Synthesis, Characterization, and Preliminary In Vitro Cytotoxic Evaluation of a Series of 2-Substituted Benzo [d] [1,3] Azoles

A series of benzo [d] [1,3] azoles 2-substituted with benzyl- and allyl-sulfanyl groups were synthesized, and their cytotoxic activities were in vitro evaluated against a panel of six human cancer cell lines. The results showed that compounds BTA-1 and BMZ-2 have the best inhibitory effects, compound BMZ-2 being comparable in some cases with the reference drug tamoxifen and exhibiting a low cytotoxic effect against healthy cells. In silico molecular coupling studies at the tamoxifen binding site of ERα and GPER receptors revealed affinity and the possible mode of interaction of both compounds BTA-1 and BMZ-2.


Introduction
Heterocyclic structures are widely distributed in nature and occur as key fragments of biomolecules, e.g., amino acids, nucleic acids, vitamins, natural products, enzymes, biological targets, etc. Besides, they constitute numerous synthetic compounds with relevant applications in different branches of chemistry and technology [1][2][3][4].
On the other hand, cancer is considered a complex group of diseases that can originate anywhere in the body, characterized by uncontrolled cell proliferation and abnormal growth of the affected tissue (neoplasm), being able to migrate causing metastasis. Cancer is the second leading cause of death worldwide; only in 2018 9.6 million deaths were estimated, with low-and middle-income countries being the most affected with approximately 70% of deaths. Lung, colorectal, stomach, liver, and breast cancers are metastatic diseases with the highest mortality rates [16]. Globally, breast cancer affects around 2.1 million
The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J s) typical for cis, trans, and geminal couplings. The methine signal (H13) of the benzyl-substituted derivatives BTA-1, BZM-2, and BOX-3 is observed as a doublet of doublets (dd) with J = 17 and 10 Hz corresponding to the couplings with protons H14a and H14b, respectively. Conversely, in the allyl-substituted compounds BTA-4, BZM-5, and BOX-6, these signals were observed as multiplets due to the additional allylic couplings with proton H8. The latter signal for the benzyl-substituted compounds BTA-1, BZM-2, and BOX-3 appear as singlets at δ 4.38-4.58 ppm, and for the allyl-substituted compounds BTA-4, BZM-5, and BOX- 6 as doublets (J = 6.9 Hz) at about 4.0 ppm. These spectroscopic data are summarized in Table 2.  3] azoles derivatives (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6).
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX- 6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26 The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26 The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26 The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The   The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The

BOX-6
Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 research group [45], involving the substitution reactions between 2-mercaptobenzimidazole and their analogs, 2-mercaptobenzothiazole and 2-mercaptobenzoxazole, with the corresponding 1-(bromomethyl)-4-vinylbenzene or allyl bromide. Most of the benzo[d]azoles heterocycle derivatives were obtained as pure microcrystalline solids in good yields (70 to 83%) with melting points between 49 and 140 °C (BTA-4 and BOX- 6 were liquids) (Table 1).  The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The   The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The   The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The   The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The   The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The The series of compounds were fully characterized by IR and NMR ( 1 H, 13 C{ 1 H}) spectroscopies, mass spectrometry, and elemental analysis. Analysis by DART and EI-MS afforded spectra that display peaks with the molecular ions plus one mass unit (M + + 1) for the compounds BMZ-2, BTA-4, BZM-5, and BOX-6 analyzed by DART, while those analyzed by EI-MS (BTA-1 and BOX-3) exhibited the expected molecular ion for the proposed structures. Additionally, analyses by 1 H NMR of compounds BZM-2 and BZM-5 produced spectra exhibiting signals around δ 11 ppm due to the -NH-proton, while signals due to the heteroaromatic cores were observed between δ 8.0 and 7.0 ppm, where signals due to the hydrogens H4 and H7 are displaced to lower field at δ 7.91-7.42 ppm. Besides, signals due to the vinyl hydrogens appear in the region of δ 6.68-5.01 ppm, with multiplicities and couplings constants (J′s) typical for cis, trans, and geminal couplings. The Further analysis by 13 C{ 1 H} NMR yielded spectra displaying all the expected signals consistent with the proposed structures for all the compounds. In general, the signal due to the quaternary carbons attached to heteroatoms appears at lower field. While the signals due to the carbon C2 binding the three heteroatoms is located between δ 166.26 and 149.87 ppm, showing further deprotection for the BTA derivatives, and signals due to carbons C3a and C7a at the bicyclic linking bonds were observed in the range δ 153.22-139.27 ppm (see experimental section).  (BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6).
compounds BTA-4, BZM-5, and BOX-6, these signals were observed as multiplets due to the additional allylic couplings with proton H8. The latter signal for the benzyl-substituted compounds BTA-1, BZM-2, and BOX-3 appear as singlets at δ 4.38-4.58 ppm, and for the allyl-substituted compounds BTA-4, BZM-5, and BOX-6 as doublets (J = 6.9 Hz) at about 4.0 ppm. These spectroscopic data are summarized in Table 2. Further analysis by 13 C{ 1 H} NMR yielded spectra displaying all the expected signals consistent with the proposed structures for all the compounds. In general, the signal due to the quaternary carbons attached to heteroatoms appears at lower field. While the signals due to the carbon C2 binding the three heteroatoms is located between δ 166.26 and 149.87 ppm, showing further deprotection for the BTA derivatives, and signals due to carbons C3a and C7a at the bicyclic linking bonds were observed in the range δ 153. 22-139.27 ppm (see experimental section).
Interestingly, derivatives BTA-1 and BZM-2 displayed the highest cytotoxic activities of all heterocyclic derivatives against the six cancer cell lines explored. Thus, compound BTA-1 showed moderate inhibition percentages of about 15% against U-251, PC-3, K-562, and HTC-15, being better than the allyl-substituted compounds BTA-4, BZM-5, and BOX-6. For breast cancer (MCF-7), derivative BTA-1 exhibited moderate inhibition (21.4%) compared to TAM (71.3%), while the cytotoxic effect was better against SKLU-1 with 38.4% compared to 43.3% of inhibition for TAM. However, BZM-2 was the one exhibiting the highest inhibitory profile among the series of benzo [d] [1,3] azole derivatives tested against the different cancer cell lines, being at least ten times more cytotoxic than the BTA-1 derivative against the different types of carcinomas, thus indicating that the BZM core increases the cytotoxic activity compared with the BTA and BOX moieties. Hence, BZM-2 exhibited the best cytotoxic effects against PC-3 (47.0%), K-562 (37.2%), MCF-7 (46.8%), and SKLU-1 (41%) cell lines. Notably, the inhibitory effect of compound BZM-2 against PC-3 was 10% greater than the reference drug, and for SKLU-1, the inhibitory percentage is very similar, more notably the fact that it exhibited a very low inhibitory effect of only 7.7% in FGH non-cancerous cells, contrasted with the 100% observed for TAM. Regarding cytotoxicity in non-cancer cells, it can be observed that most of the heterocyclic derivatives have practically null or very low cytotoxicity, with BZM-5 being the one with the greatest inhibitory effect (12%), still very low compared to TAM. In the case of the compounds with the best cytotoxic profiles against cancer cells from the in vitro studies, BTA-1 and BZM-2, the cytotoxic effect on FGH is even lower compared to the reference drug, so that BTA-1 is not active, and in the case of BZM-2, the cytotoxicity in healthy cells is very low, with a percentage of inhibition of about 8%. These values are positive and of great relevance in the search for more selective anticancer agents with low side effects. Finally, IC 50 values for compound BZM-2 were determined on PC3 (34.79 ± 0.13), K562 (22.79 ± 3.0), MCF7 (32.21 ± 0.78), and SKLU (27.93 ± 1.8).  Interestingly, derivatives BTA-1 and BZM-2 displayed the highest cytotoxic activities of all heterocyclic derivatives against the six cancer cell lines explored. Thus, compound BTA-1 showed moderate inhibition percentages of about 15% against U-251, PC-3, K-562, and HTC-15, being better than the allyl-substituted compounds BTA-4, BZM-5, and BOX-6. For breast cancer (MCF-7), derivative BTA-1 exhibited moderate inhibition (21.4%) compared to TAM (71.3%), while the cytotoxic effect was better against SKLU-1 with 38.4% compared to 43.3% of inhibition for TAM. However, BZM-2 was the one exhibiting the highest inhibitory profile among the series of benzo [d] [1,3] azole derivatives tested against the different cancer cell lines, being at least ten times more cytotoxic than the BTA-1 derivative against the different types of carcinomas, thus indicating that the BZM core increases the cytotoxic activity compared with the BTA and BOX moieties.
Hence, BZM-2 exhibited the best cytotoxic effects against PC-3 (47.0%), K-562 (37.2%), MCF-7 (46.8%), and SKLU-1 (41%) cell lines. Notably, the inhibitory effect of compound BZM-2 against PC-3 was 10% greater than the reference drug, and for SKLU-1, the inhib- Attempts to get IC 50 values in SKLU with compound BTA-1 revealed that the response was not concentration-dependent at µM concentrations of 100, 75, 50, and 25 where the r value for cytotoxicity was practically linear, and thus, if a value could be reported for this particular species, it would be >100 µM.

Molecular Docking Studies
In order to validate the performance of the molecular docking protocol, we used the crystalized X-ray ligand of the receptors with three docking programs. The receptors used for the molecular dynamic simulations were selected, since they are important targets in the development of several types of cancer. Thus, mammalian target of rapamycin receptor (mTOR) was selected, because it regulates relevant cellular pathways such as cell proliferation, autophagy, and apoptosis [46]. Additionally, the progesterone receptor (Pr), besides being a marker of the Er activity, its overexpression is observed in the development of breast cancer [47]. These facts fully justify the use of multitarget docking to evaluate the biological activity of the series of benzo [d] [1,3] azoles derivatives. The binding energy of these receptors were used as a reference to the in silico evaluation of the biological activity for the derivatives of benzo [d] [1,3] azole. To calculate the precision of the binding mode of the ligand, we calculated the root-mean-square deviation (RMSD) comparing the position of the ligand in the best poses obtained from the three programs and the crystalized ligand (Supplementary Materials Figure S19). We found a RMSD for all receptors lesser than 1 A • ( Table 4), which is a good performance for docking simulations. In addition, the binding energy for the redocking process results in a strong biding energy with the three programs. Table 4. Docking validation for the four receptors using three different molecular docking programs. The calculated root-mean-square deviation (RMSD) respect to the crystalized ligand is also shown. To select the best poses among all the conformations explored by the three programs, we used an exponential consensus rank approach. These results are shown in Table 5 in which it can be observed a good correlation between the experimental data and the molecular docking results. Since tamoxifen (TAM) was the crystal ligand in Erα; this was used as reference. As shown in Table 5, TAM has a strong interaction with all the receptors that could be related with an inhibition of the metabolic pathway. Compounds BTA-4, BZM-5, and BOX-6 establish weak interaction with all the receptors, while compound BZM-2 prefers the interactions with Erα and mTOR receptors. In addition, compound BTA-1 interacts with the EGRF pathway and the ligand BOX-3 with Pr. In spite of the molecular similarity, the series of compounds interact in different targets of cancer pathways.  3] azole to enter into the cellular membrane, we calculated the LogP for all the derivates. The results are presented in Table 5. As can be seen from this table, TAM and compound BTA-1 presented the higher values of LogP compared with the other derivates. The sulfur atom in the azole group increases the hydrophobicity of the derivates BTA-1 and BTA-4 lowering the LogP values. From the whole group of ligands, the less hydrophobics were derivates BZM-5 and BOX-6. These low values suggest a bigger difficulty to enter into cell and generate a pharmacological activity.      As it can be observed, the molecular docking simulations described the stabilizing interactions between the derivatives and four proteins related to the development of various types of cancer. The in silico evaluation is in good agreement with the preliminary in vitro cytotoxicity bioassays. That is, compounds BTA-1, BZM-2, and BOX-3 exhibit better interactions with the proteins due to the presence of the benzyl moiety in their structures thus facilitating the formation of stabilizing pi-sigma interactions. In contrast, the vinyl group in derivatives BTA-4, BZM-5, and BOX-6 hampers the formation of any interaction with the proteins . Compounds BTA-1, BZM-2, and BOX-3 also exhibited the larger values of LogP this being in favor of a better membrane transports of these species that in turn may also favor their cytotoxic activities, as was the case for compounds BZM-2 and BTA-  As it can be observed, the molecular docking simulations described the stabilizing interactions between the derivatives and four proteins related to the development of various types of cancer. The in silico evaluation is in good agreement with the preliminary in vitro cytotoxicity bioassays. That is, compounds BTA-1, BZM-2, and BOX-3 exhibit better interactions with the proteins due to the presence of the benzyl moiety in their structures thus facilitating the formation of stabilizing pi-sigma interactions. In contrast, the vinyl group in derivatives BTA-4, BZM-5, and BOX-6 hampers the formation of any interaction with the proteins . Compounds BTA-1, BZM-2, and BOX-3 also exhibited the larger values of LogP this being in favor of a better membrane transports of these species that in turn may also favor their cytotoxic activities, as was the case for compounds BZM-2 and BTA- As it can be observed, the molecular docking simulations described the stabilizing interactions between the derivatives and four proteins related to the development of various types of cancer. The in silico evaluation is in good agreement with the preliminary in vitro cytotoxicity bioassays. That is, compounds BTA-1, BZM-2, and BOX-3 exhibit better interactions with the proteins due to the presence of the benzyl moiety in their structures thus facilitating the formation of stabilizing pi-sigma interactions. In contrast, the vinyl group in derivatives BTA-4, BZM-5, and BOX-6 hampers the formation of any interaction with the proteins. Compounds BTA-1, BZM-2, and BOX-3 also exhibited the larger values of LogP this being in favor of a better membrane transports of these species that in turn may also favor their cytotoxic activities, as was the case for compounds BZM-2 and BTA-1.

Reagents and Apparatus
All reagents used were purchased commercially from Aldrich Chemical. Co. Inc. (St Louis, MO, USA), and used without further purification. Solvents were supplied by J.T. Baker (Phillipsburg, NJ, USA), which were dried and distilled prior to use, using standard procedures established under dinitrogen atmosphere. The melting points were determined (without corrections) using a MELT-TEMP II Laboratory Devices, vibrational spectroscopy IR were performed in the range of υ 4000 to 350 cm −1 in a NICOLETMAGNA spectrometer 750 FT-IR in KBr tablet. EI-MS and DART ± MS were carried out using a JEOL JMS-SX102A and JEOL JMS-T100LC spectrometer, respectively. NMR spectra were recorded in CDCl 3 at room temperature on a JEOL spectrometer GX300 ECLIPSE at 300 MHz frequency for 1 H and 75 MHz for 13 C{ 1 H}. The chemical shifts (δ) for 1 H and 13 C are reported in ppm down field of Si (Me) 4 (δ = 0.0). The abbreviations used in the description of the NMR data are the following: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; td, triplet of doublets; and m, multiplet. Coupling constants are reported as J in Hz. The number of protons (n) for a given resonance is indicated by nH. NMR spectra, mass spectra and docking results are available in supporting material. The series of 2-substituted benzo[d] [1,3]azoles derivatives were obtained by the following general procedure: To a suspension of 2-mercaptobenzothiazole (3.53 mmol) and K 2 CO 3 (0.553 g, 4 mmol) in THF (30 mL), the corresponding 1-chloromethyl-4-vinylbenzene (or allyl bromide) (0.5 mL, 3.8 mmol) was added dropwise. The reaction mixture was stirred for 48 h at room temperature under N 2 atmosphere. The reaction was monitored by thinlayer chromatography to completion. Then, the reaction was filtered and washed with CH 2 Cl 2 (3 × 5 mL). The organic filtrate was dried over anhydrous Na 2 SO 4 and evaporated. Solid compounds were recrystallized from hexane/AcOEt and dried under vacuum to afford the desired product. adenocarcinoma (PC-3), chronic myelogenous leukemia (K-562), colorectal adenocarcinoma (HTC-15), lung (SKLU-1), mammary adenocarcinoma (MCF-7), and non-cancer gingival fibroblast cells (FGH). The different carcinogenic cell lines were removed from tissue culture flasks by trypsin treatment and diluted with fresh medium. From these cell suspensions, 100 µL containing 5000-10,000 cells per well were pipetted into 96-well microtiter plates (Costar), and the material was incubated at 37 • C for 24 h in a 5% CO 2 atmosphere. Subsequently, 100 µL of a solution of each well was added the compound obtained by diluting the reserves. The cultures were exposed for 48 h to the compound at 25 µM concentrations. After the incubation period, cells were fixed to the plastic substrate by adding 50 µL of cold 50% acidic aqueous trichloroacetic. The plates were incubated at 4 • C for 1 h, washed with tap water, and air-dried. Cells fixed with trichloroacetic acid were stained with the addition of 0.4% SRB. Free SRB solution was removed by washing with 1% aqueous acetic acid. The plates were then air-dried, and the bound dye was solubilized by the addition of tris without 10 mM base buffer (100 µL). The plates were placed and shaken for 10 min, and the absorption was determined at 515 nm using an ELISA plate reader (Bio-Tex Instruments).

Methodology and Computational Details
Electronic structure calculations were carried out by using the B3LYP density functional. The initial structure of ligands was drawn with Avogadro Software [52]. For all atoms, we used the Pople 6 -31 + g basis set. All ligands were fully optimized in their geometries, and all stationary points were characterized as minima through frequency calculations. Solvent effects (water) were included with the SMD continuum method [53]. Atomic charges to be used in the molecular docking simulations were calculated with the NPA scheme. All calculations were carried out with gaussian16 suite of programs [54].
To improve the Docking outcomes and reduce the effects of force fields and system dependence, we used an exponential rack consensus (ECR) proposed by Palacio-Rodriguez et al. [55]. The ECR of each molecule was described using a consensus score P(i) corresponding to the sum of the exponential rank of j programs, by following the following equation: where σ is the expected value of the exponential distribution respect to the number of molecules i, and r j i is the rank of molecule i predicted with the program j. We used this consensus for three molecular docking programs. AutoDock 4 (AD4) [56], AutoDock Vina (Vina) [57], and Smina (vinardo scoring function) [58]. Current cancer therapies seek to target the hormone receptors. The receptors considered in this work were Erα, PR, EGFR, and mTOR. These tridimensional structures were taken from PDB codes 3ERT [59], 4OAR [60], 2J6M [61], and 4DRH [62]. These structures were used as a target in previous computational works related with anticancer activity [63][64][65]. The receptors files were prepared in the graphic interface Auto-Dock Tools 1.4.5 (ADT) [56] where the water molecules were removed, and all hydrogens atoms were properly added, and nonpolar hydrogen were merged to carbon atoms. The Gasteiger charges were added in the receptors to prepare the pdbqt files. The charges of the ligands were taken from the NPA population analysis based on density functional theory calculations. In all docking experiments, a grid box of size 60 × 60 × 60 • A 3 in X, Y, and Z coordinates with a spacing of 0.375 • A was used. The ligand-receptor complexes were analyzed with LigPlot + [66] PyMol [67] and Maestro Schrodinger programs [68].

Conclusions
We have presented a small library of 2-substituted benzo[d] [1,3]azoles derivatives; these species can be synthesized in a simple and good yield manner. Preliminary in vitro cytotoxicity bioassays with the series of compounds BTA-1, BZM-2, BOX-3, BTA-4, BZM-5, and BOX-6 has been complemented with in silico molecular docking studies, these results suggesting that molecules containing the aromatic vinyl moieties are anchoring better to ERα and GPER than those including the allylic fragments. While the higher affinity of BTA-1 and BZM-2 may be due to the atoms with a higher electron density that they present (S and N), compared to BOX-3, which includes a harder, more electronegative O in its structure. The major bioactivity in vitro of BZM-2 and its affinity for ERs may be due to the additional interaction by hydrogen bonds that it establishes with the nitrogenous unit -NH-. The latter compound BMZ-2 is especially interesting for future studies and structural modifications, since it was not only the most active compound of the series, but also the one with the lower activity against healthy cells. Hence, further studies may allow us to shed further light in the design of other species with enhanced activities aimed to develop new, specific, yet safer antineoplastic agents, related to the breast cancer estrogen receptors (ERα and GPER).