Synthesis and influenza virus sialidase inhibitory activity of analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) modified in the glycerol side-chain

Article abstract of DOI:10.1016/S0223-5234(00)80026-4

Analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) have been prepared containing carbamate substituents at the 7-hydroxy position. (4S,5R,6R)-5- Acetylamino-6-{1R-[(6-aminohexyl)carbamoyloxy]-2R,3-dihydroxypropyl}-4- guanidino-5,6-dihydro-4H-pyran-2carboxylic acid and (4S,5R,6R)-5-Acetylamino- 6-{1R-[heptylcarbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-dihydro4H- pyran2-carboxylic acid were the two analogues possessing activity comparable to Zanamivir, showing potent inhibition of influenza virus sialidases and good antiviral activity in vitro.

Full text of DOI:10.1016/S0223-5234(00)80026-4

Eur. J. Med. Chem. 34 (1999) 563−574
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
563
Original article
Synthesis and influenza virus sialidase inhibitory activity of analogues of
4-Guanidino-Neu5Ac2en (Zanamivir) modified in the glycerol side-chain
David M. Andrews*, Peter C. Cherry, David C. Humber, Paul S. Jones, Steven P. Keeling,
Paul F. Martin, Caroline D. Shaw, Stephen Swanson
Department of Medicinal Chemistry, Glaxo-Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK
(Received 11 May 1998; accepted 12 January 1999)
Abstract – Analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) have been prepared containing carbamate substituents at the 7-hydroxy
position. (4S,5R,6R)-5-Acetylamino-6-{1R-[(6-aminohexyl)carbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-dihydro-4H-pyran-2-
carboxylic acid and (4S,5R,6R)-5-Acetylamino-6-{1R-[heptylcarbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-dihydro4H-pyran-
2-carboxylic acid were the two analogues possessing activity comparable to Zanamivir, showing potent inhibition of influenza virus sialidases
and good antiviral activity in vitro. © 1999 Éditions scientifiques et médicales Elsevier SAS
influenza virus sialidase neuraminidase Zanamivir / antiviral activity
1. Introduction
Several classes of potent and selective influenza virus
sialidase inhibitors have now been reported based upon
either dihydropyrans 1 and 2 (Zanamivir, Glaxo-
Wellcome) [1–4]; cyclohexene 3 (GS4104, GS4071,
Gilead) [5–7]; or cyclopentane 4 (Biocryst) [8] core tem-
plates (figure 1). Several recent reports from our co-
workers describe structural modifications to Zanamivir
which demonstrate the important contributions to overall
binding made by each of the dihydropyran substituents
Figure 1. Structural comparison of lead classes of influenza
sialidase inhibitors.
(figure 2) [9–13]. In particular, studies have been carried
out in which the glycerol side chain has been truncated in
these more lipophilic compounds bind to the enzyme,
making interactions which are quite distinct from those
a stepwise fashion, confirming that the interaction which
characterised for the glycerol side-chain. Small variations
the 8,9-diol makes with the conserved Glu276 residue
in the position of amino acid sidechains can be used to
makes an important contribution to overall binding of
account for the separation in activity which this series
sialic acid derivatives to influenza sialidase. A previous
shows when tested against influenza A and B sialidases.
communication describes the complete oxidative cleav-
The X-ray crystal structures of sialic acid and Zan-
age of the glycerol side-chain and replacement with a
amivir bound to influenza sialidase show that the
series of carboxamide derivatives, producing a series of
7-hydroxyl group makes no direct interactions with the
compounds with exceptionally potent activity against
protein and is exposed to bulk solvent, indicating that it
influenza A, but which demonstrate lower activity against
may be possible to modify the glycerol sidechain at this
influenza B [14]. X-ray crystallography indicates that
position, whilst retaining the important 8,9-diol moi-
ety [15, 16]. 7-modification is of particular interest since
it allows the synthesis of a wide range of Zanamivir
*Correspondence and reprints

564
Figure 2. Schematic representation of interactions between
Zanamivir and influenza A sialidase.
derivatives, which, unlike 6-carboxamides, are poten-
tially A/B non-selective and could display modified
physicochemical properties. The analogue set was chosen
to probe a wide range of physicochemical properties to
see how these influence compound pharmacokinetics.
Acylation of the 7-hydroxyl group offered a potentially
quick route into testing this hypothesis, but given the
known tendency for acyl groups to migrate within sugars
onto less hindered positions [17], in this case the
9-hydroxyl group, this approach was not pursued. We
chose instead to exploit the resistance to migration which
is displayed by the carbamoyl linker, and this paper
describes the synthesis and influenza virus sialidase
inhibition of a series of 7-carbamate compounds 9, 11, 16
and 18 which are derivatives of 1 and 2 (figure 3).
Figure 3. Structure of compounds 9, 11, 16 and 18.
active ester 7. It should be noted that portionwise addition
of 4-nitrophenyl chloroformate and later addition of
DMAP are essential for efficient conversion. Treatment of
7 with suitable primary and secondary amines furnished a
range of 7-carbamates 8. Method B was adopted as the
preferred route since reaction of amines with 7 permits
synthesis of a more diverse range of analogues. The use
of commercial amines such as mono-Boc-diamino-
hexane, along with in-house synthesised amines gave
access to potentially dendrimeric compounds such as 11f
and 11g. In the case of 9i and 9j, the desired disubstituted
carbamates were more conveniently obtained by reaction
of the commercially available carbamoyl chloride with 6.
Treatment of 8 with triphenylphosphine generated an
iminophosphorane which was hydrolysed in situ at 40 °C
with aqueous triethylamine; concomitant hydrolysis of
the cyclic carbonate and methyl ester protecting groups
afforded 9 in good yield (figure 6). The azide 8 can also
be reduced by hydrogenation over Lindlar catalyst, but
2. Chemistry
The syntheses of compounds 9, 11, 16 and 18 are
outlined in figures 4–8. Initially, formation of the required
carbamate linker was by reaction of isocyanates with the
cyclic carbonate 6, which had, itself, been synthesised by
the method of Zbiral [18] from the known methyl ester
5 [11] using carbonyl diimidazole or phosgene in toluene
(figure 4, method A). In our preferred route (method B),
5 was treated with 1.2 equivalents of 4-nitrophenyl
chloroformate in dry pyridine (figure 5). TLC monitoring
first revealed formation of the intermediate cyclic carbon-
ate 6 and addition of a further portion of 4-nitrophenyl
chloroformate and DMAP afforded a good yield of the

565
Figure 4. Synthetic method A. i) COCl2, DMAP, MeCN, DCM; ii) R1NCO, DMAP, DCM.
Figure 5. Synthetic method B. i) 3 eq. p-NO2C6H4OCOCl, DMAP, pyr; ii) R1R2NH, DMAP, pyr.

566
Figure 6. Transformation of azido ester to guanidino acid. i) Ph3P, THF; ii) Et3N, H2O, ∆; iii) Bis(Boc)PCH, MeOH, Et3N; iv) TFA,
DCM.
the efficiency of this reaction is very variable, depending
on the size and bulk of the 7-carbamate substituent.
Guanidinylation of the 4-amino group in 9 with N,
N≠-bis-t-butoxycarbonyl-1H-pyrazole-1-carboxamidine
(BisBocPCH) [19] produced the protected guanidine 10.
The Boc protecting groups were removed from 10 by
treatment first with TFA, followed by water; lyophilisa-
tion of the resulting solution afforded pure compounds 11
in good yield. It is noteworthy that the product of the TFA
deprotection was significantly contaminated with its
9-trifluoroacetate ester; this proved to be very labile to
hydrolysis and allowed the generation of final products of
sufficient purity that no further purification was required.
Figure 7 describes a modification to the synthesis
which permitted access to the ethanolamine derivative
12. Under standard acylation conditions using an excess
of the amine component, very little of the desired 12 was
formed, ethanolamine being sufficiently reactive to ring-
open the initially formed carbonate 12 to generate the
dicarbamate 13. This unwanted side-reaction consistently
presented problems when more nucleophilic amines were
used. Reducing the stoichiometry of ethanolamine per-
mitted clean conversion to 12. This was a very versatile
intermediate which was converted to the bromide 15 in
three steps and could be displaced by a range of amines
and thiols to yield nitrogen- or sulfur-linked heterocyclic
compounds such as 16. In addition, ethanolamine 12
could be glycosylated under standard Koenigs-Knorr
conditions to furnish saccharide derivatives of the type 14
(figure 8). In nearly all cases, the intermediate coupled
products bearing 8,9 cyclic carbonate protection were
highly lipophilic and proved very difficult to purify.
Partial deprotection of the intermediates using sodium
methoxide in anhydrous methanol, in all cases yielded the
BisBoc guanidino esters which were more polar and
considerably easier to purify to homogeneity, although
partial loss of Boc protection complicated the recovery of
product in some cases.
A route utilising the 8,9 isopropylidene protected
intermediate 19 was considered but discounted on the
basis that a flatter, less sterically demanding protecting
group would have wider applicability (figure 9). The
carbonate protection exemplified in figures 4–8, is very
labile to the conditions used for the final basic hydrolysis
of the azide-derived iminophosphorane, permitting el-
egant final deprotection. The protection strategy can be
modified, however, to permit a final acidolytic deprotec-
tion by employing the 8,9 ethylidene acetal 20 in con-
junction with diphenyl methyl ester protection of the
carboxylic acid [20]. A further advantage which accrues
from the use of ethylidene protection is that when used in
conjunction with the more acid-stable methyl ester, it can
be isomerised under acid catalysis to a readily separable
mixture of the 8,9 (21) and 7,9 (22) acetals, allowing

567
Figure 7. Synthetic method C. i) H2NCH2CH2OH, MeCN, Et3N; ii) H2, Lindlar catalyst, MeOH; iii) Bis(Boc)PCH, MeOH; iv) CBr4,
Ph3P, DCM; v) 2-mercaptopyrimidine, DIPEA, MeCN, 50 °C vi) Et3N, H2O, THF, 50 °C; vii) TFA; viii) H2O.
access to a range of 8-functionalised Zanamivir deriva-
tives. The detail and applicability of this methodology is
the subject of a separate communication (unpublished
data).
of representative strains of influenza A and B viruses by
the previously reported method [21, 22]. The more active
inhibitors which emerged from this assay were further
evaluated for their in vitro antiviral activity in a plaque
reduction assay using MDCK cells [22, 23]. The results
obtained are shown in table I together with values for the
known inhibitors 1 and 2.
3. Results and discussion
New compounds 9, 11, 16 and 18 were evaluated for
their ability to inhibit the hydrolysis of 2≠-(4-methyl-
umbelliferyl)-α-D-N-acetylneuraminic acid by sialidases
The most active compounds in this series are highly
potent and are around an order of magnitude less active
than their glycerol counterparts 1 and 2; the approxi-

568
Figure 8. Synthetic method D. i)acetobromomannose, AgOTf, DCM –30 °C; ii) 2,4,6-collidine; iii) NaOMe, MeOH; iv) Dowex 50W
X 8; v) NaOH, MeOH, H2O; vi) 95% TFA; H2O.
mately 100 to 1 000-fold gain in activity previously seen
when the 4-amine is converted to the 4-guanidine is
maintained (9a cf. 11a). Generally, disubstituted carbam-
ates are marginally less active than monosubstituted (9b
cf. 9c), but a more important trend is that compounds
bearing bulky substituents (9i) and those which are highly
lipophilic (9d) show poorer activity than those bearing
polar or ionisable substituents (e.g. 11e–11h and 18).
These results are quite consistent with the X-ray analysis
of Zanamivir bound to influenza A sialidase [15]; the
poorer activity of the lipophilic compounds may be a
result of the fact that when bound to the enzyme, the
7-position is solvent exposed. A large lipophilic carbam-
ate substituent could experience adverse interactions with
the polar amino acid residues at the protein surface, or it
may simply exist in a highly collapsed form, minimising
the surface area which it presents to solvent, making the
7-position much more sterically crowded than is the case
for Zanamivir. Of particular importance is that unlike the
6-carboxamides previously described, none of the
7-carbamate compounds investigated shows any A/B
selectivity.
4. Conclusions
We have described the synthesis and properties of a
series of carbamate derivatives of Zanamivir. Several
compounds were shown to possess potent influenza
sialidase inhibitory activity, although in all cases, mea-
sured activity was slightly lower than that of Zanamivir.
The most potent compounds of the series are 11a and the
closely analogous 11f, which in addition to good enzyme
activity, displays potent anti-viral activity in vitro. Al-
though none of the compounds described herein are as
Figure 9. Acetal protection strategies for glycerol sidechain.

569
Table I. Influenza sialidase inhiition and plaque reduction by compounds 1, 2, 9, 11, 16 and 18a.
Compound
X
Y
Z
Method of
synthesis
Flu A Aichi
Flu B Victoria
Enzyme Plaque
Enzyme
Plaque
IC50
IC50
IC50
IC50
(µM)
(µg/mL)
(µM)
(µg/mL)
1
2
NH2
NHC(=NH)NH2
NH2
–
–
–
–
H
H
0.32
0.005
6.0
0.0037
4.2
9.8
76
48
100
0.47
0.005
1.8
0.05
1.25
2.95
–
0.41
0.004
1.95
0.0033
1.3
2.0
2.2
24
40
0.02
0.002
0.27
0.023
1.1
0.52
–
–
9a
11a
9b
9c
9d
9i
-(CH2)6CH3
A
A
A
B
B
B
B
NHC(=NH)NH2 -(CH2)6CH3
NH2
NH2
NH2
NH2
NH2
nPr
nPr
nC6H13
iPr
cC6H11
H
nPr
nC6H13
iPr
cC6H11
–
–
9j
–
11e
NHC(=NH)NH2
B
0.014
0.34
11f
NHC(=NH)NH2 -(CH2)6NH2
NHC(=NH)NH2 -(CH2)4NH2
NHC(=NH)NH2 -(CH2)4NH(CH2)2NH2
NHC(=NH)NH2
H
B
B
B
B
0.004
0.72
0.077
0.038
0.005
0.018
0.019
0.031
0.12
0.62
0.04
0.009
0.034
0.27
11g
11h
11k
-(CH2)3NH2
H
0.033
> 0.1
16
18
NHC(=NH)NH2
NHC(=NH)NH2
H
C
D
0.0062
0.013
0.0001
0.084
0.16
0.027
0.88
< 0.1
aSialidase assay: the IC50 values are calculated from the percent inhibition of enzyme activity in the presence of inhibitor, relative to a positive
(no inhibitor) control. All reactions were carried out in triplicate, and the mean values of these replicates used in the analysis of data. Plaque
assay: the percent inhibition of plaque formation relative to controls was calculated for each inhibitor concentration used. At each
concentration, data from three experiments were pooled in order to accurately determine the 50% inhibitory concentration (IC50) for each
compound. In most cases corresponding IC50s from different experiments differed by a factor of ≤ 5.
potent as Zanamivir, they demonstrate that the 7-position
is capable of tolerating groups of greatly differing size
and chemical functionality, with retention of activity. We
have synthesised a wide diversity of analogues at this
position, exemplifying the adaptability of this compound
class.

570
5. Experimental protocols
pyridine (349 mg, 2.86 mmol) and n-heptyl isocyanate
(1 g, 7.1 mmol). The solution was warmed to room
temperature and stirred for 21 h under nitrogen. The
reaction was then quenched by the addition of methanol
(1 mL) and the solution stirred for 15 min then added to
ice cold 1 M potassium dihydrogen orthophosphate
solution (100 mL) and extracted with ethyl acetate
(175 mL × 3). The combined organic extracts were
washed with concentrated aqueous NaCl (30 mL), dried
(Na2SO4) and the solvent removed under vacuum to yield
an orange solid which was purified by flash chromatog-
raphy (eluant, chloroform/methanol 20:1) to give 8a
(560 mg, 40%) as a white foam. IR (KBr): νmax 2 905
(br), 2 100, 1 805, 1 732, 1 664 cm–1. 1H-NMR (DMSO-
d6): δ 7.99 (1 H, d, AcNH), 7.33 (1 H, m, OCONH), 5.87
(1 H, d, H-3), 5.43 (1 H, m, H-8) 5.16 (1 H, m, H-7), 4.61
(1 H, t, H-6), 4.51 (1 H, t, H-9), 4.37 (1 H, dd, H-9), 4.26
(1 H, dd, H-4), 4.02 (1 H, q, H-5), 3.74 (3H, s, CO2CH3),
2.90 (2 H, q, OCONHCH2), 1.79 (3H, s, NHCOCH3),
1.23 (10 H, brs, (CH2)5), 0.85 (3 H, t, heptyl CH3); MS
498 (M + H)+.
5.1. General
FTIR spectra were recorded using a Nicolet 20SXB or
a Bio-Rad FTS-7. 1H-NMR spectra were recorded either
at 250 MHz using a Bruker AC or AM 250 or at 400 MHz
with a Varian VXR 400. Mass spectra were measured on
an HP Engine (Thermospray positive) or VG Autospec Q
(LSIMS). Routine microanalyses were performed on a
Leco CHNS-932 or Carlo-Erba instrument. Flash chro-
matography was performed with Merck Kieselgel 9385.
Flash reverse-phase chromatography was performed with
Merck C18 13900. Analytical HPLC was performed us-
ing a Rainin C18 8 µM column, eluting on a gradient of
0.1% aqueous trifluoroacetic acid in water to 90:10:0.1
acetonitrile/water/trifluoroacetic acid at a flow rate of
1 mL per min.
5.2. Syntheses
5.2.1. (4S,5R,6R)-5-Acetylamino-4-azido-6-[(S)-hydroxy-
(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-dihydro-4H-
pyran-2-carboxylic acid methyl ester 6
5.2.3. (4S,5R,6R)-5-Acetylamino-4-azido-6-[(S)-(4-nitro-
phenoxycarbonyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)-
methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester 7
To a suspension of the triol 5 [11] (8.2 g, 25 mmol) in
dry acetonitrile (100 mL) and dry dichloromethane
(200 mL) was added 4-dimethylaminopyridine (8.8 g,
72 mmol). A 20% solution of phosgene in toluene
(18 mL, 36 mmol) was added dropwise and the reaction
mixture was stirred at room temperature for 2 h. The
solution was then added to ice cold 1 M potassium
dihydrogen orthophosphate solution (400 mL) and ex-
tracted with ethyl acetate (350 mL × 3). The combined
organic extracts were washed with saturated aqueous
NaCl (30 mL), dried (Na2SO4), and the solvent removed
under vacuum to yield an orange foam which was
purified by flash chromatography (eluant, chloro-
form/methanol 15:1) to give 6 (6.01 g, 67%) as an
off-white foam. IR (KBr): νmax 2 100, 1 785, 1 735,
1 662 cm–1. 1H-NMR (DMSO-d6): δ 8.30 (1 H, d,
AcNH), 5.89 (1 H, d, H-3), 5.67 (1 H, d, OH-7), 4.60 (1
H, m, H-9), 4.57 (1 H, m, H-9), 4.99 (1 H, m, H-8), 4.44
(1 H, dd, H-4), 4.15 (1 H, m, H-6), 4.03 (1 H, m, H-7),
3.98 (1 H, m, H-5), 3.78 (3 H, s, CO2CH3), 1.94 (3 H, s,
NHCOCH3); MS 374 (M + NH4)+.
To a solution of the triol 5 (3.3 g, 10 mmol) in dry
pyridine (30 mL), under nitrogen, was added 4-nitro-
phenylchloroformate (2.42 g, 12 mmol). After stirring at
room temperature for 2.5 h a further portion of
4-nitrophenylchloroformate (2 g, 10 mmol) was added
followed by 4-dimethylaminopyridine (3.05 g, 25 mmol).
Stirring was continued at room temperature for 3.5 h. The
solution was concentrated under vacuum and the residue
was partitioned between 2 M HCl (200 mL) and ethyl
acetate (100 mL). The layers were separated and the
aqueous phase was further extracted with ethyl acetate
(100 mL × 2). The combined organic extracts were
washed with saturated aqueous NaCl (60 mL), dried
(Na2SO4) and the solvent removed under vacuum to yield
a beige foam which was purified by flash chromatography
(eluant, ethyl acetate/cyclohexane 2:1) to give 7 (3.21 g,
62%) as a white foam. IR (KBr): νmax 4 218 (br) 2 101,
1
1 792, 1 784, 1 664 cm–1. H-NMR (DMSO-d6): δ 8.36
(2H, d, Ar), 8.22 (1H, d, AcNH), 7.61 (2H, d, Ar), 5.94
(1H, d, H-3), 5.50 (1H, t, H-7), 5.35 (1H, m, H-8), 4.72
(1H, t, H-9), 4.60 (1H, t, H-9), 4.40–4.50 (2H, m, H-6,
H-4), 4.23 (1H, t, H-5), 3.78 (3H, s, CO2CH3), 1.86 (3H,
s, NHCOCH3); MS 522 (M + H)+; anal C20H19N5O12 (C,
H, N).
5.2.2. (4S,5R,6R)-5-Acetylamino-4-azido-6-[(S)-heptyl-
carbamoyloxy-(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-
dihydro-4H-pyran-2-carboxylic acid methyl ester 8a
To an ice-cold solution of 6 (1 g, 2.81 mmol) in dry
dichloromethane (10 mL) was added 4-dimethylamino-

571
5.2.4. (4S,5R,6R)-5-Acetylamino-4-azido-6-{(S)-[(2-oxo-
[1,3]dioxolan-4R-yl)-(6-tert-butoxycarbonylamino-
hexyl)-carbamoyloxy]-methyl}-5,6-dihydro-4H-pyran-2-
carboxylic acid methyl ester 8f
NHCH2(CH2)4CH2NH), 1.38 (9H, s, Boc); MS 533 (M +
H)+; anal C23H40N4O10.TFA.0.25Et3N (C, H, N).
5.2.6. (4S,5R,6R)-5-Acetylamino-4-[2,3-bis(tert-butoxy-
carbonyl]guanidino-6-{1R-[(6-tert-butoxycarbonylami-
nohexyl)-carbamoyloxy]-2,3-dihydroxypropyl}-5,6-
dihydro-4H-pyran-2-carboxylic acid 10f
To a solution of 7 (1.15 g, 2.2 mmol) in dry pyridine
(10 mL) was added 4-dimethylaminopyridine (648 mg,
5.3 mmol) and N-Boc-1,6-diaminohexane hydrochloride
(670 mg, 2.65 mmol). The solution was stirred at room
temperature for 18 h. 2 M aqueous hydrochloric acid
(150 mL) was added to the solution which was then
extracted with ethyl acetate (50 mL × 2). The combined
organic extracts were washed with saturated aqueous
NaCl (10 mL), dried (Na2SO4) and the solvent removed
under vacuum to yield a yellow foam which was purified
by flash chromatography (eluant, ethyl acetate/cyclo-
hexane 4:1) to give 8f (1.155 g, 90%) as a pale yellow
foam. IR (KBr): νmax 3 326 (br), 2 099, 1 799, 1 737,
A solution of 9f (0.75 g, 1.16 mmol), triethylamine
(0.5 mL, 3.6 mmol) and N,N≠-bis-t-butoxycarbonyl-
1H-pyrazole-1-carboxamidine Bis(Boc)PCH (0.54 g,
1.74 mmol) [15] in a mixture of dry tetrahydrofuran
(10 mL) and dry methanol (3 mL) was stirred under
nitrogen for 20 h and then evaporated under vacuum. The
residue was dissolved in ethyl acetate (100 mL), stirred
vigorously with water (20 mL) and the pH of the mixture
was adjusted to pH 2 with 2 M hydrochloric acid. The
organic layer was washed with saturated aqueous NaCl
(20 mL), dried (Na2SO4), and the solvent removed under
vacuum to yield a yellow foam which was purified by
flash chromatography (eluant, chloroform/methanol 7:1)
to give 10f (0.37 g, 42%) as a white solid. IR (KBr): νmax
1
1 693 cm–1. H-NMR (CDCl3) δ 6.31 (1 H, br.d, NH-
Boc), 5.95 (3 H, d, H-3), 5.42 (1 H, dd, AcNH), 5.10 (2
H, m, H-7, H-4), 4.95 (1 H, m, H-8) 4.5–4.8 (3 H, m, H-5,
H-9), 3.80 (3H, s, CO2CH3), 3.0–3.4 (4 H, m
NHCH2(CH2)4CH2NH), 2.06 (3H, s, NHCOCH3), 1.50
(4H, m, NHCH2(CH2)4CH2NH), 1.44 (9H, s, NHBoc),
1
3 291 (br), 1 686, 1 610 cm–1. H-NMR (DMSO-d6): δ
11.42 (1 H, s, guanidine NHBoc), 8.25 (1 H, d, NH-4),
7.95 (1 H, d, AcNH), 7.16 (1 H, t, OCONH), 6.75 (1 H,
t, heptyl NHBoc) 5.80 (1 H, s, H-3), 4.82 (1 H, d, H-7),
4.74 (1 H, t, H-4), 4.43 (1 H, d, H-6), 4.0 (4 H, m,
H-5,-8,-9), 3.25 (1 H, m, OH), 2.90 (4 H, m,
NHCH2(CH2)4CH2NH) 1.76 (3 H, s, NHCOCH3), 1.49
(9 H, s, Boc), 1.38 (18 H, s, Boc), 1.2–1.5 (8 H, m,
NHCH2(CH2)4CH2NH); MS 775 (M + H)+; anal
C34H58N6O14 (C, H, N).
1.34
(4H,
m,
NHCH2(CH2)4CH2NH);
anal
C25H38N6O11.0.35 EtOAc (C, H, N).
5.2.5. (4S,5R,6R)-5-Acetylamino-4-amino-6-{2R,3-dihy-
droxy-1R-[(6-tert-butoxy-carbonylaminohexyl)-carba-
moyloxy]-propyl}-5,6-dihydro-4H-pyran-2-carboxylic
acid trifluoroacetate 9f
5.2.7. (4S,5R,6R)-5-Acetylamino-6-{1R-[(6-aminohexyl)-
carbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-
dihydro-4H-pyran-2-carboxylic acid 11f
To a solution of 8f (1.05 g, 1.75 mmol) in dry tetrahy-
drofuran (40 mL) was added triphenylphosphine
(610 mg, 2.32 mmol) and the solution was stirred at room
temperature for 18 h. To this was added triethylamine
(12 mL, 86 mmol) and water (31 mL) and the solution
was heated at 40 °C for a further 28 h. The reaction
mixture was evaporated to yield a yellow gum which was
purified by flash reverse-phase chromatography (eluant,
water (containing trifluoroacetic acid 0.1%)/acetonitrile
8:2). The gum obtained by freeze-drying was triturated
vigorously with diethyl ether for 1 h at room temperature
to give 9f (0.79 g, 70%) as a buff powder. IR (KBr): νmax
A solution of 10f (325 mg, 0.42 mmol) in trifluoroace-
tic acid (10 mL) was stirred under nitrogen for 1 h and
evaporated under vacuum. The residue was dissolved in
water (10 mL), stirred for 1 h and the water removed by
lyophilisation to give 11f (255 mg, 86%) as a white solid.
IR (KBr): νmax 3 307 (br), 1 735 cm–1. 1H-NMR
(DMSO-d6): δ 7.97 (1 H, d, AcNH), 7.2–7.8 (6 H, m,
guanidine, hexylamine NH2s), 7.6 (1 H, d, NH-4), 5.72 (1
H, s, H-3), 5.07 (1 H, d, OH), 4.85 (1 H, d, H-7), 4.4 (2
H, m, H-6, OH), 4.33 (1 H, t, H-4), 4.02 (1 H, q, H-5),
3.83 (1 H, m, H-8), 3.3 (1 H, m, H-9), 3.4 (1 H, m, H-9),
2.91 (2 H, m, OCONHCH2), 2.78 (2 H, m, CH2NH2),
1.81 (3 H, s, NHCOCH3), 1.2–1.6 (8 H, m,
NHCH2(CH2)4CH2NH); MS 475 (M + H)+; anal: found
C 38.2; H 5.1; N 11.5. C19H34N6O8.2TFA.H2O requires
C 38.3; H 5.3; N 11.6.
1
3 310 (br), 1 685 cm–1. H-NMR (DMSO-d6): δ 7.92 (1
H, d, AcNH), 7.13, 6.75 (2 H, t, OCONH, NHBoc), 5.70
(1 H, s, H-3) 4.85 (1 H, d, H-7), 4.34 (1 H, d, H-6), 4.02
(1 H, q, H-5), 3.85 (1 H, t, H-8), 3.77 (1 H, d, H-4), 3.25,
3.44 (2 H, m, H-9), 2.90 (4 H, m, NHCH2(CH2)4-
CH2NH), 1.82 (3H, s, NHCOCH3), 1.18–1.42 (8H, m,

572
5.2.8. (4S,5R,6R)-5-Acetylamino-4-azido-6-[(S)-(2-hydro-
xyethylcarbamoyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)-
methyl]-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester 12
1.40 (9 H, 9 H, s, s, Boc, Boc); HRMS: found MH+
660.273025, calc for C27H42N5O14 660.272827.
5.2.10. (4S,5R,6R)-5-Acetylamino-4-[2,3-bis(tert-butoxy-
carbonyl)-guanidino]-6-[(S)[(2-bromoethyl)-carbamoy-
loxy]-(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-dihydro-
4H-pyran-2-carboxylic acid methyl ester 15
To a solution of 7 (7.60 g, 14.58 mmol) in dry aceto-
nitrile (60 mL) was added triethylamine (0.86 mL,
14.42 mmol), followed by ethanolamine (0.86 mL,
14.3 mmol). The solution was stirred at room temperature
for 1 h and the solvent removed under vacuum to yield an
orange foam which was purified by flash chromatography
(eluant, ethyl acetate) to yield 12 as a yellow foam
(5.78 g, 90%). IR (KBr): νmax 3 300 (br), 2 100, 1 792,
1 733, 1 653 cm–1. 1H-NMR (DMSO-d6): δ 8.09 (1 H, d,
AcNH), 7.35 (1 H, t, OCONH), 5.88 (1 H, d, H-3), 5.41
(1 H, s, H-7), 5.16 (1 H, m, H-8), 4.6 (1 H, m, OH), 4.6
(1 H, m, H-9), 4.5 (1 H, m, H-9), 4.37 (1 H, m, H-6), 4.29
(1 H, m, H-4), 4.07 (1 H, m, H-5), 3.76 (3 H, s,
COOCH3), 3.39 (2 H, m, CH2CH2OH), 3.05, 2.92 (2 H,
m, CH2CH2OH), 1.82 (3 H, s, NHCOCH3); MS 461 (M
+ NH4)+; anal C16H21N5O10 (C, H, N).
To a solution of 14 (533 mg, 0.81 mmol) in dry
dichloromethane (6 mL) was added tetrabromomethane
(407 mg, 1.23 mmol) and triphenylphosphine (318 mg,
1.2 mmol) and the solution was stirred at room tempera-
ture for 30 min. The reaction mixture was evaporated to
yield a colourless foam which was purified by flash
chromatography (eluant, ethyl acetate/cyclohexane 1:1)
to yield 15 as a colourless foam (395 mg, 68%). IR
1
(KBr): νmax 3 310 (br), 1 807, 1 732, 1 642, cm–1. H-
NMR (DMSO-d6): δ 11.4 (1 H, s BocNH), 8.18 (1 H, d,
AcNH), 7.97 (1 H, d, NH-4), 7.76 (1 H, t, OCONH), 5.85
(1 H, d, H-3), 5.46 (1 H, m, H-7), 5.18 (1 H, m, H-8), 4.8
(1 H, m, H-4), 4.64 (1 H, m, H-9), 4.53 (1 H, m, H-6),
4.46 (1 H, m, H-9), 4.02 (1 H, q, H-5), 3.75 (3 H, s,
COOCH3), 3.45 (2 H, m, CH2CH2Br), 3.3 (2 H, m,
CH2CH2Br), 1.75 (3 H, s, NHCOCH3), 1.46, 1.40 (9 H,
9 H, s, s, Boc, Boc); HRMS: found MH+ 722.188633 calc
for C27H41N5O13Br 722.188423 (for 79Br).
5.2.9. (4S,5R,6R)-5-Acetylamino-4-[2,3-bis(tert-butoxy-
carbonyl)-guanidino]-6-[(S)[(2-hydroxyethyl)-carbamoy-
loxy]-(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-dihydro-
4H-pyran-2-carboxylic acid methyl ester 14
A solution of 12 (5.7 g 12.9 mmol) in methanol
(250 mL) was added to Degussa CE407 Pb/Pd on CaCO3
R/D 5 + 5% (i.e. 5% Pd on CaCO3, poisoned with Pb 5%)
dry catalyst and stirred under hydrogen for 2 h. The
resulting mixture was filtered through celite, washed with
methanol (500 mL) and the combined filtrate and wash-
ings evaporated. The yellow foam obtained was dis-
solved, with heating, in ethanol and evaporated to a slurry
which was triturated with ethyl acetate (20 mL) to yield
the intermediate amine as an off-white solid. A mixture of
the intermediate amine (3.64 g, 8.72 mmol) and Bis-
BocPCH (3.39 g, 10.9 mmol) in methanol (150 mL) was
warmed briefly to give a solution which was allowed to
cool to room temperature and stirred for 5 h. A further
portion of BisBocPCH (0.68 g, 2.19 mmol) was added
and the solution stirred for 18 h; solvent was removed
under vacuum to give a white amorphous solid which was
purified by flash chromatography (eluant, ethyl
acetate/cyclohexane 4:1 to 9:1) to yield 14 as a white
foam (4.32 g, 57%). IR (KBr): νmax 3 290 (br) 1 800,
5.2.11. (4S,5R,6R)-5-Acetylamino-6-{2R,3-dihydroxy-1R-
[2-(pyrimidin-2-ylsulfanyl)-ethylcarbamoyloxy]-propyl}-
4-guanidino-5,6-dihydro-4H-pyran-2-carboxylic acid bis
(trifluoroacetate) 16
To a solution of 15 (373 mg, 0.52 mmol) in dry
acetonitrile (5 mL) was added 2-mercaptopyrimidine
(984 mg, 0.75 mmol) and diisopropylethylamine
(130 µL, 0.75 mmol) and the solution was stirred at 50 °C
for 2 h. The reaction mixture was evaporated to yield a
yellow foam which was purified by flash chromatography
(eluant, ethyl acetate/cyclohexane 3:1) to yield the
coupled product as
a colourless foam (354 mg,
0.47 mmol, 91%). The foam (340 mg, 0.45 mmol) was
dissolved in dry THF (1 mL), triethylamine (1 mL) and
water (0.5 mL) and heated at 50 °C for 30 h; the solvent
was evaporated to yield a cream foam (334 mg) which
was dissolved in TFA (2 mL) and stirred for 4 h. Solvent
was evaporated and the residue stirred in water (2 mL) for
2 h. This aqueous solution was purified directly by flash
reverse-phase chromatography (eluant, water (containing
trifluoroacetic acid 0.1%)/acetonitrile 9:1). Fractions of
interest were pooled and lyophilised to yield 16 as an
amorphous white powder (155 mg, 0.3 mmol, 52%). IR
1
1 738, 1 646 cm–1. H-NMR (DMSO-d6): δ 11.4 (1 H, s
BocNH), 8.18 (1 H, d, AcNH), 8.05 (1 H, d, NH-4), 7.35
(1 H, t, OCONH), 5.84 (1 H, d, H-3), 5.43 (1 H, m, H-7),
5.16 (1 H, m, H-8), 4.8 (1 H, m, H-4), 4.62 (2 H, m, H-9),
4.62 (1 H, m, H-6), 4.52 (1 H, t, OH), 4.03 (1 H, q, H-5),
3.64 (3 H, s, COOCH3), 3.41 (2 H, m, CH2CH2OH), 2.98
(2 H, m, CH2CH2OH), 1.75 (3 H, s, NHCOCH3), 1.46,
1
(KBr): νmax 3 388 (br), 1 615 cm–1. H-NMR (DMSO-
d6): δ 8.62 (2 H, d, Ar 3,5-H), 7.98 (1 H, d, AcNH), 7.57

573
(1 H, d, NH-4), 7.48 (1 H, t, OCONH), 7.21 (1 H, m, Ar
4-H), 7.2 (H, br s, HNC(=NH)NH2), 5.71 (1 H, d, H-3),
4.9 (1 H, d, H-7), 4.39 (1 H, m, H-6), 4.31 (1 H, m, H-4),
4.02 (1 H, m, H-5), 3.81 (1 H, m, H-8), 3.43 (1 H, m,
H-9), 3.23 (1 H, m, H-9), 3.34–3.06 (4 H, m,
NHCH2CH2S), 1.8 (3 H, s, NHCOCH3); HRMS: found
MH+ 514.171687 calc for C19H28N7O8S 514.172008;
anal C19H27N7O8S. 2.5TFA. H2O (C, H, N, S).
5.2.13.
(4S,5R,6R)-5-Acetylamino-6-{2R,3-dihydroxy-
1R-[2-(3S,4S,5S-trihydroxy-6R-hydroxymethyl-
tetrahydro-pyran-2S-yloxy)-ethylcarbamoyloxy]-propyl}-
4-guanidino-5,6-dihydro-4H-pyran-2-carboxylic
bis(trifluoroacetate) 18
acid
A solution of 17 in methanol (20 mL) was treated with
water (20 mL) and 0.1 M sodium hydroxide solution
(3.25 mL, 0.325 mmol). After 1 h, the solution was
evaporated and the residue triturated with diethyl ether
(10 mL) to yield a white amorphous powder which was
stirred in 95% trifluoroacetic acid (20 mL) at room
temperature for 1 h and evaporated to dryness. The
residue was stirred in water (5 mL) for 1 h, concentrated
and purified by flash reverse-phase chromatography (elu-
ant, water (containing trifluoroacetic acid 0.1%)). Frac-
tions of interest were pooled and lyophilised to a gum
which was dissolved in ethanol, concentrated and tritu-
rated with diethyl ether (10 mL) and filtered to yield 18 as
a white amorphous ethanol-solvated trifluoroacetate salt
5.2.12. (4S,5R,6R)-5-Acetylamino-4-[2,3-bis(tert-butoxy-
carbonyl)-guanidino]-6-{1R-[2-(3S,4S,5S-trihydroxy-6R-
hydroxymethyl-tetrahydropyran-2S-yloxy)-ethylcarba-
moyloxy]-(2-oxo-[1,3]dioxolan-4R-yl)-methyl}-5,6-
dihydro-4H-pyran-2-carboxylic acid methyl ester 17
To a solution of 14 (1.32 g, 2 mmol) in dry dichlo-
romethane (15 mL) was added silver triflate (0.74 g,
2.88 mmol) and the solution cooled to –30 °C. To this
solution was added dropwise over 30 s, a solution of
acetobromomannose (1.18 g, 2.9 mmol) in dry dichlo-
romethane (10 mL). The mixture was stirred in the dark
at –30 °C for 6 h and quenched by the addition of
2,4,6-collidine (400 µL, 3 mmol) and diethyl ether
(150 mL). This mixture was filtered through a celite pad,
washed with diethyl ether (100 mL) and the combined
filtrate and washings were evaporated to yield a colour-
less foam which was redissolved in ethyl acetate
(100 mL) and washed with 0.5 M HCl (20 mL). The
aqueous layer was separated and washed with ethyl
acetate (50 mL). The combined organic extracts were
washed with brine, dried (Na2SO4) and evaporated to
yield a colourless foam which was purified by flash
chromatography (eluant, chloroform/methanol 25:1) to
yield the coupled product as an impure colourless foam
(1.323 g, 68%). This foam was dissolved in anhydrous
methanol (100 mL) and the solution adjusted to pH 10.8
by dropwise addition of sodium methoxide (25% solution
in methanol). After 2 h, the solution was adjusted to pH 7
with Dowex 50W X 8 (H+) resin, filtered and evaporated
to yield a white solid which was purified by flash
chromatography (eluant chloroform/methanol 10:1) to
yield 17 as a white amorphous solid (0.27 g, 28%, loss of
Boc group). 1H-NMR (DMSO-d6): δ 11.4 (1 H, s,
NHBoc), 8.18 (1 H, d, NH-4), 8.0 (1 H, d, AcNH), 7.31
(1 H, t, OCONH), 5.83 (1 H, d, H-3), 5.07 (1 H, d,
2≠OH), 4.82 (1 H, d, H -1≠), 4.76 (1 H, t, H-4), 4.65 (1
H, m, H-7), 4.49 (1 H, d, H-6), 4.40–4.80 (5 H, m,
OH-8,9,3≠,4≠,6≠), 4.05 (1 H, q, H-5), 3.75 (3 H, s,
COOCH3), 3.10–3.80 (13 H, m, H-8,9,2≠-6≠,
NHCH2CH2O), 1.86 (3 H, s, NHCOCH3), 1.46, 1.41 (9
H, 9 H, s, s, Boc, Boc); MS: 813 (M + NH4)+.
1
(198 mg, 84%, corrected). H-NMR (DMSO-d6 + D2O):
δ 5.72 (1 H, d, H-3), 4.84 (1 H, d, H-1≠), 4.63 (1 H, m,
H-7), 4.39 (1 H, m, H-6), 4.31 (1 H, m, H-4), 4.0 (1 H, q,
H-5), 3.10–3.70 (13 H, m, H-8,9,2≠-6≠, NHCH2CH2O),
1.81 (3 H, s, NHCOCH3); HRMS: found MH+
582.224769 calc for C21H36N5O14 582.225876; anal
C21H35N5O14. EtOH. TFA (C, H, N).
Acknowledgements
We wish to thank R. Bethell, S. Madar, and P. Smith for
their contributions to this work.
References
[1] Hart G.J., Bethell R.C., Biochem. Mol. Biol. Int. 36 (1995) 695–703.
[2] Hayden F.G., Rollins B.S., Madren L.K., Antiviral Res. 25 (1994)
123–131.
[3] Hayden F.G., Treanor J.J., Betts R.F., Lobo M., Esinhart J.D.,
Hussey E.K., JAMA (J. Am. Med. Assoc.) 275 (1996) 295–299.
[4] Holzer C.T., VonItzstein M., Jin B., Pegg M.S., Stewart W.P., Wu
W.Y., Glycoconj. J. 10 (1993) 40–44.
[5] Gilead Sciences Inc., WO 9626933 (1997).
[6] Kim C.U., Lew W., Williams M.A., Zhang L., Liu H., Swaminathan
S. et al., J. Am. Chem. Soc. 119 (1997) 681–690.
[7] Kim C.U., Lew W., Williams M.A., Wu H., Zhang L., Chen X. et al.,
J. Med. Chem. 41 (1998) 2451–2460.
[8] Biocryst Pharmaceuticals Inc., WO 9747194 (1997).
[9] VonItzstein M., Wu W., Jin B., Carbohydr. Res. 259 (1994) 301–305.
[10] Bamford M.J., Pichel J.C., Husman W., Patel B., Storer R., Wier
N.G., J. Chem. Soc. Perkin Trans. 1 (1995) 1181–1187.
[11] Chandler M., Bamford M.J., Conroy R., Lamont B., Patel B., Patel
V.K. et al., J. Chem. Soc. Perkin Trans. 1 (1995) 1173–1180.

574
[12] Starkey I.D., Mahmoudian M., Noble D., Smith P.W., Cherry P.,
Howes P.D., Sollis S.L., Tetrahedron Lett. 36 (1995) 299–302.
[18] Schreiner E., Christian R., Zbiral E., Liebigs Ann. Chem. (1990)
93–97.
[13] Smith P.W., Starkey I.D., Howes P.D., Sollis S.L., Keeling S.P.,
Cherry P.C. et al., Eur. J. Med. Chem. 31 (1996) 143–150.
[19] Bernatowicz M.S., Wu Y., Matsueda G.R., Tetrahedron Lett. 34
(1993) 3389–3392.
[14] Sollis S.L., Smith P.W., Howes P.D., Cherry P.C., Bethell R.C.,
Bioorg. Med. Chem. Lett. 6 (1996) 1805–1808.
[20] Biota Sci. Management Ltd. WO9706157-A1 (1997).
[21] Potier M., Mameli L., Belisle M., Dallaire L., Melancon S.B., Anal.
Biochem. 94 (1979) 287–296.
[15] VonItzstein M., Wu W.Y., Kok G.B. et al., Nature (Lond.) 20 (1993)
418–423.
[22] Woods J.M., Bethell R.C., Coates J.A.V., Healy N., Hiscox S.A.,
Pearson B.A., Ryan D.M., Ticehurst J., Tilling J., Antimicrob.
Agents Chemother. 37 (1993) 1473–1479.
[16] Varghese J.N., Epa V.C., Colman P.M., Protein Sci. 4 (1995)
1081–1087.
[17] Yoshimoto K., Tahara K., Suzuki S., Sasaki K., Nishikawa Y., Tsuda
Y., Chem. Pharm. Bull. 27 (1979) 2661–74.
[23] Hayden F.G., Cote K.M., Douglas R.G. Jr., Antimicrob. Agents
Chemother. 17 (1980) 865–870.