A Microwave-Enhanced Synthesis and Biological Evaluation of N-Aryl-5 , 6 , 7 , 8-tetrahydrobenzo [ 4 , 5 ] thieno [ 2 , 3-d ] pyrimidin-4-amines

A series of N-aryl-5,6,7,8-tetra-hydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amines were synthesized in moderate to good yield by using a microwave-enhanced conditions. The selected compounds were evaluated for their cytotoxic effects (IC50 values) on human pulmonary carcinoma (A549), murine BALB/c spontaneous colon adenocarcinoma (CT26) and human hepatocellular liver carcinoma (HepG2) cell lines in vitro. Amongst these compounds, one compound was found to have the better cytotoxic activity with reference to the standard Erlotinib hydrochloride (Tarceva) against A549 (IC50 = 16.06 ± 0.09 μM) and HepG2 (IC50 = 15.01 ± 0.31 μM) cell lines. Especially, two compounds showed best cytotoxic effects against CT26 (IC50 = 11.38 ± 0.44 μM) and HepG2 (IC50 = 8.51 ± 0.52 μM) cell lines, respectively. The preliminary structure-activity relationships were disclosed and the thieno[2,3-d]pyrimidine skeleton could be exploited to potential antitumor agents in the future.

Herein, we take into consideration that the compounds of thieno[2, 3-d]pyrimidine skeleton express an important biological cytotoxic activity, and intend to synthesize a series of thieno[2, 3-d]pyrimidine derivatives.
Microwave irradiation technology in organic synthesis is a powerful tool for drug synthesis as it can speed up the reactions under mild conditions allowing the rapid preparation of compounds. 30As mentioned above, microwave irradiation technology has been successfully applied to the synthesis of N-arylthieno[2, 3-d]pyrimidine derivatives.Our recent endeavors involving green chemical techniques have focused on the synthesis of small organic molecular compounds.Then, in earlier work from our lab, we have reported the synthesis of 6-bromopyrido[2,3-d]pyrimidine derivatives (XXI), 31 pyrido[2, 3-d]pyrimidine derivatives (XXII), 32 cyclopenta [4,5]thieno[2, 3-d]pyrimidine derivatives (XXIII) 33 by microwave irradiation technology (Figure 4).
Interestingly, a compound of similar structure (Figure 4, compound XXIV) 34 was evaluated to inhibit EGFR (epidermal growth factor receptor) kinase activity with the enzyme inhibition IC 50 = 2.6 μM.Herein, thieno[2, 3-d]pyrimidine skeleton of compounds XXIII and XXIV were used as a template, and the structure was modified: (Figure 4a) hexatomic ring was linked to thiophene ring instead of five membered ring (compound XXIII); (Figure 4b) substituted aromatic group was linked to secondary amine and -CH 2 group was subtracted (compound XXIV).Subsequently, compounds 3a-x (Scheme 1) were obtained.

Synthesis
The synthetic strategy used in this study is outlined in Scheme 1. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile was prepared using a modified Gewald procedure via the reaction of cyclohexanone, malononitrile and sulfur in the presence of potassium carbonate (K 2 CO 3 ), which serves as a heterogeneous solid basic catalyst, in ethanol.][37][38][39][40] It was found that our method can achieve the target product in 95% yield in 15 min of reaction.Compared to the results in the literature, [35][36][37][38][39][40] our method has a shorter reaction time and high yield.So, under the assistance of the microwave irradiation, K 2 CO 3 can act as a cheap, highly efficient, and green catalyst used to prepare compound 1 in high yield over a short reaction time.
As for the synthesis of the intermediate compound 2 The synthesis of compounds 3a-x (Table 1) involved the acid-catalyzed Dimroth rearrangement between compound 2 and an appropriate aromatic amine under microwave irradiation.It was found that the reaction between m, p-substituted aromatic amines and compound 2 can easily occur in high yield with a short reaction time.However, the reaction between o-substituted aromatic  amines and compound 2 only formed trace amounts of the desired compounds under the same reaction conditions.This new synthetic route involves only three steps making this procedure simple and efficient.Hence, this method is an efficient method for the synthesis of N-arylthieno- The structures of all compounds 1, 2 and 3a-x were confirmed by infrared (IR), 1 H nuclear magnetic resonance (NMR), 13 C NMR spectroscopy, and high-resolution mass spectrometry (HRMS).The IR spectrum of compound 1 shows bands at 3444 and 2193 cm -1 corresponding to the -NH and -C≡N groups, respectively.For compound 2, the IR spectrum shows bands at 2199, 1627 and 1106 cm -1 corresponding to the -C≡N, -C=N and -C-N groups, respectively.For the sake of the assignment of 1 H NMR spectra, we chose compound 3h as the representative example.The 1 H NMR spectrum of the compound 3h recorded in DMSO-d 6 shows the multiplet peaks at d 7.74-7.60,7.54-7.43,7.42-7.30,and 6.96-6.80assignable to the aryl protons (-CH) and the other multiplets at d 3. 24-3.04,2.96-2.74,and 1.96-1.75correspond to alicyclic portions protons (-CH 2 ).However, there are two peaks at d 8.38 and 8.13, probably assigned to 2-position hydrogen on the pyrimidine ring (-CH) and the 4-position at pyrimidine ring (-NH), respectively.In order to solve this assignment problem, D-H exchange technology was applied.One drop of D 2 O was added into the NMR sample of the compound 3h and it was found that the peak at d 8.13 disappeared.Thus, the peak at d 8.13 was assigned to the hydrogen of -NH and the other peak at d 8.38 assigned to the -CH signal.
The present study investigated the effect of several different structures and from the results of cytotoxic activities of the compounds, the following preliminary structure-activity relationships can be derived: (i) The compound 3q showed the most outstanding cytotoxic activity (IC 50 = 16.06 ± 0.09 μM) and the next most promising compound was 3x, which displayed good cytotoxic activity (IC 50 = 21.38 ± 0.69 μM) against A549 cell line.Compound 3l showed moderate cytotoxic effects (IC 50 = 70.90± 0.22 μM).Based on this (Figure 7), it is possible to surmise: when introducing a group with strong electron-donating (3q, Ar = p-t-BuPh) or strong electron-withdrawing (3x, Ar = p-CF 3 Ph) into the 4-position, the cytotoxic activity (A549) of compounds could be boosted.It was also observed that the presence of π-π conjugate effect (3l, Ar = m-AcPh) played an important role in the cytotoxic effect.
(iv) To sum up, all of the synthesized compounds exhibited cytotoxic activities against HepG2 cell line with drug concentration of 25 μM, most of target compounds were shown to have cytotoxic activities against CT26 cell line, while only seven compounds demonstrated cytotoxic activities against A549 cell line.In general, it was found that all synthesized compounds showed significant selective cytotoxic effect against the HepG2.
The effects of selected compounds (IC 50 values of selected compounds > IC 50 values of Erlotinib against A549, CT26 and HepG2) on cell viability were assessed.The detailed results are provided in Figures 10, 11 and 12,  respectively.In relation to selectivity index (SI), Erlotinib showed SI > 1.92 and 2.32 for CT26 and HepG2 cells (Table 3, entry 1), respectively.Compound 3v showed moderate SI = 2.75 for CT26 cell line (Table 3, entry 4) and

Conclusions
In conclusion, we have synthesized and evaluated a library of thieno[2, 3-d]pyrimidine derivatives.Amongst these compounds (3a-x), 3q was found to have better cytotoxic activity with reference to the standard Erlotinib hydrochloride (Tarceva TM ) against A549 (IC 50 = 16.06 ± 0.09 μM) and HepG2 (IC 50 = 15.01 ± 0.31 μM) cell lines.Especially, 3w and 3x showed best cytotoxic effects against CT26 (IC 50 = 11.38 ± 0.44 μM) and HepG2 (IC 50 = 8.51 ± 0.52 μM) cell lines, respectively.Compound 3x also showed good selectivity index (SI > 11.75).Furthermore, additional preliminary structure-activity relationships could be responsible for the found potent and selective activity of new thieno[2, 3-d]pyrimidine compounds.Besides, the synthesized compounds (3a-x) would be evaluated for their enzyme activities and protein activities in following work and the more complete structure-activity relationships would be disclosed.

General remarks
All melting points were measured on an MP90 apparatus and are uncorrected.IR spectra were recorded in a VERTEX 80/Raman II (Bruker, Switzerland, KBr pellets).The 1 H NMR (400 MHz) and 13   The MS spectra were obtained on a Triple-TOF™ 5600+ spectrometer (AB SCIEX, USA).An X-ray crystallographic analysis was performed with a Bruker SMART APEX II (Bruker, Switzerland).The structure of the single crystals was refined by a Shelx-97. 44All reactions were monitored by thin layer chromatography (TLC) with ethyl acetate:hexane (1:4, v/v) as the eluent.All reagents and solvents had been purchased from commercial sources and commonly purified before their usage.

Figure 4 .
Figure 4. Structures of compounds XXI, XXII, XXIII, XXIV.(a) Hexatomic ring was linked to thiophene ring instead of five membered ring (compound XXIII); (b) substituted aromatic group was linked to secondary amine and -CH 2 group was subtracted (compound XXIV).

Figure 10 .
Figure 10.Cell viability of selected compounds toward A549 cell line.

Figure 11 .
Figure 11.Cell viability of selected compounds toward CT26 cell line.

Figure 12 .
Figure 12. Cell viability of selected compounds toward HepG2 cell line.
C NMR (100 MHz) spectra were recorded in an Avance III HD (Bruker, Switzerland) spectrometer with tetramethylsilane (TMS) as internal reference in CDCl 3 or dimethyl sulfoxide (DMSO-d 6 ) as the solvent.Only discrete or characteristic signals for the 1 H NMR are reported.
a Isolated yield; b new compounds.Scheme 2. Proposed mechanism for the Dimroth rearrangement.5

Table 2 .
Cytotoxic activities of the compounds 3a-x aAML-12: murine non-transformed hepatocyte cell line; b NA: no cytotoxic activity (at 25 μM); c NT: IC 50 value was not measured; d used as a positive control (standard Erlotinib hydrochloride).A549: human pulmonary carcinoma cell line; CT26: murine BALB/c spontaneous colon adenocarcinoma cell line; HepG2: human hepatocellular liver carcinoma cell line.

Table 3 .
Selectivity index (SI) a for selected compounds a SI, selectivity index: IC 50 on normal cells / IC 50 on cancer cells; b positive control: standard Erlotinib hydrochloride of AML-12 cell; c NC: not calculated.AML-12: murine non-transformed hepatocyte cell line; A549: human pulmonary carcinoma cell line; CT26: murine BALB/c spontaneous colon adenocarcinoma cell line; HepG2: human hepatocellular liver carcinoma cell line.