5.2
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
View/Download PDF

Translate this page into:

Original article
12 (
8
); 2721-2731
doi:
10.1016/j.arabjc.2015.05.015

Synthesis, characterization and cytotoxic investigations of novel bis(indole) analogues besides antimicrobial study

, , , , , , ,
a Department of Organic Chemistry, Foods, Drugs & Water, Andhra University, Visakhapatnam 530 003, India
b Department of Science & Humanities, Raghu Engineering College, Dakamarri, Visakhapatnam 531 162, India
c Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India
d Department of Marine Living Resources, Andhra University, Visakhapatnam 530 003, India

⁎Corresponding author. Tel.: +91 9849229804; fax: +91 891 2713813. murthyyln@gmail.com (Y.L.N. Murthy)

Received: , Accepted: ,
© The Author(s) 2015
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

Two series of novel bis(indole) analogues viz., N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) and N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f) were synthesized and characterized by spectral analysis. The target molecules were screened for their antimicrobial, anticancer activities and structure and activity relationship (SAR) was investigated. Compounds 7a, 7c and 8a were found to be active in antimicrobial screening. Anticancer screening reveals that Compound 7c was active against HeLa cell line with an IC50 of 43.1 μM and compound 7d was found to be interesting candidate with an IC50 of 26.0 and 30.2 μM against Colo-205 and Hep G2 cell lines respectively.

Keywords

Indole-3-acylhydrazides
Indole-3-carboxaldehyde
N-prenylated indole-3-carboxaldehyde
Bis(indole) analogues
Antimicrobial activity
Anticancer activity
1

1 Introduction

The indole skeleton is the interesting class of heterocyclic compounds which has been stimulated by both their unique chemical structure and the wide range of biological properties (Barraja et al., 2011). In particular, C-3-substituted indoles are important building blocks for the synthesis of many biologically active compounds which possess antimalarial (Agarwal et al., 2005), inhibitors of HIV-1 (Meanwell et al., 2009), antimicrobial (Lakshmi et al., 2010; Reddy et al., 2011), antioxidant (Lakshmi et al., 2010), anticancer (Lakshmi et al., 2010), cytotoxic (Gu et al., 1999), inhibitors of hepatitis C virus (Jin et al., 2014), anti-diabetic (Rajesh et al., 2007), and neuro protective (Mohareb et al., 2011) activities. On the other hand, N-1 and C-3-substituted indole derivatives have been found to play an important role in many biologically active compounds especially with anti-inflammatory (Hall et al., 2008; Singh et al., 2008), anticancer (Singh et al., 2008; Madadi et al., 2014), anti-nociceptive (Adam et al., 2010) and antipsychotic (Madadi et al., 2013) activities. Marine indole alkaloids have emerged as an important structural class exhibiting antiviral, antimicrobial and antitumor activity (Dembitsky et al., 2005; Bao et al., 2005; Oh et al., 2005, 2006). Among these biologically active scaffolds, bis(indole) alkaloids with a broad spectrum of biological activities are being discovered from marine invertebrates such as sponges, bryozoans, coelenterates and tunicates (Yang and Cordell, 1997; Tsuda et al., 2005; Shin et al., 1999; Ryan and Drennan, 2009; Diana et al., 2010). Dragmacidin D and topsentins and their analogues exhibited activities in vitro and in vivo against P388 murine and human tumor cells (Wright et al., 1992; Tsujii and Rinehart, 1988). Coscinamides A–C were isolated from Coscinoderma sps a marine sponge and exhibit cycloprotection against HIV in the NCI assay (Bokesch et al., 2000; Bacher et al., 2001). Asterriquinone B1 was isolated from Aspergillus terreus which is a bis(indole) derivative with 2,5-dimethoxy benzoquinone as a spacer and is effective in inhibiting the growth of several transplantable animal tumors in vivo (Liu et al., 1999). Nortopsentins A–C were isolated from a marine sponge Spongosorites ruetzleri having a 2,4-bis(3-indolyl)imidazole skeleton and exhibited in vitro cytotoxicity against P388 cell line (Alvarez and Salas, 1991; Sakem and Sun, 1991; Kawasaki et al., 1996). The structures of the naturally occurring bis(indole) molecules are presented in Fig. 1.

Naturally occurring bis(indole) molecules.
Figure 1 Naturally occurring bis(indole) molecules.

A great limitation in the use of the reservoir of marine organisms for therapy is that only very small amounts of the biologically active substances are isolated from the natural material. Due to the interesting biological activities, different analogues of the marine nortopsentins have been synthesized in which the imidazole moiety of the nortopsentin was replaced by five- or six-membered heterocyclic rings such as thiazole (Parrino et al., 2015; Carbone et al., 2015, 2013; Carbone et al., 2014), pyrazinone (Jiang and Gu, 2000), pyrimidines, pyrazines (Jiang et al., 2001), thiophenes (Diana et al., 2007a), pyridines (Xiong et al., 2001), pyrazoles (Diana et al., 2007b) and 1,2,4-thiadiazoles (Kumar et al., 2011) which were reported for their cytotoxicity against several cancer cell lines.

The acylhydrazones [(hydrazide–hydrazone (—CO—NH—N⚌CH—)] are classified as important functional group which exhibit significant role in anti-inflammatory (Todeschini et al., 1998; Radhwan et al., 2007; Gaston et al., 1996; Almasirad et al., 2005; Murineddu et al., 2001), antimalarial (Gemma et al., 2006), antimicrobial (Masunari and Tavares, 2007; Kuçukguzel et al., 2002), anticonvulsant (Dimmock et al., 2000; Ragavendran et al., 2007), antitumor (Bernhardt et al., 2009; Whitnall et al., 2006; Vicini et al., 2006; Terzioglu and Gursoy, 2003; Abadi et al., 2003), analgesic (Lima et al., 2000), antiplatelet (Silva et al., 2004) and antitubercular (Vavrikova et al., 2011a; Manvar et al., 2008; Vavrikova et al., 2011b) activities. In recent years, researchers have turned their interest by replacing the central heterocyclic spacer of the bis(indole) alkaloids with biologically active long chain linkers such as glyoxylamide derivatives (Gupta et al., 2007), and acylhydrazones and their cytotoxic activity was evaluated against human cancer cell lines (Kumar et al., 2012). Keeping the literature survey in view, we plan to synthesize the novel bis(indole) analogues by replacing the central spacer (heterocyclic and long chain) with alkane hydrazone spacer. All the synthesized compounds were screened for antimicrobial and anticancer activities.

2

2 Results and discussion

2.1

2.1 Chemistry

Synthesis of two series of novel bis(indole) analogues viz., N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) and N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f) has been accomplished by adopting known synthetic routes. Indole-3-carboxylic acids namely, indole-3-acetic (1a), indole-3-propionic (1b) and indole-3-butyric acid (1c) were selected as precursors, which on reflux with 4 N HCl in the presence of MeOH for 2–3 h to yield Indole-3-carboxylic methyl esters (2a–c), and these methyl esters on reaction with hydrazine hydrate in the presence of MeOH under refluxing conditions for 2–3 h yield three different acylhydrazides chain linkers (3a–c) (Scheme 1).

Synthesis of indole 3-substituted acylhydrazides (3a-c).
Scheme 1 Synthesis of indole 3-substituted acylhydrazides (3a-c).

Indoles (4) were formylated by Vilsmeier-Haack reaction to yield 3-Formyl Indoles (5), that were prenylated by the reaction with Prenyl bromide in the presence of NaH, DMF at 0 °C for 15 min to yield N-prenylated 3-formyl indoles (6) (Scheme 2).

Synthesis of 5-substituted-1H-indole-3-carbaldehyde (5a,b) and 5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6a,b).
Scheme 2 Synthesis of 5-substituted-1H-indole-3-carbaldehyde (5a,b) and 5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6a,b).

The series of N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) were achieved by the condensation of indole-3-substituted acylhydrazides (3a–c) with 5-substituted-1H-indole-3-carbaldehyde (5a,b) in the presence of EtOH and catalytic amount of Glac. acetic acid under refluxing conditions for 20–45 min. Similarly the condensation of indole-3-substituted acylhydrazides (3a–c) with 5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6a,b) in the presence of EtOH and 1–2 drops of Glac. acetic acid under refluxing conditions for 20–45 min yields novel N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f) (Scheme 3).

Synthesis of N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) and N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f).
Scheme 3 Synthesis of N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) and N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f).

All the synthesized compounds were well characterized by advanced spectroscopic techniques (1H, 13C, Mass, IR and elemental analysis).

2.2

2.2 Bio evaluation

2.2.1

2.2.1 Antimicrobial activity

The newly synthesized and well characterized compounds (7a–f) and (8a–f) were screened for in vitro antibacterial activity against Gram positive bacterium (Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa) and antifungal activity against Aspergillus flavus, Aspergillus niger, Candida albicans using agar well diffusion assay and zones of inhibition of the test compounds were expressed in mm.

2.2.1.1
2.2.1.1 Antibacterial activity

The antibacterial activity of the test compounds (7a–f) and (8a–f) was compared with standard “ciprofloxacin” and the results are presented in Table 1.

Table 1 Zones of inhibition (mm)a of compounds 7a–f and 8a–f against tested bacterial strains.
Entry Compound Bacterial strains
B. subtilis E. coli K. pneumonia P. auregenosa
1 7a 22 19 21 24
2 7b 19 20 19 16
3 7c 18 21 20 20
4 7d 20 20 18 20
5 7e 19 14 15 23
6 7f 21 14 19 16
7 8a 18 16 20 19
8 8b 18 16 19 21
9 8c 16 14 14 18
10 8d 19 20 19 18
11 8e 20 17 20 20
12 8f 20 14 20 16
13 Standarda 24 27 22 26

Ciprofloxacin was used as standard.

a 100 μg/mL of compound in each well.

As indicated in Table 1, most of the synthesized compounds generally showed potent antibacterial activity against all the tested bacterial strains. Compound 7a shows good antibacterial activity against B. subtilis, K. pneumonia and P. aeruginosa with zones of inhibition of 22, 21 and 24 mm respectively. In case of E. coli, compound 7c is more active with a zone of inhibition of 21 mm. On bacterial strains, compound 7a is potent against P. auregenosa with a zone of 24 mm. Considering the activity of compounds 8, with the exception of compound 8e, showed a decreased bactericidal activity suggesting that the presence of the hydrophobic prenyl group does not improve the biological activity.

2.2.1.2
2.2.1.2 Antifungal activity

The antifungal activity of tested compounds (7a–f) and (8a–f) was compared with ketoconazole (standard) and the results are tabulated in Table 2.

Table 2 Zones of inhibition (mm)a of compounds 7a–f and 8a–f against tested fungal strains.
Entry Compound A. flavus A. niger C. albicans
1 7a 16 18 14
2 7b 13 14 12
3 7c 12 16 13
4 7d 12 14 13
5 7e 15 12 14
6 7f 15 16 15
7 8a 14 13 16
8 8b 10 12 15
9 8c 12 10 9
10 8d 10 11 14
11 8e 9 10 12
12 8f 10 12 9
13 Standarda 21 23 19

Ketoconazole was used as standard.

a 100 μg/mL of compound in each well.

The results tabulated in Table 2 infer that compound 7a was found to be interesting molecule with good antifungal activity against A. flavus and A. niger with zone of inhibition of 16 and 18 mm respectively. Compound 8a is potent against C. albicans with a zone of 16 mm. Compounds 7a and 8a are acetohydrazide analogues, in which compound (7a) belongs to unprenylated series, whereas compound 8a comes under prenylated series.

2.2.2

2.2.2 In vitro anti cancer activity

The anti-proliferative/cytotoxic activities of the newly synthesized bis(indole) analogues (7a–f) and (8a–f) were evaluated against three different types of human cancer cell lines, viz., human colorectal cancer (Colo-205), human hepatocellular liver carcinoma (Hep G2), and human cervical cancer (HeLa) using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the method of Mossman (Mosmann, 1983). The cytotoxic potency of the compounds varied between the cell lines suggesting a structural property of these compounds as possible determinants of their biological activity. The cytotoxic investigations were evaluated at IICT, Hyderabad, in association with Dr. J.V. Rao, Scientist, Biology division.

It is evident from the results that all the target compounds have shown significant cytotoxic activity against all the tested cell lines (Table 3). However, 8e and 8f are found to be inactive against Colo-205 and Hep G2 cell lines at 300 μM concentration. Etoposide (a standard drug molecule) was used as a positive control in these assays and the IC50 values were recorded for Colo-205, Hep G2, and HeLa as 0.45, 4.75 and 23.3 μM respectively. Among the derivatives, about 45–55% of the test compounds have shown effective inhibition of growth in all the cell lines at less than 100 μM concentration.

Table 3 In vitro cytotoxicity of bis (indole) derivatives (7a–f) and (8a–f) against Colo-205, Hep G2 and HeLa human cancer cells by MTT assay expressed in IC50 (μM).
Entry Compound aIC50 (μM)
Colo-205 Hep G2 HeLa
1 7a 58.1 247 232
2 7b 136 274 248
3 7c 35.6 34.7 43.1
4 7d 26.0 30.2 61.4
5 7e 34.9 37.5 101
6 7f 88.7 68.1 65.2
7 8a 42.1 34.3 64.3
8 8b 216 153 229
9 8c 177 103 157
10 8d 142 154 121
11 8e NA NA 187
12 8f NA NA 62.7
13 StandardA 0.45 4.75 23.3

NA indicates that the compound is inactive at 300 μM.

a IC50 is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control cultures in the absence of an inhibitor and was calculated using the respective regression analysis. The values represent the mean of three individual observations.
A Etoposide was used as positive control.

Compound 7c exhibited an excellent anti-proliferative activity against all the cell lines (IC50 values are less than 50 μM conc.) followed by 7d, 8a, 7e and 7f. Furthermore, results indicated based on the IC50 values, the Hep G2 cell lines appear to be more sensitive than Colo-205 and HeLa cell lines. Nevertheless, the order of relative sensitivity among the cell lines differs, when compared to the activity of etoposide. All the bis(indole) analogues (7a–f) and (8a–f) were found to be effective anti-proliferative agents against HeLa cells, with 2 to 10-fold less active than the IC50 value of etoposide. Similarly, the analogues 7c, 7d, 7e, 7f and 8a, have exhibited potent anti-proliferative activity against Hep G2 cell lines. Likewise, these analogues (excluding 7f) also showed moderate cytotoxicity against Colo-205 cell lines.

2.2.3

2.2.3 Structure Activity Relationship (SAR)

The compounds, 5-bromo substituted aceto and propanehydrazides (7d and 7e) were found to be active against all the three cell lines than the corresponding unsubstituted aceto and propanehydrazides (7a and 7b) which indicates that hydrophobic bromine increases the activity. But there is an exception from the above hypothesis that the presence of bromine on position 5 in butanehydrazide (7f) slightly decreases the cytotoxicity than its corresponding unsubstituted butanehydrazide (7c).

Introduction of prenyl group increases the activity of 8a against Colo-205, 8a and 8b against Hep-G2 and 8a, 8b and 8f against HeLa cell lines. In prenylated series unsubstituted aceto and propanehydrazides (8a and 8b) exhibited potent cytotoxicity than their corresponding 5-bromo substituted aceto and propanehydrazides (8d and 8e) which indicates that hydrophobic bromine decreases the activity. In contrast, bromine on 5th position in butanehydrazide (8f) increases the cytotoxicity than its corresponding 5-bromo substituted aceto and propanehydrazides (8d and 8e). The relative cytotoxicity of prenylated series (8a–f) against the three cell lines is in the order of HeLa > Hep-G2 > Colo-205.

In unprenylated series (7a–f), 5-bromo substituted aceto and propanehydrazides (7d and 7e) and unsubstituted butanehydrazide (7c) exhibited potent cytotoxicity. In contrast the prenyl moiety alters the activity that unsubstituted aceto and propanehydrazides (8d and 8e) and 5-bromo substituted butanehydrazide (8f) were the interesting candidates in prenylated series (8a–f).

3

3 Conclusion

Among the novel series N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) and N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f) compounds 7a (B. subtilis, K. pneumonia and P. aeruginosa) and 7c (E. coli) are potent for antibacterial activity and compounds 7a (Aspergillus sps) and 8a (C. albicans) exhibit good antifungal activity.

Anticancer screening reveals that compound (7c) was active against HeLa cell line with an IC50 of 43.1 μM and compound (7d) was found to be interesting candidate with an IC50 of 26.0 and 30.2 μM against Colo-205 and Hep G2 cell lines.

4

4 Experimental

4.1

4.1 General

All chemical reagents were obtained from Sigma Aldrich and were used without further purification. Melting points were determined in open capillaries and are uncorrected. Infrared (IR) spectra were recorded using FT-IR Bruker Alpha spectrometer, ESI-Mass spectra were recorded on Finnigan Matt Mass spectrometer and NMR (1H and 13C) spectra were recorded with a Bruker Ascend-400 and 200 MHz and Bruker Ascend-400 and Jeol JNM EX-90 spectrometer.

4.2

4.2 General procedure for preparation of indole-3-carboxylates (2a–c)

The indole-3-carboxylic acids (1a–c) (10 mmol) were esterified in a classical manner with methanol and 4 N HCl under reflux for 2–3 h. After completion, the reaction mixture was cooled and methanol was evaporated under reduced pressure. Then the residue was poured onto crushed ice and treated with 10% aq. NaHCO3 solution dropwise till the reaction mixture became slightly basic in nature. The mixture was extracted with EtOAc and the organic layer was dried over anhydrous Na2SO4 and filtered and the solvent was evaporated under reduced pressure to get crude indole-3-carboxylates (2a–c). Recrystallization of the crude products was carried in ethanol.

4.2.1

4.2.1 Methyl 2-(1H-indol-3-yl)acetate (2a)

Yield: 92%., Brown viscous liquid., 1H NMR (CDCl3, 400 MHz): δ 3.75 (s, 3H), 3.84 (s, 2H), 7.08 (s, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 8.22 (s, 1H)., 13C NMR (CDCl3, 22.4 MHz): δ 31.0, 51.9, 108.1, 111.1, 118.6, 119.5, 122.0, 123.2, 127.1, 136.1, 172.6. ESI-MS: m/z 212.06 [M+Na]+, IR (KBr, cm−1): 1221, 1732, 3335., Anal. Calcd. for C11H11NO2: C, 69.83, H, 5.86, N, 7.40%, Found: C, 69.82, H, 5.90, N, 7.39%.

4.2.2

4.2.2 Methyl 3-(1H-indol-3-yl)propanoate (2b)

Yield: 90%, Pale yellow solid, Mp: 50 °C, 1H NMR (CDCl3, 400 MHz): δ 2.42 (t, J = 7.6 Hz, 2H), 3.89 (t, J = 7.6 Hz, 2H), 3.69 (s, 3H), 7.01 (s, 1H), 7.14 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 8 Hz, 1H), 7.65 (d, J = 8 Hz, 1H), 7.99 (s, 1H). 13C NMR (CDCl3, 22.4 MHz): δ 20.1, 34.3, 50.9, 111.0, 113.2, 117.8, 118.1, 121.4, 121.5, 126.6, 136.1, 173.0. ESI-MS: m/z 204 [M+H]+. IR (KBr, cm−1): 1240, 1724, 3329. Anal. Calcd. for C12H13NO2: C, 70.92, H, 6.45, N, 6.89%, Found: C, 70.90, H, 6.47, N, 6.86%.

4.2.3

4.2.3 Methyl 4-(1H-indol-3-yl)butanoate (2c)

Yield: 93%., Pale cream fluffy flakes, Mp: 68 °C. 1H NMR (CDCl3, 400 MHz): δ 2.01–2.08 (m, 2H), 2.39 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 3.65 (s, 3H), 6.98 (s, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.94 (s, 1H)., 13C NMR (CDCl3, 22.4 MHz): δ 24.4, 25.2, 33.6, 51.4, 111.0, 115.4, 118.7, 119.1, 121.4, 121.8, 127.3, 136.3, 174.1., ESI-MS: m/z 240.09 [M+Na]+., IR (KBr, cm−1): 1248, 1714, 3336., Anal. Calcd. for C13H15NO2: C, 71.87, H, 6.96, N, 6.45%, Found: C, 71.86, H, 6.99, N, 6.44%.

4.3. General procedure for preparation of indole-3-carboxylic acid hydrazides (3a–c): (Rapolu et al., 2013)

4.4

4.4 General procedure for the synthesis of 5-substituted 1H-indole-3-carbaldehyde (5a,b): (James and Snyder, 1959; Choppara et al., 2019)

To a solution of substituted indoles (4a,b) (42.6 mmol) in dry DMF (187.4 mmol) in an ice-salt bath, POCl3 (47.1 mmol) is subsequently added with stirring over a period of 30 min. After completion of addition, raise the temperature to 40 °C and stir the syrup for 1.5 h at that temperature. At the end of the reaction (as indicated by TLC) 25 g crushed ice was added to the reaction mixture. The obtained solution is transferred into 250 mL RB flask and added with NaOH (470 mmol) dissolved in 50 mL water with constant stirring and the resultant suspension is heated rapidly to the boiling point and allowed to cool to room temperature, after which it is placed in refrigerator overnight. The precipitate was filtered off and washed thrice with 100 mL water, yielding 5-substituted 1H-indole-3-carbaldehyde (5a,b).

4.4.1

4.4.1 1H-indole-3-carbaldehyde (5a): (James and Snyder, 1959; Choppara et al., 2019)

Yield: 92%., Brownish yellow solid., Mp: 196–198 °C., 1H NMR (DMSO-d6, 400 MHz): δ 7.14 (t, J = 7.2 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 8 Hz, 1H), 7.52 (s, 1H), 7.62 (d, J = 8 Hz, 1H), 8.12 (s, 1H), 9.52 (s, 1H). 13C NMR (DMSO-d6, 22.4 MHz): δ 111.4, 118.0, 119.4, 120.5, 122.4, 127.7, 131.82, 137.2, 182.7. ESI-MS: m/z 146.20 [M+H]+. IR (KBr, cm−1): 1229, 1632, 3442. Anal. Calcd. for C9H7NO: C, 74.47, H, 4.86, N, 9.65%, Found: C, 74.44, H, 4.91, N, 9.64%.

4.4.2

4.4.2 5-bromo-1H-indole-3-carbaldehyde (5b): (Choppara et al., 2019)

Yield: 90%., Cream coloured solid., Mp: 192 °C. 1H NMR (DMSO-d6, 400 MHz): δ 7.34 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 8.25 (s, 1H), 8.32 (s, 1H), 9.94 (s, 1H). 13C NMR (DMSO-d6, 22.4 MHz): δ 113.0, 114.8, 117.3, 123.1, 125.6, 135.2, 136.7, 144.4, 183.9. ESI-MS: m/z 245.95 [M+Na]+. IR (KBr, cm−1): 1229, 1643, 3312. Anal. Calcd. for C9H6BrNO: C, 48.25, H, 2.70, N, 6.25%, Found: C, 48.22, H, 2.76, N, 6.24%.

4.5

4.5 General procedure for the synthesis of substituted 1-(3-methylbut-2-enyl)- 1H-indole-3-carbaldehyde (6a,b)

To a solution of substituted 1H-indole-3-carbaldehydes (5a,b) (2.20 mmol) in dry DMF (5 mL) was added NaH (2.64 mmol, 60% oil dispersion) and the resulting mixture was stirred for 10 min in an ice bath. 3,3-dimethylallyl bromide (2.20 mmol) was added and the resulting mixture stirred for 15 min at 0 °C. The mixture was diluted with EtOAc (20 mL) and washed five times with distilled water (50 mL). The organic layer was dried over anhydrous Na2SO4 and filtered and the solvent was removed under reduced pressure to get crude residue which was purified by column chromatography (silica gel 100–200 mesh) using 8:2 (Hexane: EtOAc) as eluents affording 6a,b.

4.5.1

4.5.1 1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6a)

Yield: 87%., Brownish yellow crystals., Mp: 79–81 °C., 1H NMR (CDCl3, 400 MHz): δ 1.85 (s, 3H), 1.86 (s, 3H), 4.74 (d, J = 7.2 Hz, 2H), 5.44 (t, J = 7.2 Hz, 1H), 7.28–7.41 (m, 4H) 7.76 (s, 1H), 9.99 (s, 1H)., 13C NMR (CDCl3, 22.4 MHz): δ 17.9, 25.4, 44.6, 110.0, 117.7, 117.9, 121.8, 122.7, 123.6, 125.5, 137.3, 137.5, 138.7, 184.3., ESI-MS: m/z 214.20 [M+H]+., IR (KBr, cm−1): 1224, 1640., Anal. Calcd. for C14H15NO: C, 78.84, H, 7.09, N, 6.57%, Found: C, 78.80, H, 7.16, N, 6.55%.

4.5.2

4.5.2 5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6b)

Yield: 89%., Pale pink solid., Mp: 95–98 °C., 1H NMR (CDCl3, 400 MHz): δ 1.85 (s, 3H), 1.86 (s, 3H), 4.71 (d, J = 7.2 Hz, 2H), 5.42 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 9.6 Hz, 1H), 7.43 (dd, J = 2.0, 8.8 Hz, 1H), 7.73 (s, 1H), 8.47 (d, J = 2.0 Hz, 1H), 9.96 (s, 1H)., 13C NMR (CDCl3, 22.4 MHz): δ 12.7, 20.1, 39.4, 106.0, 110.8, 111.7, 119.0, 121.1, 121.4, 130.3, 132.6, 132.8, 133.8, 178.6., ESI-MS: m/z 314.01 [M+Na]+., IR (KBr, cm−1): 1162, 1654., Anal. Calcd. for C14H14BrNO: C, 57.55, H, 4.83, N, 4.79%, Found: C, 57.53, H, 4.90, N, 4.77%.

4.6

4.6 General procedure for preparation of N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f)

A mixture of indole-3-carboxylic acid hydrazides (3) (1 mmol) and 5-substituted-1H-indole-3-carbaldehyde (5) in ethanol and glacial acetic acid (2 drops) was refluxed for 20–30 min. As the reaction progresses, N′-((5-substituted-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)alkanehydrazides (7a–f) separate out as a solid in the reaction mixture. After completion of the reaction, the solid product was collected by simple filtration. Then the solid was dried and purified by column chromatography (silica gel 100–200 mesh) using 6:4 (Hexane: EtOAc) as eluents affording pure compounds (7a–f).

4.6.1

4.6.1 N′-((1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)acetohydrazide (7a)

Yield: 87%., Brown solid., Mp: 152 °C., 1H NMR (DMSO-d6, 400 MHz): δ 2.87 (s, 2H), 7.02–7.83 (m, 10), 8.12 (s, 1H), 8.52 (s, 1H), 10.21 (s, 1H), 10.89 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 30.1, 108.1, 111.5, 114.3, 116.6, 116.9, 118.6, 119.7, 122.3, 123.1, 123.5, 127.5, 132.2, 132.5, 136.5, 137.5, 137.9, 140.6, 173.1. ESI-MS: m/z 317 [M+H]+., IR (KBr, cm−1): 1222, 1608, 1649, 3354, 3397, 3541., Anal. Calcd. for C19H16N4O: C, 72.13, H, 5.10, N, 17.71%, Found: C, 72.11, H, 5.14, N, 17.69%.

4.6.2

4.6.2 N′-((1H-indol-3-yl)methylene)-3-(1H-indol-3-yl)propanehydrazide (7b)

Yield: 84%., Pale yellow solid., Mp: 300 °C., 1H NMR (DMSO-d6, 200 MHz): δ 2.64 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H), 6.98–7.62 (m, 10H), 8.07 (s, 1H), 8.90 (s, 1H), 10.12 (s, 1H), 10.31 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 26.3, 39.5, 111.7, 113.2, 116.8, 118.1, 119.0, 119.5, 120.2, 122.4, 123.2, 123.8, 126.1, 127.8, 128.7, 131.8, 136.0, 137.2, 140.1, 178.5., ESI-MS: m/z 331 [M+H]+., IR (KBr, cm−1): 1241, 1610, 1650, 3392, 3410, 3572., Anal. Calcd. for C20H18N4O: C, 72.71, H, 5.49, N, 16.96%, Found: C, 72.70, H, 5.51, N, 16.95%.

4.6.3

4.6.3 N′-((1H-indol-3-yl)methylene)-4-(1H-indol-3-yl)butanehydrazide (7c)

Yield: 92%., Yellow solid., Mp: 178 °C., 1H NMR (DMSO-d6, 200 MHz): δ 2.01–2.08 (m, 2H), 2.25 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 6.93–7.73 (m, 10H), 8.12 (s, 1H), 8.28 (s, 1H), 10.76 (s, 1H), 10.87 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 25.4, 26.3, 32.9, 108.3, 110.6, 115.9, 117.1, 118.0, 118.7, 119.6, 120.5, 121.2, 121.8, 122.3, 124.0, 125.7, 136.2, 137.8, 138.9, 140.3, 174.2., ESI-MS: m/z 367.15 [M+Na]+., IR (KBr, cm−1): 1244, 1623, 1646, 3329, 3396, 3542., Anal. Calcd. for C21H20N4O: C, 73.23, H, 5.85, N, 16.27%, Found: C, 73.20, H, 5.87, N, 16.26%.

4.6.4

4.6.4 N′-((5-bromo-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)acetohydrazide (7d)

Yield: 91%., Yellow solid., Mp: 156 °C., 1H NMR (DMSO-d6, 400 MHz): δ 2.74 (s, 2H), 7.19–7.71 (m, 9H), 8.19 (s, 1H), 8.41 (s, 1H), 9.92 (s, 1H), 10.14 (s, 1H)., 13C NMR (DMSO-d6, 100 MHz): δ 29.7, 108.0, 110.9, 114.7, 116.9, 117.3, 118.9, 119.7, 122.4, 123.4, 123.9, 126.1, 132.1, 133.2, 136.8, 137.4, 138.0, 149.7, 174.3., ESI-MS: m/z 395 [M]+, 397 [M+2]+., IR (KBr, cm−1): 1229, 1621, 1657, 3382, 3410, 3527., Anal. Calcd. for C19H15BrN4O: C, 57.74, H, 3.83, N, 14.17%, Found: C, 57.72, H, 3.84, N, 14.15%.

4.6.5

4.6.5 N′-((5-bromo-1H-indol-3-yl)methylene)-3-(1H-indol-3-yl)propanehydrazide (7e)

Yield: 89%., Pale pink solid., Mp: 186 °C., 1H NMR (DMSO-d6, 400 MHz): δ 2.55 (t, J = 7.2 Hz, 2H), 3.01 (t, J = 8.0 Hz, 2H), 6.94–7.80 (m, 9H), 8.14 (s, 1H), 8.37 (s, 1H), 10.80 (s, 1H), 11.01 (s, 1H)., 13C NMR (DMSO-d6, 100 MHz): δ 21.3, 35.5, 111.7, 113.4, 114.3, 114.4, 118.6, 118.8, 122.4, 122.7, 124.5, 125.4, 126.4, 127.5, 131.6, 136.2, 136.7, 139.8, 143.0, 173.6. ESI-MS: m/z 409 [M]+, 411 [M+2]+., IR (KBr, cm−1): 1251, 1616, 1654, 3293, 3423, 3540., Anal. Calcd. for C20H17BrN4O: C, 58.69, H, 4.19, N, 13.69%, Found: C, 58.70, H, 4.17, N, 13.72%.

4.6.6

4.6.6 N′-((5-bromo-1H-indol-3-yl)methylene)-4-(1H-indol-3-yl)butanehydrazide (7f)

Yield: 90%., Greenish yellow solid., Mp: 218 °C., 1H NMR (DMSO-d6, 400 MHz): δ 2.06–2.13 (m, 2H), 2.17 (t, J = 7.6 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H), 6.89–7.82 (m, 9H), 8.28 (s, 1H), 8.36 (s, 1H), 10.54 (s, 1H), 10.91 (s, 1H)., 13C NMR (DMSO-d6, 100 MHz): δ 24.8, 27.1, 33.1, 108.6, 111.2, 115.8, 117.4, 118.1, 118.5, 119.2, 120.9, 121.1, 122.3, 122.9, 123.6, 125.5, 136.7, 137.3, 138.7, 140.1, 173.2., ESI-MS: m/z 437 [M+], 439 [M+2]+., IR (KBr, cm−1): 1250, 1631, 1657, 3381, 3408, 3496., Anal. Calcd. for C21H19BrN4O: C, 59.58, H, 4.52, N, 13.24%, Found: C, 59.60, H, 4.49, N, 13.25%.

4.7

4.7 General procedure for preparation of N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-n-(1H-indol-3-yl)acetohydrazide (8a–f)

A mixture of indole-3-carboxylic acid hydrazides (3) (1 mmol) and 5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (6) (1 mmol) in ethanol and glacial acetic acid was refluxed for 20–45 min. As the reaction progresses, N′-((5-substituted-1-(3-methylbut-2-enyl)-1H-indol-3-yl) methylene)-n-(1H-indol-3-yl) acetohydrazide (8a–f) separates out as a solid in the reaction mixture. After completion of the reaction, the solid product was collected by simple filtration. The product was purified by column chromatography (silica gel 100–200 mesh) using 6:4 (Hexane: EtOAc) as eluents affording pure products (8a–f).

4.7.1

4.7.1 2-(1H-indol-3-yl)-N′-((1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)acetohydrazide (8a)

Yield: 95%., Yellow solid., Mp: 180 °C. 1H NMR (DMSO-d6, 200 MHz): δ 1.58 (s, 3H), 1.79 (s, 3H), 3.32 (s, 2H), 4.52 (d, J = 7.2 Hz, 2H), 5.31 (t, J = 7.2 Hz, 1H), 6.96–8.14 (m, 10H), 8.34 (s, 1H), 8.97 (s, 1H), 10.21 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 17.9, 25.8, 31.3, 45.8, 108.5, 111.4, 111.6, 113.0, 116.2, 118.6, 118.8, 119.9, 120.8, 121.1, 122.1, 122.9, 124.7, 124.9, 127.8, 132.3, 132.1, 136.1, 140.5, 174.4., ESI-MS: m/z 385 [M+H]+., IR (KBr, cm−1): 1224, 1615, 1651, 3272, 3452., Anal. Calcd. for C24H24N4O: C, 74.97, H, 6.29, N, 14.57%, Found: C, 74.94, H, 6.33, N, 14.55%.

4.7.2

4.7.2 3-(1H-indol-3-yl)-N′-((1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene) propane hydrazide (8b)

Yield: 87%., Cream coloured solid., Mp: 184 °C., 1H NMR (DMSO-d6, 200 MHz): δ 1.82 (s, 3H), 1.85 (s, 3H), 2.31 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H), 4.71 (d, J = 7.2 Hz, 2H), 5.39 (t, J = 7.2 Hz, 1H), 6.92–7.83 (m, 10H), 8.09 (s, 1H), 8.80 (s, 1H), 10.50 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 17.5, 24.7, 26.1, 37.8, 45.4, 110.2, 111.0, 115.2, 117.4, 118.0, 118.9, 119.5, 120.5, 121.8, 122.1, 122.6, 124.1, 125.7, 127.4, 136.7, 137.2, 137.6, 139.0, 140.1, 175.7. ESI-MS: m/z 399 [M+H]+., IR (KBr, cm−1): 1071, 1620, 1671, 3321, 3467., Anal. Calcd. for C25H26N4O: C, 75.35, H, 6.58, N, 14.06%, Found: C, 75.33, H, 6.60, N, 14.05%.

4.7.3

4.7.3 4-(1H-indol-3-yl)-N′-((1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)butane hydrazide (8c)

Yield: 87%., Cream coloured solid., Mp: 172–174 °C. 1H NMR (DMSO-d6, 200 MHz): δ 1.79 (s, 3H), 1.81 (s, 3H), 2.04–2.10 (m, 2H), 2.27 (t, J = 7.6 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H), 4.63 (d, J = 7.2 Hz, 2H), 5.39 (t, J = 7.2 Hz, 1H), 6.97–7.85 (m, 10H), 8.20 (s, 1H), 8.36 (s, 1H), 10.81 (s, 1H)., 13C NMR (DMSO-d6, 22.4 MHz): δ 17.9, 24.6, 25.7, 26.1, 33.8, 45.1, 110.1, 110.8, 115.5, 117.6, 117.9, 118.7, 119.2, 120.7, 121.7, 121.9, 122.4, 123.7, 125.3, 127.2, 136.6, 137.2, 137.4, 138.9, 140.1, 175.1., ESI-MS: m/z 451.18 [M+K]+., IR (KBr, cm−1): 1231, 1624, 1669, 3331, 3472., Anal. Calcd. for C26H28N4O: C, 75.70, H, 6.84, N, 13.58%, Found: C, 75.69, H, 6.85, N, 13.57%.

4.7.4

4.7.4 N′-((5-bromo-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)acetohydrazide (8d)

Yield: 89%., White solid., Mp: 178 °C., 1H NMR (DMSO-d6, 400 MHz): δ 1.52 (s, 3H), 1.71 (s, 3H), 3.29 (s, 2H), 4.52 (d, J = 7.2 Hz, 2H), 5.28 (t, J = 7.2 Hz, 1H), 7.02–8.17 (m, 9H), 8.41 (s, 1H), 8.99 (s, 1H), 10.35 (s, 1H)., 13C NMR (DMSO-d6, 100 MHz): δ 16.3, 24.2, 31.6, 44.9, 108.1, 111.2, 111.7, 113.2, 116.6, 118.7, 119.2, 119.5, 120.8, 121.3, 122.5, 123.6, 124.8, 125.8, 127.6, 132.6, 132.2, 135.0, 140.7, 173.2., ESI-MS: m/z 463 [M]+, 465 [M+2]+., IR (KBr, cm−1): 1212, 1624, 1658, 3325, 3487., Anal. Calcd. for C24H23BrN4O: C, 62.21, H, 5.00, N, 12.09%, Found: C, 62.20, H, 5.02, N, 12.06%.

4.7.5

4.7.5 N′-((5-bromo-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-3-(1H-indol-3-yl)propanehydrazide (8e)

Yield: 94%., Cream coloured solid., Mp: 194 °C., 1H NMR (DMSO-d6, 200 MHz): δ 1.57 (s, 3H), 1.76 (s, 3H), 2.34 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 4.67 (d, J = 7.2 Hz, 2H), 5.43 (t, J = 7.2 Hz, 1H), 6.99–7.91 (m, 9H), 8.27 (s, 1H), 8.71 (s, 1H), 10.42 (s, 1H)., 13C NMR (DMSO-d6, 50 MHz): δ 16.9, 24.4, 27.1, 37.4, 45.9, 110.3, 111.5, 115.1, 117.9, 118.3, 119.3, 119.8, 120.3, 121.7, 122.3, 122.8, 124.8, 125.6, 127.2, 136.3, 137.1, 137.8, 139.3, 140.3, 176.2., ESI-MS: m/z 477 [M]+, 479 [M+2]+., IR (KBr, cm−1): 1190, 1628, 1671, 3401, 3563., Anal. Calcd. for C25H25BrN4O: C, 62.90, H, 5.28, N, 11.74%, Found: C, 62.88, H, 5.29, N, 11.73%.

4.7.6

4.7.6 N′-((5-bromo-1-(3-methylbut-2-enyl)-1H-indol-3-yl)methylene)-4-(1H-indol-3-yl)butanehydrazide (8f)

Yield: 90%., White solid., Mp: 182 °C., 1H NMR (DMSO-d6, 400 MHz): δ 1.65 (s, 3H), 1.74 (s, 3H), 2.02–2.11 (m, 2H), 2.26 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 4.51 (d, J = 7.6 Hz, 2H), 5.31 (t, J = 7.6 Hz, 1H), 7.12–7.85 (m, 9H), 8.13 (s, 1H), 8.28 (s, 1H), 10.72 (s, 1H)., 13C NMR (DMSO-d6, 100 MHz): δ 16.8, 25.2, 25.9, 27.4, 33.9, 45.3, 110.9, 111.4, 116.2, 117.7, 118.1, 118.8, 119.5, 120.8, 121.9, 122.4, 122.9, 123.8, 125.2, 127.9, 136.9, 137.5, 137.9, 138.7, 140.5, 174.7., ESI-MS: m/z 514 [M+Na]+, 512 [M+Na+2]+., IR (KBr, cm−1): 1239, 1619, 1674, 3291, 3463., Anal. Calcd. for C26H27BrN4O: C, 63.55, H, 5.54, N, 11.40%, Found: C, 63.57, H, 5.57, N, 11.39%.

4.8

4.8 Antibacterial assay

100 mL sterile conical flask of nutrient broth was inoculated with the test organisms and incubated at 37 °C overnight. By using a sterile pipette, 0.6 mL of the broth culture of each test organism was added to 60 mL of molten agar, mixed well and maintained at 45 °C. Sterile agar test plates of each test organism were prepared by pouring inoculated medium with uniform thickness. The agar was allowed to set and harden and wells of 4 mm diameter were cut at equidistant using a sterile cork borer. Agar plugs were removed. 100 μg/mL of test solutions (7a–f) and (8a–f) were prepared in DMSO and were introduced into the wells using micropipette. The plates were kept at room temperature for 2 h for better diffusion of solution into the medium. The plates were incubated for 24 h at 37 °C. After incubation the diameter of inhibitory zones formed around each well was measured in millimetre (mm) using antibiotic zone scale. The assay was carried out in duplicate. DMSO was used as control and the antibacterial activity of the test compounds was compared with standard “ciprofloxin”.

4.9

4.9 Antifungal assay

Sterile molten potato dextrose agar (PDA) medium was inoculated with 50 μL of fungal spore suspension aseptically and maintained at 45 °C temperature. The inoculated medium was mixed well and poured immediately in sterilized petri plates. Then five wells of 6 mm diameter were punched using sterile borer and filled with 100 μg/mL of test compounds (7a–f) and (8a–f) as well as sterile DMSO 100% as negative control. Plates were incubated for 24 h at 37 °C. Antifungal activity was determined by measuring the zone of inhibition. The zones produced by the test compounds were compared with the ketoconazole (standard).

4.10

4.10 Anticancer activity

4.10.1

4.10.1 Cell lines and cell culture

The cell lines are (Colo 205) Human colon cancer cell line, Hep G2, hepatocellular carcinoma cell line which was derived from the liver tissue of a 15-year-old Caucasian American male, and HeLa (Hela or HeLa cell) the oldest and most commonly used human cell line (Rahbari et al., 2009). The cell line was derived from cervical cancer cells taken on February 8, 1951 (Scherer et al., 1953) from Henrietta Lacks, a patient who eventually died of cancer on October 4, 1951. George Gey was able to isolate one specific cell, multiply it, and start a cell line. Gey named the sample HeLa, after the initial letters of Henrietta Lack’s name. Colo 205 (Human colon cancer cell line), Hep G2 (Human liver carcinoma cell line) and HeLa (Cervical cancer cell line) cell lines were obtained from the National Centre for Cellular Sciences (NCCS), Pune, India. Cells were cultured in RPMI-1640 media, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM NaHCO3, 2 mM -glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. All cell lines were maintained in culture at 37 °C in an atmosphere of 5% CO2.

4.10.2

4.10.2 Test concentrations

Initially, stock solutions of each test substance were prepared in 100% Dimethyl Sulfoxide (DMSO, Sigma Aldrich) with a final concentration of 8 mg/mL. Exactly 50 μL of stock was diluted to 1 mL in culture medium to obtain experimental stock concentration of 400 μg/mL. This solution was further serially diluted with media to generate a dilution series of 10 μg to 200 μg/mL. Precisely, 100 μL of each test concentration was added to 100 μL of cell suspension (total assay volume of 200 μL, and efficacy of the derivatives was evaluated with three different set of experiments) and incubated for 24 h at 37 °C in 5% CO2.

4.10.3

4.10.3 Cytotoxicity

The cytotoxicity of novel bis(indole) analogues (7a–f) and (8a–f) was screened on human colorectal cancer (Colo-205), human hepatocellular liver carcinoma (Hep G2), and human cervical cancer (HeLa) cell lines using etoposide as positive control by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the method of Mosmann (1983). Briefly, the cells (2 × 104) were seeded in each well containing 100 μL of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO2, exactly 100 μL of different test concentrations (10 μg–200 μg/mL) was added to the cell suspension, which is equivalent to 2–40 μg per 200 μL of assay volume. The viability of cells was assessed after 24 h, by adding 10 μL of MTT (5 mg/mL) per well and incubated at 37 °C for additional three hours. The medium was discarded and the formazan blue, which formed in the cells, was dissolved in 100 μL of DMSO. The intensity of colour formation was measured at 570 nm in a spectrophotometer (Spectra MAX Plus, Molecular Devices, supported by SOFTmax PRO-5.4). The percent inhibition of cell viability was determined with reference to the control values (without test compound). The data were subjected to linear regression analysis and the regression lines were plotted for the best straight-line fit. The IC50 (inhibition of cell viability) concentrations were calculated using the respective regression equation and expressed in μM.

Acknowledgement

We are grateful to DRDO, New Delhi, for providing financial assistance through a project ERIP/ER/1003916/M/01/1354 and UGC, New Delhi, for the award of UGC-BSR JRF to the author P.C. The authors BM and JVR are grateful to the Director, Indian Institute of Chemical Technology, Hyderabad, India, for the encouragement.

References

  1. , , , . Synthesis of novel 1,3,4-trisubstituted pyrazole derivatives and their evaluation as antitumor and antiangiogenic agents. Chem. Pharm. Bull.. 2003;51:838-844.
    [Google Scholar]
  2. , , , , , , , , , , , , , , , , , , , , , . Design, synthesis, and structure–activity relationships of indole-3-carboxamides as novel water soluble cannabinoid CB1 receptor agonists. Med. Chem. Commun.. 2010;1:54-60.
    [Google Scholar]
  3. , , , , . Synthesis of substituted indole derivatives as a new class of antimalarial agents. Bioorg. Med. Chem. Lett.. 2005;15:3133-3136.
    [Google Scholar]
  4. , , , , , , , , . Synthesis and analgesic activity of N-arylhydrazone derivatives of mefenamic acid. J. Pharm. Pharm. Sci.. 2005;8:419-428.
    [Google Scholar]
  5. , , . Marine, nitrogen-containing heterocyclic natural products-structures and syntheses of compounds containing indole units. Heterocycles. 1991;32:1391-1429.
    [Google Scholar]
  6. , , , , , , , , . D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity. Cancer Res.. 2001;61:392-399.
    [Google Scholar]
  7. , , , , , , , , . Cytotoxic bisindole alkaloids from a marine sponge Spongosorites sp. J. Nat. Prod.. 2005;68:711-715.
    [Google Scholar]
  8. , , , , , , , , , , . Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents. Bioorg. Med. Chem.. 2011;19:2326-2341.
    [Google Scholar]
  9. , , , , , , . Iron chelators of the dipyridylketone thiosemicarbazone class: precomplexation and transmetalation effects on anticancer activity. J. Med. Chem.. 2009;52:407-415.
    [Google Scholar]
  10. , , , , . Coscinamides A, B and C, three new bis indole alkaloids from the marine sponge Coscinoderma sp. Tetrahedron Lett.. 2000;41:6305-6308.
    [Google Scholar]
  11. , , , , , , , , , , , . Synthesis and anti-proliferative activity of Thiazolyl-bis-pyrrolo[2,3-b]pyridines and Indolyl-thiazolyl-pyrrolo[2,3-c]pyridines, Nortopsentin analogues. Mar. Drugs. 2015;13:460-492.
    [Google Scholar]
  12. , , , , , , , , , , , , . Synthesis and anti-proliferative activity of substituted 3[2-(1H-indol-3-yl)-1,3-Thiazol-4-yl]-1H-Pyrrolo[3,2-b]Pyridines, marine alkaloid Nortopsentin analogues. Curr. Med. Chem.. 2014;21:1654-1666.
    [Google Scholar]
  13. , , , , , , , , , , , , , . Novel 1H-Pyrrolo[2,3-b]pyridine derivative Nortopsentin analogues: synthesis and antitumor activity in peritoneal mesothelioma experimental models. J. Med. Chem.. 2013;56:7060-7072.
    [Google Scholar]
  14. , , , , , , , , , . Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX inhibition activities. Arab. J. Chem.. 2019;12:2328-2335.
    [CrossRef] [Google Scholar]
  15. , , , . Novel antitumor agents: marine sponge alkaloids, their synthetic analogs and derivatives. Mini-Rev. Med. Chem.. 2005;5:319-336.
    [Google Scholar]
  16. , , , , , , . Synthesis and antitumor activity of 2,5-bis(3′-indolyl)-furans and 3,5-bis(3′-indolyl)-isoxazoles, nortopsentin analogues. Bioorg. Med. Chem.. 2010;18:4524-4529.
    [Google Scholar]
  17. , , , , , , , , , . Synthesis and antitumor properties of 2,5-bis(3′-indolyl)thiophenes: analogues of marine alkaloid nortopsentin. Bioorg. Med. Chem. Lett.. 2007;17:2342-2346.
    [Google Scholar]
  18. , , , , , , , . 3,5-Bis(3′-indolyl)pyrazoles, analogues of marine alkaloid nortopsentin: synthesis and antitumor properties. Bioorg. Med. Chem. Lett.. 2007;17:6134-6137.
    [Google Scholar]
  19. , , , . Anticonvulsant properties of various acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and unsaturated carbonyl compounds. Eur. J. Med. Chem.. 2000;35:241-248.
    [Google Scholar]
  20. , , , , , . Synthesis and analgesic properties of new 4-arylhydrazone 1-H pyrazole [3,4-b] pyridine derivatives. Pharm. Acta Helv.. 1996;71:213-219.
    [Google Scholar]
  21. , , , , , , , , . Synthesis of N1-arylidene-N2-quinolyl- and N2-acrydinylhydrazones as potent antimalarial agents active against CQ-resistant P. falciparum strains. Bioorg. Med. Chem. Lett.. 2006;16:5384-5388.
    [Google Scholar]
  22. , , , . Syntheses and biological activities of bis(3-indolyl)thiazoles, analogues of marine bis(indole)alkaloid nortopsentins. Bioorg. Med. Chem. Lett.. 1999;9:569-572.
    [Google Scholar]
  23. , , , , , , . Synthesis of marine alkaloid: 8,9-Dihydrocoscinamide B and its analogues as novel class of antileishmanial agents. Bioorg. Med. Chem. Lett.. 2007;17:4075-4079.
    [Google Scholar]
  24. , , , , , , , , , , , . Discovery of a novel indole series of EP1 receptor antagonists by scaffold hopping. Bioorg. Med. Chem. Lett.. 2008;18:2684-2690.
    [Google Scholar]
  25. , , . Indole-3-aldehyde. Org. Synth.. 1959;39:30-33.
    [Google Scholar]
  26. , , . Syntheses and cytotoxicity evaluation of bis(indolyl)thiazole, bis(indolyl)pyrazinone and bis(indolyl)pyrazine: analogues of cytotoxic marine bis(indole) alkaloid. Bioorg. Med. Chem.. 2000;8:363-371.
    [Google Scholar]
  27. , , , , . Synthesis and cytotoxicity evaluation of novel indolylpyrimidines and indolylpyrazines as potential antitumor agents. Bioorg. Med. Chem.. 2001;9:1149-1154.
    [Google Scholar]
  28. , , , , , , , , . Chemical genetics-based discovery of indole derivatives as HCV NS5B polymerase inhibitors. Eur. J. Med. Chem.. 2014;75:413-425.
    [Google Scholar]
  29. , , , . Total synthesis of nortopsentins A-D, marine alkaloids. Chem. Pharm. Bull.. 1996;44:1831-1839.
    [Google Scholar]
  30. , , , , , . Synthesis and biological activities of diflunisal hydrazide–hydrazones. Eur. J. Med. Chem.. 2002;38:1005-1013.
    [Google Scholar]
  31. , , , , . Synthesis and in-vitro anticancer activity of 3,5-bis(indolyl)-1,2,4-thiadiazoles. Bioorg. Med. Chem. Lett.. 2011;21:5897-5900.
    [Google Scholar]
  32. , , , , . Novel bis(indolyl)hydrazide–hydrazones as potent cytotoxic agents. Bioorg. Med. Chem. Lett.. 2012;22:212-215.
    [Google Scholar]
  33. , , , , . InCl3 mediated one-pot multicomponent synthesis, antimicrobial, antioxidant and anticancer evaluation of 3-pyranyl indole derivatives. Bioorg. Med. Chem. Lett.. 2010;20:5054-5061.
    [Google Scholar]
  34. , , , , , , , . Synthesis and analgesic activity of novel N-acylarylhydrazones and isosters, derived from natural safrole. Eur. J. Med. Chem.. 2000;35:187-203.
    [Google Scholar]
  35. , , , . Total synthesis of asterriquinone B1. Tetrahedron Lett.. 1999;40:5119-5122.
    [Google Scholar]
  36. , , , , , , . Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors. Bioorg. Med. Chem. Lett.. 2013;23:2019-2021.
    [Google Scholar]
  37. , , , , . Synthesis and anti-proliferative activity of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs against human tumor cell lines. Bioorg. Med. Chem. Lett.. 2014;24:601-603.
    [Google Scholar]
  38. , , , , , , , , , . Synthesis, antitubercular activity and 3D-QSAR study of coumarin-4-acetic acid benzylidene hydrazides. Eur. J. Med. Chem.. 2008;43:2395-2403.
    [Google Scholar]
  39. , , . A new class of nifuroxazide analogues: Synthesis of 5-nitrothiophene derivatives with antimicrobial activity against multidrug-resistant Staphylococcus aureus. Bioorg. Med. Chem.. 2007;15:4229-4236.
    [Google Scholar]
  40. , , , , , , , , , , , , , , . Inhibitors of HIV-1 attachment. Part 3: A preliminary survey of the effect of structural variation of the benzamide moiety on antiviral activity. Bioorg. Med. Chem. Lett.. 2009;19:5136-5139.
    [Google Scholar]
  41. , , , , , . Development of new indole-derived neuroprotective agents. Bioorg. Med. Chem.. 2011;19:2966-2974.
    [Google Scholar]
  42. , . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983;65:55-63.
    [Google Scholar]
  43. , , , , , , . Synthesis and analgesic-anti-inflammatoryactivities of novel acylarylhydrazones with a 5-phenyl-4-R-3-pyrrolyl-acyl moiety. Arch. Pharm.. 2001;334:393-398.
    [Google Scholar]
  44. , , , , , , , , , . Antimicrobial activity and cytotoxicity of bis (indole) alkaloids from the sponge Spongosorites sp. Biol. Pharm. Bull.. 2006;29:570-573.
    [Google Scholar]
  45. , , , , , , , , , . Bis(indole) alkaloids as sortase A inhibitors from the sponge Spongosorites sp. Bioorg. Med. Chem. Lett.. 2005;15:4927-4931.
    [Google Scholar]
  46. , , , , , , , , , , , , . 3-[4-(1H-Indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines, Nortopsentin analogues with anti-proliferative activity. Mar. Drugs. 2015;13:1901-1924.
    [Google Scholar]
  47. , , , , . Synthesis and biological evaluation of new 3-substituted indole derivatives as potential anti-inflammatory and analgesic agents. Bioorg. Med. Chem.. 2007;15:3832-3841.
    [Google Scholar]
  48. , , , , , , , . Design and synthesis of anticonvulsants from a combined phthalimide–GABA–anilide and hydrazone pharmacophore. Eur. J. Med. Chem.. 2007;42:146-151.
    [Google Scholar]
  49. , , , , , , . A novel L1 retrotransposon marker for HeLa cell line identification. Biotechniques. 2009;46:277-284.
    [Google Scholar]
  50. , , , , , , , , . Design, synthesis, and biological evaluation of substituted-N-(thieno[2,3-b]pyridin-3-yl)-guanidines, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidines, and N-(1H-indol-3-yl)-guanidines. Bioorg. Med. Chem.. 2007;15:3248-3265.
    [Google Scholar]
  51. , , , , , , , . Synthesis and biological screening of 5-(alkyl(1H-indol-3-yl))-2-(substituted)-1,3,4-oxadiazoles as anti-proliferative and anti-inflammatory agents. Eur. J. Med. Chem.. 2013;66:91-100.
    [Google Scholar]
  52. , , , , , , , . Iodine-catalyzed conjugate addition of indoles onto en-1,4-dione: a novel synthesis of 3-(1-(1H-indol-3-yl)-2-oxo-2-phenylethyl)indolin-2-ones as antibacterial and antifungal agents. Bioorg. Med. Chem. Lett.. 2011;21:6510-6514.
    [Google Scholar]
  53. , , . Divergent pathways in the biosynthesis of bisindole natural products. Chem. Biol.. 2009;16:351-364.
    [Google Scholar]
  54. , , . Nortopsentins A, B, and C. Cytotoxic and antifungal imidazolediylbis[indoles] from the sponge Spongosorites ruetzleri. J. Org. Chem.. 1991;56:4304-4307.
    [Google Scholar]
  55. , , , . Studies on the propagation in vitro of poliomyelitis viruses: iv. Viral multiplication in a stable strain of human malignant epithelial cells (strain hela) derived from an epidermoid carcinoma of the cervix. J. Exp. Med.. 1953;97:695-710.
    [Google Scholar]
  56. , , , , , . New Bis(Indole) alkaloids of the Topsentin class from the Sponge Spongosorites genitrix. J. Nat. Prod.. 1999;62:647-649.
    [Google Scholar]
  57. , , , , , , . New class of potent anti-nociceptive and antiplatelet 10H-phenothiazine-1-acylhydrazone derivatives. Bioorg. Med. Chem.. 2004;12:3149-3158.
    [Google Scholar]
  58. , , , , , . 1-Toluene-sulfonyl-3-[(3′-hydroxy-5′-substituted)-γ-butyrolactone]-indoles: Synthesis, COX-2 inhibition and anticancer activities. Bioorg. Med. Chem. Lett.. 2008;18:85-89.
    [Google Scholar]
  59. , , . Synthesis and anticancer evaluation of some new hydrazone derivatives of 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide. Eur. J. Med. Chem.. 2003;38:781-786.
    [Google Scholar]
  60. , , , , , . Synthesis and evaluation of analgesic, anti-inflammatory and antiplatelet properties of new 2-pyridylarylhydrazone derivatives. Eur. J. Med. Chem.. 1998;33:189-199.
    [Google Scholar]
  61. , , , , , . Dendridine A, a Bis-indole alkaloid from a marine sponge Dictyodendrilla Species. J. Nat. Prod.. 2005;68:1277-1278.
    [Google Scholar]
  62. , , . Topsentin, bromotopsentin, and dihydrodeoxybromotopsentin: antiviral and antitumor bis(indolyl)imidazoles from Caribbean deep-sea sponges of the family Halichondriidae. Structural and synthetic studies. J. Org. Chem.. 1988;53:5446-5453.
    [Google Scholar]
  63. , , , , , , , , , . New series of isoniazid hydrazones linked with electron-withdrawing substituents. Eur. J. Med. Chem.. 2011;46:5902-5909.
    [Google Scholar]
  64. , , , , , , , , . New fluorine-containing hydrazones active against MDR-tuberculosis. Eur. J. Med. Chem.. 2011;46:4937-4945.
    [Google Scholar]
  65. , , , , , , . Synthesis and antiproliferative activity of benzo[d]isothiazole hydrazones. Eur. J. Med. Chem.. 2006;41:624-632.
    [Google Scholar]
  66. , , , , . A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. Proc. Natl. Acad. Sci. USA. 2006;103:14901-14906.
    [Google Scholar]
  67. , , , , . A new bis-(indole) alkaloid from a deep-water marine sponge of the genus Spongosorites. J. Org. Chem.. 1992;57:4772-4775.
    [Google Scholar]
  68. , , , . Synthesis of novel analogues of marine indole alkaloids: mono(indolyl)-4-trifluoromethylpyridines and bis(indolyl)-4-trifluoromethylpyridines as potential anticancer agents. Bioorg. Med. Chem.. 2001;9:1773-1780.
    [Google Scholar]
  69. , , . Metabolism studies of Indole derivatives using a staurosporine producer, Streptomyces staurosporeus. J. Nat. Prod.. 1997;60:44-48.
    [Google Scholar]

Fulltext Views
641

PDF downloads
225
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles: