Stereoselective synthesis of phosphoramidate α(2-6)sialyltransferase transition-state analogue inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00672-3

The asymmetric synthesis of novel, potent phosphoramidate α(2-6)sialyltransferase transition-state analogue inhibitors such as (R) -9 (K_i=68 μM) is described, via condensation of cytidine phosphitamide 6 with key chiral, non-racemic α-aminophosphonates, prepared in >98% ee by Mitsunobu azidation followed by Staudinger reduction of the corresponding chiral, non-racemic α-hydroxyphosphonates.

Full text of DOI:10.1016/S0960-894X(03)00672-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3351–3354
Stereoselective Synthesis of Phosphoramidate
ꢀ(2-6)Sialyltransferase Transition-State Analogue Inhibitors
Danielle Skropeta,y Ralf Schworer and Richard R. Schmidt*
Fachbereich Chemie, Universitaet Konstanz, Fach M 725, D-78457 Konstanz, Germany
Received 23 February 2003;accepted 29 April 2003
Abstract—The asymmetric synthesis of novel, potent phosphoramidate a(2-6)sialyltransferase transition-state analogue inhibitors
such as (R)-9 (Ki=68 mM) is described, via condensation of cytidine phosphitamide 6 with key chiral, non-racemic a-aminophos-
phonates, prepared in >98% ee by Mitsunobu azidation followed by Staudinger reduction of the corresponding chiral, non-race-
mic a-hydroxyphosphonates.
# 2003 Elsevier Ltd. All rights reserved.
Sialyltransferases catalyse the addition of sialic acid
residues from the b-glycoside donor cytidine monopho-
sphate N-acetylneuraminic acid (CMP-Neu5Ac) to the
terminal position of growing oligosaccharide chains of
glycoconjugate acceptors, to produce a-sialosides
(Scheme 1) that influence a variety of physiologically
and pathologically important processes.1 Inhibitors of
sialyltransferases are thus valuable tools in elucidating
the role of sialyl residues in biological systems, in partic-
ular in light of the recent correlation between a(2-6)-
sialylation of N-acetyllactosamine and B lymphocyte
activation and immune function, which could find
medicinal application.2 As shown by us,3À7 transition
and p-substituted benzyl (Scheme 2), quinolinyl, naph-
thyl, and pyridyl rings, to produce readily accessible,
potent aromatic inhibitors of rat liver a(2-6)-sialyl-
transferase. Furthermore, we have recently developed
an asymmetric route to the most promising benzyl
a(2-6)-ST inhibitors ([a]: R=H, OPh, CF3, Scheme 2).11
To further study the influence of heteroatoms on bind-
ing affinity, we have introduced a phosphoramidate
moiety12 into our inhibitors ([b], Scheme 2). Extending
our work on the development of stereoselective routes
to sialyltransferase inhibitors, we report here our pre-
liminary results on the asymmetric synthesis and bio-
¼
¼
state (TS ) analogues based on the recently sup-
activity of novel, non-racemic phosphoramidate TS
analogue inhibitors, (R)- and (S)-9.
ported3,4,8 SN1 type mechanism (Scheme 1) involving
partial dissociation of the CMP moiety and con-
comitant formation of a planar oxocarbenium structure
It was envisioned that the desired phosphoramidate
inhibitors (R)- and (S)-9 could be obtained by modify-
ing our procedure11 for the asymmetric synthesis of
substituted benzylphosphonate derived inhibitors ([a],
Scheme 2), and employing chiral, non-racemic a-amino-
phosphonates with standard phosphitamide coupling
methodology ([b], Scheme 2).
¼
in the TS , which comprise (i) a planar anomeric car-
bon, (ii) an increased distance between the anomeric
carbon and the CMP leaving group, and (iii) at least
two negative charges close to the glycosylation cleavage
site, exhibit high affinity to sialyltransferases.3À7,9,10
In previous studies,6,11 we have shown that the neur-
¼
aminyl residue of TS analogues can be replaced by a
wide range of aryl and hetaryl moieties, including m-
a-Aminophosphonic acids are important isosteres of
a-amino acids showing a variety of biological effects.13
Thus several routes for the synthesis of chiral, non-
racemic a-aminophosphoryl compounds have already
been reported, including enzymatic14 or chemical reso-
lution,15 addition of phosphite anions to N-glycosylni-
trones,16 and via a-azidophosphonates derived from
chiral, non-racemic a-hydroxyphosphonates.17 We
*Corresponding author. Tel.: +49-7531-88-2538;fax: +49-7531-
88-3135;e-mail: richard.schmidt@uni-konstanz.de
yCurrent address: University of Sydney, School of Chemistry, NSW,
2006, Australia. Tel.: +61-2-9351-5747;fax: +61-2-9351-3329;e-mail:
d.skropeta@chem.usyd.edu.au
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00672-3

3352
D. Skropeta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3351–3354
stabilised anions of the benzylphosphonates 1a–c, with
camphorsulfonyloxaziridine (+)-2 as described pre-
viously (Scheme 3).18 Mitsunobu inversion reaction of
the a-hydroxyphosphonates (S)-3a–c furnished the cor-
responding a-azidophosphonates (R)-4a–c.20 The azides
(R)-4a–c were subjected to a Staudinger reaction21 and
converted to the corresponding iminophosphoranes and
subsequently hydrolysed with water in situ, to give the
desired a-aminophosphonates (R)-5a–c in high yield
and excellent enantiomeric excess (ee) (Scheme 3).22 The
enantiomers (S)-5a–c (not shown) were prepared in the
analogous manner employing (À)-2.
The enantiomeric purity and absolute configuration of
the a-aminophosphonates (R)- and (S)-5a–c were
established by the chemical shift differences of the 31P
1
and H NMR spectral resonances of the corresponding
Scheme 1. Mechanism of sialyltranferase catalysed sialylation.
amides derived from (S)-methyl mandelic acid (MMA)
as described by Trost et al.23 Employing the accepted
model for the conformation of the (S)-N-MMA amides
of a-aminophosphonates,23 the amides derived from
(S)-a-aminophosphonates have the phosphorus atom
shielded by the mandelate phenyl ring and are thus
shifted upfield relative to the (R)-amides.24 Thus, for the
observed chemical shift differences obtained the phos-
phorus atom of the (S)-N-MMA amides of (S)- and
(R)-5a–c (Table 1), confirmed that the Mitsunobu reac-
tion proceeded stereoselectively with inversion of con-
figuration at the chiral centre.
Employing the conditions described by Abraham and
Wagner for the synthesis of phosphoramidate amino
acid diesters of antiviral nucleosides,25 cytidine phos-
phitamide 6 was converted to its methyl phosphite by
treatment with methanol and tetrazole, and then con-
densed with chiral a-aminophosphonate (R)-5a in the
presence of iodine, to give, after work up and flash
chromatographic purification, (R)-7 in 42% yield
(Scheme 4).
Scheme 2. Asymmetric routes to sialyltranferase inhibitors.
employed the latter approach for the asymmetric
synthesis of a-aminophosphonates 5a–c (Scheme 3),
taking advantage of our recently reported method18 for
the synthesis of non-racemic a-hydroxyphosphonates
and phosphonic acids via stereoselective hydroxyl-
ation19 of diallyl benzylphosphonates.
The requisite chiral diallyl a-hydroxyphosphonates
(S)-3a–c were prepared by reaction of the phosphoryl
In our previous syntheses5À7,10 of sialyltransferase inhi-
bitors (e.g., [a], Scheme 2) we have employed triethyl-
amine for the base-catalysed cleavage of the cyanoethyl
phosphate protecting group, however, in the case of the
phosphoramidate (R)-7, this led to degradation. This
problem was solved by employing TBAF/THF for the
phosphoramidate deprotection,26 furnishing the triethyl-
ammonium salt (R)-8,27 in good yield and high ee,
although some racemisation was observed. Elaboration
to the target compound (R)-928 was performed using
our standard procedure,5À7,10 comprising Pd-catalysed
deallylation, followed by deacetylation, purification by
reverse-phase flash chromatography, and conversion to
Table 1. NMR shifts of the (S)-N-MMA amides of (S)-5a–c and (R)-5a
Compd
[a]D
31P NMR d
(major)
31P NMR d
(minor)
Á
(ppm)
%
ee
(S)-5a
(S)-5b
(S)-5c
(R)-5a
À11
À9
22.73 (>99)
22.15 (>99)
21.86 (96)
23.04 (<1)
22.48 (<1)
22.17 (4)
0.31
0.33
0.31
0.31
>98
>98
92
Scheme 3. Reaction conditions: (i) 1.5 equiv NaHMDS, THF,
À78 ꢀC, 20 mn then 2 equiv (+)-(2), THF, À78 ꢀC, 3 h, 3a–c: 36%,
46%, 42%;(ii) PPh 3, DEAD, HN3, 0 ꢀC–rt, 20 h, 4a–c: 89, 88, 98%;
(iii) PPh3, benzene, rt, 2 h, then H2O, 50–55 ꢀC, 5 h, 5a–c: 85, 84, 75%.
À17
+11
23.04 (>99)
22.73 (<1)
>98

D. Skropeta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3351–3354
3353
Scheme 4. Reagents and conditions: (i) 1 equiv cytidine-phosphitamide 6, 4 equiv tetrazole, 2 equiv MeOH, CH3CN, 2 h then 4.05 equiv (R)-(5a),
1 equiv I2, THF, 3 h, 42%;(ii) 2 equiv TBAF, THF, 1 h, 40%;(iii) 20 mol.% Pd(Ph 3)4, 10 equiv dimedone, THF, 18 h;(iv) RP-FC (1:3 EtOH–H 2O);
(v) 25% NH3–H2O, 18 h;(vi) RP-FC (95:5 H O–CH3CN);(vii) IR 120 Na + (61% over five steps).
2
References and Notes
the trisodium salt through ion-exchange chromato-
graphy (IR-120 Na+).
1. Morgenthaler, J.;Kemner, J.;Brossmer, R. Biochem. Bio-
phys. Res. Commun. 1990, 171, 860. Rosenberg, A. Biology of
Sialic Acids;Plenum: New York, 1995. Reutter, W.;Sta sche,
R.;Stehling, P.;Baum, O. In Glycosciences, Gabius, H. J.,
Gabius, S., Eds.;Chapman and Hall: Weinheim, 1997;pp 245–
259. Mammen, M.;Choi, S.-K.;Whitesides, G. M. Angew.
Chem. 1998, 110, 2908. Angew. Chem. Int. Ed. 1998, 37, 2754.
von Itzstein, M.;Colman, P. Curr. Opin. Struct. Biol. 1996, 6,
703.
The competitive inhibition of a(2-6)-ST by the phos-
phoramidates was investigated employing a previously
reported assay3 and gave (R)-9: Ki=68Æ24 mM, and
(S)-9: Ki=140Æ30 mM. Phosphoramidate (R)-9 binds
with similar affinity to a(2-6)-ST from rat liver (E.C.
2.4.99.1) as the natural substrate CMP-Neu5Ac
(KM=46Æ7 mM).3 However, the analogous (R)-phos-
phodiester ([a], R=H, Scheme 2) previously prepared by
us,6 differing only by the nature of the linkage (phospho-
diester vs phosphoramidate), shows approximately two
orders of magnitude higher affinity. Thus, it appears that
the phosphate ester linkage may be of importance in
binding to a(2-6)-ST.12,29 Furthermore, in the case of
phosphodiester inhibitors, replacement of the carboxyl-
ate group by a phosphonate group, generally improves
binding affinity to a(2-6)-ST by around one order of
magnitude.6 This may also contribute to the improved
binding affinities seen for our N-(a-phosphoryl)phos-
phoramidate inhibitors (R)- and (S)-9 compared to simi-
lar N-(a-carboxy)phosphoramidate inhibitors reported
recently,12 however, comparison of a greater number of
inhibitors will be required before a clear trend emerges.
2. Hennet, T.;Chui, D.;Paulson, J. C.;Marth, J. D.
Natl. Acad. Sci. U.S.A. 1998, 95, 4504.
Proc.
3. Schaub, C.;Mu ller, B.;Schmidt, R. R. Glycoconjugate J.
1998, 15, 345.
4. Muller, B.;Martin, T. J.;Schaub, C.;Schmidt, R. R. Tetra-
hedron Lett. 1998, 39, 509.
5. Amann, F.;Schaub, C.;Mu ller, B.;Schmidt, R. R. Chem.
Eur. J. 1998, 4, 1106.
6. Muller, B.;Schaub, C.;Schmidt, R. R. Angew. Chem. 1998,
110, 3021; Angew. Chem. Int. Ed. 1998, 37, 2893.
7. Schworer, R.;Schmidt, R. R. J. Am. Chem. Soc. 2002, 124,
1632.
8. Bruner, M.;Horenstein, B. A. Biochemistry 2000, 39, 2261.
Horenstein, B. A.;Bruner, M. Biochemistry 1998, 37, 289.
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11. Skropeta, D.;Schwo rer, R.;Haag, T.;Schmidt, R. R.
Manuscript in preparation.
12. During preparation of this article phosphoramidate
amino-acid based a(2-6)ST inhibitors with Ki values in the
range 0.73–3.8 mM were reported: Whalen, L. J.;McEvoy,
K. A.;Halcomb, R. L. Bioorg. Med. Chem. Lett. 2003, 13,
301.
13. Kafarski, P.;Lejczak, B. Phosphorus, Sulfur, Silicon Relat.
Elem. 1991, 63, 193.
14. Solodenko, V. A.;Kasheva, T. N.;Kukhar, V. P.;
Kozlova, E. V.;Mironenko, D. A.;Svedas, V. K. Tetrahedron
1991, 47, 3989.
In conclusion, we have described the stereoselective
synthesis of novel phosphoramidate a(2-6)-ST inhibitors
such as (R)-9, via condensation of cytidine phosphit-
amide with key chiral, non-racemic a-aminophosphonates
obtained in >98% ee by Mitsunobu azidation followed
by Staudinger reduction of the corresponding non-race-
mic a-hydroxyphosphonates, providing further exam-
¼
ples of TS analogues exhibiting high affinity to
sialyltransferases. This asymmetric route provides the
first access to novel N-(a-phosphoryl)-phosphoramidate
nucleosides, which may prove valuable in the area of
phosphoramidate nucleoside prodrugs,25 as well as use-
ful alternatives to the difficult construction of the N-acyl
phosphoramidate linkage found in several nucleotide
antibiotics.26
15. Lammerhofer, M.;Zarbl, E.;Lindner, W.;Simov, B. J.;
Hammerschmidt, F. Electrophoresis 2001, 22, 1182.
16. Huber, R.;Vasella, A. Helv. Chim. Acta 1987, 70, 1461.
17. Gajda, T. Tetrahedron: Asymmetry 1994, 5, 1965.
18. Skropeta, D.;Schmidt, R. R. Tetrahedron: Asymmetry
2003, 14, 265.
Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinschaft and the Fonds der Chemischen
Industrie.
19. (a) Pogatchnik, D. M.;Wiemer, D. F. Tetrahedron Lett.
1997, 38, 3495. (b) Cermak, D. M.;Yanming, D.;Wiemer,
D. F. J. Org. Chem. 1999, 64, 388.

3354
D. Skropeta et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3351–3354
20. Selected data for 4a: 1H NMR (CDCl3, 250 MHz) d
61, 9223. Moriguchi, T.;Yanagi, T.;Kunimori, M.;Wada, T.;
Sekine, M. J. Org. Chem. 2000, 65, 8229.
4.30–4.57 (m, 4H, H10), 4.75 (d, J(H,P)=16.6 Hz, 1H, CHP),
2
5.15–5.33 (m, 4H, H30), 5.72–5.95 (m, 2H, H20), 7.35–7.50 (m,
27. Selected data for (R)-8: H NMR (MeOH-d4, 250 MHz) d
1
1
3
5H, ArH); 31P NMR (CDCl3, 162 MHz) d 20.0; 4b: H NMR
1.25 (t, J=7.3 Hz, 9H, NCH2CH3), 2.05 (s, 3H, OAc), 2.08,
(CDCl3, 250 MHz) d 4.33–4.58 (m, 4H, H10), 4.72 (d,
2J(H,P)=16.6 Hz, 1H, CHP), 5.18–5.35 (m, 4H, H30), 5.75–5.95
2.09y (s, 3H, OAc), 2.19, 2.20y (s, 3H, NHAc), 3.06 (q, 3J=7.3
Hz, 6H, NCH2CH3), 3.65–4.50 (m, 3H, H40, H5a0, H5b0), 4.37–
4.80 (m, 5H, CH2–CH¼CH2, CHN), 5.10–5.42 (m, 6H, H20,
0
1
(m, 2H, H2 ), 6.92–7.38 (m, 9H, ArH); 4c: H NMR (CDCl3,
250 MHz) d 4.50 (m, 4H, H10), 4.84 (d, J(H,P)=16.5 Hz, 1H,
H30 and CH¼CH2), 5.72–5.87 (m, 1.8H, CH¼CH2), 5.87–6.03y
2
CHP), 5.18–5.32 (m, 4H, H30), 5.75–5.92 (m, 2H, H20),
7.45–7.68 (m, 4H, ArH); 31P NMR (CDCl3, 162 MHz) d 19.1.
21. Staudinger, M. Helv. Chim. Acta 1919, 2, 635.
(m, 0.2H, CH=CH2), 6.08 (d, J(H1 ,H2 )=4.6 Hz, 0.9H, H10),
3
0
0
6.14y (d, J(H1 ,H2 )=5.7 Hz, 0.1H, H10), 7.24–7.32 (m, 3H, o-
3
0
0
and p-ArH), 7.45–7.49 (m, 3H, H50 and m-ArH), 8.24 (d,
22. Selected data for 5a: H NMR (CDCl3, 250 MHz) d 1.83
J(H6 ,H5 )=7.6 Hz, 0.9H, H60), 8.36y (d, J(H6 ,H5 )=7.6 Hz,
0 0 0 0
1
3
3
(brs, 2H, NH2), 4.27 (d, 2J(H,P)=16.3 Hz, 1H, CHP), 4.28–4.49
(m, 4H, H10), 5.11–5.32 (m, 4H, H30), 5.72–5.93 (m, 2H, H20),
7.22–7.46 (m, 5H, ArH); 31P NMR (CDCl3, 162 MHz) d 26.0;
MALDIMS (DHB) m/z 306 ([MK]+, 100%), 294 (34), 290
0.1H, H60), ysignals belonging to minor (S)-diastereomer
(15%);MALDIMS (DHB) m/z 698 ([MÀEt3NH]À, 100%), 658
([MÀEt3NÀAll]À, 16), 799.7 for C34H51N5O13P2.
28. Selected data for (R)-9: 1H NMR (D2O, 600 MHz) d 3.20y
([MNa]+, 30), 267.3 for C13H18NO3P; 5b: H NMR (CDCl3,
(m, 0.1H, H30), 3.39 (t, J(H3 ,H2 )ꢁ J(H3 ,H4 )=5.4 Hz, 0.9H,
1
3
3
0
0
0
0
250 MHz) d 1.72 (br.s, 2H, NH2), 4.23 (d, J(H,P)=17.7 Hz,
H30), 3.67–3.73 (m, 3H, H20, H5a0, H5b0), 3.87 (m, 1H, H40),
2
1H, CHP), 4.38–4.52 (m, 4H, H10), 5.12–5.30 (m, 4H, H30),
5.72–5.93 (m, 2H, H20), 6.88–7.35 (m, 9H, ArH);MALDIMS
(DHB) m/z 398 ([MK]+, 50%), 382 ([MNa]+, 100), 370 (52),
360 ([MH]+, 16), 358 ([MÀAll+H+K]+, 24), 359.4 for
C19H22NO4P; 5c: 1H NMR (CDCl3, 250 MHz) d 1.70 (brs,
4.12 (dd, J(H,P)=9.7 Hz, J(H,P)=21.9 Hz, 1H, H10), 5.58 (d,
3
2
J(H1 ,H2 )=3.5 Hz, 0.9H, H10), 5.75 (d, J(H1 ,H2 )=5.8 Hz,
3
3
0
0
0
0
0.1H, H10),y 5.93y (d, J(H5,H6)=7.6 Hz, 0.1H, H5), 5.99 (d,
3J(H5,H6)=7.6 Hz, 0.9H, H5), 7.07–7.29 (m, 5H, ArH), 7.56 (d,
3J(H6,H5)=7.6 Hz, 0.9H, H6), 7.66y (d, 3J(H6,H5)=7.6 Hz, 0.1H,
3
2H, NH), 4.30–4.52 (m, 4H, H10), 4.45 (d, J(H,P)=16.4 Hz,
H6) signals belonging to minor (S)-diastereomer (15%); 13C
2
y
1H, CHP, overlapping signal), 5.12–5.30 (m, 4H, H30),
5.70–5.92 (m, 2H, H2), 7.42–7.72 (m, 4H, ArH).
23. Trost, B. M.;Bunt, R. C.;Polley, S. R. J. Org. Chem.
1994, 59, 4202.
24. In contrast to a-hydroxyphosphonates, where a different
conformation of the O-MMA esters, results in an upfield shift
of (R)-esters relative to (S)-esters. See: Kozlowski, J. K.;Rath,
N. P.;Spilling, C. D. Tetrahedron 1995, 51, 6385, and ref 18.
25. Abraham, T. W.;Wagner, C. R. Nucleosides Nucleotides
1994, 13, 1891.
NMR (D2O, 63 MHz) d 56.4 (d, 1J(C,P)=141.8 Hz, CHP), 63.5
(C50), 69.7 (C30), 75.3 (C20), 84.0 (1J(C,P)=14.2 Hz, C40), 89.8
(C10), 97.4 (C5), 127.9, 128.9, 129.0, 129.2, 129.3 (ArCH),
141.6 (ArC), 142.7 (C6), 158.7 (C2), 167.3 (C4); 31P NMR
3
(D2O, 162 MHz) d 8.27 (d, J(P,P)=42.1 Hz, NHPO3), 18.58
3
(d, J(P,P)=42.1 Hz, PO3);MALDIMS (CHCA) m/z 535
([MÀNa]À, 19%), 513 ([MÀ2Na+H]À, 100), 513
([MÀ3Na+2H]À, 56), 558.3 for C16H19N4Na3O10P2.
29. Compare also the phosphoramidate (S)-5d in ref 12
(Ki=3.8 mM), with the analogous phosphodiester (S)-7
(Ki=23 mM) in ref 6, prepared by us.
26. Moriguchi, T.;Wada, T.;Sekine, M. J. Org. Chem. 1996,