Aromatic sialic acid analogues as potential inhibitors of influenza virus neuraminidase

Article abstract of DOI:10.1016/S0014-827X(01)01081-3

The influenza virus neuraminidase (NA) is an enzyme essential for viral infection and offers a potential target for antiviral drug development. We aimed our research at the synthesis of non-carbohydrate molecules able to inhibit NA as transition-state ana

Full text of DOI:10.1016/S0014-827X(01)01081-3

www.elsevier.nl/locate/farmac
Il Farmaco 56 (2001) 305–309
Aromatic sialic acid analogues as potential inhibitors of influenza
virus neuraminidase
Armandodoriano Bianco a,*, Mario Brufani b, Cristiana Melchioni a
a Dipartimento di Chimica, Uni6ersita` ‘La Sapienza’, Piazzale Aldo Moro n. 5, I-00185, Rome, Italy
b Dipartimento di Scienze Biochimiche, Uni6ersita` ‘La Sapienza’, Rome, Italy
Received 28 July 2000; accepted 8 January 2001
Abstract
The influenza virus neuraminidase (NA) is an enzyme essential for viral infection and offers a potential target for antiviral drug
development. We aimed our research at the synthesis of non-carbohydrate molecules able to inhibit NA as transition-state
analogues. Aromatic sialic acid analogues (compound 5 and compound 10) were synthesised in good yields starting from
commercially available benzoic acids using a suitable synthetic strategy. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Sialic acid; Influenza; Neuraminidase
simulation of the enzyme–substrate complex [2–5].
Janakiraman et al. [6] proposed a mechanism of influ-
enza virus NA reaction in which the driving force
comes solely from the induction and stabilisation of the
oxocarbonium ion intermediate (see Fig. 1). According
to this mechanism it is possible to inhibit the enzyme by
structurally analogue molecules to the hypotetic transi-
tion state of considered reaction.
The concept of structural similarity to the transition
state has found wide application in drug design over the
years. The multitude of enzyme-inhibitor interactions
are governed by steric as well as electronic factors. In
theory, compounds that closely resemble the transition
state structure should give high binding affinity towards
the target enzyme. These potential inhibitors, besides,
having a high affinity for stable bond with the protein,
and they do not need to be transformed in the reaction
products.
One of the most potent synthetic inhibitors for NA
(Ki of 4 mM [7]), commercially available 2-deoxy-2,3-
didehydro-N-acetylneuraminic acid (DANA, see Fig.
1), was obtained by the simple dehydration of sialic
acid. According to the NA mechanism action described
above, DANA is considered a transition state-like ana-
logue binding to the active site of NA. Several NA
inhibitor analogues have been synthesised using DANA
as a base molecule. An extremely potent influenza NA
1. Introduction
Influenza viruses are enveloped by a host cell-derived
lipid membrane which is penetrated by numerous
copies of two distinct types of virally coded molecules:
haemagglutinin (HA) and neuraminidase (NA). The
HA of influenza viruses binds to terminal sialic acid
residues on the cell membrane as the first step of viral
infection. Viral attachment is followed by receptor me-
diated endocytosis, after which the viral and cell mem-
branes fuse themselves, allowing the nucleocapsid to
enter the cytoplasm. The NA (EC 3.2.1.18) is an exo-
glycosidase that catalyses the hydrolysis of the a-(2,3)
and a-(2,6) glycosidic linkage between a terminal sialic
acid and its adjacent carbohydrate moiety on a variety
of glycoconjugates. NA activity is thought to be essen-
tial for the maintenance of virus mobility, e.g. by means
of the prevention of self-aggregation, to facilitate re-
lease of the virus from the cell surface, to prevent virus
activity from being destroyed by the mucins which are
rich in sialic acids, and it is also involved in mediating
membrane fusion [1]. The enzyme mechanism of NA
from influenza virus has been investigated by kinetic
isotope methods, NMR, and a molecular dynamics
* Corresponding author.
E-mail address: armandodoriano.bianco@uniroma1.it (A. Bianco).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01081-3

306
A. Bianco et al. / Il Farmaco 56 (2001) 305–309
Fig. 1.
inhibitor, with Ki value of 10−10 M, is 2,3-didehydro-
2,4-dideoxy-4-guanyl-N-acetylneuraminic acid,
Zanamivir (GG167, see Fig. 1) [8–10]. GG167 is a
transition state analogue, representing the optimal con-
formational state imposed on the sialic acid unit by the
enzyme during the catalytic cleavage of the glycosidic
linkage.
in the active site correctly thank to its similar size to the
DANA sugar ring, but the planarity near the carboxy-
late mimics correctly that found in the transition-state-
like structure of DANA. Furthermore, the benzene ring
scaffold gives advantages of non-chirality, chemical and
metabolic stability and increased lipophilicity compared
with the dihydropyran ring. These factors may be im-
portant to improve the deficient pharmacokinetic pro-
files observed for GG167. Recently, simple aromatic
influenza NA inhibitors have been synthesised [12].
These compounds have a comparable affinity for the
NA with the sialic acid.
GG167 exhibited potent antiviral activity against a
variety of influenza A and B strains in the cell culture
assay; it is currently being evaluated in human clinical
trials and has shown efficacy in phase III challenge
studies in both prophylaxis and treatment of influenza
virus infections [11]. However, poor oral bioavailability
and rapid excretion precluded GG167 as a potential
oral agent against influenza infection and GG167 has to
be administered by either intranasal or inhaled routes in
clinical trials. In the case of an influenza epidemic, oral
administration may be a more convenient and econom-
ical method for treatment and prophylaxis. Therefore,
it would be desirable to have a new class of orally
active NA inhibitors as potential agents against influ-
enza infection. Moreover, the relative chemical sensitiv-
ity (e.g. to acid, base, and heat) and the complex
stereochemistry (five chiral centres) for this class of
compound seems to make the production of next gener-
ation agents problematic. The general approach to the
structure-based design of NA inhibitors uses the struc-
ture of the DANA–NA complex as a starting point
and is based on the development of new classes of lead
compounds by using chemically simpler cyclic tem-
plates instead of the dihydropyran ring of DANA. It
has been proposed that simple non-carbohydrate ana-
logues containing the carboxylate and the acetylamino
groups attached to a cyclic backbone ‘spacer’ would be
sufficient to generate lead compounds for further elabo-
ration as NA inhibitors. The spacer would need to
correctly orient these groups as found in bound
DANA. It has been also required that such compounds
adopt a planar structure near the carboxylate to mimic
the transition state and be able to present additional
side-chain functionality for interaction with other con-
served amino acid residues in the sialic acid binding
site. Among several chemical classes considered, the
benzoic acid seems to be the most suitable starting
target; in fact, not only it positions the carboxyl group
2. Results and discussion
We synthesised two aromatic analogues, compounds
5 and 10. In these molecules we conserved the func-
tional groups, the carboxylic and the acetamido ones,
necessary for the interaction with the NA active site;
also we introduced the guanidino group at C-5 and we
substituted an acetyl group for the glyceric chain. These
products were prepared as illustrated in Scheme 1 (part
A and B). Compound 5 has been synthesised utilising
as a starting material commercially available 4-amino-
3-hydroxybenzoic acid 1 (Scheme 1, part A).
Compound 1 was acetylated with acetic anhydride
and an aqueous solution of sodium acetate in presence
of hydrochloric acid to give the product 2. Compound
2 was treated with a solution of the nitrating mixture
made from acetic anhydride and nitric acid in dioxane
affording nitro derivative 3, with unsatisfactory yields.
1
The H NMR spectrum of compound 3 shows the
de-shielding of the protons at C-2 and at C-6 that give
two doublets at l 7.92 and 8.55, with coupling constant
of 1.8 Hz, confirming that the nitration of aromatic
ring has occurred in meta position as regards the car-
boxylic group and in ortho position as to the acetamido
group.
The catalytic reduction of nitro derivative 3, carried
out with hydrazine in ethanol in the presence of palla-
dium on calcium carbonate, affords compound 4. The
guanidino group at C-5 was introduced by reaction of
compound 4 with cyanamide in solution of hydrochlo-
ric acid affording the objective molecule 5.

A. Bianco et al. / Il Farmaco 56 (2001) 305–309
307
Scheme 1. (i) Ac2O, AcONaaq, 2 N HCl; (ii) Ac2O, HNO3, dioxane; (iii) N2H4, Pd–C, EtOH; (iv) NH2CN, 6 N HCl.
Compound 10 was prepared by using a similar syn-
thetic strategy (Scheme 1, part B).
to room temperature as the ice bath melted. After 4 h
a light brown precipitate had formed. This was filtered,
washed with water (25 ml), and air-dried to provide 2
(450 mg, 58% yield); it is pure enough to be utilised in
The main difference between two syntheses regards
the nitration step: in fact the compound 2 nitration
affords the nitro derivative 3 in very low yields whereas
compound 8 was obtained as the only reaction product
in quantitative yield.
1
the following synthetic passages. H NMR (DMSO-d6):
l 2.12 (s, 3H, NHAc), 2.33 (s, 3H, OAc), 7.67 (d, 1H,
arom., J=1.9 Hz), 7.77 (dd, 1H, arom., J=1.9, 8.4
Hz), 8.15 (d, 1H, arom., J=8.4 Hz), 9.62 (s, 1H, NH).
Anal. Calc. for C11H11NO5: C, 55.70; H, 4.67; N, 5.90.
Found: C, 55.60; H, 4.80; N, 5.81%.
3. Experimental
3.2. 3-Acetoxy-4-(acetylamino)-5-nitrobenzoic acid (3)
1H NMR spectra were measured with a Varian Gem-
ini 200 MHz spectrometer and chemical shifts are ex-
pressed in ppm from Me4Si. 4-Amino 3-hydroxybenzoic
acid (1) and 3-amino-4-hydroxybenzoic acid (6) were
obtained from Aldrich Chemical Co.
Product purification was obtained by solid–liquid
column chromatography on Merck 0.063–0.20 nm sil-
ica gel. The elution mixtures were determined case by
case. Merck TLC plates coated with Kiesel-Gel 60 F254
were employed to monitor the reactions, sprinkling
with 2 N H2SO4 and then heating at 120°C.
Compound 2 (500 mg) was suspended with stirring in
a mixture of Ac2O (4 ml) and dioxane (3 ml). This was
cooled to 0°C, and a cold solution of the nitrating
mixture made from Ac2O (1.5 ml) and concentrated
HNO3 (1.5 ml) was added slowly to the mixture con-
taining 2. The reaction mixture was then warmed to
30–35°C until the reaction was complete as evidence by
TLC (9:1 CHCl3+CH3OH). The reaction mixture was
poured onto ice/water (100 ml), extracted with EtOAc
(4×20 ml), dried on Na2SO4, and concentrated to
dryness on a rotary evaporator. The residue was
purified by chromatography eluting with chloroform+
methanol (9:1) to give compound 3 (262 mg, 44% yield)
3.1. 3-Acetoxy-4-(acetylamino)-benzoic acid (2)
1
To a stirred solution of commercially available com-
pound 1 (500 mg) in 2 N HCl (10 ml) at 0°C (ice bath)
a solution of NaOAc (5 g) in water was added. To this
Ac2O (2.5 ml) was added. The mixture was stirred at
0°C for 5 min, and it was then allowed to warm slowly
as an oil. H NMR (CD3OD): l 2.10 (s, 3H, NHAc),
2.30 (s, 3H, OAc), 8.05 (d, J=1.5 Hz, 1 H, arom.), 8.35
(d, J=1.5 Hz, 1H, arom.). Anal. Calc. for C11H10N2O7:
C, 46.82; H, 3.57; N, 9.93. Found: C, 46.71; H, 3.68; N,
9.81%.

308
A. Bianco et al. / Il Farmaco 56 (2001) 305–309
3.3. 3-Acetoxy-4-(acetylamino)-5-aminobenzoic acid (4)
cooled to 0°C, and a cold solution of the nitrating
mixture made from Ac2O (1.5 ml) and concentrated
HNO3 (1.5 ml) was added slowly to the mixture con-
taining 7. The reaction was then warmed to 30–35°C
until the reaction was complete as shown by TLC (9:1
CHCl3+CH3OH). The reaction mixture was poured
onto ice/water (100 ml), extracted with EtOAc (4×20
ml), dried on Na2SO4, and concentrated to dryness on
a rotary evaporator to give crude 8 (510 mg, 86%
yield). Compound 8 is pure enough to be utilised in the
following synthetic passages. For its characterisation, a
crude sample (30 mg) was purified by chromatography
eluting with chloroform+methanol (9:1) to give com-
Compound 3 (300 mg) was dissolved in EtOH (20
ml) and catalytic quantity of Pd–C was added to it. To
this mixture hydrazine hydrate (0.1 ml) was added
dropwise. The reaction mixture was heated at 80°C for
3 h. The Pd–C was filtered and the ethanol was concen-
trated under vacuum to give free amine 4 as a pale
yellow oil (260 mg, 97% yield). For the characterisation
of 4, 30 mg was purified by chromatography eluting
1
with chloroform+methanol (8:2). H NMR (D2O): l
2.22 (s, 3H, NHAc), 2.30 (s, 3H, OAc), 7.40–7.45 (m,
2H, aromatic). Anal. Calc. for C11H12N2O5: C, 52.38;
H, 4.80; N, 11.11. Found: C, 52.27; H, 4.88; N, 11.03%.
1
pound 8 (23 mg) as an oil. H NMR (CD3OD): l 2.13
(s, 3H, NHAc), 2.35 (s, 3H, OAc), 8.10 (d, J=1.5 Hz,
1H, arom.), 8.42 (d, J=1.5 Hz, 1H, arom.). Anal. Calc.
for C11H10N2O7: C, 46.82; H, 3.57; N, 9.93. Found: C,
47.70; H, 3.65; N, 9.82%.
3.4. 3-Acetoxy-4-(acetylamino)-5-guanidinobenzoic acid
(5)
Compound 4 (200 mg) was dissolved in 6 N HCl (4
ml); NH2CN (140 mg) was added. The reaction mixture
was heated at 100°C with stirring for 30 min. The
mixture was cooled in an ice bath, diluted with water (4
ml), acidified with concentrated HCl (4 ml) and cooled
at −20°C. After 3 h the solid crystallised, was filtered
on gooch and was washed with 2 N HCl (5 ml). After
chromatographic purification using as eluent chloro-
form+methanol (8:2) compound 5 (160 mg, 67% yield)
3.7. 3-(Acetylamino)-4-acetoxy-5-aminobenzoic acid (9)
Compound 8 (500 g) was dissolved in EtOH (20 ml)
and a catalytic quantity of Pd–C was added to it. To
this mixture hydrazine hydrate (0.1 ml) was added
dropwise. The reaction mixture was heated at 80°C for
3 h. The Pd–C was filtered and the ethanol was concen-
trated under vacuum to give the free amine 9 as a pale
yellow oil (422 mg, 94% yield). For the characterisation
of 9, 30 mg was purified by chromatography eluting
1
was obtained as a white solid. H NMR (DMSO-d6): l
2.14 (s, 3 H, NCOCH3), 2.30 (s, 3 H, AcO), 7.50 (br s,
2 H, NH2), 7.89 (m, 2 H, aromatic), 9.1 (s, 1H, NH),
10.01 (s, 1H, NH). Anal. Calc. for C12H14N4O5: C,
48.98; H, 4.80; N, 19.04. Found: C, 48.77; H, 4.95; N,
18.95%.
1
with chloroform+methanol (8:2). H NMR (D2O): l
2.25 (s, 3H, NHAc), 2.30 (s, 3H, OAc), 7.37–7.42 (m,
2H, arom.). Anal. Calc. for C11H12N2O5: C, 52.38; H,
4.80; N, 11.11. Found: C, 52.25; H, 4.88; N, 11.01%.
3.5. 3-(Acetylamino)-4-acetoxybenzoic acid (7)
To a stirred solution of commercially available com-
pound 6 (500 mg) in 2 N HCI (10 ml) at 0°C (ice bath)
a solution of NaOAc (5 g) in water was added. To this
Ac2O (2.5 ml) was added. The mixture was stirred at
0°C for 5 min, and it was then allowed to warm slowly
to room temperature as the ice bath melted. After 4 h
a light brown precipitate had formed. This was filtered,
washed with water (25 ml), and air-dried to provide 7
(462 mg, 60% yield); it is pure enough to be utilised in
3.8. 3-(Acetylamino)-4-acetoxy-5-guanindinobenzoic
acid (10)
Compound 9 (300 g) was dissolved in 6 N HCl (4.5
ml); NH2CN (150 mg) was added. The reaction mixture
was heated at 100°C with stirring for 30 min. The
mixture was cooled in an ice bath, diluted with water (5
ml), acidified with concentrated HCl (5 ml) and cooled
at −20°C. After 3 h the solid crystallised, was filtered
on gooch and was washed with 2 N HCl (5 ml). After
chromatographic purification using as eluent chloro-
form+methanol (8:2) compound 10 (232 mg, 66%
1
the following synthetic passages. H NMR (DMSO-d6):
l 2.17 (s, 3H, NHAc), 2.35 (s, 3H, OAc), 7.69 (d, 1H,
arom., J=1.8 Hz), 7.79 (dd, 1H, arom., J=1.8, 7.4
Hz), 8.17 (d, 1H, arom., J=7.4 Hz), 9.30 (s, 1H, NH).
Anal. Calc. for C11H11NO5: C, 55.70; H, 4.67; N, 5.90.
Found: C, 55.59; H, 4.77; N, 5.94%.
1
yield) was obtained as a white solid. H NMR (DMSO-
d6): l 2.17 (s, 3H, NCOCH3), 2.30 (s, 3H, OAc), 7.52
(br s, 2H, NH2), 7.78 (m, 2H, arom.), 8.9 (s, 1H, NH),
10.0 (s, 1H, NH). Anal. Calc. for C12H14N4O5: C, 48.98;
H, 4.80; N, 19.04. Found: C, 48.80; H, 4.87; N,
18.96%.This work is part of a project supported by
Istituto Pasteur–Fondazione Cenci Bolognetti, Univer-
sita` di Roma, ‘La Sapienza’.
3.6. 3-(Acetylamino)-4-acetoxy-5-nitrobenzoic acid (8)
Compound 7 (500 g) was suspended with stirring in a
mixture of Ac2O (4 ml) and dioxane (3 ml). This was

A. Bianco et al. / Il Farmaco 56 (2001) 305–309
309
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