Targeting HER2/VEGFR2 dual tyrosine kinases with novel Lapatinib and Neratinib hybrid analogues lead to potential apoptotic induction in HER2 positive breast cancers: Design, synthesis, in-vitro, in-vivo and molecular docking studies.

doi:10.21203/rs.3.rs-3241973/v1

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Targeting HER2/VEGFR2 dual tyrosine kinases with novel Lapatinib and Neratinib hybrid analogues lead to potential apoptotic induction in HER2 positive breast cancers: Design, synthesis, in-vitro, in-vivo and molecular docking studies.

https://doi.org/10.21203/rs.3.rs-3241973/v1

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A high percentage of women worldwide will develop breast cancer during their lifetime, and there will always be a need to look for novel breast cancer treatment possibilities. The co-expression of HER2 and VEGFR2 in some breast cancers has been associated with a more aggressive tumour phenotype and poorer prognosis. As part of continuing research focusing on the possibility of simultaneously targeting HER2 and VEGFR2, we describe the design and synthesis of new lapatinib and neratinib hybrid analogues and their in vitro and in vivo evaluation for anti-cancer activity. We used the drug extension strategy to tailor the designed compounds to fit the RTKs, such as EGFR VEGFR2 and HER2 hydrophobic subpocket and cleft regions. The designed lapatinib and neratinib derivatives were successfully synthesized using established synthetic procedures and characterized using ¹H, ¹³C-NMR, HRMS, and elemental analysis. The synthesized compounds were initially tested for their RTK inhibition capabilities, and compounds 15i and 15g were found to possess potential HER2 and VEGFR2 kinase inhibition abilities in-vitro with an IC₅₀ less than the standards lapatinib and sorafenib used. The anti-proliferative capability of all derivatives demonstrated that compounds 15i and 15g potentially suppressed the growth of HER2 positive T-47D and BT-474 cells having a differential expression of HER2 and VEGFR2 with superior activity than lapatinib and sorafenib. SAR revealed that the trifluoromethyl group on the pyridinyl moiety of the side chain at the ^fourth position of the scaffold made compound 15i the most promising candidate among the other candidates. Flowcytometric apoptotic evaluation of compound 15i demonstrated potential induction of apoptosis at its IC₅₀ in both T-47D and BT-474 cells, which was proved by examining the caspases (Caspase-3, 8, and 9) and Cytochrome-c release. Western blot analysis further determined HER2, VEGFR2, and their downstream signalling partner’s inhibition by the treatment of 15i. Further in-vivo tumour growth reduction by 15i was assessed in the T-47D xenograft mice model stating its potential anti-tumour capability. Based on docking studies, compound 15i was confirmed as a new lead candidate for the dual inhibition of HER2 and VEGFR2.

HER2

VEGFR2

Lapatinib

Neratinib

Apoptosis

Anti-cancer activity

etc

A breast cancer diagnosis accounts for 12.5% of all new cancer cases yearly, making it the most common malignancy in women and a significant cause of cancer-related deaths worldwide [1]. Various factors contribute to the heterogeneity of breast cancer, including histopathology, epidemiology, clinical, biological, and molecular characteristics. Contrary to other solid tumours, human breast cancer exhibits very distinctive clinicopathologic characteristics and more established patterns of therapeutic susceptibility and resistance to various targeted therapies. The treatment regimen for breast cancer depends largely on human epidermal growth factor receptor 2 (HER2) and hormone receptor status, as well as disease status and patients' clinical characteristics. It is estimated that about 25–30% of breast cancers exhibit overexpression or amplified expression of the proto-oncogene c-erbB2, which encodes the HER2 [2]. Vascular endothelial growth factor receptor 2 (VEGFR2) is a membrane-bound tyrosine kinase receptor critical to tumour angiogenesis which was first identified in endothelial cells. VEGFR2 promotes endothelial cell proliferation, migration, and survival and enhances vascular permeability [3]. Several studies have demonstrated that VEGFR2 can also be located in tumour cells of colon cancer [4], pancreatic cancer [5], small cell lung cancer [6], cervical adenosquamous carcinoma [7], Medullary Thyroid Carcinoma [8], glioma [9], oral squamous cell carcinoma [10], and breast cancer [11–15]. Up-regulation of VEGFR-2 mRNA was found earlier in invasive primary and metastatic breast cancers [16].

VEGFR-2 activation and expression in HER2-positive cancers has been the subject of much research in recent years. VEGFR2 expression is positively regulated by HER2 signalling, and VEGFR2 expression is associated with poor prognosis and decreased survival in patients with HER2-positive breast cancer [17–20]. A recent study revealed that the forced expression of HER2 upregulates VEGF, a key ligand of VEGFR2. In contrast, inhibition of HER2 signalling with trastuzumab or HER2 siRNA decreases VEGF levels by downregulating the PI3K activity [21]. Furthermore, VEGF levels have correlated with HER2 expression in clinical specimens of breast cancer, and patients with high levels of VEGF tend to have worse clinical outcomes [22, 23]. Thus, preclinical and clinical studies have linked VEGFR2 expression and HER2 signalling in human breast cancers. High levels of VEGFR2 may contribute to the poor prognosis and aggressive behaviour associated with breast cancers that overexpress HER2.

Anti-HER2 therapy works by blocking the activity of the HER2, which helps to suppress the growth and spread of the tumour. Several types of anti-HER2 therapy exist, including monoclonal antibodies such as trastuzumab, Pertuzumab and successful small-molecule tyrosine kinase inhibitors such as Lapatinib, Neratinib, Pyrotinib, and Tucatinib, etc., have shown promising outcomes in patients with HER2 + ve breast cancers [24–26]. Combination therapy involves combining different anti-HER2 treatments, such as monoclonal antibodies and chemotherapy, to achieve better outcomes. While current anti-HER2 therapy is effective in some patients, it can also be associated with specific challenges, including drug resistance, severe side effects, economic constraints, etc. In addition, bevacizumab, a recombinant monoclonal antibody to VEGF, has demonstrated an improved response rate and progression-free survival in combination with paclitaxel but has not improved overall survival in patients with metastatic breast cancer [27]. Although targeting EGFR, HER2, VEGFR2, and their ligands alone does not provide adequate tumour control in many patients [28, 29], preclinical studies suggest the potential for increased efficacy with combination therapy [30–32]. Furthermore, clinical trials of various doublet combinations of trastuzumab, lapatinib, and bevacizumab and newly evolved drugs suggest increased efficacy and that combined anti-HER2 and anti-VEGF treatment may overcome resistance to anti-HER2 monotherapy [33–37].

With the ultimate objective to target key oncological signalling events regulated by HER2 and VEGFR2 and taking advantage of the heterogeneity of breast cancers with HER2 positivity, and overcome the clinical challenges associated with the current anti-HER2 therapies outlined above, we designed and developed novel derivatives as HER2 & VEGFR2 dual inhibitors by considering structural features of lapatinib and neratinib essential for successful binding of growth receptor tyrosine kinases. The new compounds were in-vitro evaluated for cytotoxic potential, apoptotic induction abilities, and both HER2 and VEGFR2 inhibitory activities. Only the most active compounds were further subjected to an in-vivo anti-tumour efficacy using xenograft mice models. In addition, the molecular docking studies of the most potent inhibitors within HER2 and VEGFR2 active sites were applied to explore their binding mode and justify their high affinity. This study presents a new therapeutic approach for patients with HER2 + ve breast cancer to improve their clinical outcomes.

2.1 Rational design

Hybrid molecular scaffolds containing artificial assemblies of two or more successful pharmacophores could assist in developing novel derivatives capable of overcoming drug resistance, intensifying activity, and enhancing binding affinity. Lapatinib is composed of a central pharmacophore region with aromatic quinazoline occupying the hinge region of the kinase and hydrophobic head portion chloro-2-[(3-fluorophenyl) methoxy] benzene connected to the central region via a -NH spacer contributes for kinase activity by interacting through the back hydrophobic region of the receptor. The hydrophobic tail N-[(furan-2-yl) methyl]-2-(methanesulfonyl) ethan-1-amine moiety connected to the central region at the ^sixth position is responsible for occupying the front hydrophobic region of the receptor. Structural characteristics of lapatinib enable it to interact reversibly with receptors through noncovalent interactions (Fig. 1a). Neratinib comprises central quinoline with four substitutions responsible for irreversible covalent interactions with kinase receptors. A 2-[(2-chlorophenoxy)methyl] pyridine attached to the central region with a -NH spacer acts as a hydrophobic head that interacts with the back hydrophobic region of the receptor. A hydrophilic moieties (2E)-4-(dimethylamino) but-2-enamide at the ^sixth position and cyanide at the ^third position are necessary for increasing the binding strength of the central core ring through surrounding interactions. In order to enhance kinase selectivity and binding strength, a novel series of quinazolines with improved chemistry has been designed, taking advantage of these structural features of both lapatinib and neratinib (Fig. 1b)

The rational design of the new series comes with four substitutions at the C-2, C-4, C-6, and C-7 positions of the quinazoline core (Fig. 1c). To attain higher affinity and potency of the newly synthesized small molecules (14a-q), the C4 position was subjected to a variety of different polar/solubilizing functional groups. The oxygen of the phenoxymethyl pyridine moiety of neratinib was replaced with sulphur to increase the conformational flexibility of the terminal substituted pyridine. 2-[(phenylsulfanyl)methyl] pyridine with variable substitutions at C-4 and 3-(morpholin-4-yl) propoxy side chain at C-6 were designed to be directed into the deep hydrophobic pockets located at the back and the front side of receptor binding sites respectively. As a matter of fact, the gefitinib methoxy moiety at C-7 was introduced into all new derivatives to enhance their solubilizing properties. The hydrophilic moiety of methyl 3-aminopropanoate at C-2 contributes to increasing the binding strength of the central core ring through surrounding interactions. The substitution patterns at C-4 of the core quinazoline in the new series were designed to enhance potency and selectivity by integrating the substituted 4-anilino moiety and terminal substituted pyridine moiety with sulphur (Fig. 1c). We assessed the effectiveness of the new small molecules by using a dose-dependent percent inhibition model over a panel of tyrosine kinases. Further evaluation of the most active compound was conducted regarding its IC₅₀ values over target isoforms. The most potent compound was tested for selectivity against a small panel of kinases. Furthermore, detailed docking studies were performed to understand the binding affinities and alignment of the new compounds at both kinase's ATP binding sites.

2.2 Chemistry

The synthetic pathway employed for preparing target compounds methyl 4,6,7-trisubstituted quinazolin-2-yl)amino]propanoate derivatives is outlined in Scheme 1. The synthesis was initiated with the nitration of methyl 3-hydroxy-4-methoxybenzoate (1) with nitric acid in a mixture of acetic acid and acetic anhydride to yield a compound methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (2). Next, compound 2 was alkylated with 1-bromo-3-chloropropane in the presence of potassium carbonate in DMF to afford intermediate 3, which was reduced by powdered iron in acetic acid to give compound 4 in satisfactory yield. Cyclization of compound 4 with guanidine acetate (5) in ethanol for longer reflux affords compound 6, which was then condensed with methyl 3-chloropropanoate (7) to yield a methyl propanoate ester compound 8 on further chlorination with thionyl chloride, compound 8 yields compound 9. On the other hand, the amino group of various substituted 4-aminobenzene-1-thiols was protected using the tert-butyloxycarbonyl (Boc) anhydride in the presence of DMAP and THF to yield analogues 10a-e. As a result of further condensation of thiols (10a-e) with substituted 2-(chloromethyl) pyridine analogues (11a-o) under basic conditions, compounds 12a-q were easily prepared. The condensation of compound 9 with compounds 12a-q, which have been deprotected from the BOC group following substitution, produces compound 13a-q. Target compounds 15a-q were prepared by reacting 13a-q with morpholine (14) in a methanolic solution of potassium iodide. All compounds were structurally confirmed using ¹H and ¹³C- NMR spectral data, elemental analysis, and HRMS data.

2.3 Anti-cancer evaluation

2.3.1 In-vitro kinase inhibition

As part of the analysis of the potential inhibitory effects of the newly synthesized compounds on EGFR, HER2, and VEGFR2 kinases, we used the Z’-Lyte Kinase Kit (for EGFR & HER2) assay as well as the Kinase-Glo® MAX assay (for VEGFR2) assays in a 10-dose IC₅₀ mode with 3-fold serial dilution starting at 10 nM for each compound. Activation data were expressed as a percentage of residual kinase activity in test samples compared to vehicle (dimethyl sulfoxide). Table 1 shows the kinase inhibition data (IC₅₀ values) of the tested compounds against EGFR, HER2, and VEGFR2 using lapatinib and sorafenib as reference compounds. Seven of the seventeen compounds tested for in-vitro kinase inhibition demonstrated moderate to significant inhibitory activity (nanomolar IC₅₀ values on EGFR and HER2 and/or VEGFR2). In Table 1, compounds 15b, 15c, 15f, 15g, 15i, 15j, and 15m exhibited inhibitory activity against EGFR, HER2, and VEGFR2 with IC₅₀ values less than 100 nM (Fig. 2). From the results, it was evident that all of these compounds displayed lower IC₅₀ values against both HER2 and VEGFR2 in comparison with EGFR, indicating a higher inhibitory potency against HER2 and VEGFR2. Among the compounds studied, compound 15i exhibited the most potent inhibitory effect on HER2 and VEGFR2, with IC₅₀ values of 17.2 ± 1.3 and 21.5 ± 1.7 nM, respectively, which are better than those of lapatinib (17.9 ± 1.5 nM for HER2) and sorafenib (23.5 ± 1.6 nM)—in addition to 15i, compound 15g also demonstrated dual inhibition of both HER2 and VEGFR2 with IC₅₀ of 20.6 ± 1.7 and 29.7 ± 2.7 nM, respectively which are comparable to those of both the reference standards. Moreover, 15f and 15m showed considerable inhibitory activity against HER2 (IC₅₀ of 28.5 to 29.5 nM) and VEGFR2 (32.1 to 33.8 nM). Based on these studies, 15i and 15g demonstrated significantly higher dual inhibitory effects against HER2 and VEGFR2 than the reference standards lapatinib and sorafenib.

Table 1

The IC₅₀ values of the newly designed lapatinib and neratinib hybrid analogues (15a-q) toward EGFR, HER2, and VEGFR2 kinases.
Title	R¹	R²	Enzymatic IC₅₀ (nM)
Title	R¹	R²	EGFR	HER2	VEGFR2
15a	-H	-H	> 500	> 500	357.2 ± 21.3
15b	-H	3-Cl	88.5 ± 5.3	49.3 ± 3.3	53.1 ± 4.4
15c	-H	4-Cl	75.2 ± 4.6	33.2 ± 2.5	43.6 ± 3.9
15d	3-OCH₃	-H	54.9 ± 3.8	129.8 ± 11.6	> 500
15e	-H	3-COCH₃	172.3 ± 12.9	201.3 ± 15.2	121.9 ± 10.4
15f	-H	3-Br	86.4 ± 5.7	29.5 ± 2.3	32.1 ± 2.5
15g	-H	3-CF₃	49.3 ± 3.2	20.6 ± 1.7	29.7 ± 2.7
15h	-H	2-CH₃	> 500	412.3 ± 16.6	391.2 ± 23.9
15i	-H	4-CF₃	78.1 ± 5.8	17.2 ± 1.3	21.5 ± 1.7
15j	3-CH₃	2-Cl	93.6 ± 6.6	82.3 ± 7.2	85.1 ± 6.4
15k	3-OCH₃	2-Br	125.8 ± 8.7	53.6 ± 4.4	221.9 ± 11.3
15l	-H	3-CH₃	123.4 ± 9.3	88.1 ± 5.9	> 500
15m	-H	4-NO₂	42.1 ± 2.9	28.5 ± 2.2	33.8 ± 2.9
15n	3-CH₃	4-Br	89.3 ± 13.7	55.4 ± 5.7	121.9 ± 7.6
15o	-H	3-OCH₃	152.9 ± 9.6	172.8 ± 12.1	41.3 ± 10.5
15p	3-CH₃	3-NO₂	425.7 ± 18.4	112.6 ± 7.3	87.2 ± 4.7
15q	3-OCH₃	4-Br	365.4 ± 17.9	129.4 ± 9.5	185.9 ± 13.8
Lapatinib	-	-	27.1 ± 1.3	17.9 ± 1.5	-
Sorafenib	-	-	-	-	23.5 ± 1.6

2.3.2 In-vitro cytotoxic activity

The cytotoxic abilities of the test compounds 15a-q were evaluated in-vitro using MTT assay on breast cancer cell lines having differential HER2 and VEGFR2 over expressions such as MCF-7, T-47D, BT-474, MDAMB-231, and SKBR-3 cells. We used lapatinib as a reference compound to determine the IC₅₀ of the test compounds. The prepared compounds 15a-q exhibited differential cytotoxic effects against a panel of breast cancer cells having a heterogeneous expression of HER2 and VEGFR2. The results indicated that the prepared compounds 15a-q showed poor growth inhibitory activity (IC₅₀) against MCF-7 cells lacking HER2 and VEGFR2 receptors. In contrast, some compounds showed moderate to good growth suppression against HER2-negative MDAMB-231 cells with significant VEGFR2 expression. Most compounds also demonstrated moderate to good growth inhibitory effects against HER2-positive SKBR3 cells lacking overexpression of VEGFR2. Most compounds displayed superior growth inhibitory effects against T-47D and BT-474 cells expressing HER2 and VEGFR2. According to these results, these newly synthesized lapatinib and neratinib hybrid analogues demonstrated dual inhibitory properties against HER2 and VEGFR2. In support of the in-vitro kinase inhibitory results, compounds 15b, 15g, and 15i showed substantially superior growth inhibitory capabilities with an IC₅₀ ranges between 1.91 to 3.24 µM against both T-47D and BT-474 cells (Fig. 2). Compound 15i displayed significantly lower IC₅₀ of 1.91 ± 0.11 and 1.57 ± 0.13 µM against T-47D, and BT-474 cells having higher overexpression of HER2 and VEGFR2 respectively. The growth inhibitory effect of compound 15i is found to be superior to the standard lapatinib (IC₅₀: 2.06 ± 0.14 µM against T-47D; 1.79 ± 0.11 µM against BT-474 cells). In addition to 15i, compound 15g also showed significant growth retardation efficacy against both T-47D and BT-474 with an IC₅₀ of 2.25 ± 0.16 µM and 1.81 ± 0.17 µM, which is comparable to the standard lapatinib. In addition to these two compounds, 15b demonstrated considerable cytotoxic effects against T-474D and BT-474 cells. These results demonstrated the potential cytotoxic abilities of compounds 15i and 15g against T-474D and BT-474 cells, indicating that these compounds substantially exhibit the dual HER2 and VEGFR2 inhibitory abilities, which are significantly higher than the established EGFR/HER2 dual inhibitor Lapatinib (Fig. 2).

Table 2

The cell growth inhibition (IC₅₀) of the novel lapatinib and neratinib hybrid analogues (15a-q) against MCF-7, T-47D, BT-474, SKBR-3, and MDAMB-231 cells.
Title	Cell inhibition IC₅₀ (µM)
Title	MCF-7	T-47D	BT-474	SKBR-3	MDAMB-231
15a	23.98 ± 1.52	11.24 ± 0.87	8.56 ± 0.63	9.82 ± 0.72	13.65 ± 1.03
15b	19.51 ± 1.14	3.24 ± 0.28	2.11 ± 0.15	9.68 ± 0.81	5.33 ± 0.31
15c	22.33 ± 1.66	5.66 ± 0.41	4.17 ± 0.46	7.52 ± 0.62	2.48 ± 0.19
15d	15.24 ± 1.25	6.58 ± 0.59	12.34 ± 0.91	15.48 ± 1.25	9.35 ± 0.68
15e	8.26 ± 0.61	15.24 ± 1.29	11.89 ± 0.69	8.51 ± 0.51	12.35 ± 0.94
15f	25.42 ± 1.76	9.54 ± 0.82	7.13 ± 0.62	10.5 ± 1.33	11.97 ± 1.23
15g	31.96 ± 1.32	2.25 ± 0.16	1.81 ± 0.17	5.92 ± 0.42	7.81 ± 0.54
15h	13.69 ± 1.17	7.24 ± 0.62	5.96 ± 0.43	13.47 ± 1.04	15.69 ± 1.11
15i	35.78 ± 1.53	1.91 ± 0.11	1.57 ± 0.13	6.73 ± 0.58	5.24 ± 0.39
15j	23.69 ± 2.2	4.69 ± 0.33	6.55 ± 0.58	12.84 ± 1.11	12.44 ± 0.95
15k	16.35 ± 1.33	8.57 ± 0.69	6.54 ± 0.55	13.24 ± 1.09	12.34 ± 1.47
15l	15.90 ± 1.36	9.55 ± 0.75	7.23 ± 0.69	11.47 ± 1.05	15.36 ± 1.38
15m	16.52 ± 1.47	9.26 ± 0.81	7.22 ± .069	13.6 ± 1.26	21.33 ± 1.42
15n	18.33 ± 1.62	14.69 ± 1.25	9.48 ± 0.71	11.72 ± 0.92	12.35 ± 1.01
15o	11.98 ± 1.05	8.42 ± 0.69	6.35 ± 0.52	12.24 ± 1.04	14.68 ± 1.26
15p	13.36 ± 1.16	7.56 ± 0.54	8.36 ± 0.76	14.33 ± 1.33	10.24 ± 0.85
15q	15.95 ± 1.37	9.15 ± 0.63	7.12 ± 0.58	12.48 ± 1.16	12.32 ± 0.93
Lapatinib	17.3 ± 1.2	2.06 ± 0.14	1.79 ± 0.11	2.31 ± 0.16	21.47 ± 1.54

2.3.3 Examination of Cell Morphology

Metabolic activity & growth enhancement were both characterized by maintaining their original shape and cell density, respectively. An indication of metabolic inactivity is the loss of their original size and shape and the deformation of their shape. Cell death was characterized by rounding of cells and floating on growth media for adherent cells and loss of shining and loss of spreading ability for suspension type. Small particles of cellular remnants floating on growth media revealed severe cell damage. Examining the cells under a microscope revealed that compound 15i showed morphological deformation of T-47D and BT-474 cells by making long polygonal-shaped cells rounded off and increasing intracellular spaces. This effect was significant compared to control cells, where cells retained their original shape. At double the IC₅₀ concentration, the morphological changes were highly evident in both cells compared with the effect observed for standard lapatinib after 48 hr of treatment. These results demonstrated that compound 15i showed a potential morphological deformation in T-47D and BT-474 cells, supporting cytotoxic and kinase inhibition studies.

2.3.4 SAR studies

Based on the in-vitro kinase inhibition and cellular cytotoxic profiles of seventeen compounds (Table 1 and Table 2), the structure-activity relationship (SAR) was developed. It was observed that the substitutions on the 4-[[(pyridin-2-yl)methyl]sulfanyl]anilino moiety of the central scaffold at C-4 defined the differential cytotoxic and kinase inhibitory activity of lapatinib and neratinib hybrid analogues. Specifically, the substation on the aniline moiety and the pyridinyl ring influence cytotoxicity and cell growth inhibition. As a means of understanding the effect of different substitutions on anilino and pyridinyl rings, other structural features such as methyl 3-aminopropanoate at C-2, 3-(morpholin-4-yl)propan-1-ol at C-6, and the methoxy moiety at C-7 are held constant, and all these structural features are designed based on the binding requirements for the growth receptors. According to the activity profiles, substituting polar groups such as -CF₃ (15g & 15i) and -NO₂ (15m) significantly increased in-vitro kinase inhibition and cytotoxic effects regardless of whether they are positioned at C-3 or C-4. There is also considerable cytotoxic and kinase inhibitory activity associated with replacing electronegative groups like chlorine and bromine (15b, 15c, and 15f) in either the C-3 or C-4 position of the pyridinyl ring. More specifically, the C-4 substitution of the high polar group -CF₃ (15i) on the pyridinyl ring contributed superior cytotoxic and kinase inhibition abilities, which is more significant than the standards lapatinib and sorafenib used. According to these results, the electron-withdrawing properties of -CF₃, -NO₂, -Cl, and -Br groups contribute to stabilizing the electron-rich pyridine ring that might contribute to the hydrophobic interaction between the receptor and the molecule. On the other hand, electron-releasing groups like methyl and methoxy groups on anilino moiety destabilize the entire C-4 substitution and make the molecule more redundant to the inhibitory responses. Substitutions on the anilino moiety might cause electron crowding around the ring, resulting in a failure of the penetration into the receptor core. As a result of these findings, it was concluded that the electron-withdrawing nature of -CF₃ at the ^fourth position on the pyridinyl ring led to the potential behaviour for inhibiting both HER2 and VEGFR2 in HER2-positive breast cancer cells.

2.3.5 Compound 15i potentially induced apoptosis in both T-47D, and BT-474 cells

The most potent compound, 15i, was selected based on the in-vitro kinase inhibition and cytotoxic abilities against HER2-positive cells, T-47D, and BT-474 cells to determine their effect on apoptotic induction. T-47D and BT-474 cells were treated with compound 15i at two different concentrations such as IC₅₀ and 2xIC_50, respectively, for 48 h. As a result, flow cytometry was used to analyze the apoptotic events in test samples by comparing them with control samples (Fig. 4a). A deep sight of flowing cytometric analysis showed that treatment with compound 15i substantially increased the cell population in apoptotic phases (Q2 and Q4) in both T-47D and BT-474 cells (Fig. 4a). Compound 15i showed a superior effect than Lapatinib which was quantitatively measured in terms of % cells undergone apoptosis over the treatment. When compound 15i was treated at an IC₅₀, the early apoptotic cell population increased to 54.87% from the 4.86% observed in control. In contrast, at a double IC₅₀, the cell distribution in the late apoptotic phase increased to 56.58% from 0.78% observed in control after 48 hr of treatment in T-47D. It was significantly higher than that observed for lapatinib treatment, in which 19.68% of cells entered into the late apoptosis phase, whereas 67.27% entered into the early apoptosis phase in T-47D cells after 48 hr (Fig. 4b). Similarly with compound 15i at an IC₅₀, the early apoptotic cell population escalated to 77.24% from 4.62% observed in control, while at the double the IC₅₀ concentration, the cell distribution in the late apoptotic phase raised to 52.76% from 0.39% in control after 48 hr treatment in BT-474 cells. It is important to note that this effect was substantially higher than the effect observed for lapatinib, resulting in an increase in early apoptotic cell population to 68.05% and 18.68% of late apoptotic cell population after 48 hr of treatment in BT-474 cells (Fig. 4c). These results have been demonstrated that compound 15i induces apoptosis effectively in HER2 positive T-47D and BT-474 cells which supports the fundamental criteria for being considered to be an effective anti-cancer molecule.

2.3.6 Compound 15i significantly increased the activation of caspases-3, 8 and 9

To outline the ability of compound 15i to activate caspases 3, 8, and 9 through the inhibition of the intrinsic or the extrinsic pathway or both, a cell-based caspase fluorometric assay was developed and evaluated. The assay also evaluated the selectivity of caspase activation over the treatment in both T-47D and BT-474 cells. The assay utilizes DEVD-ProRed™, IETD-R110, and LEHD-AMC as fluorogenic indicators for detecting caspase-3, caspase-8, and caspase-9, respectively. By cleaving the synthetic substrate, cell-based caspase-3, as well as proapoptotic caspase-8 and 9, which were activated during the treatment with 15i were able to release ProRed™ (red fluorescence), R110 (green fluorescence) and AMC (blue fluorescence), which can then be quantified by fluorometry. At two different concentrations, ie. IC₅₀ and 2xIC₅₀, compound 15i, can be directly incubated with T-47D and BT-474 cells. Caspase-3 activation can be determined by comparing the fluorescence intensity of samples with and without staurosporine, a standard caspase activator. The results of this study indicated that compound 15i at its IC₅₀ concentration significantly increased caspase-3 activity by 1.9 fold, while compound 15i at double the IC₅₀ showed a 2.5 fold increase in caspase-3 activity relative to control which was normalized to 1 in T-47D cells (Fig. 6a). Similarly 15i at its IC₅₀ concentration significantly increased the breakdown of procaspase 8 and 9 into caspase 8 and 9 by 1.4 and 1.6 folds respectively, while compound 15i at double the IC₅₀ exhibited the activation of caspase 8 and 9 by 2.1 and 2.3 folds respectively relative to control in T-47D cells. In T-47D cells, the effect of caspase activation was significantly higher than that of staurosporine, which increased activation of caspase-3 by 1.6 fold, caspase 8 activation by 1.2 fold, and caspase 9 activation by 1.8 fold (Fig. 6a). Caspase activation in BT-474 cells revealed that compound 15i at its IC₅₀ concentration significantly increased active caspase-3 activity by 1.7 fold, while at double the IC₅₀ showed a 2.4 fold increase in caspase-3 activity relative to control cells in BT-474 cells (Fig. 6b). Similarly 15i at its IC₅₀ concentration significantly increased the breakdown of procaspase 8 and 9 into caspase 8 and 9 by 1.2 and 1.7 folds respectively, while at double the IC₅₀ exhibited the activation of caspase 8 and 9 by 2.5 and 2.8 folds respectively relative to control in BT-474 cells (Fig. 6b). The effect of caspase activation was more significant than the effect of staurosporine, which increased the activation of caspase-3 by 1.4 fold, caspase 8 and caspase 9 by 1.5 and 1.8 fold, respectively, in BT-474 cells (Fig. 6b). The data indicated that 15i is a putative activator of both intrinsic and extrinsic caspase pathways with more selectivity and potency effect on caspase-3 enzyme.

2.3.7 Compound 15i increases the Cytochrome-c release in both T-47D, and BT-474 cells

A cell-based cytochrome C was assayed for more proof of caspase-3, caspase-8, and cassapse-9 activation by compound 15i. It was reported that cytochrome C concentration in the cell is critical in activating caspases and the intrinsic apoptosis pathway. Apoptotic proteins can activate apoptosis by enhancing cytochrome C's release, while proapoptotic members act as activators of its release. When cytochrome C is released into mitochondria, which leads to the activation of caspase-8 and 9, eventually leading to caspase-3 activation, which in turn results in apoptosis induction and drives the cell toward the programmed death. The compound 15i was evaluated for cytochrome C against T-47D and BT-474 cells; the results are listed in Fig. 7. Compound 15i at its IC₅₀ concentration caused cytochrome C release about 3.14 and 2.73 folds increase in T-47D and BT-474 cells, respectively, compared with the control cells, comparable to standard staurosporine with a 2.92 and 3.15 folds increase in T-47D and BT-474 cells. Similarly, 15i, at its double the IC₅₀ concentration, enhanced the cytochrome-c release substantially by 4.23 and 3.15 folds increase in T-47D and BT-474 cells compared to the control, which is significantly higher than the effect shown by staurosporine. Considering the results displayed by compound 15i, it could be concluded that apoptotic activity may be attributed to the activation of cytochrome C and, consequently, the activation of the intrinsic apoptotic pathway in both HER2 [positive T-47D and BT-474 cells.

2.3.8 Compound 15i successfully inhibited HER2 and VEGFR2 in both T-47D and BT-474 cells

Given its successful anti-proliferative and apoptotic induction abilities in HER2 positive cells T-47D, BT-474 cells panel of breast cancer cells, compound 15i was selected for further analysis to understand dual inhibition of HER2, VEGFR2, and their key downstream signalling partners. Using western blot analysis, we used a candidate-based approach to explore the expression and phosphorylation status of HER2, VEGFR2, and their signalling members in both T-47D and BT-474 cells (Fig. 8a). The cells were incubated with 15i at its IC₅₀ for 48 h and then stimulated with ligand VEGF. As a result, the dual RTK members HER2/VEGFR2 and their key signalling regulators were downregulated over the treatment of 15i in both T-47D and BT-474 cells (Fig. 8a). Compared with the reference compound Lapatinib, 15i significantly inhibited band intensities of Phosphorylated forms of HER2, VEGFR2 and their signalling partners such as p-ERK1/2 and p-AKT in both T-47D (Fig. 8b) and BT-474 cells (Fig. 8c). Besides, 15i treatment made an absolute observation that T-47D cells were more sensitive than BT-474 cells having a differential expression of HER2 and VEGFR2.

2.3.9 In vivo anti-tumor Efficacy of 15i in T-47D Xenograft Models

To determine the in-vivo tumour suppression capabilities of compound 15i, the T-47D xenograft mice model was used. This study considered vehicle treatment as a control, and lapatinib was used as a reference standard. Implanted tumours were established with parent cells on each flank, and tumour growth and inhibition after each treatment from 0 to 59 days after tumour implantation are plotted in Fig. 9. Compound 15i, and Lapatinib was well tolerated and displayed significant in vivo anti-tumour efficacy when compared with water as vehicle control, and compound 15i presented remarkably higher tumour growth inhibition than Lapatinib throughout the study period of 60 days (P < 0.05) (Fig. 9a). Initially the treatment with compound 15i produced a significant reduction (P ≤ 0.05) in tumour growth on day 15th of post-inoculation as compared with vehicle control. It was observed that the tumour growth inhibition was significantly dropped on the 27th day of post-tumour inoculation when compared to the vehicle control and lapatinib-treated group in the study. The results also revealed no significant body weight change during the study (Fig. 9b). The results revealed a significant anti-tumour effect in tumour growth inhibition with compound 15i in HER2 and VEGFR2 differentially expressed T-47D breast cancer xenograft models. These observations undoubtedly displayed the anti-tumour potential of 15i superior to Lapatinib efficacy and proved potential in treating HER2-positive advanced breast cancers.

2.3.10 Molecular docking studies revealed compound 15i, potentially blocked HER2 and VEGFR2 kinases

To better understand how our promising candidate 15i interacted with the ATP-binding cleft of HER2 (3RCD) and VEGFR2 (5ew3), with conformationally constrained form, we determined a putative binding mode within the active binding pocket of both VEGFR2 and HER2 using molecular docking studies (Fig. 10). The docking results 15i with both HER2 and VEGFR2 displayed that the central scaffold occupied the ATP-binding pocket of two receptors. Three highly conserved hydrogen bonds are formed between compound 15i and the hinge region. The molecule 15i is oriented in HER2 receptor such that 4-({[4-(trifluoromethyl) pyridin-2-yl] methyl} sulfanyl) aniline branch extends toward the solvent, which strongly contributed to the hydrophobic interactions with the receptor surface and the methyl 3-aminopropanoate at C-2 position of quinazoline ring is directed into the back pocket of HER2 kinase. The docking result of compound 15i forms three hydrogen bonds with Val 797, Asp 845, and Gly 805 with a measured distance in the range of 2.51–2.59 Å. A docking score of -10.67 kcal/mol was obtained for compound 15i in the Gibbs free energy calculation with the HER2 interaction. The molecule 15i is oriented in the VEGFR2 receptor such that 4-({[4-(trifluoromethyl) pyridin-2-yl] methyl} sulfanyl) aniline branch completely buried into the core of VEGFR2 with strong hydrophobic interactions, and the 3-(morpholin-4-yl)propan-1-ol at C-6 position of quinazoline ring is directed into the terminal hydrophobic interactions and the central quinazoline region occupies the hinge region of VEGFR2 kinase. The docking result of compound 15i forms three hydrogen bonds with Lys-887, Ile-892, and Leu 902 with a measured distance in the range of 2.50–2.55 Å. A docking score of -10.19 kcal/mol was obtained for compound 15i in the Gibbs free energy calculation with the VEGFR2 interaction. As a result, our comprehensive investigation of desirable cellular anti-cancer activity, Target selectivity, and in-vivo efficacy yielded a promising candidate for the clinical development of a dual HER2/VEGFR2 inhibitor.

3.1 General Synthesis

3.1.1 synthesis of methyl 5-hydroxy-4-methoxy-2-nitrobenzoate(2): Nitric acid (9 ml, 69%) was added dropwise at 0–5°C to a solution of methyl 3-hydroxy-4-methoxybenzoate (1, 7.3 g, 40 mmol) in a mixture of acetic acid (30 mL) and acetic anhydride (10 mL). This mixture was stirred at room temperature for 6 h, then slowly poured into ice water (200 mL) and extracted with ethyl acetate (4 x 20 mL). The combined organic layer was washed with saturated sodium bicarbonate (2 x 20 mL) and brine (2 x 10 mL) and dried (Na₂ SO₄). The ethyl acetate was then removed by letting it stand under a vacuum to give a yellow oil that solidified after standing in a refrigerator for 12 h and was then recrystallized from ethyl acetate/ petroleum ether to afford the product as light yellow crystals (7.9 g, 83% yield, 99.3% HPLC purity); mp: 87–89°C; ¹H NMR (500 MHz, DMSO-d₆) δ 9.221 (s, 1H), 7.636 (d, J = 17.951 Hz, 1H), 3.874 (d, J = 6.107 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.02, 151.05, 149.48, 141.00, 121.28, 116.13, 108.31, 56.56, 53.30.

3.1.2 synthesis of methyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate(3): A mixture of methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (2, 7.1 g, 31.4 mmol), 1-bromo-3-chloro- propane (5.9 g, 37.4 mmol), and potassium carbonate (9.1 g, 65.8 mmol) in DMF (50 mL) was heated at 70°C for 4 h. The reaction mixture was cooled to room temperature, then poured slowly into ice water (300 mL) while stirring constantly. The solid formed was filtered off and washed with cold water. The off-white product was recrystallized from ethyl acetate (20 mL) to afford 10.3 g of 17 (95% yield, 99.3% HPLC purity); mp: 55 to 58°C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.305 (s, 0H), 4.107 (t, J = 5.009, 5.009 Hz, 1H), 3.865 (d, J = 15.140 Hz, 3H), 3.756 (t, J = 3.785, 3.785 Hz, 1H), 2.154 (tt, J = 5.130, 5.130, 3.784, 3.784 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 165.96, 150.87, 150.82, 141.88, 121.55, 113.82, 108.38, 66.86, 56.19, 53.30, 41.66, 31.86.

3.1.3 synthesis of methyl 2-amino-5-(3-chloropropoxy)-4-methoxybenzoate(4): Powdered iron (2.8 g, 50.1 mmol) was added to acetic acid (50 mL). The resulting suspension was stirred for 15 min at 50°C under an atmosphere of N₂, then a solution of methyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate (3, 9.5 g, 31.2 mmol) in methanol (30 mL) was added dropwise. The mixture was stirred for 30 min at 50 ~ 60°C. The catalyst was filtered and washed with methanol, and the volatiles evaporated from the combined filtrate and washes. The residue was poured into water (40 mL) and extracted with ethyl acetate (4 x 20 mL). The organic phase was washed with a saturated solution of Na₂CO₃ (2 x 10 mL) and brine (2 x 10 mL) and then dried (Na₂ SO₄). The solvent was removed under vacuum, and the brown solid residue was recrystallized from ethyl acetate/petroleum ether to afford the product as light brown crystals (6.9 g, 79% yield, 98.8% HPLC purity); mp: 96–98°C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.335 (s, 0H), 6.267 (s, 0H), 6.213 (s, 1H), 4.118 (t, J = 4.995, 4.995 Hz, 1H), 3.853 (s, 1H), 3.782–3.730 (m, 2H), 2.153 (tt, J = 4.887, 4.887, 3.783, 3.783 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 167.83, 153.99, 144.81, 141.91, 117.16, 108.45, 100.28, 66.79, 55.94, 51.94, 41.66, 31.86.

3.1.4 synthesis of 2-amino-6-(3-chloropropoxy)-7-methoxyquinazolin-4(3H)-one(6): A solution of methyl 2-amino-5-(3-chloropropoxy)-4-methoxybenzoate (4, 6.5 g, 22.9 mmol) and guanidine acetate (5, 9.4 g, 78.9 mmol) in ethanol (80 mL) was refluxed for 6 h with overhead stirring. The mixture was allowed to stand in the refrigerator overnight. The precipitate was collected by filtration, washed with ethanol, and air dried to give compound 5 as a white power (9.2 g, 93% yield, 99.1% HPLC purity); mp: 218–219°C; ¹H NMR (500 MHz, DMSO-d₆) δ 9.851 (s, 1H), 7.286 (s, 1H), 6.832 (s, 1H), 5.918 (s, 2H), 4.118 (t, J = 4.991, 4.991 Hz, 2H), 3.852 (s, 3H), 3.756 (t, J = 3.785, 3.785 Hz, 2H), 2.153 (tt, J = 4.888, 4.888, 3.783, 3.783 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 160.85, 155.51, 154.20, 147.54, 145.11, 113.42, 109.40, 102.92, 66.79, 56.09, 41.66, 31.84.

3.1.5 synthesis of methyl 3-{[6-(3-chloropropoxy)-7-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl]amino}propanoate(8): A 5.5 g (44.8 mmol) of methyl 3-chloropropanoate was dissolved in 25 mL of CH₃CN. This was followed by the addition of a trace amount of DMF. This solution was heated in an oil bath at 60°C for 30 min after gas evolution began. Then the orange reaction solution was concentrated to about 1/4 of its volume in vacuo without applying heat. This solution was then cooled in an ice bath and 8.7 g (30.6 mmol) of 2-amino-6-(3-chloropropoxy)-7-methoxyquinazolin-4(3H)-one (6) in 25 mL of N-methylpyrrolidinone was added dropwise. Cooling and stirring were continued for 2 h, and then the reaction mixture was poured into a saturated aqueous NaHCO₃ solution. The resulting brown precipitate was collected and chromatographed on silica gel. The column was eluted with a gradient of EtOAc to 1:9: 0.2 MeOH-CH₂Cl₂-Et₃N. Finally, the product was washed with ether to give compound 8 as a white power (8.7 g, 91% yield, 98.7% HPLC purity); m.p: 123 to 126 ^oC; ¹H NMR (500 MHz, DMSO-d₆) δ 8.246 (t, J = 4.396, 4.396 Hz, 1H), 7.995 (s, 1H), 7.299 (s, 1H), 6.929 (s, 1H), 4.118 (t, J = 4.991, 4.991 Hz, 2H), 3.852 (s, 3H), 3.756 (t, J = 3.785, 3.785 Hz, 2H), 3.570 (td, J = 5.467, 5.417, 4.363 Hz, 2H), 2.585 (d, J = 10.988 Hz, 1H), 2.153 (tt, J = 4.888, 4.888, 3.783, 3.783 Hz, 2H).; ¹³C NMR (125 MHz, DMSO-d₆) δ 172.66, 161.30, 154.44, 154.18, 147.61, 141.95, 115.00, 109.61, 104.23, 66.79, 56.09, 51.83, 41.66, 37.87, 33.47, 31.84.

3.1.6 synthesis of methyl 3-{[4-chloro-6-(3-chloropropoxy)-7-methoxyquinazolin-2-yl]amino}propanoate(9): Methyl 3-{[6-(3-chloropropoxy)-7-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl] amino} propanoate (8, 7.7 g, 20.8 mmol) was added to thionyl chloride (15 mL) with magnetic stirring, then DMF (10 mL) was slowly added dropwise, and the reaction flask was heated to reflux for 4 h. Most of the excess thionyl chloride was then removed under reduced pressure, and the yellow residue was dissolved in chloroform (50 mL), then washed with a saturated solution of sodium carbonate (2 x 10 mL) and water (2 x 10 mL) and dried (Na₂SO₄ ). The chloroform was then removed under reduced pressure to give an off-white power, which was recrystallized from ethyl acetate to afford the product 9 (8.1 g, 81.7% yield, 98.9% HPLC purity); mp: 156–159°C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.685 (t, J = 4.274, 4.274 Hz, 1H), 6.946 (s, 1H), 6.898–6.849 (m, 1H), 6.480 (s, 1H), 5.543 (d, J = 6.350 Hz, 1H), 4.117 (t, J = 5.006, 5.006 Hz, 2H), 3.852 (s, 3H), 3.756 (t, J = 3.785, 3.785 Hz, 2H), 3.520 (td, J = 5.403, 5.361, 4.147 Hz, 2H), 2.589 (d, J = 10.750 Hz, 1H), 2.154 (tt, J = 5.130, 5.130, 3.784, 3.784 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 172.66, 151.84, 151.11, 148.99, 138.09, 119.38, 110.70, 100.76, 66.79, 66.70, 56.09, 51.83, 41.66, 37.89, 33.40, 31.87.

3.1.7 synthesis of disubstituted di-tert-butyl [4-(pyridin-2-yl)methyl sulfanyl phenyl]-2-imidodicarbonate analogues(12 a-q): A mixture of substituted di-tert-butyl (4-sulfanylphenyl)-2-imido-dicarbonate analogues (10a-e, 10 mmol) and substituted 2-(chloromethyl)pyridine analogues (11a-o, 12.5 mmol) were dissolved in DMF in the presence of cesium carbonate (Cs₂CO₃) and refluxed for 3 h at 25 ^oC. After filtration, the filtrate was evaporated to a residue partitioned between EtOAc and saturated KHCO₃ solution. The organic layer was evaporated to give a light-yellow solid which was recrystallized with ether-hexane (1:1) to give disubstituted di-tert-butyl [4-(pyridin-2-yl) methyl sulfanyl phenyl]-2-imidodicarbonate analogues title compounds (12 a-q; 83–89%)

3.1.8 synthesis of methyl 3-{[4-(4-{[(substituted pyridin-2-yl) methyl] sulfanyl} substituted anilino) 6-(3-chloropropoxy)-7-methoxyquinazolin-2-yl] amino} propanoate derivatives13 (a-q): A stirred mixture of compound 9 (9.5 mmol) and compounds 12(a-q) (10 mmol) 2.56 g (22.2 mmol) of pyridine hydrochloride, and 35 mL of methoxyethanol was refluxed for 1 h. The cooled mixture was stirred with dilute aqueous K₂CO₃ (pH ∼9.3). The resulting precipitate was filtered, washed well with water, and dried to give a 3.27 g brown solid. The solid was stirred with 75 mL of CH₂Cl₂ at 0°C and treated with 30 mL of TFA. The resulting solution was warmed to 25°C, stirred for 15 min, diluted with toluene, and concentrated. The residue was partitioned between 20:1 EtOAc-MeOH and aqueous K₂CO₃. The organic layer was washed with brine, dried, and concentrated. Short column chromatography of the residue on silica gel with final elution by 25:5:1 EtOAc-MeOH-Et₃N gave 2.13 g (65.7%) of brown colored solid; mp: 247–250 ^oC

3.1.9 synthesis of methyl 3-{[4-(4-{[(substituted pyridin-2-yl) methyl] sulfanyl} substituted anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate derivatives(15a-q): Methyl 3-{[4-(4-{[(substituted pyridin-2-yl) methyl] sulfanyl} substituted anilino) 6-(3-chloropropoxy)-7-methoxyquinazolin-2-yl] amino} propanoate derivatives (13 (a-q), 20 mmol) and potassium iodide (1.0 g) were added to the solution of morpholine (14, 3.7 g, 42.4 mmol) in DMF (30 mL). The solution was stirred at 60°C for 30 min, then poured into ice water (250 mL) and extracted with chloroform (3 x 30 mL). The organic layers were combined, washed with a saturated solution of sodium carbonate (2 x 20 mL) and brine (1 x 10 mL), and then dried (Na₂SO₄). The solvent was removed under a vacuum. The crude product was crystallized from ethyl acetate to afford target compounds 15a-q (73 to 81% yield; 98.4 to 99.2% HPLC purity).

3.1.9.1 Methyl 3-{[4-(4-{[(pyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15a): ¹H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.90 (dd, J = 4.1, 1.7 Hz, 1H), 8.33 (s, 1H), 8.04 (td, J = 7.1, 1.6 Hz, 1H), 7.71–7.65 (m, 3H), 7.62 (dq, J = 6.9, 1.0 Hz, 1H), 7.49–7.43 (m, 3H), 7.29 (t, J = 4.7 Hz, 1H), 4.54 (d, J = 0.9 Hz, 2H), 4.44 (t, J = 6.6 Hz, 2H), 4.26 (s, 2H), 4.11–4.04 (m, 6H), 3.99 (s, 2H), 3.68 (s, 4H), 3.19 (d, J = 14.8 Hz, 1H), 3.10 (t, J = 6.5 Hz, 2H), 2.93–2.88 (m, 4H), 2.24 (p, J = 6.5 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d6) δ 171.02, 161.04, 157.31, 156.68, 152.01, 148.99, 146.60, 146.01, 138.86, 136.76, 129.76, 128.80, 123.29, 121.39, 120.17, 107.34, 106.41, 101.59, 67.32, 63.92, 55.28, 53.46, 53.23, 51.05, 39.53, 39.40, 39.36, 36.53, 33.00, 26.38.

3.1.9.2 Methyl 3-{[4-(4-{[(3-chloro pyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15b): ¹H NMR (500 MHz, DMSO) δ 9.41 (s, 1H), 8.85 (d, J = 2.0 Hz, 1H), 8.31 (s, 1H), 7.86 (dd, J = 7.9, 1.8 Hz, 1H), 7.68–7.63 (m, 2H), 7.56 (dt, J = 8.1, 0.8 Hz, 1H), 7.46–7.40 (m, 3H), 7.26 (t, J = 4.7 Hz, 1H), 4.63 (d, J = 0.7 Hz, 2H), 4.42 (t, J = 6.6 Hz, 2H), 4.24 (s, 2H), 4.09–4.01 (m, 6H), 3.96 (s, 2H), 3.65 (s, 5H), 3.17 (d, J = 14.8 Hz, 1H), 3.08 (t, J = 6.5 Hz, 2H), 2.91–2.85 (m, 4H), 2.22 (p, J = 6.5 Hz, 2H); ¹³C NMR (125 MHz, Common NMR Solvents) δ 171.18, 161.20, 156.84, 154.99, 152.17, 147.28, 146.76, 146.17, 139.02, 135.57, 129.93, 128.96, 127.14, 125.10, 120.33, 107.50, 106.58, 101.75, 67.48, 64.08, 55.44, 53.62, 53.40, 51.21, 36.69, 33.16, 26.54.

3.1.9.3 Methyl 3-{[4-(4-{[(4-chloropyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15c): ¹H NMR (500 MHz, DMSO) δ 8.41 (d, J = 3.4 Hz, 0H), 7.98 (s, 0H), 7.32 (d, J = 7.9 Hz, 0H), 7.16–7.08 (m, 2H), 6.93 (t, J = 4.7 Hz, 0H), 6.60 (d, J = 2.1 Hz, 0H), 4.58 (d, J = 0.7 Hz, 1H), 4.09 (t, J = 6.6 Hz, 1H), 3.91 (s, 1H), 3.84 (s, 1H), 3.76–3.68 (m, 3H), 3.63 (s, 1H), 2.84 (t, J = 7.4 Hz, 1H), 2.75 (t, J = 6.5 Hz, 1H), 2.58–2.52 (m, 2H), 2.15 (s, 3H), 1.89 (p, J = 6.5 Hz, 1H); ¹³C NMR (125 MHz, Common NMR Solvents) δ 171.17, 161.19, 158.26, 157.94, 156.87, 152.16, 149.46, 146.75, 146.16, 141.43, 140.79, 129.26, 123.80, 121.50, 118.31, 113.97, 107.50, 106.57, 104.20, 101.74, 67.47, 64.07, 55.43, 53.61, 53.39, 51.20, 38.88, 36.68, 33.15, 26.53.

3.1.9.4 Methyl 3-{[4-(4-{[(pyridin-2-yl) methyl] sulfanyl} 3-methoxy anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15d): ¹H NMR (500 MHz, DMSO) δ 8.67 (s, 1H), 8.55 (dd, J = 4.1, 1.7 Hz, 1H), 7.98 (s, 1H), 7.68 (td, J = 7.1, 1.6 Hz, 1H), 7.35–7.26 (m, 3H), 7.13 (dd, J = 8.0, 2.1 Hz, 1H), 7.11 (s, 1H), 6.93 (t, J = 4.7 Hz, 1H), 6.60 (d, J = 2.1 Hz, 1H), 4.29 (d, J = 0.7 Hz, 2H), 4.09 (t, J = 6.6 Hz, 2H), 3.91 (s, 2H), 3.84 (s, 2H), 3.76–3.68 (m, 5H), 3.63 (s, 2H), 2.84 (t, J = 7.4 Hz, 2H), 2.75 (t, J = 6.5 Hz, 2H), 2.58–2.52 (m, 3H), 2.15 (s, 5H), 1.89 (p, J = 6.5 Hz, 2H); ¹³C NMR (125 MHz, Common NMR Solvents) δ 171.13, 161.15, 158.71, 156.79, 152.12, 149.42, 146.71, 146.12, 141.39, 138.97, 129.92, 128.91, 123.71, 121.46, 120.28, 107.45, 106.53, 101.70, 67.43, 64.03, 55.39, 53.57, 53.35, 51.16, 36.64, 33.11, 26.49.

3.1.9.5 Methyl 3-{[4-(4-{[(3-acetylpyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15e): ¹H NMR (500 MHz, DMSO) δ 9.21 (d, J = 2.0 Hz, 1H), 9.08 (s, 1H), 8.09 (dd, J = 7.4, 1.9 Hz, 1H), 7.98 (s, 1H), 7.35–7.25 (m, 3H), 7.13–7.07 (m, 2H), 6.93 (t, J = 4.7 Hz, 1H), 4.30 (d, J = 0.7 Hz, 2H), 4.09 (t, J = 6.6 Hz, 2H), 3.91 (s, 2H), 3.76–3.68 (m, 5H), 3.63 (s, 2H), 2.84 (t, J = 7.4 Hz, 2H), 2.75 (t, J = 6.5 Hz, 2H), 2.65 (s, 2H), 2.58–2.52 (m, 3H), 2.15 (s, 5H), 1.89 (p, J = 6.5 Hz, 2H); ¹³C NMR (125 MHz, Common NMR Solvents) δ 194.88, 171.18, 161.20, 159.53, 156.84, 152.17, 149.45, 146.76, 146.17, 139.02, 135.87, 130.84, 129.93, 128.96, 123.85, 120.33, 107.51, 106.58, 101.75, 67.48, 64.08, 55.44, 53.62, 53.40, 51.21, 39.71, 39.56, 39.52, 36.69, 33.16, 26.54, 25.55.

3.1.9.6 Methyl 3-{[4-(4-{[(3-bromopyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15f): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 0H), 8.50 (d, J = 2.0 Hz, 0H), 7.97 (s, 1H), 7.74 (dd, J = 7.9, 1.8 Hz, 1H), 7.41–7.36 (m, 0H), 7.36–7.26 (m, 2H), 7.13–7.07 (m, 2H), 4.28 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 5H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.13, 159.82, 154.60, 154.14, 151.36, 149.81, 146.23, 145.84, 138.88, 138.05, 129.43, 128.71, 125.43, 120.02, 116.98, 107.22, 106.32, 101.93, 67.32, 64.23, 55.45, 53.05, 52.81, 51.19, 39.52, 39.51, 39.48, 36.82, 32.85, 26.58.

3.1.9.7 Methyl 3-{[4-(4-{[(3-trifluoromethylpyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15g): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 1H), 8.69 (dq, J = 2.4, 1.3 Hz, 1H), 7.97 (s, 1H), 7.88 (dq, J = 6.1, 1.5 Hz, 1H), 7.45 (dt, J = 6.5, 1.1 Hz, 1H), 7.36–7.31 (m, 2H), 7.29 (t, J = 4.6 Hz, 1H), 7.13–7.07 (m, 3H), 4.28 (d, J = 0.8 Hz, 2H), 4.07 (t, J = 6.5 Hz, 2H), 3.89 (s, 3H), 3.64–3.55 (m, 9H), 2.83 (t, J = 7.4 Hz, 2H), 2.69 (t, J = 6.5 Hz, 2H), 2.51–2.46 (m, 4H), 1.90 (p, J = 6.6 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 170.57, 159.26, 156.78, 154.04, 150.80, 145.67, 145.49, 145.43, 145.37, 145.32, 145.28, 138.32, 131.43, 131.40, 131.36, 131.32, 128.86, 128.16, 125.99, 123.82, 123.65, 123.39, 123.14, 122.90, 122.88, 122.87, 122.83, 122.80, 121.64, 119.46, 106.66, 105.76, 101.37, 66.77, 63.67, 54.89, 52.49, 52.25, 50.63, 39.52, 38.96, 38.92, 36.26, 32.30, 26.02.

3.1.9.8 Methyl 3-{[4-(4-{[(2-methylpyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15h): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 0H), 7.97 (s, 0H), 7.36–7.31 (m, 1H), 7.31–7.23 (m, 1H), 7.13–7.07 (m, 1H), 4.24 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 2.43 (d, J = 0.7 Hz, 1H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.04, 159.73, 156.71, 154.87, 154.51, 151.27, 146.14, 145.75, 138.79, 136.32, 129.21, 128.66, 128.62, 120.98, 120.13, 119.96, 119.93, 107.13, 106.23, 101.84, 67.23, 64.20, 64.14, 55.36, 52.96, 52.72, 51.10, 40.59, 39.52, 39.41, 36.73, 32.76, 26.49, 23.40.

3.1.9.9 Methyl 3-{[4-(4-{[(4-trifluoromethylpyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15i): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 0H), 8.57 (d, J = 3.5 Hz, 1H), 7.97 (s, 0H), 7.60–7.54 (m, 1H), 7.36–7.26 (m, 2H), 7.13–7.07 (m, 2H), 4.26 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 5H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.17, 159.86, 156.09, 156.06, 156.03, 156.00, 154.64, 151.40, 148.87, 148.83, 148.80, 148.76, 146.27, 145.88, 138.92, 137.05, 136.79, 136.53, 136.28, 129.49, 128.75, 126.52, 124.35, 122.17, 120.47, 120.43, 120.40, 120.37, 120.06, 120.00, 117.02, 117.02, 107.25, 106.36, 101.97, 67.36, 64.26, 55.49, 53.09, 52.85, 51.23, 40.12, 39.56, 39.52, 36.86, 32.89, 26.62.

3.1.9.10 Methyl 3-{[4-(4-{[(2-chloropyridin-2-yl) methyl] sulfanyl} 3-methyl anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15j): ¹H NMR (500 MHz, DMSO-d₆) δ 9.23 (s, 0H), 7.97 (s, 0H), 7.58 (dd, J = 7.8, 7.1 Hz, 0H), 7.34–7.21 (m, 2H), 7.16 (d, J = 7.8 Hz, 0H), 7.12 (s, 0H), 7.03 (dd, J = 7.8, 2.2 Hz, 0H), 4.32 (d, J = 0.8 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 2.30 (s, 1H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.15, 159.84, 156.32, 154.49, 151.38, 150.33, 146.25, 145.86, 138.47, 138.17, 137.60, 129.26, 128.35, 122.21, 121.24, 119.20, 117.32, 107.24, 106.34, 101.95, 67.35, 64.31, 64.25, 55.47, 53.07, 52.83, 51.21, 39.63, 39.62, 39.52, 36.84, 32.87, 26.60, 19.97.

3.1.9.11 Methyl 3-{[4-(4-{[(2-bromopyridin-2-yl) methyl] sulfanyl} 3-methoxy anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15k): ¹H NMR (500 MHz, DMSO-d₆) δ 9.31 (s, 0H), 7.97 (s, 0H), 7.54 (dd, J = 7.9, 7.0 Hz, 0H), 7.37–7.26 (m, 2H), 7.18–7.10 (m, 1H), 6.67 (d, J = 2.2 Hz, 0H), 4.57 (d, J = 0.8 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.83 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 160.46, 158.10, 156.87, 155.60, 152.00, 146.86, 146.47, 141.43, 141.35, 139.32, 129.70, 125.90, 122.26, 118.96, 114.47, 107.91, 106.96, 104.68, 102.57, 67.96, 64.86, 56.09, 56.09, 53.69, 53.45, 51.83, 40.16, 40.12, 40.04, 37.46, 33.49, 27.22.

3.1.9.12 Methyl 3-{[4-(4-{[(3-methylpyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15l): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 1H), 8.37 (dd, J = 2.0, 1.0 Hz, 1H), 7.97 (s, 1H), 7.56–7.50 (m, 1H), 7.36–7.31 (m, 2H), 7.29 (t, J = 4.6 Hz, 1H), 7.22–7.16 (m, 1H), 7.13–7.07 (m, 3H), 4.24 (d, J = 0.8 Hz, 2H), 4.07 (t, J = 6.5 Hz, 2H), 3.89 (s, 3H), 3.64–3.55 (m, 9H), 2.83 (t, J = 7.4 Hz, 2H), 2.69 (t, J = 6.5 Hz, 2H), 2.51–2.46 (m, 4H), 2.25 (d, J = 1.7 Hz, 1H), 2.25 (s, 2H), 1.90 (p, J = 6.6 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 160.46, 155.24, 155.17, 152.00, 149.33, 146.86, 146.48, 139.52, 136.83, 131.15, 129.96, 129.38, 129.35, 123.39, 120.68, 120.66, 107.85, 106.96, 102.57, 67.96, 64.93, 64.86, 56.09, 53.69, 53.45, 51.83, 40.25, 40.14, 39.97, 37.46, 33.49, 27.22, 17.93.

3.1.9.13 Methyl 3-{[4-(4-{[(4-nitropyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15m): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 0H), 8.50 (d, J = 4.8 Hz, 1H), 8.13 (dd, J = 4.9, 2.2 Hz, 1H), 8.01–7.95 (m, 1H), 7.36–7.26 (m, 2H), 7.13–7.07 (m, 2H), 4.27 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 5H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 160.46, 158.65, 155.55, 155.24, 152.00, 149.21, 146.86, 146.48, 139.52, 129.78, 129.35, 120.66, 117.37, 117.11, 107.85, 106.96, 102.57, 67.96, 64.86, 56.09, 53.69, 53.45, 51.83, 40.16, 40.12, 40.05, 37.46, 33.49, 27.22.

3.1.9.14 Methyl 3-{[4-(4-{[(4-bromopyridin-2-yl) methyl] sulfanyl} 3-methyl anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15n): ¹H NMR (500 MHz, DMSO-d₆) δ 9.23 (s, 0H), 8.47 (d, J = 3.9 Hz, 0H), 7.97 (s, 0H), 7.48 (dd, J = 2.2, 1.2 Hz, 0H), 7.32–7.26 (m, 1H), 7.16 (d, J = 7.8 Hz, 0H), 7.12 (s, 0H), 7.03 (dd, J = 7.8, 2.2 Hz, 0H), 4.35 (d, J = 0.8 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 2.30 (s, 1H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 160.46, 157.59, 155.10, 152.00, 149.15, 146.86, 146.48, 138.78, 138.22, 130.83, 129.87, 128.96, 126.69, 124.66, 119.81, 117.94, 107.85, 106.96, 102.57, 67.96, 64.93, 64.86, 56.09, 53.69, 53.45, 51.83, 40.25, 40.14, 39.62, 37.46, 33.49, 27.22, 20.58.

3.1.9.15 Methyl 3-{[4-(4-{[(3-methoxypyridin-2-yl) methyl] sulfanyl} anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15o): ¹H NMR (500 MHz, DMSO-d₆) δ 9.90 (s, 0H), 8.04 (d, J = 2.0 Hz, 0H), 7.97 (s, 1H), 7.44 (dd, J = 7.5, 1.2 Hz, 1H), 7.36–7.26 (m, 2H), 7.15–7.07 (m, 2H), 4.28 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.88 (d, J = 15.9 Hz, 3H), 3.64–3.55 (m, 5H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 160.46, 155.24, 153.98, 152.00, 150.74, 146.86, 146.48, 139.52, 136.61, 130.06, 129.35, 124.33, 120.66, 119.44, 107.85, 106.96, 102.57, 67.96, 64.86, 56.09, 55.38, 53.69, 53.45, 51.83, 40.20, 40.16, 40.12, 37.46, 33.49, 27.22.

3.1.9.16 Methyl 3-{[4-(4-{[(3-nitropyridin-2-yl) methyl] sulfanyl} 3-methyl anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15p): ¹H NMR (500 MHz, DMSO-d₆) δ 9.23 (s, 0H), 9.15 (d, J = 2.0 Hz, 0H), 8.39 (dd, J = 7.7, 1.8 Hz, 1H), 7.97 (s, 0H), 7.61 (dd, J = 7.6, 1.1 Hz, 0H), 7.29 (t, J = 4.6 Hz, 1H), 7.16 (d, J = 7.8 Hz, 0H), 7.12 (s, 0H), 7.03 (dd, J = 7.8, 2.2 Hz, 1H), 4.33 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 2.30 (s, 1H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.77, 162.03, 160.46, 155.10, 152.00, 146.86, 146.48, 144.46, 143.07, 138.78, 138.22, 131.28, 129.87, 128.96, 123.95, 119.81, 117.94, 107.85, 106.96, 102.57, 67.96, 64.86, 56.09, 53.69, 53.45, 51.83, 40.16, 40.12, 39.34, 37.46, 33.49, 27.22, 20.58.

3.1.9.17 Methyl 3-{[4-(4-{[(4-bromopyridin-2-yl) methyl] sulfanyl} 3-methoxy anilino) 7-methoxy-6-[3-(morpholin-4-yl) propoxy quinazolin-2-yl] amino} propanoate (15q): ¹H NMR (500 MHz, DMSO-d₆) δ 9.31 (s, 0H), 8.47 (d, J = 3.9 Hz, 0H), 7.48 (dd, J = 2.2, 1.2 Hz, 0H), 7.35–7.26 (m, 1H), 7.18–7.10 (m, 1H), 6.67 (d, J = 2.2 Hz, 0H), 4.57 (d, J = 1.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.89 (s, 1H), 3.83 (s, 1H), 3.64–3.55 (m, 4H), 2.83 (t, J = 7.4 Hz, 1H), 2.69 (t, J = 6.5 Hz, 1H), 2.51–2.46 (m, 2H), 1.90 (p, J = 6.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 171.15, 159.84, 157.49, 156.91, 154.98, 151.38, 148.49, 146.25, 145.86, 140.81, 130.21, 129.09, 126.09, 124.05, 118.34, 113.86, 107.29, 106.34, 104.07, 101.95, 67.35, 64.31, 64.25, 55.48, 55.47, 53.07, 52.83, 51.21, 39.63, 39.52, 38.82, 36.84, 32.88, 26.60.

3.2 Anti-cancer evaluation

3.2.1 In-vitro Enzymatic Activity Assay: EGFR^WT, HER2, and the Z’-Lyte Kinase Kit were purchased from Invitrogen. The experiments were performed according to the instructions of the manufacturer. Briefly, the respective final concentrations of different kinases were determined: EGFR (PV3872, Invitrogen), 0.200 µg/mL; HER2 (PV3366, Invitrogen), 0.192 µg/mL. Six concentration (0.0001-10 µM) gradients were set for all the tested compounds in DMSO, and a 4 x compound solution was prepared. An ATP solution and a kinase/peptide mixture were prepared before use. The 10 µL kinase reaction consisted of 2.5 µL of the compound solution, 5 µL of kinase/peptide mixture, and 2.5 µL of ATP solution. A 100% phosphorylation control was provided by 5 µL of phosphopeptide solution instead of the kinase/peptide mixture. A 100% inhibition control was prepared using 2.5 µL of kinase buffer instead of the ATP solution, and 2.5 µL of 4% DMSO instead of the compound solution was used as a 0% inhibition control. The solutions on the plate were mixed thoroughly and incubated for 1 h at room temperature. After 5 µL of development solution was added to each well, the plate was incubated for 1 h at room temperature; the non-phosphopeptides were cleaved at this time. Finally, 5 µL of stop reagent was added to stop the reaction. The activity was measured with an Infinite M100 Pro multilabel reader. Curve fitting and data presentations were performed using Graph Pad Prism 5.0. Every experiment was repeated at least three times. Data represented as means ± SD from three independent experiments.

3.2.2 VEGFR-2 inhibition assay: The kinase activity of VEGFR-2 was quantified with Kinase-Glo® MAX detection reagent. This kit contains purified recombinant VEGFR-2 enzyme, VGFR-2 substrate, ATP, and kinase assay buffer in 96-well plates. To the control and test wells, diluted VEGFR-2 enzyme was added for 40 minutes at 30°C after the positive control, inhibitor, and blank wells had been set up. Each well was then incubated at room temperature for 45 minutes with Kinase-Glo® MAX reagent. Microplate readers are used to reading luminescence after 15 minutes of incubation at room temperature with aluminium foil over the plate.

Cell culture

Human breast cancer cells having differential HER2 overexpression MCF-7, BT-474, T-47D, and MDA-MB-231 cells and human hepatocellular carcinoma cell line HepG2 cells were grown at 37 ^oC in an atmosphere containing 5% CO₂, with Dulbecco’s modified Eagle Medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 2% L -glutamine, 2.7% sodium bicarbonate, 1% Hepes buffer, 40 mg/L gentamicin, and 500 mg/L ampicillin.

3.2.3 Cytotoxicity assay: The in-vitro cytotoxic activity of all synthesized compounds 15a-q were evaluated against human breast cancer cells MCF-7, BT-474, T-47D, and MDA-MB-231 cells and hepatocellular carcinoma HepG2 cells using MTT colorimetric assay according to the previously published protocol (20). Each cell line in the log growth phase was harvested by trypsinization and resuspended in a complete growth medium to give a total cell count of 5x10⁴ cells/mL. Then, 190 µl of the cell suspension was seeded into the wells of 96-well plates (Corning, Falcon). The plates were incubated overnight in a humidified air atmosphere at 37°C with 5% CO₂. After overnight incubation, 10 µl of the media containing various concentrations of the compounds were added per well in triplicate (final concentration 0.1, 0.5, 1.0, 2.5, 5, 10, and 25 µM). Then, the plates were incubated for a further 24 hr. The final concentration of DMSO in the highest concentration of the applied compounds was 0.1%. Each plate had three control wells (cells without test compounds) and three blank wells (the medium with 0.1% DMSO) for cell viability. Doxorubicin was used as a positive control for cytotoxicity. After treatment, the medium was removed, and 200 µL phenol red-free medium containing MTT (1 mg/ml) was added to wells, followed by 4 hr of incubation. After incubation, the culture medium was replaced with 100 µL of DMSO, and each well's absorbance was measured using a microplate reader at 570 nm. The concentration causing 50% cell growth inhibition (IC50) for each compound was compared to the control and calculated from concentration-response curves by non-linear regression analysis.

3.2.4 Observation of morphology of T-47D and BT-474 cells: When the cell confluence (80%) was confirmed by microscopic observation, the cell morphological studies were performed using HER2-positive T-47D and BT-474 cells by adding the test sample at two different concentrations as indicated. The experiment was performed 24 hr post-seeding to prevent cell differentiation. Both T-47D and BT-474 cells were treated for 48 hr with the compound 15i at its IC₅₀ and doubled the IC₅₀ concentrations, and Lapatinib was used as a standard drug to examine morphological changes induced. Cells were observed for 48 hr after the treatment of test samples. Images were taken by Axiovert 200M phase contrast microscope at the magnification 10x. Axiovision Rel.4.2 software was used to acquire the images.

3.2.5 Measurement of apoptosis using Annexin-V-FITC/PI staining: To assess apoptosis induction and necrosis capabilities of promising lapatinib & neratinib hybrid candidate 15i, in HE2 + ve T-47D, and BT-474 cells, a flowcytometry assisted procedure was followed using Annexin V-FITC/PI apoptosis detection kit. The apoptosis of cells was determined initially by staining them with Annexin V-fluorescein isothiocyanate (FITC) and counterstaining them with propidium iodide (PI). 5 x 10⁵ Cells were exposed to compound 15i at IC₅₀ and double the IC₅₀ for 48 h. Then, the cells were trypsinized and washed in cold phosphate-buffered saline (PBS) and stained with Annexin V-FITC and PI for 15 min at 37 ^oC in the dark. Finally, the samples were analyzed using a flow cytometer (FACS Caliber, Becton Dickinson Biosciences & Co., Franklin Lakes, NJ). For each sample, the number of analyzed cells was 30000.

3.2.6 Caspase-3, 8, and 9 activation assay: The Caspase-3, Caspase-8, and Caspase-9 multiplex activity assay fluorometric Kit (Catalog # ab219915; Abcam, USA) provides an effective means for examining the caspase activation capability using fluorometric methods. The assay utilizes DEVD-ProRed™, IETD-R110, and LEHD-AMC as fluorogenic indicators for detecting caspase-3, caspase-8, and caspase-9, respectively. Upon caspase cleavage, three distinct fluorophores are released: ProRed™ (red fluorescence), R110 (green fluorescence), and AMC (blue fluorescence), which can be readily monitored in a single assay due to their nice spectral separation. Compound 15i was added to both T-47D and BT-474 cells at IC50, double the IC50 concentrations, and incubated for 48 hr. Later the activation levels of caspase-3 8 and 9 can be determined by comparison of the fluorescence intensity in samples with and without the standard activator, i.e., staurosporine.

3.2.7 Cytochrome-C release assay: T-47D and BT-474 cells were grown in DMEM medium containing 10% fetal bovine serum at 37 ◦C for 24 hr and then treated with compound 15i, at IC50 and double IC50 for 48 hr. The quantitative determination of cytochrome C release over the treatment was evaluated using Human Cytochrome C ELISA Kit (ab221832) per the manufacturer’s instructions.

3.2.8 Western blot analysis: Western blotting was used to determine the expression of regulation of HER2 and VEGFR-2 and their downstream signalling partners over the treatment of compound 15i. T-417D and BT-474 cells (5 x 10⁴/well) were incubated with IC₅₀ concentration of compound 15i for 48 h, and the cells were lysed using the lysis buffer. The BCA assay quantified the amount of protein in the cell extracts. Equal amounts of proteins were loaded onto a 12% sodium SDS-PAGE (dodecyl sulfate-polyacrylamide gel electrophoresis), separated electrophoretically, and transferred onto a PVDF membrane (GE Health Care Life Sciences, Buckinghamshire, UK) using the standard protocol. The membranes were appropriately cut considering the molecular weight of the target protein and then probed with the anti-HER2 and anti-p-HER2 antibodies (1:2000; Abcam UK) anti-VEGFR2, anti-p-VEGFR2 antibodies (1:2000; Abcam, UK), anti-ERK1/2, anti-p-ERK 1/2 (1:3000; Abcam, UK), anti-AKTand anti-p-AKT antibody (1:3000; Abcam UK) and anti-GAPDH (1:1000; Abcam, UK) at 4°C overnight. After washing the membranes, they were incubated with the secondary antibody conjugated with horseradish peroxidase at 1:8000 dilution for two hours at room temperature. GAPDH was used to confirm equal loading in the wells. The blots were visualized with the ECL advance Western blotting detection kit (GE Health Care Life Sciences, Buckinghamshire, UK) to compare the amount of proteins in each lane. Protein levels were determined with ImageJ software.

3.2.9 In vivo anti-cancer studies of compound 15i: This study was conducted following the guidelines for the regulation of scientific experiments on animals outlined in the CPCSEA guidelines, with the Institutional animal ethics committee (IEAC) at PGP Life Sciences, Hyderabad approving all animals and protocols used in the study (PGP/077/PO/2022/CPCSEA). First, 17-β-estradiol 60-day release pellets (Innovative Research of America) were transplanted into the left flank of six-week-old athymic BALB/c nude female mice 01 day before tumour inoculation. Following this procedure, 5x10⁶ T-47D cells were subcutaneously implanted into the right flank of mice in a mixture of 50% matrigel (Corning cat. 356231, New York, NY, USA). When tumour sizes reached approximately 150–170 mm³, mice were divided into three groups, each with six mice randomized into three treatment groups: vehicle_treated, 15i_treated, and Lapatinib_treated. An intraperitoneal injection of compound 15i was administered three times per week at a dose of 10 mg/kg body weight. Throughout the 60-day study period, lapatinib was administered orally twice weekly at a dose of 60 mg/kg.bw and intraperitoneally twice weekly at a dose of 60 mg/kg.bw for five weeks. We recorded the body weight and tumour size every week using digital callipers. The size of the tumour was calculated using the following formula:

Tumour Volume (mm³) = (length [mm] x (width [mm]²)/2.

3.2.10 Molecular docking studies: Human growth factor receptors HER2 (3RCD) and VEGFR2 (5ew3) crystal structures were retrieved from the Protein Data Bank (PDB). The discovery studio Biovia 21.1.1.0 module prepared receptor and ligand molecules by addressing major structural issues, protonation, eliminating extraneous water molecules, and analyzing protein structural aspects. Each of the prepared ligands was submitted separately to dock with the target. Docking calculations were conducted with AutoDock 4.2; the auxiliary program AutoGrid generated the grid maps centred at X21.33, Y19.54, and Z32.71 and the radius 12. AutoDock performs an automated docking of the ligand with the target. The program performs several runs in each docking experiment, providing one predicted binding mode. The docking scoring function is calculated and reported. Finally, we sorted the generated poses based on their docking ratings.

In summary, a series of novel lapatinib and neratinib hybrid analogues as HER2/VEGFR2 dual inhibitors were synthesized and subjected to pharmacological evaluation to establish their anti-cancer potential. The results showed that compound 15i, among seventeen derivatives synthesized, exhibited potential dual inhibition of HER2 and VEGFR2. Protein tyrosine kinase inhibitions assay indicated that compounds 15i and 15g exerted potent activity on both HER2 and VEGFR2 kinases. Moreover, these derivatives demonstrated high anti-proliferation potencies against T-474D and BT-474 cells, having a differential expression of HER2 and VEGFR2 cells. Among them, compound 15i exhibited more potent inhibition activity than the reference compound Lapatinib which merited further evaluation. Especially compound 15i also exhibited significant morphological changes against T-47D and BT-474 cells compared with Lapatinib. Furthermore, compound 15i presented remarkably higher apoptotic induction efficacy in both T-47D and BT-474 cells which was further supported by evaluating caspases (Caspase-3, 8, and 9) and cytochrome-c releasing tendencies from both T-47D and BT-474 cells, which was significantly higher than the effect shown by staurosporine. In addition, compound 15i substantially reduced the tumour growth in T-47D xenograft mice mode than Lapatinib. The docking studies revealed that compound 15i showed strong binding patterns with both HER2 and VEGFR2 kinase domains which eventually inhibits the activation of these receptors. In conclusion, these findings presented herein showed that introducing a high polar -CF₃ group at the ^fourth position on the pyridinyl moiety of the designed lapatinib and neratinib hybrid scaffold leads to generating the potential anti-cancer candidate would act by inhibiting HER2/VEGFR2 dual inhibition.

Acknowledgements

The authors thank Anurag University for providing technical facilities and support to accomplish this research work.

Author Credits

Dr Gangarapu Kiran: Conceptualization, supervision, and guidance; Mr Varikalla Rajashakar: Investigation, Work execution, Writing-original draft, Writing- Review & Editing.

Funding

Non-available

Ethical statement

This article contains no studies with human participants or animals performed by authors.

Conflict of interest

It is stated that none of the authors have any competing financial interests or personal relationships that might have appeared to influence their work.

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Scheme 1 is available in the Supplementary Files section

Scheme1.png
Scheme 1. Synthesis of novel lapatinib and neratinib hybrid analogues: Reagents and conditions: (a) Ac₂O, AcOH, HNO₃, 0-5 ^oC; (b) 1-bromo-3-chloropropane, K₂CO₃, 60 ^oC; (c) Fe, AcOH, MeOH; (d) EtOH, NaOEt, 130 ^oC, 5h; (e) methyl 3-chloropropanoate, DMF, 50 ^oC, 4hrs; (f) SOCl₂, DMF reflux; (g) KOH, Xylenol, Reflux; (h) Pyridine. HCl, 50 ^oC, 3hrs; (i) KI, 50 ^oC, 3hrs.
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Editorial decision: Major Revision
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Targeting HER2/VEGFR2 dual tyrosine kinases with novel Lapatinib and Neratinib hybrid analogues lead to potential apoptotic induction in HER2 positive breast cancers: Design, synthesis, in-vitro, in-vivo and molecular docking studies.

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Abstract

Figures

1. INTRODUCTION

2. RESULTS AND DISCUSSION

2.1 Rational design

2.2 Chemistry

2.3 Anti-cancer evaluation

2.3.1 In-vitro kinase inhibition

2.3.2 In-vitro cytotoxic activity

2.3.3 Examination of Cell Morphology

2.3.4 SAR studies

2.3.5 Compound 15i potentially induced apoptosis in both T-47D, and BT-474 cells

2.3.6 Compound 15i significantly increased the activation of caspases-3, 8 and 9

2.3.7 Compound 15i increases the Cytochrome-c release in both T-47D, and BT-474 cells

2.3.8 Compound 15i successfully inhibited HER2 and VEGFR2 in both T-47D and BT-474 cells

2.3.9 In vivo anti-tumor Efficacy of 15i in T-47D Xenograft Models

2.3.10 Molecular docking studies revealed compound 15i, potentially blocked HER2 and VEGFR2 kinases

3. EXPERIMENTAL

3.1 General Synthesis

3.2 Anti-cancer evaluation

CONCLUSION

Declarations

References

Scheme 1

Supplementary Files

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