Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a–2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI50 concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.


Introduction
Regardless the great number of research studies worldwide to improve our understanding and effort to develop new therapies, breast cancer is still the most frequent diagnosed cancer and the main cause of cancer death in women [1]. Nowadays, due to a recent histopathologic classification, breast cancer is divided into different subtypes depending on the expression of key proteins such as human epidermal growth factor receptor 2 (HER-2) estrogen and progesterone receptors; Ki67; claudins; epidermal growth factor receptor (EGFR) and others. Thus, the subtypes of breast cancer are designated as luminal A, luminal B, HER2-enriched (+), basal-like and claudin-low [2]. The most aggressive, invasive and with poor prognosis type of breast cancer is the triple negative breast cancer

Cell Growth Inhibitory Effect of the Synthesized Compounds on Two Different TNBC Cell Lines
The antitumor potential of the synthesized compounds 2a-2h was evaluated in vitro using the sulforhodamine B (SRB) assay [19,20], which allows to determine the GI 50 concentration of each compound, i.e., the drug concentration that inhibits 50% of the cell growth.
Thus, the compounds were tested in two different triple negative breast cancer (TNBC) cells, MDA-MB-231 and MDA-MB-468. Unfortunately, it was not possible to determine the GI 50 values for some of the compounds synthesized, since these compounds formed crystals in the culture medium at higher concentrations. Therefore, in these cases, the GI 50 values were considered higher than the maximum concentration tested without crystal formation. Results presented in Table 2 demonstrated that three of the synthesized compounds (2e, 2f, 2h) tested significantly inhibited the growth of both TNBC cell lines with a GI 50 relatively low. Indeed, compound 2h presented the lowest GI 50 value in the MDA-MB-468 TNBC cell line. Even though it was not possible to establish structure-activity relationships (SARs), the furan derivative 2h, with an electron-rich ring, seems more selective for the MDA-MB-468 cell line. Moreover, the same was observed for compound 2f with an EWG (p-CN). Taken together compounds 2e, 2f and 2h, showed promising anticancer activity against TNBC cell lines. When not possible to determine the GI 50 due to the formation of crystals, the highest concentration tested in which crystals were not observed is presented (GI 50 higher than that concentration).

Evaluation of the Cytotoxicity of the Most Promising Compounds against a Non-Tumorigenic Cell Line
The most promising compounds 2e, 2f and 2h, were then evaluated regarding their toxicity against the non-tumorigenic mammary epithelial MCF-12A cell line. For that, the SRB assay was performed using the GI 50 concentrations of each of the three compounds obtained in the corresponding TNBC cell lines. The results (Table 3) demonstrated that compounds 2e, 2f and 2h, at the GI 50 concentrations previously obtained in the most sensitive TNBC cancer cell line (Table 2), caused a small or no effect in the growth of the non-tumorigenic MCF-12A cells. These results suggest that the three compounds are selective for cancer cells, not affecting the non-tumorigenic cell line, at least at the concentrations tested. It should be noted that the duration of the SRB assay carried out in the non-tumorigenic cells is longer than the SRB assay carried out in cancer cells, since the non-tumorigenic cells have a slower cell growth rate. This longer duration of the assay allows evaluation of possible delayed toxic effects of the compounds in the non-tumorigenic cells. Table 3. Evaluation of the cytotoxicity of the three most promising compounds in the non-tumorigenic cell line MCF-12A, using the SRB assay.

Compounds
Concentrations

Effect of Compound 2e on Number of Viable Cells and on the Proliferation of TNBC MDA-MB-231 Cells
Compound 2e has the particularity of having part of its hydrophobic tail (the phenyl ring bearing a Cl atom) similar to Sorafenib [21], which is an antiangiogenic drug known as an active multikinase inhibitor, currently approved for the treatment of different types of cancers [22]. However, Sorafenib has not been included in the clinical practice for breast cancer treatment given its high toxicity [23,24]. Therefore, we decided to conduct some preliminary activity studies on compound 2e. Its effect on TNBC viable cell number and cell proliferation was assessed after 48 h treatment, using the trypan blue exclusion assay and the bromodeoxyuridine (BrdU) incorporation assay, respectively. For that, the MDA-MB-231 cell line was treated with the GI 50 concentration of compound 2e, which corresponds to 13 µM, with the vehicle DMSO (control; at the same concentration used for the compound), and doxorubicin, Moreover, as expected, doxorubicin significantly reduced MDA-MB-231 cellular proliferation. Furthermore, the GI50 concentration of compound 2e also significantly decreased the % of proliferating MDA-MB-231 cells, when compared to the control ( Figure  2). These results suggested that compound 2e interferes with the TNBC viable cell number and proliferation. Moreover, as expected, doxorubicin significantly reduced MDA-MB-231 cellular proliferation. Furthermore, the GI 50 concentration of compound 2e also significantly decreased the % of proliferating MDA-MB-231 cells, when compared to the control (Figure 2). These results suggested that compound 2e interferes with the TNBC viable cell number and proliferation. exclusion assay. Cells were treated for 48 h with medium (Blank), control (DMSO; vehicle), compound 2e (at 13 μM) and Doxorubicin (positive control; 50 nM). Results represent the mean ± S.E.M. of at least three independent experiments. ** p ≤ 0.01 and *** p ≤ 0.001 of Control vs. Treatments.
Moreover, as expected, doxorubicin significantly reduced MDA-MB-231 cellular proliferation. Furthermore, the GI50 concentration of compound 2e also significantly decreased the % of proliferating MDA-MB-231 cells, when compared to the control ( Figure  2). These results suggested that compound 2e interferes with the TNBC viable cell number and proliferation.

Effect of Compound 2e on Cell Cycle Profile in TNBC MDA-MB-231 Cells
Considering the previous results, we further analyzed the possible effect of compound 2e on TNBC MDA-MB-231 cell cycle profile, assessed after 48 h treatment, using the flow cytometry analysis with propidium iodide (PI). For that, the MDA-MB-231 cell line was treated with 13 μM of compound 2e (GI50 concentration), the vehicle DMSO (control) and doxorubicin. As predictable, doxorubicin (used as positive control) significantly increased the G0/G1 phase and decreased S and G2/M phases in the MDA-MB-231 cells. The results demonstrated that the GI50 concentration of compound 2e, although not statistically significant, increased G0/G1 phase and decreased S phase, when compared to control cells (Figure 3). These results suggested that compound 2e interferes with the cell cycle profile of TNBC MDA-MB-231 cells. The above results indicate that compound 2e is the most promising compound

Antitumor Effect of Compound 2e in Ovo Grafted with MDA-MB-231 Cells Using the Chick Chorioallantoic Membrane (CAM) Assay
The above results indicate that compound 2e is the most promising compound against the MDA-MD-231 cell line. In addition, this compound decreased the number of viable and proliferating tumor cells. Since compound 2e has a structural similarity with the antiangiogenic drug Sorafenib, the study of the antiangiogenic and antitumor potential of this compound was attempted using the chick embryo chorioallantoic membrane (CAM) assay [25]. Thus, CAM assay was performed by grafting TNBC MDA-MB-231 cells in ovo, in order to create tumors in the eggs, which was followed by testing the effect of the GI 50 concentration (13 µM) of compound 2e on tumor formation.
Results showed that ( Figure 4A) treatment of grafted eggs for 48 h with the GI 50 concentration of compound 2e efficiently decreased tumor formation (tumor size), when compared with the control group (vehicle). Unfortunately, the antiangiogenic potential was not possible to analyze due to the high vascularization induced by breast cancer cells, which led to some degree of inflammation. The reduction in tumor size was well visible ( Figure 4B). In addition, using hematoxylin and eosin (H&E) staining, viability of tumors at both conditions was confirmed ( Figure 4B). Remarkably, the CAM assay confirmed a promising anti-tumorigenic effect of compound 2e, although no information regarding the antiangiogenic potential was possible to obtain.

Discussion
From the eight methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a-2h synthesized (Table 1), the compounds obtained with the best yields were 2a (Ph), 2b (p-Me), 2c (p-OMe) without substitution or with EDGs, and 2e (p-Cl). Although for phenyl rings bearing EWGs (p-CF3 and p-CN) the yields were only moderate, for compound 2e the yield was excellent (82%) may be due to the positive mesomeric effect of the chlorine. Despite the different electron character of the heterocyclic rings, pyridine and furan, the resulting compounds 2g and 2h were isolated in good yields.
The evaluation of the effect of this series of compounds on cell growth (by the SRB assay) allowed to find the three most promising compounds, namely compounds 2e, 2f and 2h, which under the concentrations tested showed antitumor effect in vitro against the TNBC cell lines. Importantly, the three compounds had little or no effect against the non-tumorigenic cancer cell line MCF-12A, at the concentrations tested (which inhibited the growth of the cancer cells by 50%).
Since compound 2e has the particularity of having part of its hydrophobic tail (the phenyl ring coupled to a p-Cl atom) similar to Sorafenib, an approved anticancer agent

Discussion
From the eight methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a-2h synthesized (Table 1), the compounds obtained with the best yields were 2a (Ph), 2b (p-Me), 2c (p-OMe) without substitution or with EDGs, and 2e (p-Cl). Although for phenyl rings bearing EWGs (p-CF 3 and p-CN) the yields were only moderate, for compound 2e the yield was excellent (82%) may be due to the positive mesomeric effect of the chlorine. Despite the different electron character of the heterocyclic rings, pyridine and furan, the resulting compounds 2g and 2h were isolated in good yields.
The evaluation of the effect of this series of compounds on cell growth (by the SRB assay) allowed to find the three most promising compounds, namely compounds 2e, 2f and 2h, which under the concentrations tested showed antitumor effect in vitro against the TNBC cell lines. Importantly, the three compounds had little or no effect against the non-tumorigenic cancer cell line MCF-12A, at the concentrations tested (which inhibited the growth of the cancer cells by 50%).
Since compound 2e has the particularity of having part of its hydrophobic tail (the phenyl ring coupled to a p-Cl atom) similar to Sorafenib, an approved anticancer agent for other cancer types [21], it was decided to conduct preliminary studies on the antitumor activity of this novel compound. The results demonstrated that compound 2e decreased viable cell number and cell proliferation in the TNBC MDA-MB-231 cells. Interestingly, compound 2e did not cause alterations in the levels of two important apoptotic markers, PARP or caspase-3, therefore suggesting that apoptosis is not involved in the mechanism of action of this compound (data not shown). Remarkable, this work showed that compound 2e reduced tumor size of grafted MDA-MB-231 cells in ovo CAM model. The CAM assay allows to study the effect of compounds on the angiogenesis process, as well as to evaluate the effect on tumor growth (tumorigenic response), being suitable as an in vivo model for the screening of potential novel drugs, as an alternative to mouse models [26].
Interestingly, studies conducted in TNBC cells demonstrated that Sorafenib is able to inhibit cell proliferation and tumor growth [27][28][29]. Similar effects were observed in the present study, with MDA-MB-231 cells treated with compound 2e, both in vitro and in ovo. It is possible that the hydrophobic tail might be partly responsible for the anticancer activity of these molecules, but this hypothesis requires evaluation. Further studies should be conducted to better understand the mechanism of action of compound 2e.

General Procedure for the Suzuki-Miyaura Cross-coupling Products 2a-h
In a round flask, DME (3 mL) and water (1 mL), compound 1, (het)aryl boronic acids, pinacol esters, or potassium trifluoroborates (1.2-1.6 equiv.), PdCl 2 (dppf).CH 2 Cl 2 (1:1) (2 mol% unless stated), K 2 CO 3 (6 equiv.) were added and the mixture was heated with stirring at 100 • C for 3-4.5 h. The reactions were monitored by TLC. After cooling, water was added (10 mL) and extraction with ethyl acetate (3 × 10 mL) was performed. The organic phases were collected, dried (MgSO 4 ), filtered and the solvent removal under reduced pressure gave a solid that was submitted to column chromatography or to dry flash using solvent gradient from neat petroleum ether to mixture of ether/petroleum ether, increasing 10% of ether each time, to give compounds 2a-2h. . These media were supplemented with 5% FBS for the cell growth inhibition assay (SRB) or with 10% FBS for the remaining experiments. The non-tumorigenic cell line MCF-12A was cultured in RPMI 1640 medium supplemented with hEGF (human epidermal growth factor; 20 ng/µL), hydrocortisone (500 ng/µL), 5% FBS and 1% Penicillin-Streptomycin (Gibco), as previously described [30]. All the cell lines were maintained as adherent cell cultures and kept at 37 • C in a humidified incubator containing 5% of CO 2 . Regularly, all cells were observed using an inverted light microscope (Leica DMi1). All the experiments were carried out with cells at the exponential phase of growth and with more than 90% viability.

Trypan Blue Exclusion Assay
The cell number and viability were assessed by counting the cells with a hemocytometer (Neubauer Chamber) using the trypan blue reagent, which distinguishes alive (bright) from dead cells or non-viable cells (blue ones).

Cell Growth Inhibition Assay (SRB Assay)
The potential cytotoxicity of the compounds was tested using an assay based on a protein-binding dye, the sulforhodamine B (SRB), according to the protocol [31]. Briefly, cells were seeded at an appropriate concentration previously optimized (5.0 × 10 4 cells per mL for MDA-MD-231 and MDA-MB468 cells) by adding 100 µL of cells per well and then incubated at 37 • C for 24 h. All experiments were performed in two 96 well-plates, a T0 plate to be analyzed at 0 h (time of treatment with the compounds) and a T48 plate to be analyzed 48 h later. Then, cells were treated with five serial dilutions of each compound (ranging from 150 µM to 9.4 µM) by adding 100 µL of compound per well, and incubated at 37 • C for further 48 h. The effect of the vehicle (DMSO) on cell growth was also evaluated (control) by exposing untreated cells to the maximum concentration of DMSO (always lower than 0.25%). The maximum concentration tested for each compound was 150 mM. However, for some compounds, precipitation was detected within the tested range of concentrations. In those cases, the GI 50 concentration was not possible to determine and was indicated as "higher than" the highest concentration tested without causing precipitation. Doxorubicin (ranging from 100 nM to 6.25 nM) was used as positive control. Following 48 h incubation of cells with the compounds in the T48 plate (or 0 h in the T0 plate), cells were fixed by adding 10% (w/v) of cold TCA (w/v, final concentration) during 1 h at 4 • C. Subsequently, after washing with water, air-dried cells were stained with 0.4% of SRB (in acetic acid, w/v) for 30 min in the dark at room temperature (RT). At the end, cells were washed with 1% (v/v) of acetic acid and the bound dye was solubilized with 10 mM Tris base solution.
The absorbance was measured at 510 nm in a microplate reader (BioTek's SynergyTM Mx) using the Gen5 software (BioTek) [19,20]. The GI 50 concentrations for each compound (concentrations that inhibited cell growth by 50%) were assessed from the dose-response curves, determined in each cell line and presented as mean ± standard error (S.E.M) from at least three independent experiments. Compounds 2e, 2f, 2h were also tested against the non-malignant MCF-12A human breast epithelial cells. For that, cells (seed at 5.0 × 10 4 cells per mL) were incubated with the GI 50 concentrations of each of the three compounds obtained in the corresponding tumor cell lines for 48 h, followed by removal of the compound, addition of new medium to the cells and then 5 more days in culture. At the end of the 7 days in total, the sulforhodamine B (SRB) assay was performed, as previously described [30].

Cell Proliferation Analysis (BrdU Assay)
Cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay, according to the previously described protocol [19]. The MDA-MB-231 cells were plated at 7 × 10 4 cells per mL in 6-well plates. After 24 h, cells were treated with the GI 50 (13 µM) of compound 2e, medium (blank), the vehicle corresponding to DMSO (control; in a concentration equivalent to the one used in the treatment) or 50 nM of doxorubicin (positive control), for 48 h. After that, cells were incubated with 10 µM BrdU (Sigma-Aldrich) for 4 h. Cells were washed and fixed with 4% paraformaldehyde, PFA (Panreac) in PBS. After the preparation of cytospins, cells were treated with 2 M HCl for 20 min (DNA denaturation), followed by blocking with a PBS 0.5% Tween and 0.05% Bovine Serum Albumin solution. Cells were then incubated with a mouse anti-BrdU antibody (1:10; Dako), followed by the incubation with anti-mouse-Ig-FITC (1:100; Dako). After that, slides were prepared with Vectashield mounting medium containing 4 ,6-diamidino-2-phenylindole, DAPI (Vector Laboratories Inc, Burlingame, CA, USA). The detection of BrdU incorporation (green nuclei) was possible using a Zeiss Axio Imager Z1 (Carl Zeiss, Jena, Germany) microscope and the Axiovision 4.9 (Carl Zeiss, Jena, Germany) software. A semi-quantitative evaluation was performed by counting a minimum of 500 cells per slide using the ImageJ 2.1.0 software. The results were from three independent experiments, and are presented as mean ± S.E.M.

Cell Cycle Analysis
For cell cycle distribution analysis, MDA-MB-231 cells were plated at 7 × 10 4 cells per mL in 6-well plates for 24 h. Cells were then treated with the GI 50 (13 µM) of the compound 2e, medium (blank), the vehicle DMSO (control) and 50 nM of doxorubicin (positive control). Following 48 h incubation, cells were collected, centrifuged (1200 rpm for 5 min) and then fixed with ice-cold 70% ethanol and stored at 4 • C for at least 12 h. Cells were then centrifuged at 1200 rpm for 5 min, at 4 • C, and the pellets resuspended in a solution of PBS containing 5 µg/mL propidium iodide (PI) and 0.1 mg/mL RNase A, and incubated for at least 30 min in the dark on ice, prior to each analysis on the flow cytometer. Cellular DNA content for cell cycle distribution was analyzed by flow cytometry using the BD Accuri™ C6 Flow Cytometer (BD Biosciences). The exclusion of cell debris and aggregates was performed for each analysis, and at least 20,000 events per sample were plotted for all the acquisitions. Data was analyzed using the FlowJo software (version 7.6.5, Tree Star, Inc., Ashland, OR, USA) [19]. The results were from at least three independent experiments, and are presented as mean ± S.E.M.

In Ovo Evaluation
In Vivo Chick Egg Chorioallantoic Membrane (CAM) Model The chick embryo chorioallantoic membrane (CAM) model was used to evaluate the tumorigenic response of compound 2e. Fertilized chick (Gallus gallus) eggs were obtained from commercial sources, and incubated horizontally at 37.8 • C, in a humidified atmosphere and referred to the embryonic development day (EDD). Ethical approval is not required for experiments using embryonic chicken according to the European Directive 2010/63/EU and the Portuguese law on animal welfare does not restrict the use of chicken eggs for experimental purposes. Briefly, on EDD3, a square window was opened in the shell after removal of 1.5-2 mL of albumin to allow detachment of the developing CAM.
The window was sealed with a transparent adhesive tape and the eggs returned to the incubator. The MDA-MB-231 cells were resuspended in matrigel (1:1) and grafted on top of the same EDD9 growing CAM, into two independent 3-mm nylon rings, under sterile conditions (7.5 × 10 5 cells per ring). The eggs were resealed and returned to the incubator for an additional 24 h. At EDD10, tumor xenografts were treated with either vehicle (DMSO) or with compound 2e pairwise. Eggs were resealed and returned to the incubator for additional 3 days. On EDD14, embryos were euthanized by adding 2 mL of fixative in the top of the CAM, rings were removed, the CAM was excised from embryos and photographed ex-ovo under a stereoscope, using a 20× magnification (Olympus, SZX16 coupled with a DP71 camera). The area of CAM tumors was determined using the Cell A (Olympus) software. The experiment was performed in a blind fashion way, since the identity of the treatment (vehicle or compound 2e) was not known by the person performing the analysis. The area of CAM tumors was determined using the Olympus cell Sens Standard 1.14 program. Excised CAMs were fixed in 10% neutral-buffered formalin, paraffin-embedded for slide sections and stained with hematoxylin-eosin for histological examination.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism Software v5 (GraphPadtrial version, San Diego, CA, USA). For SRB assay, the GI 50 concentrations were assessed from the dose-response curves and determined for each compound, in each cell line. The GI 50 values presented are the mean ± standard error (S.E.M.) from at least three independent experiments. For BrdU and cell cycle analysis, the % of proliferating cells or cells in each cell cycle phase was determined, and the results presented are the mean ± S.E.M. of at least three independent experiments. In the CAM assay, the data (n = 19) were analyzed for Gaussian distribution and passed the D'Agostino and Pearson normality tests. To test the hypothesis that compound 2e treated tumors are different from the vehicle, a paired t-test was used. Statistical significance was achieved when p value < 0.05.

Conclusions
Eight novel methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a-2h were prepared in moderate to high yields by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with different boronated compounds. These were tested against TNBC cell lines and the potential of three novel thieno [3,2b]pyridine derivatives 2e, 2f, 2h as anticancer agents in TNBC cell lines was demonstrated. Importantly, no toxicity was observed in non-tumorigenic breast cells. One of the most promising compounds, 2e, presented activity against TNBC both in vitro (by decreasing viable cell number and cell proliferation and interfering with the cell cycle profile) and in ovo (by reducing grafted tumor size). However, further studies must be carried out to unravel the underlying mechanisms of action of this compound. Furthermore, the anticancer effect of compound 2e should be also assessed in vivo, using xenografted mouse models of human TNBC cell lines. Although the present work is focused on compound 2e, the other promising compounds (2f and 2h) will be also explored in the future. Therefore, this work highlights the antitumor potential of a novel thieno[3,2-b]pyridine derivative against an aggressive type of cancer, TNBC. Funding: This research was funded by Fundação para a Ciência e Tecnologia (FCT)-Portugal that financially supports CQUM (UID/QUI/686/2019), also financed by European Regional Development Fund (ERDF), COMPETE2020 and Portugal2020, the PTNMR network also supported by Portugal2020. C.P.R.X. is supported through the post-doc grant SFRH/BPD/122871/2016 and J.M.R. through the doctoral grant SFRH/BD/115844/2016, by FCT, ESF (European Social Fund) and HCOP (Human Capital Operational Programme).

Data Availability Statement:
The data presented in this study are available in article, including in the Supplementary Materials.