Sialidase inhibitors related to zanamivir. Further SAR studies of 4-amino-4H-pyran-2-carboxylic acid-6-propylamides

Article abstract of DOI:10.1016/S0960-894X(01)00019-1

SAR investigations of the 4- and 5-positions of a series of 4-amino-4H-pyran-2-carboxylic acid 6-carboxamides are reported. Potent inhibitors of influenza A sialidase with marked selectivity over the influenza B enzyme were obtained when the basic 4-amino substituent was replaced by hydroxyl or even deleted. Modifications at the 5-position exhibited a tight steric requirement, with trifluoroacetamide being optimal.

Full text of DOI:10.1016/S0960-894X(01)00019-1

Bioorganic& Medicinal Chemistry Letters 11 (2001) 669±673
Sialidase Inhibitors Related to Zanamivir.
Further SAR Studies of 4-Amino-4H-pyran-2-carboxylic
Acid-6-propylamides
Paul G. Wyatt,a,* Barry A. Coomber,a Derek N. Evans,a Torquil I. Jack,a
Heather E. Fulton,a Alan J. Wonacott,b Peter Colmanc and Jose Varghesec
aDepartment of Medicinal Chemistry, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage,
Herts SG1 2NY, UK
bDepartment of Molecular Recognition, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage,
Herts SG1 2NY, UK
cBiomolecular Research Institute, Parkville, Victoria, Australia
Received 8 December 1999; revised 6 December 2000; accepted 4 January 2001
AbstractÐSAR investigations of the 4- and 5-positions of a series of 4-amino-4H-pyran-2-carboxylic acid 6-carboxamides are
reported. Potent inhibitors of in¯uenza A sialidase with marked selectivity over the in¯uenza B enzyme were obtained when the
basic 4-amino substituent was replaced by hydroxyl or even deleted. Modi®cations at the 5-position exhibited a tight steric
requirement, with tri¯uoroacetamide being optimal. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
in¯uenza B enzyme. In this paper we describe further
development of the SAR of the carboxamide series,
looking at potency and selectivity. During these studies
we have focused on the 4- and 5-positions on the pyran
ring, structures 4 and 5, respectively. The in vitro and
in vivo antiviral properties of representatives of these
series are reported.
Zanamivir 11 and GS 4071 2 (R=H)2,3 are potent
selective inhibitors of in¯uenza A and B sialidases and
in¯uenza A and B in vitro. The e€ectiveness of both
zanamivir and the prodrug GS 4104 2 (R=Et) during
clinical trials has demonstrated the utility of sialidase
inhibitors as anti-in¯uenza agents.4 Previously, as part
of the SAR investigations into zanamivir, we reported
on a series of 4H-pyran carboxamide in¯uenza sialidase
inhibitors 3.5,6 In these compounds the glycerol side
chain of zanamivir was replaced by a lipophilic carbox-
amide moiety. Although some of this series were more
potent inhibitors of in¯uenza A sialidase than zanami-
vir, they were signi®cantly less potent against the
Results and Discussion
Although some of the previously described 4-amino-4H-
pyran-2-carboxylic acid 6-carboxamides are potent
inhibitors of in¯uenza A sialidase, they are signi®cantly
*Corresponding author. Tel.: +44-1438-764232; fax: +44-1438-763624; e-mail: pw29967@glaxowellcome.co.uk
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00019-1

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P. G. Wyatt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 669±673
less inhibitory of in¯uenza B sialidase.5,6 To expand
the SAR studies of the carboxamide series, the 4- and
5-substituents were modi®ed to investigate the e€ect on
potency and selectivity.
yielded 4a,b.7 Using established chemistry5 b-azide 98
was converted to the b-amino derivative 4c.
The syntheses of the 4-a- and b-hydroxy epimers 4d and
4e, from the glycerol-protected 4-a- and b-hydroxy
derivatives 10a9 and 10b,10 respectively, are outlined in
Scheme 2. The acid-stable t-butyldiphenylsilyl ether was
used to protect the 4-hydroxy moiety of 10a and 10b,
allowing the synthesis of acids 11a and 11b. TBTU11
mediated coupling of acids 11a,b with phenethyl-propyl-
amine gave the corresponding amides in higher yields
than the previously used two-step process via the
penta¯uorophenyl esters.5
Modi®cation of the 4-position
Previous studies of the SAR of the 4-position of zana-
mivir suggested that modi®cation of the 4-guanidino
substituent was not tolerated. Also taking in considera-
tion the potency of some of the 4-amino carboxamides14
so far described, we decided not to investigate 4-guani-
dino derivatives but to use amines 3a,b as our starting
point. 4-Alkylamino derivatives 4a,b were synthesised
using the route shown in Scheme 1. Whereas alkylation
of the 4-amino derivative 65 a€orded gross mixtures, cae-
sium carbonate mediated alkylation of the 4-tri-
¯uoroacetamide 7 gave good yields of the 4-N-alkylamino
derivatives 8a,b. Deprotection under standard conditions5
The 4-desoxy derivative 4f was synthesised using a
sodium borohydride reduction of the palladium com-
plex of b-acetate 12 (Scheme 2).12 Acetate 12 was
obtained in 92% yield by acetylating the methyl ester of
4e using acetic anhydride in pyridine.
Scheme 1. (a) (CF3CO)2O (93%); (b) Cs2CO3, RBr, DMF (85% R=CH3); (c) (i) TFA, CH2Cl2; (ii) K2CO3, MeOH, H2O (85% R=CH3);
(d) NaOMe, MeOH (94%); (e) (i) NaIO4 MeOH, H2O; (ii) NaClO2, tBuOH (58%); (f) CF3CO2C6F5, pyridine; (g) (nC3H7)2NH, THF (30% over
2 steps); (h) PPh3, THF, then Et3N, H2O; (i) Et3N, H2O (74% over 2 steps).
Scheme 2. (a) tBuPh2SiCl, DMAP, imidazole, DMF (77% 4-b-OH); (b) 80% aq AcOH (83% from 10a); (c) NaOMe, MeOH (99%); (d) (i) NaIO4,
MeOH, H2O; (ii) NaClO2, tBuOH (89% 4-b-OH); (e) TBTU, [Ph(CH2)2](nC3H7)NH, THF (42% 4-b-OH); (f) TBAF, THF (90% 4-b-OH); (g) Et3N,
H2O (43% 4-b-OH); (h) Pd(Ph3)4, NaBH4, THF (41%); (i) Et3N, H2O (69%).

P. G. Wyatt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 669±673
671
The 4-N-a-methyl- and ethylamino derivatives 4a,b
(Table 1) exhibited at least a 10-fold loss of activity
when compared to 3a. These results mirrored the ®nd-
ings in the 6-glycerol series.15 The 4-b-amino derivative
4c proved to be approximately 10-fold less active than
the corresponding 4-a-amino epimer 3a both against
in¯uenza A and B sialidase.
the 6-glycerol series. More surprisingly, the 4-desoxy
derivative 4f had similar activity to 3b and 4d. This was
an important ®nding that could aid the design of further
in¯uenza sialidase inhibitors. Even against in¯uenza B
sialidase, where the carboxamide moiety is less favour-
ably bound, removal of the basicamine of 3b did not
signi®cantly a€ect the binding, as 4f had a similar
potency to 3b. Overall, the modi®cations carried out did
not reduce the di€erence in potency against in¯uenza A
and B and in some cases increased it.
To investigate whether the need for a basicgroup in the
4-position for potent activity still held for the carbox-
amide series, we synthesised the epimeric4- a- and
b-hydroxy derivatives 4d and 4e, respectively. Unex-
pectedly, the compounds exhibited similar activity
against in¯uenza A sialidase to 3b, indicating this group
was not as important for binding in this series as for
Comparison of the crystal structures of 3b and 4e bound
into in¯uenza A sialidase (Fig. 1) or in¯uenza B siali-
dase (data not shown) did not give any obvious ration-
ale for the above observations, as the whole crystal
Table 1. Sialidase inhibitory and plaque reduction activities of 4a±f. Comparison with 1 and 3a,ba
X
R
Sialidase inhibition IC50
Plaque reduction IC50
A Aichi (mM)
B Victoria (mM)
Flu A (mg/mL)
Flu B (mg/mL)
1
0.002
0.012
0.003
0.24
0.42
0.14
0.007
0.017
0.007
0.004
2.0
3.6
140
4.5
7.4
51
>400
14
0.005
0.002
0.03
0.002
0.7
0.28
3a
3b
4a
4b
4c
4d
4e
4f
a-NH2
a-NH2
a- NHMe
a-NHEt
b-NH2
a-OH
n-Propyl
Ph(CH2)2
n-Propyl
n-Propyl
n-Propyl
Ph(CH2)2
Ph(CH2)2
Ph(CH2)2
0.005
>100
b-OH
H
aSialidase inhibitory and plaque reduction activities of compounds were determined by previously reported methods.5,16
Figure 1. Stereo ®gure showing a superimposition of the crystal structure of the active site of in¯uenza sialidase with the inhibitors 3b and 4e. Only
the residues involved in interactions around the 4-position of the ligands are shown. Protein and ligand are in atom colours for 3b, ligand in yellow
for 4e with oxygens of protein and water molecules in pink. Hydrogen bonds are shown as dotted lines.
Scheme 3. (a) (i) NaIO4, MeOH, H2O; (ii) NaClO2, tBuOH; (b) TBTU, Pr(R1)NH, THF; (c) 4 M HCl in dioxan; (d) RCOCl or CH3SO2Cl, pyri-
dine; (e) PPh3, THF, then Et3N, H2O; (f) Et3N, H2O, 50 ꢀC.

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P. G. Wyatt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 669±673
Table 2. Sialidase inhibitory and plaque reduction activities of 5a±h. Comparison with 3a,ba
R
R1
Sialidase inhibition IC50
Plaque reduction IC50
A Aichi (mM)
B Victoria (mM)
Flu A (mg/mL)
Flu B (mg/mL)
3a
5a
5b
5c
5d
3b
5e
5f
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
Ph(CH2)2
Ph(CH2)2
Ph(CH2)2
Ph(CH2)2
Ph(CH2)2
CH3CO
C2H5CO
(CH3)2CHCO
CF3CO
CH3SO2
CH3CO
C2H5CO
C3H7CO
cC3H5CO
CF3CO
0.012
0.005
17
0.0003
3.0
0.003
0.0007
43
0.05
0.003
2.0
7.0
>540
0.2
92
3.0
6.0
>500
90
0.002
0.0002
0.7
3.0
0.00001
0.03
0.3
0.3
5g
5h
0.1
0.004
0.056
aSialidase inhibitory and plaque reduction activities of compounds were determined by previously reported methods.5,16
structures including the bound ligands were essentially
superimposable. As can be seen in Figure 1 the only
di€erences observed were slight movements of aspartic
acid 151 and glutamic acid 119 and the accompanying
structural water molecules near to the 4-position. These
side-chain movements are due to the loss of hydrogen
bonds to the amino group of 3b. The small shifts of the
water molecules occur as a consequence of the replacement
of the a-amino group of 3b with the b-hydroxy of 4e.
moiety with a sulphonamide group 5d gave a 300-fold
loss of activity, whereas this modi®cation was well tol-
erated in the 6-glycerol series.13
Conclusion
In contrast to the 6-glycerol substituted series related to
zanamivir, the 6-carboxamide series does not require a
basicgroup in the 4-position for potent sialidase activity.
In fact, no functionality at all is required for nanomolar
activity against in¯uenza A sialidase. This surprising
®nding could be used to design further in¯uenza siali-
dase inhibitors. Modi®cations at the 5-position demon-
The similarity of the activity of 3b compared to 4c±f
suggests that within the carboxamide series the overall
binding of the compounds is dominated by the lipo-
philic interactions of the carboxamide moiety. There-
fore, any binding energy gained from hydrogen bonding
of the 4-substituent with the enzyme and the structural
water molecules is balanced by the desolvation of the
native enzyme and unbound ligand.17
strated
a very tight stericrequirement for this
substituent; tri¯uoroacetamide was optimal. As with the
previous examples of the carboxamides, a signi®cant
selectivity for in¯uenza A over in¯uenza B sialidase was
observed for all the compounds synthesised.
Modi®cation of the 5-position
The synthesis of 5-modi®ed derivatives 5a±f from 1313 is
outlined in Scheme 3.
Acknowledgements
The authors wish to thank Richard C. Bethell, Sa®a H.
Madar and Jackie M. Barnett for their contributions to
this work.
The activities of a range of 5-modi®ed compounds are
summarised in Table 2. Consistent with previous stu-
dies13 there was an extremely tight stericrequirement
for the 5-substituent. Replacement of acetamide with
propionamide 5a,e was tolerated with little change in
activity against in¯uenza A or B sialidase. However,
further homologation to either methylpropionamide 5b
or 2-butyramide 5f resulted in a 1000-fold loss of activ-
ity. Remarkably, the cyclopropyl derivative 5g was only
25-fold less active than the acetamide 3b. The tri-
¯uoroacetamide derivative of the dipropylamide 5c was
100-fold more potent against in¯uenza A in both
enzyme and plaque assays. The 10-fold increase in
potency in the in¯uenza B enzyme assay did not trans-
late into signi®cantly improved in¯uenza B plaque
activity.
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