Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent

Article abstract of DOI:10.1021/jm980707k

On the basis of the lead compound 4-(N-acetylamino)-3-guanidinobenzoic acid (BANA 113), which inhibits influenza A sialidase with a K(i) of 2.5 μM, several novel aromatic inhibitors of influenza sialidases were designed. In this study the N-acetyl group of BANA 113 was replaced with a 2- pyrrolidinone ring, which was designed in part to offer opportunities for introduction of spatially directed side chains that could potentially interact with the 4-, 5-, and/or 6-subsites of sialidase. While the parent structure 1-(4-carboxy-2-guanidinophenyl)pyrrolidin-2-one (8) was only a modest inhibitor of sialidase, the introduction of a hydroxymethyl or bis(hydroxymethyl) substituent at the C5' position of the 2-pyrrolidinone ring resulted in inhibitors (9 and 12, respectively) with low micromolar activity. Crystal structures of these inhibitors in complex with sialidase demonstrated that the substituents at the 5'-position of the 2-pyrrolidinone ring interact in the 4- and/or 5-subsites of the enzyme. Replacement of the guanidine in 12 with a hydrophobic 3-pentylamino group resulted in a large enhancement in binding to produce an inhibitor (14) with an IC₅₀ of about 50 nM against influenza A sialidase, although the inhibition of influenza B sialidase was 2000-fold less. This represents the first reported example of a simple, achiral benzoic acid with potent (low nanomolar) activity as an inhibitor of influenza sialidase.

Full text of DOI:10.1021/jm980707k

2332
J . Med. Chem. 1999, 42, 2332-2343
P oten t In h ibition of In flu en za Sia lid a se by a Ben zoic Acid Con ta in in g a
2-P yr r olid in on e Su bstitu en t
Venkatram R. Atigadda,† Wayne J . Brouillette,*,†,‡ Franco Duarte,† Shoukath M. Ali,† Yarlagadda S. Babu,§
Shanta Bantia,§ Pooran Chand,§ Naiming Chu,§ J ohn A. Montgomery,§ David A. Walsh,§ Elise A. Sudbeck,‡
J ames Finley,‡ Ming Luo,‡ Gillian M. Air, and Graeme W. Laver|
Department of Chemistry and Center for Macromolecular Crystallography, University of Alabama at Birmingham,
Birmingham, Alabama 35294, BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive, Birmingham, Alabama 35244,
Department of Biochemistry and Molecular Biology, University of Oklahoma, Oklahoma City, Oklahoma 73190,
and J ohn Curtin School of Medical Research, Australian National University, Canberra 2601, Australia
Received December 16, 1998
On the basis of the lead compound 4-(N-acetylamino)-3-guanidinobenzoic acid (BANA 113),
which inhibits influenza A sialidase with a Ki of 2.5 µM, several novel aromatic inhibitors of
influenza sialidases were designed. In this study the N-acetyl group of BANA 113 was replaced
with a 2-pyrrolidinone ring, which was designed in part to offer opportunities for introduction
of spatially directed side chains that could potentially interact with the 4-, 5-, and/or 6-subsites
of sialidase. While the parent structure 1-(4-carboxy-2-guanidinophenyl)pyrrolidin-2-one (8)
was only a modest inhibitor of sialidase, the introduction of a hydroxymethyl or bis-
(hydroxymethyl) substituent at the C5′ position of the 2-pyrrolidinone ring resulted in inhibitors
(9 and 12, respectively) with low micromolar activity. Crystal structures of these inhibitors in
complex with sialidase demonstrated that the substituents at the 5′-position of the 2-pyrroli-
dinone ring interact in the 4- and/or 5-subsites of the enzyme. Replacement of the guanidine
in 12 with a hydrophobic 3-pentylamino group resulted in a large enhancement in binding to
produce an inhibitor (14) with an IC50 of about 50 nM against influenza A sialidase, although
the inhibition of influenza B sialidase was 2000-fold less. This represents the first reported
example of a simple, achiral benzoic acid with potent (low nanomolar) activity as an inhibitor
of influenza sialidase.
In tr od u ction
process is mediated by HA. After viral replication, the
progeny virions must be released from the cell to repeat
the cell cycle of infection. Influenza neuraminidase
mediates the release of virus from the cell surface by
cleavage of R-glycosidic bonds to the cell surface sialic
acids.1-3 This prevents viral aggregation and allows
viral release from infected cells. This action of NA may
also facilitate viral mobility through the mucus of the
respiratory tract.
Influenza is a major respiratory tract disease affecting
millions of people each year. Despite considerable
knowledge of viral infectivity, current therapeutic mea-
sures do not control the disease. Vaccination has
provided only limited control because of the ease with
which the virus can mutate to escape the immune
system, and the vaccines must be reformulated each
year because of this high antigenic drift. Options for the
therapeutic treatment of influenza are currently limited
to amantadine and rimantadine, which act by interfer-
ing with the M2 protein ion channel function that is
found only in influenza A. Besides the insensitivity of
influenza B viruses, clinical use of these agents is also
limited because of the rapid emergence of resistance.
New influenza drugs with broad-spectrum activity are
needed.
Influenza is an RNA virus that contains two major
surface glycoproteins, neuraminidase (NA), or sialidase,
and hemagglutinin (HA). These proteins are essential
for infection. The virus infects the epithelial cells of the
upper respiratory tract by binding to the cell surface
receptor sialic acid, and it subsequently undergoes
endocytosis into the cell. This binding and endocytosis
The life cycle of the influenza virus provides several
targets for drug development, and neuraminidase offers
one attractive site for therapeutic intervention in in-
fluenza infections. For influenza A, nine subtypes of
neuraminidase have been identified, whereas only one
subtype is known for influenza B. Despite this diversity,
the catalytic site for all influenza A and B neuramini-
dases is completely conserved, even though overall
amino acid sequence identities may be only 30%.4-6 The
neuraminidase is a tetramer with C-4 symmetry and
has an approximate molecular weight of 250 000. It
consists of a globular head anchored to the membrane
through a central stalk. Each of the four subunits of the
head is a glycosylated polypeptide and contains a
catalytic site. Proteolytic cleavage of the stalk has
produced catalytically and antigenically active heads.1
Heads from several viruses have been crystallized and
the X-ray crystal structures determined.5 These struc-
tures reveal that the catalytic site is strain-invariant
and contains 18 conserved amino acid residues. Muta-
tion of these conserved amino acid residues in the active
* Corresponding author.
† Department of Chemistry, University of Alabama.
‡ Center for Macromolecular Crystallography, University of Ala-
bama.
§ BioCryst Pharmaceuticals, Inc.
University of Oklahoma.
| Australian National University.
10.1021/jm980707k CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/04/1999

Potent Benzoic Acid Inhibitor of Influenza Sialidase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2333
Ch a r t 1
F igu r e 1. Depiction of the interactions of 2c (GG167) in the
NA binding site (N9 numbering). Note that Tyr 406 lies under
2c and is not shown.
site inactivates the enzyme, suggesting that the virus
may not easily escape therapeutic intervention through
mutation.1
Most of the known inhibitors of neuraminidases are
analogues of sialic acid (1) (Neu5Ac) (Chart 1), a weak
inhibitor (IC50 ) 10 mM) which is the product of the
enzyme reaction. Over 25 years ago the dehydrated
analogue, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid
(Neu5Ac2en) (2a ), was reported to be a much more
effective inhibitor (IC50 ) 10 µM) than sialic acid.7,8 In
recent years several potent inhibitors of neuraminidase
have been developed based on the insights provided by
its X-ray crystal structure in complex with 2a .4,5,9
Compound 2c, a guanidino analogue of 2a that is a
potent inhibitor of both influenza A and B NA, is cur-
rently being evaluated in clinical trials and is effective
in both the prevention and the treatment of influenza
when administered intranasally.10 However compound
2c is inactive when administered orally as a result of
poor oral bioavailability and rapid elimination.10,11 More
recently, analogues of 2c containing hydrophobic groups
in place of the glycerol side chain, such as carboxamides
3a and 3b, have been described.12-14 While 3a and 3b
are highly selective inhibitors of influenza A NA,
cyclohexene 4 was reported14 to exhibit potent activity
against both influenza A and B, and the ethyl ester of
4 is orally active.
For convenience we have divided the catalytic site of
NA into four subsites named according to the atom
number of the pyran ring in sialic acid that bears
interacting substituents (Figure 1). Using this descrip-
tion for 2c, the carboxylic acid at C2 interacts in subsite
2, and this interaction is dominated by three arginine
residues (Arg 119, Arg 372, Arg 294). The guanidine
interacts in subsite 4 with Asp 152 and Glu 229.5f
Subsite 5 accommodates the N-acetyl methyl group; this
contains a small hydrophobic pocket created by Trp 180
and Ile 224 into which extends the methyl group, the
amide carbonyl accepts a H-bond from Arg 153, and the
N-H donates a H-bond to an ordered water molecule.
Finally, the C7 and C8 glycerol hydroxyl groups interact
with Glu 278 within subsite 6.
F igu r e 2. Depiction of the interactions of 5 (BANA 113) in
the binding site (N9 numbering). Note that Tyr 406 lies under
5 and is not shown.
In an effort to develop new classes of inhibitors using
structure-based drug design, we have described aro-
matic lead compounds as neuraminidase inhibitors.15,16
The most potent of these early inhibitors was compound
5 (BANA 113), which has a Ki value of 2.5 µM.
Compound 5 binds to the NA enzyme in a unique
manner in relation to 2c (Figure 2). Comparison of the
X-ray crystal structures of the NA complex with 5 and
2c revealed that the guanidino functionalities interact
in a completely different manner, while the carboxylate
and N-acetyl groups occupy the same subsites. In
compound 2c, the C8 and C9 hydroxyls of the glycerol
moiety occupy subsite 6, making hydrogen bonds to Glu
278, whereas the guanidino functionality occupies sub-
site 4 and interacts with Asp 152 and Glu 229 through
salt bridges.5f However, in the case of inhibitor 5, which
lacks the glycerol moiety, the guanidine functionality
occupies subsite 6 (the glycerol subsite of 2c) instead of
subsite 4 (guanidine subsite of 2c) and forms a salt
bridge with Glu 278. In compound 5 the guanidine has
the opportunity to occupy either subsite 4 or subsite 6,
requiring only a 180° rotation of the benzene ring to

2334 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Atigadda et al.
Sch em e 1a
interconvert these orientations, suggesting more favor-
able interactions with subsite 6.
Given the large enhancement in potency in going from
2a to 2c, we decided to incorporate a second guanidino
grouping in benzoic acid 5, to generate 6, to force a
guanidino group into subsite 4.17 Indeed, the crystal
structure of the complex for 6/NA revealed that the
second guanidino grouping occupied the 4-subsite in a
manner similar to that for 2c, although the inhibitory
activity for 6 (IC50 ) 250 µM) was much worse than that
for 5 (Ki ) 2.5 µM). Also, incorporation by others of a
glycerol side chain at C5, as in 7, resulted in the loss of
inhibitory activity.18 Several factors may be responsible
for the weaker activity of 6. (1) The desolvation energy
for a second guanidinium group might be much greater
than anticipated. (2) The first atom of substituents
attached to the benzene ring must be coplanar with that
ring, resulting in steric congestion of 3,4,5-trisubstituted
benzoic acids. These steric interactions may result in
an energetically unfavorable conformation for the guani-
dine of bound 6.
We next investigated the possibility of replacing the
N-acetyl grouping with a substituent containing side
chains for interaction with the 4-subsite. Here we report
that a properly substituted 2-pyrrolidinone as replace-
ment for the N-acetyl grouping on benzoic acid, when
coupled with a hydrophobic substituent, provides a
simple, achiral compound that is a low nanomolar
inhibitor of NA.
a
Reagents: (a) ethyl 4-bromobutyrate, DMF; (b) methanol, H+;
(c) HNO3, Ac2O, dioxane or NO2BF4, CH2Cl2; (d) H2, Pd/C,
methanol; (e) N,N-bis(tert-butoxycarbonyl)thiourea, HgCl2, NEt3,
DMF; (f) HCl; (g) NaH, HMPA.
Sch em e 2a
Ch em istr y
The first goal for modifying 5 was to replace the
N-acetyl grouping with a compact, cyclic substituent
that would offer opportunities for incorporation of
spatially directed side chains that could potentially
interact with the 4-, 5-, and/or 6-subsites. Computer
modeling studies suggested the 2-pyrrolidinone ring for
this purpose,27 and compound 8 was selected as the
initial target. The synthesis of 8 is shown in Scheme 1
and began with the alkylation of 4-aminobenzoic acid
(15) with ethyl 4-bromobutyrate, accompanied by cy-
clization, in a one-pot procedure in refluxing DMF, to
give pyrrolidinone 16 in 66% yield. The use of a base
such as triethylamine in this reaction resulted primarily
in esterification of the carboxylic acid. Also, attempts
to alkylate methyl 4-aminobenzoate in a similar fashion
resulted in much poorer yields of 17. Esterification of
the acid 16 followed by nitration using either HNO3/
Ac2O or NO2BF4 resulted in the desired regioisomer 18
in 90% overall yield. Later, 18 was also synthesized in
two steps by nucleophilic displacement of commercially
available 4-fluoro-3-nitrobenzoic acid (21) with 2-pyr-
rolidinone (22), in the presence of NaH, followed by
esterification. Catalytic reduction of the nitro group
followed by reaction of the amine 19 with N,N′-bis(tert-
butoxycarbonyl)thiourea in the presence of mercury
chloride provided the guanylated intermediate 20.19
Removal of the methyl ester and the tert-butoxycarbonyl
groups was achieved in one step by refluxing in 1 N HCl
to provide the target 8.
a
Reagents: (a) NaH, HMPA; (b) ethanol, H+; (c) NaBH4,
ethanol, THF; (d) H2, Pd/C, ethanol; (e) 1,3-bis(tert-butoxycarbo-
nyl)-2-methyl-2-thiopseudourea, HgCl2, NEt3, DMF; (f) CF3CO2H;
(g) NaOH.
between intermediates 23 and 25 suggested that, as a
first approach, compound 9 could be synthesized by
coupling 21 with commercially available 5-(hydroxym-
ethyl)-1-pyrrolidinone. However, coupling attempts us-
ing the dianion resulted in alkylation of the alcohol.
Therefore, pyrrolidinone carboxylic acid 24 was directly
coupled with 21 in the presence of NaH to give the
diacid 25 in 50% yield. Fischer esterification of 25 to
give diester 26 followed by selective reduction of the
aliphatic ester with NaBH4 resulted in the alcohol 27
in 50% yield. Catalytic reduction of the nitro group
provided amine 28 which reacted with N-N′-bis(tert-
butoxycarbonyl)thiourea to yield the guanylated inter-
mediate 29. Acidic removal of the tert-butoxycarbonyl
groups followed by saponification gave the target 9.
Compounds 9 and 10 were designed as analogues of
8 that contain a side chain on the pyrrolidinone ring
capable of interacting with the 4- or 5-subsite. The
preparation of 9 is described in Scheme 2. Similarities

Potent Benzoic Acid Inhibitor of Influenza Sialidase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2335
Sch em e 3a
Sch em e 5a
a
Reagents: (a) NaN3, DMF; (b) NaH, 3-nitro-4-fluorobenzoic
acid, DMF; (c) methanol, H+; (d) H2, Pd/C, di-tert-butyl dicarbon-
ate, ethyl acetate; (e) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-
thiopseudourea, HgCl2, NEt3, DMF; (f) HCl.
a
Reagents: (a) 3-pentanone, TiCl4, NEt3, CH2Cl2; (b) NaOH;
(c) 3-pentanone, NaCNBH4, acetic acid, dichloroethane.
mopropionic acid and PCl3 to provide 39.20 This under-
went cyclization in the presence of NaH to yield lactam
40 in 80% overall yield. The esters of 40 were reduced
to the bis(hydroxymethyl) derivative 41, which under-
went saponification to provide target 11. Initial at-
tempts to nitrate 40 using HNO3/Ac2O resulted in poor
yields; however, subsequent nitration with nitronium
tetrafluoroborate or HNO3/H2SO4 proceeded in greater
than 90% yield to give 42. Reduction of the nitro
functionality in intermediate 42 to the amine using
catalytic hydrogenation gave only lactam as a result of
intramolecular cyclization. Therefore, the aliphatic es-
ters in 42 were first selectively reduced to the alcohols
using NaBH4 to give 43. Reduction of the nitro group
then gave amine 44, which reacted with S-methylben-
zyloxycarbonylthiourea in the presence of mercuric
chloride to provide the guanylated intermediate 45. The
saponification of 45 provided 12.
Sch em e 4a
Initial attempts to introduce lipophilic side chains by
reaction of the amine 19 or 44 with alkyl halides failed
to provide the desired compounds. However, as shown
in Scheme 5, reductive coupling of the amine 19 with
3-pentanone in the presence of NaCNBH3 and TiCl4
followed by saponification resulted in 13 in good yields.21
Unfortunately, the reductive coupling of 44 with 3-pen-
tanone under identical conditions promoted the in-
tramolecular cyclization of the amine with the pyrroli-
dinone carbonyl to give an undesired benzimidazole
product. But the reductive coupling of amine 44 with
3-pentanone proceeded smoothly in the presence of
NaCNBH3 and acetic acid to give the alkylamine 47.
Saponification of the methyl ester provided the target
molecule 14.
a
Reagents: (a) diethyl bromomalonate; (b) 3-bromopropionic
acid, PCl3, benzene; (c) NaH, DMF; (d) NaBH4, methanol, THF;
(e) NaOH; (f) NO2BF4, CH2Cl2; (g) H2, Pd/C, methanol; (h) 1,3-
bis(benzyloxycarbonyl)-S-methylisothiourea, HgCl2, NEt3, DMF.
Compound 10 was prepared in an analogous method
as shown in Scheme 3. The coupling of 21 with 5-(azi-
domethyl)-2-pyrrolidinone (32), prepared from mesylate
31, gave intermediate 33. Fischer esterification to give
34 followed by catalytic reduction using H2/Pd-C in the
presence of di-tert-butyl dicarbonate resulted in the
reduction of both the nitro and the azide accompanied
by protection of the aliphatic amine as the t-Boc car-
bamate to give 35 in quantitative yield. Reaction of the
amine 35 with N,N′-bis(tert-butoxycarbonyl)thiourea
resulted in the guanylated intermediate 36. Acidic
hydrolysis of the methyl ester and the tert-butoxycar-
bonyl groups provided compound 10.
Biologica l Assa ys
All compounds were evaluated for in vitro inhibitory
activity using purified NA from the H1N9 (influenza A)
and B/Lee/40 strain. The assay was based upon a
published method.22 The substrate employed was 2′-(4-
methylumbelliferyl)-R-D-N-acetylneuraminic acid (MUN),
whose hydrolysis provides a fluorescent product used
for quantitation.
The synthesis of bis(hydroxymethyl)lactam 12 was
accomplished as summarized in Scheme 4. Methyl
4-aminobenzoate (37) was reacted with diethyl bromo-
malonate to give 38, which was acylated with 3-bro-
X-r a y Cr ysta llogr a p h y
Purified N9 neuraminidase was crystallized using
the hanging drop method, and the resulting crystals

2336 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Atigadda et al.
Ta ble 1. In Vitro Inhibitory Effects of Benzoic Acid Analogues
on Influenza A and B Neuraminidases
cated 11 (IC50 ) 0.8 mM) is considerably more active
than 16 (10% inhibition at 1 mM). In view of these
results, compound 12 was synthesized and evaluated
in vitro (the results are summarized in Table 1). The
addition of the guanidino grouping in 12 resulted in a
100-fold increase in activity over 11. However, in
comparison to 9 only a modest improvement (2-fold) in
activity was achieved by this additional interaction. The
crystal structure of the complex was determined by
soaking compound 12 into N9 crystals (see Figure 3c).
It clearly demonstrates that the additional hydroxym-
ethyl group hydrogen bonds to an ordered water mol-
ecule located in the 5-subsite.
IC50 (µM)a
compd
N9
250
20
2600
760
5
B/Lee/40
8
9d
1000b
10
500
10d
11
12
13
14
>8000
8
222
0.048
>5000
104c
a
IC50 values are the mean of duplicate experiments. In all cases
each IC50 value differed from its duplicate by less than 2-fold.
b
d
Data for B/Mem/89. c Value for B/Mem/89 was 94 µM. Tested
as racemic mixtures.
As described earlier, one of the most potent inhibitors
of neuraminidase is 4-guanidino-Neu5Ac2en (2c). More
recent studies demonstrated that the replacement of the
glycerol functionality with lipophilic side chains, as in
3b, resulted in potent inhibitors selective for influenza
A. Compound 4, which is a carbocyclic analogue of 3a ,
inhibits influenza A NA with an IC50 of 1 nM, and it is
nearly as effective for influenza B. The ethyl ester of
inhibitor 4, which is orally active, is currently in clinical
evaluation. Since the guanidino functionality of 12
occupies the same subsite as the glycerol of inhibitor
2c, it was anticipated that replacement of the guanidine
in inhibitor 12 with lipophilic groups would result in
effective inhibitors.
diffracted strongly to at least 2 Å resolution. Inhibitor
complexes with N9 neuraminidase were prepared by
soaking the crystal in a 1-2 mM solution of the inhibitor
in stabilization buffer for at least 24 h.
Resu lts a n d Discu ssion
As shown in Table 1 pyrrolidinone 8, not surprisingly,
was 10-15-fold less active than its N-acetyl counterpart
5, which is consistent with the loss of the hydrogen-
bonding interaction of the acetamido group with the
ordered water molecule in the 5-subsite. To determine
molecular interactions of inhibitor 8, it was soaked into
an N9 crystal of NA, and the structure of the complex
was determined (see Figure 3a). In a manner similar
to 5, the carboxylic acid interacts with Arg 119, Arg 294,
and Arg 372, and the 3-guanidine forms a salt bridge
interaction in subsite 6 with Glu 278. The pyrrolidinone
ring occupies subsite 5 and snugly fits into the hydro-
phobic pocket created by Trp 180 and Ile 224. This ring
is oriented roughly perpendicular to the planar benzene
ring, and the carbonyl oxygen is located within hydrogen-
bonding distance of Arg 153.
Modeling suggested that a hydrogen bond-donating
side chain, such as hydroxymethyl or aminomethyl, at
the 5′-position of the pyrrolidinone ring in 8 could reach
the 4-subsite and replace an ordered water, which may
enhance binding. To evaluate this hypothesis 9 and 10
were synthesized. As shown in Table 1 inhibitor 9 was
10 times more effective than the parent compound 8.
However, inhibitor 10 was slightly less active than 8.
To explore structural reasons for the binding differences
between these two compounds, 9 and 10 were soaked
into N9 crystals and the complex structures were
determined. As shown in Figure 3b, these revealed that
9 and 10 interact with the active site of NA in identical
fashion. The carboxylate and the guanidine interactions
of 9 and 10 with NA are the same as those described
for the parent compound 8, and the hydroxymethyl and
aminomethyl groups each replace an ordered water
molecule located in the 4-subsite. Although the crystal
structures of the complexes do not explain why com-
pound 10 is less active than 9, a contribution could be
the extra energy cost required for desolvation of the
charged ammonium group in 10 as compared to the
uncharged hydroxy group in 9.
To evaluate this proposal compounds 13 and 14,
which contain a hydrophobic side chain, were synthe-
sized. As shown in Table 1 compound 13 was a relatively
modest inhibitor of NA, while 14 exhibited a dramatic
improvement in activity against influenza A but had
little effect on activity against influenza B. Compound
14 was 100-fold more active than its guanidino coun-
terpart, analogue 12, and over 15 000-fold more active
than 11, which implies an energetically significant
hydrophobic interaction of the N-3-pentyl group within
the active site. Compound 14 was soaked into an N9
crystal, and the complex structure was determined (see
Figure 3d). Inhibitor 14 binds to influenza A neuramini-
dase in much the same manner as observed for 12.
However, significant differences in the binding of these
two compounds were observed for the 6-subsite, where
the guanidino group and the pentyl group undergo
interaction. For 12 the guanidino group makes a biden-
tate hydrogen-bonding interaction with Glu 278. For 14
these interactions are not possible for the pentyl group,
and the Glu 278 side chain adopts a different conforma-
tion to make an intramolecular salt bridge with Arg 226.
This interaction creates a small hydrophobic pocket into
which one branch of the pentyl side chain binds and the
other branch binds in the preexisting, larger hydropho-
bic pocket created by Ala 246 and Ile 224. The entropy
gains as a result of these hydrophobic interactions are
likely to be the major contribution for the enhanced NA
inhibitory activity (compound 14 is 15 000-fold more
active than 11 for the inhibition of influenza A NA).
Despite the essentially identical location of amino acid
side chains for the conserved amino acids that form the
first amino acid shell in influenza A and B sialidases,
compound 14 selectively inhibits (more than 2000-fold)
sialidase A. This result is consistent with earlier re-
ports12,13,24,25 on a class of sialic acid derivatives con-
taining a hydrophobic carboxamide which also oriented
the hydrophobic groups in the 6-subsite of neuramini-
Further modeling suggested that an additional hy-
droxymethyl substituent at the C5′ position of inhibitor
9 would be able to interact with, or possibly replace, a
water molecule located in the 5-subsite. To evaluate this
hypothesis compound 11, a simple model, was synthe-
sized and evaluated for its biological activity. As indi-

Potent Benzoic Acid Inhibitor of Influenza Sialidase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2337
F igu r e 3. X-ray crystal structures of the active site region for complexes formed between selected pyrrolidinone benzoic acids
and N9 neuraminidase: (a) compound 8 (green) and, for comparison, the lead compound 5 (BANA 113; orange); (b) compound 9
(green) and the analogous aminomethyl compound 10 (orange); (c) bis(hydroxymethyl) compound 12; (d) bis(hydroxymethyl)
compound 14.
dase. For example, structure-activity relationship stud-
ies for carboxamide 3a revealed that these compounds
exhibit a magnitude of selectivity for influenza A over
influenza B25similar to that observed for 14. The crystal
structures of 3c with both influenza A and B sialidases
were determined in order to investigate structural
reasons for the selectivity. In the complex of 3c with
both influenza A and B sialidases, the movement of Glu
278 (Glu 276 in influenza B) was observed to form a
salt bridge with Arg 226 (as also observed for 14 in
complex with influenza A NA). In influenza A sialidase,
this salt bridge formation resulted in minimal disrup-
tion of the surrounding protein structure (second amino
acid shell). However, in influenza B sialidase the
movement of Glu 278 (type A numbering) to form the
new salt bridge produced distortions in the protein
backbone near Glu 278 and in the second amino acid
shell, since this region contains differences among
nonconserved amino acids as compared to type A. Thus
salt bridge formation in influenza B sialidase, in com-
parison to influenza A, is believed to be associated with
additional energy penalties, resulting in a much poorer
inhibition constant. Similar selectivity for influenza A
neuraminidase has also been observed for selected
examples of potent hydrophobic inhibitors based on a
cyclohexene template,26 although structural reasons for
the selectivity were not presented. We anticipate that
the factors discussed above account for the type A
selectivity observed for compound 14.
In summary, we have developed a highly novel, low
nanomolar inhibitor of influenza A neuraminidase. This
compound is unique among known potent inhibitors of
influenza neuraminidase for several reasons. (1) Com-
pound 14 is the first reported benzoic acid with low
nanomolar inhibition. (2) It is the first achiral molecule
exhibiting low nanomolar inhibition to be reported from

2338 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Atigadda et al.
This solution was dried (Na2SO4) and concentrated to give a
yellow oil. The product was purified by flash chromatography
on silica gel (10% ethanol/ether) to give 18 (800 mg, 67.0%).
any structural class. (3) It is the first low nanomolar
inhibitor that does not contain a basic side chain (amino
or guanidino). The high selectivity of 14 for type A over
type B neuraminidase is consistent with the higher
energy cost, in type B NA, associated with the move-
ment of Glu 278 to form a new salt bridge in the
6-subsite.
1-(4-Met h oxyca r b on yl-2-a m in op h en yl)p yr r olid in -2-
on e (19). A mixture of 18 (1.00 g, 3.78 mmol) and Pd/C (500
mg) in 50 mL of methanol was shaken on a Parr hydrogenator
at 30 psi for 1 h. The reaction mixture was diluted with 50
mL of methanol, and the Pd/C was removed by filtration. The
filtrate was evaporated under reduced pressure to give 19 (840
mg, 95.0%) as a white solid: mp 136-138 °C; MS (electrospray)
235 (M + 1); 1H NMR (300 MHz, CDCl3) δ 7.49 (d, 1H, J ) 1.8
Hz), 7.47 (dd, 1H, J ) 1.8 & 8.1 Hz), 7.1 (d, 1H, J ) 8.1 Hz),
4.1 (br, 2H), 3.89 (s, 3H), 3.84 (t, 2H, J ) 7.0 Hz), 2.6 (t, 2H,
J ) 7.9 Hz), 2.2 (m, 2H); 13C NMR (CD3OD) δ 177.49, 168.33,
145.57, 131.27, 129.81, 128.12, 119.67, 118.97, 52.79, 51.12,
32.42, 19.96. Anal. (C12H14N2O3) C, H, N.
Exp er im en ta l Section
Melting points were obtained on an Electrothermal melting
point apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded on a Bruker ARX 300 spectrometer unless
otherwise noted. Infrared spectra were recorded on a Bruker
infrared spectrophotometer. Combustion analyses were pro-
vided by Atlantic Microlabs of Atlanta, GA. Mass spectra were
obtained on a Perkin-Elmer SCIEX API triple-quadrupole
mass spectrometer using electrospray ionization. Analytical
chromatography was performed on Whatman PE Sil G/UV
silica gel plates (250 µm). Flash chromatography was per-
formed using J .T. Baker silica gel (40 µm). Tetrahydrofuran
was distilled from sodium metal/benzophenone ketyl. Dichlo-
romethane and benzene were distilled from calcium hydride.
Anhydrous N,N′-dimethylformamide was purchased from Al-
drich in Sure-Seal containers. Methanol was distilled from Mg-
(OMe)2. All other commercially obtained reagents were used
as received.
1-[4-Meth oxyca r bon yl-2-(N,N′-bis(ter t-bu tyloxyca r bo-
n yl)gu a n id in o)p h en yl]p yr r olid in -2-on e (20). An ice-cold
mixture of 19 (75 mg, 0.28 mmol) in 1 mL of dry DMF was
treated with N,N′-bis(tert-butoxycarbonyl)thiourea (95 mg,
0.34 mmol), triethylamine (116 mg, 1.14 mmol), and HgCl2
(100 mg, 0.36 mmol). The resulting mixture was stirred for 4
h at 0-10 °C. DMF was removed under high vacuum, and the
crude mixture was purified by flash chromatography on silica
gel (ether) to give 20 (95 mg, 72.0%) as an oil which solidified
upon addition of hexane: mp 163-164 °C (ether); MS (elec-
1
trospray) 477 (M + 1); H NMR (300 MHz, CDCl3) δ 11.5 (s,
1H), 10.41 (s, 1H), 8.9 (d, 1H, J ) 1.8 Hz), 7.8 (dd, 1H, J ) 1.8
& 8.3 Hz), 7.2 (d, 1H, J ) 8.3 Hz), 3.91 (s, 3H), 3.75 (t, 2H, J
) 6.9 Hz), 2.59 (t, 2H, J ) 8.0 Hz), 2.23 (m, 2H), 1.5 (s, 9H),
1.4 (s, 9H); 13C NMR (CDCl3) δ 175.14, 166.21, 163,25, 153.5,
153.09, 134.64, 133.78, 129.86, 126.45, 126.33, 126.26, 83.85,
79.71, 77.64, 77.21, 76.79, 52.22, 49.86, 31.05, 28.11, 28.06,
19.06. Anal. (C23H32N4O7) C, H, N.
1-(4-Ca r boxy-2-gu a n id in op h en yl)p yr r olid in -2-on e (8).
P r oced u r e A: A suspension of 20 (25 mg, 0.050 mmol) in 1.5
mL of 1 N HCl was stirred at reflux for 3.5 h. The solvent was
removed under vaccum to give an oily material which solidified
upon the addition of ether to give 8 (14 mg, 92.0%) as a white
hygroscopic solid: 1H NMR (300 MHz, DMSO-d6) δ 9.1 (s, 1H),
7.9 (dd, 1H, J ) 1.8 & 8.2 Hz), 7.8 (d, 1H, J ) 1.8 Hz), 7.6 (d,
1H, J ) 8.2 Hz), 7.5 (br, 4H), 3.8 (t, 2H, J ) 7.0 Hz), 2.5 (t,
2H, J ) 8.0 Hz), 2.15 (m, 2H).
P r oced u r e B: A solution of 20 (200 mg, 0.420 mmol) in
0.5 mL of CH2Cl2 was treated with 0.5 mL of trifluoroacetic
acid. The mixture was stirred at room temperature for 12 h
and concentrated to dryness on high vacuum to give a colorless
oil which was dissolved in 2 mL of 1 N NaOH solution. The
mixture was stirred at room temperature for 14 h. The pH of
the mixture was adjusted to 6 with careful addition of glacial
acid and cooled to give a white precipitate which was filtered
and dried to give 8 (95 mg, 86.0%): mp 242-243 °C; MS
(electrospray) 263 (M + 1); 1H NMR (300 MHz, CD3OD) δ 8.00
(dd, 1H, J ) 1.8 & 8.3 Hz), 7.93 (d, 1H, J ) 1.8 Hz), 7.48 (d,
1H, J ) 8.3 Hz), 3.95 (t, 2H, J ) 7.0 Hz), 2.61 (t, 2H, J ) 7.9
Hz), 2.30 (m, 2H). Anal. (C12H14N4O3‚H2O) C, H, N.
1-(4-Ca r boxyp h en yl)p yr r olid in -2-on e (16). A solution of
15 (3.00 g, 21.9 mmol) and ethyl 4-bromobutyrate (6.40 g, 32.8
mmol) in 20 mL of DMF was stirred at reflux for 24 h. The
DMF was removed under vacuum to give a crude solid which
was suspended in 15 mL of water, filtered, and dried. The solid
was suspended in a small amount of ether (5 mL), filtered,
and dried to give 16 (3.00 g, 66.0%) as a white solid: mp 244-
246 °C (ether/ethanol); IR (KBr) 2924-2500, 1709, 1648, 1603
cm-1; MS (electrospray) 204 (M - 1); 1H NMR (300 MHz,
CDCl3) δ 8.1 (d, 2H, J ) 8.9 Hz), 7.7 (d, 2H, J ) 8.9 Hz), 3.9
(t, 2H, J ) 7.0 Hz), 2.6 (t, 2H, J ) 8.0 Hz), 2.2 (m, 2H); 13C
NMR (CDCl3) δ 173.61, 166.77, 142.30, 129.54, 129.44, 125.25,
117.63, 47.49, 31.87, 16.79. Anal. (C11H11NO3) C, H, N.
1-(4-Meth oxyca r bon ylp h en yl)p yr r olid in -2-on e (17). A
mixture of 16 (3.00 g, 14.6 mmol) and H2SO4 (0.2 mL) in 100
mL of methanol was stirred at reflux for 18 h. The reaction
mixture was concentrated to dryness, and the crude residue
was dissolved in 100 mL of ethyl acetate. The organic layer
was washed with saturated NaHCO3 (3 × 15 mL) and water
(10 mL), dried (Na2SO4), and evaporated to give 17 (3.00 g,
94.0%) as a white solid: mp 118-120 °C (chloroform/ether);
1
MS (electrospray) 252 (M + 1); H NMR (300 MHz, CDCl3) δ
8.03 (d, 2H, J ) 8.9 Hz), 7.73 (d, 2H, J ) 8.8 Hz), 3.91 (s, 3H),
3.89 (t, 2H, J ) 7.1 Hz), 2.62 (t, 2H, J ) 8.0 Hz), 2.15 (m, 2H);
13C NMR (CDCl3) δ 174.54, 166.49, 143.29, 130.32, 125.29,
118.46, 51.90, 48.34, 32.76, 17.71. Anal. (C12H13NO3) C, H, N.
1-(4-Me t h oxyca r b on yl-2-n it r op h e n yl)p yr r olid in -2-
on e (18). P r oced u r e A: An ice-cold mixture of 17 (2.00 g,
9.10 mmol) in 30 mL of CH2Cl2 was treated with NO2BF4 (2.50
g, 18.8 mmol). The resulting mixture was stirred at 10 °C for
2 h and then at room temperature for 4 h. The reaction mixture
was quenched with water (20 mL), and the organic layer was
evaporated under vacuum. The white solid which separated
was filtered, washed with water, and dried to give 18 (2.20 g,
92.0%): mp 159-161 °C (chloroform/ether); MS (electrospray)
265 (M + 1); 1H NMR (300 MHz, CDCl3) δ 8.5 (d, 1H, J ) 1.9
Hz), 8.25 (dd, 1H, J ) 1.9 & 8.3 Hz), 7.4 (d, 1H, J ) 8.3 Hz),
3.96 (s, 3H), 3.95 (t, 2H, J ) 6.8 Hz), 2.5 (t, 2H, J ) 7.9 Hz),
2.3 (m, 2H); 13C NMR (CD3OD/DMSO) δ 174.62, 164.73,
144.64, 135.73, 134.31, 127.86, 126.11, 125.68, 52.89, 49.35,
31.34, 18.95. Anal. (C12H12N2O5) C, H, N.
1-(4-Ca r b oxy-2-n it r op h en yl)-5-ca r b oxyp yr r olid in -2-
on e (25). A suspension of 60% NaH (1.14 g, 28.4 mmol) in
HMPA (3 mL) was treated with a solution of 24 (2.09 g, 16.2
mmol) in HMPA (12 mL). The reaction mixture was heated at
90-95 °C for 15 min and cooled to 45 °C, and then fluoroni-
trobenzoic acid 21 (1.50 g, 8.10 mmol) in HMPA (8 mL) was
added. The reaction mixture was stirred at 160 °C for 23 h.
Excess NaH was quenched with cold 1 N HCl (30 mL). The
aqueous layer was extracted with ethyl acetate (3 × 60 mL).
The combined organic layers were washed with cold water (2
× 20 mL), saturated sodium chloride (2 × 20 mL), and water
(2 × 20 mL) and dried (Na2SO4), and the solvent was removed
in vacuo. The residue was purified by flash chromatography
on silica gel (80% ethyl acetate/hexane) to provide 25 (0.965
g, 40.0%) as a light-yellow solid: mp 235-237 °C; IR (KBr)
3300-2750, 1675, 1600, 1350, 1225 cm-1; MS (electrospray)
P r oced u r e B: An ice-cold mixture of 17 (1.00 g, 4.57 mmol)
in dioxane (6.5 mL) and acetic anhydride (10 mL) was treated
with an ice-cold solution of nitrating reagent (4 mL of Ac2O
and 4 mL of HNO3). The resulting solution was stirred at 35
°C for 5.5 h, after which it was diluted with ice water (50 mL),
and the product was extracted into ethyl acetate (3 × 50 mL).
1
295 (M + 1); H NMR (300 MHz, DMSO-d6) δ 2.15-2.20 (m,
1H), 2.40-2.60 (m, 3H), 5.05 (m, 1H), 7.60 (d, 1H), 8.25 (dd,

Potent Benzoic Acid Inhibitor of Influenza Sialidase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2339
1H), 8.40 (d, 1H), 9.60 (br s, 2H); 13C NMR (DMSO-d6) δ 173.90,
171.89, 165.16, 144.26, 134.65, 134.05, 128.97, 125.91, 125.55,
60.92, 29.75, 23.22. Anal. (C12H10N2O7) C, H, N.
(m, 4H), 3.54 (dd, 2H), 4.21 (br s, 1H), 4.38 (q, 2H), 7.27 (d,
2H), 7.90 (d, 1H), 10.15 (br s, 1H), 11.50 (br s, 1H).
1-(4-Eth oxyca r bon yl-2-gu a n id in op h en yl)-5-(h yd r oxy-
m eth yl)p yr r olid in -2-on e (30). An ice-cold solution of 29
(0.23 g, 0.44 mmol) in dichloromethane (8 mL) was treated
with trifluoroacetic acid (4.65 mL, 60.4 mmol). The resulting
mixture was stirred at room temperature for 12 h. The light
red mixture was concentrated to dryness, redissolved in
dichloromethane (5 mL), and reconcentrated; this process was
repeated twice to give the trifluoroacetate salt of 30 (0.20 g,
100%) as an oil: MS (electrospray) 321 (M + 1); 1H NMR (300
MHz, D2O) δ 1.41 (t, 3H), 2.10-2.22 (m, 1H), 2.40-2.52 (m,
1H), 2.60-2.80 (m, 2H), 3.60 (dd, 2H), 4.38 (m, 1H), 4.44 (q,
3H), 7.60 (d, 1H), 8.15 (m, 2H). For the free base 30: 1H NMR
(300 MHz, D2O) δ 1.40 (t, 3H), 2.10-2.20 (m, 1H), 2.33-2.48
(m, 1H), 2.53-2.78 (m, 2H), 3.60 (m, 2H), 4.27 (m, 1H), 4.42
(q, 2H), 7.43 (d, 1H), 7.83 (d, 1H), 7.88 (dd, 1H).
1-(4-Eth oxyca r bon yl-2-n itr op h en yl)-5-(eth oxyca r bon -
yl)p yr r olid in -2-on e (26). A mixture of 25 (0.390 g, 1.33
mmol) in dry ethanol (4 mL) and H2SO4 (0.2 mL) was stirred
at reflux for 24 h. The ethanol was removed under vacuum,
the residue was suspended in 5% NaHCO3 (4 mL), and the
product was extracted into ethyl acetate (3 × 10 mL). The
combined extracts were dried (Na2SO4) and filtered, and the
solvent was removed in vacuo to give 26 (0.425 g, 91.0%) as a
yellow oil: IR (neat) 3000-2875, 1750, 1625, 1562 cm-1; MS
(electrospray) 351 (M + 1); 1H NMR (300 MHz, CDCl3) δ 1.22
(t, 3H, J ) 7.2 Hz), 1.42 (t, 3H, J ) 7.1 Hz), 2.5-2.8 (m, 3H),
2.3-2.4 (m, 1H), 4.17 (dq, 2H, J ) 3.0 & 7.1 Hz), 4.46 (q, 2H,
J ) 7.1 Hz), 4.70 (m, 1H), 7.58 (d, 1H, J ) 8.3 Hz), 8.28 (dd,
1H, J ) 2.0 & 8.3 Hz), 8.68 (d, 1H, J ) 1.9 Hz); 13C NMR
(CDCl3) δ 174.80, 170.80, 163.81, 145.56, 134.95, 134.30,
130.47, 129.45, 126.71, 62.17, 61.92, 60.29, 29.47, 23.93, 14.15,
13.93. Anal. (C16H19N2O7) C, H, N.
1-(4-Eth oxyca r bon yl-2-n itr op h en yl)-5-(h yd r oxym eth -
yl)p yr r olid in -2-on e (27). An ice-cold solution of 26 (2.00 g,
5.70 mmol) in dry ethanol (12.5 mL) and tetradydrofuran (12.5
mL) was treated with sodium borohydride (0.240 g, 6.27 mmol)
in portions over a period of 5 min. The reaction mixture was
then stirred at room temperature for 5 h. Excess NaBH4 was
quenched with cold 1 N HCl (11.0 mL). The yellow mixture
was extracted with ethyl acetate (3 × 45 mL). The combined
organic layers were washed with saturated sodium chloride
(2 × 45 mL), dried (Na2SO4), and filtered, and the solvent was
removed in vacuo. The crude mixture was purified by flash
chromatography on silica gel (80% ethyl acetate/hexane) to
provide 27 (0.906 g, 52.0%) as a yellow oil: IR (neat) 3500-
3250, 1718, 1615, 1540 cm-1; MS (electrospray) 309 (M + 1);
1H NMR (300 MHz, CDCl3) δ 1.42 (t, 3H), 2.22-2.78 (m, 4H),
3.74 (dq, 2H), 4.42 (q, 2H), 4.44 (m, 1H), 7.42 (d, 1H), 8.30
(dd, 1H), 8.58 (d, 1H); 13C NMR (CDCl3) δ 175.04, 171.12,
163.76, 145.87, 134.06, 129.50, 127.12, 126.43, 62.63, 61.76,
60.25, 30.91, 20.96, 14.04.
1-(4-Ca r boxy-2-gu a n id in op h en yl)-5-(h yd r oxym eth yl)-
p yr r olid in -2-on e (9). A solution of 30 (0.19 g, 0.44 mmol) in
1 N sodium hydroxide (5 mL) was stirred at room temperature
for 2 h. The pH of the reaction mixture was adjusted to 7.5
with acetic acid. The mixture was concentrated to one-half
volume and cooled to give 9 (0.060 g, 47%) as a white solid:
mp 253-257 °C (H2O); IR (KBr) 3500-3150, 1650, 1634 cm-1
;
MS (electrospray) 293 (M + 1); 1H NMR (300 MHz, DMSO) δ
1.92-2.44 (m, 4H), 4.10 (m, 1H), 7.30 (d, 1H), 7.76 (m, 2H),
1
7.80 (m, 2H), 7.90-8.65 (m, 2H); H NMR (300 MHz, D2O) δ
2.10-2.20 (m, 1H), 2.38-2.52 (m, 1H), 2.58-2.80 (m, 2H), 3.60
(dd, 2H), 4.33 (m, 1H), 7.52 (d, 1H), 7.94 (m, 1H), 7.97 (dd,
1H). Anal. (C12H16N4O4) C, H, N.
5-(Azid om eth yl)p yr r olid in -2-on e (32). A mixture of 31
(140 mg, 0.72 mmol) and sodium azide (100 mg, 1.5 mmol) in
1.5 mL of dry DMF was stirred at 80 °C for 5 h. The solvent
was evaporated to dryness under high vacuum, the residue
was suspended in chloroform (25 mL), the mixture was filtered,
and the filtrate was evaporated to dryness to give an oil. The
oil was purified by flash chromatography on silica gel (10%
ethanol in chloroform) to give 32 (95 mg, 95%) as an oil: MS
1
(electrospray) 141 (M + 1); H NMR (300 MHz, CDCl3) δ 7.1
(br, 1H), 3.83 (m, 1H), 3.5 (dd, 1H, J ) 4.5 & 12.3 Hz), 3.3 (dd,
1H, J ) 6.7 & 12.3 Hz), 2.36 (m, 2H), 2.28 (m, 1H), 1.8 (m,
1H). Anal. (C5H8N4O‚0.25H2O) C, H, N.
1-(4-Eth oxyca r bon yl-2-a m in op h en yl)-5-(h yd r oxym eth -
ylen e)p yr r olid in -2-on e (28). A mixture of 27 (0.914 g, 2.97
mmol), ethanol (114 mL), concentrated H2SO4 (1.5 mL), and
10% Pd/C (0.32 g) was shaken under 50 psi of hydrogen gas
for 1 h. The mixture was filtered through a bed of Celite,
washed with hot ethanol (20 mL), and concentrated to dryness
on a rotary evaporator to give a semisolid (0.745 g, 95.0%):
mp 90-95° C. The material was dissolved in 5% sodium
bicarbonate (40 mL), the solution was adjusted to pH 8.0, and
sodium chloride (0.50 g) and chloroform (40 mL) were added.
The aqueous layer was extracted with chloroform (2 × 40 mL),
and the combined organic layers were dried (Na2SO4) and
concentrated in vacuo to give 28 (0.667 g, 81.0%) as a white,
hygroscopic solid: MS (electrospray) 279 (M + 1); 1H NMR
(300 MHz, CDCl3) δ 1.38 (t, 3H), 2.20-2.80 (m, 4H), 3.50 (dd,
2H), 4.10 (m, 1H), 4.35 (q, 2H), 7.15 (d, 1H), 7.60 (d, 1H), 7.62
(d, 1H).
1-[4-Eth oxyca r bon yl-2-(N,N′-bis(ter t-bu tyloxyca r bon -
yl)gu a n id in o)p h en yl]-5-(h yd r oxym et h yl)p yr r olid in -2-
on e (29). An ice-cold solution of 28 (0.315 g, 1.13 mmol) in
dry DMF (12 mL) was treated with 1,3-bis(tert-butoxycarbo-
nyl)-2-methyl-2-thiopseudourea (0.493 g, 1.70 mmol), triethy-
lamine (0.358 g, 3.50 mmol), and mercuric chloride (0.369 g,
1.36 mmol). The reaction mixture was stirred under nitrogen
for 20 h at room temperature. The reacion mixture was diluted
with ethyl acetate (95 mL) and filtered through a bed of Celite.
The filtrate was washed with water (50 mL), saturated sodium
chloride (50 mL), and water (50 mL). The combined aqueous
layers were extracted with ethyl acetate (2 × 25 mL), and the
combined organic layers were dried (Na2SO4) and concentrated
to dryness on a rotary evaporator. The crude yellow oil was
purified by flash chromatography on silica gel (80% ethyl
acetate/hexane) to give 29 (0.360 g, 61.0%) as a foamy yellow
solid: mp 104-110 °C; MS (electrospray) 521 (M + 1); 1H NMR
(300 MHz, CDCl3) δ 1.38 (t, 3H), 1.50 (m, 18 H), 2.10-2.75
1-(4-Car boxy-2-n itr oph en yl)-5-(azidom eth yl)pyr r olidin -
2-on e (33). To a suspension of 60% NaH (900 mg, 37.5 mmol)
in 2 mL of dry DMF was added azide 32 (1.10 g, 7.80 mmol)
in 10 mL of DMF. The resulting mixture was stirred for 15
min at 75 °C and cooled to room temperature, and a solution
of 4-fluoro-3-nitrobenzoic acid (1.50 g, 8.10 mmol) in 8 mL of
DMF was added. The reaction mixture was stirred at room
temperature for 20 h. Excess NaH was quenched with 2 N HCl
(2 mL), and the solvent was removed under high vaccum below
50 °C. The crude residue was suspended in 10 mL of 1 N HCl,
and the product was extracted into ethyl acetate (3 × 50 mL).
The extracts were dried (Na2SO4) and concentrated to give an
oil which was purified by flash chromatography on silica gel
(ether) to give 33 (600 mg, 25%) as a pale-yellow oil. This was
crystallized from ether/ethanol: mp 119-121 °C; MS (elec-
trospray) 304 (M - 1); 1H NMR (300 MHz, acetone-d6) δ 8.52
(d, 1H, J ) 1.9 Hz), 8.33 (dd, 1H, J ) 1.8 & 8.4 Hz), 7.79 (d,
1H, J ) 8.4 Hz), 4.7 (m, 1H), 3.72 (d, 2H, J ) 12.6 Hz), 2.5 (m,
3H), 2.1 (m, 1H); 13C NMR (300 MHz, acetone-d6) δ 174.66,
165.39, 146.78, 135.72, 134.99, 130.27, 128.41, 127.12, 60.11,
54.47, 30.44, 23.20. Anal. (C12H11N5O5‚0.25H2O) C, H, N.
1-(4-Meth oxyca r bon yl-2-n itr op h en yl)-5-(a zid om eth yl)-
p yr r olid in -2-on e (34). A mixture of 33 (520 mg, 1.70 mmol)
and H2SO4 (0.1 mL) in 40 mL of methanol was stirred at reflux
for 12 h. Methanol was removed under vacuum, and the
resulting oil was dissolved in 100 mL of ethyl acetate. The
organic layer was washed with saturated NaHCO3 (3 × 10 mL)
and water (10 mL), dried (Na2SO4), and evaporated to give an
oil which was purified by flash chromatography on silica gel
(ether) to give 34 (520 mg, 96%) as a colorless oil. The oil was
crystallized from ether/ethanol: mp 68-70 °C; MS (electro-
spray) 320 (M + 1); 1H NMR (300 MHz, CDCl3) δ 8.64 (d, 1H,

2340 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Atigadda et al.
J ) 1.9 Hz), 8.32 (dd, 1H, J ) 1.9 & 8.3 Hz), 7.46 (d, 1H, J )
8.3 Hz), 4.36 (m, 1H), 3.98 (s, 3H), 3.57 (dd, 2H, J ) 5.1 &
12.8 Hz), 2.5 (m, 3H), 2.1 (m, 1H); 13C NMR (acetone-d6) δ
174.81, 164.77, 146.23, 134.88, 134.67, 129.9, 128.33, 127.15,
59.93, 54.12, 53.23, 30.48, 23.01. Anal. (C13H13N5O5) C, H, N.
1-(4-Meth oxycar bon ylph en yl)-5,5-bis(eth oxycar bon yl)-
p yr r olid in -2-on e (40). A suspension of 60% NaH (70 mg, 2.9
mmol) was washed with hexane (3 × 1 mL) to remove mineral
oil. The NaH in 10 mL of dry DMF was treated with 39 (517
mg, 1.10 mmol) in 7 mL of DMF. The reaction mixture was
stirred at room temperature for 2 h. Excess NaH was quenched
with 1 mL of 1 N HCl. The solvent was removed under high
vacuum, the resulting crude oil was suspended in 50 mL of
ethyl acetate, and the organic layer was washed with water
(3 × 10 mL), dried (Na2SO4), and evaporated to give pure 40
1-(4-Met h oxyca r b on yl-2-a m in op h en yl)-5-(ter t-b u t yl-
oxyca r bon yla m in om eth yl)p yr r olid in -2-on e (35). A sus-
pension of 10% Pd/C (210 mg) in 5 mL of ethyl acetate was
shaken on a Parr shaker for 5 min using 30 psi of H2 gas.
Compound 34 (220 mg, 0.69 mmol) and di-tert-butyl dicarbon-
ate (155 mg, 0.700 mmol) in 5 mL of ethyl acetate were added,
and the resulting mixture was shaken for 2.5 h at 30 psi. The
reaction mixture was diluted with 50 mL of methanol, filtered,
and evaporated to dryness under vacuum to give 35 (240 mg,
100%) as a colorless oil which was used in the next reaction
without further purification: 1H NMR (300 MHz, CDCl3) δ 7.55
(m, 2H), 7.12 (d, 1H), 5.58 (br, 1H), 4.28 (m, 1H), 4.15 (br s,
2H), 3.90 (s, 3H), 3.45 (m, 1H), 2.85 (t, 0.5H), 2.8 (t, 0.5H), 2.6
(t, 2H), 2.35 (m, 1H) 2.1 (m, 1H), 1.4 (s, 9H).
1-[4-Meth oxyca r bon yl-2-(N,N′-bis(ter t-bu tyloxyca r bo-
n yl)gu an idin o)ph en yl]-5-(ter t-bu tyloxycar bon ylam in om -
eth yl)p yr r olid in -2-on e (36). An ice-cold solution of 35 (245
mg, 0.70 mmol) in 3 mL of dry DMF under nitrogen was
treated with 1,3-bis(tert-butoxycarbonyl)-2-thiopseudourea (232
mg, 0.840 mmol), triethylamine (215 mg, 2.10 mmol), and
HgCl2 (230 mg, 0.84 mmol). The reaction mixture was then
stirred at 0-10 °C for 4 h. The DMF was removed under
vacuum, and the crude product was purified by flash chroma-
tography on silica gel (ether) to give 36 (280 mg, 60%) as a
colorless oil. The oil was crystallized from ether/hexane: mp
134-136 °C; MS (electrospray) 606 (M + 1); 1H NMR (300
MHz, CDCl3) δ 11.5 (br, 1H), 10.05 (br, 1H) 8.55 (br, 1H), 7.9
(d, 1H, J ) 8.0 Hz), 7.25 (d, 1H, J ) 8.2 Hz),5.35 (br, 1H), 4.4
(m, 1H), 3.95 (s, 3H), 3.75 (m, 1H), 3.3 (m, 1H), 2.6 (m, 2H),
2.4 (m, 1H), 1.95 (m, 1H), 1.55 (s, 9H), 1.45 (s, 9H), 1.35 (s,
9H). Anal. (C29H43N5O9) C, H, N.
1
(415 mg, 98%) as an oil: MS (electrospray) 364 (M + 1); H
NMR (300 MHz, CDCl3) δ 8.0 (d, 2H, J ) 8.4 Hz), 7.4 (d, 2H,
J ) 8.4 Hz), 4.2 (qq, 4H), 3.9 (s, 3H), 2.7 (m, 2H), 2.6 (m, 2H)
1.2 (t, 6H); 13C NMR δ 174.64, 167.75, 165.95, 140.82, 129.65,
128.56, 126.81, 74.11, 62.25, 51.79, 29.04, 28.94, 13.36. Anal.
(C18H21NO7) C, H, N.
1-(4-Meth oxyca r bon ylp h en yl)-5,5-bis(h yd r oxym eth yl)-
p yr r olid in -2-on e (41). To an ice-cold solution of 40 (500 mg,
1.37 mmol) in 6 mL of dry THF and 6 mL of dry methanol
was added NaBH4 (200 mg, 5.20 mmol) in small portions over
a period of 30 min. The reaction mixture was then stirred at
0-10 °C for 4 h. Additional NaBH4 (75 mg, 1.98 mmol) was
added, and the stirring continued for 6 h more at 0-10 °C.
The reaction mixture was quenched with 5 mL of 0.5 N HCl,
organic solvent was evaporated, and the product was extracted
into ethyl acetate (5 × 25 mL). The extracts were dried (Na2-
SO4) and evaporated to give 450 mg of crude oil, which was
purified by flash chromatography on silica gel (10% ethanol
in ether) to give 41 (330 mg, 86%) as a solid: mp 133-135 °C;
MS (electrospray) 280 (M + 1); 1H NMR (300 MHz, CD3OD) δ
8.10 (d, 2H, J ) 8.3 Hz), 7.43 (d, 2H, J ) 8.4 Hz), 3.92 (s, 3H),
3.38 (dd, 4H, J ) 7.2 & 11.9 Hz), 2.58 (t, 2H, J ) 8.5 Hz), 2.29
(t, 2H, J ) 8.5 Hz). Anal. (C14H17NO5) C, H, N.
1-(4-Car boxyph en yl)-5,5-bis(h ydr oxym eth yl)pyr r olidin -
2-on e (11). A solution of 41 (60 mg, 0.21 mmol) in 2 mL of 1
N NaOH was stirred at room temperature for 3.5 h. The pH
of the reaction mixture was adjusted to 2 with 1 N HCl; the
product was extracted into ethyl acetate (5 × 10 mL), dried,
and evaporated to provide a solid residue. The residue was
suspended in ether and filtered to give 11 (40 mg, 70%): mp
195-197 °C (ether/ethanol); MS (electrospray) 264 (M - 1);
1H NMR (300 MHz, DMSO-d6) δ 12.99 (br, 1H), 7.97 (d, 2H, J
) 8.4 Hz), 7.35 (d, 2H, J ) 8.4 Hz), 5.05 (br, 2H), 3.25 (dd,
4H, J ) 11.3 & 16.1 Hz), 2.4 (t, 2H, J ) 8.6 Hz), 2.1 (t, 2H, J
) 8.5 Hz). Anal. (C13H15NO5) C, H, N.
1-(4-Car boxy-2-gu an idin oph en yl)-5-(am in om eth yl)pyr -
r olid in -2-on e (10). A suspension of 36 (33 mg, 0.050 mmol)
in 1.5 mL of 1 N HCl was stirred at reflux for 4 h. The solvent
was removed under vacuum to give an oil which was crystal-
lized from ethanol/ether to give 10 (16 mg, 84%) as a white
hygroscopic solid: mp 205 °C (foamed); MS (electrospray) 292
(M + 1); 1H NMR (300 MHz, D2O) δ 8.18 (m, 2H), 7.62 (d, 1H),
4.6 (m, 1H), 3.15 (m, 2H), 2.75 (m, 2H), 2.65 (m, 1H), 2.15 (m,
1H). Anal. (C13H19N5O3Cl2‚0.5H2O‚0.5CH3CH2OH) C, H, N.
1-(4-Met h oxyca r b on yl-2-n it r op h en yl)-5,5-b is(et h oxy-
ca r bon yl)p yr r olid in -2-on e (42). An ice-cold solution of 40
(450 mg, 1.23 mmol) in 5 mL of CH2Cl2 was treated with
nitronium tetrafluoroborate (550 mg, 4.10 mmol). The result-
ing mixture was stirred at 5-10 °C for 3 h. The reaction
mixture was evaporated to dryness, and the crude mixture was
dissolved in 50 mL of ethyl acetate. The organic layer was
washed with water (3 × 15 mL), dried (Na2SO4), and evapo-
rated to give 42 (440 mg, 95%) as an oil which solidified upon
standing: mp 81-82 °C (ether); MS (electrospray) 409 (M +
1); 1H NMR (300 MHz, CDCl3) δ 8.7 (d, 1H, J ) 1.9 Hz), 8.35
(dd, 1H, J ) 1.9 & 8.4 Hz), 7.85 (d, 1H, J ) 8.4 Hz), 4.35 (m,
2H), 4.1 (m, 2H), 3.95 (s, 3H), 3.15 (m, 1H), 2.7 (m, 2H), 2.5
(m, 1H), 1.3 (t, 3H), 0.98 (t, 3H). Anal. (C18H20N2O9) C, H, N.
1-(4-Meth oxyca r bon yl-2-n itr op h en yl)-5,5-bis(h yd r oxy-
m eth yl)p yr r olid in -2-on e (43). An ice-cold solution of 42
(4.10 g, 10.0 mmol) in a mixture of 50 mL of dry THF and 50
mL of dry methanol was treated with small portions of NaBH4
(1.80 g, 47.0 mmol) over a period of 1 h. Stirring continued for
4 h after which additional NaBH4 (0.500 g, 13.2 mmol) was
added. After another 4 h of stirring more NaBH4 (0.500 g, 13.2
mmol) was added, and the stirring continued for 6 more h at
0° C. The reaction mixture was then quenched with 1 N HCl
(25 mL) and concentrated to about 20 mL volume under
vaccum. The product was extracted into chloroform (5 × 50
mL). The combined organic layers were dried (Na2SO4) and
evaporated to give an oil which was purified by flash chroma-
tography on silica gel (10% ethanol in ether) to give 43 (2.00
g, 62.0%) as an off-white solid: mp 144-145 °C; MS (electro-
Diet h yl 2-(4-Ca r b oxym et h ylp h en yla m in o)m a lon a t e
(38). A mixture of 37 (3.10 g, 20.5 mmol) and diethyl bromo-
malonate (2.45 g, 10.2 mmol) was heated at 115 °C for 16 h in
an oven. The reaction mixture was cooled, suspended in
benzene (20 mL), and filtered. The filtrate was diluted with
additional benzene (50 mL) and washed with 2 N HCl (3 × 10
mL) and water (10 mL). The organic layer was dried (Na2SO4)
and evaporated to give an oil, which solidified upon standing.
The crude product was recrystallized from ether/hexane to give
38 (2.7 g, 85%): mp 66-69 °C (ether/hexane); MS (electro-
1
spray) 310 (M + 1); H NMR (300 MHz, CDCl3) δ 7.9 (d, 2H,
J ) 8.8 Hz), 6.6 (d, 2H, J ) 8.8 Hz), 5.2 (br d, 1H, J ) 7.2 Hz),
4.8 (d, 1H, J ) 7.2 Hz), 4.3 (q, 4H), 3.8 (s, 3H), 1.3 (t, 6H); 13
C
NMR (CDCl3) δ 166.93, 166.86, 149.04, 131.42, 120.04, 112.19,
62.51, 59.80, 51.48, 13.87. Anal. (C15H19NO6) C, H, N.
N-[Bis(eth oxycar bon yl)m eth yl]-N-(3-br om opr opion yl)-
4-(m eth oxyca r bon yl)a n ilin e (39). A mixture of 38 (1.10 g,
3.50 mmol), 3-bromopropionic acid (1.10 g, 7.20 mmol), and
phosphorus trichloride (1 mL) in 12 mL of benzene was stirred
at reflux for 30 h. The reaction mixture was diluted with 50
mL of benzene and washed with saturated NaHCO3 (3 × 15
mL), water (10 mL), 1 N HCl (10 mL), and water (10 mL).
The organic layer was dried (Na2SO4) and evaporated to give
pure 39 (1.4 g, 89%) as an oil which was used in the next
reaction without further purification: MS (electrospray) 444
(M + 1); 1H NMR (300 MHz, CDCl3) δ 8.10 (d, 2H, J ) 8.3
Hz), 7.54 (d, 2H, J ) 8.3 Hz), 5.46 (s, 1H), 4.2 (m, 4H), 3.95 (s,
3H), 3.56 (t, 2H, J ) 6.7 Hz), 2.69 (t, 2H, J ) 6.9 Hz), 1.2 (t,
6H).

Potent Benzoic Acid Inhibitor of Influenza Sialidase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2341
spray) 325 (M + 1); 1H NMR (300 MHz, CD3OD) δ 8.58 (d,
1H, J ) 1.9 Hz), 8.29 (dd, 1H, J ) 1.9 & 8.3 Hz), 7.73 (d, 1H,
J ) 8.3 Hz), 3.95 (s, 3H), 3.8 (dd, 2H, 11.9 & 52 Hz), 3.6 (dd,
2H, 11.3 & 29.5 Hz), 2.55 (m, 2H), 2.3 (m, 1H), 2.0 (m, 1H,);
13C NMR (CD3OD) δ 179.73, 166.42, 149.62, 137.01, 135.49,
133.08, 132.28, 127.81, 73.79, 66.41, 64.98, 53.72, 31.44, 27.44.
Anal. (C14H16N2O7) C, H, N.
The reaction mixture was acidified with excess glacial acetic
acid, and the product precipitated. The mixture was filtered,
and the collected solid was washed with water and dried to
give pure 13 (125 mg, 87.0%): mp 178-180 °C (water); MS
(electrospray) 291 (M + 1); 1H NMR (300 MHz, CDCl3) δ 7.44
(d, 1H, J ) 1.2 Hz), 7.37 (dd, 1H, J ) 1.2 & 8.1 Hz), 7.09 (d,
1H, J ) 8.1 Hz), 3.75 (t, 2H, J ) 6.9 & 7.0 Hz), 3.34 (m, 1H),
2.65 (t, 2H, J ) 8.0 & 8.0 Hz), 2.23 (m, 2H), 1.55 (m, 4H), 0.92
(t, 6H, J ) 7.4 & 7.4 Hz); 13C NMR (CDCl3) δ 175.64, 170.00,
143.49, 129.99, 128.57, 126.19, 118.05, 113.73, 54.86, 50.31,
31.34, 26.34, 19.14, 9.29. Anal. (C16H22N2O3‚0.5H2O) C, H, N.
1-(4-Me t h oxyc a r b on yl-2-a m in op h e n yl)-5,5-b is(h y-
d r oxym eth yl)p yr r olid in -2-on e (44). A suspension of 43
(0.80 g, 2.5 mmol) in 25 mL of methanol was treated with 10%
Pd/C (500 mg), and the resulting mixture was placed on a Parr
shaker at 50 psi of H2 gas for 1 h. The reaction mixture was
diluted with 50 mL of methanol, filtered, and evaporated to
dryness under vacuum to give an oil which was solidified upon
the addition of ether to give pure 44 (630 mg, 87%): mp 192-
1-[4-Met h oxyca r b on yl-2-(3-p en t yla m in o)p h en yl]-5,5-
bis(h yd r oxym eth yl)p yr r olid in -2-on e (47). A solution of 44
(100 mg, 0.340 mmol) in 1 mL of dichloroethane and 0.5 mL
of acetic acid was treated with 3-pentanone (200 mg, 2.30
mmol) and NaCNBH3 (75.0 mg, 1.20 mmol). The resulting
mixture was stirred at room temperature for 6 h, and 3-pen-
tanone (200 mg, 2.30 mmol), NaCNBH3 (75.0 mg, 1.20 mmol),
and methanol (0.5 mL) were added. Stirring was continued
for 12 h, and additional 3-pentanone (300 mg, 3.40 mmol) and
NaCNBH3 (150 mg, 2.30 mmol) were added. Stirring was then
continued for an additional 16 h. The reaction mixture was
quenched with 10 mL NaHCO3 (saturated), and the product
was extracted into ethyl acetate (3 × 50 mL). The organic
layers were dried (Na2SO4) and evaporated to give an oil which
was purified by flash chromatography on silica gel (5% ethanol
in ether) to give 47 (105 mg, 82.2%) as a white solid: mp 183-
1
194 °C; MS (electrospray) 295 (M + 1); H NMR (300 MHz,
CD3OD) δ 7.52 (d, 1H, J ) 1.9 Hz), 7.34 (dd, 1H, J ) 1.9 & 8.1
Hz), 7.23 (d, 1H, J ) 8.2 Hz), 3.9 (s, 3H), 3.63-3.32 (m, 4H),
2.67-2.5 (m, 2H), 2.4-2.3 (m, 2H); 13C NMR (CD3OD) δ
179.43, 168.55, 148.19, 132.37, 131.90, 126.75, 119.74, 119.07,
73.14, 64.07, 64.02, 52.77, 31.94, 25.60. Anal. (C14H18N2O5‚
0.25CH3OH) C, H, N.
1-[4-Meth oxyca r bon yl-2-(N,N′-bis(ben zyloxyca r bon yl)-
gu a n id in o)p h en yl]-5,5-bis(h yd r oxym eth yl)p yr r olid in -2-
on e (45). An ice-cold solution of 44 (254 mg, 0.860 mmol) in 3
mL of dry DMF was treated with bis(benzyloxycarbonyl)-S-
methylthiourea (425 mg, 1.20 mmol), HgCl2 (330 mg, 1.20
mmol), and triethylamine (250 mg, 2.40 mmol). The resulting
mixture was stirred at 0 °C for 3 h and the temperature slowly
raised to room temperature, where the mixture stirred for 12
h. DMF was removed under vacuum, and the crude mixture
was purified by flash chromatography on silica gel (ether) to
give 45 (370 mg, 71%) as a colorless oil: MS (electrospray)
605 (M + 1); 1H NMR (300 MHz, CDCl3) δ 11.8 (br, 1H), 10.1
(br, 1H), 7.9 (d, 1H), 7.5 (d, 1H), 7.35 (m, 11H), 5.2 (m, 4H),
3.9 (s, 3H), 3.75 (m, 1H), 3.5 (m, 1H), 3.35 (m, 2H), 2.6 (m,
2H), 2.25 (m, 1H), 2.0 (m, 1H).
1
185 °C; MS (electrospray) 365 (M + 1); H NMR (300 MHz,
CD3OD) δ 7.31 (d, 1H, J ) 1.7 Hz), 7.25 (d, 1H, J ) 8.0 Hz),
7.2 (dd, 1H, J ) 1.7 & 8.0 Hz), 3.9 (s, 3H), 3.45 (m, 4H), 3.27
(m, 1H), 2.6 (m, 1H), 2.5 (m, 1H), 2.3 (m, 2H), 1.6 (m, 3H),
1.45 (m, 1H), 0.97 (t, 3H), 0.93 (t, 3H); 13C NMR (CD3OD) δ
179.44, 169.05, 148.40, 132.45, 132.43, 125.68, 116.90, 112.88,
73.02, 64.06, 63.89, 56.34, 52.79, 32.16, 27.79, 27.69, 25.82,
11.11, 10.74. Anal. (C19H28N2O5) C, H, N.
1-[4-Ca r b oxy-2-(3-p e n t yla m in o)p h e n yl]-5,5-b is(h y-
d r oxym eth yl)p yr r olid in -2-on e (14). A suspension of 47 (70
mg, 0.19 mmol) in 1 N NaOH (1 mL) was stirred at room
temperature for 12 h. Acidification of the reaction mixture with
glacial acetic acid resulted in a white precipitate, which was
filtered and dried to give pure 14 (50 mg, 75%): mp 220-221
°C (water); MS (electrospray) 351 (M + 1); 1H NMR (300 MHz,
DMSO-d6) δ 7.02 (d, 1H, J ) 1.5 Hz), 7.01 (d, 1H, J ) 8.0 Hz),
6.93 (dd, 1H, J ) 1.5 & 8.0 Hz), 5.3 (br, 3H), 3.2-3.0 (m, 5H),
2.3 (m, 1H), 2.2 (m, 1H), 2.05 (m, 2H), 1.4 (m, 3H), 1.2 (m,
1H), 0.8 (t, 3H, J ) 7.3 Hz), 0.73 (t, 3H, J ) 7.3 Hz). Anal.
(C18H26N2O5) C, H, N.
1-(4-Ca r boxy-2-gu a n id in op h en yl)-5,5-bis(h yd r oxym e-
th yl)p yr r olid in -2-on e (12). A solution of 45 (350 mg, 0.57
mmol) in 1 mL of methanol was treated with 1 N NaOH (2.5
mL). The resulting suspension was stirred at room tempera-
ture for 16 h. The reaction mixture was adjusted to pH 3 with
1 N HCl, and the product was chromatographed on an ion-
exchange column (Dowex; 1.4 N NH4OH) to give 12 (110 mg,
59.0%) as the ammonium salt: MS (electrospray) 323 (M +
1
1); H NMR (300 MHz, D2O) δ 7.95 (d, 1H, J ) 1.8 Hz), 7.93
(dd,1H, J ) 1.9 & 8.2 Hz), 7.45 (d, 1H, J ) 8.2 Hz), 3.7 (dd,
2H, J ) 8.1 & 12.0 Hz), 3.55 (d, 1H, J ) 12.0 Hz), 3.29 (d, 1H,
J ) 12 Hz), 2.7 (m, 2H), 2.35 (m, 2H). Anal. (C14H21N5O5‚H2O)
C, H, N.
X-r a y Cr ysta llogr a p h y. Purified N9 neuraminidase was
crystallized using the hanging drop technique in which
droplets of protein solution on siliconized cover slips are
inverted on a linbro plate. The droplet consisted of equal
volumes of protein solution (10-15 mg/mL in water) and
potassium phosphate buffer (1.7 M, pH 6.6). This mixture was
equilibrated through vapor phase with a reservoir of 1.9 M
potassium phosphate buffer at pH 6.8. Large rhombic dodeca-
hedral crystals grew in a few days. The space group has been
identified as cubic, I432, with cell dimension R ) 183.8 Å. The
crystals diffract strongly to at least 2 Å resolution on a rotating
anode source.
1-[4-Met h oxyca r b on yl-2-(3-p en t yla m in o)p h en yl]p yr -
r olid in -2-on e (46). A solution of 19 (500 mg, 2.10 mmol),
3-pentanone (190 mg, 2.20 mmol), and triethylamine (440 mg,
4.30 mmol) in 10 mL of dry CH2Cl2 was treated with TiCl4
(3.5 mL, 3.50 mmol). The resulting mixture was stirred at room
temperature for 18 h, and NaCNBH3 (410 mg, 6.50 mmol) was
added. Stirring was continued for 3 more h. The reaction
mixture was quenched with 1 N NaOH (2 mL), and the CH2-
Cl2 was evaporated under vacuum. The product was extracted
into ethyl acetate (3 × 30 mL), dried (Na2SO4), and evaporated.
The resulting oil was purified by flash chromatography on
silica gel (10% ethanol in ether) to give 46 (160 mg, 25.0%):
The complexes of the inhibitor with neuraminidase were
prepared by diffusing the compound into N9 crystals in a
stabilization buffer which was the same as the reservoir
solution used in crystallization. The final concentration of the
inhibitor in the solution was 1-2 mM. The N9 crystals were
allowed to equilibrate with the inhibitor compound for at least
24 h before data collection.
1
MS (electrospray) 305 (M + 1); H NMR (300 MHz, CDCl3) δ
7.39 (d, 1H, J ) 1.8 Hz), 7.33 (dd, 1H, J ) 1.8 & 8.0 Hz), 7.08
(d, 1H, J ) 8.0 Hz), 3.95 (br d, NH, J ) 7.6 Hz), 3.89 (s, 3H),
3.74 (t, 2H, J ) 6.9 & 6.9 Hz), 3.35 (m, 1H), 2.60 (t, 2H, J )
7.8 Hz), 2.22 (m, 2H), 1.57 (m, 4H), 0.91 (t, 6H); 13C NMR δ
174.65, 166.83, 143.42, 129.64, 128.53, 125.93, 117.33, 113.10,
54.67, 51.79, 50.04, 31.18, 26.25, 19.05, 9.75. Anal. (C17H24N2O3‚
0.75H2O) C, H, N.
All X-ray intensity measurements were recorded with a
Nicolet/Siemens X-100 multiwire area detector and a Rigaku
RU-300 rotating anode X-ray generator operating at 100 mA
and 50 kV with 0.3 × 0.3 focus and a Cu anode. The data
collection parameters were crystal-to-detector distance of 16
cm, swing angle of 28°, frame width of 0.1°, and exposure time
1-[4-Ca r b oxy-2-(3-p en t yla m in o)p h en yl]p yr r olid in -2-
on e (13). A solution of 46 (150 mg, 0.490 mmol) in 1 mL of
methanol was treated with 1 N NaOH (2 mL), and the
resulting mixture was stirred at room temperature for 16 h.

2342 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Atigadda et al.
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6-Carboxamides. Bioorg. Med. Chem. Lett. 1996, 24, 2931-2936.
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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.
(15) J edrzejas, M. J .; Singh, S.; Brouillette, W. J .; Laver, G. W.; Air,
G. M.; Luo, M. Structures of Aromatic Inhibitors of Influenza
Virus Neuraminidase. Biochemistry 1995, 34, 3144-3151.
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Brouillette, W. J . Structure-Based Inhibitors of Influenza Virus
Sialidase. A Benzoic Acid Lead with Novel Interaction. J . Med.
Chem. 1995, 38, 3217-3225.
(17) Sudbeck, E. A.; J edrzejas, M. J .; Singh, S.; Brouillette, W. J .;
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N.; Montgomery, J . A.; Walsh, D. A.; Luo, M. Guanidinobenzoic
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2251-2254.
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of 240-300 s. Each crystal provided about 800-900 frames of
data before radiation damage deteriorated the diffraction
quality.
The X-ray intensity data were processed using the XENGEN
suite of programs. The integrated intensities were then scaled
and merged to produce a final data set containing only unique
reflections. The completeness of data to 2.1 Å is generally about
90%. The refinement of the inhibitor complexes was performed
using the X-PLOR simulated annealing protocol. Diffraction
data in the 10-2.1 Å resolution range were used in the
refinement with a 2σ cutoff on Fo’s. The starting model for the
refinement of the inhibitor complexes is the 1.9 Å refined
uncomplexed neuraminidase native structure. The water
molecules in the active site of the native structure were
removed prior to the refinement of the complex structure. A
full cycle of the X-PLOR simulated annealing protocol was
carried out on this model. Fo - Fc and 2(Fo - Fc) difference
Fourier maps were calculated, and a model of the inhibitor
molecule was fit into the electron density in the active site.
Water molecules were also placed in the active site based on
the difference electron density maps, and these positions were
compared to the active site water structure in the native
neuraminidase model. At the end of the simulated annealing
protocols and positional and temperature factor refinement,
the crystallographic R-factor converged to 17-19% at 2 Å
resolution for the inhibitor complex structures.
En zym e Assa ys. All compounds were evaluated for in vitro
inhibitory actions using the method reported by von Itzstein
et al.22 The neuraminidase from the H1N9 strain of influenza
was obtained by the method described by Laver et al.23 The
assay employed a spectrofluorometric technique that uses 2′-
(methylumbelliferyl)-R-D-acetylneuraminic acid as substrate.
Cleavage of this substrate by neuraminidase produces a
fluorescent product which can quantified. The assay mixture
contained inhibitors at various concentrations (4-6 points) and
enzyme in 32.5 mM MES (2-(N-morpholino)ethanesulfonic
acid) buffer and 4 mM CaCl2 at pH 6.5 (total volume ) 80
µL). The reaction was started by the addition of 20 µL of the
substrate to a final concentration of 75 µM. After 10 min at
37 °C, 2.4 mL of 0.1 M glycine/NaOH (pH 10.2) was added to
0.1 mL of the reaction mixture to terminate the reaction. A
blank was run with the same substrate solution excluding the
enzyme. Fluorescence was read using an Aminco-Bowman
fluorescence spectrophotometer (excitation, 360 nm; emission,
450 nm), and substrate blanks were subtracted from the
sample readings. The IC50 value was calculated by plotting
percent inhibition versus the inhibitor concentration, and the
determination of each point was performed in duplicate. In
all cases each IC50 value differed from its duplicate by less
than 2-fold.
Ack n ow led gm en t. This work was supported in part
by NIH Grant U01 AI31888 (M.L., W.J .B., G.M.A.) and
by a research grant from BioCryst Pharmaceuticals, Inc.
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