The de-guanidinylated derivative of peramivir remains a potent inhibitor of influenza neuraminidase

Article abstract of DOI:10.1016/j.bmcl.2011.09.076

The guanidine function in the potent neuraminidase inhibitor peramivir was included early on in the drug design process, and examination of X-ray structural data for the enzyme-inhibitor complex would seem to indicate that the guanidine plays a critical r

Full text of DOI:10.1016/j.bmcl.2011.09.076

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7137–7141
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The de-guanidinylated derivative of peramivir remains a potent inhibitor
of influenza neuraminidase
Caleb M. Bromba a, Jeremy W. Mason a,b, Michael G. Brant a, Tracy Chan c, Martine D. Lunke b, Martin Petric c,
Martin J. Boulanger b, Jeremy E. Wulff a,
⇑
a Department of Chemistry, University of Victoria, Victoria, BC, Canada V8W 3V6
b Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6
c British Columbia Centre for Disease Control, 655 West 12th Avenue, Vancouver, BC, Canada V5Z 4R4
a r t i c l e i n f o
a b s t r a c t
Article history:
The guanidine function in the potent neuraminidase inhibitor peramivir was included early on in the drug
design process, and examination of X-ray structural data for the enzyme–inhibitor complex would seem to
indicate that the guanidine plays a critical role in promoting binding. However, this functional group may
also contribute to the poor oral availability of the drug. Given that the relative stereochemistry on the gua-
nidine-bearing carbon in peramivir is opposite to that in zanamivir (a related neuraminidase inhibitor, for
which the guanidine function is known to contribute substantially to the potency), we sought to determine
the importance of the guanidine group to peramivir’s overall potency. Here we report that the de-guanid-
inylated analogue of peramivir is only ca. 1-order of magnitude less potent than peramivir itself in two
in vitro inhibition assays. This suggests that next-generation inhibitors designed to improve on peramivir’s
properties might profitably dispense with the guanidine function.
Received 1 September 2011
Revised 17 September 2011
Accepted 19 September 2011
Available online 24 September 2011
Keywords:
Neuraminidase
Inhibitor
Oral availability
Influenza
Ó 2011 Elsevier Ltd. All rights reserved.
Peramivir
Guanidine
The influenza virus infects approximately 600,000,000 people
globally each year, resulting in 250,000–500,000 ‘excess deaths’.1
Notwithstanding the implementation of a World Health Organiza-
tion-monitored vaccine program, influenza remains a significant
global health issue. Influenza mutates rapidly, leading to the con-
tinual emergence of new strains. Because of the long lead-time re-
quired to produce the trivalent influenza vaccine (TIV) each year,
vaccinated individuals remain susceptible to these new strains.2
Treatment of influenza infections has historically relied on two
classes of molecules. The first class includes the adamantanes
rimantadine and amantadine, and works by blocking the M2 pro-
ton channel.3 Unfortunately, resistance to the adamantanes is
now widespread.4
The second, more successful, class of drugs targets viral neur-
aminidase (also called sialidase). Zanamivir (see Fig. 1 for structures)
was developed as a structural mimic of the boat-shaped sialic acid-
hydrolysis transition structure, and is effective in limiting viral rep-
lication.5 However, the large number of heteroatoms in zanamivir’s
structure limits its oral bioavailability.6 As a result, zanamivir must
be administered by aerosol inhalation, and has therefore seen re-
duced clinical use.6 Oseltamivir is a second-generation neuramini-
dase inhibitor with substantially improved oral bioavailability.7
This drug dominates the influenza market, with sales of ꢀ$1 billion
per year. Oseltamivir is a prodrug, which is hydrolyzed in the liver to
form the biologically active carboxylate.8
An attempt to access neuraminidase inhibitors with a core
structure distinct from oseltamivir led to development of the substi-
tuted cyclopentane peramivir. A guanidine function (as in zanami-
vir) was included from the beginning of the drug design process,
but no effort was made to control the absolute stereochemistry at
OH
OH
RO
HO2C
HO
HO2C
H
H
O
OH
O
OH
neuraminidase
NH OH
O
NH OH
O
HO
HO
R = oligosaccharides
sialic acid
OH
H
HO2C
OH
H
H
HO2C
HN
O
OH
OH
EtO2C
O
NH
*
NH
NH
NH
NH
HN
H2N
O
O
O
NH2
zanamivir
NH2
peramivir
oseltamivir
Figure 1. Function of neuraminidase, and structures of the three clinically used
inhibitors. Colors indicate functionally related substituents.
⇑
Corresponding author.
E-mail address: wulff@uvic.ca (J.E. Wulff).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.076

7138
C. M. Bromba et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7137–7141
2007, with the report of substantial oseltamivir resistance in H1N1
strains (a common subtype of the virus) circulating in the United
States (11%),15 Canada (26%), Europe (25%) and Hong Kong
(12%).16 Even more alarming, data from the first half of the 2008/
2009 flu season (prior to the emergence of the ‘swine flu’ H1N1
strain) showed that nearly all circulating cases of H1N1 influenza
A were resistant to oseltamivir.17 Fortunately, most cases of ‘swine
flu’ H1N1 (e.g. A/Mexico/InDRE4487/2009) responded to treatment
with oseltamivir.18 Although not yet formally approved by the FDA,
peramivir was also used successfully as an injectable antiviral
agent for the treatment of the 2009 influenza A H1N1 virus.19
Oseltamivir resistance is largely the result of a single second-
shell mutation, wherein His274 in group 1 neuraminidases (N1,
N4, N5 and N8) is mutated to tyrosine. This in turn pushes a nearby
carboxylate residue (Glu276) into the active site, where it suffers
unfavorable interactions with the lipophilic 3-pentyl sidechain of
oseltamivir, resulting in complete resistance to clinically used
doses of oseltamivir. Because peramivir incorporates the same
lipophilic 3-pentyl sidechain, it is likewise ineffective against the
H274Y variant.20 Strains of N1 H274Y influenza remain susceptible
to zanamivir, since the triol sidechain of that drug can engage in
productive hydrogen bonding interactions with Glu276 (see
Fig. 2A).21 Other mutations are known to confer resistance to
zanamivir,22 although these are currently much less common than
mutations leading to oseltamivir and peramivir resistance.
The widespread prevalence of the H274Y mutation in circulat-
ing strains of influenza suggests that next-generation neuramini-
dase inhibitors might do well to incorporate relatively polar
sidechains to bind to Glu276. This will necessarily result in an
increasing number of hydrogen bond donors and acceptors, and
will further decrease the logP to the point where such compounds
are less likely to be orally active. In the hopes of regaining oral
availability, it is important to understand which functional groups
in the existing drugs are critically important for potency, and
which groups might be replaced with less polar functionality. In
this context, the carboxylic acid function common to all three
drugs would seem to be required for binding to the Arg118/
Arg371/Arg292 triad (see Fig. 2A), but the guanidine groups in
zanamivir and peramivir represent potentially attractive sites in
which to engineer improvements.
Figure 2. Comparison of binding modes and key interactions for oseltamivir (cyan;
PDB 2HT8), zanamivir (magenta; PDB 2HTQ), and peramivir (green; PDB 2HTU) to a
representative N8 neuraminidase (shown as lighter variants of the same color).10
(A) top view of all three molecules in the active site. Key interactions include the
carboxylate binding to the Arg118/Arg371/Arg292 triad, the peramivir hydroxyl
group binding to Asp151, and the zanamivir triol binding to Glu276. A lipophilic
interaction with the N-acetyl group is also known to be important for binding,29 but
is not shown. (B) Bottom view showing the different binding modes for the
zanamivir and peramivir guanidinium groups. All distances are reported in
angstroms. Images were prepared using PyMol (DeLano Scientific).
OH
OH
HO2C
HO2C
OH
H
HO2C
HN
NH
H2N
NH
H2N
1
2
3
O
NH2
Figure 3. Structures of the three synthetic compounds evaluated in this work.
the guanidine-bearing carbon atom (indicated with an asterisk in
Fig. 1).9 The most potent compound in early development, selected
from a mixture of diastereomers by co-crystallization with the en-
zyme, contained the opposite stereochemistry to zanamivir or osel-
tamivir at this centre.9 This serendipitous discovery led to a final
optimized compound (BCX-1812, which was subsequently given
the name peramivir) that could more effectively probe a polar pock-
et at the base of the active site; as illustrated in Figure 2B for an N8
neuraminidase, the R guanidine function in peramivir can extend
deeper into the pocket comprised of Glu119, Glu227 and the back-
bone carbonyl of Trp178 than can the S guanidine of zanamivir.10
In other isotypes of neuraminidase, the guanidine groups in the
two drugs come closer to occupying the same space in the binding
site.11 Although originally intended as an oral antiviral agent, per-
amivir displayed poor bioavailability in early trials, and is now being
studied in formulations suitable for intravenous and intramuscular
injection.12
The degree to which the various functional groups on oseltamivir
and zanamivir can be modified has been extensively explored in the
published literature.23–27 For example, it is known that replacement
of the guanidine function in zanamivir with an amine leads to a >60-
fold loss in potency (IC50 of 320 nM for amino-zanamivir, vs 5 nM for
the drug itself)27; on the other hand, replacement of the amine func-
tion in oseltamivir with a guanidine leads to only a two-fold increase
in potency (IC50 of 0.5 vs 1 nM).23 Much less is known about the con-
tributions to binding of the individual functional groups on perami-
vir. Given that theguanidine function occupiesa somewhatdifferent
space for neuraminidase-bound peramivir relative to neuramini-
dase-bound zanamivir, and given that the guanidine binding pocket
is known to accommodate a water molecule in the bound structure
Resistance of circulating influenza strains to neuraminidase
inhibitors was once thought to be unlikely13; indeed, data prior
to the 2007/2008 flu season showed resistance levels of ꢀ1%, with
somewhat higher levels in children.14 That changed dramatically in
Table 1
Collected data for peramivir and analogues 1–3
Property
Peramivir
1
IC50 versus inactivated virus (10 min preincubation)
IC50 versus inactivated virus (2 h preincubation)
IC50 versus recombinant soluble neuraminidase
Ki versus recombinant soluble neuraminidase
Calculated LogP38,39
0.86 0.14 nM
0.12 0.02 nM
3.4 0.4 nM
0.83 0.13 nM
ꢁ1.37 0.45
8
7.3 0.7 nM
2.2 0.9 nM
17.7 2.3 nM
7.5 0.6 nM
+0.33 0.40
6
H-bond acceptors40
H-bond donors40
7
5
2
3
IC50 versus recombinant soluble neuraminidase
4.0 0.5 mM
37 6 mM

C. M. Bromba et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7137–7141
7139
Figure 4. Inhibition data for amino-peramivir 1 (red) and peramivir (blue).37 (A) Dose–response curve for both compounds against recombinant viral neuraminidase. (B)
Dose–response curve for both compounds against neuraminidase activity in inactivated influenza virus (10 min preincubation). (C) Lineweaver–Burk representation of
kinetic data at various concentrations of inhibitors; circles = 0 nM inhibitor; triangles = 0.75 nM peramivir or 3.75 nM 1; squares = 1.5 nM peramivir or 7.5 nM 1;
diamonds = 3 nM peramivir or 15 nM 1. (D) Michaelis–Menten representation of kinetic data at various concentrations of inhibitors. Error bars for (A) and (B) are equal to the
standard error for each measurement. Error bars are not included for (C) and (D).
of oseltamivir10 (implying that the truncation of a guanidine to an
amine is well-compensated), we sought to document the activity
of the amino variant of peramivir. Although this compound (1,
Fig. 3) was reported in the original peramivir patent,28 no detailed
enzyme inhibition data was available. The extent to which the ami-
no variant is tolerated from the standpoint of inhibitory potency
may have a significant bearing on the future use of the cyclopentane
scaffold to design new anti-influenza agents that better accommo-
date the neuraminidase H274Y mutation while maintaining or
enhancing oral availability.
Amino-peramivir 1 was synthesized in six steps from
commercially available (1R)-(ꢁ)-2-azabicyclo[2.2.1]hept-5-en-3-
one30 using a known protocol.9b,31,32 Its ability to inhibit viral neur-
aminidase was evaluated relative to peramivir,33 against a soluble
recombinant neuraminidase enzyme adapted from influenza A/
Brevig Mission/1/1918(H1N1),34,35 and also against the native neur-
aminidase protein present in NP40-inactivated influenza A/Bris-
bane/59/2007(H1N1).36
Surprisingly, compound 1 remained a very potent neuramini-
dase inhibitor, with an IC50 of 7.3 nM against the inactivated virus

7140
C. M. Bromba et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7137–7141
compared with 0.86 nM for peramivir, when the two test com-
pounds were incubated with the viral solution for 10 min prior
to analysis. Because zanamivir,41 oseltamivir,42 peramivir,43 and
other potent anti-neuraminidase agents44 have all been reported
as slow-binding inhibitors, we also compared the change in activ-
ity for peramivir and 1 upon lengthening of the incubation time to
2 h. This had the effect of increasing the apparent potency of both
inhibitors. The decrease in IC50 for peramivir upon lengthening of
the incubation period appears somewhat more substantial than
the corresponding decrease for 1 (although not to a statistically
significant extent), perhaps indicating that peramivir is a some-
what slower binder than is 1 (refer to Table 1 for tabulated results
and estimated uncertainties at the 95% confidence interval). Mea-
sured IC50 values for both peramivir and 1 were slightly higher
when the compounds were evaluated against the recombinant en-
zyme (18 nM for 1 vs 3.4 nM for peramivir), but even in this system
1 retained a Ki value in the single-digit nanomolar range (7.5 nM).
Both compounds displayed a full-range dose–response inhibi-
tory relationship (Fig. 4A and B), and kinetic analysis revealed both
molecules to be competitive inhibitors of the neuraminidase en-
zyme (Fig. 4C and D).45
A reduction in potency of a single order of magnitude (at least
from such an abundantly potent inhibitor as peramivir) may be
acceptable when weighed against a probable improvement in
pharmacokinetic properties—particularly in cases where the
remainder of the molecule is modified to include more polar
functionality intended to bind Glu276. To determine whether this
1-order of magnitude loss extends to other cyclopentanes bearing
an S-guanidine function, simplified analogues 346,47 and 248,47 were
synthesized (also from (1R)-(ꢁ)-2-azabicyclo[2.2.1]hept-5-en-3-
one). Once again, a roughly 10-fold loss in activity was observed
in moving from the guanidine-substituted 2 to the amine-substi-
tuted 3, despite the fact that these compounds are both far less
potent than peramivir and amino-peramivir 1.
References and notes
1. (a) Influenza. WHO Fact Sheet No. 211.; (b) Englund, J. A. Semin. Pediatr. Infect.
Dis. 2002, 13, 120.
2. Russell, C. A.; Jones, T. C.; Barr, I. G.; Cox, N. J.; Garten, R. J.; Gregory, V.; Gust, I.
D.; Hampson, A. W.; Hay, A. J.; Hurt, A. C.; de Jong, J. C.; Kelso, A.; Klimov, A. I.;
Kageyama, T.; Komadina, N.; Lapedes, A. S.; Lin, Y. P.; Mosterin, A.; Obuchi, M.;
Odagiri, T.; Osterhaus, A. D. M. E.; Rimmelzwaan, G. F.; Shaw, M. W.; Skepner,
E.; Stohr, K.; Tashiro, M.; Fouchier, R. A. M.; Smith, D. J. Vaccine 2008, 26, D31.
3. Cady, S. D.; Luo, W.; Hu, F.; Mei, H. Biochemistry 2009, 48, 7356.
4. Deyde, V. M.; Xu, X.; Bright, R. A.; Shaw, M.; Smith, C. B.; Zhang, Y.; Shu, Y.;
Gubareva, L. V.; Cox, N. J.; Klimov, A. I. J. Infect. Dis. 2007, 196, 249.
5. von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Phan, T.
V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese, J. N.;
Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron, J. M.; Penn, C.
R. Nature 1993, 363, 418.
6. Ryan, D. M.; Ticehurst, J.; Dempsey, M. H.; Penn, C. R. Antimicrob. Agents
Chemother. 1994, 38, 2270.
7. Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.;
Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R.
C. J. Am. Chem. Soc. 1997, 119, 681.
8. Li, W.; Escarpe, P. A.; Eisenberg, E. J.; Cundy, K. C.; Sweet, C.; Jakeman, K. J.;
Merson, J.; Lew, W.; Williams, M.; Zhang, L.; Kim, C. U.; Bischofberger, N.; Chen,
M. S.; Mendel, D. B. Antimicrob. Agents Chemother. 1998, 42, 647.
9. (a) Babu, Y. S.; Chand, P.; Bantia, S.; Kotian, P.; Dehghani, A.; El-Kattan, Y.; Lin,
T.-H.; Hutchison, T. L.; Elliott, A. J.; Parker, C. D.; Ananth, S. L.; Horn, L. L.; Laver,
G. W.; Montgomery, J. A. J. Med. Chem. 2000, 43, 3482; (b) Chand, P.; Kotian, P.
L.; Dehghani, A.; El-Kattan, Y.; Lin, T.-H.; Hutchison, T. L.; Babu, Y. S.; Bantia, S.;
Elliott, A. J.; Montgomery, J. A. J. Med. Chem. 2001, 44, 4379.
10. For a direct comparison of the binding modes of oseltamivir, zanamivir and
peramivir, see data from: Russel, R. J.; Haire, L. F.; Stevens, D. J.; Collins, P. J.;
Lin, Y. P.; Blackburn, G. M.; Hay, A. J.; Gamblin, S. J.; Skehel, J. J. Nature 2006,
443, 45. Protein Data Bank structures 2HT8, 2HTQ and 2HTU can be accessed at
www.pdb.org.
11. The guanidine functions in peramivir and zanamivir are more closely aligned in
structures wherein the inhibitors are bound to an N9 neuraminidase; e.g.
compare PDB 1L7F with PDB 1NNC. For crystallization conditions, see: (a)
Smith, B. J.; McKimm-Breshkin, J. L.; McDonald, M.; Fernley, R. T.; Varghese, J.
N.; Colman, P. M. J. Med. Chem. 2002, 45, 2207; (b) Varghese, J. N.; Epa, V. C.;
Colman, P. M. Protein Sci. 1995, 4, 1081.
12. (a) Bantia, S.; Arnold, C. S.; Parker, C. D.; Upshaw, R.; Chand, P. Antiviral Res.
2006, 69, 39; (b) Mancuso, C. E.; Gabay, M. P.; Steinke, L. M.; VanOsdol, S. J. Ann.
Pharmacother. 2010, 44, 1240.
13. Ives, J. A. L.; Carr, J. A.; Mendel, D. B.; Tai, C. Y.; Lambkin, R.; Kelly, L.; Oxford, J.
S.; Hayden, F. G.; Roberts, N. A. Antiviral Res. 2002, 55, 307.
The relatively high concentration of hydrogen bond donors and
acceptors present within a single guanidine function makes these
groups a potential liability in the development of orally active
drugs.49 For example, the guanidine-containing analogue of osel-
tamivir showed negligible oral availability in an animal model.8
This suggests that although both the amine and guanidine func-
tions will be charged at physiological pH, the amine is better-toler-
ated from the standpoint of oral availability, at least when the
carboxylate function is masked as an uncharged ester. The need
for next-generation inhibitors that are able to bind within the more
polar active site found in H274Y neuraminidases (as for zanamivir)
while maintaining oral activity (as for oseltamivir) may therefore
require that compounds built on cyclopentane scaffolds (as for per-
amivir) forgo the inclusion of the guanidine function. The results
presented here suggest that this molecular deletion is only moder-
ately detrimental, and can likely be overcome through improve-
ments elsewhere in the inhibitor’s structure. We anticipate that
this observation will be useful for the development of future neur-
aminidase inhibitors by our group50 and by others in the field.51
14. Sheu, T. G.; Deyde, V. M.; Okomo-Adhiambo, M.; Garten, R. J.; Xu, X.; Bright, R.
A.; Butler, E. N.; Wallis, T. R.; Klimov, A. I.; Gubareva, L. V. Antimicrob. Agents
Chemother. 2008, 52, 3284.
15. Centers for Disease Control and Prevention (CDC). MMWR Morb. Mortal. Wkly.
Rep. 2008, 57, 692.
16. World Health Organization. Table: Influenza A(H1N1) Virus Resistance to
Oseltamivir: Last Quarter 2007 to 2 June 2008 (Update 13 June 2008).
17. (a) Weinstock, D. M.; Zuccotti, G. J. Am. Med. Assoc. 2009, 301, 1066; (b) Dharan,
N. J.; Gubareva, L. V.; Meyer, J. J.; Okomo-Adhiambo, M.; McClinton, R. C.;
Marshall, S. A.; St. George, K.; Epperson, S.; Brammer, L.; Klimov, A. I.; Bresee, J.
S.; Fry, A. M. J. Am. Med. Assoc. 2009, 301, 1034.
18. (a) Shinde, V.; Bridges, C. B.; Uyeki, T. M.; Shu, B.; Balish, A.; Xu, X.; Lindstrom,
S.; Gubareva, L. V.; Deyde, V.; Garten, R. J.; Harris, M.; Gerber, S.; Vagasky, S.;
Smith, F.; Pascoe, N.; Martin, K.; Dufficy, D.; Ritger, K.; Conover, C.; Quinlisk, P.;
Klimov, A.; Bresee, J. S.; Finelli, L. N. Eng. J. Med. 2009, 360, 2616; (b)
Wiwanitkit, V. Clin. Ther. 2009, 160, 243.
19. Hernandez, J.; Adiga, R.; Armstrong, R.; Bazan, J.; Bonilla, H.; Bradley, J.; Dretler,
R.; Ison, M. G.; Mangino, J. E.; Maroushek, S.; Shetty, A. K.; Wald, A.; Ziebold, C.;
Elder, J.; Hollister, A. S.; Sheridan, W. Clin. Infect. Dis. 2011, 52, 695.
20. (a) Wang, M. Z.; Tai, C. Y.; Mendel, D. B. Antimicrob. Agents Chemother. 2002, 46,
3809; (b) Abed, Y.; Baz, M.; Boivin, G. Antiviral Ther. 2006, 11, 971.
21. Collins, P. J.; Haire, L. F.; Lin, Y. P.; Liu, J.; Russel, R. J.; Walker, P. A.; Skehel, J. J.;
Martin, S. R.; Hay, A. J.; Gamblin, S. J. Nature 2008, 453, 1258.
22. (a) Hurt, A. C.; Holien, J. K.; Parker, M.; Kelso, A.; Barr, I. G. J. Virol. 2009, 83,
10366; (b) Gubareva, L. V.; Matrosovich, M. N.; Brenner, M. K.; Bethell, R. C.;
Webster, R. G. J. Infect. Dis. 1998, 178, 1257.
23. Kim, C. U.; Lew, W.; Williams, M. A.; Wu, H.; Zhang, L.; Chen, X.; Escarpe, P. A.;
Mendel, D. B.; Laver, W. G.; Stevens, R. C. J. Med. Chem. 1998, 41, 2451.
24. Holzer, C. T.; Itzstein, M.; Jin, B.; Pegg, M. S.; Stewart, W. P.; Wu, W.-Y.
Glycoconjugate J. 1993, 10, 40.
25. Honda, T.; Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M. Bioorg.
Med. Chem. Lett. 2002, 12, 1925.
Acknowledgments
We gratefully acknowledge the Canadian Institutes of Health
Research and the University of Victoria for funding.
26. Smith, P. W.; Starkey, I. D.; Howes, P. D.; Sollis, S. L.; Keeling, S. P.; Cherry, P. C.;
von Itzstein, M.; Wu, W. Y.; Jin, B. Eur. J. Med. Chem. 1996, 31, 143.
27. Bamford, M. J.; Pichel, J. C.; Husman, W.; Patel, B.; Storer, R.; Weir, N. G. J. Chem.
Soc., Perkin Trans. 1 1995, 1181.
Supplementary data
28. (a) Sudhakar, Y.; Babu, J. A. M., Pooran Chand. U.S. Patent 6,562,861, 2003.; (b)
Babu, Y. S.; Chand, P.; Montgomery, J. A. International Patent WO99/33781,
1999.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.076.

C. M. Bromba et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7137–7141
7141
29. Williams, M. A.; Lew, W.; Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Laver, W. G.;
Stevens, R. C.; Kim, C. U. Bioorg. Med. Chem. Lett. 1997, 7, 1837.
30. Purchased from Aldrich Chemicals.
31. Mineno, T.; Miller, M. J. J. Org. Chem. 2003, 68, 6591.
32. Treatment of the N-Boc methyl ester of 1 (14 mg, 0.035 mmol; reported in
enantiopure form in Ref. 9b, and in racemic form in Ref. 31) with aqueous HCl
41. Hart, G. J.; Bethell, R. C. Biochem. Mol. Biol. Int. 1995, 36, 695.
42. Kati, W. M.; Saldivar, A. S.; Mohamadi, F.; Sham, H. L.; Laver, W. G.;
Kohlbrenner, W. E. Biochem. Biophys. Res. Commun. 1988, 244, 408.
43. Baum, E. Z.; Wagaman, P. C.; Ly, L.; Turchi, I.; Le, J.; Bucher, D.; Bush, K. Antiviral
Res. 2003, 59, 13.
44. Kati, W. M.; Montgomery, D.; Carrick, R.; Gubareva, L.; Maring, C.; McDaniel, K.;
Steffy, K.; Molla, A.; Hayden, F.; Kempf, D.; Kohlbrenner, W. Antimicrob. Agents
Chemother. 2002, 46, 1014.
45. For further evidence of peramivir’s ability to act as a competitive inhibitor of
neuraminidase, see: Bantia, S.; Upshaw, R.; Babu, Y. S. Antiviral Res. 2011, 91,
288.
46. (1R,4S)-(ꢁ)-2-azabicyclo[2.2.1]hept-5-en-3-one (70 mg, 0.64 mmol) was
heated to reflux for 18 h in methanolic hydrochloride (10 mL). The solvent
was removed in vacuo to yield methyl (1S,4R)-4-aminocyclopent-2-
enecarboxylate hydrochloride as a white solid. To a mixture of the crude
hydrochloride salt and sodium carbonate (207 mg, 1.95 mmol) in 10 mL
distilled water at 0 °C was added benzyl chloroformate dropwise via syringe
(1.0 M, 110 lL, 0.11 mmol) in diethyl ether (3 mL) for 24 h led to simultaneous
removal of the Boc protecting group and hydrolysis of the methyl ester, to
afford the hydrochloride salt of 1 in quantitative yield. 1H NMR (300 MHz, D2O)
d 4.52 (dd, J = 5.1, 1.5 Hz, 1H), 4.38 (dd, J = 10.5, 2.3 Hz, 1H), 3.61–3.72 (m, 1H),
3.02–3.08 (m, 1H), 2.54–2.77 (m, 1H), 2.36–2.47 (m, 1H), 2.05 (s, 3H), 1.36–
1.57 (m, 4H), 0.82–0.98 (m, 8H).
33. Purchased from D-L Chiral Chemicals.
34. Xu, X.; Zhu, X.; Dwek, R. A.; Stevens, J.; Wilson, I. A. J. Virol. 2008, 82, 10493.
35. A synthetic gene codon-optimized for expression in insect cells was designed
(Genescript) encoding a hexahistidine tag, a tetramerization domain from the
human vasodilator-stimulated phosphoprotein (as described in Ref. 34) and a
thrombin cleavage site, followed by amino acid residues 82–467 of the
ectodomain of the neuraminidase from the A/Brevig Mission/1/1918 flu virus
strain. The synthetic gene was cloned in a modified pAcGP67B vector (BD
Biosciences) and co-transfected into Sf9 cells with Sapphire bacculovirus
(Orbigen) in the presence of Cellfectin (Invitrogen) according to the
manufacturer’s instructions. After two rounds of amplification in SF9 cells,
(100 lL, 0.70 mmol). The solution was stirred for 30 min at 0 °C and 30 min at
room temperature. The aqueous layer was extracted with 3 ꢂ 20 mL
dichloromethane and the organic layers were combined and dried over
anhydrous sodium sulfate. The solvent was removed in vacuo to yield a
colorless oil. Flash-column chromatography (silica, hexanes–ether 2:1, Rf 0.22)
afforded
O-methyl,
N-carboxybenzoyl
(1S,4R)-4-aminocyclopent-2-
the recombinant virus was used to infect Hi5 cells.
A
small-scale pilot
enecarboxylate as a white solid (105 mg, 0.382 mmol, 60%). MP 155 °C; IR
(cmꢁ1, film) 3428, 1731, 1716, 1506; 1H NMR (500 MHz, CDCl3) d 7.27–7.36 (m,
5H), 5.84–5.90 (m, 2H), 5.16 (d, J = 9.7 Hz, 1H), 5.08 (s, 2H), 4.80–4.87 (m, 1H),
3.68 (s, 3H), 3.43–3.49 (m, 1H), 2.48 (dt, J = 14.2, 8.4 Hz, 1H), 1.88 (dt, J = 14.2,
3.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 175.4, 155.8, 136.8, 134.7, 131.8,
128.7, 128.3, 128.3, 66.8, 56.5, 52.4, 49.4, 34.7. A solution of the alkene
(151 mg, 0.547 mmol) and selenium dioxide (74 mg, 0.67 mmol) were heated
in 15 mL anhydrous dichloromethane to 100 °C in a microwave reactor for 8
hours. The solvent was removed in vacuo to yield a brown oil. Flash-column
chromatography (silica, hexanes–ether 1:2, Rf 0.10) afforded O-methyl, N-
carboxybenzoyl derivative of 3 as a colourless oil (99 mg, 0.34 mmol, 62%). IR
(cmꢁ1, film) 3363, 1721; 1H NMR (500 MHz, CDCl3) d 7.26–7.39 (m, 5H), 6.04
(d, J = 5.4 Hz, 1H), 5.74 (d, J = 5.4 Hz, 1H), 5.05–5.10 (m, 4H), 3.80 (s, 3H), 3.30
(br s, 1H), 2.43 (dd, J = 14.3, 7.6 Hz, 1H), 2.15 (dd, J = 14.3, 2.5 Hz, 1H); 13C NMR
(125 MHz, CDCl3) d 175.8, 156.1, 137.3, 136.6, 135.6, 128.8, 128.4, 128.4, 84.8,
67.0, 56.4, 53.7, 44.7. To this intermediate in 15 mL of anhydrous methanol
was added 10% palladium on carbon. The mixture was stirred vigorously under
an atmosphere of hydrogen gas (300 psi) for 21 h. The catalyst was removed by
filtration through cotton and the filtrate was concentrated in vacuo to afford
the methyl ester of 3 as a white solid (43 mg, 0.27 mmol, 97%). Mp 111 °C; IR
(cmꢁ1, film) 3365, 1719; 1H NMR (500 MHz, D2O) d 3.90–3.97 (m, 1H), 3.80 (s,
3H), 2.31–2.41 (m, 3H), 2.26 (dd, J = 14.1, 7.8 Hz, 1H), 1.86–2.00 (m, 2H); 13C
NMR (125 MHz, D2O) d 176.7, 81.2, 53.4, 50.8, 42.8, 36.8, 28.9.
experiment was performed to determine the optimal amount of virus to use. In
a typical preparation, 4–5 L of Hi5 cells at a density of 1.8 ꢂ 106 cells/mL were
used. After 3 days of incubation at 28 °C, the cells were removed by
centrifugation and 10
per liter of culture. The supernatant was then filtered through 3 Millipore
filters (5, and 0.45 pores) and concentrated by tangential flow/
lL of protease inhibitor cocktail (Roche) was added
1
lm
diafiltration (10 kDa cut-off). The sample was buffer-exchanged to 20 mM
HEPES, 1 M NaCl and 30 mM imidazole, pH 8. The soluble neuraminidase was
recovered by metal affinity chromatography using Ni-charged Chelating
Sepharose Fast Flow Beads (Amersham Pharmacia). Following elution with
imidazole, the samples were concentrated and buffer-exchanged with a spin
column to the Neuraminidase Assay Buffer (50 mM Tris–HCl pH 7.5, 200 mM
NaCl, 5 mM CaCl2). Aliquots were flash-frozen on liquid nitrogen and stored at
ꢁ70 °C.
36. Virus propagation: Influenza A/Brisbane/59/2007 (H1N1) virus was propagated
in Madin-Darby Canine Kidney (MDCK) cells. Seed virus was inoculated into 10
confluent 75 cm2 monolayers which were incubated at 37 °C for 2–3 days and
harvested when full cytopathic effect was observed. Virus purification: The
pooled cell lysates were frozen and thawed and clarified by centrifugation at
3000g for 20 min. The supernatant was subjected to ultracentrifugation at
25,000 rpm for 90 min and the virus containing pellet was resuspended in 2 mL
of MegaVir medium (Hyclone). Virus concentration was assessed by
hemagglutination (HA) at 40,960 HA units. Virus inactivation: NP40 (Fluka)
was added to the purified influenza virus at a final concentration of 0.2% and
the mixture was incubated at room temperature for a total of 3 hours and at
4 °C for 6 h. The inactivation of the virus was confirmed by the Tissue Culture
Infectious Dose assay. This virus preparation had a titre of 107 TCID50 per
47. To prepare samples for biological assays, the corresponding methyl ester was
dissolved in 5% aqueous sodium hydroxide. After stirring for 4 h at room
temperature, the solution was neutralized with hydrochloric acid and used
directly for enzyme inhibition experiments.
48. To the methyl ester of compound 3 (22 mg, 0.14 mmol) and triethylamine
100
l
L before inactivation and <102 TCID50 per 100
lL after NP40 treatment.
(85 lL, 0.61 mmol) in 2 mL of anhydrous dimethylformamide was added 1,3-
37. The following solutions were prepared for the enzyme assays: (1) Assay buffer:
50 mM Tris, 5 mM CaCl2, 200 mM NaCl, pH 7.5; (2) Protein stock solution:
recombinant neuraminidase was diluted in assay buffer to 1000 ng/mL, or
inactivated virus suspension was diluted to obtain a similar activity; (3)
bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea (54 mg, 0.15 mmol) and
mercury (II) chloride (41 mg, 0.15 mmol). After stirring for 18 h at room
temperature, ethyl acetate was added and the solution was filtered through
cotton. The solvent was removed in vacuo to yield a brown oil. Flash-column
chromatography (silica, hexanes–ether 1:2, Rf 0.19) afforded the O-methyl,
Substrate stock solution: 20-4(methylumbelliferyl)-
acid (Aldrich) was dissolved in DMSO to
Substrate working solution: 40 L of the substrate stock solution was diluted
to 1000 L with assay buffer, for a final concentration of 400 M (4% DMSO);
a
-
D
-N-acetylneuraminic
a
concentration of 10 mM; (4)
N,N0-bis(carboxybenzoyl) derivative of
2 as a colourless oil (30 mg,
l
0.064 mmol, 46%). IR (cmꢁ1, neat) 3325, 3064, 1730, 1691, 1638, 1617; 1H
NMR (500 MHz, CDCl3) d 11.8 (s, 1H), 8.56 (d, J = 8.04 Hz, 1H), 7.25–7.39 (m,
10H), 5.16 (s, 2H), 5.11 (s, 2H), 4.74–4.82 (m, 1H), 3.82 (s, 3H), 3.18 (br s, 1H),
2.18–2.33 (m, 3H), 2.08 (dd, J = 14.5, 5.6 Hz, 1H), 1.82–1.88 (m, 1H), 1.72–1.80
(m, 1H); 13C NMR (125 MHz, CDCl3) d 177.2, 163.9, 155.5, 153.9, 137.0, 134.8,
128.1–129.0 (overlapping signals), 80.6, 68.5, 68.3, 53.3, 52.1, 45.9, 38.5, 32.5.
To this intermediate (30 mg, 0.064 mmol) in 10 mL of anhydrous methanol
was added 10% palladium on carbon. The mixture was stirred vigorously under
an atmosphere of hydrogen gas (300 psi) for 24 h. The catalyst was removed by
filtration through cotton and the filtrate was concentrated in vacuo to afford
the methyl ester of compound 2 as a yellow oil (11 mg, 0.053 mmol, 83%). 1H
NMR (300 MHz, D2O) d 4.10–4.25 (m, 1H), 3.80 (s, 3H), 2.25–2.41 (m, 3H), 2.20
(dd, J = 14.3, 7.3 Hz, 1H), 1.75–1.94 (m, 2H).
l
l
(5) Inhibitor solutions: Inhibitors were diluted in assay buffer to provide a
range of working concentrations. For IC50 measurements with recombinant
neuraminidase, sample wells of a black 96-well plate (Nunc, optical bottom)
were charged with 40
10 L of inhibitor solution and 50
substrate, 4% DMSO). The samples (each containing 100
400 ng/mL enzyme, 200 M substrate, and 1.5% total DMSO, in 100
l
L of protein stock solution (1000 ng/mL), followed by
L of substrate working solution (400
total volume,
L total
l
l
lM
l
L
l
l
sample volume) were mixed briefly by pipetting. Fluorescence was monitored
over 5 min (kexc = 365 nm; kem = 445 nm). Experiments with inactivated virus
were conducted similarly, except that the inhibitor solutions were added to
wells containing inactivated virus, and these mixtures were allowed to
incubate for either 10 min or 2 h at room temperature prior to the addition
of working solution. For kinetic data, the working solution was subjected to
serial dilution in assay buffer containing 4% DMSO. Progress of the reaction was
measured over 10 min at various concentrations of substrate and inhibitor, as
indicated in Figure 4. Control experiments (substrate buffer only) showed no
significant background reaction. Data was plotted using XLfit (IDBS software).
38. Calculated using Advanced Chemistry Development (ACD/Labs) Software
V8.19 (Ó 1994-2011 ACD/Labs).
49. For a useful review on the challenges to oral availability associated with the
presence of guanidine functionality, and various strategies to mitigate these
liabilities, see: Sun, J.; Miller, J. M.; Beig, A.; Rozen, L.; Amidon, G. L.; Dahan, A.
Expert Opin. Drug Metab. Toxicol. 2011, 7, 313.
50. (a) Brant, M. G.; Bromba, C. M.; Wulff, J. E. J. Org. Chem. 2010, 75, 6312; (b)
Wulff, J. E.; Brant, M. G.; Bromba, C. M.; Boulanger, M. J. U.S. Patent Appl. 61/
304,738, 2010; PCT application PCT/CA2011/000174, 2011.
51. Interestingly, when the scaffold is changed from
a cyclopentane to a
39. LogP values calculated by the method of Villar: ꢁ1.80 for peramivir, ꢁ0.32 for
1; LogP values calculated by the method of Ghose and Crippen: ꢁ0.25 for
peramivir, ꢁ0.39 for 1.
pyrrolidine, much less polar hydrocarbon functionality is tolerated at the
position corresponding to the guanidine in peramivir; see: Ref. 44.
40. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.