Design, synthesis, and neuraminidase inhibitory activity of GS-4071 analogues that utilize a novel hydrophobic paradigm.

Article abstract of DOI:10.1016/S0960-894X(02)00732-1

Structure-based design has led to the synthesis of a novel analogue of GS-4071, an influenza neuraminidase inhibitor, in which the basic amino group has been replaced by a hydrophobic vinyl group. An X-ray co-crystal structure of the new inhibitor (K(i)=4

Full text of DOI:10.1016/S0960-894X(02)00732-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3425–3429
Design, Synthesis, and Neuraminidase Inhibitory Activity of GS-4071
Analogues that Utilize a Novel Hydrophobic Paradigm
Stephen Hanessian,a,* Jianchio Wang,a Debra Montgomery,b Vincent Stoll,b
Kent D. Stewart,b Warren Kati,b Clarence Maring,b Dale Kempf,b
Charles Hutchinsb andW. Graeme Laver c
a
´
´
´
Department of Chemistry, Universite de Montreal, PO Box 6128, Station Centre-ville, Montreal, QC, Canada H3C 3J7
bAbbott Laboratories, Abbott Park, IL 60064, USA
cThe Australian National University, Canberra 260, Australia
Received7 May 2002; accepted22 August 2002
Abstract—Structure-based design has led to the synthesis of a novel analogue of GS-4071, an influenza neuraminidase inhibitor, in
which the basic amino group has been replacedby a hydrophobic vinyl group. An X-ray co-crystal structure of the new inhibitor
(Ki=45 nM) boundto the active site shows that the vinyl group occupies the same subsite as the amino group in GS-4071.
# 2002 Elsevier Science Ltd. All rights reserved.
Figure 1.
The catalytic action of influenza neuraminidase1 is
responsible for infectivity andpropagation of the influ-
enza RNA virus.2 This enzyme cleaves the terminal
N-acetylneuraminic acidresidues of epithelial cell glyco-
conjugates in the upper respiratory tract, a process that
spares the virus from being entrappedby aggregation,
andprolongs its survival in the host. The development
of inhibitors of neuraminidase continues to be an active
area of research as this strategy has been clinically vali-
dated in efforts to treat respiratory influenza infection.3
Utilizing X-ray crystallographic data on the enzyme4
and N-acetylneuraminic acid(NANA) 1 as a weak
inhibitor, extensive research has already produced two
drugs: zanamivir (2),5 a guanidino analogue of dehydro-
NANA, andoseltamivir carboxylate ( 3),6 a carbocyclic
analogue of dehydro-NANA (GS-4071) administered as an
ethyl ester prodrug (3a) (Fig. 1). Elegant X-ray studies
*Corresponding author. Tel.: +1-514-343-6738; fax: +1-514-343-
5728; e-mail: stephen.hanessian@umontreal.ca
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00732-1

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3425–3429
andan acetylamino group are requiredfor effective
binding within subsites 1 and 3.4À7 The replacement of
a hydrophilic interaction in subsite 5 of the enzyme
generatedby the triol group of 1 with a hydrophobic
3-pentyl ether substituent projecting into subsites 4
and5 from the carbocyclic core of 3 exploitedthe
first new hydrophobic paradigm in neuraminidase
inhibitor design resulting in orally bioavailable inhib-
itors.6 Utilizing similar binding interactions as the six-
memberedinhibitor 3, a five-memberedinhibitor 4,
BCX-1812, has been described.7 Recently, a new class
of inhibitors exploiting a novel secondhyrdophobic
paradigm, exemplified by 5, has replacedthe hydro-
philic/chargedinteraction in subsite 2 that is usually
occupiedby a basic group such as a primary amine or a
guanidine. A-315675 (5), a potent inhibitor discovered
by Abbott scientists9 revealedthat a basic group nor-
mally requiredto interact with the amino acidresidues
Glu119, Glu227, andAsp151 in neuraminidase couldbe
replacedby a hydrophobic
cis-propenyl group. Total
syntheses of 5 have been reportedby the Abbott group, 10
11
andindependently by Hanessian andcoworkers.
Figure 2. Crystal structure of compound 6 (blue) in neuraminidase
A/Tern/75 (green) overlaidwith GS-4071 (orange, PDB entry 2QWK).
The side chains of Arg 118, Arg 371, and Arg 292 form subsite 1. The
side chains of Glu 119 and Glu 227 form subsite 2. The side chains of
Trp178 andIle 222 form subsite 3. Subsites 4 and5 form a contiguous
region with the more solvent exposedsite 4 near Ile 222 side chain and
the hydrophobic (in this case) site 5, generated by Glu 276 in its bent
conformation.
The X-ray co-crystal structure of the proline-based
inhibitor A-315675 with neuraminidase12 ledus to con-
sider alternative core structures that also capitalized on a
hydrophobic interaction. Molecular modeling studies
indicated that six-membered analogues of GS-4071 could
be designed with hydrophobic olefinic substituents
which wouldreplace the primary amino group. With this
premise in mind, we proceeded to synthesize 6 (Fig. 2),
and closely related compounds to test this paradigm shift
in our understanding of subsite 2 interactions.
have also facilitatedthe idscovery of potent carbo-
9
cyclic7,8 andheterocyclic inhibitors.
The active site of neuraminidase can be divided into five
subsites into which polar andhydrophobic groups pro-
ject from inhibitor core structures. A carboxyl group
Considering the carbocyclic nature and functional
characteristics of the intended targets, it was obvious
Scheme 1. (a) PhCHO, TsOH, toluene, reflux, 84%; (b) NBS, AIBN, CCl4, reflux, 80%; (c)
, AIBN, benzene, reflux, 82%; (d) MsCl,
Et3N, CH2Cl2, 99%; (e) MeOH, K2CO3, 99%; (f) BzCl, pyr, 85%; (g) DBU, THF, reflux; (h) NaN3, NH4Cl, MeOH, H2O, 67% (two steps); (i) Ph3P,
Et3N, H2O; (j) TrCl, Et3N, CH2Cl2, 92% (two steps); (k) BF3Et2O, ROH; (l) Ac2O, Pyr. 60% (two steps); (m) LiOH, THF, H2O, 98%.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3425–3429
Table 1. Neuraminidase (type B/Lee) inhibitory activity
3427
Ki (mM)
0.045
that as in the case of GS-4071,6 quinic acid 713 wouldbe
a logical chiron to buildupon as shown in Scheme 1.
The readily available lactone 8 was subjectedto a
regioselective ring opening reaction with NBS14 to give
the bromobenzoate 9 in high yieldandexcellent selec-
tivity. Radical-induced allylation15 afforded the C-allyl
product 10 with retention of configuration, presumably
as a result of a greater shielding by the axially disposed
benzoate. Mesylation, hydrolysis, selective benzoylation
Entry
CompdNumber
1
6
anda secondmesylation ledto
11 in excellent overall
2
3
(ref 18)
69
yield. Treatment with potassium carbonate in methanol
followedby DBU at reflux in THF gave the epoxide 12.
Upon treatment with sodium azide,5,16 the epoxide ring
was regioselectively openedto affordthe azido alcohol
13. Mesylation andtreatment with triphenylphosphine
led to the corresponding aziridine which was N-tri-
tylatedto give 14. Following an establishedprotocol,
treatment of 14 with 2-propanol or 2-pentanol in the
.
presence of BF3 Et2O followedby N-acetylation gave
15
(ref 18)
16
1.1
6
the intended C-allyl analogues of GS-4071, 15 and 16,
respectively.
4
5
14
Rather than explore an alternative route to the desired
C-vinyl analogue 6, we decided to prepare it from 15 by
a degradative protocol as shown in Scheme 2. Thus,
dihydroxylation of the trimethylsilylethyl ester of 15,
followed by oxidation gave the aldehyde 17. Further
oxidation to the acid by the Pinnick procedure,17 and
functional group manipulation ledto 18, a C-methoxy-
carbonylmethyl analogue of GS-4071.
1.3
6
7
8
18
19
2.6
Reduction of the methyl ester of 15 to the alcohol 19
followedby elimination via the 2-nitrophenylselenate
gave the ester 20 which was hydrolyzed to the desired
analogue 6. A co-crystal X-ray structure of 6 with
neuraminidase validates the proposed binding mode in
our initial modeling studies (Scheme 2). The vinyl group
projects into the region occupiedby an amino group in
GS-4071.
4.1
(ref 20)
0.18
The type B neuraminidase inhibition Ki data for 6, 15,
andother analogues are shown in Table 1. The most
Scheme 2. (a) , 2-chloro-1-methylpyridinium chloride, Et3N, 67%; (b) OsO4, NMO, aq acetone; (c) NalO4 aq MeOH, 73% (two
steps); (d) NaClO2, 2-butene, pH 3–4; then CH2N2, 40%; (e) TBAF, CH2Cl2, 97%; (f) R=Me, NaBH4, MeOH; (g) Bu3P, p-nitrophenylselenyl
cyanide, (two steps, 95%); (h) H2O2, THF, 89%; (i) LiOH, aq MeOH, 96%.

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3425–3429
active compoundis the anti/anti vinyl analogue 6 (Table
1, entry 1). The anti/anti allyl analogue 15 (entry 3) was
significantly less active. The epimeric anti/syn C-vinyl
and C-allyl analogues (entries 2 and4) were relatively
inactive.18 Elongation of the 2-propyl ether in 6 to
3-pentyl as in 16 (entry 5) did not alter the inhibitory
potency. The introduction of polar substituents such as
in the ester 18 andhydroxyl analogue 19 was slightly
detrimental to the activity (entries 6 and 7). The crystal
structure of compound 6 boundto the neuraminidase
active site was determined and confirmed that the bind-
ing mode is similar to GS4071 as shown in Figure 2.
Importantly, the vinyl group of compound 6 overlays
with the basic amine of GS4071 as designed. The two
carbons of the vinyl group are in goodVan der Waals
contact (3.5–4.5 A) with the methylenes of Asp 151 and
Glu 119 andthe p-system of Glu 119 carboxylate. There
is a longer Van der Waals contact (4.8 A) between the
p-system of Glu 227 carboxylate andthe terminal vinyl
carbon.
4. (a) Burmeister, W. P.; Ruigrok, R. W. H.; Cusack, S.
EMBO J. 1992, 11, 49. (b) Bossart-Whitaker, P.; Carson, M.;
Babu, Y. S.; Smith, C. D.; Laver, W. G.; Air, G. M. J. Mol.
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Penn, C. R. Nature 1993, 363, 418.
6. (a) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H. T.;
Zhang, L. J.; 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. (b) Kim, C. U.; Lew, W.; Williams,
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Bioorg. Med. Chem. Lett. 2000, 10, 1257.
The nanomolar activity of 6 (Ki=45 nM) represents a
significant finding, but falls short of the subnanomolar
potencies reportedpreviously for highly advancedand
optimizedcompounsd 2–5. The 2-propyl ether sub-
stituent of 6 is likely to be suboptimal for interaction
7. Babu, Y. S.; Chand, P.; Bantia, S.; Kotian, P.; Dehghani,
A.; El-Kattan, Y.; Liu, T.-H.; Hutchinson, T. L.; Elliott, A. J.;
Parker, C. D.; Ananth, S. L.; Horn, L. L.; Laver, G. W.;
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within subsites 4 and5 andshouldbe more properly
comparedto a corresponding analogue of GS-4071.
19
The replacement of the amino group in GS-4071 by a
vinyl group, while maintaining its anti/anti spatial dis-
position, leads to an inhibitor of comparable activity to
the parent series. The potential for enhancement of
activity by further optimization of these two hydro-
phobic interactions in subsites 2 and4 in this carbo-
cyclic inhibitor has sufficient precedent to warrant
further investigation.20 Our results further validate the
importance of hydrophobic interactions in structure-
based drug design.21
8. Atigadda, V. R.; Brouilette, W. J.; Duarte, F.; Ali, S. M.;
Babu, Y. S.; Bantia, S.; Chand, P.; Chu, N.; Montgomery,
J. A.; Walsh, D. A.; Sudbeck, E. A.; Finley, J.; Luo, M.; Air,
G. M.; Laver, G. W. J. Med. Chem. 1999, 42, 2332.
9. Kati, W. M.; Montgomery, D.; Carrick, R.; Larisa, G.;
Maring, C.; McDaniel, K.; Steffy, K.; Molla, A.; Hayden, F.;
Kempf, D.; Kohlbrenner, W. Antimicrob. Agents Chemother.
2002, 46, 1014.
10. (a) DeGoey, D. A.; Flosi, W. J.; Grampovnik, D.-J.;
Yeung, C. M.; Klein, L.; Kempf, D. J. Enantioselective Synth-
esis of Anti-Influenza Compound ABT-675; Presentedat the
221st National Meeting of the American Chemical Society,
San Diego, CA, April 2001; Paper ORGN 320. (b) DeGoey,
D. A.; Chen, H.-J.; Flosi, N. J.; Grampovnik, D. J.; Yeung,
C. M.; Klein, L. L.; Kempf, D. J. J. Org. Chem. 2002, 67,
5445.
Acknowledgements
Generous financial assistance from Abbott Laboratories
andthe NSERC Medicinal Chemistry Chair Program is
acknowledged.
11. Hanessian, S.; Bayrakdarian, M.; Luo, X. J. Am. Chem.
Soc. 2002, 124, 4716.
12. Molla, A.; Kati, W.; Carrick, R.; Steffy, K.; Shi, Y.;
Montgomery, D.; Gusick, N.; Stoll, V. S.; Stewart, K. D.; Ng,
T. I.; Maring, C.; Kempf, D. J.; Kohlbrenner, W. J. Virol.
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References and Notes
13. For examples of using quinic acidin natural proudct
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Pollini, G.; Zanirato, V. Tetrahedron: Asymmetry 1997, 8, 3515.
14. Hanessian, S. Org. Syn. 1987, 65, 243. Hanessian, S. Carbo-
hydr. Res. 1966, 2, 86.
15. Keck, G. E.; Yates, J. B. J. Am. Chem. Soc. 1982, 104,
5829.
16. For an ‘azide-free’ method, see: Karpf, M.; Trussardi, R.
J. Org. Chem. 2001, 66, 2044.
17. (a) Bal, B. S.; Childers, W. E.; Pinnick, H. W. Tetrahedron
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Chem. Scand. 1973, 27, 888. (c) Kraus, G. A.; Tashner, M. J.
J. Org. Chem. 1980, 45, 1175.
18. The anti/syn C-vinyl and C-allyl analogues in Table 1 were
synthesizedby a protocol analogous to the one in Scheme 1
starting with quinic acid. Details available upon request.
1. Colman, P. M. In The Influenza Viruses: Influenza Virus
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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3425–3429
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19. The ethylether/amine analogue (entry 8) has been reported
to exhibit a Ki of 185 nM under identical assay conditions; see:
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.
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R. J. Tetrahedron Lett. 2001, 42, 7687.
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