Synthesis of new imidazopyridine nucleoside derivatives designed as maribavir analogues

Article abstract of DOI:10.3390/molecules25194531

The strong inhibition of Human Cytomegalovirus (HCMV) replication by benzimidazole nucleosides, like Triciribine and Maribavir, has prompted us to expand the structure-activity relationships of the benzimidazole series, using as a central core the imidazo[4,5-b]pyridine scafflold. We have thus synthesized a number of novel amino substituted imidazopyridine nucleoside derivatives, which can be considered as 4-(or 7)-aza-d-isosters of Maribavir and have evaluated their potential antiviral activity. The target compounds were synthesized upon glycosylation of suitably substituted 2-aminoimidazopyridines, which were prepared in six steps starting from 2-amino-6-chloropyridine. Even if the new compounds possessed only a slight structural modification when compared to the original drug, they were not endowed with interesting antiviral activity. Even so, three derivatives showed promising cytotoxic potential.

Full text of DOI:10.3390/molecules25194531

molecules
Article
Synthesis of New Imidazopyridine Nucleoside
Derivatives Designed as Maribavir Analogues
1
1
2
1
2
Georgios Papadakis , Maria Gerasi , Robert Snoeck , Panagiotis Marakos , Graciela Andrei
,
1
,
1
Nikolaos Lougiakis * and Nicole Pouli
1
Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of
Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece; gpapadakis@pharm.uoa.gr (G.P.);
mairager@pharm.uoa.gr (M.G.); marakos@pharm.uoa.gr (P.M.); pouli@pharm.uoa.gr (N.P.)
Laboratory of Virology & Chemotherapy, Rega Institute, KU Leuven, 3000 Leuven, Belgium;
robert.snoeck@kuleuven.be (R.S.); graciela.andrei@kuleuven.be (G.A.)
2
*
Correspondence: nlougiak@pharm.uoa.gr
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Katherine Seley-Radtke
Received: 10 September 2020; Accepted: 29 September 2020; Published: 3 October 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The strong inhibition of Human Cytomegalovirus (HCMV) replication by benzimidazole
nucleosides, like Triciribine and Maribavir, has prompted us to expand the structure–activity
relationships of the benzimidazole series, using as a central core the imidazo[4,5-b]pyridine scaffold.
We have thus synthesized a number of novel amino substituted imidazopyridine nucleoside
derivatives, which can be considered as 4-(or 7)-aza-d-isosters of Maribavir and have evaluated
their potential antiviral activity. The target compounds were synthesized upon glycosylation of
suitably substituted 2-aminoimidazopyridines, which were prepared in six steps starting from
2
-amino-6-chloropyridine. Even if the new compounds possessed only a slight structural modification
when compared to the original drug, they were not endowed with interesting antiviral activity. Even
so, three derivatives showed promising cytotoxic potential.
Keywords: benzimidazole; nucleosides; imidazo[4,5-b]pyridine; HCMV; antiviral activity;
antiproliferative activity
1
. Introduction
Human cytomegalovirus (HCMV) is a prevalent herpesvirus, with IgG antibodies indicating past
infection found in approximately 60% of adults in developed countries and almost 100% in developing
ones [ ]. Although HCMV infection rarely leads to clinical manifestations in immunocompetent hosts,
there is an increasing amount of data associating lifelong viral persistence with vascular diseases
atherosclerosis [ ], hypertension [ ]) and the progression of some cancer types [ ]. In addition, HCMV
is a major opportunistic pathogen in immunocompromised individuals, posing a serious threat to
neonates, allograft recipients and AIDS patients [ ]. Perinatal infection can cause irreversible hearing
1
(
2
3
4
5
loss, blindness and mental retardation, while immunosuppressed adults may develop multi-organ
failure syndrome, which is a life-threatening condition [1,5].
Ganciclovir (GCV) and its orally bioavailable prodrug, Valganciclovir, have served as gold
standards for pre-emptive therapy and prophylaxis against HCMV in solid organ transplant patients [
as well as for the treatment of CMV retinitis [ ] for almost 30 years. However, their myelosuppressive
potential precludes their prophylactic use in stem cell transplant recipients, which has led to their
administration, mostly upon engraftment [ ]. In addition, mutations mapping in the gene encoding
6]
7
8
for the UL97 kinase (responsible for the first phosphorylation of GCV towards its active triphosphate
form) and in the UL54 gene encoding for the DNA polymerase (target of ganciclovir triphosphate),
have led to the emergence of drug resistant strains [
9]. Furthermore, the DNA polymerase inhibitors
Molecules 2020, 25, 4531; doi:10.3390/molecules25194531
www.mdpi.com/journal/molecules

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Cidofovir and Foscarnet, which are currently approved as second line agents, have several drawbacks
that limit their clinical use, namely severe side effects and poor pharmacokinetic properties [10].
It has become clear that there is an urgent need for better tolerated and more effective antiviral
drugs, in order to fully address the health risks posed by HCMV. There are four compounds that
have been considered or are at an advanced stage of clinical development for this purpose (Figure 1).
Brincidofovir is a per os administered hexadecyloxypropylester of Cidofovir aimed at addressing the
parent compound’s dose-limiting renal toxicity [11]. The development of Brincidofovir for therapy of
HCMV has been halted because of increased gastrointestinal toxicity of the oral formulation in adult
hematopoietic cell transplant recipients. The non-nucleoside guanosine analogue Cyclopropavir, which
shares the same mechanism of action with GCV, has proven to be more potent in HCMV inhibition
in vitro [12] and Phase I trials have been recently completed. Furthermore, the search for novel
molecular targets within the viral life cycle has led to the fast track approval of the terminase inhibitor
Letermovir in late 2017, for the prophylaxis of HCMV infection and disease in adult HCMV-seropositive
recipients of an allogeneic human stem cell transplant (HSCT) [13]. Phase II clinical trials are also
about to be launched for the use of Letermovir in paediatric patients who underwent an HSCT.
Figure 1. Recently developed antiviral agents against HCMV.
At the same time, the benzimidazole l-riboside Maribavir is about to be evaluated in Phase III
trials involving transplant recipients with HCMV infections that are refractory or resistant to the
currently approved drugs as well as for its potential superiority over Valganciclovir in HSCT patients.
Maribavir was developed in the late 1990s and has been proven to inhibit viral DNA synthesis as well
as nucleocapsid egress from the nucleus via the inhibition of the viral kinase UL97 [14]. However,
initial Phase III clinical trials failed to prove sufficient benefits for post-transplant patients. This has
later on been accredited to the pre-emptive therapy with GCV that several patients had received prior
to surgery and to the low daily administered dose [15].
As a continuation of our previous involvement with the synthesis and evaluation of purine
isosteric bioactive nucleosides [16–19], we designed and synthesized a number of novel nucleoside
derivatives, which can be considered as 4-(or 7)-aza-d-isosters of Maribavir, having in mind that the
d-enantiomer of Maribavir possesses interesting anti-HCMV properties as well [20]. Our goal was to

Molecules 2020, 25, 4531
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expand the structure–activity relationships of the benzimidazole series to the less-studied and more
“
purine-like” imidazo[4,5-b]pyridine scaffold, retaining the pattern of the 5,6-dichlorosubstitution in
the new compounds. The diverse nature of the 2-amino groups in each of the final pair of isomeric
nucleosides was at the core of our attempt to explore the spatial limitations of possible target enzymes
as well as to gain some insight on potential interactions developed. Within this context, we disclose
herein the preparation and pharmacological evaluation of the 1- and 3-regioisomeric β-d-ribosides of
5
,6-dichloroimidazo[4,5-b]pyridine, introducing various amino substituents at the vacant position of
the imidazole ring.
2
. Results and Discussion
2
.1. Chemistry
The synthetic route we envisaged in order to gain access to the target nucleosides involved the
direct glycosylation of the suitably substituted imidazo[4,5-b]pyridines 7a
intermediate for the synthesis of these derivatives is 5,6-dichloropyridine-2,3-diamine (
–
d
(Scheme 1). A common
), which
6
was prepared following a five-step procedure, previously described by our group, starting from the
pyridinamine 1 [21].
◦
Scheme 1. Reagents and conditions: (
8 h; ( ) conc. HCl, H O, EtOH, reflux, 2 h; (
conc. HCl, 60 C, 40 min; (
0 min for 7a–c, or BrCN, MeOH, H O, 24 h for 7d.
a
) pivaloyl chloride, Et N, DCM, r.t., 22 h; (
b
) NCS, DMF, 100 C,
) conc. HNO , conc. H SO , r.t., 45 min; ( ) SnCl2 2H O,
) (i) N-alkylisothiocyanate, dry THF, reflux, 36 h, (ii) HgO, dry THF, reflux,
3
1
c
d
e
·
2
3
2
4
2
◦
f
4
2
Amino substituted imidazopyridine derivatives 7a
procedure, which involves an initial treatment of
N-alkylisothiocyanates in refluxing THF. The resulting mixture of isomeric thioureas undergoes rapid
cyclodesulfurization in the presence of mercury oxide [22], yielding compounds 7a . Concerning the
–c
were prepared using a one-pot-two-stage
6
with appropriately substituted
–
c
preparation of the primary aminoderivative 7d, we implemented a different approach, which had been
previously reported by Townsend [23], for the synthesis of the corresponding benzimidazole analogue.
Thus, the addition of cyanogen bromide in a suspension of diamine 6 in a 1:1 mixture of MeOH and
water provided 7d as the sole reaction product in very good yield.
The target N1- and N3-ribonucleoside acetates were prepared under modified Vorbrüggen
conditions. The in situ formation of the persilylated heterocyclic bases by treatment of the corresponding
heterocyclic compounds 7a
of peracetylated -d-ribofuranose and trimethylsilyltrifluoromethanesulfonate (TMSOTf) (Scheme 2).
Thus, concerning the glycosylation reaction of compounds 7a , the major products isolated were the
-d-ribosides 8a , as a mixture with a small amount of the corresponding -anomers 9a . The N1
with their corresponding -d
–d with N,O-bistrimethylsilylacetamide (BSA) was followed by the addition
β
–c
3-
β
–c
α
–c
regioisomers were also obtained as mixtures of the 1-
β
-d ribosides 10a
–
c
α
anomers 11a–c.

Molecules 2020, 25, 4531
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Scheme 2. Reagents and conditions: (
a) (i) N,O-bis(trimethylsilyl)acetamide, ACN, reflux, 2h,
(ii) tetra-O-acetyl-β-d-ribofuranose, TMSOTf, reflux, 3h; (b) NH3(g), MeOH, rt, 18h.
Regarding the N1 regioisomers, we were able to isolate pure N1-
as well as their corresponding N1- -d anomers 11a , upon chromatographic purification. The site
of ribosylation as well as the anomeric configuration were unambiguously determined on the basis
of NOE spectroscopy. Taking into consideration the NOE spectra of compounds 10a and 11a–c,
β-d nucleoside acetates 10a–c
α
–c
–
c
we observed clear correlation peaks between the aromatic proton and protons of the furanose ring,
determinant of the N1-ribosylation pattern. In addition, we also noticed cross correlation peaks
0
0
between 1 -H and 4 -H of the sugar moiety in the spectra of compounds 10a
observed on the NOE spectra of 11a , thus clearly concluding that 11a were the N1-
products of the reaction, while 10a were their corresponding -d anomers. Deacetylation of 10a
with methanolic ammonia provided the final compounds 13a–c.
Isolation of the pure 3- -d nucleoside acetates proved to be difficult at this stage, so the mixtures
of 8a with their corresponding -anomers 9a were subjected to ammoniolysis, to provide the
–
c
. Such peaks were not
–
c
–
c
α
-d nucleoside
–
c
β
–c
β
–
c
α
–c
deprotected nucleosides. The ethylamino (12a) and isopropylamino (12b) derivatives were isolated in
pure form by recrystallization, whereas an analytically pure sample of the benzylamino compound 12c
was obtained upon purification with semi-preparative HPLC. Anomeric purity and configuration of
1
compounds 12a
–
c
were determined on the basis of H-NMR and NOE spectra, respectively. In the
0
0
latter ones, we observed clear correlation peaks between 1 -H and 4 -H of the ribofuranose moiety,
while there was a profound absence of correlation peaks between the aromatic and sugar protons on
the NOE spectra of each of the aforementioned compounds. The close examination of 1D and 2D
NMR spectra reveals that a simple differentiation between each pair of regio isomers can be easily
made upon inspection of the chemical shift of the aromatic proton. This proton appears upfield in the
case of the N3-isomers 12a
N1-isomers 13a–d.
–d (7.7–7.8 ppm) whereas it shifts downfield (8.0–8.1 ppm) concerning the
Applying the same reaction conditions for the glycosylation of the primary aminoderivative 7d
we isolated from the reaction mixture the 3- -d nucleoside acetate (8d), as well as the 1- -d isomer
10d), without detectable amounts of the corresponding -anomers. Deprotection of 8d and 10d with
,
β
β
(
α
methanolic ammonia provided the respective final ribosides 12d and 13d. However, the major product
of the glycosylation reaction of 7d turned out to be the di-ribosylated compound 14 (Figure 2), whose
1
13
structure was elucidated on the basis of H-NMR, C-NMR, NOE and mass spectra. Two sets of
sugar carbon peaks were observed on the 13C-NMR spectrum of 14, in the presence of only one set of
aromatic carbon peaks. The sites of substitution as well as the anomeric configuration were assigned
as exemplified in Figure 2 (namely N,1-β-d). Upon careful examination of the NOE spectrum of 14,
we observed a set of correlation peaks between the aromatic proton and protons of one sugar moiety
as well as correlation peaks between the anomeric proton of each furanose with its corresponding
0
4
-proton.

Molecules 2020, 25, 4531
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Figure 2. Structure of di-ribosylated by-product formed upon glycosylation of the amino derivative 7d
.
2
.2. Biological Evaluation
Compounds 12a–d and 13a–d were evaluated for their activity against Vaccinia virus, Adeno
virus-2, Human Coronavirus (229E), HSV-1, HSV-2, VZV, and HCMV (AD-169 and Davis strains) in
human embryonic lung cell cultures (HEL) (Tables S1–S3). Unfortunately, the new derivatives proved
to lack antiviral activity against all viruses tested. In particular, the substitution of the benzimidazole
pharmacophore present in Maribavir by the imidazo[4,5-b]pyridine scaffold, in combination with the
replacement of the l-sugar configuration with its corresponding d-riboside, resulted in total loss of
the anti-HCMV activity. Nevertheless, upon determination of the cytotoxic properties of the new
derivatives, compounds 12c, 12d and 13c, proved to possess moderate antiproliferative activity against
the three cancer cell lines tested, namely human T-lymphocyte cells (CEM), human cervix carcinoma
cells (HeLa) and human dermal microvascular endothelial cells (HMEC-1) (Table S4). Among these
compounds, both 2-benzylamino-substituted derivatives 12c and 13c showed antiproliferative activity
against CEM cell-line, with equipotent IC50 values (37
proved active in inhibiting the growth of HeLa and HMEC-1 cell lines, possessing IC values of
±
3 and 39
±
8µM) and at the same time, 12c
50
3
6
±
7
µ
M and 20
±
2 µM, respectively. The increased antiproliferative effects of 12c and 13c are worth
investigating further and are currently under active search in our laboratories.
3
. Materials and Methods
3
.1. General Information
Tetrahydrofuran (THF) was distilled from sodium/benzophenone ketyl immediately prior to use.
1
2
Melting points were determined on a Büchi apparatus and are uncorrected. H-NMR spectra and D
COSY, NOESY, HSQC, HMBC) NMR spectra were recorded on a Bruker Avance III 600 or a Bruker
(
13
Avance (Bruker, Karlsruhe, Germany) DRX 400 instrument, whereas C-NMR spectra were recorded
on a Bruker Avance III 600 in deuterated solvents and were referenced to TMS ( scale). The signals
δ
1
13
1
1
of H and C spectra were unambiguously assigned by using 2D NMR techniques: H H COSY,
NOESY, HSQC and HMBC. Mass spectra were recorded with a LTQ Orbitrap Discovery instrument,
possessing an Ionmax ionization source. Column chromatography was performed on Merck (Merk,
Darmstadt, Germany) silica gel 60 (0.040–0.063 mm), unless specified otherwise. Analytical thin
layer chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel F-254 plates.
Preparative HPLC was performed on a system equipped with two Prep LabAlliance pumps (ASI,
Richmond, CA, USA), a Fortis C-18 (5
detector (ECOM, Praha, Czech Republic). Optical rotations were obtained on a Perkin-Elmer 341
Polarimeter (Perkin Elmer, Shelton, CT, USA).
µ
m) column (i.d. 10
×
250 mm) and a FLASH 06S DAD 600
3
.2. Synthesis
,6-Dichloro-N-ethyl-3H-imidazo[4,5-b]pyridin-2-amine(7a): Ethylisothiocyanate (1 mL, 11.24
mmol) was added to a solution of diamine (500 mg, 2.81 mmol) in anhydrous THF (10 mL) and the
5
6
reaction mixture was refluxed under argon for 36 h. The mixture was then cooled to room temperature
and HgO (1.22 g, 5.65 mmol) was added, leading to an orange suspension. Refluxing the reaction
mixture resulted progressively in a black coloured suspension and within 40 min the reaction was

Molecules 2020, 25, 4531
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completed. The mixture was filtered warm through a celite pad, which was thoroughly washed with
warm MeOH. The solvent was then vacuum-evaporated and the residue was purified by column
chromatography using a mixture of CHCl /MeOH (95/5 to 85/15, v/v) as the eluent, to result in 340 mg
3
◦
1
of 7a (52% overall yield). Beige solid, mp > 300 C (dec), (MeOH). H-NMR (DMSO-d , 600MHz)
δ 1.16
6
(
t, 3H, CH , J = 7.2 Hz), 3.33 (m, 2H, CH ), 7.37 (br s, 1H, D O exchangeable, NH), 7.57 (s, 1H, H-7).
3
2
2
1
3
C-NMR (151 MHz, DMSO-d6)
δ 14.87 (CH ), 36.82 (CH ), 116.40 (C-6), 116.58 (C-7, major tautomeric
3 2
form), 121.73 (C-7, minor tautomeric form), 126.92 (C-7a), 137.46 (C-5), 156.20 (C-3a), 158.92 (C-2).
+
HR-MS (ESI) m/z: Calcd for C H Cl N : [M + H] = 231.0199, found 231.0202.
8
9
2
4
5,6-Dichloro-N-isopropyl-3H-imidazo[4,5-b]pyridin-2-amine (7b):This compound was prepared
by a procedure analogous to that described for 7a, upon reaction of diamine (500 mg, 2.81 mmol) with
6
isopropyl isothiocyanate (0.90 mL, 8.43 mmol). Purification was effected by column chromatography,
using a mixture of DCM/EtOAc (70/30 to 20/80, v/v) and then EtOAc/MeOH (99/1 to 97/3, v/v) as eluents.
◦
1
Overall yield 55%. White solid, mp 235–236 C (acetone). H-NMR (600 MHz, DMSO-d )
δ
1.20 (d,
6
6
H, 2xCH , J = 6.1 Hz), 3.92 (m, 1H, CH), 7.34 (br s, 1H, D O exchangeable, NH), 7.56 (s, 1H, H-7),
3
2
1
1.06 (br s, 1H, D O exchangeable, imidazole NH of the major tautomeric form), 11.66 (br s, 1H, D O
2
2
13
exchangeable, imidazole NH of the minor tautomeric form). C-NMR (151 MHz, DMSO-d6)
δ
22.58
(
CH ), 43.97 (CH), 116.37 (C-6), 116.61 (C-7 of the major tautomeric form), 121.62 (C-7 of the minor
3
tautomeric form), 126.86 (C-7a), 137.41 (C-5), 156.22 (C-3a), 158.24 (C-2). HR-MS (ESI) m/z: Calcd for
+
C H Cl N : [M + H] = 245.0355, found 245.0352.
9
11
2
4
N-Benzyl-5,6-dichloro-3H-imidazo[4,5-b]pyridin-2-amine (7c): This compound was prepared by
a procedure analogous to that described for 7a, upon reaction of diamine (500 mg, 2.81 mmol) with
6
benzyl isothiocyanate (0.80 mL, 6.03 mmol). Purification was effected by column chromatography,
using a mixture of DCM/EtOAc (90/10 to 50/50, v/v) and then EtOAc/MeOH (99/1 to 90/10, v/v) as
◦
1
eluents. Overall yield 51%. White solid, mp: 277–278 C (MeOH). H-NMR (600 MHz, DMSO-d )
δ
6
0 0 0
.55 (d, 2H, CH , J = 6.0 Hz), 7.24 (t, 1H, phenyl H-4 ’, J = 7.2 Hz), 7.32 (t, 2H, phenyl H-3 ’, H-5 ’, J = 7.6
2
4
0
0
Hz), 7.36 (d, 2H, phenyl H-2 ’, H-6 ’, J = 7.3 Hz), 7.60 (s, 1H, H-7), 7.94 (br s, 1H, D O exchangeable,
NH), 11.38 (br s, 1H, D O exchangeable, imidazole NH). C-NMR (151 MHz, DMSO-d )
CH ), 116.89 (C-6), 117.05 (C-7), 126.97 (phenyl C-4 ’), 127.13 (C-7a), 127.25 (phenyl C-3 ’, C-5 ’), 128.33
phenyl C-2 ’, C-6 ’), 137.50 (C-5), 139.34 (phenyl C-1 ’), 155.88 (C-3a), 158.81 (C-2). HR-MS (ESI) m/z:
2
13
δ 45.36
2
6
0
0
0
(
(
2
0
0
0
+
Calcd for C H Cl N : [M + H] = 293.0355, found 293.0358.
13
11
2
4
5,6-Dichloro-3H-imidazo[4,5-b]pyridin-2-amine (7d): To a suspension of the diamine 6 (570 mg,
3
.20 mmol) in MeOH (12 mL) and H O (12 mL) was added BrCN (1.60 mL, 5M solution in acetonitrile,
2
8
mmol) and the reaction mixture was stirred at room temperature for 48 h. MeOH was concentrated
under vacuo and the pH of the resulting aqueous phase was adjusted to 8 with a saturated NaHCO3
solution. The aqueous layer (150 mL) was extracted with EtOAc (5 200 mL) and the combined organic
layers were dried (anhydrous Na SO ) and concentrated to dryness. The residue thus obtained was
×
2
4
1
triturated with diethyl ether to afford 510mg of 7d as an amorphous solid (79%). H-NMR (DMSO-d ,
6
6
13
00MHz) δ7.05 (br s, 2H, D O exchangeable, NH ), 7.58 (s, 1H, H-7). C-NMR (151 MHz, DMSO-d6)
2 2
δ
117.54 (C-6), 118.24 (C-7), 129.57 (C-7a), 136.73 (C-5), 153.60 (C-3a), 158.85 (C-2). HR-MS (ESI) m/z:
+
Calcd for C H Cl N : [M + H] = 202.9886, found 202.9887.
6
5
2
4
0
0 0
5
,6-Dichloro-N-ethyl-3-(2 ,3 ,5 -tri-O-acetyl-
β
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (8a),
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (9a),
-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (10a) and
,6-dichloro-N-ethyl-1-(2 ,3 ,5 -tri-O-acetyl- -d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (11a):
0
0 0
5
,6-dichloro-N-ethyl-3-(2 ,3 ,5 -tri-O-acetyl-α
0
0 0
0 0
5,6-dichloro-N-ethyl-1-(2 ,3 ,5 -tri-O-acetyl-β
5
0
α
To a suspension of the imidazopyridine 7a (250 mg, 1.08 mmol) in dry CH CN (10 mL) was added
3
N,O-bis-(trimethylsilyl)acetamide (291 mg, 1.43 mmol) and the reaction mixture was refluxed for 2 h. The
suspension was then cooled to room temperature and 1,2,3,5-tetra-O-acetyl-
mmol) was added, followed by the dropwise addition of trimethylsilyltriflate (0.3 mL, 1.54 mmol) at 0 C.
The mixture was refluxed for 3 h, the solvent was vacuum evaporated, the residue was dissolved in EtOAc
β
-d-ribofuranose (413 mg, 1.30
◦
(
100 mL) and washed with a saturated NaHCO solution (100 mL). The aqueous phase was extracted once
3

Molecules 2020, 25, 4531
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more with EtOAc (100 mL) and the combined organic layers were washed with brine (200 mL), dried
Na SO ) and evaporated to dryness. The resulting oil was purified by column chromatography, using a
(
2
4
mixture of CHCl /MeOH (99/1 to 97/3, v/v) as the eluent, to afford 8a, as a mixture with its corresponding
3
1
α-anomer 9a (280 mg, total yield 53% for two anomers, 8a:9a (3-β:α) ratio 12:1, as estimated by H-NMR),
1
0a (115 mg, yield 22%) and 11a (27 mg, yield 5%).
Data for 10a: Oil, [ ] +30.55 (c=0.475, CHCl ). H-NMR (600 MHz, CDCl )
J = 7.1 Hz), 2.04 (s, 3H, CH CO), 2.17 (s, 3H, CH CO), 2.20 (s, 3H, CH CO), 3.59 (m, 1H, CH ), 3.67 (m,
1
α
δ 1.32 (t, 3H, CH3,
D
3
3
3
3
3
2
0
0 0
0
= 12.7 Hz), 4.41 (m, 1H, H-4 ), 4.65 (dd, 1H, H-5 ,
5 ,5
1
J5
5
H, CH ), 4.34 (dd, 1H, H-5 , J
0
0
= 2.2 Hz, J
0
2
5 ,4
0
0
0
= 6.2 Hz), 5.35 (m, 1H, H-2 ),
3 ,2
0
0
= 3.8 Hz, J
0
0
0
= 12.7 Hz), 5.31 (dd, 1H, H-3 , J
0
0
= 3.6 Hz, J
0
,4
5 ,5
3 ,4
13
.98 (d, 1H, H-1 , J1
0
0
= 6.5 Hz), 6.13 (br s, 1H, D O exchangeable, NH), 7.52 (s, 1H, H-7). C-NMR
,2
2
(
(
(
151 MHz, CDCl ) δ 15.03 (CH ), 20.42 (CH CO), 20.69 (CH CO), 20.93 (CH CO), 38.92 (CH ), 62.85
3 3 3 3 3 2
0
0
0
0
0
C-5 ), 69.67 (C-3 ), 71.61 (C-2 ), 81.18 (C-4 ), 86.35 (C-1 ), 117.71 (C-7), 120.20 (C-6), 125.61 (C-7a), 141.57
C-5), 153.55 (C-3 ), 156.43 (C-2), 169.49 (CO), 169.78 (CO), 170.26 (CO). HR-MS (ESI) m/z: Calcd for
α
+
C H Cl N O Na: [M + Na] = 511.0758, found 511.0762.
19
22
2
4
7
1
Data for 11a: Oil, [α] +61.23 (c=0.684, CHCl ). H-NMR (600 MHz, CDCl ) δ 1.28 (t, 3H, CH3,
D 3 3
J = 7.2 Hz), 1.82 (s, 3H, CH CO), 2.11 (s, 3H, CH CO), 2.15 (s, 3H, CH CO), 3.57 (m, 2H, CH ), 4.29 (dd,
3
3
3
2
0
0
1
H, H-5 , J
0
0
= 4.9 Hz, J
0
0
= 12.3 Hz), 4.33 (dd, 1H, H-5 , J
0
0
= 3.5 Hz, J
0
0
= 12.3 Hz), 4.66 (m, 1H,
5
,4
5 ,5
5 ,4
5 ,5
0
0
0
H-4 ), 5.27 (t, 1H, D O exchangeable, NH, J = 5.3 Hz), 5.45 (t, 1H, H-3 , J = 5.3 Hz), 5.73 (t, 1H, H-2 ,
J = 5.1 Hz), 6.19 (d, 1H, H-1 , J1
CH ), 20.28 (CH CO), 20.55 (CH CO), 20.94 (CH CO), 38.71 (CH ), 63.31 (C-5 ), 71.08 (C-3 ), 71.55
C-2 ), 78.96 (C-4 ), 85.40 (C-1 ), 119.37 (C-7), 119.69 (C-6), 125.67 (C-7a), 141.09 (C-5), 154.24 (C-3a),
2
0
13
0
0
= 4.9 Hz), 7.55 (s, 1H, H-7). C-NMR (151 MHz, CDCl ) δ 14.96
,2
3
0
0
(
(
3
0
3
0
3
3
2
0
1
+
57.01 (C-2), 169.26 (CO), 169.55 (CO), 170.51 (CO). HR-MS (ESI) m/z: Calcd for C H Cl N O Na: [M
19 22 2 4 7
+
Na] = 511.0758, found 511.0764.
0
0
0
5
,6-Dichloro-N-isopropyl-3-(2 ,3 ,5 -tri-O-acetyl-
β
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-
0
0
0
amine
b]pyridin-2-amine
imidazo[4,5-b]pyridin-2-amine
(
8b), 5,6-dichloro-N-isopropyl-3-(2 ,3 ,5 -tri-O-acetyl- -d-ribofuranosyl)-3H-imidazo[4,5-
α
0 0 0
(
9b), 5,6-dichloro-N-isopropyl-1-(2 ,3 ,5 -tri-O-acetyl-β-d-ribofuranosyl)-1H-
0 0 0
10b) and 5,6-dichloro-N-isopropyl-1-(2 ,3 ,5 -tri-O-acetyl-α-D-
(
ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (11b): These derivatives were prepared by a
procedure analogous to that described for 8a,9a,10a,11a, starting from imidazopyridine 7b (280 mg,
1
.14 mmol). Purification was effected by column chromatography, using a mixture of CHCl /MeOH
3
(
99.5/0.5 to 98/2, v/v) as the eluent, to afford 8b, as a mixture with its corresponding α-anomer 9b (350
1
mg, 61% total yield for two anomers, 8b
:9b (3-
β
:α
) ratio 24:1, as estimated by H-NMR), 10b (140 mg,
2
4% yield) and 11b (50 mg, 9% yield).
◦
1
Data for 10b: Beige solid, mp: 165-166 C (EtOAc/n-pentane). [
α
] +37.77 (c=0.495, CHCl ). H
D
3
NMR (600 MHz, CDCl3)
δ 1.30 (d, 3H, CH , J = 6.5 Hz), 1.31 (d, 3H, CH , J = 6.5 Hz), 2.04 (s, 3H,
3
3
0
CH CO), 2.15 (s, 3H, CH CO), 2.19 (s, 3H, CH CO), 4.27 (m, 1H, CH), 4.32 (dd, 1H, H-5 , J
0
0
= 2.2 Hz,
3
3
3
5 ,4
0
0
0
0
= 12.7 Hz), 5.30 (dd, 1H, H-3 ,
5 ,5
J5
0
0
= 12.7 Hz), 4.39 (m, 1H, H-4 ), 4.62 (dd, 1H, H-5 , J
0
0
= 4.0 Hz, J
0
,5
5 ,4
0
0
= 6.1 Hz), 5.33 (m, 1H, H-2 ), 5.47 (br s, 1H, D O exchangeable, NH), 5.86 (d, 1H,
3 ,2
2
J3
0
0
= 3.6 Hz, J
0
,4
0
13
H-1 , J1
0
0
= 6.7 Hz), 7.53 (s, 1H, H-7). C-NMR (151 MHz, CDCl )
δ
20.41 (CH CO), 20.67 (CH CO),
,2
3
3
3
0
0
0
0
2
8
1.04 (CH CO), 22.76 (CH ), 23.03 (CH ), 46.04 (CH), 62.80 (C-5 ), 69.59 (C-3 ), 71.87 (C-2 ), 81.06 (C-4 ),
6.26 (C-1 ), 117.74 (C-7), 119.70 (C-6), 125.49 (C-7α), 141.38 (C-5), 154.37 (C-3α), 156.10 (C-2), 169.44
3
0
3
3
+
(CO), 169.72 (CO), 170.29 (CO). HR-MS (ESI) m/z: Calcd for C H Cl N O Na: [M + Na] = 525.0914,
20 24 2 4 7
found 525.0917.
1
Data for 11b: Oil, [α] +57.75 (c = 0.561, CHCl ). H NMR (400 MHz, CDCl ) δ 1.25 (d, 3H, CH3,
D 3 3
J = 6.5 Hz), 1.26 (d, 3H, CH , J = 6.5 Hz), 1.81 (s, 3H, CH CO), 2.09 (s, 3H, CH CO), 2.14 (s, 3H, CH CO),
3
3
3
3
0
0
4
J5
.21 (m, 1H, CH), 4.26 (dd, 1H, H-5 , J
0
0
= 5.2 Hz, J
0
0
= 12.5 Hz), 4.32 (dd, 1H, H-5 , J = 3.3 Hz,
0 0
5
.4
5 ,5
5 ,4
0 0
0
= 12.3 Hz), 4.65 (m, 1H, H-4 ), 5.16 (d, 1H, D O exchangeable, NH, J = 7.0 Hz), 5.42 (t, 1H, H-3 ,
,5
2
0
0
0
13
J = 5.4 Hz), 5.74 (t, 1H, H-2 , J = 5.0 Hz), 6.23 (d, 1H, H-1 , J
0
0
= 4.7 Hz), 7.55 (s, 1H, H-7). C-NMR
1
,2
(
(
151 MHz, CDCl )
δ
20.26 (CH CO), 20.53 (CH CO), 20.88 (CH CO), 22.67 (CH ), 23.00 (CH ), 45.87
3
3
3
3
3
3
0
0
0
0
0
CH), 63.29 (C-5 ), 70.98 (C-3 ), 71.48 (C-2 ), 78.84 (C-4 ), 85.35 (C-1 ), 119.32 (C-7), 119.43 (C-6), 125.58

Molecules 2020, 25, 4531
8 of 13
(
C-7a), 140.92 (C-5), 154.52 (C-3a), 156.45 (C-2), 169.20 (CO), 169.55 (CO), 170.49 (CO). HR-MS (ESI) m/z:
+
Calcd for C H Cl N O Na: [M + Na] = 525.0914, found 525.0917.
2
0
24
2
4
7
0
0 0
N-Benzyl-5,6-dichloro-3-(2 ,3 ,5 -tri-O-acetyl-
β
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine
-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine
10c) and N-benzyl-5,6-dichloro-1-(2 ,3 ,5 -tri-O-acetyl- -d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2
amine (11c): These derivatives were prepared by a procedure analogous to that described for
a,9a,10a,11a, starting from imidazopyridine 7c (350 mg, 1.20mmol). Purification was effected by
column chromatography, using a mixture of cyclohexane/EtOAc (70/30 to 20/80, v/v) as the eluent,
0
0 0
(8c), N-benzyl-5,6-dichloro-3-(2 ,3 ,5 -tri-O-acetyl-α
0
0 0
(9c), N-benzyl-5,6-dichloro-1-(2 ,3 ,5 -tri-O-acetyl-β
0
0 0
(
-
8
α
to afford 8c, as a mixture with its corresponding
α
-anomer 9c (350 mg, 53% total yield for two
1
anomers, 8c
:9c (3-
β
:α
) ratio 12:1, as estimated by H-NMR), 10c (100 mg, crude) and 11c (40 mg, 6%
yield). Fractions containing 10c were pooled and subjected to column chromatography eluted with
CHCl /MeOH (99.5/0.5, v/v), yielding 80 mg of pure 10c (12% yield).
3
Data for 10c: Oil, [
CH CO), 1.99 (s, 3H, CH CO), 2.15 (s, 3H, CH CO), 4.21 (dd, 1H, H-5 , J
.37 (m, 1H, H-4 ), 4.57 (dd, 1H, H-5 , J
Hz, JCH2,CH2 = 14.8 Hz), 4.82 (dd, 1H, CH , J
α
]D +46.29 (c = 0.337, CHCl3). 1H NMR (600 MHz, CDCl )
δ
1.91 (s, 3H,
= 12.6 Hz),
0
= 12.6 Hz), 4.72 (dd, 1H, CH , J = 5.2
5 ,5
2 CH2,NH
3
0
0
0
= 2.0 Hz, J
0
0
3
3
3
5 ,4
5 ,5
0
0
4
0
0
= 3.4 Hz, J
0
5
,4
0
= 5.9 Hz, J = 14.8 Hz), 5.30 (dd, 1H, H-3 ,
2
CH2,NH
CH2,CH2
0
0
J3
0
0
= 3.1 Hz, J
0
0
= 6.1 Hz), 5.37 (m, 1H, H-2 ), 5.87 (d, 1H, H-1 , J
0
0
= 7.6 Hz), 5.88 (br s, 1H, D O
,4
3 ,2
1 ,2
2
0
0
0
exchangeable, NH), 7.27 (m, 1H, phenyl H-4 ’), 7.32 (t, 2H, phenyl H-3 ’, H-5 ’, J = 7.5 Hz), 7.37 (d,
H, phenyl H-2 ’, H-6 ’, J = 7.4 Hz), 7.52 (s, 1H, H-7). C-NMR (151 MHz, CDCl )
0.52 (CH CO), 20.70 (CH CO), 47.62 (CH ), 62.91 (C-5 ), 69.83 (C-3 ), 71.64 (C-2 ), 81.41 (C-4 ), 86.04
C-1 ), 117.40 (C-7), 120.01 (C-6), 126.03 (C-7a), 127.89 (phenyl C-2 ’, C-6 ’), 128.01 (phenyl C-4 ’), 128.96
phenyl C-3 ’, C-5 ’), 137.97 (phenyl C-1 ’), 141.60 (C-5), 154.26 (C-3a), 156.81 (C-2), 169.27 (CO), 169.71
CO), 170.16 (CO). HR-MS (ESI) m/z: Calcd for C H Cl N O Na: [M + Na] = 573.0914, found
0
0
13
2
2
(
(
(
δ
20.34 (CH CO),
3
3
0
0
0
0
3
3
2
0
0
0
0
0
0
0
+
24
24
2
4
7
5
73.0919.
Data for 11c: Oil, [
α
]D +53.17 (c = 0.600, CHCl ). 1H NMR (600 MHz, CDCl )
δ
1.72 (s, 3H,
= 12.4 Hz),
= 5.0
3
3
0
CH CO), 2.00 (s, 3H, CH CO), 2.12 (s, 3H, CH CO), 4.27 (dd, 1H, H-5 , J
0
0
= 4.8 Hz, J
0
0
3
3
3
5 ,4
5 ,5
0
0
4.30 (dd, 1H, H-5 , J5
0
0
= 3.5 Hz, J
0
0
= 12.3 Hz), 4.62 (m, 1H, H-4 ), 4.69 (dd, 1H, CH , J
,4
5 ,5
2
CH2,NH
0
Hz, JCH2,CH2 = 14.5 Hz), 4.76 (dd, 1H, CH , J
J = 5.2 Hz), 5.60 (br s, 1H, D O exchangeable, NH), 5.71 (t, 1H, H-2 , J = 5.2 Hz), 6.19 (d, 1H, H-1 ,
J1
H-6 ’), 7.53 (s, 1H, H-7). C-NMR (151 MHz, CDCl )
4
= 5.5 Hz, J
= 14.5 Hz), 5.44 (t, 1H, H-3 ,
2
CH2,NH
CH2,CH2
0
0
0
2
0
0
0
0
0
0
= 5.0 Hz), 7.28 (m, 1H, phenyl H-4 ’), 7.32 (m, 2H, phenyl H-3 ’, H-5 ’), 7.40 (m, 2H, phenyl H-2 ’,
,2
13
δ 20.08 (CH CO), 20.40 (CH CO), 20.94 (CH CO),
3 3 3
3
0 0 0 0 0
7.73 (CH ), 63.24 (C-5 ), 71.03 (C-3 ), 71.39 (C-2 ), 79.20 (C-4 ), 85.46 (C-1 ), 118.91 (C-7), 119.87 (C-6),
2
0
0
0
0
0
125.89 (C-7a), 128.02 (phenyl C-4 ’), 128.23 (phenyl C-2 ’, C-6 ’), 128.90 (phenyl C-3 ’, C-5 ’), 138.03
0
(
phenyl C-1 ’), 141.44 (C-5), 154.31 (C-3a), 157.13 (C-2), 169.33 (CO), 169.53 (CO), 170.39 (CO). HR-MS
+
(ESI) m/z: Calcd for C H Cl N O Na: [M + Na] = 573.0914, found 573.0920.
24
24
2
4
0
7
0
0
5
,6-Dichloro-N,1-bis-(2 ,3 ,5 -tri-O-acetyl-
β
-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine
-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (8d
β-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (10d):
0
0
0
(14), 5,6-dichloro-3-(2 ,3 ,5 -tri-O-acetyl-
β
)
0
0
0
and 5,6-dichloro-1-(2 ,3 ,5 -tri-O-acetyl-
These derivatives were prepared by a procedure analogous to that described for 8a and 10a, starting
from 7d (245 mg, 1.21 mmol). Purification was effected by column chromatography, using a mixture of
CHCl /MeOH (99.5/0.5 to 95.5/4.5, v/v) as the eluent, to afford 14 (300 mg, 35% yield), 8d (150 mg, 27%
3
yield) and 10d (40 mg, 7% yield).
1
Data for 14: Oil, [
α
]D
−
25.60 (c=0.703, CHCl ). H NMR (600 MHz, CDCl )
δ
2.07 (s, 3H, CH CO),
3
3
3
2
.07 (s, 3H, CH CO), 2.11 (s, 3H, CH CO), 2.13 (s, 3H, CH CO), 2.14 (s, 3H, CH CO), 2.23 (s, 3H,
3 3 3 3
0
0
0
0
CH CO), 4.30–4.33 (m, 3H, 2
×
H-4 , 1
×
H-5 ), 4.36 (dd, 1H, H-5 , J
= 12.1 Hz), 4.50 (m, 1H, H-5 ), 5.44 (dd, 1H, H-3 , J
0
0
= 5.1 Hz, J
0
0
= 12.1 Hz), 4.42
3
5 ,4
5 ,5
0
0
(
dd, 1H, H-5 , J5
0
0
= 3.4 Hz, J
0
0
0
0
= 3.6 Hz,
,4
5 ,5
3 ,4
0 0 0 0
0
= 6.2 Hz), 5.56 (br s, 1H, H-2 ), 5.71 (br s, 1H, H-3 ), 5.97 (br s, 1H, H-1 ), 6.03 (br s, 1H, H-2 ), 6.07
,2
br s, 1H, H-1 ), 7.39 (s,1H, H-7). C-NMR (151 MHz, CDCl3)
J3
(
0
0
13
δ 20.50 (CH CO), 20.60 (CH CO), 20.68
3 3
0
(
CH CO), 20.69 (CH CO), 20.90 (CH CO), 21.06 (CH CO), 63.35 (2xC-5 ), 70.04, 70.10, 70.49, 70.77 (2
×
3
3
3
3

Molecules 2020, 25, 4531
9 of 13
0
0
0
0
0
0
C-2 , 2
C-7a), 139.26, 142.47 (C-3a, C-5), 149.92 (C-2), 169.67 (3
HR-MS (ESI) m/z: Calcd for C H Cl N O Na: [M + Na] = 741.1184, found 741.1185.
×
C-3 ), 79.73 (C-4 ), 80.33 (C-4 ), 85.06 (C-1 ), 85.13 (C-1 ), 117.37 (C-7), 122.62, 123.67 (C-6 and
×
CO), 169.76 (CO), 170.37 (CO), 170.65 (CO).
+
28
32
2
4
14
1
Data for 8d: Oil, [α] +52.57 (c=0.675, CHCl ). H NMR (600 MHz, CDCl ) δ 2.03 (s, 3H, CH CO),
D 3 3 3
0
2
(
J3
.11 (s, 3H, CH CO), 2.16 (s, 3H, CH CO), 4.35 (dd, 1H, H-5 , J
m, 1H, H-4 ), 4.59 (dd, 1H, H-5 , J
0
0
= 2.7 Hz, J
0
0
= 12.2 Hz), 4.38
3
3
5 ,4
5 ,5
0
0
0
0
0
= 4.0 Hz, J
0
0
= 12.2 Hz), 5.50 (dd, 1H, H-3 , J
0
0
= 4.3 Hz,
5
,4
5 ,5
3 ,4
0
0
0
0
= 6.2 Hz), 5.75 (m, 1H, H-2 ), 5.87 (br s, 2H, D O exchangeable, NH ), 6.23 (d, 1H, H-1 , J
0
0
= 6.5
,2
2
2
1 ,2
13
Hz), 7.63 (s, 1H, H-7). C-NMR (151 MHz, CDCl ) δ 20.48 (CH CO), 20.70 (CH CO), 20.83 (CH CO),
3 3 3
3.24 (C-5 ), 70.27 (C-3 ), 71.19 (C-2 ), 80.73 (C-4 ), 85.04 (C-1 ), 123.94 (C-6), 125.17 (C-7), 135.02, 138.38
3
0
0
0
0
0
6
(
C-5 and C-7a), 144.67 (C-3a), 155.30 (C-2), 169.85 (CO), 169.86 (CO), 170.24 (CO). HR-MS (ESI) m/z:
+
Calcd for C H Cl N O Na: [M + Na] = 483.0445, found 483.0449.
1
7
18
2
4
7
Data for 10d: Oil, [
α
]D +22.70 (c = 0.185, CHCl3). 1H NMR (600 MHz, CDCl )
δ
2.02 (s, 3H,
= 12.5 Hz),
3
0
CH CO), 2.16 (s, 3H, CH CO), 2.35 (s, 3H, CH CO), 4.31 (dd, 1H, H-5 , J
4
J3
NH ), 7.52 (s, 1H, H-7). C-NMR (151 MHz, CDCl )
6
0
0
= 2.1 Hz, J
0
0
3
3
3
5 ,4
5 ,5
0
0
0
.43 (m, 1H, H-4 ), 4.65 (dd, 1H, H-5 , J
0
0
= 1.9 Hz, J
0
0
= 12.5 Hz), 5.39 (dd, 1H, H-3 , J
= 7.5 Hz), 7.47 (br s, 2H, D O exchangeable,
0
0
= 3.2 Hz,
5
,4
5 ,5
3 ,4
0
0
0
0
= 6.2 Hz), 5.47 (m, 1H, H-2 ), 5.89 (d, 1H, H-1 , J
0
0
,2
1 ,2
2
1
3
δ 20.36 (CH CO), 20.65 (CH CO), 21.23 (CH CO),
3 3 3
2
3
0 0 0 0 0
3.01 (C-5 ), 70.07 (C-3 ), 71.20 (C-2 ), 81.20 (C-4 ), 86.11 (C-1 ), 117.54 (C-7), 119.80 (C-6), 125.35 (C-7a),
1
41.36 (C-5), 153.77 (C-3a), 157.58 (C-2), 169.30 (CO), 169.71 (CO), 170.43 (CO). HR-MS (ESI) m/z: Calcd
+
for C H Cl N O Na: [M + Na] = 483.0445, found 483.0440.
17
18
2
4
7
5
,6-Dichloro-N-ethyl-3-(β-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (12a): A mixture of
8a and 9a (80 mg, 0.16 mmol) was treated for 18 h with a saturated methanolic ammonia solution (15
mL). Upon evaporation of the solvent, the residue was purified by column chromatography using a
mixture of EtOAc/MeOH: 99/1 (v/v), as the eluent and the desired product was recrystallized from
◦
methanol to provide pure 12a (35 mg, 59% yield). White solid, mp: 191–192 C (MeOH). [
α
]D +31.88
1
(
c = 0.367, MeOH). H NMR (600 MHz, DMSO-d )
δ
1.19 (t, 3H, CH , J = 7.2 Hz), 3.41 (m, 2H, CH ,
overlapping with water of DMSO-d ), 3.66 (m, 2H, H-5 ), 3.99 (m, 1H, H-4 ), 4.12 (dd, 1H, H-3 ,
= 7.3 Hz), 5.05–5.55 (br s, 2H, D O
OH), 5.75 (br s, 1H, D O exchangeable, OH), 5.96 (d, 1H, H-1 , J
br s, 1H, D O exchangeable, NH), 7.78 (s, 1H, H-7). C-NMR (151 MHz, DMSO-d )
7.08 (CH ), 61.42 (C-5 ), 70.29 (C-2 ), 71.04 (C-3 ), 85.74 (C-4 ), 86.37 (C-1 ), 121.03 (C-6), 122.82 (C-7),
6
3
2
0
0
0
6
0
J3
0
0
= 1.5 Hz, J
0
0
= 5.2 Hz), 4.59 (dd, 1H, H-2 , J
0
0
= 5.2 Hz, J
0
0
,4
3 ,2
2 ,3
2 ,1
2
0
exchangeable, 2
×
0
0
= 7.5 Hz), 7.64
2
1 ,2
13
(
δ
14.50 (CH3),
2
6
0
0
0
0
0
3
2
1
34.65, 136.07 (C-5 and C-7a), 145.96 (C-3a), 155.48 (C-2). HR-MS (ESI) m/z: Calcd for C H Cl N O :
13 17 2 4 4
+
[
M + H] = 363.0621, found 363.0615.
,6-Dichloro-N-isopropyl-3-( -d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (12b): This
compound was prepared by a procedure analogous to that described for 12a starting from 8b (90
mg, 0.18mmol, containing also the corresponding -anomer 9b). Purification was effected by column
5
β
α
chromatography using a mixture of DCM/MeOH: 98/2 to 95/5 (v/v) as the eluent, to afford 12b (55 mg,
◦
1
8
1% yield). White solid, mp: 127–128 C (EtOAc/n-pentane). [
α
] +30.77 (c = 0.494, MeOH). H NMR
D
0
(600 MHz, DMSO-d6)
δ
1.21 (d, 3H, CH , J = 6.5 Hz), 1.22 (d, 3H, CH , J = 6.5 Hz), 3.65 (dd, 1H, H-5 ,
= 11.7 Hz), 3.68 (dd, 1H, H-5 , J
.11–4.18 (m, 2H, H-3 , CH), 4.56 (dd, 1H, H-2 , J
OH), 5.72 (br s, 1H, D O exchangeable, OH), 5.97 (d, 1H, H-1 , J
d, 1H, D O exchangeable, NH, J = 7.5 Hz), 7.78 (s, 1H, H-7). C-NMR (151 MHz, DMSO-d )
CH ), 22.37 (CH ), 44.36 (CH), 61.42 (C-5 ), 70.25 (C-2 ), 71.03 (C-3 ), 85.71 (C-4 ), 86.26 (C-1 ), 121.00
C-6), 122.72 (C-7), 134.60, 135.99 (C-5 and C-7a), 145.91 (C-3a), 154.84 (C-2). HR-MS (ESI) m/z: Calcd
3
3
0
0
J5
4
0
0
= 2.8 Hz, J
0
0
0
0
= 2.0 Hz, J
0
0
= 11.7 Hz), 3.99 (m, 1H, H-4 ),
= 7.6 Hz), 5.02–5.52 (br s, 2H, D O
,4
5 ,5
5 ,4
5 ,5
0
0
0
0
= 5.4 Hz, J
0
0
2
,3
2 ,1
2
0
exchangeable, 2
×
0
0
= 7.6 Hz), 7.44
2
1 ,2
1
3
(
(
(
δ
22.23
2
6
0
0
0
0
0
3
3
+
for C H Cl N O : [M + H] = 377.0778, found 377.0786.
14
19
2
4
4
N-Benzyl-5,6-dichloro-3-(
compound was prepared by a procedure analogous to that described for 12a starting from 8c (130
mg, 0.23 mmol, containing also the corresponding -anomer 9c). Purification was effected by column
chromatography using a mixture of DCM/MeOH: 98/2 to 90/10 (v/v) to afford an anomeric mixture
, in a 12/1 ratio (80 mg). A second column chromatography was performed (DCM/MeOH: 97.5/2.5
β-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (12c): This
α
β/α

Molecules 2020, 25, 4531
10 of 13
to 90/10, v/v) and the fractions pooled (30 mg) were enriched in the desired β-anomer 12c (β/αratio:
1
7/1). From this mixture, pure 12c (12 mg) was obtained by semi-preparative HPLC, eluted with H O
2
(
(
1
+0.2% Acetic Acid)/ACN (70/30 to 60/40, v/v, over a period of 40 min); t =23.96 min. Oil, [
α
]D +6.44
R
1
0
0
c=0.652, MeOH). H NMR (600 MHz, DMSO-d6)
δ
3.67 (br s, 2H, H-5 ), 4.02 (m, 1H, H-4 ), 4.14 (m,
0
H, H-3 ), 4.58 (dd, 1H, CH , J
= 5.8 Hz, J
= 15.6 Hz), 4.63 (dd, 1H, CH , J
= 6.4
CH2,NH
2
CH2,NH
CH2,CH2
2
0
0
Hz, JCH2,CH2 = 15.6 Hz), 4.67 (m, 1H, H-2 ), 5.22 (d, 1H, D O exchangeable, OH-3 , J = 4.1 Hz), 5.40 (d,
1
1
2
0
0
H, D O exchangeable, OH-2 , J = 6.3 Hz), 5.72 (t, 1H, D O exchangeable, OH-5 , J = 4.3 Hz), 6.01 (d,
2
2
0
0 0 0
0
= 7.5 Hz), 7.23 (t, 1H, phenyl H-4 ’, J = 7.1 Hz), 7.32 (t, 2H, phenyl H-3 ’, H-5 ’, J = 7.6
,2
H, H-1 , J1
0
0
0
Hz), 7.35 (d, 2H, phenyl H-2 ’, H-6 ’, J = 7.2 Hz), 7.78 (s, 1H, H-7), 8.25 (t, 1H, D O exchangeable, NH,
2
13
0 0 0
δ 45.14 (CH ), 61.45 (C-5 ), 70.53 (C-2 ), 71.05 (C-3 ), 85.80
2
J = 6.1 Hz). C-NMR (151 MHz, DMSO-d )
6
0 0 0 0 0
C-4 ), 86.46 (C-1 ), 121.08 (C-6), 123.09 (C-7), 126.79 (phenyl C-4 ’), 126.99 (phenyl C-2 ’, C-6 ’), 128.26
(
(
0
0
0
phenyl C-3 ’, C-5 ’), 134.73, 136.10 (C-5 and C-7a), 139.25 (phenyl C-1 ’), 146.01 (C-3a), 155.69 (C-2).
−
HR-MS (ESI) m/z: Calcd for C H Cl N O : [M − H] = 423.0632, found 423.0632.
18
17
2
4
4
5,6-Dichloro-3-(β-d-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-2-amine (12d): This compound was
prepared by a procedure analogous to that described for 12a, starting from 8d (100 mg, 0.22 mmol).
Purification was effected by column chromatography using a mixture of DCM/MeOH: 97/3 to 90/10
◦
(
+
v/v) as the eluent, to result in 12d (70 mg, 96% yield). Beige solid, mp: 142–143 C (toluene). [
α
]
D
1
0
0
16.50 (c = 0.594, MeOH). HNMR (600 MHz, DMSO-d )
δ
3.64 (brs, 2H, H-5 ), 3.98 (m, 1H, H-4 ),
.12 (m, 1H, H-3 ), 4.62 (m, 1H, H-2 ), 5.20 (br s, 1H, D O exchangeable, OH-3 ), 5.35 (brs, 1H, D O
6
0
0
0
0
4
2
2
0
0
exchangeable, OH-2 ), 5.58 (brs, 1H, D O exchangeable, OH-5 ), 5.94 (d, 1H, H-1 , J
0
0
= 7.4 Hz), 7.39
2
1 ,2
1
3
0
(
7
brs, 2H, D O exchangeable, NH ), 7.71 (s, 1H, H-7). C-NMR (151 MHz, DMSO-d6)
0.31 (C-2 ), 70.95 (C-3 ), 85.73 (C-4 ), 86.37 (C-1 ), 121.12 (C-6), 122.58 (C-7), 134.39, 136.37 (C-5 and
C-7a), 145.71 (C-3a), 156.45 (C-2). HR-MS (ESI) m/z: Calcd for C H Cl N O : [M + H] = 335.0308,
δ
61.44 (C-5 ),
2
2
0
0
0
0
+
11
13
2
4
4
found 335.0317.
,6-Dichloro-N-ethyl-1-(
compound was prepared by a procedure analogous to that described for 12a starting from 10a (50 mg,
5
β
-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine
(13a):
This
0
9
.10 mmol). Purification was effected by column chromatography using a mixture of DCM/MeOH:
◦
8/2 to 85/15 (v/v) as the eluent to afford 13a (30 mg, 81% yield). White solid, mp: 226-227 C (MeOH).
1
[
α
] +47.23 (c = 0.271, MeOH). HNMR (600 MHz, DMSO-d )
δ
1.18 (t, 3H, CH , J = 7.2 Hz), 3.41 (m,
D
6
3
0
2H, CH , overlapping with water of DMSO-d ), 3.66 (dd, 1H, H-5 , J
0
0
= 2.0 Hz, J
0
0
= 11.8 Hz), 3.71
2
6
5 ,4
5 ,5
0
0
0
0 0
= 5.5 Hz), 4.28 (m, 1H, H-2 ),
3 ,2
(
m, 1H, H-5 ), 4.00 (m, 1H, H-4 ), 4.08 (dd, 1H, H-3 , J
0
0
= 1.6 Hz, J
0
3
,4
5
.22–5.37 (brs, 2H, D O exchangeable, 2
×
OH), 5.73 (brs, 1H, D O exchangeable, OH), 5.77 (d, 1H,
2
2
0
13
H-1 , J1
MHz, DMSO-d )
0
0
= 7.7 Hz), 7.61 (t, 1H, D O exchangeable, NH, J = 5.3 Hz), 8.04 (s, 1H, H-7). C-NMR (151
,2
2
0
0
0
0
δ
14.55 (CH ), 37.52 (CH ), 61.09 (C-5 ), 70.26 (C-3 ), 71.49 (C-2 ), 86.00 (C-4 ), 87.55
6
3
2
0
(
C-1 ), 117.00 (C-6), 118.13 (C-7), 126.68 (C-7a), 138.26 (C-5), 154.74 (C-3a), 157.22 (C-2). HR-MS (ESI)
+
m/z: Calcd for C H Cl N O : [M + H] = 363.0621, found 363.0630.
1
3
17
2
4
4
5
,6-Dichloro-N-isopropyl-1-(
compound was prepared by a procedure analogous to that described for 12a starting from 10b
90 mg, 0.18 mmol). Purification was effected by column chromatography, using a mixture of
DCM/MeOH: 97/3 to 88/12 (v/v) as the eluent, to afford 13b (65 mg, 96% yield). White solid, mp:
β-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (13b):This
(
◦
1
1
(
(
91–192 C (EtOAc/n-pentane). [
d, 6H, 2 CH , J = 6.6 Hz), 3.65 (ddd, 1H, H-5 , J
m, 1H, H-5 ), 4.00 (m, 1H, H-4 ), 4.08 (m, 1H, H-3 ), 4.11 (m, 1H, CH), 4.27 (m, 1H, H-2 ), 5.27 (d, 1H,
D O exchangeable, OH-3 , J = 3.9 Hz), 5.30 (d, 1H, D O exchangeable, OH-2 , J = 7.6 Hz), 5.70 (t, 1H,
D O exchangeable, OH-5 , J = 4.4 Hz), 5.78 (d, 1H, H-1 , J
NH, J = 7.7 Hz), 8.02 (s, 1H, H-7). C-NMR (151 MHz, DMSO-d ) δ 22.23 (CH ), 22.33 (CH ), 44.87
3 3
CH), 61.11 (C-5 ), 70.26 (C-3 ), 71.49 (C-2 ), 86.02 (C-4 ), 87.53 (C-1 ), 116.90 (C-6), 117.96 (C-7), 126.73
α] +45.80 (c = 0.559, MeOH). H NMR (600 MHz, DMSO-d ) δ 1.22
D
6
0
×
0
0
= 2.5 Hz, J
0
= 4.4 Hz, J
0
0
= 11.9 Hz), 3.71
3
5 ,4
5 ,OH
5 ,5
0
0
0
0
0
0
0
2
2
0
0
0
= 7.8 Hz), 7.37 (d, 1H, D O exchangeable,
2
1 ,2
2
13
6
0
0
0
0
0
(
(
C-7a), 138.22 (C-5), 154.76 (C-3a), 156.63 (C-2). HR-MS (ESI) m/z: Calcd for C H Cl N O : [M +
14
19
2
4
4
+
H] = 377.0778, found 377.0785.

Molecules 2020, 25, 4531
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N-Benzyl-5,6-dichloro-1-(
β
-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (13c): This
compound was prepared by a procedure analogous to that described for 12a starting from 10c
60 mg, 0.11 mmol). Purification was effected by column chromatography, using a mixture of
DCM/MeOH: 97.5/2.5 to 85/15 (v/v) as the eluent to afford 13c (40 mg, 90% yield).White solid, mp:
(
◦
1
1
36-137 C (toluene). [
α
] +17.37 (c = 0.570, MeOH). H NMR (600 MHz, DMSO-d )
δ
3.66 (dd, 1H,
0
= 11.9 Hz), 4.03 (m, 1H,
5 ,5
D
6
0
0
H-5 , J
0
0
= 2.5 Hz, J
0
0
= 11.9 Hz), 3.72 (dd, 1H, H-5 , J
0
0
= 1.8 Hz, J
0
5
,4
5 ,5
5 ,4
0
0
0
H-4 ), 4.09 (dd, 1H, H-3 , J
0
0
= 1.6 Hz, J
0
0
= 5.4 Hz), 4.32 (dd, 1H, H-2 , J
0
0
= 5.5 Hz, J
0
0
= 7.8 Hz),
3
,4
3 ,2
2 ,3
2 ,1
4
.59 (d, 2H, CH , J
= 6.1 Hz), 5.07–5.57 (br s, 2H, D O exchangeable, 2
×
OH), 5.77 (br s, 1H,
2
CH2,NH
2
0
0
0
= 7.8 Hz), 7.24 (t, 1H, phenyl H-4 ’, J = 7.2 Hz), 7.32 (t,
1 ,2
D O exchangeable, OH), 5.83 (d, 1H, H-1 , J
0
2
0 0 0 0
2H, phenyl H-3 ’, H-5 ’, J = 7.6 Hz), 7.36 (d, 2H, phenyl H-2 ’, H-6 ’, J = 7.1 Hz), 8.09 (s, 1H, H-7), 8.26
13
(
(
(
t, 1H, D O exchangeable, NH, J
C-5 ), 70.26 (C-3 ), 71.67 (C-2 ), 86.10 (C-4 ), 87.55 (C-1 ), 117.22 (C-6), 118.50 (C-7), 126.64 (C-7a), 126.87
phenyl C-4 ), 127.21 (phenyl C-2 ’, C-6 ’), 128.26 (phenyl C-3 ’, C-5 ’), 138.40 (C-5), 139.24 (phenyl
= 6.1 Hz). C-NMR (151 MHz, DMSO-d ) δ 45.71 (CH ), 61.11
NH,CH2 6 2
2
0
0
0
0
0
0
0
0
0
0
0
+
C-1 ’), 154.43 (C-3a), 157.31 (C-2). HR-MS (ESI) m/z: Calcd for C H Cl N O : [M + H] = 425.0778,
18
19
2
4
4
found 425.0784.
,6-Dichloro-1-(β-d-ribofuranosyl)-1H-imidazo[4,5-b]pyridin-2-amine (13d): This compound was
5
prepared by a procedure analogous to that described for 12a starting from 10d (40 mg, 0.09 mmol).
Purification was effected by column chromatography, using a mixture of DCM/MeOH: 97/3 to 88/12
◦
(
(
1
v/v) as the eluent, to give 13d (25 mg, 86% yield). Beige solid, mp: 194–195 C (toluene). [
α
]D +19.44
1
0
0
c = 0.360, MeOH). H NMR (600 MHz, DMSO-d )
δ
3.64 (m, 1H, H-5 ), 3.70 (m, 1H, H-5 ), 3.99 (m,
= 5.5 Hz), 4.29 (m, 1H, H-2 ), 5.25 (br s, 1H, D O
exchangeable, OH-3 ), 5.30 (br s, 1H, D O exchangeable, OH-2 ), 5.60 (br s, 1H, D O exchangeable,
6
0
0
0
H, H-4 ), 4.08 (dd, 1H, H-3 , J
0
0
= 1.5 Hz, J
0
0
3
,4
3 ,2
2
0
0
2
2
0
0
OH-5 ), 5.76 (d, 1H, H-1 , J
0
0
= 7.7 Hz), 7.45 (br s, 2H, D O exchangeable, NH ), 8.08 (s, 1H, H-7).
,2
2 2
1
1
3
0 0 0 0 0
C-NMR (151 MHz, DMSO-d6) δ 61.01 (C-5 ), 70.07 (C-3 ), 71.56 (C-2 ), 85.96 (C-4 ), 87.60 (C-1 ), 116.70
(
C-6), 118.29 (C-7), 126.13 (C-7a), 138.41 (C-5), 154.85 (C-3a), 158.06 (C-2). HR-MS (ESI) m/z: Calcd for
+
C H Cl N O : [M + H] = 335.0308, found 335.0318.
11
13
2
4
4
3
.3. Antiviral Evaluation
The compounds were evaluated against different herpesviruses, including herpes simplex virus
–
type 1 (HSV-1) strain KOS, a thymidine kinase-deficient (TK ) HSV-1 KOS strain that is resistant
r
to ACV (ACV ), herpes simplex virus type 2 (HSV-2) strain G, adeno virus-2, human coronavirus,
+
–
varicella-zoster virus (VZV) TK strain Oka, TK VZV strain 07-1, and human cytomegalovirus (HCMV)
strains AD-169 and Davis. The antiviral assays were based on inhibition of virus-induced cytopathicity
or plaque formation (for VZV) in human embryonic lung (HEL) fibroblasts. Confluent cell cultures in
microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to
infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (for VZV) and the cell cultures
were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or
plaque formation (VZV) was recorded as soon as it reached completion in the control virus-infected cell
cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or
compound concentration required, to reduce virus-induced cytopathicity or viral plaque formation by
5
0%. Cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (MCC)
or the compound concentration that caused a microscopically detectable alteration of cell morphology.
Alternatively, the cytostatic activity of the test compounds was measured based on inhibition of cell
3
growth. HEL cells were seeded at a rate of 5
×
10 cells/well into 96-well microtiter plates and allowed
to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was
added. After 3 days of incubation at 37 C, the cell number was determined with a Coulter counter.
◦
The cytostatic concentration was calculated as the CC50, or the compound concentration required to
reduce cell proliferation by 50% relative to the number of cells in the untreated controls.

Molecules 2020, 25, 4531
12 of 13
3
.4. Proliferation Assay
The human cell lines used for the proliferation were Hela (ATCC #CCL-2, cervical carcinoma),
CEM (T-lymphoblastoid cells) and HMEC-1 (human microvascular endothelialcells). First, (5
−
7.5)
×
4
10 cells were seeded onto standard 96-well microtiter plates and left to attach for 24 h. On the next
day, test compounds were added in five serial 10-fold dilutions. The cell growth rate was evaluated
after 72 h of incubation, using MTT assay. Obtained results are expressed as an IC50 value, which
stands for the concentration of the compound necessary for 50% growth inhibition. The IC50 values
are calculated from concentration–response curve using linear regression analysis. Each test was
performed in quadruplicate in at least two individual experiments.
Supplementary Materials: The following are available online: a figure indicating the numbering of the
1
13
imidazopyridine nucleosides (Figure S1); H- and C-NMR spectra of the target nucleosides 12a
–d and 13a–d,
and of the di-ribosylated by-product 14 (Figures S2–S10); the NOE spectra of target compounds 12a and 13a
Figures S11 and S12); tables with the results of the antiviral (Tables S1–S3) and the cytotoxic (Table S4) evaluation
(
of the target nucleosides.
Author Contributions: Project conception and supervision, N.L., P.M. and N.P.; Synthesis and characterization of
the compounds, G.P. and M.G.; Biological evaluation of the compounds, R.S. and G.A.; Data analysis, G.P., N.L.,
P.M., N.P., R.S. and G.A.; Writing of the manuscript, G.P., N.L., P.M., G.A. and N.P. All authors have read and
agreed to the published version of the manuscript.
Funding: The scientific work reported in this paper was supported by the Onassis Foundation, through a
postgraduate scholarship awarded to G.P. (Scholarship ID: G ZN 045-1/2017-2018).
Acknowledgments: The authors are grateful to Leentje Persoons and Brecht Dirix for excellent technical assistance
in the antiviral and antiproliferative assays.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the target compounds are available from the authors.
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