Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies

Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies

Accepted Manuscript Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies Muhammad Taha,...

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Accepted Manuscript Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies Muhammad Taha, Hayat Ullah, Laode Muhammad Ramadhan Al Muqarrabun, Muhammad Naseem Khan, Fazal Rahim, Norizan Ahmat, Muhammad Ali, Shahnaz Perveen PII:

S0223-5234(17)30873-5

DOI:

10.1016/j.ejmech.2017.10.071

Reference:

EJMECH 9863

To appear in:

European Journal of Medicinal Chemistry

Received Date: 3 August 2017 Revised Date:

1 October 2017

Accepted Date: 26 October 2017

Please cite this article as: M. Taha, H. Ullah, L.M.R. Al Muqarrabun, M.N. Khan, F. Rahim, N. Ahmat, M. Ali, S. Perveen, Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.10.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies

Muhammad Taha∗a, Hayat Ullah,b Laode Muhammad Ramadhan Al Muqarrabun c ,d , Muhammad Naseem Khane, Fazal Rahim,b Norizan Ahmatc,d, Muhammad Alie, Shahnaz Perveenf

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a Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), University of Dammam, Dammam 31441, Saudi Arabia. b Department of Chemistry, Hazara University, Mansehra-21300, Pakistan c Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia d Faculty of Applied Science Universiti Teknologi MARA (UiTM), 40450, Shah Alam, Selangor, Malaysia. e Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan f PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. SalimuzzamanSiddiqui, Karachi-75280, Pakistan

H N

O

O

NH

NH N

OH

HO HO

OH

IC50 = 0.10 ± 0.01 uM Potent B-glucuronidase inhibitor Standard Inhibitor D-saccharic acid 1,4 lactone

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N NH

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Compound 11

OH

IC50 = 48.30 ± 1.20 uM

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EP

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OH



Correspondence and reprints E-mail: E-mail: [email protected] and [email protected]: 00966502057370

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Synthesis of bis-indolylmethanes as new potential inhibitors of β-glucuronidase and their molecular docking studies

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Muhammad Taha∗a, Hayat Ullah,b Laode Muhammad Ramadhan Al Muqarrabun c ,d , Muhammad Naseem Khane, Fazal Rahim,b Norizan Ahmatc,d, Muhammad Alie, Shahnaz Perveenf a

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Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), University of Dammam, Dammam 31441, Saudi Arabia. b Department of Chemistry, Hazara University, Mansehra-21300, Pakistan c Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia d Faculty of Applied Science Universiti Teknologi MARA (UiTM), 40450, Shah Alam, Selangor, Malaysia. e Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan f PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. SalimuzzamanSiddiqui, Karachi-75280, Pakistan Abstract:

Thirty-two (32) bis-indolylmethane-hydrazone hybrids 1-32 were synthesized and characterized 13

CNNMR and HREI-MS. All compounds were evaluated in vitro for β-

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by 1HNMR,

glucuronidase inhibitory potential. All analogs showed varying degree of β-glucuronidase inhibitory potential ranging from 0.10 ± 0.01 to 48.50 ± 1.10 µM when compared with the standard drug D-saccharic acid-1,4-lactone (IC50 value 48.30 ± 1.20 µM). Derivatives 1-32

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showed the highest β-glucuronidase inhibitory potentials which is many folds better than the standard drug D-saccharic acid-1,4-lactone. Further molecular docking study validated the

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experimental results. It was proposed that bis-indolylmethane may interact with some amino acid residues located within the active site of β-glucuronidase enzyme. This study has culminated in the identification of a new class of potent β-glucuronidase inhibitors.

Keywords: Synthesis, Bis-indolylmethanes, β-glucuronidase activity, Molecular Docking, SAR.



Correspondence and reprints E-mail: E-mail: [email protected] and [email protected]: 00966502057370

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1. Introduction: Bis-indolylmethanes (BIMS) have been identified to possess wide range of applications in pharmacology, biochemistry and medicinal chemistry [1]. BIMS and their derivatives are found in terrestrial and marine metabolites [2] and are known to exhibit wide range pharmacological

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activities such as antimicrobial [3], antifungal and antitumor [4], antibacterial [5], HIV-1 integrase inhibitor [6] and aromatase inhibitor for breast cancer [7]. Some of the the bisindolylmethane derivatives are used in estrogen metabolism in humans [8], in the treatment of fibromyalgia, chronic fatigue and irritable bowel syndrome [9].

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The present work is related to the synthesis and evaluation of in vitro β-glucuronidase inhibition of a series of bis-indolylmethanes analogs 1–32. β-Glucuronidase is an exoglycosidase enzyme

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which catalyzes the cleavage of glucuronosyl-O-bonds [10]. The enzyme is present in many organs and body fluids such as kidney, spleen, bile, serum, urine, respectively [11, 12]. Enhanced activity of this enzyme has been reported in a variety of pathological conditions, including urinary tract infection [13-16], renal diseases [17], transplantation rejection [18], epilepsy [19], neoplasm of bladder [20], testes, larynx and breast [21]. Furthermore, βglucuronidase is reported to be released into the synovial fluid in inflammatory joint diseases

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such as rheumatoid arthritis [22, 23]. The over-expression of the enzyme is also reported in some hepatic diseases and AIDS [24]. β-Glucuronidase is also found to be involved in the etiology of colon cancer and higher intestinal level of the enzyme associated with higher incidence of colon carcinoma [25, 26]. Previously our research group has reported simple bis-indolylmethanes as

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potent β-glucuronidase inhibitors [27]. Keeping in view that report we planned to bring some structural modifications in bis-indolylmethane skeleton by introducing hydrazone moeties with

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the hope to enhance their β-glucuronidase inhibitory potential. After experimental results we have found outstanding β-glucuronidase inhibitory potentials of our newly designed bisindolylmethane analogues 1-32. These reports clearly suggest that the development of specific inhibitors of β-glucuronidase has great pharmacological importance. In continuation of our ongoing research on the chemistry and bioactivity of new heterocyclic compounds [28-34], herein, we report the synthesis of bis-indolylmethane-hydrazone analogs to evaluate their β-glucuronidase inhibitory potentials.

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2. Results and Discussion: 2.1. Chemistry: Methyl 1H-indole-5-carboxylate (I), 20 mmol, and 4-methyl benzaldehyde (II), 10.5 mmol, were reacted in 50 mL of refluxing acetic acid for 3 hours. The reaction mixture was poured into

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crushed ice. Crude bis-indolylmethane-bis-methyl ester (III) was formed as solid which was filtered, washed with water to remove excess acetic acid, and then dried.

Intermediate (III) was converted into hydrazide by reacting it with 50 mL hydrazine hydrate in 50 mL MeOH. The mixture was refluxed for 6 hours and then solvents were evaporated in

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vacuo. The crude product (IV) was washed with water and then dried to give 89.6% yield.

The synthesis of novel bis-indolylmethane based Schiff base derivatives (1-32) was

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accomplished by reacting different aldehydes with hydrazide (IV) in methanol.

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N

O

N H

N H N H

N

R

N H

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Table-1: Different substituents of bis-indolylmethane analogs and their β-glucuronidase activity (1-32) Compound No.

Reagent (Aldehyde)

IC50 ± SEMa 2.348±0.444

2

0.30 ±0.04

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SC

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1

7.571 ± 0.5

3

32.80 ± 0.7

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4

2.98± 0.10

6

5.874±0.05

7

0.1 0 ± 0.05

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EP

5

4

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42.75 ± 1.10

9

1.14 ± 0.01

10

0.3 ± 0.01

SC

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8

0.10 ± 0.01

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11

0.20 ± 0.01

EP

13

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12

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14

15

3.448± 0.10

2.1 ± 0.10

2.67 ± 0.10

5

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43.50 ± 1.05

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16

1.50 ± 0.1

17

48.50 ± 1.10

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SC

18

22.20 ± 0.50

19

33.50 ± 0.70

EP

21

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20

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22

23

12.50 ± 0.50

2.80 ± 0.1

6.90 ± 0.3

6

24

5.80 ± 0.20

25

1.10 ±0.10

26

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SC

1.20 ±0.1

2.20 ±0.10

27

EP

29

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28

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30

2.8 ± 0.1

15.8 ± 0.20

32.90 ± 0.60

31

23.4 ± 0.5

32

13.10 ± 0.2

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St Drug

D-saccharic acid 1,4 lactone 48.30 ± 1.20

2.2. In vitro β-Glucuronidase inhibitory Potential:

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We have synthesized thirty-two (32) bis-indolylmethane analogs (1-32) with varying degree of β-glucuronidase inhibition potential ranging in between 0.10 ± 0.01to 48.50 ± 1.10 µM when compared with the standard drug D-saccharic acid 1,4-lactone (IC50 value 48.30 ± 1.20 µM). All of thirty-two analogs 1-32, showed outstanding β-glucuronidase inhibitory potentials. The

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structure activity relationship was mainly based upon by bringing different substituents on phenyl part.

Analogs 7 and 11 with IC50 values of 0.1 0 ± 0.05 and 0.10 ± 0.01 µM were found to be the most

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potent and powerful inhibitors among the series having di- and tri- hydroxy substituents on the both sides phenyl rings. Similarly, other di- hydroxyl groups containing analogs such as 2 (IC50 = 0.30 ± 0.04 µM), 9 (IC50 = 1.14 ± 0.01 µM), 10 (IC50 = 0.3 ± 0.01 µM) and with tri- hydroxyl substitutions such as in the compound 12 (IC50 = 0.20 ± 0.01 µM); the activity pattern in all these compounds is somewhat similar. Minute differences of inhibition between these compounds are

the potential of inhibition.

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difficult to explain, however, the obvious thing is that the position of substitutions slightly effects

If we compare analog 27 (IC50 = 1.10 ± 0.10 µM) having di-chloro substituent at ortho and para positions on both side phenyl ring with analog 24 (IC50 = 2.80 ± 0.1µM), analog 25 (IC50 = 6.90 ± 0.3 µM) and analog 26 (IC50 = 5.80 ± 0.20 µM) having mono-chloro substituent on both side

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phenyl ring but position of the substituents are different. The slight difference in the activity of these analogs showed that the positions, as well as the number of substituents greatly, affect the

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inhibition.

To understand the binding interaction of the most active analogs molecular docking study was performed.

2.3. Docking Studies

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In vitro inhibition potential studies of synthesized compounds against Human-β-glucuronidase were supported by performing molecular docking studies. Molecular docking has vast applications in drug finding and development. All the synthesized compounds were docked against target enzyme and all of them show different interactions with different residues of the

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active site. The active site of the protein was selected based on targeting the key residues i.e. Glu451 and Glu540 [35]. X-ray crystal structure of Human-β-glucuronidase (PDB ID: 1BHG)

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downloaded from PDB [36] and its active site is shown in Fig.4.

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Figure-4: Structure of Human-β-glucuronidase (PDB ID: 1BHG) and its active site (red sphere and zoomed in).

First, interactions of the most active compound 11 (IC50 = 0.10 ± 0.01 µM) were analyzed with

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the residues of the target protein. Graphical investigation of the lowest energy pose illustrated that except one hydroxyl group at the phenyl groups all the remaining five hydroxyl groups mediated hydrogen bond interactions with the side chain residues of the active site. In these interactions, the residues acting as hydrogen bond donors were Asn450, Glu540, Trp528 and Lys606 respectively. While, amino acids acting as hydrogen bond acceptors were His385, Asn502 and Gln524. Also, another hydrogen bond interaction was found between the amide nitrogen atom and the hydroxyl group of Tyr508 acting as hydrogen bond donor. Other side chain residues involved in hydrophobic contacts were Asp207, His385, Asn484, His509, Tyr508, Trp587 and Thr599 as shown in Fig. 5a. 9

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Binding mode of compound12, another active derivative (IC50 = 0.20 ± 0.01 µM) showed that three hydrogen bond interactions occurred between the ligand and side chain amino acids including Glu451. All the interacting amino acids acted as a hydrogen bond acceptors. Carbonyl group of Val410 accepted hydrogen from NH group of indole ring, the carboxyl group of Glu451

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accepted hydrogen from NH of amide group whereas, carbonyl oxygen of Asn484 accepted hydrogen from the hydroxyl group attached at the ortho position of the phenyl ring. Other major

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residues involved in hydrophobic interactions were Glu451 and Asp207 as shown in Fig. 5b.

Figure-5: Models of the interaction of 11 (a) and 12 (b) with the binding site of Human-β-glucuronidase generated by poseview. Dashed lines indicate hydrogen bond interactions and a green line indicating hydrophobic interactions.

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Binding modes of another highly active compound 7 (IC50 = 0.10 ± 0.05 µM) showed that the compound was involved in various interactions. Strong to medium hydrogen bond interactions

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were found between the ligand and receptor residues. Residues acting as hydrogen bond acceptors were His385, Asn484 and Tyr508 while, residues acting as hydrogen bond donors were Asp207 and Asn450 respectively. Amino acids involved in hydrophobic interactions with the ligands were Tyr205, Phe206, Glu451, Asn502, Tyr504 and Trp587 as shown in Fig. 6a. Compound 2 is the fourth most active compound (IC50 = 0.30 ± 0.04 µM) among the synthesized derivatives. The predicted binding modes of this compound showed strong to medium hydrogen bond interactions with the side chain amino acids, His385, Val410, Glu451 and Trp528 were involved in making hydrogen bonds with the hydroxyl groups attached to phenyl rings, while, 10

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Asn484, Tyr504 and His509 were involved in making hydrogen bonds with carbonyl groups of amides. Also, another hydrogen bond occurred between Asn484 and -NH- of amide group respectively. Other amino acids involved in hydrophobic interactions were Phe206, Asn486,

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SC

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Asn502 and Tyr508 as shown in Fig. 6b.

Figure-6: (a) Two-dimensional scheme of the interactions between 7 and Human β-glucuronidase; (b) and +

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interactions between 2 and Human β-glucuronidase generated by Ligplot . Only more important residues for binding are shown.

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Along with two-dimensional analysis, the predicted binding modes of these selected compounds with the active site of Human-β-D-glucoridase were also visualized in three-dimensions. And the graphical analysis showed that these compounds fit well into the active site of the protein as shown in Fig. 7. Also, these studies showed that the substituted phenyl groups specifically containing hydroxyl groups are generally involved in hydrogen bond interactions and the indole groups of these synthesized compounds are involved in hydrophobic interactions resulting in minimizing the free energy of binding (FEB) score and making them able to fit well into the active site.

11

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Figure-7: Stereoview of simulated docking poses of compounds (a) 11; (b) 7; (c) 12 and (d) 2 to Human-βglucuronidase. Compounds 11, 7, 12 and 2 are shown as stick models with carbon colored in bright yellow; nitrogen

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colored in bright blue and oxygen atoms colored in dark red respectively. Important parts of the enzyme for interaction were shown as a stick model colored in dark red.

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3. Conclusion:

With the aim to synthesize more potent β-glucuronidase inhibiting agents, a new series of bisindolylmethanes derivatives was designed. Herein a multi-step route was adopted to achieve the desired product. In vitro β-glucuronidase inhibition activity of these molecules helped in introducing some new β-glucuronidase inhibitors. These derivatives have displayed excellent efficacy results as compared to standard drug D-saccharic acid 1,4 lactone. The most probable binding modes of these derivatives with enzyme’s active sites were described through molecular docking.

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4. Materials and Methods: 4.1.

Synthesis of dimethyl-3,3'-(p-Tolylmethylene)bis(1H-indole-5-carboxylate)

Two equimolar of methyl 1H-indole-5-carboxylate (I) (20 mmol) was treated with one equimolar of 4-methyl benzaldehyde (II) (10.5 mmol) in 50 mL of acetic acid. The mixture was refluxed at

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200 °C for 3 hours. The reaction completion was monitored by TLC. After completion of the reaction, the mixture was poured into crushed ice. Derivative (III) formed was filtered, washed with water to remove excess acetic acid, and then dried. 4.2.

Synthesis of 3,3'-(p-Tolylmethylene)bis(1H-indole-5-carbohydrazide)

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The ester group on compound (III) was converted into hydrazide by reacting it with 50 mL hydrazine hydrate in 50 mL MeOH. The mixture was refluxed at 200 °C for 6 hours and then

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rotavaped. The reaction completion was monitored by TLC. After completion of the reaction, the product (IV) was washed with water and then dried to give 89.6% yield. 4.3.

Synthesis of the library of novel bis-indolylmethanes based Schiff base

derivatives (1-32)

The synthesis of novel bis-indolylmethanes based schiff base derivatives was accomplished by reacting 2 equimolar (0.25 mmol) of different aldehydes with compound (IV) (0.1 mmol) in 10

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mL methanol.

4.3.1. 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-hydroxybenzylidene)-1H-indole-5carbohydrazide) (1)

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Yield: 84%. 1H NMR (500 MHz, DMSO-d6): δ 11.70 (s; 2H), 10.81 (s; 2H), 9.71 (s; 2H), 8.40 (s; 4H), 7.92 (d; J = 8.0 Hz, 2H), 7.69 (dd, J = 8.5, 1.5 Hz; 4H), 7.67 (d, J = 8.4 Hz; 2H), 7.16 (dd, J = 8.2, 1.1 Hz; 2H), 7.08 (dd, J = 8.6, 1.2 Hz; 2H), 6.88 (dd, J = 8.0, 1.3 Hz; 4H), 6.68 (s, 2H), 5.50 (s, 1H), 2.22 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 160.6, 160.6, 146.5, 146.5, 139.8, 139.8, 135.0, 135.3, 130.5, 130.5, 130.5, 130.5, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.6, 126.6, 126.2, 126.2,123.2, 123.2, 119.3, 119.3, 116.1, 116.1,116.1, 116.1,112.3, 112.3, 111.3, 111.3, 111.2, 111.2, 54.5, 21.2. HREI-MS: m/z Calcd for C40H32N6O4 [M]+660.2485; Found: 660.2474. 4.3.2. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dihydroxybenzylidene)-1H-indole-5carbohydrazide) (2) Yield: 81%. 1H NMR (500 MHz, DMSO-d6): δ 11.71 (s; 2H), 11.69 (s; 2H), 10.83 (s; 2H), 10.13 (s; 2H), 8.83 (s; 2H), 8.39 (s; 2H), 7.94 (d, J= 8.0 Hz, 2H), 7.71 (d, J = 8.4 Hz; 2H), 7.57 (d, J = 8.3 Hz; 2H), 7.54 (s, 2H), 6.36 (d, J = 8.2 Hz; 2H),7.17 (dd, J = 8.0, 1.2 Hz; 2H), 7.09 (dd, J = 8.1, 1.1 Hz; 2H), 6.67 (s, 2H), 5.46 (s, 1H), 2.24 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 13

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163.0, 163.0, 162.3, 162.3, 162.0, 162.0, 146.2, 146.2, 139.7, 139.7, 135.0, 135.3, 133.6, 133.6, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.6, 126.6, 123.2, 123.2, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 111.0, 111.0, 108.5, 108.5, 103.6, 103.6, 54.5, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found: 692.2374.

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4.3.3. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-nitrobenzylidene)-1H-indole-5carbohydrazide) (3)

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Yield: 82%. 1H NMR (500 MHz, DMSO-d6): δ 11.74 (s; 2H), 10.83 (s; 2H), 8.52 (s; 2H), 8.49 (s; 2H), 8.40 (s; 2H), 8.12 (d, J= 8.5 Hz, 4H), 7.95 (d, J = 8.7 Hz; 2H), 7.75 (dd, J = 8.0, 8.3 Hz; 2H), 7.63 (d, J = 8.6 Hz; 2H), 7.21 (dd, J = 8.4, 1.4 Hz; 2H), 7.11 (dd, J = 8.5, 1.5 Hz; 2H), 6.62 (s, 2H), 5.44 (s, 1H), 2.25 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 148.1, 146.5, 146.5, 139.8, 139.8, 135.2, 135.0, 134.4, 133.6, 132.3, 131.3, 129.5, 129.0, 129.0, 129.0, 128.5, 128.7, 128.7, 128.7, 128.6, 127.3, 127.3, 126.5, 126.5, 126.0, 123.2, 123.2, 121.5, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.5, 21.2. HREI-MS: m/z Calcd for C40H30N8O6 [M]+ 718.2288; Found: 718.2299. 4.3.4. 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methoxybenzylidene)-1H-indole-5carbohydrazide) (4)

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Yield: 80%. 1H NMR (500 MHz, DMSO-d6): δ 11.77 (s; 2H), 10.72 (s; 2H), 8.44 (s; 4H), 7.97 (d, J = 8.4 Hz; 2H), 7.73 (d, J = 8.3 Hz; 6H), 7.20 (dd, J = 8.6, 1.2 Hz; 2H), 7.17 (d, J = 8.3 Hz; 4H), 7.11 (dd, J = 8.8, 1.7 Hz; 2H), 6.75 (s, 2H), 5.55 (s, 1H), 3.85 (s; 6H), 2.27 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 162.7, 162.7, 146.6, 146.6, 139.8, 139.8, 135.2, 135.0, 130.0, 130.0, 130.0, 130.0, 128.6, 128.6, 128.6, 128.6, 127.4, 127.4, 126.5, 126.5, 126.2, 126.2, 123.1, 123.1, 119.1, 119.1, 114.2, 114.2, 114.2, 114.2,112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 55.7, 55.7, 54.4, 21.2. HREI-MS: m/z Calcd for C42H36N6O4 [M]+ 688.2798; Found: 688.2786. 4.3.5. 3,3'-(p-Tolylmethylene)bis(N'-((1E,2E)-3-phenylallylidene)-1H-indole-5carbohydrazide) (5)

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Yield: 86%. 1H NMR (500 MHz, DMSO-d6): δ 10.92 (s; 2H), 10.84 (s; 2H), 8.48 (s; 2H), 7.98 (d, J = 6.7 Hz; 2H), 7.92 (d, J = 8.7 Hz; 2H), 7.66 (d, J = 8.6 Hz; 2H), 7.58 (d, J = 8.2; 4H), 7.44 (m; 4H), 7.40 (m; 2H), 7.27 (d, J = 6.9; 2H), 7.21 (dd, J = 8.9, 1.8 Hz; 2H), 7.11 (dd, J = 8.4, 1.9 Hz; 2H), 6.88 (d, J = 7.1 Hz; 2H), 6.76 (s, 2H), 5.42 (s, 1H), 2.28 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 139.5, 139.5, 137.0, 137.0, 135.3, 135.1, 135.1, 135.0, 134.0, 134.0, 128.6, 128.6, 128.6, 128.6, 128.3, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.2, 127.7, 127.7, 127.3, 127.3, 126.4, 126.4, 126.1, 126.1, 123.3, 123.3, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,54.4, 21.2. HREI-MS: m/z Calcd for C44H36N6O2 [M]+ 680.2900; Found: 680.2899. 4.3.6. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxybenzylidene)-1H-indole-5carbohydrazide) (6) 14

ACCEPTED MANUSCRIPT

RI PT

Yield: 88%. 1H NMR (500 MHz, DMSO-d6): δ 11.83 (s; 2H), 10.90 (s; 2H), 9.55 (s; 2H), 8.47 (s; 2H), 8.41 (s; 2H), 7.88 (d, J = 8.1 Hz; 2H), 7.75 (d, J = 8.2 Hz; 2H), 7.35 (d, J = 8.5 Hz; 2H), 7.29 (d, J = 1.9Hz; 2H), 7.20 (dd, J = 8.2, 8.0Hz; 2H), 7.17 (dd, J = 8.3, 1.4 Hz; 2H), 7.12 (dd, J = 8.7, 1.7 Hz; 2H), 6.96 (d, J = 7.8 Hz; 2H), 6.76 (s, 2H), 5.53 (s, 1H), 2.29 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 158.3, 158.3, 146.5, 146.5, 139.5, 139.5, 138.6, 138.6, 135.2, 135.0, 130.0, 130.0, 128.5, 128.5, 128.5, 128.5, 127.4, 127.4, 126.6, 126.6, 123.3, 123.3, 121.7,121.7, 119.3, 119.3, 118.0, 118.0, 114.7, 114.7, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O4 [M]+ 660.2485; Found: 660.2494. 4.3.7. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,3-dihydroxybenzylidene)-1H-indole-5-

SC

carbohydrazide) (7)

M AN U

Yield: 78%. 1H NMR (500 MHz, DMSO-d6): δ 13.82 (s; 2H), 11.84 (s; 2H), 10.90 (s; 2H), 9.60 (s; 2H), 8.88 (s; 2H), 8.48 (s; 2H), 7.96 (d, J = 8.8 Hz; 2H), 7.66 (d, J = 8.7 Hz; 2H), 7.19 (dd, J = 8.9, 1.9 Hz; 2H), 7.13 (dd, J = 8.9, 1.8 Hz; 2H), 7.05 (d, J = 7.9 Hz; 2H), 6.78 (d, J = 7.5 Hz; 2H), 6.76 (dd, J = 7.5, 7.8 Hz; 2H), 6.69 (s, 2H), 5.58 (s, 1H), 2.29 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 151.6, 151.6, 146.3, 146.3, 146.1, 146.1, 139.7, 139.7, 135.2, 135.0, 128.5, 128.5, 128.5, 128.5, 127.3, 127.3, 126.4, 126.4, 124.5, 124.5, 123.1, 123.1, 122.5, 122.5, 119.6, 119.6, 119.3, 119.3, 119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found: 692.2380. 4.3.8. 3,3'-(p-Tolylmethylene)bis(N'-((E)-benzylidene)-1H-indole-5-carbohydrazide) (8)

EP

TE D

Yield: 79%. 1H NMR (500 MHz, DMSO-d6): δ 11.79 (s; 2H), 10.80 (s; 2H), 8.40 (s; 4H), 7.96 (m; 4H), 7.93 (d, J = 8.9 Hz; 2H), 7.64 (d, J = 8.4 Hz; 2H), 7.56 (m; 6H), 7.15 (dd, J = 8.6, 1.5 Hz; 2H), 7.06 (dd, J = 8.4, 1.6 Hz; 2H), 6.62 (s, 2H), 5.44 (s, 1H), 2.21 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7, 139.7, 139.7, 135.2, 135.0, 133.5, 133.5,131.1, 131.1, 129.0, 129.0,129.0, 129.0,128.7, 128.7, 128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 127.2, 127.2, 126.3, 126.3, 123.1, 123.1, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O2 [M]+ 628.2587; Found: 628.2579.

AC C

4.3.9. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,5-dihydroxybenzylidene)-1H-indole-5carbohydrazide) (9)

Yield: 83%. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s; 2H), 11.69 (s; 2H), 10.74 (s; 2H), 9.42 (s; 2H), 8.73 (s; 2H), 8.34 (s; 2H), 7.87 (d, J = 8.5 Hz; 2H), 7.63 (d, J = 8.8 Hz; 2H), 7.11 (dd, J = 8.2, 1.1 Hz; 2H), 7.06 (s; 2H), 7.02 (dd, J = 8.1, 1.3 Hz; 2H), 6.71 (d, J = 7.6 Hz; 2H), 6.63 (d, J = 7.8 Hz; 2H), 6.60 (s, 2H),5.41 (s, 1H), 2.22 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 153.5, 153.5, 151.0, 151.0, 146.2, 146.2, 139.7, 139.7, 135.3, 135.0, 128.8, 128.8, 128.8, 128.8, 127.3, 127.3, 126.5, 126.5,123.1, 123.1, 120.3, 120.3, 119.8, 119.8, 119.4, 119.4, 119.2, 119.2, 116.1, 116.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found: 692.2374.

15

ACCEPTED MANUSCRIPT

4.3.10. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3,4-dihydroxybenzylidene)-1H-indole-5carbohydrazide) (10)

SC

RI PT

Yield: 87%. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 10.82 (s; 2H), 9.46 (s; 4H), 8.44 (s; 2H), 8.34 (s; 2H), 7.87 (d, J = 8.8 Hz; 2H), 7.62 (d, J = 8.6 Hz; 2H), 7.21 (d, J = 1.4 Hz; 2H), 7.19 (dd, J = 7.6, 0.9 Hz; 2H), 7.15 (dd, J = 7.9, 1.3 Hz; 2H), 7.09 (dd, J = 8.2, 1.2 Hz; 2H),6.74 (d, J = 7.3 Hz; 2H), 6.60 (s, 2H), 5.40 (s, 1H), 2.25 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 149.4, 149.4,146.5, 146.5, 146.0, 146.0, 139.6, 139.6, 135.2,135.0, 131.2, 131.2, 128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.5, 126.5, 123.1, 123.1,123.0, 123.0, 119.2, 119.2, 117.1, 117.1, 116.1, 116.1, 112.0, 112.0,111.1, 111.1,111.0, 111.0,54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O6 [M]+ 692.2383; Found: 692.2375. 4.3.11. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,5-trihydroxybenzylidene)-1H-indole-5carbohydrazide) (11)

TE D

M AN U

Yield: 77%. 1H NMR (500 MHz, DMSO-d6): δ 11.70 (s; 2H), 11.69 (s; 2H), 10.82 (s; 2H), 9.51 (s; 4H), 8.77 (s; 2H), 8.36 (s; 2H), 7.87 (d, J = 8.3 Hz; 2H), 7.65 (d, J = 8.2 Hz; 2H), 7.12 (dd, J = 8.1, 1.5 Hz; 2H), 7.04 (dd, J = 8.3, 1.3 Hz; 2H), 6.90 (s, 2H), 6.64 (s, 2H), 6.14 (s, 2H), 5.47 (s, 1H), 2.18 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 155.0, 155.0, 152.3, 152.3,146.1, 146.1, 139.7, 139.7, 138.6, 138.6, 135.2, 135.0, 128.8, 128.8, 128.8, 128.8,127.2, 127.2, 126.3, 126.3, 123.3, 123.3,119.1, 119.1, 117.4, 117.4, 112.4, 112.4, 112.0, 112.0,111.1, 111.1,111.0, 111.0,105.0, 105.0,54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O8 [M]+ 724.2282; Found: 724.2273. 4.3.12. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4,6-trihydroxybenzylidene)-1H-indole-5carbohydrazide) (12)

AC C

EP

Yield: 81%. 1H NMR (500 MHz, DMSO-d6): δ 11.64 (s; 2H), 10.75 (s; 2H), 10.37 (s; 2H), 10.24 (s; 4H), 8.35 (s; 2H), 8.33 (s; 2H), 7.85 (d, J = 8.8 Hz; 2H), 7.61 (d, J = 8.5 Hz; 2H), 7.11 (dd, J = 8.2, 1.6 Hz; 2H), 7.07 (s; 4H),7.02 (dd, J = 8.4, 1.5 Hz; 2H), 6.67 (s, 2H), 5.42 (s, 1H), 2.17 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.8, 163.8, 163.8, 163.8,163.4, 163.4, 163.0, 163.0, 143.1, 143.1,139.6, 139.6, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 126.4, 126.4, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0,106.1, 106.1, 96.1, 96.1, 96.1, 96.1, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O8 [M]+ 724.2282; Found: 724.2274. 4.3.13. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-4-methoxybenzylidene)-1H-indole5-carbohydrazide) (13)

Yield: 85%. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s; 2H), 10.81 (s; 2H),10.23 (s; 2H), 8.83 (s; 2H), 8.41 (s; 2H), 7.94 (d, J = 8.3 Hz; 2H), 7.79 (d, J = 8.5 Hz; 2H), 7.69 (d, J = 8.8 Hz; 2H), 7.18 (dd, J = 8.4, 1.7 Hz; 2H), 7.07 (dd, J = 8.1, 1.8 Hz; 2H), 6.72 (s, 2H), 6.57 (d, J = 7.8 Hz; 2H), 6.50 (s, 2H), 5.50 (s, 1H), 3.87 (s; 6H), 2.23 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 164.2, 164.2, 163.0, 163.0, 162.0, 162.0, 146.1, 146.1, 139.7, 139.7, 135.2, 135.0, 133.3, 133.3, 16

ACCEPTED MANUSCRIPT

128.7, 128.7, 128.7, 128.7,127.3, 127.3, 126.6, 126.6, 123.1, 123.1,119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 110.7, 110.7, 107.1, 107.1, 103.3, 103.3, 55.5, 55.5, 54.4, 21.2. HREI-MS: m/z Calcd for C42H36N6O6 [M]+ 720.2696; Found:720.2687. 4.3.14. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxy-5-methoxybenzylidene)-1H-indole-

RI PT

5-carbohydrazide) (14)

M AN U

SC

Yield: 76%. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 11.35 (s; 2H), 10.81 (s; 2H), 8.75 (s; 2H), 8.41 (s; 2H), 7.91 (d, J = 8.6 Hz; 2H), 7.70 (d, J = 8.5 Hz; 2H), 7.20 (s; 2H), 7.10 (dd, J = 8.9, 1.3 Hz; 2H), 7.02 (dd, J = 8.6, 1.4 Hz; 2H), 6.80 (d, J = 8.0 Hz; 4H), 6.60 (s, 2H), 5.50 (s, 1H), 3.75 (s; 6H), 2.23 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 153.2, 153.2, 153.1, 153.1, 146.1, 146.1, 139.7, 139.7, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.6, 126.6, 123.1, 123.1, 119.2, 119.2, 119.3, 119.3, 118.1, 118.1, 117.1, 117.1, 113.3, 113.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 55.6, 55.6, 54.4, 21.2.HREI-MS: m/z Calcd for C42H36N6O6 [M]+ 720.2696; Found: 720.2686. 4.3.15. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-4-methoxybenzylidene)-1H-indole5-carbohydrazide) (15)

TE D

Yield: 84%. 1H NMR (500 MHz, DMSO-d6): δ 11.71 (s; 2H), 10.76 (s; 2H), 9.30 (s; 2H), 8.43 (s; 2H), 8.33 (s; 2H), 7.86 (d, J = 8.8 Hz; 2H), 7.62 (d, J = 8.7 Hz; 2H), 7.37 (s; 2H), 7.16 (dd, J = 8.4, 1.6 Hz; 2H), 7.12 (d, J = 7.7 Hz; 2H), 7.01 (d, J = 8.1 Hz; 4H), 6.63 (s, 2H), 5.44 (s, 1H), 3.88 (s; 6H), 2.26 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0,152.1, 152.1, 147.1, 147.1, 146.4, 146.4, 139.6, 139.6, 135.3, 135.0, 131.1, 131.1, 128.7, 128.7, 128.7, 128.7, 127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 122.7, 122.7, 119.2, 119.2, 115.7, 115.7, 112.1, 112.1, 112.0, 112.0, 111.1, 111.1,111.0, 111.0, 56.0, 56.0, 54.4, 21.2.HREI-MS: m/z Calcd for C42H36N6O6 [M]+ 720.2696; Found: 720.2687.

EP

4.3.16. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-hydroxy-2-iodo-4-methoxybenzylidene)-1Hindole-5-carbohydrazide) (16)

AC C

Yield: 75%. 1H NMR (500 MHz, DMSO-d6): δ 11.60 (s; 2H), 10.72 (s; 2H), 9.55 (s; 2H), 8.35 (s; 2H), 8.33 (s; 2H), 7.85 (d, J = 9.0 Hz; 2H), 7.65 (d, J = 8.9 Hz; 2H), 7.19 (dd, J = 8.6, 1.2 Hz; 2H), 7.15 (d, J = 7.9 Hz; 2H), 7.07 (dd, J = 8.3, 1.1 Hz; 2H), 6.81 (d, J = 7.8 Hz; 2H), 6.61 (s, 2H), 5.41 (s, 1H), 3.85 (s; 6H), 2.27 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 155.2, 155.2, 151.7, 151.7, 143.1, 143.1,139.7, 139.7, 135.2, 135.0, 133.6, 133.6, 128.8, 128.8, 128.8, 128.8, 127.3, 127.3, 126.4, 126.4, 124.3, 124.3, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.1, 111.1, 111.0, 111.0, 86.1, 86.1, 56.0, 56.0, 54.4, 21.2. HREI-MS: m/z Calcd for C42H34I2N6O6 [M]+ 972.0629; Found: 972.0639. 4.3.17. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-hydroxybenzylidene)-1H-indole-5carbohydrazide) (17)

17

ACCEPTED MANUSCRIPT

RI PT

Yield: 79%. 1H NMR (500 MHz, DMSO-d6): δ 11.58 (s; 2H), 11.03 (s; 2H), 10.71 (s; 2H), 8.75 (s; 2H), 8.31 (s; 2H), 7.83 (d, J = 8.5 Hz; 2H), 7.60 (d, J = 8.7 Hz; 2H), 7.49 (d, J = 8.0 Hz; 2H), 7.30 (m, 2H), 7.13 (dd, J = 8.3, 1.4 Hz; 2H), 7.09 (dd, J = 8.0, 1.3 Hz; 2H), 6.97 (d, J = 7.9 Hz; 2H), 6.94 (m, 2H), 6.68 (s, 2H), 5.54 (s, 1H), 2.24 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 157.0, 157.0,146.1, 146.1,139.6, 139.6, 135.2, 135.0, 132.1, 132.1,128.7, 128.7, 128.7, 128.7,127.3, 127.3, 127.3, 127.3, 126.6, 126.6, 123.1, 123.1,121.3, 121.3, 119.2, 119.2, 118.2, 118.2, 117.4, 117.4, 112.0, 112.0, 111.1, 111.1,111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H32N6O4 [M]+ 660.2485; Found: 660.2476. 4.3.18. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-nitrobenzylidene)-1H-indole-5-

SC

carbohydrazide) (18)

M AN U

Yield: 78%. 1H NMR (500 MHz, DMSO-d6): δ 11.56 (s; 2H), 10.73 (s; 2H), 8.56 (s; 2H), 8.36 (s; 2H), 8.07 (d, J = 8.7 Hz; 2H), 7.94 (d, J = 8.9 Hz; 2H), 7.91 (d, J = 8.6 Hz; 2H), 7.68 (m, 2H), 7.65 (d, J = 8.2 Hz; 2H),7.58 (m, 2H), 7.12 (dd, J = 8.5, 1.7 Hz; 2H), 7.04 (dd, J = 8.5, 1.5 Hz; 2H), 6.64 (s, 2H), 5.48 (s, 1H), 2.18 (s; 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 147.7, 147.7, 143.1, 143.1, 139.6, 139.6, 135.2, 135.0, 134.5, 134.5, 131.6, 131.6,130.0, 130.0, 128.7, 128.7, 128.7, 128.7, 128.2, 128.2, 127.4, 127.4, 126.6, 126.6, 124.2, 124.2, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.2, 21.2. HREI-MS: m/z Calcd for C40H30N8O6 [M]+ 718.2288; Found: 718.2275. 4.3.19. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-methylbenzylidene)-1H-indole-5-

TE D

carbohydrazide) (19)

AC C

EP

Yield: 82%. 1H NMR (500 MHz, DMSO-d6): δ 11.54 (s; 2H), 10.69 (s; 2H), 8.34 (s; 2H), 8.32 (s; 2H), 7.83 (d, J = 8.7 Hz; 2H), 7.73 (d, J = 8.1 Hz; 2H), 7.62 (d, J = 8.4 Hz; 2H), 7.25 (m, 6H), 7.09 (dd, J = 8.7, 1.4 Hz; 2H), 7.01 (dd, J = 8.3, 1.2 Hz; 2H), 6.58 (s, 2H), 5.41 (s, 1H), 2.45 (s, 6H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 143.1, 143.1,139.6, 139.6, 135.2, 135.2, 135.2, 135.0, 131.0, 131.0, 130.8, 130.8,129.1, 129.1, 128.8, 128.8, 128.8, 128.8,127.4, 127.4, 126.6, 126.6, 126.3, 126.3, 125.6, 125.6, 123.1, 123.1, 119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2, 18.8, 18.8. HREI-MS: m/z Calcd for C42H36N6O2 [M]+ 656.2900; Found: 656.2889. 4.3.20. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-methylbenzylidene)-1H-indole-5carbohydrazide) (20)

Yield: 82%. 1H NMR (500 MHz, DMSO-d6): δ 11.58 (s; 2H), 10.80 (s; 2H), 8.48 (s; 2H), 8.38 (s; 2H), 7.92 (d, J = 8.4 Hz; 2H), 7.75 (s, 2H), 7.67(d, J = 8.5 Hz; 4H), 7.46 (dd, J = 8.4, 8.1 Hz; 2H), 7.28 (d, J = 8.0 Hz; 4H), 7.14 (dd, J = 8.5, 1.1 Hz; 2H), 7.06 (dd, J = 8.5, 1.3 Hz; 2H), 6.66 (s, 2H), 5.49 (s, 1H), 2.43 (s, 6H), 2.20 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7,139.7, 139.7, 138.3, 138.3, 135.2, 135.0, 133.5, 133.5, 131.2, 131.2, 129.3, 129.3, 128.6, 128.6, 128.6, 128.6,128.6, 128.6, 127.4, 127.4, 126.4, 126.4, 126.0, 126.0, 123.1, 123.1,

18

ACCEPTED MANUSCRIPT

119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2, 21.2, 21.2. HREI-MS: m/z Calcd for C42H36N6O2 [M]+ 656.2900; Found: 656.2904. 4.3.21. 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-methylbenzylidene)-1H-indole-5carbohydrazide) (21)

SC

RI PT

Yield: 83%. 1H NMR (500 MHz, DMSO-d6): δ 11.60 (s; 2H), 10.79 (s; 2H), 8.39 (s; 4H), 7.92 (d, J = 8.8 Hz; 4H), 7.91 (d, J = 8.9 Hz; 2H), 7.68(d, J = 8.7 Hz; 2H),7.41 (d, J = 8.2 Hz; 4H), 7.13 (dd, J = 8.2, 1.3 Hz; 2H), 7.07 (dd, J = 8.3, 1.1 Hz; 2H), 6.67 (s, 2H), 5.51 (s, 1H), 2.44 (s, 6H), 2.14 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.7, 146.7,140.5, 140.5, 139.4, 139.4, 135.2, 135.0, 130.2, 130.2, 129.0, 129.0, 129.0, 129.0, 128.6, 128.6, 128.6, 128.6, 127.3, 127.3, 126.3, 126.3, 126.0, 126.0, 126.0, 126.0, 123.1, 123.1, 119.1, 119.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2, 21.2, 21.2. HREI-MS: m/z Calcd for C42H36N6O2 [M]+ 656.2900; Found: 656.2898.

carbohydrazide) (22)

M AN U

4.3.22. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-chlorobenzylidene)-1H-indole-5-

TE D

Yield: 81%. 1H NMR (500 MHz, DMSO-d6): δ 11.61 (s; 2H), 10.80 (s; 2H), 8.97 (s; 2H), 8.38 (s; 2H), 7.94 (d, J = 8.7 Hz; 2H), 7.76 (d, J = 8.3 Hz; 2H), 7.69 (d, J = 8.2 Hz; 2H), 7.52 (m, 4H), 7.40 (m, 2H), 7.19 (dd, J = 8.4, 1.5 Hz; 2H), 7.10 (dd, J = 8.6, 1.3 Hz; 2H), 6.71 (s, 2H), 5.52 (s, 1H), 2.16 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 138.5, 138.5, 139.6, 139.6, 135.2, 135.0, 134.4, 134.4, 133.6, 133.6, 132.1, 132.1,130.0, 130.0, 128.7, 128.7, 128.7, 128.7, 127.3, 127.3, 127.0, 127.0, 126.7, 126.7, 126.5, 126.5, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30Cl2N6O2 [M]+ 696.1807; Found: 696.1819. 4.3.23. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-chlorobenzylidene)-1H-indole-5-

EP

carbohydrazide) (23)

AC C

Yield: 81%. 1H NMR (500 MHz, DMSO-d6): δ 11.62 (s; 2H), 10.81 (s; 2H), 8.48 (s; 2H), 8.39 (s; 2H), 7.95 (d, J = 8.6 Hz; 2H), 7.77 (s, 2H), 7.70(d, J = 8.4 Hz; 2H), 7.66 (d, J = 8.1 Hz; 2H),7.56 (d, J = 8.3 Hz; 2H), 7.50 (dd, J = 8.7, 8.5 Hz; 2H), 7.20 (dd, J = 8.7, 1.8 Hz; 2H), 7.11 (dd, J = 8.4, 1.6 Hz; 2H), 6.72 (s, 2H), 5.52 (s, 1H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSOd6): δ 163.0, 163.0, 146.7, 146.7, 139.8, 139.8, 135.2, 135.0, 135.0, 135.0, 134.3, 134.3, 131.0, 131.0, 130.0, 130.0,128.7, 128.7, 128.7, 128.7,127.4, 127.4, 127.1, 127.1, 127.0, 127.0, 126.6, 126.6, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREIMS: m/z Calcd for C40H30Cl2N6O2 [M]+ 696.1807; Found: 696.1817. 4.3.24. 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-chlorobenzylidene)-1H-indole-5carbohydrazide) (24) Yield: 82%. 1H NMR (500 MHz, DMSO-d6): δ 11.63 (s; 2H), 10.86 (s; 2H), 8.42 (s; 4H), 7.98 (d, J = 8.8 Hz; 2H), 7.85 (d, J = 8.7 Hz; 4H), 7.70 (d, J = 8.6 Hz; 2H), 7.48 (d, J = 8.9 Hz; 4H), 19

ACCEPTED MANUSCRIPT

RI PT

7.21 (dd, J = 8.4, 1.5 Hz; 2H), 7.12 (dd, J = 8.6, 1.9 Hz; 2H), 6.76 (s, 2H), 5.56 (s, 1H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 146.6, 146.6,1397, 139.7, 1365, 136.5,135.2, 135.0, 131.7, 131.7, 130.5, 130.5, 130.5, 130.5, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.5, 126.5, 123.2, 123.2, 119.2, 119.2, 112.0, 112.0, 111.1, 111.1,111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30Cl2N6O2 [M]+ 696.1807; Found: 696.1815. 4.3.25. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2,4-dichlorobenzylidene)-1H-indole-5carbohydrazide) (25)

M AN U

SC

Yield: 80%. 1H NMR (500 MHz, DMSO-d6): δ 11.64 (s; 2H), 10.87 (s; 2H), 8.97 (s; 2H), 8.44 (s; 2H), 8.01 (d, J = 9.0 Hz; 2H), 7.99 (d, J = 8.7 Hz; 2H), 7.71 (s; 2H), 7.70 (d, J = 8.2 Hz; 2H), 7.53 (d, J = 9.1 Hz; 2H), 7.26 (dd, J = 8.6, 1.4 Hz; 2H), 7.13 (dd, J = 8.7, 1.8 Hz; 2H), 6.77 (s, 2H), 5.59 (s, 1H), 2.25 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 139.6, 139.6, 138.5, 138.5,135.2, 135.0, 132.5, 132.5, 131.2, 131.2, 129.2, 129.2, 129.1, 129.1, 128.7, 128.7, 128.7, 128.7, 128.0, 128.0, 127.4, 127.4, 127.1, 127.1, 126.6, 126.6, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H28Cl4N6O2 [M]+ 764.1028; Found: 764.1038. 4.3.26. 3,3'-(p-Tolylmethylene)bis(N'-((E)-2-fluorobenzylidene)-1H-indole-5carbohydrazide) (26)

EP

TE D

Yield: 80%. 1H NMR (500 MHz, DMSO-d6): δ 11.69 (s; 2H), 10.89 (s; 2H), 8.45 (s; 2H), 8.39 (s; 2H), 7.89 (d, J = 8.8 Hz; 2H), 7.80 (d, J = 8.6 Hz; 2H), 7.75 (d, J = 8.5 Hz; 2H), 7.53 (m, 2H), 7.40 (dd, J = 8.2, 1.1 Hz; 2H), 7.30 (m, 2H), 7.21 (dd, J = 8.3, 1.3 Hz; 2H), 7.14 (dd, J = 8.6, 1.6 Hz; 2H), 6.72 (s, 2H), 5.60 (s, 1H), 2.20 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 159.5, 159.5, 143.1, 143.1, 139.6, 139.6, 135.2, 135.0, 132.3, 132.3, 130.7, 130.7, 128.7, 128.7, 128.7, 128.7, 127.2, 127.2, 126.4, 126.4, 124.2, 124.2, 123.1, 123.1, 119.3, 119.3, 118.0, 118.0, 115.5, 115.5, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30F2N6O2 [M]+ 664.2398; Found: 664.2407.

AC C

4.3.27. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-fluorobenzylidene)-1H-indole-5carbohydrazide) (27)

Yield: 79%. 1H NMR (500 MHz, DMSO-d6): δ 11.67 (s; 2H), 10.78 (s; 2H), 8.47 (s; 2H), 8.37 (s; 2H), 7.90 (d, J = 8.5 Hz; 2H), 7.80 (d, J = 8.2 Hz; 2H), 7.66 (d, J = 8.4 Hz; 2H), 7.63 (m, 2H), 7.53 (m, 2H), 7.44 (dd, J = 8.0, 1.2 Hz; 2H), 7.13 (dd, J = 8.5, 1.4 Hz; 2H), 7.05 (dd, J = 8.4, 1.5 Hz; 2H), 6.65 (s, 2H), 5.48 (s, 1H), 2.19 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 162.9, 162.9, 146.7, 146.7, 139.7, 139.7, 135.2, 135.2, 135.2, 135.0,130.3, 130.3, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.6, 126.6, 124.7, 124.7, 123.1, 123.1, 119.1, 119.1, 117.7, 117.7, 114.1, 114.1, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30F2N6O2 [M]+ 664.2398; Found: 664.2388.

20

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4.3.28. 3,3'-(p-Tolylmethylene)bis(N'-((E)-4-fluorobenzylidene)-1H-indole-5carbohydrazide) (28)

SC

RI PT

Yield: 88%. 1H NMR (500 MHz, DMSO-d6): δ 11.72 (s; 2H), 10.73 (s; 2H), 8.34 (s; 4H), 7.90 (d, J = 8.7 Hz; 2H), 7.77 (dd, J = 8.5, 1.9 Hz; 4H), 7.69 (d, J = 8.6 Hz; 2H), 7.30 (dd, J = 8.5, 8.2 Hz; 4H), 7.19 (dd, J = 8.2, 1.2 Hz; 2H), 7.09 (dd, J = 8.5, 1.4 Hz; 2H), 6.67 (s, 2H), 5.49 (s, 1H), 2.17 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 165.0, 165.0, 163.0, 163.0, 146.6, 146.6, 139.8, 139.8, 135.2, 135.0, 130.7, 130.7, 130.7, 130.7, 129.2, 129.2, 128.7, 128.7, 128.7, 128.7, 127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 119.3, 119.3, 115.5, 115.5,115.5, 115.5, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H30F2N6O2 [M]+ 664.2398; Found: 664.2387. 4.3.29. 3,3'-(p-Tolylmethylene)bis(N'-((E)-3-bromo-4-fluorobenzylidene)-1H-indole-5carbohydrazide) (29)

TE D

M AN U

Yield: 76%. 1H NMR (500 MHz, DMSO-d6): δ 11.73 (s; 2H), 10.71 (s; 2H), 8.45 (s; 2H), 8.36 (s; 2H), 7.89 (d, J = 8.8 Hz; 2H), 7.87 (m, 2H), 7.84 (d, J = 8.3 Hz; 2H), 7.64 (d, J = 8.7 Hz; 2H), 7.23 (dd, J = 8.7, 8.4 Hz; 2H), 7.15 (dd, J = 8.1, 1.6 Hz; 2H), 7.03 (dd, J = 8.3, 1.4 Hz; 2H), 6.62 (s, 2H), 5.47 (s, 1H), 2.16 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 167.6, 167.6, 163.0, 163.0, 146.6, 146.6, 139.7, 139.7, 135.2, 135.0, 134.0, 134.0, 131.4, 131.4,129.6, 129.6, 128.8, 128.8, 128.8, 128.8, 127.4, 127.4, 126.4, 126.4, 123.1, 123.1, 119.2, 119.2, 117.7, 117.7, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 110.0, 110.0, 54.4, 21.2. HREI-MS: m/z Calcd for C40H28Br2F2N6O2 [M]+ 820.0609; Found: 820.0620. 4.3.30. 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-3-ylmethylene)-1H-indole-5carbohydrazide) (30)

AC C

EP

Yield: 89%. 1H NMR (500 MHz, DMSO-d6): δ 10.89 (s; 2H), 10.77 (s; 2H), 9.07 (s; 2H), 8.72 (d, J = 8.6 Hz; 2H), 8.41 (d, J = 8.5 Hz; 2H), 8.36 (s; 2H), 8.33 (s; 2H), 7.84 (d, J = 8.5 Hz; 2H), 7.60 (d, J = 8.8 Hz; 2H), 7.43 (dd, J = 8.5, 8.7 Hz; 2H), 7.12 (dd, J = 8.0, 1.3 Hz; 2H), 7.02 (dd, J = 8.2, 1.5 Hz; 2H), 6.59 (s, 2H), 5.41 (s, 1H), 2.23 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 151.8, 151.8,149.1, 149.1,143.2, 143.2, 139.7, 139.7, 135.2, 135.0, 133.6, 133.6, 130.3, 130.3, 128.6, 128.6, 128.6, 128.6, 127.2, 127.2, 126.6, 126.6, 123.7, 123.7, 123.1, 123.1, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C38H30N8O2 [M]+ 630.2492; Found: 630.2481. 4.3.31. 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-4-ylmethylene)-1H-indole-5carbohydrazide) (31) Yield: 89%. 1H NMR (500 MHz, DMSO-d6): δ 10.86 (s; 2H), 10.74 (s; 2H), 8.61 (d, J = 8.9 Hz; 4H), 8.33 (s; 2H), 8.30 (s; 2H), 7.96 (d, J = 8.8 Hz; 4H), 7.93 (d, J = 8.7 Hz; 2H), 7.58 (d, J = 8.9 Hz; 2H), 7.20 (dd, J = 8.3, 1.6 Hz; 2H), 7.11 (dd, J = 8.4, 1.2 Hz; 2H), 6.72 (s, 2H), 5.56 (s, 1H), 2.27 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 149.1, 149.1, 149.1, 149.1,146.6, 21

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146.6,144.1, 144.1, 139.7, 139.7, 135.2, 135.0, 128.8, 128.8, 128.8, 128.8,127.4, 127.4, 126.6, 126.6, 123.1, 123.1, 120.3, 120.3, 120.3, 1203, 119.3, 119.3, 112.0, 112.0, 111.1, 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C38H30N8O2 [M]+ 630.2492; Found: 630.2484. 4.3.32. 3,3'-(p-Tolylmethylene)bis(N'-((E)-pyridin-2-ylmethylene)-1H-indole-5-

RI PT

carbohydrazide) (32)

M AN U

SC

Yield: 87%. 1H NMR (500 MHz, DMSO-d6): δ 10.83 (s; 2H), 10.73 (s; 2H), 8.53 (d, J = 9.2 Hz; 2H), 8.36 (s; 2H), 7.90 (s; 2H), 7.87 (d, J = 8.6 Hz; 2H), 7.82 (d, J = 8.9 Hz; 2H), 7.79 (m, 2H), 7.69 (d, J = 8.8 Hz; 2H), 7.40 (m, 2H), 7.17 (dd, J = 8.5, 1.5 Hz; 2H), 7.07 (dd, J = 8.1, 1.5 Hz; 2H), 6.67 (s, 2H), 5.47 (s, 1H), 2.17 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 163.0, 163.0, 153.5, 153.5, 149.0, 149.0, 144.6, 144.6,139.7, 139.7, 136.0, 136.0, 135.2, 135.0, 128.7, 128.7, 128.7, 128.7, 127.3, 1273, 126.5, 126.5, 126.0, 126.0, 123.1, 123.1, 120.1, 120.1,119.2, 119.2, 112.0, 112.0, 111.1 111.1, 111.0, 111.0, 54.4, 21.2. HREI-MS: m/z Calcd for C38H30N8O2 [M]+ 630.2492; Found: 630.2482. 4.4. Molecular Docking

Molecular docking was carried out to study the interactions between the synthesized compounds and the active site of the enzyme using Auto dock Vina [37]. Keeping in view the previous molecular docking study importance [38] here in this study, receptor was treated as rigid while ligands flexible with its active rotatable bonds ranging from 9 to 13 keeping the amide bond non-

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rotatable. Three-dimensional X-ray crystal structure of Human-β-glucuronidase was downloaded from protein data bank [www.rcsb.org/pdb] (PDB ID 1bhg). Heteroatoms and chain B were removed from the dimer using Discovery Studio Visualizer [39] and was saved as pdb file. Prior to autodocking the pdb file was converted into pdbqt format containing gasteiger charges, polar

EP

hydrogen atoms and merged nonpolar hydrogen atoms using Autodock Tools [40]. Twodimensional structures of ligands were sketched and optimized using Chembi3D Ultra (Version;

AC C

14.0.0.117) and were saved in pdb format. After that gasteiger charges were computed for the entire ligand and ‘auto dock type’ was assigned to each atom using ADT. The search space was defined by constructing a grid box of size 20 × 20 × 20 Å centered at X = 81.733, Y= 82.591 and Z = 89.436. These parameters along with pdbqt files of receptor and ligand and with a number of modes (20) were included in the configurational file. The docking results were analyzed using +

Lig Plot [41], Poseview [42] and Discovery Studio Visualizer. Acknowledgements: The author (Dr. M.A.) is also grateful to HEC Pakistan for providing a research grant vide 201933/NRPU/R&D/HEC/12/5017. 22

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ACCEPTED MANUSCRIPT

Synthesized of bis-indolylmethanes analogs



Evaluated in vitro β-glucuronidase activity



Identified a new c of β-glucuronidase inhibitors



established Structure Activity Relationship



Performed Molecular docking

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