Design, synthesis and biological evaluation of 4-bromo-N-(3,5-dimethoxyphenyl)benzamide derivatives as novel FGFR1 inhibitors for treatment of non-small cell lung cancer

Abstract A series of 4-bromo-N-(3,5-dimethoxyphenyl)benzamide derivatives were designed and synthesised as novel fibroblast growth factor receptor-1 (FGFR1) inhibitors. We found that one of the most promising compounds, C9, inhibited five non-small cell lung cancer (NSCLC) cell lines with FGFR1 amplification, including NCI-H520, NCI-H1581, NCI-H226, NCI-H460 and NCI-H1703. Moreover, the IC50 values for the compound C9 were 1.36 ± 0.27 µM, 1.25 ± 0. 23 µM, 2.31 ± 0.41 µM, 2.14 ± 0.36 µM and 1.85 ± 0.32 µM, respectively. The compound C9 arrested the cell cycle at the G2 phase in NSCLC cell lines. The compound C9 also induced cellular apoptosis and inhibited the phosphorylation of FGFR1, PLCγ1 and ERK in a dose-dependent manner. In addition, molecular docking experiments showed that compound C9 binds to FGFR1 to form six hydrogen bonds. Taken together, our data suggested that the compound C9 represented a promising lead compound-targeting FGFR1.


Introduction
Fibroblast growth factor receptors (FGFRs), a type of receptor tyrosine kinases (RTKs), are key regulators in cellular signalling that is related to cell proliferation, survival, differentiation, migration and angiogenesis 1-3 . When fibroblast growth factors (FGFs) are bound to specific receptors, they induce FGFR dimerisation and autophosphorylation, resulting in activation of downstream signalling pathways, including the MAPK and PLCc signalling pathways 3,4 . Previous studies explained that the FGF signalling pathway plays fundamental roles not only in embryogenesis, tissue repair and wound healing, but also in tumour formation and progression 5,6 . Preclinical studies in which gene knockout and pharmaceutical inhibition of FGFRs were used, have confirmed that FGFRs are promising targets in cancer therapy 7,8 . In recent years, several small molecules that were reported to target the FGFR have entered clinical development, for example, regorafenib, ponatinib, intedanib and lenvatinib [9][10][11] . Due to the fact that members of the RTK family have a significant sequence homology, it was found that most of these compounds were multi-targeted, thereby leading to undesired side effects in anticancer therapies [12][13][14] . Thus, the development of effective FGFR inhibitors with a higher selectivity is of utmost importance for medical treatment. After the 3D structure of the FGFR protein was reported, it was easier to design selective and potent inhibitors-targeting the FGFR. Numerous representative inhibitors exist, including AZD4547 15 , NVP-BGJ398 16 , CH5183284 17 , LY2874455 18 and JNJ-42756493 19 (Figure 1).
FGFR1 is the main member of the FGFR family and known to be a common target of deregulation by gene amplification in several human cancers, including cancer of the breast, bladder and lung 8 . Our goal was to design a batch of novel tyrosine kinase inhibitors that simulate adenosine triphosphate (ATP) and suppress the constitutive activation of FGFR1, thereby blocking the FGFR1driven cancer signalling pathway. According to the mechanism of action of the above-mentioned FGFR competitive inhibitors, we used the 3D structure of the FGFR1 protein as a template. The interaction area of competitive small-molecule inhibitors and proteins can be divided into four main parts 20,21 : (1) a hinge region, (2) an inward hydrophobic pocket of the hinge region (pocket 1), (3) a downward pocket of the hinge region (pocket 2) and (4) the outward extending near solvent domain. In general, the parental nucleus of all small-molecule inhibitors can interact with the amino acid residues in the hinge region through 1-3 hydrogen bonds. Based on this, the activity and selectivity can be changed by introducing appropriate pharmacophores to the side chain of the molecule. Given the fact that the patent space will become increasingly crowded, different pharmacophores may result in different pharmacological properties 22,23 . In addition, based on our previous studies on the FGFR 24,25 , we decided to develop novel pharmacophore-based inhibitors for treating FGFR1-driven cancers using a hybridisation strategy.
Bono et al. 26 reported that compound SSR128129E is an allosteric FGFR inhibitor, the IC 50 of FGFR1 is 1.9 mM, and there is little activity to other tyrosine kinase. In brief, SSR128129E was bound in advance to FGFR1 protein ( Figure 2). The molecular docking model showed that the structure of sodium benzoate formed two hydrogen bonds with Glu486 and Ala488 in the hinge region, while the methoxyl group only formed a hydrogen-bonding interaction with Ala564 in pocket 1, and amino group formed two hydrogen-bond interactions with Glu571 in pocket 2. To develop novel small-molecule inhibitors that bind more closely to the FGFR1 kinase domain, we modified its side chain but retained the parent nucleus that strongly binds to the hinge region. Furthermore, pocket 1 is considered an important distinguishing site for small molecules to exhibit selectivity to FGFR1 enzymes, and the group that can be inserted into this hydrophobic pocket is primarily 3,5-dimethoxyphenyl 20,21,27 . Thus, as the starting scaffold, commercially available 4-bromo-2-nitrobenzoic acid was selected, to which 3,5-dimethoxybenzene was introduced at the 1position using amide condensation to create intermediate 1.
Subsequently, intermediate 1 was reduced to produce intermediate 2. Then, we modified the two-position to synthesise three series of compounds. As a result, a new class of FGFR1 inhibitors was obtained ( Figure 3).

Chemistry
Melting points were determined using a SGWX-4 microscopic melting point metre and were reported in an uncorrected form. 1 H and 13 C NMR spectra were recorded on a Bruker 600 MHz NMR spectrometer, using CDCl 3 -d 6 or DMSO-d 6 as solvents. Chemical shifts are expressed in ppm with TMS as internal reference. J values are provided in hertz. Mass spectra were recorded on a Waters Xevo TQ-S micro mass spectrometer. Reactions were monitored by thin layer chromatography (TLC) on silica gel GF-254coated glass plates. Column chromatography was performed with 200-300 mesh silica gel.
General procedure for preparation of intermediate 1 The following components were added to a reaction vessel: 4bromo-2-nitrobenzoic acid (5.788 g, 0.024 mol), EDC HCl (4.518 g, 0.024 mol) and ethanol (30 ml). After the mixture was activated for 30 min at room temperature, 3,5-dimethoxyaniline (3 g, 0.020 mol) was added. The resulting solution was stirred for 5 h at 80 C, then cooled to room temperature. Water (30 ml) was added, filtered and dried to give the intermediate 1 (5.349 g). The reaction yield was 71.6%. Physical and chemical data for intermediate 1, please refer to the supplementary material.
General procedure for preparation of intermediate 2 Iron powder (2.292 g, 0.041 mol) and ammonium chloride (0.365 g, 0.007 mol) were added to a 100-ml flask. Next, water (40 ml) was added and the mixture was heated to at 85 C for 10 min. Then,   General procedure for the preparation of A1-9 Intermediate 2 (50 mg, 0.142 mol) and different anhydride (2 ml) were added to a 25 ml flask and allowed to react at room temperature for 2 h. The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The residue was purified by silica gel chromatography to produce the desired product. The purity of the target compound, please refer to the supplementary material.
General procedure for the preparation of B1-15 A mixture of intermediate 2 (50 mg, 0.142 mol), different substituted benzoyl chlorides (200 mL) and DMAP (10 mg, 0.066 mol) as a catalyst in pyridine (2 ml) was stirred overnight at room temperature. After completion of the reaction, the mixture was concentrated in vacuo. The residue was added to ethanol (10 ml), refluxed for 30 min at 90 C, cooled to room temperature, filtered and dried to result the desired compound. The purity of the target compound, please refer to the supplementary material.
General procedure for the preparation of C1-11 A solution of 2 ml of intermediate 2 in Pridine was added substituted benzenesulfonyl chloride (0.284 mol) and DMAP as a catalyst (10 mg, 0.066 mol). After completion of the reaction, the reaction mixture was concentrated in vacuo. Methanol (10 ml) was added and refluxed for 30 min at 90 C, cooled to room temperature, filtered and dried to produce the desired product. The purity of the target compound, please refer to the supplementary material.    13
Antiproliferative activity in vitro NCI-H520, NCI-H1581, NCI-H226, NCI-H460, NCI-H1703, A549, PC-9, A431, HCC827 and BEAS-2B cells were cultured until cells were in the logarithmic phase, after which 5000-10 000 cells/well were seeded in 96-well plates in growth medium containing 10% of FBS, and grown overnight. Cells were treated with various concentrations of each compound in triplicate and cultured in 10% of FBS medium for 48 h. Cells treated with dimethyl sulfoxide (DMSO) served as the control. Next, tetrazolium dye (MTT) solution (5 mg/ mL, 20 mL/well) was added and incubated for 4 h. After removing the supernatant, the generated formazan crystals were dissolved in 150 mL of DMSO and the absorbance was read using a spectrophotometer at a wavelength of 490 nm using an enzyme-linked immunosorbent assay plate reader. The data were calculated using GraphPad Prism version 5.0 software (GraphPad, San Diego, CA). The IC 50 values were calculated and fitted using a non-linear regression model with a sigmoidal dose response.
Western blot analysis NCI-H226, NCI-H520 and NCI-H1581 cells were treated with the indicated dose of C9 for 2 h and lysed in 1 Â sodium dodecyl sulphate (SDS) sample buffer. Equivalent amounts of proteins were loaded into 10% of SDS À PAGE gels and transferred onto nitrocellulose membranes. An ECL kit (Bio-Rad, Hercules, CA) was used visualise the immunoreactive bands, and the results were analysed using ImageJ software (National Institute of Health, MD). Antibodies directed against phospho-FGFR1, FGFR1, phospho-PLCc1, PLCc1, phospho-ERK and ERK (Cell Signalling Technology, Danvers, MA) were used for Western blot analysis. An antibody directed against GAPDH was used as a loading control (Bioword Technology, Louis Park, MN) [28][29][30] .
Cell apoptosis assay NCI-H226 and NCI-H1581 (2 Â 10 5 ) cells were seeded into six-well plates. After overnight culture, fresh growth media containing compound C9 (5, 10 and 20 mM) as well as a positive drug SSR128129E (20 mM) were added. RPMI 1640 medium containing 1‰ DMSO was used as a control. After culturing for 48 h, the growth medium was collected and cells were trypsinised and collected in the corresponding medium. After centrifugation at 4 C for 5 min, the supernatant was removed and the cells were washed twice with pre-cold PBS. One hundred microliter of 1 Â binding buffer, 5 mL of PI (Becton Dickinson, Franklin Lakes, NJ) and 5 mL of FITC-labelled Annexin-V (FITC-Annexin V, Becton Dickinson) were added. Cells were then gently vortexed and incubated for 15 min at 25 C in the dark. Next, 1 Â binding buffer was used to a final volume of to 500 mL. Cells were then stained with PI and Annexin-V was used as a positive control. Cells underwent flow cytometry using a BD Accuri TM C6 flow cytometer (Becton Dickinson) and the data were processed using FlowJo 7.6.1 software (Brea, CA, USA).
In vitro cell cycle effects NCI-H1581 and NCI-H1703 (2 Â 10 5 ) cells were seeded into six-well plates and cultured overnight. Fresh growth media containing C9 (2.5, 5.0, 7.5 and 10 mM) as well as a positive drug SSR128129E (10 mM) were added. Medium containing 1‰ DMSO was used as a control. After culturing for 24 h, the growth medium was collected and the cells were trypsinised and collected in the corresponding medium. After centrifugation at 4 C for 5 min, the supernatant was removed and the cells were washed twice with pre-cold PBS.
Cells were fixed in 70% of cold ethanol, incubated overnight at -20 C, and stained with PI/RNase staining buffer (BD Pharmingen, San Jose, CA). Flow cytometry was performed using a BD Accuri TM C6 flow cytometer (Becton Dickinson) and the data were processed using FlowJo 7.6.1 software.

In vitro kinase activity
The 1Â kinase reaction buffer and termination solution were prepared in advance. A total of 10 mL peptide substrate (2.5Â) was added to 5 mL of (5Â) compound liquid, incubated for 10 min at room temperature, and added to an additional 10 mL of peptide substrate (2.5Â). The mixture was allowed to react at 28 C. After a period of time, the termination solution was added to terminate the reaction. Data were collected through the Caliper. The inhibition rate of kinase activity% ¼ (maxconversion)/(maxmin) Â 100. The "max" represented DMSO without compound, "min" represented low control.

Statistical analysis
All experiments were performed in triplicate (n ¼ 3), and the data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Pro Prism 5.0 software (GraphPad, San Diego, CA). The Student's t-test and a two-way ANOVA were used to analyse the differences between treatment groups. A p value of <.05 was considered statistically significant.

Results and discussion
Chemistry As outlined in Scheme 1, we designed and synthesised three series of compounds (Table 1). In the A series, we primarily aimed to increase the hydrophobic effects by introducing chain alkanes. In the B series, our goal is to increase the binding to protein-binding sites and increase the space utilisation of compounds in hydrophobic pocket 2 and near solvent domains, so we substituted them with benzene rings to create p-p interactions. Finally, in the C series, we added a sulphur oxygen double bond to increase the formation of hydrogen bonds with other amino acid residues of FGFR1, thereby increasing the adhesion strength of the compounds with FGFR1 protein.

Biological evaluation
Antiproliferative effect of synthetic compounds Antiproliferative activities of the synthesised compounds were evaluated against five NSCLC cell lines harbouring FGFR1 amplification (NCI-H520, NCI-H1581, NCI-H226, NCI-H460 and NCI-H1703) and compared with those of DMSO and SSR128129E. The  concentration of all compounds was 10 mM. The MTT assay was performed using a standard protocol. In Figure 4(A), compounds were tested for their inhibitory effect on NCI-H520 cells and compounds A9, B13, C2, C5, C6, C7 and C9 were found to have greater than 50% inhibition. Moreover, compounds A9, B13 and C9 were more active than compound SSR128129E; Figure 4(B) shows the results of compounds treated with NCI-H1581 cells. The inhibitory effect of A9, B3, B13 and C9 was above 50%, and the activity of C9 was better than that of SSR128129E. Figure 4(C) shows the antiproliferative effect of the compounds on NCI-H226 cells. The inhibitory rates of all the compounds in the C series and compound B9 were over 50%, better than the lead compound SSR128129E. Similarly, Figure 4(D) shows the antiproliferative effect of the compounds on NCI-H460 cells. The inhibitory rates of B9, C3 and C9 were greater than 50%. Moreover, the activity of B9, C3 and C9 was better than that of SSR128129E. Finally, Figure 4(E) shows the inhibitory effect of the compounds on NCI-H1703 cells.
The inhibitory rates of C4, C5 and C9 were greater than 50%. In addition, C4, C5 and C9 are more active than SSR128129E.
The IC 50 of 10 compounds used to treat different cell lines A total of 10 compounds with a suppression rate of at least two cell lines greater than 50% were chosen, including A9, B9, B13, C2, C3, C4, C5, C6, C7 and C9. Seven concentrations (30, 20, 10, 1.0, 0.50, 0.10 and 0.01 mM) were set for these compounds. Five cell lines, including NCI-H520, NCI-H1581, NCI-H226, NCI-H460 and NCI-H1703, were treated with various concentrations of each compound, respectively. The optical density (OD) value was obtained and the inhibition rate was calculated, and the IC 50 of the different compounds was calculated by GraphPad Prism 5 software.
The results are presented in Table 2, and indicate that compound C9 affected the five cell lines with an IC 50 of 1.36 ± 0.27 mM, 1.25 ± 0.23 mM, 2.31 ± 0.41 mM, 2.14 ± 0.36 mM and 1.85 ± 0.32 mM, respectively, which is better than that of SSR128129E obviously. Furthermore, the toxic effect of the compounds on BEAS-2B cells showed that 30 mM of the target compound had no toxic side effects on normal lung cells (see supplementary material). Therefore, compound C9 was chosen for further mechanistic studies.

Structure-activity relationship analysis
In combination with the inhibitory effect of all compounds on five cell lines, we found that compounds in the A series have no effect on the activity as the alkane chain increases. Among them, compound A9, which contains more electronegative groups, shows more excellent inhibitory activity. Compounds B9 and B13 are the most prominent compounds in the B series, they are the nitro group and the p-chloromethyl group on the benzene ring, respectively. It can be concluded from the other compounds that the stronger the electronegativity of the substituent groups on the benzene ring, the poorer the activity. Due to the introduction of sulfonyl groups, we found a large number of more effective compounds in the C series of compounds, while there is a negative correlation between the electronegativity and activity of many compounds, such as compounds C8-C11. The increase of the electronegativity of benzene ring decreases the activity obviously. The activity of p-tert-butyl substituted compound C9 was the best among all the target compounds.

Inhibitory effects of C9 on FGFR1-mediated signalling pathways in cancer cells
To evaluate the inhibitory effect on FGFR1 phosphorylation and the downstream signalling transduction, Western blot analysis of compound C9 in NCI-H226, NCI-H520 and NCI-H1581 cells was performed. The data, presented in Figure 5, indicated that compound C9 inhibited the phosphorylation of FGFR1, PLCc1 and ERK in a dose-dependent manner. In addition, the effect of 20 mM of C9 on the phosphorylation of FGFR1, PLCc1 and ERK was better compared to that of SSR128129E.

Compound C9 induces cellular apoptosis
The pro-apoptotic effects of compound C9 were examined by an Annexin V/propidium iodide (PI) assay ( Figure 6). NCI-H226 and NCI-H1581 cells were treated with compound C9 for 48 h. We determined that a concentration of 10 mM of C9 exhibited a significant effect on the overall apoptosis rate. Compared to SSR128129E, compound C9 was found to promote apoptosis in a dose-dependent manner.
Compound C9 affects the cell cycle Cell cycle experiments were performed with NCI-H1581 and NCI-H1703 cells treated with 2.5 mM, 5.0 mM, 7.5 mM and 10 mM of C9 and 10 mM SSR128129E, respectively. The data are presented in Figure 7, and demonstrate that 2.5 mM of C9 significantly increased the number of NCI-H1581 cells in the G2 phase in a dose-dependent manner. Moreover, the effect of 10 mM of C9 was better compared to that of SSR128129E. In addition, 7.5 mM of C9 significantly increased the number of NCI-H1703 cells in the G2 phase at a dose-dependent manner. Moreover, the effect of 10 mM of C9 was better than that of SSR128129E. Based on the results, we concluded that C9 arrested cells with FGFR1 amplification in the G2 phase.
Kinase inhibitory activity and kinase selectivity profile Selected tyrosine kinase receptors, such as FGFR1, EGFR, SRC and RET and ATP concentrations at Km were treated with 10 mM.
Compound C9 and inhibition rates were determined using a mobility shift assay ( Table 3). The FGFR1 inhibition rate of C9 was 84.3%, and the inhibition rates of other RTK receptors were lower, including wild-type and mutant EGFR family receptors, FLT family receptors, Janus kinase (JAK) family receptors. The relative maximum inhibition rate of other kinases was only 33.7%. Therefore, our data showed that C9 is a selective FGFR1 inhibitor.   Figure 8. We found that compound C9 formed four hydrogen bonds with amino acid residues Glu571, Arg570, Asn659 and Thr658 in the hydrophobic pocket 2, formed a hydrogen bond with amino acid residues Glu486 in the hinge region, and formed a hydrogen bond with amino acid residue Asn568 in the hydrophobic pocket 1. This experiment indicated several key hydrogen bonding interactions were formed, which may explain why compound C9 has outstanding activity.

Conclusion
In this study, we designed and synthesised 35 novel 4-bromo-N-(3,5-dimethoxyphenyl)benzamide derivatives, and evaluated the activity of these FGFR1-targeting compounds. Although most compounds demonstrated a modest inhibition of FGFR1, compound C9 showed excellent inhibition of five NSCLC cell lines with FGFR1 amplification, which was significantly better than the  lead compound SSR128129E. These findings may be due to the space structure of the FGFR1 ATP pocket, which was large enough to accommodate larger molecules and form more bonds. Although SSR128129E did not exhibit similar effects, compound C9 significantly increased apoptosis in NCI-H226 and NCI-H1581 cell lines and in a dose-dependent manner. Cell cycle analysis showed that compound C9 arrested NCI-H1581 and NCI-H1703 cell lines at the G2 phase. Furthermore, compound C9 downregulated the phosphorylation of FGFR1, PLCc1 and ERK in NCI-H226, NCI-H520 and NCI-H1581 cell lines. Based on in vitro enzymatic inhibitory activities of compound C9 against different statuses of RTKs, and the antiproliferative activity of EGFRaddicted cells, it can preliminarily be concluded that compound C9 is an FGFR1 inhibitor that has some degree of selectivity. Taken together, our results suggested that compound C9 is a highly selective FGFR1 inhibitor with a novel chemical scaffold that may serve as a potential agent for further drug development in FGFR1-driven cancer therapy.  >30 a A549 is a human lung cancer cell line (WT EGFR/k-RAS dependent). b PC-9 is a human lung cancer cell line (EGFR del E746_A750). c A431 is a human epithelial carcinoma cell line (overexpressed WT EGFR). d HCC827 is a human lung cancer cell line (EGFR dependent /WT k-RAS). The value ">30" indicates that no inhibitory effect at 30 lM compound concentration.