Hydrophobic benzoic acids as inhibitors of influenza neuraminidase

Article abstract of DOI:10.1016/S0968-0896(99)00197-2

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray structu

Full text of DOI:10.1016/S0968-0896(99)00197-2

Bioorganic & Medicinal Chemistry 7 (1999) 2487±2497
Hydrophobic Benzoic Acids as Inhibitors of In¯uenza
Neuraminidase
Venkatram R. Atigadda, a Wayne J. Brouillette, a,b,* Franco Duarte, a
Yarlagadda S. Babu, c Shanta Bantia, c Pooran Chand, c Naiming Chu, c
John A. Montgomery, c David A. Walsh, c Elise Sudbeck, b James Finley, b
Gillian M. Air, d Ming Luo b and Graeme W. Laver e
aDepartment of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA
bCenter for Macromolecular Crystallography, University of Alabama at Birmingham, Birmingham, AL 35294, USA
cBioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive Birmingham, AL 35244, USA
dDepartment of Biochemistry and Molecular Biology, University of Oklahoma, Oklahoma City, OK 73190, USA
eJohn Curtin School of Medical Research, Australian National University, Canberra 2601, Australia
Received 13 January 1999; accepted 7 June 1999
AbstractÐNeuraminidase (NA) plays a critical role in the life cycle of in¯uenza virus and is a target for new therapeutic agents. A
new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray struc-
ture of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid in complex with NA revealed that the lipophilic side chain binds
in a newly created hydrophobic pocket formed by the movement of Glu 278 to interact with Arg 226, whereas the guanidine of 11
interacts in a negatively charged pocket created by Asp 152, Glu 120 and Glu 229. Compound 11 was highly selective for type A
(H2N2) in¯uenza NA (IC50 1 mM) over type B (B/Lee/40) in¯uenza NA (IC50 500 mM). # 1999 Elsevier Science Ltd. All rights
reserved.
Introduction
New and emerging strains of in¯uenza virus cause ser-
ious disease each year.1 Although vaccination is avail-
able, it is often ine€ective due to the high antigenic
changes exhibited by the in¯uenza virus. Therefore,
e€orts have focused on rational drug design to develop
new agents against potential targets within the virus.
The enzyme sialidase, or neuraminidase (NA), a surface
glycoprotein, plays a critical role in the life cycle of
in¯uenza virus and has been the focus of new drug
development. NA mediates the cleavage of terminal
sialic acid residues from the cell surface glycoproteins
and glycolipids, liberating the virus from the infected
cell's surface to facilitate spread. This process may also
enhance viral movement through the mucus of the
respiratory tract to infect epitheliel cells.2±4 NA consists
of a symmetrical tetrameric head, which projects from
Key words: Neuraminidase; in¯uenza virus; sialidase; structure-based
drug design.
* Corresponding author. Tel.: +1-205-934-8288; fax: +1-205-934-
2543; e-mail: wbrou@uab.edu
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00197-2

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V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
the viral membrane through a centrally attached stalk.
Each of the four subunits of the head contains a cata-
lytic site and a calcium binding site. The stalk of the NA
has been proteolytically cleaved to release the heads,
which can be crystallized. The released enzyme retains
catalytic activity. Based on antigenic cross reactivity,
nine subtypes of NA have been identi®ed for in¯uenza
A whereas only one subtype exists for in¯uenza B. The
crystal structures of NA's from both types A and B
reveal that the catalytic site is completely conserved
among all in¯uenza subtypes.5±7 A symmetrical folding
pattern of six four-stranded b-sheets in a propeller
fashion forms the active site, which is composed pri-
marily of charged amino acids.5,8±13 Site-speci®c muta-
genesis of these residues usually results in inactivation
of the enzyme, indicating that the virus may not easily
escape NA targeted drug therapy by mutation.14,15 In
animals administration of NA-speci®c antibodies limits
viral replication and infection, indicating that this
enzyme represents a suitable target for antiviral drug
development.16 Furthermore, a NA minus mutant was
unable to replicate. Currently three NA inhibitors have
entered clinical trials.6,28,30
simple inhibitors, we prepared several achiral benzoic
acids, among which 4-N-acetylamino-3-guanidino-
benzoic acid (BANA 113) was a modest inhibitor with
an IC50 value of 10 mM.22±24
The crystal structure of BANA 113 complexed with
NA(Fig. 2) revealed that it interacts with NA in a simi-
lar fashion to 3 with the exception of the guanidino
group (Fig. 1). In compound 3, the guanidine interacts
In earlier studies it was found that 2,3-didehydro-2-
deoxy-N-acetylneuraminic acid (1) (neu5Ac2en), an
analogue of sialic acid, is an inhibitor of NA with a
Ki of 4 mM.17,18 Based on the structural information
provided by the crystal structure of 1 complexed with
NA several potent inhibitors were designed. Both
amine 2 and guanidine 3 are more potent inhibitors
8
10
than 1 with Ki values of 10 and 10
M, respec-
Figure 2. X-ray crystal structure of the binding site region for the
complex formed between BANA 113 and N9 neuraminidase.
tively.6,19,20 However, these compounds are not orally
active, they are stereochemically complex, and their synth-
esis can be lengthy. In an attempt to develop chemically
Figure 1. Cartoon showing the interactions of 3 (GG167) in the neuraminidase binding site (N9 numbering). Note that Tyr 406 lies under 3 and is
not shown.

V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
2489
electrostatically with Asp 152, Glu 229 and Glu 120 (C4
Chemistry
subsite), and the glycerol forms hydrogen bonds with
Glu 278 (C6 subsite).21 However, in BANA 113 the
guanidine occupies the glycerol binding subsite (C6
subsite) of 3, forming a salt bridge with Glu 278.
The synthesis of hydrophobic amide 6 is described in
Scheme 1. The Fischer esteri®cation of 3,4-diamino-
benzoic acid (12) gave 13, which underwent regioselective
acylation, using 2-ethylbutyric acid, on the 3-amino group
to provide amide 14. This intermediate was then N-acety-
lated and the ester saponi®ed to provide target 6.
Recent studies indicate that the replacement of the gly-
cerol side chain of 3 with hydrophobic side chains, as in
4a, results in potent inhibitory activity, although these
compounds are highly selective for type A over type B
NA.25,26 On the other hand the cyclohexene analogue 5
displayed broad spectrum activity against both in¯uenza
A and B sialidases.28,29 The crystal structure of 4b in
complex with NA revealed that it binds to the enzyme in
essentially the same way as 3, except the hydrophobic side
chain interacts in a hydrophobic pocket (C6 subsite)
resulting from the movement of Glu 278 to interact with
Arg 226. In order to determine the e€ect of hydrophobic
substituents in place of, and in addition to, the guanidino
grouping of BANA 113, we carried out the synthesis and
in vitro evaluation of benzoic acids 6±11.
Similarly, the synthesis of hydrophobic aniline 7 is
summarized in Scheme 2. Ester 13 underwent regiose-
lective alkylation, using 3-bromopentane, on the 3-
amino group to give intermediate 16. This underwent N-
acetylation to yield 17, which was saponi®ed to form the
®nal product 7.
The synthesis of 8 and 9, as shown in Scheme 3, began
with the Fischer esteri®cation of 4-amino-3-hydroxy-
benzoic acid (18). Selective alkylation of the resulting
phenol (19) with 3-bromopentane or 4-bromoheptane in
the presence of K2CO3 provided the methyl 3-alkoxy-4-
Scheme 1.
Scheme 2.

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V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
aminobenzoates 20 and 21 in 90% yield. The N-acety-
lation of each in the presence of acetic anhydride and
DMAP to give 22 and 23, followed by saponi®cation,
provided 8 and 9.
The synthesis of 4-(N-acetylamino)-5-guanidino-3-(3-
pentyloxy)benzoic acid (11) is summarized in Scheme
4. Acetylation of methyl 4-amino-3-hydroxybenzoate
(19) in the presence of acetic anhydride and DMAP gave 24
Scheme 3.
Scheme 4.

V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
2491
in quantitative yield. The nitration of 24 using HNO3 and
acetic anhydride provided the desired regioisomer 25.
Generation of the phenol 26 in the presence of Zn and
methanol followed by O-alkylation with 3-bromopentane
provided methyl 4-(N-acetylamino)-5-nitro-3-(3-penty-
loxy)benzoate (27) in 80% yield. The reduction of the
nitro functionality using catalytic hydrogenation gave
the amine 28. Saponi®cation of the ester in 28 provided
target 10. Alternatively, compound 28 underwent gua-
nylation with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-
thiopseudourea to provide 29. The acidic removal of the
Boc groups followed by basic hydrolysis of the ester
provided the target molecule 11.
(IC50=25±37 mM for N9), while compound 9, which con-
tained a 4-heptyl group, was roughly 10 times less e€ective
(IC50=230 mM). Comparison of the activity of com-
pounds 6±8 to their parent structures (BANA 101 or
BANA 108) reveals that the hydrophobic 3-pentyl group-
ing enhances activity by at least 400 times. Also of interest
was the observation that compounds 6±9 exhibited 20-
to >100-fold selectivity for NA from in¯uenza A as
compared to in¯uenza B.
In order to con®rm the expected orientation of the
hydrophobic group in the glycerol binding subsite, the
crystal structure of the complex of 8 with NA was
determined (Fig. 3). As revealed by the crystal structure,
the interactions of the carboxylic acid and the acetami-
do groups were similar to those observed for 3. The
carboxylate interacts in a strongly positively charged
Biological Assays
All compounds were evaluated for in vitro inhibitory
activity using puri®ed NA from the H2N2 and H1N9
strains of in¯uenza A and from B/Lee/40. Since in our
experience the potency of benzoic acid inhibitors for N9
(an avian strain) correlates well with N2 (infects
humans), only selected compounds were tested against
H2N2. The assay was based upon a published method.32
Table 1. In vitro inhibitory e€ects of benzoic acid analogues on
in¯uenza A and B neuraminidases (IC50, mM),a as compared with sia-
lic acid derivatives
Compound
H2N2
H1N9
B/Lee/40
3
3
3
4
6
7
8
9
10
5Â10
1Â10
4Â10
3
0.5
>670
2300
>5000
>10,000
2000
27
25
37
230
15
3
X-ray Crystallography
Puri®ed N9 NA was crystallized using the hanging drop
method, and the resulting crystals were di€racted
strongly to at least 2 A resolution. Inhibitor complexes
with enzyme were prepared by soaking the crystal in a
1±2 mM solution of the inhibitor in stabilization bu€er
for at least 24 h.
15
8
1
11
500
BANA 101b
BANA 108b
BANA 113b
>10,000
>10,000
10
>10,000
>20,000
10c
a
IC50 values are the mean of duplicate experiments. In all cases each
IC50 value di€ered from its duplicate by less than twofold.
b
Data taken from ref 20.
Determined using B/Mem/89 neuraminidase.
Results and Discussion
c
As depicted in Figure 1, the crystal structure of 3 in com-
plex with NA21 revealed that the C-8 and C-9 hydroxyl
groups interact with Glu 278, while the carbon framework
of the glycerol side chain makes hydrophobic interactions
with Arg 226. Initially, when BANA 113 was designed, the
guanidino group was expected to mimic the interactions
of the guanidine in compound 3. The guanidine group in 3
interacts in the C-4 subsite containing Asp 152, Glu 229
and Glu 120. However, in BANA 113 the guanidino group
occupies the glycerol subsite (C-6 subsite) of 3 and inter-
acts with Glu 278 (Fig. 2). This may be due to the struc-
tural constraints of the planar benzene ring. However, the
presence of an additional substituent, such as a lipophilic
side chain at the 5-position of BANA 113, should occupy
the glycerol binding subsite of 3 and thus force the guani-
dino group into the C-4 subsite. To test the feasibility of
this approach, the simple model compounds 6±9 were
synthesized. Compounds 6±8 all contain the 3-pentyl side
chain, di€ering only in the mode of connection to the
benzene ring, while compound 9 contains the more
hydrophobic 4-heptyl group.
Figure 3. X-ray crystal structure of the binding site region for the
complex formed between benzoic acid 8 and N9 neuraminidase.
The results of enzyme inhibition assays for 6±9 are given in
Table 1. Those compounds containing the 3-pentyl group
(6±8) were equally e€ective inhibitors of in¯uenza A NA

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V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
pocket created by three arginines (Arg 119, Arg 294,
Arg 372). The carbonyl of the N-acetyl group hydrogen
bonds to Arg 153 and the methyl group occupies a
hydrophobic pocket created by Trp 180 and Ile 224.
However, the pentyloxy group interacts in a hydro-
phobic pocket lined by Glu 278, Arg 226, Ala 246 and
Ile 224. In BANA 113 the guanidine occupies this same
subsite and makes electrostatic interactions with Glu
278. In the complex with inhibitor 8 this interaction is
not available, and Glu 278 adopts a di€erent con-
formation in which it interacts with Arg 226, creating a
hydrophobic pocket into which the pentyloxy group
binds. Despite the identical location of the conserved
amino acids that form the ®rst shell in in¯uenza A and
B sialidases, these compounds selectively inhibit siali-
dase A. This result is consistent with earlier reports on
hydrophobic carboxamide analogues of sialic acid.27
For example, inhibitors 4a and 4b exhibit a magnitude
of selectivity for in¯uenza A over in¯uenza B similar to
that observed for 6±8. In the crystal structures of 4b
with in¯uenza A and B sialidases, the movement of Glu
278 (Glu 276 in in¯uenza B) was accompanied by for-
mation of a salt bridge with Arg 226 (as also observed
for 8 in complex with ¯u B). In in¯uenza A sialidase,
this rearrangement resulted in minimal disruption of the
surrounding protein structure, whereas in B sialidase
this movement created distortions in the protein back-
bone near Glu 278 and in the second amino acid shell,
since this region contains di€erences among non-
conserved amino acids as compared to type A. Thus salt
bridge formation in in¯uenza B sialidase, in comparison
to in¯uenza A, is believed to be associated with addi-
tional energy penalties, resulting in a much weaker
inhibition constant. We anticipate that the same factors
account for the type A selectivity of inhibitors 6±9.
Figure 4. X-ray crystal structure of the binding site region for the
complex formed between benzoic acid 11 and N9 neuraminidase.
example, the conformation of the guanidine required for
bound 11 may be a higher energy one.
This study clearly demonstrates that properly sub-
stituted benzoic acids interact with neuraminidase in a
similar manner to non-aromatic inhibitors, and that
benzoic acids provide an attractive structural template
for developing more e€ective agents. Since hydrophobic
groupings on this template can signi®cantly enhance
binding, the presence of a hydrophobic grouping and an
alternative polar side chain for interaction in the C-4
subsite (other than guanidine) may provide more potent
benzoic acid inhibitors.
In light of these results, compounds 10 and 11, which
contain amino and guanidino side chains, respectively,
at the 5-position were synthesized. Compound 10
inhibited in¯uenza A NA with an IC50 value of 15 mM
and compound 11 with an IC50 value of 3 mM. The
activity of 11 represents a smaller enhancement in
activity than was anticipated if the hydrophobic inter-
actions of 8 were maintained and the guanidino group
properly oriented into the comparable binding subsite
of 3. Thus the crystal structure of inhibitor 11 in com-
plex with the enzyme was determined. As shown in
Figure 4, interactions of the carboxylate, pentyloxy and
acetamido groups were preserved as also observed for
compound 8. Additionally, as anticipated, the guanidine
in compound 11 occupies the guanidine subsite (C-4
subsite) of 3, interacting electrostatically with Glu 229
and Asp 152. This result was due to the structural con-
straints imposed by the 3-pentyloxy group, which occu-
pies the large hydrophobic pocket created by Glu 278,
Arg 226, Ala 246 and Ile 224.
Experimental
General methods
Melting points were obtained on an electrothermal
melting point apparatus and are uncorrected. 1H NMR
and 13C NMR spectra were recorded on a Bruker ARX
300. Infrared spectra were recorded on a Bruker FT
infrared spectrophotometer. Combustion analyses were
provided by Atlantic Microlabs of Atlanta, GA. Mass
spectra were obtained on a Perkin±Elmer API triple-
quadrupole mass spectrometer using positive electrospray
ionization. Analytical chromatography was performed on
Whatman PE Sil G/UV silica gel plates. Flash chroma-
tography was performed using J.T. Baker silica gel (40
mM). Tetrahydrofuran (THF) was distilled from sodium
metal/benzophenone. Dichloromethane (CH2Cl2) and
benzene were distilled from calcium hydride. Anhydrous
N,N0-dimethylformamide (DMF) was purchased from
Aldrich in sure-seal containers. Methanol was distilled
from Mg(OMe)2. All other commercially obtained
reagents were used as received. The H2N2 neur-
aminidase (A/Singapore/1/57) was obtained from Dr.
We previously reported31 a similar result upon evaluat-
ing 4-(N-acetylamino)-3,5-diguanidinobenzoic acid in
comparison to BANA 113. While the reasons for the
poor improvement in binding of benzoic acids which
orient the guanidino group into the C-4 subsite of 3
remain unclear, some possibilities may be noted. For

V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
2493
Robert Webster at St. Jude Children's Hospital, Mem-
phis, TN. The H1N9 neuraminidase was obtained from
Dr. Graeme W. Laver at John Curtin School of Medical
Research, Australian National University, Canberra
2601, Australia.
product precipitated. The mixture was cooled for 30 min,
®ltered and air dried to give 9 (280 mg, 98.0%) as a white
solid: mp 165±167ꢀC (chloroform). MS m/z 294 (M+1);
1H NMR (300 MHz, CDCl3) d 12.3 (br, 1H), 8.5 (d, 1H,
J=8.5 Hz), 8.0 (br, 1H), 7.74 (dd, 1H, J=1.6 and 8.5 Hz),
7.6 (d, 1H, J=1.6 Hz), 4.4 (m, 1H), 2.2 (s, 3H), 1.68 (m,
4H), 1.4 (m, 4H), 0.95 (t, 6H, J=7.3 Hz); 13C NMR
(CD3OD) d 171.6, 169.5, 148.9, 133.7, 127.4, 123.6, 121.6,
114.9, 80.5, 37.1, 24.4, 19.7, 14.6. Anal. calcd for
4-(N-Acetylamino)-3-[N-(2-ethylbutanoyl)amino]benzoic
acid (6). Compound 15 (0.15 g, 0.49 mmol) was dissolved
in 1 N sodium hydroxide (3.5 mL) and stirred at room
temperature for 2 h. To the pale orange reaction mixture
was carefully added glacial acetic acid, to pH 5, and the
reaction mixture was then cooled to 0ꢀC. The white pre-
cipitate was ®ltered and washed with cold water to pro-
vide 0.14 g (100%) of 6 as a white solid: mp 286±291ꢀC.
MS m/z 293 (M+1); 1H NMR (300 MHz, DMSO) d 0.89
(t, 6H, J=7.3 Hz), 1.40±1.61 (m, 4H), 2.03 (s, 3H), 2.26±
2.32 (m, 1H), 7.57 (d, 1H, J=8.1 Hz), 7.54 (d, 1H J=8.1
Hz), 7.92 (s, 1H), 9.92 (s, 1H), 10.01 (s, 1H). Anal. calcd
.
C16H23NO4 0.25H2O: C, 64.50; H, 7.80; N, 4.70. Found:
C, 64.14; H, 7.71; N, 4.71.
4-(N-Acetylamino)-5-amino-3-(3-pentyloxy)benzoic acid
(10). A solution of 28 (100 mg, 0.340 mmol) in 0.5 mL
of methanol and 2 mL of 0.5 N NaOH was stirred at room
temperature for 2 h. The reaction mixture was acidi®ed
with glacial acetic acid, the methanol was removed under
vacuum, and the residual aqueous layer was cooled to give
a precipitate. This was ®ltered and dried to give 10 (60 mg,
63%): mp 165±170ꢀC (decomposed). MS m/z 281 (M+1);
1H NMR (300 MHz, CDCl3) d 7.5 (br, 1H), 7.1 (br, 1H),
7.0 (br, 1H), 5.5 (br, 3H), 4.25 (m, 1H), 2.2 (s, 3H), 1.65
.
for C15H20N2O4 0.25H2O: C, 60.71; H, 6.91; N, 9.44.
Found: C, 60.94; H, 6.94; N, 9.49.
4-(N-Acetylamino)-3-[N-(3-pentyl)amino]benzoic acid
(7). Compound 17 (0.105 g, 0.380 mmol) was dissolved
in 1 N sodium hydroxide. After 6 h the orange mixture
was carefully acidi®ed with acetic acid to pH 4.5±5 and
then cooled in the refrigerator. The precipitate was ®ltered
and washed with cold water to provide 0.080 g (80%) of 7
as a light brꢀown solid. Rf 0.16 (50% ether±chloroform);
.
(m, 4H), 0.95 (t, 6H). Anal. calcd for C14H20N2O4 0.5
H2O: C, 58.13; H, 7.27; N, 9.67. Found: C, 57.79; H, 7.12;
N, 9.42.
4-(N-Acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic
acid (11). A solution of 29 (100 mg, 0.18 mmol) and
tri¯uoroacetic acid (150 mg) in 0.5 mL of CH2Cl2 was
stirred at room temperature for 4 h. Tri¯uoroacetic acid
(500 mg) and CH2Cl2 (0.5 mL) were added and stirring
was continued for another 5 h. The solvent and excess
tri¯uoroacetic acid were removed under vacuum to give
86 mg of white solid which was dissolved in methanol (1
mL) and 1 N NaOH (2 mL). The resulting suspension was
stirred at room temperature for 16 h. The methanol was
evaporated below 40ꢀC and the aqueous layer was neu-
tralized with glacial acetic acid. The solvent was evapo-
rated to dryness, and the crude solid residue was dissolved
in 1 mL of water and cooled to give 11 (40 mg, 66%) as a
white precipitate: mp 221±223ꢀC. MS m/z 323 (M+1); 1H
NMR (300 MHz, D2O) d 7.51 (d, 1H), 7.35 (d, 1H), 4.3
(m, 1H), 2.1 (s, 3H), 1.6 (m, 4H), 0.85 (t, 6H). Anal. calcd
1
mp 145±148 C (water). MS m/z 265 (M+1). H NMR
(300 MHz, DMSO) d 0.89 (t, 6H, J=7.3 Hz), 1.45±1.58
(m, 4H), 2.09 (s, 3H), 3.24 (m, 1H), 4.76 ( d, 1H, J=7.9
Hz), 7.12 (dd, 1H, J=1.6 and 8.0 Hz), 7.17 (s, 1H), 7.37
(d, 1H, J=8.0 Hz), 12.3 (br, 1H). Anal. calcd for
C14H20N2O3: C, 61.54; H, 7.33; N, 10.26. Found: C, 61.39;
H, 7.33; N, 10.21.
4-(N-Acetylamino)-3-(3-pentyloxy)benzoic acid (8). A
suspension of 22 (130 mg, 0.460 mmol) in 0.2 mL of
methanol and 0.8 mL of 1N NaOH was stirred at room
temperature for 15 h. The methanol was evaporated
under vacuum below 40ꢀC and the reaction mixture was
diluted with 0.5 mL of 1 N NaOH and water (5 mL). The
aqueous layer was washed with 5 mL of ether and then
acidi®ed with 1 N HCl, during which time the product
precipitated. The mixture was cooled at 0ꢀC for 30 min,
®ltered, and air dried to give 8 (100 mg, 82.0%): mp 149±
152ꢀC (ether). MS m/z 266 (M+1); 1H NMR (300 MHz,
CDCl3) d 8.5 (d, 1H, J=8.4 Hz), 8.0 (br, 1H), 7.74 (dd,
1H, J=1.7 and 8.4 Hz), 7.58 (d, 1H, J=1.7 Hz), 4.33 (m,
1H), 2.24 (s, 3H), 1.7 (m, 4H), 0.99 (t, 6H); 13C NMR
(CDCl3) d 171.5, 168.9, 146.0, 133.5, 124.3, 123.9, 118.9,
113.6, 81.9, 26.0, 25.13, 9.6. Anal. calcd for
.
for C15H22N4O4 1.25H2O: C, 52.25; H, 7.11; N, 16.25.
Found: C, 52.24; H, 6.87; N, 16.15.
Methyl 3,4 - diaminobenzoate (13). 3,4-Diaminobenzoic
acid (12, 20.0 g, 0.130 mmol) was dissolved in dry
methanol (460 mL) and concentrated sulfuric acid (17
mL). The mixture was stirred at re¯ux under a nitrogen
atmosphere. After 15 h the dark reaction mixture was
cooled to room temperature and quenched with 10%
sodium bicarbonate (400 mL) to pH 7.5. Ethyl acetate
(600 mL) was added, the mixture shaken in a separatory
funnel, and the aqueous layer was further extracted with
ethyl acetate (3Â200 mL). The combined organic layers
were washed with 5% sodium bicarbonate (3Â200 mL)
and water (3Â200 mL), and then dried over sodium
sulfate. The solvent was removed in vacuo. The crude
material was puri®ed by recrystallization from benzene:
hexane to provide 14.6 g (67.0%) of 13 as a pale yellow
solid: mp 103±105ꢀC (benzene:hexane). Rf 0.50 (80%
EtOAc:hexane); MS m/z 167 (M+1); 1H NMR (300
.
C14H19NO4 0.5H2O: C, 61.31; H, 7.38; N, 5.10. Found: C,
61.73; H, 7.04; N, 5.21.
4-(N-Acetylamino)-3-(4-heptyloxy)benzoic acid (9). A
suspension of 23 (300 mg, 0.980 mmol) in 1.5 mL of
methanol and 2.0 mL of 1 N NaOH was stirred at room
temperature for 12 h. The methanol was evaporated
under vacuum below 40ꢀC, and the reaction mixture
was diluted with 1 mL of 1 N NaOH and water (5 mL).
The aqueous layer was washed with 5 mL of ether and
then acidi®ed with 1 N HCl, during which time the

2494
V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
MHz, CDCl3) 3.25±4.0 (br s, 4H), 3.85 (s, 3H), 6.68 (d,
1H), 7.41 (d, 1H), 7.46 (dd, 1H). Anal. calcd for
C8H10N2O2: C, 57.82; H, 6.07; N, 16.86. Found: C, 58.02;
H, 6.05; N, 16.92.
0ꢀC with an ice bath. To the red solution was added
acetic anhydride (1 mL) and the reaction mixture was
allowed to equilibrate to room temperature. After 18 h,
ethyl acetate (39 mL) and water (23 mL) were added to
the light-red reaction mixture. The organic layer was
washed with water (3Â23 mL) and the combined aque-
ous layers were extracted with ethyl acetate (2Â23 mL)
and dried over sodium sulfate. After ®ltration the solvent
was removed in vacuo. The residue was puri®ed by
recrystallization from ethyl acetate:hexane to provide
0.122 g (68.0%) of 17 as o€-white crystals: mp 107±
109ꢀC (EtOAc:hexane). Rf 0.50 (60% ethyl acetate:hex-
ane); MS m/z 278 (M+1); 1H NMR (300 MHz, DMSO)
d0.89 (t, 6H, J=7.4 Hz), 1.52 (m, 4H), 2.10 (s, 3H), 3.25
(m, 1H), 3.80 (s, 3H), 4.82 (d, 1H, J=7.8 Hz), 7.13 (d,
1H, J=8.1 Hz), 7.16 (s, 1H), 7.43 (d, 1H, J=8.1 Hz),
9.24 (br s, 1H). Anal. calcd for C15H22N2O3: C, 64.73; H,
7.97; N, 10.06. Found: C, 64.54; H, 7.97; N, 9.98.
Methyl 4-amino-3-[N-(2-ethylbutanoyl)amino]benzoate
(14). 2-Ethylbutyric acid (0.38 mL, 3.0 mmol) was dis-
solved in dry tetrahydrofuran (2.5 mL). N,N0-Carbo-
nyldiimidazole (0.54 g, 3.3 mmol) was then added and
bubbling was observed. After 10 min, methyl 3,4-diami-
nobenzoate (13, 0.50 g, 3.0 mmol) was added and the yel-
low reaction mixture was stirred at room temperature.
After 30 h, water (3 mL) and ethyl acetate (3 mL) were
added. The mixture was ®ltered to provide 0.64 g (80%) of
14 as a white solid: mp 200±203ꢀC (water). Rf 0.47 (50%
1
ether±chloroform); MS m/z 265 (M+1); H NMR (300
MHz, DMSO) d 0.86 (t, 6H, J=7.4 Hz), 1.49 (m, 4H), 2.25
(m, 1H), 3.75 (s, 3H), 5.63 (br s, 2H), 6.75 (d, 1H, J=8.5
Hz), 7.52 (dd, 1H, J=2.0 and 8.4 Hz), 7.86 (d, 1H, J=2.0
Hz), 9.20 (s, 1H). Anal. calcd for C14H20N2O3: C, 63.64; H,
7.58; N, 10.61. Found: C, 63.58; H, 7.67; N, 10.67.
Methyl 4-amino 3-hydroxybenzoate (19). Acid 18 (10.0
g, 65.0 mmol) was dissolved in a mixture of dry metha-
nol (700 mL) and concentrated sulfuric acid (15 mL).
The dark reaction mixture was stirred under re¯ux
under a nitrogen atmosphere. After 14 h, the dark mix-
ture was quenched with solid sodium bicarbonate until
pH 7±8, and ethyl acetate was added (600 mL). This was
separated and the aqueous layer was extracted with
additional ethyl acetate (3Â200 mL). The combined
organic layers were washed with 5% sodium bicarbonate
(2Â100 mL), water (2Â100 mL), and dried over sodium
sulfate. After ®ltration the solvent was removed in vacuo
to provide 9.06 g (83.0%) of 19 as a brown±red solid: mp
Methyl 4-(N-acetylamino)-3-[N-(2-ethylbutanoyl)amino]-
benzoate (15). Aniline 14 (0.15 g, 0.57 mmol) was dis-
solved in acetic anhydride (2.17 mL, 23.0 mmol) and the
white reaction mixture was stirred at room temperature.
After 1.5 h, ethyl acetate (38 mL) and water (24 mL) were
added. The organic layer was washed with water (3Â24
mL). The combined aqueous layers were extracted with
ethyl acetate (2Â35 mL) and the organic layers were dried
(sodium sulfate), ®ltered and the solvent was removed in
vacuo to provide 0.15 g (88%) of 15 as a white solid: mp
138±142ꢀC. Rf 0.53 (ether); MS m/z 307 (M+1); H
114±116 C. MS m/z 168 (M+1); H NMR (300 MHz,
DMSO) d 3.85 (s, 3H), 4.18 (br s, 2H), 6.68 (d, 1H), 7.50
(d, 1H), 7.58 (m, 1H). Anal. calcd for C8H9NO3: C, 57.48;
H, 5.43; N, 8.38. Found: C, 57.55; H, 5.38; N, 8.30.
ꢀ
1
1
NMR (300 MHz, DMSO) d 0.89 (t, 6H, J=7.3 Hz), 1.53
(m, 4H), 2.09 (s, 3H), 2.25 (m, 1H), 3.84 (s, 3H), 7.75 (m,
2H), 8.12 (s, 1H), 9.51 (br s, 2H). Anal. calcd for
C16H22N2O4: C, 62.74; H, 7.19; N, 9.15. Found: C, 62.58;
H, 7.25; N, 9.11.
Methyl 4-amino-3-(3-pentyloxy)benzoate (20) Method A.
A suspension of aminophenol 19 (500 mg, 3.00 mmol),
bromopentane (500 mg, 3.30 mmol) and K2CO3 (500
mg, 3.60 mmol) in 10 mL of acetone was stirred at
re¯ux for 12 h. Bromopentane (500 mg, 3.30 mmol) and
K2CO3 (250 mg, 1.80 mmol) were added and the reac-
tion continued for another 6 h, after which additional
bromopentane (500 mg, 3.30 mmol) and K2CO3 (250
mg, 1.80 mmol) were added. Stirring was continued for
another 6 at re¯ux. The reaction mixture was evapo-
rated to dryness. The crude material was suspended in
15 mL of 2 N NaOH and the product extracted into
ethyl acetate (3Â25 mL). The combined organic layers
were washed with water (3Â10 mL), dried (Na2SO4) and
evaporated to give 20 (600 mg, 85.0%) as an oil which
solidi®ed upon standing: mp 45±46ꢀC (ether:hexane).
Methyl 4 - amino - 3 - [N - (3 - pentyl)amino]benzoate (16).
Methyl 3,4-diaminobenzoate (13, 2.00 g, 12.0 mmol) was
dissolved in dry tetrahydrofuran (6 mL). To the dark
solution was added 3-bromopentane (1.82 g, 12.1 mmol)
and pyridine (2.35 g, 29.7 mmol). The dark reaction mix-
ture was stirred under re¯ux. After 14 h, the mixture was
cooled to room temperature and ethyl acetate (127 mL)
and water (127 mL) were added. The organic layer was
washed with water (3Â125 mL). The combined aqueous
layers were extracted with ethyl acetate (3Â75 mL). The
combined organic layers were dried over sodium sulfate,
®ltered and the solvent was removed in vacuo to provide a
crude brown solid. Puri®cation of the crude product by
¯ash chromatography on silica using 40% ethyl acet-
ate:hexane provided 1.17 g (41.0%) of 16 as an amber oil.
Intermediate 16 was carried forward without additional
puri®cation. Rf 0.57 (40% ethyl acetate:hexane); MS m/z
237 (M+1); 1H NMR (300 MHz, DMSO) d 0.96 (t, 6H,
J=7.3 Hz), 1.63 (m, 4H), 3.35 (br s, 1H), 3.92 (s, 3H), 6.67
(d, 1H, J=8.0 Hz), 7.48 (m, 2H).
Method B. To a suspension of 60% NaH (145 mg, 6.00
mmol) in 1 mL of dry DMF was added a solution of
aminophenol 19 (500 mg, 3.00 mmol) in 1.5 mL of
DMF followed by 3-bromopentane (453 mg, 3.00
mmol) in 1.5 mL of DMF. After 3 h of stirring addi-
tional NaH (145 mg, 6.00 mmol) and additional 3-bro-
mopentane (453 mg, 3.00 mmol) were added, and the
stirring was continued for another 9 h. The DMF was
removed under vacuum and the crude reaction mixture
Methyl 4-N-acetylamino 3-[N-(3-pentylamino)]benzoate
(17). Compound 16 (0.155 g, 0.560 mmol) was dis-
solved in dry tetrahydrofuran (2.3 mL) and cooled to

V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
2495
was suspended in 15 mL of 2 N NaOH. The basic aque-
ous layer was extracted with chloroform (3Â25 mL) and
the combined organic layers were dried (Na2SO4) and
evaporated to give 20 (600 mg, 85.0%) as an oil which
solidi®ed upon standing: mp 45±46ꢀC (ether:hexane). MS
m/z 238 (M+1); 1H NMR (300 MHz, CDCl3) d 7.5 (dd,
1H, J=1.8 and 8.5 Hz), 7.45 (d, 1H, J=1.8 Hz), 6.65 (d,
1H, J=8.5 Hz), 4.2±4.3 (m, 3H), 3.85 (s, 3H), 1.7 (m, 4H),
0.95 (t, 6H, J=7.4 Hz). Anal. calcd for C13H19NO3: C,
65.82; H, 8.01; N, 5.90. Found: C, 65.85; H, 8.06; N, 5.97.
Methyl 4-(N-acetylamino)-3-acetyloxybenzoate (24). A
suspension of phenol 19 (3.00 g, 18.0 mmol) and DMAP
(50 mg) in 40 mL of acetic anhydride was stirred at room
temperature for 12 h, during which time the product pre-
cipitated. The reaction mixture was diluted with 100 mL
of water, ®ltered, and the collected solid air dried to give
24 (4.50 g, 100%) as a white solid: mp 156±159ꢀC (ether).
MS m/z 252 (M+1); 1H NMR (300 MHz, CDCl3) d 8.4
(d, 1H, J=8.6 Hz), 7.9 (dd, 1H, J=1.9 and 8.6 Hz), 7.82
(d, 1H, J=1.9 Hz), 7.38 (br, 1H), 3.9 (s, 3H), 2.4 (s, 3H),
2.2 (s, 3H). Anal. calcd for C12H13NO5: C, 57.37; H, 5.18;
N, 5.57. Found: C, 57.61; H, 5.08; N, 5.41.
Methyl 4-amino-3-(3-heptyloxy)benzoate (21). To a sus-
pension of 60% NaH (450 mg, 18.8 mmol) in 1 mL of
dry DMF were added a solution of aminophenol 19
(1.00 g, 5.98 mmol) in 1.5 mL of DMF and 4-bromo-
heptane (1.60 g, 8.93 mmol) in 2.5 mL of DMF. After 3
h of stirring NaH (400 mg, 16.6 mmol) and 4-bromo-
heptane (500 mg, 2.79 mmol) were added, and the stir-
ring was continued for an additional 9 h. The DMF was
removed under vacuum and the crude reaction mixture
was suspended in 15 mL of 2 N NaOH. The basic aqueous
layer was extracted with chloroform (3Â25 mL) and the
combined organic layers were dried (Na2SO4) and evapo-
rated to give 21 (1.10 g, 69.0%) as an oil, which was car-
ried forward in this form without additional puri®cation.
Methyl 4-(N-acetylamino)-3-acetyloxy-5-nitrobenzoate
(25). An ice cold solution of 24 (1.00 g, 4.00 mmol) in a
mixture of dioxane (8 mL) and acetic anhydride (8 mL)
was treated slowly with ice cold nitrating reagent (4 mL
of HNO3 and 4 mL of acetic anhydride). The resulting
mixture was stirred at 0ꢀC for 15 min and at room
temperature for 5 h. The reaction mixture was then dilu-
ted with 50 mL of ice water and the product was extracted
into ethyl acetate (3Â50 mL). The combined organic lay-
ers were washed with ice cold water (3Â30 mL), dried
(Na2SO4) and evaporated under vacuum to give a yellow
oil. The oil was triturated with hexane (3Â5 mL), the
hexane was discarded, and the residue was dried on a high
vacuum pump, which resulted in partial solidi®cation.
The semisolid was triturated with ether, ®ltered, and
washed with small amounts of ether to give 25 (0.50 g,
1
MS m/z 238 (M+1); H NMR (300 MHz, CDCl3) d 7.5
(dd, 1H, J=1.7 and 8.5 Hz), 7.45 (d, 1H, J=1.7 Hz), 6.65
(d, 1H, J=8.5 Hz), 4.4 (m, 1H), 4.25 (br, 2H) 3.85 (s, 3H),
1.65 (m, 4H), 1.4 (m, 4H), 0.92 (t, 6H, J=7.5 Hz).
42.7%): mp 139±141ꢀC. MS m/z 297 (M+1); H NMR
1
Methyl 4-(N-acetylamino)-3-(3-pentyloxy)benzoate (22).
A mixture of 20 (300 mg, 1.20 mmol) and DMAP (25 mg)
in 1 mL of acetic anhydride was stirred at room tempera-
ture for 12 h. The reaction mixture was diluted with 50 mL
of CH2Cl2 and the organic layer was washed with 1 N HCl
(15 mL), H2O (15 mL) and saturated NaHCO3 solution (15
mL), dried (Na2SO4) and evaporated to give an oil. This
was puri®ed by ¯ash column chromatography on silica gel
(ether) to give 22 (325 mg, 92.0%) as an oil which solidi®ed
upon standing: mp 88±89ꢀC (hexane). MS 280 m/z (M+1);
1H NMR (300 MHz, CDCl3) d 8.45 (d, 1H, J=8.5 Hz), 8.0
(br, 1H), 7.65 (dd, 1H, J=1.8 and 8.5 Hz), 7.52 (d, 1H,
J=1.8 Hz), 4.25 (m, 1H), 3.88 (s, 3H), 2.2 (s, 3H), 1.7 (m,
4H), 0.95 (t, 6H, J=7.4 Hz); 13C NMR (CDCl3) d 168.4,
166.7, 145.9, 132.9, 124.8, 122.9, 118.7, 113.2, 81.7, 52.0,
25.9, 25.0, 9.5. Anal. calcd for C15H21NO4: C, 64.51; H,
7.52; N, 5.02. Found: C, 64.51; H, 7.53; N, 5.08.
(300 MHz, CDCl3) d 8.58 (d, 1H, J=1.6 Hz), 8.48 (br,
1H), 8.13 (d, 1H, J=1.6 Hz), 3.96 (s, 3H), 2.3 (s, 3H), 2.2
(s, 3H). Anal. calcd for C12H12N2O7: C, 48.65; H, 4.05; N,
9.46. Found: C, 48.43; H, 4.26; N, 9.21.
Methyl 4-(N-acetylamino)-3-hydroxy-5-nitrobenzoate
(26). A solution of 25 (780 mg, 2.60 mmol) in 25 mL of
methanol was treated with activated Zn (1.00 g). The
resulting mixture was then stirred at room temperature
for 1.5 h. The reaction mixture was ®ltered, the solvent
was removed under vacuum and the resulting oil was
dissolved in 100 mL of ethyl acetate. The organic layer
was then washed with 0.5 N HCl (3Â10 mL), dried
(Na2SO4) and evaporated to give a solid which was
suspended in ether and ®ltered to give 26 (600 mg,
89.7%): MS m/z 253 (M 1); 1H NMR (300 MHz,
CD3OD) d 10.25 (br, 1H), 9.45 (br, 1H), 8.4 (d, 1H,
J=1.9 Hz), 7.9 (d, 1H, J=1.9 Hz), 4.0 (s, 3H), 2.4 (s,
3H). This intermediate was carried forward without
additional puri®cation.
Methyl 4-(N-acetylamino)-3-(4-heptyloxy)benzoate (23).
A mixture of 21 (1.00 g, 3.70 mmol) and DMAP (10 mg)
in 2 mL of acetic anhydride was stirred at room tem-
perature for 2 h. The reaction was diluted with 50 mL of
ether, washed with water (3Â10 mL), dried (Na2SO4)
and evaporated to give 23 (1.10 g, 95.0%) as a white
Methyl 4-(N-acetylamino)-5-nitro-3-(3-pentyloxy)benzo-
ate (27). A solution of phenol 26 (480 mg, 1.89 mmol),
3-bromopentane (800 mg, 5.30 mmol) and K2CO3 (530
mg, 3.80 mmol) in 10 mL of acetone was stirred at
re¯ux for 2 h. Additional 3-bromopentane (260 mg, 1.72
mmol) was added and the stirring continued for another
2 h. After this time 3-bromopentane (820 mg, 5.42
mmol) and K2CO3 (450 mg, 3.25 mmol) were again
added and the stirring continued for 12 h. The acetone
was removed under vacuum, and the crude mixture was
suspended in 100 mL of ethyl acetate and washed with
water (3Â15 mL). The aqueous layer was extracted with
solid: mp 66±68ꢀC (hexane). MS m/z 308 (M+1); H
1
NMR (300 MHz, CDCl3) d 8.4 (d, 1H, J=8.5 Hz), 7.9
(br, 1H), 7.6 (dd, 1H, J=1.7 and 8.5 Hz), 7.5 (d, 1H,
J=1.7 Hz), 4.4 (m, 1H), 3.89 (s, 3H), 2.2 (s, 3H), 1.66
(m, 4H), 1.48 (m, 4H), 0.94 (t, 6H, J=7.5 Hz); 13C
NMR (CDCl3) d 168.7, 167.1, 146.3, 133.2, 125.1, 123.2,
119.0, 113.3, 79.4, 52.3, 36.3, 25.3, 18.9, 14.4. Anal.
calcd for C17H25NO4: C, 66.45; H, 8.14; N, 4.56. Found:
C, 66.52; H, 8.14; N, 4.56.

2496
V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
ethyl acetate (25 mL) and the combined organic layers
were dried (Na2SO4) and concentrated to give a solid. This
was puri®ed by ¯ash chromatography on silica gel (2:1,
ether:hexane) to give 27 (500 mg, 82.0%): mp 103±105ꢀC
(ether). MS m/z 325 (M+1); 1H NMR (300 MHz, CDCl3)
d 8.1 (d, 1H, J=1.7 Hz), 7.78 (br, 1H), 7.73 (d, 1H, J=1.7
Hz), 4.37 (m, 1H), 3.89 (s, 3H), 2.2 (s, 3H), 1.7 (m, 4H),
0.95 (t, 6H, J=7.5 Hz); 13C NMR (CDCl3) d 167.99,
164.8, 150.8, 144.1, 126.6, 124.3, 117.6, 116.6, 82.6, 52.6,
25.6, 23.4, 9.2. Anal. calcd for C15H20N2O6: C, 55.55; H,
6.17; N, 8.64. Found: C, 55.69; H, 6.17; N, 8.62.
The complexes of the inhibitor with NA were prepared
by di€using the compound into N9 crystals in a stabili-
zation bu€er which was the same as the reservoir solu-
tion used in crystallization. The ®nal concentration of
the inhibitor in the solution was 1±2 mM. The N9 crys-
tals were allowed to equilibrate with the inhibitor com-
pound for at least 24 h before data collection.
All X-ray intensity measurements were recorded with a
Nicolet/Siemens X-100 multiwire area detector on a
Rigaku RU-300 rotating anode X-ray generator oper-
ating at 100 mA and 50 kV with 0.3Â0.3 focus and a Cu
anode. The data collection parameters were crystal to
detector distance of 16 cm, swing angle of 28ꢀ, frame
width of 0.1ꢀ, and exposure time of 240±300 s. Each
crystal provided about 800±900 frames of data before
radiation damage deteriorated the di€raction quality.
Methyl 4-(N- acetylamino)- 5-amino- 3-(3 -pentyloxy)-
benzoate (28). A suspension of 27 (300 mg, 0.920 mmol)
and 10% Pd/C (275 mg) in 15 mL of methanol was shaken
in the presence of hydrogen gas (30 psi) for 1 h. The reac-
tion mixture was diluted with 25 mL of methanol, ®ltered,
and the ®lter was washed well with methanol (25 mL). The
methanol was evaporated to dryness under vacuum to
give 28 (270 mg, 99.0%) as an oil which was crystallized
from ether:hexane: mp 128±129ꢀC. MS m/z 295 (M+1);
1H NMR (300 MHz, CDCl3) d 7.39 (br, 1H), 7.10 (s,1H),
7.01 (s, 1H), 4.46 (br, 2H), 4.25 (m, 1H), 3.89 (s, 3H), 2.25
(s, 3H), 1.67 (m, 4H), 0.95 (t, 6H, J=7.4 Hz). Anal. calcd
for C15H22N2O4: C, 61.22; H, 7.48; N, 9.52. Found: C,
61.07; H, 7.52; N, 9.41.
The X-ray intensity data were processed using the
XENGEN suite of programs. The integrated intensities
were then scaled and merged to produce a ®nal data set
containing only unique re¯ections. The completeness of
data to 2.1 A was generally about 90%. The re®nement
of the inhibitor complexes was performed using the X-
PLOR simulated annealing protocol. Di€raction data in
the 10±2.1 A resolution range were used in the re®nement
with a 2s cuto€ on F0s. The starting model for the
re®nement of the inhibitor complexes was the 1.9 A
re®ned uncomplexed NA native structure. The water
molecules in the active site of the native structure were
removed prior to the re®nement of the complex struc-
ture. A full cycle of the X-PLOR simulated annealing
protocol was carried out on this model. F0 Fc and
2(F0 Fc) di€erence Fourier maps were calculated, and a
model of the inhibitor molecule was ®tted into the elec-
tron density in the active site. Water molecules were also
placed in the active site based on the di€erence electron
density maps, and these positions were compared to the
active site water structure in the native NA model. At
the end of the simulated annealing protocols and posi-
tional and temperature factor re®nement, the crystal-
lographic R-factor converged to 17±19% at 2 A
resolution for the inhibitor-complex structures.
Methyl 4-(N-acetylamino)-5-[N,N0 -di(tert-butyloxycar-
bonyl)guanidino]-3-(3-pentyloxy)benzoate (29). To an ice
cold solution of 28 (270 mg, 0.920 mmol) in 3 mL of dry
DMF was added 1,3-bis(tert-butoxycarbonyl)-2-methyl-
2-thiopseudourea (423 mg, 1.37 mmol), triethylamine
(190 mg, 1.80 mmol) and HgCl2 (374 mg, 1.37 mmol).
The resulting mixture was stirred at 0ꢀC for 3 h and at
room temperature for 3 h. The DMF was evaporated
below 50ꢀC and the crude product was suspended in 100
mL of ether, ®ltered and evaporated to give an oil. This
was puri®ed by ¯ash chromatography on silica gel (2:1,
ether:hexane) to give 29 (450 mg, 92.0%) as a colorless
oil which was crystallized from ether:hexane: mp 131-
133ꢀ C. MS m/z 537 (M+1); 1H NMR (300 MHz,
CDCl3) d 11.5 (br, 1H), 10.3 (br, 1H), 8.1 (s, 1H), 7.5 (br,
1H), 7.4 (s, 1H), 4.26 (m, 1H), 3.9 (s, 3H), 2.2 (s, 3H), 1.7
(m, 4H), 1.5 (s, 9H), 1.48 (s, 9H), 0.94 (t, 6H); 13C NMR
(CDCl3) d 168.7, 166.1, 162.8, 153.6, 152.5, 133.5, 128.3,
124.1, 118.5, 110.7, 83.3, 81.0, 79.3, 51.9, 27.7, 25.3, 22.9,
9.0. Anal. calcd for C26H40N4O8: C, 58.20; H, 7.46; N,
10.45. Found: C, 58.34; H, 7.49; N, 10.54.
Enzyme Assays
All compounds were evaluated for in vitro inhibitory
actions using the method reported by von Itzstein et
al.32 The NA from the H1N9 strain of in¯uenza was
obtained using the method described by Laver et al.33
The assay employed a spectro¯uorometric technique
that uses 20-(methylumbelliferyl)-a-d-acetylneuraminic
acid as substrate. Cleavage of this substrate by NA
produces a ¯uorescent product which can be quanti®ed.
The assay mixture contained inhibitors at various con-
centrations (4±6 points) and enzyme in 32.5 mM MES
(2-(N-morpholino)ethanesulfonic acid) bu€er, 4 mM
CaCl2 at pH 6.5 (total volume=80 mL). The reaction
was started by the addition of 20 mL of the substrate to
give a ®nal concentration of 75 mM. After 10 min at
37ꢀC, 2.4 mL of 0.1 M glycine:NaOH (pH 10.2) was
added to 0.1 mL of the reaction mixture to terminate
X-ray crystallography
Puri®ed N9 NA was crystallized using the hanging drop
technique in which droplets of protein solution on sili-
conized 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 buf-
fer (1.7 M, pH 6.6). This mixture was equilibrated
through the vapor phase with a reservoir of 1.9 M
potassium phosphate bu€er at pH 6.8. Large rhombic
dodecahedral crystals grew in a few days. The space
group has been identi®ed as cubic, I432, with cell
dimension a=183.8 A. The crystals di€ract strongly to
at least 2 A resolution on a rotating anode source.

V. R. Atigadda et al. / Bioorg. Med. Chem. 7 (1999) 2487±2497
2497
the reaction. A blank was run with the same substrate
solution excluding the enzyme. Fluorescence was read
using an Aminco-Bowman ¯uorescence spectro-
photometer (excitation, 360 nm; emission, 450 nm), and
substrate blanks were subtracted from the sample
readings. The IC50 was calculated by plotting percent
inhibition versus the inhibitor concentration, and
determination of each point was performed in duplicate.
In all cases each IC50 value di€ered from its duplicate by
less than twofold.
14. Air, G. M.; Laver, W. G. Proteins: Struct., Funct., Genet.
1989, 6, 341.
15. Ghate, A. N.; Air, G. M. Eur J. Biochem 1998, 258, 320.
16. Schulman, J. L.; Khakpour, M.; Kilbourne, E. J. Virol
1968, 2, 778.
17. Meindl, P.; Bodo, G.; Palese, P.; Schulman, J.; Tuppy, H.
Virology 1974, 58, 457.
18. Holzer, C. T.; Von Itzstein, M.; Jin, B.; Pegg, M. S.;
Stewart, W. P.; Wu, W. Y. Glycoconjugate J. 1993, 10, 40.
19. Taylor, N. R.; Von Itzstein, M. J. Med. Chem. 1994, 37, 616.
20. von Itzstein, M.; Wu, W. Y.; Jin, B. Carbohydr. Res. 1994,
259, 301.
21. Varghese, J. N.; Epa, V. C.; Colman, P. M. Protein Sci.
1995, 4, 1081.
Acknowledgements
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This work was supported in part by NIH grant U01
AI31888 (M.L., W.J.B., G.M.A.).
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