Uses of Cyclohexan-1,4-dione for the Synthesis of 2-Amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one Derivatives with Anti-proliferative and Pim-1 Kinase Activities

Abstract

The reaction of cyclohexan-1,4-dione with elemental sulfur and any of the 2-cyano-N-arylacetamide derivatives 2a–c gave the 2-amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one derivatives 3a–c to be used in some heterocyclization reactions. The multicomponent reactions of any of compounds 3a–c with aromatic aldehydes 6a–c and either of malononitrile or ethylcyanoacetate gave the 5,9-dihydro-4H-thieno[2,3-f]chromene derivatives 9a–r, respectively. The anti-proliferative evaluation of the newly synthesized compounds against the six cancer cell lines A549, HT-29, MKN-45, U87MG, SMMC-7721 and H460 showed that the nine compounds 3c, 5c, 9e, 9h, 9i, 9j, 9l, 9q, 11e and 13e with highest cytotoxcity. Toxicity of these compounds against shrimp larvae revealed that compounds 3c, 9j, 9q, and 13e showed no toxicity against the tested organisms. The c-Met kinase inhibition of the most potent compounds showed that compounds 9j, 9q, 10e, 12e and 13e have the highest activities. Compounds 9j, 9l, 9q and 11e showed high activity towards tyrosine kinases. Moreover, compounds 9j, 9q and 13e showed the highest inhibitor activity towards Pim-1 kinase.

Cancer is a group of complex and heterogeneous diseases that within the organism display deviation of cell control mechanisms which guide proliferation and differentiation.^1–3) The condition is affecting more and more people and in recent years presents accentuated mortality rates all over the world,⁴⁾ becoming a greater public health issue and causing societal concern. Among the available types of cancer treatment, chemotherapy is an important clinical choice, especially for being a systematic approach. However, chemotherapeutical drugs are nonspecific and toxic to healthy tissues which explains for the most part their collateral effects; that may include bone marrow suppression, lesions in the gastrointestinal tract, hair loss and nausea.^5,6) In many cases permanent damage to the kidneys, heart, reproductive system and lungs occurs.⁷⁾ New anticancer agents which combine effectiveness, safety and convenience remain a challenge for the international science community. Currently, anticancer chemotherapy development is conducted through cytotoxic compounds identification, in other words, identifying those compounds capable of destroying cancer cells. Such agents improve the survival rates and QOL of patients with tumors.^8,9) The traditional cell line test screening model is a widely used tool because of its easy manipulation, molecular characterization, and its high degree of specificity and reproducibility. Such trials are an exceptional means to study cellular pathways and the genes critically involved in cancer.¹⁰⁾ In organic chemistry, due to their many applications, derivatives of thiophene stand out among biomolecules used in trials to determine biological activity. They are present in natural products and are frequently incorporated into agrochemicals, dyes, and drugs in general.^11–13) Various thiophene derivatives are produced by molecular modifications through varied synthesis routes that result in increases in specificity and thus safety profiles.^14,15) Within the thiophenic family, the 2-amino-thiophenes have been well reported. In recent drugs investigations, they acquired a particular importance due to the innovations in their synthesis (Gewald reaction), availability, stability, and structural simplicity that allow them to be important scaffolds in chemical and therapeutic products; like the top selling drugs olanzepine and tinoridine.^16–18) In addition, they present a large spectrum of biological properties that include antimicrobial,^19,20) anti-inflammatory,²¹⁾ anxiolytic,²²⁾ antileishmanial,²³⁾ anti-diabetes,²⁴⁾ antifungal,²⁵⁾ antioxidant²⁶⁾ and antiplatelet activities.²⁷⁾ The antitumor evaluation of the 2-aminothiophenes has already been addressed through different reports.^28–30) Due to the biological importance of aminothiophenes especially the antitumor activity, our efforts were focused in this context as we synthesized a series of 2-amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one derivatives capable for further heterocyclization and multicomponent reactions. The newly synthesized products were screened for their anti-proliferative activities followed by measuring the toxicity, inhibition toward tyrosine kinase of the most potent compounds and Pim-1 kinase inhibition was deterimened for some selected compounds.

Results and Discussion

In the present work the reaction of cyclohexan-1,4-dione (1) with elemental sulfur and any of the 2-cyano-N-arylacetamide derivatives 2a–c gave the 2-amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one derivatives 3a–c. The structures of compounds 3a–c were confirmed on the basis of their respective analyses (see Experimental). The reaction of any of compounds 3a–c with elemental sulfur and phenylisothiocyanate (4) afforded the 7-amino-3-phenyl-4,5-dihydrothieno[2′,3′:3,4]benzo[1,2-d]thiazole-2(3H)-thione derivatives 5a–c, respectively (Chart 1). The structures of compounds 5a–c were established on the basis of their ¹H-NMR and ¹³C-NMR spectra. Thus, the ¹H-NMR spectrum of compound 5a (as an example) showed the presence of two triplets at δ 1.64–1.90 ppm due to the presence of the two CH₂ groups, a singlet at δ 4.86 ppm equivalent to the NH₂ group, a multiplet at δ 7.28–7.39 ppm indicating the two phenyl groups, thiophene and thiazole carbons and a singlet at δ 8.22 ppm due to the presence of the NH group. In addition, the ¹³C-NMR spectrum revealed, beside the expected signals, the presence of δ 164.8 eq to the CO group and δ 178.5 indicating the C=S goup.

Chart 1

It is well known that multi-component reactions (MCRs) can combine three or more components together in a single reaction vessel and produce final products with a minimum of synthetic time and effort³¹⁾ because there is no need to separate any reaction intermediate.³²⁾ Such MCRs often result in high atome conomy and high selectivity products.³³⁾ They are also applicable to the synthesis of heterocyclic systems.³⁴⁾ It is quite remarkable that many top-selling pharmaceuticals contains “4-H pyran” derivatives^35–39) this encouraged us to synthesis 4-H pyran derivative through the multi-component reactions of compounds 3a–c. Thus, the reaction of any of compounds 3a–c with any of benzaldehyde (6a), 4-chlorobenzaldehyde (6b) or 4-nitrobenzaldehyde (6c) and either of malononitrile (7a) or ethyl cyanoacetate (7b) gave the 5,9-dihydro-4H-thieno[2,3-f]chromene derivatives 9a–r, respectively (Chart 2). The reaction took place through the intermediate formation of 8a–r followed by the Micheal addition, in case of the reaction with malononitrile, and elimination of ethanol, in the case of the reaction with ethyl cyanoacetate. The analytical and spectral data of the latter compounds were consistent with their respective structures (see Experimental).

Chart 2

In addition, the reaction of any of compounds 3a–c with any of the acetophenone derivatives 10a–c in the presence of ammonium acetate in an oil bath at 120°C gave the Knoevenagel condensation products 11a–i their structure were based on their respective analytical and spectral data. Thus, the ¹H-NMR spectrum of 11a showed the presence of two triplets at δ 1.36–1.66 ppm equivalent to the two CH₂ groups, a singlet at δ 2.88 ppm for the CH₃ group, a singlet at δ 4.86 ppm indicating the NH₂ group, a multiplet at δ 7.28–7.39 ppm for the two phenyl groups and a singlet at δ 8.29 ppm due to the presence of the NH group. Moreover, the ¹³C-NMR spectrum showed beside the expected signals, δ 18.3 for the CH₃ group, two signals at δ 92.6, 98.4 eq to the C=C group and two signals at δ 163.8, 164.2 eq to the two CO groups. Finally, the reactions of any of compounds 11a, 11e or 11i and any of the diazonium salts 12a, 12b or 12b gave the corresponding arylhydrazone derivatives 13a–i, respectively (Chart 3).

Chart 3

Biology

Cell Proliferation Assay

The anti-proliferative activities of the newly synthesized compounds (Table 1) were evaluated against the six cancer cell lines A549, HT-29, MKN-45, U87MG, SMMC-7721 and H460 using the standard 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay in which the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide was reduced by metabolically active cells in vitro, with foretinib as the positive control.^40,41)

Table 1. In Vitro Growth Inhibitory Effects IC₅₀±S.E.M. (µM) of the Newly Synthesized Compounds against Cancer Cell Lines

Compound No.	IC50±S.E.M. (µM)
	A549	H460	HT29	MKN-45	U87MG	SMMC-7721
3a	4.39±1.75	6.59±2.36	3.69±1.49	6.48±2.32	5.38±1.48	8.46±2.58
3b	1.48±0.80	1.83±0.69	2.70±0.39	1.63±0.80	1.26±0.69	0.98±0.07
3c	0.23±0.19	0.86±0.36	1.36±0.87	0.85±0.03	0.26±0.05	1.46±0.48
5a	9.36±2.29	8.26±3.58	7.36±1.44	5.27±1.39	6.39±2.30	8.31±1.68
5b	1.37±0.83	0.79±0.39	0.95±0.31	0.93±0.29	0.83±0.39	1.05±0.29
5c	0.79±0.15	0.83±0.17	1.42±0.70	2.49±0.39	0.19±0.06	0.93±0.21
9a	9.40±2.32	8.92±2.45	8.74±2.52	9.31±1.40	6.09±2.38	10.58±2.83
9b	7.24±1.39	8.27±2.43	8.66±2.59	6.49±1.49	8.27±2.52	6.79±1.26
9c	4.29±1.33	6.39±1.49	8.57±2.28	8.61±2.53	9.38±2.61	8.24±2.14
9d	1.36±0.69	2.51±0.79	1.38±0.92	0.85±0.13	1.42±0.59	0.80±0.02
9e	0.33±0.05	0.59±0.04	1.22±0.60	0.93±0.36	0.88±0.09	0.61±0.08
9f	1.48±0.81	2.53±0.55	0.79±0.41	0.93±0.92	1.28±0.77	0.85±0.67
9g	6.36±1.49	8.07±2.38	7.47±1.28	6.29±1.73	8.39±2.38	6.51±1.80
9h	0.36±0.09	0.42±0.09	0.29±0.07	0.88±0.29	1.83±0.29	0.72±0.09
9i	0.29±0.04	0.83±0.05	1.28±0.36	0.24±0.08	0.73±0.25	1.39±0.32
9j	0.80±0.12	1.26±0.83	0.48±0.21	0.77±0.19	0.39±0.04	1.28±0.77
9k	2.39±0.85	1.08±0.69	1.68±0.83	0.69±0.03	2.48±0.93	1.59±0.49
9l	0.28±0.04	0.71±0.49	0.48±0.36	1.32±0.79	0.36±0.06	0.52±0.13
9m	9.32±0.14	8.30±1.69	9.08±1.58	7.39±2.42	9.24±1.69	6.22±0.79
9n	4.23±1.25	5.09±1.27	6.58±1.59	8.32±2.42	8.22±1.49	6.58±2.84
9o	9.35±2.07	8.63±3.22	7.90±2.27	6.38±1.42	8.42±2.66	5.48±1.39
9p	1.04±0.53	0.69±0.32	0.41±0.29	0.29±0.08	1.45±0.38	0.29±0.09
9q	0.73±0.26	0.39±0.05	0.61±0.17	0.82±0.31	0.36±0.16	0.53±0.29
9r	4.03±1.59	4.29±1.37	2.60±0.82	1.03±0.82	1.28±0.39	1.93±0.82
11a	8.43±2.59	9.39±2.47	10.39±2.39	8.53±4.53	9.47±2.52	10.37±2.55
11b	2.32±0.64	2.53±0.86	4.90±1.17	4.66±1.28	7.37±2.52	6.53±1.49
11c	9.68±3.43	8.49±2.53	6.40±3.39	7.44±1.58	9.46±3.65	8.57±3.57
11d	4.63±1.55	3.19±1.28	4.18±1.72	2.17±0.93	4.80±1.28	3.52±1.28
11e	0.12±0.03	0.61±0.04	0.58±0.27	1.37±0.26	0.53±0.13	1.62±0.77
11f	2.47±0.83	3.52±1.14	2.57±0.83	3.87±1.53	1.73±0.92	2.83±1.03
11g	8.78±3.62	9.39±2.51	6.31±3.69	8.51±2.74	10.26±2.39	12.39±3.62
11h	2.47±0.89	2.72±0.93	3.59±1.03	2.95±0.93	3.07±0.79	2.59±0.83
11i	9.42±2.59	8.28±3.19	10.53±3.63	8.26±2.80	10.21±2.59	8.42±2.88
13a	7.29±2.44	8.80±2.89	9.63±3.82	6.70±2.69	5.52±1.79	9.29±3.79
13b	1.08±0.93	1.37±0.69	2.69±1.39	2.45±0.85	3.68±1.62	1.76±0.80
13c	6.35±2.72	7.90±1.79	8.38±2.57	9.03±3.82	6.80±2.27	11.40±2.41
13d	6.33±2.69	5.80±1.80	6.80±1.79	4.85±1.94	5.73±2.55	4.69±1.74
13e	0.49±0.26	0.29±0.08	0.42±0.07	0.53±0.06	0.92±0.52	0.88±0.30
13f	1.28±0.94	2.72±0.96	4.69±1.70	5.09±1.69	3.48±1.73	2.90±0.88
13g	6.77±1.80	5.79±1.63	6.09±1.59	7.94±2.69	8.69±2.46	8.94±2.58
13h	5.28±2.80	6.48±1.80	5.24±2.94	4.21±1.41	5.18±2.94	5.93±1.69
13i	8.26±2.74	8.73±2.80	6.29±2.80	5.84±1.64	7.08±2.42	6.31±1.72
Foretinib	0.08±0.01	0.18±0.03	0.15±0.023	0.03±0.0055	0.90±0.13	0.44±0.062

Structure Activity Relationship

The mean values of three independent experiments, expressed as IC₅₀ values, were presented in Table 1. Most of the synthesized compounds exhibited potent anti-proliferative activity with IC₅₀ values less than 12 µM. Generally, the variations of substituents within the thienopyridine moiety together with the heterocycle ring being attached have a notable influence on the anti-proliferative activity.⁴²⁾ The nine compounds 3c, 5c, 9e, 9h, 9i, 9j, 9l, 9q, 11e and 13e showed high potencies against the six cancer cell liones.

It is clear from Table 1 that most of the newly synthesized compounds showed from moderate to high cytotoxicity against the six cancer cell lines. Considering compounds 3a–c where compound 3c with X=OCH₃ showed the highest cytotoxicity among the three compounds, it showed activity towards U87MG cell lines with IC₅₀ 0.26 µM and such activity is higher than that of the reference foretinib. On the other hand, compound 3b (X=Cl) showed moderate activity, although it showed high cytotoxicity against SMMC-7721 with the IC₅₀ 0.98 µM. The reaction of any of 3a–c with elemental sulfur and phenylisothiocyanate gave the 7-amino-3-phenyl-4,5-dihydrothieno[2′,3′:3,4]-benzo[1,2-d]thiazole-2(3H)-thione derivatives 5a–c. Through the latter compounds 5b (X=Cl) and 5c (X=OCH₃) showed the highest cytotoxicity. It is of great value to note that compound 5c showed superior inhibitory activity to the reference drug “foretinib” against U87MG cell line with IC₅₀ 0.19 µM. Among the 2-amino-9-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-8-carbonitrile derivatives 9a–r compounds 9a (X=Y=H, R=NH₂), 9m (X=OCH₃, Y=H, R=NH₂) and 9o (X=OCH₃, Y=Cl, R=NH₂) showed poor cytotoxicity. Amazingly, compounds 9e (X=H, Y=NO₂, R=NH₂), 9h (X=Cl, Y=H, R=OH), 9i (X=Y=Cl, R=NH₂), 9j (X=Y=Cl, R=OH), 9l (X=Cl, Y=NO₂, R=OH) and 9q (X=OCH₃, Y=NO₂, R=NH₂) showed the highest cytotoxicity among such series of compounds. Compound 9l showed higher cytotoxicity than foretinib against U87MG cell line with the IC₅₀ 0.36 µM. Moreover, compound 9p showed higher activity than foretinib against SMMC-7721 cell line with the IC₅₀ 0.29 µM. Through the series of compounds 9a–r we can conclude that the presence of the electronegative Cl and OH groups and/or the electron rich NH₂ and OCH₃ groups are responsible for the high cytotoxicity. Considering the condensate compounds 11a–i, where compound 11e (X=Y=Cl) with the two electronegative Cl groups showed the highest cytotoxicity among such series of compounds. For the phenyl hydrazone derivatives 13a–i, compounds 13b, 13e and 13f were the most active compounds. Compound 13b (X=Y=R=H) showed better selectivity against A549, H460 and SMMC-7721 cell lines but still displayed moderate activity against HT29 and MKN-45 cell lines but it retained potency against U87MG cell line. Compound 13e (X=Y=R=Cl) showed the highest cytotoxicity towards the six cancer cell lines among the arylhydrazone derivatives 13a–i. Compound 13f (X=Cl, Y=Cl, R=NO₂) slightly high potency against A549 cell line with IC₅₀ 1.28 µM but is showed moderate potency against MKN-45 with IC₅₀ 5.09 µM.

Toxicity

Bioactive compounds are often toxic to shrimp larvae. Thus, in order to monitor these chemicals’ in vivo lethality to shrimp larvae (Artemia salina), Brine-Shrimp Lethality Assay⁴³⁾ was used. Results were analyzed with LC₅₀ program to determine LC₅₀ values and 95% confidence intervals.^44,45) Results are given in Table 2 for the compounds which exhibited optimal cytotoxic effect against cancer cell lines which are the nine compounds 3c, 5c, 9e, 9i, 9j, 9l, 9q, 11e and 13e.

Table 2. Toxicity of the Most Potent Compounds against the Cancer Cell Lines

Compound No.	Cons. (µg/mL)	Mortalitya)	Toxicity	LC50	Upper 95% lim.	Lower 95% lim.
3c	10	0	Non toxic	880.27	—	—
	100	2
	1000	4
5c	10	0	Harmful	148.21	80.48	16.7
	100	3
	1000	8
9e	10	2	Very toxic	38.7	—	—
	100	6
	1000	10
9i	10	2	Very toxic	97.2	—	—
	100	6
	1000	10
9j	10	0	Non toxic	968.29	—	—
	100	0
	1000	4
9l	10	0	Harmful	112.35	72.38	22.79
	100	5
	1000	10
9q	10	0	Non-toxic	845.24	—	—
	100	0
	1000	4
11e	10	5	Very toxic	135.39	—	—
	100	8
	1000	10
13e	10	0	Non toxic	868.28	—	—
	100	2
	1000	4
DMSO	—	0	—	—	—	—

a) Ten organisms (A. salina) tested for each concentration.

The shrimp lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials.⁴⁶⁾ It has also been shown that, A. salina toxicity test results have a correlation with rodent and human acute oral toxicity data. Generally, a good correlation was obtained between A. salina toxicity test and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man, including A. salina toxicity test, was slightly better than the rat test for test compounds.

In order to prevent the toxicity results from possible false effects originated from solubility of compounds and dimethyl sulphoxide (DMSO)’s possible toxicity effect, compounds were prepared by dissolving in DMSO in the suggested DMSO volume ranges. The degree of toxicity whether the compound is nontoxic, harmful or toxic depends on the mortality of ten organisms. Where, if the mortality rate from 4–6 organisms the compound will be considered a non toxic. It is clear from Table 2 that compounds 3c, 9j, 9q, and 13e exhibit non toxicity against the tested organisms. On the other hand, compounds 9e, 9i and 11e are very toxic, in addition, compounds 5c and 9l are harmful.

Homogeneous Time-Resolved Fluorescence (HTRF) Kinase Assay

The c-Met kinase activity (Table 3) of the most potent compounds 3c, 5c, 9e, 9i, 9j, 9l, 9q, 11e and 13e were measured using HTRF assay as previously reported.^47,48) In addition, these compounds were further evaluated against other five tyrosine kinase (c-Kit, Flt-3, vascular endothelial growth factor receptor (VEGFR)-2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR)) using the same method (Table 4). It is clear from Table 3 that compounds 9j, 9q, 11e and 13e were the most potent compounds toward c-Met kinase activity. Compounds 9j (X=Y=Cl, R=OH), 9q (X=OCH₃, Y=NO₂, R=NH₂) and 13e (X=Y=R=Cl) showed more potencies than foretinib with IC₅₀’s 1.13, 0.89 and 0.39 nM, respectively. It is obvious that the presence of the three electronegative Cl groups responsible for the extremely high potency of compound 13e. Although compounds 3c and 5c showed high anti-proliferative activities towards the six cancer cell lines relative to foretinib but they showed low potency towards c-Met kinase. Compounds 9i, 9j and 9l showed high anti-proliferative activities towards the six cancer cell lines at the same time they showed moderate potency towards c-Met kinase. Compounds 9q and 13e showed high potencies against the cancer cell lines and c-Met kinase.

Table 3. c-Met Enzymatic Activity of the Most Potent Selected Compounds

Compound No.	X	Y	R	IC50 (nM) c-Met
3c	OCH3	—	—	6.83±2.59
5c	H	—	—	8.62±2.76
9e	H	NO2	NH2	2.93±1.18
9i	Cl	Cl	NH2	2.31±0.95
9j	Cl	Cl	OH	1.13±0.46
9l	Cl	NO2	OH	2.62±1.14
9q	OCH3	NO2	NH2	0.89±0.89
11e	Cl	Cl	—	1.38±0.82
13e	Cl	Cl	Cl	0.39±0.08
Foretinib	—	—	—	1.16±0.17

Table 4. Inhibitory Effect of Compounds 3c, 5c, 9e, 9i, 9j, 9l, 9q, 11e and 13e towards Tyrosine Kinases [Enzyme IC₅₀ (nM)]

Compound No.	c-Kit	Flt-3	VEGFR-2	EGFR	PDGFR
3c	8.42±2.11	8.63±3.64	9.80±1.80	6.49±2.63	10.25±3.92
5c	10.58±2.66	9.63±1.05	8.47±3.69	5.63±1.48	8.63±2.59
9e	4.39±1.38	6.88±2.66	5.69±3.95	7.83±1.04	8.55±3.52
9i	2.49±0.88	3.72±1.02	2.75±0.95	3.69±0.51	4.58±1.52
9j	0.39±0.06	0.84±0.10	0.69±0.23	0.94±0.36	1.07±0.62
9l	1.06±0.51	1.17±0.69	0.94±0.27	1.80±0.38	0.92±0.29
9q	1.27±0.93	1.05±0.77	0.88±0.39	1.16±0.42	0.73±0.61
11e	0.38±0.17	0.69±0.37	0.92±0.41	0.81±0.29	0.72±0.55
13e	0.36±0.19	0.25±0.06	0.61±0.19	0.59±0.33	0.19±0.05
Foretinib	0.19±0.05	0.17±0.07	0.20±0.01	0.13±0.04	0.26±0.115

Inhibitory Effect of the Most Potent Compounds towards Tyrosine Kinases

The inhibitory effect of compounds compounds 3c, 5c, 9e, 9i, 9j, 9l, 9q, 11e and 13e towards c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR tyrosine kinases was demonstrated through Table 4. It is clear that compounds 3c, 5c and 9e exhibited the lowest inhibitory effect towards the tested kinases although compound 9e showed relatively high potency against c-Met kinase. The inhibitory effect of compound 9i showed close IC₅₀’s towards c-Met kinase and c-Kit and VEGFR-2 kinases. Compound 9j exhibited higher inhibition towards the five tyrosine kinases that its inhibition towards c-Met kinase. On the other hand, compound 9q exhibited higher inhibition towards c-Met than the five tyrosine kinases. Compounds 11e and 13e showed the maximum inhibition towards the five tyrosine kinases. It is obvious from Table 4 that compound 9j showed selectively high potency towards c-Kit kinase and compound 9l showed specific activity towards VEGFR-2 and PDGFR kinases. On the other hand compound 9q exhibited high potency toward VEGFR-2 and PDGFR kinases with IC₅₀’s 0.88 and 0.73 nM. Specifically compound 12e showed the maximum inhibition among the tested compounds towards Flt-3 and PDGFR kinases with IC₅₀’s 0.25 and 0.19 nM, respectively. Foretinib was used as the positive control.

Pim-1 Kinase Inhibition

Being a member of the Pim kinase family, Pim-1 kinase has the privilege of possessing a unique structure of its active site thus paving the way for designing small molecules that confer high selectivity as Pim-1 kinase inhibitors.⁴⁹⁾ As evidenced, high Pim-1 kinase expression levels are intimately concomitant with malignancy survival and proliferation.⁵⁰⁾

The potency of the selected compounds 9j, 9l, 9q, 11e and 13e to inhibit Pim-1 kinase was evaluated at 10 and 1 µM concentrations in duplicate assays as previously described.⁵¹⁾ The percentages of residual kinase activities are reported in Table 5. IC₅₀ values were only determined when the remaining kinase activity was less than 45% when the compounds were tested at 1 µM. Our slection for 9j, 9l, 9q, 11e and 13e was based on their relative activity towards c-Met kinase, together with their inhibition towards tyrosine kinases, the more potent to inhibit Pim-1 activity 9j, 9q and 13e with IC₅₀ value of 0.41, 0.39 and 0.28 µM, while 9l and 11e were less effective (IC₅₀>10 µM). SGI-1776 was used as positive control with IC₅₀ 0.048 µM in the assay. These profiles in combination with cell growth inhibition data of compounds 9j, 9q and 13e was listed in Table 5 indicated that Pim-1 was a potential target of these compounds. Compounds 9j, 9q and 13e showed high potencies against the cancer cell lines and the Pim-1 kinase. On the other hand although compounds 9l and 11e showed high anti-proliferative against the cancer cell lines they showed reduced inhibition towards the Pim-1 kinase. It is of great value to note that among the tested compounds, 13e showed the maximum inhibition against the tyrosine kinases and Pim-1 kinase.

Table 5. The Inhibitory Effect of Compounds 9j, 9l, 9q, 11e and 13e against Pim-1 Kinase

Compound No.	Inhibition ratio at 10 µM	IC50 (µM)
9j	85	0.41
9l	36	>10
9q	82	0.39
11e	28	>10
13e	94	0.28
SGI-1776	—	0.048

Conclusion

Throughout this work the 2-amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one derivatives 3a–c were synthesized and used for many heterocyclization reactions. The anti-proliferative evaluation of the newly synthesized compounds against the six cancer cell lines A549, HT-29, MKN-45, U87MG, SMMC-7721 and H460 showed that the nine compounds 3c, 5c, 9e, 9h, 9i, 9j, 9l, 9q, 11e and 13e with highest cytotoxcity. Toxicity of these compounds against shrimp larvae revealed that compounds 3c, 9j, 9q, and 13e showed non toxicity against the tested organisms. The c-Met kinase activity of the most potent compounds showed that 9j, 9q, 11e and 13e with the highest activties. Compounds 9j, 9l, 9q, 11e and 13e showed high activity towards tyrosine kinases. Moreover, compounds 9j, 9q and 13e showed the highest potency towards Pim-1 kinase.

Experimental

General

¹³C-NMR and ¹H-NMR spectra were recorded on Bruker DPX200 instrument in DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in δ (ppm). Mass spectra were recorded on EI-MS (Shimadzu, Kyoto, Japan) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were carried out by the Microanalytical Data Unit Ludwig-Maximilians-Universitat-Munchen, Germany. All reagents were purchased from Sigma Company, U.S.A. Column chromatography (CC) was performed over silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., China). The progress of all reactions was monitored by TLC on 2×5 cm precoated silica gel 60 F254 plates of thickness of 0.25 mm (Qingdao Marine Chemical Group, Co.). Spots were visualized at 254 and 366 nm under UV and iodine. All commercially available solvents and reagents were used without further purification. The purity of all compounds was determined through used for peak purity determination in high performance HPLC. Where in this work the purity of compounds was improved through changing the eluents being used and the solvent introduced through the data was that which minimize the peak distortion and consequently indicating the best purity of the described compounds.^52,53)

Cell Proliferation Assay

The cancer cell lines were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Approximate 4×10³ cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO₂ at 37°C for 24 h. The compounds tested at the indicated final concentrations at 10 µM in DMSO were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 µg/mL, and incubated with cells at 37°C for 4 h. The formazan crystals were dissolved in 100 µL of DMSO each well, and the absorbence at 492 nm (for absorbance of MTT formazan) and 630 nM (for the reference wavelength) was measured with an enzyme-linked immunosorbent assay (ELISA) reader. All of the compounds were tested three times in each cell line. The results expressed as IC₅₀ (inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

Brine Shrimp Cytotoxicity Assay

In vitro cytotoxicity assay was performed according to the standardized protocol of Ali et al.⁵⁴⁾ Briefly, a stock solution of the test sample was prepared by dissolving 10 mg of the tested compound in 1 mL of DMSO. The eggs of the brine shrimp were hatched in a tray containing artificial sea salt water (4% NaCl) at 25°C. A total of 10 brine shrimp was collected from the fool and transferred to each vial containing sea salt water and test samples at different concentrations. The vials were incubated at 25°C for 24 h. After the specified time, percent mortality was recorded and the results were subjected to GraphPad Prism (Graphpad Prism software Version 5.0, GraphPad software Inc., CA, U.S.A.) for LC₅₀ calculation.

c-Met and Tyrosine Kinases Assay

Briefly, 20 µg/mL poly (glutamic acid (Glu), tyrosine (Tyr)) 4 : 1 (Sigma) was used as a substrate in 384-well plates. Then 50 µL of 10 mMATP (Invitrogen) solution diluted in kinase reaction buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), Ph 7.0, 1 M dithiothreitol (DTT), 1 M MgCl₂, 1 M MnCl₂, and 0.1% NaN₃) was added to each well. Various concentrations of the tested compounds diluted in 10 µL of 1% DMSO (v/v) were used as the negative control. The kinase reaction was started by the addition of purified tyrosine kinase proteins diluted in 39 µL of kinase reaction buffer solution. The incubation time for the reactions was 30 min at 25°C and ceased by the addition of 5 µL of Streptavidin-XL665 and 5 µL Tk Antibody Cryptate working solution to all of wells. The plates were read using Envision (PerkinElmer, Inc.) at 320 and 615 nM. The inhibition rate (%) was calculated using the mathematical equation: % inhibition=100−[(Activity of enzyme with tested compounds−Min)/(Max−Min)]×100 (Max: the observed enzyme activity measured in the presence of enzyme, substrates, and cofactors; Min: the observed enzyme activity in the presence of substrates, cofactors and in the absence of enzyme).

Pim-1 Kinase Inhibition Assay

A 20 mg/mL stock solution of the Pim-1 kinase inhibitor KHCARB13(PubChem CID: 54613583); each of the tested compound was prepared in DMSO and then further diluted in phosphate-buffered saline (PBS) or medium; 1×103 HCT-116 or 3×103 LS174T cells/well were seeded in a 96-well plate and incubated under standard conditions. For treatment, the medium was aspirated and replaced by 100 µL of Iscove’s modified Dulbecco’s medium (IMDM)/10% fetal calf serum (FCS) medium containing KH-CARB13 at the indicated concentrations, and cells were incubated at 37°C for 72 h.

General Procedure for the Synthesis of the 2-Amino-4,5-dihydrobenzo[b]thiophen-6(7H)-one Derivatives 3a–c

To a solution of cyclohexan-1,4-dione in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) each of elemental sulfur and any of 2-cyano-N-phenylacetamide (1.60 g, 0.01 mol), N-(4-chlorophenyl)-2-cyanoacetamide (1.94 g, 0.01 mol) or 2-cyano-N-(4-methoxyphenyl)acetamide (1.90 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 1 h then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product, in each case, was collected by filtrations.

2-Amino-6-oxo-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (3a)

HPLC purity=89% (C-18 NovaPak column; MeOH–H₂O, 80 : 20), RT=20 min; orange crystals (ethanol), yield 70% (2.00 g), mp 183–185°C; (IR (KBr) ν_max 3493, 3329, 3055, 2984, 1689, 1675, 1639, 1583; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.65–1.92 (2t, 4H, 2CH₂), 2.79 (s, 2H, CH₂), 4.85 (s, 2H, D₂O exchangeable, NH₂), 7.26–7.38 (m, 5H, C₆H₅), 8.28 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.6, 38.8, 40.0 (3CH₂), 119.5, 120.5, 124.8, 127.2, 133.5, 138.4, 140.2, 141.8 (C₆H₅, thiophene C), 164.8, 166.2 (2CO); EI-MS: m/z 286 [M]⁺ (26%); Anal. Calcd for C₁₅H₁₄N₂O₂S (286.35): C, 62.92; H, 4.93; N, 9.78; S, 11.17%. Found: C, 63.19; H, 4.69; N, 9.84; S, 11.36%.

2-Amino-N-(4-chlorophenyl)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (3b)

HPLC purity=96% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=22 min; Orange crystals (ethanol), yield 76% (2.43 g), mp 210–213°C; (IR (KBr) ν_max 3479, 3351, 3055, 2986, 1688, 1677, 1639, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.63–1.90 (2t, 4H, 2CH₂), 2.81 (s, 2H, CH₂), 4.87 (s, 2H, D₂O exchangeable, NH₂), 7.24–7.48 (m, 4H, C₆H₄), 8.26 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.5, 38.8, 40.2 (3CH₂), 119.8, 121.2, 123.8, 129.2, 133.6, 138.2, 140.7, 141.9 (C₆H₅, thiophene C), 164.4, 166.0 (2CO); EI-MS: m/z 320 [M]⁺ (28%), 322 [M⁺+2], (12 %); Anal. Calcd for C₁₅H₁₃ClN₂O₂S (320.79): C, 56.16; H, 4.08; N, 8.73; S, 10.00%. Found: C, 56.07; H, 4.17; N, 8.93; S, 10.25%.

2-Amino-N-(4-methoxyphenyl)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (3c)

HPLC purity=95% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 80% (2.52 g), mp 251–254°C; (IR (KBr) ν_max 3479–3351, 3055, 2986, 1688, 1680, 1639, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.66–1.87 (2t, 4H, 2CH₂), 2.82 (s, 2H, CH₂), 3.67 (s, 3H, OCH₃), 4.89 (s, 2H, D₂O exchangeable, NH₂), 7.22–7.45 (m, 4H, C₆H₄), 8.24 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.7, 38.6, 40.1 (3CH₂), 52.8 (OCH₃), 120.8, 122.6, 123.9, 124.8, 130.4, 136.9, 139.7, 142.3 (C₆H₅, thiophene C), 164.6, 166.2 (2CO); EI-MS: m/z 320 [M]⁺ (28%); Anal. Calcd for C₁₆H₁₆N₂O₃S (316.37): C, 60.74; H, 5.10; N, 8.85; S, 10.14%. Found: C, 60.68; H, 4.87; N, 8.79; S, 10.29%.

General Procedure for the Synthesis of the 7-Amino-4,5-dihydrothieno[2′,3′:3,4]benzo[1,2-d]thiazole-2(3H)thione Derivatives 5a–c

To a solution of any of compound 3a (2.86 g, 0.01 mol), 3b (3.20 g, 0.01 mol) or 3c (3.16 g, 0.01 mol) in 1,4-dioxane (40 mL) each of elemental sulfur (0.32 g, 0.01 mol) and phenylisothiocyanate (1.30 g, 0.01 mol) were added. The reaction mixture, in each case was heated under reflux for 45 min then left to cool and the formed solid product was collected by filtration.

7-Amino-N-3-diphenyl-2-thioxo-2,3,4,5-tetrahydrothieno[2′,3′:3,4]benzo[1,2-d]thiazole-6-carboxamide (5a)

HPLC purity=93% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; yellow crystals (ethanol), yield 76% (3.01 g), mp 205–208°C; (IR (KBr) ν_max 3472–3346, 3056, 2987, 1687, 1635, 1586, 1208; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.90 (2t, 4H, 2CH₂), 4.86 (s, 2H, D₂O exchangeable, NH₂), 7.28–7.39 (m, 10H, 2C₆H_5, thiophene, thiazole C), 8.22 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.6 (2CH₂), 120.4, 121.8, 122.2, 122.8, 123.6, 125.3, 126.5, 127.9, 128.6, 132.6, 137.6, 139.5, 140.2, 141.6 (2C₆H₅, thiophene C), 164.8 (CO), 178.5 (C=S); EI-MS: m/z 435 [M]⁺ (32%); Anal. Calcd for C₂₂H₁₇N₃OS₃ (435.58): C, 60.66; H, 3.93; N, 9.65; S, 22.08%. Found: C, 60.53; H, 4.08; N, 9.81; S, 21.83%.

7-Amino-N-(4-chlorophenyl)-3-phenyl-2-thioxo-2,3,4,5-tetrahydrothieno[2′,3′:3,4]benzo[1,2-d]thiazole-6-carboxamide (5b)

HPLC purity=94% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=222 min; orange crystals (ethanol), yield 68% (3.19 g), mp 168–171°C; (IR (KBr) ν_max 3489–3331, 3055, 2986, 1687, 1632, 1593; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.88 (2t, 4H, 2CH₂), 4.89 (s, 2H, D₂O exchangeable, NH₂), 7.23–7.49 (m, 9H, C₆H₅, C₆H₄), 8.29 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.5, 38.7 (2CH₂), 120.8, 121.0, 121.9, 122.6, 123.8, 124.2, 125.8, 127.5, 129.2, 136.4, 139.3, 140.6, 141.2, 142.3 (C₆H₅, C₆H₄, thiophene, thiazole C), 164.4 (CO), 178.6 (C=S); EI-MS: m/z 470 [M]⁺ (32%), 472 [M⁺+2] (8%); Anal. Calcd for C₂₂H₁₆ClN₃OS₃ (470.03): C, 56.22; H, 3.43; N, 8.94; S, 20.47%. Found: C, 56.42; H, 3.29; N, 9.04; S, 20.36%.

7-Amino-N-(4-methoxyphenyl)-3-phenyl-2-thioxo-2,3,4,5-tetrahydrothieno[2′,3′:3,4]benzo[1,2-d]thiazole-6-carboxamide (5c)

HPLC purity=92% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 68% (3.16 g), mp 166–168°C; (IR (KBr) ν_max 3469–3332, 3055, 2986, 1688, 1636, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.88 (2t, 4H, 2CH₂), 3.69 (s, 3H, OCH₃), 4.84 (s, 2H, D₂O exchangeable, NH₂), 7.23–7.46 (m, 9H, C₆H₅, C₆H₄), 8.26 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.5, 38.8 (2CH₂), 53.1 (OCH₃), 120.6, 121.8, 122.8, 123.6, 124.8, 125.3, 127.9, 128,5, 130.4, 132.6, 134.3, 139.7, 140.5, 142.3 (C₆H₅, C₆H₄, thiophene, thiazole C), 164.4 (CO), 178.4 (C=S); EI-MS: m/z 465 [M]⁺ (33%); Anal. Calcd for C₂₃H₁₉N₃O₂S₃ (465.61): C, 59.33; H, 4.11; N, 9.02; S, 20.66%. Found: C, 59.51; H, 4.31; N, 8.94; S, 20.83%.

Synthesis of the 5,9-Dihydro-4H-thieno[2,3-f]chromene Derivatives 9a–r

To a solution of any of compound 3a (2.86 g, 0.01 mol), 3b (3.20 g, 0.01 mol) or 3c (3.16 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) any of benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-nitrobenzaldehyde (1.51 g, 0.01 mol) and either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) were added. The whole reaction mixture was heated under reflux for 3 h then left to cool and the formed solid product, in each case, was collected by filtration.

2,7-Diamino-8-cyano-N-9-diphenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9a)

HPLC purity=95% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=20 min; orange crystals (ethanol), yield 68% (3.16 g), mp 163–165°C; (IR (KBr) ν_max 3483–3329, 3055, 2986, 2220, 1687, 1633, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.62–1.87 (2t, 4H, 2CH₂), 4.86, 5.22 (2s, 4H, D₂O exchangeable, 2NH₂), 6.02 (s, 1H, pyran H-4), 7.25–7.49 (m, 10H, 2C₆H₅), 8.29 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.7, 38.6 (2CH₂), 80.7 (pyran C-4), 115.9, 120.4, 120.9, 122.0, 123.9, 125.5, 125.5, 126.8, 128.2, 130.9, 132.8, 134.3, 138.1, 139.6, 140.4, 140.7, 141.8 (2C₆H₅, C₆H₄, thiophene, pyran C), 164.2 (CO); EI-MS: m/z 440 [M]⁺ (36%); Anal. Calcd for C₂₅H₂₀N₄O₂S (440.52): C, 68.16; H, 4.58; N, 12.72; S, 7.28%. Found: C, 68.30; H, 4.47; N, 12.92; S, 7.41%.

2-Amino-8-cyano-7-hydroxy-N-9-diphenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9b)

HPLC purity=93% (C-18 NovaPak column; MeOH–H₂O, 80 : 5), RT=18 min; orange crystals (ethanol), yield 73% (3.22 g), mp 195–198°C; (IR (KBr) ν_max 3553–3329, 3055, 2986, 2220, 1687, 1633, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.86 (2t, 4H, 2CH₂), 4.84 (s, 2H, D₂O exchangeable, NH₂), 6.05 (s, 1H, pyran H-4), 7.27–7.44 (m, 10H, 2C₆H₅), 8.32 (s, 1H, D₂O exchangeable, NH), 9.34 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.8 (2CH₂), 80.9 (pyran C-4), 116.4, 120.7, 121.3, 122.6, 122.8, 124.3, 125.6, 126.9, 127.1, 130.8, 131.6, 132.8, 134.6, 138.8, 140.3, 140.8, 141.9 (2C₆H₅, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 441 [M]⁺ (32%); Anal. Calcd for C₂₅H₁₉N₃O₃S (441.50): C, 68.01; H, 4.34; N, 9.52; S, 7.26%. Found: C, 68.26; H, 4.51; N, 9.80; S, 7.33%.

2,7-Diamino-9-(4-chlorophenyl)-8-cyano-N-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9c)

HPLC purity=94% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=15 min; orange crystals (ethanol), yield 58% (2.74 g), mp 180–182°C; (IR (KBr) ν_max 3463–3357, 3055, 2988, 2221, 1686, 1630, 1593; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.63–1.86 (2t, 4H, 2CH₂), 4.83, 5.24 (2s, 4H, D₂O exchangeable, 2NH₂), 6.03 (s, 1H, pyran H-4), 7.24–7.48 (m, 9H, C₆H₅, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.5, 38.6 (2CH₂), 80.7 (pyran C-4), 116.4, 120.3, 120.8, 121.9, 122.5, 123.6, 125.8, 126.3, 128.3, 129.4, 131.6, 132.6, 133.2, 138.4, 140.1, 140.8, 141.5 (C₆H₅, C₆H₄, thiophene, pyran C), 164.4 (CO); EI-MS: m/z 474 [M]⁺ (28%), 476 [M⁺+2] (7%); Anal. Calcd for C₂₅H₁₉ClN₄O₂S (474.96): C, 63.22; H, 4.03; N, 11.80; S, 6.75%. Found: C, 63.31; H, 4.26; N, 11.69; S, 6.83%.

2-Amino-9-(4-chlorophenyl)-8-cyano-7-hydroxy-N-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9d)

HPLC purity=94% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=22 min; yellow crystals (ethanol), yield 65% (3.08 g), mp 216–218°C; (IR (KBr) ν_max 3571–3354, 3056, 2989, 2223, 1688, 1632, 1591; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.85 (2t, 4H, 2CH₂), 4.81 (s, 2H, D₂O exchangeable, NH₂), 6.04 (s, 1H, pyran H-4), 7.22–7.45 (m, 9H, C₆H₅, C₆H₄), 8.32 (s, 1H, D₂O exchangeable, NH), 9.22 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.8 (2CH₂), 80.6 (pyran C-4), 116.4, 119.6, 120.5, 121.6, 122.8, 123.3, 124.1, 125.6, 126.8, 128.3, 131.6, 132.6, 132.9, 135.2, 140.1, 140.2, 141.9 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 475 [M]⁺ (20%), 477 [M⁺+2] (10%); Anal. Calcd for C₂₅H₁₈ClN₃O₃S (474.95): C, 63.09; H, 3.81; N, 8.83; S, 6.74%. Found: C, 63.26; H, 3.99; N, 8.67; S, 6.92%.

2,7-Diamino-8-cyano-9-(4-nitrophenyl)-N-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9e)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; yellow crystals (ethanol), yield 70% (3.39 g), mp 180–182°C; (IR (KBr) ν_max 3493–3342, 3054, 2988, 2223, 1689, 1630, 1593; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.62–1.87 (2t, 4H, 2CH₂), 4.80, 5.19 (2s, 4H, D₂O exchangeable, 2NH₂), 6.02 (s, 1H, pyran H-4), 7.26–7.43 (m, 9H, C₆H₅, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.7 (2CH₂), 80.5 (pyran C-4), 115.9, 119.8, 120.3, 120.8, 121.4, 122.5, 123.9, 124.2, 125.4, 128.6, 131.8, 132.2, 133.5, 133.8, 140.2, 140.4, 141.7 (C₆H₅, C₆H₄, thiophene, pyran C), 164.4 (CO); EI-MS: m/z 485 [M]⁺ (26%); Anal. Calcd for C₂₅H₁₉N₅O₄S (485.51): C, 61.85; H, 3.94; N, 14.42; S, 6.60%. Found: C, 62.08; H, 3.74; N, 14.39; S, 6.73%.

2-Amino-8-cyano-7-hydroxy-9-(4-nitrophenyl)-N-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9f)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; yellow crystals (ethanol), yield 73% (3.53 g), mp 138–140°C; (IR (KBr) ν_max 3490–3341, 3055, 2988, 2220, 1687, 1630, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.63–1.87 (2t, 4H, 2CH₂), 4.81 (s, 2H, D₂O exchangeable, NH₂), 6.04 (s, 1H, pyran H-4), 7.24–7.47 (m, 9H, C₆H₅, C₆H₄), 8.33 (s, 1H, D₂O exchangeable, NH), 9.24 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.5 (2CH₂), 80.4 (pyran C-4), 116.2, 120.2, 121.7, 122.3, 123.2, 123.9, 124.5, 125.8, 126.4, 128.1, 129.2, 132.4, 133.3, 135.5, 138.6, 140.2, 141.5 (C₆H₅, C₆H₄, thiophene, pyran C), 164.2 (CO); EI-MS: m/z 486 [M]⁺ (18%); Anal. Calcd for C₂₅H₁₈N₄O₅S (486.50): C, 61.72; H, 3.73; N, 11.52; S, 6.59%. Found: C, 61.93; H, 3.80; N, 11.49; S, 6.63%.

2,7-Diamino-N-(4-chlorophenyl)-8-cyano-9-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9g)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 62% (2.94 g), mp 267–269°C; (IR (KBr) ν_max 3463–3349, 3059, 2986, 2220, 1688, 1630, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.61–1.87 (2t, 4H, 2CH₂), 4.84, 5.22 (2s, 4H, D₂O exchangeable, 2NH₂), 6.03 (s, 1H, pyran H-4), 7.22–7.49 (m, 9H, C₆H₅, C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.5 (2CH₂), 80.6 (pyran C-4), 115.9, 120.1, 121.6, 121.9, 122.7, 123.6, 124.9, 125.1, 127.5, 129.1, 130.3, 131.9, 133.3, 135.7, 140.4, 140.9, 141.2 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 474 [M]⁺ (19%), 476 [M⁺+2] (4%); Anal. Calcd for C₂₅H₁₉ClN₄O₂S (474.96): C, 63.22; H, 4.03; N, 11.80; S, 6.75%. Found: C, 63.08; H, 4.17; N, 11.79; S, 6.47%.

2-Amino-N-(4-chlorophenyl)-8-cyano-7-hydroxy-9-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9h)

HPLC purity=95% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=22 min; Orange crystals (ethanol), yield 75% (3.56 g), mp 188–190°C; (IR (KBr) ν_max 3530–3363, 3055, 2983, 2221, 1687, 1630, 1592; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.62–1.88 (2t, 4H, 2CH₂), 4.82 (s, 2H, D₂O exchangeable, NH₂), 6.04 (s, 1H, pyran H-4), 7.24–7.47 (m, 9H, C₆H₅, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH), 9.22 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.7 (2CH₂), 80.4 (pyran C-4), 116.4, 120.8, 121.3, 121.9, 122.8, 124.1, 124.7, 125.6, 126.2, 128.8, 130.6, 131.6, 132.6, 134.2, 140.6, 140.3, 141.6 (C₆H₅, C₆H₄, thiophene, pyran C), 164.5 (CO); EI-MS: m/z 475 [M]⁺ (42%), 477 [M⁺+2] (12%); Anal. Calcd for C₂₅H₁₈ClN₃O₃S (475.95): C, 63.09; H, 3.81; N, 8.83; S, 6.74%. Found: C, 63.26; H, 3.61; N, 9.02; S, 6.52%.

2,7-Diamino-N-9-bis(4-chlorophenyl)-8-cyano-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9i)

HPLC purity=96% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (1,4-dioxane), yield 58% (2.95 g), mp 267–269°C; (IR (KBr) ν_max 3482–3356, 3055, 2980, 2220, 1689, 1630, 1592; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.64–1.88 (2t, 4H, 2CH₂), 4.80, 5.21 (2s, 4H, D₂O exchangeable, 2NH₂), 6.02 (s, 1H, pyran H-4), 7.26–7.52 (m, 8H, 2C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.5 (2CH₂), 80.3 (pyran C-4), 116.3, 120.5, 120.8, 121.3, 122.7, 123.2, 124.5, 126.1, 126.8, 127.3, 128.4, 130.5, 131.8, 133.9, 140.6, 140.6, 141.4 (C₆H₅, C₆H₄, thiophene, pyran C), 164.3 (CO); EI-MS: m/z 509 [M]⁺ (36%), 511 [M⁺+2] (28%), 513 [M⁺+4] (10%); Anal. Calcd for C₂₅H₁₈Cl₂N₄O₂S (509.41): C, 58.94; H, 3.56; N, 11.00; S, 6.29%. Found: C, 58.64; H, 3.70; N, 10.93; S, 6.48%.

2-Amino-N-9-bis(4-chlorophenyl)-8-cyano-7-hydroxy-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9j)

HPLC purity=95% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=20 min; orange crystals (ethanol), yield 69% (3.52 g), mp 225–228°C; (IR (KBr) ν_max 3552–3342, 3055, 2983, 2220, 1689, 1632, 1595; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.60–1.89 (2t, 4H, 2CH₂), 4.83 (s, 2H, D₂O exchangeable, NH₂), 6.04 (s, 1H, pyran H-4), 7.21–7.49 (m, 8H, 2C₆H₄), 8.32 (s, 1H, D₂O exchangeable, NH), 9.42 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.7 (2CH₂), 80.6 (pyran C-4), 116.7, 120.9, 121.4, 121.5, 123.2, 123.6, 124.1, 125.8, 127.5, 128.8, 130.8, 131.9, 133.5, 134.2, 140.2, 140.6, 141.9 (C₆H₅, C₆H₄, thiophene, pyran C), 164.2 (CO); EI-MS: m/z 510 [M]⁺ (33%), 512 [M⁺+2] (23%), 514 [M⁺+4] (16%); Anal. Calcd for C₂₅H₁₇Cl₂N₃O₃S (510.39): C, 58.83; H, 3.36; N, 8.23; S, 6.28%. Found: C, 58.68; H, 3.57; N, 8.41; S, 6.33%.

2,7-Diamino-N-(4-chlorophenyl)-8-cyano-9-(4-nitrophenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9k)

HPLC purity=96% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=22 min; orange crystals (1,4-dioxane), yield 63% (3.27 g), mp 196–198°C; (IR (KBr) ν_max 3467–3341, 3055, 2980, 2222, 1689, 1632, 1591; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.62–1.87 (2t, 4H, 2CH₂), 4.83, 5.19 (2s, 4H, D₂O exchangeable, 2NH₂), 6.03 (s, 1H, pyran H-4), 7.22–7.48 (m, 8H, 2C₆H₄), 8.33 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.1, 38.7 (2CH₂), 80.4 (pyran C-4), 116.7, 120.2, 120.6, 122.6, 122.7, 123.8, 125.1, 125.9, 126.8, 127.3, 128.8, 129.3, 131.8, 133.9, 140.9, 141.2, 141.6 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 519 [M]⁺ (42%), 521 [M⁺+2] (33%); Anal. Calcd for C₂₅H₁₈ClN₅O₄S (519.96): C, 57.75; H, 3.49; N, 13.47; S, 6.17%. Found: C, 57.82; H, 3.59; N, 13.52; S, 6.32%.

2-Amino-N-(4-chlorophenyl)-8-cyano-7-hydroxy-9-(4-nitrophenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9l)

HPLC purity=94% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=21 min; orange crystals (ethanol), yield 58% (3.02 g), mp 166–168°C; (IR (KBr) ν_max 3530–3339, 3049, 2969, 2223, 1687, 1628, 1582; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.45–1.86 (2t, 4H, 2CH₂), 4.84 (s, 2H, D₂O exchangeable, NH₂), 6.02 (s, 1H, pyran H-4), 7.24–7.54 (m, 8H, 2C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH), 9.38 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.5 (2CH₂), 80.3 (pyran C-4), 116.4, 120.6, 121.3, 122.8, 124.1, 124.8, 125.0, 125.8, 127.9, 129.3, 130.3, 131.6, 133.7, 134.9, 140.4, 140.8, 142.4 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 520 [M]⁺ (56%), 522 [M⁺+2] (41%); Anal. Calcd for C₂₅H₁₇ClN₄O₅S (520.94): C, 57.64; H, 3.29; N, 10.75; S, 6.16%. Found: C, 57.80; H, 3.42; N, 10.94; S, 6.23%.

2,7-Diamino-8-cyano-N-(4-methoxyphenyl)-9-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9m)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=19 min; Orange crystals (1,4-dioxane), yield 68% (3.19 g), mp 144–147°C; (IR (KBr) ν_max 3487–3364, 3055, 2980, 2221, 1689, 1632, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.61–1.89 (2t, 4H, 2CH₂), 3.68 (s, 3H, OCH₃), 4.80, 5.19 (2s, 4H, D₂O exchangeable, 2NH₂), 6.02 (s, 1H, pyran H-4), 7.21–7.56 (m, 9H, C₆H₅, C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.1, 38.7 (2CH₂), 80.4 (pyran C-4), 53.6 (OCH₃), 116.5, 120.6, 120.6, 122.6, 123.2, 123.8, 124.1, 124.5, 126.5, 127.1, 127.5, 128.7, 131.8, 136.5, 138.5, 140.4, 141.8 (C₆H₅, C₆H₄, thiophene, pyran C), 164.3 (CO); EI-MS: m/z 470 [M]⁺ (28%); Anal. Calcd for C₂₆H₂₂N₄O₃S (470.54): C, 66.37; H, 4.71; N, 11.91; S, 6.81%. Found: C, 66.41; H, 4.92; N, 12.19; S, 6.68%.

2-Amino-8-cyano-7-hydroxy-N-(4-methoxyphenyl)-9-phenyl-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9n)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 66% (3.12 g), mp 280–284°C; (IR (KBr) ν_max 3543–3342, 3055, 2969, 2221, 1689, 1625, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.48–1.83 (2t, 4H, 2CH₂), 3.69 (s, 3H, OCH₃), 4.86 (s, 2H, D₂O exchangeable, NH₂), 6.02 (s, 1H, pyran H-4), 7.28–7.42 (m, 9H, C₆H₅, C₆H₄), 8.28 (s, 1H, D₂O exchangeable, NH), 9.34 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.5 (2CH₂), 53.8 (OCH₃), 80.1 (pyran C-4), 116.8, 120.2, 122.9, 123.2, 124.8, 125.6, 126.3, 126.5, 127.9, 129.4, 130.6, 131.1, 133.4, 134.2, 141.1, 144.5, 142.6 (C₆H₅, C₆H₄, thiophene, pyran C), 164.5 (CO); EI-MS: m/z 471 [M]⁺ (26%); Anal. Calcd for C₂₆H₂₁N₃O₄S (471.53): C, 66.23; H, 4.49; N, 8.91; S, 6.80%. Found: C, 66.49; H, 4.51; N, 9.23; S, 6.79%.

2,7-Diamino-9-(4-chlorophenyl)-8-cyano-N-(4-methoxyphenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9o)

HPLC purity=94% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=21 min; orange crystals (1,4-dioxane), yield 71% (3.58 g), mp 177–179°C; (IR (KBr) ν_max 3468–3351, 3055, 2980, 2220, 1688, 1632, 1590; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.63–1.89 (2t, 4H, 2CH₂), 3.69 (s, 3H, OCH₃), 4.81, 5.21 (2s, 4H, D₂O exchangeable, 2NH₂), 6.02 (s, 1H, pyran H-4), 7.24–7.49 (m, 8H, 2C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.6 (2CH₂), 80.6 (pyran C-4), 53.7 (OCH₃), 116.9, 119.8, 120.3, 122.2, 123.9, 124.3, 125.4, 124.6, 126.3, 127.1, 127.9, 128.3, 131.3, 136.2, 138.7, 140.6, 141.5 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 504 [M]⁺ (18%), 506 [M⁺+2] (12%); Anal. Calcd for C₂₆H₂₁ClN₄O₃S (504.99): C, 61.84; H, 4.19; N, 11.09; S, 6.35%. Found: C, 61.80; H, 4.29; N, 11.25; S, 6.46%.

2-Amino-9-(4-chlorophenyl)-8-cyano-7-hydroxy-N-(4-methoxyphenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9p)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 78% (3.93 g), mp 154–156°C; (IR (KBr) ν_max 3522–3312, 3056, 2966, 2220, 1687, 1627, 1582; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.43–1.80 (2t, 4H, 2CH₂), 3.67 (s, 3H, OCH₃), 4.85 (s, 2H, D₂O exchangeable, NH₂), 6.03 (s, 1H, pyran H-4), 7.26–7.44 (m, 8H, 2C₆H₄), 8.26 (s, 1H, D₂O exchangeable, NH), 9.32 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.5 (2CH₂), 53.5 (OCH₃), 80.2 (pyran C-4), 116.5, 120.3, 121.6, 123.6, 125.2, 125.9, 126.1, 127.8, 128.4, 129.6, 130.8, 132.4, 133.6, 134.7, 140.8, 142.6, 142.9 (2C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 505 [M]⁺ (48%), 507 [M⁺+2] (32%); Anal. Calcd for C₂₆H₂₀ClN₃O₄S (505.97): C, 61.72; H, 3.98; N, 8.30; S, 6.34%. Found: C, 61.54; H, 4.19; N, 8.52; S, 6.51%.

2,7-Diamino-8-cyano-N-(4-methoxyphenyl)-9-(4-nitrophenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9q)

HPLC purity=97% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=20 min; orange crystals (ethanol), yield 57% (2.93 g), mp 244–247°C; (IR (KBr) ν_max 3449–3323, 3055, 2983, 2220, 1687, 1630, 1592; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.58–1.87 (2t, 4H, 2CH₂), 3.66 (s, 3H, OCH₃), 4.82, 5.19 (2s, 4H, D₂O exchangeable, 2NH₂), 6.04 (s, 1H, pyran H-4), 7.23–7.54 (m, 8H, 2C₆H₄), 8.33 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.9 (2CH₂), 80.2 (pyran C-4), 53.5 (OCH₃), 116.7, 120.2, 121.9, 122.5, 122.6, 123.4, 124.1, 125.9, 126.2, 127.8, 128.5, 129.4, 131.3, 134.8, 136.2, 139.6, 140.2 (C₆H₅, C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 515 [M]⁺ (42%); Anal. Calcd for C₂₆H₂₁N₅O₅S (515.54): C, 60.57; H, 4.11; N, 13.58; S, 6.22%. Found: C, 60.72; H, 4.32; N, 13.80; S, 6.52%.

2-Amino-8-cyano-7-hydroxy-N-(4-methoxyphenyl)-9-(4-nitrophenyl)-5,9-dihydro-4H-thieno[2,3-f]chromene-3-carboxamide (9r)

HPLC purity=96% (C-18 NovaPak column; MeOH–H₂O, 80 : 10), RT=18 min; orange crystals (ethanol), yield 69% (3.93 g), mp 144–146°C; (IR (KBr) ν_max 3538–3331, 3054, 2968, 2220, 1689, 1631, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.39–1.68 (2t, 4H, 2CH₂), 3.64 (s, 3H, OCH₃), 4.87 (s, 2H, D₂O exchangeable, NH₂), 6.04 (s, 1H, pyran H-4), 7.24–7.42 (m, 8H, 2C₆H₄), 8.27 (s, 1H, D₂O exchangeable, NH), 9.34 (s, 1H, D₂O exchangeable, OH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.7 (2CH₂), 53.4 (OCH₃), 80.4 (pyran C-4), 116.7, 120.8, 122.9, 124.8, 125.2, 125.9, 126.5, 127.6, 128.8, 129.6, 130.8, 131.9, 133.3, 137.9, 140.8, 142.6, 142.9 (2C₆H₄, thiophene, pyran C), 164.6 (CO); EI-MS: m/z 516 [M]⁺ (33%); Anal. Calcd for C₂₆H₂₀N₄O₆S (516.53): C, 60.46; H, 3.90; N, 10.85; S, 6.21%. Found: C, 60.59; H, 3.83; N, 10.93; S, 6.42%.

General Procedure for the Synthesis of the 2-Amino-7-(1-phenylethylidene)-4,5-dihydrobenzo[b]thiophene Derivatives 11a–i

To a dry solid of any of compounds 3a (2.86 g, 0.01 mol), 3b (3.20 g, 0.01 mol) or 3c (3.16 g, 0.01 mol) any of acetophenone (1.20 g, 0.01 mol), 4-chloroacetophenone (1.55 g 0.01 mol) or 4-methylacetophenone (1.35 g, 0.01 mol) and ammonium acetate (0.50 g) were added. The reaction mixture was heated in an oil bath at 120°C then left to cool. The solid product formed upon triturating the remaining product with ethanol was collected by filtration.

2-Amino-6-oxo-N-phenyl-7-(1-phenylethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11a)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=22 min; orange crystals (ethanol), yield 80% (3.10 g), mp 210–212°C; (IR (KBr) ν_max 3479–3352, 3055, 2968, 1688, 1679, 1631, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.36–1.66 (2t, 4H, 2CH₂), 2.88 (s, 3H, CH₃), 4.86 (s, 2H, D₂O exchangeable, NH₂), 7.28–7.39 (m, 10H, 2C₆H₅), 8.29 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.5, 38.9 (2CH₂), 18.3 (CH₃), 92.6, 98.4 (C=C), 120.9, 122.8, 125.0, 125.7, 126.1, 127.8, 128.8, 129.6, 130.8, 131.5, 132.2, 136.3 (2C₆H₅, thiophene C), 163.8, 164.2 (2CO); EI-MS: m/z 388 [M]⁺ (24%); Anal. Calcd for C₂₃H₂₀N₂O₂S (388.48): C, 71.11; H, 5.19; N, 7.21; S, 8.25%. Found: C, 71.08; H, 5.23; N, 7.41; S, 8.05%.

2-Amino-7-(1-(4-chlorophenyl)ethylidene)-6-oxo-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11b)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=20 min; orange crystals (ethanol), yield 67% (2.82 g), mp 170–172°C; (IR (KBr) ν_max 3493–3356, 3055, 2967, 1689, 1686, 1630, 1582; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.68 (2t, 4H, 2CH₂), 2.87 (s, 3H, CH₃), 4.85 (s, 2H, D₂O exchangeable, NH₂), 7.22–7.45 (m, 9H, C₆H₅, C₆H₄), 8.32 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.6 (2CH₂), 18.8 (CH₃), 92.9, 98.7 (C=C), 121.3, 123.6, 124.6, 125.3, 126.6, 127.3, 128.4, 129.1, 130.8, 131.5, 132.2, 136.8 (C₆H₅, thiophene C), 163.5, 164.8 (2CO); EI-MS: m/z 422 [M]⁺ (56%), 524 [M⁺+2] (38%); Anal. Calcd for C₂₃H₁₉ClN₂O₂S (422.93): C, 65.32; H, 4.53; N, 6.62; S, 7.58%. Found: C, 65.38; H, 4.61; N, 6.80; S, 7.74%.

2-Amino-6-oxo-N-phenyl-7-(1-(p-tolyl)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11c)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=22 min; orange crystals (ethanol), yield 55% (2.21g), mp 204–207°C; (IR (KBr) ν_max 3483–3339, 3055, 2967, 1688, 1686, 1630, 1583; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.31–1.65 (2t, 4H, 2CH₂), 2.59, 2.85 (2s, 6H, 2CH₃), 4.86 (s, 2H, D₂O exchangeable, NH₂), 7.28–7.36 (m, 9H, C₆H₅, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.8 (2CH₂), 18.8, 23.6 (2CH₃), 92.4, 98.9 (C=C), 121.6, 122.8, 123.1, 124.7, 126.8, 127.6, 128.6, 129.0, 130.6, 130.9, 131.6, 136.5 (C₆H₅, C₆H₄, thiophene C), 163.2, 164.4 (2CO); EI-MS: m/z 402 [M]⁺ (38%); Anal. Calcd for C₂₄H₂₂N₂O₂S (402.51): C, 71.62; H, 5.51; N, 6.96; S, 7.97%. Found: C, 71.80; H, 5.80; N, 6.77; S, 7.83%.

2-Amino-N-(4-chlorophenyl)-6-oxo-7-(1-phenylethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11d)

HPLC purity=97% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=20 min; orange crystals (ethanol), yield 59% (2.48 g), mp 195–198°C; (IR (KBr) ν_max 3458–3371, 3055, 2967, 1687, 1686, 1630, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.31–1.67 (2t, 4H, 2CH₂), 2.64 (s, 3H, CH₃), 4.85 (s, 2H, D₂O exchangeable, NH₂), 7.23–7.42 (m, 9H, C₆H₅, C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.6 (2CH₂), 18.7 (CH₃), 92.1, 99.2 (C=C), 120.2, 122.5, 123.3, 124.5, 126.8, 127.6, 128.6, 129.3, 130.2, 132.2, 133.9, 136.7 (C₆H₅, C₆H₄, thiophene C), 163.3, 164.8 (2CO); EI-MS: m/z 422 [M]⁺ (42%), 424 [M⁺+2] (32%); Anal. Calcd for C₂₃H₁₉ClN₂O₂S (422.93): C, 65.32; H, 4.53; N, 6.62; S, 7.58%. Found: C, 65.41; H, 4.63; N, 6.81; S, 7.70%.

2-Amino-N-(4-chlorophenyl)-7-(1-(4-chlorophenyl)ethylidene)-6-oxo-4,5,6,7-tetra-hydrobenzo[b]thiophene-3-carboxamide (11e)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=18 min; orange crystals (ethanol), yield 63% (2.88 g), mp 128–131°C; (IR (KBr) ν_max 3482–3321, 3055, 2967, 1689, 1687, 1632, 1588; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.36–1.63 (2t, 4H, 2CH₂), 2.63 (s, 3H, CH₃), 4.86 (s, 2H, D₂O exchangeable, NH₂), 7.22–7.53 (m, 8H, 2C₆H₄), 8.34 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.7 (2CH₂), 18.5 (CH₃), 92.0, 99.5 (C=C), 121.5, 122.5, 124.6, 124.8, 126.8, 128.0, 129.2, 129.6, 132.5, 134.6, 135.3, 137.9 (2C₆H₄, thiophene C), 163.4, 164.3 (2CO); EI-MS: m/z 456 [M]⁺ (30%), 458 [M⁺+2] (18%), 460 [M⁺+4] (13%); Anal. Calcd for C₂₃H₁₈Cl₂N₂O₂S (457.37): C, 60.40; H, 3.97; N, 6.12; S, 7.01%. Found: C, 60.58; H, 4.15; N, 6.31; S, 7.22%.

2-Amino-N-(4-chlorophenyl)-6-oxo-7-(1-(p-tolyl)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11f)

HPLC purity=95% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=24 min; Orange crystals (ethanol), yield 70% (3.05 g), mp 230–233°C; (IR (KBr) ν_max 3468–3355, 3058, 2967, 1689, 1688, 1632, 1588; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.36–1.68 (2t, 4H, 2CH₂), 2.63, 2.87 (2s, 6H, 2CH₃), 4.88 (s, 2H, D₂O exchangeable, NH₂), 7.22–7.46 (m, 8H, 2C₆H₄), 8.31 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.9 (2CH₂), 18.5, 22.8(2CH₃), 92.4, 99.7 (C=C), 121.8, 122.0, 123.8, 124.4, 126.5, 128.5, 129.0, 129.8, 132.7, 134.6, 135.2, 136.6 (2C₆H₄, thiophene C), 163.2, 164.5 (2CO); EI-MS: m/z 436 [M]⁺ (26%), 438 [M⁺+2] (16%); Anal. Calcd for C₂₄H₂₁ClN₂O₂S (436.95): C, 65.97; H, 4.84; N, 6.41; S, 7.34%. Found: C, 65.72; H, 4.90; N, 6.36; S, 7.57%.

2-Amino-N-(4-methoxyphenyl)-6-oxo-7-(1-phenylethylidene)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxamide (11g)

HPLC purity=95% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=22 min; orange crystals (ethanol), yield 55% (2.29 g), mp 193–195°C; (IR (KBr) ν_max 3485–3329, 3056, 2967, 1688, 1685, 1630, 1586; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.34–1.65 (2t, 4H, 2CH₂), 2.89, 3.67 (2s, 6H, CH₃, OCH₃), 4.86 (s, 2H, D₂O exchangeable, NH₂), 7.27–7.41 (m, 9H, C₆H₅, C₆H₄), 8.34 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.1, 38.8 (2CH₂), 18.9, 52.8 (CH₃, OCH₃), 92.7, 98.9 (C=C), 120.8, 122.5, 123.5, 124.8, 126.9, 128.0, 128.8, 129.7, 130.2, 131.5, 133.9, 136.5 (C₆H₅, C₆H₄, thiophene C), 163.2, 164.7 (2CO); EI-MS: m/z 418 [M]⁺ (53%); Anal. Calcd for C₂₄H₂₂N₂O₃S (418.51): C, 68.88; H, 5.30; N, 6.69; S, 7.66%. Found: C, 66.69; H, 5.27; N, 6.53; S, 7.80%.

2-Amino-7-(1-(4-chlorophenyl)ethylidene)-N-(4-methoxyphenyl)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (11h)

HPLC purity=97% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=20 min; orange crystals (ethanol), yield 62% (2.63 g), mp 255–259°C; (IR (KBr) ν_max 3492–3351, 3054, 2968, 1688, 1687, 1630, 1585; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.67 (2t, 4H, 2CH₂), 2.87, 3.68 (2s, 6H, CH₃, OCH₃), 4.84 (s, 2H, D₂O exchangeable, NH₂), 7.23–7.49 (m, 8H, 2C₆H₄), 8.32 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.4 (2CH₂), 18.6, 52.7 (CH₃, OCH₃), 92.5, 98.7 (C=C), 119.6, 122.8, 123.6, 125.2, 127.3, 128.6, 129.3, 129.7, 131.3, 132.8, 134.8, 135.9 (2C₆H₄, thiophene C), 163.3, 164.5 (2CO); EI-MS: m/z 452 [M]⁺ (32%), 524 [M⁺+2] (25%); Anal. Calcd for C₂₄H₂₁ClN₂O₃S (452.95): C, 63.64; H, 4.67; N, 6.18; S, 7.08%. Found: C, 63.77; H, 4.90; N, 6.28; S, 7.31%.

2-Amino-N-(4-methoxyphenyl)-6-oxo-7-(1-(p-tolyl)ethylidene)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxamide (11i)

Orange crystals (1,4-dioxane), yield 74% (3.19 g), mp>300°C; (IR (KBr) ν_max 3473–3330, 3054, 2965, 1689, 1686, 1630, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.69 (2t, 4H, 2CH₂), 2.67, 2.84, 3.69 (3s, 9H, 2CH₃, OCH₃), 4.82 (s, 2H, D₂O exchangeable, NH₂), 7.21–7.44 (m, 8H, 2C₆H₄), 8.33 (s, 1H, D₂O exchangeable, NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.31, 38.5 (2CH₂), 18.4, 19.6, 52.7 (2CH₃, OCH₃), 92.6, 98.9 (C=C), 120.8, 121.4, 122.8, 123.9, 125.6, 126.3, 127.6, 128.3, 130.2, 131.5, 133.6, 134.3 (2C₆H₄, thiophene C), 163.4, 164.8 (2CO); EI-MS: m/z 432 [M]⁺ (60%); Anal. Calcd for C₂₅H₂₄N₂O₃S (432.53): C, 69.42; H, 5.59; N, 6.48; S, 7.41%. Found: C, 69.58; H, 5.62; N, 6.33; S, 7.38%.

General Procedure for the Synthesis of the 2-(2-Arylhydrazono)ethylidene)-4,5-dihydrobenzo[b]thiophen-6(7H)-one (13a–i)

To a solution of any compounds 11a (3.88 g, 0.01 mol), 11e (4.56 g, 0.01 mol) or 11i (4.32 g, 0.01 mol) in ethanol (60 mL) containing sodium acetate (8.0 g) any of benzenediazonium chloride (0.01 mol), 4-chlorobenzenediazonium chloride (0.01 mol) or 4-nitrobenzenediazonium chloride (0.01 mol) [prepared by the addition of sodium nitrite (0.70 g, 0.01 mol) to a cold solution (0–5°C) of any of aniline (0.93 g, 0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) or 4-nitroaniline (1.38 g, 0.01 mol) in concentrated hydrochloric acid (12 mL, 18 M) was added with continuous stirring] was added drop-wise with continuous stirring. The whole reaction mixture, in each case, was stirred at room temperature for additional 2h and the formed solid product, in each case, was collected by filtration.

2-Amino-6-oxo-N-phenyl-7-(1-phenyl-2-(2-phenylhydrazono)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13a)

HPLC purity=94% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=20 min; orange crystals (ethanol), yield 72% (3.54 g), mp 207–210°C; (IR (KBr) ν_max 3469–3332, 3055, 2967, 1688, 1685, 1630, 1586; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.67 (2t, 4H, 2CH₂), 4.89 (s, 2H, D₂O exchangeable, NH₂), 5.80 (s, 1H, CH=N), 7.28–7.36 (m, 15H, 3C₆H₅), 8.28, 8.32 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.5 (2CH₂), 96.3, 98.6 (C=C), 120.5, 121.6, 122.2, 122.6, 123.8, 124.8, 125.0, 126.9, 127.4, 128.3, 128.5, 129.7, 130.8, 131.3, 133.6, 136.8 (3C₆H₅, thiophene C), 163.8, 164.4 (2CO), 172.8 (C=N); EI-MS: m/z 492 [M]⁺ (28%); Anal. Calcd for C₂₉H₂₄N₄O₂S (492.59): C, 70.71; H, 4.91; N, 11.37; S, 6.51%. Found: C, 70.83; H, 5.03; N, 11.29; S, 6.79%.

2-Amino-7-(2-(2-(4-chlorophenyl)hydrazono)-1-phenylethylidene)-6-oxo-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13b)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=24 min; orange crystals (1,4-dioxane), yield 64% (3.37 g), mp 133–135°C; (IR (KBr) ν_max 3483–3316, 3055, 2967, 1687, 1684, 1630, 1580; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.34–1.69 (2t, 4H, 2CH₂), 4.87 (s, 2H, D₂O exchangeable, NH₂), 5.83 (s, 1H, CH=N), 7.24–7.42 (m, 14H, 2C₆H₅, C₆H₄), 8.29, 8.34 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.6 (2CH₂), 96.1, 98.9 (C=C), 120.8, 121.4, 122.8, 123.2, 124.5, 124.7, 125.6, 126.3, 126.9, 128.1, 129.3, 129.8, 130.7, 131.6, 134.9, 138.8 (2C₆H₅, C₆H₄, thiophene C), 163.5, 164.2 (2CO), 172.5 (C=N); EI-MS: m/z 527 [M]⁺ (33%), 529 [M⁺+2] (21%); Anal. Calcd for C₂₉H₂₃ClN₄O₂S (527.04): C, 66.09; H, 4.40; N, 10.63; S, 6.08%. Found: C, 65.89; H, 4.28; N, 10.82; S, 5.79%.

2-Amino-7-(2-(2-(4-nitrophenyl)hydrazono)-1-phenylethylidene)-6-oxo-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13c)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=18 min; orange crystals (1,4-dioxane), yield 60% (3.22 g), mp 180–184°C; (IR (KBr) ν_max 3473–3363, 3056, 2963, 1688, 1684, 1632, 1583; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.67 (2t, 4H, 2CH₂), 4.86 (s, 2H, D₂O exchangeable, NH₂), 5.80 (s, 1H, CH=N), 7.24–7.48 (m, 14H, 2C₆H₅, C₆H₄), 8.26, 8.32 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.7 (2CH₂), 96.4, 98.7 (C=C), 120.4, 121.8, 123.1, 123.6, 124.8, 124.9, 126.1, 126.8, 127.2, 128.6, 129.5, 129.8, 130.2, 132.7, 133.2, 138.2 (2C₆H₅, C₆H₄, thiophene C), 163.6, 164.7 (2CO), 172.3 (C=N); EI-MS: m/z 537 [M]⁺ (22%); Anal. Calcd for C₂₉H₂₃N₅O₄S (537.59): C, 64.79; H, 4.31; N, 13.03; S, 5.96%. Found: C, 64.63; H, 4.42; N, 12.93; S, 5.88%.

2-Amino-N-(4-chlorophenyl)-7-(1-(4-chlorophenyl)-2-(2-phenylhydrazono)ethylidene)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13d)

HPLC purity=95% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=20 min; pale brown crystals (1,4-dioxane), yield 77% (4.32 g), mp >300°C; (IR (KBr) ν_max 3492–3321, 3056, 2966, 1687, 1686, 1631, 1587; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.34–1.68 (2t, 4H, 2CH₂), 4.88 (s, 2H, D₂O exchangeable, NH₂), 5.84 (s, 1H, CH=N), 7.22–7.49 (m, 13H, C₆H₅, 2C₆H₄), 8.22, 8.34 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.4 (2CH₂), 96.6, 98.9 (C=C), 120.6, 120.9, 122.8, 123.3, 124.2, 125.8, 126.0, 126.6, 127.4, 127.9, 129.2, 129.5, 131.8, 133.2, 134.7, 139.6 (2C₆H₅, C₆H₄, thiophene C), 163.3, 164.5 (2CO), 172.6 (C=N); EI-MS: m/z 561[M]⁺ (18%), 563 [M⁺+2] (15%), 465 [M⁺+4] (8%); Anal. Calcd for C₂₉H₂₂Cl₂N₄O₂S (561.48): C, 62.03; H, 3.95; N, 9.98; S, 5.71%. Found: C, 62.93; H, 4.02; N, 10.18; S, 5.92%.

2-Amino-N-(4-chlorophenyl)-7-(1-(4-chlorophenyl)-2-(2-(4-chlorophenyl)hydrazono)ethylidene)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13e)

HPLC purity=95% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=24 min; Yellow crystals (ethanol), yield 50% (2.85 g), mp 244–247°C; (IR (KBr) ν_max 3472–3340, 3053, 2968, 1688, 1687, 1630, 1589; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.34–1.69 (2t, 4H, 2CH₂), 4.85 (s, 2H, D₂O exchangeable, NH₂), 5.82 (s, 1H, CH=N), 7.25–7.54 (m, 12H, 3C₆H₄), 8.23, 8.32 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.1, 38.6 (2CH₂), 96.5, 98.7 (C=C), 120.2, 120.3, 121.6, 122.5, 123.7, 124.2, 125.8, 126.9, 127.1, 128.5, 129.6, 130.5, 130.9, 132.6, 134.8, 138.4 (3C₆H₄, thiophene C), 163.6, 164.8 (2CO), 172.4 (C=N); EI-MS: m/z 595 [M]⁺ (22%), 597 [M⁺+2] (16%), 596 [M⁺+4] (8%); Anal. Calcd for C₂₉H₂₁Cl₃N₄O₂S (595.93): C, 58.45; H, 3.55; N, 9.40; S, 5.38%. Found: C, 58.22; H, 3.80; N, 9.37; S, 5.42%.

2-Amino-N-(4-chlorophenyl)-7-(1-(4-chlorophenyl)-2-(2-(4-nitrophenyl)hydrazono)ethylidene)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13f)

HPLC purity=98% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=24 min; Orange crystals (ethanol), yield 63% (3.81 g), mp 188–190°C; (IR (KBr) ν_max 3459–3370, 3055, 2966, 1689, 1687, 1631, 1585; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.32–1.69 (2t, 4H, 2CH₂), 4.87 (s, 2H, D₂O exchangeable, NH₂), 5.80 (s, 1H, CH=N), 7.21–7.52 (m, 12H, 3C₆H₄), 8.26, 8.33 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.3, 38.8 (2CH₂), 96.4, 98.9 (C=C), 119.5, 120.8, 122.0, 122.9, 123.2, 125.4, 126.2, 126.9, 128.2, 129.4, 129.8, 130.2, 131.6, 132.6, 135.3, 138.9 (3C₆H₄, thiophene C), 163.3, 164.1 (2CO), 172.2 (C=N); EI-MS: m/z 606 [M]⁺ (42%), 608 [M⁺+2] (36%), 610 [M⁺+4] (14%); Anal. Calcd for C₂₉H₂₁Cl₂N₅O₄S (606.48): C, 57.43; H, 3.49; N, 11.55; S, 5.29%. Found: C, 57.19; H, 3.61; N, 11.80; S, 5.38%.

2-Amino-N-(4-methoxyphenyl)-6-oxo-7-(2-(2-phenylhydrazono)-1-(p-tolyl)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamidede (13g)

HPLC purity=95% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=23 min; pale yellow crystals (ethanol), yield 79% (4.23 g), mp 274–277°C; (IR (KBr) ν_max 3480–3349, 3057, 2966, 1687, 1685, 1631, 1586; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.67 (2t, 4H, 2CH₂), 2.68, 3.69 (2s, 6H, CH₃, OCH₃), 4.85 (s, 2H, D₂O exchangeable, NH₂), 5.82 (s, 1H, CH=N), 7.25–7.43 (m, 13H, C₆H₅, 2C₆H₄), 8.28, 8.31 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.2, 38.7 (2CH₂), 18.9, 52.1 (CH₃, OCH₃), 96.7, 98.8 (C=C), 120.8, 121.3, 122.5, 122.7, 124.6, 124.8, 125.8, 126.4, 127.8, 128.8, 129.3, 130.8, 132.3, 133.7, 134.4, 138.6 (C₆H₅, 2C₆H₄, thiophene C), 163.6, 164.5 (2CO), 172.0 (C=N); EIMMS: m/z 536 [M]⁺ (25%); Anal. Calcd for C₃₁H₂₈N₄O₃S (536.64): C, 69.38; H, 5.26; N, 10.44; S, 5.98%. Found: C, 69.42; H, 5.06; N, 10.28; S, 6.16%.

2-Amino-7-(2-(2-(4-chlorophenyl)hydrazono)-1-(p-tolyl)ethylidene)-N-(4-methoxy-phenyl)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13h)

HPLC purity=94% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=24 min; Yellow crystals (ethanol), yield 62% (3.54 g), mp 199–202°C; (IR (KBr) ν_max 3489–3329, 3055, 2963, 1688, 1686, 1630, 1583; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.31–1.68 (2t, 4H, 2CH₂), 2.65, 3.66 (2s, 6H, (CH₃, OCH₃)), 4.82 (s, 2H, D₂O exchangeable, NH₂), 5.80 (s, 1H, CH=N), 7.21–7.52 (m, 12H, 3C₆H₄), 8.26, 8.30 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.4, 38.5 (CH₂), 18.8, 52.6 (CH₃, OCH₃), 96.6, 98.9 (C=C), 119.6, 120.6, 122.2, 123.9, 124.2, 124.5, 126.1, 126.7, 127.3, 128.7, 129.5, 130.2, 131.3, 133.9, 135.8, 137.2 (3C₆H₄, thiophene C), 163.8, 164.2 (2CO), 172.3 (C=N); EIMMS: m/z 570 [M]⁺ (31%), 572 [M⁺+2] (26%); Anal. Calcd for C₃₁H₂₇ClN₄O₃S (571.09): C, 65.20; H, 4.77; N, 9.81; S, 5.61%. Found: C, 65.33; H, 4.93; N, 10.01; S, 5.80%.

2-Amino-N-(4-methoxyphenyl)-7-(2-(2-(4-nitrophenyl)hydrazono)-1-(p-tolyl)ethylidene)-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (13i)

HPLC purity=96% (C-18 NovaPak column; MeOH–EtOAc, 80 : 10), RT=22 min; yellow crystals (ethanol), yield 48% (2.78 g), mp 245–247°C; (IR (KBr) ν_max 3465–3373, 3055, 2960, 1688, 1687, 1633, 1583; ¹H-NMR (DMSO-d₆, 200 MHz) δ: 1.33–1.66 (2t, 4H, 2CH₂), 2.62, 3.66 (2s, 6H, CH₃, OCH₃), 4.84 (s, 2H, D₂O exchangeable, NH₂), 5.83 (s, 1H, CH=N), 7.24–7.57 (m, 12H, 3C₆H₄), 8.24, 8.32 (2s, 2H, D₂O exchangeable, 2NH); ¹³C-NMR (DMSO-d₆, 75 MHz) δ: 18.1, 38.6 (2CH₂), 19.2, 52.1 (CH₃, OCH₃), 96.3, 98.6 (C=C), 120.2, 120.9, 121.4, 122.6, 123.9, 124.1, 126.8, 126.7, 128.0, 128.4, 129.9, 130.8, 132.6, 134.2, 135.7, 138.5 (3C₆H₄, thiophene C), 163.3, 164.6 (2CO), 172.0 (C=N); EIMMS: m/z 581 [M]⁺ (22%); Anal. Calcd for C₃₁H₂₇N₅O₅S (581.64): C, 64.01; H, 4.68; N, 12.04; S, 5.51%. Found: C, 63.82; H, 4.47; N, 12.26; S, 5.32%.

Acknowledgments

R. M. Mohareb would like to thank the Alexander von Humboldt Foundation in Bonn, Germany, for sponsoring this research and regularly providing visiting fellowships to Germany in order to complete the experimental work.

Conflict of Interest

The authors declare no conflict of interest.

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