Synthesis, Crystal Structure and Antineoplasmic Activity of 3,6,8-Tribromo-5-hydroxy-2,7-dimethoxy- 2-phenyl-2,3-dihydrochromen-4-one①

LI Wu-Wu ZHENG Min-Yn LI Xio-Bo ZHANG Zun-Ting

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Synthesis, Crystal Structure and Antineoplasmic Activity of 3,6,8-Tribromo-5-hydroxy-2,7-dimethoxy- 2-phenyl-2,3-dihydrochromen-4-one①

LI Wu-Wua②ZHENG Min-YanaLI Xiao-BoaZHANG Zun-Tingb

a(,712000)b(710062)

The reaction of5-hydroxyl-7-methoxyflavone (tectochrysin) with bromine obtained 3,6,8-tribromo-5-hydroxy-2,7-dimethoxy-2,3-dihydrochromen-4-one (1), which was characterized by FT-IR,1H-NMR,13C-NMR, elemental analysis and X-ray single-crystal diffraction. Complex 1 belongs to the monoclinic system, space groupwith= 9.4673(8),= 17.9938(15),= 21.2004(17) ?,= 3611.5(5) ?3,= 8,= 0.6726 mm-1,D= 1.795 g/cm3,(000) = 2080, the final= 0.0358 and= 0.0644 with2(). The results show that the addition reaction occurs at the carbon-carbon double bond (C2 and C3) of tectochrysin and 1 belongs to dihydroflavone. The reaction mechanism was discussed and the structure revealed that the crystal structure of 1 is stabilized by intramolecular hydrogen bonds and C–Br···interactions. The antitumor ability of 1 was evaluated against human leukemia cells (K562), human breast cancer cells (MCF-7) and human lung cancer cells (A549). 1 exhibited potent antitumor activities against human leukemia cells (K562) with the IC50values of18.9 μmol/L.

3,6,8-tribromo-5-hydroxy-2,7-dimethoxy-2,3-dihydrochromen-4-one,spectroscopicproperty, crystal structure, reaction mechanisms, antitumor activity;

1 INTRODUCTION

Flavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegeta- bles[1]. Tectochrysin (5-hydroxy-7-methoxyflavone, Scheme 1), one of the major flavonoids of propolis[2], is contained in many plants, especially in some edible plants such as Kaempferia parviflora (Zingi- beraceae)[3], Alpinia xyphylla (Zingiberaceae)[4], Muntingia calabura (Tiliaceae)[5], and Carya genus (Juglandaceae)[6], whose fruits, rhizomes or leaves have been traditionally used for local foods and folk medicines[7]. Pharmacological studies have shown that tectochrysin possesses antioxidant[8], anti- cancer[1], antidiarrheal[9], anti-inflammatory[10]and aryl hydrocarbon receptor ligand activities[11]. It is also previously found that tectochrysin inhibits NO production in LPS-primed macrophages perhaps by suppressing the activity of iNOS[12]. Due to the presence of two benzene rings in the flavonoids structure, most of the structural modifications are concentrated in the electrophilic substitution reaction of the benzene ring. The carbon-carbon double bond (C2 and C3) of flavonoids is not easy to participate in reaction because the carbon-carbon double bond is conjugated with the carbonyl group (C4). There are only a few reports on the addition reaction of carbon-carbon double bond (C2 and C3). Daidzein and genistein are hydrogenated by using Pd/C catalysts[13]. The epoxides of flavonoids are synthe- sized by using DMDO (dimethyldioxirane) as an oxygen source[14]. Inspired by this,in order to get the new C2 and C3 addition product of tectochrysin, 3,6,8-tribromo-5-hydroxy-2,7-dimethoxy-2-phenyl-2 ,3-dihydrochromen-4-one (1) was synthesized by reacting tectochrysin with bromine in MeOH (Scheme 1). The structure of 1 was confirmedFT-IR,1H-NMR,13C-NMR and elemental analysis. The crystal structures of 1were determined by X-ray single-crystal diffraction analysis. The mechanism of addition reaction was discussed. Moreover, the anti- tumor effects of 1against human leukemia cells (K562), human breast cancer cells (MCF-7) and human lung cancer cells (A549) were evaluated by the standard MTT assay[15].

Scheme 1. Synthetic route for compound 1

2 EXPERIMENTAL

2. 1 General experimental information

NMR spectra were recorded on a Bruker AM 300 instrument using CDCl3as the solvent. IR spectra were recorded on a Nicollet 170SX FT-IR spectro- photometer with KBr pellets. Melting points were measured using X-5 melting point apparatus and uncorrected. The crystal diffraction data were collected on a Bruker Smart-1000 CCD diffracto- meter. C and H contents were analyzed using a PE- 2400 elemental analyzer.

Reagents were purchased from commercial sources and used as received unless mentioned otherwise. Reactions were monitored by thin layer chromatography using silica gel GF254plates. Column chromatography was performed on silica gel (300～400 mesh).

2. 2 Synthesis of 3,6,8-tribromo-5-hydroxy-2,7-dimethoxy-2-phenyl-2,3-dihydrochro-men-4-one

A solution of tectochrysin (0.37 mmol) in MeOH (4.0 mL) was refluxed, and then liquid bromine (1.85 mmol) was added. The reaction fluid continued to reflux for 120 min. After completion of the reaction, as indicated by TLC, the reaction mixture was poured into NaHSO4aqueous solution (5%, 50 mL) and a yellow precipitate appeared. After 5 h, the precipitate was filtered and dried at 80 ℃. The precipitate was directly subjected to flash column chromatography on silica gel (DCM/MeOH = 10:1～8:1) to furnish compound 1 as a yellow solid. Yield = 86%. m.p.: 128～130 oC. IR(cm?1, KBr): 3425, 2971, 1698, 1601, 1266, 1151, 934, 872, 608.1H- NMR (CDCl3, 300 MHz)(ppm): 11.96 (s, 1H), 7.70～7.51 (m, 5H), 4.51 (s, 1H), 4.02 (s, 3H), 3.05 (s, 3H).13C NMR(CDCl3, 75 MHz)(ppm): 190.5, 162.0 159.0, 151.8, 134.1, 129.5, 129.2, 128.3, 128.1, 126.3, 105.5, 103.0, 99.5, 97.0, 60.6, 52.2, 48.0. Anal. Calcd. for C17H13Br3O5(%): C, 38.02; H, 2.44. Found (%): C, 38.82; H, 1.93.

2. 3 Crystal structure determination

A yellow crystal of compound 1 suitable for single-crystal X-ray diffraction was obtained by the slow evaporation of the solvent from ethanol. The crystal with dimensions of 0.34mm × 0.22mm × 0.09mm was chosen for X-ray diffraction analysis. The data were collected on a Bruker Smart-1000 CCD diffractometer with graphite-monochromated Mo-radiation (= 0.71073 ?) by ascan mode within the 2.87<<27.23° range at 293(2) K. A total of 17027 reflections were collected to give 3210 independent reflections (int= 0.0676). The structure was solved by direct methods and refined by full-matrix least-squares fitting on2, employing the SHELX-97[16]. The final= 0.0358 and= 0.0644 for observed reflections with> 2(), and= 0.0758 and= 0.0761 for all data with (Δ/)max= 0.616 and (Δ/)min= –0.502 e·?-3. All non- hydrogen atoms were refined anisotropically. All hydrogen atoms were added at the calculated posi- tions and refined using a riding model.

2. 4 Bioassay of the antitumor activities

2. 4. 1 Cell culture

The leukemic cells K562 and human lung cancer cells A549 were incubated with a RPMI-1640 medium. The number of tumor cells is 5 × 103cells per hole in a 96-well plate, the tumor cells continued to incubate for 24 h at 37 oC, and the air in the incubator is humid air with 5% CO2.

2. 4. 2 Antitumor activity evaluation

DMSO (two methyl sulfoxide) solutions of com- pound 1 were prepared for reserve. When the cells were incubated for 24 h, the solution of each com- pound was added to each pore of the 96-well plates, and the concentrations of each compound were 5, 10, 20, 40 and 80 μmol/L. respectively. After incubating for 48 h, 10 μL of 5 mg/mL MTT solution was added to each hole. The tumor cells continued to incubate for 4 h at 37 oC and the air in the incubator is humid air with 5% CO2. 150 μL DMSO was added to each hole and the solution was clarified after shaking for 10 min. The absorbance of each pore sample was measured by microplate reader. Inhibition rates (IR) were calculated by using the following formula:

IR = (1 – ODEvaluated/ODContrast) × 100%

The IC50values were calculated by prohibiting regression using SPSS Statistics17.0 software and displayed as mean ± SD (standard deviation).

3 RESULTS AND DISCUSSION

3. 1 Spectral characteristic

In the IR spectra of 1, the stretching vibration of carbonyl appeared at 1698 cm-1, which is increased by 40 cm-1compared with the carbonyl absorption of tectochrysin (1658 cm?1)[17]. The carbonyl absorption peak moves to high wave numbers, which is caused by the destruction of the conjugate system. This indicates that the addition reaction occurred at the carbon-carbon double bond between C2 and C3.

Chemical shifts of tectochrysin’s ring A (C6–H and C8–H) are 6.25 and 6.40 ppm, respectively[17]. In the1H-NMR spectra of 1, these two absorption peaks disappeared, indicating that the substitution reaction appeared on C6 and C8. The chemical shift of tectochrysin’s pyrone ring (C3–H) is 6.55 ppm[17], that of this proton is 4.51 ppm in 1, and the value of methoxy group (C2) is 3.05 ppm,. These findings indicate that the addition reaction occurred at the carbon-carbon double bond between C2 and C3. Chemical shift at 11.69 ppm belongs to the proton of hydroxyl (C5–OH), and its chemical shift is higher than normal due to the existence of O–H···O intarmolecular hydrogen bond between oxygen atom from carbonyl (C4=O) and hydrogen atom from hydroxyl (C5–OH). In the13C-NMR spectra of 1,the chemical shift of carbonyl (C4) is 190.5 ppm, of C5 and C7 are 162.0 and 159.0 ppm respectively, of C6 and C8 are 99.5 and 97.0 ppm respectively, of C2 and C3 are 103.0 and 60.6 ppm respectively, and the chemical shift range of other aromatic carbon atoms is from 105.5 to 151.8 ppm.

3. 2 Description of the crystal structure of 1

The molecular structure of 1 is illustrated in Fig. 1 and the selected bond lengths and bond angles are listed in Table 1. Compound 1 is composed of three rings, three bromine atoms, two methoxyls and a hydroxy. In the three rings, two of them are benzene (ring A (C(10)～C(15)) and ring B (C(1)～C(6))), and the other one is six-membered oxygenated heterocycle (ring C (O(4)/C(7)～C(11))). The six- membered oxygenated heterocycle (ring C) exhibits an envelope conformation, in which C(7) can be seen as an envelope seal, and five other atoms (O(4)/C(8)～C(11)) are almost planar (Pa), with the distance from C(7) to the plane (Pa) to be 0.609 ?. The plane (Pa) and ring A are coplanar (Pb), and the dihedral angle between plane (Pb) and ring B is 47.70o. Coplanarity of 1 is less than tectochrysin[18], which is caused by the destruction of the conjugate system. The Br(1) on C(8) and the methoxyl on C(7) are located on different sides of the C(7)–C(8) bond, and the torsion angle of O(1)–C(7)–C(8)–Br(1) is 177.57o. The bond lengths of C(14)–Br(3) and C(14)–Br(2) are 1.881 and 1.887 ? respectively, and C(8)–Br(1) is 1.929 ?. The difference of these C–Br bond lengths is due to the different hybridization of carbon atoms. Additionally, the flavone skeleton is stabilized by intramolecular hydrogen bonds, with O(3)–H(3)···O(2) to be 2.606(5) ?.

Table 1. Geometric Parameters for 1 (?,o)

BondDist.BondDist.BondDist. Br(1)–C(8)Br(2)–C(12) Br(3)–C(14)O(1)–C(7)O(1)–C(17)C(4)–C(5)C(5)–C(6)C(6)–C(7)C(12)–C(13)1.929(5)1.887(5)1.882(5)1.395(5)1.431(5)1.381(7)1.385(6)1.514(6)1.393(6)O(2)–C(9)O(3)–C(15)O(4)–C(11)O(4)–C(7)O(5)–C(13)C(7)–C(8) C(8)–C(9)C(9)–C(10)C(13)–C(14)1.229(5)1.353(5)1.360(5)1.437(5)1.372(6)1.540(6)1.500(6)1.450(6)1.371(6)O(5)–C(16)C(1)–C(6)C(1)–C(2)C(2)–C(3)C(3)–C(4)C(10)–C(11) C(10)–C(15) C(11)–C(12)C(14)–C(15)1.438(6)1.369(6)1.379(7)1.366(8)1.367(7)1.400(6)1.411(6)1.382(6)1.388(7) Angles(o)Angles(o)Angles(o) C(7)–O(1)–C(17) C(11)–O(4)–C(7) C(13)–O(5)–C(16) C(6)–C(1)–C(2) C(3)–C(2)–C(1)C(2)–C(3)–C(4) C(3)–C(4)–C(5)C(4)–C(5)–C(6) C(1)–C(6)–C(5) C(1)–C(6)–C(7) O(3)–C(15)–C(14) 116.5(3)117.2(3)113.9(4)120.8(5)120.1(5)120.0(5)120.0(6)120.4(5)118.7(5)119.4(5)119.2(5)C(5)–C(6)–C(7) O(1)–C(7)–O(4) O(1)–C(7)–C(6) O(4)–C(7)–C(6) O(1)–C(7)–C(8) O(4)–C(7)–C(8) C(6)–C(7)–C(8) C(9)–C(8)–C(7) C(10)–C(9)–C(8)C(11)–C(10)–C(15) O(3)–C(15)–C(10)121.9(4)111.1(4)114.5(4)106.2(4)100.7(4)109.9(4)114.4(4)110.2(4)116.6(4)118.2(4)120.2(5)C(11)–C(10)–C(9) C(15)–C(10)–C(9) O(4)–C(11)–C(12) O(4)–C(11)–C(10) C(12)–C(11)–C(10) C(11)–C(12)–C(13) O(5)–C(13)–C(14)O(5)–C(13)–C(12) C(14)–C(13)–C(12) C(13)–C(14)–C(15) C(14)–C(15)–C(10) 120.0(4)121.8(4)117.3(4)121.8(4)120.9(4)119.6(4)120.1(4)118.9(5)120.9(5)119.9(4)120.6(4)

Fig. 1. Molecular structure of 1, showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 25% probability level. The dashed lines indicate hydrogen bonds

3. 3 C–Br···π interactions in crystal structure of 1

As demonstrated by Shukla[19], halogen bonding involving-type electron donors (double and triple bonds, aromatic groups) can form C–Br×××interac- tions. The molecular packing (Fig. 2) is stabilized by two different C–Br×××interactions. One is be- tween the Br(1) and the benzene ring (CgA#) of the adjacent molecules with Br(1)×××CgA# distance of 3.823 ? and C(8)–Br(1)×××CgA# angle of 154.54 o; and the other is between the Br(3) and phenyl ring (CgB*) of the neighbouring molecules with Br(3)×××CgB* distance of 3.664 ? and C(14)– Br(3)×××CgA* angle of 171.36 o (Fig. 2; CgA# and CgB* are the centroids of C(10)～C(15) phenyl at (1/2+, 1/2?, 1?)and the C(1)–C(6) benzene at (,1/2?, ?1/2+), respectively). The C–Br×××interactions lead 1 into a three-dimensional supra- molecular structure (Fig. 2).

Fig. 2. C–Br×××interactions and packing diagram of 1

3. 4 Addition reaction mechanism of tectochrysin

The electrophilic substitution reaction occurs on ring A of tectochrysin, andthe substitution activity of ring A is greater than that of ring B because of the existence of oxhydryl and methoxyl on ring A. The addition reaction mechanism of carbon-carbon double bond (C2 and C3) with MeOH is shown in Scheme 2. The addition reaction of carbon-carbon double bond (C2 and C3) with MeOH belongs to electrophilic addition reaction. First of all, the bromine reacts with carbon-carbon double bond (C2 and C3) to form bromonium ion. Next, the oxygen atom of MeOH attacks on C2 for producing oxonium ion. Finally, the title compound was obtained by the removal of a proton from the oxonium ion.

Scheme 2. Addition reaction mechanism of tectochrysin

3. 5 Antitumor activity evaluation

antitumor activities of 1 against human leukemia cells (K562), human breast cancer cells (MCF-7) and human lung cancer cells (A549) were tested with MTT assay, with cisplatin used as the positive control. The inhibitory activity of tumor cell growth was expressed as the IC50(Table 2). 1 hasgrowth inhibitory activity towards human cancer cells K562, MCF-7 and A549. The growth inhibitory activity against cancer cells K562 is higher than that of cisplatin, and that towards cancer cells A549 is comparable to cisplatin. The efficacy of inhibition against cancer cells K562 was stronger than that of Chrysin, while the efficacy of inhibition against cancer cells MCF-7 and A549 was lower than that of Chrysin.

Table 2. Anti-tumor Activities of the Target Compound

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10 January 2018;

10 April 2018 (CCDC 183584)

① This research was supported by Youth Backbone Teachers Project Funded by Xianyang Normal University (No. XSYGG201606), Scientific Research Program Funded by Shaanxi Provincial Education Department (No. 16JK1822), Natural Science Basic Research Plan Funded by Shaanxi Province of China (No. 2016JM5024), Science and Technology Projects of Xianyang City (No. 2017k02-19), University Students Research and Innovation Training Program of Xianyang Normal University (No. 2017060 & 201710722003) and University Students Research and Innovation Training Program of Shaanxi Province (No. 2490)

. Li Wu-Wu. E-mail: langyuan2012@126.com

10.14102/j.cnki.0254-5861.2011-1948