Design, Synthesis and Anti-Proliferative Activities of 2,6-Substituted Thieno[3,2-d]pyrimidine Derivatives Containing Electrophilic Warheads

Thieno[3,2-d]pyrimidine as an effective pharmacophore has been extensively studied. However, its 2,6-substituted derivatives are rarely reported. In the present study, eighteen 2,6-substituted thieno[3,2-d]pyrimidine derivatives containing electrophilic warheads were designed based on the first known Fibroblast growth factor receptor-4 (FGFR4) inhibitor Blu9931. Unexpectedly, all of the derivatives exhibited negligible activity against FGFR4. However, most of the target compounds exhibited antiproliferative activities against four human cancer cell lines, including A431, NCI-H1975, Ramos and SNU-16. Compound 12 showed the most potent antiproliferative activities on the above four cell lines with IC50 values of 1.4 μM, 1.2 μM, 0.6 μM, and 2.6 μM, respectively. Additionally, the antiproliferative activity of 12 against MDA-MB-221 proved that 12 had the selectivity towards certain tumor cell lines. Furthermore, preliminary structure-activity relationship analysis was discussed based on the experimental data.


Introduction
Cancer is one of the most formidable enemies to the public health worldwide [1]. Despite the numerous achievements in the field of cancer pharmacotherapy, there is a continuous demand for novel agents with improved therapeutic efficacy, selectivity, and safety. Over the past few decades, more and more drugs with fused bicyclic pyrimidine scaffolds have been approved by the Food and Drug Administration (FDA) with significant biological activities, especially antitumor activities. These fused bicyclic pyrimidines exhibit anticancer function by targeting different kinases, such as Epidermal growth factor receptor (EGFR), Brutons tyrosine kinase (BTK), Janus Kinase (JAK), and Phosphatidylinositol 3 kinase (PI 3 K) (Figure 1) [2][3][4][5][6]. Considering broader applications of fused bicyclic pyrimidines in anticancer drugs, chemical modifications of the bicyclic pyrimidine scaffolds have been extensively studied. Previously, various thieno [3,2-d]pyrimidine derivatives have attracted attention due to their broad spectrum activities, such as GDC-0941 and Olmutinib ( Figure 2) [7][8][9][10][11][12][13][14][15][16][17][18]. Although a large number of studies on derivatives of thieno [3,2-d]pyrimidine have been documented, the 2,6-substituted analogs are seldom reported.  Recently, therapies using covalent drugs have been proved to be successful for various indications. Although there have been concerns about the toxicity of covalent drugs, the high potencies and prolonged effects might result in less frequent drug dosing and wide therapeutic margins for patients [19][20][21][22]. Many covalent drugs have been reported for the treatment of cancer by inhibiting different targets. Among these derivatives, Afatinib, an irreversible EGFR tyrosine kinase inhibitor (TKI) [23], was approved by the FDA for the treatment of metastatic non-small cell lung cancer in 2013 [24]. Ibrutinib, a selective irreversible BTK inhibitor, has been approved by FDA for treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia [25,26]. Blu9931, which contains a quinazoline core and an acrylamide electrophilic warhead, is the first selective small molecule inhibitor of FGFR4 ( Figure 3) [27]. Evidence suggests that covalent drugs are a feasible antitumor strategy. It is worth mentioning that bicyclic pyrimidine scaffolds are widely used in covalent drug design. Based on previously published results and our continued interest in studying thieno [3,2-d] pyrimidine derivatives, we decided to design a series of 2,6-substituted thieno [3,2-d]pyrimidine compounds containing electrophilic warheads based on the first known FGFR4 inhibitor Blu9931. FGFR4 is a tyrosine kinase that regulates a wide range of important biological functions. The fibroblast growth factor 19 (FGF19) is the first FGF member exhibiting selective binding to its receptor FGFR4 [28]. Aberrant signaling through the FGF19/FGFR4 signaling complex has been shown to cause hepatocellsular carcinoma in mice and has indicated a similar role in humans [27]. Therefore, FGFR4 is a potential therapeutic target for anticancer drugs. In addition, FGFR1 is highly homologous to FGFR4. Herein, we presented the design, synthesis and antitumor activities of the compounds related to the general structure A (Figure 4). The preliminary structure-activity relationship was also discussed.

Chemistry
Detailed synthetic strategy to key intermediate 8 was illustrated in Scheme 1. Compound 4 was synthesized according to published procedures [29] and then treated with urea at 170 • C to produce compound 5. Subsequently, compound 5 was treated with POCl 3 followed by the treatment with Pd/C/NaHCO 3 to produce 7. The newly synthesized 7 was treated with NH 3 /EtOH (4 M) at 150 • C in a steel tube to afford 8. The synthetic route for the synthesis of derivatives 12-29 was depicted in Scheme 2 and the chemical structures of 12-29 were shown in Table 1. The derivative 8 was involved in a nucleophilic aromatic substitution reaction in the presence of 9a-c to obtain crude products. Furthermore, reduction of the nitro group was carried out under basic conditions to give the compound 10a-c, which was then treated with sulfonyl chloride to give 11a-c. In addition, 10a-c and 11a-c were mixed with acryloyl chloride to afford the targets 12-17. On the other hand, 10a-c and 11a-c were treated with (E)-4-bromobut-2-enoyl chloride before reacting with 1-methylpiperazine or 30% dimethylamine solution in water to afford targets 18-29.

In Vitro Enzymatic Assays-FGFR1 and FGFR4
Firstly, the FGFR1 and FGFR4 enzymatic assays of compounds 12-29 were performed, since their structures are similar to the first small molecular FGFR4 inhibitor Blu9931. Unfortunately, all of the compounds lost the enzyme activities against FGFR1 and FGFR4 with less than 50% inhibition at 17 Cl H Me

In Vitro Enzymatic Assays-FGFR1 and FGFR4
Firstly, the FGFR1 and FGFR4 enzymatic assays of compounds 12-29 were performed, since their structures are similar to the first small molecular FGFR4 inhibitor Blu9931. Unfortunately, all of the compounds lost the enzyme activities against FGFR1 and FGFR4 with less than 50% inhibition at

In Vitro Enzymatic Assays-FGFR1 and FGFR4
Firstly, the FGFR1 and FGFR4 enzymatic assays of compounds 12-29 were performed, since their structures are similar to the first small molecular FGFR4 inhibitor Blu9931. Unfortunately, all of the compounds lost the enzyme activities against FGFR1 and FGFR4 with less than 50% inhibition at 1 µM. The results summarized in Figures S1 and S2 (supplied in supplementary data) were expressed as inhibition rates, which were derived from double independent determinations (the information of positive controls can be found in supplementary data), suggesting that the interaction between Blu9931 and FGFR4 might be sensitive to the specific angle of two benzene rings.

In Vitro Cytotoxic Activities and SAR Analysis
The antiproliferative activity of compounds 12-29 against A431 (human skin squamous cell carcinoma), NCI-H1975 (human lung adenocarcinoma cell), Ramos (human B lymphoma cell) and SNU-16 (human gastric cancer cell) cell lines were tested. Using the approved drug Doxorubicin (ADR) as positive controls, the results of the mean values of experiments from three independent determinations, expressed as half-maximal inhibitory concentration (IC 50 ) values, were summarized in Table 2.
As presented in Table 2, most of the target compounds exhibited moderate antiproliferative activities against Ramos cells. The different substituents of hydrogen or methyl in benzene ring were first investigated. Compound 12 exhibited moderate activity (IC 50 = 0.6 µM), while the replacement of hydrogen with o-methyl group (compound 13) or p-methyl group (compound 14) resulted in a slight loss of activity (IC 50 = 3.9 µM and 2.7 µM, respectively), suggesting that the antiproliferative activity is sensitive to small changes on the benzene ring adjacent to the pyrimidine side. To further identify the effects of the benzene ring next to the thiophene side on the biological profiles, compounds 15-17 were synthesized and tested. Dramatically reduced antitumor activities were observed when 12-14 were transformed into their respective dichloride substituted derivatives 15-17 (IC 50 = 8.0 µM, 14.1 µM and 6.4 µM, respectively). The result suggests that the benzene ring next to the thiophene side plays an important role in antitumor activity. In addition, analogues 18-29 with different Michael acceptors displayed significantly reduced activity against Ramos cells compared with the corresponding compound with acryloyl group. Most of the compounds 12-29 also exhibited moderate to great antiproliferative activity against the other three cell lines. It is interesting to note that the structure-activity relationship analyses of 12-29 against the four tested cell lines were highly consistent. Furthermore, the antiproliferative activity of the most potent compound 12 against NCI-H1581 (Human non-small cell lung cancer cell line), MDA-MB-231 (Human breast cancer cell line) was evaluated. The derivative 12 showed loss of antiproliferative activity against MDA-MB-221 (IC 50 > 20 µM) indicating that 12 exhibits selectivity towards certain tumor cell lines (Table 3). Table 3. Antiproliferative activity of 12 against NCI-H1581, MDA-MB-231.

In Vitro Enzymatic Assay-BTK
BTK plays a crucial role in B cell lines growth and reproduction [30]. It has been well documented that most of the BTK inhibitors formed a covalent bond between its electrophilic warhead and a nucleophilic center in the BTK protein. As shown in Table 2, most of the target compounds displayed potent anti-proliferative activity on Ramos cells (B cell lines). Therefore, the inhibition of 12-29 against BTK at 10 nM, 100 nM and 500 nM were tested. Using Ibrutinib as the positive control, the results of the mean values of experiments from double independent determinations, expressed as inhibition rates, were exhibited in Figure S3 (supplied in Supplementary Data). Compounds 12-29 demonstrated less than 40% inhibition against BTK at 500 nM, and their inhibitory activities are much inferior to Ibrutinib. These results indicated that BTK is not the biological target for these compounds.

In Vitro Enzymatic Assays
To investigate the molecular mechanisms, we screened compound 12 against twenty selected tyrosine kinases. The results summarized in Table S3 (supplied in Supplementary Data) were expressed as inhibition rates, derived from three independent determinations (details related to positive controls can be found in Supplementary Data). Unfortunately, compound 12 was judged to have a negligible impact on the tested kinases.

Materials and Methods
All reagents and solvents were purchased from commercial suppliers and were further purified or dried if necessary. The 1 H-NMR spectra and 13 C-NMR spectra were recorded for CDCl 3 , MeOD, or Dimethyl sulfoxide-d 6 (DMSO-d 6 ) by a Bruker DRX-400 (400 MHz) (Bruker Biospin AG, Fällanden, Switzerland) with Tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported as δ (ppm) and spin-spin coupling constants as J (Hz) values. The mass spectra were obtained on a Waters -SDQ mass spectrometer (Manchester, UK) or Waters SYNAPT G2 ESI-TOF-MS analyzer. Melting points were determined on an SGW X-4 melting point detector (Shanghai, China), uncorrected and reported in degrees Centigrade. The derivatives synthesized were purified by column chromatography using silica gel (200-300 mesh). The purity of all tested derivatives was established by High Performance Liquid Chromatography (HPLC) to be >95%. (2) Step 1. 3,5-dimethoxybenzoyl chloride 3,5-dimethoxybenzoic acid (27.3 g, 150 mmol, compound 1) was suspended in 120 mL thionyl chloride, and the mixture was heated and held at reflux until 1 dissolved. Then, thionyl chloride was evaporated under reduced pressure conditions and the residue was diluted with 150 mL anhydrous acetonitrile.

1-(3,5-Dimethoxyphenyl)ethanone
Step 2. Ethyl 3,5-dimethoxybenzoate Ethyl potassium malonate (51 g, 300 mmol) and magnesium chloride (36 g, 375 mmol) were suspended in 600 mL anhydrous acetonitrile, and 63 mL Et 3 N was added slowly. The mixture stirred at room temperature for 2 h. Then 3,5-dimethoxybenzoyl chloride in 50 mL anhydrous acetonitrile was added dropwise. The reaction mixture stirred at room temperature overnight. Dilute hydrochloric acid added to the mixture until pH < 5. The aqueous layer was extracted three times with ethyl acetate (EA) and the combined organic layers were washed with brine and then dried over anhydrous sodium sulfate, filtered and the solution was concentrated under reduced pressure to give the corresponding crude product.

Conclusions
In summary, a series of 2,6-substituted thieno [3,2-d]pyrimidine containing electrophilic warheads derivatives were designed and synthesized. The derivatives possessed moderate antiproliferative properties against four tested cancer cell lines (A431, NCI-H1975, Ramos and SNU-16). Compound 12 showed the most potent cytotoxic activity on the four cell lines above with IC 50 values of 1.4 µM, 1.2 µM, 0.6 µM, and 2.6 µM, respectively. However, the antiproliferative activity of 12 against MDA-MB-221 indicated that 12 had the selectivity towards certain tumor cell lines. The preliminary investigation showed that the acrylamide electrophilic warhead was more active than the other two warheads. It was interesting to note that an o-Me substituent on the benzene ring attached to the electrophilic warhead weakens the activity slightly while p-Me substituent results in a significant loss of activity. Another interesting phenomenon was that a dramatic loss in antitumor activity was observed when 12-14 were transformed into their dichloride substituted derivatives 15-17. In addition, compounds 12-29 demonstrated less than 40% inhibition against BTK at 500 nM, indicating that BTK might not be the biological target for these compounds. The enzymatic screen assays suggested that the selected twenty kinases might also not be the biological targets for the synthesized compounds. Thus, future efforts will focus on investigating the molecular mechanisms of the target compounds, especially 12. In summary, compound 12 is worth further research as a new potential anticancer agent.
Supplementary Materials: The following are available online. The general HPLC method, ESI-TOF-MS spectrums of 12-29, FGFR1 and FGFR4 enzymatic assay, BTK enzymatic assay, and in vitro enzymatic assays, Figure S1. FGFR1 inhibition rate of 12-29 at the concentration of 10 nM, 100 nM and 1000 nM, Table S1: Positive controls in FGFR1 enzymatic assay, Figure S2. FGFR4 inhibition rate of 12-29 at the concentration of 10 nM, 100 nM and 1000 nM, Table S2: Positive controls in FGFR4 enzymatic assay, Figure S3. BTK inhibition rate of 12-29 at the concentration of 10 nM, 100 nM and 1000 nM, Table S3-S6: Some positive controls in in vitro enzymatic screen assays.
Author Contributions: Q.Z. and W.L. participated in designing the target compounds. Q.Z. was the chief experimenter of chemistry, analyzed the experimental data and wrote the paper. Y.C., Z.H., and Q.S. performed all of the biological assays and participated in analyzing the data. W.L. and Y.C. participated in the corrections of the final manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: