Human sialidase inhibitors: Design, synthesis, and biological evaluation of 4-acetamido-5-acylamido-2-fluoro benzoic acids

Article abstract of DOI:10.1016/j.bmc.2009.04.065

Recent advances in the sialidase biology have clarified the role of human sialidases (NEU 1 to NEU4) in the development of various disease states such as cancer, diabetes and arteriosclerosis. Isoform selective human sialidase inhibitors could be a therap

Full text of DOI:10.1016/j.bmc.2009.04.065

Bioorganic & Medicinal Chemistry 17 (2009) 4595–4603
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Human sialidase inhibitors: Design, synthesis, and biological evaluation
of 4-acetamido-5-acylamido-2-fluoro benzoic acids
a
b
c
b
Sadagopan Magesh a,d, , Vats Savita , Setsuko Moriya , Tohru Suzuki , Taeko Miyagi ,
*
Hideharu Ishida a, Makoto Kiso a,d,
*
a Department of Applied Bioorganic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Japan
b Department of Biochemistry, Miyagi Cancer Center Research Institute, Japan
c The United Graduate School of Agricultural Sciences, Gifu University, Japan
d Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent advances in the sialidase biology have clarified the role of human sialidases (NEU 1 to NEU4) in
the development of various disease states such as cancer, diabetes and arteriosclerosis. Isoform selective
human sialidase inhibitors could be a therapeutic tool or molecular probes for the exploration of the spe-
cific functions of human sialidases. In the present study, de novo design based virtual screening was per-
formed to find a new class of human sialidase inhibitors using the experimental crystal structure of NEU2
isoform. A few of nitro benzene and fluoro benzoic acid were identified and a series of 4-acetamido-5-
acylamido-2-fluoro benzoic acids were synthesized and, the inhibitory activity of all these compounds
against all human sialidase enzymes was evaluated. All these compounds were found to have a poor
inhibitory activity and only NEU2 showed more sensitivity to this series of compounds as compared to
other isoforms. Molecular docking was performed to gain insight regarding the binding mode of these
inhibitors and thereby provided valuable information for our study on the design of selective human sial-
idase inhibitors further.
Received 28 February 2009
Revised 28 April 2009
Accepted 29 April 2009
Available online 4 May 2009
Keywords:
Sialidase
De novo design
Virtual screening
Inhibitor design
Fluoro benzoic acid
Ludi/CAP
Docking
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
get for the prevention of influenza infection.5 Some of the bacterial
(e.g., Vibrio cholerae, Streptococcus pneumoniae) and protozoic sia-
Sialidase enzymes are glycoside hydrolase enzymes (EC
3.2.1.18), which cleave the glycosidic linkages of sialic acid from
various glycoproteins, glycopeptides, gangliosides, oligosaccha-
rides and polysaccharides.1 Sialidases are mainly involved in the
metabolism of sialic acids and therefore regulate the cellular pro-
cesses by modifying the sialic acid content in the cell. Sialidases
lidases (e.g., Trypanosoma cruzi, Trypanosoma brucei) have been
considered as important virulence factor and have also been dis-
cussed as potential targets for drug design.6–9
Several mammalian sialidases have been described at the
molecular level. They have been demonstrated to differ from
microbial sialidases in various aspects, especially in the presence
of multi forms even in the single cell. At least four sialidase homo-
logs have been identified in the human genome namely, intra lyso-
somal sialidase (NEU1), cytosolic (NEU2), plasma membrane
associated sialidase (NEU3) and lysosomal or mitochondrial mem-
brane associated sialidase (NEU4). Apart from their subcellular
localization, these sialidases isoform (NEUs) also differ in their sub-
strate specificities, immunological properties, and it has been sug-
gested that distinct NEUs play specific role(s) in diverse cellular
processes.10 Unregulated NEUs expression has been speculated to
be associated with various pathological conditions, for example,
tumor progression, apoptosis suppression, metastasis, hyperinsuli-
nemia, atheroma plaque formation, inflammation, etc.11–14
Although the availability of genetically modified mice has recently
allowed the functional characterization of individual NEUs in the
diseases states like cancer, diabetes and arteriosclerosis, it is al-
ways desirable to have an inhibitor that selectively inhibits the tar-
are classified into two main types namely, endo-
3.2.1.129) and exo-
-sialidase (EC 3.2.1.18), respectively.2 exo-
Sialidase is also known as -N-acylneuraminate glycohydrolase
or Neuramindase, which attacks the terminal sialic acid linkages.
exo- -Sialidases (hereafter sialidases) are widely distributed
a-sialidase (E.C.
a
a-
a
a
among the different classes of organisms such as viruses, bacteria,
protozoa and vertebrates.3 They are thought to be involved in var-
ious biological processes like cell growth, cell proliferation, cell dif-
ferentiation, cell migration, cellular transport, signal transduction,
antigen masking, inter-intra cell interactions and infection.4 The
most extensively studied sialidase is the viral sialidase, a drug tar-
* Corresponding authors. Tel./fax: +81 582932918 (S.M.).
E-mail addresses: sadagopan_magesh@yahoo.com (S. Magesh), kiso@gifu-u.ac.jp
(M. Kiso).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.065

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S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
get isoform in the presence of others, so as to minimize the cross
reactivity or potential side effects. So, there is an intense interest
in identifying the isoform-selective inhibitors for human sialidase,
and such tool inhibitors would facilitate the differential functional
analysis of human sialidases in many vital cellular functions and
potential therapeutic utilities of sialidase inhibitors.
selectivity for NEU1 over NEU2, NEU3, and NEU4 with 3 (GSC-
649) (Fig. 1b).19
In the present approach, we applied structure based virtual
screening approach to generate the novel inhibitor scaffolds, away
from sugar framework, which are readily available or synthetically
accessible and have desired molecular properties as well. The crys-
tal structure of NEU2 has been used, as it is the only human siali-
dase which is experimentally resolved among human counterparts.
First, structure based interaction sites were designed based on the
interaction map generated by the LUDI program.20 Then Ludi/CAP li-
brary was screened with selected interaction sites as three-dimen-
sional (3D) queries. A set of fluoro-benzoic acids were predicted as
potential fragments that could bind to sialidase active site. An ex-
tra amide linked various hydrophobic substituents were added to
the fluoro-benzoic acids to hold out of glycerol-binding pocket
for selectivity probing among NEUs. Here we report the design,
synthesis and biological evaluation of a series of fluoro-benzoic
acid derivatives as human sialidase inhibitors.
Before embarking on the selectivity design, it is important to
address the question of what key structurally and functionally
important sites are that differentiate the target isoform protein
from the others. Structural studies on various viral, bacterial, pro-
tozoic and human sialidases have revealed the common canonical
six-blade b-propeller 3D arrangement in spite of their lower se-
quence similarity.15 There are many conserved residues within
the active sites of various sialidases, and are thought to be impor-
tant for catalytic mechanism. These include the arginine triad and
its close neighbor Tyr, and a pair of acidic residues (Asp/Glu) above
and below the active site crevice. On the other hand, sialic acids
(Neu5Ac, 1) 5-N-acetyl or/and 6-glycerol binding groups in the ac-
tive site are variable from sialidase to sialidase (Fig. 1a). In fact, this
variation has been speculated to be responsible for the substrate/
inhibitor specificity of sialidase from various sources.16
2. Results and discussion
Free sialic acid 1 is itself the only poor inhibitor of all the sialid-
ases (mM range) and its transition-state analogue, DANA (2-deoxy-
2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, 2) is available
as a non-specific and high affinity inhibitor for most sialidases
2.1. Structure based de novo design/virtual screening
The structure directed approach is a rational starting point for
the design of compounds that inhibit the target receptor or mimics
the substrate of the receptor. This methodology uses the steric
(shape and size) and chemical (charge and non-polar interactions)
complimentaries of the target receptor. The methods include de
novo design, virtual screening and fragment-based discovery. Ludi
is a well-known de novo design program that uses a library of frag-
ments and can be subjected to virtual screening protocols.21 Ludi is
absolutely geometric and avoids time-consuming potential energy
calculations. Ludi calculates molecular interaction sites on a recep-
tor and uses them to search for complementary compounds in the
library of molecular fragments that should interact with receptor.
As a part of the search and fit computation, Ludi also estimates
the energy of each conformation searched from the fragments in
the library, in the context of the receptor site. Ludi/CAP is a 3D Ludi
library prepared from two Accelrys databases: Chemicals Available
and inhibits the NEUs with IC50 values ꢀ40–150
l
M (Fig. 1b).17
In our previous study, we have predicted structural similarities
and differences at the active site amongst human sialidases by
molecular modeling techniques using the crystal structure of
NEU2.18 Despite the high level of active site residues conservation,
they seem to have differences in the amino acid residues that form
the glycerol-binding pocket (specificity pocket). We reasoned that
structure based approach in this regard might provide hints to ex-
ploit the non-selective inhibitor substituents that extend to the
specificity pocket of the active site pocket or ‘de novo design’ for
isoform selective inhibitor design. In our initial approach, we de-
signed a series of C9 amide linked hydrophobic derivatives of
DANA modified at glycerol side chain and followed by the synthe-
sis and evaluation against all four NEUs gave us at least 100-fold
a
6-glycerol
Arg triad
1
2-carboxyl
HO
8
COOH
Specificity
region
OH
6
9
7
HO
AcHN
O
OH
2
Conserved
region
5
4
3
HO
5-N-acetyl
4-hydroxyl
1
b
NH
HO
HO
OH
OH
O
COOH
Sialidase IC50(mM)
COOH
O
O
HO
AcHN
Sialidase IC50(mM)
AcHN
NEU1
NEU2
NEU3
NEU4
0.01
>1
>1
>1
NEU1
NEU2
NEU3
NEU4
0.143
0.04
0.06
0.07
HO
HO
3
2
Figure 1. (a) Sialic acid 1 (Neu5Ac) and its four constituent groups required for binding and (b) structure of DANA 2 and GSC-649 3 with their inhibition data against human
sialidases.19

S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
4597
Table 1
for Purchase (CAP), a structural database of commercially available
compounds, and CAPScreening, a database of compounds available
from the suppliers of screening.22 Known compounds retrieved
from Ludi/CAP can be purchased as ligand reagents from the sup-
pliers and can be tested either directly or with small modifications.
The crystal structure of human cytosolic sialidase NEU2, in
complex with the inhibitor DANA (PDB code 1VCU) was taken as
a starting-point.23 Amino acid residues sites that complement DA-
NAs four constituent groups, required for binding were chosen to
represent the interaction sites. Ludi has identified several interac-
tion features (HD—hydrogen-bond donors, HA—hydrogen-bond
acceptors, and L—lipophilic areas) at the central part of the binding
pocket (Fig. 2). The various representative structural features (7HA,
4HD, 4L) of twelve residues were selected to create 3D queries with
four to eight features, which are then evaluated in an iterative pro-
cess (Table 1). The combinations of features were selected in such a
way that they could cover the active site volume and should allow
the ligand to achieve the maximum interactions at the active of the
enzyme. Instead of building all the possible combinations using the
10 available features, which would have led to a total of more than
1000 combinations, we manually designed about 25 3D queries,
keeping conserved Arg304 in the middle of the arginine triad and
specificity residue Leu217 as main residues, while maintaining
the balance between the HBA, HBD and LIPO features.
We initiated virtual screening of Ludi/CAP database (78,175
compounds) to identify the compounds that satisfy the selected
3D structural queries. A total of 3437 hits possessing chemical
matching with the 3D queries were retrieved and 914 fragments
with high scores (>300) were retained. Since many of the hits were
identified more than one 3D query, a careful manual selection was
made not to duplicate hits. This operation yielded 214 unique com-
pounds, which were chosen for a further analysis. The binding
mode of selected hits was visually examined for their fit into the
active site of NEU2 and those that bind in a convincing mode, par-
ticularly hits that had at least an interaction with the Arg triad,
were considered. This resulted in a total of 14 compounds mainly
fluoro benzoic acids and nitro benzene derivatives, which were ta-
ken to represent potential inhibitors (Fig. 3). Interestingly, benzoic
acid derivatives are already known as sialidase inhibitors for viral
List of query features from selected atoms and their corresponding amino acid
residues (HD—hydrogen-bond donors, HA—hydrogen-bond acceptors, and L—lipo-
philic areas)
Residue
Atom
Feature
Residue
Atom
Feature
Arg21
Arg41
Met85
Ile103
Ile105
Tyr179
HH22
HH22
CG
CG
CG
HA1
HA2
L1
L2
Tyr181
Leu217
Arg237
Glu270
Arg304
Tyr334
OH
CG
HH22
OE2
HH22
OH
HA4, HD2
L4
HA5
HD3
HA6
L3
OH
HA3, HD1
HA7, HD4
and bacterial counter parts, and some of them showed effective
IC50 of
molar to several hundred
molar concentrations.24,25
l
l
It is worth noting that ortho-fluoro group of the compounds
could make an extra contacts with the conserved arginine triad
of sialidase enzymes. An important fact is that the most sialidase
inhibitors known until recently including the natural products,
contain a typical carboxylic acid group and this fact reveals that
carboxylic acid group is absolutely necessary for the sialidases
inhibition. Hence, we reasoned that benzoic acid containing
ortho-fluoro group could be a logical template to explore as a hu-
man sialidase inhibitors, moreover, there is no report of human
sialidase inhibition with benzoic acid scaffold alone so far. As we
aimed at specificity probing among human sialidases, we incorpo-
rated various N-amide linked derivatives into fluoro benzoic acid
core structure, which is expected to make interactions in the glyc-
erol group binding region (specificity pocket). Since the same re-
gion is primarily flanked by hydrophobic residues, various
hydrophobic groups were designed to make hydrophobic contacts
within this region. This fluoro benzoic acid ring was also incorpo-
rated with N-acetyl at para-position to mimic the conserved inter-
actions at N-acetyl binding region.
COOH
F
O
N
R
H
NH
H3C
O
I
2.2. Synthesis
We envisaged that 4-acetamido-2-fluoro benzoic acids having
5N-linked hydrophobic derivatives I could be easily synthesized
by the following methods (Scheme 1). The commercially available
3-fluoro-4-methyl aniline 4 could be acetylated to give a 3-fluoro-
4-methyl acetamide 5, and then 5 can be oxidized to a 2-fluoro-4-
acetamido benzoic acid 6. Selective nitration at 5 position can af-
ford a 5-nitro-2-fluoro-4-acetamido benzoic acid 7, then subse-
quently it can be reduced and acylated with various hydrophobic
acid chlorides or equivalents. The expected position of nitration
in the benzene ring is 5, since it is meta- to carboxylic acid group
that is meta-director and para to F, which is largely a para director.
Accordingly, the starting material 3-fluoro-4-methyl aniline 4 was
acetylated to give 5 in 96% yield using acetic anhydride in diisopro-
pyl ether at 50 °C for 30 min. The crystalline compound 5 was then
subjected to oxidation step using potassium dichromate/H2SO4
mixture. Oxidation was carried out by heating the oxidation mix-
ture with 5 in water at 100 °C for 8 h, gave the compound 6 in
Figure 2. The interaction of map of NEU2 active site generated by the LUDI program.
Hydrogen bond acceptors and donors are presented as green-gray and blue-gray
sticks, respectively, and hydrophobic regions are displayed with gray spheres. This
map also shows the interactions of a bound hit (A) with the active sites residues and
the scope for the interactions at the glycerol binding and N-acetyl binding cavities.

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S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
NO2
NO2
NO2
COOH
NO2
CH3
NO2
COOH
F
F
F
F
OH
OH
Cl
Cl
F
Cl
OH
G
F
H3C
O
E
F
D
A
B
C
(624)
(591)
(582)
(727)
(690)
(620)
(660)
COOH
COOH
NO2
NO2
COOH
COOH
HOOC
I
Cl
N
Cl
H3C
OH
Cl
N
OCH3
OH
H3C
O
H3C
O
F
O
CH3
M
L
K
N
J
H
I
(420)
(466)
(515)
(481)
(407)
(535)
(528)
Figure 3. Hits obtained from Ludi/CAP library search. Individual Ludi score is given in the bracket.
COOH
COOH
CH3
CH3
F
F
F
F
b
c
a
NO2
H3C
NH
H3C
NH
H3C
NH
NH2
O
O
O
6
7
4
5
COOH
COOH
e
F
F
d
O
N
H
R
NH2
H3C
NH
H3C
NH
O
O
8
9a-9m
Scheme 1. Reagents and conditions: (a) (CH3CO)2O, diisopropylether, 50 °C, 30 min, 96% ; (b) KMnO4, MgSO4, H2O 100 °C, 8 h, 73%; (c) HNO3, ꢁ20 °C, 2 h, 69% ; (d) Pd/C, H2, rt,
ethylacetate/MeOH (1:1), 2d; (e) ROCl, satd NaHCO3, THF/MeOH (1:1),1 h, 50–84% (two steps).
73% yield. The following nitration step gave some trouble in the
beginning, but after some experimentation, we could achieve the
selective nitration to give 7 in 69% yield. The condition employed
for the nitration was the addition of compound in small amounts
for 30 min to neat nitric acid, cooled to ꢁ20 °C. After having the ni-
trated compound in hand, reduction, followed by acylation was
carried out. The hydrogenation of 7 in methanol and ethyl acetate
(1:1) was done using Pd/C as a catalyst, gave the amine 8, which
was then converted to the respective amides. The condition used
for the acylation is the addition of acyl chloride dissolved in THF
to the stirring solution in satd NaHCO3. This procedure gave the
various hydrophobic 4-acetamido-5-acylamido-2-fluoro benzoic
acids (9a–9m) in 50–84% yields.
2.3. Human sialidase inhibition
Inhibitory activities of a series of substituted 2-fluoro benzoic
acids (9a–9m) against all four human sialidases were investigated.
The percentage of inhibition at 1 mM concentration of inhibitors is
given in Table 2. None of the compounds inhibited NEU1 at the
tested concentrations, whereas some compounds inhibited NEU3
and NEU4 scarcely. NEU2 seems slightly more sensitive than the
other isoforms to this series of compounds, as most of the com-
pounds inhibited NEU2 in the range of 15–60% inhibition at
1 mM. The highest inhibition (%I = 60) was observed with com-
pound 9m which has N-amide linked bulky biphenyl group (IC50
of 0.552 0.124 mM and Ki value of 0.307 0.073 mM with

S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
4599
Table 2
Chemical structures of compounds 9a–9m and their inhibitory activities against human sialidases: NEU1, NEU2, NEU3 and NEU4.
COOH
F
O
N
R
H
H3C
NH
O
Compound
R
% of sialidase inhibition (at 1 mM)
NEU3
NEU1a
—
NEU2
37
NEU4
8
9a
23
9b
9c
—
—
17
17
—
—
6
—
9d
9e
9f
—
—
—
—
—
—
—
—
—
—
—
—
—
13
—
—
—
—
20
—
—
<5
—
9g
9h
9i
7
25
15
—
<5
11
<5
<5
9j
9k
23
9l
—
—
33
8
10
9m
60 (IC50 = 0.552 0.124 mM)
8 (IC50 = >5 mM)
10 (IC50 = >5 mM)
a
No inhibition.

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S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
Figure 5. The predicted binding conformation of the inhibitor 9m at NEU2 active
site. Green and pink lines represent the hydrogen bonding and steric clash,
respectively.
selectivity of 4-acetamido-5-acylamido-2-fluoro benzoic acids to
NEU2 over other isoforms. Extended hydrophobic interactions of
biphenyl carboxamido group at the active site of NEU2 can be rea-
soned for the increased binding affinity of 9m over acetamido
group (9a), as bulky groups are often known to exhibit better affin-
ity and selectivity.
Figure 4. Lineweaver–Burk plot for the inhibition of the NEU2 activity by
compound 9a (Ki value of 0.307 0.073 mM with 4MU-Neu5Ac as a substrate).
4MU-Neu5Ac as a substrate) (Fig. 4). Compound 9m hardly inhib-
ited NEU3 (%I = 8) and NEU4 (%I = 10) both with IC50 of >5 mM.
Compounds 9a (%I = 37) and 9l (%I = 33) showed the modest inhib-
itory activity against NEU2 with the IC50 of 1.2 mM and 1.37 mM,
respectively. The maximum inhibition of NEU3 was achieved with
compound 9a (%I = 23) and with the estimated IC50 of >2 mM. Ta-
ken together, about 10-fold of NEU2 selectivity was achieved with
compound 9m over other human sialidases.
3. Conclusion
Structure-based de novo design/virtual screening enabled us to
identify some nitro benzene and fluoro benzoic acids as potential
inhibitors of human sialidase from Ludi/CAP database. 2-fluoro
benzoic acid scaffold was selected and modified for the selectivity
probing among human sialidases. The simple and straightforward
method to access the various 4-acetamido-5-acylamido-2-fluoro
benzoic acid analogues was developed. The synthesized com-
pounds (9a–9m) were biologically evaluated against all four types
of human sialidases. This series of compounds showed very weak
inhibitory activities against human sialidases, except some deriva-
tives inhibited NEU2 moderately as compared to other isoforms.
Highest NEU2 inhibition (60% at 1 mM, IC50 of 0.552 0.124 mM
and Ki value of 0.307 0.073 mM) was achieved with compound
9m with ꢀ10-fold selectivity. A docking investigation revealed that
the conformational restriction of the current series of the deriva-
tives which might disrupt the conserved interaction to the arginine
triad of human sialidases could be a reason for their observed poor
inhibitory activities. The inhibitory activity and isoform selectivity
among human sialidases of current series can be improved by
addressing the flexibility of the substituents at C5 of 4-acetam-
ido-2-fluoro benzoic acid core structure. With this regard, the fur-
ther efforts on synthesis and optimization are currently underway
to expand the scope of this work. On the whole, we strongly be-
lieve that the opportunities and the limitations of the present
study might be an interesting subject for the future inhibitor
design.
2.4. Docking studies
In an attempt to understand the molecular basis of observed
affinity of this series to NEU2, automated docking was performed
using the DS LigandFit.26 However, the docking study was not able
to explain this binding affinity as binding poses were apparently in
an irrational mode. This behavior might be due to the restricted ac-
cess of bulky biphenyl group into the active site of NEU2, since
LigandFit employs flexible docking of ligand into the fixed protein
binding site. Thus, compounds (9a and 9m) were manually docked
into NEU2 active site in a proposed binding mode. A special
emphasis was given to the interaction between carboxylic acid
group of inhibitor and Arg304 of NEU2. The resulting complex
was energetically refined to reduce the steric clashes and also to
maximize the interactions. The binding poses of these compounds
suggested that amide substituents could be tolerated in the glyc-
erol-binding pocket of NEU2, but show a potential loss of a con-
served H bond interaction with Arg234 and a steric bump of the
same residue with H6 of benzoic acid core ring. This is probably
due to their conformation and restricted rotation along with the
flat benzoic acid ring structure that could cause the overturning
of the ligands, which could be an explanation for the observed
weak inhibitory activity (Fig. 5). Interestingly, amino acid residue
that located at the lip of the glycerol binding pocket is more flexi-
ble in NEU2 (Gln270), while in NEU3 and NEU4, it is substituted
with comparatively steric bulk amino acid residues His277 and
Trp274, respectively.18 In the case of NEU1, this scenario is differ-
ent as this subsite in NEU1 is majorly composed of acidic amino
acids. These relative differences in the glycerol-binding pocket of
human sialidases can be accounted for the observed marginal
4. Experimental
4.1. Synthesis
4.1.1. General
Reagents were purchased in the best quality available and the
reagent grade solvents were dried over activated molecular sieves
of appropriate size. Commercial grade solvents (CH2Cl2, hexane,

S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
4601
EtOAc, MeOH, acetone, CHCl3, toluene) were purchased from Wako
chemical company and used without any further purification.
Reactions were monitored by thin layer chromatography (TLC)
using Merck silica gel plates GF245. Compounds were detected
either with 254 nm UV light or nihydrin. Purification by normal
phase chromatography was achieved by elution through columns
of silica gel ꢀ80 or 300 mesh size. 1H, 13C and 19F nuclear magnetic
resonance (NMR) spectra were recorded using a Jeol ECX NMR
spectrometer. All NMR spectra were recorded at 400, 500 or
600 MHz. Chemical shifts are expressed as parts per million
(ppm, d) and are relative to the standard internal reference tetra
methyl silane (TMS) (0.0d). In order to confirm assignments, 2D
NMR experiments were performed using the 1H–1H correlation
spectroscopy (COSY) experiment for proton-proton interactions
confirmed by 1H–13C HMQC) ; 19F NMR (DMSO-d6) d ꢁ99.56. HRMS
(ESI) calcd for C9H6FN2O5 (MꢁH+) 241.0261; found 241.0192.
4.1.1.4. 4-Acetamido-5-amino-2-fluoro benzoic acid (8).
To
the solution of compound 7 (242 mg, 1 mmol) in the mixture of
ethyl acetate and methanol (1:1) (10 mL) was added 10% w/w pal-
ladium on carbon (25 mg) under nitrogen. The resulting suspen-
sion was hydrogenated at room temperature and ambient
pressure for 2 days. The mixture was filtered through the Celite
and the filtrate was concentrated to dryness under reduced pres-
sure. The crude was used for subsequent reactions without further
purification. Compound 8: Rf = 0.5 (5% MeOH in EtOAc). HRMS (ESI)
calcd for C9H8FN2O3 (MꢁH+) 211.0519; found 211.0723.
and the 1H–13
C
heteronuclear multiple quantum coherence
4.1.2. General procedure V for the synthesis of 4-Acetamido-5-
acylamido-2-fluoro benzoic acids (9a–9m)
To the solution of crude compound 8 in the mixture of ethyl
acetate and methanol (1:1) (10 mL) was added saturated aq NaH-
CO3 solution (10 mL) at rt. Acid chloride (1 mmol) dissolved in
(HMQC) experiment for proton–carbon interactions. Mass spectral
data were obtained on a Bruker Daltonics microTOF-II in the posi-
tive or negative ion detection modes.
4.1.1.1. N-(3-Fluoro-4-methyphenyl) acetamide (5).
To the
the THF (ꢀ500
lL) was added to the reaction, while maintaining
solution of 3-fluoro-4-methyl aniline (4) (10 g, 80 mmol) in diiso-
propyl ether (50 mL) was added acetic anhydride (9.5 mL,
100 mmol) and heated at 50 °C for 30 min. Upon cooling, white
needle shaped crystals were formed, filtered, and dried in vacuo
to give 5 (12.8 g, 96%). Compound 5: Rf = 0.5 (hexane/EtOAc 1:1).
1H NMR (DMSO-d6) d 2.16 (3H, s, Ar-CH3), 2.03 (3H, s, NAc), 7.17
(2H, m, H-5, H-4), 7.44 (1H, d, J2,F 13.0 Hz, H-2), 10.05 (1H, s, NH)
(assignments were confirmed by 1H–1H COSY); 13C NMR (DMSO-
d6) d 13.6 (Ar-CH3), 23.9 (NC(O)Me), 105.7 (C-2), 114.43 (C-6),
118.0 (C-4), 131.3 (C-5), 138.7 (C-1), 160.2 (C-3), 168.4 (NC(O)Me)
(assignments were confirmed by 1H–13C HMQC); 19F NMR (DMSO-
the basic pH of the reaction mixture. Then the reaction mixture
was stirred for 1–3 h. Then reaction mixture was adjusted to pH
5–6 using 2 N HCl and extracted with ethyl acetate (3 ꢂ 10 mL).
The organic layers were combined and washed with water
(10 mL), brine solution, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified by either
column chromatography (ethyl acetate/MeOH/H2O) or recrystal-
lized from ethyl acetate to give off-white to pure white powders.
4.1.2.1. 4,5-Diacetamido-2-fluoro benzoic acid (9a).
Pre-
pared according to General procedure V. Yield: (155 mg, 61 %).1H
NMR (DMSO-d6) d 2.07, 2.12 (2 ꢂ 3H, 2 ꢂ s, 2 ꢂ NAc), 7.81 (1H, d,
J3,F 12.8 Hz, H-3), 7.92 (1H, d, J6,F 7.3 Hz, H-6), 9.43, 9.5 (2 ꢂ 1H,
2 ꢂ s, 2 ꢂ NH), 13.1 (1H, s, COOH) (assignments were confirmed
by 1H–1H COSY); 13C NMR (DMSO-d6) d 24.1, 24.6 (2 ꢂ NC(O)Me),
110.5 (C-3), 114.05 (C-1), 124.9 (C-5), 129.4 (C-6), 137.6 (C-4),
158.6 (C-2), 164.8 (C(O)OH), 169.6 (2 ꢂ NC(O)Me) (assignments
d6)
d
ꢁ116.25. HRMS (ESI) calcd for C9H10FNNaO (M + Na)
190.0644; found 190.0714.
4.1.1.2. 4-Acetamido-2-fluoro benzoic acid (6).
To the solu-
tion of KMnO4 (29.4 g) and MgSO4 (20.4 g) in water was added
compound 5 (10 g, 60 mmol) in portion wise, and the mixture
was heated at 70–80 for 8 h. The reaction mixture was allowed
to stand overnight and solid Na2CO3 (29 g) was added. The mix-
ture was further stirred for 15 min and filtered. The colorless fil-
trate was cooled to 0 °C and acidified with 40 mL of concd HCl.
The formed precipitate was filtered, dried and recrystallized
from ethanol–water to give 6 (8.6 g, 73%) as off-white crystals.
Compound 6: Rf = 0.4 (5% MeOH in EtOAc). 1H NMR (DMSO-d6)
d 2.09 (3H, s, NAc), 7.33 (1H, dd, J5,6 8.8 Hz, J5,3 1.38 Hz, H-5),
7.66 (1H, t, J6,F and J6,5 8.8 Hz, H-6), 7.83 (1H, dd, J3,F 13.7 Hz,
J3,5 1.38 Hz, H-3), 10.43 (1H, s, NH) 12.9 (1H, s, COOH) (assign-
ments were confirmed by 1H–1H COSY); 13C NMR (DMSO-d6) d
24.2 (NC(O)Me), 106.1 (C-3), 112.8 (C-1), 114.6 (C-5), 132.8 (C-
6), 144.7 (C-4), 160.8 (C-2), 164.5 (C(O)OH), 169.2 (NC(O)Me)
were confirmed by 1H–13
C
HMQC) ; 19F NMR (DMSO-d6) d
ꢁ112.46. HRMS (ESI) calcd for C11H10FN2O4 (MꢁH+) 253.0625;
found 253.0841.
4.1.2.2. 4-Acetamido-5-benzamido-2-fluoro
benzoic
acid
(9b). Prepared according to general procedure V. Yield:
(68 mg, 215%). 1H NMR (DMSO-d6) d 2.12 (3H, s, NAc), 7.52–7.63
(3H, m, H-30, H-40, H-50), 7.83 (1H, d, J3,F 12.8 Hz, H-3), 7.98 (1H,
d, J6,F 7.3 Hz, H-6), 8.01 (2H, d, H-20, H-60), 9.76, 9.89 (2 ꢂ 1H,
2 ꢂ s, 2 ꢂ NH), 13.17 (1H, s, COOH) (assignments were confirmed
by 1H–1H COSY); 13C NMR (DMSO-d6) d 24.8 (NC(O)Me), 110.8
(C-3), 114.1 (C-1), 124.9 (C-5), 128.4 (C-20, C-60), 128.8 (C-30, C-
50), 130.7 (C-6), 132.3 (C-4), 134.6 (C-1), 138.5 (C-4), 159.1 (C-2),
166.3 (C(O)OH), 164.9 (NC(O) phenyl), 169.9 (NC(O)Me) (assign-
ments were confirmed by 1H–13C HMQC); 19F NMR (DMSO-d6) d
ꢁ111.85. HRMS (ESI) calcd for C16H12FN2O4 (MꢁH+) 315.0781;
found 315.091.
(assignments were confirmed by 1H–13
C
HMQC); 19F NMR
(DMSO-d6) d ꢁ107.73. HRMS (ESI) calcd for C9H7FNO3 (MꢁH+)
196.0410; found 196.0465.
4.1.1.3. 4-Acetamido-2-fluoro-5-nitro benzoic acid (7).
To
the fuming nitric acid (50 mL) cooled at ꢁ20 °C was added com-
pound 6 (4 g, 20 mmol) portion wise. After 2hrs the reaction mix-
ture poured over ice (50 g) and water (50 mL) with stirring. The
light yellow solid was collected by filtration and dried under re-
duced pressure to give the title compound (3.4 g, 69 %). Compound
7: Rf = 0.5 (5% MeOH in EtOAc). 1H NMR (DMSO-d6) d 2.17 (3H, s,
NAc), 7.88 (1H, d, J3,F 12.8 Hz, H-3), 8.46 (1H, d, J6,F 7.3 Hz, H-6),
10.55 (1H, s, NH) 13.72 (1H, s, COOH) (assignments were con-
firmed by 1H–1H COSY); 13C NMR (DMSO-d6) d 24.13 (NC(O)Me),
111.2 (C-3), 114.5 (C-1), 129.9 (C-6), 136.1 (C-4), 137.5 (C-5),
162.6 (C-2), 164.1 (C(O)OH), 169.2 (NC(O)Me) (assignments were
4.1.2.3. 4-Acetamido-5-cyclohexanecarboxamido-2-fluoro ben-
zoic acid (9c).
Prepared according to general procedure V.
Yield: (74 mg, 238%). 1H NMR (DMSO-d6) d 1.15–1.45 (10H,
2 ꢂ m, H-20, H-30, H-40, H-50, H-60), 2.11 (3H, s, NAc), 2.35–2.42
(1H, m, H-10), 7.72 (1H, d, J3,F 12.8 Hz, H-3), 7.94 (1H, d, J6,F
7.9 Hz, H-6), 9.48, 9.31 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.11 (1H, s, COOH)
(assignments were confirmed by 1H–1H COSY); 13C NMR (DMSO-
d6) d 24.8 (NC(O)Me), 25. 7 (C-30, C-50), 25. 9 (C-40), 29. 5 (C-20, C-
60), 44.7 (C-10), 111.0 (C-3), 114.2 (C-1), 126.6 (C-5), 129.1 (C-6),
137.1 (C-4), 158.5 (C-2), 164.9 (C(O)OH), 169.5 (NC(O)Ac), 175.4
(NC(O) cyclohexyl) (assignments were confirmed by 1H–13
C

4602
S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
HMQC) ; 19F NMR (DMSO-d6) d ꢁ112.87. HRMS (ESI) calcd for
C16H18FN2O4 (MꢁH+) 321.1251; found 321.148.
158.7 (C-2), 164.8 (C(O)OH), 169.6 (NC(O)Ac), 172.5 (NC(O) pentyl)
(assignments were confirmed by 1H–13C HMQC); 19F NMR (DMSO-
d6)
d
ꢁ112.59. HRMS (ESI) calcd for C14H16FN2O4 (MꢁH+)
4.1.2.4. 4-Acetamido-5-cyclopentanecarboxamido-2-fluoro
295.1094; found 295.1238.
benzoic acid (9d).
Prepared according to general procedure
V. Yield: (212 mg, 69%). 1H NMR (DMSO-d6) d 1.52–1.90 (8H,
3 ꢂ m, H-20, H-30, H-40, H-50), 2.11 (3H, s, NAc), 2.84 (1H, p, H-10),
7.72 (1H, d, J3,F 12.6 Hz, H-3), 7.94 (1H, d, J6,F 6.8 Hz, H-6), 9.37,
9.53 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.12 (1H, s, COOH) (assignments
were confirmed by 1H–1H COSY); 13C NMR (DMSO-d6) d 24.5
(NC(O)Me), 26. 1 (C-20, C-40), 30.4 (C-30, C-50), 45. 3 (C-10), 111.8
(C-3), 113.9 (C-1), 125.3 (C-5), 128.3 (C-6), 137.2 (C-4), 158.4 (C-
2), 164.4 (C(O)OH), 169.5 (NC(O)Ac), 175.5 (NC(O) cyclopentyl)
4.1.2.9. 4-Acetamido-5-isobutyramido-2-fluoro benzoic acid
(9i).
Prepared according to general procedure V. Yield:
1
(226 mg, 80%). H NMR (DMSO-d6) d 1.13 (6H, d, H-20, H-200), 2.11
(3H, s, NAc), 2.64 (1H, o, H-10), 7.72 (1H, d, J3,F 13.4 Hz, H-3), 7.95
(1H, d, J6,F 7.9 Hz, H-6), 9.32, 9.49 (2 ꢂ1H, 2 ꢂ s, 2 ꢂ NH), 13.09
(1H, s, COOH) (assignments were confirmed by 1H–1H COSY); 13C
NMR (DMSO-d6) d 19.9 (C-20, C-200), 24.5 (NC(O)Me), 35.0 (C-10),
111.1 (C-3), 114.4 (C-1), 125.3 (C-5), 129.2 (C-6), 137.2 (C-4),
158.3 (C-2), 164.8 (C(O)OH), 169.5 (NC(O)Ac), 176.3 (NC(O) isobu-
tyl) (assignments were confirmed by 1H–13C HMQC); 19F NMR
(DMSO-d6) d ꢁ112.74. HRMS (ESI) calcd for C13H14FN2O4 (MꢁH+)
281.0938; found 281.1315.
(assignments were confirmed by 1H–13
C HMQC) ;
19F NMR
(DMSO-d6) d ꢁ112.41. HRMS (ESI) calcd for C15H16FN2O4 (MꢁH+)
307.1094; found 307.1201.
4.1.2.5. 4-Acetamido-5-cyclopropanecarboxamido-2-fluoro
benzoic acid (9e).
Prepared according to general procedure V.
4.1.2.10. 4-Acetamido-5-pivalamido-2-fluoro
(9j). Prepared according to general procedure V. Yield:
benzoic
acid
Yield: (162 mg, 58%). 1H NMR (DMSO-d6) d 0.82–0.84 (4H, m, H-20,
H-30), 1.84 (1H, p, H-10), 2.12 (3H, s, NAc), 7.74 (1H, d, J3,F 12.8 Hz,
H-3), 8.01 (1H, d, J6,F 7.9 Hz, H-6), 9.63, 9.71 (2 ꢂ 1H, 2 ꢂ s,
2 ꢂ NH), 13.09 (1H, s, COOH) (assignments were confirmed by
(189 mg, 64%). 1H NMR (DMSO-d6) d 1.22 (9H, s, NC(O)CMe3),
2.11 (3H, s, NAc), 7.51 (1H, d, J3,F 12.2 Hz, H-3), 7.89 (1H, d, J6,F
7.9 Hz, H-6), 8.94, 9.64 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.18 (1H, s, COOH)
(assignments were confirmed by 1H–1H COSY); 13C NMR (DMSO-
d6) d 24.5 (NC(O)Me), 27.6 (NC(O)CMe3), 39.8 (NC(O)CMe3), 111.8
(C-3), 115.1 (C-1), 126.4 (C-5), 130.3 (C-6), 137.6 (C-4), 158.5 (C-
2), 164.8 (C(O)OH), 169.5 (NC(O)Ac), 177.5 (NC(O)CMe3) (assign-
ments were confirmed by 1H–13C HMQC); 19F NMR (DMSO-d6) d
ꢁ112.71. HRMS (ESI) calcd for C14H16FN2O4 (MꢁH+) 295.1094;
found 295.1342.
13
1H–1H COSY); C NMR (DMSO-d6) d 8.0 (C-20, C-30), 15.05. 7 (C-
10), 24.5 (2 ꢂ NC(O)Me), 45. 3 (C-10), 111.0 (C-3), 114.5 (C-1),
125.4 (C-5), 128.6 (C-6), 136.6 (C-4), 158.3 (C-2), 164.6 (C(O)OH),
169.6 (NC(O)Ac), 172.9 (NC(O) cyclopropyl) (assignments were
confirmed by 1H–13C HMQC); 19F NMR (DMSO-d6) d ꢁ113.1. HRMS
(ESI) calcd for C13H12FN2O4 (MꢁH+) 279.0781; found 279.0822.
4.1.2.6. 4-Acetamido-5-butyramido-2-fluoro
benzoic
acid
(9f). Prepared according to general procedure V. Yield:
4.1.2.11. 4-Acetamido-5-(2-ethylbutanamido)-2-fluoro benzoic
1
(203 mg, 72%). H NMR (DMSO-d6) d 0. 94 (3H, t, H-30), 1.63 (2H,
sex, H-20), 2.11 (3H, s, NAc), 2.34 (1H, t, H-10), 7.77 (1H, d, J3,F
12.6 Hz, H-3), 7.93 (1H, d, J6,F 6.8 Hz, H-6), 9.38, 9.50 (2 ꢂ 1H,
2 ꢂ s, 2 ꢂ NH), 13.08 (1H, s, COOH) (assignments were confirmed
acid (9k).
Prepared according to general procedure V. Yield:
1
(220 mg, 71%). H NMR (DMSO-d6) d 0. 90 (6H, t, H-30, H-300),
1.50, 1.57 (2 ꢂ 2H, 2 ꢂ p, H-20, H-200), 2.09 (3H, s, NAc), 2.23 (1H,
p, H-10), 7.68 (1H, d, J3,F 12.8 Hz, H-3), 7.93 (1H, d, J6,F 8.2 Hz, H-
6), 9.46, 9.50 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.21 (1H, s, COOH) (assign-
ments were confirmed by 1H–1H COSY); 13C NMR (DMSO-d6) d 11.6
(C-30, C-300), 23.9 (NC(O)Me), 24.9 (C-20, C-200), 49.3 (C-10), 110.7 (C-
3), 113.9 (C-1), 125.1 (C-5), 128.5 (C-6), 136.6 (C-4), 158.1 (C-2),
164.2 (C(O)OH), 169.7 (NC(O)Ac), 174.6 (NC(O) 2-ethylbutyl)
13
by 1H–1H COSY); C NMR (DMSO-d6) d 14.1 (C-30), 18.9 (C-20),
23.9 (NC(O)Me), 38.3 (C-10), 111.8 (C-3), 114.5 (C-1), 125.0 (C-5),
129.2 (C-6), 137.3 (C-4), 158.5 (C-2), 164.92 (C(O)OH), 169.6
(NC(O)Ac), 172.4 (NC(O) butyl) (assignments were confirmed by
1H–13C HMQC); 19F NMR (DMSO-d6) d ꢁ112.62. HRMS (ESI) calcd
for C13H14FN2O4 (MꢁH+) 281.0938; found 281.1019.
(assignments were confirmed by 1H–13
C
HMQC); 19F NMR
(DMSO-d6) d ꢁ112.44. HRMS (ESI) calcd for C15H18FN2O4 (MꢁH+)
4.1.2.7. 4-Acetamido-5-(2-methylbutanamido)-2-fluoro
ben-
309.1251; found 309.1491.
zoic acid (9g). Prepared according to general procedure V.
1
Yield: (175 mg, 59%). H NMR (DMSO-d6) d 0.90 (3H, t, , H-30),
1.11 (3H, d, H-100), 1.38–1.68 (2H, 2 ꢂ m, H-20), 2.10 (3H, s, NAc),
2.46 (1H, sex, H-10), 7.71 (1H, d, J3,F 12.8 Hz, H-3), 7.94 (1H, d, J6,F
7.9 Hz, H-6), 9.39, 9.47 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.17 (1H, s, COOH)
(assignments were confirmed by 1H–1H COSY); 13C NMR (DMSO-
d6) d 12.1 (C-30), 17.6 (C-100), 24.5 (NC(O)Me), 27.2 (C-20), 42.1 (C-
10), 111.3 (C-3), 114.4 (C-1), 125.4 (C-5), 129.1 (C-6), 137.2 (C-4),
158.7 (C-2), 164.8 (C(O)OH), 169.4 (NC(O)Ac), 175.8 (NC(O) 2-
methylbutyl) (assignments were confirmed by 1H–13C HMQC);
19F NMR (DMSO-d6) d ꢁ112.68. HRMS (ESI) calcd for C14H16FN2O4
(MꢁH+) 295.1094; found 295.1185.
4.1.2.12. 4-Acetamido-5-(2-naphthamido)-2-fluoro
acid (9l). Prepared according to general procedure V. Yield:
benzoic
(190 mg, 52%). 1H NMR (DMSO-d6) d 2.13 (3H, s, NAc), 7.62–7.65
(2H, m, H-60, H-70), 7.83 (1H, d, J3,F 12.8 Hz, H-3), 7.99–8.08 (5H,
m, H-6, H-30, H-40, H-50, H-80), 8.66 (1H, s, H-10), 9.83, 10.13
(2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.21 (1H, s, COOH) (assignments were
confirmed by 1H–1H COSY); 13C NMR (DMSO-d6) d 24.0 (NC(O)Me),
110.5 (C-3), 114.1 (C-1), 124.5 (C-5), 126.8 (C-30), 127.6 (C-10, C-40),
127.8 (C-50, C-80), 128.4 (C-60), 128.9 (C-70), 130.2 (C-6), 131.5 (C-
100), 132.0 (C-20), 134.3 (C-90), 137.5 (C-4), 157.8 (C-2), 163.7
(NC(O)Nap), 165.9 (C(O)OH), 169.4 (NC(O)Ac) (assignments were
confirmed by 1H–13C HMQC); 19F NMR (DMSO-d6) d ꢁ112.19.
HRMS (ESI) calcd for C20H14FN2O4 (MꢁH+) 365.0938; found
365.1213.
4.1.2.8. 4-Acetamido-5-pentanamido-2-fluoro benzoic acid
(9h).
Prepared from X according to General procedure V.
1
Yield: (228 mg, 77%). H NMR (DMSO-d6) d 0. 91 (3H, t, H-40),
1.35 (2H, sex, H-30), 1.59 (2H, p, H-20), 2.11 (3H, s, NAc), 2.36
(1H, t, H-10), 7.77 (1H, d, J3,F 13.4 Hz, H-3), 7.92 (1H, d, J6,F 7.9 Hz,
H-6), 9.37, 9.48 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH), 13.11 (1H, s, COOH)
(assignments were confirmed by 1H–1H COSY); 13C NMR (DMSO-
d6) d 14.3 (C-40), 22.3 (C-30), 24.6 (NC(O)Me), 27.6 (C-20), 36.1 (C-
10), 110.9 (C-3), 114.2 (C-1), 125.1 (C-5), 129.2 (C-6), 137.4 (C-4),
4.1.2.13. 4-Acetamido-5-(biphenyl-4-ylcarboxamido)-2-fluoro-
benzoicacid (9m).
Prepared according to general procedure
V. Yield: (196 mg, 50%). 1H NMR (DMSO-d6) d 2.13 (3H, s, NAc),
7.43 (1H, t, H-400), 7.52 (2H, t, H-200, H-600), 7.77 (2H, d, H-300, H-
500), 7.84–7.86 (3H, m, H-3, H-30, H-50), 7.99 (1H, d, J6,F 7.68 Hz,
H-6), 8.11 (2H, d, H-20, H-60), 9.79, 9.96 (2 ꢂ 1H, 2 ꢂ s, 2 ꢂ NH),

S. Magesh et al. / Bioorg. Med. Chem. 17 (2009) 4595–4603
4603
13.21 (1H, s, COOH) (assignments were confirmed by 1H–1H
COSY); 13C NMR (DMSO-d6) d 24.5 (NC(O)Me), 110.1 (C-3), 114.5
(C-1), 126.5 (C-200, C-600), 126.9 (C-5, C-30, C-50), 128. 1 (C-
100),128.5 (C-20, C-60), 129.0 (C-10), 130.2 (C-6), 132.9 (C-400), 139.0
(C-40), 143.2 (C-4), 158.2 (C-2), 164.3 (NC(O) biphenyl), 165.5
(C(O)OH), 169.4 (NC(O)Ac) (assignments were confirmed by
1H–13C HMQC); 19F NMR (DMSO-d6) d ꢁ111.88. HRMS (ESI) calcd
for C22H16FN2O4 (MꢁH+) 391.1094; found 391.1164.
mined fluorometrically. Sialidase activity with the 4MU-NeuAc
(NEU1, NEU2, and NEU4) as substrate was assayed by spectrofluo-
rometric measurement of the 4-methylumbelliferone released. In
the case of NEU3, mixed ganglioside was used as a substrate and
sialic acid released from GM3 were measured by fluorometric
HPLC with malononitrile. One unit of sialidase was defined as the
amount of enzyme cleaving 1 nmol of sialic acid/h. IC50 values from
concentration-inhibition curves were calculated by means of non-
linear regression analysis using Microsoft Excel 2000.
4.2. Molecular modeling
Acknowledgments
All computations and simulations were carried out on an Intel
P4 based Microsoft windows 2000 workstation using Discovery
Studio Modeling 1.1 and 1.2 Package (Accelrys).27 The crystal
structure of the human NEU2–DANA complex was obtained from
the Brookhaven protein Data Bank (accession code: 1VCU, Chain
B).23 4-morpholineethanesulfonic acid (MES) and all water mole-
cules were removed from the protein structure. Hydrogen atoms
were added and the atom types were assigned using the CHARMM
forcefield. The protonation states of amino acid residues were ad-
justed to the dominant ionic forms at pH 7.4. The active site was
defined as the collection of amino acids enclosed within 8 Å radius
sphere centered on bound ligand DANA. The program LUDI was ap-
plied to design the potential fragments like compounds that bind
NEU2 active site. The 15 structural features of 12 amino acid resi-
dues (Arg21, Arg41, Met85, Ile103, Ile105, Tyr179, Tyr181, Leu217,
Arg237, Gln270, Arg 304, Tyr334) were used for the selection of
structure based 3D queries (Tables 1 and 2). Ludi de novo receptor
mode of program was used to generate interaction map and then a
systematic search of Ludi/CAP library containing 78,175 com-
pounds was performed with generated 3D queries. Ludi energy
estimate3 was used to estimate the change in free energy upon
the binding of the fragment to the receptor. Automated docking
was attempted as reported elsewhere.19 For the manual docking,
structure of compounds 9a and 9m were built using Ludi/CAP
search Hit A-NEU2 model complex as a template generated during
Ludi/CAP search. The obtained complexes were then energetically
minimized with 500–1000 iterations of ‘in situ ligand minimiza-
tion algorithm’ using the SMART MINIMIZER program. A distance con-
straint was applied between carboxylic group of inhibitor and
Arg304 of the conserved Arg triad in the NEU2 active site. Then
binding poses were analyzed to understand the molecular interac-
tions between the docked inhibitor and NEU2.
The first author is grateful to Ministry of Education, Culture,
Sports, Science and Technology, Government of Japan (MEXT)
and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto
University for his past and current financial assistance,
respectively.
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of PBS, pH 7.4, containing 1 mM EDTA, 0.5 mM PMSF, 10
lg/ml leu-
peptin and 0.5 g/ml pepstatin, and centrifuged at 1000 g for
l
10 min. The supernatant (crude extract) was used for sialidase as-
says. Briefly, the routine reaction mixture contained 10–20 nmol of
substrate as bound sialic acid, 0.2 mg of BSA, 10
lmol of sodium
acetate (pH 4.6) and 0.2 mg of Triton X-100. After incubation at
37 °C for 10–30 min, the amount of sialic acid released was deter-
27. DS Modeling 1.1 and 1.2, 2003, Accelrys, San Diego, CA, USA.