Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors

Article abstract of DOI:10.1021/jm980162u

A series of influenza neuraminidase inhibitors with the cyclohexene scaffold containing lipophilic side chains have been synthesized and evaluated for influenza A and B neuraminidase inhibitory activity. The size and geometry of side chains have been modified systematically in order to investigate structure-activity relationships of this class of compounds. The X-ray crystal structures of several analogues complexed with neuraminidase revealed that the lipophilic side chains bound to the hydrophobic pocket consisted of Glu276, Ala246, Arg224, and Ile222 of the enzyme active site. The structure-activity relationship studies of this series have also demonstrated remarkably different inhibitory potency between influenza A and B neuraminidase. This indicated that the lipophilic side chains had quite different hydrophobic interactions with influenza A and B neuraminidase despite their complete homology in the active site. Influenza B neuraminidase appeared to be much more sensitive toward the increased steric bulkiness of inhibitors compared to influenza A neuraminidase. From the extensive structure-activity relationship investigation reported in this article, GS 4071 emerged as one of the most potent influenza neuraminidase inhibitors against both influenza A and B strains.

Full text of DOI:10.1021/jm980162u

J . Med. Chem. 1998, 41, 2451-2460
2451
Str u ctu r e-Activity Rela tion sh ip Stu d ies of Novel Ca r bocyclic In flu en za
Neu r a m in id a se In h ibitor s
Choung U. Kim,*,† Willard Lew,† Matthew A. Williams,† Huiwei Wu,† Lijun Zhang,† Xiaowu Chen,†
Paul A. Escarpe,† Dirk B. Mendel,† W. Graeme Laver,‡ and Raymond C. Stevens§
Gilead Sciences Inc., 333 Lakeside Drive, Foster City, California 94404, J ohn Curtin School of Medical Research,
The Australian National University, Canberra 260, Australia, and Department of Chemistry, University of California,
Berkeley, California 94720
Received March 17, 1998
A series of influenza neuraminidase inhibitors with the cyclohexene scaffold containing lipophilic
side chains have been synthesized and evaluated for influenza A and B neuraminidase inhibitory
activity. The size and geometry of side chains have been modified systematically in order to
investigate structure-activity relationships of this class of compounds. The X-ray crystal
structures of several analogues complexed with neuraminidase revealed that the lipophilic
side chains bound to the hydrophobic pocket consisted of Glu276, Ala246, Arg224, and Ile222
of the enzyme active site. The structure-activity relationship studies of this series have also
demonstrated remarkably different inhibitory potency between influenza A and B neuramini-
dase. This indicated that the lipophilic side chains had quite different hydrophobic interactions
with influenza A and B neuraminidase despite their complete homology in the active site.
Influenza B neuraminidase appeared to be much more sensitive toward the increased steric
bulkiness of inhibitors compared to influenza A neuraminidase. From the extensive structure-
activity relationship investigation reported in this article, GS 4071 emerged as one of the most
potent influenza neuraminidase inhibitors against both influenza A and B strains.
Sch em e 1
In tr od u ction
Influenza neuraminidase (NA) catalyzes the cleavage
of the terminal sialic acid attached to glycoproteins and
glycolipids.1,2 This process is believed to be necessary
for the release of newly formed virus from infected cells
and for efficient spread of virus in the respiratory
tract.3,4 Therefore, NA is recognized as a potential
target for developing agents against influenza infection.
It has been proposed that the sialic acid cleavage by NA
might proceed via the oxonium cation transition state
3 (Scheme 1).5,7 Zanamivir (5) is a transition state
analogue of 3 which exhibits the potent NA inhibitory
activity8 and is currently being evaluated in phase III
clinical trials via the topical delivery to the respiratory
tract.9,10 Aiming at developing oral agents against
influenza infection, we have designed new carbocyclic
NA inhibitors 6 with various lipophilic alkyl side chains
(R1) in order to examine NA inhibitory activity and oral
bioavailability. In this series, it was found that the size
and geometry of the alkyl side chains significantly
influenced NA inhibitory activity. In fact, X-ray crystal-
lographic studies of several analogues in this series
complexed with NA revealed that the alkyl groups (R1
in 6) bound into a hitherto unreported hydrophobic
pocket in the NA active site.11 The structure-activity
relationship (SAR) studies of this novel series identified
GS 4071 (7) as a potent NA inhibitor with IC50 of 1 nM.11
Subsequently, GS 4104 (8, the ethyl ester prodrug of 7)
was found to be highly orally bioavailable in several
animal species and efficacious in both the mouse and
ferret models of influenza infection by oral administra-
tion.12 In recent phase II clinical trials, oral efficacy of
GS 4104 has been demonstrated both in prophylaxis and
treatment of human influenza infection.13 GS 4104 is
currently in phase II/III clinical trials for the prophy-
laxis and treatment of influenza virus infection.
Herein are described the SAR studies of carbocyclic
NA inhibitors against influenza A and B viral neuramini-
dases. In addition to the series of the C3 modification,
several C2 and C6 substituted analogues of GS 4071 (7)
were examined in order to define further structural
features for optimal NA inhibitory activity.
Ch em istr y
† Gilead Sciences Inc.
The carbocyclic analogues 6 were prepared according
to Scheme 2 from a common intermediate, N-tritylaziri-
‡ Australian National University.
§ University of California.
S0022-2623(98)00162-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/09/1998

2452 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kim et al.
Sch em e 2
Sch em e 5a
Sch em e 3a
a
Reagents: (a) (Boc)2O, Et3N; (b) OsO4, NMO; (c) SO2Cl2,
pyridine; (d) DBU, THF; (e) Ac2O, pyridine; (f) Me2CuLi, Et2O; (g)
HCl, EtOAc; (h) aqueous KOH, MeOH.
a
Reagents: (a) R1MgBr, Li2CuCl4; (b) K2CO3, MeOH; (c)
MeMgBr, CuI.
Sch em e 6a
Sch em e 4a
a
Reagents: (a) N,N′-bis(tert-butoxycarbonyl)thiourea, HgCl2,
Et3N, DMF; (b) KOH, THF/H2O; (c) CF3CO2H, CH2Cl2.
dine 9 which was prepared from quinic acid by a
previously described procedure.11
Enantiomerically pure alcohols 14a -e were synthe-
sized from (2R)-(-)-glycidal tosylate 11 according to
Scheme 3. Regioselective ring opening of 11 with the
corresponding Grignard reagent (R1MgBr) under lithium
tetrachlorocuprate catalysis provided hydroxy tosylate
12 which is cyclized to epoxide 13 with potassium
carbonate in anhydrous methanol.14 Epoxide 13 is
converted to alcohol 14 by the copper(I) iodide catalyzed
addition of methylmagnesium bromide. The (3R)-enan-
tiomer of 14e was prepared from the corresponding (2S)-
(+)-glycidal tosylate in a similar manner.
The guanidino analogues 17 were prepared from the
corresponding amino methyl esters 15 as shown in
Scheme 4. Treatment of 15 with N,N′-bis(tert-butoxy
carbonyl)thiourea15 using mercury(II) chloride and tri-
ethylamine in DMF provided bis-BOC protected guani-
dine 16. Saponification of 16 followed by removal of the
BOC groups with trifluoroacetic acid furnished the
guanidino analogues 17.
a
Reagents: (a) p-TsOH, MeOH; (b) p-TsOH, 3-pentanone; (c)
MsCl, Et3N; (d) BH3‚Me2S, TMSOTf; (e) KHCO3; (f) MOMCl,
i-Pr2EtN; (g) p-TsOH, MeOH; (h) oxalyl chloride, DMSO; (i)
NaClO2; (j) EtOH, 1,3-diisopropylcarbodiimide; (k) PPh3, THF/
H2O; (l) KOH, THF.
sequence involving formation of the cyclic sulfate with
sulfuryl chloride, elimination of the cyclic sulfate to the
allylic alcohol with 1,8-diazabicyclo[5.4.0]undec-7-ene,
and acetylation with acetic anhydride. Introduction of
the C6-methyl group was accomplished by stereo- and
regioselective 1,4-addition of Me2CuLi to 20 with con-
comitant elimination of the acetate group to provide 21.
Deprotection of 21 with anhydrous hydrochloric acid in
ethyl acetate followed by saponification provided the C6-
methyl analogue 22.
The C6-methyl analogue 22 was prepared according
to Scheme 5. The 3-pentyl ethyl ester 8 was treated
with di-tert-butyl dicarbonate followed by osmium tet-
raoxide to give diol 18. Compound 18 was converted to
allylic acetate 20 stereospecifically via a three-step
The C2-fluoro analogue 30 was prepared according to
Scheme 6. Shikimic acid was converted into fluoro
alkene 23 by the procedure reported by Bartlett and

Novel Carbocyclic Influenza Neuraminidase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2453
Rich.16 Ketal exchange of 23 with 3-pentanone with
catalytic p-toluenesulfonic acid provided 24. Treatment
of 24 with methanesulfonyl chloride followed by regio-
selective reductive cleavage of the ketal ring with BH3‚
Me2S and treatment with potassium bicarbonate pro-
vided the epoxy alcohol 25. The MOM protected epoxide
26 was converted to azide 27 in a stereo- and regiose-
lective manner with sodium azide in the presence of
ammonium chloride. Conversion of 27 into ethyl ester
29 was accomplished in four steps: (1) deprotection of
the MOM group with catalytic p-toluenesulfonic acid in
methanol, (2) Swern oxidation to aldehyde 28, (3)
oxidation of the aldehyde to the carboxylic acid with
NaClO2, (4) and esterification with ethanol and diiso-
propylcarbodiimide to give 29. Reduction of the azide
group of 29 with triphenylphosphine followed by sa-
ponification furnished the C2-fluoro analogue 30.
Ta ble 1. Influenza Neuraminidase Inhibitory Activity of
Carbocyclic Analogues with Linear Alkyl Chains
neuraminidase (IC50, nM)
compound
R
influenza Aa
influenza Bb
31
32
33
34
35
36
37
38
39
40
41
H
CH3
CH3CH2
6300
3700
2000
180
300
200
150
270
180
210
600
NDc
ND
185
ND
215
ND
1450
ND
CH3(CH2)2
CH3(CH2)3
CH3(CH2)4
CH3(CH2)5
CH3(CH2)6
CH3(CH2)7
CH3(CH2)8
CH3(CH2)9
3500
ND
ND
a
b
A/PR. B/Lee. c ND ) not determined.
Resu lts a n d Discu ssion
A main objective of the present research was to
develop new, orally active NA inhibitors as anti-
influenza agents. In the case of an influenza epidemic,
oral administration may be a convenient and economical
method for treatment and prophylaxis. As reported in
our preliminary account of a new series of carbocyclic
neuraminidase inhibitors, a new hydrophobic pocket in
the region corresponding to the glycerol subsite of sialic
acid in the NA active site was discovered.11 The X-ray
crystal structures of several carbocyclic inhibitors bound
to NA revealed that this hydrophobic pocket consisted
of Glu276, Ala246, Arg224, and Ile222.11,17 The lipo-
philicity of inhibitors is an important factor for design-
ing orally active drugs since balancing lipophilicity and
water solubility could be as critical as the size of the
molecule for its absorption from the intestinal tract.18
Although the hydrophobic effect is still poorly under-
stood on the molecular level,19 hydrophobic interactions
between enzymes and inhibitors or receptors and ligands
often resulted in increased binding affinity. By explor-
ing the newly identified hydrophobic pocket in the NA
active site, we were able to increase lipophilicity and
potency of the carbocyclic NA inhibitors.
To examine the influence of lipophilic groups (R1 in
6) on the magnitude of the hydrophobic interaction
between inhibitors and NA, we undertook SAR studies
by making systematic changes in the length, size and
branching of alkyl chains. To establish the limit of the
alkyl chain length which can fit in the hydrophobic
pocket, linear alkyl analogues were prepared as shown
in Table 1. While the methyl and ethyl analogues (32
and 33) had steadily increased potency compared to the
unsubstituted analogue 31, as measured by their IC50
against influenza NA, compound 34 gave a significant
increase in inhibitory activity, which is likely a reflection
of added hydrophobic interactions by the terminal
methyl of the n-propyl group. Further extension of the
alkyl chain up to n-nonyl (34 to 40) resulted in similar
potency with IC50 values of 150-300 nM, suggesting the
additional hydrocarbons up to n-nonyl were insufficient
to impart higher potency. The X-ray crystal structure
of the n-hexyl analogue 37 bound to NA revealed that
the binding energy gained by the hydrophobic interac-
tions of the n-hexyl chain was offset by the partial
exposure of the side chain into water outside of the
active site. The poor NA inhibitory potency of 41
implies that additional hydrocarbons beyond the n-nonyl
length may result in the unfavorable hydrophobic
binding interaction. Although active-site amino acid
residues are conserved across influenza A and influenza
B NA subtypes,1 differences in the binding region of the
glycerol side chain of Zanamivir (5), particularly in the
position and side chain conformation of Glu276 have
been reported.20 The large activity difference in the
inhibitory activity of compounds 37 and 39 against
influenza A and B NA also suggests that the large alkyl
groups in these compounds had less favorable hydro-
phobic interactions in the active site of the influenza B
NA.
The geometry and conformational mobility of the alkyl
chains would also be expected to have great impact on
the hydrophobic interactions. Therefore, we next in-
vestigated various branched alkyl chain analogues as
shown in Table 2. Since the n-propyl analogue 34
displayed similar NA inhibitory activity with longer
alkyl chains (Table 1), various alkyl groups were added
on the n-propyl group as branching analogues. Addition
of the methyl at the â position of the n-propyl chain
(compound 42) did not influence the influenza NA
inhibitory activity. However, the additional methyl at
the R position (compounds 43 and 44) resulted in a 20-
fold increase in NA inhibitory activity. This indicated
that there is opportunity to increase the strength of
binding by tailoring the structure of the branched alkyl
chains around the R position of the n-propyl group.
Interestingly, both diastereomers 43 and 44 exhibited
almost equal NA inhibitory activity. This result led us
to speculate that the methyl and the ethyl groups in
the sec-butyl analogues 43 and 44 contribute almost the
same binding energy by an equal degree of interaction
with amino acids in the active site. Therefore, the
chirality of the side chains of 43 and 44 had little
influence in overall hydrophobic interactions. This
result was also consistent with the further improved NA
inhibitory activity of 7 (GS 4071), in which both ethyl
groups bound into the two different hydrophobic pockets
and contributed significantly for the hydrophobic inter-
action with NA as shown by the X-ray crystal struc-
ture.11 Relatively small activity differences between two
allylic diastereomers (45 and 46) also supported the idea

2454 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kim et al.
Ta ble 3. Influenza Neuraminidase Inhibitory Activity of
Ta ble 2. Influenza Neuraminidase Inhibitory Activity of
Carbocyclic Analogues with Branched Alkyl Chains
Carbocyclic Analogues with Aryl and Aryl Alkyl Side Chains
of two different directional hydrophobic bindings for the
chiral side chains. The almost identical influenza A and
B NA inhibitory activity of 7 and 47 agreed with the
observation that the chain length of the linear alkyl
analogues could be extended considerably without losing
the influenza A NA inhibitory activity as discussed
above. However, the potent influenza B inhibitory
activity exhibited by 47 is quite puzzling in light of the
greatly reduced influenza B NA inhibitory activity of
39, which possesses the same carbon chain length as
47. To evaluate the importance of steric bulkiness on
the alkyl chain for the hydrophobic interaction, cyclo-
hexyl analogues (48, 49, and 50) were prepared. Com-
pound 48, with the large cyclohexyl ring attached close
to the cyclohexene scaffold, exhibited significantly re-
duced NA inhibitory activity when compared to 7. The
X-ray crystal structure of 48 bound to NA revealed that
the cyclohexyl ring of 48 is far from Glu276 and resulted
in the limited hydrophobic interactions. However, when
the cyclohexyl ring was extended by an additional one
or two methylenes from the chiral center (compounds
49 and 50), influenza A NA inhibitory activity was
improved considerably. In contrast with this, com-
pounds 49 and 50 had greatly reduced influenza B NA
inhibitory activity, suggesting that the binding affinity
toward influenza B NA could be influenced significantly
by the increased steric bulkiness of the C3 side chain.
In the aryl and aryl alkyl series of compounds (Table
3), the phenyl and the benzyl analogues (51 and 52)
exhibited poor NA inhibitory activity. However, the
biphenyl analogue 57 was more potent when compared
to 51 in NA inhibition, suggesting hydrophobic interac-
tions may extend from the para position of the phenyl
ring in 51. Two diastereomers 53 and 54 exhibited very
potent influenza A NA inhibitory activity. In this case,
the (S) isomer 53 is more potent than the (R) isomer
for influenza A NA, but the influenza B NA inhibitory
activities of 53 and 54 were comparable. The X-ray
crystal structures of 53 bound to NA revealed that a
portion of the phenyl ring (yellow in Figure 1) is exposed
to water, similar to the X-ray structure of the n-hexyl
analogue 37. The decreased NA inhibitory activities of
analogues 55 and 56 are likely due to result from the
unfavorable hydrophobic interactions caused by two
phenyl rings at each terminal position of the branched
chains.
It was reported that the conversion of the C4-amino
group in 4 to the guanidino functionality (compound 5)
imparted over a 100-fold increased potency in NA
inhibitory activity.8 This increased potency is believed
to be a consequence of the additional charge-charge
interaction brought by the guanidino group with Glu227
located near the NA active site. In the series of
carbocyclic NA inhibitors, several guanidino analogues
were also prepared, and their NA inhibitory activities
were compared with those of corresponding amino
analogues (Table 4). Although the guanidino analogues
in Table 4 exhibited consistently more potent NA
inhibitory activity compared to their amino counter-
parts, activity differences ranged from 2 to 100-fold.
Amino analogues (43, 44, and 7) which exhibit already
potent NA inhibitory activity appeared to have a lesser
increase in their NA inhibitory activity upon conversion

Novel Carbocyclic Influenza Neuraminidase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2455
F igu r e 1. X-ray structure of 53 bound to influenza neuraminidase. A part of the phenyl ring (yellow) is exposed to water. Dash
lines indicate close contacts; numbers are distances in angstroms.
Ta ble 4. Influenza Neuraminidase Inhibitory Activity of
Carbocyclic Guanidino Analogues
Ta ble 5. Influenza Neuraminidase Inhibitory Activity of C2
and C6 Substituted GS 4071 (7) Analogues
neuraminidase (IC50, nM)
neuraminidase (IC50, nM)
compound
R
influenza Aa
compound
X
Y
influenza Aa
influenza Bb
58
59
60
61
62
63
H
100 (6300)b
2 (180)
3 (300)
0.5 (10)
0.5 (9)
7
64
30
22
H
CH3
F
H
H
H
CH3
1
2300
3
3
NDc
90
CH3(CH2)2
CH3(CH2)3
CH3CH2(CH3)CH* (R)
CH3CH2(CH3)CH* (S)
(CH3CH2)2CH
H
1500
36000
0.5 (1)
a
b
A/PR. B/Lee. c ND ) not determined.
a
b
A/PR. IC50 (nM) of corresponding amino compound 6 in
parentheses.
For this purpose, C2-methyl, C2-fluoro, and C6-methyl
analogues of GS 4071 (7) were prepared (Table 5). The
C2-methyl analogue 64 exhibited an over 1000-fold
decrease in the NA inhibitory activity compared to the
parent compound 7. The poor NA inhibitory activity
exhibited by 64 may be due to the steric clash between
the C2-methyl and the nearby Arg292. With the widely
held view that the fluorine atom may be regarded as
an isostere of the hydrogen atom,21 the C2-fluoro ana-
logue 30 retained potent NA inhibitory activity of 7
of the amino group to the guanidino group (61, 62, and
63). The different degree of the binding enhancement
observed in the guanidino series might suggest that the
hydrophobic interactions of the C3-alkyl groups influ-
enced the hydrogen bonding interaction of the guanidino
groups differently through the amino acid network of
the NA active site. Other objectives in this SAR study
were to determine how C2 and C6 substituents on the
cyclohexene scaffold would affect NA inhibitory activity.

2456 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kim et al.
bromide (9.2 mL of a 3.0 M solution in ether, 27.6 mmol)
dropwise. After 10 min, a solution of 13d (1.95 g, 13.7 mmol)
in dry THF (10 mL) was added via cannula. The reaction
mixture was stirred at -40 °C for 1 h, quenched with saturated
NH4Cl, and diluted with ether. The organic layer was
separated, washed sequentially with 1 M HCl, water, satu-
rated sodium bicarbonate, and brine, dried (MgSO4), and
filtered through a thin pad of silica gel. Concentration in vacuo
gave 14d (2.04 g, 94%) as an oil: 1H NMR (CDCl3) δ 3.55 (m,
1H), 1.30-1.45 (m, 14H), 0.95 (m, 6H).
against influenza A. However, to our surprise, the
influenza B NA activity of 30 was considerably lower
than that of 7. The greatly reduced NA activity of the
C6-methyl analogue 22 might suggest that the lipophilic
substituents at the C6 position could result in consider-
ably less favorable hydrophobic interactions in the NA
active site.
Con clu sion s
1,7-Dip h en ylh ep ta n -4-ol (65). To a solution of 3-phenyl-
propionaldehyde (2.2 g, 14.85 mmol) in dry THF (80 mL) at
-78 °C was added (3-phenylpropyl)magnesium bromide (25
mL of a 1.3 M solution in ether, 32.5 mmol) dropwise. The
reaction mixture was stirred for 1.5 h at -78 °C and then at
0 °C for an additional 30 min. The reaction was quenched
with saturated NH4Cl and extracted with EtOAc. The organic
layer was separated, washed with 1 N HCl, water, saturated
sodium bicarbonate, and brine, and dried (Na2SO4). Concen-
tration in vacuo followed by flash chromatography of the
residue on silica gel with EtOAc/hexane (3:1) afforded the
alcohol (2.82 g, 71%): 1H NMR (CDCl3) δ 7.20 (m, 10H), 3.60
(s, 1H), 2.64 (m, 4H), 1.32-1.80 (m, 8H).
Rep r esen ta tive P r oced u r e: Alcoh olysis of 9. Syn th e-
sis of Eth yl (3R,4R,5S)-4-Aceta m id o-5-a zid o-3-(1-eth yl-
p r op oxy)-1-cycloh exen e-1-ca r boxyla te (10, R ) 3-p en tyl).
To a solution of 9 (15 g, 34 mmol) in 3-pentanol (230 mL) was
added BF3‚Et2O (6.27 mL, 51 mmol). The solution was heated
at 70-75 °C for 2 h and then evaporated to give a residue
which was dissolved in dry pyridine (2.0 mL) and treated with
acetic anhydride (16 mL, 170 mmol) and (dimethylamino)-
pyridine (200 mg, 1.6 mmol). [Note: In cases where evapora-
tion of excess alcohol was not possible, the reaction mixture
was directly treated with excess acetic anhydride and pyridine.
Separation and purification by flash chromatography on silica
gel furnished the desired product.] The reaction was stirred
at room temperature for 18 h and evaporated, and the residue
was partitioned between ethyl acetate and 1 M HCl. The
organic phase was washed with saturated sodium bicarbonate
and brine and dried. The solvent was evaporated, and the
residue was purified by chromatography eluting with hexane/
ethyl acetate (1:1) to give 10 (R ) 3-pentyl) (7.66 g, 69%). An
analytical sample was recrystallized from hexane/ethyl acetate
to afford 10 (R ) 3-pentyl) as needles: mp 121-123 °C; 1H
NMR (CDCl3) δ 6.79 (t, 1H, J ) 2.1 Hz), 5.92 (d, J ) 7.5 Hz,
1H), 4.58 (bd, J ) 8.7 Hz, 1H), 4.35-4.25 (m, 1H), 3.77 (s,
3H), 3.36-3.25 (m, 2H), 2.85 (dd, J ) 5.7, 17.4 Hz, 1H), 2.29-
2.18 (m, 1H), 2.04 (s, 3H), 1.60-1.45 (m, 4H), 0.91 (t, J ) 3.7
Hz, 3H), 0.90 (t, J ) 7.3 Hz, 3H); 13C NMR (CDCl3) δ 171.1,
166.2, 138.4, 127.7, 82.1, 73.8, 57.5, 57.2, 52.0, 30.4, 26.1, 25.5,
23.4, 9.5, 9.1. Anal. (C15H24N4O4) C, H, N.
On the basis of the structural information available
from the X-ray crystal structures of sialic acid and its
analogues complexed with influenza NA, a new series
of potent carbocyclic NA inhibitors has been identified.
The discovery of a new hydrophobic pocket in the NA
active site allowed us to investigate various C3-alkyl and
aryl analogues for optimization of NA inhibition. X-ray
crystal structures of several analogues in this series
complexed with NA revealed that the alkyl chains could
bind in two directions for optimal hydrophobic binding
interactions. From the SAR described in this report,
compound 7 (GS 4071) emerged as one of the most
potent influenza A and B NA inhibitor in this series.
The ethyl ester prodrug of 7 (8, GS 4104) proved to be
safe and efficacious for the oral treatment and prophy-
laxis of human influenza infection in phase I and phase
II clinical trials. GS 4104 is currently being evaluated
in phase II/III human clinical trials.
Exp er im en ta l Section
Gen er a l. All reactions were conducted under a dry atmo-
sphere of argon unless otherwise noted. All reaction solvents
were anhydrous grade obtained from Aldrich Chemical Co.
Melting points were obtained on a Thomas-Hoover melting
point apparatus and are uncorrected. Elemental analyses
were performed by Robertson Microlit Laboratories, Inc.,
Madison, NJ . High-resolution mass spectra were performed
by the Mass Spectrometry Lab, University of California,
Berkeley. Column chromatography was carried out using
230-400 mesh silica gel. NMR spectra were recorded at 300
MHz unless otherwise indicated.
Alcohols used in the opening of aziridine 9 were obtained
from either commercial sources or prepared from literature
methods unless otherwise specified in the experimental.
Compounds 7, 31, 32, 33, 34, 35, 42, 43, 44, 48, 51, and 58
have been previously reported.11,17
Rep r esen ta tive P r oced u r e: Ep oxid e Op en in g of 11.
(2R)-Tolu en e-4-su lfon ic Acid 2-Hydr oxyn on yl Ester (12d).
To a solution of Li2CuCl4 (11.0 mL of a 0.10 M solution in THF,
1.1 mmol) in dry THF (132 mL) at -35 °C was added
n-hexylmagnesium bromide (11 mL of a 2.0 M solution in
ether, 22 mmol) dropwise. After 40 min, a precooled solution
of (2R)-glycidyl tosylate 11 (5.02 g, 22 mmol) in dry THF (66
mL) was added via cannula. After 3 h the reaction was
quenched with saturated NH4Cl and diluted with ether. The
organic layer was separated, washed with brine, dried (Na2-
SO4), and evaporated. The residue was purified by flash
chromatography; eluting with EtOAc/hexane (1:4) gave 12d
(4.3 g, 62%).
R ep r esen t a t ive P r oced u r e: E p oxid e F or m a t ion of
12d . (2R)-1,2-Ep oxyn on a n e (13d ). To a solution of 12d (4.3
g, 13.64 mmol) in dry methanol (70 mL) was added solid K2-
CO3 (2.2 g, 15.92 mmol). After being stirred at room temper-
ature for 1 h, the reaction mixture was filtered, concentrated,
and partitioned between ether and water. The organic layer
was separated, washed with brine, and dried (MgSO4). Con-
centration followed by distillation gave 13d (1.6 g, 82%) as an
oil.
Rep r esen ta tive P r oced u r e: Syn th esis of 7 fr om 10 (R
) 3-p en tyl). (3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(1-eth yl-
p r op oxy)-1-cycloh exen e-1-ca r boxylic Acid (7). To a solu-
tion of azide 10 (R ) 3-pentyl) (1 g, 3.1 mmol) in THF (30 mL)
were added triphenylphosphine (1.21 g, 4.6 mmol) and water
(5.6 mL). The solution was heated at 50 °C for 10 h, THF
was evaporated, and the aqueous oily residue was partitioned
between ethyl acetate and brine. The organic phase was dried,
filtered, and evaporated. Purification of the residue by chro-
matography eluting with methanol/ethyl acetate (1:1) gave 830
mg of an oil which was dissolved in THF (15 mL) and was
treated with 1 N potassium hydroxide (4 mL, 4.16 mmol). The
reaction mixture was stirred at room temperature for 40 min
and acidified to pH 6 with Dowex 50WX8. The resin was
filtered and washed with water and methanol. Solvents were
evaporated, and the residue was purified by C18 chromatog-
raphy, eluting with water and then with 5% acetonitrile in
water. Fractions containing the desired product were pooled
and lyophilized to afford 7 (600 mg, 75%) as a white solid: 1H
NMR (D2O) δ 6.50 (t, J ) 2.1 Hz, 1H), 4.30-4.26 (m, 1H), 4.03
(dd, J ) 9.0, 11.7 Hz, 1H), 3.58-3.48 (m, 2H), 2.88 (dd, J )
5.4, 16.8 Hz, 1H), 2.53-2.41 (m, 1H), 1.62-1.40 (m, 4H), 0.90
Rep r esen ta tive P r oced u r e: Ep oxid e Op en in g of 13d .
(3S)-Deca n -3-ol (14d ). To a slurry of CuI (1.05 g, 5.5 mmol)
in dry THF (25 mL) at -40 °C was added methylmagnesium

Novel Carbocyclic Influenza Neuraminidase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2457
(t, J ) 7.5 Hz, 3H), 0.85 (t, J ) 7.5 Hz, 3H). Anal.
(C14H24N2O4‚1.5H2O) C, H, N.
1H), 2.63 (m, 4H), 2.44 (m, 1H), 1.62 (m, 4H), 1.55 (m, 4H);
HRMS (FAB) calcd for C28H37N2O4 (MH+) 465.2753, found
465.2755.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(h exyloxy)-1-cyclo-
h exen e-1-ca r boxylic Acid (37). Prepared in 41% overall
yield from aziridine 9: 1H NMR (D2O) δ 6.60 (s, 1H), 4.29 (d,
J ) 8.8 Hz, 1H), 4.13 (dd, J ) 9.0, 11.0 Hz, 1H), 3.82 (m, 1H),
3.61 (m, 1H), 3.54 (m, 1H), 2.93 (dd, J ) 17.0, 5.0 Hz, 1H),
2.52 (m, 1H), 2.16 (s, 3H), 1.62 (m, 2H), 1.38 (m, 6H), 0.95 (t,
J ) 7.0 Hz, 3H). Anal. (C15H26N2O4‚1.8H2O) C, H, N.
(3R,4R,5S)-4-Acet a m id o-5-a m in o-3-(h ep t yloxy)-1-cy-
cloh exen e-1-ca r boxylic Acid (38). Prepared in 26% overall
yield from aziridine 9: 1H NMR (D2O) δ 6.64 (s, 1H), 4.33 (d,
J ) 8.2 Hz, 1H), 4.21 (t, J ) 11.0 Hz, 1H), 3.66 (m, 1H), 3.64-
3.85 (m, 2H), 2.99 (dd, J ) 17 Hz, 5 Hz, 1H), 2.60 (m, 1H),
2.19 (S, 3H), 1.67 (m, 2H), 1.40 (m, 8H), 0.97 (m, 3H); HRMS
(FAB) calcd for C16H29N2O4 (MH+) 313.2127, found 313.2131.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(octyloxy)-1-cyclo-
h exen e-1-ca r boxylic Acid (39). Prepared in 29% overall
yield from aziridine 9: 1H NMR (D2O) δ 7.02 (s, 1H), 4.37 (d,
J ) 8.0 Hz, 1H), 4.22 (dd, J ) 9.0, 12.0 Hz, 1H), 3.87 (m, 1H),
3.71 (m, 1H), 3.65 (m, 1H), 3.05 (dd, J ) 5.5, 17.0 Hz, 1H),
2.63 (m, 1H), 1.64 (m, 2H), 1.37 (m, 10H), 0.95 (t, J ) 7.0 Hz,
3H); HRMS (FAB) calcd for C17H31N2O4 (MH+) 327.2284, found
327.2279.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(n on yloxy)-1-cyclo-
h exen e-1-ca r boxylic Acid (40). Prepared in 35% overall
yield from aziridine 9: 1H NMR (D2O) δ 6.99 (s, 1H), 4.38 (d,
J ) 8 Hz, 1H), 4.21 (dd, J ) 9.0, 11.0 Hz, 1H), 3.83 (m, 1H),
3.73 (m, 1H), 3.63 (m, 1H), 3.06 (dd, J ) 5.5, 17.0 Hz, 1H),
2.62 (m, 1H), 2.19 (s, 3H), 1.64 (m, 2H), 1.37 (s, 12H), 0.90 (t,
J ) 7.0 Hz, 3H); HRMS (FAB) calcd for C18H33N2O4 (MH+)
341.2440, found 341.2434.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(d ecyloxy)-1-cyclo-
h exen e-1-ca r boxylic Acid (41). Prepared in 37% overall
yield from aziridine 9: 1H NMR (D2O) δ 6.71 (s, 1H), 4.11 (d,
J ) 8 Hz, 1H), 4.06 (dd, J ) 8.0, 10.4 Hz, 1H), 3.69 (m, 1H),
3.51 (m, 1H), 3.42 (m, 1H), 2.89 (dd, J ) 6.0, 17.0 Hz, 1H),
2.46 (m, 1H), 2.05 (s, 3H), 1.57 (m 2H), 1.32-1.38 (m, 14 H),
0.92 (t, J ) 7.0 Hz, 3H); HRMS (FAB) calcd for C19H35N2O4
(MH+) 355.2597, found 355.2599.
(3R,4R,5S)-4-Acet a m id o-5-a m in o-3-[(3S)-d ecyloxy]-1-
cycloh exen e-1-ca r boxylic Acid (47). Prepared in 34%
overall yield from aziridine 9: 1H NMR (D2O) δ 6.89 (s, 1H),
4.44 (d, J ) 8.0 Hz, 1H), 4.10 (dd, J ) 9.0, 12.0 Hz, 1H), 3.65
(m, 2H), 3.00 (dd, J ) 5.5, 17.0 Hz, 1H), 2.65 (m, 1H), 2.14 (s,
3H), 1.61 (m, 4H), 1.33 (m, 10H), 0.92 (m, 6H); HRMS (FAB)
calcd for C19H35N2O4 (MH+) 355.2597, found 355.2604.
(3R,4R,5S)-4-Acet a m id o-5-a m in o-3-(1(S)-(cycloh exyl-
m eth yl)p r op oxy)-1-cycloh exen e-1-ca r boxylic Acid (49).
Prepared in 30% overall yield from aziridine 9: 1H NMR (D2O)
δ 6.68 (s, 1H), 4.19 (d, J ) 8.0 Hz, 1H), 3.96 (dd, J ) 8.0, 11.0
Hz, 1H), 3.62 (m, 1H), 3.46 (m, 1H), 2.89 (dd, J ) 5.5, 17.4
Hz, 1H), 2.45 (m, 1H), 2.07 (s, 3H), 1.28-1.70 (m, 5H), 0.94 (t,
J ) 7.0 Hz, 3H); HRMS (FAB) calcd for C19H33N2O4 (MH+)
353.2440, found 353.2441.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-(1(S)-(2-cycloh exyl-
eth yl)pr opoxy)-1-cycloh exen e-1-car boxylic Acid (50). Pre-
pared in 35% yield from aziridine 9: 1H NMR (D2O) δ 6.64 (s,
1H), 4.15 (d, J ) 8.0 Hz, 1H), 3.96 (dd, J ) 8.0, 10.7 Hz, 1H),
3.48 (m, 1H), 3.41 (m, 1H), 2.86 (dd, J ) 5.7, 17.6 Hz, 1H),
2.44 (m, 1H), 2.05 (s, 3H), 1.18-1.76 (m, 17H), 0.94 (t, J ) 7
Hz, 3H); HRMS (FAB) calcd for C20H35N2O4 (MH+) 367.2597,
found 367.2599.
(3R,4R,5S)-4-Acet a m id o-5-a m in o-3-(b is(p h en ylm et h -
yl)m et h oxy)-1-cycloh exen e-1-ca r b oxylic Acid H yd r o-
ch lor id e (55). Prepared in 12% overall yield from aziridine
9: 1H NMR (D2O) δ 7.21-6.96 (m, 10H), 6.11 (br s, 1H), 4.12
(br d, J ) 7.5 Hz, 1H), 3.90-3.82 (m, 2H), 3.47-3.38 (m, 1H),
2.75-2.50 (m, 5H), 2.34-2.25 (m, 1H), 1.87 (s, 3H).
(3R,4R,5S)-4-Acetam ido-5-am in o-3-(bis(3-ph en ylpr opyl)-
m eth oxy)-1-cycloh exen e-1-ca r boxylic Acid (56). Prepared
in 15% overall yield from aziridine 9: 1H NMR (D2O) δ 7.15-
7.25 (m, 10H), 6.62 (s, 1H), 4.13 (d, J ) 8.0 Hz, 1H), 3.94 (dd,
J ) 8.0, 11.0 Hz, 1H), 3.60 (m, 1H), 3.38 (m, 1H), 2.85 (m,
(3R,4R,5S)-4-Acet a m id o-5-a m in o-3-(3-b ip h en ylylp r o-
p oxy)-1-cycloh exen e-1-ca r boxylic Acid (57). Prepared in
29% overall yield from aziridine 9: 1H NMR (D2O) δ 7.30-
7.60 (m, 9H), 6.74 (s, 1H), 4.15 (m, 1H), 4.10 (m, 1H), 3.73 (m,
1H), 3.57 (m, 1H), 3.44 (m, 1H), 2.89 (dd, J ) 5.5, 17.0 Hz,
1H), 2.75 (t, J ) 6.0 Hz, 2H), 2.50 (m, 1H), 2.05 (s, 3H), 1.93
(m, 2H); HRMS (FAB) calcd for C24H29N2O4 (MH+) 409.2127,
found 409.2131.
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-[[(1S)-1-eth ylbu t-3-
en yl]oxy]-1-cycloh exen e-1-ca r boxylic Acid (45). Prepared
in 26% overall yield from aziridine 9: 1H NMR (D2O) δ 6.43
(s, 1H), 5.76-5.61 (m, 1H), 5.04-4.90 (m, 2H), 4.21-4.17 (m,
1H), 3.92 (dd, J ) 8.7, 11.7 Hz, 1H), 3.56-3.50 (m, 1H), 3.48-
3.38 (m, 1H), 2.77 (dd, J ) 5.4, 17.1 Hz, 1H), 2.42-2.30 (m,
1H), 2.24-2.10 (m, 1H), 1.96 (s, 3H), 1.50-1.40 (m, 2H), 0.79
(t, J ) 7.5 Hz, 3H).
(3R,4R,5S)-4-Aceta m id o-5-a m in o-3-[[(1R)-1-eth ylbu t-3-
en yl]oxy]-1-cycloh exen e-1-ca r boxylic Acid (46). Prepared
in 24% overall yield from aziridine 9: 1H NMR (D2O) δ 6.32
(s, 1H), 5.79-5.65 (m, 1H), 5.09-4.90 (m, 2H), 4.10 (bd, J )
9.1 Hz, 1H),3.74 (dd, J ) 8.7, 11.3 Hz, 1H), 3.51-3.46 (m, 1H),
3.15-3.05 (m, 1H), 2.63 (dd, J ) 5.4, 17.3 Hz, 1H), 2.25-2.10
(m, 3H), 1.91 (s, 3H), 1.46-1.26 (m, 2H), 0.70 (t, J ) 7.5 Hz,
3H).
Rep r esen ta tive P r oced u r e: Gu a n yla tion of 15 (R )
n -p r op yl). (3R,4R,5S)-4-Aceta m id o-5-[N2,N3-Bis(ter t-bu -
toxycar bon yl)gu an idin o]-3-pr opoxy-1-cycloh exen e-1-car -
boxylic Acid (16; R ) n -p r op yl). To a solution of amine 15
(R ) n-propyl; 529 mg, 1.97 mmol), N,N′-bis(tert-butoxycar-
bonyl)thiourea (561 mg, 2.02 mmol), and triethylamine (930
mL) in dry DMF (5.0 mL) cooled to 0 °C was added HgCl2 (593
mg, 2.18 mmol) in one portion. The heterogeneous reaction
mixture was stirred for 45 min at 0 °C and then at room
temperature for 15 min, after which the reaction was diluted
with EtOAc and filtered through a pad of Celite. Concentra-
tion in vacuo followed by flash chromatography of the residue
on silica gel with EtOAc/hexane (9:1) gave 16 (R ) n-propyl;
904 mg, 90%) as a pale oil: 1H NMR (CDCl3) δ 11.39 (s, 1H),
8.63 (d, J ) 7.8 Hz, 1H), 6.89 (t, J ) 2.4 Hz, 1H), 6.46 (d, J )
8.7 Hz, 1H), 4.43-4.32 (m, 1H), 4.27-4.17 (m, 1H), 4.13-4.06
(m, 1H), 3.77 (s, 3H), 3.67-3.59 (m, 1H), 2.83 (dd, J ) 5.1,
17.7 Hz, 1H), 2.45-2.33 (m, 1H), 1.95 (s, 3H), 1.65-1.50 (m,
2H), 1.45 (s, 18H), 0.90 (t, J ) 7.5 Hz, 3H).
Rep r esen ta tive P r oced u r e: Sa p on ifica tion a n d De-
block in g of 16 (R ) n -p r op yl). (3R,4R,5S)-4-Aceta m id o-
5-gu a n id in yl-3-p r op oxy-1-cycloh exen e-1-ca r boxylic Acid
(59). To a solution of 16 (R ) n-propyl; 904 mg, 1.77 mmol)
in THF (10 mL) was added aqueous KOH (3.45 mL of a 1.039
N solution). The reaction mixture was stirred at room tem-
perature for 17 h, cooled to 0 °C, and acidified to pH 4.0 with
Amberlite IR-120 (H+) exchange resin. The resin was filtered
and washed with water and methanol. Concentration in vacuo
gave the free acid as a pale foam which was dissolved in CH2-
Cl2 (40 mL) and treated with neat trifluoroacetic acid (25 mL)
at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and
then at room temperature for 2 h. Concentration in vacuo gave
a pale orange solid which was purified by C18 chromatography
eluting with water. Fractions containing the desired product
were pooled and lyophilized to give 59 (R ) n-propyl; 495 mg,
68%) as a pale solid: 1H NMR (D2O) δ 6.66 (s, 1H), 4.29 (bd,
J ) 9.0 Hz, 1H), 4.01 (dd, J ) 10.8, 10.8 Hz, 1H), 3.87-3.79
(m, 1H), 3.76-3.67 (m, 1H), 3.60-3.50 (m, 1H), 2.83 (dd, J )
5.1, 17.4 Hz, 1H), 2.47-2.36 (m, 1H), 2.06 (s, 3H), 1.65-1.50
(m, 2H), 0.90 (t, J ) 7.2 Hz, 3H). Anal. (C15H23O6N4F3) C, H,
N.
(3R,4R,5S)-4-Acet a m id o-5-gu a n id in yl-3-b u t oxy-1-cy-
cloh exen e-1-ca r boxylic a cid (60): 1H NMR (D2O) δ 6.65 (s,
1H), 4.24 (br d, J ) 8.9 Hz, 1H), 3.99-3.92 (m, 1H), 3.81-
3.68 (m, 2H), 3.57-3.50 (m, 1H), 2.79 (dd, J ) 5.1, 17.4 Hz,
1H) 2.42-2.31 (m, 1H), 2.00 (s, 3H), 1.55-1.45 (m, 2H), 1.36-
1.24 (m, 2H), 0.85 (t, J ) 7.4 Hz, 3H).

2458 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kim et al.
(3R,4R,5S)-4-Aceta m id o-5-gu a n id in yl-3-(1(R)-m eth yl-
p r op oxy)-1-cycloh exen e-1-ca r boxylic a cid (61): 1H NMR
(D2O) δ 6.47 (s, 1H), 4.28 (bd, J ) 8.4 Hz, 1H), 3.93-3.74 (m,
2H), 3.72-3.63 (m, 1H), 2.78 (dd, J ) 4.8, 17.4 Hz, 1H), 2.43-
2.32 (m, 1H), 2.04 (s, 3H), 1.58-1.45 (m, 2H), 1.13 (d, J ) 6.0
Hz, 3H), 0.90 (t, J ) 7.4 Hz, 3H).
(3R,4R,5S)-4-Acet a m id o-5-gu a n id in yl-3-(1(S)-m et h yl-
p r op oxy)-1-cycloh exen e-1-ca r boxylic a cid (62): 1H NMR
(D2O) δ 6.81 (s, 1H), 4.31 (br d, J ) 8 Hz, 1H), 3.94-3.76 (m,
2H), 3.71-3.61 (m, 1H), 2.83 (dd, J ) 5, 17.5 Hz, 1H), 2.43-
2.32 (m, 1H), 2.06 (s, 3H), 1.58-1.45 (m, 2H), 1.19 (d, J ) 6.1
Hz, 3H), 0.82 (t, J ) 7.2 Hz, 3H).
(3R,4R,5S)-4-Acet a m id o-5-gu a n id in yl-3-(1-et h ylp r o-
poxy)-1-cycloh exen e-1-car boxylic acid (63): 1H NMR (D2O)
δ 6.72 (s, 1H), 4.23 (br d, J ) 8.4 Hz), 3.84-3.66 (m, 2H), 3.46-
3.38 (m, 1H), 2.73 (dd, J ) 4.8, 17.4 Hz, 1H), 2.33-2.23 (m,
1H), 1.91 (s, 3H), 1.48-1.27 (m, 4H), 0.76 (t, J ) 7.5 Hz, 3H),
0.71 (t, J ) 7.5 Hz, 3H).
ethylamino)pyridine (5 mg) followed by acetic anhydride (226
mL, 2.4 mmol). The mixture was stirred for 2.5 h at room
temperature, diluted with CH2Cl2, washed with water, and
dried (Mg2SO4). Concentration in vacuo followed by flash
chromatography of the residue on silica gel gave 20 (900 mg,
96%): mp 150-151 °C; 1H NMR (CDCl3) δ 7.2 (d, J ) 4.0 Hz,
1H), 5.78 (brs, 2H), 5.42 (d, J ) 9.0 Hz, NH), 4.4 (m, 2H), 4.25
(m, 2H), 3.8 (m, 1H), 3.55 (m, 1H), 2.08 (s, 3H), 1.96 (s, 3H),
1.55 (m, 4H), 1.45 (s, 9H), 1.36 (t, J ) 7.0 Hz, 3H), 0.90 (m,
6H). Anal. (C23H38N2O8) C, H, N.
Eth yl-4â-Aceta m id o-5r-(ter t-bu toxyca r ba m id o)-3r-(1-
e t h ylp r op oxy)-6â-m e t h yl-1-cycloh e xe n e -1-ca r b oxyl-
a te (21). To a suspension of copper(I) iodide (486 mg, 2.55
mmol) in dry ether (40 mL) at -10 °C was added methyl-
lithium (1.4 M, 3.64 mL, 5.1 mmol) dropwise. The mixture
was stirred between -10 and 0 °C for 15 min and cooled to
-45 to 40 °C, and a solution of 20 (800 mg, 1.70 mmol) in dry
THF (10 mL) was added. The reaction was stirred for 30 min
at room temperature, quenched with saturated ammonium
chloride, washed with water, and dried (Mg2SO4). Concentra-
tion in vacuo followed by flash chromatography of the residue
on silica gel gave 21 (180 mg, 25%) as a crystalline solid: mp
Eth yl 4â-Aceta m id o-5r-(ter t-bu toxyca r ba m id o)-1,2-d i-
h yd r oxy-3r-(1-eth ylp r op oxy)-1-cycloh exa n e-1-ca r boxyl-
a te (18). To a solution of 8 (14.0 g, 44.8 mmol) in dry THF
(120 mL) was added a solution of di-tert-butyl dicarbonate (11.7
g, 53.8 mmol) in dry THF (30 mL) followed by triethylamine
(0.3 mL, 2.15 mmol) at 0 °C. The reaction was stirred for 10
min at 0 °C and for 30 min at room temperature, concentrated,
and filtered through a plug of silica gel eluting with EtOAc.
After evaporation the residue was dissolved in 2-methyl-2-
propanol (200 mL) and treated with N-methylmorpholine
N-oxide (7.75 g, 66.2 mmol) and osmium tetraoxide (1%
solution in water, 15 mL). The mixture was stirred for 3 h at
70 °C, cooled to room temperature, and quenched with sodium
thiosulfate pentahydrate (5 g in 20 mL of water). The mixture
was concentrated in vacuo and partitioned between EtOAc and
water. The organic phase was separated and the aqueous
phase extracted with EtOAc. The combined organic layers
were washed with brine, dried (Na2SO4), and filtered through
a plug of silica gel. Recrystallization from hexane/EtOAc
afforded diol 18 (18.4 g, 92%) as a colorless solid: 1H NMR
(CDCl3) δ 6.18 (d, J ) 9.0 Hz, NH), 4.88 (d, J ) 9.0 Hz, NH),
4.3 (q, J ) 7.0 Hz, 2H), 3.96 (m, 2H), 3.88 (m, 1H), 3.8 (s, OH),
3.5 (m, 2H), 2.5 (d, J ) 5.0 Hz, OH), 2.02 (s, 3H), 1.8-2.1 (m,
2H), 1.45-1.7 (m, 4H), 1.45 (s, 9H), 1.36 (t, J ) 7.0 Hz, 3H),
0.88 (m, 6H).
Eth yl 4â-Aceta m id o-3r-(ter t-bu toxyca r ba m id o)-5r-(1-
e t h ylp r op oxy)-6â-h yd r oxy-1-cycloh e xe n e -1-ca r b oxyl-
a te (19). To a solution of 18 (8.5 g, 19.0 mmol) in pyridine
(4.2 mL, 52 mmol) and CH2Cl2 (100 mL) at -78 °C was added
sulfuryl chloride (1.9 mL, 23.8 mmol) dropwise. The reaction
was stirred for 10 min at -78 °C, slowly warmed to room
temperature, and concentrated. The residue was dissolved in
EtOAc, washed with brine, and concentrated. The crude
product was recrystallized from hexane/EtOAc to give the
cyclic sulfate as a white solid (7.2 g, 75%, 14.2 mmol). The
sulfate was dissolved in dry THF (60 mL), and 1,8-diazabicyclo-
[5.4.0]undec-7-ene (2.54 mL, 17.0 mmol) was added at 0 °C.
After 16 h of stirring at room temperature the reaction was
cooled to 0 °C, and water (0.25 mL) and H2SO4 (1 N in THF,
18 mL) were added. The mixture was stirred for 40 min at
room temperature followed by the addition of saturated sodium
bicarbonate and dilution with EtOAc. The organic phase was
separated, and the aqueous phase was extracted with EtOAc.
The combined organic layers were washed with brine and dried
(Mg2SO4). Concentration in vacuo followed by flash chroma-
tography of the residue on silica gel EtOAc/hexane (2:1) gave
18 (3.4 g, 56%) as a colorless solid: mp 118-119 °C; 1H NMR
(CDCl3) δ 7.0 (d, J ) 4.0 Hz, 1H), 6.5 (d, J ) 8.0 Hz, NH), 5.4
(d, J ) 9.0 Hz, NH), 4.5 (m, 1H), 4.25-4.42 (m, 4H), 3.8 (m,
1H), 3.45 (m, 1H), 3.28 (s, 1H), 1.98 (s, 3H), 1.5 (m, 4H), 1.44
(s, 9H), 1.38 (t, J ) 7.0 Hz, 3H), 0.9 (m, 6H). Anal.
(C21H36N2O7) C, H, N.
1
225-226 °C; H NMR (CDCl3) δ 6.63 (s, 1H), 5.8 (d, J ) 10.0
Hz, NH), 4.8 (d, J ) 10.0 Hz, NH), 4.24 (m, 2H), 4.05 (ddd, J 1
) J 2 ) J 3 ) 10.0 Hz, 1H), 3.97 (d, J ) 10.0 Hz, 1H), 3.46 (ddd,
J 1 ) J 2 ) J 3 ) 10.0 Hz, 1H), 3.35 (m, 1H), 2.56 (m, 1H), 1.99
(s, 3H), 1.52 (m, 4H), 1.45 (s, 9H), 1.3 (t, J ) 7.5 Hz, 3H), 1.2
(d, J ) 7.5 Hz, 3H), 0.9 (m, 6H). Anal. (C22H38N2O6) C, H, N.
4â-Aceta m id o-5r-a m in o-3r-(1-eth ylp r op oxy)-6â-m eth -
yl-1-cycloh exen e-1-ca r boxylic a cid (22). To a solution of
21 (80 mg, 0.188 mmol) in CH2Cl2 (500 mL) and EtOAc (1.0
mL) was added HCl (1 N in EtOAc, 3 mL), and the mixture
was stirred for 1.5 h at room temperature. Solid sodium
bicarbonate was added, and after an additional 2 h the mixture
was filtered through a plug of silica gel eluting with EtOAc/
methanol (7:3). The filtrate was then evaporated to afford the
free amine (58 mg, 95%). A portion of this material (36 mg,
0.11 mmol) was dissolved in methanol (1.0 mL) and water (500
mL) and treated with KOH (340 mL of 0.97 N solution). After
16 h of stirring at room temperature, the mixture was acidified
to pH 6 with Dowex 50WX8. The resin was filtered and
washed with water and methanol. Concentration in vacuo
gave a pale solid which was purified by C18 chromatography,
eluting with water. Fractions containing the desired product
were pooled and lyophilized to afford 22 (22 mg, 67%): 1H
NMR (D2O) δ 6.21 (s, 1H), 4.28 (d, J ) 11.0 Hz, 1H), 4.05 (dd,
J 1 ) 11.0, J 2 ) 11.5 Hz, 1H), 3.55 (m, 1H), 3.29 (dd, J ) 11.5,
11.0 Hz, 1H), 2.9 (m, 1H), 2.12 (s, 3H), 1.45-1.65 (m, 4H), 1.25
(d, J ) 7.5 Hz, 3H), 0.9 (m, 6H). Anal. (C15H26N2O4‚2.1H2O)
C, H, N.
1-[[(2,5-Dim eth oxyben zyl)oxy]m eth yl]-3r,4r-(d im eth -
ylm et h ylen ed ioxy)-2-flu or o-5â-m et h oxym et h oxycyclo-
h exen e (23). Vinyl fluoride 23 was prepared from shikimic
acid in 29% overall yield by a modified procedure of Bartlett:
16
1H NMR (CDCl3) δ 6.95 (s, 1H), 6.8 (s, 2H), 4.75 (S, 2H),
4.74 (brs, 1H), 4.5 (s, 2H), 4.3 (m, 1H), 4.2 (m, 2H), 3.96 (m,
1H), 3.77 (s, 3H), 3.76 (s, 3H), 3.4 (s, 3H), 2.6 (m, 1H), 2.32
(m, 1H), 1.52 (s, 3H), 1.45 (s, 3H); 19F NMR (CDCl3, CFCl3)
-120.3.
3r,4r-(Dieth ylm eth ylen ed ioxy)-1-[[(2,5-d im eth oxyben -
zyl)oxy]m eth yl]-2-flu or o-5â-h yd r oxycycloh exen e (24). A
mixture of 23 (6.0 g, 14.6 mmol) and p-toluenesulfonic acid
monohydrate (140 mg, 0.73 mmol) in methanol (100 mL) was
refluxed for 6 h. The reaction mixture was evaporated to
dryness and then coevaporated with 3-pentanone. The crude
residue was dissolved in 3-pentanone (60 mL), stirred over
molecular sieves (4 Å, 6 g) for 6 h at room temperature, and
filtered. Concentration in vacuo followed by flash chromatog-
raphy of the residue on silica gel with hexane/EtOAc (2:1) gave
24 (4.4 g, 77%): 1H NMR (CDCl3) δ 6.98 (s, 1H), 6.8 (s, 2H),
4.75 (m, 1H), 4.52 (d, J ) 12.5 Hz, 1H), 4.48 (d, J ) 12.5 Hz,
1H), 4.05-4.3 (m, 4H), 3.84 (s, 3H), 3.83 (s, 3H), 2.63 (m, 1H),
2.33 (m, 1H), 2.1 (d, J ) 2.5 Hz, 1H), 1.72 (m, 4H), 0.98 (m,
6H).
E t h yl 4â-Acet a m id o-6â-a cet oxy-3r-(ter t-b u t oxyca r b -
a m id o)-5r-(1-et h ylp r op oxy)-1-cycloh exen e-1-ca r b oxyl-
a te (20). To a solution of 19 (857 mg, 2.0 mmol) in CH2Cl2
(10 mL) were added pyridine (242 mL, 3.0 mmol) and (dim-

Novel Carbocyclic Influenza Neuraminidase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2459
4r,5r-Ep oxy-3r-(1-eth ylp r op oxy)-2-flu or o-1-(h yd r oxy-
m eth yl)cycloh exen e (25). To a solution of 24 (4.4 g, 11.1
mmol) in CH2Cl2 (50 mL) at 0 °C was added triethylamine (3.1
mL, 22.2 mmol) followed by methanesulfonyl chloride (1.1 mL,
14.3 mmol). The reaction mixture was stirred for 10 min at 0
°C and then for an additional 30 min at room temperature.
The reaction was diluted with CH2Cl2 and quenched with ice-
water. The organic phase was separated, washed with brine,
and dried (MgSO4). To the filtrate (ca. 250 mL) cooled to -78
°C was added a solution of BH3‚Me2S (5.5 mL of a 10 M
solution in THF, 55.0 mmol) followed by trimethylsilyl triflu-
oromethanesulfonate (4.9 mL, 27.5 mmol). The reaction
mixture was stirred for 10 min at -78 °C and then warmed
slowly to 0 °C. The reaction was then hydrolyzed by the
addition of saturated sodium bicarbonate (250 mL) and
extracted with CH2Cl2. The combined organic layers were
washed with brine and dried (MgSO4). Concentration in vacuo
gave a residue which was dissolved in methanol (50 mL) and
treated with potassium bicarbonate (2.2 g, 22 mmol) and water
(35 mL). After 2 h of stirring at 50 °C, the reaction was
evaporated and extracted with EtOAc. Concentration in vacuo
followed by flash chromatography of the residue on silica gel
gave epoxide 25 (1.43 g, 57%) as a colorless solid: mp 72-73
°C; 1H NMR (CDCl3) δ 4.38 (m, 1H), 4.1-4.35 (m, 2H), 3.45-
3.55 (m, 3H), 2.88 (m, 1H), 2.6 (m, 1H), 1.63 (m, 4H), 1.38 (t,
J ) 7.5 Hz, OH), 1.0 (m, 6H). Anal. (C12H19FO3) C, H, N, F.
4r,5r-Epoxy-3r-(1-eth ylp r opoxy)-2-flu or o-1-[(m eth oxy-
m eth oxy)m eth yl]cycloh exen e (26). To a solution of epoxide
25 (1.4 g, 6.1 mmol) in CH2Cl2 (20 mL) at room temperature
was added diisopropylethylamine (2.1 mL, 12.0 mmol) followed
by chloromethyl methyl ether (0.7 mL, 9.1 mmol). After 1 h
of refluxing, the reaction was diluted with water and extracted
with CH2Cl2. Concentration in vacuo followed by flash chro-
matography of the residue on silica gel gave 26 (1.6 g, 96%)
as an oil: 1H NMR (CDCl3) δ 4.64 (s, 2H), 4.35-4.45 (m, 2H),
3.92 (m, 1H), 3.45-3.55 (m, 3H), 3.4 (s, 3H), 2.8 (m, 1H), 2.62
(m, 1H), 1.63 (m, 4H), 1.0 (m, 6H); 19F NMR (CDCl3, CFCl3)
-125.2.
mmol), and potassium phosphate monobasic (0.84 g, 6.2 mmol)
in THF/H2O/DMSO (15:15:1 mL) was stirred for 6 h at room
temperature. The reaction mixture was poured into 2 N HCl
(10 mL) at 0 °C and extracted with EtOAc. The organic phase
was washed with brine, dried (MgSO4), and evaporated to give
the carboxylic acid. The crude carboxylic acid was dissolved
in CH2Cl2 (10 mL) and THF (10 mL). Ethanol (0.36 mL, 6
mmol), 4-(dimethylamino)pyridine (40 mg), and 1,3-diisopro-
pylcarbodiimide (0.8 mL, 5.2 mmol) were added then added
sequentially. After the mixture was stirred for 4 h at room
temperature, volatiles were evaporated and the residue was
diluted with hexane/EtOAc (1:2). The mixture was filtered and
concentrated in vacuo to give a solid which was purified by
flash chromatography on silica gel to afford 29 (0.55 g, 60%)
as colorless solid: mp 103-104 °C; 1H NMR (CDCl3) δ 5.85
(d, J ) 7.5 Hz, NH), 4.68 (m, 1H), 4.35 (m, 1H), 4.3 (q, J ) 7.2
Hz, 2H), 3.4-3.55 (m, 2H), 2.84 (m, 1H), 2.32 (m, 1H), 2.08 (s,
3H), 1.55 (m, 4H), 1.55 (t, J ) 7.2 Hz, 3H), 0.93 (t, J ) 6.6 Hz,
6H); 19F NMR (CDCl3, CFCl3) -98.2. Anal. (C16H25FN4O4) C,
H, N, F.
4â-Aceta m id o-5r-a m in o-3r-(1-eth ylp r op oxy)-2-flu or o-
1-cycloh exen e-1-ca r boxylic a cid (30). Prepared from 29
in 40% yield following the previously described procedure:11
1H NMR (D2O) δ 4.42 (m, 1H), 4.24 (m, 1H), 3.58-3.66 (m,
2H), 2.84 (m, 1H), 2.62 (m, 1H), 2.1 (s, 3H), 1.58 (m, 4H), 0.88
(m, 6H); 13C NMR 8.5, 8.7, 22.6, 25.2, 25.3, 27.8 (J ) 4.7 Hz),
48.7, 53.6 (J ) 8.0 Hz), 74.2 (J ) 23.0 Hz), 85.7, 113.4, 153.9
(J ) 267.0 Hz), 172.0, 175.3; 19F NMR (D2O, CFCl3) -114.8.
Anal. (C14H23FN2O4‚1.4H2O) C, H, N, F.
Neu r a m in id a se En zym e Assa y. Neuraminidase enzyme
activity was determined using minor modifications to the
literature method.22 Influenza A/PR/8/34 (H1N1), purified on
sucrose density gradients, was used as the source of enzyme,
and 4-methylumbelliferyl-R-D-N-acetylneuraminic acid was
used as substrate in a reaction buffer containing 33 mM MES,
pH 6.5, and 4 mM calcium chloride. Virus was mixed with
various inhibitor concentrations and incubated at room tem-
perature for 30 min before substrate was added to a final
concentration of 10 µM. Reactions were stopped after 8 min
at 37 °C with the addition of 1.5 volumes of 14 mM sodium
hydroxide in 83% ethanol. Fluorescence was quantitated in
a Perkin-Elmer fluorimeter (Model LS50B) with an excitation
wavelength of 360 nm, emission wavelength of 448 nm, and
slit widths of 2.5 nM.
4â-Aceta m id o-5r-a zid o-3r-(1-eth ylp r op oxy)-2-flu or o-1-
[(m et h oxym et h oxy)m et h yl]cycloh exen e (27). Prepared
from 26 in 58% over yield by a previously described procedure:
11
mp 98-99 °C; 1H NMR (CDCl3) δ 5.85 (d, J ) 9.5 Hz, NH),
4.65 (s, 2H), 4.5 (m, 1H), 4.2-4.35 (m, 2H), 4.95 (m, 1H), 3.58
(m, 1H), 3.48 (m, 1H), 3.42 (s, 3H), 2.6 (m, 1H), 2.2 (m, 1H),
2.08 (s, 3H), 1.55 (m, 4H), 0.95 (m, 6H); 19F NMR (CDCl3,
CFCl3) -119. Anal. (C16H27FN4O3) C, H, N, F.
Cr ysta lliza tion a n d Da ta Collection . Isolation, purifica-
tion, and crystallization of N9 neuraminidase has previously
been reported.23 Crystals of the neuraminidase-inhibitor 53
complex were obtained by soaking the neuraminidase crystals
overnight in 5 mM inhibitor 53 solution (5 mM inhibitor, 2
volumes of 1.4 M KH2PO4, 1 volume of 3 M K2HPO4). Cubic
shaped crystals with approximate dimensions 0.2 × 0.2 × 0.2
mm were used to collect room temperature data at the
Stanford Synchrotron Radiation Laboratory, beamline 7-1 (λ
) 1.08 Å) using a MAR30 image plate system. The crystals
belong to the cubic space group I432, a ) 182.9 Å. A complete
data set to 2.7 Å resolution was collected using 1° oscillations
and a total of 30°. The reflections were indexed using DENZO
1.5.11 and merged/scaled using the program SCALEPACK. A
total of 51 334 reflections were collected; 12 383 reflections
were unique. The reduced data set was 98% complete to 2.7
Å resolution with an Rmerge of 7.1%.
4â-Aceta m id o-5r-a zid o-3r-(1-eth ylp r op oxy)-2-flu or o-1-
cycloh exen e-1-ca r boxa ld eh yd e (28). A solution of 27 (1.15
g, 3.21 mmol) and p-toluenesulfonic acid monohydrate (0.61
g, 3.21 mmol) in methanol (40 mL) was refluxed for 4 h. After
evaporation the residue was dissolved with EtOAc, washed
with sodium bicarbonate, and dried (MgSO4). Concentration
in vacuo gave a solid which was recrystallized from hexane/
EtOAc to give the alcohol (0.94 g, 93%) as a white solid. In a
separate flask dimethyl sulfoxide (0.69 g, 8.8 mmol) in dry CH2-
Cl2 (30 mL) cooled to -60 to -50 °C was treated with oxalyl
chloride (2.2 mL of a 2 M solution in CH2Cl2, 4.4 mmol). The
mixture was stirred for 20 min at this temperature after which
a solution of the above alcohol (0.92 g, 2.93 mmol) in dry THF
(10 mL) was added. After the mixture was stirred for 20 min
at this temperature triethylamine (2.0 mL, 14.4 mmol) was
added, and the resultant mixture was allowed to slowly warm
to room temperature and then evaporated. The residue was
partitioned between EtOAc and water. The organic phase was
washed with water and dried (MgSO4). Concentration in
vacuo gave a solid which was recrystallized from hexane/
EtOAc acetate to afford the aldehyde 28 (0.8 g, 87%) as a white
solid: 1H NMR (CDCl3) δ 10.15 (s, 1H), 6.0 (d, J ) 9.0 NH),
4.88 (m, 1H), 4.35 (m, 1H), 3.52 (m, 1H), 3.4 (m, 1H), 2.82 (m,
1H), 2.1 (m, 1H), 2.08 (s, 3H), 1.55 (m, 4H), 0.95 (m, 6H).
Str u ctu r e Solu tion a n d Refin em en t. The neuramini-
dase complexed with inhibitor 53 structure was solved by
molecular replacement using the influenza A subtype N9
neuraminidase crystal structure containing residues 82-469
(PDB ID 1INY).24 Alternate rounds of model building with
the molecular graphics program O version 6.0.325 and refine-
ment of the atomic coordinates using X-PLOR 3.126 were
performed until convergence of the free-R and R-factor was
achieved. Examination of Fo-Fc maps showed unambiguous
density for the orientation of inhibitor 53, sugar molecules,
and calcium ion. Addition of four N-acetyl-D-glucosamine
sugars, five R-D-mannose sugars, one calcium ion, and one
E t h yl 4â-Acet a m id o-5r-a zid o-3r-(1-et h ylp r op oxy)-2-
flu or o-1-cycloh exen e-1-ca r boxyla te (29). A mixture of
aldehyde 28 (0.8 g, 2.56 mmol), sodium chlorite (0.92 g, 10.2

2460 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kim et al.
Influenza Neuraminidase Inhibitor GS 4071 Protects Mice and
Ferrets Against Influenza Infection. Antimicrob. Agents Chemo-
ther. 1998, 42, 640-646.
inhibitor 53 molecule reduced the free-R and R factor further.
The model was refined through positional and overall B factor
refinement in X-PLOR until the minimization was complete.
The final model has an R factor of 16.3% (Rfree ) 22.8) in the
6 to 2.7 Å resolution range, with rms deviations of 0.012 Å
and 1.83° for the bond lengths and angles, respectively.
Str u ctu r e An a lysis. Visual inspection of the structures
was aided with the programs O25 and INSIGHTII27 on an
INDIGO Silicon Graphics workstation. The stereochemistry
of the models were examined with PROCHECK 2.1.4.28
(13) Hayden, F. G.; Lobo, M.; Treanor, J . J .; Miller, M.; Mills, R. G.
Efficacy and Tolerability of Oral GS 4104 for Early Treatment
of Experimental Influenza in Humans. 37th ICAAC, Late
Breaker Session, Toronto, Ontario, Canada, September, 1997.
(14) Klunder, J . M.; Onami, T.; Sharpless, K. B.; Arenesulfonate
Derivatives of Homo Chiral Glycidol: Versatile Chiral Building
Blocks for Organic Synthesis. J . Org. Chem. 1989, 54, 1295-
1304.
(15) Kim, K. S.; Qian, L. Improved Method for the Preparation of
Guanidines. Tetrahedron Lett. 1993, 34, 7677-7680.
(16) Rich, R. H.; Bartlett, P. A. Synthesis of (-)-2-Fluoroshikimic
Acid. J . Org. Chem. 1996, 61, 3916-3919.
(17) Williams, M. A.; Lew, W.; Mendel, D. B.; Tai, C. Y.; Escarpe, P.
A.; Laver, W. G.; Stevens, R. C.; Kim, C. U. Structure-Activity
Relationships of Carbocyclic Influenza Neuraminidase Inhibi-
tors. Bioorg. Med. Chem. Lett. 1997, 7, 1837-1842.
(18) Belpaire, F. M.; Bogaert, M. G. In The Practice of Medicinal
Chemistry: The Fate of Xenobiotics in Living Organisms;
Wermuth, C. G., Ed.; Academic Press: New York, 1996; pp 596-
600.
(19) Hummer, G.; Garde, S.; Garcia, A. E.; Pohorille, A.; Pratt, L. R.
An Information Theory Model of Hydrophobic Interactions. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 8951-8955.
(20) Smith, P. W.; Sollis, S. L.; Howes, P. D.; Cherry, P. C.; Cobley,
K. N.; Taylor, H.; Whittington, A. R.; Scicinski, J .; Bethell, R.
C.; Taylor, N.; Skarzynski, T.; Clersby, A.; Singh, O.; Wonacott,
A.; Varghese, J .; Colman, P. Novel Inhibitors of Influenza
Sialidases Related to GG167. Bioorg. Med. Chem. Lett. 1997, 7,
1837-1842.
Refer en ces
(1) Colman, P. M. In The Influenza Viruses: Influenza Virus
Neuraminidase, Enzyme and Antigen; Krug, R. M., Ed.; Plenum
Press: New York, 1989; pp 175-218.
(2) Colman, P. M. Influenza Virus Neuraminidase: Structure,
Antibodies and Inhibitors. Protein Sci. 1994, 3, 1687-1696.
(3) Palese, P.; Tobita, K.; Ueda, M.; Compans, R. W. Characteriza-
tion of Temperature Sensitive Influenza Virus Mutants Defective
in Neuraminidase. Virology 1974, 61, 397-410.
(4) Liu, C.; Air, G. M. Selection and Characterization of a Neuramini-
dase-Minus Mutant of Influenza Virus and its Rescue by Cloned
Neuraminidase Genes. Virology 1993, 193, 1-5.
(5) J anakiraman, M. N.; White, C. L.; Laver, W. G.; Air, G. M.; Luo,
M. Structure of Influenza Virus Neuraminidase B/Lee/40 Com-
plexed with Sialic Acid and
a Dehydro Analogue at 1.8-Å
Resolution: Implication for the Catalytic Mechanism. Biochem-
istry 1994, 33, 8172-8179.
(6) Chong, A. K. J .; Pegg, M. S.; Taylor, N. R.; Von Itzstein, M.
Evidence for a Sialosyl Cation Transition-State Complex in the
Reaction of Sialidase from Influenza Virus. Eur. J . Biochem.
1992, 207, 335-343.
(21) Filler, R.; Naqvi, S. M. In Biomedicinal Aspects of Fluorine
Chemistry; Filler, R.; Kobayashi, Y., Ed.; Elsevier Biochemical:
Amsterdam, New York, Oxford, 1982; pp 1-32.
(7) Taylor, N. R.; Von Itzstein, M. Molecular Modeling Studies on
Ligand Binding to Sialidase from Influenza Virus and the
Mechanism of Catalysis. J . Med. Chem. 1994, 37, 616-624.
(8) Von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J .
C.; J in, B.; Phan, T. V.; Smyth, M. L.; White, H. F.; Oliver, S.
W.; Colman, P. M.; Varghese, J . N.; Ryan, D. M.; Woud, J . M.;
Bethell, R. C.; Hotham, V. J .; Cameron, J . M.; Penn, C. R.
Rational Design of Potent Sialidase-Based Inhibitors of Influenza
Virus Replication. Nature (London) 1993, 363, 418-423.
(9) Fromtling, R. A.; Castaner, J . Zanamivir. Drugs Future 1996,
21, 375-382.
(10) Hayden, F. G.; Osterhaus, J . J .; Treanor, D. M.; Fleming, F. Y.;
Aoki, K. G.; Nicholson, A. M.; Bohnen, H. M.; Hirst, H. M.;
Keene, O.; Wightman, K. Efficacy and Safety of the Neuramini-
dase Inhibitor Zanamivir in the Treatment of Influenza Virus
Infections. N. Engl. J . Med. 1997, 337, 874-880.
(11) 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. Influenza Neuramini-
dase Inhibitors Possessing a Novel Hydrophobic Interaction in
the Enzyme Active Site: Design, Synthesis and Structural
Analysis of Carbocyclic Sialic Acid Analogues with Potent Anti-
Influenza Activity. J . Am. Chem. Soc. 1997, 119, 681-690.
(12) Mendel, D. B.; Tai, C. Y.; Escarpe, P. A.; Li, W.; Sidwell, R. W.;
Huffman, J . H.; Sweet, C.; J akeman, K. J .; Merson, J .; Lacy, S.
A.; Lew, W.; Williams, M. A.; Zhang, L.; Chen, M. S.; Bischof-
berger, N.; Kim, C. U. Oral Administration of a Prodrug of the
(22) Potier, M.; Mameli, L.; Belisle, M.; Dallaire, L.; Melancon, S. B.
Fluorometric Assay of Neuraminidase With
a Sodium (4-
methylumbelliferyl-R-D-N-acetylneuraminate) Substrate. Anal.
Biochem. 1979, 94, 287-296.
(23) Laver, W. G.; Colman, P. M.; Webster, R. G.; Hinshaw, V. S.;
Air, G. M.; Influenza Virus Neuraminidase with Hemagglutinin
Activity. Virology 1984, 137(2), 314-323.
(24) White, C. L.; J anakiraman, M. N.; Laver, W. G.; Philippon, C.;
Vasella, A.; Air, G. M.; Luo, M. A Sialic Acid-Derived Phospho-
nate Analogue Inhibits Different Strains of Influenza Virus
Neuraminidase with Different Efficiencies. J . Mol. Biol. 1995,
245, 623-634.
(25) J ones, T. A.; Zou, J . Y.; Cowan, S. W.; Kjeldgaard, M. Improved
Methods for Building Protein Models in Electron Density Maps
and the Location of Errors in These Models. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 1991, A47, 110-119.
(26) Brunger, A. T. The X-PLDR 3.1 Software; Yale University: New
Haven, CT, 1993.
(27) Biosym Technologies, 1992.
(28) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J .
M. PROCHECK: A Program to Check the Stereochemical
Quality of Protein Structures. J . Appl. Crystallogr. 1993, 26,
283-291.
J M980162U