Alkenyl substituted bicyclic nucleoside analogues retain nanomolar potency against Varicella Zoster Virus

Article abstract of DOI:10.1016/j.bmc.2009.03.022

Novel alkenyl substituted aryl bicyclic furano pyrimidines have been prepared and evaluated in vitro against Varicella Zoster Virus (VZV). The para-substituted analogues retain the nanomolar potency we have reported for p-alkyl analogues, while the ortho-

Full text of DOI:10.1016/j.bmc.2009.03.022

Bioorganic & Medicinal Chemistry 17 (2009) 3025–3027
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alkenyl substituted bicyclic nucleoside analogues retain nanomolar
potency against Varicella Zoster Virus
a
a
b
b
b
Christopher McGuigan a, , Olivier Bidet , Marco Derudas , Graciela Andrei , Robert Snoeck , Jan Balzarini
*
a Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel alkenyl substituted aryl bicyclic furano pyrimidines have been prepared and evaluated in vitro
against Varicella Zoster Virus (VZV). The para-substituted analogues retain the nanomolar potency we
have reported for p-alkyl analogues, while the ortho- and meta- alkenyl systems lose 3–4 orders of
potency.
Received 24 November 2008
Revised 9 March 2009
Accepted 10 March 2009
Available online 14 March 2009
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords:
BCNAs
VZV
Antiviral
We have previously reported the discovery in our laboratories
of the bicyclic furano pyrimidine family of antivirals (the BCNAs),
which display unique potency and selectivity versus Varicella Zos-
ter Virus (VZV).1 We had originally reported the alkyl family but
subsequently noted substantial increases in potency for the phenyl
analogues, particularly with p-alkyl substitution (1–3).2 We believe
the p-pentyl analogue to be the most potent inhibitor of VZV
reported to date, with in vitro efficacy at ca. 0.5 nM, and little or
no detectable toxicity.3 Indeed, as its orally bioavailable 50-valyl
prodrug, FV100, (4) has entered clinical trials for VZV shingles,
and promising early clinical data have emerged.4
There are few reports of alternative substitution patterns on the
phenyl ring, although we have noted that ortho halogens are toler-
ated while meta are not.7 In this paper we report the effect of
unsaturation in the p-alkyl side chain at the position adjacent to
the phenyl ring, and also the effect of moving the alkenyl chain
to the ortho and meta positions of the phenyl.
The synthetic route to the desired target compounds was based
around the need for a regio- and stereo- selective access. We chose
to follow the one-pot method of Kabalka8 where E-1-arylalkenes
are prepared in high selectivity from ketones and aromatic
aldehydes. Kabalka suggests a tandem Aldol-Grob sequence mech-
anism for this reaction.9 Thus, the ortho- (5a) meta- (5b) and para-
(5c) bromo benzaldehydes were reacted with symmetrical ketones
pentan-3-one, heptan-4-one and nonan-5-one in the presence of
boran-trifluoride to generate the corresponding E-1-alkenylaryl
bromides (Scheme 1). Yields ranged from 12% to 81% and were
higher for longer chain homologues. Pure E stereochemistry was
confirmed by a ca. 15 Hz olefinic coupling constant.
Following poorly satisfactory attempts to convert these
bromophenyl systems to their trimethysilylacetylenes for coupling
to 5-iodo nucleoside, we decided instead to prepare the
We have described some of the structure–activity relationships
surrounding lead compound (3).5 In particular, activity increases
ca. 1000-fold on para-n-pentyl substitution of the phenyl ring.6
* Corresponding author. Tel./fax: +44 2920874537.
E-mail address: mcguigan@cardiff.ac.uk (C. McGuigan).
Scheme 1.
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.022

3026
C. McGuigan et al. / Bioorg. Med. Chem. 17 (2009) 3025–3027
R
R
O
N
HN
Pd(PPh3)4
9
CuI
O
O
O
iPr2EtN, DMF
RT, 17h
CuI
Et3N
HO
N
HN
O
13. R=Me
14. R=Et
15. R=nPr
a. ortho
O
N
Iodoarylalkene
O
N
series a:
dioxane
reflux 18h.
series b-c
MeOH
6-8, a-c
HO
OH
HO
O
O
b. meta
c. para
OH
OH
10-12, a-c
reflux 4h
Scheme 2.
Table 1
Anti-VZV activity of compounds 13–15a–c
C3 EC50 C3 ( M)
l
C4 EC50
YS
(
l
M)
C5 EC50
YS
(lM)
Compd OKA
YS
MCC (
l
M) CC50
(lM) Compd OKA
MCC (
lM) CC50
(l
M) Compd OKA
MCC (
lM) CC50 (lM)
o
m
p
13a
13b
13c
12
13
—
58
400
>200
103
>200
>200
14a
14b
14c
33
1.9
—
2.3
400
P20
102
68
>200
15a
15b
15c
8.4
1.1
—
1.3
80
P200
41
>200
>200
0.004 0.004 P50
0.0006 0.0007 P5
0.0003 0.0008 >5
p-Alkyl
1
0.011 0.003 P50
188
2
0.0016 0.0005 P50
>200
3
0.0006 0.0005 P50
>200
5-ethynyl nucleoside and couple to the iodo phenyl system. Thus,
we carried out halogen (iodine for bromine) exchange on the aryl
bromides using copper iodide in hexamethyl phosphoric acid and
elevated temperature.10 Thus, products 6–8a–c were isolated in
good yield (67–93%) after flash column chromatography. The sub-
stitution of bromine by iodine was most evident in the 13C NMR
Spectra (dc Ar-I 95 ppm, Ar-Br 120 ppm), and by changes in the
phenyl hydrogen pattern in the 1H NMR.
The necessary nucleoside synthon 5-ethynyl-20-deoxyuridine
(9) was prepared from the 5-iodo nucleoside via Sonogoshira
coupling to trimethylsilyl acetylene,11 and this was coupled to
the iodophenyl species 6–8a–c under Pd(0) and Cu(I) catalysis in
DMF to yield the intermediate 5-alkynyl species 10–12a–c, Scheme
2. These were then cyclised to give the desired bicyclic systems
13–15a–c. The products were characterised by 1H and 13C NMR,
mass spectrometry and microanalysis, all data confirming the
structure and purity of the products.12
Figure 1. Flexible alignment simulation of compounds 3 and 14c.
The target compounds were assayed for their ability to inhibit
two strains of VZV in vitro, OKA and YS, with data being shown
in Table 1.13 Data on the comparative alkyl analogues (para only)
1–3 are included for comparison.
For the para series 13–15c a very potent anti-VZV effect is noted
ranging from ca. 4 nM (C3) to ca. 0.3–0.8 nM (C4, C5). Data on this
family mirrors closely that of the parent alkyl series 1–3.
In all cases, compounds lost completely activity versus thymi-
dine kinase-deficient virus mutants (data not shown) confirming
their complete dependence on VZV TK for activation. By compari-
son to the para family the ortho- and meta series were noted to
be poorly active, being ca. 3–4 orders of magnitude less potent
than the para series. For the C3–C4 homologues the meta com-
pounds were slightly more active than the ortho and indeed were
equiactive with acyclovir in these assays but still ca. 2000-fold less
potent than their para isomers.
Figure 2. Energy minimized structures of compounds 14a–c.
Lastly, slight cytotoxicity was seen for some of the ortho and
meta compounds but not the para regioisomers.
supporting their similarly high potencies, while the ortho and the
meta derivatives showed completely different structures (Fig. 2),
supporting the poor activity for these latter compounds.
Flexible alignment simulation14 (Fig. 1) of 3 (red chain) and 15c
(green chain) showed a good alignment for these two compounds,

C. McGuigan et al. / Bioorg. Med. Chem. 17 (2009) 3025–3027
3027
Table 2
den Heurck, Mrs. Anita Camps and Mr. Steven Carmans for excel-
lent technical assistance. Financial support was provided by the
K.U.Leuven (GOA 05/19).
Inhibition of VZV TK-catalysed dThd phosphorylation by test compounds
a
Compound
IC50 (lM)
13a
13b
13c
14a
14b
14c
15a
15b
15c
1
0.31 0.04
2.6 1.0
1.9 1.5
3.8 0.4
References and notes
1. McGuigan, C.; Yarnold, C. J.; Jones, G.; Velazques, S.; Barucki, H.; Brancale, A.;
Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1999, 42, 4479.
2. McGuigan, C.; Barucki, H.; Blewett, S.; Carangio, A.; Erichsen, J. T.; Andrei, G.;
Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 2000, 43, 4993.
3. Sienaert, R.; Naesens, L.; Brancale, A.; De Clercq, E.; McGuigan, C.; Balzarini, J.
Mol. Pharmacol. 2002, 61, 249.
23
7
2.4 0.2
119 73
397 34
2.3 0.4
4. Canas, S. M.; Wasgin, B.; Boehlecke, B.; Henson, G.; Patti, J. M.; Morris, A. M.
21st ICAR, Montreal, April 13–17, 2008, p 40.
25
3
2
3
4.7 0.2
4.1 0.5
5. McGuigan, C.; Pathirana, R.; Migliore, M.; Adak, R.; Angell, A.; Henson, G.;
Snoeck, R.; Andrei, G.; De Clercq, E.; Balzarini, J. Antiviral Res. 2006, 70, A31.
6. McGuigan, C.; Pathirana, R. N.; Migliore, M.; Adak, R.; Luoni, G.; Jones, A. T.;
Diez-Torrubia, A.; Camarasa, M.-J.; Velazquez, S.; Henson, G.; Verbeken, E.;
Sienaert, R.; Naesens, L.; Snoeck, R.; Andrei, G.; Balzarini, J. J. Antimicrob.
Chemother. 2007, 60, 1316.
a
50% Inhibitory concentration required to inhibit VZV TK-catalysed dThd (1
phosphorylation by 50%.
lM)
7. McGuigan, C.; Jukes, A.; Blewett, S.; Barucki, H.; Erichsen, J. T.; Andrei, G.;
Snoeck, R.; De Clercq, E.; Balzarini, J. Antiviral Chem. Chemother. 2003, 14, 165.
8. Kabalka, G. W.; Tejedor, D.; Li, N. S.; Mallah, R. R.; Trotman, S. J. Org. Chem. 1998,
63, 6438.
9. Kabalka, G. W.; Li, N. S.; Tejedor, D.; Malladi, R. R.; Trotman, S. J. Org. Chem.
1999, 64, 3157.
The compounds have also been investigated for their affinity
against recombinant VZV thymidine kinase (TK) (Table 2). The
assays were performed by measuring the inhibition of VZV TK-cat-
alysed [3H]thymidine phosphorylation by the test compounds. The
50% inhibitory concentrations ranked between 0.33 and 416 lM
10. Suzuki, H.; Kondo, A.; Ogawa, T. Chem. Lett. 1985, 4, 11.
11. (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 50, 4467; (b)
Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854.
and did not correlate within the C3 (13), C4 (14), C5 (15) series
nor with their antiviral activity.
12. Typical procedure and data for (13b): To a stirred solution of 5-ethynyl-20-
deoxyuridine (500 mg, 1.98 mmol) in dry dimethylformamide (8 mL), at room
For example, compound 13a (ortho analogue with C3 side
temperature
under
a
nitrogen
atmosphere,
were
added
dry
chain) was more inhibitory (IC50: 0.33
counterparts (IC50: 2.7–3.2 M), whereas the para derivative in the
C5-series was more inhibitory (IC50: 2.6 M) than the ortho and
meta derivatives (IC50: 160–416 M). Lack of correlation between
the antiviral activity of the BCNAs and their affinity for VZV TK
has been noted earlier,15 and indicates a different structure–activ-
ity relationship of the compounds for VZV TK and the eventual
antiviral target.
While the precise mode of action of the BCNAs versus VZV
remains unclear, the data in this manuscript support our earlier con-
clusions3 that phosphorylation of the compounds by the VZV-en-
coded thymidine kinase is a pre-requisite for their antiviral action.
Interestingly, we have found16 that although the TK from Simian
Varicella Virus (SVV) can phosphorylate the BCNAs, the agents are
not inhibitory to SVV in vitro. This implies that the eventual target
of the BCNAs may be specific to VZV. We are now further pursuing
such a target by utilising a tritium labelled (3) prepared by the cata-
lytic addition of tritium to compound (15c) described above. The re-
sults of these studies will be reported in due course.
lM) than its meta and para
diisopropylethylamine (2.64 mmol, 0.46 mL), 1-E-(1-propenyl)-3-iodobenzene
(322 mg, 1.32 mmol), tetrakis (triphenylphosphine) palladium(0) (153 mg,
0.132 mmol) and copper(I) iodide (50 mg, 0.264 mmol). The reaction mixture
was stirred at room temperature, for 18 h. Copper(I) iodide (50 mg, 0.264 mmol),
triethylamine (10 mL) were then added to the mixture, which was heated at
80 °C, for 6 h. The reaction mixture was concentrated in vacuo, and the resulting
residue was dissolved in methanol and dichloromethane (1:1) (50 mL)
whereupon an excess of Amberlite IRA-400 (hydrogen carbonate form) was
added and stirred for 1 h at room temperature. The resin was filtered, washed
with methanol, and the combined filtrate was evaporated to dryness to obtain a
brown residue. The crude product was purified by flash column chromatography
on silica gel to offer a yellow solid. The product was recrystallised in hot
methanol to give a white solid (205 mg, 28%).1H NMR (DMSO-d6; 300 MHz) d
8.89 (1H, s, H-4), 7.82 (1H, m Ar-H), 7.65 (1H, m, Ar-H), 7.44 (2H, m, Ar-H), 7.33
(1H, s, H-5), 6.48 (2H, m, H-c1 and H-c2), 6.19 (1H, app t, J = 6.0 Hz, H-10), 5.32
(1H, d, J = 4.3 Hz, OH-30), 5.21 (1H, t, J = 5.1 Hz, OH-50), 4.26 (1H, m, H-30), 3.94
(1H, m, H-40), 3.68 (2H, m, H-50), 2.41 (1H, m, H-20), 2.15 (1H, m, H-20), 1.88 (3H, d,
J = 6.9 Hz, H-c3); 13C NMR (DMSO-d6; 75 MHz) d 171.4 (C-7a), 154.1, 153.9 (C-2,
C-6), 138.6 (C-4), 138.5 (C-c), 130.5, 129.7, 127.3, 126.9, 123.3, 122.0 (6 ꢀ C: C-b,
C-d, C-e, C-f, C-c1, C-c2), 129.0 (C-a), 107.1 (C-4a), 100.1 (C-5), 88.5 (C-40), 88.0
(C-10), 69.8 (C-30), 60.9 (C-50), 41.6 (C-20), 18.7 (C-c3); MS (ES+) m/e 391 (MNa+,
100%); Accurate mass: C20H20N2O5Na requires 391.1270; found 391.1274; Anal.
Calcd for C20H20N2O5ꢁH2O: C, 65.21; H, 5.47; N, 7.60. Found: C, 64.99; H, 5.04; N,
7.21.
l
l
l
In conclusion, p-1-alkenyl phenyl BCNAs are noted to be highly
potent anti-VZV agents, being roughly equipotent with their p-al-
kyl parents. By contrast their ortho and meta 1-akenyl analogues
are rather poorly active. There was, however, no correlation
between affinity of the compounds for VZV TK and the eventual
anti-VZV activity.
13. Confluent HEL cells grown in 96-well microtiter plates were inoculated with
VZV at an input of 20 PFU (plaque-forming units/well). After a 1–2 h incubation
period, residual virus was removed and the infected cells were further
incubated with MEM (supplemented with 2% inactivated FCS, 1% L-glutamine
and 0.3% sodium bicarbonate) containing varying concentrations of the
compounds. Antiviral activity was expressed as EC50 (50% effective
concentration), or compound concentration required to reduce viral plaque
formation after 5 days by 50% compared to the untreated control.
14. Minimisation and flexible alignment simulation were performed using MOE
2007.09 (molecular operating environment) (MOE 2007.09. Chemical
Computing Group, Inc., Montreal, Que., Canada. http://www.chemcomp.com).
15. Balzarini, J.; McGuigan, C. Biochim. Biophys. Acta 2002, 1587, 287.
16. Sienaert, R.; Andrei, G.; Snoeck, R.; De Clercq, E.; Luoni, G.; McGuigan, C.;
Balzarini, J. Antiviral Res. 2004, 62. 17th ICAR, Tucson AZ, May 2–6, abstract 94.
Acknowledgements
The authors would like to thank Helen Murphy for excellent
secretarial assistance. Mrs. Lizette van Berckelaer, Mrs. Lies Van