Identification of bisindolylmethane–hydrazone hybrids as novel inhibitors of β-glucuronidase, DFT, and in silico SAR intimations

Abstract

The present study involves the synthesis of bisindolylmethane–hydrazone hybrids, 1–30, in a three-step reaction sequence, followed by evaluation against β-glucuronidase enzyme. The IC₅₀ values for the potent compounds were in the range from 0.10 to 83.50 μM. Compound, a trihydroxy analog was found to be the most potent derivative, having an IC₅₀ value of 0.10 ± 0.001 μM. Molecular docking revealed that the active compounds could fit perfectly into the binding groove of β-D-glucuronidase. The presence of hydroxyl groups on the aromatic side chain proved to be the single most important factor that contributed toward the inhibitory potential of these compounds. On the other hand, the imino group of the hydrazone linkage displayed interactions with the side chain carboxyl oxygen (Oε2) of Asp207. The high inhibitory potential of these compounds could be associated with these strong hydrogen bonds. Structures of all the synthesized compounds were confirmed using modern spectroscopic methods.

---

1. Introduction

A number of indole alkaloids are found naturally in marine and terrestrial sources with unique chemical structures and diverse biological potentials such as antibacterial, cytotoxic,¹ and protection against DNA damage in human colon cell lines.² Some commercial drugs contain an indole moiety as either a fundamental unit or as an attached part to invoke specific biological activities.³ Bisindole is a special class of the indole family applauded as an important scaffold in pharmaceutical chemistry.^4–9 Bisindole and its derivatives have been widely identified as a pharmacophore that can exhibit antiviral,¹⁰ anticancer,¹¹ antitubercular,¹² antihypertension,¹³ free radical induced lipid peroxidation,¹⁴ antioxidant,¹⁵ Alzheimer disease, and antihyperlipidemic potentials.¹⁶ A number of bis(indolyl)methanes were reported to have antioxidant potential,¹⁷ anti-inflammatory with analgesic properties, and ulcerogenic activities in mice and rat models.¹⁸ Their interesting biological profiles, distinctive chemical structures, and low availability make the bisindole alkaloids attractive targets for use in drug discovery.

β-Glucuronides are the final metabolites of hydrophobic xenobiotics,¹⁹ and their hydrolysis is catalyzed by the β-glucuronidase enzyme, which is commonly found in animals, plants, and bacteria.²⁰ In animals, low β-glucuronidase activity is maintained through localization in lysosomes.²¹ However, β-glucuronidase produced by intestinal bacteria hydrolyzes glucuronide to liberate xenobiotics. These xenobiotics exhibit toxicity in the intestine and lower the rate of excretion of xenobiotic by reabsorption. Thus, to decrease toxicity, the inhibition of bacterial β-glucuronidase in the intestine is required to promote the excretion of xenobiotics. A number of β-glucuronidase inhibitors have been isolated from nature and categorized as terpenoids and their glucuronides,^22–25 flavonoids and their glucuronides,^26,27 and pseudo-sugars that contain nitrogen,²⁸ whereas various synthetic compounds have also been found to be potent β-glucuronidase inhibitors.²⁶

In continuation of our work on the development of new enzyme inhibitors for drug discovery, we produced ongoing work on the synthesis and biological evaluation of potent heterocyclic scaffolds.^27–29 Recently, we published bisindolylmethane analogs as a potent inhibitor of β-D-glucuronidase enzyme. This effort resulted in the identification of some novel inhibitors of β-D-glucuronidase enzyme.³⁰ However, these bisindolylmethane derivatives seriously lacked the sites for further modifications for the identification of more potential leads. For a more potent analog identification, we adopted the technique of utilizing hybrid molecules, in which we combined two or more biologically active scaffolds. This methodology proved extremely successful and led us to identifying some distinctly potent inhibitors, such as the bisindolylmethane–hydrazone hybrids 1–30 of the β-D-glucuronidase enzyme in the present study. The synthesized hybrids were evaluated in vitro against β-D-glucuronidase enzyme and an SAR was developed, whereas the mode of binding action was evaluated by molecular modeling and DFT studies.

2. Results and discussion

2.1. Chemistry

Bisindolylmethane ester I was obtained by reacting 1-methyl-1H-indole and methyl 4-formylbenzoate in refluxing acetic acid. Intermediate I was transformed into an advanced hydrazide intermediate II by refluxing it in ethanolic hydrazine hydrate solution (Scheme 1). In the present study, various methods were used (I: NaBrO₃/NaHSO₃, EAN, and BF₃·Et₂O) to synthesize the starting material methyl 4-(bis(1-methyl-1H-indol-3-yl)methyl)benzoate (I),³¹ but in very low yield. When the same reaction was carried out in refluxing acetic acid, we managed to recover up to 93% yield by handling the reaction carefully. The other conversions from ester to hydrazide and hydrazide to hydrazone are well documented, with yields up to 70–85%.

Scheme 1 Synthesis of hydrazide II.

The advanced intermediate hydrazide II was then reacted with various aryl aldehydes in the presence of acetic acid to yield the targeted bisindolylmethane–hydrazone hybrids 1–30 (Scheme 2; Table 1) in good to excellent yields. The structures of all the synthesized compounds 1–30 were confirmed using various spectroscopic techniques, such as NMR and EIMS, and were further confirmed through CHN analysis.

Scheme 2 Synthesis of the hydrazone derivatives 1–30.

Table 1 Bisindolylmethane–hydrazone hybrids 1–30 and their β-D-glucuronidase inhibitory potentials

S. no.	R	IC50 (μM ± SEM^a)	S. no.	R	IC50 (μM ± SEM^a)
a SEM is the standard error of the mean.b N.A. no activity.
1		83.50 ± 1.70	16		0.10 ± 0.001
2		N. A.^b	17		18.60 ± 0.30
3		73.50 ± 1.40	18		9.60 ± 0.16
4		0.20 ± 0.01	19		32.36 ± 0.64
5		51.10 ± 1.29	20		16.10 ± 0.29
6		23.12 ± 0.45	21		11.28 ± 0.23
7		3.50 ± 0.05	22		13.38 ± 0.22
8		0.50 ± 0.3	23		6.30 ± 0.10
9		3.80 ± 0.08	24		N. A.^b
10		88.56 ± 1.88	25		2.20 ± 0.06
11		13.60 ± 0.26	26		26.63 ± 0.38
12		12.10 ± 0.24	27		15.20 ± 0.28
13		N. A.^b	28		26.20 ± 0.39
14		22.18 ± 0.45	29		16.25 ± 0.29
15		15.26 ± 0.26	30		N. A.^b
D-Saccharic acid 1,4-lactone			48.4 ± 1.25

---

2.2. β-Glucuronidase inhibition studies

Herein, we evaluated bis-indole bearing hydrazones 1–30 for their β-glucuronidase inhibitory potential according to the protocol reported in the literature.³⁰ These compounds demonstrated varying degrees of inhibitory potentials, whereas the IC₅₀ values for the potent compounds were found to be in the range from 0.10 to 83.50 μM (Table 1) when compared with standard D-saccharic acid 1,4-lactone (IC₅₀ value 48.4 ± 1.25 μM). In addition to the determination of the inhibitory potentials, molecular dockings and DFT calculations were also performed to estimate the binding modes of the current series of compounds. The results are shown in Table 1. These compounds do not show toxicity, which may be due to their higher carbon content and are expected to have lower toxicity levels compared to compounds with small molecules as reported.³²

Compound 16, a trihydroxy derivative, with an IC₅₀ value of 0.10 ± 0.001, was found to be ∼500 times more active than the standard D-saccharic acid 1,4-lactone. Compounds 6 (IC₅₀ = 18.46 ± 0.65 μM), 7 (IC₅₀ = 34.46 ± 0.85 μM), 8 (IC₅₀ = 29.15 ± 0.75 μM), 9 (IC₅₀ = 15.14 ± 0.55 μM), and 18 (IC₅₀ = 21.14 ± 1.05 μM) were found to be more potent than the standard. Compounds 25 (IC₅₀ = 42.26 ± 1.16 μM) and 11 (IC₅₀ = 44.16 ± 1.20 μM) displayed inhibitory potentials comparable to the standard. It was shown that the presence of hydroxyl and halogen groups, in general, proved to be the decisive factor that resulted in the enhanced inhibition of β-glucuronidase, as depicted in our previous studies.^33,34 Furthermore, some stunning similarities in inhibition potential between the fluoro-, chloro-, hydroxyl, and nitro-substituted derivatives were observed. For example, all the ortho analogs, 12 with an OH group, 27 with a chloro group, 18 with fluoro substitution, and finally compound 22 with a nitro group, displayed a similar trend in their inhibitory potential (9.60 ± 0.16 to 13.38 ± 0.22 μM) toward β-glucuronidase. Similarly, the meta-hydroxy and meta-chloro analogs, 29 and 20, showed a similar potential of inhibition. These findings suggest that electronic factors are also operating and play a role in inhibition in addition to hydrogen bonding effects of the hydroxyl groups. However, it is difficult to argue the strong inhibitory potentials of the di- and trihydroxy substituted analogs and their interactions with receptor binding sites. To better understand the inhibition mechanisms and binding modes of the compounds 1–30 with β-glucuronidase, molecular modeling studies became imperative to understand the underlying phenomenon.

2.3. Molecular docking analysis

In order to obtain more insights into the binding mode of bisindolylmethane derivatives within the active site of β-D-glucuronidase and to obtain additional validations for the experimental results, molecular docking studies were performed. Herein, the X-ray crystal structure of the human β-glucuronidase enzyme at 2.6 Å resolution (PDB ID: 1BHG)³⁶ was employed to further identify the binding modes involved in the inhibition activity. Human β-D-glucuronidase 3D structure was used for our structure–activity relationship (SAR) studies due to the absence of the bovine β-D-glucuronidase structure.

Prior to the docking of the bisindolylmethane derivatives, the known substrate molecule p-nitrophenyl β-D-glucuronide was first docked into the active site of β-D-glucuronidase using the docking program AutoDock 4.2.³⁷ The modeled substrate-bound structure of human β-D-glucuronidase showed that the glycoside bond of p-nitrophenyl β-D-glucuronide was properly oriented toward the catalytic residues, Glu451 and Glu540 (Fig. 1). It has been proposed for human β-D-glucuronidase that during catalysis, Glu451 acts as the acid/base catalyst, whereas Glu540 serves as the nucleophilic residue.³⁶ As shown in Fig. 1, the substrate neatly fits in the active site, forming various hydrogen bonding interactions with the active site residues, including Asp207, His385, Glu451, Tyr507, and Glu540.

Fig. 1 Native β-D-glucuronide in the active site of β-glucuronidase surrounded by the amino acid residues Asp207, His385, Glu451, Tyr507, and Glu540.

All the residues play an important role in binding the substrate molecule in the active site of β-D-glucuronidase.³⁶ This modeled protein structure was used in the prediction of a favorable binding mode for the newly synthesized bisindolylmethane derivatives. The predicted binding models of the biologically active compounds are shown in Fig. 2. The docking studies showed that the active compounds are well accommodated in the binding cavity of the enzyme. Analysis of the predicted binding conformations of our most active compound 16 (IC₅₀ = 0.10 ± 0.001 μM) revealed that compound 16 can fit straight into the binding groove of β-D-glucuronidase. Visually inspecting the best binding position for compound 16 showed that the hydroxyl group at the C-3 position formed a strong hydrogen bond interaction with the side chain carboxyl oxygen (Oε1) of Glu451 (1.94 Å), whereas the hydroxyl group at the C-5 position formed a hydrogen bond with the side chain nitrogen (Nε2) of His385 (2.43 Å). However, the hydroxyl group at the C-4 position did not form any interaction with the surrounding amino acid residues. The amino group of the hydrazone linkage is involved in strong hydrogen bonding interaction with the side chain carboxyl oxygen (OD2) of Asp207 (1.82 Å). The high inhibition activity for compound 16 toward β-D-glucuronidase can be explained by these strong hydrogen bonds. The predicted binding mode of compound 16 is shown in Fig. 2c. His385, Asn450, Asn484, Glu451, and Tyr508 are involved in stabilizing the binding of the compound in the active site of β-D-glucuronidase through π-sigma, π-anion, and T-shaped π–π interactions.

Fig. 2 Binding position for (a) compound 4, (b) compound 8, (c) compound 16, and (d) compound 25 in the same binding cavity occupied by native β-D-glucuronide surrounded by the amino acid residues Asp207, His385, Glu451, Tyr507, and Glu540.

In contrast, the binding mode of compound 4 (IC₅₀ = 0.20 ± 0.01 μM), which is the second most active compound among the series, revealed that the hydroxyl group at the meta position is involved in hydrogen bond interaction with the side chain carboxyl oxygen (Oε1) of Glu540 (1.98 Å) (Fig. 2a). Another hydroxyl group of compound 4 at the para position was observed to form a hydrogen bond with Asn450 (2.68 Å). The binding mode of compound 8 (IC₅₀ = 0.50 ± 0.3 μM) revealed that the hydroxyl group at the ortho position forms a hydrogen bond with the side chain carboxyl of Glu451 (1.93 Å), whereas the hydroxyl at the para position did not adopt a favorable conformation for binding with the surrounding residues (Fig. 2b). The observed binding mode of compound 25 showed that it possesses three hydroxyl groups at the ortho and para positions. The docking study showed that the hydroxyl at the para position was able to interact well with Glu540 (2.03 Å), Asn450 (2.94 Å), and His385 (2.59 Å). Both hydroxyl groups at the ortho position of compound 25 formed strong interactions with the side chain hydroxyl oxygen (OD2) of both Asp207 (1.67 Å) and Glu451 (1.56 Å) (Fig. 2d). Hydrazone linkage formed weak hydrogen bonding with some important residues like Arg600 (3.03 Å), Lys606 (2.81 Å), and Tyr504 (2.36 Å).

In the present study, it was observed that the compounds are positioned in such a way that enabled the meta hydroxyl substituent to form a better interaction with the important residues, such as Glu451 and Glu540, than to the hydroxyls at the ortho and para positions.

The docking studies on the inactive compounds 1 (IC₅₀ = 83.50 ± 1.70 μM), 3 (IC₅₀ = 73.50 ± 1.40 μM), and 10 (IC₅₀ = 88.56 ± 1.88 μM) (Fig. 3) clearly demonstrated the reason for their inactivity against the enzyme. These compounds were unable to form any noteworthy hydrogen bond interactions with the surrounding amino acid residues. It was observed that their hydrazone linkage generally formed hydrogen bond interactions with Asn207 and Arg600 with the distance between 1.81 and 2.65 Å. Hence, the activity of these compounds largely depends on the maximum hydrophilic interactions with the active site amino acids of β-D-glucuronidase.

Fig. 3 Binding mode of the inactive compound 1 (a), compound 3 (b), and compound 10 (c) in the same binding cavity occupied by native β-D-glucuronide surrounded by the amino acid residues Asp207, His385, Glu451, Tyr507, and Glu540.

2.4. Density functional predictions for hydrogen bonding

In order to emphasize the role of the hydrogen bonds (intramolecular or intermolecular) of the synthesized bisindolylmethane–hydrazone hybrids in β-glucuronidase inhibition, DFT calculations were performed at the B3LYP/6-31+G(d,p) level. The potency of the β-glucuronidase inhibition of the synthesized bisindolylmethane derivatives can be related to the number of intramolecular hydrogen bonds occurring in the substrates (bisindolylmethane derivatives) and to the number of intermolecular hydrogen bonds that occur at the substrate–receptor complex. Herein, we focused on the stability and effects of the number of intramolecular hydrogen bonds of the bisindolylmethane derivatives in β-glucuronidase inhibition. A high number of hydrogen bonds mean that bisindolylmethane is more stable and the inhibition is more profound (low IC₅₀). The gap energies (eV), electronic energies, number of electronic donor groups, number of withdrawing groups, number of intramolecular hydrogen bonds, and number of intramolecular hydrogen bonds of the bisindolylmethane hybrids are reported in Table 2. The bisindolylmethane hybrids that show high β-glucuronidase inhibition (0.1 ≤ IC₅₀ ≤ 10 μM) are those with a high number of electron donating groups. For instance, bisindolylmethane derivative 16, with two intramolecular hydrogen bonds (Table 1S†) and three electron donating groups (i.e., the ability to form three intermolecular hydrogen bonds) showed potent β-glucuronidase inhibition (IC₅₀ = 0.1 μM) compared with the bisindolylmethane derivative 7, which possesses one intramolecular hydrogen bond (Table 1S, ESI†) and two electron donor groups (IC₅₀ = 3.50 μM). The bisindolylmethane derivative 16 is relatively stable compared to 7 (ΔE = 4.7 kcal mol⁻¹), which confirms the role of hydrogen bonding in the stability of the substrate and its role (i.e., via hydrogen bonding) in inhibiting β-glucuronidase. The 3D-structure of the optimized bisindolylmethane–hydrazone hybrids are reported in Fig. 1S in the ESI.† Based on the results from the docking and density functional theory studies, some discussion on the ability of these compounds to mainly interact with the target enzyme through hydrogen bonding or van der Waals interactions is included. As the results showed that the compounds interact quite well through these interactions, which involve no chemical interaction, these compounds could possibly be reversible inhibitors.

Table 2 Number of electron donating groups (EDG), number of electron withdrawing groups (EDG), number of intramolecular hydrogen bonds (HB), number of possible intermolecular hydrogen bonds (HB′), electronic energies E (kcal mol⁻¹), and gap energies of the active bisindolylmethane derivatives

Compounds	EDG	EWG	HB	HB′	OH	Electronic energy, E	Gap energy	IC50 (μM ± SEM^a)
a SEM is the standard error of the mean.b N.A. no activity.
1	1	0	0	1	0	−1 056 229.52	3.69	83.5 ± 1.70
2	1	0	0	0	0	−1 009 033.76	3.60	N. A.^b
3	0	1	0	0	0	−1 112 695.66	2.43	73.5 ± 1.40
4	2	0	1	2	2	−1 078 770.62	3.67	0.2 ± 0.01
5	1	0	0	0	0	−1 009 034.12	3.62	51.1 ± 1.29
6	1	0	0	0	0	−1 009 032.00	3.55	23.12 ± 0.45
7	2	0	1	2	2	−1 078 773.53	3.52	3.5 ± 0.05
8	2	0	1	2	2	−1 078 768.97	3.57	0.5 ± 0.3
9	0	1	1	0	0	−1 046 636.60	3.56	3.8 ± 0.08
10	0	0	0	0	0	−994 424.90	3.42	88.56 ± 1.88
11	2	0	1	2	1	−1 103 439.57	3.67	13.6 ± 0.26
12	1	0	1	1	1	−1 031 570.87	3.55	12.1 ± 0.24
13	0	1	0	0	0	−1 112 696.32	2.31	N. A.^b
14	2	0	1	2	2	−1 078 773.53	3.52	22.18 ± 0.45
15	1	0	1	1	1	−1 031 566.53	3.69	15.26 ± 0.26
16	3	0	2	3	3	−1 125 978.56	3.65	0.1 ± 0.008
17	0	0	0	0	0	−994 424.73	3.36	18.6 ± 0.30
18	0	1	0	0	0	−1 046 636.25	3.46	9.6 ± 0.16
19	2	0	1	2	1	−1 103 434.15	3.69	32.36 ± 0.64
20	0	1	0	0	0	−1 272 761.43	3.44	16.1 ± 0.29
21	2	0	1	2	1	−1 103 436.46	3.54	11.28 ± 0.23
22	0	1	0	0	0	−1 112 690.60	2.44	13.38 ± 0.22
23	2	0	1	2	2	−1 078 776.66	3.67	6.3 ± 0.10
24	1	0	0	0	0	−1 009 033.85	3.59	N. A.^b
25	3	0	1	3	3	−1 125 980.82	3.69	2.2 ± 0.06
26	0	1	0	0	0	−1 272 761.84	3.47	26.63 ± 0.38
27	0	1	0	0	0	−1 272 757.00	3.47	15.2 ± 0.28
28	0	1	0	0	0	−1 046 636.45	3.45	26.2 ± 0.39
29	1	0	1	1	1	−1 031 565.89	3.53	16.25 ± 0.29
30	0	0	0	0	0	−994 425.20	3.24	N. A.^b

---

In conclusion, the binding of the active compounds with the target site is mainly dependent on the nature and position of the polar groups, such as the hydroxyl and halogen groups. In addition to the identification of the hydroxyl and halogen substituted analogs as the most potent compounds, it was also observed that the compounds in which hydroxyl groups were positioned adjacently were found to interact exclusively with the important residues, such as Glu451 and Glu540, as compared to hydroxyl groups that were far apart from each other. Furthermore, DFT studies confirmed the role of hydrogen bonding along with the role of the electronic nature of the substituents; these factors played pivotal roles in the inhibition potential of β-glucuronidase.

3. Experimental

3.1. General

NMR experiments were performed on an UltraShield Bruker FT NMR 500 MHz; CHN analysis was performed on a Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI-MS) were obtained on a Finnegan MAT-311A, Germany. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms were visualized by UV at 254 and 365 nm.

3.2. β-Glucuronidase inhibition activity

β-Glucuronidase (E.C. 3.2.1.31 from bovine liver, G-0251) inhibition activity of the compounds was evaluated using the method reported in the ref. 30 and ³⁷. In the present study, D-saccharic acid 1,4-lactone was used as the standard drug.^38,39

3.3. Synthesis of methyl 4-(bis(1-methyl-1H-indol-3-yl)methyl)benzoate (I)

Methyl 2-hydroxybenzoate (1) (7.60 g, 53 mmol) and 20 mL of hydrazine hydrate were mixed in methanol (50 mL). The mixture was refluxed for 6 h. Methanol was then evaporated and the product formed was rinsed with plenty of water to remove excess hydrazine hydrate. The product formed was left to dry at room temperature and its yield was recorded. Yield: 9.68 g (94.9%). White solid. mp 239–241 °C; IR (cm⁻¹, ATR): 3082, 2975, 1738, 1627, 1546, 1468, 1126; ¹H NMR (500 MHz, DMSO-d₆): δ 7.97 (d, J = 7.5 Hz, 2H), 7.83 (dd, J = 7.5, 1.6 Hz, 2H), 7.76 (dd, J = 7.4, 1.5 Hz, 2H), 7.63 (s, 2H), 7.44 (d, J = 7.5 Hz, 2H), 7.32 (dtd, J = 29.3, 7.5, 1.5 Hz, 4H), 5.74 (s, 1H), 3.92 (s, 3H), 3.78 (s, 6H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ 167.45, 141.55, 141.55, 140.27, 130.69, 130.37, 130.37, 128.65, 128.65, 128.49, 128.49, 128.20, 128.20, 124.69, 124.69, 123.42, 123.42, 122.72, 122.72, 111.63, 111.63, 111.08, 111.08, 52.18, 38.43, 35.19, 35.19; HREI-MS: m/z calcd for C₂₇H₂₄N₂O₂ [M]⁺ 408.1838; found 408.1838; anal. calcd for C₂₇H₂₄N₂O₂, C, 79.39; H, 5.92; N, 6.86; O, 7.83; found C, 79.39; H, 5.92; N, 6.86; O, 7.83.

3.4. Synthesis of 4-(bis(1-methyl-1H-indol-3-yl)methyl)benzohydrazide (II)

A mixture of compound 2 (7.00 g, 46 mmol), methyl 4-formylbenzoate (7.56 g, 46 mmol), and a catalytic amount of acetic acid in methanol (50 mL) was refluxed for 3 h. The solvent was evaporated and the residue (3) was washed with diethyl ether, filtered, dried, and then crystallized from ethanol to give a brownish solid. Yield: 5.69 g, 93.0%. mp 269–271 °C; IR (cm⁻¹, ATR): 3423, 3352, 3137, 2997, 1655, 1612, 1558, 1469, 1341, 1268; ¹H NMR (500 MHz, DMSO-d₆): δ 8.33 (s, 1H), 7.94–7.84 (m, 4H), 7.76 (dd, J = 7.5, 1.4 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.42 (s, 2H), 7.34 (td, J = 7.4, 1.5 Hz, 2H), 7.26 (td, J = 7.4, 1.5 Hz, 2H), 5.73 (s, 1H), 4.08 (s, 2H), 3.76 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 167.32, 142.28, 141.57, 141.57, 135.10, 135.10, 130.34, 130.34, 129.66, 129.66, 128.20, 128.20, 126.76, 126.76, 124.79, 124.79, 123.56, 123.56, 122.85, 122.75, 111.78, 111.78, 111.28, 111.28, 38.48, 35.99, 35.99; HREI-MS: m/z calcd for C₂₆H₂₄N₄O [M]⁺ 408.1950; found 408.1950; anal. calcd for C₂₆H₂₄N₄O, C, 76.45; H, 5.92; N, 13.72; found C, 76.45; H, 5.92; N, 13.72.

3.4.1. General procedure for the synthesis of oxadiazole benzohydrazones (1–30). Equimolar quantities (1 mmol) of compound 5 and the substituted benzaldehydes (1 mmol) in methanol (25 mL) were refluxed for 3 h in the presence of a catalytic amount of glacial acetic acid. The resulting solid was filtered and recrystallized from methanol in good yields.
3.4.1.1. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-methoxybenzylidene)benzohydrazide (1). Brown solid. Yield: 75.2%. mp: 259–261 °C. IR (cm⁻¹, ATR): 3425, 3339, 3143, 1643, 1609, 1254. ¹H NMR (500 MHz, DMSO-d₆): δ 11.62 (s, 1H), 8.36 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.94 (s, 1H), 3.81 (s, 3H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.32, 160.20, 149.54, 142.36, 141.70, 141.70, 134.31, 130.25, 130.25, 129.63, 129.63, 129.22, 129.22, 128.19, 128.19, 128.09, 128.09, 127.15, 124.79, 124.79, 123.42, 123.42, 122.71, 122.71, 114.42, 114.42, 111.78, 111.78, 111.02, 111.02, 56.13, 38.32, 35.92, 35.92; HREI-MS: m/z calcd for C₃₄H₃₀N₄O₂ [M]⁺ 526.2369; found 526.2373; anal. calcd for C₃₄H₃₀N₄O₂, C, 77.54; H, 5.74; N, 10.64; found C, 77.56; H, 5.73; N, 10.65.
3.4.1.2. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-methylbenzylidene)benzohydrazide (2). Pale yellow solid. Yield: 82.9%. mp: 262–263 °C. IR (cm⁻¹, ATR): 3324, 3260, 3064, 1656, 1590, 1539, 1469, 1357, 1277, 1180, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.73 (s, 1H), 8.38 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.55 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 8.1 Hz, 1H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.38, 148.78, 142.56, 141.40, 141.40, 137.86, 136.61, 134.31, 130.45, 130.45, 129.81, 129.63, 128.74, 128.74, 128.59, 128.31, 128.09, 128.09, 124.79, 124.79, 123.51, 123.51, 123.49, 123.49, 122.73, 122.73, 111.78, 111.78, 111.15, 111.15, 38.48, 35.69, 35.69, 21.35; HREI-MS: m/z calcd for C₃₄H₃₀N₄O [M]⁺ 510.2420; found 510.2424; anal. calcd for C₃₄H₃₀N₄O, C, 79.97; H, 5.92; N, 10.97; found C, 79.98; H, 5.93; N, 10.98.
3.4.1.3. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-nitrobenzylidene)benzohydrazide (3). Red solid. Yield: 71.3%. mp: 262–264 °C. IR (cm⁻¹, ATR): 3335, 3260, 1560, 3138, 1657, 1609, 1275, 1125; ¹H NMR (500 MHz, DMSO-d₆): δ 12.03 (s, 1H), 8.55 (s, 2H), 8.26 (d, J = 7.4 Hz, 1H), 8.15 (d, J = 7.0 Hz, 1H), 7.84 (d, J = 7.8 Hz, 2H), 7.76 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.5 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.52, 148.78, 147.38, 142.71, 141.47, 141.47, 137.78, 134.11, 132.69, 130.31, 130.31, 129.96, 129.51, 129.51, 128.25, 128.25, 128.20, 128.20, 124.79, 124.79, 124.12, 123.44, 123.44, 122.72, 122.72, 122.43, 111.78, 111.78, 111.26, 111.26, 38.37, 35.92, 35.92; HREI-MS: m/z calcd for C₃₃H₂₇N₅O₃ [M]⁺ 541.2114; found 541.2118; anal. calcd for C₃₃H₂₇N₅O₃, C, 73.18; H, 5.02; N, 12.93; found C, 73.19; H, 5.00; N, 12.94.
3.4.1.4. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3,4-dihydroxybenzylidene)benzohydrazide (4). Red solid. Yield: 78.2%. mp: 278–280 °C. IR (cm⁻¹, ATR): 3423, 3299, 3072, 1636, 1617, 1545, 1277, 1180; ¹H NMR (500 MHz, DMSO-d₆): δ 11.50 (s, 1H), 9.35 (s, 1H), 9.23 (s, 1H), 8.23 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.23 (s, 1H), 7.13 (t, J = 7.6 Hz, 2H), 6.91–6.95 (m, 3H), 6.88 (s, 1H), 6.78 (d, J = 8.1 Hz, 1H), 5.94 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.38, 148.69, 148.09, 145.47, 142.76, 141.48, 141.48, 134.18, 130.32, 130.32, 129.73, 129.73, 128.49, 128.49, 128.20, 128.20, 127.87, 124.69, 124.69, 123.36, 123.36, 122.65, 122.65, 121.12, 116.73, 115.93, 111.58, 111.58, 111.18, 111.18, 38.52, 35.92, 35.92; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₃ [M]⁺ 528.2161; found 528.2161; anal. calcd for C₃₃H₂₈N₄O₃, C, 74.98; H, 5.34; N, 10.60; found C, 75.01; H, 5.32; N, 10.58.
3.4.1.5. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-methylbenzylidene)benzohydrazide (5). Dark brown solid. Yield: 79.4%. mp: 246–248 °C. IR (cm⁻¹, ATR): 3347, 3352, 3043, 1631, 1591, 1555, 1357, 1270, 1143, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.68 (s, 1H), 8.38 (s, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.27 (d, J = 7.5 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H), 2.35 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.38, 149.24, 142.56, 141.47, 141.47, 138.49, 134.15, 131.96, 130.38, 130.38, 129.43, 129.43, 129.25, 129.25, 128.28, 128.28, 128.07, 128.07, 127.30, 127.30, 124.82, 124.82, 123.36, 123.36, 122.63, 122.63, 111.67, 111.67, 111.04, 111.04, 38.38, 35.72, 35.72, 21.09; HREI-MS: m/z calcd for C₃₄H₃₀N₄O [M]⁺ 510.2420; found 510.2425; anal. calcd for C₃₄H₃₀N₄O, C, 79.97; H, 5.92; N, 10.97; found C, 79.98; H, 5.90; N, 10.95.
3.4.1.6. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-methylbenzylidene)benzohydrazide (6). Light brown solid. Yield: 89.7%. mp: 246–248 °C. IR (cm⁻¹, ATR): 3325, 3299, 3043, 1631, 1568, 1357, 1277, 1180, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.73 (s, 1H), 8.72 (s, 1H), 7.83 (t, J = 7.7 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.32–7.23 (m, 3H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H), 2.43 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.28, 145.62, 142.69, 141.45, 141.45, 136.27, 134.26, 133.49, 130.51, 130.51, 130.31, 129.50, 129.50, 128.49, 128.31, 128.31, 128.12, 128.12, 126.79, 126.27, 124.69, 124.69, 123.48, 123.48, 122.79, 122.79, 111.58, 111.58, 111.18, 111.18, 38.32, 35.91, 35.91, 20.15; HREI-MS: m/z calcd for C₃₄H₃₀N₄O [M]⁺ 510.2420; found 510.2415; anal. calcd for C₃₄H₃₀N₄O, C, 79.97; H, 5.92; N, 10.97; found C, 79.99; H, 5.90; N, 10.96.
3.4.1.7. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2,5-dihydroxybenzylidene)benzohydrazide (7). Red solid. Yield: 92.6%. mp: 284–285 °C. IR (cm⁻¹, ATR): 3353, 3139, 2994, 1657, 1611, 1556, 1495, 1466, 1259, 1166, 749; ¹H NMR (500 MHz, DMSO-d₆): δ 11.90 (s, 1H), 10.40 (s, 1H), 8.95 (s, 1H), 8.53 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.97–6.91 (m, 3H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.46, 152.63, 151.39, 149.69, 142.52, 141.47, 141.47, 134.18, 130.34, 130.34, 129.57, 129.57, 128.16, 128.16, 128.09, 128.09, 124.76, 124.76, 123.26, 123.26, 122.65, 122.65, 122.16, 121.52, 118.28, 116.47, 111.78, 111.78, 111.04, 111.04, 38.47, 35.93, 35.93; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₃ [M]⁺ 528.2161; found 528.2157; anal. calcd for C₃₃H₂₈N₄O₃, C, 74.98; H, 5.34; N, 10.60; found C, 74.99; H, 5.32; N, 10.58.
3.4.1.8. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2,3-dihydroxybenzylidene)benzohydrazide (8). Orange solid, Yield: 86.5%. mp: 257–259 °C. IR (cm⁻¹, ATR): 3381, 3062, 1613, 1611, 1269, 1205, 750; ¹H NMR (500 MHz, DMSO-d₆): δ 12.02 (s, 1H), 11.18 (s, 1H), 9.17 (s, 1H), 8.56 (s, 1H), 7.85 (d, J = 6.4 Hz, 2H), 7.52 (d, J = 6.6 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 7.7 Hz, 2H), 7.13 (t, J = 6.5 Hz, 2H), 6.94 (t, J = 7.3 Hz, 2H), 6.89 (s, 1H), 6.86 (d, J = 7.1 Hz, 1H), 6.74 (t, J = 6.6 Hz, 1H), 5.96 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.37, 149.97, 146.93, 144.72, 142.68, 141.57, 141.57, 134.19, 130.34, 130.34, 129.52, 129.52, 128.18, 128.18, 128.08, 128.08, 124.91, 124.91, 123.49, 123.49, 122.85, 122.85, 122.09, 121.42, 119.90, 119.73, 111.61, 111.61, 111.18, 111.18, 38.39, 35.94, 35.94; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₃ [M]⁺ 528.2161; found 528.2165; anal. calcd for C₃₃H₂₈N₄O₃, C, 74.98; H, 5.34; N, 10.60; found C, 74.99; H, 5.35; N, 10.58.
3.4.1.9. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-fluorobenzylidene)benzohydrazide (9). Brown solid. Yield: 85.8%. mp: 248–250 °C. IR (cm⁻¹, ATR): 3354, 3212, 3021, 1626, 1606, 1519, 1470, 1372, 1269, 1186, 760; ¹H NMR (500 MHz, DMSO-d₆): δ 11.77 (s, 1H), 8.42 (s, 1H), 7.86–7.73 (m, 4H), 7.50 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.32–7.26 (m, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.4 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.52, 160.29 (d, J = 261.5 Hz), 149.43, 142.51, 141.62, 141.62, 134.74, 130.69, 130.40, 130.40, 130.25, 130.25, 129.43, 129.43, 128.31, 128.31, 128.15, 128.15, 124.82, 124.82, 123.36, 123.36, 122.72, 122.72, 115.51, 115.51, 111.78, 111.78, 111.18, 111.18, 38.25, 35.69, 35.69; HREI-MS: m/z calcd for C₃₃H₂₇FN₄O [M]⁺ 514.2169; found 514.2172; anal. calcd for C₃₃H₂₇FN₄O, C, 77.02; H, 5.29; N, 10.89; found C, 77.01; H, 5.31; N, 10.90.
3.4.1.10. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(pyridin-3-ylmethylene)benzohydrazide (10). Light brown solid. Yield: 89.6%. mp: 258–260 °C. IR (cm⁻¹, ATR): 3373, 3159, 2835, 1609, 1558, 1470, 1445, 1257, 1186, 754; ¹H NMR (500 MHz, DMSO-d₆): δ 11.93 (s, 1H), 8.85 (s, 1H), 8.61 (s, 1H), 8.48 (s, 1H), 8.13 (s, 1H), 7.83 (d, J = 7.3 Hz, 2H), 7.51 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 7.7 Hz, 2H), 7.13 (t, J = 7.3 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 165.32, 150.15, 146.51, 145.72, 142.46, 141.70, 141.70, 135.59, 134.17, 133.82, 130.45, 130.45, 129.33, 129.33, 128.18, 128.18, 128.07, 128.07, 124.91, 124.91, 124.31, 123.45, 123.45, 122.85, 122.85, 111.72, 111.72, 111.02, 111.02, 38.52, 35.79, 35.79; HREI-MS: m/z calcd for C₃₂H₂₇N₅O [M]⁺ 497.2216; found 497.2213; anal. calcd for C₃₂H₂₇N₅O, C, 77.24; H, 5.47; N, 14.07; found C, 77.25; H, 5.48; N, 14.05.
3.4.1.11. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-hydroxy-4-methoxybenzylidene)benzohydrazide (11). Red solid. Yield: 89.6%. mp: 255–257 °C. IR (cm⁻¹, ATR): 3389, 3249, 3002, 1684, 1652, 1585, 1467, 1496, 1282, 1205, 750; ¹H NMR (500 MHz, DMSO-d₆): δ 11.90 (s, 1H), 11.64 (s, 1H), 8.51 (s, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.40 (t, J = 4.2 Hz, 2H), 7.39 (s, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 6.52 (d, J = 8.6 Hz, 1H), 6.50 (s, 1H), 5.95 (s, 1H), 3.78 (s, 3H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.41, 162.10, 161.25, 151.29, 142.69, 141.72, 141.72, 134.31, 130.92, 130.32, 130.32, 129.58, 129.58, 128.26, 128.26, 128.18, 128.18, 124.79, 124.79, 123.36, 123.36, 122.62, 122.62, 113.87, 111.69, 111.69, 111.18, 111.18, 107.39, 102.25, 56.23, 38.39, 35.92, 35.92; HREI-MS: m/z calcd for C₃₄H₃₀N₄O₃ [M]⁺ 542.2318; found 542.2323; anal. calcd for C₃₄H₃₀N₄O₃, C, 75.26; H, 5.57; N, 10.33; found C, 75.27; H, 5.56; N, 10.35.
3.4.1.12. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-hydroxybenzylidene)benzohydrazide (12). Brown solid. Yield: 79.7%. mp: 272–274 °C. IR (cm⁻¹, ATR): 3347, 3140, 3054, 1650, 1603, 1489, 1465, 1260, 1184, 750; ¹H NMR (500 MHz, DMSO-d₆): δ 12.02 (s, 1H), 11.30 (s, 1H), 8.60 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.96–6.91 (m, 3H), 6.89 (s, 2H), 5.96 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.51, 158.09, 151.32, 142.50, 141.66, 141.66, 134.34, 130.20, 130.20, 129.86, 129.43, 129.43, 128.51, 128.32, 128.32, 128.08, 128.08, 124.79, 124.79, 123.35, 123.35, 122.86, 122.86, 121.23, 120.42, 117.31, 111.64, 111.64, 111.28, 111.28, 38.45, 35.69, 35.69; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₂ [M]⁺ 512.2212; found 512.2209; anal. calcd for C₃₃H₂₈N₄O₂, C, 77.32; H, 5.51; N, 10.93; found C, 77.33; H, 5.53; N, 10.95.
3.4.1.13. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-nitrobenzylidene)benzohydrazide (13). Yellow solid. Yield: 91.7%. mp: 284–285 °C. IR (cm⁻¹, ATR): 3429, 3254, 2835, 1642, 1614, 1584, 1561, 1520, 1470, 1440, 1275, 1236, 1180, 755; ¹H NMR (500 MHz, DMSO-d₆): δ 12.06 (s, 1H), 8.52 (s, 1H), 8.30 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 6.7 Hz, 2H), 7.84 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.34, 149.22, 148.66, 142.14, 141.95, 141.95, 139.64, 134.35, 130.26, 130.26, 129.21, 129.21, 128.52, 128.52, 128.12, 128.12, 127.87, 127.87, 124.95, 124.95, 124.58, 124.58, 123.47, 123.47, 122.65, 122.65, 111.78, 111.78, 111.06, 111.06, 38.52, 35.78, 35.78; HREI-MS: m/z calcd for C₃₃H₂₇N₅O₃ [M]⁺ 541.2114; found 541.2117; anal. calcd for C₃₃H₂₇N₅O₃, C, 73.18; H, 5.02; N, 12.93; found C, 73.19; H, 5.01; N, 12.95.
3.4.1.14. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3,5-dihydroxybenzylidene)benzohydrazide (14). Brown solid. Yield: 86.2%. mp: 262–263 °C. IR (cm⁻¹, ATR): 3143, 2962, 3062, 1603, 1583, 1490, 1462, 1266, 1164, 756; ¹H NMR (500 MHz, DMSO-d₆): δ 11.61 (s, 1H), 9.41 (s, 2H), 7.81 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.40 (s, 1H), 7.40–7.27 (m, 4H), 7.13 (t, J = 7.3 Hz, 2H), 6.93 (t, J = 7.3 Hz, 2H), 6.88 (s, 2H), 6.59 (s, 2H), 6.26 (s, 1H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.30, 158.28, 158.28, 147.66, 142.91, 141.52, 141.52, 138.42, 134.29, 130.25, 130.25, 129.52, 129.52, 128.29, 128.29, 128.12, 128.12, 124.89, 124.89, 123.46, 123.46, 122.45, 122.45, 111.78, 111.78, 111.28, 111.28, 107.45, 107.45, 104.27, 38.32, 35.49, 35.49; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₃ [M]⁺ 528.2161; found 528.2158; anal. calcd for C₃₃H₂₈N₄O₃, C, 74.98; H, 5.34; N, 10.60; found C, 75.01; H, 5.32; N, 10.58.
3.4.1.15. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-hydroxybenzylidene)benzohydrazide (15). Dark brown solid. Yield: 86.2%. mp: 276–278 °C. IR (cm⁻¹, ATR): 3394, 3296, 3064, 1607, 1587, 1507, 1462, 1266, 1205, 1157, 736; ¹H NMR (500 MHz, DMSO-d₆): δ 11.53 (s, 1H), 9.88 (s, 1H), 8.31 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.4 Hz, 2H), 6.88 (s, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.94 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.28, 158.34, 149.44, 142.56, 141.32, 141.32, 134.41, 130.25, 130.25, 129.72, 129.72, 129.43, 129.43, 128.25, 128.25, 128.18, 128.18, 126.72, 124.69, 124.69, 123.75, 123.75, 122.46, 122.46, 115.68, 115.68, 111.59, 111.59, 111.23, 111.23, 38.89, 35.42, 35.42. HREI-MS: m/z calcd for C₃₃H₂₈N₄O₂ [M]⁺ 512.2212; found 512.2215; anal. calcd for C₃₃H₂₈N₄O₂, C, 77.32; H, 5.51; N, 10.93; found C, 77.34; H, 5.48; N, 10.90.
3.4.1.16. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2,4,5-trihydroxybenzylidene)benzohydrazide (16). Light brown solid. Yield: 85.3%. mp: 291–293 °C. IR (cm⁻¹, ATR): 3352, 3182, 3075, 1633, 1591, 1276, 1246, 1171, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.70 (s, 1H), 10.65 (s, 1H), 9.51 (s, 1H), 8.52 (s, 1H), 8.41 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.4 Hz, 2H), 6.88 (s, 2H), 6.85 (s, 1H), 6.33 (s, 1H), 5.94 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.51, 153.78, 150.93, 149.71, 142.51, 141.78, 141.78, 140.25, 134.35, 130.53, 130.53, 129.32, 129.32, 128.72, 128.72, 128.18, 128.18, 124.83, 124.83, 123.31, 123.31, 122.85, 122.85, 117.22, 112.87, 111.64, 111.64, 111.06, 111.06, 102.76, 38.37, 35.86, 35.86; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₄ [M]⁺ 544.2111; found 544.2107; anal. calcd for C₃₃H₂₈N₄O₄, C, 72.78; H, 5.18; N, 10.29; found C, 72.79; H, 5.20; N, 10.31.
3.4.1.17. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(pyridin-2-ylmethylene)benzohydrazide (17). Light red solid. Yield: 86.3%. mp: 251–252 °C. IR (cm⁻¹, ATR): 3433, 3253, 2915, 1660, 1557, 1466, 1277, 755; ¹H NMR (500 MHz, DMSO-d₆): δ 11.94 (s, 1H), 8.61 (d, J = 4.3 Hz, 1H), 8.45 (s, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.86 (T, J = 8.9 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.4 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.32, 154.70, 148.07, 146.05, 142.66, 141.50, 141.50, 137.60, 134.21, 130.35, 130.35, 129.53, 129.53, 128.21, 128.21, 128.10, 128.10, 124.89, 124.89, 123.46, 123.46, 123.00, 122.75, 122.75, 119.09, 111.68, 111.68, 111.08, 111.08, 38.42, 35.89, 35.89; HREI-MS: m/z calcd for C₃₂H₂₇N₅O [M]⁺ 497.2216; found 497.2221; anal. calcd for C₃₂H₂₇N₅O, C = 77.24; H = 5.47; N = 14.07; found C = 77.25; H = 5.49; N = 14.05.
3.4.1.18. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-fluorobenzylidene)benzohydrazide (18). Light pink solid, Yield: 80.7%. mp: 243–245 °C. IR (cm⁻¹, ATR): 3432, 3183, 3065, 1625, 1542, 1469, 1253, 743; ¹H NMR (500 MHz, DMSO-d₆): δ 11.88 (s, 1H), 8.68 (s, 1H), 7.95 (s, 1H), 7.83 (d, J = 7.9 Hz, 2H), 7.50 (t, J = 8.7 Hz, 3H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.32–7.26 (m, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.95 (s, 1H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.41, 161.35, 149.37, 142.76, 141.41, 141.41, 134.27, 130.83, 130.55, 130.55, 129.63, 129.32, 129.32, 128.18, 128.18, 128.09, 128.09, 125.35, 124.75, 124.62, 124.54, 123.41, 123.41, 122.85, 122.85, 117.38, 111.52, 111.52, 111.16, 111.16, 38.38, 35.86, 35.86; HREI-MS: m/z calcd for C₃₃H₂₇FN₄O [M]⁺ 514.2169; found 514.2172; anal. calcd for C₃₃H₂₇FN₄O, C, 77.02; H, 5.29; N, 10.89; found C, 77.03; H, 5.26; N, 10.86.
3.4.1.19. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-hydroxy-4-methoxybenzylidene)benzohydrazide (19). Dark brown solid. Yield: 86.4%. mp: 279–281 °C. IR (cm⁻¹, ATR): 3427, 3126, 2947, 1609, 1464, 1270, 749; ¹H NMR (500 MHz, DMSO-d₆): δ 11.56 (s, 1H), 9.26 (s, 1H), 8.27 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.26 (s, 1H), 7.13 (t, J = 7.3 Hz, 2H), 7.03 (t, J = 8.4 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.94 (s, 1H), 3.81 (s, 3H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.48, 149.80, 148.58, 146.65, 142.46, 141.32, 141.32, 134.28, 130.25, 130.25, 129.76, 129.31, 129.31, 128.34, 128.34, 128.09, 128.09, 124.93, 124.93, 123.41, 123.41, 122.62, 122.62, 120.75, 115.34, 115.21, 111.78, 111.78, 111.26, 111.26, 56.69, 38.46, 35.79, 35.79; HREI-MS: m/z calcd for C₃₄H₃₀N₄O₃ [M]⁺ 542.2318; found 542.2321; anal. calcd for C₃₄H₃₀N₄O₃, C, 75.26; H, 5.57; N, 10.33; found C, 75.27; H, 5.56; N, 10.31.
3.4.1.20. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-chlorobenzylidene)benzohydrazide (20). Red solid. Yield: 81.7%. mp: 275–277 °C. IR (cm⁻¹, ATR): 3406, 3293, 3153, 1623, 1543, 1467, 1280, 757; ¹H NMR (500 MHz, DMSO-d₆): δ 11.89 (s, 1H), 8.41 (s, 1H), 7.82 (d, J = 7.7 Hz, 2H), 7.77 (s, 1H), 7.68 (s, 1H), 7.54–7.47 (m, 4H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 1H), 7.13 (t, J = 7.2 Hz, 2H), 6.93 (t, J = 7.2 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.31, 148.58, 142.54, 141.68, 141.68, 135.32, 134.18, 134.14, 130.45, 130.45, 130.26, 129.57, 129.57, 128.65, 128.29, 128.29, 128.15, 128.15, 127.68, 125.23, 124.91, 124.91, 123.59, 123.59, 122.65, 122.65, 111.51, 111.51, 111.16, 111.16, 38.46, 35.72, 35.72; HREI-MS: m/z calcd for C₃₃H₂₇ClN₄O [M]⁺ 530.1873; found 530.1877; anal. calcd for C₃₃H₂₇ClN₄O, C, 74.64; H, 5.12; N, 10.55; found C, 74.65; H, 5.09; N, 10.53.
3.4.1.21. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-hydroxy-5-methoxybenzylidene)benzohydrazide (21). Brown solid. Yield: 91.4%. mp: 263–265 °C. IR (cm⁻¹, ATR): 3273, 3120, 3021, 1608, 1589, 1309, 1256, 747; ¹H NMR (500 MHz, DMSO-d₆): δ 11.99 (s, 1H), 10.71 (s, 1H), 8.59 (s, 1H), 7.84 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.15–7.11 (m, 3H), 6.95–6.92 (m, 2H), 6.89 (s, 2H), 6.86 (d, J = 9.0 Hz, 1H), 5.95 (s, 1H), 3.74 (s, 3H), 3.73 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.26, 154.31, 154.34, 149.71, 142.56, 141.13, 134.25, 130.53, 129.35, 128.47, 128.21, 124.96, 123.56, 122.43, 121.78, 116.67, 116.23, 112.86, 111.38, 111.18, 56.25, 38.48, 35.94; HREI-MS: m/z calcd for C₃₄H₃₀N₄O₃ [M]⁺ 542.2318; found 542.2314; anal. calcd for C₃₄H₃₀N₄O₃, C, 75.26; H, 5.57; N, 10.33; found C, 75.27; H, 5.58; N, 10.31.
3.4.1.22. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-nitrobenzylidene)benzohydrazide (22). Light yellow solid. Yield: 72.5%. mp: 232–233 °C. IR (cm⁻¹, ATR): 3422, 3382, 3242, 1609, 1546, 1466, 1273, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 12.12 (s, 1H), 8.85 (s, 1H), 8.14 (d, J = 6.9 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 7.8 Hz, 2H), 7.71–7.64 (m, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.4 Hz, 2H), 6.94 (t, J = 7.4 Hz, 2H), 6.89 (s, 1H), 5.96 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): δ 164.39, 148.03, 142.84, 142.62, 141.47, 141.47, 134.18, 133.79, 130.49, 130.31, 130.31, 129.43, 129.43, 129.38, 129.38, 128.16, 128.16, 128.07, 128.07, 125.16, 124.69, 124.69, 123.42, 123.42, 122.71, 122.71, 111.69, 111.69, 111.18, 111.18, 38.32, 35.81, 35.81; HREI-MS: m/z calcd for C₃₃H₂₇N₅O₃ [M]⁺ 541.2114; found 541.2110; anal. calcd for C₃₃H₂₇N₅O₃, C, 73.18; H, 5.02; N, 12.93; found C, 73.17; H, 5.03; N, 12.91.
3.4.1.23. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2,4-dihydroxybenzylidene)benzohydrazide (23). Brown solid. Yield: 72.5%. mp: 262–264 °C. IR (cm⁻¹, ATR): 3431, 3255, 3023, 1613, 1589, 1543, 1519, 1468, 1274, 755; ¹H NMR (500 MHz, DMSO-d₆): δ 11.82 (s, 1H), 11.49 (s, 1H), 9.93 (s, 1H), 8.47 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 6.36 (d, J = 8.3 Hz, 1H), 6.32 (s, 1H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): 164.36, 160.58, 160.52, 151.66, 142.09, 141.53, 141.53, 134.86, 130.35, 130.21, 130.21, 129.21, 129.21, 128.53, 128.53, 128.34, 128.34, 124.46, 124.46, 123.89, 123.89, 122.85, 122.85, 113.19, 111.58, 111.58, 111.18, 111.18, 109.25, 103.14, 38.43, 35.94, 35.94; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₃ [M]⁺ 528.2161; found 528.2161; anal. calcd for C₃₃H₂₈N₄O₃, C, 74.98; H, 5.34; N, 10.60; found C, 74.98; H, 5.34; N, 10.60.
3.4.1.24. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-methylbenzylidene)benzohydrazide (24). Dark brown solid. Yield: 84.8%. mp: 254–256 °C. IR (cm⁻¹, ATR): 3539, 3252, 3075, 3143, 1640, 1564, 1469, 1342, 1274, 1145, 732; ¹H NMR (500 MHz, DMSO-d₆): δ 11.76 (s, 1H), 8.40 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.28 (s, 1H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 1H), 5.95 (s, 1H), 3.81 (s, 3H), 3.73 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.52, 148.78, 142.56, 141.52, 141.52, 137.89, 136.76, 134.31, 130.28, 130.28, 129.61, 129.33, 129.33, 128.47, 128.47, 128.13, 128.13, 128.09, 127.89, 124.53, 124.53, 123.51, 123.51, 123.26, 122.71, 122.71, 111.58, 111.58, 111.04, 111.04, 38.39, 35.86, 35.86, 21.18; HREI-MS: m/z calcd for C₃₄H₃₀N₄O [M]⁺ 510.2420; found 510.2423; anal. calcd for C₃₄H₃₀N₄O, C, 79.97; H, 5.92; N, 10.97; found C, 79.98; H, 5.94; N, 10.96.
3.4.1.25. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2,4,6-trihydroxybenzylidene)benzohydrazide (25). Dark red solid. Yield: 89.4%; mp: 313–315 °C, IR (cm⁻¹, ATR): 3413, 1466, 1341, 3051, 1657, 1519, 1269, 1113, 756; ¹H NMR (500 MHz, DMSO-d₆): δ 11.77 (s, 1H), 11.07 (s, 1H), 9.77 (s, 1H), 8.77 (s, 1H), 7.83 (d, J = 7.8 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.4 Hz, 2H), 6.88 (s, 1H), 5.94 (s, 1H), 5.84 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 163.92, 163.12, 161.47, 161.47, 144.68, 142.35, 141.47, 141.47, 134.18, 130.25, 130.25, 129.50, 129.50, 128.38, 128.38, 128.09, 128.09, 124.69, 124.69, 123.56, 123.56, 122.85, 122.85, 111.69, 111.69, 111.18, 111.18, 106.29, 95.87, 95.87, 38.32, 35.86, 35.86; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₄ [M]⁺ 544.2111; found 544.2108; anal. calcd for C₃₃H₂₈N₄O₄, C, 72.78; H, 5.18; N, 10.29; found C, 72.79; H, 5.15; N, 10.26.
3.4.1.26. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(4-chlorobenzylidene)benzohydrazide (26). Red solid. Yield: 88.3%; mp: 237–239 °C, IR (cm⁻¹, ATR): 3434, 3232, 3019, 1633, 1514, 1469, 1233, 1187, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.82 (s, 1H), 8.41 (s, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.51 (t, J = 8.0 Hz, 4H), 7.40 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.4 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.42, 149.37, 142.56, 141.51, 141.51, 135.23, 134.82, 134.20, 130.37, 130.37, 129.51, 129.51, 129.48, 129.48, 129.16, 129.16, 128.31, 128.31, 128.13, 128.13, 124.97, 124.97, 123.47, 123.47, 122.85, 122.85, 111.58, 111.58, 111.09, 111.09, 38.43, 35.83, 35.83; HREI-MS: m/z calcd for C₃₃H₂₇ClN₄O [M]⁺ 530.1873; found 530.1877; anal. calcd for C₃₃H₂₇ClN₄O, C, 74.64; H, 5.12; N, 10.55; found C, 74.66; H, 5.13; N, 10.57.
3.4.1.27. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(2-chlorobenzylidene)benzohydrazide (27). Red solid. Yield: 79.4%; mp: 247–249 °C, IR (cm⁻¹, ATR): 3119, 3026, 2828, 1658, 1539, 1449, 1269, 745; ¹H NMR (500 MHz, DMSO-d₆): δ 11.99 (s, 1H), 8.84 (s, 1H), 8.02 (s, 1H), 7.84 (d, J = 7.7 Hz, 2H), 7.52 (t, J = 7.3 Hz, 2H), 7.45 (d, J = 4.0 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.4 Hz, 2H), 6.94 (t, J = 7.4 Hz, 2H), 6.89 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.48, 149.92, 142.32, 141.58, 141.58, 134.31, 132.59, 131.20, 130.93, 130.37, 130.37, 129.73, 129.73, 128.69, 128.69, 128.41, 128.41, 128.20, 128.20, 127.73, 124.92, 124.92, 123.51, 123.51, 122.79, 122.79, 111.58, 111.58, 111.28, 111.28, 38.38, 35.72, 35.72; HREI-MS: m/z calcd for C₃₃H₂₇ClN₄O [M]⁺ 530.1873; found 530.1871; anal. calcd for C₃₃H₂₇ClN₄O, C, 74.64; H, 5.12; N, 10.55; found C, 74.62; H, 5.10; N, 10.56.
3.4.1.28. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-fluorobenzylidene)benzohydrazide (28). Light orange solid. Yield: 81.9%; mp: 263–264 °C, IR (cm⁻¹, ATR): 3421, 3282, 3061, 1660, 1608, 1519, 1466, 1360, 1272, 752; ¹H NMR (500 MHz, DMSO-d₆): δ 11.86 (s, 1H), 8.43 (s, 1H), 7.82 (d, J = 7.5 Hz, 2H), 7.52 (t, J = 13.1 Hz, 3H), 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.51, 162.78 (d, J = 261.5 Hz), 148.79, 142.46, 141.51, 141.51, 138.63, 134.31, 130.37, 130.37, 129.87, 129.43, 129.43, 128.31, 128.31, 128.12, 128.12, 124.91, 123.51, 123.51, 122.74, 122.05, 117.14, 116.92, 114.35, 114.19, 111.58, 111.58, 111.09, 111.09, 38.54, 35.91, 35.91; HREI-MS: m/z calcd for C₃₃H₂₇FN₄O [M]⁺ 530.1873; found 530.1869; anal. calcd for C₃₃H₂₇FN₄O, C, 77.02; H, 5.29; N, 10.89; found C, 77.01; H, 5.31; N, 10.90.
3.4.1.29. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(3-hydroxybenzylidene)benzohydrazide (29). Brown solid. Yield: 72.4%; mp: 233–234 °C, IR (cm⁻¹, ATR): 3312, 3179, 2833, 1633, 1609, 1505, 1467, 1253, 1141, 747; ¹H NMR (500 MHz, DMSO-d₆): δ 11.69 (s, 1H), 9.92 (s, 1H), 9.60 (s, 1H), 8.33 (s, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 7.19 (s, 1H), 7.16–7.09 (m, 3H), 6.93 (t, J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.95 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.52, 156.37, 148.69, 142.51, 141.40, 141.40, 137.07, 134.31, 130.72, 130.38, 130.38, 129.52, 129.52, 128.31, 128.31, 128.15, 128.15, 124.91, 124.91, 123.56, 123.56, 122.73, 122.73, 120.07, 119.35, 114.82, 111.48, 111.48, 111.18, 111.18, 38.40, 35.92, 35.92; HREI-MS: m/z calcd for C₃₃H₂₈N₄O₂ [M]⁺ 512.2212; found 512.2216; anal. calcd for C₃₃H₂₈N₄O₂, C, 77.32; H, 5.51; N, 10.93; found C, 77.30; H, 5.52; N, 10.94.
3.4.1.30. (E)-4-(Bis(1-methyl-1H-indol-3-yl)methyl)-N′-(pyridin-4-ylmethylene)benzohydrazide (30). Red solid. Yield: 82.6%; mp: 253–254 °C, IR (cm⁻¹, ATR): 3423, 3352, 3138, 1657, 1611, 1269; ¹H NMR (500 MHz, DMSO-d₆): δ 12.02 (s, 1H), 8.65 (d, J = 4.2 Hz, 2H), 8.42 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.66 (s, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.13 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H), 3.72 (s, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 164.52, 150.19, 150.19, 149.46, 142.58, 141.79, 141.79, 140.75, 134.31, 130.38, 130.38, 129.63, 129.63, 128.32, 128.32, 128.20, 128.20, 124.93, 124.93, 123.56, 123.56, 122.71, 122.71, 122.45, 122.45, 111.98, 111.98, 111.18, 111.18, 38.52, 35.79, 35.79; HREI-MS: m/z calcd for C₃₂H₂₇N₅O [M]⁺ 497.2216; found 497.2221; anal. calcd for C₃₂H₂₇N₅O, C, 77.24; H, 5.47; N, 14.07; found C, 77.25; H, 5.45; N, 14.06.

3.5. Cytotoxicity assays using 3T3-L1 and CC-1 cell lines

In vitro cytotoxicity assays were performed as described in Taha et al. 2015,³⁵ using the 3T3-L1 mouse embryo fibroblast cell line (American Type Culture Collection ‘ATCC’, Manassas, VA 20108, USA), and CC-1 cells, a rat Wistar hepatocyte cell line (European Collection of Cell Cultures, Salisbury, UK). The CC-1 cells were suspended in Minimum Essential Medium Eagle (MEM) supplemented with 10% FBS, 2 mM glutamine, 1% non-essential amino acids, and 20 mM HEPES. The 3T3-L1 cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) formulated with 10% FBS. Using flat-bottomed plates, both cell lines were plated at a concentration of 6 × 104 cells per mL and incubated for 24 h at 37 °C in a 5% CO₂ environment. After removal of the media, the cells were challenged with three different concentrations (1.0, 5.0, and 20 μg mL⁻¹) of compounds in triplicates and were then further incubated for 48 h at 37 °C in a CO₂ incubator. Following exposure to each compound, the cells' viability was assessed using 0.5 mg mL⁻¹ of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h, followed by removal of the supernatant and the addition of DMSO to solubilize the formazan complex. Plates were read at 540 nm after one minute shaking and the readings were processed using the MS Excel software. The results were expressed as means ± SD of the triplicate readings.

3.6. Docking studies

The structures of all the compounds were prepared using Chem3D by CambridgeSoft. The human β-D-glucuronidase crystal structure was first retrieved from the protein data bank (PDB code: 1BHG).³⁶ Docking was carried out using AutoDock 4.2.³⁷ Using the Genetic Algorithm (GA) search parameter, the number of runs was set at 150, whereas the other settings were left as the default. The docking parameter was also left at its default settings. The 3D docking results were visualized using Discovery Studio Visualizer 4.1.

3.7. DFT studies

The geometry optimization and frequency calculations of the synthesized novel bisindolylmethane derivatives were carried out at the B3LYP/6-31+G(d,p) level of theory,⁴⁰ as implemented in the Gaussian 09 package.⁴¹ The optimized minima were confirmed by the absence of imaginary frequencies. The solvent effects were taken into account implicitly using an integral equation formalism (IEF) version of the polarizable continuum model (PCM). In PCM, the molecule (e.g., the bisindolylmethane derivative) is embedded into a cavity surrounded by the solvent, which is described by its dielectric constant ε.⁴²

4. Conclusions

We synthesized novel bisindolylmethane derivatives containing a benzohydrazone moiety. In conclusion, molecular docking studies evidently demonstrated the interaction pattern of the synthetic compounds in correspondence with a biological inhibitory assay. Analysis of the binding mode clearly showed the presence of a hydrophilic group, i.e., that the hydroxyl moiety plays a key role in the activity profile. It was also observed that the compounds are positioned in the binding site in such a way that two or more hydroxyl groups have to be substituted on the carbon adjacent to each other for good interaction to take place with important residues like Glu451 and Glu540, compared to hydroxyls that are substituted far apart.

Acknowledgements

The authors would like to acknowledge Universiti Teknologi MARA for the financial support under the Research Intensive Faculty grant scheme with reference number UiTM 600-RMI/DANA 5/3/RIF (347/2012).

References

1. X.-H. Gu, X.-Z. Wan and B. Jiang, Bioorg. Med. Chem. Lett., 1999, 9, 569.
2. C. Bonnesen, I. M. Eggleston and J. D. Hayes, Cancer Res., 2001, 61, 6120.
3. Indoles: Part I, ed. W. Houlihan, W. Remers and R. Brown, 1992.
4. R. J. Sundberg, The Chemistry of Indoles, New York, Academic Press, 1996.
5. A. Casapullo, G. Bifulco, I. Bruno and R. Riccio, J. Nat. Prod., 2000, 63, 447.
6. B. Bao, Q. Sun, X. Yao, J. Hong, C.-O. Lee, C. J. Sim, K. S. Im and J. H. Jung, J. Nat. Prod., 2005, 68, 711.
7. L. Gupta, A. Talwar, S. Palne, S. Gupta and P. M. Chauhan, Bioorg. Med. Chem. Lett., 2007, 17, 4075.
8. K. Kaniwa, M. A. Arai, X. Li and M. Ishibashi, Bioorg. Med. Chem. Lett., 2007, 17, 4254.
9. S.-L. Zhu, S.-J. Ji, X.-M. Su, C. Sun and Y. Liu, Tetrahedron Lett., 2008, 49, 1777.
10. G. La Regina, A. Coluccia, F. Piscitelli, A. Bergamini, A. Sinistro, A. Cavazza, G. Maga, A. Samuele, S. Zanoli and E. Novellino, J. Med. Chem., 2007, 50, 5034.
11. S. Routier, P. Peixoto, J.-Y. Mérour, G. Coudert, N. Dias, C. Bailly, A. Pierré, S. Léonce and D.-H. Caignard, J. Med. Chem., 2005, 48, 1401.
12. A. Andreani, S. Burnelli, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, L. Varoli, L. Landi and C. Prata, J. Med. Chem., 2008, 51, 4563.
13. V. S. Velezheva, P. J. Brennan, V. Y. Marshakov, D. V. Gusev, I. N. Lisichkina, A. S. Peregudov, L. N. Tchernousova, T. G. Smirnova, S. N. Andreevskaya and A. E. Medvedev, J. Med. Chem., 2004, 47, 3455.
14. K. E. Knott, S. Auschill, A. Jäger and H.-J. Knölker, Chem. Commun., 2009, 1467.
15. M. I. Rodríguez-Franco, M. I. Fernández-Bachiller, C. Pérez, B. Hernández-Ledesma and B. Bartolomé, J. Med. Chem., 2006, 49, 459.
16. K. V. Sashidhara, A. Kumar, M. Kumar, A. Srivastava and A. Puri, Bioorg. Med. Chem. Lett., 2010, 20, 6504.
17. S. H. Benabadji, R. Wen, J.-B. Zheng, X.-C. Dong and S.-G. Yuan, Acta Pharmacol. Sin., 2004, 25, 666.
18. K. Sujatha, P. T. Perumal, D. Muralidharan and M. Rajendran, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2009, 48, 267.
19. R. Kato and T. Kamataki, Drug Metabolism, Tokyo Kagaku Dojin, Tokyo, 1996, in Japanese.
20. G. A. Levvy and J. Conchie, β-glucuronidase and the hydrolysis of glucuronides, glucuronic acid, ed. G. J. Dutton, Academic Press, New York, 1966, pp. 301–364.
21. S. Yasuoka, Rinsho Kensa Mook, 1982, 11, 183.
22. H. R. Nawaz, A. Malik, P. M. Khan, S. Shujaat and A. Rahman, Chem. Pharm. Bull., 2000, 48, 1771.
23. S. B. Shim, N. J. Kim and D. H. Kim, Planta Med., 2000, 66, 40.
24. D. H. Ki, B. Shim, N. J. Kim and I. S. Jang, Biol. Pharm. Bull., 1999, 22, 162.
25. T. Hayashi, M. Kawasaki, K. Okamura, Y. Tamada and N. Morita, J. Nat. Prod., 1992, 55, 1748.
26. D. B. I. Cenci, P. Dorling, L. Fellows and B. Winchester, FEBS Lett., 1984, 176, 61.
27. J. Waqas, P. Shagufta, A. A. S. Syed, T. Muhammad, H. I. Nor, P. Shahnaz, A. Nida, M. K. Khalid and I. C. Muhammad, Molecules, 2014, 19, 8788.
28. S. Imran, M. Taha, N. H. Ismail, S. Fayyaz, K. M. Khan and M. I. Choudhary, Bioorg. Chem., 2015, 62, 83.
29. M. K. Khan, F. Rahim, N. Ambreen, M. Taha, M. Khan, H. Jahan, A. Shaikh, S. Iqbal, S. Perveen and I. M. Choudhary, Med. Chem., 2013, 9, 588.
30. K. M. Khan, M. Taha, M. Ali and S. Perveen, Lett. Org. Chem., 2009, 6, 319.
31. K. M. Khan, F. Rahim, A. Wadood, M. Taha, M. Khan, S. Naureen, N. Ambreen, S. Hussain, S. Perveen and M. I. Choudhary, Bioorg. Med. Chem. Lett., 2014, 24, 1825.
32. Bioactive Natural Products Part E, ed. Atta-ur-Rahman, Elsevier, Amsterdam, 2000, vol. 24, p. 333.
33. (a) B. P. Bandgar and K. A. Shaikh, Tetrahedron Lett., 2003, 44, 1959; (b) K. M. Khan, M. Taha, F. Rahim, W. Jamil, S. Perveen and M. I. Choudhary, J. Iran. Chem. Soc., 2012, 9, 81; (c) S. A. Mulla, A. Sudalai, M. Y. Pathan, S. A. Siddique, S. M. Inamdar, S. S. Chavan and R. S. Reddy, RSC Adv., 2012, 2, 3525; (d) X. F. Xu, Y. Xiong, X. G. Ling, X. M. Xie, J. Yuan, S. T. Zhang and Z. R. Song, Chin. Chem. Lett., 2014, 25, 406.
34. K. M. Khan, A. Karim, S. Saied, N. Ambreen, X. Rustamova, S. Naureen, S. Mansoor, M. Ali, S. Perveen, M. I. Choudhary and G. A. Morales, Mol. Diversity, 2014, 18, 295.
35. M. Taha, N. H. Ismail, S. Imran, A. Wadood, F. Rahim, M. Ali and A. U. Rehman, MedChemComm, 2015, 6, 1826.
36. S. Jain, W. B. Drendel, Z. W. Chen, F. S. Mathews, W. S. Sly and J. H. Grubb, Nat. Struct. Biol., 1996, 3, 375.
37. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput. Chem., 2009, 16, 2785.
38. K. M. Khan, F. Rahim, S. A. Halim, M. Taha, M. Khan, S. Perveen, Z. -ul-Haq, M. A. Mesaik and M. I. Choudhary, Bioorg. Med. Chem., 2011, 19, 4286.
39. (a) O. Lauren and M. H. Court, J. Pharmacogenomics Pharmacoproteomics, 2008, 60, 1175; (b) K. M. Khan, S. M. Saad, N. N. Shaikh, S. Hussain, M. I. Fakhri, S. Perveen, M. Taha and M. I. Choudhary, Bioorg. Med. Chem., 2014, 22, 3449; (c) K. M. Khan, N. Ambreen, M. Taha, S. A. Halim, Z. -ul-Haq, S. Naureen, S. Rasheed, S. Perveen, S. Ali and M. I. Choudhary, J. Comput.-Aided Mol. Des., 2014, 28, 577; (d) N. K. N. Z. Abdullah, M. Taha, N. Ahmat, A. Wadood, N. H. Ismail, F. Rahim, M. Ali, N. Abdullah and K. M. Khan, Bioorg. Med. Chem., 2015, 23, 3119.
40. A. D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys., 1993, 98, 5648–5652.
41. M. Andersson and P. Uvdal, New scale factors for harmonic vibrational frequencies using the B3LYP density functional method with the triple-ζ basis set 6-311+G(d,p), J. Phys. Chem. A, 2005, 109, 2937–2941.
42. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. F. J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision A.02, 2009.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra19513f

---