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Title A small oxazine compound as an anti-tumor agent : A novel pyranoside mimetic that binds to VEGF, HB-EGF, andTNF-α

Author(s) Basappa; Murugan, Sengottuvelan; Kavitha, Chandagirikoppal V.; Purushothaman, Anurag; Nevin, Kottayath G.;Sugahara, Kazuyuki; Rangappa, Kanchugarakoppal S.

Citation Cancer Letters, 297(2), 231-243https://doi.org/10.1016/j.canlet.2010.05.016

Issue Date 2010-11-28

Doc URL http://hdl.handle.net/2115/49204

Type article (author version)

Additional Information There are other files related to this item in HUSCAP. Check the above URL.

File Information Supplementary_material.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

Basappa et al., Supplemental Material

Experimental Procedures

Chemistry

Supplemental Scheme 1. General procedure for the synthesis of FMBO, CMBO, and

PMBO. Equimolar solution of aldehydes (R1-CHO) and 1-[2-amino-1-(4-methoxy-

phenyl)-ethyl]-cyclohexanol monoacetate were mixed in 5.0 ml of methanol in the

presence of anhydrous K2CO3 and the reaction mixture was stirred at room temperature

for 10-12 h. After the reaction complete, evaporated the methanol, water was added and

the reaction mass was extracted three times with ethyl acetate. After drying over Na2SO4

and evaporation of the organic solvent in vacuo, purified the compounds using column

chromatography (solvent system: chloroform: methanol (9:1).

OCH3

NH2

OH

OCH3

O

NH

R1

K2CO3/ Methanol / RT

R1-CHO

Acetate Salt Oxazine Derivatives

A. Synthesis of FMBO. FMBO was obtained from an amino alcohol (1g, 3.23 mmol), 4-

fluoro benzaldehyde (0.48g, 3.88mmol) and anhydrous Na2CO3 (1.37g, 12.94 mmol) as

amorphous white solid (0.8g, 80%). Mp 130 0C. IR νmax (KBr): 3212, 2945, 1520, 1240

cm-1. 1H NMR (CDCl3 400 MHz) δ: 7.61 (2H, d, J=5.5, Ar-H), 7.15 (5H, m, J=6, Ar-H),

6.86 (2H, d, J=6.5, Ar-H), 5.31 (1H, d, NH), 3.75 (3H, s, OCH3), 3.50 (1H, d, CH2-CH),

3.32 (3H, s, Ar-CH), 2.41-2.92 (2H, m, CH-CH2), 1.30-1.75 (10H, m, cyclohexyl). Anal.

(C22H26FNO2) C, H, N.

B. Synthesis of CMBO. CMBO was obtained from an amino alcohol (1g, 3.23 mmol), 4-

chloro benzaldehyde (0.54g, 3.88mmol) and anhydrous Na2CO3 (1.37g, 12.94 mmol) as

colourless crystalline solid (0.78g, 78%). Mp 80 0C. IR νmax (KBr): 3209, 2933, 1523,

1238, 1008 cm-1. 1H NMR (CDCl3 400 MHz) δ: 7.58 (2H, d, J=14.1, Ar-H), 7.45 (2H, d,

J=15.6, Ar-H), 7.12 (2H, d, J =8.4, Ar-H), 6.88 (2H, d, J=8.7, Ar-H), 5.33 (1H, d, NH),

3.73 (3H, s, OCH3), 3.50 (1H, d, CH), 3.32 (3H, s, Ar-CH), 2.51-2.72 (2H, m, CH-CH2),

1.30-1.75 (10H, m, cyclohexyl). Anal. (C22H26ClNO2) C, H, N.

C. Synthesis of PMBO. PMBO was obtained from an amino alcohol (1g, 3.23 mmol),

pyridin-3-aldehyde (0.41g, 3.88 mmol) and anhydrous Na2CO3 (1.37g, 12.94mmol) as

amorphous white solid (0.78g, 78%). Mp 840C. IR νmax (KBr): 3089, 2868, 1627, 1508,

1232 cm-1. 1H NMR (CDCl3 400 MHz) δ: 8.91 (1H, s, Ar-H), 8.65 (1H, d, J=7, Ar-H),

7.95 (1H, d, J=7.6 Ar-H), 7.42 (1H, t, J=7.6, Ar-H), 7.15 (2H, d, Ar-H), 5.45 (1H, s,

NH), 3.80 (3H, s, OCH3), 3.62 (1H, t, CH-CH2), 3.34 (3H, s, Ar-CH), 2.51-2.90 (m, 1H,

CH-CH2), 0.91-1.85 (10H, m, cyclohexyl). Anal. (C21H26N2O2) C, H, N.

Biology

Hyaluronidase Digestion Assay: FITC-labelled hyaluronic acid (HA) (20 μg) was incubated

with 2 mIU of hyaluronidase (from Streptomyces hyalurolytics) in the presence or absence of

DMBO (100 μM) in a total volume of 50 μl of 20 mM acetate–Na buffer, pH 6.0 at 60°C for

4h. The digests were subjected to gel filtration HPLC on a Superdex 200 (GE Healthcare) at a

flow rate of 0.4 mL/min using 0.2 M NH4HCO3 as the eluent. Eluates were monitored by

measuring fluorescence with excitation and emission wavelengths of 490 and 520 nm,

respectively (1).

Chondroitinase ABC digestion Assay: Chondrotin sulfate (CS)-A (2 μg) from whale

cartilage, was digested with chondroitinase (CSase) ABC (10 mIU) (2) in the presence or

absence of DMBO (100 μM) and labeled with 2-Aminobenzamide (2AB) (3). The excess

2AB was removed by extraction with chloroform (4). The 2AB-labeled digest was analyzed

by anion exchange HPLC on a PA-03 silica column (YMC-Pack PA, Kyoto, Japan) with a

linear gradient of NaH2PO4 from 16 to 538 mM over 60 min at a flow rate of 1.0 mL/min at

room temperature. Identification and quantification of the resulting disaccharides were

achieved by comparison with the elution of authentic CS-derived unsaturated disaccharides

(3).

References

1. Li F., Yamada, S., Basappa,. Shetty, A. K., Sugiura, M., Sugahara, K. (2008).

Glycoconj J. 25, 603-610.

2. Saito, H., Yamagata, T., Suzuki, S. (1968). J Biol Chem. 243, 1536–1542.

3. Kinoshita A, Sugahara K. (1999). Anal Biochem. 269, 367–378.

4. Kawashima, H., Atarashi, K., Hirose, M., Hirose, J., Yamada, S., Sugahara, K.,

Miyasaka, M. (2002). J Biol Chem. 277, 12921–12930.

Figure legends

Supplemental Fig. 1. The structure of the photo-affinity linker and the dummy thiol linker.

Supplemental Fig. 2. The SPR signal image for VEGF bound with immobilized DMBO and

its location on PGSs. The interaction between DMBO and VEGF (arrows) was quantified

using an SPR analyzer. The IUPAC names of the other small molecules are as follows:

CMBO: 2-(4-Chlorophenyl)-4a,5,6,7,8,8a-hexahydro-4a-(4-methoxyphenyl)-4H-

benzo[e][1,3]oxazine; PMBO: 2-(3-Pyridyl)-4a,5,6,7,8,8a-hexahydro-4a-(4-methoxyphenyl)-

4H-benzo[e][1,3] oxazine; KSB110: (2-Ethoxyphenyl)(4-(6-fluorobenzo[d]isoxazol-3-

yl)piperidin-1-yl) methanone; RS01: 2-Butyl-4-chloro-5-(2,5-diphenylisoxazolidin-3-yl)-1H-

imidazole; KSB76: 1-(2-Bromo-4,5-dimethoxybenzyl)-2-butyl-4-chloro-1H-imidazole-5-

carbaldehyde; KSB75: 2-Butyl-4-chloro-1-((5-methylbenzo[d][1,3]dioxol-6-yl)methyl-1H-

imidazole-5-carbaldehyde; KSB58: 4’-(2-Butyl-4-chloro-5-formyl-imidazol-1-ylmethyl)-

biphenyl-2-carbonitrile; KSB78: 2-Butyl-4-chloro-1-((4,5-dihydro-5-methylisoxazol-4-

yl)methyl)-1H-imidazole-5-carbaldehyde; KSB28: 4-(2,6-Difluorobenzylideneamino)-5-

ethyl-4H-1,2,4-triazole-3-thiol; KSB29: 4-(2,6-Difluorobenzylideneamino)-5-phenyl-4H-

1,2,4-triazole-3-thiol; KSB07: 4-(2,6-Difluorobenzylideneamino)-5-(4-chlorophenyl-4H-

1,2,4-triazole-3-thiol; KSB52: 4-(2,6-Difluorobenzylideneamino)-5-ethyl-4H-1,2,4-triazole-

3-thiol; KSB34: 2-Butyl-1,5,6,7a-tetrahydro-5-methylimidazo[4,5-e][1,3]thiazin-(2-methyl-

3,4-triazolo)-7(3aH)-imine.

Supplemental Fig. 3. The SPR signal image for TNF-α bound with immobilized DMBO and

its location on PGSs. The interaction between DMBO and TNF (arrows) was quantified using

an SPR analyzer. The IUPAC names of the other small molecules are described in the legend

to Supplemental Fig. 2.

Supplemental Fig. 4. The SPR signal for HB-EGF bound with immobilized DMBO and its

location on PGSs. The interaction between DMBO and HB-EGF (arrows) was quantified

using an SPR analyzer. The IUPAC names of the other small molecules are described in the

legend to Supplemental Fig. 2.

Supplemental Fig. 5. Interaction of FGF-2 with DMBO and other oxazine molecules. Bar

graph depicts the maximum SPR signal strength for FGF-2 binding with immobilized oxazine

molecules. Oxazine molecules (10 mM) were immobilized on the PGSs and their interactions

with FGF-2 were detected in solution by SPR imaging.

Supplemental Fig. 6. Interaction of midkine with DMBO and other oxazine molecules. Bar

graph depicts the maximum SPR signal strength for midkine binding with immobilized

oxazine molecules. Oxazine molecules (10 mM) were immobilized on the PGSs and their

interactions with midkine were detected in solution by SPR imaging. The location of DMBO

and other oxazine molecules on PGSs is presented.

Supplemental Fig. 7. Interaction of pleotrophin with DMBO and other oxazine molecules.

Bar graph depicts the maximum SPR signal strength for pleotrophin binding with

immobilized oxazine molecules. Oxazine molecules (10 mM) were immobilized on the PGSs

and their interactions with pleotrophin were detected in solution by SPR imaging.

Supplemental Fig. 8. Interaction of BSA with DMBO and other oxazine molecules. Bar

graph depicts the maximum SPR signal strength for BSA binding with immobilized oxazine

molecules. Oxazine molecules (10 mM) were immobilized on the PGSs and their interactions

with BSA were detected in solution by SPR imaging.

Supplemental Fig. 9. Histopathological studies of liver and kidney in normal and DMBO

treated mice. The toxic effects of DMBO ((1.5 mg/kg, the highest dose) were evaluated by

histopathological examination of the liver and kidney section by hematoxylin & eosin (H&

E) staining. Representative photomicrographs of normal and DMBO treated liver (A and B),

and kidney (C and D) respectively. Normal and DMBO treated animals show normal-

appearing hepatocyte parenchyma (A and B), kidney glomerulai and tubules (C and D).

Magnification 20X.

Supplemental Fig. 10. DMBO does not inhibit hyaluronidase enzymatic degradation of

FITC-HA. Chromatogram of FITC-labelled HA alone (20 μg) (top panel), digested by the

hyaluronidase (2 mIU) in the presence (last panel) and absence (middle panel) of DMBO

(100 μM) as inhibitor. The mixture was incubated at 60°C for 4h and the samples solutions

were subjected to gel filtration chromatography on a Superdex 200 column equilibrated with

0.2 M NH4HCO3. Eluates were monitored by measuring fluorescence with excitation and

emission wavelengths of 490 and 520 nm, respectively. The undegraded FITC-HA (top

panel) was eluted near the void volume or the degraded FITC-HA by the enzyme

hyaluronidase in the absence (middle panel) or in the presence of DMBO (bottom panel), had

a longer retention time than the control, undegraded FITC-HA. The results shows that FITC-

HA was specifically degraded, indicating that hyaluronidase is active even in the presence of

DMBO.

Supplemental Fig. 11. Anion-exchange HPLC analysis of CSase ABC digests of the CS-A.

The CS-A was digested with CSase ABC in the presence or absence of DMBO (100 μM).

After 2AB-labeling, each digest was analyzed by HPLC on an amine-bound silica PA-03

column using a linear gradient of NaH2PO4 as indicated by the dashed line. The peaks before

10 min were derived from 2AB-derivatizing reagents. Upper panel, CS-A was digested with

CSase ABC with DMBO (100 μM); lower panel, CS-A was digested with CSase ABC

without DMBO. Arrows indicate the elution positions of unsaturated disaccharide standards:

(1) Δ4,5HexUA-GlcNAc; (2) Δ4,5HexUA-GalNAc; (3) Δ4,5HexUA-GalNAc(6S); (4)

Δ4,5HexUA-GalNAc(4S); (5) Δ 4,5HexUA(2S)-GalNAc(6S); (6) Δ4,5HexUA-GalNAc(4S, 6S).

.

Basappa et al.,

Suppl. Figure 1

HSC11H22OO

O

HN

OO

NH

O

CF3

NN4

HSC11H22OO

OOH

Photo-affinity thiol linker

Dummy thiol

Basappa et al.,

Suppl. Figure 2

KSB46 KSB43 DMBO KSB110 RS01

KSB76 KSB75 KSB58 KSB78 DMSO

KSB46 KSB43 DMBO RS01 KSB110

KSB28 KSB29 KSB07 KSB52 KSB34

Basappa et al.,

Suppl. Figure 3

KSB46 KSB43 DMBO KSB110 RS01

KSB76 KSB75 KSB58 KSB78 DMSO

KSB46 KSB43 DMBO RS01 KSB110

KSB28 KSB29 KSB07 KSB52 KSB34

Basappa et al.,

Suppl. Figure 4

KSB46 KSB43 DMBO KSB110 RS01

KSB76 KSB75 KSB58 KSB78 DMSO

KSB46 KSB43 DMBO RS01 KSB110

KSB28 KSB29 KSB07 KSB52 KSB34

Basappa et al.,

Suppl. Figure 5

Basappa et al.,

Suppl. Figure 6

Basappa et al.,

Suppl. Figure 7

Basappa et al.,

Suppl. Figure 8

Basappa et al., Suppl. Figure 9

C D

A B

Basappa et al., Suppl. Figure 10

Time (min)

Basappa et al., Suppl. Figure 11

1 2 3 5 64

1 2 3 5 64

Supplementary Table 1: Inhibition of the proliferation of tumor cells by MBO, FMBO, CMBO or

PMBO.

Anti-proliferative activity (IC50 in μM) Compound R1

LM8 LM8G7 SKOV-3 OVSAHO

1 H NSa NS NS NS

2 4-Fluoro-phenyl 64 ± 0.5 86 ± 0.1 74 ± 0.6 94 ± 0.4

3 4-Chloro-phenyl NS >100 >100 21 ± 2

4 pyridine-3-yl 95 ± 6 NS 65 ± 0.5 NS

aNot significant