Exploring the purine core of 3′-C-ethynyladenosine (EAdo) in search of novel nucleoside therapeutics

Article abstract of DOI:10.1016/j.bmcl.2016.03.005

A series of new nucleoside analogues based on a C-3 branched ethynyl sugar derivative as present in 3′-C-ethynylcytidine (ECyd) and -adenosine (EAdo), combined with modified purine bases was synthetized and evaluated against a broad array of viruses and t

Full text of DOI:10.1016/j.bmcl.2016.03.005

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1970–1972
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring the purine core of 30-C-ethynyladenosine (EAdo) in search
of novel nucleoside therapeutics
Fabian Hulpia a, Jan Balzarini b, Dominique Schols b, Graciela Andrei b, Robert Snoeck b,
Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
b KU Leuven, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
A series of new nucleoside analogues based on a C-3 branched ethynyl sugar derivative as present in 30-C-
ethynylcytidine (ECyd) and -adenosine (EAdo), combined with modified purine bases was synthetized
and evaluated against a broad array of viruses and tumour cell lines. The pronounced cytostatic activity
of EAdo was confirmed. EAdo and its 2,6-diaminopurine analogue showed inhibitory activity against vac-
Article history:
Received 4 February 2016
Revised 1 March 2016
Accepted 2 March 2016
Available online 3 March 2016
cinia virus (EC50: 0.31 and 51
lM, respectively). Derivative 10 on the other hand was found active against
varicella zoster virus (EC50: 4.68
l
M).
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
Nucleosides
30-C-Ethynyladenosine
Vorbrüggen coupling
Nucleoside analogues have been used as therapeutics for the
treatment of cancer and viral infections for over 50 years. Despite
this fact, new agents with improved efficacy and tolerability have
still been discovered over the past decade.1
Recently, a strategy relying on building nucleosides with a fixed
sugar and varying heterocyclic bases has shown to reveal intrigu-
ing biological activity.2,3 Additionally, previous focussed profiling
(e.g., only HIV, HCV or anticancer) might have overlooked starting
points for other indications (e.g., cytotoxicity in antiviral assays or
activity against other viruses). Furthermore, re-investigating cer-
tain sugar modifications has already shown to be a fruitful
approach with some derivatives ultimately being advanced into
clinical trials.4
With this in mind, research was initiated to explore the
untapped potential of certain nucleoside scaffolds. We therefore
became interested in a C-3 branched sugar motif, as is present in
a former Phase-II candidate, 30-C-ethynylcytidine (2, ECyd). After
the discovery in the mid-90s by Matsuda et al.,5 SAR of its sugar6
as well as its heterocyclic7 part has been reported, but failed to pro-
duce analogues with improved activity. The adenosine (1, EAdo)
congener of ECyd however, which also showed antitumour proper-
ties,5 only received scarce follow-up8,9 (see Fig. 1).
egy mentioned above. In this study we concentrated on the poorly
explored purine analogues.
In order to prioritize, we focused on substituents present in
FDA-approved nucleosides as well as clinical phase analogues. This
translated into modifications of EAdo at both the C-2 and C-6 posi-
tion of the purine ring to give a heterocycle pattern as is, for exam-
ple, found in the two FDA-approved nucleosides nelarabine 3 and
clofarabine 4.1 Additionally, two modifications (cyclopentylamine,
3-chlorobenzylamine) were included as they are preferred in
purinergic receptor ligands.10
The synthesis of the key ribofuranose building block 4 was
accomplished using known literature procedures.7,11 Next, three
different purines were subjected to ‘classical’ Vorbrüggen condi-
tions to achieve glycosylation with 412 (Scheme 1). Deprotection
or nucleophilic aromatic substitution with either NH3 in MeOH
or NaOMe in MeOH yielded the corresponding target nucleosides
1,5 7, 8, 12 and 13. Compounds 9 and 10 were obtained by nucle-
ophilic aromatic substitution with the appropriate amine, immedi-
ately followed by deprotection. Initially, synthesis of 14 and 15 was
also attempted from 11 and 6, respectively. However, due to pro-
longed exposure and elevated temperature to force nucleophilic
aromatic ring substitution, the desired product could not be iso-
lated. Instead, the product formed was the enol ether resulting
from a 5-exo dig cyclization of the 50-OH onto the alkyne (16 and
17). Therefore, ring substitution was performed before glycosyla-
tion (Scheme 2), resulting in 20 and 21. Final deprotection of these
intermediates furnished 14 and 15.
Based on these findings, the C-3 ethynyl ribofuranose unit was
believed a good sugar starting point for the ‘mix-and-match’ strat-
⇑
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: Serge.VanCalenbergh@ugent.be (S. Van Calenbergh).
http://dx.doi.org/10.1016/j.bmcl.2016.03.005
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

F. Hulpia et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1970–1972
1971
Figure 1.
Scheme 1. Reagents and conditions: (a) (1) N-benzoyladenine, HMDS, cat. (NH4)2SO4, reflux, (2) TMSOTf, MeCN, reflux; (b) 7 N NH3 in MeOH (40% over 2 steps (a + b)); (c) (1)
2,6-dichloropurine, HMDS, cat. (NH4)2SO4, reflux, (2) TMSOTf, 1,2-dichloroethane, reflux (72%); (d) 7 N NH3 in MeOH for 7 (31%), NaOMe in MeOH for 8 (72%), for 9: (1) c-
pentylamine, EtOH, reflux, (2) 7 N NH3 in MeOH (85%); for 10: (1) 3-chlorobenzylamine, EtOH, reflux, (2) 7 N NH3 in MeOH (77%); (e) (1) 2-amino-6-chloropurine, HMDS, cat.
(NH4)2SO4, reflux, (2) TMSOTf, 1,2-dichloroethane, reflux (43%); (f) 7 N NH3 in MeOH for 12 (25%) and NaOMe in MeOH for 13 (63%); (g) NaOMe in MeOH, rt to reflux, 38% for
16 and 29% for 17.
All final compounds were investigated for their potential activ-
ity against a broad array of viruses including herpes simplex virus-
1 and -2 (HSV), cytomegalovirus (CMV), varicella zoster virus
(VZV), vaccinia virus (VV), adenovirus-2, influenza A virus (H1N1,
H3N2), influenza B virus, feline corona virus, feline Herpes virus,
para-influenza virus, reovirus-1, sindbis virus, Coxsackie virus B4,
Punta Toro virus, vesicular stomatitis virus, respiratory syncytial
virus (RSV), HIV-1 and HIV-2. Additionally, inhibition of cell prolif-
eration of murine leukemia (L1210), human CD+4 T-lymphocyte
(CEM) and human cervix carcinoma cells (HeLa) was also evaluated
(Table 1).
EAdo 1 was confirmed to exhibit potent cytostatic activity,
while all other derivatives were found to be poorly cytostatic
(IC50: 104 > 250
cytotoxic (22–50
30 times less potent then EAdo. All nucleosides were found to be
inactive in the antiviral assays up to a concentration of 100 M,
l
l
M). Compound 10 was found to be moderately
M) in the three cell lines tested, but at least
Scheme 2. Reagents and conditions: (a) Na, MeOH, reflux, 91% for 18; 83% for 19;
(b) (1) 2-amino-6-methoxypurine (18) or 2,6-dimethoxypurine (19), HMDS, cat.
(NH4)2SO4, reflux, (2) TMSOTf, 1,2-dichloroethane, reflux; 29% for 20; 36% for 21; (c)
NaOMe in MeOH; 62% for 14; 66% for 15.
l

1972
F. Hulpia et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1970–1972
Table 1
Inhibition of proliferation
Acknowledgements
L1210
CEM
IC50
HeLa
IC50
F.H. is indebted to the Flanders Research Fund (FWO) for a PhD-
IC50
(l
M)
(lM)
(lM)
scholarship and D.S., G.A., R.S. and J.B. to the KU Leuven for finan-
cial support (GOA 15/19 TBA). The authors wish to thank Prof. K.
Jacobson for performing the adenosine A3-receptor assays on
derivatives 9 and 10.
1
7
8
9
10
12
13
14
15
0.73 0.14
167 37
>250
170 28
50 14
193 80
>250
0.61 0.08
>250
210 56
104 33
22 10
P250
>250
>250
>250
0.29 0.11
>250
175 106
150
43 20
1
123
7
Supplementary data
>250
>250
>250
>250
>250
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03.
005.
except for three. EAdo 1 showed potent anti-vaccinia virus activity
at subtoxic concentrations [EC50: 0.35 0.05 M; MCC (minimal
cytotoxic concentration): 20 M]. Diaminopurine derivative 13,
showed weak activity against vaccinia virus (EC50: 51 M;
MCC: >100 M). Finally, m-chlorobenzylamino derivative 10
showed moderate activity against VZV (EC50: 4.68 M; MCC:
References and notes
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obtained in either TK+ or TKꢀ strains.
Compounds 9 and 10 were also evaluated for their agonistic
behaviour at the adenosine A3-receptor. Both were found to bind
only weakly (70 9% and 48 6% inhibition at 10 lM for 9 and
10, respectively), which is in line with previous observations.13
Interestingly, compound 7 did not show any cytostatic activity,
even though the C-2 chloro substituent should make it more resis-
tant towards adenosine deaminase,14 the enzyme responsible for
the breakdown of EAdo.9 Furthermore, lack of phosphorylation
by cellular kinase(s) could also be a contributor to the observed
results, and further investigation on a prodrug approach that
allows intracellular release of the monophosphate form might be
more promising.
In conclusion, a subset of purine-modified nucleosides based on
C-3 branched chain sugar matched with different purines was syn-
thetized and evaluated against a broad array of viruses and tumour
cell lines. The potent cytostatic activity of EAdo was confirmed.
This compound was found inhibitory to vaccinia virus at subtoxic
concentrations. Two of the newly synthetized compounds were
found active antivirally. While their activity is only moderate, they
could serve as a starting point for further structural elaboration to
improve antiviral activity. Furthermore, these results indicate the
usefulness of the ‘mix-and match’ approach in finding novel bio-
logically active nucleosides.
13. Cappellacci, L.; Franchetti, P.; Pasqualini, M.; Petrelli, R.; Vita, P.; Lavecchia, A.;
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