Synthesis of N-heteroaroyl aminosaccharide derivatives as fibroblast growth factor 2 signaling modulators

Article abstract of DOI:10.1248/cpb.58.1210

Fibroblast growth factor 2 (FGF2) signaling plays an important role in angiogenesis. Heparin/heparan sulfate (HS) is required for FGF2 signaling but heparin mimics either promotes or inhibits FGF2 signaling. To take advantage such properties of heparin mimics, a series of N-heteroaroyl aminosaccharide derivatives were designed and synthesized as FGF2 signaling modulators. The bioactivity was determined in a FGF2 and heparin-dependent cell proliferation assay using FGFR1c expressing BaF3 cells. We found that most of the compounds inhibited heparin- and FGF2-dependent BaF3 cell proliferation while three compounds promoted the cell proliferation. These results suggest that the small molecular heparin mimics approach might be useful in developing novel anti-angiogenic agents.

Full text of DOI:10.1248/cpb.58.1210

1
210
Note
Chem. Pharm. Bull. 58(9) 1210—1215 (2010)
Vol. 58, No. 9
Synthesis of N-Heteroaroyl Aminosaccharide Derivatives as Fibroblast
Growth Factor 2 Signaling Modulators
a
a
b
b
,a
Jin DONG, Shuowei YAO, Xiaodong ZHOU, Lijuan ZHANG, and Yungen XU*
a
Department of Medicinal Chemistry, China Pharmaceutical University; 24 Tongjiaxiang, Nanjing 210009, China: and
Department of Pathology and Immunology, Washington University School of Medicine; 660 S. Euclid Avenue, St. Louis,
b
MO 63110, U.S.A. Received December 15, 2009; accepted May 4, 2010; published online June 10, 2010
Fibroblast growth factor 2 (FGF2) signaling plays an important role in angiogenesis. Heparin/heparan sul-
fate (HS) is required for FGF2 signaling but heparin mimics either promotes or inhibits FGF2 signaling. To take
advantage such properties of heparin mimics, a series of N-heteroaroyl aminosaccharide derivatives were de-
signed and synthesized as FGF2 signaling modulators. The bioactivity was determined in a FGF2 and heparin-
dependent cell proliferation assay using FGFR1c expressing BaF3 cells. We found that most of the compounds
inhibited heparin- and FGF2-dependent BaF3 cell proliferation while three compounds promoted the cell prolif-
eration. These results suggest that the small molecular heparin mimics approach might be useful in developing
novel anti-angiogenic agents.
Key words fibroblast growth factor 2; fibroblast growth factor receptor; angiogenesis; N-heteroaroyl aminosaccharide; heparin
mimic
12)
Canonical fibroblast growth factor (FGF) signals are not as drugs for patients with bleeding complications.
only essential in mammalian embryogenesis, but also play FGF2 is a major inducer of tumor angiogenesis. He-
important roles in tissue repair, tumor biology, aging, and parin/HS and their derivatives are required for FGF signaling
13)
1,2)
3)
4
)
14)
diabetic control throughout adult life. FGF2, the most ex- at both cell and animal levels. The chain length was found
tensively studied member of the FGF family, exerts its bio- to be critical for the bioactivity of heparin/HS. While he-
logical activities including angiogenesis through binding to parin/HS possessing anticoagulation activity must have a size
5)
high-affinity tyrosine kinase FGF receptors and heparin/he- equal or larger than a pentasaccharide, heparin-derived di-,
paran sulfate (HS). Heparin/HS is required to form a ternary tri- and tetra-saccharides were found to promote FGF signal-
complex with FGF and FGFR to stabilize and activate FGF ing in cell-based assays
1
5,16)
which indicate that smaller
signaling for a long enough period of time to elicit a specific oligosaccharides can be developed to modulate FGF signal-
6)
response. It has been demonstrated that the heparin se- ing without affecting coagulation properties of the system.
quences that bind to FGF and FGFR are different from that The sulfation pattern is another important determinant of
of required for ternary complex formation and different he- heparin/HS biological activity. It is emerging that different
7)
8)
parin sequences either inhibits or promotes FGF signaling.
sulfation patterns modulate different biological activities.
Therefore, it is possible to develop heparin mimics-based Crystallography studies have shown that interactions between
FGF signaling modulators by taking advantage of complex FGF, FGFR and heparin are mediated by sulfates and other
17)
sugar structures.
functional groups such as carboxylate and ring oxygens.
Heparin and the structurally related heparan sulfate (HS) Similarly, the formation of antithrombin·thrombin·heparin
are glycosaminoglycans (GAGs) constituted by repeating dis- ternary complex largely depends on the hydrogen bonds par-
1
8,19)
accharide units of a-D-glucosamine (GlcNAc or D-glu- ticipated by sulfates.
Moreover, only fully sulfated disac-
cosamine N-sulfate (GlcNSO )) and an uronic acid (b-D-glu- charide with eight sulfates, but not disaccharides with less
curonic acid (GlcA) or a-L-iduronic acid (IdoA)). Clinical than eight sulfates form a ternary complex and promote FGF
trials have revealed that treatment with various low-molecu- signaling. In contrast, the non-sulfated di- and tri-saccha-
lar-weight heparins improves the survival time of tumor pa- rides mentioned above bind to FGF2 with high affinity lig-
tients. The anti-angiogenic effects of these heparin deriva- ands and promote FGF signaling through an unknown mech-
3
20)
9)
16)
tives likely play a central role in their anti-tumor activities.
anism. Both N- and O-desulfated heparin have been re-
Preclinical studies have demonstrated that heparin/HS are ported to be non-anticoagulants but remain some other bio-
8
,21—24)
able to modulate the activities of various angiogenic growth logical activities such as anti-angiogenic activities.
factors, most of which are heparin-binding proteins, such as Thus, while the anti-coagulant effect and FGF/FGFR/heparin
10)
FGF and vascular endothelial growth factor (VEGF). PI- ternary complex formation requires a high level of sulfation,
8, a mixture of highly sulfated, monophosphorylated man- non-sulfated sugars also modulate FGF signalings through an
nose oligosaccharides, derived from the extracellular phos- unidentified mechanism.
8
phomannan of the yeast Pichia holstii which inhibits tumor
angiogenesis via antagonism of the interactions of angio-
genic growth factors and their receptors with heparin/HS, has
Indeed, Murphy and his co-workers reported that mono-
11)
entered phase III clinical trial as an anti-tumor agent. How-
ever, the use of heparin/HS and their derivatives as therapeu-
tic agents against tumor can be limited due to their inhibition
of the blood-coagulation cascade. Non-anticoagulant he-
parins, which can be used at a higher dose, may be developed Fig. 1. The Structure of Compounds 1 and 2
∗
To whom correspondence should be addressed. e-mail: xu_yungen@hotmail.com
© 2010 Pharmaceutical Society of Japan

September 2010
1211
saccharide-based glycoconjugates such as compound 1 (Fig. the protecting group by acidification with HCl in acetone
) exhibited potent bioactivities in both a FGF2 binding gave the compound 4 as HCl salt. Then aminogalactose 4Ј
1
25)
assay and an endothelial cell survival assay. Further, they was obtained through treatment of sodium acetate.
found per-O-acetylated glucosamine derivatives such as Except Compound 24, all other target compounds were
compound 2 possessing significantly improved inhibitory po- obtained by coupling aminosaccharides with diverse hetero-
tency to their corresponding polyhydroxylated deriva- cyclic acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
2
6,27)
tives.
However, it remains to be demonstrated whether imide hydrochloride/1-hydroxybenzotriazole (EDCI/HOBt)
these glycoconjugates reduced endothelial cell proliferation as condensing agent (Chart 1). In this reaction, in order to
25,26)
was through their inhibition of FGF signaling.
To under- avoid undesirable deacetylation, the amount of triethylamine
stand the molecular mechanism and to discover novel small required for the conversion of the HCl salts to their corre-
non-sulfated heparin mimics, we reported the design and sponding free amines should be limited to between 1 and
synthesis of a series of N-heteroaroyl aminosaccharides de- 2 eq. But this method was unsuccessful for the synthesis of
rivatives. Their FGF2 signaling modulating abilities were compound 24. Therefore, compound 24 was obtained
evaluated by using an established FGF2-dependent cell pro- through the reaction of 3Ј with the corresponding acyl chlo-
8)
liferation assay.
-Amino-2-deoxy galactose 4Ј was prepared from D-galac-
ride derived from 5-methylisoxazole-4-carboxylic acid.
All the compounds were evaluated in a FGF2 and heparin-
2
tosamine hydrochloride, according to a reported procedure dependent cell proliferation assay using FGFR1c expressing
28)
for the preparation of 2-amino-2-deoxy glucose 3Ј. This BaF3 cells. Heparin- and FGF-dependent BaF cell prolifera-
29)
method involves amino protection by benzaldehyde, which tion assay was established in 1992. BaF3 cells are a lym-
followed by acetylation with Ac O. Subsequent removal of phoid cell line, which are dependent on cytokine IL-3 for
2
Compound
R1
R2
Ar
Compound
R1
R2
Ar
1
1
1
6
7
8
OAc
OAc
OAc
H
H
H
2
2
6
7
H
H
OAc
OAc
2
2
8
9
H
H
OAc
OAc
1
2
9
0
OAc
OAc
H
H
3
0
H
OAc
3
3
3
1
2
3
H
H
H
OAc
OAc
OAc
2
2
1
2
OAc
OAc
H
H
2
3
OAc
H
3
4
H
OAc
2
2
4
5
OAc
H
H
OAc
Chart 1. Synthesis of N-Heteroaroyl Aminosaccharide Derivatives (Compounds 16—34)

1
212
Vol. 58, No. 9
growth and have no intrinsic response to FGF. When stably BaF3 cells proliferation comparing to galactosamine deriva-
transfected to express FGFR, BaF3 cells die if only FGF or tives 25—34. However, most unexpectedly, while galac-
heparin is added to the IL-3 free cell culture media. Proper tosamine derivatives 32, 33, and 34 showed modest in-
BaF3 cells growth can be achieved in the presence of both hibitory effects, their corresponding glucosamine derivatives
heparin and FGF. Because BaF3 cells express no endogenous 22, 23, and 24 were partial agonists in this assay. These re-
FGF, the background activity in the absence of added FGF is sults indicated that the carbohydrate moiety is the primary
very low regardless of whether the cells express an FGFR, pharmacophore. Given that a change of the configuration of
which makes this system idea for testing compounds that can the hydroxyl group at position 4 may either reduce or reverse
regulate FGF2/FGFR/heparin signaling in this unique cell the bioactivity of these compounds, the binding site of these
based system.
We used resazurin to replace previously used H-thymi- ceptor which is sensitive to stereo-configuration.
dine to measure FGF- and GAG-dependent BaF cell prolifer- The inhibition of heparin- and FGF2-dependent BaF3 cell
glycoconjugates may share common characteristics with a re-
3
8)
ation. Resazurin is a metabolic indicator dye. In its normal proliferation observed in this assay are not likely due to cyto-
oxidized state it shows very little fluorescence. Once the dye toxic effects for the following reasons: 1) the cytotoxic ef-
penetrates the cell it becomes reduced to the highly fluores- fects should be more potent when the concentrations of
cent resorufin. The rate of resazurin reduction directly corre- tested compounds are increased. However, most of com-
lates with cell numbers and thus reflects overall cell growth pounds had higher relative fluorescence unit (RFU) values at
induced by heparin, FGFs, and testing compounds.
In this assay, all of the compounds exhibited modulating tion of higher RFU values and highest concentration
200 mg/ml than those at 40 mg/ml (Fig. 2B); 2) the correla-
activities on heparin- and FGF2-dependent BaF3 cell prolif- (200 mg/ml) for the same compound was consistently ob-
eration stimulated by 1 mg/ml heparin and 8 nM FGF2.
served when repeated in independent BaF3 cell proliferation
Heparin promoted FGF2/FGFR1c signaling-dependent assays including testing the compounds in the absence of he-
8)
BaF cell proliferation as reported previously (Fig. 2A). We parin or performing the same experiments with FGF1 (data
tested how these compounds regulated FGF2/FGFR1c/he- not shown); and 3) the inhibition effect observed was de-
parin-dependent BaF cell proliferation in the presence of pendent on the structures of the tested compounds. Such de-
1
mg/ml heparin and 8 nM FGF2 (Fig. 2B). The glucosamine pendency indicates the observation was not an artifact of the
derivatives 16—21 showed more potent inhibitory effects on assay.
Fig. 2. The Effects of N-Heteroaroyl Aminosaccharide Derivatives on BaF3 Cells Proliferation
(A) The effect of heparin on FGF2-dependent BaF3 FGFR1c cell proliferation. (B) The effect of compounds 16—34 on FGF2-dependent BaF3 FGFR1c cell proliferation (The
FGF2 used for testing compounds 22, 23, and 24 was from a different batch. This FGF2 lot at the same concentration (8 nM) had higher activities in the presence of 1 mg/ml heparin
than the rest). RFU, relative fluorescence unit.

September 2010
1213
1
Because the heparin/HS possessing anticoagulant effects boxamide (18): Isolated yield, 47.7%; mp 220—221 °C (decomp.). H-NMR
(
3
300 MHz, CDCl ) d: 2.02, 2.07, 2.09, 2.12 (each 3H, each s, each CH ),
must contain a domain structure with the size larger than
pentasaccharide, it is expected that our target compounds are
FGF2 signaling modulators but are not anticoagulants.
However, further tests are required to confirm the claim.
3
3
.86—3.88 (1H, m, H-5), 4.15—4.18 (1H, m, H-6b), 4.31 (1H, dd, H-6a,
Jϭ4.5, 12.3 Hz), 4.53 (1H, m, H-2), 5.20—5.32 (2H, m, H-3,4), 5.83 (1H, d,
H-1, Jϭ8.7 Hz), 6.57 (1H, d, NH, Jϭ9.3 Hz), 7.57 (2H, d, ArH, Jϭ4.5 Hz),
8.76 (2H, d, ArH, Jϭ3.6 Hz). IR (KBr) cm : 3357 (NH), 2964, 2883 (CH),
3
0—33)
Ϫ1
In summary, we have described the synthesis of a series of 1749 (CϭO, ester), 1663 (CϭO, amide), 1598, 1531, 1492, 1220, 1119,
ϩ
25
1
079, 1038, 710. MS (ESI(ϩ)70 V, m/z): 453.2 [MϩH] . [a]D ϩ24.3
N-heteroaroyl aminosaccharides. This series of compounds
modulated FGF2/FGFR1c signaling in a structure-dependent
manner. Our studies demonstrated that monosaccharide-
based glycoconjugates have effect on FGF2 signaling, which methylpyridine-3-carboxamide (19): Isolated yield, 42.3%; mp 227—229 °C
suggests that it may be feasible to search for small non-sul- (decomp.). H-NMR (300 MHz, DMSO-d
fate heparin mimics to develop novel anti-angiogenic agents.
Compounds 16—21 could be considered as lead compounds
for development such agents. Since it is still challenging to
synthesize a “sequence-pure” polysaccharide, low molecular (KBr) cm : 3468 (OH), 2950 (CH), 1754 (CϭO, ester), 1672 (CϭO,
inhibitors/promoters are more likely to be accepted by the
pharmaceutical industry. Further investigation on glycocon-
jugates will significantly aid the exploration of new potential
anti-angiogenic agents. In addition, non-anticoagulant he-
parin mimics could lead to a better anti-tumor drug by reduc- 240—241 °C (decomp.). H-NMR (300 MHz, DMSO-d ) d: 1.86, 1.98,
ing unwanted side effects.
(
cϭ0.065, CHCl ). Anal. Calcd for C H N O : C, 53.10; H, 5.35; N, 6.19.
3
20 24
2
10
Found: C, 52.97; H, 5.25; N, 5.96.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-4-hydroxyl-6-
1
) d: 1.84, 1.97, 1.97, 2.02 (each
6
3
4
H, each s, each CH ), 2.24 (3H, s, CH ), 3.99 (1H, d, H-6b, Jϭ10.8 Hz),
3
3
.18—4.23 (3H, m, H-2, H-5, H-6a), 4.92 (1H, t, H-4, Jϭ9.6 Hz), 5.50 (1H,
t, H-3, Jϭ9.9 Hz), 6.07 (1H, d, H-1, Jϭ8.7 Hz), 6.22 (1H, s, ArH), 8.29 (1H,
d, ArH, Jϭ6.3 Hz), 10.49 (1H, d, NH, Jϭ9.0 Hz), 12.16 (1H, br s, OH). IR
Ϫ1
amide), 1499, 1224, 1082, 1039, 709. MS (ESI(Ϫ)70 V, m/z): 481.2
Ϫ
25
[
5
MϪH] . [a]D ϩ23.2 (cϭ0.185, CHCl ). Anal. Calcd for C H N O : C,
3
21 26
2
11
2.28; H, 5.43; N, 5.81. Found: C, 52.24; H, 5.50; N, 5.57.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-4-hydroxyl-5-
nitro-6-methylpyridine-3-carboxamide (20): Isolated yield, 37.3%; mp
1
6
1
.99, 2.02 (each 3H, each s, each CH ), 2.35 (3H, s, aromatic CH ), 4.00
3
3
(
4
1H, dd, H-6b, Jϭ1.5, 12.3 Hz), 4.09 (1H, ddd, H-5, Jϭ1.5, 3.9, 9.9 Hz),
.22—4.31 (2H, m, H-6a, H-2), 4.93 (1H, t, H-4, Jϭ9.6 Hz), 5.54 (1H, t, H-
Experimental
3, Jϭ9.9 Hz), 6.09 (1H, d, H-1, Jϭ8.7 Hz), 8.46 (1H, s, ArH), 9.72 (1H, d,
NH, Jϭ9.6 Hz), 13.03 (1H, s, OH). IR (KBr) cm : 3453 (OH), 3252 (NH),
Ϫ1
Chemistry Melting points were determined on a RDCSY-I capillary ap-
paratus and were uncorrected. The IR spectra (in KBr pellets) were recorded 2953 (CH), 1753 (CϭO, ester), 1680 (CϭO, amide), 1536, 1227, 1076,
1
13
Ϫ
25
on a Nicolet Impact 410 spectrophotometer. The H- and C-NMR spectra 1039, 585. MS (ESI(Ϫ)70 V, m/z): 526.2 [MϪH] . [a]D ϩ50.4 (cϭ0.10,
were recorded on a Brucker AV-300 or AV-500 NMR spectrometer using CHCl ). Anal. Calcd for C H N O : C, 47.82; H, 4.78; N, 7.97. Found: C,
3
21 25
3
13
tetramethylsilane (TMS) as an internal standard and chemical shifts were 47.74; H, 5.13; N, 7.88.
given in d ppm with TMS. Mass spectra were recorded on an Agilent 1100
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-thiazole-4-car-
1
series LC/MSD Tarp (SL). The Elemental analysis data were obtained using boxamide (21): Isolated yield, 56.9%; mp 232—234 °C (decomp.). H-NMR
an Elementar Vario EL III instrument. All solvents were purchased from (300 MHz, CDCl ) d: 1.96, 2.05, 2.06, 2.11 (each 3H, each s, each CH ),
3
3
commercial sources and used as received unless otherwise stated.
3.88 (1H, ddd, H-5, Jϭ2.1, 4.5, 9.6 Hz), 4.15 (1H, dd, H-6b, Jϭ2.1,
General Procedure for Synthesis of Compounds 16—22 and 25—34 12.6 Hz), 4.33 (1H, dd, H-6a, Jϭ4.5, 12.6 Hz), 4.41—4.47 (1H, m, H-2),
Aminosaccharide 3 (or 4) (0.50 g, 1.30 mmol) was suspended in anhydrous
5.19 (1H, t, H-4, Jϭ9.6 Hz), 5.36 (1H, t, H-3, Jϭ9.3 Hz), 5.87 (1H, d, H-1,
CH Cl (20 ml). To this mixture, Et N (0.17 ml, 1.30 mmol) was added while Jϭ8.7 Hz), 7.44 (1H, d, NH, Jϭ9.6 Hz), 8.18 (1H, d, ArH, Jϭ1.8 Hz), 8.75
2
2
3
Ϫ1
cooling in an ice bath, and the resulting solution was stirred at room temper-
ature for 1 h. To this solution, HOBt (0.175 g, 1.30 mmol), heterocyclic acids
1.3 mmol), and EDCI (0.25 g, 1.30 mmol) were added successively, and the (ESI(Ϫ)70 V, m/z): 457.1 [MϪH] . [a]D ϩ43.9 (cϭ0.14, CHCl ). Anal.
reaction mixture was stirred at room temperature for 24 h. Then the mixture
(1H, d, ArH, Jϭ2.1 Hz). IR (KBr) cm : 3365 (NH), 2963, 2872 (CH), 1749
(CϭO, ester), 1660 (CϭO, amide), 1544, 1490, 1231, 1072, 1048. MS
Ϫ
25
(
3
Calcd for C H N O S: C, 47.16; H, 4.84; N, 6.11. Found: C, 47.10; H,
1
8
22
2
10
was washed with water (20 mlϫ2), saturated aqueous NaHCO (20mlϫ2)
4.73; N, 6.08.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-2-chloro-pyri-
3
and brine (20 mlϫ2), dried over anhydrous Na SO , filtered and concen-
2
4
trated under reduced pressure. The residue was chromatographed on a silica dine-3-carboxamide (22): Isolated yield, 26.9%; mp 228—230 °C (de-
gel column by use of dichloromethane–methanol (100 : 1—80 : 1, v/v) to
give compounds 16—22 and 25—34.
1
comp.). H-NMR (300 MHz, CDCl ) d: 2.06, 2.06, 2.11, 2.15 (each 3H,
3
each s, each CH ), 3.89 (1H, ddd, H-5, Jϭ2.1, 4.2, 9.6 Hz), 4.16 (1H, dd, H-
3
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-thiophene-3-car-
6b, Jϭ1.8, 12.6 Hz), 4.31 (1H, dd, H-6a, Jϭ4.5, 12.3 Hz), 4.43—4.46 (1H,
m, H-2), 5.19 (1H, t, H-4, Jϭ9.6 Hz), 5.34 (1H, t, H-3, Jϭ9.6 Hz), 5.89 (1H,
d, H-1, Jϭ8.7 Hz), 6.33 (1H, d, NH, Jϭ9.3 Hz), 7.34 (1H, dd, ArH, Jϭ4.8,
1
boxamide (16): Isolated yield, 26.9%; mp 230—231 °C (decomp.). H-NMR
(
3
300 MHz, CDCl ) d: 2.00, 2.06, 2.09, 2.11 (each 3H, each s, each CH ),
3
3
.84—3.86 (1H, m, H-5), 4.16 (1H, dd, H-6b, Jϭ1.8, 12.3 Hz), 4.30 (1H, dd, 7.8 Hz), 7.86 (1H, dd, ArH, Jϭ1.8, 7.8 Hz), 8.47 (1H, dd, ArH, Jϭ1.8,
Ϫ1
H-6a, Jϭ4.5, 12.6 Hz), 4.46—4.55 (1H, m, H-2), 5.19—5.25 (2H, m, H- 4.8 Hz). IR (KBr) cm : 3307 (NH), 2951, 2897 (CH), 1745 (CϭO, ester),
,4), 5.78 (1H, d, H-1, Jϭ8.7 Hz), 6.08 (1H, d, NH, Jϭ9.3 Hz), 7.33—7.34 1662 (CϭO, amide), 1582, 1535, 1223, 1086, 1043, 698. MS (ESI(ϩ)70 V,
3
(
2
1
4
Ϫ1
ϩ
Ϫ
25
D
2H, m, ArH), 7.82 (1H, d, ArH, Jϭ1.8 Hz). IR (KBr) cm : 3360 (NH),
m/z): 487.1 [MϩH] ; MS (ESI(Ϫ)70 V, m/z): 485.0 [MϪH] . [a] ϩ23.2
956 (CH), 1749 (CϭO, ester), 1653 (CϭO, amide), 1536, 1511, 1254, (cϭ0.185, CHCl ). Anal. Calcd for C H ClN O : C, 49.34; H, 4.76; N,
3 20 23 2 10
224, 1080, 1050, 745, 602. MS (electrospray ionization (ESI)(ϩ)70 V, m/z): 5.75. Found: C, 49.44; H, 4.77; N, 5.66.
ϩ
25
57.5 [M] . [a] ϩ45.3 (cϭ0.09, CHCl ). Anal. Calcd for C H NO S:
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-thiophene-2-
D
3
19 23
10
1
C, 49.89; H, 5.07; N, 3.06. Found: C, 49.89; H, 5.25; N, 2.79.
carboxamide (25): Isolated yield, 37.2%; mp 174—175 °C. H-NMR
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-3-methylthio-
(300 MHz, CDCl ) d: 1.98, 2.07, 2.11, 2.20 (each 3H, each s, each CH ),
3
3
phene-2-carboxamide (17): Isolated yield, 51.5%; mp 228—230 °C (de-
4.07—4.21 (3H, m, H-6a, H-6b, H-5), 4.61—4.71 (1H, m, H-2), 5.20 (1H,
1
comp.). H-NMR (300 MHz, CDCl ) d: 2.02, 2.06, 2.10, 2.10, (each 3H, dd, H-3, Jϭ3.0, 11.4 Hz), 5.43 (1H, d, H-4, Jϭ3.0 Hz), 5.81 (1H, d, H-1,
3
each s, each CH ), 2.47 (3H, s, CH ), 3.86 (1H, ddd, H-5, Jϭ2.4, 4.8,
Jϭ8.7 Hz), 5.93 (1H, d, NH, Jϭ9.0 Hz), 7.07—7.10 (1H, m, ArH), 7.43
3
3
Ϫ1
9
1
.9 Hz), 4.15 (1H, dd, H-6b, Jϭ2.1, 12.3 Hz), 4.30 (1H, dd, H-6a, Jϭ4.5,
2.3 Hz), 4.46—4.49 (1H, m, H-2), 5.19 (1H, t, H-4, Jϭ9.6 Hz), 5.31 (1H, t,
(1H, d, ArH, Jϭ3.6 Hz), 7.51 (1H, d, ArH, Jϭ4.8 Hz). IR (KBr) cm : 3353
(NH), 2961 (CH), 1749 (CϭO, ester), 1646 (CϭO, amide), 1536, 1512,
Ϫ
25
H-3, Jϭ9.3 Hz), 5.82 (1H, d, H-1, Jϭ8.7 Hz), 5.95 (1H, d, NH, Jϭ9.3 Hz), 1421, 1218, 1076, 1044. MS (ESI(Ϫ)70 V, m/z): 456.0 [MϪH] . [a]D
Ϫ1
6
3
1
.88 (1H, d, ArH, Jϭ5.1 Hz), 7.27 (1H, d, ArH, Jϭ4.8 Hz). IR (KBr) cm
365 (NH), 2960, 2876 (CH), 1751 (CϭO, ester), 1645 (CϭO, amide), N, 3.06. Found: C, 49.91; H, 5.13; N, 3.11.
512, 1250, 1221, 1076, 1040, 763, 601. MS (ESI(ϩ)70 eV, m/z): 494.2
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-thiophene-3-
:
ϩ21.3 (cϭ0.1250, CHCl ). Anal. Calcd for C H NO S: C, 49.89; H, 5.07;
3 19 23 10
ϩ
Ϫ
25
1
[
MϩNa] , MS (ESI(Ϫ)70 V, m/z): 470.1 [MϪH] . [a] ϩ39.4 (cϭ0.105, carboxamide (26): Isolated yield, 47.2%; mp 187—189 °C. H-NMR
D
CHCl ). Anal. Calcd for C H NO S: C, 50.95; H, 5.34; N, 2.97. Found: C, (300 MHz, CDCl ) d: 1.98, 2.06, 2.10, 2.20 (each 3H, each s, each CH ),
0.69; H, 5.35; N, 2.99.
3
20 25
10
3
3
5
4.04—4.25 (3H, m, H-6a, H-6b, H-5), 4.62—4.72 (1H, m, H-2), 5.20 (1H,
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-pyridine-4-car-
dd, H-3, Jϭ3.0, 11.1 Hz), 5.43 (1H, d, H-4, Jϭ2.7 Hz), 5.81 (1H, d, H-1,

1
214
Vol. 58, No. 9
Jϭ9.0 Hz), 5.91 (1H, d, NH, Jϭ9.6 Hz), 7.32—7.36 (2H, m, ArH), 7.81— 1584, 1554, 1402, 1370, 1222, 1078, 1043. MS (ESI(Ϫ)70 V, m/z): 485.0
Ϫ1
Ϫ
24
7
.82 (1H, m, ArH). IR (KBr) cm : 3355 (NH), 2957 (CH), 1749 (CϭO, [MϪH] . [a]D ϩ10.5 (cϭ0.065, CHCl ). Anal. Calcd for C H ClN O :
3 20 23 2 10
ester), 1650 (CϭO, amide), 1537, 1511, 1432, 1231, 1083, 1044. MS C, 49.34; H, 4.76; N 5.75. Found: C, 49.04; H, 4.81; N, 5.51.
Ϫ
25
(
ESI(Ϫ)70 V, m/z): 456.0 [MϪH] . [a]D ϩ32.0 (cϭ0.065, CHCl ). Anal.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-5-bromo-pyri-
3
1
Calcd for C H NO S: C, 49.89; H, 5.07; N, 3.06. Found: C, 49.95; H, dine-2-carboxamide (33): Isolated yield, 78.3%; mp 202—204 °C. H-NMR
19
23
10
5
.06; N, 2.95.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-3-methyl-thio-
(300 MHz, CDCl ) d: 1.93, 2.06, 2.11, 2.20, (each 3H, each s, each CH ),
3
3
4.10—4.21 (3H, m, H-6a, H-6b, H-5), 4.56—4.63 (1H, m, H-2) 5.30 (1H,
1
phene-2-carboxamide (27): Isolated yield, 35.9%; mp 179—181 °C. H-
dd, H-3, Jϭ3.0, 11.1 Hz), 5.45 (1H, d, H-4, Jϭ3.0 Hz), 5.88 (1H, d, H-1,
NMR (300 MHz, CDCl ) d: 2.00, 2.06, 2.12, 2.19, (each s, each 3H, each Jϭ9.0 Hz), 7.86 (1H, d, NH, Jϭ9.6 Hz), 7.96—8.07 (2H, m, ArH), 8.60
3
Ϫ1
CH ), 2.47 (3H, s, CH ), 4.04—4.24 (3H, m, H-5, H-6a, H-6b), 4.57—4.67 (1H, s, ArH). IR (KBr) cm : 3354 (NH), 2969 (CH), 1752 (CϭO, ester),
3
3
(
1H, m, H-2), 5.22 (1H, dd, H-3, Jϭ3.3, 11.1 Hz), 5.42 (1H, d, H-4,
1671 (CϭO, amide), 1579, 1531, 1465, 1372, 1220, 1078, 1046. MS
Ϫ
24
Jϭ2.7 Hz), 5.66 (1H, d, NH, Jϭ9.3 Hz), 5.83 (1H, d, H-1, Jϭ8.7 Hz), 6.89
(ESI(Ϫ)70 V, m/z): 528.8 [MϪH] . [a]D ϩ31.4 (cϭ0.13, CHCl ). Anal.
3
1
3
(1H, d, ArH, Jϭ5.1 Hz), 7.28 (1H, d, ArH, Jϭ5.1 Hz). C-NMR (75 MHz,
Calcd for C H BrN O : C, 45.21; H, 4.36; N, 5.27. Found: C, 45.22; H,
2
0
23
2
10
CDCl ) d: 15.60 (1C, CH ), 20.60 (4C, each CH ), 20.61, 20.67, 20.86, 4.47; N, 5.13.
3
3
3
5
1
0.26 (6C, carbohydrate ring carbons), 61.35, 66.57, 70.42, 72.14, 93.17,
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-5-methylisoxa-
1
27.06 (4C, ArC), 132.19, 142.08, 163.08 (1C, CϭO, amide), 169.51 (4C, zole-4-carboxamide (34): Isolated yield, 50.8%; mp 165—167 °C. H-NMR
Ϫ1
CϭO, ester), 170.14, 170.37, 170.69. IR (KBr) cm : 3325 (NH), 2958 (300 MHz, CDCl ) d: 2.00, 2.06, 2.11, 2.20 (each 3H, each s, each CH ),
3
3
(CH), 1743 (CϭO, ester), 1646 (CϭO, amide), 1553, 1522, 1438, 1229, 2.70 (3H, s, CH ), 4.07—4.20 (3H, m, H-5, H-6a, H-6b), 4.58—4.68 (1H,
3
Ϫ
25
1
085, 1040. MS (ESI(Ϫ)70 V, m/z): 470.0 [MϪH] . [a]D ϩ21.2 (cϭ0.10,
m, H-2), 5.18 (1H, dd, H-3, Jϭ3.0, 11.1 Hz), 5.41 (1H, d, H-4, Jϭ2.7 Hz),
CHCl ). Anal. Calcd for C H NO S: C, 50.95; H, 5.34; N, 2.97. Found: C, 5.79 (1H, d, H-1, Jϭ8.7 Hz), 6.41 (1H, d, NH, Jϭ9.6 Hz), 8.35 (1H, s, ArH).
3
20 25
10
Ϫ1
5
0.86; H, 5.25; N, 2.63.
IR (KBr) cm : 3366 (NH), 2979 (CH), 1752 (CϭO, ester), 1677 (Cϭ
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-pyridine-3-car-
O, amide), 1617, 1540, 1484, 1370, 1220, 1042. MS (ESI(Ϫ)70 V, m/z):
boxamide (28): Isolated yield, 42.6%; mp 154—157 °C. H-NMR 454.9 [MϪH] . [a]D ϩ15.4 (cϭ0.065, CHCl3). Anal. Calcd for
1
Ϫ
24
(
4
300 MHz, CDCl ) d: 1.99, 2.06, 2.11, 2.21 (each 3H, each s, each CH ), C H N O ·0.5H O: C, 49.03; H, 5.41; N, 6.02. Found: C, 49.17; H, 5.39;
3 3 19 24 2 11 2
.07—4.23 (3H, m, H-6a, H-6b, H-5), 4.68—4.78 (1H, m, H-2), 5.27 (1H, N, 6.02.
dd, H-3, Jϭ3.0, 11.1 Hz), 5.45 (1H, d, H-4, Jϭ3.0 Hz), 5.87 (1H, d, H-1,
Jϭ8.7 Hz), 6.76 (1H, br s, NH), 7.43 (1H, dd, ArH, Jϭ4.8, 8.4 Hz), 8.10
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-5-bromo-pyri-
dine-2-carboxamide (23) HOBt (0.19 g, 1.41 mmol), 5-bromopicolinic
acid (0.29 g, 1.44 mmol), and EDCI (0.28 g, 1.46 mmol) were added succes-
sively into a solution of aminosaccharide 3Ј (0.50 g, 1.44 mmol) in anhy-
drous CH Cl (20 ml) while cooling in an ice bath, and the reaction mixture
(
1H, d, ArH, Jϭ8.1 Hz), 8.75 (1H, d, ArH, Jϭ3.9 Hz), 8.96 (1H, s, ArH). IR
Ϫ1
(KBr) cm : 3366 (NH), 2961 (CH), 1747 (CϭO, ester), 1657 (CϭO,
amide), 1529, 1529, 1420, 1219, 1081, 1044. MS (ESI(ϩ)70 V, m/z): 453.1
2
2
ϩ
25
[
MϩH] . [a] ϩ24.1 (cϭ0.19, CHCl ). Anal. Calcd for C H N O : C, was stirred at room temperature for 17 h. Then the mixture was washed with
D 3 20 24 2 10
5
3.10; H, 5.35; N, 6.19. Found: C, 52.72; H, 5.14; N, 5.81.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-pyridine-4-car-
water (20 mlϫ2), saturated aqueous NaHCO3 (20 mlϫ2) and brine
(20 mlϫ2), dried over anhydrous Na SO , filtered and concentrated under re-
2
4
1
boxamide (29): Isolated yield, 39.1%; mp 179—180 °C. H-NMR duced pressure. The residue was chromatographed on a silica gel column by
(
4
300 MHz, CDCl ) d: 1.99, 2.07, 2.11, 2.21 (each 3H, each s, each CH ), use of petroleum ether–acetic ether (3 : 2—1 : 1, v/v) to give compound 23 as
3
3
1
.06—4.25 (3H, m, H-6a, H-6b, H-5), 4.64—4.74 (1H, m, H-2), 5.25 (1H, a white solid. Isolated yield, 45.8%; mp 202—203 °C. H-NMR (300 MHz,
dd, H-3, Jϭ3.3, 11.4 Hz), 5.44 (1H, d, H-4, Jϭ2.7 Hz), 5.85 (1H, d, H-1,
CDCl ) d: 1.94, 2.05, 2.05, 2.11 (each 3H, each s, each CH ), 3.89 (1H, ddd,
3
3
Jϭ8.7 Hz), 6.50 (1H, br s, NH), 7.56 (2H, d, ArH, Jϭ5.1 Hz), 8.76 (2H, d, H-5, Jϭ2.1, 4.2, 9.6 Hz), 4.15 (1H, dd, H-6b, Jϭ2.1, 12.3 Hz), 4.33 (1H, dd,
Ϫ1
ArH, Jϭ4.8 Hz). IR (KBr) cm : 3363 (NH), 2958 (CH), 1745 (CϭO, H-6a, Jϭ4.5, 12.3 Hz), 4.42—4.45 (1H, m, H-2), 5.19 (1H, t, H-4,
ester), 1663 (CϭO, amide), 1533, 1492, 1434, 1226, 1083, 1044. MS Jϭ9.6 Hz), 5.38 (1H, t, H-3, Jϭ9.3 Hz), 5.89 (1H, d, H-1, Jϭ8.7 Hz),
ϩ
25
(
ESI(ϩ)70 V, m/z): 453.2 [MϩH] . [a]D ϩ25.2 (cϭ0.21, CHCl ). Anal.
7.95—7.99 (3H, m, NH, ArH), 8.60 (1H, d, ArH, Jϭ1.8 Hz). IR (KBr)
3
Ϫ1
Calcd for C H N O : C, 53.10; H, 5.35; N, 6.19. Found: C, 53.10; H, cm : 3366 (NH), 2952 (CH), 1753 (CϭO, ester), 1671 (CϭO, amide),
20
24
2
10
5
.40; N, 6.19.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-6-methyl-5-
nitro-4-hydroxy-pyridine-3-carboxamide (30): Isolated yield, 41.4%; mp C, 45.21; H, 4.36; N, 5.27. Found: C, 45.46; H, 4.44; N, 5.23.
1525, 1219, 1085, 1041, 855, 693, 600. MS (ESI(Ϫ)70 V, m/z): 528.9
Ϫ
25
[MϪH] . [a]D ϩ49.7 (cϭ0.175, CHCl ); Anal. Calcd for C H BrN O :
3
20 23
2
10
1
1
2
2
94—195 °C (decomp.). H-NMR (300 MHz, CDCl ) d: 2.01, 2.05, 2.12,
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-glucopyranosyl)-5-methylisoxa-
3
.18 (each 3H, each s, each CH ), 2.49 (3H, s, CH ), 3.79—3.88 (1H, m, H- zole-4-carboxamide (24) 5-Methylisoxazole-4-carboxylic acid (0.25 g,
3
3
), 4.17—4.18 (3H, m, H-5, H-6a, H-6b), 5.41 (1H, d, H-4, Jϭ3.3 Hz), 6.07
1.97 mmol) were suspended in SOCl2 (1 ml, 13.8 mmol), and refluxed for
(
1H, dd, H-3, Jϭ3.3, 11.1 Hz), 6.40 (1H, d, H-1, Jϭ8.7 Hz), 8.76 (1H, d, 4 h. After the completion of the reaction, excess SOCl was removed under
2
ArH, Jϭ6.3 Hz), 10.01 (1H, d, NH, Jϭ7.2 Hz), 10.77 (1H, br s, OH). IR
reduced pressure to give acyl chloride as yellow oil which was used without
Ϫ1
(KBr) cm : 3480 (OH), 3264 (NH), 2961 (CH), 1751 (CϭO, ester), 1679 further purification. The oil was dissolved in CH Cl (1.4 mLl and the solu-
2
2
(
5
CϭO, amide), 1538, 1434, 1370, 1221, 1074, 1041. MS (ESI(Ϫ)70 V, m/z): tion was added into a biphasic mixture of aminosaccharide 3Ј (0.68 g,
Ϫ
25
26.2 [MϪH] . [a]D ϩ22.0 (cϭ0.295, CH OH). Anal. Calcd for 1.97 mmol) in anhydrous CH Cl (9.6 ml) and Na CO (0.21 g, 1.97 mmol)
3
2
2
2
3
C H N O ·0.5H O: C, 47.02; H, 4.89; N, 7.83. Found: C, 47.14; H, 4.83; in H O (9.6 ml). The reaction mixture was stirred at room temperature for
21
25
3
13
2
2
N, 7.72.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-thiazole-4-car-
12 h. Then the organic layer was removed and the aqueous layer was ex-
tracted with CH Cl (27 mlϫ2). The combined organic layer was washed
2
2
1
boxamide (31): Isolated yield, 55.0%; mp 224—226 °C (decomp.). H-NMR saturated aqueous NaHCO (27 mlϫ2) and brine (27 mlϫ2), dried over an-
3
(
4
300 MHz, CDCl ) d: 1.94, 2.06, 2.08, 2.20 (each 3H, each s, each CH ),
hydrous Na SO , filtered and concentrated under reduced pressure. The
.08—4.24 (3H, m, H-6a, H-6b, H-5), 4.54—4.64 (1H, m, H-2), 5.20 (1H, residue was chromatographed on a silica gel column by use of petroleum
3
3
2 4
dd, H-3, Jϭ3.3, 11.1 Hz), 5.44 (1H, d, H-4, Jϭ2.7 Hz), 5.88 (1H, d, H-1,
Jϭ8.7 Hz), 7.32 (1H, d, NH, Jϭ9.6 Hz), 8.17 (1H, d, ArH, Jϭ2.1 Hz), 8.74
ether–acetic ether (3 : 2, v/v) to give compound 24 as a yellowish solid. Iso-
1
lated yield, 54.8%; mp 170—172 °C. H-NMR (300 MHz, CDCl ) d: 2.02,
3
Ϫ1
(
(
(
1H, d, ArH, Jϭ2.1 Hz). IR (KBr) cm : 3358 (NH), 2963 (CH), 1750 2.07, 2.10, 2.11 (each 3H, each s, each CH ), 2.69 (3H, s, CH ), 3.84 (1H,
3
3
CϭO, ester), 1662 (CϭO, amide), 1544, 1489, 1371, 1226, 1073, 1044. MS ddd, H-5, Jϭ2.1, 4.5, 8.1 Hz), 4.16 (1H, dd, H-6b, Jϭ2.4, 12.6 Hz), 4.29
Ϫ
25
ESI(Ϫ)70 V, m/z): 457.0 [MϪH] . [a]D ϩ19.7 (cϭ0.065, CHCl ). Anal.
(1H, dd, H-6a, Jϭ4.8, 12.6 Hz), 4.43—4.47 (1H, m, H-2), 5.19—5.22 (2H,
3
Calcd for C H N O S: C, 47.16; H, 4.84; N, 6.11. Found: C, 47.37; H, m, H-3, H-4), 5.77 (1H, d, H-1, Jϭ8.7 Hz), 5.95 (1H, d, NH, Jϭ9.0 Hz),
18
22
2
10
Ϫ1
4
.87; N, 6.10.
N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-2-chloro-pyri-
8.28 (1H, s, ArH). IR (KBr) cm : 3328 (NH), 2957, 2891 (CH), 1745
(CϭO, ester), 1658 (CϭO, amide), 1609, 1537, 1249, 1224, 1087, 1037,
1
Ϫ
25
dine-3-carboxamide (32): Isolated yield, 19.1%; mp 147—150 °C. H-NMR
936, 593. MS (ESI(Ϫ)70 V, m/z): 455.0 [MϪH] . [a]D ϩ25.6 (cϭ0.075,
(
300 MHz, CDCl ) d: 2.03, 2.06, 2.16, 2.21 (each 3H, each s, each CH ), CHCl ). Anal. Calcd for C H N O : C, 50.00; H, 5.30; N 6.14. Found: C,
3
3
3
19 24
2
11
4
.10—4.20 (3H, m, H-6a, H-6b, H-5), 4.56—4.65 (1H, m, H-2), 5.28 (1H, 50.08; H, 5.50; N, 5.92.
Bioassay In brief, 96-well plates were dispersed with compounds 16—
Jϭ8.7 Hz), 6.43 (1H, d, NH, Jϭ9.0 Hz), 7.31—7.35 (1H, m, ArH), 7.84 34 at 0, 1.6, 8, 40, and 200 mg/ml in cell culture media containing 1 mg/ml
1H, dd, ArH, Jϭ7.5, 1.5 Hz), 8.45 (1H, dd, ArH, Jϭ1.8, 4.8 Hz). IR (KBr) heparin (final concentration). BaF3 cells expressing FGFR1c cells (30000
cm : 3426 (NH), 2923 (CH), 1751 (CϭO, ester), 1659 (CϭO, amide), live cells/well) were added to compounds 16—34. FGF2 was then added to
dd, H-3, Jϭ3.3, 11.4 Hz), 5.43 (1H, d, H-4, Jϭ3.3 Hz), 5.90 (1H, d, H-1,
(
Ϫ1

September 2010
1215
each plate at a final concentration of 8 nM. Cells were cultured for 40 h, fol-
14) Hassan H. H., Mini Rev. Med. Chem., 7, 1206—1235 (2007).
lowed by adding 20 ml resazurin (2 mg/ml dissolved in water, Cat. # R7017, 15) Delehedde M., Lyon M., Gallagher J. T., Rudland P. S., Fernig D. G.,
Sigma Aldrich, St. Louis, MO, U.S.A.) to the media for 16 h. The fluores- Biochem. J., 366, 235—244 (2002).
cent signal was monitored using 544 nm excitation wavelength and 595 nm 16) Ornitz D. M., Herr A. B., Nilsson M., Westman J., Svahn C. M., Waks-
emission wavelength by Spectramax M2 plate reader (Molecular Devices). man G., Science, 268, 432—436 (1995).
The relative fluorescence unit (RFU) generated from the assay was propor- 17) Schlessinger J., Plotnikov A. N., Ibrahimi O. A., Eliseenkova A. V.,
tional to the number of living cells in each well. RFU represented heparin
and target compounds stimulated or inhibited cell proliferation. Data are
meanϮrange of duplicates.
Yeh B. K., Yayon A., Linhardt R. J., Mohammadi M., Mol. Cell, 6,
743—750 (2000).
18) Jin L., Abrahams J. P., Skinner R., Petitou M., Pike R. N., Carrell R.
W., Proc. Natl. Acad. Sci. U.S.A., 94, 14683—14688 (1997).
Acknowledgements The project is sponsored by the Scientific Research 19) Li W., Johnson D. J., Esmon C. T., Huntington J. A., Nat. Struct. Mol.
Foundation for the Returned Overseas Chinese Scholars, State Education Biol., 11, 857—862 (2004).
Ministry, Qing Lan Project of Jiangsu Province, and a research fund from 20) Yeh B. K., Eliseenkova A. V., Plotnikov A. N., Green D., Pinnell J.,
Children’s Discovery Institution in St. Louis, Missouri in the U.S.A. We ap-
preciate the support from Center for Instrumental Analysis, China Pharma-
ceutical University, for structural analysis of all synthesized compounds.
Polat T., Gritli-Linde A., Linhardt R. J., Mohammadi M., Mol. Cell.
Biol., 22, 7184—7192 (2002).
21) Fryer A., Huang Y. C., Rao G., Jacoby D., Mancilla E., Whorton R.,
Piantadosi C. A., Kennedy T.,Hoidal J., J. Pharmacol. Exp. Ther., 282,
208—219 (1997).
References
1)
2)
3)
Desnoyers L. R., Pai R., Ferrando R. E., Hotzel K., Le T., Ross J., 22) Lapierre F., Holme K., Lam L., Tressler R. J., Storm N., Wee J., Stack
Carano R., D’Souza A., Qing J., Mohtashemi I., Ashkenazi A., French
D. M., Oncogene, 27, 85—97 (2008).
Pollock P. M., Gartside M. G., Dejeza L. C., Powell M. A., Mallon M.
R. J., Castellot J., Tyrrell D. J., Glycobiology, 6, 355—366 (1996).
23) Lundin L., Larsson H., Kreuger J., Kanda S., Lindahl U., Salmivirta
M., Claesson-Welsh L., J. Biol. Chem., 275, 24653—24660 (2000).
A., Davies H., Mohammadi M., Futreal P. A., Stratton M. R., Trent J. 24) Purkerson M. L., Tollefsen D. M., Klahr S., J. Clin. Invest., 81, 69—74
M., Goodfellow P. J., Oncogene, 26, 7158—7162 (2007). (1988).
Lanske B., Razzaque M. S., Curr. Opin. Nephrol. Hypertens., 16, 25) Pitt N., Duane R. M., O’Brien A., Bradley H., Wilson S. J., O’Boyle
3
11—318 (2007).
K. M., Murphy P. V., Carbohydr. Res., 339, 1873—1887 (2004).
26) Rawe S. L., Doyle D., Zaric V., Rozas I., McMahon K., Tosin M.,
Bunz H. M., Murphy E. P., Km O. B., Murphy P. V., Carbohydr. Res.,
341, 1370—1390 (2006).
4
5
)
)
Kharitonenkov A., Shanafelt A. B., BioDrugs, 22, 37—44 (2008).
Presta M., Dell’Era P., Mitola S., Moroni E., Ronca R., Rusnati M.,
Cytokine Growth Factor Rev., 16, 159—178 (2005).
6
7
)
)
Ornitz D. M., Bioessays, 22, 108—112 (2000).
Wu Z. L., Zhang L., Yabe T., Kuberan B., Beeler D. L., Love A.,
Rosenberg R. D., J. Biol. Chem., 278, 17121—17129 (2003).
27) Rawe S. L., Zaric V., O’Boyle K. M., Murphy P. V., Bioorg. Med.
Chem. Lett., 16, 1316—1319 (2006).
28) Xu J.-F. F., Z.-J., Ju C.-L., Chin. J. Syn. Chem., 11, 379—433 (2003).
8)
McDowell L. M., Frazier B. A., Studelska D. R., Giljum K., Chen J., 29) Ornitz D. M., Yayon A., Flanagan J. G., Svahn C. M., Levi E., Leder
Liu J., Yu K., Ornitz D. M., Zhang L., J. Biol. Chem., 281, 6924—
930 (2006).
Mousa S. A., Cardiovasc. Drug Rev., 22, 121—134 (2004).
P., Mol. Cell. Biol., 12, 240—247 (1992).
6
30) Liu J., Shriver Z., Pope R. M., Thorp S. C., Duncan M. B., Copeland
R. J., Raska C. S., Yoshida K., Eisenberg R. J., Cohen G., Linhardt R.
J., Sasisekharan R., J. Biol. Chem., 277, 33456—33467 (2002).
9
)
1
0) Norrby K., APMIS, 114, 79—102 (2006).
11) Ferro V., Dredge K., Liu L., Hammond E., Bytheway I., Li C., John- 31) Atha D. H., Lormeau J. C., Petitou M., Rosenberg R. D., Choay J., Bio-
stone K., Karoli T., Davis K., Copeman E., Gautam A., Semin.
Thromb. Hemost., 33, 557—568 (2007).
2) Kragh M., Loechel F., Int. J. Oncol., 27, 1159—1167 (2005).
chemistry, 24, 6723—6729 (1985).
32) Maccarana M., Casu B., Lindahl U., J. Biol. Chem., 268, 23898—
23905 (1993).
1
1
3) Cross M. J., Claesson-Welsh L., Trends Pharmacol. Sci., 22, 201—207 33) Petitou M., van Boeckel C. A., Angew. Chem. Int. Ed. Engl., 43,
2001). 3118—3133 (2004).
(