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Int. J. Mol. Sci. 2011, 12(9), 6104-6115; doi:10.3390/ijms12096104

Article
Structure–Activity Relationship of Halophenols as a New Class of Protein Tyrosine Kinase Inhibitors
Xiu E. Feng 1,2, Wan Yi Zhao 1, Shu Rong Ban 1, Cheng Xiao Zhao 1, Qing Shan Li 1,2,3,* and Wen Han Lin 3
1
School of Pharmaceutical Science, Shanxi Medical University, Taiyuan 030001, Shanxi, China; E-Mails: xiuefeng@163.com (X.E.F.); zhaowanyi@yahoo.cn (W.Y.Z.); shurongban@163.com (S.R.B.); zhaochengxiao1982@163.com (C.X.Z.)
2
School of Public Health, Shanxi Medical University, Taiyuan 030001, Shanxi, China
3
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China; E-Mail: whlin@bjmu.edu.cn
*
Author to whom correspondence should be addressed; E-Mail: qingshanl@yahoo.com; Tel.: +86-351-4690322; Fax: +86-351-4690322.
Received: 25 May 2011; in revised form: 14 September 2011 / Accepted: 15 September 2011 /
Published: 19 September 2011

Abstract

: A series of new benzophenone and diphenylmethane halophenol derivatives were prepared. Their structures were established based on 1H NMR, 13C NMR and HRMS data. All prepared compounds were screened for their in vitro protein tyrosine kinase (PTK) inhibitory activities. The effects of modification of the linker, functional groups and substituted positions at the phenyl ring on PTK inhibitory activity were investigated. Twelve halophenols showed significant PTK inhibitory activity. Among them, compounds 6c, 6d, 7d, 9d, 10d, 11d and 13d exhibited stronger activities than that of genistein, the positive reference compound. The results gave a relatively full and definite description of the structure–activity relationship and provided a foundation for further design and structure optimization of the halophenols.
Keywords:
protein tyrosine kinase; halophenol; structure-activity relationship; benzophenone; diphenylmethane

1. Introduction

Protein tyrosine kinases (PTKs), which are members of a large family of oncoproteins and proto-oncoproteins, play a major role in mitogenic signal transduction, and are involved in the control of cell proliferation, differentiation and transformation. Continuing activation of PTK is associated with proliferative disorders such as cancer; hence, PTK inhibitors have been developed as molecular-targeting cancer therapeutic agents. The discovery and development of PTK inhibitors as new cancer therapeutic agents have now attracted much attention [19]. To date, many PTK inhibitors with potent activities have already passed or are currently in clinical trials to investigate their applicability as anti-cancer drugs [10].

Various structural halophenols isolated from biologically active natural products such as various marine algae, ascidians and sponges present a wide spectrum of bioactivities including protein tyrosine phosphatase (PTP1B) inhibitory [11], antioxidative [12,13], antithrombotic [14], antimicrobial [15,16], anti-inflammatory [17], enzyme inhibitory [18], cytotoxic [19], and appetite suppressant [20], PTK inhibitory activities [21]. However, to the best of our knowledge, very little is known about the inhibitory activity of benzophenone and diphenylmethane halophenols against PTK, and their corresponding structure–activity relationships (SARs) have been rarely reported, despite the fact that several natural diphenylmethane bromophenols isolated from the brown alga Leathesia nana have been reported to show moderate inhibitory activity against PTK with over-expression of c-kit [21], which reveals that diphenylmethane halophenols may possess potential significant PTK inhibitory activity.

We therefore designed and synthesized a series of new diphenylmethane and benzophenone halophenol derivatives by modification of the linker (illustrated in Table 1), functional groups, and substituted positions at the phenyl ring to find novel structural halophenol derivatives with strong PTK inhibitory activity, and tried to establish the SAR on the basis of this new compound library. In our previous study [22], a series of bromo- and chloro- substituted halophenols were reported for their significant in vitro antioxidative and cytoprotective activities. However, the PTK inhibitory activity has not been evaluated. In the continued efforts towards discovering potent PTK inhibitors, a series of new fluoro- and iodo- functionalized benzophenone and diphenylmethane halophenols derivatives were also prepared and screened for their in vitro PTK inhibitory activity with genistein as positive control [23,24], in accordance with the fact that inclusion of F or I atoms in a compound may have profound effects on drug disposition [2530]. The results provide some clear and useful information about recognition of the SAR.

2. Results and Discussion

2.1. Chemistry

A series of new benzophenone and diphenylmethane halophenol derivatives with several groups substituted at different positions on the phenyl ring were synthesized to determine how the substituents affect the PTK inhibitory activity. Halophenol compounds 5c-13c and 5d-13d were prepared in our previous study [22]. In the present study, 12 fluoro- and four iodo- functionalized halophenol derivatives (1a-4d) were also prepared (Scheme 1), including 11 new compounds 1b, 1c, 1d, 2b, 2c, 2d, 3b, 3d, 4b, 4c and 4d.

2.2. In vitro PTK Inhibitory Activity

The in vitro PTK inhibitory activity of the prepared compounds listed in Table 1 was tested by ELISA with genistein as a positive reference compound. As shown in Table 1, 12 halophenols exhibited strong activities, which in some cases, were identical to, or even higher than, that of genistein in the same model. Among these, seven compounds, 6c, 6d, 7d, 9d, 10d, 11d and 13d, showed the strongest activities with IC50 values of 2.97–12.9 μM, which were stronger than that of genistein with an IC50 value of 13.6 μM. Compound 8d with an IC50 value of 14.8 μM exhibited identical activity to genistein. Compounds 8c, 9c and 11c showed lower activities with IC50 values of 17.7, 17.8 and 16.0 μM, respectively. Compound 10c exhibited weak activity with an IC50 of 41.6 μM.

2.3. SAR Analysis

Diphenylmethane halophenols 7d, 8d, 9d, 10d, 11d and 13d displayed higher activities with IC50 values of 6.34, 14.8, 12.9, 6.97, 6.26 and 5.05 μM than those of corresponding benzophenone halophenols 7c, 8c, 9c, 10c, 11c and 13c. Bromophenols 7c and 13c, which are isomers, showed no activity. Isomers of chlorophenols 8c, 9c, 10c and bromophenol 11c exhibited moderate activity with IC50 values of 17.7, 17.8, 41.6 and 16.0 μM, respectively. Replacement of the methylene group by a carbonyl group, except 6c which showed similar activity to 6d, led to an obvious decrease, even complete disappearance of the activity, which suggested that the methylene group may greatly contribute to the PTK inhibitory activity.

Meanwhile, substitution of the hydroxyl groups by methoxyl groups resulted in the disappearance of activity, and indeed, none of the compounds with methoxyl groups on the phenyl ring showed any activity with IC50 value higher than 50 μM. This indicated that the methoxyl group exerted a great negative effect on the PTK inhibitory activity, and also illustrated that the hydroxyl groups were essential. It is implied that these active halophenols as hydrogen donors could have key interactions with PTK.

By comparing the activities of the halogen-substituted compounds 5c, 6c, 5d and 6d, which possessed five hydroxyls and two halogen atoms at the same positions, we found that the chlorophenol compounds 6c and 6d exhibited the strongest activities with IC50 values of 2.97 μM and 3.96 μM, respectively. However, the bromophenols 5c and 5d showed no activity. Moreover, for all of the fluoro- and iodo- functionalized halophenols, no activity was observed. Hence, the halogen atoms on the phenyl ring contributed to the activity in the order of Cl > Br > F (or I), which suggested that the chloro atom may play a pivotal role between the interaction of active halophenols and PTK. The results also showed that an increased number of hydroxyl groups and chloro atoms may be beneficial to the activity.

Compounds 8c and 9c with a chloro atom at the ortho- and meta- position of the carbonyl group exhibited moderate activities, with IC50 values of 17.7 μM and 17.8 μM, respectively. Compound 10c with a chloro atom at the para-position of the carbonyl group showed weak activity, with an IC50 value of 41.6 μM. Compounds 10d and 9d, with a chloro atom at the para- and meta- position of the methene group, showed high activities with IC50 values of 6.97 μM and 12.9 μM, respectively. Compound 8d with a chloro atom at the ortho-position of the methene group exhibited identical activity with an IC50 value of 14.8 μM, compared to that of the positive control compound (IC50 = 13.6 μM). To the isomers of these chlorophenols, the chloro atom substituted at different position on the phenyl ring had significant effects on the activity. In addition, the same substituted position of the chloro atom on the phenyl ring had an entirely different effect on the activity of benzophenone and diphenylmethane halophenols. The results indicated that the linker and halogen had a combined influence on the activity.

The activities of compounds 12c and 12d were not enhanced by the increased number of halogen atoms. The bromophenols 11c and 11d substituted by one bromo atom obviously showed better activities than those of bromophenols 12c and 12d with two bromo atoms at the same phenyl ring.

3. Experimental Section

3.1. General

Melting points were taken on a micromelting point apparatus, which were uncorrected. 1H and 13C NMR spectra were recorded with a Bruker-AV 400 spectrometer at 400 and 100 MHz respectively, in CDCl3, DMSO-d6 or CD3OD with TMS as reference. Chemical shifts (δ values) and coupling constants (J values) were given in ppm and Hz, respectively. ESI mass spectra were obtained on an API QTRAP 3200 MS spectrometer, and HRMS were recorded on a Bruker Daltonics Apex IV 70e FTICR-MS (Varian 7.0T).

Ether was distilled from sodium benzophenone ketyl. Dichloromethane was distilled from calcium hydride. Other reagents and solvents were commercially available unless otherwise indicated.

3.2. Typical Procedures for the Preparation of Halophenol Derivatives

The general procedures for compounds 5c–13c, and 5d–13d have been reported in our earlier study [22]. A series of new fluoro- and iodo- functionalized compounds were prepared according to the following general procedures.

3.2.1. Preparation of compounds 1a–4a

2-fluoro-3′,4′-dimethoxyl benzophenone (1a)

2-Fluorobenzoyl chloride (10 mL, 82.2 mmol) was added at 0 °C to a solution that contained 1,2-dimethoxybenzene (10 mL, 78.5 mmol) in dried CH2Cl2 (60 mL). Anhydrous AlCl3 (11.1 g, 83.2 mmol) was added by portion wise. The mixture was allowed to warm to room temperature and stirred for 4 h, then ice-water (100 mL) was added to the mixture. The organic phase was separated, washed with water (50 mL) and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the crude product was recrystallized from methanol to give compound 1a as a white powder in 73% yield. m.p. 77–79 °C; 1H NMR (CDCl3) δ: 7.58 (d, 1H, Ph-6-H), 7.15–7.55 (m, 5H, PhH), 6.90 (d, 1H, Ph-5′-H), 3.97 (s, 3H, OCH3), 3.96 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 192.0, 161.0, 158.5, 153.8, 149.1, 132.5, 132.4, 130.5, 130.4, 130.3, 127.5, 127.4, 125.9, 124.2, 124.1, 116.3, 116.1, 111.0, 109.9, 56.1, 56.0; MS (ESI) m/z: 261.0 ([M + H]+, 100).

Compounds 2a–4a were prepared in the similar manner as described for 1a.

3-fluoro-3′,4′-dimethoxyl benzophenone (2a)

White powder, Yield 80%, mp 103–105 °C; 1H NMR (CDCl3) δ: 7.46–7.56 (m, 4H, Ph-2,4,5,6-H), 7.39 (dd, J = 2.0, 8.4 Hz, 1H, Ph-6′-H), 7.26–7.30 (m, 1H, Ph-2′-H), 6.93 (d, J = 8.4 Hz, 1H, Ph-5′-H), 3.96 (s, 3H, OCH3), 3.98 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 194.1, 163.6, 161.2, 153.3, 149.2, 140.4, 140.3, 129.9, 129.8, 129.6, 125.6, 125.4, 118.9, 118.7, 116.6, 116.4, 112.0, 109.8, 56.1, 56.1; MS (ESI) m/z: 261.0 ([M + H]+, 100).

4-fluoro-3′,4′-dimethoxyl benzophenone (3a)

White powder, Yield 86%, mp 101–103 °C; 1H NMR (CDCl3) δ: 7.80–7.84 (m, 2H, Ph-2,6-H), 7.48 (d, J = 2.0 Hz, 1H, Ph-2′-H), 7.37 (dd, J = 2.0, 8.4 Hz, 1H, Ph-6′-H), 7.15–7.19 (m, 2H, Ph-3,5-H), 6.93 (d, J = 8.4 Hz, 1H, Ph-5′-H), 3.98 (s, 3H, OCH3), 3.96 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 194.2, 166.3, 163.8, 153.1, 149.1, 134.4, 132.3, 132.2, 130.1, 125.2, 115.4, 115.2, 112.1, 109.8, 56.1, 56.0; MS (ESI) m/z: 261.0 ([M + H]+, 100).

2-iodo-3′,4′-dimethoxyl benzophenone (4a)

White powder, Yield 85%, mp 143–145 °C; 1H NMR (CDCl3) δ: 7.93 (d, J = 8.0 Hz, 1H, Ph-3-H), 7.60 (d, J = 2.0 Hz, 1H, Ph-6-H), 7.45 (m, 1H, Ph-5-H), 7.31 (dd, J = 2.0, 8.0 Hz, 1H, Ph-4-H), 7.16–7.21 (m, 2H, Ph-2′,6′-H), 6.86 (d, J = 8.4 Hz, 1H, Ph-5′-H), 3.96 (s, 3H, OCH3), 3.95 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 196.1, 154.0, 149.4, 144.7, 139.5, 130.9, 128.6, 128.2, 127.7, 126.8, 111.1, 109.9, 92.4, 56.2, 56.1; MS (ESI) m/z: 369.0 ([M + H]+, 100).

3.2.2. The Preparation of Compounds 1b–4b

2-fluoro-3′,4′-dimethoxyl diphenylmethane (1b)

Anhydrous AlCl3 (2.00 g, 15.0 mmol) and LiAlH4 (1.00 g, 26.4 mmol) were slowly added to dried ether (50 mL). After stirring for 5 min, compound 1a (2.00 g, 7.69 mmol) was dissolved in dried ether (10 mL) and dropwisely added to the solution. The mixture was warmed to 40 °C, refluxed for 2 h, then cooled and quenched with 50 mL 18% aqueous HCl. The organic phase was separated. The water layer was extracted twice with ethyl acetate (2 × 30 mL). The combined organics were washed to neutral with water, dried over anhydrous Na2SO4, and then concentrated via rotary evaporation. The crude product was purified by silica gel chromatography with ethyl acetate-petroleum ether (1/9) as eluent to afford compound 1b. The product was recrystallized from methanol to give a white powder in 40% yield. mp 51–52 °C; 1H NMR (CDCl3) δ: 7.04–7.24 (m, 4H, Ph-3,4,5,6-H), 6.77–6.84 (m, 3H, Ph-2′,5′,6′-H), 3.97 (s, 2H, CH2), 3.88 (s, 3H, OCH3), 3.87 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 162.1, 159.7, 149.0, 147.5, 132.4, 130.9, 130.8, 128.4, 128.3, 127.9, 127.8, 124.1, 124.0, 120.8, 115.4, 115.2, 112.1, 111.2, 55.9, 55.8, 34.4; MS (ESI) m/z: 247.0 ([M + H]+, 100).

Compounds 2b-4b were prepared in the similar manner as described for 1b.

3-fluoro-3′,4′-dimethoxyl diphenylmethane (2b)

Yellow liquid, Yield 62%; 1H NMR (CDCl3) δ: 7.26–7.30 (m, 1H, Ph-5-H), 7.01 (d, 1H, J = 7.2 Hz, Ph-6-H), 6.89–6.94 (m, 2H, Ph-2, 4-H), 6.85 (d, 1H, J = 8.0 Hz, Ph-5′-H), 6.72–6.77 (m, 2H, Ph-2′,6′-H), 3.95 (s, 2H, CH2), 3.89 (s, 3H, OCH3), 3.87 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 164.2, 161.8, 149.0, 147.6, 144.1, 144.0, 132.8, 129.9, 129.8, 124.4, 121.0, 115.8, 115.5, 113.1, 112.9, 112.2, 111.3, 55.9, 55.8, 41.2; MS (ESI) m/z: 247.0 ([M + H]+, 100).

4-fluoro-3′,4′-dimethoxyl diphenylmethane (3b)

Yellow liquid, Yield 40%; 1H NMR (CDCl3) δ: 7.15–7.18 (m, 2H, Ph-2,6,-H), 6.98–7.02 (m, 2H, Ph-3,5-H), 6.84 (d, 1H, J = 8.0 Hz, Ph-5′-H), 6.71–6.75 (m, 2H, Ph-2′,6′-H), 3.93 (s, 2H, CH2), 3.89 (s, 3H, OCH3), 3.86 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 162.6, 160.2, 149.0, 147.5, 137.1, 137.0, 133.5, 130.2, 130.1, 120.8, 115.3, 115.1, 112.1, 111.3, 55.9, 55.8, 40.6; MS (ESI) m/z: 247.0 ([M + H]+, 100).

2-iodo-3′,4′-dimethoxyl diphenylmethane (4b)

White powder, Yield 23%, mp 48–49 °C; 1H NMR (CDCl3) δ: 7.90 (dd, J = 1.2, 8.0 Hz, 1H, Ph-3-H), 7.27–7.31 (m, 1H, Ph-5-H), 7.13 (dd, J = 1.2, 7.6 Hz, 1H, Ph-6-H), 6.91–6.96 (m, 1H, Ph-4-H), 6.82 (d, J = 8.0 Hz, 1H, Ph-5′-H), 6.72–6.74 (m, 2H, Ph-2′,6′-H), 4.06 (s, 2H, CH2), 3.89 (s, 3H, OCH3), 3.86 (s, 3H, OCH3); 13C NMR (CDCl3) δ: 149.0, 147.6, 143.9, 139.5, 132.1, 130.2, 128.4, 128.0, 121.1, 112.4, 112.2, 101.2, 55.9, 55.8, 46.2; MS (ESI) m/z: 355.0 ([M + H]+, 100).

3.2.3. The Preparation of Compounds 1c–4c

2-fluoro-3′,4′-dihydroxyl benzophenone (1c)

Ten milliliters BBr3 solution (BBr3/CH2Cl2, v/v, 1/9) was dropwisely added to a cooled (−78 °C) solution of compound 1a (500 mg, 1.92 mmol) in dried CH2Cl2 (20 mL). The mixture was warmed to room temperature, stirred for 2 h, and poured into ice-water (50 mL). The organic phase was separated. The water layer was extracted twice with ethyl acetate (2 × 20 mL). The combined organics were washed with 20 mL water, dried over anhydrous Na2SO4 and concentrated via rotary evaporation. The crude product was purified by silica gel chromatography with ethyl acetate-petroleum ether (1/4, containing 1 mL acetic acid in 100 mL elution solvent) as eluent to afford compound 1c as a white powder in 53% yield. mp 149–150 °C; 1H NMR (DMSO-d6) δ: 7.58–7.63 (m, 1H, Ph-6-H), 7.46–7.50 (m, 1H, Ph-4-H), 7.32–7.38 (m, 2H, Ph-3,5-H), 7.26 (d, 1H, J = 1.6 Hz, Ph-2′-H), 7.12 (dd, 1H, J = 1.2, 8.4 Hz, Ph-6′-H), 6.87 (d, 1H, J = 8.4 Hz, Ph-5′-H), 3.40 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 191.5, 160.4, 158.0, 152.1, 145.8, Int. J. Mol. Sci. 2011, 12 6111 132.9, 132.8, 130.3, 130.2, 128.8, 128.1, 127.9, 125.1, 125.0, 123.9, 116.6, 116.4, 115.8; MS (ESI) m/z: 231.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H9F1O3 [M − H]: 231.0463; Found: 231.0463.

Compounds 2c-4c were prepared in the similar manner as described for 1c.

3-fluoro-3′,4′-dihydroxyl benzophenone (2c)

White powder, Yield 48%, mp 104–106 °C; 1H NMR (DMSO-d6) δ: 7.44–7.59 (m, 4H, Ph-2,4,5,6-H), 7.27 (d, 1H, J =2.0 Hz, Ph-2′-H), 7.15 (dd, 1H, J = 2.0, 8.0 Hz, Ph-6′-H), 6.89 (d, 1H, J = 8.4 Hz, Ph-5′-H), 5.00 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 193.4, 163.4, 161.0, 151.5, 145.7, 141.8, 141.2, 141.1, 131.0, 130.9, 128.1, 125.6, 124.1, 117.3, 115.7; MS (ESI) m/z: 231.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H9F1O3 [M − H]: 231.0463; Found: 231.0463.

4-fluoro-3′,4′-dihydroxyl benzophenone (3c)

White powder, Yield 55%, mp 155–157 °C; 1H NMR DMSO-d6) δ: 7.73–7.76 (m, 2H, Ph-2,6-H), 7.35–7.39 (m, 2H, Ph-3,5-H), 7.24 (d, 1H, J = 2.0 Hz, Ph-2′-H), 7.11 (dd, 1H, J = 2.0, 8.0 Hz, Ph-6′-H), 6.87 (d, 1H, J = 8.0 Hz, Ph-5′-H), 3.34 (br, 2H, Ph-OH); 13C NMR (CD3OD) δ: 194.3, 164.0, 158.1, 146.0, 133.4, 133.3, 133.2, 125.2, 123.1, 118.0, 116.4, 116.2, 115.8; MS (ESI) m/z: 231.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H9F1O3 [M − H]: 231.0463; Found: 231.0464.

2-iodo-3′,4′-dihydroxyl benzophenone (4c)

White powder, Yield 60%, mp 186–188 °C; 1H NMR (DMSO-d6) δ: 7.89–7.95 (m, 1H, Ph-3-H), 7.46–7.54 (m, 1H, Ph-6-H), 7.16–7.31 (m, 2H, Ph-4,5-H), 7.00 (dd, 1H, J = 1.6, 8.4 Hz, Ph-6′-H), 6.77 (s, 1H, Ph-2′-H), 6.41 (d, 1H, J = 8.4 Hz, Ph-5′-H); 13C NMR (CD3OD) δ: 198.4, 153.2, 146.7, 146.6, 140.6, 131.9, 130.4, 129.0, 128.9, 126.2, 117.7, 116.0, 92.8; MS (ESI) m/z: 339.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H9I1O3 [M − H]: 338.9524; Found: 338.9514.

3.2.4. The Preparation of Compounds 1d–4d

2-fluoro-3′,4′-dihydroxyl diphenylmethane (1d)

To a cooled (−78 °C) solution of compound 1b (400 mg, 1.63 mmol) in dried CH2Cl2 (5 mL) was dropwisely added 5 mL BBr3 solution (BBr3/CH2Cl2, v/v, 1/9). The mixture was warmed to room temperature, stirred for 15 min, and poured into ice-water (50 mL). The organic layer was separated. The water layer was extracted twice with ethyl acetate (2 × 10 mL). The combined organics were washed with 10 mL water and dried over anhydrous Na2SO4, then concentrated via rotary evaporation. The crude product was purified by silica gel chromatography with ethyl acetate/petroleum ether (3/7, containing 1 mL acetic acid in 100 mL elution solvent) as eluent to afford compound 1d as pale yellow powder in 40% yield. mp 91–92 °C; 1H NMR (DMSO-d6) δ: 8.80 (dd, 1H, J = 4.8, 41.6 Hz, Ph-6-H), 7.10–7.27 (m, 3H, Ph-3,4,5-H), 6.67 (d, 1H, J = 8.0 Hz, Ph-6′-H), 6.59 (s, 1H, Ph-2′-H), 6.50 (d, 1H, J = 8.0 Hz, Ph-5′-H), 3.79 (s, 2H, CH2), 3.42 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 162.0, 159.5, 145.6, 144.1, 131.7, 131.6, 131.1, 129.1, 128.9, 128.5, 128.4, 124.8, 124.7, 119.7, 116.4, 116.0, 115.7, 115.5, 33.9; MS (ESI) m/z: 217.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H11F1O2 [M − H]: 217.0670; Found: 217.0672.

Compounds 2d–4d were prepared in the similar manner as described for 1d.

3-fluoro-3′,4′-dihydroxyl diphenylmethane (2d)

Pale yellow powder, Yield 66%, mp 66–68 °C; 1H NMR (DMSO-d6) δ: 7.28–7.34 (m, 1H, Ph-5-H), 6.99–7.04 (m, 3H, Ph-2,4,6-H), 6.68 (d, 1H, J = 8.0 Hz, Ph-5′-H), 6.60 (d, 1H, J = 2.0 Hz, Ph-2′-H), 6.51 (dd, 1H, J = 2.0, 8.0 HZ, Ph-6′-H), 3.78 (s, 2H, CH2), 3.42 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 163.9, 161.5, 145.6, 145.5, 145.4, 144.1, 131.9, 130.6, 130.5, 125.1, 119.9, 116.6, 116.0, 115.7, 115.5, 113.1, 112.9, 40.5; MS (ESI) m/z: 217.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H11F1O2 [M − H]: 217.0670; Found: 217.0672.

4-fluoro-3′,4′-dihydroxyl diphenylmethane (3d)

White powder, Yield 59%, mp 59–61 °C; 1H NMR (DMSO-d6) δ: 7.32–7.86 (m, 2H, Ph-2,6-H), 6.95–7.23 (m, 2H, Ph-3,5-H), 6.66 (d, 1H, J = 8.0 Hz, Ph-5′-H), 6.55 (d, 1H, J = 2.0 Hz, Ph-2′-H), 6.45 (dd, 1H, J = 2.0, 8.0 Hz, Ph-6′-H), 3.88 (s, 2H, CH2), 3.38 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 162.3, 159.9, 145.6, 143.9, 138.6, 138.5, 132.5, 130.8, 130.7, 119.8, 116.5, 116.0, 115.5, 115.3, 40.0; MS (ESI) m/z: 217.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H11F1O2 [M − H]: 217.0670; Found: 217.0672.

2-iodo-3′,4′-dihydroxyl diphenylmethane (4d)

White powder, Yield 40%, mp 130–132 °C; 1H NMR (DMSO-d6) δ: 7.86 (dd, 1H, J = 1.2, 8.0 Hz, Ph-3-H), 7.36 (m, 1H, Ph-5-H), 7.23 (dd, 1H, J = 1.6, 7.6 Hz, Ph-6-H), 6.99 (m, 1H, Ph-4-H), 6.66 (d, 1H, J = 8.0 Hz, Ph-5′-H), 6.55 (d, 1H, J = 2.0 Hz, Ph-2′-H), 6.47 (dd, 1H, J = 2.0, 8.0 Hz, Ph-6′-H), 3.88 (s, 2H, CH2), 3.38 (br, 2H, Ph-OH); 13C NMR (DMSO-d6) δ: 144.4, 139.6, 130.9, 130.8, 128.9, 128.7, 120.0, 116.5, 116.4, 116.0, 115.9, 101.8, 45.3; MS (ESI) m/z: 325.0 ([M − H], 100); HRMS (ESI): Calcd. for C13H11I1O2 [M − H]: 324.9731; Found: 324.9726.

3.3. Pharmacology

After removal of the male mouse (SD) meninges, the brain was weighted and homogenized with a glass homogenizer in four volumes of cold medium (containing 20 mmol/L Tris–HCl, pH 7.5, 0.25 mol/L sucrose, 2 mmol/L DDT, 2 mmol/L EDTA, 2 mmol/L Na3VO4, 1 mmol/L DMSF, 215 mg/L aprotinin, 1 mg/L PTIFtatin, 5 mg/L leupeptin) at high speed. The mixture was centrifuged for 10 min at 1000 × g at 4 °C; the supernatant was collected and re-centrifuged for 10 min at 10,000 × g at 4 °C to obtain the target supernatant. The target supernatant that contained cytoplasmic tyrosine kinase was collected, separately packed and stored at −70 °C.

PTK activity was determined by the ELISA method. In brief, the concentrations of PTKs used to construct calibration curves were as follows: 600, 500, 341, 200 and 100 × 10−7 U/mL for PTK. 100 μL of 20 mg/L PGT (Sigma Aldrich) in 20 mmol/L PBS solution was added to 96-well microtiter plates at 37 °C overnight. After removing excess substrate solution, PBS–Tween 20 (PBST) was used to wash the wells one time, which were dried for 2 h at 37 °C and kept at 4 °C. Subsequently, 50 μL of the above PTK extraction (50 mmol/L HEPES, pH 7.4, 20 mmol/L MgCl2, 0.1 mmol/L MnCl2, 0.2 mmol/L Na3VO4, 0.6 mmol/L ATP) was added to the 96-well plates. Then, a certain concentration of the tested compounds (10 μL) and tyrosine kinase tissue extract solution were incubated at 37 °C for 1 h. The plate was washed three times with PBST at the end of the treatment, and 100 μL of the diluted horseradish-peroxidase-labeled mouse anti-phosphotyrosine monoclonal antibody IgG2bk (Sigma Aldrich) was added to each well, and incubated at 37 °C for 30 min. After removal of the antibody complex, and then washing three times with PBST, 100 μL freshly prepared TMB horseradish peroxidase color development solution (Beijing 4A Biotech Co., Ltd, China) was added and protected from light for 10 min. The reaction was terminated by addition of 100 μL/well 1 N sulfuric acid. Absorbance was measured at 450 nm in a microplate reader (SpectraMax M5) [4].

4. Conclusions

In this study, we prepared a series of new benzophenone and diphenylmethane halophenol derivatives and investigated the influence of the linker, substituted groups, number and positions of halogen atoms on PTK inhibitory activities.

All of the chlorophenols exhibited strong activities, and the substituted position of the chloro atom also markedly affected the activity. Unfortunately, all of the fluoro- and iodo- functionalized compounds were inactive. Our findings confirmed that the hydroxyls and halogens (chloro or bromo) seem to be essential to the activity, and the chloro atom and methylene group play a crucial role in the activity. Hence, these compounds with high activities represent a new paradigm for halophenols and appear to offer great potential as new PTK inhibitors. The results also gave a relatively full recognition of the SARs, and provided a foundation for further structural optimization of the halophenols in the search for suitable clinical candidates.

Acknowledgments

We would like to thank Guanhua Du, Lianhua Fang, Guorong He, for their help with the in vitro assay of the compounds. This work was financially supported by the “Innovative Drugs Development” State Key Science and Technology Projects of China (No.2009ZX09302-003), from the National “863” Program of China (No. 2011), from the State Key laboratory of Natural and Biomimetic Drugs, Peking University (NO.20080210) and Shanxi Foundation for overseas returned (2008-51), by the Program for the Top Science and Technology Innovation Teams of Higher Learning Institutions of Shanxi Province (2011), by the Program for the Top Young and Middle-aged Innovative Talents of Higher Institutions of Shanxi Province and by High School Technology Fund Projects of Shanxi Province (200811011).

  • Conflict of InterestThe authors have declared no conflict of interest.

References

  1. Bolla, M; Rostaing-Puissant, B; Bottari, SP; Chedin, M; Marron-Charriere, J; Colonna, M; Berland, E; Chambaz, E. Protein tyrosine kinase activity in 350 T1/T2, N0/N1 breast cancer. Preliminary results. Breast Cancer Res Treat 1996, 39, 327–334. [Google Scholar]
  2. Hirabayashi, A; Mukaiyama, H; Kobayashi, H; Shiohara, H; Nakayama, S; Ozawa, M; Tsuji, E; Miyazawa, K; Misawa, K; Ohnota, H; et al. Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors. Bioorg Med Chem 2008, 16, 9247–9260. [Google Scholar]
  3. Jung, KJ; Lee, EK; Yu, BP; Chung, HY. Significance of protein tyrosine kinase/protein tyrosine phosphatase balance in the regulation of NF-κB signaling in the inflammatory process and aging. Free Radic Biol Med 2009, 47, 983–991. [Google Scholar]
  4. Shi, L; Feng, XE; Cui, JR; Fang, LH; Du, GH; Li, QS. Synthesis and biological activity of flavanone derivatives. Bioorg Med Chem Lett 2010, 20, 5466–5468. [Google Scholar]
  5. Hori, H; Nagasawa, H; Uto, Y; Ohkura, K; Kirk, KL; Uehara, Y; Shimamura, M. Design of hypoxia-targeting protein tyrosine kinase inhibitor using an innovative pharmacophore 2-methylene-4-cyclopentene-1,3-dione. Biochim Biophys Acta 2004, 1697, 29–38. [Google Scholar]
  6. Liechti, C; Sequin, U; Bold, G; Furet, P; Meyer, T; Traxler, P. Salicylanilides as inhibitors of the protein tyrosine kinase epidermal growth factor receptor. Eur J Med Chem 2004, 39, 11–26. [Google Scholar]
  7. Li, HQ; Zhu, TT; Yan, T; Luo, Y; Zhu, HL. Design, synthesis and structure–activity relationships of antiproliferative 1,3-disubstituted urea derivatives. Eur J Med Chem 2009, 44, 453–459. [Google Scholar]
  8. Kilic, Z; Isgor, YG; Olgen, S. Synthesis and pp60c-Src tyrosine kinase inhibitory activities of novel indole-3-imine and amine derivatives substituted at N1 and C5. Arch Pharm Chem Life Sci 2009, 342, 333–343. [Google Scholar]
  9. Olgen, S; Isgor, YG; Coban, T. Synthesis and activity of novel 5-substituted pyrrolo[2,3-d]pyrimidine analogues as pp60c-Src tyrosine kinase inhibitors. Arch Pharm Chem Life Sci 2008, 341, 113–120. [Google Scholar]
  10. Mazitschek, R; Giannis, A. Inhibitors of angiogenesis and cancer-related receptor tyrosine kinases. Curr Opin Chem Biol 2004, 8, 432–441. [Google Scholar]
  11. Li, J; Guo, SJ; Su, H; Han, LJ; Shi, DY. Total synthesis of bis-(2,3-dibromo-4,5- dihydroxyphenyl)-methane as potent PTP1B inhibitor. Chin Chem Lett 2008, 19, 1290–1292. [Google Scholar]
  12. Li, K; Li, XM; Ji, NY; Gloer, JB; Wang, BG. Urceolatin, a structurally unique bromophenol from Polysiphonia urceolata. Bioorg Med Chem 2007, 15, 6627–6631. [Google Scholar]
  13. Duan, XJ; Li, XM; Wang, BG. Highly brominated mono- and bis-phenols from the marine red alga symphyocladia latiuscula with radical-scavenging activity. J Nat Prod 2007, 70, 1210–1213. [Google Scholar]
  14. Shi, DY; Li, J; Guo, SJ; Han, LJ. Antithrombotic effect of bromophenol, the alga-derived thrombin inhibitor. J Biotechnol 2008, 136, S579. [Google Scholar]
  15. Popplewell, WL; Northcote, PT. Colensolide A: A new nitrogenous bromophenol from the New Zealand marine red alga Osmundaria colensoi. Tetrahedron Lett 2009, 50, 6814–6817. [Google Scholar]
  16. Oh, KB; Lee, JH; Lee, JW; Yoon, KM; Chung, SC; Jeon, HB; Shin, J; Lee, HS. Synthesis and antimicrobial activities of halogenated bis(hydroxyphenyl)methanes. Bioorg Med Chem Lett 2009, 19, 945–948. [Google Scholar]
  17. Wiemer, DF; Idler, DD; Fenical, W. Vidalols A and B, new anti-inflammatory bromophenols from the Caribbean marine alga Vidalia obtusiloba. Experientia 1991, 47, 851–853. [Google Scholar]
  18. Wang, W; Okada, Y; Shi, HB; Wang, YQ; Okuyama, T. Structures and aldose reductase inhibitory effects of bromophenols from the red alga Symphyocladia latiuscula. J Nat Prod 2005, 68, 620–622. [Google Scholar]
  19. Xu, XL; Song, FH; Wang, SJ; Li, S; Fan, X; Zhao, JL; Yang, YC; Shang, SQ; Yang, L; Shi, JG. Dibenzyl bromophenols with diverse dimerization patterns from the brown alga Leathesia nana. J Nat Prod 2004, 67, 1661–1666. [Google Scholar]
  20. Kurata, K; Taniguchii, K; Takashima, K; Hayashi, I; Suzuki, M. Feeding-deterrent bromophenols from Odonthalia corymbifera. Phytochemistry 1997, 45, 485–487. [Google Scholar]
  21. Shi, DY; Li, J; Guo, SJ; Su, H; Fan, X. The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo. Chin J Oceanol Limnol 2009, 27, 277–282. [Google Scholar]
  22. Zhao, WY; Feng, XE; Ban, SR; Lin, WH; Li, QS. Synthesis and biological activity of halophenols as potent antioxidant and cytoprotective agents. Bioorg Med Chem Lett 2010, 20, 4132–4134. [Google Scholar]
  23. Jager, W; Winter, O; Halper, B; Salamon, A; Sartori, M; Gajdzik, L; Hamilton, G; Theyer, G; Graf, J; Thalhammer, T. Modulation of liver canalicular transport processes by the tyrosine- kinase inhibitor genistein: Implications of genistein metabolism in the rat. Hepatology 1997, 26, 1467–1476. [Google Scholar]
  24. Majewska, E; Paleolog, E; Baj, Z; Kralisz, U; Feldmann, M; Tchorzewski, H. Role of tyrosine kinase enzymes in TNF-a and IL-1 induced expression of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells. Scand J Immunol 1997, 45, 385–392. [Google Scholar]
  25. Han, XJ; Civiello, RL; Mercer, SE; Macor, JE; Dubowchik, GM. Synthesis of aza and fluorine-substituted 3-(piperidin-4-yl)-4,5-dihydro-1H-benzo[d][1,3]diazepin-2(3H)-ones. Tetrahedron Lett 2009, 50, 386–388. [Google Scholar]
  26. Xue, J; Kumar, V; Khaja, SD; Chandrasekaran, EV; Locke, RD; Matta, KL. Syntheses of fluorine-containing mucin core 2/core 6 structures using novel fluorinated glucosaminyl donors. Tetrahedron 2009, 65, 8325–8335. [Google Scholar]
  27. Tsirkone, VG; Tsoukala, E; Lamprakis, C; Manta, S; Hayes, JM; Skamnaki, VT; Drakou, C; Zographos, SE; Komiotis, D; Leonidas, DD. 1-(3-Deoxy-3-fluoro-β-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic, crystallographic and modelling studies. Bioorg Med Chem 2010, 18, 3413–3425. [Google Scholar]
  28. Fehér, C; Urbán, B; Urge, L; Darvas, F; Bakos, J; Skoda-Foldes, R. Facile synthesis of 6-iodo-2,2′-dipivaloyloxy-1,1′-binaphthyl, a key intermediate of high reactivity for selective palladium-catalyzed monofunctionalization of the 1,1′-binaphthalene core. Tetrahedron Lett 2010, 51, 3629–3632. [Google Scholar]
  29. Castro, MD; Marzabadi, CH. Preparation and reactions of iodo sugars. Tetrahedron 2010, 66, 3395–3404. [Google Scholar]
  30. Mphahlele, MJ; Lesenyeho, LG; Makelane, HR. Synthesis of 1H-pyrrolo[3,2-c]quinoline derivatives via palladium-catalyzed heteroannulation of 2-aryl-3-iodo-4-(phenylamino)quinolines and 4-(N,N-allylphenylamino)-2-aryl-3-iodoquinolines. Tetrahedron 2010, 66, 6040–6046. [Google Scholar]
Ijms 12 06104f1 1024
Scheme 1. Synthesis of benzophenone and diphenylmethane halophenol derivatives. Reagents and conditions: (a) AlCl3/CH2Cl2, rt, 4 h; (b) LiAlH4/AlCl3/ether, 40 °C, refluxed for 2 h; (c) BBr3/CH2Cl2(1/9, v/v) was added to the solution at −78 °C, warmed to room temperature and stirred for 2 h.

Click here to enlarge figure

Scheme 1. Synthesis of benzophenone and diphenylmethane halophenol derivatives. Reagents and conditions: (a) AlCl3/CH2Cl2, rt, 4 h; (b) LiAlH4/AlCl3/ether, 40 °C, refluxed for 2 h; (c) BBr3/CH2Cl2(1/9, v/v) was added to the solution at −78 °C, warmed to room temperature and stirred for 2 h.
Ijms 12 06104f1 1024
Table Table 1. Structures and in vitro protein tyrosine kinase (PTK) inhibitory activities of the prepared halophenols. PTK activity was determined by the ELISA method with genistein as positive control.

Click here to display table

Table 1. Structures and in vitro protein tyrosine kinase (PTK) inhibitory activities of the prepared halophenols. PTK activity was determined by the ELISA method with genistein as positive control.
Ijms 12 06104f2
Compd.Substituted group
IC50a (μM)
LinkerR1R2R3R4R5R6R7R8R9
1c *C=OFHHHHHHOHOH>50
1d *CH2FHHHHHHOHOH>50
2c *C=OHFHHHHHOHOH>50
2d *CH2HFHHHHHOHOH>50
3cC=OHHFHHHHOHOH>50
3d *CH2HHFHHHHOHOH>50
4c *C=OIHHHHHHOHOH>50
4d *CH2IHHHHHHOHOH>50
5cC=OBrOHOHOHBrHHOHOH>50
5dCH2BrOHOHOHBrHHOHOH>50
6cC=OClOHOHOHClHHOHOH2.97
6dCH2ClOHOHOHClHHOHOH3.96
7cC=OHBrOHHHBrHOHOH>50
7dCH2HBrOHHHBrHOHOH6.34
8cC=OClHHHHHHOHOH17.7
8dCH2ClHHHHHHOHOH14.8
9cC=OHClHHHHHOHOH17.8
9dCH2HClHHHHHOHOH12.9
10cC=OHHClHHHHOHOH41.6
10dCH2HHClHHHHOHOH6.97
11cC=OHHHHHBrHOHOH16.0
11dCH2HHHHHBrHOHOH6.26
12cC=OHHHHHBrBrOHOH>50
12dCH2HHHHHBrBrOHOH>50
13cC=OOHHHBrHBrHOHOH>50
13dCH2OHHHBrHBrHOHOH5.05
Control (Genistein)13.6

*New compound;aThe IC50 values were determined in triplicate.

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