Dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including H5N1 avian influenza

Article abstract of DOI:10.1021/jm040891b

The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13 are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along with dimer 3, are being investigated as potential second generation inhale

Full text of DOI:10.1021/jm040891b

2964
J. Med. Chem. 2005, 48, 2964-2971
Dimeric Zanamivir Conjugates with Various Linking Groups Are Potent,
Long-Lasting Inhibitors of Influenza Neuraminidase Including H5N1 Avian
Influenza
Simon J. F. Macdonald,*,† Rachel Cameron,‡ Derek A. Demaine,† Rob J. Fenton,† Graham Foster,† David Gower,†
J. Nicole Hamblin,† Stephanie Hamilton,‡ Graham J. Hart,† Alan P. Hill,† Graham G. A. Inglis,† Betty Jin,‡
Haydn T. Jones,† Darryl B. McConnell,‡ Jennifer McKimm-Breschkin,O Gail Mills,† Van Nguyen,‡ Ian J. Owens,†
Nigel Parry,† Stephen E. Shanahan,† Donna Smith,† Keith G. Watson,‡,§ Wen-Yang Wu,‡ and Simon P. Tucker‡
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom, Biota Holdings,
Level 4, 616 St. Kilda Road, Melbourne, 3004, Victoria, Australia, and CSIRO Health Sciences and Nutrition,
343 Royal Parade Parkville, 3052, Victoria, Australia
Received October 25, 2004
The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13
are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along
with dimer 3, are being investigated as potential second generation inhaled therapies both for
the treatment of influenza and for prophylactic use. They show outstanding activity in a 1
week mouse influenza prophylaxis assay, and compared with ZMV, high concentrations of 8
and 13 are found in rat lung tissue after 1 week. Retention of compounds in rat lung tissue
correlated both with molecular weight (excluding 3 and 15) and with a capacity factor K′ derived
from immobilized artificial membrane (IAM) chromatography (including 3 and 15). Pharma-
cokinetic parameters for 3, 8 and 13 in rats show the compounds have short to moderate plasma
half-lives, low clearances and low volumes of distribution. Dimer 3 shows NA inhibitory activity
against N1 viruses including the recent highly pathogenic H5N1 A/Chicken/Vietnam/8/2004.
In plaque reduction assays, 3, 8 and 13 show good to outstanding potency against a panel of
nine flu A and B virus strains. Consistent with its shorter and more rigid linking group, dimer
8 has been successfully crystallized.
Introduction
Periodically, new strains of influenza develop which
can result in pandemics leading to the deaths of millions
of people.1 Few effective therapies were available until,
in 1999, a neuraminidase inhibitor called “Relenza”
(zanamivir) 1 (Figure 1) was launched as a new inhaled
treatment for influenza by Biota and GlaxoWellcome
(now GlaxoSmithKline). Zanamivir (ZMV) is a highly
potent inhibitor of influenza A and B sialidases2 and
operates by inhibiting viral replication, and more specif-
ically, an enzyme called neuraminidase (NA) which
cleaves terminal sialic acids from cell surface glycocon-
jugates. NA is thought to be necessary for the release
of the virus from cell surfaces and thus for the move-
ment of the virus through mucus.
As part of a follow-up program aimed at developing
second generation therapies for the treatment of influ-
enza and for prophylactic use, polymeric,3 tetrameric,4
trimeric4 and dimeric5 derivatives linked through the
C7 hydroxyl (as in 2) were prepared and shown to
possess outstanding antiviral potency and the ability
to remain in the lung for prolonged periods. A series of
straight chain alkyl-linked dimers proved of significant
interest, and, despite exhibiting comparatively weak
inhibition of diverse influenza NA, dimers with the
appropriate length linking group are remarkably potent
Figure 1. Zanamivir 1 and the “C14” and “C13” dimers 3 and
15, respectively.
inhibitors of viral replication in tissue culture and in
animal models. In particular, dimer 3 is 100 times more
potent than ZMV in a mouse in vivo influenza prophy-
laxis model, and concentrations remaining in a rat lung
after 1 week as determined by a rat lung residence
model are over a 100 times greater than ZMV.5 We have
previously presented our hypothesis for the mechanism
of action and reason for the remarkable in vivo activity
* To whom correspondence should be addressed. E-mail:
simon.jf.macdonald@gsk.com.
† GlaxoSmithKline.
O CSIRO.
‡ Biota Holdings.
§ Current address: Walter
& Eliza Hall Institute of Medical
Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
10.1021/jm040891b CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/16/2005

Dimeric Zanamivir Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2965
Scheme 1. Synthesis of 8 and 13a
a Reagents and conditions: (a) O2NC6H4OCOCl, DMAP, pyridine; (b) 6, 57% or 11, 49%; (c) anisole, CF3CO2H, CH2Cl2; (d) Et3N, H2O,
MeOH then preparative HPLC 52% for 8, 50% for 13; (e) BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate),
Et3N, BocNH(CH2)3NH2, DMF, 90%; (f) TFA, DCM, 98%.
of these dimers.5 We decided to investigate a range of
alternative linking groups which might provide dimers
with different physicochemical properties and therefore
give us various options for development of a long-acting
inhaled therapy. We pursued a strategy with the objec-
tives of discovering compounds approaching a similar
biological profile to that displayed by 3 but with ZMV
linking groups representing different structural classes.
It was recognized that the entropy associated with the
14-carbon linker (that is the fragment between the two
ZMVs) in 3 may reduce its propensity for crystallinity.
To address this, we decided to rigidify the linker and to
introduce functionality capable of forming hydrogen
bonds and also aromatic rings capable of π stacking,
while maintaining an approximately similar chain
Figure 2. Crystal packing of 8. Note the π stacking of the
bibenzyl rings and the intricate network of H-bonds between
the different carbohydrate units. Black, red and blue spheres
represent carbon, oxygen and nitrogen atom, respectively.
Hydrogen atoms are not shown.
length between the ZMV units.
Data are presented for two new compounds (8 and
13) that fulfill these criteria from the 1 week mouse
influenza prophylaxis assay, a rat lung residency assay,
along with antiviral effectiveness for all three dimers
(3, 8 and 13) against a panel of nine flu A and B strains
in plaque reduction assays. A representative compound
(3) is shown to be effective against the recently isolated
and highly pathogenic H5N1 A/Chicken/Vietnam/8/
2004. Pharmacokinetic parameters for the three dimeric
compounds are also presented.
Finally, details are provided of correlations between
the residence time for compounds made during this
program in rat lung tissue with molecular weight and
with a “capacity factor” K′ derived from compound
retention times obtained from immobilized artificial
membrane chromatography.
with diisocyanates.6,7 However, the limited range of
available diisocyanates and their associated toxicity
make them unattractive as “dimerizing” reagents. The
use of diamines, in contrast, is more appealing, and new
chemistry was developed to allow their use. This is best
exemplified by the syntheses of two analogues that met
the criteria outlined above (Scheme 1). The starting
material 4 was readily prepared from a known precur-
sor.8 The key step is activation of the sterically hindered
7-hydroxyl for the dimerization reaction. After azeotro-
ping the alcohol 4 with toluene, treatment with p-
nitrophenyl chloroformate and 4-(dimethylamino)pyri-
dine (DMAP) in pyridine gave the activated carbonate
5 primed for coupling with diamines. Bis-carbamate
Chemistry. Previously the synthesis of dimers such
as 3 required coupling of a protected ZMV intermediate

2966 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Macdonald et al.
Table 1. Data for 8 and 13 in the 7-Day Mouse Efficacy Model
and Rat Lung Retention Assay Relative to 1 (ZMV)
infection and comparing this to the level of virus in the
lungs of untreated mice. Both 8 and 13 showed excellent
activity although neither compound appears to be as
effective as dimer 3. 3, 8 and 13 were tested using virus
A/Victoria/3/75 and ZMV 1 using virus A/Singapore/1/
57. It is reasonable to compare these, as the dodecyl
linked analogue of 3 was tested using both viruses and
gave similar results.5
The in vivo retention of dimers 8 and 13 in lung tissue
was determined by treating rats with an intratracheal
dose of an aqueous solution of compound, followed by
removal of the lungs at 48 h and 168 h (three rats per
timepoint). Concentrations of the dimers in lung tissue
were measured by mass spectroscopy, and the results
are presented alongside those for 3 and with all three
dimers measured relative to zanamivir (Table 1).5
Substantially greater concentrations of the dimers were
observed compared with zanamivir after 2 days and
particularly after a week where about 30 times greater
concentration of dimers 8 and 13 was observed.
Compounds 3, 8 and 13 were tested alongside zan-
amivir 1 in plaque reduction assays against a wide
range of influenza A and B strains (Table 2). Dimers 3
and 13 both exhibited broad spectrum activity and were
between 10 and 1000-fold more potent than 1. The
activity of 8 ranged from equipotent to substantially
more potent than 1 depending on the viral strain.
IC50 values against neuraminidase from influenza
strains Mem/Bel, PR8 and an H5N1 avian virus
A/Chicken/Vietnam/8/2004 were generated by enzyme
inhibition assays10 for 1 and 3 (Table 3). The IC50 values
are approximately 10 fold less potent for 3 than for those
observed with 1.
rat lung retention assayd
compound concentration
in lung relative to 1
7-day mouse efficacy model
compd
ED90 (mg/kg)a
48 h
168 h
1
2.92b
1
1
160
33.1
33.7
3
0.03c
34.5
14.2
8.3
8
13
0.016,c 0.74,c 0.39c
1.15,c 0.566c
a Compounds were dosed 7 days prior to infection with a
nonlethal strain of virus. Efficacy was determined by reductions
in lung virus titer. Lungs were removed 1 day following infection,
and viral load was compared to titers of virus in vehicle-treated
mice. b Dosed with influenza A/Singapore/1/57. Average value from
numerous assays. c Dosed with influenza A/Victoria/3/75. d Rats
were treated with an intratracheal dose of compound (0.4 mg/kg)
followed by removal of the lungs at set times and determination
of compound concentration. Mean values from three rats per time
point are quoted relative to 1 (see ref 5).
formation with diamines 6 and 11 gave the carbamates
7 and 12 in reasonable yield. Two-stage deprotection,
first with trifluoroacetic acid and then with aqueous
triethylamine followed by preparative HPLC purifica-
tion, gave the dimers 8 and 13 in around 50% yield. This
sequence allows rapid access to a broad range of dimers.
The biphenyl-linked dimer 8 proved crystalline when
isolated from a slowly evaporating solution in aqueous
ethanol, and the X-ray structure was subsequently
determined (Figure 2).
Biological Evaluation and Results. Dimers 8 and
13 were tested in a 7 day mouse influenza prophylaxis
model9 and a rat lung retention assay, and Table 1
shows the results, together with comparative data for
3. The mouse efficacy model involves administration of
a single intranasal dose of compound, 7 days prior to
infection of the animals with influenza virus. The
effectiveness of the compounds in preventing virus
replication is determined by measuring the amount of
virus remaining in the lungs 24 h after the viral
Plasma pharmacokinetic parameters were obtained
for 1, 3, 8 and 13 (Table 4) after intravenous, oral and
intratracheal (it) dosing in rats. In general, all the
compounds have short to moderate plasma half-lives,
low clearances and low volumes of distribution. They
Table 2. Plaque Reduction Data Across Different Strains of Influenza A and B (EC50 ng/mL)a
compd
A/WSNb
A/Vicc
A/Sydd
A/Newe
A/Panf
A/Bayg
B/Vich
B/HKi
B/Yamj
1
56, >100
5.5
2.4
0.27
<0.0001
0.003
2.7
35
11.5
0.05, 0.1
ntk
21
0.2, 3.1
<0.0001
0.006
3
<0.0001
<0.001
0.032
3.7
0.032
<0.0001
>1
0.37
0.23
0.32
8
13
<0.0001, 0.001
0.02
0.0001, 0.038
0.003
1.8, >10
0.03
0.05
0.06
0.009
ntk
0.022
a Values are from separate experiments. There are insufficient replicate data available for meaningful standard deviations to be quoted.
b A/WSN/33 (H1N1). c A/Victoria/7/95 (H3N2). d A/Sydney/5/97 (H3N2). e A/New Caledonian/20/99 (H1N1). f A/Panama/2007/99 (H3N2).
g A/Bayern/7/95 (H1N1). h B/Victoria/1/67. i B/Hong Kong/5/72. j B/Yamanashi/166/98. k Not tested.
Table 3. Enzyme Inhibition Assay of N1 Virusesa
A/Mem/Bel/71
(H3N1) (nM)
A/PR/8/34
(H1N1) (nM)
A/Chicken/Vietnam/8/2004
(H5N1) (nM)
compd
1
3
0.77
7.86
2.48
21.7
3.25
61
a Mean IC50 values generated according to ref 10. All values are at least n ) 5 and are within 60% of the mean.
Table 4. Plasma Pharmacokinetic Parameters for 1, 3, 8, and 13 in Rat
Cld
(mL/min/kg)
Vd
F (%)f
po
F (%)f
it
e
t1/2 (h)
t1/2 (h)
t1/2 (h)
compd
iva
pob
itc
(L/kg)
1
0.4
2.2
0.45
0.4
0.25
4.9
2
ntg
5
1.42
0.7
7
0.03
0.2
0.2
0.2
2.7
<1
nt
67
100
67
3
8
13
3
<1
nch
2
0.7
<1
a Dosed intravenously (iv) at 2 mg/kg. b Dosed orally (po) at 10 mg/kg unless otherwise stated. c Dosed intratracheally (it) at 0.4 mg/kg.
d Clearance (Cl). e Volume of distribution (Vd). f Percentage bioavailability (F (%)). g Not tested. h Not calculated.

Dimeric Zanamivir Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2967
Figure 3. Normalized data for concentration in rat lung tissue (as a ratio to zanamivir) against molecular weight at 168 h
postdose for a series of monomers (blue), dimers (magenta), trimers (yellow) and tetramers (red). Compounds 8 and 13 are in the
middle of the magenta cluster (omitted for clarity). Note the exceptionally high concentrations of compounds 3 and 15.
have no measurable bioavailability after oral dosing but
have high bioavailability after it dosing.
pathogenic H5N1 avian virus A/Chicken/Vietnam/8/
2004.11,12 This strain caused outbreaks of disease in
poultry in Asia and caused human fatalities in Vietnam
and Thailand. These data, combined with those detailed
in Table 1 and the established broad spectrum of
activity of 1, suggest that the dimeric compounds are
likely to be effective against most, if not all, known
influenza A and B strains and may prove useful for the
management of outbreaks with pandemic potential.
Given these compounds have short to moderate plas-
ma half-lives, low clearances, low volumes of distribu-
tion and after it dosing have high bioavailability (Table
4), it is likely that the quantity of compound present in
the lung after 168 h and having an antiviral effect, is
very low. Further, these data suggest that the com-
pounds are rapidly removed from the systemic circula-
tion as the half-lives are short. Although it appears that
the levels of drug present in the lung after 168 h for 8
in the rat lung retention assay is inconsistent with the
100% bioavailability after it dosing in the pharmacoki-
netic study, this difference can be explained as follows.
The concentration of 8 in lung tissue after 168 h in the
lung retention assay is approximately 1600 ng/g (with
the limit of detection in this assay at 20 ng/g) which is
actually less than 3% of the administered dose. (Rats
were dosed at 400 µg/kg which is approximately equiva-
lent to a 100 µg it dose). These data are consistent with
a very small but measurable amount of drug remaining
in the lung in both experiments, long after almost all
of the dose has been absorbed into the circulation as
determined by the plasma concentrations in the phar-
macokinetic study (leading to the 100% bioavailability
after it dosing). The 1600 ng/g of compound in lung
tissue in the lung retention assay is below the 50 ng/
mL of plasma limit of quantification for compound in
the it bioavailability pharmacokinetic study.
Discussion
We have previously shown5 that certain dimeric
derivatives of ZMV are extremely potent inhibitors of
influenza when tested in both in vitro and in vivo
experiments. Here we have extended our study of
dimeric derivatives of ZMV to show that variation of
the structure of the linking group can still allow
retention of antiviral potency while also affording op-
portunities to improve the crystallinity and fine-tune
the lung residence time, thereby providing alternative
structures for development.
The dimers 3, 8 and 13 in many cases demonstrate
extraordinary potency (in some cases approaching pi-
comolar potency) in the plaque reduction assays (Table
2). These exceptional levels of potency have been
discussed elsewhere in the case of 3 and may be
rationalized in terms of a multivalent binding effect via
intervirion and interneuraminidase linkages rather
than improved inhibition of NA per se.5 Interestingly,
compound 8 which has the most rigid, but also a slightly
shorter, linking group has the most variable activity in
the plaque reduction assays. While we have no direct
proof, we believe the variability in the activity of 8
reflects differences in the accessibility of the NA active
site in different influenza strains, possibly due to steric
constraints on the outer surface of the protein and
accentuated by the rigid linker. The substantially
increased drug levels of 8 and 13 seen after a week in
the rat lung retention assays, when compared with
zanamivir 1, built confidence that these compounds
would be suitable for prophylactic use.
The IC50 values for 1 and 3 against neuraminidase
strains Mem/Bel and PR8 (Table 3) are consistent with
data generated previously from enzyme kinetic experi-
ments.5 Significantly, both 1 and 3 are also active
against a Vietnamese isolate of the recent highly
Correlation of Compound Concentration in Rat
Lung Tissue with Molecular Weight and the Ca-
pacity Factor K′. A large number of zanamivir conju-

2968 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Macdonald et al.
Figure 4. Log of the rat lung concentration for a range of dimers at 168 h (relative to the standard) against the IAM (immobilized
artificial membrane) capacity factor K′. K′ is the retention time (tr) minus the column void volume time (to) divided by the column
void volume time ie K′ ) (tr - to)/to. Compounds 3, 8 and 15 are labeled. (13 was not tested.)
gates were prepared during the project (from tetramers
to monomers) featuring a wide variety of linkers. These
include lipophilic and hydrophilic linkers, neutral, basic
and acidic linkers, linkers with a range of embedded
functionality such as acids, tertiary amines, amides and
sulfonamides and linkers with differing rigidity.3-7,17
The concentrations of these zanamivir conjugates in rat
lung tissue were determined using the assay described
above. As a general trend, increased concentration in rat
lung after 1 week correlated with increased molecular
weight (Figure 3).13 Additionally, at a structural level,
it is likely that the increased lung retention is derived,
at least in part, from the compounds being too hydro-
philic for transcellular absorption and not being sub-
strates for active transport through the lung epithelium.
Dimers 3 and 155 were found to remain in rat lung
tissue at far higher concentrations than would be
expected from their molecular weights (Figure 3). One
factor that may contribute to these much higher con-
centrations is the significantly decreased aqueous solu-
bility of 3 and 15 compared with most other dimers
synthesized (which are readily soluble in water). The
structures of these compounds with their flexible lipo-
philic linkers capped by highly hydrophilic moieties
(structurally akin to glycolipids) may cause the forma-
tion of insoluble aggregates.
Another factor may be the partitioning of 3 and 15
into phospholipids (or glycolipids) in the epithelial lining
fluid or epithelium itself.14 To explore this we used im-
mobilized artificial membrane chromatography (IAM)
which has been used extensively to model in vitro drug
interactions with phospholipids.15 We determined the
capacity factors K′ (a derivative of the retention time)
for a range of dimers (including 3 and 15) made during
this program and found that the IAM K′s values cor-
relate well with the rat lung concentrations (Figure 4).
The exception to this is 8. A potential contributing factor
that might explain this is that while 8, 3 and 15 are
likely to have grossly similar lipophilicities (the log P/D
values were unmeasurable), their structural flexibilities
are profoundly different, with 8 being almost rigid and
3 and 15 highly flexible. Despite 8 being an outlier, IAM
chromatography proved a useful in vitro tool for predict-
ing lung concentration in this series with the significant
advantages of speed and reduction in animal usage.
The successful crystallization of dimer 8 raises the
prospect that within the family of zanamivir dimers
there is the option of developing a compound which may
be able to be isolated and purified without the need for
chromatography and which could also be readily for-
mulated for use as an inhaled powder.
Conclusion
The potency of 8 and 13 in the described biological
assays and their ability to have a prolonged antiviral
effect can be explained in terms of their polarity,
molecular weight and dimeric structure which leads to
a multivalent binding effect via intervirion and inter-
neuraminidase linkages.5 The properties of dimers 8 and
13 are commensurate with a superior treatment for
influenza A or B and long-acting prophylaxis, and the
compounds are potential candidates for further develop-
ment. They are likely to be effective against strains of
neuraminidase that may yet develop.
Experimental Section
1H NMR spectra were obtained using a Bruker DPX 400
spectrometer working at 400.13 MHz and 9.4 T using as
internal standard the signal from the residual protonated
solvents.
LC/MS Details. Orange Method. This method used a
Micromass Platform II instrument in electrospray positive
ionization mode. The range was 100-1000 amu. The injection
volume was 5 µL. The diode array detector range was 220-
300 nm. The mobile phase was water (solvent A) buffered with
0.05% v/v TFA. The organic phase (solvent B) was acetonitrile
buffered with 0.05% v/v TFA. The gradient ran from 0% B to
95% B over 8 min.
LC/MS Details. Green Method. This method used a
Micromass Platform II instrument in electrospray positive
ionization mode. The range was 100-1000 amu. The column
was a 3.3 cm × 4.6 mm ID 3 µm ABZ+ with a flow rate of 3.0
mL/min. The injection volume was 5 µL. The diode array
detector range was 220-300 nm. The mobile phase was 95%

Dimeric Zanamivir Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2969
acetonitrile buffered with 0.05% formic acid (solvent A). The
aqueous phase was 0.1% formic acid containing 10 mM
ammonium acetate (solvent B). The gradient was 0% A/0.7
min, 0-100% A/3.5 min, 100% A/1.1 min, 100-0% A/0.2 min.
LC/MS Details. Red Method. This method used a Micro-
mass Platform LC instrument in electrospray positive ioniza-
tion mode. The range was 500-1500 amu. The column was a
3.3 cm × 4.6 mm ID 3 µm ABZ+ with a flow rate of 3.0 mL/
min. The injection volume was 5 µL. The diode array detector
range was 220-300 nm. The mobile phase was 95% acetoni-
trile containing 0.05% formic acid (solvent A) and 0.1% formic
acid containing 10 mM ammonium acetate (solvent B). The
gradient was 0% A/0.7 min, 0-100% A/3.5 min, 100% A/1.1
min, 100-0% A/0.2 min.
LC/MS Details. Black Method. This method used a
Micromass Platform LC TOF mass spectrometer in electro-
spray positive ionization mode. The range was 100-1000 amu.
The column was a 3.3 cm × 4.6 mm ID 3 µm ABZ+ with a
flow rate of 3.0 mL/min. The injection volume was 5 µL. The
mobile phase was 95% acetonitrile containing 0.05% formic
acid (solvent A) and 0.1% formic acid (solvent B). The gradient
was 0% A/0.7 min, 0-100% A/3.5 min, 100% A/1.1 min, 100-
0% A/0.2 min.
Preparative HPLC Method for 8. The prep column used
was a Kromasil C18 (20 cm × 5 cm). The UV wavelength was
230 nM with a flow rate of 80 mL/min. Solvent A was 1% TFA
in water and solvent B 80% acetonitrile containing 1% TFA.
The gradient was 0-100%B/70 min. Following HPLC, ap-
propriate fractions were combined and volatile components
removed by evaporation under reduced pressure. The aqueous
was applied to a column of Amberchrom CG-161 resin (10 ×
2.5 cm) which was eluted with water (500 mL), then a 2:2:1
mixture of acetonitrile:MeOH:water (500 mL).
biphenyldiylbis(methanediylimino(oxomethanediyl)oxy{(S)-
[(4R)-2-oxo-1,3-dioxolan-4-yl]methanediyl})]bis(3-(acetylamino)-
4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-
carboxylate) trifluoroacetate as a white solid (1.59 g, 94%
yield). 1H NMR (d4 - MeOH) δ 7.39 (d, 4H), 7.17 (d, 4H), 5.71
(s, 2H), 5.45 (s, 2H), 4.98 (m, 2H), 4.60-4.50 (m, 4H), 4.32-
4.12 (m, 6H), 4.09-3.95 (m, 4H), 3.60 (s, 6H), 1.78 (s, 6H). LC/
MS (Green Method) showed (M + 2H+)/2 ) 505; tRET ) 2.27
min.
The above diester dicarbonate (1.59 g, 1.3 mmol) was
dissolved in a mixture of water (28.5 mL) and methanol (28.5
mL). To this was added triethylamine (2.85 mL) and the
solution stirred for 4 h. Volatile organic components were
removed in vacuo, and the residual solution was adjusted to
pH 2 by addition of TFA. Reverse phase preparative HPLC
gave the title compound 8 (0.70 g; 57% yield). 1H NMR (D2O)
(rotamers) δ 7.65-7.49 (m, 4H), 7.27 (m, 4H), 5.05 (bs, 1.4H),
5.45 (bs, 0.6H), 4.87-4.75 (m, 2H), 4.45-3.92 (m, 12H), 3.60-
3.27 (m, 3.4H), 3.02 (m, 0.6H), 1.82 (m, 6H). LC/MS (Green
Method) showed (M + 2H+)/2 ) 465; tRET ) 2.00 min.
Microanalysis: Calc. for C40H52N10O16‚6H2O C 46.3%, H 6.2%;
N 13.5%. Found: C 46.3%; H 5.9%; N 13.2%
Crystals of 8 were grown by dissolving 8 (20 mg) in water
(0.23 mL) in an A vial. This vial was then placed in a D vial
containing ethanol (1 mL) and the vial sealed with a screw
cap. After 4 months, crystals of 8 were present in the A vial.
Dimethyl (2R,3R,4S,2′R,3′R,4′S)-2,2′-[Benzene-1,4-diyl-
bis((oxomethanediyl)imino-3,1-propanediylimino-
(oxomethanediyl)oxy{(S)-[(4R)-2-oxo-1,3-dioxolan-4-yl]-
methanediyl})]bis(3-(acetylamino)-4-{[bis({[(1,1-
dimethylethyl)oxy]carbonyl}amino)methylidene]amino}-
3,4-dihydro-2H-pyran-6-carboxylate) 12. Prepared similarly
to 7 using N,N′-bis-(3-aminopropyl)-1,4-benzenedicarboxamide
11.17 1H NMR (CDCl3) δ 11.35 (s, 2H), 8.49 (d, 2H), 8.12 (m,
2H), 8.00 (m, 2H), 7.95 (s, 4H), 6.90 (m, 2H), 6.76 (d, 2H), 5.91
(s, 2H), 5.52 (m, 2H), 5.05 (m, 6H), 4.72 (t, 2H), 4.65 (t, 2H),
4.25 (m, 4H), 3.79 (s, 6H), 3.67 (m, 2H), 3.45 (m, 4H), 3.13 (m,
Immobilized Artifical Chromatography. This chroma-
tography was carried out using a Regis DD.PC IAM column
(PC ) phosphatidyl choline) with 5 µM 300 Å packing and 3
cm by 4.6 mm ID at 37 °C. The mobile phase was Dulbecco’s
PBS with a flow rate of 1 mL/min. Compounds were dissolved
in the eluent at ∼0.5 mg/mL (10 µL injected).
2H), 1.80 (s, 6H), 1.45 (s, 36H). LC-MS (Red Method): tRET
)
3.76 min; MH+ ) 1474.
(2R,3R,4S,2′R,3′R,4′S)-2,2′-(Benzene-1,4-diylbis{(oxo-
methanediyl)imino-3,1-propanediylimino(oxometh-
anediyl)oxy[(1R,2R)-2,3-dihydroxy-1,1-propanediyl]})-
bis(3-(acetylamino)-4-{[amino(imino)methyl]amino}-3,4-
dihydro-2H-pyran-6-carboxylic acid) 13. The tert-butyl
carbamate groups of 12 were removed similarly to 8 to afford
dimethyl (2R,3R,4S,2′R,3′R,4′S)-2,2′-[benzene-1,4-diylbis((ox-
omethanediyl)imino-3,1-propanediylimino(oxomethanediyl)-
oxy{(S)-[(4R)-2-oxo-1,3-dioxolan-4-yl]methanediyl})]bis(3-(acety-
lamino)-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-
6-carboxylate). 1H NMR (MeOH-d4) δ 7.88 (s, 4H), 5.89 (s, 2H),
5.56 (s, 2H), 5.15 (m, 2H), 4.70 (m, 4H), 4.43 (m, 4H), 4.14 (m,
2H), 3.80 (s, 6H), 3.43 (m, 4H), 3.16 (m, 4H), 1.92 (s, 6H), 1.80
(m, 2H). LC-MS: (Red Method) tRET ) 2.13 min; MH+ ) 1075.
The bis-trifluoroacetic acid salt was first converted into the
bis-hydrochloride. The bis-trifluoroacetic acid salt (10 g) was
dissolved in MeOH/water (1:1v/v) (150 mL) and applied to a
prewashed Amberlite IRA-410 (chloride form) ion exchange
column (4 × 50 cm). The dihydrochloride was eluted from this
column with MeOH/water (1:1v/v). Fractions containing prod-
uct were concentrated in vacuo to give a white foam. The bis-
hydrochloride (10.4 g) was dissolved in methanol (144 mL) and
water (144 mL) and cooled in ice. Triethylamine (7.75 mL) was
added in three portions over 10 min. The reaction was
monitored by reverse phase HPLC. When the reaction was
complete the reaction mixture was evaporated to dryness in
vacuo.
Dimethyl (2R,3R,4S,2′R,3′R,4′S)-2,2′-[4,4′-Biphenyl-
diylbis(methanediylimino(oxomethanediyl)oxy{(S)--[(4R)-
2-oxo-1,3-dioxolan-4-yl]methanediyl})]bis(3-(acetylamino)-
4-{[bis({[(1,1-dimethylethyl)oxy]carbonyl}amino)methyli-
dene]amino}-3,4-dihydro-2H-pyran-6-carboxylate) 7. The
alcohol 418 (2.74 g, 4.79 mmol) was dried by azeotroping four
times from anhydrous toluene and then dissolved in anhydrous
pyridine (13.75 mL). To this were added DMAP (1.46 g, 11.98
mmol) and p-nitrophenyl chloroformate (1.06 g, 5.27 mmol),
and the mixture was stirred at room temperature for 3 h. To
the mixture was then added more pyridine (8.25 mL), followed
by [1,1′-biphenyl]-4,4′-dimethanamine16 6 (0.51 g, 2.4 mmol),
and stirring was continued for a further 16 h. The mixture
was concentrated in vacuo and applied as a solution in DCM
to a 90 g, silica Biotage cartridge. This was eluted with diethyl
ether, followed by Et2O:EtOAc (1:1), then Et2O:EtOAc (1:2) and
finally EtOAc to afford the title compound as a white solid
(1.92 g, 57% yield). 1H NMR (CDCl3) δ 11.34 (s, 2H), 8.63 (m,
2H), 7.46 (d, 4H), 7.30 (d, 4H), 5.83 (s, 2H), 5.50 (s, 2H), 5.38
(bs, 2H), 5.15 (bs, 2H), 4.95 (m, 2H), 4.66-4.53 (m, 4H), 4.46-
4.54 (m, 4H), 4.30-4.15 (m, 6H), 3.72 (s, 6H), 1.84 (s, 6H), 1.45
(s, 36H). LC/MS (Green Method) showed (M + 2H+)/2 ) 705;
tRET ) 3.96 min.
(2R,3R,4S)-3-(Acetylamino)-2-{(1R,2R)-1-[({[(4′-{[({[(1R,-
2R)-1-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)me-
thyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-2,3-
dihydroxypropyl]oxy}carbonyl)amino]methyl}-1,1′-
biphenyl-4-yl)methyl]amino}carbonyl)oxy]-2,3-
dihydroxypropyl}-4-{[amino(imino)methyl]amino}-3,4-
dihydro-2H-pyran-6-carboxylic Acid 8. The above dimer
7 (1.92 g, 1.36 mmol) was dissolved in a 10:1 mixture of DCM:
anisole (27.5 mL) and treated with TFA (25 mL). The resulting
solution was stirred at room temperature for 2 h then
concentrated in vacuo. Trituration of the residue with diethyl
ether afforded dimethyl (2R,3R,4S,2′R,3′R,4′S)-2,2′-[4,4′-
Purification. The impure product (10.5 g) was dissolved
in water (80 mL) and orthophosphoric acid (5 mL) was added
to ensure the sample solution was pH 2. This solution (ca. 100
mL) was subjected to preparative HPLC using a 7 µm Kromasil
C8 column (25 cm × 5 cm id.). Chromatographic resolution of
the product was achieved using ion-pair gradient elution where
the start mobile phase A was H2O to which had been added 8
g/L of sodium lauryl sulfate (SLS) and 2 mL/L H3PO4. The final

2970 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Macdonald et al.
mobile phase B was 60%CH3CN/H2O to which had been added
8 g/liter SLS and 2 mL/L H3PO4. Gradient time was 0 to 100%
B in 70 min holding at 100% B for 20 min. Flow was set at 80
mL/min. A total of four injections at 25 mL each were done.
The detector was set at 260 nm. The fractions, 50 mL each,
were bulked according to their analytical purity as measured
by HPLC, the specification being >97.5%. This bulked volume
was approximately 1800 mL for four chromatographic separa-
tions. To remove the majority of the phosphate and the lauryl
sulfate ions, the aqueous bulk was added to 1200 mL of IRA
410 ion-exchange resin in the chloride form and the solution
stirred for 1 h. The resin was removed by filtration and washed
with 400 mL of H2O, and this was combined with the filtrate.
The aqueous was then passed through a column of IRA 410
in the chloride form (15 cm × 2.5 cm id.) to remove any
remaining phosphate or lauryl sulfate ion contamination, the
column was washed with 50 mL of H2O, and this was combined
with the filtrate. After removal of the CH3CN by rotary
evaporation at 40 °C, the pH was adjusted to 7.0. The aqueous
was split into two portions. The first portion was passed
through a column of Amberchrom CG 161 (25 cm × 2.5 cm) in
order to adsorb the product. This column is a polystyrene
divinyl benzene resin, which acts as a reversed phase packing
material. The column was washed with 300 mL of H2O until
the wash was clear of chloride ion, this being checked by the
absence of a reaction with AgNO3 solution. The column was
eluted with 30% CH3CN/H2O (500 mL). This process was
repeated with the second portion, and the eluents were
combined. The solvents (1 liter) were removed by rotary
evaporation to yield 5.7 g of title product. The purified material
was taken up in water (300 mL) and washed with dichlo-
romethane (3 × 200 mL). The aqueous solution was then
freeze-dried to give the title compound. 1H NMR (D2O) δ 7.85
(s, 4H, 4 × ArH), 5.67, 5.57 (2 × d, 2H, J ) 2.1 Hz, 2 × CH),
4.95 (dd, 2H, J ) 9.0,1.6 Hz, 2 × CH), 4.52 (dd, 2H, J ) 10.5,
1.6 Hz, 2 × CH), 4.42 (dd, 2H, J ) 9.3, 2.2 Hz, 2 × CH), 4.14-
4.04 (m, 4H, 4 × CH), 3.67 (dd, 2H, J ) 12.3 Hz, 2 × CH),
3.54-3.44 (m, 6H, 2 × CH +2 × CH2), 3.21 (t,4H, J ) 6.7 Hz,
2 × CH2), 1.96 (s, 6H, 2 × CH3), 1.85 (m, 4H, 2 × CH2). LC/
MS (Black Method): tRET ) 1.67 min; (M + 2H+)/2 ) 498.
Microanalysis: Calc. for C40H58N12O18‚4H2O C 45.02%; H
6.23%; N 15.75%. Found: C 44.80%; H 6.68%; N 14.89%
Lung Retention Assay and Pharmacokinetics for it
Dosing. Male Sprague-Dawley rats (weighing 220-280 g)
were anesthetizd with isoflurane. The rats were then placed
in a supine position on a board inclined at an angle of 45° from
the horizontal and were secured in position by hooking the
upper incisors to a band secured to the upper part of the board.
A halogen-fiber light was positioned close to the neck to guide
the intubation. When the lower jaw was pulled down and the
tongue pulled to one side of the muzzle, the trachea was
brought into view, and a tracheal cannula was inserted until
just above the bifurcation point. Compound, at a concentration
of 0.5 mg/mL in PBS, pH 7.4, was administered as bolus at a
dose volume of 0.8 mL/kg. The cannula was immediately
withdrawn after dosing and the animal held in the upright
position until full recovery from the anesthetic was achieved.
This prevented the dose from refluxing back up the trachea.
At 48 h and 168 h postdose, animals (two per timepoint) were
placed under terminal anesthesia and the lungs were then
removed for sample analysis.
Lung tissue was homogenized in 5 mL of CH3CN/H2O (5/
95) using a PRO200 homogenizer with 10 mm cutting tools
attached. A 200 µL aliquot of the homogenate was placed into
a 1.5 mL eppendorf tube, and protein was precipitated by the
addition of 600 µL of CH3CN and 100 µL of 3% acetic acid.
Samples were then vortex mixed and centrifuged at high speed
in a Microfuge for 10 min. Using a Tecan GenesisOˆ robotic
system, a 700 µL aliquot of the resulting supernatant was
transferred to a 96-well plate. The samples were then evapo-
rated to dryness under nitrogen heated to 40 °C and recon-
stituted in 200 µL of CH3CN/H2O (5/95). Prior to analysis, the
sample plate was centrifuged at 2000g for 15 min. For
quantitative analysis, 50 µL of the sample were injected onto
an LC-MS/MS system. The LC system consisted of a CTC
autosampler, an HP series 1100 quaternary pump, and a Luna
C18, 4.6 cm × 2.1 mm ID, 5 µm particular size, analytical
column. Mobile phases were 1% aqueous formic acid and 1%
formic acid in CH3CN. Analysis was performed by gradient
elution starting from 5% organic to 95% organic phase over 5
min at a flow rate of 0.4 mL/min. Mass detection was
performed by Sciex API365 with turbo-ion spray interface with
the voltage set at 5 kV and the temperature set to 350 °C.
Positive multiple reaction monitoring transitions were moni-
tored after optimization by infusion, and quantification was
performed using MacQuan (Sciex) software.
Biology Experimental. In Vivo 7-Day Mouse Efficacy
Model. Experimental details are provided in ref 9.
iv and Oral Phamacokinetic Dosing. Male Sprague
Dawley rats were used to determine kinetic profile after iv
and oral dosing with compounds. For iv administration, a bolus
dose formulated in water was dosed in the lateral tail vein
and serial blood samples removed from a previously inserted
indwelling tail cannula at various time points post-dose. For
oral administration, a bolus dose formulated in water was
given using a ball ended gavage needle. As for iv, serial blood
samples were removed from previously inserted indwelling tail
cannula. As soon as possible after blood collection, the blood
was centrifuged to obtained plasma fraction for analysis.
Plaque Reduction Assay. MDCK cells were seeded into
six-well tissue culture plates and grown to confluence using
standard methods. Influenza viruses were diluted in a minimal
volume of phosphate buffered saline (PBS) supplemented with
0.2% bovine serum albumin (BSA) to yield an estimated titer
of 50-100 plaque forming units (pfu) per well. After adsorption
onto the MDCK cells for 1 h at 37 °C in a 5% CO2 atmosphere,
viral inocula were aspirated and replaced with viral growth
media (minimal Eagle’s medium supplemented with BSA,
trypsin, and insulin/transferrin/selenium at optimal concen-
trations) containing sufficient agar or agarose (generally
1-2%) to cause the media to gel at room temperature. The
plates were incubated at 37 °C in a CO2 atmosphere until
plaques developed (generally 2-4 days). Plaques were visual-
ized with a suitable stain (e.g. 0.4% crystal violet in formal
saline) before counting. Antiviral potency (EC50) was deter-
mined as the concentration of compound in the media which
reduced plaque numbers by 50% of the untreated control value.
Enzyme Inhibition Assay. Enzyme inhibition assays for
A/Mem/Bel/71(H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42
(H1N1)], A/PR/8/34 (H1N1) and A/Chicken/Vietnam/2004
(H5N1) were based as previously described.10 Briefly, the
dilution of each virus which gave enzyme activity in the linear
portion of the dose-response curve (excess substrate) was
incubated for 45 min with half log dilutions of the inhibitors.
2′-(4-Methylumbelliferyl)-R-D-N-acetylneuraminic acid (0.2 mM)
was then added, and samples were incubated for 90 min, after
which the reaction was stopped by the addition of 200 mM
sodium carbonate, pH 9.5, and fluorescence was read in a
Perkin-Elmer fluorimeter.
Supporting Information Available: Details of the X-ray
crystallographic determination of 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
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