Structure–Activity Relationship of Halophenols as a New Class of Protein Tyrosine Kinase Inhibitors

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.


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 [1][2][3][4][5][6][7][8][9]. 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 [25][26][27][28][29][30]. The results provide some clear and useful information about recognition of the SAR.

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 IC 50 values of 2.97-12.9 μM, which were stronger than that of genistein with an IC 50 value of 13.6 μM. Compound 8d with an IC 50 value of 14.8 μM exhibited identical activity to genistein. Compounds 8c, 9c and 11c showed lower activities with IC 50 values of 17.7, 17.8 and 16.0 μM, respectively. Compound 10c exhibited weak activity with an IC 50 of 41.6 μM.
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 IC 50 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 IC 50 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 IC 50 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 IC 50 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 IC 50 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 IC 50 value of 14.8 μM, compared to that of the positive control compound (IC 50 = 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.

General
Melting points were taken on a micromelting point apparatus, which were uncorrected. 1 H and 13 C NMR spectra were recorded with a Bruker-AV 400 spectrometer at 400 and 100 MHz respectively, in CDCl 3 , DMSO-d 6 or CD 3 OD 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.

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.

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 Na 3 VO 4 , 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 MgCl 2 , 0.1 mmol/L MnCl 2 , 0.2 mmol/L Na 3 VO 4 , 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 IgG 2bk (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