MENG Qing-Hua LIU Qi-Feng ZHANG Zun-Ting

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Synthesis, Crystal Structure and Neuroprotection of a Novel Isoflavone Sulfonamide Compound①

MENG Qing-Huaa, c②LIU Qi-FengbZHANG Zun-Tingc

a(712046)b(061100)c(,,,710062)

4?,7-Dimethoxy-N-methylisoflavone-3?-sulfonamide (3) was synthesized by a two step synthesis method and characterized via IR and1H NMR. Its chemical structure was determined byX-ray single-crystaldiffraction. It crystallizes in monoclinic, space group2/with= 27.677(13),= 10.490(5) ,= 13.982(7) ?,= 118.781(9)o,= 3558(3) ?3,= 8,(000) = 1568,D= 1.401 Mg/m3,M= 375.39,= 0.217 mm–1,= 0.71073?, the final= 0.0436 and= 0.0910 for 3168 observed reflections with> 2(). Thecompound was studied subsequently for the potent neuroprotective activity, and showed effects against oxygen-glucose deprivation injury in PC12 cells in a dose-dependent manner (protection: 42.5% at 100 μg·mL?1, 35.1% at 10 μg·mL?1and 22.5% at 1 μg·mL?1).

4?,7-dimethoxy-N-methylisoflavone-3?-sulfonamide, crystal structure, neuroprotection;

1 INTRODUCTION

Daidzein(4?,7-dihydroxy-isoflavone), one of the effective principals of isoflavone, shows many biological activities such as inhibiting the growth of cancer cells[1–3], accelerating the formation of bone cells[4], playing the role of female hormone[5],. However, the biological utilization rate is low and the dose is high for its poor solubility of isoflavone. In order to study its possible biological effects, we had already synthesized more derivatives of iso- flavone and studied their structures and biological activities, such as sodium 7-methoxy-4?-hydroxy- isoflavone-3?-sulfonate[6], sodium 4?,7-dihydro- xyisoflavone-3?-sulfonate[7, 8], and 7,4?-bimeth- oxyisoflavone-3?-sulfonate[9]. The results showed that they possess better biological activity than daidzein. Furthermore, as is well-known, isoflavone sulfonamide compounds have been representing an important class of organic heterocycles[10, 11]. They have received considerable attention because of their widespread biological activities[12, 13], inclu- ding anticancer, antimicrobial, antiviral, anti-HIV, and aromatase inhmitors. In this paper, the crystal structure and a two step synthesis method for an isoflavone sulfonamide 4?,7-dimethoxy-N-methyli- soflavone-3?-sulfonamide (3) using 4?,7-dimethoxy- isoflavone as a starting material is reported (Fig. 1). The preliminary bioassay results showed that 3 exhibits potent neuroprotective activity.

Fig. 1. Synthetic route for compound 3

2 EXPERIMENTAL

2. 1 Apparatus and materials

All the chemicals were commercially available and used without further purification. IR spectra (400～4000 cm–1) were measured on a Nicolet 170SX FT-IR spectrometer.1H NMR spectra were obtained using a Bruker AM-300. Elemental ana- lyses were determined using a PE-2400 elemental analyzer. Melting point was measured on a WRS- 113 digital melting point instrument and the ther- mometer was uncorrected. Fetal bovine serum (FBS) was purchased fromSijiqing Biological Engi- neering Institute, (Hangzhou).Trypsinase and DMEM were obtained from Gibco BRL (Div. of Invitrogen, Gaithers-burg, MD, USA). 3-(4,5- Di- methylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide (MTT) was purchased from Sigma Chemical Co. (St.Louis, MO, USA).

2. 2 Synthesis and characterization of compound 3

Chlorosulfonic acid (8 mmol) was added dropwise to 4?,7-dimethoxyisoflavone[14](8 mmol) with strong stirring for 0.5 h at 273 K. Then the temperature was raised at 338 K and kept for 3 h. After cooling to room temperature, the mixture was poured into ice water (60 mL) and a white pre- cipitate appeared. The precipitate was filtered and washed with water until the pH of the filtrate was 7. It was dissolved in acetone, then the methylamine solution (2 mL, 18%) was added, followed by stirring at room temperature. After 0.5 h, the mixture was poured into water (50 mL) and a yellow precipitate came into being. Finally, the precipitate was filtered, dried, and recrystallized from methanol, leading to compound 3 as yellow powder.After recrystallization from methanol, yellow single crystals suitable for X-ray diffraction analysis were obtained by slow evaporation 3 days letter.

m.p.: 535～537 K. Anal. Calcd. for C18H17NO6S (%): C, 57.59; H, 4.56; N, 3.73. Found: C, 57.29; H, 4.64; N, 3.75. IR (KBr pellet cm-1): 3293, 3081, 2942,1635, 1569, 1499, 1442, 1320, 1258, 1065,1046 cm-1.1H NMR (DMSO-): 8.55 (s, 1H, H-C2), 8.05 (d, 1H,= 8.3 Hz, H-C5),7.56 (dd, 1H,= 8.6 Hz,= 2.4 Hz, H-C6), 7.20 (d, 1H,= 2.4 Hz, H-C8), 3.93 (s, 3H, C7-OCH3), 8.00 (s, 1H, H-C2?), 7.09 (d, 1H,= 8.4 Hz, H-C5?),7.13 (d, 1H,= 8.4 Hz, H-C6?), 3.92 (s, 3H, C4?-OCH3),2.43 (d, 3H, N-CH3).

2. 3 Crystal structure determination

A yellow crystal of3 with approximate di- mensions of 0.34mm × 0.24mm × 0.06mm was selected and mounted on a glass fiber. The intensity data were collected on a Bruker Smart-1000 CCD diffractometer using graphite-monochromated Moradiation (= 0.71073 ?) by a-scan mode within the range of 1.68<<25.10°. A total of 8887 reflections were collected, with 3168 inde- pendent ones (int= 0.0593).

The structure was solved by direct methods using SHELXS-97[15]and refined on2by full-matrix least-squares with the SHELXL-97[16]. The re- finement converged to the final(> 2())= 0.0436,= 0.0910,(all data) = 0.1074 and= 0.1172, (Δ)min= –0.228 and (Δ)max= 0.183 e/?3. Nitrogen H atoms were located in a difference Fourier map and refined with N–H restrained to 0.86(2) ?. Others H atoms were placed at the calculated positions and treated as riding, with the C–H distance falling in the 0.93～0.96 ? range andiso(H) = 1.2eqof the attached C atom (1.5eq(C) for methyl H atoms).

2. 4 Neuroprotective effects

In order to study the potential neuroprotective activity of the title compound, a preliminary screening was performed to investigate the neuro- protection on impairment induced by sodium dithionite (Na2S2O4) in PC12 cells, as evaluated by MTT assay[17]. PC12 cells were grown in DMEM supplemented 10% FBS, 5% HS in a humidified incubator at 37 °Cand 5% CO2. Trypsinization (0.25%) was done when 80～90% cells were fused. PC12 cells were inoculated in 96-well plate with the density of 5×104for 24-hour wall adhering. The medium was changed to Earle?s liquor without sugar. After 30 min,Na2S2O4(2 mmol·L?1) balanced by D-Hanks was added for 1 h. Prior to treatments, PC12 cells were rinsed by DMEM for three times (5 min each), and then covered with serum-free DMEM in which compound 3 (1, 10, 100 μg·mL?1) was added. After incubation in a CO2incubator for 24 h, PC12 cells were subsequently tested by MTT assay.

3 RESULTS AND DISCUSSION

3. 1 Molecular structure

The1H NMR and IR for 3 were in good agreement with 4?,7-dimethoxy-N-methyli-sofla- vone-3?-sulfonamide. In IR spectra of 3, the characteristic absorption at 1635 cm-1belongs to the C=O vibration of the group in the isoflavone skeleton. And two strong absorptions were in- creased at 1258and 1165 cm-1by contrast with IR spectra of 4?,7-dimethoxyisoflavone, validating the existence of sulfo-group in3. In the1H NMR spectra of 3, the shapes, chemical shifts and coupling constants of protons in the benzopyranone moiety had no obvious alterations, while for ring B, the chemical shifts and coupling constants of 3 indicated that a sulfo-group was introduced to C(3?) position of ring B. In order to confirm the con- figuration of3, a single crystal of the title com- pound was cultured for X-ray diffraction analysis.

The crystal structure of compound 3is given in Fig. 2.The selected bond lengths, bond angles and partial values of torsional angles are listed in Table 1.The atoms of the benzopyranone moiety including rings A (C(9)/C(10)/C(12)～C(15)) andC(C(7)～C(11)/O(4)) are essentially coplanar, with the mean deviation form the least-squares planes to be 0.0073(21)? and their dihedral angle of 0.74(9)°. To avoid steric conflict, the phenyl rings B (C(1)～C(6)) and benzopyranone moieties are rotated by 46.50(8)° witheach other. An independent N(1)–H(1A)···O(3)intramolecular hydrogen bond of 3forms a characteristic S(6) motif.

Table 1. Selected Bond Lengths (?), Bond Angles (°) and Torsional Angles (°) for the Title Compound 3

Fig. 2. Molecular structure of compound 3

Table 2. Typical Hydrogen Bond Lengths and Bond Angles for the Title Compound 3

Symmetry codes: (i) –+3/2,+1/2, –+3/2; (ii) –+1,+1, –+1/2; (iii) –+1,, –+1/2; (iv)–1/2, –+1/2,–1/2; (v) –+1, –, –+1

Fig. 3. Part of the crystal structure of 3, showing the formation of sheet [01]C–H···O and N–H···O hydrogen bonds. (Symmetry codes: (vi)+ 1/2, –+1/2,+1/2 (vii) –x+3/2,–1/2, –+3/2)

Fig. 4. π-π stacking interactions and hydrogen bonds along the-axis

Fig. 5. Crystal packing viewed approximately along the-axis with hydrogen bonding pattern as dashed lines

Moleculesare linked into a chain by N(1)–H(1A)···O(2)hydrogen bonds,and these chains are linked into the sheet [01] by theC(13)–H(13)···O(1)hydrogen bonds (Fig. 3).

Along the-axis,stacking interactions (CgA···CgCV=CgC···CgAV= 3.773(2) ?, CgAC···

CgACiii= 3.708(2) ?) and hydrogen bonds(C(4)– H(4)···O(5), C(16)–H(16)···O(5)) (Fig. 4) linked the sheets into a neat three-dimensional supra- molecular structure (Fig. 5). The hydrogen bonds are presented in Table 2.

3. 2 Neuroprotective effects

As shown in Table 3, remarkably, compound3 showed potent neuroprotective effectsagainst oxy- gen-glucose deprivation injury in PC12 cellsin a dose-dependent manner (protection: 42.5% at 100 μg·mL?1, 35.1% at 10 μg·mL?1and 22.5% at 1 μg·mL?1). Effects of 3 against sodium hydro- sulfite-induced neurotoxicity in PC12 cells indi- cated that it can attenuate neurotoxicity.

Table 3. Effects of Compound 3 against Sodium Hydrosulfite-induced Neurotoxicity in PC12 Cells

*< 0.05the model group

(1) Jing, Y. K.; Han, R. Differentiation of B16 melanoma cells induced by daidzein.1992, 6, 278-278.

(2) Sarthyamoorhy, N.; Wang, T.T.Y. Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells.1997, 33, 2384-2389.

(3) Jing, Y.; Nakaya, K.; Han, R. Differentiation of promyelocytic leukemia cells HL-60 induced by daidzein in vitro and in vivo.1992, 13, 1049-1054.

(4) Sugimoto, E.; Yamaguchi, M. Stimulatory effect of daidzein in osteoblastic MC3T3-E1 cells.2000, 59, 471-475.

(5) Miksicek, R. J. Commonly occurring plant flavonoids have estrogenic activity.1993, 44, 37-43.

(6) Zhang, Z. Z.; Liu, Q. G.; Liu, X. H.; Yang, B. L.; Duan, Y. F.; Gao, Z. W.; Yu, K. B. Synthesis, crystal structure and biological activity of monomethylated daidzein sulfonates.2002, 60, 1846-1853.

(7) Liu, Q. G.; Zhang, Z. Z.; Xue, D. Synthesis, crystal structure and activity of sulfated daidzein.2003, 24, 820-825.

(8) Zhang, Z. T.; Wang, Q. Y.; Li, W. W.; Meng, Q. H.; Zhang, X. L. Four 3D metal-organic frameworks derived from barium(II) and isoflavonesulfonate ligands.. 2012, 14, 5042-5052.

(9) Zhang, Z. T.; Wang, Q. Y.; He, Y.; Wang, X. B.; Xue, D.; Zheng, J. B. Synthesis, crystal structure and biological activity of bimethylation daidzein sulfonates.2005, 26, 2247-2253.

(10) Sardari, S.; Mori, Y.; Horita, K.; Micetich, R. G.; Nishibe, S.; Daneshtalab, M. Synthesis and antifungal activity of coumarins and angular furanocoumarins.1999, 7, 1933-40.

(11) Ojala, T.; Remes, S.; Haansuu, P.; Vuorela, H.; Hiltunen, R.; Haahtela, K.; Vuorela, P. Antimicrobial activity of some coumarin containing herbal plants growing in Finland.2000, 73, 299-305.

(12) Guo, P.; Zhou, Z.; Dong, Y.; Li, Z.; Guo, Y.; Pan, Y.; Li, Y.; Bu, Q. Preparation of polymethoxyflavone derivatives as antitumor agents.CN200610037942.2 [P] 2006.

(13) Kurono, M.; Baba, Y.; Iwata, N.; Oonishi, O.; Kakubuchi, M.; Isogawa, Y.; Mitani, T.; Ishiwatari, Y.; Oowaki, H.; Sawai, K. Preparation of flavone derivatives as virucides. JP07002826 [P] 1995.

(14) Wang, Q. Y.; Zhang, Z. T. Hexaaqua-nickel(II) bis-(4?,7-dimethoxy-isoflavone-3?-sulfonate) octa-hydrate.2005,61, m215-m217.

(15) Sheldrick, G. M.

(16) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(17) Taro, T.; Takashi, D.; Masahiro, Y.; Akiharu, K.; Tetsuhiro, N.; Ryota, T.; Toshiaki, K.; Akinori, A.; Hachiro, S. Synthesis and neuroprotective effects of serofendic acid analogues.2006, 16, 5080-5083.

① This work was supported by grants from the project of the education department of Shaanxi Province (No.2013JK0839)

② Corresponding author. E-mail: mengqinghua66@126.com

10.14102/j.cnki.0254-5861.2011-0741

30 March 2015; accepted 23 June 2015 (CCDC 640737for 3)