Synthesis and biological evaluation of gem-diamine 1-N-iminosugars related to L-iduronic acid as inhibitors of heparan sulfate 2-O-sulfotransferase

Article abstract of DOI:10.1016/j.bmcl.2005.10.055

A variety of gem-diamine 1-N-iminosugars related to L-iduronic acid were synthesized and evaluated as inhibitors of heparan sulfate uronyl 2-O-sulfotransferase using an in vitro enzyme assay. Two iminosugars containing guanidino groups acted as potent in

Full text of DOI:10.1016/j.bmcl.2005.10.055

Bioorganic & Medicinal Chemistry Letters 16 (2006) 532–536
Synthesis and biological evaluation of gem-diamine
1-N-iminosugars related to L-iduronic acid as inhibitors of
heparan sulfate 2-O-sulfotransferase
Jillian R. Brown,a Yoshio Nishimurab and Jeffrey D. Eskoa,*
aDepartment of Cellular and Molecular Medicine, Glycobiology Research and Training Center,
University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA
bMicrobial Chemistry Research Center, 3-14-23, Kamiosaki Sinagawa-ku, Tokyo 141-0021, Japan
Received 30 September 2005; revised 15 October 2005; accepted 18 October 2005
Available online 4 November 2005
Abstract—A variety of gem-diamine 1-N-iminosugars related to L-iduronic acid were synthesized and evaluated as inhibitors of hep-
aran sulfate uronyl 2-O-sulfotransferase using an in vitro enzyme assay. Two iminosugars containing guanidino groups acted as
potent inhibitors of the enzyme.
Ó 2005 Elsevier Ltd. All rights reserved.
Heparan sulfate plays important roles in development
and normal physiologic processes. Many of their func-
tions depend on specific sequences of sulfated saccha-
rides, which act as recognition motifs to bind and
regulate the activities of protein ligands.1 Heparan sul-
fate assembles by the copolymerization of N-acetyl-D-
glucosamine (GlcNAc) and D-glucuronic acid (GlcA).
The chain undergoes a series of modifications that in-
clude N-deacetylation and N-sulfation of GlcNAc units,
epimerization of D-GlcA to L-iduronic acid (D-IdoA),
and multiple sulfation reactions at C-6 and C-3 of gluco-
samine units and C-2 of IdoA and GlcA units.2 These
modifications occur in contiguous sections of the chain,
creating binding sites for adhesion proteins, enzymes
and enzyme inhibitors, and a variety of growth factors
involved in early development.
donor,
adenosine
30-phosphate-50-phosphosulfate
(PAPS), to IdoA residues and with lesser efficiency to
GlcA.4 Only a single isoform of the enzyme is known,5
making it an attractive drug target. In theory, inhibitors
might block the action of key growth factors required
for tumorigenesis.3,6
In this report, a series of gem-diamine 1-N-iminosugar
analogs were prepared that are structurally similar to
IdoA (Fig. 1). gem-Diamine 1-N-iminosugars are cyclic
monosaccharides with a nitrogen atom in place of the
O
OH
OH
CO H
2
HO C
2
O
OH
OH
OH
OH
OH
Altering heparan sulfate biosynthesis could be of thera-
peutic benefit for treating disorders related to aberrant
growth, such as tumor growth and metastasis.3 To this
end, we have synthesized and tested a variety of gem-
diamine 1-N-iminosugars related to L-iduronic acid as
inhibitors of heparan sulfate uronyl 2-O-sulfotransferase
(2OST). 2OST catalyzes sulfate transfer from the sulfate
OH
L-Iduronic acid
HO C
2
OH
NR
HO C
2
HO
NH
RHN
HO
NR'R"
NHR
L-Alturonic acid-type iminosugars
Uronic acid-type iminosugars
Keywords: Synthesis; Iminosugars; Inhibitors; Glycosyltransferase;
Heparan sulfate.
Figure 1. L-Iduronic acid, uronic acid-type gem-diamine 1-N-imino-
sugars, and L-alturonic acid-type gem-diamine 1-N-iminosugars
(R,R0,R00 = functional groups).
*
Corresponding author. Tel.: +1 858 822 1100; fax: +1 585 534
5611; e-mail: jesko@ucsd.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.055

J. R. Brown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 532–536
533
involved an intramolecular Michael addition of O-imi-
date to a,b-unsaturated ester 11 through cis-oxiamina-
tion.13 Compound 11 was easily obtainable from
siastatin B (8) and smoothly underwent cis-oxiamina-
tion by treatment with trichloroacetonitrile to give the
desired oxazoline 12. Hydrolysis of the oxazoline ring
of 12 was achieved by treatment with p-toluenesulfonic
acid in a mixture of pyridine and water to afford the tri-
chloroacetamide 13 in 77% yield. Reductive cleavage of
the trichloroacetyl group with sodium borohydride gave
the amine 14 in 57% yield. Compound 14 can be then
utilized for guanidine formation by use of N,N0-bis(-
tert-butoxycarbonyl)thiourea in the presence of mercu-
ric chloride. The reaction efficiently proceeded to
afford the bis-Boc-protected guanidine 15 in 95% yield.
Compound 15 was transformed into 3 by removal of
the protecting groups with hydrogen chloride in dioxane
in 90% yield. Compound 4 was also obtained by remov-
al of the protecting groups from amine 14 with hydrogen
chloride in dioxane in 93% yield.
OH
1
NR
HO C
R HN
2
2
3
NHR
1
2
3
1: R =R =H, R =COCH
3
1
2
3
2: R =H, R =C(=NH)NH , R =-COCH
2
3
1
2
3
3: R =COCH(CH CH ) , R =C(=NH)NH , R =COCH
3
2
3 2
2
1
2
3
4: R =COCH(CH CH ) , R =H, R =-COCH
3
2
3 2
1
2
3
5: R =H, R =C(=NH)NH , R =-COCF
2
3
OH
OH
7
NH
HO C
NH
NHCOCF
2
HO C
2
3
NHCOCF
HO
3
HO
6
CO H
2
HO
CO H
2
HO
HO
NH
NHCOCF
HO
NH
NHR
3
9
8: R=-COCH
3
10: R=-COCF
3
Scheme 1. L- and D-Uronic acid-type gem-diamine 1-N-iminosugars.
We tested 2-O-desulfated heparin (2-DSH) an acceptor
substrate using assay conditions described previously.14
The assays were proportional to time up to 30 min at
37 °C and showed conventional saturation kinetics with
2-O-desulfated heparin (Km ꢀ 4 lM, 80 lg/ml) as sub-
strate. In each assay, the iminosugar inhibitor was add-
ed at 1 mM (ꢀ250 times Km for 2-O-desulfated heparin).
Assays of gem-diamine 1-N-iminosugars 1–10 revealed
that 2 and 3 inhibited sulfate transfer by 76% and
84%, respectively (Fig. 2A). A dose–response curve
showed that 2 and 3 caused 80% inhibition at the lowest
concentration tested (25 lM) (Figs. 2B and C). In con-
trast to these results, addition of 2 or 3 to cultured Chi-
nese hamster ovary cells did not affect sulfation of
heparan sulfate in vivo. The lack of activity most likely
anomeric carbon.7 Two of the compounds containing
positively charged guanidinium groups act as inhibi-
tors of 2OST. This directed approach complements
more general high throughput screening methods of
chemical libraries directed at inhibitors of other
sulfotransferases.8
1-N-Iminosugars of 1, 2, and 5,7 6, 7, and 99 and 1010
were prepared by methods previously reported (Scheme
1). Natural siastatin B (8) was obtained from Streptomy-
ces culture.11 The synthetic method for 1-N-iminosugars
3 and 4 is outlined in Scheme 2.12 Synthesis of 3 and 4
O
O
O
CO CHPh
2
2
CO CHPh
2
2
N
CO CHPh
2
2
N
O
N
AcHN
AcHN
AcHN
b
OH
a
HO
N
NHCCCl
11
3
CCl
O
3
13
12
O
OH
N
HO C
2
2
H N
d
NHAc
c
4
O
CO CHPh
2
2
O
N
OH
e
O
AcHN
CO CHPh
2
2
d
N
HO C
OH
2
HN
N
NH
2
AcHN
14
NBoc
NHAc
OH
HN
NH
NH
2
NHBoc
3
15
Scheme 2. Synthesis of iminosugars 3 and 4. Reagents and conditions: (a) CCl3CN, DBU, CH2Cl2, rt, 30 min, 64%; (b) p-TsOH, Py/H2O, 80 °C, 2 h,
77%; (c) NaBH4, EtOH, rt, 2 h, 57%; (d) 4 M HCl/dioxane, rt, 12 h, 90–93%; (e) (BocNH)2CS, HgCl2, Et3N, DMF, rt, 2 h, 95%.

534
J. R. Brown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 532–536
A
5000
4000
3000
2000
1000
0
3000
3000
2500
2000
1500
1000
500
B
C
O
OH
.
NH 4HCl
2500
2000
1500
1000
500
OH
HO C
2
HN
HO C
2
HN
N
3HCl
NHAc
HN
NH
2
NHAc
HN
NH
2
25 µM
25 µM
0
0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
Iminosugar 3 (mM)
Iminosugar 2 (mM)
Figure 2. (A) gem-Diamine 1-N-iminosugars (1–10) screened as inhibitors of HS 2-O-ST. Iminosugar 2 (B) and iminosugar 3 (C) were mixed at
various concentrations with acceptor substrate, 2-O-desulfated heparin (2DSH).
reflects poor uptake by cells due to the positively
charged guanidine moiety.
of an enzyme involved in heparan sulfate synthesis. Fur-
ther modification of 2 and 3 may render them as poten-
tial inhibitors of glycosaminoglycan chain biosynthesis
in vivo.
Iminosugars 2 and 3 both contain a guanidine and an
acetamide group at C-4 and C-2, respectively. Iminosug-
ar 3 also contains 1-N-2-ethyl-butyramide present in the
starting material 1. Since iminosugar 4 also contained
the 1-N-2-ethyl-butyramide but lacked activity, this moi-
ety does not appear to be responsible for the inhibitor
activity of iminosugar 3. Iminosugar 5, which also con-
tains the guanidine moiety at C-4, lacked activity, but
the large bulky –NHCOCF3 moiety at C-2 may have
blocked binding. The guanidine moiety may bind to
the heparin substrate through charge–charge interac-
tions or form a salt bridge with an appropriately posi-
tioned carboxylate in the active site of the enzyme.
The guanidine moiety is an important feature of many
biologically active compounds, for example, 4-guanidi-
no-Neu5Ac-2-en is a potent inhibitor of influenza virus
sialidase.15
Acknowledgments
This work was supported by Grant CA112278 from the
National Institutes of Health (to J.D.E.) and by Grants-
in-Aid for Scientific Research from Japan Society for the
Promotion of Science (JSPS) (KAKENHI 14370761)
and MPS Research grants from National MPS Society
(to Y.N). J.R.B was supported in part through a Univer-
sity Biotechnology Research and Training Grant.
References and notes
1. Esko, J. D.; Selleck, S. B. Annu. Rev. Biochem. 2002, 71,
435.
2. Esko, J. D.; Lindahl, U. J. Clin. Invest. 2001, 108, 169.
3. Fuster, M. M.; Esko, J. D. Nat. Rev. Cancer 2005, 5, 526.
4. Rong, J.; Habuchi, H.; Kimata, K.; Lindahl, U.; Kusche-
Gullberg, M. Biochemistry 2001, 40, 5548.
5. Kobayashi, M.; Habuchi, H.; Habuchi, O.; Saito, M.;
Kimata, K. J. Biol. Chem. 1996, 271, 7645.
In conclusion, we have identified two gem-diamine 1-N-
iminosugars that inhibit a central sulfotransferase in-
volved in heparan sulfate biosynthesis. To our knowl-
edge, this is the first evidence that gem-diamine 1-N-
iminosugars related to L-iduronic acid act as inhibitors

J. R. Brown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 532–536
535
6. Guimond, S.; Maccarana, M.; Olwin, B. B.; Lindahl, U.;
Rapraeger, A. C. J. Biol. Chem. 1993, 268, 23906;
Maccarana, M.; Casu, B.; Lindahl, U. J. Biol. Chem.
1993, 268, 23898.
C15H27N5O5Æ2HCl: C, 41.86; H, 6.79; N, 16.28. Found: C,
42.36; H, 7.03; N, 16.43.
Compound 4 as its hydrochloride: ½aꢁ2D5 +24.5 (c 0.56, H2O);
1H NMR (D2O, 400 MHz) d 0.78 and 0.82 (total 3H, each t,
J = 7.5 Hz, –CH(CH2CH3)2), 0.91 and 0.92 (total 3H, each
t, J = 7.5 Hz, –CH(CH2CH3)2), 1.5–1.7 (4H, m,
–CH(CH2CH3)2), 2.04 and 0.07 (total 3H, each s,
NHCOCH3), 2.85 and 2.91 (total 2H, quintet, J = 6.6 Hz,
–CH(CH2CH3)2), 3.00–3.08 (m, a part of H-3 and a part of
H-2ax), 3.13 (dt, J = 4.8 and 12 Hz, a part of H-3), 3.48 (dd,
J = 12 and 15 Hz, a part of H-2ax), 3.50–4.20 (total 1H, m,
H-4), 4.21 and 4.22 (total 1H, each t, J = 3 Hz, H-5), 4.55
(dd, J = 15 and 3.6 Hz, a part of H-2eq), 4.92 (br d,
J = 12 Hz, a part of H-2eq) and 6.08 and 6.51(total 1H, each
d, J = 2.4 Hz, H-6); 13C NMR (D2O, 150 MHz) d 10.73,
11.09 and 11.18 (–CH(CH2CH3)2), 21.79 (NCOCH3),
25.11, 25.43 and 25.47 (–CH(CH2CH3)2), 37.78 and 41.32
(C-2), 39.06 and 40.00 (C-3), 44.25 and 44.37
(–CH(CH2CH3)2), 49.00 and 49.14 (C-4), 59.35 and 63.44
(C-6), 65.18 and 65.75 (C-5), 173.16, 173.69, 173.75 and
173.84 (NCOCH3 and NCOCH(CH2CH3)2), 179.64 and
180.33 (CO2H); HRMS calcd for C14H25N3O5 (M+Na):
338.16919. Found: 338.16773; Anal. Calcd for
C14H25N3O5HCl: C, 47.79; H, 7.45; N, 11.94. Found: C,
48.01; H, 7.27; N, 12.15.
7. Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeu-
chi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y. J. Am.
Chem. Soc. 1996, 118, 3051; Nishimura, Y.; Satoh, T.;
Adachi, H.; Kondo, S.; Takeuchi, T.; Azetaka, M.;
Fukuyasu, H.; Iizuka, Y. J. Med. Chem. 1997, 40, 2626.
8. Armstrong, J. I.; Portley, A. R.; Chang, Y. T.; Nieren-
garten, D. M.; Cook, B. N.; Bowman, K. G.; Bishop, A.;
Gray, N. S.; Shokat, K. M.; Schultz, P. G.; Bertozzi, C. R.
Angew. Chem., Int. Ed. 2000, 39, 1303; Best, M. D.; Brik,
A.; Chapman, E.; Lee, L. V.; Cheng, W. C.; Wong, C. H.
Chembiochem 2004, 5, 811.
9. Nishimura, Y.; Shitara, E.; Adachi, H.; Toyoshima, M.;
Nakajima, M.; Okami, Y.; Takeuchi, T. J. Org. Chem.
2000, 65, 2.
10. Nishimura, Y.; Kudo, T.; Kondo, S.; Takeuchi, T.;
Tsuruoka, T.; Fukuyasu, H.; Shibahara, S. J. Antibiot.
(Tokyo) 1994, 47, 101; (b) Satoh, T.; Nishimura, Y.;
Kondo, S.; Takeuchi, T. Carbohydr. Res. 1996, 286, 173.
11. Umezawa, H.; Aoyagi, T.; Komiyama, T.; Morishima, H.;
Hamada, M. J. Antibiot. (Tokyo) 1974, 27, 963.
25
1
12. Compound 12: ½aꢁD ꢂ59.4 (c 0.28, MeOH); a part of H
NMR (CDCl3, 400 MHz) d 3.23 and 3.54 (total 1H, dt
each, J = 13.2 and 4.2 Hz, H-3), 3.35 and 3.36 (total 1H, t
each, J = 13.2 Hz, H-2ax), 3.94 and 4.11 (total 1H, dd
each, J = 13.2 and 4.2 Hz, H-2eq), 5.12 and 5.39 (total 1H,
dd each, J = 9.7 and 4.2 Hz, H-4), 5.26 and 5.69 (total 1H,
dd each, J = 9.8 and 2.5 Hz, H-5), 5.69 and 6.08 (total 1H,
dd each, J = 5.8 and 2.5 Hz, H-6).
13. Shitara, E.; Nishimura, Y.; Nerome, K.; Hiramoto, Y.;
Takeuchi, T. Org. Lett. 2000, 2, 3837.
14. 2-O-Sulfotransferase assays: A sequence encoding Chinese
hamster ovary (CHO) heparan sulfate 2OST amino acids
28–353 (lacking the presumptive transmembrane domain
at the amino terminus) was cloned into pRK5F10PROTA
(Glycomed, Inc.). This plasmid was designed to express a
secreted fusion protein containing protein A and 2OST.
COS-7 cells were transiently transfected with
pRK5F10PROTA-2-O-ST using LipofectAMINE (Invit-
rogen) according to the manufacturerꢀs instructions. After
72 h of incubation, the fusion protein was recovered from
the cell culture supernatant by affinity chromatography
using IgG-agarose beads. The conditioned media and
beads were mixed end-over-end overnight at 4 °C and
centrifuged for 5 min, and the supernatant was aspirated.
The beads were washed twice with 10 ml of 20% (v/v)
glycerol in 50 mM Tris–HCl, pH 7.4, and resuspended in
the same buffer containing protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 1 lg/ml leupeptin, and
1 lg/ml pepstatin A) to achieve a 50% (v/v) slurry. The
immobilized enzyme was stable at 4 °C for at least 4
months. The HS 2-O-ST activity was assayed as
described.16 Briefly, the assay (25 ll) contained: 50 mM
MES (pH 6.5), 1% TX-100, 10 mM MgCl2, 10 mM
MnCl2, 5 mM CaCl2, 87.5 lM NaF, ꢀ0.1 lCi [35S]PAPS,
1 lg 2-O-desulfated heparin as an acceptor substrate and
5 ll protein A bead slurry (50%) containing immobilized
enzyme. The reaction was incubated for 30 min at 37 °C
with occasional mixing and stopped by adding 475 ll of
0.1 M EDTA (pH 7.4) containing 0.25 mg heparin. The
35S-labeled products were separated from unreacted
[35S]PAPS by anion exchange chromatography on 0.25-
ml columns of DEAE–Sephacel packed in disposable
polypropylene tips as described.17 The column was washed
with 15 ml of 0.25 M NaCl, 20 mM sodium acetate (pH
6.0) and eluted with 2.5 ml of a 1 M NaCl, 20 mM sodium
acetate (pH 6.0). An aliquot (1 ml) was counted by liquid
scintillation (Ultima Gold X-R, Packard BioScience).
Penicillium chrysogenum APS kinase was a kind gift from
Dr. Irwin Segel, University of California, and was used to
prepare [35S]PAPS as described.18
23
1
Compound 13: ½aꢁD ꢂ27.6 (c 0.16, MeOH); a part of H
NMR (CDCl3, 400 MHz) d 2.99 (1H, t, J = 12.0 Hz, H-
2ax), 3.11 (1H, dt, J = 12.0 and 3.4 Hz, H-3), 3.95 (1H, br
s, H-5), 4.44 (1H, br m, H-2eq), 4.69 (1H, dt, J = 12.0 and
2.5 Hz, H-4), 4.97 (1H, dd, J = 8.8 and 2.5 Hz, H-6).
23
Compound 14: ½aꢁD +23.9 (c 2.3, MeOH); a part of 1H
NMR(CDCl3, 400 MHz) d 2.70 (1H, t, J = 12.0 Hz, H-2ax),
2.84 (1H, dt, J = 12.0 and 3.9 Hz, H-3), 2.80–2.90 (1H, m,
–CH(CH2CH3)2), 3.28 (1H, dd, J = 12.0 and 2.5 Hz, H-4),
3.87 (1H, br s, H-5), 4.06 (1H, br d, J = 12.0 Hz, H-2eq),
4.95 (1H, dd, J = 7.0 and 3.7 Hz, H-6).
23
Compound 15: ½aꢁD ꢂ25.4 (c 0.7, MeOH); a part of 1H
NMR (CDCl3, 400 MHz) d 2.80–3.00 (2H, m, a part of H-3
and –CH(CH2CH3)2), 3.13 (1H, dt, J = 11.7 and 3.9 Hz, H-
3), 3.91 (1H, br s, H-5), 4.71 (1H, t, J = 11.0 Hz, H-4), 4.88
(1H, dd, J = 11.7 and 3.9 Hz, H-2), 6.17 (1H, d, J = 8.8 Hz,
H-6).
Compound 3 as its hydrochloride: ½aꢁ2D5 +19.9 (c 0.16, H2O);
1H NMR(D2O, 400 MHz) d 0.62 and 0.65 (total 3H, each t,
J = 7.3 Hz, –CH(CH2CH3)2), 0.75 and 0.76 (total 3H, each
t, J = 7.3 Hz, –CH(CH2CH3)2), 1.3–1.7 (4H, m,
s
–
CH(CH2CH3)2), 1.87 and 1.91 (total 3H, each,
NHCOCH3), 2.55–2.75 (3H, m, H-2eq, H-3 and –
CH(CH2CH3)2), 2.96 and 3.28 (total 1H, each t,
J = 13.0 Hz, H-2ax), 3.91 (1H, br s, H-5), 3.99 and 4.22
(total 1H, dd each, J = 13.0 and 4.0 Hz, H-4), 5.89 and 6.29
(1H, br s, H-6); 13C NMR (D2O, 150 MHz) d 10.71, 10.91,
11.04, 11.12 and 11.22 (–CH(CH2CH3)2), 21.81 and 21.84
(NCOCH3),
25.016,
25.25,
25.38
and
25.42
(–CH(CH2CH3)2), 38.80 and 42.27 (C-2), 42.93 and 44.31
(C-3), 44.05 and 44.22 (–CH(CH2CH3)2), 50.54 and 50.79
(C-4), 59.92 and 64.04 (C-6), 66.64 and 67.18 (C-5), 156.64
and 156.72 (–NHC(@NH)NH2), 173.81 (NCOCH3 or
NCOCH(CH2CH3)2),
175.68
and
175.74
(NCOCH(CH2CH3)2) or NCOCH3), 179.39 and 180.28
(CO2H); HRMS calcd for C15H27N5O5 (M+Na):
380.19099. Found: 380.18834; Anal. Calcd for
15. Lednicer, D.; Mitscher, L. A. The Organic Chemistry of
Drug Synthesis; Wiley: New York, 1997; von Itzstein, M.;

536
J. R. Brown et al. / Bioorg. Med. Chem. Lett. 16 (2006) 532–536
Wu, W. Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, 17. Bame, K. J.; Esko, J. D. J. Biol. Chem. 1989, 264,
B.; Van Phan, T.; Smythe, M. L.; White, H. F.; Oliver, S.
W., et al. Nature 1993, 363, 418.
16. Bai, X.; Esko, J. D. J. Biol. Chem. 1996, 271, 17711.
8059.
18. MacRae, I.; Segel, I. H. Arch. Biochem. Biophys. 1997,
337, 17.