Discovery of 3,3a,4,5-tetrahydro-2H-benzo[g]indazole containing quinoxaline derivatives as novel EGFR/HER-2 dual inhibitors

Abstract

In the present study, twenty-five pyrazole–quinoxaline derivatives (4a–4y) were designed and synthesized, and their biological activity as potential EGFR or HER-2 kinase inhibitors was evaluated. Among them, compound 4l displayed better antiproliferative activity against A549 and MCF-7 cell lines than Eroltinib. Further kinase inhibitory activity assay results indicated that compound 4l demonstrated the most potent enzyme inhibitory activity. Docking simulations were then performed to position compounds 4l and 4x into the active binding site of EGFR to determine the probable binding model. 3D-QSAR models were built to aid in the effective design of the presently studied and future EGFR inhibitors. These discoveries suggested that the title compounds are potential EGFR/HER-2 dual inhibitors and compound 4l may be a promising lead compound for the development of novel antitumor agents, potentially via inhibiting EGFR/HER-2.

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1. Introduction

Cancer is a major problem in human health and remains the second highest cause of mortality worldwide, with millions of cases every year.¹ The use of molecularly targeted therapeutics, which are highly selective and do not have the serious toxicity associated with conventional cytotoxic drugs, has become an attractive approach for cancer chemotherapy.² Protein kinases catalyze the phosphorylation of tyrosine and serine/threonine residues in various proteins,³ thus, protein kinases are extensively targeted in order to discover inhibitors suitable as potential cancer-treatment drugs.^4–7 In recent years, a variety of novel anticancer lead compounds have been developed that target mutant or aberrantly expressed oncogenic growth factor receptor and non-receptor tyrosine kinases in the mitogenic or proliferative signal transduction pathways of cancer cells.⁸

As a transmembrane protein tyrosine kinase (PTK), the epidermal growth factor receptor (EGFR) family plays a crucial role in regulating cell proliferation, survival, adhesion, migration, differentiation and apoptosis, and has been identified to play a critical role in cancer.^9,10 The EGFR family consists of the human epidermal growth factor receptor (EGFR/ErbB-1), human epidermal growth factor receptor 2 (HER-2/ErbB-2), human epidermal growth factor receptor 3 (HER-3/Erb-3), and human epidermal growth factor receptor 4 (HER-4/Erb-4).¹¹ Among these, as the targets in current cancer research, EGFR and HER-2 have been implicated in the development and malignancy of many human cancers,⁷ because their overexpression or abnormal activation often causes the malignant transformation of cells, such as non-small cell lung cancer (NSCLC), and prostate, breast, stomach, colon, and ovarian cancers.^12–15

Recently, thousands of small organic molecules with different structures have been developed as multi-target inhibitors.^16–18 Compounds that inhibit the kinase activity of EGFR and/or HER-2 after binding to their ATP binding sites are of potential interest as new therapeutic antitumor agents.¹⁹ A number of tyrosine kinase inhibitors (TKIs) targeting EGFR and HER-2 have been approved for the clinical treatment of cancer by the FDA. As shown in Fig. 1, the EGFR inhibitors Gefitinib, Erlotinib, Lapatinib, Vandetanib, Icotinib and Afatinib have been marketed and launched successfully to treat various kinds of cancer in clinical practice. There are also several EGFR kinase inhibitors, such as Dacomitinib, that are already showing promise in phase III clinical trials.

Fig. 1 Chemical structures of EGFR and HER-2 inhibitors.

In previous studies, several pyrazole derivatives have been reported to possess bioactivity in a wide range of applications. The pyrazole moiety makes up the core structure of numerous biologically active compounds, such as antiviral/antitumor,^20–22 antibacterial,^23,24 anti-inflammatory,²⁵ analgesic,²⁶ fungistatic,²⁷ and antihyperglycemic²⁸ agents. Recently, special attention has been paid to the usage of pyrazole to develop anticancer agents. Lv et al. reported a series of novel pyrazole derivatives (PD, Fig. 1) which showed modest to potent EGFR TK inhibitory activity.²⁹ In addition, a series of novel EGFR and HER-2 inhibitors (N1, N2, Fig. 1) with a thiazolyl-pyrazoline scaffold have been discovered.^30,31 Moreover, it was demonstrated that quinoxaline derivatives display a broad spectrum of biological activity; some of these compounds have been described as potential candidates for the treatment of cancer.^32–34

However, to our knowledge, few reports have been dedicated to the design and synthesis of a type of antitumor compound that contains pyrazole and quinoxaline moieties simultaneously. A perusal of the literature revealed that the B ring of pyrazole derivatives (PD, N1 and N2) has a great influence on their activity. In our continued search for potential EGFR and HER-2 inhibitory agents with novel scaffolds, we constrained the spatial location of the B ring by replacing the 3,5-diphenyl-4,5-dihydro-1H-pyrazole moiety with a 3-phenyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazole moiety, then combined the novel tricyclic pyrazole derivative with a thiazolo quinoxaline ring. The two combined substructures were expected to exhibit a synergistic anticancer effect (Fig. 2).

Fig. 2 Design strategy and modification of novel EGFR/HER-2 inhibitors.

2. Results and discussion

2.1. Chemistry

The protocol for the synthesis of the novel tricyclic heterocycles containing thiazolo[4,5-b]quinoxaline derivatives (4a–4y) is summarized in Scheme 1. To explore the influence of the substituted group at the 4-position of the phenyl and 8-position of the tricyclic heterocycles on the compounds’ activity, compounds 4a–4o were synthesized. We also designed compounds 4p–4y to expand the structural diversity of the target compounds. Firstly, the diverse substituted chalcones (2a–2y) were afforded by treating tetralone derivatives with the appropriate aldehyde in the presence of a solution of sodium hydroxide in methanol. Secondly, cyclization of the different chalcones with thiosemicarbazide in the presence of excess sodium hydroxide gave tricyclic heterocycles containing a thiourea group (3a–3y). Finally, the target compounds 4a–4y were obtained by reacting 3a–3y with 2,3-dichloroquinoxaline in ethanol. All of the target compounds gave satisfactory analytical and spectroscopic data.

Scheme 1 Synthetic route for the preparation of the target compounds 4a-4y. Reagents and conditions: (a) ArCHO, 8 N NaOH, ethanol, 2 h, r.t. 65–83%; (b) thiosemicarbazide, NaOH, ethanol, 8 h, reflux, 62–75%; (c) 2,3-dichloroquinoxaline, ethanol, 8 h, reflux, 31–57%.

2.2. Biological activity

2.2.1. Antiproliferation assay. Newly synthesized derivatives 4a–4y were assessed for their antiproliferative activity by employing the MTT-based assay, using Erlotinib and Gefitinib as positive controls against four cultured cancer cell lines including A549, MCF-7, Hela and HepG2 (Table 1). Most target compounds were found to possess broad antiproliferative activity with IC₅₀ values ranging from 2.02 to 31.54 μM. Among them, compounds 4k and 4v displayed the most broad-spectrum antiproliferative activity; their corresponding IC₅₀ values were all below 7.00 μM against the four cancer cell lines. It is noticeable that several compounds exerted antiproliferative activity against the A549 and MCF-7 cell lines comparable to that of the positive control drugs, such as 4b, 4k, 4l, 4o, 4q, 4t, 4v and 4y. In particular, the most significant inhibition was achieved by compound 4l with IC₅₀ = 1.91 μM toward the MCF-7 cell line, which was better than that of Erlotinib (IC₅₀ = 2.32 μM) and Gefitinib (IC₅₀ = 6.26 μM). In addition, compound 4l (IC₅₀ = 3.04 μM) also showed antiproliferative activity against the A549 cell line that was higher than Erlotinib (IC₅₀ = 4.28 μM) and comparable to Gefitinib (IC₅₀ = 2.96 μM).

Table 1 In vitro antiproliferative activity (IC₅₀, μM) of compounds 4a–4y against four human tumor cell lines

Compounds	R1	R2	X	IC50^a (μM)
				A549	MCF-7	Hela	HepG2
a Antiproliferative activity was measured using the MTT assay. The values are the average of three independent experiments run in triplicate. The variation was generally 5–10%.
4a	H	H	C	8.32	6.88	2.98	4.38
4b	H	4-OCH3	C	4.38	3.28	8.25	2.58
4c	H	4-F	C	28.26	15.26	5.38	10.08
4d	H	4-Cl	C	15.26	11.33	15.24	17.18
4e	H	4-CH3	C	4.62	5.86	8.89	5.58
4f	H	2,4-Cl	C	15.34	28.26	12.25	19.56
4g	H	3-Cl	C	15.26	18.26	9.28	10.08
4h	H	2-Cl	C	8.88	8.23	10.33	5.36
4i	H	3-CH3	C	15.36	10.16	16.28	8.99
4j	H	2-CH3	C	13.22	14.87	18.27	19.23
4k	OCH3	H	C	5.38	3.48	6.28	4.18
4l	OCH3	4-OCH3	C	3.04	1.91	7.38	5.76
4m	OCH3	4-F	C	14.19	12.28	10.25	12.61
4n	OCH3	4-Cl	C	24.73	8.23	7.46	15.25
4o	OCH3	4-CH3	C	9.59	2.53	16.23	14.57
4p	H	H	O	4.12	6.83	5.88	12.56
4q	H	4-OCH3	O	4.26	3.23	7.63	6.12
4r	H	4 F	O	10.92	28.22	15.66	18.59
4s	H	4-Cl	O	24.96	13.36	31.54	9.88
4t	H	4-CH3	O	6.82	3.99	6.81	7.26
4u	CH3	H	O	4.47	12.8	2.02	8.29
4v	CH3	4-OCH3	O	6.38	2.02	5.38	5.66
4w	CH3	4 F	O	18.65	21.34	26.98	24.45
4x	CH3	4-Cl	O	28.26	16.26	17.64	17.05
4y	CH3	4-CH3	O	13.67	3.28	7.46	11.38
Erlotinib				4.28	2.32	1.93	3.02
Gefitinib				2.96	6.26	4.12	3.18

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2.2.2. EGFR and HER-2 inhibitory activity. The aforementioned compounds were designed to assess the EGFR/HER-2 inhibitory activity of novel tricyclic heterocycle-containing thiazolo[4,5-b]quinoxaline derivatives. Therefore, all of the compounds were assayed for their inhibitory activity against EGFR and HER-2 tyrosine kinases, using Erlotinib as a positive control.

As shown in Table 2, the pyrazole–quinoxaline derivatives (4a–4y) have demonstrated fairly good EGFR and HER-2 inhibitory activity. Subsequent structure–activity relationship (SAR) analysis indicated that the substituted group on ring A (R₂) played important roles in inhibiting kinase activity; compounds with an electron-donating substituent at the 4-position of ring A exhibited better potency than those with an electron-withdrawing group. For example, 4b (IC₅₀ = 2.26 μM) > 4e (IC₅₀ = 8.26 μM) > 4a (IC₅₀ = 9.82 μM) > 4d (IC₅₀ = 15.67 μM) > 4c (IC₅₀ = 21.24 μM), which was illustrated by the potency order: OCH₃ > CH₃ > H > Cl > F. In addition, compounds (4d and 4e) with methyl or chlorine at the 4-position of ring A displayed more potent activity than compounds (4g–4j) with methyl or chlorine at the 2-position and 3-position of ring A. Compounds (4k–4o) with an electron-donating methoxy group at the 4-position of ring B (R₁) displayed higher inhibitory activity than those (4p–4y) with a methyl or hydrogen substituent. Therefore, the biological data demonstrated that an electron-donating substituent at the R₁ and/or R₂ position might increase the inhibitory activity against EGFR/HER-2. Replacing the 3,3a,4,5-tetrahydro-2H-benz[g]indazole ring with a 2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]pyrazole ring only resulted in a slight decrease in activity (4a–4e vs. 4p–4t). For the given compounds, it was observed that the IC₅₀ values of the inhibitors against EGFR were consistent with those against HER-2, which is possibly attributable to the high sequence homology in the catalytic domains of these two kinases. Among these twenty-five pyrazole–quinoxaline derivatives, 4l seemed to be the most potent EGFR and HER-2 inhibitor with IC₅₀ values of 0.28 μM and 1.26 μM, respectively, compared with the positive control Erlotinib’s IC₅₀ values of 0.08 μM and 0.23 μM.

Table 2 Enzyme inhibition activities of compounds 4a–4y against EGFR and HER-2

Compounds	IC50^a (μM)
	EGFR^b	HER-2^b
a Errors were in the range of 5–10% of the reported values, from three different assays.b Human recombinant enzymes, by the esterase assay (4-nitrophenylacetate as substrate).
4a	9.82	11.43
4b	2.26	3.68
4c	21.24	18.43
4d	15.67	26.58
4e	8.26	12.38
4f	15.83	17.46
4g	14.63	11.06
4h	15.82	3.04
4i	9.26	13.98
4j	15.28	20.88
4k	4.68	5.34
4l	0.28	1.26
4m	8.16	15.24
4n	18.23	25.46
4o	0.96	3.23
4p	3.32	8.68
4q	1.23	5.25
4r	18.26	30.46
4s	15.34	28.4
4t	2.36	5.67
4u	5.23	11.34
4v	0.86	2.35
4w	15.28	33.56
4x	6.8	12.8
4y	1.23	4.66
Erlotinib	0.08	0.23

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Taking the biological data as a whole, we could reach the preliminary conclusion that some of the target compounds are potent EGFR/HER-2 inhibitors, and are helpful for the further research of EGFR inhibitors and expanding the types of compounds that act as EGFR inhibitors.

2.2.3. Cytotoxicity test. As shown in Table 3, all of the target compounds (4a–4y) were evaluated for their toxicity against the human kidney epithelial 293T cell line using the MTT assay; these compounds were tested at multiple doses to study the viability of 293T cell. The median cytotoxic concentration (CC₅₀) showed that all of the tested compounds displayed almost no cytotoxicity in vitro against 293T cells.

Table 3 The median cytotoxic concentration (CC₅₀) data of all compounds

Compounds	CC50^a (μmol)
a The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (CC50).
4a	51.28
4b	55.43
4c	46.78
4d	50.36
4e	53.28
4f	55.33
4g	46.72
4h	49.38
4i	52.68
4j	51.47
4k	61.33
4l	78.44
4m	55.56
4n	51.42
4o	68.45
4p	48.28
4q	56.12
4r	41.56
4s	48.33
4t	52.67
4u	48.16
4v	59.26
4w	51.38
4x	48.34
4y	56.25
Erlotinib	68.03

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2.3. Molecular docking

To further investigate the interactions between these compounds and EGFR, molecular docking of the 3,3a,4,5-tetrahydro-2H-benz[g]indazole derivative 4l and 2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]pyrazole derivative 4x into the ATP binding site of EGFR kinase were performed on the binding model based on the EGFR complex structure (1M17.pdb).³⁵

The docking pose of 4l in its complex with EGFR is shown in Fig. 3A and B. In the binding mode, the oxygen of the methoxy moiety forms hydrogen bonding interactions with Met769 and Gly697. This may explain why compound 4l, with a methoxy substituent at R₁ and R₂, could display increased inhibitory activity against EGFR kinase. In addition, a hydrogen bonding interaction is observed between the nitrogen of the quinoxaline moiety and Asp831. Simultaneously, the terminal phenyl moiety of compound 4l forms a π–π interaction with Phe699. As shown in Fig. 3C and D, a similar π–π interaction also occurs between the terminal phenyl moiety of compound 4x and Phe699. Furthermore, the oxygen of the tricyclic moiety forms a hydrogen bond with Met769 and the nitrogen of the quinoxaline moiety forms hydrogen bonds with Lys721 and Asp831. Comparing these models (Fig. 3E and F), it is obvious that compounds 4l and 4x are able to nicely occupy the active binding pocket of EGFR, as well as Erlotinib. Interestingly, the similar hydrogen bonding interactions with Met769 all occurred in the binding pocket of 4l, 4x and Erlotinib in complex with EGFR.

Fig. 3 Docking modes of compounds in the binding pockets of EGFR (PDB ID: 1M17). Interactions between the protein and the ligand are shown as yellow dotted lines, the residues are shown as orange sticks, and the ligands are shown as stick models in green (4l), cyan (4x) and magenta (Erlotinib). (A) The predicted binding mode of 4l with EGFR; (B) the surface of the binding site of 4l with EGFR; (C) the predicted binding mode of 4x with EGFR; (D) the surface of the binding site of 4x with EGFR; (E) the predicted binding mode of Erlotinib with EGFR; (F) the docking modes of compounds 4l, 4x and Erlotinib in the active site of EGFR.

In summary, the docking analysis strongly suggests that the substituents on the two different rings and the quinoxaline moiety form hydrogen bonding interactions and π–π interactions with the protein residues in the ATP binding site, thus resulting in dramatic EGFR inhibition activity.

2.4. 3D-QSAR model

To study the bioactivity of the pyrazole–quinoxaline derivatives in greater depth, a 3D-QSAR model was built according to the compounds synthesized and their corresponding capabilities.³⁶ In doing so, we intended to explain the mechanism of the SAR, and aid in the discovery of more potent novel antagonists of EGFR. This model was executed by the built-in QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd), with all of the molecules converted to their active conformation and the corresponding IC₅₀ (μM) values transformed from an Indian medicinal chemistry lab (http://www.sanjeevslab.org/tools-IC50.html). By a random selection, these compounds were divided into a test set containing 5 compounds and a training set comprising the rest of the 20 compounds.

By default, the alignment conformation of each molecule possessed the lowest CDOCKER_INTERACTION_ENERGY of all of the docked poses. The critical regions (steric or electrostatic) affecting the binding affinity were identified by this 3D-QSAR model. Using a CHARMM force field and PLS regression, the model was set up with a conventional R² of 0.95, indicating this model possesses a good predictive capability. The relationship between the observed and predicted values has been shown graphically in Fig. 4.

Fig. 4 Plot of the experimental vs. predicted values for the EGFR inhibitory activity of the training set and test set.

Also, the molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on electrostatic potential grids (Fig. 5a) and van der Waals grids (Fig. 5b) were listed. The electrostatic map displayed the favorable (in blue) or unfavorable (in red) electrostatic field regions in a contour plot, while the energy grids corresponding to the favorable (in green) or unfavorable (in yellow) steric effects were also marked out. For compounds based on the 3D-QSAR model, possessing strong van der Waals attraction forces in the green areas and a polar group in the blue electrostatic potential areas means achieving potent bioactivity. The results yielded by the model were accordant with the actual findings, in terms of identifying the potent compounds. Thus, this promising model could provide guidelines for the design and optimization of more effective tubulin inhibitors and pave the way for further study.

Fig. 5 (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients and red represents negative coefficients. (b) 3D-QSAR model coefficients on van der Waals grids. Green represents positive coefficients and yellow represents negative coefficients.

3. Conclusions

In this work, a series of pyrazole–quinoxaline derivatives that potentially function as inhibitors of EGFR and HER-2 kinases have been synthesized, and most of them exhibited potent affinity for EGFR or HER-2 kinase as well as excellent antiproliferative activity. Compound 4l showed excellent EGFR/HER-2 inhibitory activities and better antiproliferative activity against A549 and MCF-7 cell lines than Erlotinib. After analysis of the binding models of compounds 4l and 4x with EGFR, it was found that the models of compounds 4l or 4x in complex with the ATP binding site were similar to that of Erlotinib. The quinoxaline moiety might play a crucial role in the compounds’ EGFR inhibition activity by forming π–π interactions and hydrogen bonding interactions with the residues in the binding pocket. Finally, QSAR models were built with the activity data and binding conformations to begin our work in this paper, as well as to provide a reliable tool for the reasonable design and synthesis of potent tyrosine kinase inhibitors. In conclusion, the preliminary evaluation results demonstrated that the newly developed tricyclic heterocycle-containing pyrazoles combined with a thiazolo quinoxaline ring, acting as potent dual EGFR and HER-2 inhibitors, may possess therapeutic potential for cancer treatment.

4. Experiments

4.1. Materials and measurements

All chemicals and reagents used in this study were analytical grade. Thin-layer chromatography (TLC) was performed on silica gel plates with a fluorescent indicator. All of the analyzed samples were homogeneous when assessed using TLC in at least two different solvent systems. All of the ¹H NMR spectra were recorded on a Bruker DPX 300 model spectrometer in DMSO-d₆ and the chemical shifts (δ) were reported in parts per million (ppm). ESI-MS spectra were recorded on a Mariner System 5304 mass spectrometer. Elementary analyses were performed on an Elementar Vario EL III instrument.

4.2. Synthesis

4.2.1. General synthetic procedure of chalcones (2a–2y). To a solution of tetralone derivatives 1a–1d (50 mmol) and appropriate aldehyde (50 mmol) in ethanol (20 mL) was slowly added an aqueous solution of NaOH (0.012 mol, 8 N) at room temperature. The mixture was stirred for 2 h. The solid precipitate was collected by filtration and then washed with cold ethanol (30 mL) three times. The solid was dried in vacuo to give each chalcone (2a–2y).

4.2.2. General synthetic procedure of pyrazole derivatives (3a–3y). A mixture of chalcones 2a–2y (10 mmol), thiosemicarbazide (10 mmol) and NaOH (25 mmol) in ethanol (25 mL) was refluxed for 8 h. The solution was poured into ice water and then filtered. The residue was washed with a small amount of methanol and dried in vacuo to give each pyrazole derivative (3a–3y).

4.2.3. General synthetic procedure of dihydropyridin containing thiazolinone derivatives (4a–4y). A mixture of pyrazole derivatives 3a–3y (10 mmol) and 2,3-dichloroquinoxaline (10 mmol) in absolute ethanol (15 mL) was refluxed for 8 h. The progress of the reaction was monitored using TLC. After the completion of the reaction, the solvent was removed under reduced pressure and the residue was recrystallized from methanol to give 4a–4y.
4.2.3.1. 2-(3-Phenyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4a). White solid. Yield 51%; m.p. 201–203 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 7.94 (d, J = 6.4 Hz, 1H, Ar-H); 7.90 (d, J = 7.8 Hz, 1H, Ar-H); 7.84–7.81 (m, 2H, Ar-H); 7.63–7.53 (m, 2H, Ar-H); 7.49–7.32 (m, 2H, Ar-H); 7.30–7.28 (m, 1H, Ar-H); 7.03–6.89 (m, 4H, Ar-H); 6.08 (d, J = 10.7 Hz, 1H, CH); 4.10–4.03 (m, 1H, CH); 3.01–2.82 (m, 2H, CH₂); 1.74–1.60 (m, 1H, CH₂); 0.96–0.81 (m, 1H, CH₂). ESI-MS m/z: 434.2 [M + H]⁺. Anal. calcd for C₂₆H₁₉N₅S (%): C, 72.03; H, 4.42; N, 16.15. Found: C, 72.43; H, 4.33; N, 16.28.
4.2.3.2. 2-(3-(4-Methoxyphenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4b). White solid. Yield 47%; m.p. 186–187 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.04 (d, J = 7.4 Hz, 1H, Ar-H); 7.98 (d, J = 7.7 Hz, 1H, Ar-H); 7.86–7.84 (m, 1H, Ar-H); 7.73–7.62 (m, 2H, Ar-H); 7.48–7.36 (m, 2H, Ar-H); 7.31–7.29 (m, 1H, Ar-H); 7.12 (d, J = 8.2 Hz, 2H, Ar-H); 6.91 (d, J = 8.5 Hz, 2H, Ar-H); 6.10 (d, J = 10.6 Hz, 1H, CH); 4.14-4.02 (m, 1H, CH); 3.70 (s, 3H, CH₃); 3.02–2.86 (m, 2H, CH₂); 1.84–1.81 (m, 1H, CH₂); 1.06–0.96 (m, 1H, CH₂). ESI-MS m/z: 464.3 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅OS (%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.85; H, 4.49; N, 15.28.
4.2.3.3. 2-(3-(4-Fluorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4c). White solid. Yield 48%; m.p. 202–204 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.08 (d, J = 7.5 Hz, 1H, Ar-H); 7.99–7.95 (m, 1H, Ar-H); 7.82–7.79 (m, 1H, Ar-H); 7.40–7.28 (m, 3H, Ar-H); 7.23–7.20 (m, 1H, Ar-H); 7.15–7.09 (m, 3H, Ar-H); 7.05–7.00 (m, 2H, Ar-H); 6.03 (d, J = 10.6 Hz, 1H, CH); 3.87–3.37 (m, 1H, CH); 2.92–2.76 (m, 2H, CH₂); 1.78–1.74 (m, 1H, CH₂); 0.80–0.66 (m, 1H, CH₂). ESI-MS m/z: 452.2 [M + H]⁺. Anal. calcd for C₂₆H₁₈FN₅S (%): C, 69.16; H, 4.02; N, 15.51. Found: C, 69.33; H, 4.09; N, 15.41.
4.2.3.4. 2-(3-(4-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4d). Yellow solid. Yield 37%; m.p. 211–213 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.05–7.97 (m, 2H, Ar-H); 7.89–7.84 (m, 1H, Ar-H); 7.72–7.63 (m, 2H, Ar-H); 7.58–7.48 (m, 2H, Ar-H); 7.44–7.35 (m, 3H, Ar-H); 7.31–7.23 (m, 2H, Ar-H); 6.17 (d, J = 10.7 Hz, 1H, CH); 4.06–4.00 (m, 1H, CH); 2.98–2.87 (m, 2H, CH₂); 1.72–1.70 (m, 1H, CH₂); 0.97–0.93 (m, 1H, CH₂). ESI-MS m/z: 468.2 [M + H]⁺. Anal. calcd for C₂₆H₁₈ClN₅S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.63; H, 3.69; N, 14.78.
4.2.3.5. 2-(3-(P-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4e). Yellow solid. Yield 40%; m.p. 191–193 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.12–8.03 (m, 1H, Ar-H); 7.97 (d, J = 7.7 Hz, 1H, Ar-H); 7.82–7.78 (m, 1H, Ar-H); 7.42–7.30 (m, 2H, Ar-H); 7.33–7.21 (m, 2H, Ar-H); 7.15–7.10 (m, 2H, Ar-H); 7.08–7.01 (m, 1H, Ar-H); 6.95–6.80 (m, 2H, Ar-H); 6.13 (d, J = 10.3 Hz, 1H, CH); 3.57–3.37 (m, 1H, CH); 2.82–2.66 (m, 2H, CH₂); 2.41 (s, 3H, CH₃); 1.80–1.73 (m, 1H, CH₂); 0.85–0.79 (m, 1H, CH₂). ESI-MS m/z: 448.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅S (%): C, 72.46; H, 4.73; N, 15.65. Found: C, 72.63; H, 4.69; N, 15.58.
4.2.3.6. 2-(3-(2,4-Dichlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4f). White solid. Yield 57%; m.p. 196–198 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.04–7.99 (m, 2H, Ar-H); 7.89–7.81 (m, 2H, Ar-H); 7.74–7.67 (m, 2H, Ar-H); 7.47–7.30 (m, 4H, Ar-H); 7.00 (d, J = 8.3Hz, 1H, CH); 6.41 (d, J = 11.3 Hz, 1H, CH); 4.36–4.30 (m, 1H, CH); 3.04–2.87 (m, 2H, CH₂); 1.94–1.90 (m, 1H, CH₂); 1.10–1.07 (m, 1H, CH₂). ESI-MS m/z: 502.2 [M + H]⁺. Anal. calcd for C₂₆H₁₇Cl₂N₅S (%): C, 62.16; H, 3.41; N, 13.94. Found: C, 62.32; H, 3.35; N, 14.04.
4.2.3.7. 2-(3-(3-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4g). Yellow solid. Yield 32%; m.p. 186–187 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.01–7.94 (m, 2H, Ar-H); 7.88–7.82 (m, 1H, Ar-H); 7.70–7.53 (m, 3H, Ar-H); 7.48–7.43 (m, 1H, Ar-H); 7.40–7.32 (m, 3H, Ar-H); 7.30–7.24 (m, 2H, Ar-H); 6.13 (d, J = 10.6 Hz, 1H, CH); 4.03–3.98 (m, 1H, CH); 2.95–2.82 (m, 2H, CH₂); 1.70–1.65 (m, 1H, CH₂); 0.96–0.90 (m, 1H, CH₂). ESI-MS m/z: 468.3 [M + H]⁺. Anal. calcd for C₂₆H₁₈ClN₅S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.63; H, 3.96; N, 14.86.
4.2.3.8. 2-(3-(2-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4h). Yellow solid. Yield 42%; m.p. 187–189 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.11 (d, J = 8.1 Hz, 1H, Ar-H); 8.07–7.95 (m, 2H, Ar–H); 7.72–7.69 (m, 1H, Ar–H); 7.66–7.59 (m, 1H, Ar-H); 7.51 (d, J = 7.9 Hz, 1H, Ar-H); 7.43 (d, J = 9.1 Hz, 1H, Ar-H); 7.34–7.29 (m, 3H, Ar-H); 7.21–7.13 (m, 2H, Ar-H); 6.20 (d, J = 10.5 Hz, 1H, CH); 4.11–4.05 (m, 1H, CH); 2.99–2.83 (m, 2H, CH₂); 1.85–1.71 (m, 1H, CH₂); 1.07–1.03 (m, 1H, CH₂). ESI-MS m/z: 468.2 [M + H]⁺. Anal. calcd for C₂₆H₁₈ClN₅S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.62; H, 3.86; N, 16.89.
4.2.3.9. 2-(3-(M-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4i). Yellow solid. Yield 48%; m.p. 178–180 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.08 (d, J = 7.6 Hz, 1H, Ar-H); 7.99 (d, J = 8.3 Hz, 1H, Ar-H); 7.82–7.77 (m, 1H, Ar-H); 7.42–7.31 (m, 2H, Ar-H); 7.30–7.21 (m, 2H, Ar-H); 7.15–7.10 (m, 2H, Ar-H); 7.08–7.01 (m, 1H, Ar-H); 6.95–6.80 (m, 2H, Ar-H); 6.13 (d, J = 10.2 Hz, 1H, CH); 3.58–3.47 (m, 1H, CH); 2.85–2.76 (m, 2H, CH₂); 2.48 (s, 3H, CH₃); 1.80–1.72 (m, 1H, CH₂); 0.86–0.75 (m, 1H, CH₂). ESI-MS m/z: 448.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅S (%): C, 72.46; H, 4.73; N, 15.65. Found: C, 72.61; H, 4.69; N, 15.58.
4.2.3.10. 2-(3-(M-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4j). White solid. Yield 42%; m.p. 188–190 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.12–7.96 (m, 2H, Ar–H); 7.80 (d, J = 7.7 Hz, 1H, Ar–H); 7.35 (d, J = 7.6 Hz, 2H, Ar-H); 7.30–7.22 (m, 2H, Ar-H); 7.13–7.10 (m, 2H, Ar-H); 7.08–6.92 (m, 3H, Ar-H); 6.03 (d, J = 10.7 Hz, 1H, CH); 3.56–3.41 (m, 1H, CH); 2.82–2.69 (m, 2H, CH₂); 2.39 (s, 3H, CH₃); 1.81–1.75 (m, 1H, CH₂); 0.85–0.73 (m, 1H, CH₂). ESI-MS m/z: 448.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅S (%): C, 72.46; H, 4.73; N, 15.65. Found: C, 72.61; H, 4.66; N, 15.68.
4.2.3.11. 2-(8-Methoxy-3-phenyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4k). White solid. Yield 43%; m.p. 196–198 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 7.98 (d, J = 6.8 Hz, 1H, Ar-H); 7.84 (d, J = 7.3 Hz, 1H, Ar-H); 7.71–7.64 (m, 2H, Ar-H); 7.50–7.42 (m, 1H, Ar-H); 7.34–7.29 (m, 3H, Ar-H); 7.21–7.20 (m, 3H, Ar-H); 7.07–7.05 (m, 1H, Ar-H); 6.16 (d, J = 10.7 Hz, 1H, CH); 4.16–4.08 (m, 1H, CH); 3.85 (s, 3H, CH₃); 2.89–2.81 (m, 2H, CH₂); 1.85–1.82 (m, 1H, CH₂); 0.86–0.79 (m, 1H, CH₂). ESI-MS m/z: 464.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅OS (%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.69; H, 4.49; N, 15.28.
4.2.3.12. 2-(8-Methoxy-3-(4-methoxyphenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4l). White solid. Yield 34%; m.p. 184–186 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.01 (d, J = 7.2 Hz, 1H, Ar-H); 7.96 (d, J = 7.7 Hz, 1H, Ar-H); 7.86–7.83 (m, 1H, Ar-H); 7.73–7.66 (m, 2H, Ar-H); 7.45–7.34 (m, 2H, Ar-H); 7.31–7.28 (m, 1H, Ar-H); 7.12 (d, J = 8.2 Hz, 2H, Ar-H); 6.90 (d, J = 8.5 Hz, 1H, Ar-H); 6.11 (d, J = 10.6 Hz, 1H, CH); 4.14–4.08 (m, 1H, CH); 3.70 (s, 3H, CH₃); 3.59 (s, 3H, CH₃); 3.04–2.83 (m, 2H, CH₂); 1.84–1.80 (m, 1H, CH₂); 1.02–0.94 (m, 1H, CH₂). ESI-MS m/z: 494.2 [M + H]⁺. Anal. calcd for C₂₈H₂₃N₅O₂S (%): C, 68.13; H, 4.70; N, 14.19. Found: C, 68.28; H, 4.59; N, 14.38.
4.2.3.13. 2-(3-(4-Fluorophenyl)-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4m). White solid. Yield 47%; m.p. 181–183 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.10–8.01 (m, 1H, Ar-H); 7.97–7.92 (m, 1H, Ar-H); 7.65 (d, J = 2.6 Hz, 1H, Ar-H); 7.55–7.51 (m, 1H, Ar-H); 7.28–7.20 (m, 1H, Ar-H); 7.16–7.09 (m, 3H, Ar-H); 7.04–6.94 (m, 3H, Ar-H); 6.01 (d, J = 10.6 Hz, 1H, CH); 3.80 (s, 3H, CH₃); 3.80–3.67 (m, 1H, CH); 2.81–2.73 (m, 2H, CH₂); 1.77–1.73 (m, 1H, CH₂); 0.76–0.61 (m, 1H, CH₂). ESI-MS m/z: 482.2 [M + H]⁺. Anal. calcd for C₂₇H₂₀FN₅OS (%): C, 67.34; H, 4.19; N, 14.54. Found: C, 67.63; H, 4.29; N, 14.48.
4.2.3.14. 2-(3-(4-Chlorophenyl)-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4n). Yellow solid. Yield 33%; m.p. 203–205 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.03–7.91 (m, 2H, Ar-H); 7.88–7.67 (m, 3H, Ar-H); 7.58–7.48 (m, 2H, Ar-H); 7.44–7.38 (m, 1H, Ar-H); 7.35–7.32 (m, 1H, Ar-H); 7.31–7.22 (m, 2H, Ar-H); 6.13 (d, J = 10.7 Hz, 1H, CH); 4.12–4.07 (m, 1H, CH); 3.81 (s, 3H, CH₃); 2.87–2.81 (m, 2H, CH₂); 1.66–1.61 (m, 1H, CH₂); 0.95–0.88 (m, 1H, CH₂). ESI-MS m/z: 498.2 [M + H]⁺. Anal. calcd for C₂₇H₂₀ClN₅OS (%): C, 65.12; H, 4.05; N, 14.06. Found: C, 65.33; H, 4.09; N, 14.28.
4.2.3.15. 2-(8-Methoxy-3-(p-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4o). Yellow solid. Yield 32%; m.p. 202–204 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 7.92–7.89 (m, 1H, Ar-H); 7.97 (d, J = 7.7 Hz, 1H, Ar-H); 7.80 (d, J = 8.7 Hz, 1H, Ar-H); 7.35 (d, J = 5.4 Hz, 1H, Ar-H); 7.30–7.21 (m, 2H, Ar-H); 7.13–7.07 (m, 2H, Ar-H); 7.05–7.01 (m, 1H, Ar-H); 6.93–6.77 (m, 2H, Ar-H); 6.10 (d, J = 10.9 Hz, 1H, CH); 3.81 (s, 3H, CH₃); 3.57–3.35 (m, 1H, CH); 2.85–2.62 (m, 2H, CH₂); 2.38 (s, 3H, CH₃); 1.82–1.71 (m, 1H, CH₂); 0.88–0.82 (m, 1H, CH₂). ESI-MS m/z: 478.2 [M + H]⁺. Anal. calcd for C₂₈H₂₃N₅OS (%): C, 70.42; H, 4.85; N, 14.66. Found: C, 70.63; H, 4.59; N, 14.48.
4.2.3.16. 3-Phenyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4p). White solid. Yield 49%; m.p. 193–195 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.04 (d, J = 6.3 Hz, 1H, Ar-H); 7.97 (d, J = 7.8 Hz, 1H, Ar-H); 7.88–7.81 (m, 2H, Ar-H); 7.66–7.55 (m, 2H, Ar-H); 7.51–7.37 (m, 3H, Ar-H); 7.30–7.23 (m, 2H, Ar-H); 7.10–6.95 (m, 2H, Ar-H); 5.98 (d, J = 10.7 Hz, 1H, CH); 4.63–4.55 (m, 1H, CH); 4.14–4.12 (m, 1H, CH); 4.07–4.03 (m, 1H, CH). ESI-MS m/z: 436.2 [M + H]⁺. Anal. calcd for C₂₅H₁₇N₅OS (%): C, 68.95; H, 3.93; N, 16.08. Found: C, 68.73; H, 3.79; N, 16.28.
4.2.3.17. 3-(4-Methoxyphenyl)-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4q). White solid. Yield 43%; m.p. 207–209 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.03 (d, J = 7.3 Hz, 1H, Ar-H); 7.97 (d, J = 7.5 Hz, 1H, Ar-H); 7.86–7.74 (m, 3H, Ar-H); 7.46–7.34 (m, 2H, Ar-H); 7.32–7.25 (m, 1H, Ar-H); 7.12–6.99 (m, 4H, Ar-H); 6.20 (d, J = 10.4 Hz, 1H, CH); 4.44–4.41 (m, 1H, CH₂); 4.24–4.12 (m, 1H, CH); 3.86–3.76 (m, 1H, CH₂); 3.78 (s, 3H, CH₃); . ESI-MS m/z: 466.2 [M + H]⁺. Anal. calcd for C₂₆H₁₉N₅O₂S (%): C, 67.08; H, 4.11; N, 15.04. Found: C, 67.32; H, 4.22; N, 15.15.
4.2.3.18. 3-(4-Fluorophenyl)-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4r). White solid. Yield 37%; m.p. 211–213 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.06 (d, J = 7.3 Hz, 1H, Ar-H); 7.99–7.97 (m, 1H, Ar-H); 7.82–7.76 (m, 1H, Ar-H); 7.40–7.33 (m, 1H, Ar-H); 7.30–7.28 (m, 2H, Ar-H); 7.23–7.20 (m, 1H, Ar-H); 7.15–7.09 (m, 3H, Ar-H); 7.05–7.00 (m, 2H, Ar-H); 6.02 (d, J = 10.9 Hz, 1H, CH); 4.66–4.58 (m, 1H, CH₂), 4.11–4.08 (m, 1H, CH₂); 3.87–3.37 (m, 1H, CH). ESI-MS m/z: 454.2 [M + H]⁺. Anal. calcd for C₂₅H₁₆FN₅OS (%): C, 66.21; H, 3.56; N, 15.44. Found: C, 66.53; H, 3.49; N, 15.53.
4.2.3.19. 3-(4-Chlorophenyl)-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4s). Yellow solid. Yield 31%; m.p. 174–176 °C;¹H NMR (DMSO-d₆, 300 MHz); δ: 8.01–7.95 (m, 2H, Ar-H); 7.88–7.82 (m, 1H, Ar-H); 7.720–7.66 (m, 2H, Ar-H); 7.54–7.49 (m, 2H, Ar-H); 7.43–7.37 (m, 3H, Ar-H); 7.31–7.25 (m, 2H, Ar-H); 6.07 (d, J = 10.1 Hz, 1H, CH); 4.41–4.33 (dd, J = 10.1 and 12.2 Hz, 1H, CH₂); 4.06–3.97 (m, 1H, CH); 3.89–3.77 (dd, J = 6.1 and 10.2 Hz, 1H, CH₂). ESI-MS m/z: 470.1 [M + H]⁺. Anal. calcd for C₂₅H₁₆ClN₅OS (%): C, 63.89; H, 3.43; N, 14.90. Found: C, 63.63; H, 3.49; N, 14.78.
4.2.3.20. 2-(Thiazolo[4,5-b]quinoxalin-2-yl)-3-(p-tolyl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4t). Yellow solid. Yield 32%; m.p. 197–199 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.10 (d, J = 7.7 Hz, 1H, Ar-H); 7.97 (d, J = 4.4 Hz, 1H, Ar-H); 7.82–7.78 (m, 1H, Ar-H); 7.42–7.22 (m, 4H, Ar-H); 7.13–7.09 (m, 2H, Ar-H); 7.03–6.93 (m, 3H, Ar-H); 6.33 (d, J = 10.6 Hz, 1H, CH); 4.42–4.37 (m, 1H, CH₂); 3.88–3.78 (m, 1H, CH₂); 3.47–3.37 (m, 1H, CH); 2.41 (s, 3H, CH₃). ESI-MS m/z: 450.2 [M + H]⁺. Anal. calcd for C₂₆H₁₉N₅OS (%): C, 69.47; H, 4.26; N, 15.58. Found: C, 69.63; H, 4.39; N, 15.48.
4.2.3.21. 8-Methyl-3-phenyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4u). White solid. Yield 42%; m.p. 187–189 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 7.99 (d, J = 8.7 Hz, 2H, Ar-H); 7.82–7.77 (d, J = 6.7 Hz, 1H, Ar-H); 7.42–7.21 (m, 4H, Ar-H); 7.16–7.11 (m, 2H, Ar-H); 7.08–7.03 (m, 1H, Ar-H); 6.93–6.83 (m, 2H, Ar-H); 6.03 (d, J = 10.1 Hz, 1H, CH); 4.51–4.42 (m, 1H, CH₂); 3.80–3.77 (m, 1H, CH₂); 3.55–3.43 (m, 1H, CH); 2.48 (s, 3H, CH₃). ESI-MS m/z: 450.2 [M + H]⁺. Anal. calcd for C₂₆H₁₉N₅OS (%): C, 69.47; H, 4.26; N, 15.58. Found: C, 69.63; H, 4.33; N, 15.48.
4.2.3.22. 3-(4-Methoxyphenyl)-8-methyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4v). Yellow solid. Yield 36%; m.p. 192–194 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.02–7.95 (m, 1H, Ar–H); 7.88–7.82 (m, 1H, Ar-H); 7.77 (d, J = 8.7 Hz, 1H, Ar-H); 7.45–7.30 (m, 3H, Ar-H); 7.22–7.17 (m, 2H, Ar-H); 7.10–6.88 (m, 3H, Ar-H); 6.25 (d, J = 10.4 Hz, 1H, CH); 4.44–4.33 (dd, J = 10.3 and 12.8 Hz, 1H, CH₂); 3.81 (s, 3H, CH₃); 3.65–3.59 (dd, J = 6.0 and 10.2 Hz, 1H, CH₂); 3.55–3.42 (m, 1H, CH); 2.33 (s, 3H, CH₃). ESI-MS m/z: 480.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅O₂S (%): C, 67.62; H, 4.41; N, 14.60. Found: C, 67.38; H, 4.49; N, 14.48.
4.2.3.23. 3-(4-Fluorophenyl)-8-methyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4w). White solid. Yield 42%; m.p. 199–201 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.12–8.01 (m, 1H, Ar-H); 7.96–7.92 (m, 1H, Ar-H); 7.62 (d, J = 3.6 Hz, 1H, Ar-H); 7.54–7.50 (m, 1H, Ar-H); 7.28–7.21 (m, 1H, Ar-H); 7.16–7.09 (m, 3H, Ar-H); 7.04–6.94 (m, 3H, Ar-H); 6.06 (d, J = 10.9 Hz, 1H, CH); 4.55–4.49 (dd, J = 11.1 and 13.2 Hz, 1H, CH₂); 3.80–3.69 (m, 1H, CH); 3.55–3.49 (dd, J = 5.1 and 10.2 Hz, 1H, CH₂); 2.34 (s, 3H, CH₃). ESI-MS m/z: 468.2 [M + H]⁺. Anal. calcd for C₂₆H₁₈FN₅OS (%): C, 66.79; H, 3.88; N, 14.98. Found: C, 66.63; H, 3.79; N, 14.73.
4.2.3.24. 3-(4-Chlorophenyl)-8-methyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4x). White solid. Yield 51%; m.p. 205–207 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.04–7.96 (m, 2H, Ar-H); 7.85–7.80 (m, 2H, Ar-H); 7.75–7.63 (m, 2H, Ar-H); 7.47–7.30 (m, 4H, Ar-H); 6.94 (d, J = 8.3 Hz, 1H, CH); 6.31 (d, J = 10.2 Hz, 1H, CH); 4.60–4.51 (m, 1H, CH₂); 4.26–4.20 (m, 1H, CH); 3.88–3.79 (m, 1H, CH₂); 2.41 (s, 3H, CH₃). ESI-MS m/z: 484.1 [M + H]⁺. Anal. calcd for C₂₆H₁₈ClN₅OS (%): C, 64.52; H, 3.75; N, 14.47. Found: C, 64.63; H, 3.59; N, 14.38.
4.2.3.25. 8-Methyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-3-(p-tolyl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4y). Yellow solid. Yield 43%; m.p. 196–198 °C; ¹H NMR (DMSO-d₆, 300 MHz); δ: 8.03 (d, J = 7.2 Hz, 1H, Ar-H); 7.92 (d, J = 7.3 Hz, 1H, Ar-H); 7.82–7.72 (m, 1H, Ar-H); 7.42–7.21 (m, 4H, Ar-H); 7.13–7.02 (m, 2H, Ar-H); 6.92–6.80 (m, 2H, Ar-H); 6.19 (d, J = 10.8 Hz, 1H, CH); 4.49–4.39 (dd, J = 10.3 and 12.8 Hz, 1H, CH₂); 3.77–3.69 (dd, J = 5.1 and 10.2 Hz, 1H, CH₂); 3.53–3.44 (m, 1H, CH); 2.48 (s, 3H, CH₃); 2.44 (s, 3H, CH₃); ESI-MS m/z: 464.2 [M + H]⁺. Anal. calcd for C₂₇H₂₁N₅OS (%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.63; H, 4.49; N, 15.28.

4.3. Preparation and purification of EGFR and HER-2 and the inhibitory assay

A 1.6 kb cDNA encoding the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186) and 1.7 Kb cDNA encoding the human HER-2 cytoplasmic domain (HER-2-CD, amino acids 676–1245) were cloned into the baculoviral expression vectors pBlueBacHis2B and pFASTBac HTc (Huakang Company, China), separately. A sequence that encodes (His)₆ was located at the 5′ upstream of the EGFR and HER-2 sequences. Sf-9 cells were infected for 3 days for protein expression. Sf-9 cell pellets were solubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10 μM ammonium molybdate, 100 lM sodium vanadate, 10 μg mL⁻¹ aprotinin, 10 μg mL⁻¹ leupeptin, 10 μg mL⁻¹ pepstatin, and 16 μg mL⁻¹ benzamidine HCl for 20 min followed by 20 min centrifugation. The crude extract supernatant was passed through an equilibrated Ni-NTA superflow packed column and washed with 10 mM and then 100 mM imidazole to remove nonspecifically bound material. Histidine tagged proteins were eluted with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1 μg mL⁻¹ each of aprotinin, leupeptin, and pepstatin for 2 h. The entire purification procedure was performed at 4 °C or on ice.

The EGFR and HER-2 kinase assays were both set up to assess the level of autophosphorylation based on DELFIA/time-resolved fluorometry. Compounds 4a–4y were dissolved in 100% DMSO and diluted to the appropriate concentrations with 25 mM HEPES at pH 7.4. In each well, 10 μL of each compound was incubated with 10 μL (12.5 ng for HER-2 or 5 ng for EGFR) recombinant enzyme (1 : 80 dilution in 100 mM HEPES) for 10 min at room temperature. Then, 10 μL of 5 mM buffer (containing 20 mM HEPES, 2 mM MnCl₂, 100 μM Na₃VO₄, and 1 mM DTT) and 20 μL of 0.1 mM ATP–50 mM MgCl₂ were added for 1 h. Positive and negative controls were included in each plate by incubation of the enzyme with or without ATP–MgCl₂. At the end of incubation, the liquid was aspirated, and plates were washed three times with wash buffer. A 75 μL (400 ng) sample of europium labeled anti-phosphotyrosine antibody was added to each well for another 1 h of incubation. After washing, an enhancement solution was added and the signal was detected using a Victor multilabel reader (Wallac Inc.) with excitation at 340 nm and emission at 615 nm. The percentage of auto-phosphorylation inhibition by the compounds was calculated using the following formula: 100% − [(negative control)/(positive control − negative control)]. The IC₅₀ was obtained from curves of the percentage inhibition with eight concentrations of the compound. As the amount of contamination in the enzyme preparation is fairly low, the majority of the signal detected by the anti-phosphotyrosine antibody is from EGFR or HER-2.

4.4. Antiproliferative activity

The antiproliferative activities of the prepared compounds were evaluated using a standard (MTT)-based colorimetric assay with some modifications. Cell lines were grown to log phase in DMEM supplemented with 10% fetal bovine serum, under a humidified atmosphere of 5% CO₂ at 37 °C. Cell suspensions were prepared and 100 μL per well was dispensed into 96-well plates giving 10⁵ cells per well. The plates were returned to the incubator for 24 h to allow the cells to reattach. Subsequently, cells were treated with the target compounds at increasing concentrations in the presence of 10% FBS for 48 h. Then, cell viability was assessed using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and carried out strictly according to the manufacturer’s instructions (Sigma). The absorbance (OD₅₇₀) was read on an ELISA reader (Tecan, Austria).

4.5. Experimental protocol of the docking study

Molecular docking of compounds 4l and 4x into the three dimensional X-ray structure of EGFR (PDB code: 1M17) was carried out using the Discovery Studio (version 3.5) as implemented through the graphical user interface DS-CDOCKER protocol. The three-dimensional structures of the aforementioned compounds were constructed using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2013)], then they were energetically minimized by using MMFF94 with 5000 iterations and a minimum RMS gradient of 0.10. The crystal structures of the protein complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters and ligands were eliminated from the protein. The molecular docking was performed by inserting compounds 4l and 4x into the binding pocket of EGFR based on the binding mode. The types of interactions of the docked protein with the ligand-based pharmacophore model were analyzed after the end of molecular docking.

4.6. 3D-QSAR

The ligand-based 3D-QSAR approach was performed using the QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The training sets were composed of inhibitors with the corresponding pIC₅₀ values, which were converted from the obtained IC₅₀ (μM) values, and test sets comprised compounds of data sets, as listed in Table 4.

Table 4 Experimental and predicted inhibitory activity of compounds 4a-4y against EGFR protein using the 3D-QSAR model

Compounds	EGFR
	Actual-pIC50	Predicted-pIC50
4a	5.01	5.08
4b	5.65	5.70
4c	4.67	4.56
4d	4.80	4.64
4e	5.08	5.14
4f	4.97	5.12
4g	5.33	5.24
4h	5.74	5.61
4i	5.03	5.26
4j	4.82	5.00
4k	5.25	5.31
4l	6.55	6.54
4m	5.09	4.99
4n	4.74	4.92
4o	6.02	6.01
4p	5.48	5.41
4q	5.91	5.83
4r	4.74	4.71
4s	4.81	4.75
4t	5.63	5.58
4u	5.28	5.47
4v	6.07	6.04
4w	4.82	4.76
4x	5.17	5.10
4y	5.91	5.811

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All of the definition of the descriptors can be seen in the “Help” of the DS 3.5 software and they were calculated using the QSAR protocol of DS 3.5. The alignment conformation of each molecule was the one with the lowest interaction energy in the docking results of CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on the cross-validated correlation coefficient, which qualifies the predictive ability of the models. A Y scrambling procedure was performed to investigate the risk of chance correlations. The inhibitory potencies of compounds were randomly reordered 30 times and subjected to leave-one-out validation tests. The models were also validated by test sets, in which the compounds were not included in the training sets.

Acknowledgements

This work is supported by The Fundamental Research Funds for the Central Universities (2242014R30019), Technology Supporting Program of Jiangsu province (BE2012657), and National Basin Research Program of China (no. 2011CB933503). We thank Pro. Hai-liang Zhu (State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University) for a license to use their software.

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