Synthesis and anti-HBV activity of carbocyclic nucleoside hybrids with salient features of entecavir and aristeromycin

Article abstract of DOI:10.1039/d0md00059k

Modified carbocyclic nucleosides (4a-g) constituting 7-deazapurine, 4′-methyl, exocyclic double bond and 2′,3′-hydroxy were synthesized. NOE and X-ray studies of4cconfirmed the α-configuration of 4′-methyl. The anti-HBV assay demonstrated4e(IC₅₀

Full text of DOI:10.1039/d0md00059k

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RESEARCH ARTICLE
Synthesis and anti-HBV activity of carbocyclic
nucleoside hybrids with salient features of
entecavir and aristeromycin†
Cite this: DOI: 10.1039/
d0md00059k
Ramakrishnamraju Samunuri,ab Masaaki Toyama,c Renuka Sivasankar Pallaka,b
a
Seshubabu Neeladri,b Ashok Kumar Jha,b Masanori Babac and Chandralata Bal
*
Received 22nd February 2020,
Accepted 22nd April 2020
Modified carbocyclic nucleosides (4a–g) constituting 7-deazapurine, 4′-methyl, exocyclic double bond and
2′,3′-hydroxy were synthesized. NOE and X-ray studies of 4c confirmed the α-configuration of 4′-methyl.
The anti-HBV assay demonstrated 4e (IC50 = 3.4 μM) without notable cytotoxicity (CC50 = 87.5 μM) as a
promising lead for future exploration.
DOI: 10.1039/d0md00059k
rsc.li/medchem
Hepatitis B is one of the common viral diseases. As per
estimation, 350 million people are chronically infected with
hepatitis B virus (HBV) worldwide and are at the risk of
developing liver cancer.1,2 Chronic HBV patients require long-
term treatment, which only suppresses the infection and is
not efficient in eliminating the virus. Drug-resistant viruses
emerging due to the long-term regimen mandates synthesis
and efforts to be directed towards finding more potent and
less toxic novel anti-HBV agents. Entecavir (I, Fig. 1) has
become one of the most prescribed anti-HBV drugs for the
treatment of chronically infected patients.3,4 It comprises a
carbocyclic framework with a 6′-exo double bond, which
seems to be an essential pharmacophore.5 Aristeromycin (II,
Fig. 1) is a naturally occurring carbocyclic purine nucleoside
and its modified derivatives are reported to exhibit a wide
range of pharmacological activities against viral infection,
cancer etc.6,7 Recently, 4′-substituted nucleosides have
attracted consideration as balapiravir (for HCV) and festinavir
(for HIV) reached the advanced phase of drug development.8
Moreover, there are a few reports on 4′-substituted carbocyclic
nucleosides analogs (CNAs) in literature.9,10
(CC50 > 100 μM). These results motivated us for further
chemical exploration of this new class of CNAs towards
improving their medicinal properties. Recently, base-
modified nucleosides containing 7-deazapurine have attracted
considerable attention.15,16 Therefore, in this study, we
designed a novel class of modified carbocyclic nucleosides
(IV, Fig. 1) by replacing the adenine moiety of III with
7-deazapurine. Herein, we report our efforts toward the
synthesis, anti-HBV activity, and cytotoxicity profiles of newly
designed molecules.
The carbocyclic sugar intermediate (1) as
a single
diastereomer was achieved in eight synthetic steps from
D-ribose with an overall yield of 17–20%.5 The Cl/Br/I group
was successfully substituted at the C-7 position of 6-chloro-7-
deazapurine (2a) by treating with NCS/NBS/NIS in DMF.12
The fluoro derivative (2b) was obtained by heating 2a with
selectfluor in a acetonitrile : acetic acid (80 vol, 5 : 1) mixture
at 70 °C.17 The coupling of 1 with 6-chloro-7-deazapurine (2a)
or its 7-halo derivatives (2b–e) under Mitsunobu reaction
conditions afforded the corresponding protected coupled
products. The deprotection was performed under acidic
conditions without further purification to yield 3a–e
(Scheme 1).
From the last decade, our group has been involved in the
synthesis of biologically significant novel CNAs.11–14 Recently,
we described the synthesis of aristeromycin analogs (III,
Fig. 1) with novel features: the 6′-exocyclic double bond and
4′-α-methyl group.5 Although none of them demonstrated
significant anti-HBV activity, none were strongly cytotoxic
The treatment of 3a–e with methanolic ammonia at 100
°C under a sealed condition yielded the desired carbocyclic
nucleosides (4a–e).18
The 7-vinyl/ethynyl analogs of 4a were synthesized from
the iodo derivative 4e by palladium-catalyzed cross-coupling
reaction to give the desired compounds (4f–g) in good yield
(Scheme 2).18 In brief, the 7-ethynyl derivative (4f) was
synthesized in two steps from 4e by treating with
trimethylsilyl acetylene (TMS-acetylene) and PdIJPPh3)4 in
DMF at 50 °C under the sealed condition, followed by
treatment with K2CO3 in methanol at ambient temperature.
The vinyl derivative (4g) was synthesized from 4e by treating
a Department of Chemistry, Birla Institute of Technology, Mesra, Ranchi,
Jharkhand, 835215, India. E-mail: cbal@bitmesra.ac.in
b Chemistry Services, GVK Biosciences Pvt. Ltd, IDA Nacharam, Hyderabad, India
c Division of Antiviral Chemotherapy, Joint Research Center for Human Retrovirus
Infection, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima, 890-8544, Japan
† Electronic supplementary information (ESI) available. CCDC 1969219. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
d0md00059k
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Fig. 1 Chemical structures entecavir (I), aristeromycin (II), aristeromycin analogs (III), and modified carbocyclic nucleosides (IV) for investigation in
the present study.
with tributyl vinyl tin and PdIJPPh3)4 in DMF at 110 °C. All
compounds and key intermediates were characterized via
spectral analyses.
4a–b and 4g were inactive towards HBV. It is interesting to
note that the introduction of the bulkier halo groups at C-7
of the base (4e) remarkably increased the activity.
The NOE study of 4c (Fig. 2) indicated the α-configuration
of the methyl group at 4′-position in the carbocyclic sugar
ring,19 which was further confirmed via single-crystal X-ray
diffraction studies (Fig. 3).
In summary, we synthesized a new series of modified
carbocyclic nucleosides (4a–g) from commercially available
starting materials in 10–12 synthetic steps. These compounds
were evaluated for their antiviral activity against the hepatitis
B virus and cytotoxicity properties in the HepG2.2.15.7 cells.
Among the screened compounds, 4e exhibited a noteworthy
antiviral activity (IC50 = 3.4 μM) without notable cytotoxicity.
From these studies, it is evident that these novel carbocyclic
nucleosides might be valuable in designing new drugs for the
HBV treatment.
HepG2.2.15.7 cells (1 × 104 cells per well) were inoculated
into a microtiter plate. After incubation for 24 h, the cells
were cultured in the presence of various concentrations of
test compounds (4a–g). Then, every three days, the culture
medium was replaced by
appropriate concentration of 4a–g.
After nine days of incubation, the culture supernatants
a fresh one containing an
were collected and examined for HBV DNA levels using real- Experimental and spectral data of
time PCR. The cells were tested for their viability by the
tetrazolium dye method. IC50: 50% effective concentration
based on the inhibition of the HBV DNA levels in culture
final compounds
General method for synthesis of 4a–e
supernatants and CC50: 50% cytotoxic concentration based
on the reduction of viable cells was calculated. The results
A screw-cap vial equipped with a magnetic stirrer bar was
charged with NH3 in methanol (7 M, 7 ml) and appropriate
3a–e (0.80 mmol), and then sealed with a screw cap. The vial
was heated up to 100 °C and stirred for 24 h. The reaction
mixture was concentrated under a reduced pressure, and
are summarized in Table
1 and Fig. 4. Interestingly,
structural modification resulted in a noteworthy antiviral
activity (4c–f) with IC50 ranging from 3.4 to 22.9 μM without
considerable cytotoxicity. From the above-mentioned results,
Scheme 2 Synthesis of 7-ethynyl/vinyl derivatives (4f–g). Reagents
and conditions: i) a) trimethylsilyl acetylene, CuI, Et3N, PdIJPPh3)4, DMF,
50 °C, 3 h; b) K2CO3, MeOH, rt, 30 min; ii) tri-n-butyl vinyl tin,
PdIJPPh3)4, DMF, 110 °C, 3 h.
Scheme 1 Synthesis of 4a–e. Reagents and conditions: (i) PPh3, DIAD,
THF, 10 °C–rt, 1 h; (ii) TFA : H2O (8 : 2 ratio), rt, 30 min; (iii) NH3 in
MeOH, 100 °C, sealed tube, 24 h.
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Table 1 Anti-HBV and cytotoxicity of 4a–g in HepG2.2.15.7 cells
Compound
IC50 (μM)
CC50 (μM)
4a
4b
4c
4d
>100
>100
22.9
8.3
3.4
>100
>100
>100
>100
87.5
4e
4f
4g
6.3
>100
>3
>100
>100
>3
Aristeromycin
Entecavir
0.18 (nM)
>100 (nM)
Fig. 2 Compound 4c showed NOE between 4′-methyl with 1′-H, as
shown in red arrow to demonstrate α-configuration.
1H), 8.02 (s, 1H); 19F NMR (376 MHz, DMSO-d6) δ: −168.25;
13C NMR (100 MHz, DMSO-d6) δ: 18.9, 49.1, 62.0, 69.1, 73.4,
74.2, 91.9, 105.2, 107.8, 140.3, 146.4, 152.3, 154.2, 155.7;
HRMS (ESI-Orbitrap) m/z: exact mass calculated for
C14H18FN4O3 [M + H]+: 309.1285, found: 309.1325.
(1S,2R,3R,5R)-5-(4-Amino-5-chloro-7H-pyrroloij2,3-d]-
p y r i m i d i n - 7 - y l ) - 3 - ( h y d r o x y m e t h y l ) - 3 - m e t h y l - 4 -
methylenecyclopentane-1,2-diol (4c): purified yield: 80%, off-
white solid, (TLC: Rf 0.2, 10% MeOH in CH2Cl2); [α]2D0: −30.41
(c = 0.25, DMSO); mp: 226–229 °C; UV (MeOH) λmax: 281.25
nm; 1H NMR (300 MHz, CD3OD) δ: 1.18 (s, 3H), 3.51 (d, J =
11.1 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.99 (d, J = 4.8 Hz,
1H), 4.59 (d, J = 2.7 Hz, 1H), 4.68 (dd, J = 4.8 and 9.9 Hz, 1H),
5.08 (d, J = 3.0 Hz, 1H), 5.46–5.51 (m, 1H), 7.24 (s, 1H), 8.05
(s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 18.9, 49.2, 62.4,
69.1, 73.5, 74.2, 99.4, 101.4, 107.9, 120.0, 149.6, 152.2, 154.7,
156.7; HRMS (ESI-Orbitrap) m/z: exact mass calculated for
C14H18ClN4O3 [M + H]+: 325.0989, found: 325.1031.
(1S,2R,3R,5R)-5-(4-Amino-5-bromo-7H-pyrroloij2,3-d]-
p y r i m i d i n - 7 - y l ) - 3 - ( h y d r o x y m e t h y l ) - 3 - m e t h y l - 4 -
methylenecyclopentane-1,2-diol (4d): purified yield: 75%, off-
white solid, (TLC: Rf 0.2, 10% MeOH in CH2Cl2); [α]2D0: +6.14
(c = 0.25, DMSO); mp: 228–232 °C; UV (MeOH) λmax: 283.25
nm; 1H NMR (300 MHz, CD3OD) δ: 1.18 (s, 3H), 3.51 (d, J =
11.1 Hz, 1H), 3.65 (d, J = 11.1 Hz, 1H), 3.98 (d, J = 4.5 Hz,
1H), 4.58 (d, J = 2.7 Hz, 1H), 4.69 (dd, J = 4.8 and 9.9 Hz, 1H),
5.08 (d, J = 2.7 Hz, 1H), 5.48–5.51 (m, 1H), 7.30 (s, 1H), 8.04
(s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 18.9, 49.2, 62.5,
69.1, 73.5, 74.2, 85.4, 100.6, 107.9, 122.5, 150.0, 152.0, 154.7,
156.8; HRMS (ESI-Orbitrap) m/z: exact mass calculated for
C14H18BrN4O3 [M + H]+: 369.0484, found: 369.0522.
crude was purified by flash chromatography on a silica gel
(230–400 mesh, elution gradient 0–9% MeOH in CH2Cl2).
(1S,2R,3R,5R)-5-(4-Amino-7H-pyrroloij2,3-d]pyrimidin-7-yl)-3-
(hydroxymethyl)-3-methyl-4-methylenecyclopentane-1,2-diol
(4a): purified yield: 78%, off-white solid, (TLC: Rf 0.2, 10%
MeOH in CH2Cl2); [α]2D0: +2.73 (c = 0.25, DMSO); mp: 210–220
1
°C; UV (MeOH) λmax: 274.25 nm; H NMR (400 MHz, CD3OD)
δ: 1.19 (s, 3H), 3.51 (d, J = 10.8 Hz, 1H), 3.66 (d, J = 10.8 Hz,
1H), 4.01 (d, J = 5.2 Hz, 1H), 4.55 (d, J = 3.2 Hz, 1H), 4.73 (dd,
J = 4.4 and 9.6 Hz, 1H), 5.07 (d, J = 3.2 Hz, 1H), 5.50–5.53 (m,
1H), 6.70 (d, J = 3.2 Hz, 1H), 7.27 (d, J = 3.2 Hz, 1H), 8.08 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 18.9, 49.2, 62.7, 69.2,
73.5, 74.3, 99.8, 102.0, 107.8, 123.8, 148.3, 149.5, 155.0, 155.3;
HRMS (ESI-Orbitrap) m/z: exact mass calculated for
C14H19N4O3 [M + H]+: 291.1457, found: 291.1422.
(1S,2R,3R,5R)-5-(4-Amino-5-fluoro-7H-pyrroloij2,3-d]-
p y r i m i d i n - 7 - y l ) - 3 - ( h y d r o x y m e t h y l ) - 3 - m e t h y l - 4 -
methylenecyclopentane-1,2-diol (4b): purified yield: 55%, off-
white solid, (TLC: Rf 0.2, 10% MeOH in CH2Cl2); [α]2D0: −6.43
(c = 0.25, DMSO); mp: 243–247 °C; UV (MeOH) λmax: 280.25
nm; 1H NMR (300 MHz, CD3OD) δ: 1.18 (s, 3H), 3.50 (d, J =
10.8 Hz, 1H), 3.63 (d, J = 10.8 Hz, 1H), 3.98 (d, J = 4.8 Hz,
1H), 4.59 (d, J = 3.2 Hz, 1H), 4.63 (dd, J = 4.5 and 9.6 Hz, 1H),
5.07 (d, J = 3.3 Hz, 1H), 5.47–5.51 (m, 1H), 7.00 (d, J = 2.1 Hz,
(1S,2R,3R,5R)-5-(4-Amino-5-iodo-7H-pyrroloij2,3-d]pyrimidin-
7-yl)-3-(hydroxymethyl)-3-methyl-4-methylenecyclopentane-1,2-
diol (4e): purified yield: 80%, off-white solid, (TLC: Rf 0.2,
10% MeOH in CH2Cl2); [α]2D0: +1.92 (c = 0.25, DMSO); mp:
1
227–228 °C; UV (MeOH) λmax: 290.25 nm; H NMR (300 MHz,
CD3OD) δ: 1.17 (s, 3H); 3.51 (d, J = 11.1 Hz, 1H), 3.65 (d, J =
10.8 Hz, 1H), 3.99 (d, J = 4.8 Hz, 1H), 4.57 (d, J = 2.4 Hz, 1H),
4.70 (dd, J = 4.8 and 9.9 Hz, 1H), 5.07 (d, J = 3.0 Hz, 1H),
5.40–5.50 (m, 1H), 7.37 (s, 1H), 8.04 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 18.9, 49.2, 50.4, 62.6, 69.2, 73.6, 74.3,
102.8, 107.9, 127.9, 150.7, 151.6, 154.8, 157.1; HRMS (ESI-
Orbitrap) m/z: exact mass calculated for C14H18IN4O3 [M +
H]+: 417.0345, found: 417.0377.
Fig.
3 Single-crystal X-ray structure of 4c, showing a thermal
displacement ellipsoid (50% probability) plot [CCDC no. 1969219].
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Fig. 4 The anti-HBV activity of 4c–f. The cell viability and activity are shown in the bar diagram. IC50: 50% effective concentration based on the
inhibition of the HBV DNA levels in culture supernatants. CC50: 50% cytotoxic concentration based on the reduction of viable cells.
Synthetic procedure for 4f
Synthetic procedure for 4g
A suspension of 4e (0.60 mmol), trimethylsilyl acetylene
(3.0 mmol), CuI (0.06 mmol), Et3N (3.0 mmol) and (PPh3)4-
Pd (0.06 mmol) in DMF was stirred at 50 °C under sealed
condition for 3 h. The reaction mixture was concentrated
under reduced pressure and crude was purified by silica
gel (100–200 mesh) column chromatography, elution
gradient 0–6% MeOH in CH2Cl2 to afford trimethylsilyl
protected compound. The deprotection was carried out by
stirring in methanol and K2CO3 (3.0 mmol) at rt for 30
min. The reaction mixture was concentrated under reduced
pressure and purified by flash chromatography on silica
gel (230–400 mesh), eluting gradient 0–7% MeOH in
CH2Cl2.
To a suspension of 4e (0.6 mmol), (PPh3)4Pd (0.06 mmol) in
anhydrous DMF under argon atmosphere, tri-n-butyl(vinyl)tin
(1.8 mmol) was added. The resulting mixture was heated at
110 °C for 3 h under sealed condition. Upon completion of
reaction, concentrated the volatile under reduced pressure
and crude was purified by flash chromatography on silica gel
(230–400 mesh), elution gradient 0–7% MeOH in CH2Cl2.
(1S,2R,3R,5R)-5-(4-Amino-5-ethynyl-7H-pyrroloij2,3-d]-
p y r i m i d i n - 7 - y l ) - 3 - ( h y d r o x y m e t h y l ) - 3 - m e t h y l - 4 -
methylenecyclopentane-1,2-diol (4g): purified yield: 60%, off
white solid, (TLC: Rf 0.1, 10% MeOH in CH2Cl2); [α]2D0: −14.28
(c = 0.25, DMSO); mp: 194–198 °C; UV (MeOH) λmax: 294.25
nm; 1H NMR (300 MHz, CD3OD) δ: 1.19 (s, 3H), 3.52 (d, J =
11.1 Hz, 1H), 3.67 (d, J = 11.1 Hz, 1H), 4.00 (d, J = 4.8 Hz, 1H),
4.57 (d, J = 2.7 Hz, 1H), 4.78 (dd, J = 4.8 and 9.9 Hz, 1H), 5.07
(d, J = 3.3 Hz, 1H), 5.24 (dd, J = 1.5 and 10.8 Hz, 1H), 5.44–
5.48 (m, 1H), 5.58 (dd, J = 1.8 and 17.4 Hz, 1H), 7.05 (dd, J =
10.8 and 11.1 Hz, 1H), 7.35 (s, 1H), 8.02 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 18.9, 49.2, 62.3, 69.2, 73.5, 74.1, 100.2,
107.7, 112.1, 113.3, 120.0, 129.2, 151.1, 155.0, 157.5; HRMS
(ESI-Orbitrap) m/z: exact mass calculated for C16H21N4O3 [M +
H]+: 317.1614, found: 317.1575.
(1S,2R,3R,5R)-5-(4-Amino-5-vinyl-7H-pyrroloij2,3-d]pyrimidin-
7-yl)-3-(hydroxymethyl)-3-methyl-4-methylenecyclopentane-1,2-
diol (4f): purified yield: 62%, off white solid. (TLC: Rf 0.1,
10% MeOH in CH2Cl2); [α]2D0: −2.17 (c = 0.25, DMSO); mp:
1
183–187 °C; UV (MeOH) λmax: 283.25 nm; H NMR (400 MHz,
CD3OD) δ: 1.18 (s, 3H), 3.51 (d, J = 10.8 Hz, 1H), 3.66 (d, J =
10.8 Hz, 1H), 3.70 (s, 1H), 3.99 (d, J = 4.4 Hz, 1H), 4.58 (d, J =
2.8 Hz, 1H), 4.72 (dd, J = 4.4 and 10.0 Hz, 1H), 5.08 (d, J = 3.2
Hz, 1H), 5.48–5.44 (m, 1H), 7.48 (s, 1H), 8.06 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ: 18.9, 49.3, 62.8, 69.2, 73.5, 74.2, 77.7,
82.7, 93.0, 102.1, 108.0, 128.5, 150.0, 152.4, 154.8, 157.4;
HRMS (ESI-Orbitrap) m/z: exact mass calculated for
C16H19N4O3 [M + H]+: 315.1457, found: 315.1418.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
12 A. Thiyagarajan, M. T. Salim, T. Balaraju, C. Bal, M. Baba
and A. Sharon, Bioorg. Med. Chem. Lett., 2012, 22,
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13 M. Kasula, R. Samunuri, H. Chakravarty, C. Bal, M. Baba,
A. K. Jha and A. Sharon, Nucleosides, Nucleotides Nucleic
Acids, 2016, 35, 43–52.
The authors are thankful to CIF, BIT Mesra, Ranchi, and
GVK's analytical team for spectroscopic characterization data.
The authors thank Dr. Ashoke Sharon, Department of
Chemistry, BIT Mesra, for X-ray structure solution.
14 M. Kasula, M. Toyama, R. Samunuri, F. Rozy, M. Yadav, C.
Bal, A. K. Jha, M. Baba and A. Sharon, Bioorg. Med. Chem.
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19 C14H17ClN4O3, M = 324.77, monoclinic, space group: P2IJ1), a
= 8.6168(3), b = 6.7974(3), c = 13.2395(4) Å, α = 90.000(0), β =
99.3435IJ8), γ = 90.000(0), V = 765.17(6) Å3, T = 293(2) K, Z =
2, μ = 0.268 mm−1, FIJ000) = 340.0, Dc = 1.410 Mg m−1, crystal
size 0.35 × 0.25 × 0.20 mm, 15 852 reflections measured,
3621 unique, R1 = 0.0323 for 2861 Fo > 4σ(Fo), and 0.0370 for
all 3143 data and 226 parameters. Unit cell determination
and intensity data collection was performed at 293(2) K.
Structure solutions by direct methods and refinements by
full-matrix least-squares methods on F2 [CCDC NO:
1969219].
11 M. Kasula, T. Balaraju, M. Toyama, A. Thiyagarajan, C. Bal,
M. Baba and A. Sharon, ChemMedChem, 2013, 8, 1673–1680.
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