Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Article abstract of DOI:10.1016/j.bmc.2012.01.026

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC₅₀ of 14.6 ± 3.0 nM (oseltamivir 25 ± 4 nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC₅₀ of 0.1 ± 0.03 nM (oseltamivir 0.2 ± 0.02 nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.

Full text of DOI:10.1016/j.bmc.2012.01.026

Bioorganic & Medicinal Chemistry 20 (2012) 2152–2157
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro study of novel neuraminidase inhibitors against
avian influenza virus
Jarinrat Kongkamnerd a,d, Luca Cappelletti b, Adolfo Prandi b, Pierfausto Seneci b,c,
,
⇑
Thanyada Rungrotmongkol d, Nutthapon Jongaroonngamsang d, Pornchai Rojsitthisak d,
h
h
h
h
Vladimir Frecer e,f,g, , Adelaide Milani , Giovanni Cattoli , Calogero Terregino , Ilaria Capua ,
⇑
i
e
Luca Beneduce i, Andrea Gallotta i, Paolo Pengo i, , Giorgio Fassina , Stanislav Miertus ,
⇑
Wanchai De-Eknamkul a,
⇑
a Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
b Centro Interdipartimentale Studi biomolecolari e Industriali applicati (CISI), Via Fantoli 16/15, 20138 Milan, Italy
c Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milan, Italy
d Department of Pharmaceutical Chemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Patumwan, Bangkok 10330, Thailand
e ICS-UNIDO, International Centre for Science and High Technology, United Nations Industrial Development Organization, AREA Science Park, Padriciano 99, 34149 Trieste, Italy
f Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, Odbojarov 10, Bratislava SK-83232, Slovakia
g Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, Bratislava SK-83391, Slovakia
h OIE, FAO and National Reference Laboratory for Avian Influenza and Newcastle Disease, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro,
PD, Italy
i Xeptagen SpA, VEGA Science Park Building Auriga, Via delle industrie 9, 30175 Marghera-Venezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In
this study, five oseltamivir analogs PMC-31–PMC-36, synthesised according to the outcomes of a rational
design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of
oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and
N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neur-
aminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus
expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir
carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibi-
tion on N1 with an IC50 of 14.6 3.0 nM (oseltamivir 25 4 nM), while PMC-35 showed a significant
inhibitory effect on N3 with an IC50 of 0.1 0.03 nM (oseltamivir 0.2 0.02 nM). The analysis of the inhib-
itory properties of this panel of compounds allowed a preliminary assessment of a structure–activity
relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The sub-
stitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the
observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of
the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both
N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was
observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate intro-
ducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases,
displaying a more pronounced effect against N1.
Received 27 September 2011
Revised 11 January 2012
Accepted 16 January 2012
Available online 26 January 2012
Keywords:
Neuraminidase inhibitors
Avian influenza
Screening assay
Ó 2012 Published by Elsevier Ltd.
1. Introduction
avian influenza virus in suitable ‘mixing vessels’ thus generating
novel viral strains raised the concern about the next pandemic.
The spreading of avian influenza in birds increased the proba-
bility of direct infection in humans. To date, person to person
transmission has been very rare and associated with close human
contact.1 Nonetheless, the possibility of genetic reassortment of
Currently, anti-influenza medications consist of two classes of
drugs: M2 protein inhibitors (amantadine and rimantadine) and
neuraminidase inhibitors (oseltamivir and zanamivir).2 M2 protein
inhibitors are specifically active against influenza A; they interfere
with the viral uncoating process through a direct interaction with
the matrix (M2) protein, which functions as a channel for hydrogen
ions.3 Neuraminidase inhibitors prevent the removal of the sialic
acid (N-acetylneuraminic acid) residue from the glycopeptide
⇑
Corresponding authors.
E-mail addresses: pierfausto.seneci@unimi.it (P. Seneci), frecer@fpharm.uniba.sk
(V. Frecer), pengo@xeptagen.com (P. Pengo), dwanchai@chula.ac.th (W. DeEknamkul).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2012.01.026

J. Kongkamnerd et al. / Bioorg. Med. Chem. 20 (2012) 2152–2157
2153
receptor by the viral neuraminidase, which would otherwise allow
the virus particles to be released from the infected cell thus spread-
ing to neighboring cells.4 As a class, neuraminidase inhibitors are
effective against all neuraminidase subtypes and, therefore, against
all strains of influenza. This is a key point in epidemic and pandemic
preparedness and an important advantage over the M2 protein
inhibitors which are effective only against sensitive strains of influ-
enza A.2 Among all antiviral drugs, oseltamivir is the recommended
antiviral to treat patients infected with avian influenza (H5N1)
including chemoprophylaxis in high-risk populations.5
Alarming oseltamivir resistances, such as the one associated to
the N294S mutation of the neuraminidase detected in an influenza
A (H5N1) infected patient in Egypt6 or the H274Y mutation in
influenza A (H5N1) reported in Vietnamese patients during treat-
ment,7,8 are important examples stressing the present need of
novel putative neuraminidase inhibitors.
Synthetic oseltamivir analogs were dissolved in water in order
to obtain a stock solution of the compounds. All the stock solutions
were aliquoted and stored at ꢀ20 °C until further use. For PMC-35
(5) the use of 10% DMSO in water was necessary in order to obtain
a clear solution.
2.2. Reagents
20-(4-Methylumbelliferyl)-
a-D-acetyl neuraminic acid (MUN-
ANA) was purchased from Gold-Biotechnology Inc., 4-methylum-
belliferone (4-MU) was purchased from Alfa Aesar GmbH & Co.
KG. 2-N-Morpholino-ethanesulfonic acid (MES) and Glycine were
purchased from Sigma, CaCl2 was purchased from Sigma–Aldrich.
2.3. Neuraminidase
The available X-ray crystallographic data suggest that the strat-
egy of designing neuraminidase inhibitors binding to the highly con-
served region of the neuraminidase, achieves inhibitors effective
against all influenza neuraminidase subtypes, N1–N9 and influenza
B viruses.9–13 However, X-ray crystallographic studies of neuramin-
idase from type A influenza viruses, also revealed that the neuramin-
idase structures can be divided in two different classes, known as
group-1 (comprising N1, N4, N5, N8) and group-2 neuraminidases
(comprising N2, N3, N6, N7, N9), according to distinctive structural
features near the oseltamivir binding site. In fact, the major differ-
ence between the two groups is the presence of a large cavity, known
as the ‘150-cavity’, adjacent to the active site in group-1 but not
present in group-2 neuraminidases.14 Since currently available
neuraminidase inhibitors were targeted against the structures of
group-2 neuraminidases, the discovery of the ‘150-cavity’ in
group-1 neuraminidases prompted the design the novel neuramin-
idase inhibitorsin order to improve the efficiency of current antiviral
treatments. In fact, many studies have been focused on using com-
putational drug design for modifying the structure of oseltami-
vir.10–15 However, these data are limited only to in silico screening.
In this study, we report a preliminary in vitro assessment of the
neuraminidase inhibition activity of five novel putative inhibitors
synthesised so far that contain extensions of the 4-amido and
5-amino groups intended to fill the 150-cavity of the group-1 neu-
raminidases. These analogs were proposed by computer-assisted
combinatorial design of more potent N1 inhibitors17 taking advan-
tage of the aforementioned recent X-ray crystallographic studies
of neuraminidase N1 co-crystallized with oseltamivir14 and struc-
ture-based in silico screening of a virtual library of oseltamivir ana-
logs. The compounds studied in this work do not contain structural
modifications of the 3-ether group and thus are not expected to dis-
play inhibitory activity against the oseltamivir-resistant N294S and
H247Y variants of N1. The mutated residues are located near the 3-
ether group and thus will most probably not be significantly affected
by the 4-amido and 5-amino groups extensions. The neuraminidas-
es used in this study were expressed in baculovirus and found on the
viral membrane capsule, the sequences were taken from the genome
of influenza A H7N1 and H7N3 viruses.21,22 Our study has identified
oseltamivir analogs with elevated binding affinity to N1 and N3
neuraminidase subtypes.
Lyophilized aliquots of baculovirus supernatant expressing re-
combinant Neuraminidase (N1) and (N3) were provided by OFFLU
Network on Avian Influenza. The lyophilized material was dis-
solved in double distilled water. These solutions were aliquoted
and stored at ꢀ80 °C until further use. The enzymatic activity of
the supernatant was assessed as previously described.18,19
2.4. Neuraminidase inhibition assay
2.4.1. Analysis of inhibition against N1
In a microtitre plate, 90 ll of 33 mM MES buffer pH 6.5 contain-
ing 4 mM CaCl2 were dispensed in a suitable number of wells.
Since PMC-35 was insoluble in 33 mM MES buffer pH 6.5, 10%
DMSO was added in this case. Ten microliter of 1 mM oseltamivir
carboxylate and its analogs were added in a first well, and the solu-
tion was thoroughly mixed obtaining a 0.1 mM inhibitor concentra-
tion. A 10 ll aliquot of this solution was transferred in a second well
and thoroughly mixed, reaching after dilution an inhibitor concen-
tration of 0.01 mM. The process was continued until a seventh,
0.1 nM inhibitor concentration was obtained, at the end of the dilu-
tion process. Each well contained a final volume of 90 ll. Twenty-
five microliter of enzyme stock solution were added to each well,
and the mixtures were incubated for 2 h at 37 °C; after the incuba-
tion time, 25
final concentrations of oseltamivir carboxylate or its analogs span-
ning the 0.064 nM–64.2 M range. After incubating the plate for 5 h
at 37 °C, 50 l of stop solution (0.1 M glycine in 25% aqueous EtOH,
ll of a 20 lM MUNANA solution were added, with
l
l
pH 10.7) were added. The fluorescence of the solutions contained in
each well was read with the Victor3 multilabel counter. The 50%
inhibitory concentration (IC50) was determined from the dose–
response curve using the GraphPad Prism 5 software (GraphPad,
San Diego, CA). All measurements were replicated in 3–5 indepen-
dent experiments.
2.4.2. Analysis of inhibition against N3
2.4.2.1. Procedure for PMC-34 (4), PMC-35 (5), PMC-36 (6) and
oseltamivir free acid.
pH 6.5 containing 4 mM CaCl2 were dispensed in a suitable number
of wells in a microtiter plate. Namely, 10 l of 10 M inhibitor
Ninety microliter of 33 mM MES buffer
l
l
solutions (PMC-34, PMC-35 and commercial oseltamivir) were
added in a first well and the mixture was thoroughly mixed obtain-
ing a 1
was transferred in a second well and thoroughly mixed obtaining a
0.1 M inhibitor solution. The process was continued until a sev-
enth 0.001 nM solution was obtained, at the end of the dilution
process. Each well contained a final volume of 90 l. Twenty-five
microliter of enzyme stock solution were added to each well, and
the mixtures were incubated for 2 h at 37 °C; after the incubation
lM inhibitor concentration. A 10 ll aliquot of this solution
2. Materials and methods
l
2.1. Oseltamivir and its analogs
l
Oseltamivir carboxylate was purchased from Toronto Research
Chemical Inc. The compound was dissolved in distilled water
obtaining a stock solution with concentration 17.58 mM. The stock
solution was aliquoted and stored at ꢀ20 °C until further use.
time, 25
ll of a 20 lM MUNANA solution were added, with final
concentrations of oseltamivir carboxylate or its analogs spanning

2154
J. Kongkamnerd et al. / Bioorg. Med. Chem. 20 (2012) 2152–2157
Table 1
Kinetic parameters of N1 and N3
Recombinant protein
Neuraminidase activity (mU/mg)
Km SDa
400 60
87
(l
M)
IC50 SDa,b (nM)
25
0.2 0.02
N1
N3
0.04
0.4
4
4
a
Km, IC50 values were obtained from three independent experiments.
IC50 is a concentration of oseltamivir which inhibits by 50% the neuraminidase activity.
b
Figure 1. Structures of the compounds tested in the study.
the 0.00064–642 nM range. After incubating the plate for 5 h at
37 °C 50 ll of stop solution (0.1 M glycine in 25% aqueous EtOH,
enzyme required to convert one micromole of substrate per min-
ute at 37 °C. The enzymes were further characterized by determin-
ing their Michaelis–Menten constant against the substrate; the
pH 10.7) were added. The fluorescence of the solutions contained
in each well was read with the Victor3 multilabel counter. The
50% inhibitory concentration (IC50) was determined from dose–re-
sponse curve using a GraphPad Prism 5 software (GraphPad, San
Diego, CA). All measurements were replicated in 3–5 independent
experiments.
Michaelis constant of N1 was 400 60
lM, while that of N3 was
87 M. The inhibitory activity of oseltamivir carboxylate
4
l
against the two enzymes was assessed and used as reference to
compare the different inhibitors synthesized. The IC50 of commer-
cially available oseltamivir carboxylate on N3 and N1 were found
to be 0.2 0.02 nM and 25 4 nM, respectively, the obtained data
are reported in Table 1.
2.4.2.2. Procedure for PMC-31 (1), PMC-32 (2), PMC-33
(3).
For PMC-31 (1), PMC-32 (2), PMC-33 (3), the same proce-
dure described for the N1 study was used. The 50% inhibitory con-
centration (IC50) was determined from dose–response curves using
the GraphPad Prism 5 software (GraphPad, San Diego, CA). All mea-
surements were replicated in 3–5 independent experiments.
3.2. Synthesis of oseltamivir carboxylate (PMC 34) and of
oseltamivir analogs PMC 31–33, PMC 35,36
The structures of oseltamivir carboxylate PMC-34/compound 4
and of five oseltamivir analogs PMC 31–33, and PMC 35–36 (com-
pounds 1–3, 5 and 6), prepared and biologically characterized in
this study as potassium salts, are reported in Figure 1.
3. Results and discussion
Their synthesis was achieved through the adaptation of some
3.1. Characterization of recombinant neuraminidase
published experimental protocols,14–26 and required at first the
preparation of
(Scheme 1).
a common, key amino-azide intermediate 15
Both N1 and N3 containing supernatants were examined to as-
sess their enzymatic activity as previously described.23,24 The con-
centration of active enzyme in both the supernatants was
determined by incubating each preparation with excess substrate
and monitoring the extent of substrate cleavage over time. The
amount of released product was monitored fluorimetrically, the
concentration of 4-methylumbelliferone was determined by com-
parison with a calibration curve obtained by using a reference sam-
ple of 4-methylumbelliferone. This analysis revealed that the N3
containing supernatant displayed a neuraminidase activity 10
times higher than the N1 containing supernatant. The measured
concentrations of active enzyme were 0.4 mU/mg in the case of
N3 and 0.04 mU/mg in the case of N1 where one unit of enzyme
activity was defined as the amount of active neuraminidase
Key intermediate 15 was acylated (PMC 31 and 32), acylated
and reduced (PMC-33–35), or acylated, reduced and alkylated
(PMC-36) to provide, respectively, the six carboxylate esters 16,
17, 19, 21, 24 and 26. The six esters were hydrolyzed with KOH
to give the six potassium carboxylates PMC 31–36 (Scheme 2).
3.3. Neuraminidase Inhibition assay
3.3.1. Inhibitory activity of oseltamivir analogs on N1
All the inhibitors were tested at different concentrations, span-
ning the range 0.064 nM–64.2
lM. A dose–response curve was
obtained for each compound. Fitting of the experimental data

J. Kongkamnerd et al. / Bioorg. Med. Chem. 20 (2012) 2152–2157
2155
O
O
O
HO
HO
HO
HO
O
O
OCH CH
2
OH
OCH CH
2
b
a
3
3
c
O H
H
OH
O
7
8
9
O
O
O
O
O
O
O
O
OCH CH
OCH CH
OCH CH
2
2
3
2
3
3
d
e
f
H
O
OSO CH
OSO CH
3
2
3
2
10
11
12
O
O
O
O
O
O
OCH CH
OCH CH
OCH CH
2
g
h
2
3
2
3
3
HO
H2N
14
N
H
15
N
N
3
3
13
Scheme 1. Reagents and conditions: (a) Ethanol, SOCl2, reflux 3 h; (b) 3-pentanone, p-TSA, toluene, 100 °C, MW, 15 min; (c) MsCl, TEA, DCM, rt, 2 h; (d) Et3SiH, TiCl4, DCM,
ꢀ35 °C, 1 h; (e) KHCO3, H2O, EtOH, 60 °C, 1 h; (f) NaN3, NH4Cl, H2O, EtOH, 68 °C, 14 h; (g) PMe3, THF, CH3CN, 25 °C, 10 min; (h) NaN3, NH4Cl, DMF, 70 °C, overnight.
allowed the determination of the IC50 for each compound; the
experimental data are graphically reported in Figure 2.
and PMC-31 (1) had a limited inhibitory activity with an IC50 of
138.9 21 M and 51.6 3.0 M, respectively (Table 2).
l
l
The analysis of the experimental data indicated PMC-36 (6) as
the best inhibitor of N1 activity within the panel of tested com-
pounds, displaying an IC50 of 14.6 3.0 nM, while commercial osel-
tamivir displayed an IC50 of 24.9 3.9 nM; a synthetically prepared
sample of oseltamivir, PMC-34 (4), was also included in this anal-
ysis and it displayed an IC50 of 39.3 3.2 nM (Table 2). Compound
PMC-35 (5) displayed an IC50 of 31.8 6.9 nM, while PMC-32 (2)
was less active with an IC50 of 84.4 20 nM. Both PMC-33 (3)
The variations in the measured inhibition for the different com-
pounds revealed that an isobutyl group installed on the 5-amino
group site to form a secondary amine is well tolerated and appears
to increase the affinity for the enzyme, as shown in the case of
PMC-36 (6). This might be because the alkyl group affects the
strengths of hydrogen bonds of the amine group with acidic resi-
dues of the N1 neuraminidase, or more probably due to favorable
van der Waals contacts in the 150-loop cavity, which is located
Scheme 2. Reagents and conditions: (a) Acyl chloride, NMM, DCM, rt; (b) PMe3, DCM, 1 M HCl, rt; (c) isobutyraldehyde, DCM, then NaBH(OAc)3, 0 °C to rt; (d) 1 M aq KOH,
dioxane, 4–23 h, rt.

2156
J. Kongkamnerd et al. / Bioorg. Med. Chem. 20 (2012) 2152–2157
Figure 4. Titration curves of oseltamivir and its analogs against N3.
Figure 2. Titration curves of oseltamivir and its analogs against N1.
benchmark. The IC50 of commercial oseltamivir on N1 did not per-
fectly match that of synthesized oseltamivir (PMC-34), but its var-
iability is well within the acceptable limits. The titration curves
and the IC50 values of all the tested compounds on N1 are reported
in Figure 2 and Table 2, respectively.
Table 2
The IC50 of oseltamivir analogs on N1 and N3
Compounds
IC50 SD on N1 (nM)
IC50 SD on N3 (nM)
PMC-31
PMC-32
51,576 2904
84.4 20
99,295 32,416
2.2 1.4
PMC-33
PMC-34 (oseltamivir)
PMC-35
138,962 21,589
39.3 3.2
4512 1172
0.1 0.08
0.1 0.03
3.3.2. Inhibitory activity of oseltamivir analogs on N3
31.8 6.9
For neuraminidase N3, all the inhibitors were tested at different
concentrations either spanning the 0.64 pM–642.8 nM range
(PMC-34 (4); PMC-35 (5), commercial oseltamivir), or the
PMC-36
14.6 3.0
28.1 9.7
Commercial oseltamivir
24.9 3.9
0.2 0.02
Oseltamivir carboxylatea
36.1b ꢀ 53.2c
2.9d ꢀ 3.3e
0.064 nM–64.2 lM range (PMC-31 (1), PMC-32 (2), PMC-33 (3);
a
b
c
Taken from Govorkova et al., 2001.21
PMC-36 (6)). A dose–response curve was obtained for each com-
pound, and fitting of the experimental data allowed the determina-
tion of the IC50 of each putative inhibitor. The dose–response
curves for each compound are reported in Figure 4.
Influenza virus: A/teal/Hong Kong/W312/97 (H6N1).
Influenza virus: A/duck/Alberta/35/76 (H1N1).
Influenza virus: A/duck/Singapore/3/97 (H5N3).
Influenza virus: A/duck/Germany/1215/73 (H2N3).
d
e
Among the tested compounds PMC-35 (5), PMC-34 (4) and com-
mercial oseltamivir showed a potent inhibitory activity on neur-
aminidase subtype
3 with IC50 of 0.1 0.03, 0.1 0.08 and
0.2 0.02 nM, respectively. All the other compounds displayed a
lower inhibitory potency, with IC50 values ranging from
2.2 1.4 nM for PMC-32 (2) to 99.3 32.4 lM for PMC-31 (1). The
data for the whole set of tested compounds is reported in Table 2.
The differences in the observed inhibitory properties of the
investigated species indicated that the primary 5-amino group is
essential for inhibitory activity on neuraminidase subtype 3. Mod-
ification of the amine seems to decrease the activity, as shown for
PMC-32 (2) and PMC-36 (6); this observation infers a different
trend in comparison with N1, where PMC-36 (6) outperformed
PMC-35 (5). Adding a small group on the 4-amido function as for
PMC-35 (5) does not affect the activity. In contrast, the presence
of a bulky group on the amide as in PMC-31 (1) and PMC-33 (3)
greatly decreases the ability to inhibit the N3 neuraminidase. The
IC50 of commercial oseltamivir is similar to that of synthesized
oseltamivir (PMC-34). Higher inhibitory potency of oseltamivir to-
wards the group-2 neuraminidase N3 over the group-1 neuramin-
idase subtype N1 as reported also by other groups.9
Figure 3. Oseltamivir binding site in the crystal structure of neuraminidase N1.14
Partially transparent molecular surface (colored according to interpolated atomic
charge) shows the 150-loop and the associated 150-cavity specific for the group-1
neuraminidases. Oseltamivir is shown in a stick representation, hydrogen atoms are
omitted for better clarity. Coloring scheme: hydrogen: white, carbon: green,
nitrogen: blue, oxygen: red).
4. Conclusion
The modification at both the 5-amino and 4-amido group of
oseltamivir carboxylate is needed in order to make more efficient
neuraminidase inhibitors based on this scaffold. In this paper, we
reported a preliminary activity screening of novel putative neur-
aminidase inhibitors obtained by computer-assisted combinatorial
design on the basis of the oseltamivir structure as well as the crys-
tal structure of N1 neuraminidase. The collected data suggest that
the introduction of small substituents on the 5-amino group and 4-
acetamido group of oseltamivir increases the affinity of the new
analogs against N1, but it seems to be rather ineffective with re-
spect to N3. In addition, the introduction of large substituents on
in the vicinity of the amino and acetamido groups of bound osel-
tamivir (Fig. 3).14
Moreover, a small increase in size for the acyl group on the 4-
amido group from the original acetyl group of oseltamivir also
seems to enhance the inhibitory activity of the novel species, as ob-
served in the case of PMC-36 (6) and PMC-35 (5). However, this
position seems to be intolerant to the introduction of larger groups,
since the presence of a 2-butenyl amide dramatically reduces the
affinity of compound PMC-33 (3) with respect to the oseltamivir

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Acknowledgments
Jarinrat Kongkamnerd would like to acknowledge financial sup-
port from ICS-UNIDO within a collaborative framework of activities
with the Chulalongkorn University and from the Italian Ministry of
Foreign Affairs.
This work was partially supported by the Grant from The Slovak
Research and Development Agency under the contracts VVCE-
0001-07 and by the VEGA Grant 2/0153/11.
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Supplementary data
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Supplementary data (synthetic procedures and spectral data of
the described compounds) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2012.01.026.
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