M. J. Di Grandi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3483–3486
3485
ð6Þ
In this report, we have described a modified synthesis of
tetrazines 1 and 2, their usefulness as CMV protease
inhibitors, and proposed a mechanism for their antiviral
properties.
Figure 1. ORTEP representation of the X-ray crystal structure of
compound 1d.
Table 2. Summary of the biological activity of tetrazines 2 against
the HCMV UL80 protease
Entry
Compd
R
IC50 (mM)
References and Notes
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
2g
2i
2j
–H
–CH3
–nBu
–tBu
–CH(Ph)2
Ph
4-MePh
4-MeOPh
4-NO2Ph
0.1
0.3
0.4
0.6
1.7
0.4
0.3
0.5
1.7
1. Mocarski, E. S. In Virology; Fields, B. N., Knipe, D. M.,
Howley, P. M., Eds; Lippincott-Raven: Philadelphia, PA,
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N., Knipe, D. M., Howley, P. M., Eds, Lippincott-Raven:
Philadelphia, PA, 1996; p 2493.
2. More recently, valganciclovir, a valine ester prodrug of
ganciclovir has been demonstrated to have improved bio-
availability and is at least as efficacious as intravenous ganci-
clovir, see: Martin, D. F.; Sierra-Madero, J.; Walmsey, S.;
Wolitz, R. A.; Macey, K.; Georgiou, P.; Robinson, C. A.;
Stempien, M. J. New Engl. J. Med. 2002, 346, 1119.
3. HCMV protease is presumed to be required on the basis
that the analogous HSV protease is known to be essential; see:
Preston, V. G.; Coates, J. A.; Rixon, F. J. J. Virol. 1983, 45,
1056. Gao, M.; Matusick-Kumar, L.; Hurlburt, W.; Ditusa,
S.; Newcomb, W.; Brown, J. R.; McCann, P. J.; Devkman, I.;
Colonno, R. J. J. Virol. 1994, 68, 3702.
4. For a recent review, see: Olgilvie, W. W. Curr. Med. Chem.
Anti-Infective Agents 2002, 1, 177. Also see: Borthwick, A. D.;
Exall, A. M.; Haley, T. M.; Jackson, D. L.; Mason, A. M.;
Weingarten, G. G. Bioorg. Med. Chem. Lett. 2002, 12, 1719.
Borthwick, A. D.; Crame, A. J.; Ertl, P. F.; Exall, A. M.;
Haley, T. M.; Hart, G. G.; Mason, A. M.; Pennell, A. M. K.;
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Chem. 2002, 45, 1. Borthwick, A. D.; Angier, S. J.; Crame,
A. J.; Exall, A. M.; Haley, T. M.; Hart, G. H.; Mason, A. M.;
Pennell, A. M. K.; Weingarten, G. G. J. Med. Chem. 2000, 43,
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L.; Mansour, T. Bioorg. Med. Chem. Lett. 1998, 8, 365. Yoa-
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6. These compounds demonstrated selectivity for HCMV
protease over other known serine proteases—data not shown.
7. Prepared by condensing 1,3-diaminoguanidine with ethyl 2-
methylacetoacetate; see: Hlavka, J. J.; Bitha, P.; Lin, Y.;
Strohmeyer, T. J. Heterocyclic Chem. 1984, 21, 1537.
With several potent inhibitors of HCMV UL80 protease
in hand, we next examined their mode of action and
discovered tetrazines 2 undergo redox chemistry via a
mechanism reminiscent of flavins. In particular, mass
spectrometry determined that exposure of HCMV pro-
tease to compound 2a afforded a chemically modified
enzyme in which two pairs of Cys residues (Cys84–
Cys87 and Cys138–Cys161) were oxidized to disulfides
(eq 5).5 On the basis of these results and molecular
orbital calculations, we propose the following mechan-
ism. The nucleophilic sulfur of a Cys residue attacks at
C-10 of 2, pushing electrons into the highly conjugated
p system. Subsequent attack by a neighboring Cys sul-
fur generates a disulfide, releasing the dihydrotetrazine.
ð5Þ
That this redox mechanism is plausible is supported by
several pieces of evidence. Firstly, dihydrotetrazines 1
are completely inactive in the scintillation proximity
assay. Secondly, riboflavin and flavin mononucleotide
are equally potent inhibitors of this protease5 and
finally, the reduction of tetrazines 2 to dihydrotetrazine
1 was easily accomplished with thiophenol. The addi-
tion of PhSH to a CDCl3 solution of 2f (R=Ph) resul-
ted in
precipitation of 1f (eq 6).16
a
slow discoloration with concomitant