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I. Stansfield et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5085–5088
retains both a hydrogen bond donor at C6 and a nitrogen
lone pair orientated in similar fashion to the C5 O–Mg
bond in the proposed chelate) were devoid of activity.
Assuming that metal-binding does indeed involve a six-
membered chelate as depicted in Figure 2, these results,
together with the observation that N-methylpyrimidones9
such as 24 retain activity (IC50 4.2lM) suggest that pyrim-
idines may interact with the enzyme as the keto-tautomer
4b rather than in the enol form 4a, and that hydrogen
bond acceptor functionality is required at C6.
In conclusion, we have described a series of 2-thiophene
substituted 5,6-dihydroxypyrimidine-4-carboxylic acids
that show a 10-fold improvement in activity over the
original lead compound in this series 2. Tight SAR
around the pyrimidine ring was delineated, and struc-
tural changes at the C6 OH groupwere not permitted.
In contrast, a trifluoromethyl acyl sulfonamide replace-
ment for the C4 carboxylic acid groupof 4 was toler-
ated, providing an alternative structurally related series
for future development.
The effects of structural changes at the C4 carboxylic
acid groupare summarised in Table 3. A charged group
at this position was essential; carboxylic esters (com-
pound 10) showed no inhibition of the polymerase en-
zyme. Surprisingly, charged acid isosteres such as the
phosphonic acid 11, the tetrazole 12, and the acylsulfon-
amide derivative 13 were also inactive. While this may re-
flect an inability of these groups to satisfy the geometric
requirements for interaction with Mg2+ at the active site,
the electronic nature of the acidic groupis also likely to
be important. Changes in pKa at C4 likely affect the
Mg2+ interaction of the C4 acidic groupitself and may
also have an impact on the pKa/metal interaction of the
C5 (or C6) phenolic OH groups. To further explore the
role of pKa at C4 the trifluoromethyl acylsulfonamide
derivative 13 was prepared, and provided the first struc-
ture in which modification of the b,c-dihydroxy carboxy-
lic acid motif did not seriously compromise potency.
Acknowledgement
This work was funded in part by a grant from the
MIUR.
References and notes
1. Beaulieu, P. L.; Llinas-Brunet, M. Current Med. Chem.:
Anti-Infective Agents 2002, 1, 163.
2. Zeuzem, S.; Feinman, S. V.; Rasenack, J.; Heathcote, E.
J.; Lai, M.-Y.; Gane, E.; OꢀGrady, J.; Reichen, J.; Diago,
M.; Lin, A.; Hoffman, J.; Brunda, M. J. N. Engl J. Med.
2000, 343, 1666.
3. Monto, A.; Wright, T. L. Semin. Oncol. 2001, 441.
4. Walker, M. P.; Hong, Z. Current Opin. Pharmacol. 2002,
2, 534.
5. Behrens, S.-E.; Tomei, L.; De Francesco, R. EMBO J.
1996, 15, 12; Lesburg, C. A.; Cable, M. B.; Ferrari, E.;
Hong, Z.; Mannarino, A. F.; Weber, P. C. Nature Struct.
Biol. 1999, 6, 937.
The improved potency of 14 (IC50 6.2lM) relative to 13
(IC50 > 50lM) most likely stems from its lower acylsul-
fonamide pKa rather than alleviation of an unfavour-
13
6. Joyce, C. M.; Steitz, T. A. J. Bacteriol. 1995, 177, 6321.
7. Chan, L.; Reddy, T. J.; Proulx, M.; Das, S. K.; Pereira, O.;
Wang, W.; Siddiqui, A.; Yannopoulos, C. G.; Poisson, C.;
Turcotte, N.; Drouin, A.; Alaoui-Ismaili, M. H.; Bethell,
R.; Hamel, M.; LꢀHeureux, L.; Bilimoria, D.; Nguyen-Ba,
N. J. Med. Chem. 2003, 46, 1283.
8. Summa, V.; Petrocchi, A.; Pace, P.; Matassa, V. G.; De
Francesco, R.; Altamura, S.; Tomei, L.; Koch, U.;
Neuner, P. J. Med. Chem. 2004, 47, 14.
9. Summa, V.; Petrocchi, A.; Matassa, V. G.; Taliani, M.;
Laufer, R.; De Francesco, R.; Altamura, S.; Pace, P. J.
Med. Chem., submitted for publication.
10. Bougie, I.; Charpentier, S.; Bisaillon, M. J. Biol. Chem.
2003, 278, 3868.
able steric interaction (for the benzyl groupof 13)
since methyl acylsulfonamides were inactive in a closely
related series.14 Compound 13 retained the metal
dependent inhibition profile9 shown by the correspond-
ing dihydroxypyrimidine carboxylic acid 4, suggesting
that the binding modes of these compounds are similar.
This was confirmed by a kinetic competition study15 in
which the IC50 of 4 was measured in the presence of
three fixed concentrations of 14. The expected linear
dose-dependent shift in the inhibition curve of 4 (Dixon
plot) was indeed observed.
11. Culbertson, T. P. J. Het. Chem. 1979, 16, 1423.
12. A full account of the role of heterocyclic groups at the C2
position of the dihydroxy pyrimidine ring and of the
tautomeric equilibrium in this series will be published
elsewhere.
Table 3. Structural modifications at the C4 position in the
dihydroxypyrimidine series
OH
OH
N
13. The Hammet rp values for a benzyl and CF3 substituent
are ꢀ0.19 and +0.54, respectively. The measured pKa (in
MeOH) of a methyl acylsulfonamide of a benzoic acid
derivative was found to be more than 4 log units higher
than the corresponding trifluoromethyl acylsulfonamide:
Talanov, V. S.; Talanova, G. G.; Gorbunova, M. G.;
Bartsch, R. A. J. Chem. Soc., Perkin Trans. 2 2002, 209;
For a further account of the acidifying effect of the
NHSO2CF3 substituent see: Yagupolskii, L. M.; Petrik, V.
N.; Kondratenko, N. V.; Soovali, L.; Kaljurand, I.; Leito
Koppel, I. A. J. Chem. Soc., Perkin Trans. 2 2002, 1950.
14. Unpublished observation.
S
N
X
Compound
X
IC50 (lM)
4
COOH
2.6
n/aa
n/a
n/a
n/a
6.2
10
11
12
13
14
COOMe
PO3H2
Tetrazole
CONHSO2Bn
CONHSO2CF3
a Compounds designated n/a showed less than 30% inhibition of NS5B
at an inhibitor concentration of 50lM.
15. Dixon, M. Biochem. J. 1953, 55, 1.