Synthesis and Anti-Proliferative Assessment of Triazolo-Thiadiazepine and Triazolo-Thiadiazine Scaffolds

A series of triazolo-thiadiazepines 4a–k were synthesized with excellent yields using dehydrated PTSA as a catalyst in toluene. Two triazolo-thiadiazines were obtained; 8a was formed directly by reflux in ethanol, whereas, PTSA promoted the formation of 8b. The molecular structure of the formed triazolo-thiadiazepines is identical to the imine-form 4a–k and not the enamine-tautomer 6a–k. The structures of the newly synthesized triazolo-thiadiazepines 4a–k and triazolo-thiadiazines 8a–b were elucidated using NMR (1H, and 13C), 2D NMR, HRMS, and X-ray single crystal. Furthermore, 4a was deduced using X-ray single crystal diffraction analysis. These new thiadiazepine hits represent an optimized series of previously synthesized indole-triazole derivatives for the inhibition of EGFR. The cytotoxicity activity against two cancer cell lines including human liver cancer (HEPG-2) and breast cancer (MCF-7) was promising, with IC50 between 12.9 to 44.6 µg/mL and 14.7 to 48.7 µg/mL for the tested cancer cell lines respectively, compared to doxorubicin (IC50 4.0 µg/mL). Docking studies revealed that the thiadiazepine scaffold presented a suitable anchor, allowing good interaction of the various binding groups with the enzyme binding regions and sub-pockets.


Introduction
Indole and 1,2,4-triazoles heterocycles exhibit important biological activity and are present in many drugs [1,2]. The search for model drugs for different targets has led to the identification of ribavirin (an anti-viral drug), rizatriptan (used in the treatment of migraine headaches), and letrozole (for the treatment of hormonally-responsive breast cancer after surgery) [3][4][5].
Seven-membered heterocycles, like thiazepines, have occupied a unique place in pharmaceutical chemistry as they act as calcium channel blockers and are known to be applicable as cardiovascular drugs; these include diltiazem, clentiazem, and siratiazem [6][7][8][9]. Conversely, many examples of heterocycles have been reported with the benzodiazepine motif, such as estazolam, used for treatment of insomnia; alprazolam, commonly used for the treatment of panic disorder and anxiety disorders; disorders; flurazepam, which is anticonvulsant, anxiolytic and sedative, and enhanced the skeletal muscle relaxant responses; and nevirapine, used to treat HIV-1 infection and AIDS (Figure 1) [10,11].
Additionally, thiadiazepines are expected to play a vital role in the biological system. Recently, researchers have increased the focus towards the synthesis and biological importance of thiadiazepines [12][13][14][15][16]. The synthesis of annulated heterocyclic systems, which incorporate biologically active nuclei, is expected to show interesting new pharmacological and biological activities which are highly challenging.
Recently, Boraei, A.T.A., et al., reported the cytotoxicity activity of a set of indole triazole derivatives, as potential EGFR inhibitors [17]. Therefore, we decided to combine the two previously discussed privileged structures as a potential scaffold to optimize the interaction of the indoletriazole series with the EGFR receptor.

Results
The synthesis of annulated heterocyclic systems, which incorporate biologically active nuclei, is expected to show interesting new pharmacological and biological activities which are highly challenging.
Recently, Boraei, A.T.A., et al., reported the cytotoxicity activity of a set of indole triazole derivatives, as potential EGFR inhibitors [17]. Therefore, we decided to combine the two previously discussed privileged structures as a potential scaffold to optimize the interaction of the indole-triazole series with the EGFR receptor.

Synthesis of 4a-k
The required precursor chalcones were synthesized via the reaction of aromatic aldehydes and ketones in the presence of either aqueous KOH or NaOH in ethanol [18]. Chalcones 2a-k were reacted with 4-amino-1,2,4-triazole-3-thione having indole moiety 1, using p-toluenesulfonic acid (pTsOH or PTSA) as a reaction promoter, in dry toluene for 30 min under reflux, to yield the target compounds 4a-k with high yield (Scheme 1).

Synthesis of 4a-k
The required precursor chalcones were synthesized via the reaction of aromatic aldehydes and ketones in the presence of either aqueous KOH or NaOH in ethanol [18]. Chalcones 2a-k were reacted with 4-amino-1,2,4-triazole-3-thione having indole moiety 1, using p-toluenesulfonic acid (pTsOH or PTSA) as a reaction promoter, in dry toluene for 30 min under reflux, to yield the target compounds 4a-k with high yield (Scheme 1). The mechanism of the reaction is expected to follow one of the following pathways: route (i), which involved Michael addition of the sulfhydryl group of the 4-amino-1,2,4-triazole-3-thione derivative 1 to the α,β-unsaturated carbonyl compounds 2a-k [18] to form the intermediate thia-Michael adducts 3a-k, followed by condensation and dehydration due to the intramolecular nucleophilic attack of the amino group on the carbonyl carbon, or route (ii), which involves, firstly, the removal of a water molecule due to the condensation of the amino and carbonyl groups to form the intermediate aza-dienes 5a-k, and then a subsequent intramolecular conjugate addition of the sulfhydryl group to produce triazolo-thiadiazepine [19].
Triazolo-thiadiazines 8a, and 8b were obtained via the reaction of 4-amino-1,2,4-triazole-3thione derivative 1 with phenacyl bromide and m-bromophenacyl bromide, respectively. We noticed that in the case of phemacyl bromide, 8a was obtained directly by reflux in ethanol for 4 h whereas, in the case of m-bromophenacyl bromide at the same conditions, the intermediate 7b was isolated which then cyclized by reflux in the presence of PTSA in ethanol for 20 min to afford product 8b. The formation of the intermediate 7b is suggested to be due to the deactivation caused by the electron withdrawing effect of bromine atom. Scheme 1. Synthesis and substrate scope of the target molecules 4a-k.
The mechanism of the reaction is expected to follow one of the following pathways: route (i), which involved Michael addition of the sulfhydryl group of the 4-amino-1,2,4-triazole-3-thione derivative 1 to the α,β-unsaturated carbonyl compounds 2a-k [18] to form the intermediate thia-Michael adducts 3a-k, followed by condensation and dehydration due to the intramolecular nucleophilic attack of the amino group on the carbonyl carbon, or route (ii), which involves, firstly, the removal of a water molecule due to the condensation of the amino and carbonyl groups to form the intermediate aza-dienes 5a-k, and then a subsequent intramolecular conjugate addition of the sulfhydryl group to produce triazolo-thiadiazepine [19].
Triazolo-thiadiazines 8a, and 8b were obtained via the reaction of 4-amino-1,2,4-triazole-3-thione derivative 1 with phenacyl bromide and m-bromophenacyl bromide, respectively. We noticed that in the case of phemacyl bromide, 8a was obtained directly by reflux in ethanol for 4 h whereas, in the case of m-bromophenacyl bromide at the same conditions, the intermediate 7b was 2.1.1. Structural Analysis 1 H NMR of the triazole-thiadiazepines 4a-k showing that the three protons of the thiadiazepine ring are not equivalent and appeared mostly as doublet of doublets at different chemical shifts. For example, one of the methylene protons (described as trans proton) appeared as doublet of doublets around 3.45 ppm, with coupling constants 2 J ≈ 13.5 Hz and 3 J ≈ 13.2 Hz, whereas, the second methylene proton (described as cis proton) appeared around 3.74 ppm with 2 J ≈ 13.5 Hz, and 3 J ≈ 4.7 Hz; and the methine proton (CH) attached to the asymmetric carbon was identified between δ 5.33 and 5.60 ppm with a 3 J around 13.0 and 4.7 Hz respectively. The remaining aromatic protons were observed between δ 7.00 and 8.30 ppm, while the indole NH was observed around δ 12.00 ppm. 13 C NMR displayed the thiadiazepine methylene carbon (CH2) around δ 36.6 ppm and methine carbon (CH) around δ 53.5 ppm. The quaternary carbon resulted from the carbonyl condensation, appeared around δ 171.0 ppm, and the remaining aromatic carbons appeared between δ 104.0, and 148.0 ppm.
The NMR of 7b showed the methylene protons (S-CH2-) at δ 4.30 ppm, and the amino group protons at δ 6.31 ppm. The methylene carbon (S-CH2-) appeared at δ 39.2 ppm, whereas the carbonyl carbon was found at δ 192.8 ppm. For example, one of the methylene protons (described as trans proton) appeared as doublet of doublets around 3.45 ppm, with coupling constants 2 J ≈ 13.5 Hz and 3 J ≈ 13.2 Hz, whereas, the second methylene proton (described as cis proton) appeared around 3.74 ppm with 2 J ≈ 13.5 Hz, and 3 J ≈ 4.7 Hz; and the methine proton (CH) attached to the asymmetric carbon was identified between δ 5.33 and 5.60 ppm with a 3 J around 13.0 and 4.7 Hz respectively. The remaining aromatic protons were observed between δ 7.00 and 8.30 ppm, while the indole NH was observed around δ 12.00 ppm. 13 C NMR displayed the thiadiazepine methylene carbon (CH 2 ) around δ 36.6 ppm and methine carbon (CH) around δ 53.5 ppm. The quaternary carbon resulted from the carbonyl condensation, appeared around δ 171.0 ppm, and the remaining aromatic carbons appeared between δ 104.0, and 148.0 ppm.
The NMR of 7b showed the methylene protons (S-CH 2 -) at δ 4.30 ppm, and the amino group protons at δ 6.31 ppm. The methylene carbon (S-CH 2 -) appeared at δ 39.2 ppm, whereas the carbonyl carbon was found at δ 192.8 ppm.

X-Ray Diffraction Analysis
Compound 4a fortunately was obtained in a single crystal, which was solved using X-ray single crystal diffraction [20,21]. Table S1 lists the crystallographic data collection and structure refinement features. The later compound crystalized in a space group, a = 10.1159 (11)  The structural feature of 4a as depicted in Figure 2 shows an imine functionality, and the C13=N5 bond is [1.296 (5) (15) Å]. In the molecular packing ( Figure S1), molecules were connected via strong classical intermolecular hydrogen bonds N1-H1N1···N2 i (Table S3). NMR of triazolo-thiadiazines 8a-b showed -SCH2-protons as singlets around δ 4.50 ppm, whereas the respective carbon was observed at δ 22.9 ppm. The thiadiazine quaternary carbon resulted from condensation appeared at δ 156.0 ppm in 8a and δ 154.8 ppm in 8b. The two triazole carbons in 8a-b were assigned around δ 142.0, and 146.5 ppm. The indole NH proton was identified around δ 12.00 ppm.

X-Ray Diffraction Analysis
Compound 4a fortunately was obtained in a single crystal, which was solved using X-ray single crystal diffraction [20,21]. Table S1 lists the crystallographic data collection and structure refinement features. The later compound crystalized in a space group, a = 10.1159 (11) Figure 2 shows an imine functionality, and the C13=N5 bond is [1.296 (5) (15) Å ]. In the molecular packing ( Figure S1), molecules were connected via strong classical intermolecular hydrogen bonds N1-H1N1···N2 i (Table S3).

Anticancer Activity
The cytotoxic activity of the synthesized indolo-triazolo-thiadiazepines was tested in vitro against two cancer cell lines namely human liver cancer (HEPG-2), and breast cancer (MCF-7). The hits showed IC50 between 12.9 to 44.6 µg/mL and 14.7 to 48.7 µg/mL for the tested cancer cell lines respectively, compared to doxorubicin (IC50 4.0 µg/mL) ( Table 1). The triazolo-thiadiazepine ring notably improved the binding properties and subsequent antiproliferative activity, as observed from the low activity of compounds 8a and 8b compared to that of compounds such as 4b and 4c, which possessed the triazolo-thiadiazepine nucleus. Compounds having para-substituted phenyl with an electron withdrawing group, such as 4c, 4e, and 4b were shown to have better activity than the ortho 4g or meta 4f or para 4d with electron donating analogues. Interestingly, compounds bearing a heterocyclic substituent showed slightly better activity against HEPG-2 than their phenyl counterparts (Compounds 4i, and 4j compared to compounds 4c, and 4d), an observation that was not noted against the MCF-7 cell line, which could be explained by the difference in compound

Anticancer Activity
The cytotoxic activity of the synthesized indolo-triazolo-thiadiazepines was tested in vitro against two cancer cell lines namely human liver cancer (HEPG-2), and breast cancer (MCF-7). The hits showed IC 50 between 12.9 to 44.6 µg/mL and 14.7 to 48.7 µg/mL for the tested cancer cell lines respectively, compared to doxorubicin (IC 50 4.0 µg/mL) ( Table 1). The triazolo-thiadiazepine ring notably improved the binding properties and subsequent antiproliferative activity, as observed from the low activity of compounds 8a and 8b compared to that of compounds such as 4b and 4c, which possessed the triazolo-thiadiazepine nucleus. Compounds having para-substituted phenyl with an electron withdrawing group, such as 4c, 4e, and 4b were shown to have better activity than the ortho 4g or meta 4f or para 4d with electron donating analogues. Interestingly, compounds bearing a heterocyclic substituent showed slightly better activity against HEPG-2 than their phenyl counterparts (Compounds  4i, and 4j compared to compounds 4c, and 4d), an observation that was not noted against the MCF-7 cell line, which could be explained by the difference in compound uptake by the two cancer cell types.
The chemaxon-calculated LogP was found to be approximately 4.65 for the heterocyclic derivatives, and 5.62 for the phenyl derivatives, with no big difference in the pKa compounds. The possible difference in LogP compounds could have impacted the penetration through the cancer cells and favored the HEPG-2 cells in the case of the heterocyclic derivatives.

Molecular Modeling
We have previously reported the design and synthesis of indolyl-1,2,4-triazole-3-thione and its 3-benzylsulfanyl analogues as potent anticancer agents [17]. Herein, we are presenting a lead optimization through ring variation, ring expansion, and functional group extension techniques for EGFR inhibition. Ring expansion and variation with a triazolo-thiadiazepine ring was performed to increase the flexibility of the molecule and allow better interaction of the important binding groups with the enzyme sub pockets. The triazolo-thiadiazepine ring allowed better positioning of the substituents in the hydrophobic sub pocket and the cleft region. The triazolo-thiadiazepine ring had the advantage of being a spider-like scaffold, which enables the exploration and optimization of the substituents around the scaffold for optimum enzyme binding. It also allows for pharmacokinetic optimization and preclinical development.
All active compounds showed poses that are comparable to the co-crystallized inhibitor and occupied the enzyme's three main binding pockets (Figures 3 and 4). The triazolo-thiadiazepine ring acts as an anchor that positions the compounds tightly in the active site through hydrogen bonding with key residue Met 793. The bicyclic, slightly flexible system also allowed for better interaction of the aryl and heteroaryl substituents, with the residues in the cleft and hydrophobic regions (Figures 3 and 4). The para-halo substituted derivatives demonstrated better interaction through dipole-dipole bonds, while the meta-substituted, and ortho-substituted compounds were not able to form such an interaction. This partially explained the lower activity of the ortho-, and meta-substituted derivatives, as well as the para-methyl substituted derivatives. The binding scores were slightly better in the case of the phenyl derivatives than for the heterocyclic ones; however, this was not conclusive, and the difference in activity could not be explained based on the binding energy calculation.      Compound 4j docked well in the active site and showed an important hydrogen bond with the key Met 793 residue, with the triazole anchor at a distance of 2.8 angstrom. The p-chlorophenyl ring occupied the hydrophobic pocket and formed a dipole-dipole interaction with Lys 745 side chain. The indole ring was positioned well in the cleft region and formed a hydrogen bond with Cys 797 (Figure 5a). Compound 4b showed a similar pose at the active site but with the indole ring occupying the hydrophobic pocket, and exhibited potential hydrogen bonding with Ile 744 with the possible induced fit observed in biological systems. The p-fluorophenyl interacted with residues in the cleft region, such as Met 1002 (Figure 5b).
Compound 8a had only one phenyl substituent in the thiadiazepine ring and showed comparable poses to other compounds, but with less interaction. The indole ring occupied the hydrophobic region and the triazole ring coordinated to Met 793, but lacked the interaction with the cleft region (Figure 5c).
In conclusion, the triazolo-thiadiazepine ring represents a good scaffold for EGFR inhibition, yielded active compounds that occupied all enzyme sub pockets, and has good potential for further optimization and development. Compound 4b showed a similar pose at the active site but with the indole ring occupying the hydrophobic pocket, and exhibited potential hydrogen bonding with Ile 744 with the possible induced fit observed in biological systems. The p-fluorophenyl interacted with residues in the cleft region, such as Met 1002 (Figure 5b).
Compound 8a had only one phenyl substituent in the thiadiazepine ring and showed comparable poses to other compounds, but with less interaction. The indole ring occupied the hydrophobic region and the triazole ring coordinated to Met 793, but lacked the interaction with the cleft region (Figure 5c).
In conclusion, the triazolo-thiadiazepine ring represents a good scaffold for EGFR inhibition, yielded active compounds that occupied all enzyme sub pockets, and has good potential for further optimization and development.

Synthesis of Chalcones
Chalcones were synthesized by stirring the appropriate aldehydes and active hydrogen ketones in ethanol, with a few drops of aq. NaOH (50%). The target chalcones were precipitated and subsequently underwent filtration and recrystallization from EtOH to give pure compounds [18]. 4-Amino-1,2,4-triazole-3-thione derivative 1 was prepared according to the procedures [22].

Conclusions
PTSA were found to be a useful catalyst for the synthesis of indolo-triazolo-thiadiazepine-targeted molecules in high yields. The obtained compounds were fully characterized, and the X-ray crystal structure for 4a was resolved. The triazolo-thiadiazepine scaffold represents a very promising scaffold, being spider-like, and is suitable for the parallel synthesis and subsequent development of highly selective EGFR inhibitors, as well as for selective multi-targeting to improve efficacy and reduce toxicity. We successfully prepared compounds demonstrating good antiproliferative activity, compared to that of the standard drug doxorubicin, including compounds 4b, and 4j. The molecular modeling studies demonstrated that the scaffold acts as an anchor binding the key residue Met793 and allowing the substituents to interact with the substrate access channel and the hydrophobic sub pocket. This work represents a good starting point to utilize triazolo-thiadiazepine as a promising scaffold for the optimization and preclinical development of EGFR inhibitors.
Supplementary Materials: NMR spectrum, X-ray crystallographic analysis, cytotoxicity assay, and molecular docking study associated with this article can be found in the online version.

Conflicts of Interest:
The authors declare no conflict of interest.