Synthesis of new indanyl nucleoside analogues and their biological evaluation on hepatitis C virus (HCV) replicon

Article abstract of DOI:10.3390/molecules24050990

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

Full text of DOI:10.3390/molecules24050990

molecules
Article
Synthesis of New Indanyl Nucleoside Analogues
and their Biological Evaluation on Hepatitis C Virus
(HCV) Replicon
Matías E. Gómez 1,†, Emiliano A. Gentile , M. Florencia Martini 1,3, María L. Cuestas ,
2,†
2
2
4
1,3,
Verónica L. Mathet , Graciela Y. Moltrasio and Albertina G. Moglioni
*
1
Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires,
Ciudad Autónoma de Buenos Aires C1113AAD, Argentina; matgmz@gmail.com (M.E.G.);
flormartini1@gmail.com (M.F.M.)
2
Instituto de Investigaciones en Microbiología y Parasitología Médica,
CONICET-Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires C1121ABF, Argentina;
emilianogentile@gmail.com (E.A.G.); marilu_cuestas@yahoo.com.ar (M.L.C.); vmathet@gmail.com (V.L.M.)
Instituto de la Química y Metabolismo del Fármaco, CONICET-Universidad de Buenos Aires,
Ciudad Autónoma de Buenos Aires C1113AAD, Argentina
Cátedra de Química Orgánica II, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires,
Ciudad Autónoma de Buenos Aires C1113AAD, Argentina; gmoltra@gmail.com
Correspondence: amoglio@ffyb.uba.ar or bmoglio2015@gmail.com; Tel.: +54-11-5287-4852
These authors contributed equally to this work.
3
4
*
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 15 January 2019; Accepted: 2 March 2019; Published: 11 March 2019
Abstract: Here, we report a convenient synthetic procedure for the preparation of four novel indanyl
carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C
virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations,
all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication.
Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8.
Several biological experiments and computational studies were performed to elucidate the effect of
these compounds on HCVg1b replication. Based on all the experiments performed, we propose that
the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that
of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG
cells was indirectly determined for the first time in this work.
Keywords: nucleoside analogues; cis-2-amino-1-indanol; HCV replicon; molecular modeling
1
. Introduction
Interest in the design and synthesis of modified nucleosides has increased steadily over the past
few decades [1–5]. The introduction of structural diversity into the nucleoside scaffold for use
as potential chemotherapeutic agents has long been considered an important approach to drug
design. In this sense, the substitution of the furan ring with a cyclopentane ring, and the subsequent
replacement of both at positions 2’ and 3’ by an aromatic or heteroaromatic cycle (Figure 1: I and II,
respectively), lead to differences in the physicochemical properties of the synthesized carbanucleosides,
which may influence the biological activity due to higher lipophilicity, conformational rigidity,
and steric hindrance than their natural nucleosides [6].
Molecules 2019, 24, 990; doi:10.3390/molecules24050990
www.mdpi.com/journal/molecules

Molecules 2019, 24, 990
2 of 13
Figure 1. Carbanucleosides with an aromatic or heteroaromatic cycle in their structure.
In this regard, we have previously designed and synthesized numerous carbanucleosides derived
from the four commercially available stereoisomers of 1-amino-2-indanol [ ] using the short, efficient,
], which showed interesting results
7
and linear synthetic route proposed by Shealy and Clayton [
as antiviral agents [9].
8
Persistent infection with hepatitis C virus (HCV) is associated with chronic hepatitis (CHC),
cirrhosis, and the development of hepatocellular carcinoma (HCC). The current therapeutic approach
is based on the combined use of direct-acting antiviral agents targeting the viral polymerase, the viral
protease, and NS5A, with or without ribavirin [10
against hepatitis C continues to evolve at a rapid pace.
Taking into account our previous results [ ], the literature available [17–20] regarding the activity
–16]. Therefore, the study of direct-acting antivirals
7,
9
observed for other indanyl nucleosides against different viruses, the fact that carbanucleosides derived
from 2-amino-1-indanol have not been previously synthesized and evaluated as antiviral agents,
and the persistent need to find effective new drugs for the treatment of hepatitis C, we decided
to synthesize this type of derivative that contains triazole, purine, or azapurine moiety in their
structures. Once synthesized, we evaluated their anti-HCV activity in vitro, by using the replicon
cell line, Huh7.5 SG, to find new agents against this infection. The results obtained fell short of our
expectations, as in all the cases, the synthesized compounds increased HCV replication. In this context
and considering that the molecular analysis of these nucleoside analogues is relevant for explaining
the effect observed, the aim of this work was expanded to search for the atomic grouping present in
these derivatives, which are responsible for this unexpected effect.
2
. Results and Discussion
2
.1. Chemistry
Based on our previous experience with the synthesis of indanyl nucleosides [7,9] and considering
that 2-amino-1-indanol is not commercially available in any possible stereoisomeric form, we attempted
a synthetic method that would lead to 2-amino-1-indanol with a defined cis stereochemistry and good
yield. For this purpose, commercially available indene was treated with N-bromosuccinimide (NBS)
to give (+/
to yield a mixture of products that could not be identified; however, the treatment of compound
sodium azide led to the isolation of the corresponding racemic azide 2 [23] with a very good yield
Scheme 1).
−
) trans-bromohydrin
1
[21
,
22]. Thus, compound
1
was treated with ammonium hydroxide
1
with
(

Molecules 2019, 24, 990
3 of 13
Scheme 1. Synthesis of indanyl nucleosides. Reagents and conditions: (i) NaN , (ii) H /Pd/C, 40 psi,
3
2
3
h, (iii) n-BuOH, triethylamine, 5-amino-4,6-dichloropyrimidine, reflux, 48 h, (iv) triethyl orthoformate,
◦
HCl 12N, 12 h, (v) NaNO /H O/HCl 1N, 0 C, (vi) heat, (vii) NH OH (aq), reflux, (viii) CuSO .5H O,
2
2
4
4
2
sodium ascorbate, propargyl alcohol, rt.
The complexity of the observed signals for the aliphatic H atoms of compound
2,
despite corresponding to the chemical displacements already described [23 24], did not allow
,
the expected cis stereochemistry to be ensured, which could be defined through the bidimensional
NOESY (Nuclear Overhauser SpectroscopY) spectra (Figure S1), where the nuclear Overhauser
effect (nOe) between H1 and H2 of the indanyl ring was observed, indicating that both are
on the same face of the molecule and confirming the cis stereochemistry. Then, compound
was reduced by catalytic hydrogenation to the desired (+/ )-cis -2-amino-1-indanol 24].
Indanyl carbonucleosides were prepared using the classical approach to carbocyclic nucleosides
proposed by Shealy and Clayton [ 20]. In this way, the condensation of compound with
-amino-4,6-dichloropyrimidine in refluxing n-butanol containing triethylamine afforded compound
To obtain the imidazole ring of the purinyl analogues, compound was treated with triethyl
orthoformate, using a catalytic amount of hydrochloric acid, to render compound in a 73% yield.
The cis configuration between the hydroxyl group in C1, of the indane ring, and the 6-chloropurine
residue of compound was determined using HSQC (Heteronuclear Single Quantum Coherence)
and NOESY correlation spectra (Figures S2–S4). A triazole ring was formed from compound
by intramolecular reaction of the diazonium salt of the primary amino group with sodium nitrite,
in an acidic medium, to render a highly unstable compound (not isolated), which was converted into
the 8-aza purine derivative by heating. Compound treated with ammonium hydroxide afforded
compound . Finally, triazole was synthesized by treating compound with propargyl alcohol
through the Huisgen dipolar cycloaddition of azides and alkynes, a powerful class of the concerted
2
−
3 [
8,
3
5
4.
4
5
5
4
6
7
6
8
9
2

Molecules 2019, 24, 990
4 of 13
click reaction, which were widely applied [25]. This reaction was catalyzed by Cu (II) in the presence
of sodium ascorbate. All the compounds obtained were characterized by spectroscopic methods.
2
.2. Biological Results
2
.2.1. Cytotoxicity of Indanyl Derivatives
Huh7.5 SG cells were incubated at different concentrations (1.56 to 100.00 µM) of the four newly
synthesized carbanucleoside analogues (
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner
salt)/phenazine methosulfate) assay. Results showed that the exposure of Huh7.5 SG cells to 25.00
of compound and to 100.00 M of nucleoside analogues , for 96 h did not significantly decrease
5, 7–9) and cytotoxicity was estimated using the MTS/PMS
(
µM
5
µ
7–9
cell viability when compared to their corresponding controls. In contrast, 50.00 and 100.00 µM of
compound 5 caused a significant cell viability loss in the HCV replicon cell line (Figure 2).
Figure 2. Effect of the four newly synthesized carbanucleoside analogues (5, 7–9) on the viability of
Huh7.5 SG cells.
2
.2.2. HCVg1b Replication Assay
Compounds
observed: 25.00 M (for compound
Figure 3, there was a significant increase in the RNA levels of HCV in those Huh7.5 SG cells treated
with all the derivatives: Compound and (more than 2-fold); compound (approximately 5-fold);
and compound 9 (nearly 6-fold), after 72 h post-incubation.
5,
7–
9
were assayed at the highest concentration in which no cytotoxicity was
µ
5
) and 100.00 M (for compounds ). Surprisingly, as shown in
µ
7–9
5
7
8

Molecules 2019, 24, 990
5 of 13
Figure 3. Effect of carbanucleosides
Huh7.5 SG cells.
5 (25.00 µM) and 7–9 (100.00 µM) on the viral load of HCV in
Since none of the tested compounds yielded the expected antiviral activity, we found it was not
worthwhile to assay the activity as a function of the concentration of these compounds.
Given these results, our investigations were redirected to the search of the structural
motif that was responsible for the biological activity observed. Thus, considering the fact that
the cis-2-amino-1-indanol moiety (
the 6-chloropurine residue, only contained in compound
was conducted by testing the action of compounds
Although it was observed that compound
3) is common to all the nucleoside analogues tested, and that
5, is commercially available, a new experiment
3,
5
, and 6-chloropurine on viral replication.
3
promotes an increase in viral load replication, its effect
was lower than in all the nucleoside analogues assayed. Moreover, 6-chloropurine promoted viral
replication, but in a lower proportion than cis-2-amino-1-indanol (Figure 4).
Figure 4. Effect of compound
3 (25.00 µM), 5 (25.00 µM), and 6-chloropurine (25.00 µM) on the viral
load of HCV in Huh7.5 SG cells at 48 and 72h.
Similar increases in the viral loads to those observed in this work were previously reported in
the literature by Li et al. [26 27]. They reported that Huh7.0, Huh.8, and FCA-1 cells express the -opioid
,
µ
receptor and it was proposed that the increase in HCV replication in morphine addicts is due to
the interaction between this drug and the abovementioned receptor. Since Li et al. [26] showed that
the effect on viral replication is time-dependent, we also decided to measure the HCV RNA at 48 h
post-incubation for compounds 3, 5, and 6-chloropurine (Figure 4). Just as these authors had previously
reported, the viral loads in our cell system were also higher at 48 h post-incubation, with respect to our
observations at 72 h.

Molecules 2019, 24, 990
6 of 13
2
.2.3. Indanyl Nucleoside Activity Related to the Opioid Receptor
As was mentioned in 2.2.2, some abuse drugs, such as morphine, have been described
as compounds that enhance HCV replicon expression [26 27]. In addition, naltrexone and naloxone,
opioid receptor antagonists, abrogate the enhancing effect of that abuse drug on HCV replicon
expression [26 27]. To elucidate whether the Huh7.5 SG replicon system responds to morphine
in a similar way as the mentioned cells, an assay with morphine at 1.00 M was conducted at 48 h
and 72 h (Figure 5). The morphine treatment increased viral replication, and the effect was greater
at 48 h. However, pretreating the cell culture with naloxone (0.01 M) for 30 min inhibited the ability of
,
,
µ
µ
morphine to exert its effect. These results suggest the presence of an opioid receptor in this subgenomic
HCV replicon system.
Figure 5. Effect of morphine (1.00 µM), naloxone (0.01 µM), and the combination of both compounds
on the viral load of HCV in Huh7.5 SG cells at 48 and 72 h.
Based on these results and considering that morphine-related compounds
[28],
such as phenylpiperidines (prodines), share the pharmacophore group that could be present
in the assayed indanyl nucleosides (Figure 6), Huh7.5 SG cells were pretreated for 30 min with
naloxone and then compound
5 was added to elucidate whether this compound can upregulate
HCVg1b replication in a similar manner to morphine.
Figure 6. Structures of morphine, prodine, naloxone, and compound 5 showing the pharmacophore
group in bold letters.
A drop in HCVg1b replication was observed when the Huh7.5 SG cells were preincubated with
naloxone for 30 min and then compound 5 was added (Figure 7).

Molecules 2019, 24, 990
7 of 13
Figure 7. Effect of naloxone (0.01
µM), compound 5 (25.00 µM), and the combination of both on
the viral load of HCV in the Huh7.5 SG cells at 48 and 72 h.
Taken together, these results led us to conclude that the increase in viral replication in the presence
of nucleoside analog could be mediated, at least partly, by the opioid receptor in the Huh7.5 SG cells,
5
since a decrease in viral replication was observed when preincubation with naloxone was performed.
A similar mechanism could be responsible for the action of compounds 7–9.
To further evaluate if compound
treatment with morphine was assayed. Unexpectedly, the effect of the combined treatment displayed
a lower HCVg1b replication level compared to compound alone, but higher than that observed
when cells were treated only with morphine. A possible explanation of this phenomenon could be
that although morphine seems to have a higher affinity for the opioid receptor than compound
5 exerts its function through the opioid receptor, a combined
5
5,
the latter compound induces higher levels of HCVg1b replication (Figure 8), which reaffirms its effect,
as previously mentioned.
Figure 8. Effect of the co-administration of morphine (1.00 µM) and compound 5 (25.00 µM) on HCVg1b
replication in the Huh7.5 SG cells at 48 and 72 h.
An assay demonstrating the activity of a racemic mixture does not assure or rule out the activity of
both enantiomers. Nevertheless, given the unexpected activity of compound on HCVg1b replication,
regardless of whether either of the two enantiomers or both were responsible for the observed activity,
we attempted to establish a possible structural similarity between each enantiomer of compound
and morphine. In this way, molecular modeling studies for the two enantiomers were carried
out. Both enantiomers of compound and morphine were structurally optimized according to
5
5
5
the procedure described in the Materials and Methods section. Each one of the 10 conformers obtained
was superimposed on the structure of morphine, considering the atoms involved in the pharmacophore
group (Figure 6). The best fitting structures of both enantiomers in compound 5 and the morphine
structure are shown in Figure 9.

Molecules 2019, 24, 990
8 of 13
Figure 9. Molecular modeling of compound
5 and morphine: (A) Morphine optimized structure.
(
B
) 1R,2S enantiomer of compound
) 1S,2R enantiomer of compound
5
5
superimposed on the pharmacophore of morphine (RMSD: 0.275).
superimposed on the pharmacophore of morphine (RMSD: 0.274).
(C
Since both enantiomers of compound
5 had a low RMSD value (0.274 for 1S,2R and 0.275
for 1R,2S), we were able to verify that their overlap with the morphine pharmacophore was adequate.
This fact could lead us to the preliminary conclusion that both enantiomers are capable of exerting
the observed effect.
3
. Materials and Methods
3
.1. Chemical Experimental Section
Melting points were determined on a Thomas Hoover apparatus (Arthur H. Thomas Company,
1
13
Philadelphia, PA, USA). H and C NMR spectra were recorded on Bruker 600 or a Bruker 300
spectrometers (Bruker Biospin, Rheinstetten, Germany), and reported in parts per million ( ) relative
to tetramethylsilane (TMS: 0.00 ppm). Data are reported as follows: Chemical shift, multiplicity
s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublet, ddd = doublet of doublets
δ
δ
(
of doublets, bs = broad singlet), coupling constants (J, Hz), and integration. High resolution mass
spectra (HRMS) were recorded on a micrOTOF-Q mass spectrophotometer (BRUKER DALTONIK,
Bremen, Germany). Preparative thin layer chromatography (p-TLC) and thin layer chromatography
analyses (TLC) were performed on Kieselgel 60 F254 (Merck KGaA, Darmstadt, Germany) plates.
3
.1.1. (+/−)-trans-2-bromo-1-indanol (1)
A solution of indene (0.4 mL, 3.4 mmol), NBS (0.67 g, 3.8 mmol), water (3.5 mL),
and tetrahydrofurane (THF) (3.5 mL) was stirred for 12 h at room temperature. Product
as a white solid (0.68 g, 94%), which was filtered and crystallized from ethanol; m.p.: 127–129 C;
1
was obtained
◦
◦
m.p. lit.: 132–133 C [21,22]. Compound 1 was used in the next step without any further purification.
1
H NMR (300 MHz, CDCl3)
δ
2.45 (d, J = 5.6 Hz, 1H, OH); 3.20 (dd, J = 7.4 Hz, J = 16.2 Hz, 1H, CHH);
.58 (dd, J = 7.2 Hz, J = 16.2 Hz, 1H, CHH); 4.28–4.38 (m, 1H, CHBr); 5.32 (t, J = 5.5 Hz, 1H, CHOH);
.22–7.33 (m, 3H, ArH); 7.40–748 (m, 1H, ArH-7) ppm. 13C NMR (75.4 MHz, CDCl3)
41.1 (CH ), 55.1
3
7
δ
2
(CHBr), 84.0 (CHOH), 124.8, 125.2, 128.3, 129.7, 140.4, and 142.3 ppm.

Molecules 2019, 24, 990
9 of 13
3
.1.2. (+/−)-cis-2-azido-1-indanol (2)
NaN (0.16 g, 2.5 mmol) was added to a stirred solution of
2.5 mL). The reaction mixture was stirred during 6 h at 80 C and then diluted with water (8.0 mL).
1
(0.5 g, 2.35 mmol) in anhydrous DMF
3
◦
(
The aqueous solution was extracted with diethyl ether (3
×
10 mL). The organic phases were combined
and dried over Na SO , filtrated, and the solvent was removed under reduced pressure until dryness.
2
4
The residue was purified by p-TLC (n-hexane/diethyl ether; 1:1), affording compound
2
δ
(0.330 g, 80%)
2.40 (bs., 1H,
CHOH), 3.17 (m, 2H, CH ), 4.36 (m, 1H, CHN ), 5.17 (d, J = 4.6 Hz, 1H, CHOH), 7.26–7.35 (m, 3H,
◦
◦
1
as a white solid; m.p.: 130–132 C; mp lit.: 129–131 C [23]. H NMR (600 MHz, CDCl )
3
2
3
13
ArH), 7.46 (d, J = 6.8 Hz, ArH-7); C NMR (150 MHz, CDCl3)
24.7, 125.1, 127.6, 129.0, 139.1, 141.8.
δ 35.2 (CH ), 65.7 (CHN ), 76.4 (CHOH),
2 3
1
3
.1.3. (+/−)-cis-2-amino-1-indanol (3)
Palladium/activated charcoal (10% Pd/C; 11 mg) was added to a stirred solution of compound
90.5 mg, 0.5 mmol) prepared from compound , as previously described [23 24] in absolute ethanol
10 mL)). The mixture was hydrogenated in a Parr hydrogenation apparatus at 40 psi of hydrogen
2
(
(
1
,
pressure for 3 h. When the reduction was completed (TLC), the reaction was stopped, the reaction
mixture was filtered, and the solvent was removed under reduced pressure until it reached dryness.
◦
1
Compound
CDCl3) 2.76 (dd, J = 5.1 Hz, J = 15.9 Hz, 1H, CHH), 3.13 (dd, J = 6.7 Hz, J =15.9, 1H, CHH), 3.61–3.65
m, 1H, CHNH), 4.83 (d, J = 5.4 Hz, 1H, CHOH), 7.20–7.26 (m, 3H, ArH), 7.39–7.42 (m, 1H, ArH-7)
3 was obtained (77.6 mg, 99%) as a yellow solid; m.p.: 130–132 C. H NMR (300 MHz,
δ
(
13
ppm. C NMR (75.4 MHz, CDCl )
δ 39.2 (CH2), 54.1 (CHNH ), 75.3 (CHOH), 125.1, 125.3, 127.0, 128.4,
2
3
1
41.0, 143.4 ppm.
3
.1.4. (+/−)-2-(5-amino-6-chloropyrimidin-4-yl-amino)-2,3-dihydro-1H-inden-1-ol (4)
A mixture of compound (200 mg, 1.34 mmol) and 5-amino-4,6-dichloropyrimidine (220 mg,
.34 mmol) in dry triethylamine (1.5 mL) and n-butanol (5.3 mL) was heated at reflux for 24 h
under argon atmosphere. Then, the reaction mixture was cooled and the solid obtained was
filtered and washed with EtOAc (2 5 mL) to afford compound as a grey solid (150 mg, 40%);
m.p.: 172–174 C. H NMR (600 MHz, DMSO-d6) 3.03 (dd, J = 8.5 Hz, J = 15.4 Hz, 1H, CHH),
.12 (dd, J = 7.5 Hz, J = 15.4 Hz, 1H, CHH), 4.54–4.64 (m, 1H, CHNH), 5.02 (t, J = 5.3 Hz, 1H, CHOH),
.18 (brs, 2H, NH ), 5.25 (d, J = 5.4 Hz, 1H, OH), 6.83 (d, J = 6.3 Hz, 1H, CHNH), 7.21–7.28 (m, 3 H,
3
1
×
4
◦
1
δ
3
5
2
13
ArH), 7.39 (d, 1H, J = 7.2 Hz, ArH-7), 7.77 (s. 1H, N=CH–N) ppm; C NMR (150 MHz, DMSO-d6)
δ
35.9 (CH ), 55.6 (CHNH), 72.4 (CHOH), 124.3, 125.1, 125.9 (C7), 127.1, 128.6, 137.4, 141.5, 144.4, 146.1
2
+
(N=CH–N), 152.5 ppm. HRMS-ESI m/z [M + H] calcd for C H N ClO: 277.0856, found: 277.0859.
13
14
4
3
.1.5. (+/−)-2-(6-chloro-9H-purin-9-yl)-2,3-dihydro-1H-inden-1-ol (5)
A mixture of compound (80 mg, 0.29 mmol), triethylorthoformate (1.8 mL), and HCl 12 N
0.1 mL) was stirred at room temperature under argon atmosphere for 2 h. Then, the solvent
4
(
was removed under reduced pressure until it reached dryness, and the product was purified by
◦
p-TLC (eluant EtOAc:Hexane 1:1) to afford compound
with decomposition. H NMR (600MHz, DMSO-d6)
5
as a solid (60 mg, 73%); m.p.: >220 C
1
δ 3.46 (dd, J = 7.8 Hz, J = 16.3 Hz, 1H, CHH),
3
5
8
.73 (dd, J = 8.4 Hz, 15.8 Hz, 1H, CHH), 5.14 (t, J = 5.7 Hz, 1H, CHOH), 5.40–5.44 (m, 1H, CHN),
.54 (bs, 1H, OH), 7.29–7.40 (m, 3H, ArH), 7.43 (d, J = 7.4 Hz, 1H, ArH-7), 8.60 (s, 1H, (N=CH–N),
.81 (s, 1H, (N=CH–N) ppm, 1 C NMR (150 MHz, DMSO-d6)
3
δ
35.1 (CH ), 58.2 (CHN), 73.1 (CHOH),
2
+
125.2, 126.0, 127.7, 129.3, 140.5, 143.0, 147.4, 149.2, 151.9, 153.1 ppm. HRMS-ESI m/z [M + H] calcd for
C H ClN O: 287.0700, found: 287.0695.
14
12
4

Molecules 2019, 24, 990
10 of 13
3
.1.6. 3-((+/−)1-hydroxy-2,3-dihydro-1H-inden-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ol (7)
◦
A cooled solution (0 C) of compound
4 (100 mg, 0.36 mmol) in HCl 1N (1.3 mL) was treated
with a solution of sodium nitrite (36 mg, 0.48 mmol) in water (3.3 mL). The mixture was stirred
and allowed to warm up to room temperature, and then heated at reflux for 1 h. Finally, the solvent
was removed under reduced pressure until it reached dryness. The solid residue was purified by
◦
1
p-TLC (eluant EtOAc) to afford compound
600 MHz, DMSO-d6) 3.49 (dd, J = 7.8 Hz, J = 16.0 Hz, 1H, CHH), 3.98 (dd, J = 6.3 Hz, J = 16.0 Hz,
H, CHH), 5.34 (t, J = 6.2 Hz, 1H, CHOH), 5.39 (d, J = 6.3 Hz, 1H, OH), 5.45–5.55 (m, 1H, CHN),
7 as a white solid (70 mg, 72%), m.p.: 178–180 C. H NMR
(
1
7
δ
.26–7.41 (m, 4H, ArH), 8.26 (s, 1H, N=CH–N), 12.66 (s, 1H, N=C–OH) ppm; 13C NMR (150 MHz,
DMSO-d6)
δ
34.1 (C ), 61.4 (CHN), 74.3 (CHOH), 125.0, 125.6, 127.4, 128.9, 129.9, 140.6, 143.0, 149.6,
2
+
1
49.9, 155.9 ppm. HRMS-ESI m/z [M + Na] calcd for C H N NaO : 292.0810, found: 292.0807.
13
11
5
2
3
.1.7. (+/−)2-(7-amino-3H-[1,2,3]triazolo[4,5–d]pyrimidin-3-yl)-2,3-dihydro-1H-inden-1-ol (8)
A solution of sodium nitrite (33 mg, 0.48 mmol) in water (3.7 mL) was added to a cooled
◦
(
0 C) solution of compound
4
(100 mg, 0.36 mmol) and 1N HCl (1 mL) The mixture was stirred
◦
at 0 C for 15 min, and then NH OH (2 mL) was added and the mixture was heated under reflux
4
for 5 min. The solvent was removed under reduced pressure, and the solid residue was purified
◦
by p-TLC (eluant EtOAc) to afford compound
decomposition. H NMR (600 MHz, DMSO-d6)
8
as a white solid (80 mg, 82%); m.p.: >250 C with
1
δ 3.49 (dd, J = 8.0 Hz, J = 16.0 Hz, 1H, CHH),
4
.05 (dd, J = 6.7 Hz, J = 16.0 Hz, 1H, CHH), 5.30–5.37 (m, 2H, CHOH and OH), 5.35–5.60 (m, 1H, CHN),
.26–7.42 (m, 4H, ArH), 8.00 (bs, 1H, N=CH–NH), 8.30 (s, 1H, N=CH–NH), 8.34 (bs, 1H, C=NH) ppm;
7
13
C NMR (150 MHz, DMSO-d6),
δ
34.0 (CH ), 61.0, (CHN), 74.1, (CHOH), 124.3, 125.0, 125.7, 127.3
2
+
128.9, 140.8, 143.2, 150.1, 156.6, 156.8 ppm. HRMS-ESI m/z [M + Na] calcd for C H N NaO :
13
12
6
2
2
91.0973, found: 291.0965.
3
.1.8. (+/−)-2-(4-hydroxymethyl-1H-1,2,3-triazol-1-yl)-2,3-dihydro-1H-inden-1-ol (9)
Propargyl alcohol (66
in 2 mL of a water/tert-butanol mixture (1:1). Sodium ascorbate (0.3 mmol, 300
prepared 1 M solution in water) was added, followed by copper (II) sulfate pentahydrate
7.5 mg, 0.03 mmol, in 100 L of water). The heterogeneous mixture was stirred vigorously overnight,
µL, 1.14 mmol) and compound
2
(100 mg, 0.57 mmol) were suspended
µL of freshly
(
µ
at which point it cleared and the TLC analysis indicated total consumption of the reactants. The reaction
mixture was diluted with 10 mL of water and the brown precipitate was collected by filtration.
After being washed with cold water (2
compound as a brown solid (70 mg, 53%); m.p.: 131–133 C. H NMR (600 MHz, DMSO-d6)
dd, J = 7.5 Hz, J = 15.9 Hz, 1H, CHH), 3.45 (dd, J = 6.7 Hz, J = 15.4 Hz, 1H, CHH), 4.50 (m, J = 5.42 Hz,
×
5 mL), the precipitate was dried under vacuum to afford
◦
1
9
δ 3.40
(
2
6
7
7
H CH OH), 5.14 (t, J = 5.7 Hz, 1H, CH OH), 5.18 (bs, J = 5.6 Hz, 1H, CHOH), 5.37 (ddd, J = 5.6Hz,
2 2
.7 Hz, 7.5 Hz, 1H, CHN), 5.58 (bs, 1H, CHOH), 7.28–7.36 (m, 3H, ArH), 7.40 (d, J = 7.1 Hz, 1H, ArH),
.68 (s, 1H, N–CH=C); 13C NMR (150 MHz, DMSO-d6)
δ
36.0 (CH ), 55.6 (CH OH) 63.8, (HCN),
2
2
4.2, (HCOH), 122.7 (NCH=C), 125.1, 125.5, 127.6 129.0, 140.2, 143.3, 147.8 (N–C=C). HRMS-ESI m/z
+
[M + H] calcd for C H N O : 232.1086, found: 232.0988.
12
14
3
2
3
3
.2. Evaluation of Biological Activity
.2.1. Cells
The HCV genotype 1b replicon cell line, Con1/SG-Neo (I)—Huh7.5, cells containing
the subgenomic (SG) HCV replicon (Huh 7.5 SG, Catalogue number APC50) was donated by
◦
APATH (New York, NY, USA). These cells were maintained at 37 C in a humidified atmosphere
of 5% CO in a subconfluent state in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented
with 10% FBS (Life Technologies Corp., Carlsbad, CA, USA) and 750
Carlsbad, CA, USA).
µg/mL G418 (Life Technologies,

Molecules 2019, 24, 990
11 of 13
3
.2.2. Drug Preparation
Compounds
3
,
5,
7
,
8,
9
, 6-chloropurine, and morphine (Sigma-Aldrich, St. Louis, MO, USA)
were solubilized in DMSO. Naloxone (Denver Farma S.A., Buenos Aires, Argentina) was solubilized in
physiological solution.
Unless described in detail elsewhere, the different compounds and drugs were used in
the following final concentrations: (i)
iii) morphine: 1.00 µM; and (iv) naloxone 0.01 µM.
The drug concentrations used for the assays were defined according to: (i) The cytotoxicity effect
exerted on the Huh 7.5 SG cell line for the four newly synthesized carbanucleosides (compounds );
ii) the equivalent amount of the precursor on the final structure of carbanucleoside for 6-chloropurine
and compound 3; and (iii) the previous results reported by Li et al. [26] for morphine and naloxone.
3, 5, and 6-chloropurine: 25.00 µM; (ii) 7–9: 100.00 µM;
(
5, 7–9
(
5
3
.2.3. Cytotoxic Assays
4
The Huh7.5 SG cells were seeded in 96-well plates (2.0
×
10 cells/well), grown overnight
and treated with the carbanucleoside analogues synthesized at different concentrations
1.56 to 100.00 M). The cytotoxic activity was determined after 96 h of incubation for the HCV
replicon cells using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)
2H-tetrazolium/phenazine methosulfate (MTS/ PMS) assay Promega, Madison, WI USA).
(
µ
-
Absorbance at
λ
= 495 nm was determined using FlexStation 3 (Molecular Devices, San Jose, CA, USA).
3
.2.4. HCVg1b Replication Assay
5
The Huh7.5 SG cells were incubated for 96 h in 6-well plates (5.0
×
10 cells/well) in the presence
of different drugs, alone or combined. Samples were collected every 24 h and the medium of each well
was completely renewed using fresh medium supplemented with 5% FBS without G418 (to eliminate
the potential loss of cells due to the reduction of the HCV replicon copy number and G418 resistance).
HCVg1b replication was determined by the quantification of viral RNA at 48 and 72 h post-incubation
®
®
®
in the supernatants of cell cultures by RT-qPCR using the COBAS AmpliPrep/COBAS TaqMan
HCV Quantitative Test (Roche, Basel, Switzerland). Untreated cultures were included in each assay
as negative controls (100% of HCV viral load). Viral RNA levels were normalized to each untreated
control (100% of HCV viral load).
3
.3. Molecular Modelling and Superimpose Structures
Morphine and both enantiomers of compound
5 were optimized with the density functional
theory (DFT) using the B3LYP functional and 6-31G* basis set. The quantum calculations included full
optimization of the geometric structures and the scan of the dihedral angle, which connects both bicyclic
rings of compound 5. Then, the 10 conformational minima for each enantiomer were superimposed
on the morphine pharmacophore group. For this purpose, the Chimera v1.12 software (Resource for
Biocomputing, Visualization, and Informatics—University of California, San Francisco, CA, USA)
was used for obtaining the root mean square deviation (RMSD) of the 10 superimposed atoms. The best
RMSD was obtained for the conformer of the second minimum potential energy for each enantiomer.
4
. Conclusions
We report here a convenient synthetic procedure for the preparation of novel
three purinyl- and 8-azapurinyl-carbanucleoside derivatives, obtained as a racemic mixture from
cis-2-amino-1-indanol, as well as one triazolyl analogue obtained from racemic cis-2-azido-1-indanol.
The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon
cell line, Huh7.5 SG. All the nucleoside analogues studied here promoted viral replication.
Several biological experiments and computational studies were performed to elucidate the effect of
these compounds on HCVg1b replication. Based on the results of all the experiments performed,

Molecules 2019, 24, 990
12 of 13
we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar
mechanism to that of morphine on the enhancement of this replication. The presence of opioid
receptors in the Huh7.5 SG cells was indirectly determined in this work for the first time.
The findings of this work impact on the design of nucleoside analogues because, surprisingly,
the compounds of a family that is commonly known for its antiviral effect could be inactive
ones. Nevertheless, it is unlikely that they show the opposite effect as reported in this work.
Our results provide relevant molecular information to gain further knowledge about this system.
Moreover, these open the possibility of considering other compounds that enhance viral replication in
the recurrence of HCV infection, such as morphine in addiction cases.
Supplementary Materials: The following are available online, Figure S1: NOESY spectrum of compound 2,
Figure S2: HSQC of compound 5, Figure S3: NOESY spectrum of compound 5, Figure S4: Selective nOe of
compound 5.
Author Contributions: A.G.M., G.Y.M. and V.L.M. designed the study and experiments. M.E.G. designed
the novel compound syntheses. V.L.M., M.L.C. and E.A.G. design and executed the biological assays. M.F.M.
designed and executed the molecular modeling and superimposition computational studies. A.G.M., G.Y.M.,
M.F.M. and V.L.M. analyzed and interpreted the experimental data. A.G.M., G.Y.M. and M.F.M. wrote,
reviewed and edited the manuscript.
Funding: This work was financially supported by Agencia Nacional de Promoción Científica y Tecnológica grant
PICT 2012-0032, Universidad de Buenos Aires grants B033 and 20020130100257BA and Consejo Nacional de
Investigaciones Científicas y Tecnológicas grants PIP 0086 and PIP 11220120100074CO (Argentina).
Acknowledgments: The authors acknowledge APATH for the donation of the cells containing the subgenomic
(SG) HCV replicon.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
De Clerc, E. Toward improved anti-HIV chemotherapy: Therapeutic strategies for intervention with HIV
infections. J. Med. Chem. 1995, 38, 2491–2517. [CrossRef]
Huang, P.; Farquhar, D.; Plunkett, W. Selective action of 3 -azido-3 -deoxythymidine 5 -triphosphate on viral
reverse transcriptases and human DNA polymerases. J. Biol. Chem. 1990, 265, 11914–11918.
0
0
0
Bressi, J.C.; Choe, J.; Hough, M.T.; Buckner, F.S.; van Voorhis, W.C.; Verlinde, C.L.; Hol, W.G.J.; Gelb, M.H.
Adenosine analogues as Inhibitors of Trypanosoma brucei phosphoglycerate kinase: Elucidation of a novel
binding mode for a 2-amino-N6-substituted adenosine. J. Med. Chem. 2000, 43, 4135–4150. [CrossRef]
[
PubMed]
Johnston, M.I.; Hoth, D.F. Present status and future prospects for HIV therapies. Science 1993, 260, 1286–1293.
CrossRef] [PubMed]
4
5
.
.
[
Norbeck, D.W.; Kern, E.; Hayashi, S.; Rosenbrook, W.; Sham, H.; Herrin, T.; Plattner, J.J.; Erickson, J.;
Clement, J. Cyclobut-A and cyclobut-G: Broad-spectrum antiviral agents with potential utility for the therapy
of AIDS. J. Med. Chem. 1990, 33, 1281–1285. [CrossRef]
6
.
Fernandez, F.; García-Mera, X.; Morales, M.; Vilariño, L.; Caamaño, O.; De Clercq, E. Synthesis of new
0
0
6-substituted purinyl-5 -nor-1 -homocarbanucleosides based on indanol. Tetrahedron 2004, 60, 9245–9253.
[CrossRef]
7.
8.
9.
Ugliarolo, E.A.; Lantaño, B.; Moltrasio, G.Y.; Moglioni, A.G. An efficient approach to homochiralindane
nucleosides. Tetrahedron Asymmetry 2009, 20, 1848–1853. [CrossRef]
Shealy, Y.F.;
Clayton, J.D. 9-[β-DL-2α,3α-Dihydroxy-4β-(hydroxymethyl)-cyclopentyl]adenine,
the carbocyclic analog of adenosine. J. Am. Chem. Soc. 1966, 88, 3885–3887. [CrossRef]
Ugliarolo, E.A.; Gagey, D.; Lantaño, B.; Moltrasio, G.Y.; Campos, R.H.; Cavallaro, L.V.; Moglioni, A.G.
Synthesis and biological evaluation of novel homochiral carbocyclic nucleosides from 1-amino-2-indanols.
Bioorg. Med. Chem. 2012, 20, 5986–5991. [CrossRef] [PubMed]
1
0. Zampino, R.; Marrone, A.; Restivo, L.; Guerrera, B.; Sellitto, A.; Rinaldi, L.; Romano, C.; Adinolfi, L.E.
Chronic HCV infection and inflammation: Clinical impact on hepatic and extra-hepatic manifestations.
World J. Hepatol. 2013, 5, 528–540. [CrossRef] [PubMed]

Molecules 2019, 24, 990
13 of 13
1
1. Jacobson, I.M.; Lim, J.K.; Fried, M.W. American Gastroenterological Association Institute clinical practice
update-expert review: Care of patients who have achieved a sustained virologic response after antiviral
therapy for chronic hepatitis C infection. Gastroenterology 2017. [CrossRef] [PubMed]
1
1
1
2. Li, G.; De Clercq, E. Current therapy for chronic hepatitis C: The role of direct-acting antivirals. Antivir. Res.
2
017, 442, 83–122. [CrossRef] [PubMed]
3. Karayiannis, P. Interferon-based combination treatment for chronic hepatitis C in the era of direct-acting
antivirals. Ann. Gastroenterol. 2015, 28, 55–65.
4. Sherman, K.E.; Flamm, S.L.; Afdhal, N.H.; Nelson, D.R.; Sulkowski, M.S.; Everson, G.T.; Fried, M.W.;
Adler, M.; Reesink, H.W.; Martin, M.; et al. Response-guided telaprevir combination treatment for hepatitis
C virus infection. N. Engl. J. Med. 2011, 365, 1014–1024. [CrossRef]
1
1
1
5. Wilby, K.J.; Partovi, N.; Ford, J.A.; Greanya, E.; Yoshida, E.M. Review of boceprevir and telaprevir for
the treatment of chronic hepatitis C. Can. J. Gastroenterol. 2012, 26, 205–210. [CrossRef] [PubMed]
6. Greig, S. Sofosbuvir/Velpatasvir: A review in chronic hepatitis C. Drugs 2016, 76, 1567–1578. [CrossRef]
[PubMed]
7. Balo, C.; López, C.; Caamaño, O.; Fernández, F.; García-Mera, X.; Rodríguez-Borges, J.E. Synthesis of novel
0
purinyl-1 -homocarbanucleosides based on a cyclopenta[b]pyrazine system. Chem. Pharm. Bull. 2008
,
5
6, 654–658. [CrossRef] [PubMed]
1
8. Yao, S.W.; Lopes, V.H.C.; Fernández, F.; García-Mera, X.; Morales, M.; Rodríguez-Borges, J.E.;
Cordeiro, M.N.D.S. Synthesis and QSAR study of the anticancer activity of some novel indane carbocyclic
nucleosides. Bioorg. Med. Chem. 2003, 11, 4999–5006. [CrossRef] [PubMed]
1
9. Hergueta, A.R.; Fernandez, F.; López, C.; Balzarini, J.; De Clercq, E. Novel carbocyclic nucleosides containing
a cyclobutyl ring: Adenosine analogues. Chem. Pharm. Bull. 2001, 49, 1174–1177. [CrossRef]
0. Borges, J.E.; Fernández, F.; García, X.; Hergueta, A.R.; López, C.; Andrei, G.; Snoeck, R.; Witvrounw, M.;
Balzarini, J.; De Clercq, E. Novel carbocyclic nucleosides containing a cyclobutyl ring. Guanosine
and adenosine analogues. Nucleosides Nucleotides 1998, 17, 1237–1253. [CrossRef] [PubMed]
1. Imuta, M.; Ziffer, H. Synthesis and absolute stereochemistry of cis- and trans-1,2-indandiols: Metabolites of
indene and 2-indanone. J. Org. Chem. 1978, 43, 4540–4552. [CrossRef]
2
2
2
2
2
2
2
2
2
2. Kozhushkov, S.I.; Yufit, D.S.; de Meijere, A. Convenient and Inexpensive Synthesis of
(1R,2R)-trans-1-Amino-6-nitroindan-2-ol. Adv. Synth. Catal. 2005, 347, 255–265. [CrossRef]
3. Iacazio, G.; Réglier, M. Chemo-enzymatic synthesis of all four diastereoisomers of 1-fluoro-2-amino-indane.
Tetrahedron Asymmetry 2005, 16, 3633–3639. [CrossRef]
4. Mitrochkine, A.; Gil, G.; Règlier, M. Synthesis of enantiomerically pure cis and trans-2-amino-1-indanol.
Tetrahedron Asymmetry 1995, 6, 1535–1538. [CrossRef]
5. Kolb, H.C.; Finn, M.G. and Sharpless, K.B. Click Chemistry: Diverse Chemical Function from a Few Good
Reactions. Angew. Chem. Int. Ed. 2001, 40, 2004–2021. [CrossRef]
6. Li, Y.; Zhang, T.; Douglas, S.D.; Lai, J.P.; Xiao, W.D.; Pleasure, D.E.; Ho, W.Z. Morphine enhances hepatitis C
virus (HCV) Replicon Expression. Am. J. Pathol. 2003, 163, 1167–1175. [CrossRef]
7. Wang, C.Q.; Li, Y.; Douglas, S.D.; Wang, X.; Metzger, D.S.; Zhang, T.; Ho, W.Z. Morphine withdrawal
enhances hepatitis C virus replicon expression. Am. J. Pathol. 2005, 167, 1333–1340. [CrossRef]
8. Delgado Cirilo, A.; Minguillón Llombart, C.; Joglar Tamargo, J. Introducción a la Química Terapéutica, 2nd ed.;
Ediciones Díaz de Santos: Madrid, Spain, 2003; p. 307.
Sample Availability: Samples of the compounds 7, 8 and 9 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(