Building a successful structural motif into sialylmimetics - Cyclohexenephosphonate monoesters as pseudo-sialosides with promising inhibitory properties

Article abstract of DOI:10.1016/j.bmc.2005.09.025

A variable synthesis of a new class of sialylmimetics which provides access to pseudo-sialosides containing the successful cyclohexene motif in the sialic acid mimicking part has been developed. The D- and L-xylo cyclohexenephosphonate scaffolds allow att

Full text of DOI:10.1016/j.bmc.2005.09.025

Bioorganic & Medicinal Chemistry 14 (2006) 1047–1057
Building a successful structural motif into
sialylmimetics—cyclohexenephosphonate monoesters as
pseudo-sialosides with promising inhibitory properties
b
Hansjorg Streichera,b, and Heike Busse
*
¨
aChemistry Department, University of Sussex, Brighton BN1 9QG, UK
bDepartment of Chemistry, University of Konstanz, D-78457 Konstanz, Germany
Received 12 April 2005; revised 2 September 2005; accepted 9 September 2005
Available online 17 October 2005
Dedicated, with respect and gratitude, to Professor Nathan Sharon on the occasion of his 80th birthday.
Abstract—A variable synthesis of a new class of sialylmimetics which provides access to pseudo-sialosides containing the successful
cyclohexene motif in the sialic acid mimicking part has been developed. The D- and L-xylo cyclohexenephosphonate scaffolds allow
attachment of selected aglycons or aglycon mimetics via mixed phosphonate diester strategies and some target compounds thus
synthesized displayed promising inhibitory properties when tested with parasitic or bacterial sialidases.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
This fact, the persistent influenza threat and newly
emerged targets such as parasitic trans-sialidases,6,7
paramyxoviral hemagglutinin-neuraminidases,7–9 and
other sialic acid modifying enzymes, prompted us to
search for an approach which allows the incorporation
of a successful structural motif, namely the cyclohexene
ring as a transition state mimetic of glycoside hydrolysis,
into more elaborated structures. The diabetes drug acar-
bose, containing the cyclohexene valienamine or the
anti-influenza drug and neuraminidase inhibitor GS-
4071, containing a L-xylo-configured cyclohexenecarb-
oxylic acid as the bioactive sialylmimetic, may serve as
examples (Fig. 1B).
Sialic acids are a family of naturally occurring deriva-
tives of 3-deoxy-D-glycero-D-galacto-2-nonulosonic
acids abundant on cell surfaces in higher animals
(Fig. 1A).1 At their terminal position at the non-reduc-
ing end of oligosaccharide chains in cell wall glycoconju-
gates, sialic acids represent a crucial first encounter and
receptor for attachment of exogenous, often pathogenic,
agents such as viruses, bacteria or parasites and their
respective lectins and toxins. In addition, important
endogenous events such as a cellÕs metastatic potential
are mediated by density as well as degree and kind of
modification of their cell surface sialic acids.2,3
Consequently, a variety of these interactions have been
targeted by small molecule inhibitor design and synthe-
sis, with varying success, leading to nearly nanomolar
inhibition of sialyltransferases or of the sialidase form
influenza virus A and B but yet failing, for instance, to
provide the same potency for sialic acid binding lectins
or other microbial sialidases.4,5
2. Results and discussion
2.1. Inhibitor design and retrosynthetic approach
Replacement of the carboxylate in the L-xylo-configured
cyclohexene scaffold of GS-4071 by a phosphonate leads
to L-xylo-configured cyclohexenephosphonates which al-
low attachment of aglycon mimetics or natural aglycons
of sialic acids such as galactoses thus leading to pseudo-
disaccharides containing a carbocyclic sialylmimetic
(Fig. 1C). In such cyclohexenephosphonate monoesters,
both the negative charge under physiological conditions
as well as the flattened half-chair conformation are
Keywords: Phosphonates; Sialic acid; Sialidases; Sialyl mimetics.
*
Corresponding author. Tel.: +44 1273 876678; fax: +44 1273
677196; e-mail: H.Streicher@sussex.ac.uk
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.025

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H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
Figure 1. (A) N-Acetylneuraminic acid (Neu5Ac, R1–R6 = H) and locations of modifications leading to the structural variety in the sialic acid family,
indicated by spheres and ellipsoids. (B) Drugs acarbose and GS-4071 and classical sialidase inhibitor DANA, which mimic carbohydrates by means
of a cyclohexene moiety. (C) Example for a pseudo-sialoside based on a xylo-configured cyclohexenephosphonate monoester.
retained, properties at least difficult to achieve if a
cyclohexenecarboxylic acid was to be functionalized. If
required, simple hydrogenation then leads to chair con-
formations in the carbocycle, thus significantly increas-
ing the number and type of accessible structures.
In our retrosynthetic approach, we deliberately included
both the GS-4071-like double bond orientation and the
orientation corresponding to the long-known transition-
state analogous sialidase inhibitor DANA in order to
ensure upmost variability (Scheme 1).
Starting from D- or L-xylose, respectively, the corre-
sponding xylo-configured cyclohexenephosphonates
with or without azide moiety at C-4 are obtained
via Horner–Wadsworth–Emmons type cyclization of
suitably modified xylofuranoses (Scheme 1).10 This
approach, previously reported by us for diethyl phos-
phonates,11,12 is extended to dimethyl and dibenzyl
phosphonates. These allow, following protection and
partial saponification, synthesis of mixed diesters con-
taining aglycon mimetics through alkylation or Mitsun-
obu esterification which, after deprotection, furnish
pseudo-sialosides such as 1 and 2 (Scheme 1).
2.2. Syntheses
We have previously reported the conversion of diethyl
phosphonates 3 and 4 to obtain cyclohexenephospho-
nates 5 and 6 carrying modified sidechains (Scheme
2).13 In order to obtain the corresponding monoethyl es-
ters, we partially saponified intermediates 7–9,13 with
sodium hydroxide solution and obtained, after neutral-
ization and purification on Biogel P2 columns, mono-
ethyl esters 10–12 in literally quantitative yield
(Scheme 2).
The synthesis of benzyl cyclohexenephosphonates started
from 3-azido-3-deoxy-1,2-O-isopropylidene-5-O-trifluo-
romethanesulfonyl-L-xylofuranose,11 which was reacted
with the tetrabenzyl methylenediphosphonate anion to
give the C-elongated xylofuranose diphosphonate 13
Scheme 1. Retrosynthetic analysis of L-xylo-configured pseudo-sialo-
side 1 mimicking Neu5Ac and D-xylo-configured pseudo-sialoside 2
mimicking KDN.

H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
1049
configured dimethyl cyclohexenephosphonate 24, which
was readily acetylated to yield 25. Thiophenol/triethyl-
amine treatment of 25 gave monomethyl ester 26, which
could both be alkylated with sugar triflate 20 to give
mixed diester 27h,l and be condensed with 1.2,3.4-di-
O-isopropylidene-a-D-galactopyranose under Mitsun-
obu conditions,15,16 to yield the same compound. This
reaction increases the range of accessible pseudo-sialo-
sides significantly. The selective benzyl protection at
C-4 in compounds 24–27 now offers selective access
for modifications of all three hydroxy groups with the
chemical differentiation of the hydroxy groups at posi-
tions 3 and 5 having been demonstrated by us previous-
ly. In this synthesis, however, the benzyl group had to be
selectively removed in presence of the double bond by
transfer hydrogenation with cyclohexadiene which, after
acetylation, gave 28h,l as an inseparable mixture of dia-
stereomers. The three-step deprotection via protected
monoesters 29 and 30 followed essentially the same
methodology as in the benzyl series. Thus, fully depro-
tected pseudo-sialoside 2, with the carbocycle being an
analogue of 3-deoxy-D-glycero-D-galacto-2-nonulosonic
acid (KDN) rather than of its 5-deoxy-5-acetamido
counterpart Neu5Ac (Scheme 4), was obtained.
Scheme 2. Synthesis of sidechain-functionalized cyclohexenephospho-
nates and their corresponding monoethyl esters. Reagents: (i) TMSBr/
CHCl3;11,12 (ii) NaOH (0.4 M); (iii) Biogel P2, NH4HCO3 (0.1 M).
2.3. Conformation of cyclohexenephosphonates
(Scheme 3). Removal of the isopropylidene group under
acidic conditions liberated the aldehyde, which was cyc-
lized with lithium bis(trimethylsilyl)amide as a base to
yield dibenzyl cyclohexenephosphonate 14, which was
readily converted to 15. Monobenzyl ester 16 was then
obtained by partial cleavage with thiophenol/triethyl-
amine, ready for alkylation with alkyl triflates. Deacety-
Sialidase inhibitor DANA (Fig. 1B) occupies a half-
chair conformation leaving the acetamido substituent
in a pseudo-equatorial position with the vicinal cou-
plings of the corresponding pseudo-axial proton (H-5)
being 8.9 and 10.9 Hz, respectively. This simulates the
situation around the equatorial acetamide in sialic acids
(10.4 and 10.9 Hz) (Fig. 1A).17 Whenever examined, we
find a similar setting for the C-4 substituent in D- and
L-xylo-configured cyclohexenephosphonates, for in-
stance in compound 17 (8.8 Hz, 10.8 Hz). Slightly lower
values (ꢀ7 Hz and ꢀ9 Hz) are typically found when the
compounds are protected (see Section 4). A pseudo-
equatorial position of the three substituents, especially
the acetamide or hydroxy group at C-4, can therefore
be assumed.
lation
and
purification
by
gel
permeation
chromatography yields 17, which was included in inhibi-
tion assays. Mitsunobu condensation of 16 with suitable
alcohols such as sugar hydroxy groups gave only margin-
al yields of desired mixed diesters. We therefore alkylated
monoester 16 with octyl triflate to give an inseparable
mixture of diastereomeric benzyl octyl phosphonates
18h,l. Selective removal of the benzyl group with thiophe-
nol/triethylamine followed by deacetylation and purifica-
tion gave octyl phosphonate 19 in high yield.
Introduction of a sugar moiety rather than a hydrophobic
group was achieved by reacting monoester 16 with
1.2,3.4-di-O-isopropylidene-5-O-trifluoromethanesulfo-
nyl-a-D-galactopyranoside 2014 as electrophile to give the
diastereomeric mixed diesters 21h,l in nearly quantitative
yield, based on consumed phosphonate. The sequence of
the final deprotection steps proved to be crucial, starting
with debenzylation to give the monoester, which was
deacetylated in the same step to furnish 22. Isopropylid-
ene cleavage with diluted trifluoroacetic acid gave fully
deprotected pseudo-sialoside 1 (Scheme 3).
3. Sialidase inhibition
In order to demonstrate the usefulness of the structural
concept, we have tested the inhibition of selected bacte-
rial sialidases and a trypanosomal sialidase by our
xylo-configured cyclohexenephosphonate monoesters.
Compounds 1, 17 and 19 all showed improved inhibi-
tion (0.2, 0.09 and 0.2 mM, respectively) of Salmonella
typhimurium sialidase when compared to the parent
cyclohexenephosphonate.12,13,18 Moreover, although
lacking an optimized side chain mimetic, monobenzyl
phosphonate 17 exceeds benchmark sialidase inhibitor
DANA in inhibitory potency by a factor of 4 and comes
close to the so far best inhibitor of the latter enzyme, iso-
carba-DANA (IC50 = 40 lM).19 In addition, these
monoesters display a significant preference for the S.
typhimurium enzyme, with the enzyme from Clostridium
perfringens, for instance, not being inhibited below
4 mM inhibitor concentration. Compound 2, which
The dimethyl phosphonate methodology for the synthe-
sis of cyclohexenephosphonate dimethyl esters was
introduced in the D-xylo series (Scheme 4). 3-O-Ben-
zyl-1,2-O-isopropylidene-D-xylofuranose-5-triflate11 was
substituted with the tetramethyl methylenediphospho-
nate anion to give xylofuranose diphosphonate 23.
Isopropylidene cleavage and cyclization gave D-xylo-

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H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
Scheme 3. Synthesis of pseudo-sialosides via dibenzyl phosphonates. Reagents: (i) CH2(PO3Bn2)2, LiN(SiMe3)2; (ii) H+/H2O; (iii) LiN(SiMe3)2;
(iv) PMe3/Ac2O; (v) H2/Pd/C; (vi) NaOH; (vii) C8H17OTf, DMF; (viii) H2/Pd/C; (ix) NH3/MeOH; (x) 20, DMF; (xi) H2/Pd/C then NH3/MeOH;
(xii) CF3COOH (50%).
lacks the acetamide and is therefore expected to be inac-
tive,12 will be tested for inhibition of KDNases in due
course (Table 1).
was inactive but inhibition, albeit moderate, was
found with unsubstituted monoethyl D-xylo-cyclo-
hexenephosphonate,12 and its derivative 11 (5.7 and
4.7 mM, respectively) carrying a hydroxyethyl moiety
as the glycerol side chain mimetic. Very little inhibi-
tion (<20% at 8 mM) was detected with the mixture
of diastereomers 10 and the 4-azido compound 12.
The trans-sialidase from Trypanosoma cruzi was tested
in a fluorescence-based trans-sialidase assay as recently
described by Schauer and co-workers20 Interestingly, 1

H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
1051
Scheme 4. Synthesis of pseudo-sialosides via dimethyl phosphonates. Reagents: (i) CH2(PO3Me2)2, LiN(SiMe3)2; (ii) H+/H2O; (iii) LiN(SiMe3)2; (iv)
Ac2O/pyridine; (v) PhSH/NEt3; (vi) 20, DMF; (vii) DIAD/Ph3P; (viii) C6H8/EtOH; (ix) Ac2O/pyridine; (x) PhSH/NEt3; (xi) NaOH; (xii) CF3COOH
(50%).
The inhibition data obtained with monoethyl esters
are highlighted by the fact that the parent
cyclohexenephosphonate without ethyl ester group,12
as well as the corresponding derivatives 5 and 6 with
side chain mimetics displayed no detectable inhibition
in the concentration range tested (Table 2).
It is, however, too early to propose a structure–activ-
ity relationship before more substituents as well as
both double bond regioisomers have been compared.
In conclusion, we have developed a synthetic ap-
proach towards carbocyclic sialylmimetics which al-
lows exploration and mimicking of the full
structural space displayed by sialoconjugates in phys-
iological recognition events. The methodology can be
applied in design and synthesis of inhibitors or inhib-
itor libraries of virtually any sialic acid binding lectin
or enzyme, some of which are currently being under
investigation in our laboratory.
Taking into account that it has been difficult to find
donor substrate analogous inhibitors for T. cruzi
trans-sialidase, with inhibitor DANA itself being only
a weak inhibitor (Ki > 14 mM),6,21 the data obtained
indicate a beneficial effect of the monoethyl phospho-
nate group and a possible entry to such inhibitors.

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H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
Table 1. Inhibition of bacterial sialidases by cyclohexenephosphonate
monoesters
to the residual proton resonance of the respective deu-
terated solvents, CDCl3 (7.24 ppm), D2O (4.63 ppm)
and D2O in CD3OD (4.88 ppm). For 31P NMR spectra
H3PO4 was used as external standard (0 ppm). In some
cases, 13C chemical shifts were deduced from heteronu-
clear multiple quantum correlation (HMQC) spectra.
In pseudo-disaccharidic systems, the cyclohexene ring
is indicated by the suffix ÔaÕ, the sugar by the suffix ÔbÕ.
In cases where diastereomeric mixtures of mixed diesters
were obtained, this is indicated by the suffixes h (higher
moving) and l (lower moving) but no attempts of sepa-
ration were made. HR-ESI-MS spectra were recorded
on a Bruker Daltonics Apex III in positive mode with
MeOH/H2O as solvent. MALDI-MS spectra were
recorded on a Bruker Biflex III spectrometer in positive,
linear mode with a delayed extraction MALDI source or
on a Kratos Analytic Kompact Maldi 2 using 3,5-
dihydroxybenzoic acid (DHB), a-hydroxy-a-cyano-cin-
namic acid (HCCA) or azidothymidine (ATT) as ma-
trix. Gel permeation chromatography was carried out
in the 1–5 mg scale on a XK 16/70 column (bed volume
130 mL), from Amersham packed with Biogel P2 fine
(particle size 45–90 lm) and 0.1 M NH4HCO3 as buffer.
Detection was achieved with a differential refractometer
from Knauer, Berlin, Germany.
Compound
S.t. sialidase C.p. sialidase
IC50 (mM)
IC50 (mM)
0.3
2
HO
AcHN
--
+
PO3
2NH4
ref.12
HO
1
17
0.2
0.09
0.2
0.4
n.i.
n.i.
n.i.
0.04
19
DANA
Inhibition of bacterial sialidases by cyclohexenephosphonate
monoesters.
S.t.: Salmonella typhimurium.
C.p.: Clostridium perfringens.
Values are means of three independent experiments with a deviation of
less than 20%. n.i.: non-inhibitory.
4. Experimental
4.1. General
Compounds 3–9 were prepared as described previous-
ly.11–13
Reaction solvents were purchased anhydrous and used
as received. Solvents for chromatography were distilled
before use. Reactions were monitored by TLC using
precoated silica gel 60 F254 plates. Compounds were
detected by UV absorption and/or by staining with a
molybdenum phosphate reagent (20 g ammonium
molybdate and 0.4 g cerium(IV) sulfate in 400 mL of
10% aq sulfuric acid) and subsequent heating at
120 °C for 5 min. Silica gel 60 M (particle size 40–
4.2. Ammonium [ethyl (3S,4S,5R)-4-acetamido-3-[(R,S)-
20,30-dihydroxypropyloxy]-5-hydroxy-1-cyclohexene-
phosphonate] (10h,l)
The diastereomeric mixture of protected diethylphosph-
onates 7h,l (6 mg, 11.8 lmol) was dissolved in dioxane
(1.5 mL) and NaOH solution (1.5 mL, 0.4 M) was add-
ed. The solution was stirred at room temperature for
24 h, diluted with H2O (2 mL) and neutralized with
Amberlite IR-120 (H+). The resin was filtered off, the
solvent was evaporated in vacuo and the residue was dis-
solved in 1 mL of aqueous NaHCO3-solution (0.1 M).
63 lm) from Macherey-Nagel, Duren, Germany, was
¨
1
used for flash chromatography. H, 13C and 31P NMR
spectra were recorded on a Bruker DRX 600 spectrom-
eter at 600, 150.9 and 242.9 MHz, respectively. Chemi-
1
cal shifts in H and 13C NMR spectra were referenced
Table 2. Comparison of cyclohexenephosphonate monoethyl esters (column 1), the corresponding free phosphonates (column 2) and more
elaborated monoalkyl esters (column 3) for inhibition of Trypanosomal cruzi trans-sialidase
R2O
R2O
HO
-
+
NH4
-
+
R3
AcHN
--
PO3 2NH4
+
AcHN
PO2
PO3Et
NH4
OR
R1O
R1O
HO
OH
OH
10: R1 = H, R2 = CH2-CH-CH2OH, R3 = NHAc
11: R1 = H, R2 = CH2-CH2OH, R3 = NHAc
12: R1 = H, R2 = H, R3 = N3
5: R1 = H, R2 = CH2-CH-CH2OH
6: R1 = H, R2 = CH2-CH2OH
ref12: R1 = H, R2 = H, R3 = NHAc
1: R = 6-Gal
17: R = benzyl
19: R = n-octyl
ref12: R1 = H, R2 = H, R3 = NHAc
10: IC20 = >8 mM
11: IC50 = 4.7 mM
12: IC20 = >8 mM
Ref. 12: IC50 = 5.7 mM
non-inhibitory in the concentration
range tested
non-inhibitory in the concentration
range tested
Inhibition of Trypanosoma cruzi TS by cyclohexenephosphonate monoesters. Values are means of three independent experiments with a deviation of
less than 20%.

H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
1053
Gel permeation chromatography on a Biogel P2 column
followed by lyophilization gave 10h,l (3.7 mg,
tetrabenzyl methylenediphosphonate) (15.8 mmol) in
dry DMF (30 mL). 100 lL of crown ether (15-crown-
5) was added, the mixture was stirred at room tempera-
ture for 4 h and the reaction was quenched by addition
of solid NH4Cl. The solvent was evaporated in vacuo,
the residue was dissolved in CH2Cl2 and extracted twice
with saturated NaCl-solution. The organic layer was
dried over MgSO4, filtered and evaporated. The residue
was chromatographed (toluene/EtOAc, 1:3) to give 13
(1.7 g, 2.31 mmol) in 24% yield. Rf = 0.14 (toluene/
EtOAc, 1:10). [a]D +5.3 (c 1, CHCl3). 1H NMR (CDCl3)
d 7.36–7.25 (m, 20H, 4C6H5) 5.80 (d, 1H, 3J1,2 = 3.7 Hz,
H-1) 5.13–5.01 (m, 8H, 4CH2Ph) 4.62 (d, 1H, H-2) 4.58
(m, 1H, H-4) 3.68 (d, 1H, H-3) 2.75 (m, 1H, H-6) 2.37
(m, 1H, H-5a) 2.16 (m, 1H, H-5b) 1.37, 1.26 (2s, 6H,
C(CH3)2). 13C NMR (CDCl3) d 104.2 (C-1) 83.9 (C-2)
77.2 (C-4) 66.6 (C-3), 34.1 (C-6), 26.5, 26.4 (C(CH3)2)
25.5 (C-5). 31P NMR (CDCl3) d 22.42, 22.05 (2s,
2PO3Bn2). C37H41N3O9P2 (M 733.23) MALDI-MS
(HCCA) 756 (M+Na)+ 772 (M+K)+.
1
10.6 mmol) in 90% yield. H NMR (D2O) d 6.04 (d,
3
3
1H, JH,P = 19.6 Hz, H-2), 4.10 (d, 1H, J3,4 = 11.2 Hz,
H-3), 3.67–3.35 (m, 10H), 2.61 (ddd, 1H, J = 17.6,
ꢀ8 Hz, ꢀ8 Hz, H-6a), 2.04 (m, 1H, H-6b), 1.88 (s, 3H,
COCH3), 1.07 (m, 3H, CH2CH3). 13C NMR (D2O) d
137.2 (C-2) 76.4 (C-5) 70.9 (C-3) 70.8–62.8 (C-10, C-20,
C-30) 61.9 (CH2CH3) 56.9 (C-4) 31.1 (C-6) 22.3
(COCH3). 31P NMR (D2O) d 15.88 (s, PO3Etꢁ).
C13H24NO8P (acid form) (M 353.3) MALDI-MS
(DHB) 376 (M+Na)+ 392 (M+K)+. HR-ESI-MS (m/z)
[M+Na]+ calcd for C13H24NNaO8P: 376.1131. Found:
376.1135.
4.3. Ammonium [ethyl (3S,4S,5R)-4-acetamido-3-
[20hydroxyethyloxy]-5-hydroxy-1-cyclohexenephospho-
nate] (11)
Diethyl cyclohexenephosphonate 8 (4 mg, 9.2 lmol) was
deprotected and purified as described for 7h,l. After
lyophilization, monoethyl ester 11 (2.5 mg, 7.8 lmol)
was obtained in 85% yield. 1H NMR (D2O) d 6.05
4.6. Dibenzyl (3R,4R,5S)-4-azido-3,5-dihydroxy-1-cyclo-
hexenephosphonate (14)
3
(d, 1H, JH,P = 19.5 Hz, H-2), 4.10 (d, 1H, H-3), 3.72
(dd, 1H, J = ꢀ9 Hz, ꢀ9 Hz, H-4), 3.67 (m, 2H,
CH2CH3), 3.55–3.50 (m, 5H, H-5, H10a, H-10b, H-20a,
H-20b), 2.59 (ddd, 1H, J = 17.4 Hz, ꢀ8.5 Hz, ꢀ8.5 Hz,
H-6a), 2.04 (m, 1H, H-6b), 1.88 (s, 3H, COCH3), 1.07
(t, 3H, CH2CH3). 13C NMR (D2O) d 137.3 (C-2) 76.1
(C-5) 71.0 (C-3) 70.9 (C-10), 61.5 (CH2CH3), 61.1 (C-
20), 31.3 (C-6), 22.3 (COCH3) 16.0 (CH2CH3). 31P
NMR (D2O) d 15.90 (s, PO3Etꢁ). C12H22NO7P (acid
form) (M 323.3) MALDI-MS (DHB) 346 (M+Na)+
362 (M+K)+. HR-ESI-MS (m/z) [M+Na]+ calcd for
C12H22NNaO7P: 346.1026. Found: 346.1035.
Bisphosphonate 13 (0.92 g, 1.25 mmol) was dissolved in
dioxane/5%H2SO4 (60 mL) and the solution was stirred
at 80 °C until TLC indicated the absence of starting
material (ca. 2 h). Following neutralization with saturat-
ed NaHCO3-solution the mixture was extracted with
CH2Cl2, the organic layer was dried over MgSO4, fil-
tered and evaporated. The residue was chromato-
graphed (EtOAc/toluene, 10:1, Rf = 0.5) to give the
furanose as a colourless oil (480 mg, 0.69 mmol) in
55% yield. The product was used in the subsequent cycli-
zation step without detailed characterization. The de-
isopropylidenated furanose obtained from 13 (130 mg,
0.19 mmol) was dissolved in dry dioxane (6 mL) and
cooled to 0 °C. Lithium bis(trimethylsilyl)amide in hex-
ane (1.5 M, 300 lL) was added and the solution was stir-
red for 4 h at room temperature. The reaction was
neutralized with Amberlite IR-120 (H+) and filtered.
The filtrate was concentrated in vacuo and the residue
was chromatographed (EtOAc/toluene, 1:5) to furnish
14 (50 mg, 0.12 mmol) as a colourless syrup in 64%
yield. Rf = 0.4 (EtOAc/toluene, 10:1). [a]D ꢁ26.8 (c 1,
CHCl3). 1H NMR (CDCl3) d 7.28–7.17 (m, 10H,
4.4. Ammonium [ethyl (3S,4S,5R)-4-azido-3,5-dihydroxy-
1-cyclohexenephosphonate] (12)
Diethyl 4-azido-cyclohexenephosphonate
9
(5 mg,
17.2 lmol) was deprotected and purified as described
for 7h,l. After lyophilization, monoethyl ester 12
(3.7 mg, 14.1 lmol) was obtained in 82% yield. 1H
3
NMR (D2O) d 6.02 (d, 1H, JH,P = 19.5 Hz, H-2), 4.08
3
(d, 1H, J3,4 ꢀ 10 Hz, H-3), 3.70–3.65 (m, 3H, H-5,
3
3
CH2CH3), 3.30 (dd, J3,4, J4,5 = 8.8 Hz, 10.6 Hz, H-4),
2.51 (m, 1H, J = 17.3 Hz, 6.9 Hz, ꢀ7.1 Hz, H-6a), 2.07
(m, 1H, H-6b), 1.91 (s, 3H, COCH3), 1.09 (m, 3H,
CH2CH3). 13C NMR (D2O) d 137.1 (C-2) 71.2 (C-5)
70.6 (C-4) 68.9 (C-5), 61.6 (CH2CH3), 33.6 (C-6), 22.0
(COCH3) 16.0 (CH2CH3). 31P NMR (D2O) d 15.53 (s,
PO3Etꢁ). C8H14N3O5P (acid form) (M 263.2) MAL-
DI-MS (DHB) 264 (M+H)+ 286 (M+Na)+ 302 (M+K)+.
3
4C6H5) 6.43 (d, 1H, J2,P = 21.8 Hz, H-2) 4.99–4.92
(m, 4H, 2CH2Ph) 4.05 (m, 1H, H-3) 3.51 (m, 1H, H-5)
3.25 (dd, 1H, H-4, 3J = 9.9 Hz, 3J = 8.1 Hz) 2.51 (m,
1H, H-6a) 2.05 (m, 1H, H-6b). 13C NMR (CDCl3) d
142.7 (C-2) 71.7 (C-3) 69.3 (C-4) 68.1 (C-5) 32.2 (C-6).
31P NMR (CDCl3) d 18.25 (s, PO3Bn2). C20H22N3O5P
(M 415.13) MALDI-MS (HCCA) 438 (M+Na)+ 454
(M+K)+.
4.5. Tetrabenzyl [3-azido-1,2-O-isopropylidene-3,5,6-tri-
deoxy-a-L-glucofuranos-6,60-diyl]bisphosphonate (13)
4.7. Dibenzyl (3R,4R,5S)-4-acetamido-3,5-di-acetoxy-1-
cyclohexenephosphonate (15)
To a solution of 3-azido-3-deoxy-1,2-O-isopropylidene-
5-O-trifluoromethanesulfonyl-a-L-xylofuranose12 (3.43 g,
9.88 mmol) in dry DMF (34 mL) under argon atmo-
sphere at 0 °C was added, with stirring, a freshly pre-
pared solution of the monosodium anion of
tetrabenzyl methylenediphosphonate22 (from NaH and
Compound 14 (70 mg, 0.17 mmol), dissolved in dry
THF (1 mL), was added dropwise to a solution of acetic
anhydride (64 lL, 0.67 mmol) and trimethyl phosphine
(1 mmol) in dry THF (2.5 mL). The mixture was stirred
at room temperature until TLC (EtOAc/toluene, 10:1)

1054
H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
indicated the absence of starting material (ca. 24 h). The
mixture was evaporated to dryness and the residue was
chromatographed (EtOAc/toluene, 10:1 ! EtOAc/
MeOH, 10:1) to give 15 (53 mg, 0.11 mmol) in 61%
yield. Rf = 0.21 (EtOAc/toluene, 10:1). [a]D ꢁ49.5 (c 1,
CHCl3). 1H NMR (CDCl3) d 7.38–7.26 (m, 10H,
added, the mixture was stirred at room temperature
for 12 h and another 20 mg (0.04 mmol in 0.4 mL dry
DMF) octyl triflate was added. After 12 h of stirring,
the solvent was evaporated in vacuo and the residue
was chromatographed (EtOAc/toluene, 10:1) to give
mixed diesters 18h,l (32 mg, 0.06 mmol) in 75% yield.
Unreacted starting material (monoester 16) could be
recovered by elution with EtOAc/MeOH, 4:1, 1%
NEt3. Rf = 0.28 (EtOAc/toluene, 10:1). 1H NMR
(CDCl3) d 7.40–7.36 (m, 5H, C6H5) 6.40, 6.34 (2d, 1H,
3J2,P = 21.4 Hz, H-2(h,l)) 5.48, 5.40 (2m, 1H, H-3(h,l))
5.09–5.00 (m, 3H, H-5, CH2Ph) 4.15 (m, 1H, H-4) 4.00
(m, 2H, H-10a, H-10b) 2.67 (m, 1H, H-6a) 2.24 (m,
1H, H-6b) 2.05, 2.03, 1.90 (3s, 9H, 3COCH3) 1.63 (m,
2H, H-20), 1.33–1.28 (m, 12H, H-30–H-70) 0.88 (s, 3H,
H-80). 13C NMR (CDCl3) d 72.6 (C-3) 69.5 (C-5) 67.7
(C-10) 53.3 (C-4) 31.1 (C-20) 30.9 (C-6) 22.5
(NHCOCH3) 20.4 (COCH3). 31P NMR (CDCl3) d
18.47, 18.45 (2s, 2PO3BnOct). C27H40NO8P (M
537.25) MALDI-MS (DHB) 560 (M+Na)+ 576
(M+K)+.
3
4C6H5) 6.38 (d, 1H, J2,P = 21.4 Hz, H-2) 5.49 (d, 1H,
3
3JNH,4 = 9.8 Hz, NH) 5.39 (d, 1H, J3,4 = 9.3 Hz, H-3)
5.06–5.00 (m, 4H, 2CH2Ph) 4.85 (m, 1H, H-5) 4.26
3
(ddd, 1H, J4,5 = ꢀ9 Hz, H-4) 2.59 (m, 1H, H-6a) 2.31
(m, 1H, H-6b). 13C NMR (CDCl3) d 139.6 (C-2) 71.8
(C-3) 68.7 (C-5) 53.3 (C-4) 31.1 (C-6). 31P NMR
(CDCl3) d 15.04 (s, PO3Bn2). C26H30NO8P (M 515.50)
MALDI-MS (HCCA) 539 (M+Na)+ 555 (M+K)+.
4.8. Triethylammonium [benzyl (3R,4R,5S)-4-acetamido-
3,5-di-acetoxy-1-cyclohexenephosphonate] (16)
Dibenzyl ester 15 (100 mg, 0.19 mmol) was treated with
thiophenol (139 lL, 1.36 mmol) and NEt3 (376 lL,
2.66 mmol) in 2.25 mL THF for 48 h. The solvent was
evaporated in vacuo and the residue was chromato-
graphed (MeOH/EtOAc, 1:4, 1% NEt3) to give 16
(100 mg) in nearly quantitative yield. Rf = 0.16 (EtOAc/
MeOH, 4:1). [a]D ꢁ36.8 (c 1, CHCl3). 1H NMR (CD3OD)
d 7.38–7.26 (m, 5H, C6H5) 6.21 (d, 1H, 3J2,P = 18.8 Hz, H-
2) 5.45 (1H, H-3) 4.96 (m, 1H, H-5) 4.82, 4.81 (2d, 2H,
CH2Ph) 4.16 (dd, 1H, H-4) 3.19 (q, 6H, N(CH2CH3)3)
2.76 (m, 1H, H-6a) 2.28 (m, 1H, H-6b) 2.02, 1.98, 1.87
(3s, 9H, 3COCH3) 1.29 (t, 9H, N(CH2CH3)3). 13C
NMR (CD3OD) d 134.0 (C-2) 73.3 (C-3) 70.4 (C-5) 67.2
(CH2Ph) 53.7 (C-4) 47.6 (N(CH2CH3)3) 31.9 (C-6). 31P
NMR (CDCl3) d 12.55 (s, PO3Bnꢁ). C19H24NO8P (free
acid) (M 425.37) MALDI-MS (DHB) 448 (M+Na)+
464 (M+K)+ 486 (M+Na+KꢁH)+.
4.11. Ammonium [1-octyl (3R,4R,5S)-4-acetamido-3,5-
dihydroxy-1-cyclohexenephosphonate] (19)
Mixed diester 18h,l (15 mg, 0.028 mmol) in MeOH
(2 mL) containing Pd/C (10%, 5 mg) was debenzylated
under a hydrogen atmosphere for 5 min (TLC-monitor-
ing). The mixture was filtered through Celite, concen-
trated ammonia was added (25%, 2 mL) and the
mixture was stirred overnight at room temperature. Fol-
lowing evaporation to dryness the residue was purified
on a Biogel P2 column (0.1 M NH4HCO3) and repeated-
ly lyophilized to give 19 (11 mg, 0.028 mmol) in literally
quantitative yield. 1H NMR (D2O) 6.09 (d, 1H,
3J2,P = 19.4 Hz, H-2) 4.12 (m, 1H, H-3) 3.73–3.56 (m,
4H, H-4, H-5, H-10a, H-10b) 2.54 (m, 1H, H-6a) 2.12
(m, 1H, H-6b) 1.93 (s, 3H, COCH3) 1.47 (m, 2H, H-
20), 1.36–1.13 (m, 12H, H-30–H-70) 0.72 (s, 3H, H-80).
13C NMR (CDCl3) d 136.8 (C-1) 70.2 (C-3) 69.0–57.5
(C-5, C-4, C-10) 32.7 (C-6) 30.5–21.4 (C-20–C-70) 21.5
(COCH3) 12.7 (C-80). 31P NMR (CDCl3) d 15.81 (s,
PO3BnOct). C16H30NO8P (M 363.39) MALDI-MS
(DHB) 364 (M+H)+ 386 (M+Na)+. HR-ESI-MS (m/z)
[M+Na]+ calcd for C12H30NaNO6P: 386.1702. Found:
386.1713.
4.9. Ammonium [benzyl (3R,4R,5S)-4-acetamido-3,5-di-
hydroxy-1-cyclohexenephosphonate] (17)
Monoester 16 (100 mg, 0.24 mmol) was dissolved in
H2O/dioxane (20 mL), concentrated ammonia (25%,
2 mL) was added and the mixture was stirred at room
temperature for 4 d. Following lyophilization inhibitor
17 was purified by gel permeation chromatography on
a Biogel P2 column (0.1 M NH4HCO3) to yield 17
(80 mg), in almost quantitative yield. [a]D ꢁ21.7 (c 1,
1
D2O). H NMR (CD3OD) d 7.38–7.23 (m, 5H, C6H5)
3
6.31 (d, 1H, J2,P = 19.2 Hz, H-2) 4.82, 4.81 (2d, 2H,
4.12. 1,2:3,4-Di-O-isopropylidene-6-O-trif-
luormethanesulfonyl-a-D-galactopyranose (20)
3
CH2Ph) 4.09 (d, 1H, H-3) 3.74 (dd, 1H, J = ꢀ9 Hz,
ꢀ10.5 Hz, H-4) 3.56 (m, 1H, H-5) 2.69 (m, 1H, H-6a)
2.20 (m, 1H, H-6b) 2.00 (s, 3H, COCH3). 13C NMR
(CD3OD) d 138.7 (C-2) 72.7 (C-3) 69.3 (C-5) 67.0
(CH2Ph) 60.2 (C-4) 35.3 (C-6). 31P NMR (CDCl3) d
14.68 (s, PO3Bnꢁ). C15H20NO6P (free acid) (M 341.27)
MALDI-MS (DHB) 364 (M+Na)+ 386 (M+2NaꢁH)+.
HR-ESI-MS (m/z) [M+Na]+ calcd for C15H20NNaO6P:
364.0920. Found: 364.0924.
Triflate 20 was synthesized according to literature
procedures.14
4.13. Benzyl 1,2:3,4-di-O-isopropylidene-a-D-galactopyr-
anos-6-yl [(3R,4R,5S)-4-acetamido-3,5-di-acetoxy-1-
cyclohexenephosphonate] (21h,l)
Monobenzyl ester 16 (39 mg, 0.074 mmol) and galactose
triflate 20 (95.3 mg, 0.24 mmol) were dissolved in dry
DMF (0.7 mL) and stirred for 12 h. More triflate
(39 mg, 0.074 mmol) was added and, after additional
12 h of stirring, the solvent was evaporated and the res-
idue was chromatographed (EtOAc/toluene, 10:1) to
4.10. Benzyl 1-octyl (3R,4R,5S)-4-acetamido-3,5-di-acet-
oxy-1-cyclohexenephosphonate (18h,l)
Monoester 16 (20 mg, 0.04 mmol) was dissolved in dry
DMF (0.4 mL), octyl triflate23 (42 mg, 0.16 mmol) was

H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
1055
give 21h,l (52 mg, 0.078 mmol) in 56% yield. Unreacted
monoester could be recovered by elution of the column
with MeOH/EtOAc, 1:4, 1%NEt3. Rf = 0.22 (EtOAc/tol-
PO3Rꢁ(a,b)). C14H24NO11P (acid form) (M 413.32)
MALDI-MS (DHB) 414.7 (M+H)+ 436.7 (M+Na)+
452.7 (M+K)+. FAB-MS (glycerol) 436 (M+Na)+.
HR-ESI-MS (m/z) [M+Na]+ calcd for C14H24NNaO11P:
436.0979. Found: 436.1047.
1
uene, 10:1). H NMR (CD3OD) d 7.43–7.36 (m, C6H5)
6.40 (2d, J2a,P = 21.5 Hz, H-2a(h,l)) 5.52–5.48 (m, 2H,
3
H-3a(h,l), H-1b) 5.14–5.06 (m, 2.5H, H-5a(h), CH2Ph)
4.97 (m, 0.5H, H-5a(l)) 4.64 (1H, H-3b) 4.38–4.36 (m,
1.5H, H-4a(l), H-2b) 4.25–4.03 (m, 4.5H, H-4a(h), H-
4b, H-5b, H-6b, H-6b0) 2.74 (m, 0.5H, H-6a(h)) 2.64
(m, 0.5H, H-6a(l)) 2.46 (m, 0.5H, H-6a0(l)) 2.27 (m,
0.5H, H-6a0(h)) 2.05, 2.00, 1.98 (3s, 9H, 3COCH3)
1.51–1.28 (12H, 2C(CH3)2). 13C NMR (CDCl3) d
140.5, 140.4 (C-2a(h,l)) 97.3 (C-1b) 72.6 (C-3a(h,l))
71.9 (C-3b) 71.7 (C-4b) 71.5 (C-2b) 70.0 (C-5a(l)) 69.4,
68.8 (C-5a(h), CH2Ph) 68.6 (C-5b) 67.4, 66.5 (C-
6b(h,l)) 53.2 (C-4a(h)) 52.9 (C-4a(l)) 30.7 (C-6a(h,l)).
31P NMR (CDCl3) d 19.64, 18.41 (2s, 2 PO3R2(h,l)).
C31H44NO13P (M 667.26) MALDI-MS (DHB) 690
(M+Na)+ 706 (M+K)+.
4.16. Tetramethyl [3-O-benzyl-5,6-dideoxy-1.2-O-isopro-
pylidene-a-D-glucofuranos-6,60-diyl]bisphosphonate (23)
To a solution of 3-O-benzyl-1,2-O-isopropylidene-5-O-
(450 mg,
trifluormethanesulfonyl-a-D-xylofuranose11
1.1 mmol) in dry DMF (7.5 mL) under argon atmo-
sphere at 0 °C was added, with stirring, a freshly pre-
pared solution of the monosodium anion of
tetramethyl methylenediphosphonate24 (from NaH and
tetramethyl methylenediphosphonate) (1.75 mmol) in
dry DMF (7.5 mL). The mixture was stirred at room
temperature for 4 h and the reaction was quenched by
addition of solid NH4Cl (1 g). The solvent was evaporat-
ed in vacuo, the residue suspended in EtOAc/MeOH
(10:1) and chromatographed to give 23 (290 mg,
0.59 mmol) in 54% yield. Rf = 0.59 (EtOAc/MeOH,
3:1). [a]D ꢁ21.5 (c 1, CHCl3). 1H NMR (CDCl3) d
4.14. Triethylammonium [1,2:3,4-di-O-isopropylidene-a-
D-galactopyranos-6-yl (3R,4R,5S)-4-acetamido-3,5-dihy-
droxy-1-cyclohexenephosphonate] (22)
3
7.30–7.19 (m, 5H, C6H5) 5.82 (d, 1H, J1,2 = 3.7 Hz,
The mixture of diesters 21h,l (20 mg, 0.03 mmol) was
dissolved in MeOH (4 mL) containing Pd/C (10%,
8 mg) and debenzylated under a hydrogen atmosphere
for 5 min (TLC-monitoring). The mixture was filtered
through Celite, concentrated ammonia was added
(25%, 2 mL) and the mixture was stirred overnight at
room temperature. Repeated lyophilization from diox-
H-1) 4.54 (d, 1H, H-2) 4.61 (d, CH2Ph) 4.44–4.42 (m,
2H, H-4, CH2Ph) 3.77–3.70 (m, 13H, H-3, 4OCH3)
2.61 (m, 1H, H-6) 2.36 (m, 1H, H-5a) 2.05 (m, 1H, H-
5b) 1.41, 1.24 (2s, 6H, C(CH3)2). 13C NMR (CDCl3) d
104.7 (C-1) 82.6 (C-2) 81.9 (C-3) 77.5 (C-4) 71.9
(CH2Ph), 53.6–53.1 (OCH3), 32.3 (C-6), 27.0 C(CH3)2
24.6 (C-5). 31P NMR (CDCl3) d 27.66, 27.65 (2s,
2PO3Me2). C20H32O10P2 (M 494.41) MALDI-MS
(DHB) 517 (M+Na)+.
1
ane/H2O gave 22 (17 mg, 0.03 mmol). H NMR (D2O)
3
d 6.13 (d, J2a,P = 19.7 Hz, H-2a) 5.54 (d, 1H, H-1b,
3J1b,2b = 4.8 Hz) 4.63 (1H, H-3b) 4.41 (1H, H-2b) 4.35
(1H, H-4b) 4.13 (m, 1H, H-3a) 3.97 (m, 1H, H-5b)
3.77–3.66 (m, 4H, H-4a, H-5a, H-6b, H-6b0) 2.54 (m,
1H, H-6a) 2.13 (m, 1H, H-6a0) 1.94 (s, 3H, COCH3)
1.46–1.34 (12H, 2C(CH3)2). 13C NMR (D2O) d 138.8
(C-2a) 97.0 (C-1b) 72.0 (C-3a) 71.4 (C-4b) 71.1 (C-2b)
68.9 (C-5a) 59.3 (C-4a) 34.5 (C-6a) 23.3 (COCH3). 31P
NMR (D2O) d 16.19 (s, PO3Rꢁ). C20H32NO11P (M
493.45) MALDI-MS (DHB) 515.8 (M+Na)+.
4.17. Dimethyl (3S,4S,5R)-4-benzyloxy-3,5-dihydroxy-1-
cyclohexenephosphonate (24)
To a solution of bisphosphonate 23 (600 mg, 1.21 mmol)
in dioxane/H2O (1:1, 40 mM) was added toluenesulfonic
acid monohydrate (100 mg) and the reaction mixture
was stirred at 60 °C for 4 d. Following neutralization
with NaHCO3 (s) the solvent was evaporated in vacuo
and the residue was chromatographed (EtOAc/MeOH,
3:1, Rf = 0.3–0.4) to give the furanose as a colourless
oil (490 mg, 1.08 mmol) in 93% yield. The product was
used in the subsequent cyclization step without detailed
characterization. The de-isopropylidenated furanose ob-
tained from 23 (140 mg, 0.31 mmol) was dissolved in dry
dioxane (5 mL), 32 mg sodium methoxide (s) was added
and the solution was stirred for 2 h at room tempera-
ture. Another 16 mg of NaOMe (s) was added and, fol-
lowing 2 h of stirring, the reaction was neutralized with
Amberlite IR-120 (H+) and filtered. The filtrate was con-
centrated in vacuo and the residue was chromato-
graphed (EtOAc/MeOH, 10:1) to furnish 24 (45 mg,
0.14 mmol) in 45% yield. Rf = 0.59 (EtOAc/MeOH,
3:1). [a]D ꢁ4.6 (c 1, CHCl3). 1H NMR (CDCl3) d
4.15. Ammonium [a,b-D-galactopyranos-6-yl (3R,4R,5S)-
4-acetamido-3,5-dihydroxy-1-cyclohexenephosphonate] (1)
A solution of 22 (5 mg, 9.8 lmol) in trifluoroacetic acid
(50%) was stirred for 2 h at room temperature and
immediately lyophilized. Purification on a Biogel P2 col-
umn (0.1 M NH4HCO3) gave, after lyophilization, pseu-
1
do-sialoside 1 in 71% yield (3 mg, 7 lmol). H NMR
3
(D2O) d 6.12 (d, 1H, J2a,P = 19.7 Hz, H-2a) 5.12 (d,
0.4H, H-1b(a), 3J1ba,2ba = 3.8 Hz) 4.45 (d, 0.6H, H-
1b(b), 3J1bb,2bb = 7.9 Hz) 4.12 (m, 1H, H-3a) 4.04
(0.4H, H-5b(a)) 3.87 (0.4H, H-4b(a)) 3.83 (0.6H, H-
4b(b)) 3.77–3.66 (m, 6.4H) 3.50 (0.6H, H-3b(b)) 3.34
(dd, 0.6H, H-2b(b)) 2.54 (m, 1H, H-6a) 2.13 (m, 1H,
H-6a0) 1.92 (s, 3H, COCH3). 13C NMR (D2O) d 139.0
(C-2a) 97.6 (C-1b(b)) 93.5 (C-1b(a)) 74.9 (C-4a) 73.8
(C-3b(b)) 72.9 (C-2b(b)) 71.9 (C-3a) 70.5 (C-5b(a))
70.2 (C-4b(a),C-3b(a)) 69.5 (C-4b(b)) 68.9 (C-5a, C-
2b(a)) 64.6, 64.3 (C-6b(a,b)) 59.2 (C-5b(b)) 34.3 (C-6a)
23.5 (COCH3). 31P NMR (D2O) d 16.28, 16.24 (2s,
3
7.38–7.30 (m, 5H, C6H5) 6.59 (d, 1H, J2,P = 21.7 Hz,
H-2) 4.93, 4.78 (2d, 2H, CH2Ph) 4.33 (br d, 1H, H-3)
3
3.91 (ddd, 1H, J5,6a,b = ꢀ6 Hz, ꢀ9 Hz, H-5) 3.75–3.71
3
(m, 6H, 2OCH3) 3.51 (dd, 1H, J4,3 = 6.6 Hz,
3J4,5 = 8.9 Hz, H-4) 2.63 (m, 1H, H-6a) 2.24 (m, 1H,
H-6b). 13C NMR (CDCl3) d 143.7 (C-2) 83.4 (C-4)

1056
H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
74.3 (CH2Ph) 71.7 (C-3) 68.3 (C-5) 52.9 (OCH3), 31.6
(C-6). 31P NMR (CDCl3) d 22.09 (s, PO3Me2).
C15H21O6P (M 328.3) MALDI-MS (DHB) 329
(M+H)+ 351 (M+Na)+ 367 (M+K)+.
ilization. The acid (25 mg, 0.063 mmol) was dissolved in
dry THF (1 mL), 1,2:3,4-di-O isopropylidene-a-D-galac-
topyranose21 (33 mg, 0.126 mmol), triphenyl phosphine
(33 mg, 0.126 mmol) and diisopropyl azodicarboxylate
(25 lL, 0.126 mmol) were added and the mixture was
stirred at 70 °C overnight. Evaporation of the solvent
4.18. Dimethyl (3S,4S,5R)-4-benzyloxy-3,5-di-acetoxy-1-
cyclohexenephosphonate (25)
followed
by
chromatography
(toluene/EtOAc,
1:1 ! 1:10) gave 27h,l (23 mg, 0.036 mmol) in 57% yield.
Compound 24 (68 mg, 0.21 mmol) was acetylated in
pyridine/acetic anhydride (2:1, 2 mL) overnight and
the solvent was evaporated in vacuo. Chromatography
of the residue yielded 25 (85 mg, 0.21 mmol) in quantita-
tive yield. Rf = 0.62 (EtOAc/MeOH, 10:1). [a]D +40.1 (c
1, CHCl3). 1H NMR (CDCl3) d 7.35–7.25 (m, 5H, C6H5)
Rf = 0.18 (toluene/EtOAc, 1:1). 1H NMR (CDCl3) d
7.36–7.27 (m, C6H5) 6.48 (d, J2a,P = 21.2 Hz, H-2a)
3
5.54–5.53 (m, 2H, H-3a, H-1b) 5.16 (m, 1H, H-5a)
4.72–4.67 (m, 2H, CH2Ph) 4.61 (m, 1H, H-3b) 4.31
(1H, H-2b) 4.22 (dd, 1H, H-4b) 4.18–4.15 (m, 2H, H-
6b, H-6b0) 4.03 (m, 1H, H-5b) 3.78 (dd, 1H, H-4a)
3.76–3.74 (m, 3H, OCH3) 2.80 (m, 1H, H-6a) 2.34 (m,
1H, H-6a0)2.04–2.00 (6H, 2COCH3) 1.55–1.32 (12H,
2C(CH3)2). 13C NMR (CDCl3) d 138.6 (C-2a) 96.7 (C-
1b) 78.1 (C-4a) 74.3 (CH2Ph) 72.6 (C-3a) 70.9 (C-4b,
C-3b) 70.6 (C-2b) 70.1 (C-5a) 67.4 (C-5b) 65.6 (C-6b)
53.0 (OCH3) 29.6 (C-6a). 31P NMR (CDCl3) d 20.28,
19.58 (2s, 2PO3R2(h,l)). C30H41O13P (M 640.63) MAL-
DI-MS (DHB) 663 (M+Na)+ 679 (M+K)+.
3
6.46 (d, 1H, J2,P = 21.2 Hz, H-2) 5.51 (1H, H-3) 5.14
3
(ddd, 1H, J5,6a,b = ꢀ8.5 Hz, ꢀ6 Hz, H-5) 4.72, 4.68
3
(2d, 2H, CH2Ph) 3.80 (dd, 1H, J4,3 = 6.2 Hz,
3J4,5 = 8.9 Hz, H-4) 3.74–3.69 (m, 6H, 2OCH3) 2.77
(m, 1H, H-6a) 2.30 (m, 1H, H-6b). 13C NMR (CDCl3)
d 138.4 (C-2) 77.4 (C-4) 74.0 (CH2Ph) 71.8 (C-3) 69.6
(C-5) 52.6 (OCH3) 29.1 (C-6). 31P NMR (CDCl3) d
20.77 (s, PO3Me2). C19H25O8P (M 412.38) MALDI-
MS (DHB) 435 (M+Na)+ 451 (M+K)+.
4.21. Methyl 1,2:3,4-di-O-isopropylidene-a-D-galactopyr-
anos-6-yl [(3S,4S,5R)-3,4,5-tri-acetoxy-1-cyclo-
hexenephosphonate] (28h,l)
4.19. Triethylammonium [methyl (3S,4S,5R)-4-benzyl-
oxy-3,5-di-acetoxy-1-cyclohexenephosphonate] (26)
Dimethyl ester 25 (84 mg, 0.2 mmol) in THF (2 mL) was
treated with thiophenol (168 lL) and triethylamine
(420 lL) for 16 h at room temperature. Following evap-
oration of the solvent the residue was chromatographed
(EtOAc/MeOH, 1:1, 1% NEt3) to give 26 (90 mg,
0.18 mmol) in 90% yield. Rf = 0.4 (EtOAc/MeOH, 1:1,
Benzyl ether 27h,l (24 mg, 37,5 lmol) was hydrogenated
with cyclohexadiene (0.75 mL) and Pd/C (10 mg) in
EtOH (1.5 mL) for 3 d at 55 °C. The mixture was filtered
through Celite, the solvent was evaporated in vacuo and
the residue was acetylated in pyridine/acetic anhydride
(2:1) overnight. Following evaporation of the solvent
the residue was chromatographed (toluene/EtOAc, 1:4)
to give 28h,l (22 mg, 37 lmol) in 98% yield. Rf = 0.12
(toluene/EtOAc, 1:1). 1H NMR (CDCl3) d 6.48 (d,
3J2a,P = 21.2 Hz, H-2a) 5.58 (1H, H-3a) 5.53 (d, 1H,
1
1% NEt3). [a]D +47.9 (c 1, MeOH). H NMR (CD3OD)
3
d 7.34–7.25 (m, 5H, C6H5) 6.17 (d, 1H, J2,P = 18.7 Hz,
3
H-2) 5.48 (m, 1H, H-3) 5.11 (ddd, 1H, J5,6a,b = ꢀ6 Hz,
ꢀ9 Hz, H-5) 4.73, 4.68 (2d, 2H, CH2Ph) 3.84 (dd, 1H,
3
3
3J4,3 = 7.0 Hz, J4,5 = 9.6 Hz, H-4) 3.48 (d, 3H,
H-1b, J1b,2b = 5 Hz) 5.28 (m, 1H, H-4a) 5.17 (m, 1H,
3JP,H = 7.1 Hz, OCH3) 3.17 (q, 6H, N(CH2CH3)3) 2.77
(m, 1H, H-6a) 2.35 (m, 1H, H-6b) 2.02, 1.98 (2s, 6H,
2COCH3) 1.29 (t, 9H, N(CH2CH3)3). 13C NMR
(CD3OD) d 133.3 (C-2) 79.7 (C-4) 74.9 (CH2Ph) 74.2
(C-3) 71.7 (C-5) 51.7 (OCH3) 47.9 (N(CH2CH3)3) 31.3
(C-6) 21. 31P NMR (CDCl3) d 14.44 (s, PO3Meꢁ).
C18H23O8P (free acid) (M 398.35) MALDI-MS (DHB)
421 (M+Na)+ 443 (M+2NaꢁH)+.
H-5a) 4.60 (m, 1H, H-3b) 4.32 (1H, H-2b) 4.22 (dd,
1H, H-4b) 4.18–4.15 (m, 2H, H-6b, H-6b0) 4.01 (m,
1H, H-5b) 3.76–3.74 (m, 3H, OCH3) 2.83 (m, 1H, H-
6a) 2.41 (m, 1H, H-6a0) 2.08–2.00 (9H, 3COCH3)
1.54–1.31 (12H, 2C(CH3)2). 13C NMR (CDCl3) d
138.6 (C-2a) 96.4 (C-1b) 71.7 (C-4a) 71.5 (C-3a) 70.9
(C-3b) 70.8 (C-4b) 70.6 (C-2b) 68.3 (C-5a) 67.2 (C-5b)
64.9 (C-6b) 52.6 (OCH3) 29.9 (C-6a). 31P NMR (CDCl3)
d 19.66, 18.91 (2s, 2PO3R2(h,l)). C25H37O14P (M 592.54)
MALDI-MS (DHB) 593 (M+H)+ 615 (M+Na)+ 631
(M+K)+.
4.20. Methyl 1,2:3,4-di-O-isopropylidene-a-D-galactopyr-
anos-6-yl [(3S,4S,5R)-3-benzyloxy-3,5-di-acetoxy-1-
cyclohexenephosphonate] (27h,l)
4.22. Triethylammonium [1,2:3,4-di-O-isopropylidene-a-
D-galactopyranos-6-yl (3S,4S,5R)-3,4,5-tri-acetoxy-1-
cyclohexenephosphonate] (29)
Via alkylation: monoester 26 (45 mg, 0.11 mmol) and tri-
flate 20 (140 mg, 0.38 mmol) were stirred in 1 mL of dry
DMF for 16 h. The solvent was evaporated in vacuo and
the residue was chromatographed (toluene/EtOAc, 1:4)
to give mixed diester (35 mg, 0.055 mmol) in 50% yield.
Unreacted monoester could be quantitatively reisolated
by eluting the column with EtOAc/MeOH (1:1, 1%
NEt3). The 31P NMR spectra indicate the formation of
both diastereomers in comparable amounts. Via Mitsun-
obu condensation: triethylammonium salt 26 was con-
verted into the free acid by treatment with Amberlite
IR-120 (H+) in dioxane/H2O (1:1), filtration and lyoph-
Compound 28h,l (25 mg, 42.2 lmol) was demethylated
by stirring with thiophenol (100 lL) and triethylamine
(250 lL) in THF (1 mL) for 3 d at room temperature.
Following evaporation to dryness the residue was chro-
matographed (EtOAc/MeOH, 1:1, 1% NEt3) to give
monoester 29 (24 mg, 35 lmol) in 80% yield. Rf = 0.5
1
(EtOAc/MeOH, 1:1, 1% NEt3). H NMR (D2O) d 6.07
3
(d, J2a,P = 19.1 Hz, H-2a) 5.53 (1H, H-3a) 5.52 (d,
1H, H-1b) 5.16 (m, 1H, H-4a) 5.15 (m, 1H, H-5a) 4.66

H. Streicher, H. Busse / Bioorg. Med. Chem. 14 (2006) 1047–1057
1057
(m, 1H, H-3b) 4.40 (1H, H-2b) 4.32 (m, 1H, H-4b) 3.94
(m, 1H, H-5b) 3.77–3.68 (m, 2H, H-6b, H-6b0) 3.06 (q,
6H, N(CH2CH3)3) 2.68 (m, 1H, H-6a) 2.34 (m, 1H, H-
6a0) 1.99–1.94 (9H, 3COCH3) 1.44–1.25 (12H,
2C(CH3)2) 1.13 (t, 9H, N(CH2CH3)3). 13C NMR
(D2O) d 132.2 (C-2a) 95.1 (C-1b) 72.3 (C-4a) 71.4 (C-
3a) 69.6 (C-4b) 69.2 (C-3b, C-2b) 68.5 (C-5a) 66.4 (C-
5b) 62.4 (C-6b) 45.9 (N(CH2CH3)) 29.2 (C-6a). 31P
NMR (D2O) d 14.56 (s, PO3Rꢁ). C24H35O14P (acid
form) (M 578.52) MALDI-MS (HCCA) 579 (M+H)+
601 (M+Na)+ 617 (M+K)+ 623 (M+2NaꢁH)+.
395.0783. [M+2NaꢁH]+ calcd for C12H20Na2O11P:
417.0533. Found: 417.0573.
Acknowledgments
We thank the Deutsche Forschungsgemeinschaft for
financial support. We are indebted to Professor R.
Schauer and Dr. S. Schrader, University of Kiel, for
making the TcTS assay available to us, and to Dr. C.
Ro¨hrig and Dr. A. Abdul-Sada for recording the mass
spectra.
4.23. Triethylammonium [1,2:3,4-di-O-isopropylidene-a-
D-galactopyranos-6-yl (3S,4S,5R)-3,4,5-trihydroxy-1-
cyclohexenephosphonate] (30)
References and notes
1. Schauer, R.; Kamerling, J. P. In Glycoproteins II; Mon-
treuil, J., Vliegenthardt, J. F. G., Schachter, H., Eds.;
Elsevier: Amsterdam, 1997, pp 241–400.
2. Angata, T.; Varki, A. Chem. Rev. 2002, 102, 439.
3. Schauer, R. Glycoconj. J. 2000, 17, 485.
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3, 149.
Compound 29 (10 mg, 14.7 lmol) was deacetylated by
stirring in aqueous ammonia (10%) for 3 h and repeated
lyophilization from NH4HCO3 (0.1 M). Yield: 90%
(6 mg, 13.2 lmol). 1H NMR (D2O)
d 6.06 (d,
(d, 1H, H-1b,
3J2a,P = 19.6 Hz,
H-2a) 5.51
3J1b,2b = 5.0 Hz) 4.65 (m, 1H, H-3b) 4.40 (1H, H-2b)
4.32 (br d, 1H, J4b,5b = 8.2 Hz, H-4b) 4.07 (m, 1H, H-
3a) 3.95 (m, 1H, H-5b) 3.77–3.67 (m, 2H, H-6b, H-
3
3
6b0) 3.62 (m, 1H, H-5a) 3.34 (m, 1H, J4a,3a 8.2 Hz,
3J4a,5a = 10.2 Hz, = H-4a) 2.51 (m, 1H, H-6a) 2.04 (m,
1H, H-6a0) 1.45–1.26 (12H, 2C(CH3)2). 13C NMR
(D2O) d 137.3 (C-2a) 95.1 (C-1b) 76.2 (C-4a) 71.9 (C-
3a) 69.7 (C-4b) 69.4 (C-3b, C-2b) 68.5 (C-5a) 66.9 (C-
5b) 62.3 (C-6b) 32.3 (C-6a). 31P NMR (D2O) d 16.35
(s, PO3Rꢁ). C18H29O11P (acid form) (M 452.40) MAL-
DI-MS (HCCA) 453 (M+H)+ 475 (M+Na)+ 491
(M+K)+ 497 (M+2NaꢁH)+ 513 (M+Na+KꢁH)+.
8. Crennell, S.; Takimoto, T.; Portner, A.; Taylor, G. Nat.
Struct. Biol. 2000, 7, 1068.
9. Alymova, I. V.; Taylor, G.; Takimoto, T.; Lin, T.-H.;
Chand, P.; Babu, Y. S.; Li, C.; Xiong, X.; Portner, A.
Antimicrob. Agents Chemother. 2004, 48, 1495.
10. Streicher, H. Monatsh. Chem.—Chem. Mon. 2002, 133,
1263.
4.24. Ammonium [a,b-D-galactopyranos-6-yl (3S,4S,5R)-
3,4,5-trihydroxy-1-cyclohexenephosphonate] (2)
11. Streicher, H.; Meisch, J.; Bohner, C. Tetrahedron 2001, 57,
8851.
12. Streicher, H.; Bohner, C. Tetrahedron 2002, 58, 7573.
13. Streicher, H. Bioorg. Med. Chem. Lett. 2004, 14, 361.
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2001, 20, 31.
15. Campbell, D. A.; Bermak, J. C. J. Org. Chem. 1994, 59,
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16. Borodkin, V. S.; Milne, F. C.; Ferguson, M. A. J.;
Nikolaev, A. V. Tetrahedron Lett. 2002, 43, 7821.
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son, S. B. Anal. Biochem. 1977, 94, 287.
A solution of 30 (5 mg, 10.6 lmol) in aqueous trifluoro-
acetic acid (50%) was stirred for 3 h and lyophilized. The
product was purified by gel permeation chromatography
on a Biogel P2 column (0.1 M NH4HCO3) and subse-
quently lyophilized to give 2 in 72% yield (3 mg,
7.7 lmol). 1H NMR (D2O)
d
6.05 (d, 1H,
3
3J2a,P = 19.7 Hz, H-2a) 5.10 (d, 0.3H, H-1b(a), J1ba,2-
3
ba = 3.7 Hz) 4.44 (d, 0.7H, H-1b(b), J1bb,2bb = 7.9 Hz)
4.08 (m, 1H, H-3b(a,b)) 4.02 (0.3H, H-5b(a)) 3.87
(0.3H, H-4b(a)) 3.81 (0.7H, H-4b(b)) 3.75–3.64 (m,
3.3H), 3.50 (0.7H, H-3b(b)) 3.34–3.33 (m, 1.7H, H-
4b(a,b), H-2b(b)) 2.52 (m, 1H, H-6a) 2.03 (m, 1H, H-
6a0). 13C NMR (D2O) d 139.2 (C-2a) 97.5 (C-1b(b))
93.6 (C-1b(a)) 77.9, 75.0, 73.7, 73.1, 70.4, 70.2, 69.6
(C-3a, -4a, -5a, -2b, -3b, -4b, -5b(a,b)) 64.6 (C-6b(a,b))
34.3 (C-6a). 31P NMR (D2O) d 16.38, 16.35 (2s,
PO3Rꢁ(a,b)). C12H21O11P (acid form) (M 372.27)
MALDI-MS (DHB) 395 (M+Na)+. HR-ESI-MS (m/z)
[M+Na]+ calcd for C12H21NaO11P: 395.0713. Found:
19. Vorwerk, S.; Vasella, A. Angew Chem. 1998, 110, 1765; .
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