C-7), 87.79 (C-4’), 83.17 (C-2’),
81.66 (C-3’), 60.98 (C-5’), 26.75 and 25.01 ((CH3)2C), 20.79 (Ph-CH3). HRMS [M+H]+ calcd
for C20H24N5O4: 398.18283; found: 398.18213.
4.2.20.
4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(4-fluorophenyl)-1H-
[1,2,3]triazole[4,5-c]pyridine (15c)
1H NMR (DMSO-d6) δ 8.15 (dd, J=5.8 Hz, J=8.6 Hz, 2H, Ph), 7.66 (s, 1H, 7-H), 7.31-
7.23 (m, 4H, Ph, NH2), 6.60 (s, 1H, 1’-H), 5.63 (d, J2’,3’=6.0 Hz, 1H, 2’-H), 5.05 (dd, J3’,2’
=
6.0 Hz, J3’,4’=1.6 Hz, 1H, 3’-H), 4.96 (t, J5’OH, 5’H=5.4 Hz, 1H, 5’-OH), 4.24 (m, 1H, 4’-H),
3.31 (m, 2H, 5’-H), 1.57 i 1.38 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 162.58 (d,
JC,F=246.14 Hz, C-4Ph), 151.38 (C-4, C-6), 139.37 (C-7a), 135.46 (d, JC,F=2.97 Hz, C-1Ph),
130.71 (C-3a), 128.90 (d, JC,F=8.42 Hz, C-2Ph, C-6Ph), 115.18 (d, JC,F=21.26 Hz, C-3Ph, C-5Ph),
112.87 ((CH3)2C), 91.03 (C-1’), 90.50 (C-7), 87.76 (C-4’), 83.17 (C-2’), 81.62 (C-3’), 60.97
19
(C-5’), 26.74 and 25.00 ((CH3)2C). F NMR (DMSO d6) δ -38.02. HRMS [M+H]+ calcd for
C19H21N5O4F: 402.15776; found: 402.15771.
4.2.21. 4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(4-trifluoromethylphenyl)-1H-
[1,2,3]triazole[4,5-c]pyridine (15d)
1H NMR (DMSO-d6) δ 8.32 (d, J=8.0 Hz, 2H, Ph), 7.83 (m, 3H, Ph, 7-H), 7.41 (s, 2H,
NH2), 6.64 (d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.65 (dd, J2’,1’=1.6 Hz, J2’,3’=6.0 Hz, 1H, 2’-H), 5.06
(dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.97 (t, J5’OH,5’H=5.4 Hz, 1H, 5’-OH), 4.25 (m, 1H,
4’-H), 3.31 (m, 2H, 5’-H), 1.57 and 1.38 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 151.59
and 150.66 (C-4, C-6), 142.90 (Ph), 139.27 (C-7a), 131.16 (C-3a), 128.76 (q, JC,F=30.72 Hz,
C-4Ph), 127.47 (Ph), 125.34 (q, JC,F=3.08 Hz, C-3Ph, C-5Ph), 124.35 (q, JC,F=271.51 Hz, CF3),
112.87 ((CH3)2C), 91.95 and 91.13 (C-1’, C-7), 87.89 (C-4’), 83.24 (C-2’), 81.64 (C-3’),
17

60.97 (C-5’), 26.74 and 25.00 ((CH3)2C). 19F NMR (DMSO-d6): 14.79. HRMS [M+H]+ calcd
for C20H21N5O4F3: 452.15456; found: 452.15393.
4.2.22.
4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(naphth-2-yl)-1H-
[1,2,3]triazole[4,5-c]pyridine (15e)
1
Crystalline material, mp 167-170 ºC. H NMR (DMSO-d6) δ 8.68 (s, 1H, naphthyl), 8.28
(dd, J=1.8 Hz, J=8.6 Hz, 1H, naphthyl), 8.00 (d, J=8.8 Hz, 2H, naphthyl) ,7.95 (m, 1H,
naphthyl), 7.85 (s, 1H, 7-H), 7.55 (m, 2H, naftyl-H), 7.36 (s, 2H, NH2), 6.66 (d, J1’,2’=1.6 Hz,
1H, 1’-H), 5.68 (dd, J2’,1’=1.6 Hz, J2’,3’=6.0 Hz, 1H, 2’-H), 5.07 (dd, J3’,2’=6.4 Hz, J3’,4’=2.0
Hz, 1H, 3’-H), 4.99 (t, J5’OH,5’H=5.4 Hz, 1H, 5’-OH), 4.26 (m, 1H, 4’-H), 3.33 (m, 2H, 5’-H),
1.58 and 1.39 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 152.27 (C-6), 151.49 (C-4),
139.48 (C-7a), 136.43, 133.09 and 132.96 (naphthyl), 130.93 (C-3a), 128.43, 127.81, 127.51,
126.47, 126.41, 125.99 and 124.84 (naphthyl), 112.87 ((CH3)2C), 91.18 (C-7), 91.04 (C-1’),
87.87 (C-4’), 83.21 (C-2’), 81.68 (C-3’), 60.99 (C-5’), 26.77 and 25.02 ((CH3)2C). HRMS
[M+H]+ calcd for C23H24N5H4: 434.18283; found: 434.18195.
4.2.23. 4-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(6-methoxynaphth-2-yl)-1H-
[1,2,3]triazole[4,5-c]pyridine (15f)
Crude material after chromatography was crystallized from hexane-EtOAc to give white
crystals, mp 140 ºC. 1H NMR (DMSO-d6) δ 8.59 (s, 1H, naphthyl), 8.22 (dd, J=1.6 Hz, J=8.8
Hz, 1H, naphthyl), 7.89 (dd, J=5.6 Hz, J=8.8 Hz, 2H, naphthyl), 7.78 (s, 1H, 7-H), 7.36 (d,
J=2.0 Hz, 1H, naphthyl), 7.30 (s, 2H, NH2), 7.20 (dd, J=2.4 Hz, J=8.8 Hz, 1H, naphthyl), 6.64
(d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.67 (dd, J2’,1’=1.4 Hz, J2’,3’=6.2 Hz, 1H, 2’-H), 5.07 (dd, J3’,2’=6.0
Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.98 (t, J5’OH,5’H=5.4 Hz, 1H, 5’-OH), 4.26 (m, 1H, 4’-H), 3.90 (s,
13
3H, OCH3), 3.32 (m, 2H, 5’-H), 1.58 and 1.39 (2×s, 6H, C(CH3)2). C NMR (DMSO-d6) δ
157.75 (C-6naphthyl), 152.50 (C-6), 151,39 (C-4), 139.48 (C-7a), 130.78 (C-3a), 134.47,
134.18, 129.96, 128.33, 126.68, 125.85, 125.28 and 118.92 (naphthyl), 112.86 ((CH3)2C),
105.84 (naphthyl), 91.02 (C-1’), 90.56 (C-7), 87.79 (C-4’), 83.16 (C-2’), 81.65 (C-3’), 60.97
(C-5’), 55.21 (OCH3), 26.75 and 25.01 ((CH3)2C). HRMS [M+H]+ calcd for C24H26N5O5:
464.19340; found: 464.19342.
4.2.24. General procedure for sulfamoylation reactions
To a stirred solution of compound 15a–f (dried in vacuo over P2O5 at room temperature)
in MeCN (15 ml/mmol) containing DMA (50 equiv) was added 1.33 M sulfamoyl chloride
18

[35] in MeCN (2.5 equiv) at 0 ºC. The mixture was stirred on ice-cooling for 15 min, then at
room temperature overnight. It was evaporated and the resulting residue was
chromatographed on silica gel column with CH2Cl2-MeOH (98:2→95:5) to obtain product
1
16a–f contaminated with DMA as solid foam (yields 51-71% calculated from H NMR
spectra).
4.2.25.
4-Amino-6-phenyl-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-1H-
[1,2,3]triazole[4,5-c]pyridine (16a)
1H NMR (DMSO-d6) δ 8.11 (m, 2H, Ph), 7.68 (s, 1H, 7-H), 7.55 (brs, 2H, SO3NH2), 7.43
(m, 3H, Ph), 7.31 (brs, 2H, NH2), 6.77 (d, J1’,2’=1.2 Hz, 1H, 1’-H), 5.65 (dd, J2’,1’=1.2 Hz,
J2’,3’=6.0 Hz, 1H, 2’-H), 5.13 (dd, 1H, J3’2’=6.0 Hz, J3’,4’=2.4 Hz, 1H, 3’-H), 4.48 (dt, J4’,3’=2.3
Hz, J4’,5’= 6.5 Hz, 1H, 4’-H), 4.06 and 3.91 (2×dd, J5’,4’=6.6 Hz, J5’a,5’b=10.6 Hz, 2H, 5’-H),
1.58 and 1.40 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 152.83 and 151.44 (C-4, C-6),
139.56 (C-7a), 138.94 (Ph), 130.66 (C-3a), 128.76, 128.41 and 126.91 (Ph), 113.29
((CH3)2C), 90.38 and 90.31 (C-1’, C-7), 84.80 (C-4’), 83.51 (C-2’), 81.50 (C-3’), 67.63 (C-
5’), 26.68 and 25.00 ((CH3)2C). HRMS [M+H]+ calcd for C19H23N6O6S: 463.1394; found:
463.1413.
4.2.26.
4-Amino-6-(4-methylphenyl)-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-
ribofuranosyl)-1H-[1,2,3]triazole[4,5-c]pyridine (16b)
1H NMR (DMSO-d6) δ 8.02 (d, J=8.0 Hz, 2H, Ph), 7.65 (s, 1H, 7-H), 7.55 (s, 2H,
SO3NH2), 7.27 (m, 4H, NH2, Ph), 6.75 (d, J1’,2’=1.2 Hz, 1H, 1’-H), 5.65 (dd, J2’,1’=1.4 Hz,
J2’,3’=6.2 Hz, 1H, 2’-H), 5.13 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.48 (dt, J4’,3’=2.1 Hz,
J4’,5’=6.6 Hz, 1H, 4’-H), 4.06 and 3.91 (2×dd, J5’,4’=6.7 Hz, J5’a,5’b=10.7 Hz, 2H, 5’-H), 2.35 (s,
3H, CH3), 1.58 and 1.40 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 152.80 and 151.35 (C-4,
C-6), 139.58 (C-7a), 138.21 and 136.13 (Ph), 130.53 (C-3a), 128.99 and 126.80 (Ph), 113.26
((CH3)2C), 90.33 and 89.73 (C-1’, C-7), 84.76 (C-4’), 83.48 (C-2’), 81.49 (C-3’), 67.61 (C-
5’), 26.67 and 24.99 ((CH3)2C), 20.78 (CH3). HRMS [M+H]+ calcd for C20H25N6O6S:
477.1556; found: 477.1563.
4.2.27.
4-Amino-6-(4-fluorophenyl)-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-
ribofuranosyl)-1H-[1,2,3]triazole[4,5-c]pyridine (16c)
1H NMR (DMSO-d6) δ 8.16 (m, 2H, Ph), 7.68 (s, 1H, 7-H), 7.56 (s, 2H, SO3NH2), 7.31
(m, 4H, NH2, Ph), 6.75 (d, J1’,2’=1.2 Hz, 1H, 1’-H), 5.65 (dd, J2’,1’=1.2 Hz, J2’,3’=6.0 Hz, 1H,
19

2’-H), 5.13 (dd, J3’,2’=6.0 Hz, J3’,4’=2.4 Hz, 1H, 3’-H), 4.48 (dt, J4’,3’=2.1 Hz, J4’,5’=6.6 Hz, 1H,
4’-H), 4.07 and 3.92 (2×dd, J5’,4’=6.5 Hz, J5’a,5’b=10.5 Hz, 2H, 5’-H), 1.58 and 1.40 (2×s, 6H,
C(CH3)2). 13C NMR (DMSO-d6) δ 162.66 (d, JC,F=245.6 Hz, C-4Ph), 151.73 and 151.44 (C-4,
C-6), 139.58 (C-7a), 135.41 (Ph), 130.57 (C-3a), 128.97 (d, JC,F=8.22 Hz, C-2Ph, C-6Ph),
115.24 (d, JC,F=21.47 Hz, C-3Ph, C-5Ph), 113.30 ((CH3)2C), 90.40 and 90.12 (C-1’, C-7), 84.81
(C-4’), 83.51 (C-2’), 81.48 (C-3’), 67.63 (C-5’), 26.67 and 24.99 ((CH3)2C). 19F NMR
(DMSO-d6) δ: -37.94. HRMS [M+H]+ calcd for C19H22N6O6SF: 481.1300; found: 481.1308.
4.2.28.
4-Amino-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-6-(4-
trifluoromethylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine (16d)
1H NMR (DMSO-d6) δ 8.32 (d, J=8.0 Hz, 2H, Ph), 7.84 (s and d, 3H, Ph, 7-H), 7.54 (brs,
2H, SO3NH2), 7.44 (s, 2H, NH2), 6.78 (d, J1’,2’=1.2 Hz, 1H, 1’-H), 5.66 (dd, J2’,1’=1.1 Hz,
J2’,3’=6.2 Hz, 1H, 2’-H), 5.14 (dd, J3’,2’=6.0 Hz, J3’,4’=2.4 Hz, 1H, 3’-H), 4.49 (dt, J4’,3’=2.0 Hz,
J4’,5’=6.4 Hz, 1H, 4’-H), 4.07 and 3.92 (2×dd, J5’,4’=6.5 Hz, J5’a,5’b=10.5 Hz, 2H, 5’-H), 1.58
13
and 1.40 (2×s, 6H, C(CH3)2). C NMR (DMSO-d6) δ 151.61 and 150.94 (C-4, C-6), 142.80
(Ph), 139.42 (C-7a), 130.98 (C-3a), 128.80 (q, JC,F=31.70 Hz, C-4Ph), 127.49 (Ph), 125.34 (q,
JC,F=3.53 Hz, C-3Ph, C-5Ph), 124.34 (q, JC,F=272.05, CF3), 113.28 ((CH3)2C), 91.52 (C-7),
90.48 (C-1’), 84.85 (C-4’), 83.52 (C-2’), 81.43 (C-3’), 67.60 (C-5’), 26.67 and 24.99
((CH3)2C). 19F NMR (DMSO-d6) δ14.78. HRMS [M+H]+ calcd for C20H22N6O6SF3:
531.1268; found: 531.1288.
4.2.29. 4-Amino-6-(naphth-2-yl)-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-
1H-[1,2,3]triazole[4,5-c]pyridine (16e)
1H NMR (DMSO-d6) δ 8.68 (s, 1H, naphthyl), 8.28 (dd, J=1.6 Hz, J=8.8 Hz, 1H,
naphthyl), 7.98 (m, 3H, naphthyl), 7.87 (s, 1H, 7-H), 7.55 (m, 4H, naphthyl, SO3NH2), 7.39
(s, 2H, NH2), 6.80 (d, J1’,2’=1.2 Hz, 1H, 1’-H), 5.69 (dd, J2’,1’=1.2 Hz, J2’,3’=6.0 Hz, 1H, 2’-H),
5.15 (dd, J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.51 (dt, J4’,3’=2.3 Hz, J4’,5’=6.6 Hz, 1H, 4’-H),
4.08 and 3.93 (2×dd, J5’,4’=6.6 Hz, J5’a,5’b=10.6 Hz, 2×1H, 5’-H), 1.60 and 1.41 (2×s, 6H,
C(CH3)2). 13C NMR (DMSO-d6) δ 152.58 and 151.51 (C-4, C-6), 139.63 (C-7a), 136.35,
133.12 and 132.95 (naphthyl), 130.75 (C-3a), 128.43, 127.83, 127.52, 126.52, 126.43, 126.04
and 124.85 (naphthyl), 113.28 ((CH3)2C), 90.78 and 90.43 (C-1’, C-7), 84.84 (C-4’), 83.50
(C-2’), 81.51 (C-3’), 67.62 (C-5’), 26.69 and 25.00 ((CH3)2C). HRMS [M+H]+ calcd for
C23H25N6O6S: 513.1556; found: 513.1550.
20

4.2.30.
4-Amino-6-(6-methoxynaphth-2-yl)-1-(5-O-sulfamoyl-2,3-O-isopropylidene-β-D-
ribofuranosyl)-1H-[1,2,3]triazole[4,5-c]pyridine (16f)
1H NMR (DMSO-d6) δ 8.60 (s, 1H, naphthyl), 8.23 (dd, J=1.8 Hz, J=8.6 Hz, 1H,
naphthyl), 7.90 (m, 2H, naphthyl), 7.80 (s, 1H, 7-H), 7.56 (s, 2H, SO3NH2), 7.35 (m, 3H,
naphthyl, NH2), 7.20 (dd, J=2.4 Hz, J=8.8 Hz, 1H, naphthyl), 6.78 (d, J1’,2’=1.2 Hz, 1H, 1’-H),
5.68 (dd, J2’,1’=1.4 Hz, J2’,3’=6.2 Hz, 1H, 2’-H), 5.15 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-
H), 4.50 (dt, J4’,3’=2.1 Hz, J4’,5’=6.6 Hz, 1H, 4’-H), 4.08 (dd, J5a’,4’=6.8 Hz, J5’a,5’b=10.8 Hz,
2H, 5’a-H), 3.93 (m, 4H, 5’b-H, OCH3), 1.60 and 1.41 (2×s, 6H, C(CH3)2). 13C NMR
(DMSO-d6) δ 157.79 (C-6naphthyl), 152.84 and 151.45 (C-4, C-6), 139.66 (C-7a), 134.54,
134.13, 130.60, 130.00, 128.34, 126.72, 125.93, 125.32 and 118.98 (naphthyl, C-3a), 113.29
((CH3)2C), 105.86 (naphthyl), 90.42 and 90.18 (C-1’, C-7), 84.79 (C-4’), 83.48 (C-2’), 81.51
(C-3’), 67.61 (C-5’), 55.24 (OCH3), 26.69 and 25.00 ((CH3)2C). HRMS [M+H]+ calcd for
C24H27N6O7S: 543.1656; found: 543.1668.
4.2.31. General procedure for the preparation of compounds 17a–f
To a solution of compound 16a–f (contaminated with DMA) in MeCN (20 ml/mmol) was
added 1,8-diazabicyclo[5.4.0]undec-7-ene (1.5 equiv) and the mixture was stirred at room
temperature for 15 min. In a separate flask 1,1'-carbonyldiimidazole (1.4 equiv) and salicylic
acid (1.4 equiv) were dissolved in MeCN (20 ml/mmol of substrate) and the resulting solution
was stirred at 60 ºC for 2 h, then it was cooled to room temperature. Both solutions were
combined and stirred at room temperature overnight. After evaporation the residue was
chromatographed on silica gel column with CH2Cl2-MeOH (98:2→9:1). Crude product
obtained was rechromatographed on silica gel column using EtOAc to give pure 17a–f as
solid foam in 56-86% yield.
4.2.32.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-isopropylidene-β-D-
ribofuranosyl}-6-phenyl-1H-[1,2,3]triazole[4,5-c]pyridine (17a)
1
UV (MeOH): λmax 246 nm (32500), 309.5 nm (13700). H NMR (DMSO-d6) δ 13.49 (s,
1H, OH), 8.12 (d, J=7.2 Hz, 2H, Ph), 7.77 (dd, J=1.8 Hz, J=8.2 Hz, 1H, sal), 7.66 (s, 1H, 7-
H), 7.43 (m, 3H, Ph), 7.25 (m, 3H, NH2, sal), 6.71 (m, 3H, sal, 1’-H), 5.59 (dd, J2’,1’=1.6 Hz,
J2’,3’=6.0 Hz, 1H, 2’-H), 5.16 (dd, J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.47 (dt, J4’,3’=2.1 Hz,
J4’,5’=6.5 Hz, 1H, 4’-H), 4.07 (dd, J5’a,4’=7.0 Hz, J5’a,5’b=11.0 Hz, 1H, 5’a-H), 3.91 (dd,
J5’b,4’=6.0 Hz, J5’b,5’a=10.8 Hz, 1H, 5’b-H), 1.56 and 1.36 (2×s, 6H, C(CH3)2). 13C NMR
(DMSO-d6) δ 171.11 (CO), 160.69 (C-OHsal), 152.76 and 151.43 (C-4, C-6), 139.41 (C-7a),
21

138.96 (Ph), 132.57 (sal), 130.67 (C-3a), 129.81 (sal), 128.65, 128.35 and 126.96 (Ph),
119.72, 117.50 and 116.52 (sal), 113.17 ((CH3)2C), 90.66 and 90.36 (C-1’, C-7), 84.94 (C-4’),
83.31 (C-2’), 81.61 (C-3’), 67.18 (C-5’), 26.69 and 24.96 ((CH3)2C). HRMS [M+H]+ calcd
for C26H27N6O8S: 583.1606; found: 583.1607.
4.2.33.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-isopropylidene-β-D-
ribofuranosyl}-6-(4-methylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine (17b)
1
UV (MeOH): λmax 249.5 nm (31400), 309.5 nm (15900). H NMR (DMSO-d6) δ 13.49 (s,
1H, OH), 8.03 (d, J=8.4 Hz, 2H, Ph), 7.77 (dd, J=2.0 Hz, J=8.0 Hz, 1H, sal), 7.62 (s, 1H, 7-
H), 7.24 (m, 5H, NH2, sal, Ph), 6.72 (m, 2H, sal), 6.67 (d, J1’,2’=2.0 Hz, 1H, 1’-H), 5.58 (dd,
J2’,1’=1.8 Hz, J2’,3’=6.2 Hz, 1H, 2’-H), 5.16 (dd, J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.46 (m,
1H, 4’-H), 4.06 (dd, J5’a,4’=6.8 Hz, J5’a,5’b=10.8 Hz, 1H, 5’a-H), 3.91 (dd, J5’b,4’=6.4 Hz,
13
J5’b,5’a=10.8 Hz, 1H, 5’b-H), 2.35 (s, 3H, CH3), 1.56 and 1.36 (2×s, 6H, C(CH3)2). C NMR
(DMSO-d6) δ 171.19 (CO), 160.72 (C-OHsal), 152.80 and 151.38 (C-4, C-6), 139.49 (C-7a),
138.16 and 136.18 (Ph), 132.65 (sal), 130.60 (C-3a), 129.85 (sal), 129.01 and 126.89 (Ph),
119.72, 117.56 and 116.57 (sal), 113.21 ((CH3)2C), 90.67 and 89.84 (C-1’, C-7), 84.94 (C-4’),
83.32 (C-2’), 81.63 (C-3’), 67.24 (C-5’), 26.71 and 24.98 ((CH3)2C), 20.82 (CH3). HRMS
[M+H]+ calcd for C27H29N6O8S: 597.1762; found: 597.1760.
4.2.34.
4-Amino-6-(4-fluorophenyl)-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-
isopropylidene-β-D-ribofuranosyl}-1H-[1,2,3]triazole[4,5-c]pyridine (17c)
1
UV (MeOH): λmax 246.5 nm (33300), 311.5 nm (14400). H NMR (DMSO-d6) δ 13.47 (s,
1H, OH), 8.17 (m, 2H, Ph), 7.77 (dd, J=1.8 Hz, J=8.2 Hz, 1H, sal), 7.65 (s, 1H, 7-H), 7.27 (m,
5H, NH2, sal, Ph), 6.72 (m, 2H, sal), 6.66 (d, J1’,2’=1.6 Hz, 1H, 1’-H), 5.58 (dd, J2’,1’=1.6 Hz,
J2’,3’=6.0 Hz, 1H, 2’-H), 5.16 (dd, J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.47 (dt, J4’,3’=2.0 Hz,
J4’,5’=6.2 Hz, 1H, 4’-H), 4.07 (dd, J5’a,4’=6.6 Hz, J5’a,5’b=10.6 Hz, 1H, 5’a-H), 3.92 (dd,
J5’b,4’=6.0 Hz, J5’b,5’a=10.8 Hz, 1H, 5’b-H), 1.56 and 1.36 (2×s, 6H, C(CH3)2). 13C NMR
(DMSO-d6) δ 171.09 (CO), 162.60 (d, JC,F=244.93 Hz, C-4Ph), 160.68 (C-OHsal), 151.65 and
151.42 (C-4, C-6), 139.38 (C-7a), 135.40 (d, JC,F=2.18 Hz, C-1Ph), 132.57 (sal), 130.60 (C-
3a), 129.79 (sal), 129.04 (d, JC,F=8.27 Hz, C-2Ph, C-6Ph), 119.71, 117.49 and 116.51 (sal),
115.14 (d, JC,F=21.31 Hz, C-3Ph, C-5Ph), 113.20 ((CH3)2C), 90.75 and 90.18 (C-1’, C-7), 84.87
(C-4’), 83.25 (C-2’), 81.52 (C-3’), 67.18 (C-5’), 26.69 and 24.95 ((CH3)2C). 19F NMR
(DMSO-d6) δ -38.08. HRMS [M+H]+ calcd for C26H26N6O8SF: 601.1511; found: 601.1518.
22

4.2.35.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-isopropylidene-β-D-
ribofuranosyl}-6-(4-trifluoromethylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine (17d)
1
UV (MeOH): λmax 243.5 nm (45100), 312 nm (17200). H NMR (DMSO-d6) δ 13.47 (s,
1H, OH), 8.34 (d, J=8.4 Hz, 2H, Ph), 7.79 (m, 4H, Ph, 7-H, sal), 7.41 (s, 2H, NH2), 7.24 (m,
1H, sal), 6.71 (m, 3H, sal, 1’-H), 5.57 (dd, J2’,1’=2.0 Hz, J2’,3’=6.0 Hz, 1H, 2’-H), 5.17 (dd,
J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.49 (dt, J4’,3’=2.1 Hz, J4’,5’=6.2 Hz, 1H, 4’-H), 4.09 and
13
3.95 (2×dd, J5’,4’=6.3 Hz, J5’a,5’b=10.9 Hz, 2H, 5’-H), 1.57 and 1.36 (2×s, 6H, C(CH3)2). C
NMR (DMSO-d6) δ 171.15 (CO), 160.69 (C-OHsal), 151.62 and 150.91 (C-4, C-6), 142.81
(Ph), 139.20 (C-7a), 132.60 (sal), 131.08 (C-3a), 129.80 (sal), 128.74 (q, JC,F=31.80 Hz, C-
4Ph), 127.60 (Ph), 125.28 (q, JC,F=3.77 Hz, C-3Ph, C-5Ph), 124.37 (q, JC,F=272.19 Hz, CF3),
119.70, 117.52 and 116.53 (sal), 113.27 ((CH3)2C), 91.65 and 90.97 (C-1’, C-7), 84.87 (C-4’),
19
83.27 (C-2’), 81.44 (C-3’), 67.22 (C-5’), 26.71 and 24.96 ((CH3)2C). F NMR (DMSO-d6) δ
14.78. HRMS [M+H]+ calcd for C27H26N6O8SF3: 651.1479; found: 651.1479.
4.2.36.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-isopropylidene-β-D-
ribofuranosyl}-6-(naphth-2-yl)-1H-[1,2,3]triazole[4,5-c]pyridine (17e)
1
UV (MeOH): λmax 251 nm (29600), 317.5 nm (9400). H NMR (DMSO-d6) δ 13.50 (s,
1H, OH), 8.70 (s, 1H, naphthyl), 8.29 (dd, J=1.6 Hz, J2=8.4 Hz, 1H, naphthyl), 7.98 (m, 3H,
naphthyl), 7.84 (s, 1H, 7-H), 7.77 (m, 1H, sal), 7.54 (m, 2H, naphthyl), 7.36 (s, 2H, NH2),
7.24 (m, 1H, sal), 6.72 (m, 3H, sal, 1’-H), 5.62 (dd, J2’,1’=1.6 Hz, J2’,3’=6.4 Hz, 1H, 2’-H), 5.18
(dd, J3’,2’=6.2 Hz, J3’,4’=2.2 Hz, 1H, 3’-H), 4.50 (m, 1H, 4’-H), 4.09 (dd, J5’a,4’=6.8 Hz,
J5’a,5’b=10.8 Hz, 1H, 5’a-H), 3.94 (dd, J5’b,4’=6.0 Hz, J5’b,5’a=10.8 Hz, 1H, 5’b-H), 1.58 and
1.37 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 171.14 (CO), 160.70 (C-OHsal), 152.54 and
151.51 (C-4, C-6), 139.47 (C-7a), 136.39, 133.10 and 132.98 (naphthyl), 132.58 (sal), 130.79
(C-3a), 129.81 (sal), 128.50, 127.76, 127.47, 126.43, 126.33, 126.08 and 124.96 (naphthyl)
119.72, 117.50 and 116.52 (sal), 113.19 ((CH3)2C), 90.85 and 90.78 (C-1’, C-7), 84.94 (C-4’),
83.27 (C-2’), 81.59 (C-3’), 67.19 (C-5’), 26.71 and 24.97 ((CH3)2C). HRMS [M+H]+ calcd
for C30H29N6O8S: 633.1762; found: 633.1767.
4.2.37.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-2,3-O-isopropylidene-β-D-
ribofuranosyl}-6-(6-methoxynaphth-2-yl)-1H-[1,2,3]triazole[4,5-c]pyridine (17f)
1
UV (MeOH): λmax 247 nm (58700), 323.5 nm (27300). H NMR (DMSO-d6) δ 13.48 (s,
1H, OH), 8.61 (s, 1H, naphthyl), 8.25 (dd, J=1.8 Hz, J=8.6 Hz, 1H, naphthyl), 7.90 (dd, 2H,
naphthyl), 7.77 (m, 2H, 7-H, sal), 7.33 (m, 3H, NH2, naphthyl), 7.25 (m, 1H, sal), 7.19 (dd,
23

J=2.6 Hz, 1H, naphthyl), 6.71 (m, 3H, sal, 1’-H), 5.61 (dd, J2’,1’=1.8 Hz, J2’,3’=6.2 Hz, 1H, 2’-
H), 5.18 (dd, J3’,2’=6.0 Hz, J3’,4’=2.0 Hz, 1H, 3’-H), 4.49 (dt, J4’,3’=2.3 Hz, J4’,5’=6.3 Hz, 1H,
4’-H), 4.09 (dd, J5’a,4’=6.8 Hz, J5’a,5’b=10.8 Hz, 1H, 5’a-H), 3.93 (m, 4H, 5’b-H, OCH3), 1.58
and 1.37 (2×s, 6H, C(CH3)2). 13C NMR (DMSO-d6) δ 171.17 (CO), 160.71 (C-OHsal), 157.76
(C-6naphthyl), 152.78 and 151.45 (C-4, C-6), 139.51 (C-7a), 134.52 and 134.14 (naphthyl),
132.62 (sal), 130.68 (C-3a), 130.08, 129.84, 128.38, 126.69, 125.99, 125.44, 119.72, 118.89,
117.54 and 116.54 (sal, naphthyl), 113.22 ((CH3)2C), 105.83 (naphthyl), 90.80 and 90.28 (C-
1’, C-7), 84.89 (C-4’), 83.25 (C-2’), 81.59 (C-3’), 67.22 (C-5’), 55.24 (OCH3), 26.73 and
24.98 ((CH3)2C). HRMS [M+H]+ calcd for C31H31N6O9S: 663.1868; found: 663.1870.
4.2.38. General procedure for the preparation of compounds 6a–f
Compound 17a–f was treated with cooled 80% aq trifluoroacetic acid (10 ml/mmol) and,
if necessary to dissolve the starting material, MeOH was added. The resulting mixture was
stirred at 5 ºC until deprotection was complete. Then it was evaporated and co-evaporated
several times with MeOH. The residue was chromatographed on silica gel column with
EtOAc-MeOH-Et3N (90:10:0.5→70:30:0.5) to obtain product 6a–f as triethylammonium salt
(solid foam, 65-75% yield).
4.2.39. 4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-ribofuranosyl}-6-phenyl-1H-
[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6a)
UV (MeOH): λmax 245.5 nm (37700), 309 nm (16200). UV (H2O): λmax 245 nm (30800),
303.5 nm (12700). Fluorescence emission in MeOH: λmax 419 nm, ΦF = 0.37; in H2O: λmax
433 nm, ΦF = 0.0015. 1H NMR (DMSO-d6) δ 13.57 (s, 1H, OH), 9.09 (brs, 1H, Et3NH+), 8.16
(m, 2H, Ph), 7.79 (dd, J=2.0 Hz, J=7.6 Hz, 1H, sal), 7.57 (s, 1H, 7-H), 7.39 (m, 3H, Ph), 7.25
(m, 3H, NH2, sal), 6.72 (m, 2H, sal), 6.27 (d, J1’,2’=5.2 Hz, 1H, 1’-H), 5.62 (d, J2’-OH,2’-H=6.0
Hz, 1H, 2’-OH), 5.46 (d, J3’-OH,3’-H=5.2 Hz, 1H, 3’-OH), 4.71 (m, 1H, 2’-H), 4.36 (m, 1H, 3’-
H), 4.25 (m, 2H, 4’-H, 5’a-H), 4.16 (m, 1H, 5’b-H), 3.04 (brs, 6H, (CH3CH2)3NH+), 1.15 (t,
J=7.4 Hz, 9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ 170.92 (CO), 160.67 (C-OHsal),
152.47 and 151.45 (C-4, C-6), 139.05 (C-7a), 138.88 (Ph), 132.57 (sal), 130.89 (C-3a),
129.83 (sal), 128.58, 128.33 and 127.07 (Ph), 119.85, 117.51 and 116.55 (sal), 90.48 and
90.33 (C-1’, C-7), 82.97 (C-4’), 73.03 (C-2’), 70.55 (C-3’), 68.66 (C-5’), 45.71
((CH3CH2)3NH+), 8.62 ((CH3CH2)3NH+). HRMS [M-H]- calcd for C23H21N6O8S: 541.1147;
found: 541.1141.
24

4.2.40.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-ribofuranosyl}-6-(4-
methylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6b)
UV (MeOH): λmax 250 nm (33700), 311.5 nm (16400). UV (H2O): λmax 249.5 nm (27400),
306.5 nm (14000). Fluorescence emission in MeOH: λmax 412 nm, ΦF = 0.06; in H2O: λmax
1
433 nm, ΦF = 0.0018. H NMR (DMSO-d6) δ 13.56 (s, 1H, OH), 8.06 (d, J=8.0 Hz, 2H, Ph),
7.80 (dd, J=1.8 Hz, J=7.8 Hz, 1H, sal), 7.52 (s, 1H, 7-H), 7.24 (m, 5H, Ph, sal, NH2), 6.72 (m,
2H, sal), 6.25 (d, J1’,2’=5.6 Hz, 1H, 1’-H), 5.61 (d, J2’OH,2’H=6.0 Hz, 1H, 2’-OH), 5.46 (d,
J3’OH,3’H=5.2 Hz, 1H, 3’-OH), 4.70 (m, 1H, 2’-H), 4.35 (m, 1H, 3’-H), 4.24 (m, 2H, 4’-H, 5’a-
H), 4.17 (m, 1H, 5’b-H), 3.02 (m, 6H, (CH3CH2)3NH+), 2.32 (s, 3H, CH3), 1.15 (t, J=7.2 Hz,
9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ 171.37 (CO), 160.64 (C-OHsal), 152.15 and
151.27 (C-4, C-6), 139.02 (C-7a), 138.01 and 135.81 (Ph), 132.48 (sal), 130.74 (C-3a),
129.79 (sal), 128.93 and 126.93 (Ph), 119.84, 117.43 and 116.48 (sal), 90.34 and 90.06 (C-1’,
C-7), 82.96 (C-4’), 72.97 (C-2’), 70.51 (C-3’), 68.63 (C-5’), 45.50 ((CH3CH2)3NH+), 20.73
(CH3), 8.46 ((CH3CH2)3NH+). HRMS [M-H]- calcd for C24H23N6O8S: 555.1304; found:
555.1294.
4.2.41.
4-Amino-6-(4-fluorophenyl)-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-
ribofuranosyl}-1H-[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6c)
UV (MeOH): λmax 246 nm (39800), 308.5 nm (16000). UV (H2O): λmax 245 nm (30000),
304.5 nm (13400). Fluorescence emission in MeOH: λmax 420 nm, ΦF = 0.11; in H2O: λmax
1
442 nm, ΦF = 0.002. H NMR (DMSO-d6) δ 13.56 (s, 1H, OH), 8.21 (m, 2H, Ph), 7.79 (m,
1H, sal), 7.56 (s, 1H, 7-H), 7.23 (m, 5H, NH2, Ph, sal), 6.72 (m, 2H, sal), 6.26 (d, J1’,2’=5.6
Hz, 1H, 1’-H), 5.61 (d, J2’-OH,2’H=6.4 Hz, 1H, 2’-OH), 5.46 (d, J3’-OH,3’-H=4.8 Hz, 1H, 3’-OH),
4.70 (m, 1H, 2’-H), 4.36 (m, 1H, 3’-H), 4.24 (m, 2H, 4’-H, 5’a-H), 4.18 (m, 1H, 5’b-H), 3.08
(m, 6H, (CH3CH2)3NH+), 1.17 (t, J=7.4 Hz, 9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ
170.90 (CO), 162.56 (d, JC,F=244.88 Hz, C-4Ph), 160.65 (C-OHsal), 151.45 and 151.35 (C-4,
C-6), 138.93 (C-7a), 135.33 (Ph), 132.57 (sal), 130.85 (C-3a), 129.81 (sal), 129.18 (d,
JC,F=8.42 Hz, C-2Ph, C-6Ph), 119.82, 117.50 and 116.54 (sal), 115.06 (d, JC,F=21.47 Hz, C-3Ph,
C-5Ph), 90.43 and 90.37 (C-1’, C-7), 83.07 (C-4’), 72.92 (C-2’), 70.49 (C-3’), 68.65 (C-5’),
19
45.68 ((CH3CH2)3NH+), 8.60 ((CH3CH2)3NH+). F NMR (DMSO-d6) δ -38.30. HRMS [M-
H]- calcd for C23H20N6O8SF: 559.1053; found: 559.1051.
4.2.42.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-ribofuranosyl}-6-(4-
trifluoromethylphenyl)-1H-[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6d)
25

UV (MeOH): λmax 244 nm (46900), 310 nm (18700). UV (H2O): λmax 244 nm (46900),
309 nm (18000). Fluorescence emission in MeOH: λmax 409 nm, ΦF = 0.32; in H2O: λmax 422
nm, ΦF = 0.0019. 1H NMR (DMSO-d6) δ 13.54 (s, 1H, OH), 8.39 (d, J=8.4 Hz, 2H, Ph), 7.76
(m, 4H, Ph, sal, 7-H), 7.37 (s, 2H, NH2), 7.24 (m, 1H, sal), 6.71 (m, 2H, sal), 6.28 (d,
J1’,2’=6.0 Hz, 1H, 1’-H), 5.62 (d, J2’-OH,2’-H=6.4 Hz, 1H, 2’-OH), 5.47 (d, J3’-OH,3’-H=5.2 Hz, 1H,
3’-OH), 4.69 (m, 1H, 2’-H), 4.36 (m, 1H, 3’-H), 4.24 (m, 3H, 4’-H, 5’-H), 3.09 (m, 6H,
(CH3CH2)3NH+), 1.17 (t, J=7.2 Hz, 9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ 171.00
(CO),160.66 (C-OHsal), 151.64 and 150.57 (C-4, C-6), 142.67 (Ph), 138.62 (C-7a), 132.55
(sal), 131.35 (C-3a), 129.78 (sal), 128.62 (q, JC,F=32.12 Hz, C-4Ph), 127.70 (Ph), 125.17 (q,
JC,F=3.85 Hz, C-3Ph, C-5Ph), 124.39 (q, JC,F=276.08 Hz, CF3), 119.77, 117.46 and 116.52 (sal),
91.84 and 90.67 (C-1’, C-7), 83.27 (C-4’), 72.92 (C-2’), 70.43 (C-3’), 68.65 (C-5’), 45.68
((CH3CH2)3NH+), 8.60 ((CH3CH2)3NH+). 19F NMR (DMSO-d6) δ 14.74. HRMS [M-H]- calcd
for C24H20N6O8SF3: 609.1021; found: 609.1013.
4.2.43. 4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-ribofuranosyl}-6-(napht-2-yl)-
1H-[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6e)
UV (MeOH): λmax 251 nm (46300), 315 nm (14800). UV (H2O): λmax 250 nm (48200),
311.5 nm (13600). Fluorescence emission in MeOH: λmax 412 nm, ΦF = 0.38; in H2O: λmax
420 nm, ΦF = 0.0025. 1H NMR (DMSO-d6) δ 13.58 (s, 1H, OH), 8.73 (s, 1H, naphthyl), 8.34
(dd, J=1.8 Hz, J=8.6 Hz, 1H, naphthyl), 8.06 (m, 1H, naphthyl), 7.92 (m, 2H, naphthyl), 7.80
(dd, J=1.8 Hz, J=7.8 Hz, 1H, sal), 7.74 (s, 1H, 7-H), 7.51 (m, 2H, naphthyl), 7.32 (s, 2H,
NH2), 7.24 (m, 1H, sal), 6.72 (m, 2H, sal), 6.30 (d, J1’,2’=6.0 Hz, 1H, 1’-H), 5.64 (d,
J2’OH,2’H=6.0 Hz, 1H, 2’-OH), 5.48 (d, J3’OH,3’H=5.2 Hz, 1H, 3’-OH), 4.75 (m, 1H, 2’-H), 4.38
(m, 1H, 3’-H), 4.28 (m, 2H, 4’-H, 5’a-H), 4.20 (m, 1H, 5’b-H), 3.07 (brs, 6H,
(CH3CH2)3NH+), 1.16 (t, J=7.2 Hz, 9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ 170.98
(CO), 160.68 (C-OHsal), 152.22 and 151.51 (C4, C6), 139.02 (C-7a), 136.29, 133.05 and
133.01 (naphthyl), 132.52 (sal), 131.01 (C-3a), 129.83 (sal), 128.65, 127.68, 127.33, 126.34,
126.14, 126.05 and 125.11 (naphthyl), 119.82, 117.46 and 116.51 (sal), 90.92 and 90.40 (C-
1’, C-7), 83.05 (C-4’), 72.92 (C-2’), 70.53 (C-3’), 68.63 (C-5’), 45.65 ((CH3CH2)3NH+), 8.62
((CH3CH2)3NH+). HRMS [M-H]- calcd for C27H23N6O8S: 591.1304; found: 591.1295.
4.2.44.
4-Amino-1-{5-O-[N-(2-hydroxybenzoyl)sulfamoyl]-β-D-ribofuranosyl}-6-(6-
methoxynapht-2-yl)-1H-[1,2,3]triazole[4,5-c]pyridine triethylammonium salt (6f)
26

UV (MeOH): λmax 247.5 nm (47000), 322.5 nm (21200). UV (H2O): λmax 243 nm (26600),
318.5 nm (12600). Fluorescence emission in MeOH: λmax 418 nm, ΦF = 0.5; in H2O: λmax 425
nm, ΦF = 0.007. 1H NMR (DMSO-d6) δ 13.57 (s, 1H, OH), 8.65 (d, J=1.6 Hz, 1H, naphthyl),
8.30 (dd, J=1.8 Hz, J=8.6 Hz, 1H, naphthyl), 7.96 (d, J=8.8 Hz, 1H, naphthyl), 7.82 (m, 2H,
naphthyl, sal), 7.68 (s, 1H, 7-H), 7.27 (m, 4H, naphthyl, sal, NH2), 7.15 (dd, J=2.6 Hz, J=9.0
Hz, 1H, naphthyl), 6.72 (m, 2H, sal), 6.29 (d, J1’,2’=6.0 Hz, 1H, 1’-H), 5.64 (d, J2’OH,2’H=6.0
Hz, 1H, 2’-OH), 5.48 (d, J3’OH,3’H=5.2 Hz, 1H, 3’-OH), 4.74 (m, 1H, 2’-H), 4.38 (m, 1H, 3’-
H), 4.24 (m, 3H, 4’-H, 5’-H), 3.89 (s, 3H, OCH3), 3.04 (q, J=7.2 Hz, 6H, (CH3CH2)3NH+),
1.16 (t, J=7.4 Hz, 9H, (CH3CH2)3NH+). 13C NMR (DMSO-d6) δ 171.01 (CO), 160.69 (C-
OHsal), 157.68 (C-6naphthyl), 152.47 and 151.45 (C-4, C-6), 139.05 (C-7a), 134.46 and 134.08
(naphthyl), 132.56 (sal), 130.90 (C-3a), 130.23 (naphthyl), 129.85 (sal), 128.42, 126.61,
125.94 and 125.59 (naphthyl), 119.83 (sal), 118.69 (naphthyl), 117.49 and 116.53 (sal),
105.70 (naphthyl), 90.41 and 90.35 (C-1’, C-7), 83.05 (C-4’), 72.89 (C-2’), 70.53 (C-3’),
68.66 (C-5’), 55.20 (OCH3), 45.62 ((CH3CH2)3NH+), 8.71 ((CH3CH2)3NH+). HRMS [M-H]-
calcd for C28H25N6O9S: 621.1409; found: 621.1402.
4.3. MbtA Enzyme Assay
MbtA was expressed in E. coli and purified as described. MbtA concentration was
determined by active site titration with 2 as described [34]. The inhibition assays were
performed as reported in duplicate under initial velocity conditions. In brief the reaction was
initiated by adding 10 µL [32P]PPi with 7 nM MbtA in 90 µL reaction buffer (278 µM
salicylic acid, 11.1 mM ATP, 1.11 mM PPi, 83.3 mM Tris-HCl, pH 7.5, 11.1 mM MgCl2,
2.22 mM DTT) at 37 °C in the presence of eight different concentrations of the inhibitor (1.5-
fold dilution from 100 nM down to 8.78 nM and a 3-fold dilution to 2.93 nM). The reaction
was terminated at 20 min by the addition of 200 µL of quenching buffer (350 mM HClO4, 100
mM PPi, 1.8 % w/v activated charcoal). The charcoal was pelleted by centrifugation and
washed once with 500 µL H2O and analyzed by liquid scintillation counting as described.
Fractional initial velocities were fit by nonlinear regression analysis to the Morrison equation
(eq 1) using GraphPad Prism 5.0
(eq 1)
where, I represents the concentration of inhibitor, Kiapp is the apparent inhibition constant, E is
27

the active enzyme concentration (determined by active-site titration), vi is the initial rate at
each [I], and v0 is the initial rate of the DMSO control. A fitted curve for compound 6e is
shown (Fig. 3) as an example.
Figure 3. Dose-response of fractional initial velocity of [32P]-ATP formation catalyzed by
MbtA as a function of inhibitor 6e concentration.
4.4. M. tuberculosis H37Rv MIC Assay
All compounds minimum inhibitory concentrations (MICs) were experimentally
determined as previously described. MICs were determined in triplicate in iron-deficient
GAST according to the broth microdilution method using compounds from DMSO stock
solutions or with control wells treated with an equivalent amount of DMSO. Isoniazid was
used as a positive control while DMSO was employed as a negative control. All
measurements reported herein used an initial cell density of 104–105 cells/assay and growth
monitored at 10–14 days, with the untreated and DMSO-treated control cultures reaching an
OD620 0.2–0.3. Plates were incubated at 37 C (100 µL/well) and growth was recorded by
measurement of optical density at 620 nm.
Acknowledgments
This work was supported by the Polish Ministry of Science and Higher Education grant
no. N N405 2516 33 (J.Z.), under statutory financing, and under the KNOW program. It was
also supported by a grant from the NIH (AI070219 to C.C.A.) and the Intramural Research
28

Program of the NIAID, NIH (C.E.B.). We wish to thank Dr. Lukasz Marczak and Dr. Barbara
Swarcewicz for MS spectra measurements.
Supplementary data
Supplementary data associated with this article can be found in the online version at
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