Design, Synthesis and Biological Assessment of N′-(2-Oxoindolin-3-ylidene)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazides as Potential Anti-Proliferative Agents toward MCF-7 Breast Cancer

Breast cancer is a serious threat to the health and lives of women. Two novel series of N′-(2-oxoindolin-3-ylidene)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazides and 1-(aryl)-3-(6-methylimidazo[2,1-b]thiazol-5-yl)ureas were designed, synthesized and investigated for their anticancer efficacy against the MCF-7 breast cell line. Three compounds of the first series showed potent activity toward MCF-7 with IC50 in the range 8.38–11.67 µM, respectively, as compared to Sorafenib (IC50 = 7.55 µM). N′-(1-butyl-2-oxoindolin-3-ylidene)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide inhibited VEGFR-2 with IC50 = 0.33 µM when compared with Sorafenib (IC50 = 0.09 µM). Furthermore, this compound was introduced to PCR assessment, where it increased Bax, caspase 8, caspase 9 and cytochrome C levels by 4.337-, 2.727-, 4.947- and 2.420-fold, respectively, while it decreased levels of Bcl-2, as the anti-apoptotic gene, by 0.359-fold when compared to the untreated control MCF-7. This compound was also arrested in the G2/M phase by 27.07%, compared with 11.31% for the control MCF-7. Furthermore, it induced early and late apoptosis in MCF-7. In addition, a molecular docking study in the VEGFR-2 active site was performed to assess the binding profile for the most active compounds. Moreover, ADME parameters of the targeted compounds were also evaluated.


Introduction
Cancer is one of the most lethal diseases, and it is rapidly evolving into one of the world's most serious health challenges.Furthermore, the unfavorable side effects of classic non-selective chemotherapies, as well as the growth of resistance to existing chemotherapy medications, make the search for more selective novel cancer fighters a top goal.However, it is difficult to design a drug that prevents the production of aberrant cells with no influence on the exerting of normal cells [1,2].Pathological angiogenesis, on the flip side, occurs not just in the growth of tumors but additionally in a variety of non-neoplastic conditions [3].Angiogenesis is also suggested to be linked to a variety of disorders, including cancer [4,5].The inhibition of angiogenesis may decrease the tumor development because it can slow tumor growth without creating significant negative or resistant effects over time [6].The VEGF (vascular endothelial growth factor) family members are well-known angiogenic agents that govern blood and lymphatic vessel development and balance in both normal and pathological angiogenesis.known angiogenic agents that govern blood and lymphatic vessel development and balance in both normal and pathological angiogenesis.
VEGF-A assumes a crucial role in directing vascular development and angiogenesis by interacting with VEGFR-2.The nuanced functions of VEGFR-2 in blood vessel development are supported by adaptor proteins.In the realm of cancer, the secretion of VEGF by cancer cells is instrumental in activating the VEGFR-2 pathway in neighboring endothelial cells, thereby participating in the phenomenon of cancer-related angiogenesis.Notably, the activation of VEGFR-2 signaling has been discerned in breast cancer cells [7][8][9].
A new class of imidazo [2,1-b]thiazoles were developed for anticancer activity and they showed potential activity toward MCF-7 cell line and elicited apoptotic properties such as caspase-9 upregulation; for example, imidazo [2,1-b]thiazole derivatives IX showed IC 50 equal to 0.794 µM on the MCF-7 cell line and were further used for comprehensive biological investigations [22] (Figure 1).
As illustrated in Figure 1, isatin in Sunitinib (III) and lead compounds VI-VIII, in addition to the imidazo [2,1-b]thiazole system in the potent anticancer compound IX, are important in the synthesis of potent VEGFR-2 inhibitors and/or MCF-7 anticancer agents.On the other hand, the hydrazine linker in compounds VI-VIII and urea bridge in Sorafenib (I), Lenvatinib (II), IV and V are critical for the same purpose.
In continuation to our research, which deals with the discovery of novel potent anticancer agents [23-28], herein we utilized the fragment linking strategy to construct novel anti-proliferative agents for MCF-7 breast cancer therapy and evaluated their effectiveness on VEGFR enzyme inhibition.
The Sorafenib-VEGFR-2 crystal structure showed the role of the pyridine ring of Sorafenib (I) with certain amino acids in the VEGFR-2 hydrophobic pocket [29,30] (Figure 2).However, it is important to understand the SAR of synthetic VEGFR-2 kinase inhibitors [31], which may be a critical factor in determining the efficiency of the agent and its tolerability.
The binding mode of VEGFR-2 inhibitors is characterized by a "head" component that binds to the hinge region and possesses crucial H-bond donor and/or acceptor capabilities for interacting with Cys919.Additionally, they have a "linker" spanning three to four chemical bonds with H-bonding moiety to establish the interactions with Asp1046.Furthermore, they have a "tail" segment containing a hydrophobic moiety that occupies the allosteric hydrophobic back pocket [32][33][34].
In this context, we developed two sets of N -(2-oxoindolin-3-ylidene)-6-methylimidazo thiazole-5-carbohydrazides 6a-l and 1-(aryl)-3-(6-methylimidazo[2,1-b]thiazol-5-yl)ureas 8a-g (Figures 1 and 2).Regarding the binding pocket of Sorafenib (I), the pyridine residue was specifically linked with Leu1035, Phe1047, Val848, Val916 and Cys1045 through various forms of interactions in the hinge region, whereas the H interaction was preserved via the urea linker with Asp1046.Furthermore, Leu889 and Glu885 maintained the link between the substituted phenyl ring through distinct interactions (Figure 2).Therefore, the aimed structures 6a-l were designed according to the fragment combination approach, as follows: the 6-methylimidazo[2,1-b]thiazole system replaced the 4-phenoxypyridine moiety in the left side of Sorafenib (I), whereas the isatin moiety with several substitution patterns in position 1 and/or 5 replaced the aryl group of the right side of Sorafenib (I) with a carbohydrazide linker (H-bond acceptor-H-bond donor linker) similar to the urea bridge in Sorafenib (I) in order to obtain an activity similar or higher to that of Sorafenib (I) (Figure 2).We also followed the same approach to design urea derivatives 8a-g (Figure 2).The latter two sets were synthesized and evaluated toward MCF-7 cell cytotoxicity.The most potent compounds were introduced to examine their activity toward VEGFR-2, PCR assessment, apoptosis and cell cycle analysis.

Chemistry
The reaction of 2-chloroethylacetoacetate (1) with 2-aminothiazole (2), in 1,2-dimethoxyethane at 90 • C, yielded the key ester 3.Under reflux circumstances, the latter ester was transformed into acid hydrazide 4 using 99% hydrazine hydrate [29].Subsequently, compound 4 was reacted with several substituted indoline-2,3-dione derivatives 5a-l to yield the target 6-methyl-N - On the other hand, compound 4 was subjected to a Citrous rearrangement process by its reaction with NaNO2 and HCl to obtain azide 7 [30] which was then refluxed in dioxane with various anilines to yield urea derivatives 8a-g (Scheme 2).The 1 H NMR spectra of compounds 8a-g revealed three signals, one around 2.12 ppm for CH3 protons and the remaining two signals around 8.16 ppm and 9.02 ppm for two D2O exchangeable NH protons of the urea linker.Compound 8g was characterized with an additional D2O exchangeable signal of sulphonamide NH2 at 7.17 ppm.The existence of two separate signals On the other hand, compound 4 was subjected to a Citrous rearrangement process by its reaction with NaNO 2 and HCl to obtain azide 7 [30] which was then refluxed in dioxane with various anilines to yield urea derivatives 8a-g (Scheme 2).The 1 H NMR spectra of compounds 8a-g revealed three signals, one around 2.12 ppm for CH 3 protons and the remaining two signals around 8.16 ppm and 9.02 ppm for two D 2 O exchangeable NH protons of the urea linker.Compound 8g was characterized with an additional D 2 O exchangeable signal of sulphonamide NH 2 at 7.17 ppm.The existence of two separate signals around 13.37 ppm and 154.20 ppm for the CH 3 and carbonyl groups was seen in 13 C NMR spectra of 8a-g.The 1 H NMR of compounds 8b and 8c showed the signals of -CH 3 and -OCH 3 protons, respectively, and the appearance of their carbon signals in 13 C NMR (Figures S20-S33, Supplementary Data).around 13.37 ppm and 154.20 ppm for the CH3 and carbonyl groups was seen in 13 C NMR spectra of 8a-g.The 1 H NMR of compounds 8b and 8c showed the signals of -CH3 and -OCH3 protons, respectively, and the appearance of their carbon signals in 13  ii

Apoptotic Assay for Compound 6i
In comparison to the control cells, the apoptotic assay employing Annexin V/PI analysis for compound 6i generated a substantial amount of early and late apoptosis in MCF-7.Compound 6i induced early apoptosis in MCF-7 at rate of 22.05, compared with the control MCF-7 (0.43) (Table 6 and Figure 5).Furthermore, 6i demonstrated late apoptosis at rate 7.61 compared with the control MCF-7 (0.11).

Apoptotic Assay for Compound 6i
In comparison to the control cells, the apoptotic assay employing Annexin V/PI analysis for compound 6i generated a substantial amount of early and late apoptosis in MCF-7.Compound 6i induced early apoptosis in MCF-7 at rate of 22.05, compared with the control MCF-7 (0.43) (Table 6 and Figure 5).Furthermore, 6i demonstrated late apoptosis at rate 7.61 compared with the control MCF-7 (0.11).

Molecular Modeling Study
The computational docking method is used for approximating two fundamental concepts.The main objective is to determine the correct spot and orientation of a particular chemical pose in the binding site of the enzyme.The second concept is the calculation of docking score (the strength of protein-ligand interactions) [37].In an attempt to explain the cytotoxic action of 6b, 6i and 6j, a docking study in the VEGFR-2 active site was performed.Redocking of sorafenib (I) in the active site of VEGFR-2 (PDB: 4ASD) was nearly in the exact position as the co-crystalized ligand with d. s. = −10.6 kcal/mol) (Figure S1, Supplementary Data).
The docking investigation of 6b, 6i and 6j in the VEGFR-2 active site revealed binding mechanisms that were similar to the lead medicine and they showed docking scores that were higher or comparable to the docked lead.The hot areas were satisfied by targeted compounds, which established H-bonds with Cys919, Glu885 and Asp1046 residues which helped to explain the substantial VEGFR-2 inhibitory effect, as shown in Figure 6.

Molecular Modeling Study
The computational docking method is used for approximating two fundamental concepts.The main objective is to determine the correct spot and orientation of a particular chemical pose in the binding site of the enzyme.The second concept is the calculation of docking score (the strength of protein-ligand interactions) [37].In an attempt to explain the cytotoxic action of 6b, 6i and 6j, a docking study in the VEGFR-2 active site was performed.Redocking of sorafenib (I) in the active site of VEGFR-2 (PDB: 4ASD) was nearly in the exact position as the co-crystalized ligand with d. s. = −10.6 kcal/mol) (Figure S1, Supplementary Data).
The docking investigation of 6b, 6i and 6j in the VEGFR-2 active site revealed binding mechanisms that were similar to the lead medicine and they showed docking scores that were higher or comparable to the docked lead.The hot areas were satisfied by targeted compounds, which established H-bonds with Cys919, Glu885 and Asp1046 residues which helped to explain the substantial VEGFR-2 inhibitory effect, as shown in Figure 6.S1, Supplementary Data).
As shown in the interactions of 6b, 6i and 6j on the VEGFR-2 active site, they interacted with the same pattern of sorafenib (I) with a hydrogen-bond interaction with amino acid Asp1046 via the linker hydrazide moiety, while imidazothiazole bonded with Val916  S1, Supplementary Data).
As shown in the interactions of 6b, 6i and 6j on the VEGFR-2 active site, they interacted with the same pattern of sorafenib (I) with a hydrogen-bond interaction with amino acid Asp1046 via the linker hydrazide moiety, while imidazothiazole bonded with Val916 and Cys1045 as the pyridine side; on the other hand, phenyl isatin bonded with Leu889 via pi-sigma as the substituted phenyl in the Sorafenib (I) structure (Figure S2, Supplementary Data).

Computational ADME Study
Three rules, at least, of the following five rules are requested for oral bioavailability: There are no more than five H-bond donors, no more than 500 Da MWt, no more than five log P, only one violation and no more than ten H-bond acceptors, according to Lipinski's rule of five [38].The Veber rule predicts that molecules with less than 10 rotatable bonds and at least 140.2 of polar surface area will have appropriate oral bioavailability [39].ADME parameters of the targeted compounds 6a-l and 8a-g such as the proportion of human intestinal absorption and blood-brain barrier penetration were evaluated using the SWISSADME method.Their BBB penetration was expected to be minimal, which prevents these candidates from passing the BBB or having harmful effects on the central nervous system (Figure S3 and Table S2, Supplementary Data).

Chemistry
The specifications of instruments used in the chemistry part are listed in the Supplementary Data, page 42.

PCR Assay
The gene expression levels of apoptosis genes were evaluated in MCF-7 cells using the following approaches in the Supplementary Data, page 43.

Cell Cycle Analysis and Apoptosis
These were performed using the approach described in the Supplementary Data, page 44 [46].

In Silico Predictive ADME Study
The ADME study was performed using the SWISSADME server [47] (Supplementary Data, page 45).

Conclusions
Three derivatives, 6b, 6i and 6j, of isatin-imidazo[2,1-b]thiazole hybrids with a carbohydrazide function as a linker showed significant anti-cancerous activity using the MTT assay, with the most active candidates being submitted for further investigation to test their invitro VEGFR-2 inhibitory efficacy compared to Sorafenib as a reference drug.The potent candidate 6i was then subjected to a PCR study to measure Bax, Bcl2, caspase 8, caspase 9 and cytochrome C to understand the potency of the most active candidate 6i on malignant cells.Ultimately, for the full investigation regarding its activity, 6i was utilized for the cell cycle and apoptosis.It additionally arrested the cell cycle in the G2/M phase and induced apoptosis at rates higher than a standard drug.A docking protocol and ADME study were also performed and our candidates had the same binding mode of Sorafenib through interactions with the amino acid residues Lys868, Cys1045, Glu885 and Asp1046.As a consequence, 6i was specified as a promising lead for additional research to design efficient anti-cancer agents.

Figure 1 .
Figure 1.Structure of clinically approved VEGFR-2 inhibitors and some reported anticancer agents (I-IX) with their corresponding IC 50 values, in addition to our targeted hybrids 6a-l and 8a-g.

Figure 2 .
Figure 2. Docking of Sorafenib (I) with VEGFR-2 binding site and the assumed possible modifications of the targeted structures 6a-l and 8a-g.
1 H NMR data of compounds 6a-d revealed three distinct signals, one around 2.65 ppm for CH3 of imidazo[2,1-b]thiazole and two D2O exchangeable signals around 11.30 and 13.26 ppm for isatin NH and carbohydrazide NH, respectively.Compounds 6e-h with n-propyl substitution in position 1 showed three aliphatic signals of the -CH2-CH2-CH3 group around 0.90,

Figure 2 .
Figure 2. Docking of Sorafenib (I) with VEGFR-2 binding site and the assumed possible modifications of the targeted structures 6a-l and 8a-g.

Figure 3 .
Figure 3. Quantitative RT-PCR results analysis of the apoptosis-related genes, cytochrome, Bax, Caspases 8, 9 and Bcl-2, in 6i-treated MCF-7 cells.Dashed line represents the gene expression for potent 6i and blue line represents untreated control.

Table 4 .
Fold change of apoptosis genes in treated MCF-7 cells with 6i.