Synthesis of functionalized cyclohexenephosphonates and their inhibitory activity towards bacterial sialidases

Article abstract of DOI:10.1016/S0040-4020(02)00873-6

We have synthesized a series of cyclohexenephosphonates derived from D- and L-xylose, designed as core structures for the development of high affinity mimics of sialic acid and of the sialidase reaction transition state. Extension of our syntheses to both xylose enantiomers has given us access to two series of cyclohexenephosphonates with regioisomeric double bonds. We have demonstrated the selective functionalization of the hydroxyl groups towards introduction of a glycerol side chain mimic and immobilization via a silyl linker. The inhibitory activity of a selected set of compounds towards three bacterial sialidases has been tested and moderate activity was found.

Full text of DOI:10.1016/S0040-4020(02)00873-6

Tetrahedron 58 (2002) 7573–7581
Synthesis of functionalized cyclohexenephosphonates and their
inhibitory activity towards bacterial sialidases
*
Hansjorg Streicher and Christoph Bohner
¨
Department of Chemistry, University of Konstanz, D-78457 Konstanz, Germany
Received 17 May 2002; accepted 19 July 2002
Abstract—We have synthesized a series of cyclohexenephosphonates derived from D- and L-xylose, designed as core structures for the
development of high affinity mimics of sialic acid and of the sialidase reaction transition state. Extension of our syntheses to both xylose
enantiomers has given us access to two series of cyclohexenephosphonates with regioisomeric double bonds. We have demonstrated the
selective functionalization of the hydroxyl groups towards introduction of a glycerol side chain mimic and immobilization via a silyl linker.
The inhibitory activity of a selected set of compounds towards three bacterial sialidases has been tested and moderate activity was
found. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
The crucial role played by sialic acids in many physiological
recognition events has led to increased interest in sialic acid
metabolizing enzymes.1,2 Great achievements have been
made in the development of high affinity inhibitors of both
the sialidase from influenza virus,3–6 an enzyme that
cleaves terminal sialic acids from the host cell glycocalix
in the early stage of viral infection, and of sialyl
transferases, enzymes which transfer the sialosyl cation
from the natural donor CMP–Neu5Ac to the glycan chain
terminus during biosynthesis.7–9
Inhibitors of comparable potency targeted towards siali-
dases from bacteria are still lacking even though some of
them have been identified to be involved in infection
processes.2,10,11 Compared to viruses, bacteria have more
means of interaction with the host cell glycan chains, which
is also reflected in the bifunctionality of some bacterial or
protozoal sialidases. These contain, as has been shown by
both X-ray crystallography and sequence alignment
methods, additional carbohydrate binding sites apart from
the active site.12,13
Scheme 1. Structures and double bond positions in fully functionalized
cyclohexenephosphonates (2)-1 and (þ)-1 with respect to well-known
sialidase inhibitors GS 4104 and Neu5Ac2en.
1.1. Synthetic approach
We have previously synthesized cyclohexenephosphonate
(2)-1,14 which can be seen as an analog of the well known
influenza drug GS 4104 containing a phosphonate group
instead of a carboxylate group (Schemes 1 and 2).
This could, at least in principle, be exploited by designing
inhibitors that allow attachment of a spacer molecule or an
aglycon mimic while keeping in mind that the negative
charge, known to be required for effective recognition,
should be retained.14
In this paper we report the synthesis of the enantiomer (þ)-1,†
†
Numbering of D- and L-xylose derived cyclohexenephosphonates.
Whenever determined, the cyclohexenephosphonates derived from
D-xylose showed positive optical rotation and were therefore termed
(þ)-1, (þ)-10, etc. Consequently, their enantiomers derived from
L-xylose showed negative optical rotation and were numbered (2)-1,
(2)-10, etc.
Keywords: sialidase inhibitors; sialic acid; cyclohexenephosphonates;
Vibrio cholerae sialidase; bacterial sialidases.
*
Corresponding author. Tel.: þ49-7531-884403; fax: þ49-7531-883135;
e-mail: hansjoerg.streicher@uni-konstanz.de
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00873-6

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H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
Scheme 4. Synthesis of acetamido-cyclohexenephosphonate monoethyl
ester (þ)-13. (i) NaOEt, EtOH then NaOH, Biogel P2.
of 4 into the triflate 5 and nucleophilic substitution to give
the xylo-configured azide 6.
Desilylation of 6 gave the known alcohol 7,19 which was
activated as the triflate 8 to allow substitution with the
tetraethyl methylenediphosphonate anion to give the
diphosphonate 9. Removal of the isopropylidene group
followed by base-mediated cyclization gave cyclohexene-
phosphonate (þ)-10, which, as well as its enantiomer,
serves as a crucial intermediate for selective modifications
at the hydroxyl groups (see below). To complete the
synthesis of (þ)-1, the azido-cyclohexenephosphonate (þ)-
10 was acetylated ((þ)-11), the azide moiety was reduced
Scheme 2. Synthetic approach to enantiomeric cyclohexenephosphonates
(2)-1 and (þ)-1, respectively.
Scheme 3. Synthesis of cyclohexenephosphonate (þ)-1, employing the same strategy we reported previously for (2)-1. (i) (COCl)2, DMSO (ii) NaBH4, EtOH,
(90% for two steps); (iii) Tf2O, pyridine, CH2Cl2; (iv) NaN3, EtOH, (85% for two steps); (v) TBAF, THF, (96%); (vi) Tf2O, pyridine, CH2Cl2, (86%);
(vii) CH2((P(O)(OEt)2)2, LiN(SiMe3)2, DMF, (40%); (viii) IR-120 (Hþ), dioxane, H2O, then LiN(SiMe3)2 (46%), (ix) Ac2O, pyridine, (qu); (x) H2S, pyridine,
H2O then Ac2O, pyridine, (70%); (xi) TMSBr, CHCl3, then H2O, Biogel P2.
having the double bond in an orientation comparable to that
in the classical sialidase inhibitor Neu5Ac2en15 (Scheme 1).
To further optimize both series of inhibitors we focused on
(a) the selective introduction of an alkyl side chain
mimicking the glycerol moiety in Neu5Ac, which offers
possibilities for further optimization and (b) the selective
silylation of the position equivalent to the 4-OH in Neu5Ac
or Neu5Ac2en thus simulating immobilization on a suitable
resin via a silyl linker (compounds 22 and 27, respectively,
Scheme 1). The synthesis of (þ)-1 was accomplished in a
straightforward manner by simply shifting the method-
ology14,16–18 of the cyclohexenphosphonate synthesis from
L-xylose to D-xylose (Scheme 2).
and acetylated to give the acetamide (þ)-12. Finally the
phosphonate diester was cleaved by treatment with
bromotrimethylsilane and hydrolysis which, after gel
filtration, led to the ammonioum salt (þ)-1 (Scheme 3).
2.1. Synthesis of a phosphonate–monoester
It is of importance for our concept14 to show that
2. Results and discussion
Cyclohexenephosphonate (þ)-1 was synthesized from 1,2-
O-isopropylidene-5-triisopropylsilyl-D-xylofuranose 2 as
described previously for the enantiomeric compound (2)-
1 (Scheme 3).14,19–21 In brief, the absolute configuration at
C-3 was inverted by Swern-oxidation to give the ketone 3
followed by reduction with NaBH4, which furnished
D-ribofuranose 4. The azide was introduced by conversion
Scheme 5. Synthesis of deprotected azido-cyclohexenephosphonates (2)-
14 and (þ)-14.

H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
7575
2.3. Differentiation of the hydroxyl groups in the (1)-
and (2)-series
To pave the way to optimized inhibitors starting from the
azides (þ)-10 and (2)-10, reaction sequences had to be
established that would allow the selective introduction of an
alkyl side chain at the position corresponding to C-6 in
Neu5Ac and a silyl group simulating a linker22 at the
position corresponding to C-4 in Neu5Ac.
In Scheme 6, the modifications necessary to facilitate
inhibitor optimization are depicted: in phosphonate (þ)-10,
the allylic hydroxyl group at C-3 corresponds to 4-OH in
Neu5Ac, it therefore should be selectively silylated to allow
alkylation at C-5. In the enantiomeric phosphonate (2)-10,
the allylic hydroxyl group at C-3 occupies the position of
the glycerol moiety in Neu5Ac, it therefore should be
selectively alkylated followed by silylation (Scheme 6).
Finally, reduction and acylation of the azide group as well as
ester cleavage and, if required, esterification leads to fully
optimized inhibitors. In this report, all reactions were
performed in solution to find suitable reaction conditions.
Scheme 6. Regioselective modifications of key compounds (2)-10 and
(þ)-10 required to obtain potential sialidase inhibitors.
phosphonate monoesters display at least some inhibition
of sialidases. We therefore deprotected and partially
de-esterified acetamide (þ)-12 to give monoethyl ester
(þ)-13 in almost quantitative yield (Scheme 4).
2.2. Azide-containing inhibitors
2.4. Modification of azido-cyclohexenephosphonate (1)-
10
In order to assess the significance of the acetamide group,
azide containing diphosphonates (þ)-10 and (2)-10 were
hydrolyzed with bromotrimethylsilane and converted into
the corresponding ammonium salts (þ)-14 and (2)-14
(Scheme 5).
Initially, we intended to employ a linker system based on an
alkyldiphenylsilyl group and therefore we selectively
silylated derivative (þ)-10 with tert-butyldiphenylsilyl
Scheme 7. Selective modifications and deprotection reactions in the (þ)-series. (i) TBDPSCl, imidazole, (58%); (ii) Ac2O, pyridine, (92%); (iii) allyl bromide,
Ag2O, (45%); (iv) TIPSCl, imidazole, (51%); (v) Ac2O, pyridine, (94%); (vi) allyl bromide, Ag2O, TBAI, (85%); (vii) H2S, pyridine, H2O then Ac2O, pyridine
(71%); (viii) TMSBr, CHCl3, then H2O, HPLC-purification.

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H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
Scheme 8. Selective modifications and deprotection reactions in the (2)-series. (i) allyl bromide, Ag2O, TBAI, (70%); (ii) Ac2O, pyridine, (94%); (iii) TIPSCl,
imidazole, (69%); (iv) H2S, pyridine, H2O then Ac2O, pyridine (81%); (v) TMSBr, CHCl3, then H2O, HPLC-purification.
chloride to give 15, which was acetylated to confirm the
position of the silyl group (Scheme 7). Allylation of 15 to
give 17 proceeded in only moderate yield due to migration
of the silyl group from C-3 to C-5, which led to a mixture of
regioisomers (only the desired compound 17 is shown in
Scheme 7).
Vibrio cholerae, Salmonella typhimurium and Clostridium
perfringens. Even though the substances tested still lack a
glycerol side chain mimic optimized towards the binding to
the respective sialidase we wanted to address several
important questions immediately. (1) Does this kind of
4-acetamido cyclohexenephosphonates show activity
against sialidases? (2) Is the acetamido group required for
recognition by the enzymes? (3) Do the monoethyl- and
diethyl esters of the phosphonate group display activity? We
performed a well established sialidase inhibition assay23,24
according to a modification by Schauer and co-workers25
and extracted IC50 values (in M) from the inhibition curves
(see Section 4).
A similar effect was observed by us previously.14 To
overcome this problem, we enhanced base stability by using
the triisopropylsilyl group: silylation of (þ)-10 with
triisopropylsilyl chloride gave 18 which again was acetyl-
ated to elucidate the position of the silyl group (19). No silyl
migration was observed when 18 was allylated to give fully
functionalized cyclohexenenphosphonate 20. After
reduction of the azide followed by acetylation, acetamide
21 was obtained in good yield and then further treated with
bromotrimethylsilane in chloroform and then water. After
purification by HPLC, target compound 22 was obtained as
its triethylammonium salt.
The data obtained are given in Table 1.
3. Conclusions
3.1. Syntheses
2.5. Modification of azido-cyclohexenephosphonate (2)-
10
xylo-Configured cyclohexenephosphonates containing an
acetamide group have been synthesized starting from D- and
L-xylose, respectively. The molecules are designed to
mimic the sialosyl cation intermediate and could therefore
serve as scaffolds for the generation of optimized sialidase
inhibitors. The D-xylose derived series has its double bond
in a position equivalent to the well known inhibitor
Neu5Ac2en while in the L-xylose derived series the double
bond is in a regioisomeric position, equivalent to the
carbocyclic influenza drug GS 4104. Selective alkylations,
silylations and deprotection reactions of both series have
been demonstrated. We therefore have, after modification of
the allyl group, introduction of virtually any acylamide,
ester cleavage and aglycon attachment26 via a monoester,
access to an unlimited variety of inhibitors.
As described above, selective alkylation should proceed
silylation in the (2)-series. Fortunately, mono-allylation of
(2)-10 to give 23 was accomplished in good yield (Scheme
8), again the regioselectivity of the reaction was confirmed
by acetylation of the product to furnish 24. Although target
compound 27 is, of course, directly accessible from 23 or
24, we silylated 23 and obtained fully derivatized
cyclohexenephosphonate 25. Reduction and acetylation
gave acetamide 26 which was, under the conditions
described above for the (þ)-series, converted into the target
compound 27 and isolated as its triethylammonium salt.
2.6. Biological evaluation
We have tested a selected set of our cyclohexene-
phosphonates towards their ability to inhibit three com-
mercially available sialidases from the pathogenic bacteria
3.2. Sialidase inhibition
Both enantiomeric xylo-cyclohexenephosphonates (þ)-1

H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
7577
Table 1. Inhibition of three bacterial sialidases by a selected set of xylose-derived cyclohexenephosphonates and reference inhibitor Neu5Ac2en
Compound
Sialidase
V. cholerae
S. typhimurium
C. perfringens
3£1023
3£1024
2£1023
(.1022
)
n.t.
n.t.
n.i.
n.i.
n.i.
n.i.
2£1023
1£1023
3£1023
3£1023
3£1024
6£1024
4£1025
Neu5Ac2en
1024
IC50 values are given in M; n.i., non-inhibitory; n.t., not tested.
and (2)-1 inhibit the sialidases from V. cholerae and S.
typhimurium in the millimolar range, they can therefore be
regarded as useful scaffolds for the further development of
high affinity sialidase inhibitors. Only the L-xylo derived
compound (2)-1 showed activity towards the sialidase from
C. perfringens in the concentration range testet. It is,
however, in view of the lacking side chain too early to
speculate about the reason for this selectivity. More
importantly, the very weak respectively the lacking activity
of azide (2)-14 and the corresponding 4-hydroxy deriva-
tive12 indicate that the acetamide group is an important
recognition determinant, a well established fact in the field
of sialic acid biology.1 Another finding important for our
concept is the activity of monoethyl ester (þ)-13, thus
allowing attachment of aglyca. The importance of a
negative charge at this position is underlined by the fact
that the diethyl ester corresponding to 13 does not show any
inhibition (data not shown).
MS were recorded on a Kratos Analytic Kompact Maldi 2,
using DHB: 3,5-dihydroxybenzoic acid; HCCA:
a-hydroxy-a-cyano-cinnamic acid; or ATT: azidothymi-
dine as matrix. FAB-MS were recorded on a modified
Finnigan MAT 312/AMD-5000. Reactions were monitored
with plastic plates coated with silica gel 60F254. Solvents for
flash chromatography (EE: ethyl acetate, Tol: toluene) were
distilled before use. HPLC chromatography devices were
from Knauer GmbH, Berlin, Germany equipped with an
Shimadzu RI-detector, Shimadzu GmbH, Duisburg,
Germany. Chemicals: TIPSCl, triisopropylsilyl chloride;
TBDPSCl, tert-butyldiphenylsilyl chloride; TMSBr,
bromotrimethylsilane; TBAF, tetrabutylammonium
fluoride; TBAI, tetrabutylammonium iodide.
4.2. Compounds 2–9
D-Xylofuranose derivatives 2–9 were synthesized via the
same sequence of reactions previously reported for the
corresponding L-xylofuranose derivatives.14 Conditions and
yields are given in Scheme 3.
4. Experimental
4.1. General
4.2.1. Diethyl (3S,4S,5R )-4-azido-3,5-dihydroxy-1-cyclo-
hexenephosphonate (1)-10, diethyl (3S,4S,5R )-3,5-di-
acetoxy-4-azido-1-cyclohexenephosphonate (1)-11 and
diethyl (3S,4S,5R )-3,5-di-acetoxy-4-acetamido-1-cyclo-
hexenephosphonate (1)-12. Compounds (þ)-10, (þ)-11
and (þ)-12 were synthesized according to our previously
reported procedures,14 conditions and yields are given in
Scheme 3. NMR- and MS-data were identical to those
1H, 13C and 31P NMR spectra were recorded on a Bruker
DRX-600 and a Bruker AC-250 spectrometer, taking the
chemical shift of deuterated solvent as standard, except for
31P-spectra, where 85% phosphoric acid was used (0 ppm).
13C chemical shifts were deduced from heteronuclear
multiple quantum correlation (HMQC)-spectra. MALDI-

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H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
previously reported for the (2)-enantiomers. (þ)-10:
[a ]D20¼15 (c¼1, CHCl3). (þ)-11: [a ]2D0¼53 (c¼0.2,
CHCl3). (þ)-12: [a ]2D0¼60 (c¼1, CHCl3).
529.3) MALDI-MS (pos. mode, DHB): 568.5 (MþK)þ,
552.5 (MþNa)þ, 530.5 (MþH)þ.
4.2.5. Diethyl (3S,4S,5R)-5-acetoxy-4-azido-3-tert-butyl-
diphenylsilyloxy-1-cyclohexenephosphonate (16). Mono-
silylated compound (15) (20 mg, 38 mmol) is stirred over-
night in acetic anhydride/pyridine (1:1), concentrated to
dryness and the residue is chromatographed (EE) to give
(16) (20 mg, 92%). Rf¼0.75 (EE); [a ]2D0¼40 (c¼1, CHCl3);
1H NMR (600 MHz, CDCl3): d¼¼7.70–7.37 (2m, 10H,
4.2.2. Ammonium [ethyl (3S,4S,5R )-4-acetamido-3,5-
dihydroxy-1-cyclohexenephosphonate] (1)-13. Diethyl
ester (þ)-12 (18 mg, 46 mmol) is dissolved in dry EtOH
(3 mL) and a solution of sodium ethanoate in EtOH is added
(1 M, 50 mL). The mixture is stirred overnight, neutralized
with Amberlite IR-120 (Hþ-form), filtered and concentrated
in vacuo. The residue is dissolved in a solution of NaOH in
water (3 mL, 0.1 M), strirred for another 3 h, neutralized
with Amberlite IR-120 (Hþ-form) and the solvent is
evaporated. The residue is purified by gel filtration (Biogel
P2, 0.1 M NH4HCO3) and lyophilized to give (þ)-13
3
2C6H5), 6.25 (bd, 1H, H-2, J2,P¼21.4 Hz), 4.83 (ddd, 1H,
H-5, 3J5,4¼,10 Hz, 3J5,6¼,10, ,6 Hz), 4.17 (m, 1H, H-3),
3
4.03–3.97 (m, 4H, 2CH2CH3), 3.72 (dd, 1H, H-4, J¼8.2,
10.9 Hz), 2.73 (m, 1H, H-6a), 2.26 (m, 1H, H-6b), 2.10 (s,
3H, COCH3), 1.28–1.23 (2t, 6H, 2CH2CH3), 1.08 (s, 9H,
SiC(CH3)3). 13C NMR (150.9 MHz, CDCl3): d¼141.0
(C-2), 72.3 (C-3), 70.3 (C-5), 67.4 (C-4), 30.0 (C-6). 31P
NMR (242.9 MHz, CDCl3): d¼17.08 (s, 1P, P(O)(OEt)2).
C28H38N3O6PSi (M 571.4) MALDI-MS (pos. mode, DHB):
594.6 (MþNa)þ, 572.4 (MþH)þ.
1
(10 mg, 78%) as its ammonium salt. H NMR (600 MHz,
3
D2O): d¼6.12 (bd, 1H, H-2, J2,P¼19.6 Hz), 4.14 (m, 1H,
H-3), 4.15–3.62 (m, 4H, H-4, H-5, CH2CH3), 2.52 (m, 1H,
H-6a), 2.11 (m, 1H, H-6b), 1.95 (s, 3H, COCH3), 1.14–1.12
(m, 3H, CH2CH3). 13C NMR (150.9 MHz, D2O): d¼142.3
(C-2), 71.2 (C-3), 67.9 (C-5), 61.2 (CH2CH3), 58.1 (C-4),
33.8 (C-6). 31P NMR (242.9 MHz, D2O): d¼15.23 (s, 1P,
PO3Et2). C10H18NO6P (M 279.1) MALDI-MS (pos. mode,
DHB): 302.3 (MþNa)þ.
4.2.6. Diethyl (3S,4S,5R)-4-azido-5-(prop-20-enyloxy)-3-
tert-butyldiphenylsilyloxy-1-cyclohexenephosphonate
(17). Compound (15) (30 mg, 57 mmol) is dissolved in DMF
(2 mL), allyl bromide (26 mL, 285 mmol), Ag2O (66 mg,
285 mmol) and TBAI (5 mg) are added. The mixture is
stirred overnight at rt, filtered, diluted with saturated NH4Cl
solution (5 mL) and extracted with CH2Cl2 (3£5 mL). The
solvent is evaporated and the residue is chromatographed
(Tol/EE 1:1) to give (17) (15 mg, 45%). Rf¼0.70 (EE);
[a ]2D0¼32 (c¼1, CHCl3); 1H NMR (600 MHz, CDCl3):
d¼7.73–7.37 (2m, 10H, 2C6H5), 6.20 (bd, 1H, H-2,
3J2,P¼21.4 Hz), 5.90 (m, 1H, H-20), 5.30–5.16 (m, 2H,
H-30a, H-30b), 4.13–3.95 (m, 7H, H-3, H-10a, H-10b,
2CH2CH3), 3.60 (dd, 1H, H-4), 3.37 (m, 1H, H-5), 2.71 (m,
1H, H-6a), 2.18 (m, 1H, H-6b), 1.28–1.22(2t, 6H, 2CH2CH3),
1.10–1.02 (s, 9H, SiC(CH3)3). 13C NMR (150.9 MHz,
CDCl3): d¼141.3 (C-2)76.2 (C-5), 72.6 (C-3), 69.3 (C-4),
30.5 (C-6). 31P NMR (242.9 MHz, CDCl3): d¼17.73 (s, 1P,
P(O)(OEt)2). C29H40N3O5PSi (M 569.4) MALDI-MS (pos.
mode, DHB): 608.2 (MþK)þ, 592.2 (MþNa)þ.
4.2.3. Ammonium (3R,4R,5S)-4-azido-3,5-dihydroxy-1-
cyclohexenephosphonate ((2)-14) and ammonium
(3S,4S,5R )-4-azido-3,5-dihydroxy-1-cyclohexenephos-
phonate ((1)-14). Diethyl esters (2)-1012 or (þ)-10
(15 mg, 51 mmol) are dissolved in dry CHCl3 (3 mL) and
TMSBr (300 mL) is added. The mixture is stirred for 2 d at
rt, evaporated and the residue is taken up in water (3 mL),
stirred for additional 2 h and then lyophilized. Compounds
(2)-14 and (þ)-14 are purified by gel filtration (Biogel P2,
0.1 M NH4HCO3) to give the respective ammonium salts
1
(,10 mg, 80%). H NMR (600 MHz, D2O): d¼5.92 (bd,
3
1H, H-2, J2,P¼18.6 Hz), 4.08 (m, 1H, H-3), 3.68 (m, 1H,
H-5), 3.32 (dd, 1H, H-4, 3J¼9.5, 10.2 Hz), 2.60 (m, 1H, H-
6a), 2.14 (m, 1H, H-6b). 13C NMR (150.9 MHz, D2O):
1
d¼133.95 (C-1, J1,P¼168 Hz), 131.7 (C-2), 70.9 (C-3),
69.4 (C-4), 68.43 (C-5), 33.0 (C-6). 31P NMR (242.9 MHz,
D2O): d¼11.50 (s, 1P, PO232). C6H8N3O5P (M 235.2)
MALDI-MS (pos. mode, DHB): 257.2 (MþNa)þ, (neg.
mode, ATT): 234.2 (M2H)2.
4.2.7. Diethyl (3S,4S,5R )-4-azido-5-hydroxy-3-triiso-
propylsilyloxy-1-cyclohexenephosphonate (18). Cyclo-
hexenephosphonate (þ)-10 (55 mg, 0.19 mmol) is
dissolved in CH2Cl2 (5 mL), imidazole (129 mg,
1.9 mmol) and TIPSCl (204 mL, 0.95 mmol) are added
and the mixture is refluxed for 16 h. The solvent is
evaporated and the residue is chromatographed (Tol/EE
1:2) to yield (18) (43 mg, 51%). Excess starting material
(10 mg, 18%) can be recovered by elution with EE:MeOH
(10:1). Rf¼0.55 (EE); [a ]2D0¼39.5 (c¼1, CHCl3); 1H NMR
4.2.4. Diethyl (3S,4S,5R )-4-azido-5-hydroxy-3-tert-
butyldiphenylsilyloxy-1-cyclohexenephosphonate (15).
Cyclohexenephosphonate (þ)-10 (37 mg, 0.13 mmol) is
dissolved in CH2Cl2 (3 mL) and cooled to 08C. Imidazole
(35 mg, 0.65 mmol) and TBDPSCl (36 mL, 0.14 mmol) are
added, and the mixture is stirred for 1 h, another 1.1 equiv.
of TBDPSCl is added and, after additional stirring for
30 min the reaction is quenched by addition of saturated
NH4Cl solution (5 mL). The layers are separated, and the
aqueous layer is extracted twice with CH2Cl2 (5 mL). The
combined organic layers are dried (MgSO4), evaporated and
the residue is chromatographed (EE) to yield (15) (40 mg,
3
(250 MHz, CDCl3): d¼6.47 (bd, 1H, H-2, J2,P¼21.4 Hz),
4.32 (m, 1H, H-3), 4.13–4.00 (m, 4H, 2CH2CH3), 3.69 (m,
1H, H-5), 3.44 (dd, 1H, H-4, 3J¼9.5, 7.9 Hz), 2.66 (m, 2H,
H-6a, OH), 2.29 (m, 1H, H-6b), 1.33–1.27 (2t, 6H,
2CH2CH3), 1.22–0.99 (m, 19H, 3SiCH(CH3)2). C22H42N3-
O5PSi (M 447.4) MALDI-MS (pos. mode, DHB): 486.3
(MþK)þ, 470.3 (MþNa)þ.
1
58%). Rf¼0.58 (EE); [a ]2D0¼34 (c¼1, CHCl3); H NMR
(250 MHz, CDCl3): d¼7.73–7.33 (2m, 10H, 2C6H5), 6.18
(bd, 1H, H-2, 3J2,P¼21.5 Hz), 4.19 (m, 1H, H-3), 4.02–3.85
(m, 4H, 2CH2CH3), 3.66–3.48 (m, 2H, H-5, H-4), 2.60 (m,
1H, H-6a), 2.24 (m, 1H, H-6b), 1.21, 1.22 (2t, 6H,
2CH2CH3), 1.07 (s, 9H, SiC(CH3)3). C26H36N3O5PSi (M
4.2.8. Diethyl (3S,4S,5R )-5-acetoxy-4-azido-3-triiso-
propylsilyloxy-1-cyclohexenephosphonate (19). Mono-
silylated compound (18) (20 mg, 38 mmol) is stirred

H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
7579
overnight in acetic anhydride/pyridine (1:1), concentrated to
dryness and the residue is chromatographed (EE) to give
(19) (21 mg, 94%). Rf¼0.70 (EE); [a]2D0¼65 (c¼1, CHCl3);
1H NMR (600 MHz, CDCl3): d¼6.47 (bd, 1H, H-2,
3J2,P¼21.4 Hz), 4.96 (m, 1H, H-5), 4.31 (m, 1H, H-3),
indicates complete conversion of the ester moieties. The
solvent is evaporated, water (3 mL) is added, the mixture is
stirred for 2 h and lyophilized. The product is purified by
HPLC (acetonitrile/50 mM triethylammonium hydrogen
carbonate 95:5) on an Phenomenex C18, Aqua 5m column.
1H NMR (600 MHz, D2O): d¼6.15 (bd, 1H, H-2,
3J2,P¼21.5 Hz), 5.87 (m, 1H, H-20), 5.27–5.16 (m, 2H,
H-300a, H-30b), 4.19 (m, 1H, H-3), 4.14, 4.02 (2m, 2H, H-30a,
H-5), 3.15 (q, 12H, 2 N(CH2CH3)3), 2.79 (m, 1H, H-6a),
2.19 (m, 1H, H-6b), 2.01 (s, 3H, COCH3), 1.23 (t, 18H, 2
N(CH2CH3)3). 13C NMR (150.9 MHz, D2O): d¼134.3
(C-2), 75.7 (C-5), 71.3 (C-3), 57.3 (C-4), 31.6 (C-6). 31P
NMR (242.9 MHz, D2O): d¼12.49 (s, 1P, PO232).
C11H18NO6P (M 291.2) MALDI-MS (neg. mode, ATT):
291.1 (M2H)2.
3
4.10–4.07 (m, 4H, 2CH2CH3), 3.59 (dd, 1H, H-4, J¼9.5,
10.5 Hz), 2.79 (m, 1H, H-6a), 2.26 (m, 1H, H-6b), 2.13 (s,
3H, COCH3), 1.35–1.30 (2t, 6H, 2CH2CH3), 1.12–1.00 (m,
19H, 3SiCH(CH3)2). 13C NMR (150.9 MHz, CDCl3):
3
H-3 b), 3.83 (dd, 1H, H-4, J¼9.3, 10.4 Hz), 3.60 (m, 1H,
1
d¼142.3 (C-2), 125.7 (C-1, J1,P¼185 Hz), 72.0 (C-3),
70.3 (C-5), 67.9 (C-4), 30.1 (C-6). 31P NMR (242.9 MHz,
CDCl3): d¼17.29 (s, 1P, P(O)(OEt)2). C24H44N3O6PSi (M
489.4) MALDI-MS (pos. mode, DHB): 528.4 (MþK)þ,
512.3 (MþNa)þ.
4.2.9. Diethyl (3S,4S,5R )-4-azido-5-(prop-20-enyloxy)-3-
triisopropylsilyloxy-1-cyclohexenephosphonate
(20).
Mono-silylated compound (18) (56 mg, 0.125 mmol), allyl
bromide (106 mL, 1.25 mmol), Ag2O (290 mg, 1.25 mmol)
and TBAI (23 mg, 62.5 mmol) are suspended in CH2Cl2
(5 mL) and the mixture is stirred overnight at rt. After
filtration the solvent is evaporated and the residue is
chromatographed (Tol/EE 1:4) to give (20) (52 mg, 85%).
4.2.12. Diethyl (3R,4R,5S )-4-azido-5-hydroxy-3-(prop-
20-enyloxy)-1-cyclohexenephosphonate (23). Cyclo-
hexenephosphonate (2)-10 (98 mg, 0.34 mmol), allyl
bromide (32 mL, 0.38 mmol) and Ag2O (395 mg,
1.7 mmol) are suspended in CH2Cl2 (10 mL), the mixture
is cooled to 08C and TBAI (63 mg, 0.17 mmol) is added.
After 3 h of stirring EtOH (0.5 mL) is added, insoluble
material is filtered off, solvent is evaporated and the residue
is chromatographed (Tol/EE 1:2) to give mono-allylated
compound (23) (70 mg, 70%). Rf¼0.25 (Tol/EE 1:2);
1
Rf¼0.50 (Tol/EE 1:1); [a]2D0¼63 (c¼1, CHCl3); H NMR
3
(600 MHz, CDCl3): d¼6.42 (bd, 1H, H-2, J2,P¼21.4 Hz),
5.95 (m, 1H, H-20), 5.35–5.20 0(m, 2H, H-30a, H-30b), 4.22–
4.07 (m, 8H, H-3, H-10a, H-1 b, 2CH2CH3), 3.88 (m, 1H,
H-3), 3.82 (m, 1H, H-5), 3.50–3.46 (m, 2H, H-3, H-4), 2.77
(m, 1H, H-6a), 2.19 (m, 1H, H-6b), 1.35–1.30 (2t, 6H,
2CH2CH3), 1.16–1.03 (m, 19H, 3SiCH(CH3)2). 13C NMR
(150.9 MHz, CDCl3): d¼134.4 (C-2), 76.6 (C-5), 72.5
(C-3), 69.9 (C-4), 30.9 (C-6). 31P NMR (242.9 MHz,
CDCl3): d¼18.01 (s, 1P, P(O)(OEt)2). C25H46N3O5PSi (M
487.4) MALDI-MS (pos. mode, DHB): 526.5 (MþK)þ,
510.5 (MþNa)þ.
1
[a ]2D0¼240 (c¼1, CHCl3); H NMR (250 MHz, CDCl3):
d¼6.59 (bd, 1H, H-2, J2,P¼21.7 Hz), 5.93 (m, 1H, H-20),
3
5.34–5.20 (m, 2H, H-30a, H-30b), 4.23 (dd, 1H, H-10a),
4.14–3.95 (m, 6H, H-10b, H-3, 2CH2CH3), 3.66 (m, 1H,
H-5), 3.50 (dd, 1H, H-4, 3J¼8.4, 10.3 Hz), 2.83 (d, 1H, OH,
3JOH,5¼2.2 Hz), 2.67 (m, 1H, H-6a), 2.19 (m, 1H, H-6b),
1.30 (2t, 6H, 2CH2CH3). C13H22N3O5P (M 331.3) MALDI-
MS (pos. mode, DHB): 370.4 (MþK)þ, 354.4 (MþNa)þ.
4.2.10. Diethyl (3S,4S,5R )-4-acetamido-5-(prop-20-enyl-
oxy)-3-triisopropylsilyloxy-1-cyclohexenephosphonate
(21). Azide (20) (120 mg, 0.25 mmol) is dissolved in
pyridine/water (4:1), the solution is saturated with hydrogen
sulfide and stirred until TLC indicates the absence of
starting material. The solvent is evaporated and the residue
is taken up in pyridine/water (1:1, 5 mL), stirred for 3 h, and
concentrated to dryness. Flash chromatography (EE/MeOH
10:1) yields acetamide (21) (85 mg, 71%). Rf¼0.50 (EE);
[a]2D0¼29 (c¼1, CHCl3); 1H NMR (600 MHz, CDCl3):
4.2.13. Diethyl (3R,4R,5S )-5-acetoxy-4-azido-3-(prop-20-
enyloxy)-1-cyclohexenephosphonate (24). Compound
(23) (15 mg, 44 mmol) is stirred overnight in acetic
anhydride/pyridine (1:1), concentrated to dryness and the
residue is chromatographed (EE) to give (24) (21 mg, 94%).
Rf¼0.75 (EE/MeOH 10:1); [a ]2D0¼235 (c¼0.4, CHCl3); 1H
NMR (600 MHz, CDCl3): d¼6.60 (bd, 1H, H-2,
3J2,P¼21.7 Hz), 5.92 (m, 1H, H-20), 5.34–5.24 (m, 2H,
H-300a, H-30b), 4.96 (ddd, 1H, H-5), 4.22, 4.17 (2m, 2H,
H-1 a, H-10b), 4.10–4.05 (m, 4H, 2CH2CH3), 3.98 (m, 1H,
d¼6.50 (bd, 1H, H-2, J2,P¼21.5 Hz), 5.86 (m, 1H, H-20),
H-3), 3.68 (dd, 1H, H-4, J¼8.5, 11.1 Hz), 2.76 (m, 1H,
3
3
5.48 (bs, 1H, NH), 5.27–5.14 (m, 2H, H-30a, H-30b), 4.82
(m, 1H, H-3), 4.13–4.05 (m, 5H, H-10a, 2CH2CH3), 4.02–
3.95 (m, 2H, H-4, H-10b), 3.52 (m, 1H, H-4), 2.74 (m, 1H,
H-6a), 2.20 (m, 1H, H-6b), 1.98 (s, 3H, COCH3), 1.35–1.30
H-6a), 2.21 (m, 1H, H-6b), 2.13 (s, 3H, COCH3), 1.35–1.30
(2t, 6H, 2CH2CH3). 13C NMR (150.9 MHz, CDCl3):
d¼139.2 (C-2), 77.7 (C-3), 69.5 (C-5), 64.9 (C-4), 30.1
(C-6). 31P NMR (242.9 MHz, CDCl3): d¼17.10 (s, 1P,
P(O)(OEt)2). C15H14N3O6P (M 373.3) MALDI-MS (pos.
mode, DHB): 412.3 (MþK)þ, 396.4 (MþNa)þ, 374.4
(MþH)þ.
(2t, 6H, 2CH2CH3), 1.12–1.00 (m, 19H, 3SiCH(CH3)2). 13
C
NMR (150.9 MHz, CDCl3): d¼143.5 (C-2), 73.5 (C-5),
71.1 (C-3), 60.0 (C-4), 31.3 (C-6). 31P NMR (242.9 MHz,
CDCl3): d¼18.52 (s, 1P, P(O)(OEt)2). C27H50NO6PSi (M
503.4) MALDI-MS (pos. mode, DHB): 542.3 (MþK)þ,
526.3 (MþNa)þ.
4.2.14. Diethyl (3R,4R,5S )-4-azido-3-(prop-20-enyloxy)-
5-triisopropylsilyloxy-1-cyclohexenephosphonate (25).
Compound (23) (90 mg, 272 mmol) is dissolved in CH2Cl2
(5 mL), imidazole (185 mg, 2.72 mmol) and TIPSCl
(291 mL, 1.36 mmol) are added and the mixture is refluxed
for 12 h. Following evaporation of the solvent the residue is
chromatographed (Tol/EE 1:1) to give (25) (91 mg, 69%).
4.2.11. Triethylammonium (3S,4S,5R )-4-acetamido-3-
hydroxy-5-(prop-20-enyloxy)-1-cyclohexenephosphonate
(22). Diethyl ester (21) (25 mg, 50 mmol) is dissolved in
CHCl3 (3 mL), TMSBr is added (300 mL) and the mixture is
stirred until the NMR spectrum of an analytical sample
1
Rf¼0.75 (EE/MeOH 10:1); [a ]2D0¼214 (c¼1, CHCl3); H

7580
H. Streicher, C. Bohner / Tetrahedron 58 (2002) 7573–7581
NMR (600 MHz, CDCl3): d¼6.57 (bd, 1H, H-2,
3J2,P¼21.5 Hz), 5.93 (m, 1H, H-20), 5.34–5.20 (m, 2H,
H-30a, H-30b), 4.23–4.05 (m, 6H, H-10a, H-10b, 2CH2CH3),
3.88 (m, 1H, H-3), 3.82 (m, 1H, H-5), 3.47 (dd, 1H, H-4,
3J¼8.8, 9.9 Hz), 2.67 (m, 1H, H-6a), 2.21 (m, 1H, H-6b),
1.35–1.30 (2t, 6H, 2CH2CH3), 1.14–0.87 (m, 19H,
3SiCH(CH3)2). 13C NMR (150.9 MHz, CDCl3): d¼139.7
(C-2), 78.5 (C-3), 70.2 (C-5), 68.7 (C-4), 34.3 (C-6). 31P
NMR (242.9 MHz, CDCl3): d¼17.69 (s, 1P, P(O)(OEt)2).
C25H46N3O5PSi (M 487.4) MALDI-MS (pos. mode, DHB):
525.7 (MþK)þ, 509.8 (MþNa)þ.
4.3. Inhibition assay
The inhibitory potencies (IC50 values) of the different
inhibitors were determined in a spectrofluorimetric assay
using (4-methylumbelliferyl-)-a-D-N-acetylneuraminic
acid (4-MU-NANA) as substrate according to Schauer and
co-workers.25 The sialidases from S. typhimurium and C.
perfringens were from Sigma (Taufkirchen, Germany) and
the sialidase from V. chloreae was from Boehringer
Mannheim (Mannheim, Germany). 4-MU-NANA was
from Fluka (Buchs, Switzerland). The assay in brief:
Incubations were carried out in a final volume of 100 mL
containing 0.2 mU of sialidase (20 mL of a stock solution of
0.1 U in 10 mL of 0.1 M acetate buffer of pH 5.5, 0.5 mM
CaCl2, 0.01% (w/v) NaN3, 0.1 mg/mL bovine serum
albumine (Serva)), a final CaCl2 concentration of 0.5 mM,
0.1 M acetate buffer (pH 5.5), 20 mL of a solution of
inhibitor in water (50–0.5 mM) by finally (after 10 min of
incubation) adding 20 mL of 4-MU-NANA in water
(1 mM). Enzymatic reactions were stopped by rapidly
adding 900 mL of cold glycine buffer of pH 10 (0.06 M
NaCl, 0.042 M Na2CO3, 0.133 M glycine) and liberated
4-methylumbelliferone (MU) determined on Wallac Victor2
fluorescence spectrophotometer using a wavelength of
460 nm for emission and 365 nm for excitation. Blanks
were run without enzyme, controls were run without
inhibitor, benchmark inhibitor Neu5Ac2en15 (ammonium
salt) was included in every assay for comparison.
4.2.15. Diethyl (3R,4R,5S)-4-acetamido-3-(prop-20-enyl-
oxy)-5-triisopropylsilyloxy-1-cyclohexenephosphonate
(26). Azide (25) (30 mg, 0.06 mmol) is dissolved in
pyridine/water (4:1), the solution is saturated with hydrogen
sulfide and stirred until TLC indicates the absence of
starting material. The solvent is evaporated and the residue
is taken up in pyridine/water (1:1, 5 mL), stirred for 3 h, and
concentrated to dryness. Flash chromatography (EE/MeOH
10:1) yields acetamide (26) 24 mg, 81%). Rf¼0.3
(EE/MeOH 10:1); [a ]2D0¼221 (c¼1, CHCl3); 1H NMR
3
(600 MHz, CDCl3): d¼6.64 (bd, 1H, H-2, J2,P¼21.5 Hz),
5.87 (m, 1H, H-20), 5.48 (bs, 1H, NH), 5.29–5.17 (m, 2H,
H-30a, H-30b), 4.34 (m, 1H, H-3), 4.22–4.00 (m, 7H, H-5,
H-10a, H-10b, 2CH2CH3), 3.77 (m, 1H, H-4), 2.66 (m, 1H,
H-6a), 2.25 (m, 1H, H-6b), 1.99 (s, 3H, COCH3), 1.35–1.30
(2t, 6H, 2CH2CH3), 1.13–0.90 (m, 19H, 3SiCH(CH3)2). 13
C
NMR (150.9 MHz, CDCl3): d¼140.8 (C-2), 76.3 (C-3),
68.2 (C-5), 57.5 (C-4), 34.6 (C-6). 31P NMR (242.9 MHz,
CDCl3): d¼18.08 (s, 1P, P(O)(OEt)2). C27H50NO6PSi (M
503.4) MALDI-MS (pos. mode, DHB): 542.3 (MþK)þ,
526.3 (MþNa)þ.
Acknowledgments
The authors thank the Deutsche Forschungsgemeinschaft
and the University of Konstanz for financial support.
4.2.16. Triethylammonium (3R,4R,5S )-4-acetamido-5-
hydroxy-3-(prop-20-enyloxy)-1-cyclohexenephosphonate
(27). Diethyl ester (26) (15 mg, 30 mmol) is dissolved in
CHCl3 (2 mL), TMSBr is added (200 mL) and the mixture is
stirred until the NMR spectrum of an analytical sample
indicates complete conversion of the ester moieties. The
solvent is evaporated, water (3 mL) is added, the mixture is
stirred for 2 h and lyophilized. The product (27) is purified
by HPLC (acetonitrile/50 mM triethylammonium hydrogen
carbonate 95:5) on an Phenomenex C18, Aqua 5m column.
1H NMR (600 MHz, D2O): d¼6.17 (bd, 1H, H-2,
3J2,P¼19.6 Hz), 5.80 (m, 1H, H-20), 5.23–5.13 (m, 2H,
H-300a, H-30b), 4.10–4.06 (m, 2H, H-10a, H-3), 3.96 (dd, 1H,
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