Novel Synthesis and Antitumor Evaluation of Polyfunctionally Substituted Heterocyclic Compounds Derived from 2-Cyano-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide

The reaction of 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene with ethyl cyanoacetate gave 2-cyano-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide. The latter was used to synthesize different heterocyclic derivatives comprising thiophene, thiazole, pyrazole, pyridine, pyrimidine, and coumarin rings. The mechanistic and synthetic pathways depended on regioselective attack and/or cyclization by the cyanoacetamido moiety in the key precursor on various chemical reagents. The competition of the reaction pathways including dipolar cyclization, dinucleophilic-bielectrophilic attack, β-attack, Gewald-type attack, and condensation reactions led to the diversity of the synthesized products. The antitumor activities of the synthesized products were studied and evaluated. Most of the compounds revealed high inhibitory effects when screened in vitro for their antiproliferative activity. Three human cancer cell lines, namely, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) were used in the screening tests. The simplicity of the synthetic procedures which mainly involved one-pot reactions under mild reaction conditions, the convenience of yield production and the diversity of the reactive sites in the produced systems play a valuable role for further heterocyclic transformations and further biological investigations.


Introduction
Benzothiophene systems and their substituted derivatives have attracted a great deal of interest over the years. Their aromatic character contributes to their reactivity, stability and chemical and electronic properties. A vast number of heterocyclic derivatives observed in natural products have been reported [1][2]. On the other side, they find increasing application as superconductors [3][4], optoelectronics [5][6], light emission diodes LEDs, and non-linear optical (NLO) chromophores [7,8].

Chemistry
The synthetic strategies adopted to obtain the newly synthesized compounds 3-17 depended on the regioselective attack on the cyanoacetamido moiety of the key precursor 2 by different reagents, which, in one or two steps added a highly functionalized substituent or heterocyclic ring to the molecule. The mechanistic pathways for our protocol are outlined in Schemes 1-9. CHNS microanalytical data, IR, 1 H-NMR and MS spectral data are indicated in the Experimental section.
The reaction of 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene (1) [32] with ethyl cyanoacetate gave 2-cyano-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide 2 ( Scheme 1). The IR spectrum of 2 revealed two CN stretching bands at 2,262 and 2,196 cm −1 , and a characteristic C=O stretching at 1,696 cm −1 . Moreover, the 1 H-NMR spectrum exhibited a multiplet due to four CH 2 groups at δ 1.70-2.60, a singlet at δ 4.11 for the acetamido CH 2 and a singlet at The absence of one of the two CN functions (IR) and of δ singlet CH 2 ( 1 H NMR) observed in compound 2 as well as the appearance of a NH 2 singlet at δ 3.61 ppm ( 1 H-NMR) confirmed the fused structure 3. It is noteworthy that the IR spectrum of compound 3 showed a C=O stretching band at 1,621 cm −1 and its 1 H-NMR showed a D 2 O exchangeable singlet at  6.97 ppm corresponding to the NH group, confirming that 3 exists in both keto and enol forms. Compound 3 revealed a [M + ] (m/z 245) which is also the base peak.
The reactivity of compound 2 towards various chemical reagents was investigated with the aim to producing thiophene systems with potential biological activities. Thus, the reaction of 2 with salicylaldehyde gave the coumarin derivative 4. On the other hand, the reaction of 2 with either benzaldehyde or acetophenone gave the benzylidene derivatives 5 and 6, respectively (Scheme 2).

Scheme 2.
Synthesis of the coumarin derivative 4 and benzal derivatives 5 and 6. When compound 2 was reacted with acetophenone in the presence of ammonium acetate in an oil bath at 140 °C the Knoevenagel condensation product 6 was obtained (Scheme 2). The structure of compound 6 was based on analytical and spectral data (see Experimental section). Moreover, the GC-MS spectrum of compound 6 revealed a molecular ion peak [M + − 1] at m/z 346, a base peak at m/z 150, resulting from the fragmentation of [CNHC(=O)C(CN)=C(CH 3 Ph)] + (m/z 197) from the [M + ] ion, and a fragment ion peak at m/z 178 due to the fragmentation of [C(=O)C(CN)=C(CH 3 Ph)] + (m/z 170) from the [M + + 1] ion peak.
When compound 4 reacted with either hydrazine hydrate or phenyl hydrazine, the respective pyrazole systems 7a,b were obtained as the major products (Scheme 3). The reaction involves β-attack on the C(=O)C=C moiety in 4 with subsequent 1,5-intramolecular dipolar cyclization and concomitant aromatization.  Minor products 8a,b were also obtained through the first condensation with the amide C=O followed by cyclization. Microanalysis and spectral data of 7a,b were fully consistent with the proposed structures. The mass spectrum of the pyrazole system 7b exhibited a molecular ion peak [M + ] (m/z 439) corresponding to molecular formula C 25 H 21 N 5 OS.
On the other hand, treatment of the benzylidene derivative 4 with methylene carbonitrile reagents (XCH 2 CN; X = CN, X = CO 2 Et) afforded the respective pyridone derivatives 9a,b (Scheme 4). The reaction took place via β-attack on the benzylidene moiety in 4 followed by 1,6-intramolecular dipolar cyclization with concomitant aromatization. The IR spectrum of 9a revealed the presence of three CN stretching bands at  2,253, 2,223 and 2,209 cm −1 . Moreover, the 1 H-NMR spectra of 9a and 9b showed the presence of one singlet for each at δ 3.61 and δ 3.41 ppm, respectively, due to the presence of the NH 2 group. Compound 9b showed a triplet at  1.21 for the ester CH 3 group and a quartet at  4.30 corresponding to the ester CH 2 group. Moreover, in the mass spectrum of 9a the existing [M + ] ion (m/z 397) corresponding to the formula C 22 H 15 N 5 OS and representing the base peak was observed, whereas the mass spectrum of 9b exhibited a molecular ion peak [M + ] at m/z = 444 confirming its molecular formula C 24 H 20 N 4 O 3 S.
Further confirmation of the reaction products 9a,b was achieved through an alternative synthetic route involving treatment of the starting compound 2 with benzylidene carbonitrile reagents (PhCH=C(CN)X; X = CN; X = CO 2 Et) to afford the same pyridone derivatives 9a,b (verified by IR fingerprint, m.p. and mixed m.p.) with better yields (80%, 86%) than in their formation by the reaction of compound 4 and either malononitrile (75% yield) or ethyl cyanoacetate (73% yield) (Scheme 4). By subjecting the starting material 2 to reaction with active methylene reagents (X CH 2 Y; X = Y = CN; X = CN, Y = CO 2 Et; X = Y = COCH 3 or X = COCH 3 , Y = CO 2 Et) the respective 2-pyridone derivatives 10a-d were obtained (Scheme 5).
All data for compounds 10a-d were consistent with the proposed structures. Thus, the absence of the δ-1 H CH 2 cited for the acetamido methylene protons observed with 2 at 4.11 ppm and the appearance of the pyridine C5-H protons at δ 7.89, 6.92, 6.51 and 6.88 ppm in the respective 1 H-NMR spectra of 10a-d confirmed the proposed structures. Moreover, the CH 3 proton singlets were recorded at δ 2.11 and 2.36 ppm with compound 10c and at δ 2.55 ppm with 10d, whereas the δ-1 H signals for OH were integrated at 11.89 and 12.10 ppm in the respective 1 H-NMR spectra of compounds 10b,d. At the other extreme, when compound 2 reacted with elemental sulfur and either of the methylene carbonitrile reagents (X-CH 2 -CN or X = CN; X = CO 2 Et) it gave the thiophene derivatives 11a,b, respectively. The reaction of compound 2 with phenyl isothiocyanate and elemental sulfur gave the thiazole-2-thione derivative 12. Formation of 11a,b took place through intermediate formation of A and B, while the formation of 12 occurred through intermediacy of A and C (Scheme 6). Scheme 6. Synthesis of the highly functionalized thiophenes 11a,b and thiazole 12. The 1 H-NMR spectrum of compound 11a revealed the existence of two singlets at  3.34 and 3.38 ppm corresponding to two NH 2 groups, while compound 11b showed two singlets at 3.31 and 3.35 ppm corresponding to the two NH 2 groups, a triplet at  1.12 for the ester CH 3 group and a quartet at Next, we moved to the studying of the the reaction of compound 2 with phenyl isothiocyanate in 1,4-dioxane containing triethylamine. The reaction involved a nucleophilic attack by the amidic NH function in 2 on the C=S terminal of the isocyanate reagent to produce the acyclic intermediate A. The latter then underwent 1,6-dipolar cyclization to afford the 6-thioxo-2-pyrimidone derivative 13 as the major product (Scheme 7). On the other hand, we are involved in a comprehensive programme studying the reactivity of active methylene reagents towards phenyl isothiocyanate in basic dimethylformamide followed by heterocyclization with α-halocarbonyl compounds. These reactions lead to the formation of either thiophene or thiazole systems or both, depending on reaction conditions and the nature of the α-halocarbonyl reagent [33]. Thus, subjecting the active methylene key precursor 2 to the aforementioned reaction using α-halocarbonyl reagents XCH 2 C(=O)R (X = Cl, R = OEt; X = Br, R = Ph; X = Cl, R = CH 3 ) afforded the functionalized thiophene and thiazole derivatives 14a,b and 15, respectively (Scheme 8). The reaction took place through the intermediacy of the potassium sulphide salt A. The disappearance of δ-1 H CH 2 singlet observed in the precursor 2, and the appearance of D 2 O exchangeable NH 2 singlets at δ 4.37 and δ 4.80 ppm for compounds 14a and 14b, respectively, as well as the appearance of a δ-1 H singlet at 6.67 ppm assigned to a thiazole C 5  On the other hand, compound 2 reacted with benzenediazonium chloride to give the phenylhydrazo derivative 16. The latter compound reacted with either malononitrile or ethyl cyanoacetate to give the 3-phenylazo-pyridone derivatives 17a and 17b, respectively (Scheme 9). The structural assignments of 16, 17a,b were based on analytical and spectral data. Thus, the 1 H-NMR spectrum of compound 16 revealed two singlets at δ 9.14 and 10.88 ppm (D 2 O exchangeable) due to the two NH groups.

In vitro evaluation of antiproliferative activity of the synthesized compounds
The tumor cell growth inhibition activities of the newly synthesized thiophene systems (22 compounds in total) were assessed in vitro [34] on three human tumor cell lines, namely, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) after a continuous exposure of 48 h. The results were compared to the antiproliferative effects of the reference control doxorubicin [35]. All the compounds were dissolved in DMSO at 1 mg/mL immediately before use and diluted just before addition to the cell culture.
The data (Table 1) represents means ± SEM of three independent experiments performed in duplicate. The results indicated that most compounds demonstrated substantial growth inhibitory effects against the human tumor cells at the concentrations tested. The antiproliferative activity of the test compounds against each of the title tumor cell lines may be arranged in a descending order due to measured concentration required to inhibit tumor cell proliferation by 50% (GI 50 µ/M).  In general, compounds 17a, 7a, 14b, 4, and 10a showed significant activity on the three tumor cell lines tested. The inhibitory effect of the other systems on tumor cell growth varied, according to the tested tumor cell, from high to medium or marginal effects. Some compounds had no impact on a specific tumor cell proliferation, while exhibited some specificity to the other. Thus compound 11a revealed GI 50 ~ 66.6 µ/M towards MCF-7 tumor cell versus GI 50 ~ 12.0 µ/M for NCI-H460. Similarly, compound 12 had no effect on NCI-H460 tumor cell proliferation (GI 50 ~ 146 µ/M) while it showed high selectivity towards breast derived cells MCF-7 (GI 50 ~ 10.9 µ/M).
It is of interest that the pyrazole derivative 7a, comprising one phenyl substituent, showed significant growth inhibition activity on the three tumor cell lines, compared to its counterpart 7b with two phenyl functions. Also, comparing the 5-cyano pyridone derivative 17a and its 5-ethoxy-carbonyl counterpart 17b, it is obvious that the former has the highest inhibitory activity towards adenocarcinoma (MCF-7), while 17b showed the lowest effect on the same tumor cell line.

General
All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on a FTIR plus 460 or Pye Unicam SP-1000 spectrophotometer. 1 H-NMR spectra were recorded with Varian Gemini -200 (200 MHz) and Jeol AS 500 MHz instruments in DMSO-d6 as solvent using TMS as internal standard and chemical shifts are expressed as δ ppm. The mass spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. Analytical data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental CHNS analyzer.