One-Pot Synthesis of 1-Thia-4-azaspiro[4.4/5]alkan-3-ones via Schiff Base: Design, Synthesis, and Apoptotic Antiproliferative Properties of Dual EGFR/BRAFV600E Inhibitors

In this investigation, novel 4-((quinolin-4-yl)amino)-thia-azaspiro[4.4/5]alkan-3-ones were synthesized via interactions between 4-(2-cyclodenehydrazinyl)quinolin-2(1H)-one and thioglycolic acid catalyzed by thioglycolic acid. We prepared a new family of spiro-thiazolidinone derivatives in a one-step reaction with excellent yields (67–79%). The various NMR, mass spectra, and elemental analyses verified the structures of all the newly obtained compounds. The antiproliferative effects of 6a–e, 7a, and 7b against four cancer cells were investigated. The most effective antiproliferative compounds were 6b, 6e, and 7b. Compounds 6b and 7b inhibited EGFR with IC50 values of 84 and 78 nM, respectively. Additionally, 6b and 7b were the most effective inhibitors of BRAFV600E (IC50 = 108 and 96 nM, respectively) and cancer cell proliferation (GI50 = 35 and 32 nM against four cancer cell lines, respectively). Finally, the apoptosis assay results revealed that compounds 6b and 7b had dual EGFR/BRAFV600E inhibitory properties and showed promising antiproliferative and apoptotic activity.


Introduction
The development of new drugs in anti-cancer research depends on a better understanding of druggable targets. According to this strategy, changing particular cancer biomarkers will produce beneficial therapeutic effects [1]. Selective anti-cancer medications must be more effective at destroying tumors while having fewer side effects on normal cells [2]. Single-target therapy has been recognized as causing chemotherapeutic resistance [3]. Recently, combination therapy (drug cocktails combining two or more therapeutic agents with different modes of action and non-overlapping toxicities) was authorized as an alternative to single-target chemotherapy for cancer [4]. Despite the possibility of additive and synergistic effects, combination therapy frequently results in unexpected adverse effects, such as increased toxicity. Alternatives to combination therapy include drugs with two or more targets, a reduced risk of drug interactions, enhanced pharmacokinetics (PK), and improved safety profiles [5]. Additionally, a dual-target kinase may inhibit drug interactions, harmful off-target impacts, poor patient compliance, and elevated costs of production [5].
Combining tyrosine kinase (TK) and BRAF inhibitors has proven useful in preventing tumor growth and minimizes resistance in clinical trials. Combining vemurafenib and EGFR inhibitors in thyroid carcinoma may help overcome resistance to BRAF inhibitors [6]. This combination has also been effective in BRAF V600E colorectal cancer [7]. Additionally, several in vitro substances have been developed, including EGFR/VEGFR-2 and BRAF, which contain the essential pharmacophoric groups necessary to suppress tyrosine kinase [8,9]. In summary, dual/multi-targeted kinase inhibitors can be turned into effective anti-cancer medications [10].
In medicinal chemistry, nitrogen heterocycles are crucial structural components. The chemical and biological uses of quinoline, one of many heterocyclic compounds, have been studied by numerous research groups [11,12]. The FDA recently approved quinazoline derivatives, such as gefitinib and erlotinib (Figure 1), as EGFR inhibitors for treating nonsmall cell breast and lung cancers [13][14][15]. In addition, Thr766 in the EGFR pocket forms a water-mediated hydrogen bond with the nitrogen atom at position 3 of the quinazoline core [16,17]. New quinoline derivatives, such as pelitinib and neratinib, which are potent EGFR inhibitors, were developed through the bioisosteric replacement of the quinazoline core with a quinoline ring ( Figure 1) [18][19][20][21][22]. The bioisosteric substitution did not require water molecules to facilitate the interaction with amino acid residue Thr766 [23]. We previously reported synthesizing two novel series of quinoline-2-one-based derivatives as potential apoptotic antiproliferative agents targeting the EGFR inhibitory pathway [24]. Compounds I and II (Figure 1) inhibited EGFR effectively, with IC 50 values of 0.18 and 0.09 µM, respectively. Furthermore, the two compounds significantly increased the apoptotic markers caspase-3, caspase-8, and the Bax levels while decreasing antiapoptotic Bcl2. In another study [25], compound III, a quinoline-based derivative, was developed as a dual EGFR and BRAF V600E inhibitor with IC 50 values of 1.30 and 3.8 µM, respectively.
The Spiro scaffold is another building material with potential applications in medicinal chemistry. Chemists have introduced a ring for rigidity when designing new drugs to potentially reduce the entropic penalties upon binding to target proteins. Conformational restriction can also be achieved by spiro ring fusion. They are frequently used in the design and discovery of drugs due to their inherent three-dimensionality and novel structural characteristics. Previously, spiro ring systems were efficiently integrated into enzyme inhibitors, with kinases topping the list, as well as protein-protein interaction inhibitors [26][27][28].
On the other hand, the thiazole moiety has piqued researchers' interest due to its robust biological properties [29,30]. Regarding anti-cancer activity, thiazole and its derivatives rank among the most active compounds [31][32][33][34][35][36]. Furthermore, several clinically available anti-cancer drugs contain thiazole-containing compounds, such as dabrafenib (IV, Figure 2) (BRAF inhibitor) [37]. Abdel-Maksoud et al. also developed a novel series of thiazole-based derivatives for testing as BRAF V600E inhibitors. Compound V (Figure 2) exhibited the most potent antiproliferative activity, with potent BRAF V600E inhibitory activity and an IC 50 value of 0.05 µM [38]. We recently reported the synthesis of novel thiazole-based derivatives that act as dual EGFR and BRAF V600E inhibitors [39]. Compound VI ( Figure 2) demonstrated significant antiproliferative activity against EGFR and BRAF V600E , with IC 50 values of 74 and 107 nM, respectively.
Following our previous study on dual targeting strategies and motivated by the findings above [40][41][42], we synthesized new spiro-compounds that combine quinolinone, thiazolidinone, and spiro-cyclic in a single molecule via the reaction of thioglycolic acid and 4-(2-cyclodenehydrazinyl)4uinoline-2(1H)-one. As a result, two new compounds, 6a-e (Scaffold A) and 7a and 7b (Scaffold B), were developed to generate new potent antiproliferative agents targeting EGFR and/or BRAF V600E , as shown in Figure 3.
The new compounds underwent a cell viability assay test to determine their influence on the vitality of normal cell lines. They were also tested against a panel of four cancer cell lines as antiproliferative agents. Furthermore, the most active compounds were tested for antiproliferative activity against EGFR and BRAF V600E . Finally, we tested the most active compounds for apoptotic potential against caspase 3, caspase 8, Bax, and the antiapoptotic Bcl2.
The possibility of an isomeric structure in compound 6b ( Figure 4) was ruled out by 1 H NMR, which revealed an NH chemical shift at 10.07 ppm due to hydrogen bonding with the carbonyl group (C-3 ). Furthermore, the 15 N NMR spectrum revealed a signal at δ N = 131.7 ppm, which produced an HSQC correlation with the proton at δ H = 10.07 ppm. An HMBC correlation with two protons at δ H = 10.07 and 6.11 ppm, assigned as NH-4b and H-3, was also indicated ( Figure 5). The latter applies to structure 6b but not 6b . Furthermore, the two signals at δ N = 142.1 ppm, designated as NH-1, show an HSQC correlation with the proton at H 6.11 ppm, designated as H-3. These correlations are not possible for structure 6b . We also investigated the reaction of thioglycolic acid with 4-(2-(2,2,6,6-tetramethylpiperidin-4-ylidene)hydrazinyl)quinolin-2(1H)-one (5f) and 4-(2-(1-benzylpiperidin-4-ylidene) hydrazineyl)quinolin-2(1H)-one (5g). The reaction followed the same pattern and produced similar products, 7a and 7b, in high yields (Scheme 2). For example, consider compound 7b, which was designated as 8-benzyl-4-((2-oxo-1,2-dihydroquinolin-4-yl)amino)-1-thia-4,8-diazaspiro [4.5]decan-3-one with the molecular formula C 23 H 24 N 4 O 2 S ( Figure 6, Table 1). The 1 H NMR of compound 7b revealed that H-3 was distinctive as the vinylic singlet at δ H 6.08 ppm, with its attached carbon appearing at δ C 92.33 ppm. Furthermore, H-3 exhibits an HMBC correlation with both protonated nitrogens at δ N 141.9 and 132.0 ppm. The latter could be N-1 and N-4b, although it is unclear which was which. Furthermore, N-1 exhibits an HMBC correlation with the proton resonance at δ H 7.28 ppm, implying that H-8 is present. Of the two carbons that exhibit an HSQC correlation with this proton, the one at δ C 115.45 ppm exhibits an HMBC correlation with the protons in the quinoline ring, designated as C-8. The only visible 1H doublet in the aromatic region appears at δ H 7.98 ppm and is assigned to H-5, whose attached carbon appears at δ C 122.44 ppm. H-5 establishes a COSY correlation with the dd at δ H 7.12 ppm, designated as H-6. The attached carbon appears at δ C 120.31 ppm in H-6, establishing a COSY correlation with the other dd, at δ H 7.46, designated as H-7. Attached carbon appears at δ C 130.13 ppm in H-7. In turn, H-7 establishes a COSY correlation with H-8. The carbon at δ C 139.23 establishes an HMBC correlation with H-5 and H-7, designated as C-8a. The carbon at δ C 112.26 establishes a strong HMBC correlation with H-3, NH-4b, H-6, and H-8, designated as C-4a. These six HMBC correlations are three-bond correlations. The carbon at δ C 162.90 establishes an HMBC correlation with H-3 and (weakly) NH-4b, designated as C-2. The carbon at δ C 149.51 establishes an HMBC correlation with H-3, NH-4b, H-5, and (weakly) H-8, designated as C-4. This arrangement constituted the quinolinone structure. Furthermore, H-8" is distinctive as the 2H singlet at δ H 3.56, whose attached carbon appears at δ C 61.36 ppm., and establishes an HMBC correlation with the signal at δ C 128.73 ppm, designated as C-o. Both C-o and C-m establish HSQC correlations with the m-signal at δ H 7.35 ppm. Furthermore, the carbon at δ C 126.93 establishes a COSY correlation with both H-o and H-m, and HSQC correlations with the signal at 7.28 ppm. This carbon must be C-p, as shown in Table 1.
Compounds 6a-e were synthesized by cyclizing the corresponding hydrazones 5a-e with thioglycolic acid according to the suggested mechanism in Scheme 3. Compounds 5a-c were catalyzed with thioglycolic acid to form tertiary carbocation 8 and a nucleophilic addition of HS-thioglycolic, followed by rearrangement with the loss of an H+-proton to produce the intermediate 9. Nucleophilic attacks from NH-2 to the carbonyl group eliminated an H 2 O molecule, yielding the corresponding products 6a-e via intermediates 9 and 10. Compounds 7a and 7b were also formed via a similar mechanism.

Cell Viability Assay
The epithelial (MCF-10A) cell line of a normal human mammary gland was used to test the viability of the new substances. Compounds 6a-e, 7a, and 7b were incubated on MCF-10A cells for four days before being tested for viability using the MTT assay [46,47]. According to Table 2, none of the tested substances revealed cytotoxic impacts, and the cell viability for the compounds tested at 50 µM was >89%.

Antiproliferative Assay
The antiproliferative role of 6a-e, 7a, and 7b was tested against the four human cancer cell lines, namely MCF-7 (breast cancer cell line), Panc-1 (pancreatic cancer cell line), A-549 (lung cancer cell line), and HT-29 (colon cancer cell line), using the MTT assay and erlotinib as the reference drug [48,49]. Table 2 shows the median inhibitory concentration of each tested compound (IC 50 ).
Overall, the recently screened derivatives 6a-e, 7a, and 7b showed encouraging antiproliferative action against the four cancer cell lines tested, with an average IC 50 (GI 50 ) ranging between 32 and 81 nM compared to the reference erlotinib, which had a GI 50 of 33 nM. Compounds 6b, 6e, and 7b had the highest antiproliferative activity, with GI 50 values of 35, 40, and 32 nM, respectively. Compound 7b (R = Ph-CH 2 , R 1 = R 2 = H, Scaffold B) was the most effective derivative, with a GI 50 value of 32 nM. It was comparable to, and even more effective than, erlotinib (GI 50 = 33 nM) against the MCF-7 cell line, as shown in Table 3. The other Scaffold B compound 7a (R = H, R 1 = R 2 = CH 3 ) was the least effective derivative, with GI 50 values of 81 nM. It was 2.5-fold less potent than 7b, suggesting the significance of the N-benzyl piperidine moiety for antiproliferative activity.
Compounds 6b (R = H, n = 1, Scaffold A) and 6e (R = OCH 3 , n = 1, Scaffold A) were the second and third most active compounds, with GI 50 values of 35 and 40 nM, respectively. They were 1.1-and 1.25-fold less potent than 7b. Additionally, compound 6c (R = CH 3 , n = 1, Scaffold A) showed modest antiproliferative activity with a GI 50 value of 73 nM against the four cancer cell lines tested. Compound 6c was 2.1-and 1.8-folds less potent than 6b and 6e, respectively.
Compound 6a (R = H, n = 0, Scaffold A) was 1.3-fold less potent than compound 6b (R = H, n = 1, Scaffold A) against the four cancer cell lines, with a GI50 value of 45 nM. The same pattern was observed when comparing compounds 6d (R = OCH3, n = 0, Scaffold A) and 6e (R = OCH3, n = 1, Scaffold A). Compound 6d had a GI50 of 52 nM, whereas 6e had a GI50 of 40 nM. These results showed that the spiro moiety's ring size and the type of substitution at the quinoline moiety's 6-position significantly impacted the antiproliferative function. Regarding the ring size, the cyclohexyl ring moiety was more tolerated for antiproliferative activity than the cyclopentyl ring moiety. Furthermore, the nature of substitution at the quinoline 6-position was associated with an increase in the antiproliferative activity, in the order H > OCH3 > CH3.

EGFR Inhibitory Assay
As potential targets for their antiproliferative activity, compounds 6a-e, 7a, and 7b were tested for EGFR inhibitory activity [50]. Table 3 displays these results as IC 50 values. The outcomes of this test align with the antiproliferative test, where the two most effective antiproliferative derivatives, compounds 7b (R = Ph-CH 2 , R 1 = R 2 = H, Scaffold B) and 6b (R = H, n = 1, Scaffold A), were the most effective EGFR inhibitors, with IC values of 78 ± 05 and 84 ± 06 nM, respectively. They were also equipotent to the reference erlotinib (IC 50 = 80 ± 05).

BRAF V600E Inhibitory Assay
The anti-BRAF V600E activity of compounds 6a-e, 7a, and 7b was also investigated in vitro [51], using erlotinib as a reference compound; the results are displayed in Table 3. According to the enzyme testing results, the investigated compounds had moderate to good BRAF V600E inhibitory activity, with IC 50 values between 96 and 187 nM. In every instance, the compounds were less effective than the standard drug erlotinib (IC 50 = 60 nM). Table 3 shows that the most potent EGFR inhibitors, compounds 7b (R = Ph-CH 2 , R 1 = R 2 = H, Scaffold B) and 6b (R = H, n = 1, Scaffold A), were also the most potent BRAF V600E inhibitors, with IC values of 96 ± 8 and 108 ± 9 nM, respectively. Compounds 7b and 6b were roughly equivalent to erlotinib as antiproliferative agents; however, as BRAF V600E inhibitors, they were 1.6-and 1.8-fold less potent . Compounds 6a, 6c, 6d, 6e, and 7a showed weak to moderate anti-BRAF activity with IC 50 values ranging between 129 and 187 nM, as shown in Table 3. The in vitro assay findings revealed that compounds 6b and 7b are potent antiproliferative agents that can act as dual EGFR/BRAF V600E inhibitors.

Apoptotic Markers Activation Assay
Numerous biochemical and morphological processes are involved in apoptosis, also called programmed cell death [52]. Antiapoptotic proteins such as Bcl-2 and Bc-W coexist with proapoptotic proteins such as Bad and Bax [53]. Proapoptotic proteins stimulate the release of cytochrome-c, whereas antiapoptotic proteins control apoptosis by inhibiting the release of cytochrome-c. The outer mitochondrial membrane becomes permeable when the ratio of proapoptotic proteins exceeds that of antiapoptotic proteins, setting off a series of events. The release of cytochrome c triggers both caspase-3 and caspase-9. Then, caspase-3 induces apoptosis by attacking several of the vital proteins that the cell requires [53].

Caspase 3 Activation Assay
Compounds 6b and 7b were tested as caspase-3 activators against the human pancreatic (Panc-1) cancer cell line [54]; the findings are shown in Table 4. Our results showed that derivatives 6b and 7b had a significant overexpression of caspase-3 protein levels (487.50 ± 4 and 544.50 ± 5 pg/mL, respectively) compared to the reference staurosporine (503.00 ± 4 pg/mL). The highest active substance, 7b, caused caspase-3 protein overexpression (544.50 ± 5 pg/mL) in the Panc-1 cancer cell line, which was 8.5-fold higher than the control with untreated cells, and even higher than staurosporine. Compound 6b (487.50 ± 4 pg/mL) showed comparable caspase-3 activation to the reference staurosporine. The above findings suggest that apoptosis may have contributed to the antiproliferative action of the tested compounds and caspase-3 overexpression.

Caspase-8, Bax and Bcl-2 Levels Assay
Compounds 6b and 7b were investigated for their impact on the Bacl-2, Bax, and caspase-8 levels against the Panc-1 cancer cell line, with staurosporine as a reference [55]. Our results are presented in Table 5. Our findings demonstrated that the investigated substances significantly increased the Bax and caspase-8 levels compared to staurosporine. Compound 7b had the highest level of caspase-8 overexpression (1.90 ng/mL), followed by compound 6b (1.50 ng/mL) and the reference staurosporine (1.80 ng/mL). Additionally, 7b showed a comparable induction of Bax (295 pg/mL) to staurosporine (280 pg/mL), which was 37-fold higher than the control with untreated Panc-1 cancer cells. Finally, compound 7b caused the equipotent downregulation of the Bcl-2 protein level (1.00 ng/mL), followed by compound 6b (1.30 ng/mL) in the Panc-1 cell line, and the reference staurosporine (1.10 ng/mL). The apoptosis assay results revealed that compounds 6b and 7b possess dual EGFR/BRAF inhibitory properties and show promising antiproliferative and apoptotic activity.

Conclusions
We designed and synthesized a small set of novel compounds, 4-((quinolin-4-yl)amino) thia-azaspiro [4.4/5] alkan-3-ones 6a-e, 7a, and 7b, in order to develop new dual-targeting antiproliferative agents. The MTT assay was used to investigate the antiproliferative effects of the new compounds against a panel of four cancer cell lines. Compounds 6b, 6e, and 7b had higher antiproliferative activity, with GI 50 values of 35, 40, and 32 nM, than erlotinib, which had a GI 50 of 33 nM. The most potent compounds were then tested for EGFR and BRAF V600E inhibition. Our results showed that compounds 6b, 6e, and 7b were the most potent EGFR inhibitors, with IC 50 values of 84, 92, and 78 nM, respectively. Furthermore, 6b, 6e, and 7b showed promising BRAF V600E inhibitory activity, with IC 50 values of 108, 129, and 96 nM, respectively. Therefore, they can be considered effective antiproliferative agents that act as dual EGFR/BRAF V600E inhibitors. The most active derivative in Panc-1 cells, 7b, induces apoptosis via caspase 3 overexpression and an increased ratio of Bax/Bcl-2 genes compared to the control cells. In the future, more in vitro and in vivo studies, as well as chemical modifications, may be required to develop highly effective antiproliferative agents.
General procedure for preparation of compounds 6a-e and 7a,b The 5a-g (1 mmol) and thioglycolic acid (20 mmol) were refluxed in 20 mL dray benzene in a round-bottom flask for 20 h. The reaction mixture was allowed to cool, and a white precipitate formed. The precipitate was suction filtrated and washed four times with 25 mL of 3% Na 2 CO 3 solution to remove unreacted thioglycolic acid before being thoroughly dried to yield the products 6a-e, 7a, and 7b.

Cell Viability Assay
The normal human mammary gland epithelial (MCF-10A) cell line was used to test the viability of the new compounds [46,47]. See Appendix SA (Supplementary File).

Antiproliferative Assay
The antiproliferative activity of 6a-e, 7a, and 7b was tested against the four human cancer cell lines, Panc-1 (pancreatic cancer cell line), MCF-7 (breast cancer cell line), HT-29 (colon cancer cell line), and A-549 (lung cancer cell line), using the MTT assay and Erlotinib as the reference drug [48,49]. See Appendix SA (Supplementary File).

EGFR Inhibitory Assay
Compounds 6a-e, 7a, and 7b were tested for EGFR inhibitory activity as a potential target for their antiproliferative activity [50]. See Appendix SA (Supplementary File).

BRAF V600E Inhibitory Assay
The anti-BRAF V600E activity of compounds 6a-e, 7a, and 7b was also investigated in vitro [51] using Erlotinib as a reference compound. See Appendix SA (Supplementary File).

Caspase-8, Bax and Bcl-2 Levels Assay
Compounds 6b and 7b were further investigated for their impact on the caspase-8, Bax, and Bacl-2 levels against the Panc-1 cancer cell line and staurosporine as a reference [55]. See Appendix SA (Supplementary File). Data Availability Statement: The data will be provided upon request.