New potent sialyltransferase inhibitors - Synthesis of donor and of transition-state analogues of sialyl donor CMP-Neu5Ac

Article abstract of DOI:10.1002/(SICI)1521-3765(19980615)4:6<1106::AID-CHEM1106>3.0.CO;2-7

Enzymatic sialyl transfer with CMP-Neu5Ac as donor can be inhibited by CDP. Therefore phosphonates 1a,b, 2 and 3 were synthesized as substrate analogues. With α(2-6)-sialyltransferase from rat liver (EC2.4.99.1) only moderate inhibition was found for these compounds. In order to obtain transition-state analogues of CMP-Neu5Ac different linkages between 2,3-dehydro-N-acetylneuraminol and CMP were generated, yielding 4, (R)-5 and (R)-6. Compound (R)-6, in which the CMP residue is attached to C-1 of 2,3-dehydro-N-acetylneuramin-1-yl phosphonate, exhibited excellent α(2-6)-sialyltransferase inhibition in the nanomolar range (K(i)=350 nM), resulting in a 130-fold higher affinity for the enzyme than CMP-Neu5Ac (K(M) = 46 υM).

Full text of DOI:10.1002/(SICI)1521-3765(19980615)4:6<1106::AID-CHEM1106>3.0.CO;2-7

FULL PAPER
New Potent Sialyltransferase InhibitorsÐSynthesis of Donor and of
Transition-State Analogues of Sialyl Donor CMP-Neu5Ac
Franz Amann, Christoph Schaub, Bernd Müller, and Richard R. Schmidt*
Abstract: Enzymatic sialyl transfer with CMP-Neu5Ac as donor can be inhibited by
CDP. Therefore phosphonates 1a,b, 2 and 3 were synthesized as substrate analogues.
With a(2 ± 6)-sialyltransferase from rat liver (EC2.4.99.1) only moderate inhibition
was found for these compounds. In order to obtain transition-state analogues of
CMP-Neu5Ac different linkages between 2,3-dehydro-N-acetylneuraminol and CMP
were generated, yielding 4, (R)-5 and (R)-6. Compound (R)-6, in which the CMP
residue is attached to C-1 of 2,3-dehydro-N-acetylneuramin-1-yl phosphonate,
exhibited excellent a(2 ± 6)-sialyltransferase inhibition in the nanomolar range
(Ki ˆ 350nm), resulting in a 130-fold higher affinity for the enzyme than CMP-
Neu5Ac (KM ˆ 46mm).
Keywords: enzyme inhibitors ´ neu-
raminic acids ´ substrate analogues
´ transferases ´ transition states
Introduction
Results and Discussion
Sialic acids play an important role in quite a few biological
processes, such as cell adhesion and inflammation.[1] Addi-
tionally, several reports indicate that there is a correlation
between cell-surface sialic acid or sialyltransferase activity
and the growth[2] or metastatic potential of tumour cells.[3, 4] In
order to elucidate the influence of sialyl residues in biological
systems it is therefore desirable to develop specific inhibitors
for sialyltransferases. Various sialyltransferases, independent
of their source and their acceptor specificity, employ cytidine
monophosphate N-acetylneuraminic acid (CMP-Neu5Ac,
Scheme 1) as the donor substrate;[5] donor analogues or
transition-state analogues (Scheme 1, CMP-Neu5Ac=) with
high enzyme affinity could therefore become particularly
versatile inhibitors. Only a few donor or acceptor analogues
serving as sialyltransferase inhibitors have been reported.[6±11]
We present here new CMP-Neu5Ac analogues (1 ± 3) which
are derived from cytidine diphosphate (CDP) A, which is a
natural sialyltransferase inhibitor.[9, 12] From the potential
transition state (CMP-Neu5Ac=) in sialyltransferase catalyzed
reactions (Scheme 1),[9, 13] new types of transition-state ana-
logues are derived (4 ± 6).[14] The synthesis of compounds 1 ± 6
and their inhibition properties of a(2 ± 6)-sialyltransferase,
obtained from rat liver (EC2.4.99.1), will be reported.
Synthesis of sialyltransferase inhibitors based on CDP: CDP
is known to be a potent sialyltransferase inhibitor which
shows competitive inhibition for a(2 ± 6)-sialyltransferase of
rat liver with a Ki value of approximately 10mm[9, 12] (Table 1).
Therefore, the corresponding methylene phosphonate ana-
logue B (Scheme 1) could result in similar inhibition proper-
ties (which have not yet been determined).[15] In order to
investigate the importance of the sialyl moiety for binding
affinity, we recently studied various CMP-quinic acid deriv-
atives as donor analogues.[9] The most potent inhibitor was
CMP-quinic acid itself (Scheme 1, C) with a Ki value of 44mm,
which is practically identical to the KM value of CMP-Neu5Ac
(45mm) in this reaction.[9] Therefore, we reasoned that an
additional negative charge at the CMP residue could be
sufficient for high enzyme affinity, yet the complete Neu5Ac
residue may not be required. Consequently, structurally much
simpler compounds were synthesized, for instance 1a, where
the distance between the two negative charges (four bonds) is
similar to that in CDP. Additionally, compounds 1b, 2 and 3
were prepared, which have a distance of five bonds between
the negative charges, as in CMP-Neu5Ac. For compounds 1 ±
3 Ki values at least in the range of CDP were expected.
For the synthesis of 1a, commercially available triethyl
phosphonoacetate 7a[16] was treated with trimethylsilyl iodide
in acetonitrile, resulting in phosphonate ester cleavage;
subsequent addition of aniline furnished anilinium salt 8a[17]
(Scheme 2). Condensation of 8a with 5'-O-unprotected cyti-
dine derivative 9[9, 10, 18] and DCC in pyridine afforded
cytidinyl phosphonate 10a. Deacylation with NaOMe/MeOH
[*] Prof. Dr. R. R. Schmidt, Dr. F. Amann, Dipl.-Chem. B. Müller,
Dipl.-Chem. C. Schaub
Fakultät Chemie, Universität Konstanz, Fach M 725
Konstanz, D-78457 Konstanz (Germany)
Fax : (‡ 49)7531-883135
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1106±1115
Scheme 1. Cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its activated form, and analogues of the two.
Table 1. Inhibition constants (Ki) of substrate analogues A, C, and 1 ± 3.[a]
followed by ester cleavage with aqueous NaOH yielded the
desired cytidinyl phosphonate 1a. Inhibition studies with
a(2 ± 6)-sialyltransferase[19] showed competitive inhibition
with Ki ˆ 2mm, which is surprisingly two orders of magnitude
less efficient than CDP (Table 1).
Molecular modelling calculations[20] indicated that cytidinyl
phosphonopropionate 1b, the homologue of 1a, is structurally
more closely related to CDP than 1a. For the synthesis of 1b
practically the same methodology was applied: commercially
available triethyl phosphonopropionate 7b[21] was trans-
formed into monoester 8b, which was condensed with 9[9, 18]
by means of 2,4,6-triisopropylbenzenesulfonyl chloride
(TIBS-Cl) in pyridine as condensing agent, to furnish 10b in
high yield; deacylation and then ester cleavage gave target
molecule 1b (Scheme 2). Substrate 1b exhibited competitive
inhibition of a(2 ± 6)-sialyltransferase with Ki ˆ 270mm, thus
showing markedly higher enzyme affinity than 1a.
Replacement of the carboxylate group in 1b by a phos-
phonate group led to 2, which was readily obtained from
ethane-1,2-diphosphonate 11 (Scheme 3).[22] Reaction of the
bis-tetrabutylammonium salt of 11 with 5'-O-tosyl-cytidine
derivative 12, which was readily obtained from 9 by treatment
with p-toluenesulfonic anhydride in the presence of DMAP,
gave ethane-1,2-diphosphonate monocytidinyl ester 13 in high
yield. Deacylation with NaOMe in MeOH gave target
molecule 2. Its Ki value was determined as 750mm, suggesting
Ki [mm]
Ref.
A (CDP)
C
1a
1b
2
10 Æ 2
44 Æ 7
[9,12]
[9]
±
±
±
2000 Æ 200
270 Æ 20
750 Æ 70
250 Æ 20
3
±
[a] For details, see Experimental Section.
Abstract in German: Die enzymatische Übertragung von
Sialylgruppen mit Hilfe von CMP-Neu5Ac als Donor kann mit
CDP inhibiert werden. Deshalb wurden als Substratanaloga
die Phosphonate 1a,b, 2 und 3 synthetisiert. Sie wirkten jedoch
bei der a(2 ± 6)-Sialyltransferase aus Rattenleber (EC2.4.99.1)
nur mäûig inhibierend. Um Übergangszustandsanaloga von
CMP-Neu5Ac zu erhalten, wurden verschiedene Verknüp-
fungen zwischen 2,3-Dehydro-N-acetylneuraminol und CMP
erzeugt und so die Verbindungen 4, (R)-5 und (R)-6 erhalten.
Verbindung (R)-6, bei welcher der CMP-Rest an C-1 von 2,3-
Dehydro-N-acetylneuramin-1-ylphosphonat geknüpft ist, zeig-
te Inhibition der a(2 ± 6)-Sialyltransferase im nanomolaren
Bereich (Ki ˆ 350 nM); (R)-6 weist somit eine 130mal höhere
Affinität zum Enzym auf als das natürliche Substrat CMP-
Neu5Ac (KM ˆ 46mm).
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R. R. Schmidt et al.
FULL PAPER
charges as in CMP-Neu5Ac should be favoured (Scheme 4).
To this end, a-methylene-b-phosphonopropionate 14[23] was
condensed with 9 in the presence of DCC in pyridine as
Scheme 4. Synthesis of cytidin-5'-yl (2-carboxy-2-propenyl)phosphonate
salt 3.
Scheme 2. Synthesis of cytidinyl phosphonate 1a and cytidinyl phosphono-
propionate 1b.
condensing agent to afford cytidinyl phosphonate 15. Deacy-
lation with NaOMe in MeOH, transformation into the sodium
salt (!16) and then ethyl ester cleavage with NaOH in
MeOH/water gave target molecule 3 which exhibited a Ki
value of 250mm.
Evidently, readily accessible but structurally simplified
CMP-Neu5Ac analogues 1 ± 3 are able to inhibit a(2 ± 6)-
sialyltransferase; however, they do not reach the values of
CDP or CMP-quinic acid (Scheme 1, A, C), despite being
structurally similar to CDP. Therefore, a new approach was
considered.
that the phosphonate group did not contribute significantly to
enzyme binding affinity. Therefore, the effect of introduction
of an a-methylene group into 1b was studied, leading to 3 as
target molecule. Thus, a spatial orientation of the negative
Synthesis of transition-state analogues of CMP-Neu5Ac: The
proposed transition state developing from sialyltransferases
with CMP-Neu5Ac in the sialyl transfer to acceptors is shown
in Scheme 1 (CMP-Neu5Ac=). From this model, which is
based on the recently supported SN1-type mechanism,[9, 13]
phosphorus derivatives 4 ± 6 were considered potentially
efficient inhibitors. In compounds 4 and 6 the distance
between the anomeric center (C-2 of the Neu5Ac residue)
and the leaving group (CMP) is increased, compared with
those expected in the transition state (CMP-Neu5Ac=). In
particular 6 was of interest, because it contains two negative
charges separated by five bonds, as in CMP-Neu5Ac, and in
addition the anomeric center is trigonal planar, the confor-
mation assumed for an SN1-type transition state.
Compounds 4 ± 6 were prepared from 2,3-dehydroneura-
minic acid. For the synthesis of 4, Neu5Ac was transformed
into 2,3-dehydro derivative 17,[24] which, with p-thiocresol in
the presence of carbonyl diimidazole (CDI) as condensing
agent, gave the thioester derivative 18. Reduction with
NaBH4 in EtOH afforded neuraminol derivative 19
(Scheme 5). Condensation with cyanoethoxy-bis(diisopropyl-
amino)phosphane[25] in the presence of the activator system
Scheme 3. Synthesis of cytidin-5'-yl ethylenediphosphonate salt 2.
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Sialyltransferase Inhibitors
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diisopropylamine/tetrazole led to phosphitamide derivative
20 in high yield. Subsequent condensation with 9 in the
presence of tetrazole afforded a phosphite triester intermedi-
ate, which on oxidation with tert-butylhydroperoxide and then
treatment with triethylamine as base led to loss of the
cyanoethyl group to afford phosphorus diester 21, but only in
moderate yield. Therefore, 9 was treated with cyanoethoxy-
bis(diisopropylamino)phosphane with diisopropylammonium
tetrazolide to give phosphitamide derivative 22.[26] Reaction
of neuraminol derivative 19 with 22 in the presence of
tetrazole, then oxidation with tert-butylhydroperoxide and
base-catalyzed cleavage of the cyanoethyl group afforded 21
in very good overall yield. Deacylation with NaOMe in
MeOH, chromatography over RP-18 with triethylammonium
carbonate buffer as eluent, and then ion exchange afforded
target molecule 4. Compound 4 exhibited competitive inhib-
ition with Ki ꢀ 2mm, thus showing unexpectedly low affinity
for the active site of a(2 ± 6)-sialyltransferase (Table 2).
For the synthesis of target molecules 5 and 6, neuraminol
derivative 19 was oxidized with Dess ± Martin periodinane[27]
leading to aldehyde 23 in high yield (Scheme 6). Reaction
with dibenzyl phosphite in the presence of NEt3 as base
Table 2. Affinity of CMP-Neu5Ac (KM) and comparison of inhibition
constants (Ki) of transition-state analogues 4 ± 6 and 6'.[a]
KM [mm]
Ki [mm]
Ref.
Scheme 6. Synthesis of 24 and 25, precursors of 5 and 6, and of a-
hydroxyphosphonate (R)-26 from neuraminol derivative 19.
CMP-Neu5Ac
4
(R)-5
(E)-6'
(R)-6
46 Æ 7
±
[9,28]
±
±
±
±
< 2000
±
±
±
±
400 Æ 40
6 Æ 0.5
furnished epimeric a-hydroxyphosphonates (R)-24 and (S)-24
in a 4:1 ratio, which could be separated by chromatography.
Only (R)-24 was used in ensuing reactions because the minor
0.35 Æ 0.05
[a] For details, see Experimental Section.
Scheme 5. Synthesis of cytidin-5'-yl phosphate 4 from 2,3-dehydroneuraminic acid.
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R. R. Schmidt et al.
FULL PAPER
epimer (S)-24 was not available in sufficient quantity. The
structural assignment was based on the Mosher method as
discussed below. Full O-acetylation was required in order to
carry out hydrogenolytic debenzylation of 24 successfully;
thus, (R)-24 treated with acetic anhydride in pyridine gave
(R)-25; subsequent hydrogenolytic debenzylation with palla-
dium on carbon as catalyst and then O-deacylation with
NaOMe in MeOH afforded a-hydroxyphosphonate (R)-26 in
high yield; in the hydrogenation step reduction of the enol
ether moiety was not observed. Hydrogenolytic debenzyla-
tion of (R)-25 and immediate coupling with cytidine deriva-
tive 9 in the presence of DCC in pyridine containing DMAP
gave condensation product (R)-27 (Scheme 7). Deacylation of
(R)-27 with NaOMe in MeOH afforded target molecule (R)-
5, which also exhibited competitive inhibition with a Ki value
of 400mm.
Scheme 8. Synthesis of phosphate 6.
Scheme 7. Synthesis of phosphonate 5.
We reasoned that introduction of a second negative charge
at the glycosylation site should greatly increase binding
affinity to sialyltransferases. In order to prove this hypothesis,
(R)-24 was condensed with cytidinyl phosphitamide 22 in the
presence of tetrazole as catalyst; then oxidation of the
intermediate phosphite triester with tert-butylhydroperoxide
gave the corresponding phosphate, which on treatment with
NEt3 as base led to phosphate diester (R)-28 in good yield
(Scheme 8). Hydrogenolytic debenzylation with Pd/C as
catalyst followed by deacylation with NaOMe in MeOH
furnished target molecule (R)-6 in very good yield. As a minor
by-product diene derivative (E)-6' was isolated, which ob-
viously originates from a base-promoted deacetoxyphospho-
nylation. The configurational assignment was based on NMR
data (two-dimensional ROESY spectrum); the assignment is
also in accordance with the generally observed influence of
stereoelectronic effects.[28, 29]
The observed deacetoxyphosphonylation is quite a facile
reaction when there is no electron-withdrawing moiety in this
system. Thus, transformation of neuraminol derivative 19 into
bromide 30 via mesylate 29 and then Michaelis ± Arbuzov
reaction with tris(trimethylsilyl)phosphite gave phosphonate
31 in quantitative yield (Scheme 9). Treatment of 31 with
TBAF in THF gave, on formal acetoxybis(trimethylsilyl)-
phosphonate elimination, diene 32, which turned out to be a
quite labile compound.
Scheme 9. Synthesis of diene 32 from neuraminol derivative 19.
value of 6mm for (E)-6' and 0.35mm for (R)-6. Thus, the
transition-state analogue (R)-6 exhibits excellent inhibition
results; it shows a 130-fold increased affinity to the active site
of a(2 ± 6)-sialyltransferase compared with the natural sub-
strate CMP-Neu5Ac (KM ˆ 46mm).[9, 30] Obviously, a planar C-
2 of the Neu5Ac residue in combination with two negative
charges in close proximity, which are separated by five bonds
and presumably in a syn-arrangement, leads to high affinity
for the active site of a(2 ± 6)-sialyltransferase. Additionally,
the cytidine residue is a basic requirement for active-site
recognition, as shown by Kleineidam et al.[8] Further studies
with this powerful substance are in progress.
Configurational assignment of (R)- and (S)-24: The configuration of the
new stereocenter in 24 was based on chemical correlation and Mosherꢁs
method.[31, 32] To this end, (R)-26 was treated with ozone; the resulting ester
was saponified without isolation to give ( )-a-hydroxyphosphonoacetic
acid, which by comparison was found to have (R) configuration.[18, 32]
Hydroxyphosphonoacetic acid was also obtained from a new galactal
derivative whose configuration could be assigned by 31P NMR of a
corresponding Mosher ester.[18]
( )-a-
For both compound (R)-6 and by-product (E)-6' the
inhibition of a(2 ± 6)-sialyltransferase was investigated. Com-
petitive inhibition was found for both compounds with a Ki
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Sialyltransferase Inhibitors
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dark red solution, to which cytidine 9 (554 mg, 1.5 mmol) was added in
portions. After 15 h at room temperature the reaction was quenched by
addition of water (5 mL) and filtered over Celite, and the solvents were
removed under reduced pressure. The residue was purified by flash
chromatography (ethyl acetate/methanol 5:1 ‡ 1% to 3:1 ‡ 1% triethyl-
amine) and RP-18 chromatography (water/methanol 2:1) to afford the
triethylammonium salt 10b (475 mg, 50%) contaminated with minor
quantities of phosphonic acid 8b, which could be removed by preparative
HPLC (CH3CN/water 5:95 to 30:70). Rf ˆ 0.11 (ethyl acetate/methanol
3:1 ‡ 1% triethylamine); [a]D ˆ ‡21.7 (c ˆ 1 in methanol); 1H NMR
(250 MHz, CD3OD): d ˆ 1.22 (t, J ˆ 7.2 Hz, 3H; OCH2CH3), 1.32 (t, J ˆ
7.1 Hz, 9H; 3NCH2CH3), 1.8 ± 2.0 (m, 2H; 1a,b''-H), 2.07, 2.10, 2.18 (3s,
9H; 3COCH3), 2.50 ± 2.63 (m, 2H; 2a,b''-H), 3.19 (q, 6H; NCH2CH3), 4.10
(q, 2H; OCH2CH3), 4.06 ± 4.25 (m, 2H; 5a,b'-H), 4.38 ± 4.42 (m, 1H; 4'-H),
5.40 ± 5.49 (m, 2H; 2'-, 3'-H), 6.16 (d, J(1',2') ˆ 4.2 Hz, 1H; 1'-H), 7.52 (d,
J(6,5) ˆ 7.6 Hz, 1H; 6-H), 8.41 (d, 1H; 5-H); 31P NMR (161.7 MHz,
CD3OD): d ˆ 24.46 (s, phosphonate); MS (FAB, negative mode, matrix: 3-
Experimental Section
Solvents were purified according to the standard procedures. Melting
points are reported in degrees Celsius (uncorrected). NMR measurements
were recorded at 228C on a Bruker AC250 Cryospec, Bruker DRX600 or a
Jeol JNM-GX400. TMS or the resonance of the deuterated solvent was
used as internal standard; solvents: CDCl3, d ˆ 7.24; CD3OD, d ˆ 3.315;
D2O, d ˆ 4.63. For 31P NMR phosphoric acid was used as an external
1
standard; 31P NMR spectra were broadband H-decoupled. MALDI-mass
spectra were recorded on a Kratos Kompact Maldi 1 and 2,5-dihydrox-
ybenzoic acid (DHB) or 6-aza-2-thiothymine (ATT) were used as matrices.
FAB mass spectra were measured on a Finnigan MAT312/AMD5000
(70 eV, 708C). Optical rotations were measured on a Perkin ± Elmer
polarimeter 241/MS in a 1 dm cell at 228C. Thin-layer chromatography was
performed on Merck silica gel plastic plates 60F254 or Merck glass plates
RP-18; compounds were visualised by treatment with a solution of
(NH4)6Mo7O24 ´ 4H2O (20 g) and Ce(SO4)2 (0.4 g) in 10% sulfuric acid
(400 mL). Flash chromatography was performed on J.T. Baker silica gel 60
(0.040 ± 0.063 mm) at a pressure of 0.3 bar. Preparative HPLC separations
were performed on an Autochrom System with a Shimadzu LC8A
preparative pump and a Rainin Dynamax UV 1 detector at 254 nm. The
‡
‡
nitrobenzyl alcohol): m/z ˆ 532 [(M NHEt3 ) ], 554 [(M NHEt3
‡
‡
H ‡Na ) ], 634.3 for C26H43N4O12P.
Disodium cytidin-5'-yl carboxymethylphosphonate (1a): A solution of
sodium methoxide (0.5m) in dry methanol (0.2 mL) was added to a solution
of ethyl ester 10a (50 mg, 0.08 mmol) in dry methanol (5 mL). After 2 h of
stirring at room temperature an additional 0.5m solution of sodium
methoxide in dry methanol (0.5 mL) was added. After 8 h of stirring the
column used was
a
Lichrosorb RP-18, 7 mm, 250 Â 16 mm (Knauer,
Germany). Mixtures of acetonitrile and 0.05m triethylammonium bicar-
bonate (TEAB) (pH 7.2 ± 7.5) were used as the mobile phase.
‡
solution was neutralized with Amberlite IRC176 (H ), filtered and
Anilinium (ethoxycarbonylmethyl)phosphonate (8a) was synthesized from
7a as previously described.[17]
evaporated. The residue was dissolved in water/methanol (1:1, 3 mL) and
NaOH (1m, 0.5 mL) was added. After stirring overnight, the solution was
‡
Anilinium (2-ethoxycarbonylethyl)phosphonate (8b): The triethyl ester 7b
(3 g, 12.59 mmol) and potassium iodide (4.18 g, 25.19 mmol) were sus-
pended in dry acetonitrile (15 mL) and TMSCl (2.74 g, 25.19 mmol) was
added dropwise. After stirring for 30 min at 358C the suspension was
filtered and the residue was washed twice with dry diethyl ether. The
filtrate was concentrated under reduced pressure and the residue was
dissolved in dry methanol (15 mL) containing aniline (2.29 mL,
25.19 mmol). The solvents were removed to afford a yellow solid which
was purified by recrystallization from acetone to yield white needles of 8b
(2.59 g, 75%). M.p. 162 ± 1648C; 1H NMR (CD3OD): d ˆ 1.24 (t, J ˆ 7.2 Hz,
3H; OCH2CH3), 1.8 ± 2.0 (m, 2H; 1a,b-H), 2.50 ± 2.65 (m, 2H; 2a,b-H),
4.12 (q, 2H; OCH2CH3), 7.0 ± 7.1 (m, 3H; 3-, 4-, 5-anilinium-H), 7.25 ± 7.35
(m, 2H; 2-,6-anilinium-H); C11H18NO5P (275.24): calcd C 48.01, H 6.59, N
5.09; found C 47.89, H 6.68, N 5.06.
neutralized with Amberlite IRC176 (H ) and filtered and the pH adjusted
to 8 with NaOH (1m). Lyophilization and subsequent purification by
preparative HPLC (0.05m TEAB ‡ 0.5% CH3CN, 8 mLmin 1, tR ˆ 9 min)
yielded 1a (24 mg, 73%) as a colourless foam. Rf ˆ 0.33 (ethyl acetate/
methanol/1m NH4OAc 1:1:1); 1H NMR (250 MHz, D2O): d ˆ 2.61 (d,
2J(H,P) ˆ 21.0 Hz, 2H; 1a,b''-H), 3.9 ± 4.2 (m, 5H; 2'-, 3'-, 4'-, 5a,b'-H), 5.79
(d, 2J(1',2') ˆ 3.5 Hz, 1H; 1'-H), 6.0 (d, J(6,5) ˆ 7.6 Hz, 1H; 6-H), 7.87 (d,
1H, 5-H); MS (MALDI, negative mode, matrix: ATT): 365 [(M
‡
‡
2Na ‡H ) ], 409.2 for C11H14N3O9PNa2.
Disodium cytidin-5'-yl (2-carboxyethyl)phosphonate (1b): To a solution of
ethyl ester 10b (42 mg, 0.067 mmol) in dry methanol (5 mL) a solution of
sodium methoxide (0.5m) in dry methanol (0.5 mL) was added. After
‡
stirring for 1 h, the solution was neutralized with Amberlite IRC176 (H ),
and filtered, and the solvents were removed under reduced pressure. The
residue was dissolved in water/methanol (1:1, 3 mL), and NaOH (1m,
0.5 mL) was added. After 3 days of stirring at room temperature the
N-Acetyl-2',3'-di-O-acetylcytidine (9) was synthesized as previously de-
scribed.[9, 18]
‡
solution was neutralized with Amberlite IRC176 (H ) and filtered and the
Triethylammonium (N-acetyl-2',3'-di-O-acetylcytidin-5'-yl) (ethoxycarbo-
nylmethyl)phosphonate (10a): A solution of the anilinium salt 8a (417 mg,
1.6 mmol) in dry methanol (10 mL) was converted into the free acid by ion-
pH adjusted to a value of 8 with NaOH (1M). After lyophilization the
residue was purified by preparative HPLC (0.05m TEAB ‡ 0.5% CH3CN,
1
‡
8 mLmin
,
tR ˆ 9.2 min). Ion-exchange chromatography (Amberlite
exchange chromatography (Amberlite IR120, H ). Dry pyridine (0.5 mL)
‡
IR120, Na ) afforded colourless disodium salt 1b (17 mg, 60%). Rf ˆ
0.67 (ethyl acetate/methanol/1m NH4OAc 1:1:1); 1H NMR (250 MHz,
D2O): d ˆ 1.6 ± 1.8 (m, 2H; 1a,b''-H), 2.1 ± 2.3 (m, 2H; 2a,b''-H), 3.80 ± 4.05
(m, 2H; 5a,b'-H), 4.05 ± 4.15 (m, 3H; 2'-, 3'-, 4'-H), 5.78 (d, 3J(1',2') ˆ
3.3 Hz, 1H; 1'-H), 5.92 (d, J(6,5) ˆ 7.4 Hz, 1H; 6-H), 7.78 (d, 1H; 5-H);
31P NMR (161.7 MHz, D2O): d ˆ 28.19 (s, phosphonate); MS (FAB,
was added and the solution was evaporated. The residue was coevaporated
with toluene and dissolved in dry pyridine (3 mL). DCC (495 mg,
2.4 mmol) was added to the solution and after 10 min of stirring cytidine
9 (298 mg, 0.8 mmol) was added to the red suspension. The mixture was
stirred for 9 days at room temperature; water (15 mL) was then added and
after 30 min the urea precipitate was filtered off. The solvents were
removed under reduced pressure and the residue was purified by flash
chromatography (ethyl acetate/methanol 3:1 ‡ 1% triethylamine) to yield
the phosphonate ester 10a (207 mg, 42%) as colourless triethylammonium
salt. Rf ˆ 0.12 (ethyl acetate/methanol 3:1 ‡ 1% triethylamine); [a]D ˆ 28
(c ˆ 0.5 in methanol); 1H NMR (250 MHz, CD3OD): d ˆ 1.21 ± 1.33 (m,
12H; 3NCH2CH3, OCH2CH3), 2.06, 2.10, 2.18 (3s, 9H; COCH3), 2.83 (d, 2J
(1''P) ˆ 20.5 Hz, 2H; 1a,b''-H), 3.19 (q, J ˆ 7.3 Hz, 6H; 3NCH2CH3), 4.13
(q, J ˆ 7.2 Hz, 2H; OCH2CH3), 4.15 ± 4.30 (m, 2H; 5a'-, 5b'-H), 4.40 ± 4.45
(m, 1H; 4'-H), 5.40 ± 5.55 (m, 2H; 2'-, 3'-H), 6.21 (d, J(1',2') ˆ 4.6 Hz, 1H;
1'-H), 7.52 (d, J(5,6) ˆ 7.5 Hz, 1H; 5-H), 8.48 (d, 1H; 6-H); 31P NMR
(161.7 MHz, CD3OD): d ˆ 13.43 (s, phosphonate); MS (MALDI, negative
‡
‡
negative mode, matrix: glycerol): m/z ˆ 378 [(M 2Na ‡H ) ], 400
‡
‡
[(M Na ) ], 422 [(M H ) ] 423.2 for C12H16N3O9PNa2.
N-Acetyl-2',3'-di-O-acetyl-5'-O-tosylcytidine (12): Cytidine derivative 9
(105 mg, 284 mmol) was stirred with 4-toluenesulfonic acid anhydride
(139 mg, 427 mmol) and dimethylaminopyridine (35 mg, 427 mmol) in
dichloromethane (4 mL) for 7 days under exclusion of moisture. Triethyl-
amine (200 mL) was added before the solution was concentrated. The
residue was purified over silica gel (toluene/acetone 3:1) to obtain 12
(114 mg, 77%) as a colourless foam. Rf ˆ 0.44 (toluene/acetone 1:1), [a]D
ˆ
‡33 (c ˆ 1.0 in CHCl3); 1H NMR (250.13 MHz, CDCl3): d ˆ 2.03 (s, 3H;
OAc), 2.04 (s, 3H; OAc), 2.23 (s, 3H; NHAc), 2.43 (s, 3H; SO2C6H4CH3),
4.26 (dd, J(4',5a') ˆ 3.3 Hz, J(5a',5b') ˆ 10.9 Hz, 1H; 5a'-H), 4.34 (ddd,
J(3',4') ˆ 4.3 Hz, J(4',5b') ˆ 2.3 Hz, 1H; 4'-H), 4.41 (dd, 1H; 5b'-H), 5.24
(dd, J(1',2') ˆ J(2',3') ˆ 5.0 Hz, 1H; 2'-H), 5.28 (dd, 1H; 3'-H), 6.07 (d, 1H;
1'-H), 7.32 (d, J(5,6) ˆ 7.6 Hz, 1H; 5-H), 7.35 (d, J ˆ 8.5 Hz, 2H;
SO2C6H4CH3, meta), 7.68 (d, 1H; 6-H), 7.79 (d, 2H; SO2C6H4CH3, ortho),
9.96 (brs, 1H; NH); C22H25N3O10S (523.52): calcd C 50.47, H 4.81, N 8.03;
found C 49.63, H 4.66, N 7.97.
‡
mode, matrix: ATT): m/z ˆ 519 [(M NHEt3 ) ], 639 [(M‡H2O) ], 620.6
for C25H41N4O12P.
Triethylammonium (N-acetyl-2',3'-di-O-acetylcytidin-5'-yl) (2-ethoxycar-
bonylethyl)phosphonate (10b): The anilinium salt 8b (823 mg, 3 mmol)
was converted to the free acid by ion-exchange chromatography (Amber-
lite IR, 120H ), coevaporated twice with dry pyridine and finally dissolved
in dry pyridine (10 mL). TIBS-Cl (1.36 g, 4.5 mmol) was added to form a
‡
Chem. Eur. J. 1998, 4, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0406-1111 $ 17.50+.25/0
1111

R. R. Schmidt et al.
FULL PAPER
Trisodium N-acetyl-2',3'-di-O-acetylcytidin-5'-yl ethylene-1,2-diphospho-
nate (13): Ethylene-1,2-diphosphonic acid[22] (41 mg, 215 mmol) was mixed
with tetrabutylammonium hydroxide (0.8m) in methanol (537 mL). The
solvent was removed and the residue was coevaporated several times with
dry acetone and finally with toluene. The resulting salt 11 was stirred
together with tosylate 12 (45 mg, 86 mmol) in DMF (2 mL) overnight. The
reaction mixture was concentrated under reduced pressure, taken up with
water and stirred with Dowex W50X2 (ammonium form). The ion-
exchange resin was filtered off and the filtrate concentrated. The raw
product was purified by chromatography on cellulose (acetone/0.05m
ammonium bicarbonate 3:1). The fractions containing the product were
concentrated, lyophilized from water, taken up in water, stirred with IR120
H), 5.87 (d, J(1',2') ˆ 3.1 Hz, 1H; 1'-H), 6.09 (d, J(5,6) ˆ 7.8 Hz, 1H; 5-H),
6.19 (d, 1H; 3b''-H), 8.28 (d, 1H; 6-H).
Disodium cytidin-5'-yl (2-carboxy-2-propenyl)phosphonate (3): NaOH
(1m, 0.5 mL) was added to a solution of ethyl ester 16 (50 mg, 0.11 mmol)
in water/methanol (2:1, 3 mL). After stirring overnight, the solution was
‡
neutralized with Amberlite IR120 (H ), filtered and converted into the
‡
sodium salt with Amberlite IR120 (Na ). The filtrate was concentrated
under reduced pressure and the residue was dissolved in 1 mL of water.
Addition of 10 mL of ethanol caused 3 (30 mg, 63%) to precipitate as a
white powder. Rf ˆ 0.81 (RP-18 water/ethanol 3:1); [a]D
ˆ
12.8 (c ˆ 0.05,
water); 1H NMR (250 MHz, D2O): d ˆ 2.62 (d, 2J(CH2,P) ˆ 21.0 Hz, 2H;
CH2-P), 3.86 ± 4.08 (m, 5H; 2'-,3'-,4'-,5a',b'-H), 5.57 (d, 2J(3a'',3b'') ˆ
5.1 Hz, 1H; 3a''-H), 5.74 (d, J(1',2') ˆ 3.1 Hz, 1H; 1'-H), 5.98 (d, 1H,
3b''-H), 6.01 (d, J(5,6) ˆ 7.8 Hz, 1H, 5-H), 7.91 (d, 1H, 6-H). 31P NMR
(161.70 MHz, D2O): d ˆ 25.86 (s, phosphonate); MS (FAB, negative mode,
‡
(Na ), filtered and lyophilized again to give 13 (21 mg, 42%). Rf ˆ 0.35
(cellulose, acetone/0.05m ammonium bicarbonate 2:1); 1H NMR
(250.13 MHz, D2O): d ˆ 1.52 ± 1.64 (m, 4H; 2PCH2), 1.95 (s, 3H; OAc),
1.97 (s, 3H; OAc), 2.04 (s, 3H; NHAc), 3.90 ± 4.07 (m, 2H; 5a'-H, 5b'-H),
4.40 (brs, 1H; 4'-H), 5.25 (dd, J(2',3') ˆ J(3',4') ˆ 5.2 Hz, 1H; 3'-H), 5.32
(dd, J(1',2') ˆ 4.2 Hz, 1H; 2'-H), 5.99 (d, 1H; 1'-H), 7.18 (d, J(5,6) ˆ 7.5 Hz,
1H; 5-H), 8.16 (d, 1H; 6-H); MS (FAB, negative mode, matrix: DMF/
‡
matrix: glycerol/3-nitrobenzyl alcohol): m/z ˆ 390 [(M H ) ], 482
‡
[(M‡glycerol H ) ], 391.25 for C13H18N3O9P.
5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-
galacto-non-2-enopyranosonic acid (17): The compound was synthesized
from 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid as previously de-
‡
‡
nitrobenzylalcohol):
C17H22N3Na3P2O13
m/z ˆ 540
[(M 3Na ‡2H ) ]
607.3
for
.
scribed.[24] Rf ˆ 0.39 (ethyl acetate/methanol 7:1 ‡ 3% acetic acid); [a]D
ˆ
Disodium cytidin-5'-yl ethylenediphosphonate (2): Acetylated starting
material 13 (20 mg, 33 mmol) was taken up in methanol (5 mL) and treated
with sodium methoxide (1m) in methanol (0.5 mL). After ten minutes
‡65 (c ˆ 1.0 in CHCl3); 1H NMR (250.13 MHz, CDCl3): d ˆ 1.91 (s, 3H;
NHAc), 2.01 (s, 3H; OAc), 2.04 (s, 3H; OAc), 2.05 (s, 3H; OAc), 2.09 (s,
3H; OAc), 4.09 (dd, J(8,9a) ˆ 5.8 Hz, J(9a,9b) ˆ 11.0 Hz, 1H; 9a-H),
4.31 ± 4.41 (m, 2H; 5-H, 6-H), 4.55 (dd, J(8,9b) ˆ 3.0 Hz, 1H; 9b-H), 5.34
(ddd, J(7,8) ˆ 4.8 Hz, 1H; 8-H), 5.45 ± 5.52 (m, 2H; 4-H, 7-H), 6.03 (d,
J(3,4) ˆ 3.1 Hz, 1H; 3-H), 6.12 (d, J(5,NH) ˆ 9.1 Hz, 1H; NH), 10.71 (brs,
1H; COOH); C19H25NO12 ´ H2O (459.41): calcd C 47.80, H 5.70, N 2.93;
found C 48.05, H 5.65, N 3.21.
‡
methanol-washed IRC176 (H ) was added. The mixture was filtered and
concentrated and the residue was lyophilized from water to give 2 (15 mg,
quant.) of
a
powder. Rf ˆ 0.06 (cellulose, acetone/0.05m ammonium
bicarbonate 2:1); 1H NMR (250.13 MHz, D2O): d ˆ 1.41 ± 1.71 (m, 4H;
2PCH2), 3.88 (ddd, J(4',5a') ˆ 2.4 Hz, J(5a',5b') ˆ 11.8 Hz, J(5a',P) ˆ
5.2 Hz, 1H; 5a'-H), 3.98 (ddd, J(4',5b') ˆ 1.9 Hz, J(5b',P) ˆ 3.9 Hz, 1H;
5b'-H), 4.07 ± 4.14 (m, 3H; 2'-H, 3'-H, 4'-H), 5.77 (d, J(1',2') ˆ 3.6 Hz, 1H;
1'-H), 5.92 (d, J(5,6) ˆ 7.6 Hz, 1H; 5-H), 7.75 (d, 1H; 6-H); 31P NMR
(242.55 MHz, D2O): d ˆ 27.45 (d, 2J ˆ 73.1 Hz, phosphonate), 31.65 (d,
phosphonate); MS (FAB, negative mode, matrix: water/DMSO/acetic acid/
4-Methylphenyl
5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-di-
deoxy-d-glycero-d-galacto-non-2-enpyranosonothioate (18): Acid 17
(95 mg, 20 mmol) was dissolved in DMF (20 mL) and cooled to 08C.
Carbonyldiimidazole (35 mg, 219 mmol) was then added. The cooling bath
was removed and the solution stirred for 2 h under exclusion of moisture. 4-
Thiocresol was added to the solution, which was diluted with ethyl acetate
(100 mL) after another hour. The solution was washed with brine and
several times with water. The aqueous layers were combined and extracted
again with ethyl acetate. The combined organic layers were finally washed
with water, dried over magnesium sulfate and concentrated. The remaining
oil was purified by chromatography over silica gel (toluene/ethyl acetate/
methanol 20:4:1) and yielded 1.12 g (74%) crystals. M.p. 1398C; Rf ˆ 0.49
(toluene/acetone 1:1), [a]D ˆ ‡62 (c ˆ 1.0 in CHCl3); 1H NMR
(250.13 MHz, CDCl3): d ˆ 1.93 (s, 3H; NHAc), 2.03 (s, 3H; OAc), 2.06
(s, 3H; OAc), 2.09 (s, 3H; OAc), 2.15 (s, 3H; OAc), 2.36 (s, 3H;
SC6H4CH3), 4.23 (dd, J(8,9a) ˆ 6.0 Hz, J(9a,9b) ˆ 12.4 Hz, 1H; 9a-H), 4.38
(ddd, J(5,6) ˆ J(5,NH) ˆ 9.0 Hz, J(4,5) ˆ 7.2 Hz, 1H; 5-H), 4.51 (dd,
J(6,7) ˆ 3.5 Hz, 1H; 6-H), 4.52 (dd, J(8,9b) ˆ 3.4 Hz, 1H; 9b-H), 5.50 ±
5.56 (m, 3H; 4-H, 7-H, NH), 5.51 (ddd, J(7,8) ˆ 6.0 Hz, 1H; 8-H), 5.87 (d,
J(3,4) ˆ 3.3 Hz, 1H; 3-H), 7.19 ± 7.29 (m, 4H; SC6H4CH3); C26H31NO11S
(565.60): calcd C 55.21, H 5.52, N 2.48; found C 55.31, H 5.59, N 2.66.
‡
‡
glycerol 1:1:1:1): m/z ˆ 414 [M 2Na ‡H ], 459.2 for C11H17N3Na2P2O10
.
Monocyclohexylammonium (2-ethoxycarbonyl-2-propenyl)phosphonate
(14) was synthesized as previously described.[23]
Triethylammonium (N-acetyl-2',3'-di-O-acetylcytidin-5'yl) (2-ethoxycar-
bonyl-2-propenyl)phosphonate (15): A solution of the monocyclohexylam-
monium salt 14 (500 mg, 1.8 mmol) in dry methanol (5 mL) was converted
into the free acid by ion-exchange chromatography (Amberlite IR120,
‡
H ). Dry pyridine (0.5 mL) was added and the solution was evaporated.
The residue was coevaporated with toluene and dissolved in dry pyridine
(3 mL). DCC (1.2 g, 5.8 mmol) was added to the solution. After 10 min of
stirring, cytidine 9, (1.5 g, 4 mmol) was added to the white suspension. The
mixture was stirred for 7 days at room temperature, then water (15 mL)
was added and after 30 min the urea precipitate was filtered off. The
solvents were removed under reduced pressure and the residue was
purified by flash chromatography (ethyl acetate/methanol 5:1 ‡ 1%
triethylamine) to yield phosphonate ester 15 (500 mg, 43%) as a colourless
salt. Triethylammonium salt 15 was contaminated with minor quantities of
phosphonic acid 14. Rf ˆ 0.11 (ethyl acetate/methanol 3:1 ‡ 1% triethyl-
amine); 1H NMR (250 MHz, CDCl3): d ˆ 1.25 (m, 12H; 3 NCH2CH3,
5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-
galacto-non-2-enitol (19): Compound 18 (85 mg, 150 mmol) was dissolved
in ethanol (5 mL) and cooled to 08C under exclusion of moisture. Sodium
borohydride (144 mg, 3.81 mmol) was added under rapid stirring. After
three hours, concentrated ammonium chloride solution was poured into the
reaction mixture. When the evolution of gas ceased, water (20 mL) was
added. The solution was extracted with ethyl acetate several times. The
organic layers were collected, dried over magnesium sulfate and concen-
trated. Chromatography over silica gel (toluene/acetone 1:1) gave 19
(49 mg, 73%), a colourless foam. Rf ˆ 0.38 (toluene/ethyl acetate/methanol
5:2:1), [a]D ˆ ‡41 (c ˆ 1.0 in CHCl3); 1H NMR (250.13 MHz, CDCl3): d ˆ
1.89 (s, 3H; NHAc), 2.02 (s, 3H; OAc), 2.03 (s, 3H; OAc), 2.06 (s, 3H;
OAc), 2.09 (s, 3H; OAc), 2.98 (brs, 1H; OH), 3.88 (brd, J(1a,1b) ˆ
13.6 Hz, 1H; 1a-H), 3.97 (dd, J(8,9a) ˆ 7.2 Hz, J(9a,9b) ˆ 12.4 Hz, 1H;
9a-H), 4.09 (brd, 1H; 1b-H), 4.19 (dd, J(5,6) ˆ 9.6 Hz, J(6,7) ˆ 2.9 Hz, 1H;
6-H), 4.36 (ddd, J(5,NH) ˆ 9.7 Hz, J(4,5) ˆ 7.9 Hz, 1H; 5-H), 4.60 (dd,
J(8,9b) ˆ 2.5 Hz, 1H; 9b-H), 4.87 (d, J(3,4) ˆ 2.7 Hz, 1H; 3-H), 5.35 (ddd,
J(7,8) ˆ 7.2 Hz, 1H; 8-H), 5.37 ± 5.42 (m, 2H, 4-H; 7-H), 5.66 (d, 1H; NH);
C19H27NO11 (445.43): calcd C 51.23, H 6.11, N 3.14; found C 50.91, H 6.16, N
3.17.
2
OCH2CH3), 1.99, 2.06, 2.18, (3s, 9H; 3OAc), 2.88 (d, J(CH2,P) ˆ 21.0 Hz,
2H; CH2-P), 3.15 (q, J ˆ 7.3 Hz, 6H; 3NCH2CH3), 4.2 (m, 5H; 4'-H, 5a',b'-
H, OCH2CH3), 5.40 (m, 2H; 2'-,3'-H), 5.78 (d, 2J(3b'',3a'') ˆ 5.1 Hz, 1H;
3b''-H), 6.15 (d, J(1',2') ˆ 3.9 Hz, 1'-H), 6.21(d, 2J(3a'',3b'') ˆ 5.1 Hz, 1H;
3a''-H) , 7.41 (d, 3J(5,6) ˆ 7.5 Hz, 1H; 5-H), 8.14 (d, 1H; 6-H); MS
‡
(MALDI, negative mode, matrix: ATT): m/z ˆ 566 [(M H ) ], 567 for
C21H27N3NaO12P.
Sodium cytidin-5'-yl (2-ethoxycarbonyl-2-propenyl)phosphonate (16): To a
solution of ethyl ester 15 (100 mg, 0.16 mmol) in dry methanol (4 mL) a
solution of sodium methoxide (1m) in dry methanol (0.2 mL) was added.
After 30 min of stirring at room temperature the solution was neutralized
‡
with Amberlite IR120 (H ), and evaporated. The residue was dissolved in
‡
methanol (3 mL) and converted with Amberlite IR120 (Na ) into the
sodium salt. It was purified by crystallization from chloroform to yield 16 as
colourless crystals (50 mg, 73%). M.p. 163 ± 1668C; Rf ˆ 0.68 (RP-18,
water/ethanol 3:1); 1H NMR (250 MHz, CD3OD): d ˆ 1.25 (t, J ˆ 7.1 Hz,
3H; OCH2CH3), 2.8 (d, 2J(CH2,P) ˆ 21.0 Hz, 2H; CH2-P), 4.02 ± 4.21 (m,
7H; 2'-,3'-,4'-,5a',b'-H, OCH2CH3), 5.81 (d, 2J(3a'',3b'') ˆ 5.1 Hz, 1H; 3a''-
1112
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0406-1112 $ 17.50+.50/0
Chem. Eur. J. 1998, 4, No. 6

Sialyltransferase Inhibitors
1106±1115
(5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-
galacto-non-2-enitol-1-yl) 2-cyanoethyl phosphite diisopropylamide (20):
Alcohol 19 (117 mg, 263 mmol) was dried under reduced pressure together
with diisopropylammonium tetrazolide (23 mg, 131 mmol). This mixture
was taken up in dichloromethane (4 mL) under exclusion of moisture. 2-
Cyanoethyl phosphite bisdiisopropylamide[25] (167 mL, 525 mmol) was
added. After the mixture had been stirred for one hour, concentrated
sodium bicarbonate solution (5 mL), water and dichloromethane were
added. The organic layer was dried over magnesium sulfate and concen-
trated. The raw product was quickly filtered over silica gel (toluene/acetone
1:1) to give a mixture of the two diastereomers of 20 and 2-cyanoethyl
phosphite diisopropylamide, which does not interfere in the next step (in
total 228 mg of a colourless oil). Rf ˆ 0.6 (toluene/acetone 1:1).
triethylammonium bicarbonate buffer‡ 7% acetonitrile): tR ˆ 13.5 min;
1H NMR (250.13 MHz, D2O): d ˆ 1.12 (t, J ˆ 7.4 Hz, 9H; 3NCH2CH3), 1.90
(s, 3H; NHAc), 3.04 (q, 6H; 3NCH2CH3), 3.44 (brd, J(7'',8'') ˆ 9.5 Hz, 1H;
7-H), 3.47 (dd, J(8'',9a'') ˆ 6.6 Hz, J(9a'',9b'') ˆ 12.5 Hz, 1H; 9a''-H),
3.66 ± 3.73 (m, 2H; 8''-H, 9b''-H), 3.87 (dd, J(4'',5'') ˆ 8.6 Hz, J(5'',6'') ˆ
10.8 Hz, 1H; 5''-H), 3.86 ± 4.76 (m, 7H; 2'-H, 3'-H, 4'-H, 5a'-H, 5b'-H, 1a''-
H, 1b''-H), 4.03 (brd, 1H; 6''-H), 4.16 (d, J(3'',4'') ˆ 1.7 Hz, 1H; 4''-H), 4.83
(d, 1H; 3''-H), 5.82 (d, J(1',2') ˆ 3.5 Hz, 1H; 1'-H), 5.94 (d, J(5,6) ˆ 8.0 Hz,
1H; 5-H), 7.76 (d, 1H; 6-H); 31P NMR (242.55 MHz, D2O): d ˆ 3.71 (s,
phosphate); MS (FAB, negative mode, matrix: glycerol/water 1:1): m/z ˆ
‡
581 [(M HNEt3 ) ] 682.7 for C26H45N5O14P.
5-Acetamido-4,7,8,9-tetra-O-acetylaldehydo-2,6-anhydro-3,5-dideoxy-d-g-
lycero-d-galacto-non-2-enose (23): Alcohol 19 (235 mg, 528 mmol) was
dissolved in dichloromethane (10 mL). Periodinane (257 mg, 607 mmol)
was added and the solution stirred for 20 min. Subsequently the mixture
was diluted with dichloromethane (100 mL) and stirred for another 20 min
with a mixture of sodium thiosulfate (2.2 g), water (5 mL), and concen-
trated sodium bicarbonate solution (5 mL). The organic layer was
separated, washed with water and dried over magnesium sulfate. The
crude product was purified over silica gel (toluene/acetone 1:1) to give 23
Triethylammonium (5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-
dideoxy-d-glycero-d-galacto-non-2-enitol-1-yl) (N-acetyl-2',3'-di-O-acetyl-
cytidin-5'-yl) phosphate (21):
a) From phosphite 20: The raw product 20 (225 mg, approximately
260 mmol) and compound 9 (96 mg, 260 mmol) were coevaporated several
times with toluene and finally with dichloromethane and dried under
reduced pressure. This mixture was taken up with dichloromethane
(10 mL) under a nitrogen atmosphere. 1H-Tetrazole (27 mg, 390 mmol)
was added and dissolved slowly. The solution was cooled to 108C after
stirring overnight. Tert-butylhydroperoxide solution (3m) in toluene
(173 mL) was added. 3 h later, triethylamine (1 mL) diluted with dichloro-
methane (5 mL) was dropped into the reaction mixture, which was
concentrated after 2 h. The residue was purified over silica gel (ethyl
acetate/methanol 3:1 ‡ 2% triethylamine) to give 110 mg of the product
which could not be separated from an impurity (probably 1-phosphite of
compound 19).
(193 mg, 82%) as a colourless foam. Rf ˆ 0.39 (toluene/acetone 1:1), [a]D
ˆ
1
‡102 (c ˆ 1.0 in CHCl3); H NMR (250.13 MHz, CDCl3): d ˆ 1.93 (s, 3H;
NHAc), 2.03 (s, 3H; OAc), 2.04 (s, 3H; OAc), 2.08 (s, 3H; OAc), 2.10 (s,
3H; OAc), 4.19 (dd, J(8,9a) ˆ 6.5 Hz, J(9a,9b) ˆ 12.3 Hz, 1H; 9a-H), 4.38
(ddd, 1H; J(4,5) ˆ 6.6 Hz, J(5,6) ˆ 8.3 Hz, J(5,NH) ˆ 8.3 Hz, 5-H), 4.41 (dd,
J(6,7) ˆ 3.9 Hz, 1H; 6-H), 4.48 (dd, J(8,9b) ˆ 3.3 Hz, 1H; 9b-H), 5.38 (ddd,
J(7,8) ˆ 5.9 Hz, 1H; 8-H), 5.46 (dd, 1H; 7-H), 5.56 (dd, J(3,4) ˆ 3.2 Hz, 1H;
4-H), 5.60 (d, 1H; NH), 5.82 (d, 1H; 3-H), 9.20 (s, 1H; CHO); C19H25NO11
(443.41): calcd C 51.47, H 5.68, N 3.16; found C 51.09, H 5.76, N 3.23.
b) From phosphite 22: Phosphite 22 (63 mg, 141 mmol) and alcohol 19
(73 mg, 128 mmol) were taken up in dichloromethane, foamed and dried in
vacuum. The foam was dissolved in dry dichloromethane (5 mL) under
nitrogen. 1H-Tetrazole (13 mg, 192 mmol) and, after this had been stirred
for three hours, tert-butylhydroperoxide solution in toluene (3m, 85 mL,
256 mmol) were added. After another hour triethylamine (1 mL) was
added. After stirring overnight, the reaction mixture was carefully
concentrated and chromatographed over silica gel (ethyl acetate/methanol
5:1 ‡ 1% triethylamine) to yield 21 (122 mg, ca. 90%) of a pale yellow
syrup. Rf ˆ 0.11 (ethyl acetate/methanol 3:1); 1H NMR (250.13 MHz,
CD3OD): d ˆ 1.36 (t, J ˆ 7.3 Hz, 9H; 3NCH2CH3), 1.90 (s, 3H; NHAc), 2.03
(s, 3H; OAc), 2.04 (s, 3H; OAc), 2.07 (s, 3H; OAc), 2.12 (s, 3H; OAc), 2.13
(s, 3H; OAc), 2.15 (s, 3H; OAc), 2.23 (s, 3H; NHAc), 3.25 (q, 6H;
3NCH2CH3), 4.08 ± 4.46 (m, 9H; 4'-H, 5a'-H, 5b'-H, 1a'' 1b''-H, 5''-H, 6''-
H, 9a''-H, 9b''-H), 5.04 (d, J(3'',4'') ˆ 2.4 Hz, 1H; 3''-H), 5.23 (ddd,
J(7'',8'') ˆ 7.2 Hz, J(8'',9a'') ˆ 10.0 Hz, J(8'',9b'') ˆ 2.8 Hz, 1H; 8''-H),
5.42 ± 5.52 (m, 4H, 2'-H, 3'-H, 4''-H, 7''-H), 6.24 (d, J(1',2') ˆ 3.8 Hz, 1H;
1'-H), 7.57 (d, J(5,6) ˆ 7.5 Hz, 1H; 5-H), 8.47 (d, 1H; 6-H); MS (MALDI,
Dibenzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-D-
erythro-L-gluco-non-2-enitol-1-yl)phosphonate [(R)-24] and dibenzyl (5-
acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-erythro-l-ma-
nno-non-2-enitol-1-yl)phosphonate [(S)-24]: Aldehyde 23 (53 mg,
119 mmol) was dissolved in dichloromethane (3 mL). Dibenzyl phosphite
(40 mL, 179 mmol) and triethylamine (5 mL) were added. The solution was
stirred for one hour, concentrated and chromatographed over silica gel
(toluene/acetone 1:1) to give 83 mg (99%) of the product as a colourless
foam. The two diastereomers were formed in a ratio of R:S ˆ 4:1; they
could be separated by MPLC over silica gel (toluene/acetone 2:3).
(R)-24: Rf ˆ 0.44 (toluene/acetone 2:3), [a]D ˆ ‡56 (c ˆ 1.0 in CHCl3);1H
NMR (250.13 MHz, CDCl3): d ˆ 1.85 (s, 3H; NHAc), 1.98 (s, 6H; 2OAc),
2.00 (s, 3H; OAc), 2.04 (s, 3H; OAc), 3.86 (dd, J(8,9a) ˆ 7.4 Hz, J(9a,9b) ˆ
12.4 Hz, 1H; 9a-H), 4.13 (dd, J(1,OH) ˆ 9.4 Hz, J(OH,P) ˆ 6.5 Hz, 1H;
OH), 4.19 (dd, J(5,6) ˆ 9.1 Hz, J(6,7) ˆ 3.5 Hz, 1H; 6-H), 4.31 (dd, J(1,P) ˆ
14.6 Hz, 1H; 1-H), 4.42 (ddd, J(4,5) ˆ 7.5 Hz, J(5,NH) ˆ 9.1 Hz, 1H; 5-H),
4.61 (dd, J(8,9b) ˆ 2.6 Hz, 1H; 9b-H), 5.02 (dd, J(3,4) ˆ J(3,P) ˆ 3.1 Hz,
1H; 3-H), 5.06 ± 5.11 (m, 4H; 2CH2C6H5), 5.29 (ddd, J(7,8) ˆ 4.9 Hz, 1H; 8-
H), 5.36 (dd, 1H, 4-H), 5.40 (dd, 1H; 7-H), 5.83 (d, 1H; NH), 7.30 ± 7.34 (m,
10H; 2CH2C6H5); C33H40NO14P ´ 3/2 H2O (705.66): calcd C 54.10, H 5.91, N
1.91; found C 54.12, H 5.72, N 2.11.
(S)-24 Rf ˆ 0.44 (toluene/acetone 2:3), [a]D ˆ ‡69 (c ˆ 1.0 in CHCl3); 1H
NMR (250.13 MHz, CDCl3): d ˆ 1.85 (s, 3H; NHAc), 1.99 (s, 3H; OAc),
2.00 (s, 3H; OAc), 2.02 (s, 6H; 2 OAc), 3.55 (brs, 1H; OH), 4.08 (dd,
J(8,9a) ˆ 6.8 Hz, J(9a,9b) ˆ 12.0 Hz, 1H; 9a-H), 4.24 (dd, J(5,6) ˆ J(6,7) ˆ
4.7 Hz, 1H; 6-H), 4.40 (dd, J(8,9b) ˆ 4.0 Hz, 1H; 9b-H), 4.43 (d, J(1,P) ˆ
13.2 Hz, 1H; 1-H), 4.52 (ddd, J(4,5) ˆ 4.7 Hz, J(5,NH) ˆ 9.3 Hz, 1H; 5-H),
5.00 ± 5.17 (m, 6H; 3-H, 4-H, 2CH2C6H5), 5.40 (ddd, J(7,8) ˆ 4.1 Hz, 1H; 8-
H), 5.50 (dd, 1H; 7-H), 6.37 (d, 1H; NH), 7.25 ± 7.34 (m, 10H; 2CH2C6H5);
C33H40NO14P ´ 3/4 H2O (705.66): calcd C 55.11, H 5.82, N 1.95; found C 55.08,
H 5.94 N 2.09.
‡
negative mode, matric:ATT): m/z ˆ 875 [(M HNEt3 ) ], 976.9 for
C40H59N5O21P.
(N-Acetyl-2',3'-di-O-acetylcytidin-5'-yl) 2-cyanoethyl phosphite diisopro-
pylamide (22): Alcohol 9 (95 mg, 257 mmol) was dried under reduced
pressure together with diisopropylammonium tetrazolide. The mixture was
taken up with dichloromethane (3 mL) under exclusion of moisture. 2-
Cyanoethyl phosphite bisdiisopropylamide (163 mL, 514 mmol) was added.
After stirring overnight, the reaction mixture was concentrated and
purified over silica gel (toluene/acetone 3:1) to give 22 (108 mg, 74%), a
colourless foam containing both diastereomers. Rf ˆ 0.46 (toluene/acetone
1:1); 1H NMR (250.13 MHz, CDCl3): d ˆ 1.14 ± 1.26 (m, 12H; 4NCHCH3),
2.02 ± 2.09 (m, 6H; 2OAc), 2.24 (s, 3H; NHAc), 2.65 (m, 2H;
OCH2CH2CN), 3.54 ± 3.66 (m, 2H; 2NCHCH3), 3.72 ± 4.04 (m, 4H; 5a'-
H, 5b'-H, OCH2CH2CN), 4.32 (brs, 1H; 4'-H), 5.31 ± 5.46 (m, 2H; 2'-H, 3'-
H), 6.28 ± 6.35 (d, J(1',2') ˆ 5.0 Hz, 1H; 1'-H), 7.42 (d, J(5,6) ˆ 7.6 Hz, 1H;
5-H), 8.22 ± 8.23 (d, 1H; 6-H), 9.92 (brs, 1H; NH); C24H36N5O9P (569.56):
calcd C 50.61, H 6.37, N 12.30; found C 50.55, H 6.44, N 12.15.
Dibenzyl (5-acetamido-1,4,7,8,9-penta-O-acetyl-2,6-anhydro-3,5-dideoxy-
d-erythro-l-gluco-non-2-enitol-1-yl)phosphonate [(R)-25]: Compound
(R)-24 (153 mg, 217 mmol) was mixed with pyridine and acetic anhydride
(2 mL each). After stirring for 2 h the solution was concentrated under
vacuum and filtered over silica gel (toluene/acetone 2:1) to give (R)-25
Triethylammonium
(5-acetamido-2,6-anhydro-3,5-dideoxy-d-glycero-d-
galacto-non-2-enitol-1-yl) (cytidin-5'-yl) phosphate (4): Compound 21
(120 mg, 122 mmol) was dissolved in methanol (3 mL) and treated with
sodium methoxide (1m) in methanol (0.5 mL). After stirring overnight,
ammonium chloride (100 mg) was added. The solvent was removed and the
residue was purified by HPLC over RP-18 silica gel. Lyophilization gave 4
(56 mg, 67%). HPLC: prep. RP-18 column (flow: 7 mLmin 1, 0.05m
(150 mg, 92%) as a colourless foam. Rf ˆ 0.45 (toluene/acetone 1:1), [a]D ˆ
‡62 (c ˆ 1.0 in CHCl3); 1H NMR (250.13 MHz, CDCl3): d ˆ 1.89 (s, 3H;
NHAc), 1.90 (s, 3H; OAc), 2.00 (s, 6H; 2OAc), 2.01 (s, 6H; 2OAc), 4.09
(dd, J(8,9a) ˆ 6.2 Hz, J(9a,9b) ˆ 12.2 Hz, 1H; 9a-H), 4.17 (dd, J(5,6) ˆ
J(6,7) ˆ 6.0 Hz, 1H; 6-H), 4.33 (dd, J(8,9b) ˆ 3.9 Hz, 1H; 9b-H), 4.42 (ddd,
J(4,5) ˆ 6.0 Hz, J(5,NH) ˆ 9.5 Hz, 1H; 5-H), 5.00 ± 5.15 (m, 5H; 3-H,
Chem. Eur. J. 1998, 4, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0947-6539/98/0406-1113 $ 17.50+.25/0
1113

R. R. Schmidt et al.
FULL PAPER
2CH2C6H5), 5.21 (ddd, J(3,4) ˆ J(4,P) ˆ 6.0 Hz, 1H; 4-H), 5.36 (dd, J(7,8) ˆ
5.2 Hz, 1H; 8-H), 5.47 (dd, 1H; 7-H), 5.53 (d, J(1,P) ˆ 14.0 Hz, 1H; 1-H),
5.93 (d, 1H; NH), 7.27 ± 7.39 (m, 10H; 2CH2C6H5); C35H42NO15P (747.70):
calcd C 56.22, H 5.66, N 1.87; found C 56.01, H 5.74, N 1.91.
7.3 Hz, 9H; 3NCH2CH3), 1.88 (s, 3H; NHAc), 1.98 (s, 3H; OAc), 2.00 (s,
6H; 2OAc), 2.07 (s, 3H; OAc), 2.11 (s, 3H; OAc), 2.12 (s, 3H; OAc), 2.20
(s, 3H; NHAc), 3.21 (q, 6H; 3NCH2CH3), 4.11 (dd, J(8'',9a'') ˆ 6.0 Hz,
J(9a'',9b'') ˆ 11.1 Hz, 1H; 9a''-H), 4.17 (ddd, J(4',5a') ˆ 4.5 Hz,
J(5a',5b') ˆ 12.7 Hz, J(5a',P) ˆ 1.8 Hz, 1H; 5a'-H), 4.26 ± 4.50 (m, 4H; 4'-
H, 5b'-H, 5''-H, 6''-H), 4.46 (dd, J(8'',9b'') ˆ 2.2 Hz, 1H; 9b''-H), 5.15 (dd,
J(1'',P) ˆ 11.1 Hz, J(1'',P) ˆ 15.6 Hz, 1H; 1''-H), 5.19 ± 5.31 (m, 6H; 3''-H,
8''-H, 2CH2C6H5), 5.45 ± 5.52 (m, 4H; 2'-H, 3'-H, 4''-H, 7''-H), 6.24 (d,
J(1',2') ˆ 4.0 Hz, 1H; 1'-H), 7.34 ± 7.44 (m, 10H; 2CH2C6H5), 7.54 (d,
J(5,6) ˆ 7.5 Hz, 1H; 5-H), 8.18 (d, J(5'',NH) ˆ 9.4 Hz, 1H; NH), 8.48 (d,
1H; 6-H); MS (MALDI, positive mode, matrix: ATT): m/z ˆ 1161 [(M
Disodium (5-acetamido-2,6-anhydro-3,5-dideoxy-d-erythro-l-gluco-non-2-
enitol-1-yl)phosphonate [(R)-26]: Compound (R)-25 (48 mg, 64 mmol) was
dissolved in isopropanol (10 mL). Palladium on charcoal (5 mg, 10% Pd)
was added and the mixture was stirred vigorously under hydrogen at
normal pressure. After 40 min the catalyst was filtered off and washed with
methanol. Filtrate and washings were combined and sodium methoxide in
methanol (1m, 0.2 mL) was added. After 10 min the solution was
concentrated to a volume of about 3 mL and quickly diluted with acetone
(15 mL). The precipitate was centrifuged, washed with acetone and dried
under reduced pressure to give (R)-26 (23 mg, 90%) of an amorphous
powder. Rf ˆ 0.70 (cellulose, acetone/0.05m ammonium bicarbonate 1:1);
1H NMR (250.13 MHz, D2O): d ˆ 1.90 (s, 3H, NHAc), 3.40 (dd, J(6,7) ˆ
1.7 Hz, J(7,8) ˆ 9.8 Hz, 1H; 7-H), 3.44 (dd, J(8,9a) ˆ 6.9 Hz, J(9a,9b) ˆ
12.0 Hz, 1H; 9a-H), 3.69 (dd, J(8,9b) ˆ 2.6 Hz, 1H; 9b-H), 3.74 (ddd, 1H;
8-H), 3.87 ± 4.01 (m, 3H; 1-H, 5-H, 6-H), 4.22 (ddd, J(3,4) ˆ J(4,6) ˆ 2.7 Hz,
J(4,5) ˆ 7.0 Hz, 1H; 4-H), 4.77 (dd, J(3,P) ˆ 2.7 Hz, 1H; 3-H); MS (FAB,
negative mode, matrix: glycerol/acetic acid/DMSO/water 1:1:1:1): m/z ˆ
‡
‡
NEt3‡Na ) ], 1238.1 for C54H73N5O24P2.
Trisodium 5-acetamido-2,6-anhydro-3,5-dideoxy-1-phosphoryl-d-erythro-
l-gluco-non-2-enitol-1-yl cytidin-5'-yl phosphate [(R)-6] and sodium (1E)
5-acetamido-2,6-anhydro-3,4,5-trideoxy-d-manno-non-1,3-dienitol-1-yl cy-
tidin-5'-yl phosphate [(E)-6']: Freshly prepared compound (R)-28 (81 mg,
65 mmol) was taken up with methanol (5 mL), and palladium on charcoal
(10 mg, 10% Pd) was added. The mixture was stirred vigorously under a
hydrogen atmosphere at normal pressure. The catalyst was filtered off and
washed. Filtrate and washings were combined and sodium methoxide (1m,
0.3 mL) was added. After 15 min acetone (20 mL) was quickly added. The
precipitate was centrifuged, washed with acetone and dried under reduced
pressure. The crude product was purified by HPLC over RP-18 silica gel.
The fractions containing the product were combined and lyophilized, taken
‡
378 [(M Na ) ] 401.2 for C11H18NNa2O10P.
Triethylammonium (N-acetyl-2',3'-di-O-acetylcytidin-5'-yl) (5-acetamido-
1,4,7,8,9-penta-O-acetyl-2,6-anhydro-3,5-dideoxy-d-erythro-l-gluco-non-2-
enitol-1-yl)phosphonate [(R)-27]: The peracetylated phosphonate (R)-25
(125 mg, 167 mmol) was taken up in methanol (15 mL) and stirred for
30 min with palladium on charcoal (10 mg, 10% Pd) under a hydrogen
atmosphere at normal pressure. The mixture was filtered and washed with
methanol. Pyridine (200 mL) was added before the solvent was removed.
The residue was coevaporated with pyridine several times, dried under
reduced pressure and taken up with pyridine again. Dicyclohexylcarbodii-
mide (103 mg, 501 mmol), dimethylaminopyridine (2 mg, 17 mmol) and
compound 9 (93 mg, 251 mmol) were added under exclusion of moisture.
After stirring overnight, water (2 mL) was added and the reaction mixture
was filtered, washed with pyridine and concentrated. Chromatography over
silica gel (ethyl acetate/methanol 5:1 to 3:1 ‡ 1% triethylamine) afforded
(R)-27 (111 mg, 65%) of a colourless glass. Rf ˆ 0.22 (ethyl acetate/
‡
up in water and stirred with IR120 (Na ). Filtration and lyophilization
afforded 42 mg (88%) (R)-6. A second compound was eluted from the
column (after the product), which may have been formed from the unstable
intermediate after the hydrogenolysis. It was also transformed into the
sodium form and lyophilized to give (E)-6' (2 mg, 5%).
(R)-6: HPLC: prep. RP-18 column (flow: 8 mLmin 1, 0.1m triethylammo-
nium hydrogencarbonate buffer‡ 2% acetonitrile): tR ˆ 6.5 min; 1H NMR
(250.13 MHz, D2O): d ˆ 1.94 (s, 3H; NHAc), 3.42 (brd, J(7,8) ˆ 9.2 Hz, 1H;
7-H), 3.49 (dd, J(8'',9a'') ˆ 6.6 Hz, J(9a'',9b'') ˆ 11.8 Hz, 1H; 9a''-H), 3.74
(dd, J(8'',9b'') ˆ 2.6 Hz, 1H; 9b''-H), 3.82 (ddd, 1H; 8''-H), 3.87 (dd,
J(4'',5'') ˆ 4.0 Hz, J(5'',6'') ˆ 7.7 Hz, 1H; 5''-H), 4.05 (dd, 1H; 6''-H), 4.10 ±
4.33 (m, 7H; 2'-H, 3'-H, 4'-H, 5a'-H, 5b'-H, 1''-H, 4''-H), 4.86 (dd,
J(3'',4'') ˆ J(3'',P) ˆ 2.4 Hz, 1H; 3''-H), 6.02 (d, J(1',2') ˆ 3.2 Hz, 1H; 1'-H),
6.03 (d, J(5,6) ˆ 7.5 Hz, 1H; 5-H), 7.92 (d, 1H; 6-H); 31P NMR
(161.70 MHz, D2O): d ˆ 1.00 (d, phosphate), 11.28 (d, phosphonate); MS
(FAB, negative mode, matrix: glycerol/acetic acid/DMSO 1:1:1): m/z ˆ 683
1
methanol 2:1 ‡ 1% triethylamine); H NMR (250.13 MHz, CD3OD): d ˆ
1.35 (t, J ˆ 7.3 Hz, 9H; 3NCH2CH3), 1.90 (s, 3H; NHAc), 2.02 (s, 3H; OAc),
2.03 (s, 3H; OAc), 2.07 (s, 3H; OAc), 2.11 (s, 3H; OAc), 2.12 (s, 3H; OAc),
2.15 (s, 3H; OAc), 2.17 (s, 3H; OAc), 2.23 (s, 3H; NHAc), 3.24 (q, 6H,
3NCH2CH3), 4.23 (dd, J(8'',9a'') ˆ 6.6 Hz, J(9a'',9b'') ˆ 12.4 Hz, 1H; 9a''-
H), 4.25 ± 4.46 (m, 5H; 4'-H, 5a'-H, 5b'-H, 5''-H, 6''-H), 4.52 (dd,
J(8'',9b'') ˆ 2.8 Hz, 1H; 9b''-H), 5.14 (dd, J(3'',4'') ˆ J(3'',P) ˆ 2.8 Hz, 1H;
3''-H), 5.37 (d, J(1'',P) ˆ 14.3 Hz, 1H; 1''-H), 5.38 ± 5.55 (m, 5H; 2'-H, 3'-H,
4''-H, 7''-H, 8''-H), 6.25 (d, J(1',2') ˆ 4.5 Hz, 1H; 1'-H), 7.58 (d, J(5,6) ˆ
7.5 Hz, 1H; 5-H), 8.47 (d, 1H; 6-H); MS (MALDI, negative mode, matrix:
‡
‡
[(M 2Na ‡H ) ] 728.4 for C20H29N4Na3O17P2.
(E)-6': HPLC: prep. RP-18 column (flow: 8 mLmin 1, 0.1m triethylammo-
nium bicarbonate buffer‡ 2% acetonitrile): tR ˆ 21.6 min; 1H NMR
(250.13 MHz, D2O): d ˆ 1.95 (s, 3H; NHAc), 3.49 (dd, J(6'',7'') ꢀ 1.0 Hz,
J(7'',8'') ˆ 8.9 Hz, 1H; 7''-H), 3.57 (dd, J(8'',9a'') ˆ 6.9 Hz, J(9a'',9b'') ˆ
12.6 Hz, 1H; 9a''-H), 3.74 ± 3.79 (m, 3H; 6''-H, 8''-H, 9b''-H), 4.03 ± 4.22
(m, 5H; 2'-H, 3'-H, 4'-H, 5a'-H, 5b'-H), ꢀ 4.6 (m, 1H; 5''-H), 5.70 (ddd,
J(3'',4'') ˆ 10.2 Hz, J(1'',3'') ꢀ J(3'',5'') 1.0 Hz, 1H; 3''-H), 5.90 (d, J(1',2') ˆ
4.2 Hz, 1H; 1'-H), 6.01 (d, J(5,6) ˆ 7.5 Hz, 1H; 5-H), 6.43 (brd, J(1'',P) ˆ
4.6 Hz, 1H; 1''-H), 6.50 (dd, J(4'',5'') ˆ 2.1 Hz, 1H; 4''-H), 7.77 (d, 1H; 6-
H); 31P NMR (161.70 MHz, D2O): d ˆ 2.23 (s, phosphate); MS (FAB,
negative mode, matrix: glycerol/acetic acid/DMSO/water 1:1:1:1): m/z ˆ
‡
ATT): m/z ˆ 917 [(M HNEt3 ) ], 1019.9 for C42H62N5O22P.
Sodium cytidin-5'-yl (5-acetamido-2,6-anhydro-3,5-dideoxy-d-erythro-l-
gluco-non-2-enitol-1-yl)phosphonate [(R)-5]: Compound 27 (85 mg,
83 mmol) was dissolved in methanol (6 mL) and treated with sodium
methoxide (1m) in methanol (0.2 mL). After reaction overnight, the
solution was diluted with acetone (14 mL). Centrifugation, washing with
acetone and drying under reduced pressure afforded 5 (44 mg, 88%), an
amorphous powder. Rf ˆ 0.71 (cellulose, acetone/0.05m ammonium bicar-
bonate 1:1); 1H NMR (250.13 MHz, D2O): d ˆ 1.90 (s, 3H; NHAc), 3.43
(brd, J(7'',8'') ˆ 8.6 Hz, 1H; 7''-H), 3.46 (dd, J(8'',9a'') ˆ 6.7 Hz,
J(9a'',9b'') ˆ 13.7 Hz, 1H; 9a''-H), 3.68 (brd, 1H; 9b''-H), 3.71 (ddd, 1H;
8''-H), 3.91 (dd, J(4'',5'') ˆ 8.1 Hz, J(5'',6'') ˆ 10.8 Hz, 1H; 5''-H), 3.98 ± 4.20
(m, 7H; 2'-H, 3'-H, 4'-H, 5a'-H, 5b'-H, 1''-H, 6''-H), 4.25 (ddd, J(3'',4'') ˆ
1.0 Hz, J(4'',6'') ˆ 5.5 Hz, 1H; 4''-H), 4.83 (dd, J(3'',P) ˆ 1.0 Hz, 1H; 3''-H),
5.82 (d, J(1',2') ˆ 3.4 Hz, 1H; 1'-H), 5.92 (d, J(5,6) ˆ 7.5 Hz, 1H; 5-H), 7.83
(d, 1H; 6-H); 31P NMR (161.70 MHz, D2O): d ˆ 16.69 (s, phosphonate);
MS (FAB, negative mode, matrix: glycerol/acetic acid/DMSO 1:1:1): m/z ˆ
‡
563 [(M Na ) ] 586.4 for C20H28N4NaO13P.
5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-1-O-mesyl-d-
glycero-d-galacto-non-2-enitol (29): Alcohol 19 (354 mg, 794 mmol) was
taken up in a mixture of dichloromethane (10 mL) and triethylamine
(223 mL, 1.59 mmol). The solution was cooled to 08C and mesylchloride
(109 mL, 1.41 mmol) dissolved in dichloromethane (0.5 mL) was added. The
reaction mixture was diluted with dichloromethane (50 mL), after 1 h,
washed with water, dried over magnesium sulfate and concentrated to give
29 (422 mg, quant) as a colourless foam. Rf ˆ 0.40 (toluene/ethyl acetate/
methanol 5:2:1), [a]D ˆ ‡50 (c ˆ 1.0 in CHCl3);1H NMR (250.13 MHz,
CDCl3): d ˆ 1.92 (s, 3H; NHAc), 2.03 (s, 3H; OAc), 2.04 (s, 3H; OAc),
2.05 (s, 3H; OAc), 2.11 (s, 3H; OAc), 3.07 (s, 3H; SO2CH3), 4.10 (dd,
J(8,9a) ˆ 6.2 Hz, J(9a,9b) ˆ 12.2 Hz, 1H; 9a-H), 4.29 (dd, J(5,6) ˆ 7.9 Hz,
J(6,7) ˆ 4.5 Hz, 1H; 6-H), 4.35 (dd, J(8,9b) ˆ 2.6 Hz, 1H; 9b-H), 4.41 (ddd,
J(4,5) ˆ 6.1 Hz, J(5,NH) ˆ 9.4 Hz, 1H; 5-H), 4.48 (d, J(1a,1b) ˆ 12.4 Hz,
1H; 1a-H), 4.56 (d, 1H; 1b-H), 5.14 (d, J(3,4) ˆ 3.4 Hz, 1H; 3-H), 5.33 (dd,
1H; 4-H), 5.38 (ddd, J(7,8) ˆ 6.2 Hz, 1H; 8-H), 5.46 (dd, 1H; 7-H), 5.63 (d,
1H; NH); C20H29NO13S (523.51): calcd C 45.89, H 5.58, N 2.68; found C
45.30, H 5.64, N 2.84.
‡
581 [(M Na ) ] 604.4 for C20H30N4NaO14P.
Triethylammonium (5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-1-di-
benzylphosphoryl-3,5-dideoxy-d-erythro-l-gluco-non-2-enitol-1-yl)
(N-
acetyl-2',3'-di-O-acetylcytidin-5'-yl) phosphate [(R)-28]: Compound (R)-
24 (111 mg, 157 mmol) was treated as described for compound 21 (route b).
A colourless syrup of (R)-28 (143 mg, 73%) was obtained. Rf ˆ 0.30 (ethyl
acetate/methanol 2:1); 1H NMR (250.13 MHz, CD3OD): d ˆ 1.33 (t, J ˆ
1114
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Chem. Eur. J. 1998, 4, No. 6

Sialyltransferase Inhibitors
1106±1115
5-Acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-1-bromo-1,3,5-trideoxy-d-
glycero-d-galacto-non-2-enitol (30): Compound 29 (180 mg, 344 mmol) was
dissolved in DMF (15 mL). Powdered potassium bromide (164 mg,
1.37 mmol) was added. After stirring overnight, the reaction mixture was
concentrated under reduced pressure. Purification over silica gel (toluene/
acetone 1:1) afforded 30 (168 mg, 96%) as a colourless foam. Rf ˆ 0.43
[4] a) F. DallꢁOlio, N. Malagolini, G. DiStefanno, F. Minni, D. Marrano, F.
Serafini-Cessi, Int. J. Cancer 1989, 44, 434 ± 439; b) P. Gessner, S.
Riedl, A. Quentmaier, W. Kemmner, Cancer Lett. 1993, 75, 143 ± 149;
c) W. Kemmner, D. Kruck, P. Schlag, Clin. Exp. Metastasis 1994, 12,
245 ± 254.
[5] A. Harduin-Lepers, M. A. Recchi, P. Delannoy, Glycobiology 1995, 5,
741 ± 758.
1
(toluene/ethyl acetate/methanol 5:2:1), [a]D ˆ ‡51 (c ˆ 1.0 in CHCl3); H
NMR (250.13 MHz, CDCl3): d ˆ 1.92 (s, 3H; NHAc), 2.04 (s, 6H; 2OAc),
2.06 (s, 3H; OAc), 2.11 (s, 3H; OAc), 3.74 (d, J(1a,1b) ˆ 11.0 Hz, 1H; 1a-
H), 3.81 (d, 1H; 1b-H), 4.12 (dd, J(8,9a) ˆ 5.9 Hz, J(9a,9b) ˆ 12.4 Hz, 1H;
9a-H), 4.31 ± 4.40 (m, 2H; 5-H, 6-H), 4.44 (dd, J(8,9b) ˆ 3.0 Hz, 1H; 9b-
H), 5.03 (d, J(3,4) ˆ 3.0 Hz, 1H; 3-H), 5.38 (ddd, J(7,8) ˆ 5.9 Hz, 1H; 8-H),
5.34 ± 5.39 (m, 2H; 4-H, NH), 5.45 (dd, J(6,7) ˆ 3.1 Hz, 1H; 7-H);
C19H26BrNO10 (508.32): calcd C 44.89, H 5.16, N 2.76; found C 44.32, H
5.13, N 3.21.
[6] a) W. Korytnyk, N. Angelino, W. Klohs, R. J. Bernacki, Eur. J. Med.
Chem. 1980, 15, 77 ± 84; b) I. Kijima-Suda, Y. Migamata, S. Toyoshima,
M. Itoh, T. Osawa, Cancer Res. 1986, 46, 858 ± 862; c) S. H. Khan, K. L.
Matta, in Glycoconjugates, Composition, Structure, Function (Eds.:
H. J. Allen, E. C. Kisailus), M. Dekker, New York, 1992, pp. 361 ± 378
and references therein.
[7] a) H. Hashimoto, K. Sato, T. Wakabayashi, H. Kodama, Y. Kajihara,
Carbohydr. Res. 1993, 247, 179 ± 193; b) M. Imamoto, H. Hashimoto,
Tetrahedron Lett. 1996, 37, 1451 ± 1454.
Bistrimethylsilyl (5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-1,3,5-tri-
deoxy-d-glycero-d-galacto-non-2-enitol-1-yl)phosphonate (31): Bromide
30 (12 mg, 24 mmol) was foamed with dichloromethane and dried under
reduced pressure. Subsequently tristrimethylsilyl phosphite (100 mL) was
added. This mixture was heated to 908C under a pressure of 25 mbar for
two hours. Then excess phosphite was removed by distillation under a
pressure of approximately 1 mbar. The product (approximately 20 mg) is a
colourless oil sensitive to moisture. Rf ꢀ 0 (toluene/acetone 1:1); 1H NMR
(250.13 MHz, CDCl3): d ˆ 0.25 ± 0.29 (m, 18H; 6CH3), 1.91 (s, 3H; NHAc),
2.01 (s, 3H; OAc), 2.02 (s, 3H; OAc), 2.03 (s, 3H; OAc), 2.09 (s, 3H; OAc),
2.45 (dd, J(1a,1b) ˆ 14.9 Hz, J(1a,P) ˆ 22.0 Hz, 1H; 1a-H), 2.63 (dd,
J(1b,P) ˆ 21.8 Hz, 1H; 1b-H), 4.17 (dd, J(8,9a) ˆ 7.0 Hz, J(9a,9b) ˆ
12.0 Hz, 1H; 9a-H), 4.24 (d, J(5,6) ˆ J(6,7) ˆ 6.2 Hz, 1H; 6-H), 4.40 ± 4.49
(m, 2H; 5-H, 9b-H), 4.93 (dd, J(3,4) ˆ J(3,P) ˆ 4.3 Hz, 1H; 3-H), 5.15 (ddd,
J(4,5) ˆ 9.6 Hz, J(4,P) ˆ 3.0 Hz, 1H; 4-H), 5.42 (ddd, J(7,8) ˆ 4.6 Hz,
J(8,9b) ˆ 3.5 Hz, 1H; 8-H), 5.51 (dd, 1H; 7-H), 6.59 (d, J(5,NH) ˆ
9.1 Hz, 1H; NH).
[8] a) R. G. Kleineidam, T. Schmelter, R. T. Schwarz, R. Schauer,
Glycoconjugate J. 1997, 14, 57 ± 66; b) D. H. van den Eijnden, T. J.
Martin, R. R. Schmidt, unpublished results; c) G. Dufner, Disserta-
tion, Universität Konstanz, 1997.
[9] a) C. Schaub, B. Müller, R. R. Schmidt, Glycoconjugate J. 1998, 15,
345 ± 354; b) C. Schaub, R. R. Schmidt, Abstract C10, Second Euro-
pean Conference on Carbohydrate Mimics, La Garda (Italy), 1996,
and references therein.
[10] B. Müller, T. J. Martin, C. Schaub, R. R. Schmidt, Tetrahedron Lett.
1998, 39, 509 ± 512.
[11] Recently antisense technology has been investigated in order to
decrease sialyltransferase expression: W. Kemmner, K. Hohaus, P. M.
Schlag, FEBS Lett. 1997, 409, 347 ± 350.
[12] a) J. C. Paulson, J. L. Rearick, R. L. Hill, J. Biol. Chem. 1977, 252,
2363 ± 2371; b) W. D. Klohs, R. J. Bernacki, W. Korytnik, Cancer Res.
1987, 39, 1231 ± 1238.
[13] B. A. Horenstein, M. Bruner, J. Am. Chem. Soc. 1996, 118, 10371 ±
10379; b) Biochemistry 1998, 37, 289 ± 297 and references therein.
[14] Other examples of transition-state analogues of sugar nucleotides:
UDP-Gal: a) K. Frische, Dissertation, Universität Konstanz, 1992;
b) K. Frische, R. R. Schmidt, Bioorg. Med. Chem. Lett. 1993, 3, 1747 ±
1750; GDP-Fuc: c) S. Cai, M. R. Strond, S. Hakomori, T. Toyokuni, J.
Org. Chem. 1992, 57, 6693 ± 6696.
5-Acetamido-1,2,3-tri-O-acetyl-4,8-anhydro-5,6,7,9-tetradeoxy-d-manno-
non-6,8-dienitol (32): Ester 31 (19 mg, 24 mmol) was dissolved in dry
acetone (1 mL). The solution was cooled to 08C and treated with
tetrabutylammonium fluoride solution (1m) in THF (100 mL). After stirring
for 30 min it was concentrated and purified over silica gel (toluene/acetone
1:1). Product 32 is a colourless oil which decomposes on storage. Rf ˆ 0.48
(toluene/acetone 1:1); 1H NMR (250.13 MHz, CDCl3): d ˆ 1.96 (s, 3H;
NHAc), 2.03 (s, 3H; OAc), 2.05 (s, 3H; OAc), 2.08 (s, 3H; OAc), 3.99 (dd,
J(4,5) ˆ 7.1 Hz, J(3,4) ˆ 2.7 Hz, 1H; 4-H), 4.16 (dd, J(1a,1b) ˆ 12.5 Hz,
J(1a,2) ˆ 5.6 Hz, 1H; 1a-H), 4.26 (s, 1H; 9a-H), 4.44 (dd, J(1b,2) ˆ 2.2 Hz,
1H; 1b-H), 4.54 (s, 1H; 9b-H), 4.57 ± 4.58 (m, 1H; 5-H), 5.31 (dd, J(2,3) ˆ
6.8 Hz, 1H; 3-H), 5.36 (ddd, 1H; 2-H), 5.55 (d, J(5,NH) ˆ 8.8 Hz, 1H; NH),
5.77 (brdd, J(6,7) ˆ 3.3 Hz, J(5,7) ˆ 1.0 Hz, 1H; 7-H), 6.10 (dd, J(5,6) ˆ
2.0 Hz, 1H; 6-H).
[15] N. B. Tarusova, C. G. Zavgorodnii, T. I. Osipova, Bioorg. Khim. 1986,
11, 802 ± 807.
[16] Commercially available from Fluka.
[17] T. Morita, Y. Okamoto, H. Sakurai, Bull. Chem. Soc. Jpn. 1981, 54,
267 ± 273.
[18] F. Amann, Dissertation, Universität Konstanz, 1997.
[19] For details, see ref. [9].
[20] CSC Chem3D (MM2).
[21] Commercially available from Aldrich.
Sialyltransferase assay: The inhibitors 1a, 1b, 2, 3, 4, (R)-5, (R)-6 and (E)-6'
were tested according to the published procedure;[9] a-(2,6)-sialyltransfer-
ase from rat liver (EC2.4.99.1) was purchased from Sigma.
[22] D. J. Whelan, Aust. J. Chem. 1973, 26, 4155 ± 4156.
[23] C. L. D. Jenkins, R. L. N. Harris, A. G. McFadden, Biochem. Int. 1986,
12, 619 ± 625.
[24] a) P. Meindl, H. Tuppy, Monatsh. Chem. 1965, 96, 803 ± 815; b) ibid.
1969, 100, 1295 ± 1306.
[25] W. Bannwarth, A. Trzeciak, Helv. Chim. Acta 1987, 70, 175 ± 186.
[26] a) Y. Kajihara, K. Koseki, T. Ebata, H. Kodama, H. Matsusita, H.
Hashimoto, Carbohydr. Res. 1994, 264, C1 ± C5; b) J. Org. Chem. 1995,
60, 5732 ± 5735.
Acknowledgements: This work was supported by the Deutsche For-
schungsgemeinschaft and the Fonds der Chemischen Industrie. C.S. is
grateful for a stipend from the Fonds der Chemischen Industrie. We are
grateful to Dr. Armin Geyer for his help in the structural assignments.
Received: December 10, 1997 [F920]
[27] D. B. Dess, J. C. Martin, J. Am. Chem. Soc. 1991, 113, 7277 ± 7287.
[28] P. Deslongchamps, in Stereoelectronic Effects in Organic Chemistry,
Pergamon, Oxford, 1983.
[29] A. C. Satterthwait, F. H. Westheimer, J. Am. Chem. Soc. 1978, 100,
3197 ± 3203.
[1] a) A. Rosenberg, in Biology of Sialic Acids, Plenum, New York, 1995;
b) F. DallꢁOlio, N. Malagolini, F. Serafini-Cessi, Int. J. Cancer 1992, 50,
325 ± 330; c) J. Morgenthaler, J. Kemmner, R. Brossmer, Biochem.
Biophys. Res. Commun. 1990, 171, 860 ± 866.
[2] a) R. J. Bernacki, Science 1977, 195, 557 ± 580; b) Q. Zhou, S.
Hakomori, K. Kitamura, Y. Igarashi, J. Biol. Chem. 1994, 269,
1959 ± 1965.
[3] a) M. Fogel, P. Alvogt, V. Schirrmacher, J. Exp. Med. 1983, 157, 371 ±
376; b)S. Hakomori, Chem. Phys. Lipids 1986, 42, 209 ± 233; c) J. W.
Dennis, in Cell Surface Carbohydrates and Cell Development (Ed.: M.
Fukada), CRC, Boca Raton, 1991, pp. 174 ± 175.
[30] H. J. Gross, U. Rose, J. M. Krause, J. C. Paulson, K. Schmid, R. E.
Feeney, R. Brossmer, Biochemistry 1989, 28, 7386 ± 7392.
[31] J. A. Dale, H. S. Mosher, J. Am. Chem. Soc. 1973, 95, 512 ± 519.
[32] F. Hammerschmidt, Y.-F. Li, Tetrahedron 1994, 50, 10253 ± 10264 and
references therein.
Chem. Eur. J. 1998, 4, No. 6
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0947-6539/98/0406-1115 $ 17.50+.25/0
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