Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX inhibition activities

Praveen Choppara; Y.V. Prasad; C.V. Rao; K. Hari Krishna; G. Trimoorthulu; G.U. Maheswara Rao; J. Venkateswara Rao; M.S. Bethu; Y.L.N. Murthy

doi:10.1016/j.arabjc.2015.02.006

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Original article

12 (

); 2328-2335

doi:

10.1016/j.arabjc.2015.02.006

Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX inhibition activities

Praveen Choppara^{^a}, Y.V. Prasad^{^a}, C.V. Rao^{^b}, K. Hari Krishna^{^b}, G. Trimoorthulu^{^b}, G.U. Maheswara Rao^{^b}, J. Venkateswara Rao^{^c}, M.S. Bethu^{^c}, Y.L.N. Murthy^{^a,⁎}

a Department of Organic Chemistry, Foods, Drugs & Water, Andhra University, Visakhapatnam 530 003, India

b Resaearch & Development Centre, Laila impex, Vijayawada 520 007, India

c Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, (AP), India

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

Received: 2014-8-23, Accepted: 2015-2-11, Published: 2015-12-01

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

A series of novel N-1 and C-3 substituted indole derivatives (5a–f) were designed, synthesized and evaluated for their cytotoxic properties, viz Brine Shrimp Lethality Bioassay (BSLB) besides 5-Lipoxygenase (5-LOX) inhibitory activities through in vitro assays. Structure Activity Relation (SAR) studies showed that compound 5d with an LC₅₀ of 6.49 μM and 5c with an IC₅₀ of 33.69 μM were found to be interesting for cytotoxicity and 5-LOX inhibitory activity respectively.

Keywords

Brine shrimp lethality bioassay

Cytotoxicity

5-Lipoxygenase inhibition

N prenylated indole-3-carbazones

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1 Introduction

Indole derivatives are an important class of heterocyclic compounds with a wide range of biological activities (Bandini and Eichholzer, 2009). Indole is a substructural element of many natural products which is an important pharmacophore moiety of a large number of molecules with significant biological activities (Diana et al., 2011a,b,c; Carbone et al., 2013, 2014 ) and is widely used as a scaffold in agricultural and medicinal chemistry. In particular, 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), anti cancer (Madadi et al., 2014; Singh et al., 2008; Mashayekhi et al., 2013 ), anti nociceptive (Adam et al., 2010; Moir et al., 2010) and anti psychotic activity (Madadi et al., 2013). On the other hand, carbazones (semicarbazones, thiosemicarbazones) are compounds of considerable interest because of their important chemical properties and potentially beneficial biological activities.

Literature survey reveals that carbazone analogues attain a wide range of biological activities. Thiosemicarbazones appear to be a structural class with anti-pox virus activity (Katz, 1987; Katz et al., 1975; Rao et al., 1966 ). Methisazone (I) plays an important role as prophylactic agent against several viral diseases (Sethi, 2002). Thiosemicarbazone is the important pharmacophore in the therapy and prophylaxis of mycobacterial infections and can represent a template for the development of novel antimycobacterial drugs such as SRI-224 (II), SRI-286 (III) (Bermudez et al., 2003; Dover et al., 2007), Oxazolyl thiosemicarbazones (IV) (Sriram et al., 2006) and some S-alkylisothiosemicarbazones (V) (Cocco et al., 2002; Logu et al., 2005). Thiacetazone (VI) is a thiosemicarbazone analogue which has been widely used for the treatment of Multi Drug Resistant-Tuberculosis (MDR-TB) in many developing countries (Houston and Fanning, 1994). Thiosemicarbazide analogues possess a wide range of biological activities including anticonvulsant (Tripathi et al., 2012), antimicrobial (Zhong et al., 2011), antiviral (Garcial et al., 2003), antitrypanosomal (Moreira et al., 2014), and mushroom tyrosinase inhibitors (Yi et al., 2011). On the other hand, Semicarbazide analogues exhibit anticonvulsant (Rajak et al., 2013), antitubercular (Sriram et al., 2004), antitrypanosomal (Cerecetto et al., 2000), anti inflammatory (Vieira et al., 2012), anti amnesic, cognition enhancing and anticholinesterase (Sinha and Shrivastava, 2013) and anticancer activity (Qi et al., 2013).

Thus the importance of indole and carbazone nuclei in medicinal chemistry prompted us to synthesize novel N prenylated indole carrying carbazones at C-3 position and in order to extend the boundaries of pharmacological properties of indole and carbazone moieties and the target compounds were screened for cytotoxic (BSLB) and 5-LOX inhibitory activities.

2 Results and discussion

2.1

2.1 Chemistry

Due to various biological and pharmacological activities associated with indole and carbazone skeletons, it is worthwhile to incorporate the two moieties in a single molecular frame with an assumption to obtain more potent biologically active compounds, such as 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides (5a–f). The target molecules have been accomplished by modifying first at N–H position and later at third position (C-3) of indole. The modifications mainly include replacement of hydrogen at N–H by prenylation (2-methyl but-2-ene) and formation of carbazones at C-3 with semicarbazide/thiosemicarbazide to increase its pharmacological efficacy.

The target molecules (5a–f) were achieved by Vilsmeier–Haack reaction of 5-substituted indoles (1a–c) yields 5-substituted 3-formyl indoles (2a–c), the modification at N–H position was brought by the reaction with Prenyl bromide in presence of NaH, and DMF at 0 °C for 15 min yields N-prenylated 3-formyl indoles (3a–c). The formation of carbazones at C-3 was accompanied by the reaction of compounds (3a–c) with semicarbazide/thiosemicarbazide (4a, b). The synthetic path involved in synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3-yl)methylene) carbazides (5a–f) was presented in Scheme 1. All the synthesized compounds were well characterized by advanced spectroscopic techniques.

Synthetic scheme for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3-yl)methylene) carbazides (5a-f).

2.2

2.2 Bioevaluation

2.2.1

2.2.1 In vitro cytotoxic activity by Brine Shrimp Lethal Bioassay (BSLB)

Brine Shrimp Lethal Bioassay (BSLB) (Meyer et al., 1982), i.e. toxicity to the Artemia salina (brine shrimp) nauplii was performed and has been used for establishing the fungal toxins, plant extract toxicity, heavy metals, cyano bacteria toxins, pesticides, cytotoxicity testing of dental materials (Carballo et al., 2002), natural and synthetic organic compounds (Choudhary and Thomsen, 2001). Toxicity to A. salina has a good correlation with antitumor, pesticidal (Mc Laughlin, 1991), antitrypanosomal activities (Zani et al., 1995), rodent and human acute oral toxicity data. The brine shrimp larvae respond similarly to the corresponding mammalian systems (Solis et al., 1993). The DNA-dependent RNA polymerases of A. salina have been shown to be similar to the mammalian type (Birndorf et al., 1975). This test is not only used for predicting cytotoxicity, but also used to predict antitumor, antibacterial and pesticidal activities (Sanchez et al., 1993). The brine shrimp lethality method is widely used in the bioassay for identification of the bioactive compounds (Venkateswara Rao et al., 2007; Olowa and Nuneza, 2013). The bioassay determines lethal concentrations of newly synthesized 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides (5a–f) in brine medium adopting the microplate assay developed by Meyer et al., 1982. The Median lethal concentration (LC₅₀) values of the target compounds were compared with the LC₅₀ value of the positive control, Podophyllotoxin. The LC₅₀ values (μM) for the test compounds (5a–f) obtained from BSLB were tabulated in Table 1.

Table 1 In vitro cytotoxicity of title compounds (5a–f) by Brine Shrimp Lethal Bioassay.

Entry	Compound	^∗LC₅₀ ± SE (μM)	Logarithmic Regression equation Y = Y^bar + b (x − x^bar)	95% Confidence fiducial limits		Relative activity
Entry	Compound	^∗LC₅₀ ± SE (μM)	Logarithmic Regression equation Y = Y^bar + b (x − x^bar)	LCL	UCL
1	5a	477.14 ± 44.66	Y = −0.61 + 2.66x	114.72	149.79	70.39
2	5b	315.95 ± 16.91	Y = 0.42 + 2.34x	81.45	105.66	49.55
3	5c	206.04 ± 11.57	Y = 0.71 + 2.31x	64.79	79.21	39.29
4	5d	6.49 ± 0.30	Y = 3.59 + 3.74x	2.158	2.599	1.29
5	5e	274.02 ± 11.08	Y = −0.83 + 3.05x	74.99	87.86	44.32
6	5f	205.27 ± 9.06	Y = −0.11 + 2.83x	58.39	69.42	34.88
7	Standard^†	4.42 ± 0.28	Y = 4.10 + 3.40x	1.63	2.02	1.00

48 h old nauplii of brine shrimp (A. salina) were treated with different concentrations of indole carbazides (5a-f) for 24 h and lethal concentration was investigated through probit analysis.^∗Regression equation Y = Y^bar + b (X − x^bar) or (Y^bar − b ∗ x^bar) + b ∗ x). Y^bar = weighted avg. of working probit values; x^bar = avg. of log 10 (concentration); b = slope of the line; X = x scale concentration in Log). ^†Podophyllotoxin used as positive control. Three replicates for each concentration and the control (without test compound), were tested for lethal bio-efficacy and the mean data were used to calculate the probit regression equation. ^∗LC₅₀ is defined as the concentration of extract that can cause 50% mortality in the exposed population.

The results from Table 1 show that compound 5d exhibits good cytotoxicity with LC₅₀ of 6.49 μM and its relative active (1.29) was close-at-hand to Podophyllotoxin (1.00). Among the semicarbazides (5a,5c,5e), unsubstituted indole analogue (5a) was found to be weak than the substituted analogues (5c and 5e), bromo analogue (5c) was interesting candidate with LC₅₀ of 206.04 μM followed by cyano analogue (5e) with LC₅₀ 274.02 μM and their relative activities were 39.29 and 44.32 respectively. The results of thiosemicarbazides (5b,5d,5f) showed that bromo analogue (5d) was found to be interesting with LC₅₀ of 6.49 μM, then cyano analogue (5f) with 205.27 μM and unsubstituted indole analogue (5b) was weak with LC₅₀ of 315.95 μM. In conclusion, among the synthesized compounds, unsubstituted analogues (5a, 5b) showed poor activity than the corresponding substituted analogues (5c–f).

2.2.2

2.2.2 5-Lipoxygenase inhibition assay

Lipoxygenases (LOXs), which are widely distributed in both the plant and animal kingdoms, belong to a class of non-heme iron-containing enzymes which catalyze the hydroperoxidation reaction of fatty acids to peroxides (Peters-Golden and Henderson, 2007). 5-Lipoxygenase (5-LOX) is the key enzyme in the biosynthesis of leukotrienes (LTs) through catalyzing the initial two steps in conversion of arachidonic acid to LTs (Samuelson, 1983). LTB4 and the cysteinyl-leukotrienes are potent constrictors of human airways, and have powerful proinflammatory properties (Funk, 2001). Inhibition of 5-LOX may lead to the development of new therapeutic treatments for pathologies such as asthma, allergies, and other inflammatory disorders (Samuelson, 1983, Young, 1999, Drazen, 1998). Recent studies have implicated a role of 5-LOX products involved in a number of other diseases, including cancer (Romano and Claria, 2003), atherosclerosis (Spanbroek and Habenicht, 2003), stroke (Helgadottir et al., 2004) and osteoporosis (Werz and Steinhilber, 2005). Therapeutic potential of 5-LOX inhibition has been widely highlighted in recent years (Balkan and Berk, 2003; Funk, 2005; Yoshimura et al., 2005 ). Literature survey reveals N-1 and C-3 substituted indoles are identified as 5-LOX inhibitors (Prasher et al., 2014; Singh and pooja, 2013; Zheng et al., 2007 ). Based on the literature survey the novel 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f) were screened for their inhibitory properties against 5-LOX enzyme employing the assay described by Reddanna et al., 1990. 5-LOX inhibition of the tested compounds was determined by measuring the Inhibition Concentration; IC₅₀ (IC₅₀ represents the concentration of a drug that is required for 50% inhibition) expressed in μM. The results for test compounds (5a–) were compared with the positive control Curcumin. The IC₅₀ values for the test compounds (5a–f) obtained from 5-LOX inhibition assay were tabulated in Table 2.

Table 2 IC₅₀ values obtained from in vitro 5-Lipoxygenase inhibition assay for the compounds (5a–f).

Entry	Compound	IC₅₀ (μM)
1	5a	>100
2	5b	>100
3	5c	33.69
4	5d	36.65
5	5e	>100
6	5f	>100
7	Standard^a	27.58

IC₅₀ represents the concentration of a drug that is required for 50% inhibition expressed in μM.

a Curcumin as positive control.

The results obtained from Table 2 shows that the unsubstituted (5a, 5b) and cyano (5e, 5f) analogues were weak with an IC₅₀ value of >100 μM and bromo analogues (5c, 5d) were the engrossing compounds compared with the standard “curcumin” with an IC₅₀ of 33.69 and 36.65 μM respectively.

2.2.3

2.2.3 Structure–Activity Relationship Study (SARS)

In 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides (5a–f), substituent plays an important role to make difference in the activity. From the results of Brine Shrimp Lethal Bioassay as shown in Table 1, compound 5d (Table 1 entry-4) is more potent among the series, in which bromine is present on the 5th position of indole and thiosemicarbazone at 3rd position. From the results of 5-LOX inhibition assay as shown in Table 2, compound 5c (Table 2 entry-3) is more potent 5-LOX inhibitor. In compound 5c bromine is present on the 5th position of indole and semicarbazone at 3rd position.

Bioevaluation assays reveal that among the target compounds (5a–f), compounds 5c and 5d exhibit potent activity. In both the compounds 5c and 5d bromine is present on the 5th position of indole. The 3rd position was occupied by semicarbazone and thiosemicarbazone in compounds 5c and 5d respectively. In the SAR studies of novel 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl)methylene) carbazides (5a–f), it was observed that the presence of hydrophobic substituent (bromine) at 5th position of indole moiety makes the compounds 5c and 5d more potent than unsubstituted analogues (5a, 5b) and cyano analogues (5e, 5f) which lack hydrophobic substituent.

3 Experimental

3.1

3.1 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 Jeol JNM EX-90 spectrometer.

3.2

3.2 General procedure for the synthesis of substituted 1H-indole-3-carbaldehyde (2a–c)

To a solution of substituted indoles (1a–c) (42.6 mmol) in dry DMF (187.4 mmol) in an ice-salt bath and 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 gms crushed ice was added to the reaction mixture. The obtained solution is transferred into 250 mL RB flask, 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 substituted 1H-indole-3-carbaldehyde (2a–c).

3.2.1

3.2.1 1H-indole-3-carbaldehyde (2a): (James and Snyder, 1959)

Yield: 92%; 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, 131.8, 137.2, 182.7. ESI-MS: m/z 146.20 [M+H]⁺. IR (KBr, cm⁻¹): 1224, 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%.

3.2.2

3.2.2 5-bromo-1H-indole-3-carbaldehyde (2b)

Yield: 90%; 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]⁺; 247.95 [M+Na+2]⁺. 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%.

3.2.3

3.2.3 3-formyl-1H-indole-5-carbonitrile (2c)

Yield: 84%; Mp: 220–224 ^oC. ¹H NMR (DMSO-d₆, 400 MHz): δ 7.27 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 8.21 (s, 1H), 8.43 (s, 1H), 10.23 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 103.4, 111.3, 113.6, 116.1, 117.2, 123.8, 126.1, 134.8, 137.1, 139.8, 185.8. ESI-MS: m/z 171. [M+H]⁺. IR (KBr, cm⁻¹): 1221, 1650, 3345. Anal. Calcd. for C₁₀H₆N₂O: C, 70.58, H, 3.55, N, 16.46%; Found: C, 70.60, H, 3.52, N, 16.45%.

3.3

3.3 General procedure for the synthesis of substituted 1-(3-methylbut-2-enyl)- 1H-indole-3-carbaldehyde (3a–)

To a solution of substituted 1H-indole-3-carbaldehyde (2a–c) (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, filtered and the solvent was removed under reduced pressure to get crude residue which was purified by silica gel column chromatography (100–200 mesh) using 8:2 (Hexane:EtOAc) as eluents affording 3a–c.

3.3.1

3.3.1 1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3a)

Yield: 87%; 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⁺], IR (KBr, cm⁻¹): 1161, 1640. Anal. Calcd. for C₁₄H₁₅NO: C, 78.84, H, 7.09, N, 6.57%; Found: C, 78.80, H, 7.16, N, 6.55%.

3.3.2

3.3.2 5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3b)

Yield: 89%; 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, 8.8 Hz, 1H), 7.73 (s, 1H), 8.47 (d, J = 2 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 292.03 [M]⁺; 294.03 [M+2]⁺; 314.01 [M+Na]⁺; 316.01 [M+Na+2]⁺. 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%.

3.3.3

3.3.3 3-formyl-1-(3-methylbut-2-enyl)-1H-indole-5-carbonitrile (3c)

Yield: 79%; Mp: 90–92 °C ¹H NMR (CDCl₃, 400 MHz): δ 1.73 (s, 3H), 1.75 (s, 3H), 4.68 (d, J = 7.2 Hz, 2H), 5.44 (t, J = 7.2 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 2, 8.4 Hz, 1H), 7.81 (s, 1H), 8.32 (d, J = 2 Hz, 1H), 10.03 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ 17.7, 25.8, 40.2, 109.1, 111.4, 112.5, 116.7, 120.3, 122.2, 122.6, 129.5, 132.2, 133.0, 133.7, 179.1, ESI-MS: m/z 239 [M+H]⁺; IR (KBr, cm⁻¹): 1134, 1649. Anal. Calcd. for C₁₅H₁₄N₂O: C, 75.61, H, 5.92, N, 11.76%; Found: C, 75.62, H, 5.93, N, 11.77%.

3.4

3.4 General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) semicarbazide (5a, 5c, 5e)

To the solution of substituted 1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3a–c) (3 mmol) and KOH (4 mmol) in 10 mL absolute ethanol, solution of semicarbazide hydrochloride (4a) (4 mmol) in absolute ethanol and water (9:1 mL) was added slowly and the reaction mixture was refluxed for 1.5 h. Removal of the solvent in vacuo furnished a thick liquid which produced precipitate on addition of water. The precipitate was filtered off, washed with hot water, and dried to afford as a solid, which was purified by silica gel column chromatography (100–200 mesh) using 6:4 (Hexane:EtOAc) as eluents to afford target compounds 5a, 5c and 5e.

3.4.1

3.4.1 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-yl)methylene) semicarbazide (5a)

Yield: 79%; Mp: 148 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.70 (s, 3H), 1.81 (s, 3H), 4.76 (d, J = 7.2 Hz, 2H), 5.32 (t, J = 7.2 Hz, 1H), 6.25 (s, 2H), 7.34–7.45 (m, 4H), 7.79 (s, 1H), 8.09 (s, 1H), 8.94 (s, 1H). ¹³C NMR (DMSO-d₆, 22.4 MHz): δ 17.8, 25.4, 44.1, 109.8, 111.0, 118.8, 120.8, 121.8, 122.7, 125.2, 130.7, 137.0, 137.3, 138.8, 158.7. ESI-MS: m/z 271.29 [M+H]⁺. IR (KBr, cm⁻¹): 1165, 1694, 3148, 3469. Anal. Calcd. for C₁₅H₁₈N₄O: C, 66.64, H, 6.71, N, 20.73%; Found: C, 66.63, H, 6.75, N, 20.71%.

3.4.2

3.4.2 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) semicarbazide (5c)

Yield: 82%; Mp: 188–190 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.71 (s, 3H), 1.81 (s, 3H), 4.76 (d, J = 6.8 Hz, 2H), 5.34 (t, J = 7.2 Hz, 1H), 6.25 (s, 2H), 7.34 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 8.02 (s, 1H), 8.25 (s, 1H), 9.94 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 18.3, 25.8, 44.3, 111.0, 112.8, 113.7, 119.9, 124.2, 125.4, 126.6, 133.0, 135.8, 137.0, 137.9, 157.2. ESI-MS: m/z 349.06 [M⁺]; 351.06 [M+2]⁺; 387.02 [M+K]⁺; 389.02 [M+K+2]⁺. IR (KBr, cm⁻¹): 1196, 1623, 3142, 3473. Anal. Calcd. for C₁₅H₁₇BrN₄O: C, 51.59, H, 4.91, N, 16.04%; Found C, 51.57, H, 4.99, N, 16.01%.

3.4.3

3.4.3 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) semicarbazide (5e)

Yield: 84%; Mp: 178–180 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.69 (s, 3H), 1.74 (s, 3H), 4.69 (d, J = 7.2 Hz, 2H), 5.41 (t, J = 7.2 Hz, 1H), 6.51 (s, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 8.19 (s, 1H), 8.43 (s, 1H), 10.37 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 17.7, 26.9, 43.9, 105.1, 111.3, 113.2, 113.7, 116.9, 120.1, 125.1, 125.9, 132.6, 136.9, 137.3, 138.8, 160.1. ESI-MS: m/z 296 [M+H]⁺. IR (KBr, cm⁻¹): 1254, 1642, 3135, 3489. Anal. Calcd. for C₁₆H₁₇N₅O: C, 65.07, H, 5.80, N, 23.71%; Found C, 65.08, H, 5.81, N, 23.70%.

3.5

3.5 General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) thiosemicarbazide (5b, 5d, 5f)

To the solution of substituted 1-(3-methylbut-2-enyl)- 1H-indole-3-carbaldehyde (3a–c) (3 mmol) and KOH (3 mmol) in 10 mL absolute ethanol, solution of thiosemicarbazide (4b) (4 mmol) in absolute ethanol and water (9:1 mL) was added slowly, and the reaction mixture was refluxed for 1 h. Removal of the solvent in vacuo furnished a thick liquid which produced precipitate on addition of water. The precipitate was filtered off, washed with excess of hot water and dried to afford crude products, which was purified by silica gel column chromatography (100–200 mesh) using 6:4 (Hexane:EtOAc) as eluents to afford target compounds 5b, 5d and 5f.

3.5.1

3.5.1 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-yl)methylene)thiosemicarbazide (5b)

Yield: 85%; Mp: 162 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.75 (s, 3H), 1.78 (s, 3H), 4.51(d, J = 7.2 Hz, 2H), 5.26 (t, J = 7.2 Hz 1H), 5.90 (s, 2H), 7.20–7.29 (m, 4H), 7.78 (s, 1H), 8.04 (s, 1H), 9.99 (s, 1H). ¹³C NMR (DMSO-d₆, 22.4 MHz): δ 17.9, 25.5, 44.0, 109.9, 110.6, 118.6, 121.1, 121.8, 122.8, 125.0, 131.8, 137.0, 137.5, 140.8, 177.0. ESI-MS: m/z 287.25 [M]⁺. IR (KBr, cm⁻¹): 1161, 1613, 3148, 3413. Anal. Calcd. for C₁₅H₁₈N₄S: C, 62.91, H, 6.33, N, 19.56%; Found: C, 62.88, H, 6.39, N, 19.55%.

3.5.2

3.5.2 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) thiosemicarbazide (5d)

Yield: 83%; Mp: 220 °C. ¹H NMR (DMSO-d₆, 400 MHz): 1.71 (s, 3H), 1.81 (s, 3H), 4.76(d, J = 6.8 Hz, 2H), 5.32 (t, J = 1.2 Hz, 1H), 6.26 (s, 2H), 7.34 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 8.02 (s, 1H), 8.25 (s, 1H), 9.95 (s, 1H). ¹³C NMR (DMSO-d₆, 22.4 MHz): δ 18.4, 26.0, 44.2, 110.9, 112.7, 114.2, 120.0, 124.3, 124.5, 126.7, 133.1, 135.5, 137.5, 138.0, 179.3. ESI-MS: m/z 365.04 [M]⁺; 367.04 [M+2]⁺; 387.03 [M+Na]⁺; 389.02 [M+Na+2]⁺. IR (KBr, cm⁻¹): 1187, 1621, 3160, 3387. Anal. Calcd. for C₁₅H₁₇BrN₄S: C, 49.32, H, 4.69, N, 15.34%; Found: C, 49.31, H, 4.72, N, 15.31%.

3.5.3

3.5.3 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) thiosemicarbazide (5f)

Yield: 91%; Mp: 192–194 °C. ¹H NMR (DMSO-d₆, 400 MHz): 1.75 (s, 3H), 1.79 (s, 3H), 4.75 (d, J = 7.2 Hz, 2H), 5.21 (t, J = 1.6 Hz, 1H), 6.84 (s, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 8.14 (s, 1H), 8.46 (s, 1H), 10.41 (s, 1H). ¹³C NMR (DMSO-d₆, 22.4 MHz): δ 17.2, 26.4, 45.1, 105.2, 111.1, 112.9, 114.7, 115.7, 119.5, 125.6, 126.2, 133.7, 134.8, 136.3, 138.7, 180.1. ESI-MS: m/z 312 [M+H]⁺; IR (KBr, cm⁻¹): 1192, 1626, 3178, 3421. Anal. Calcd. for C₁₆H₁₇N₅S: C, 61.71, H, 5.50, N, 22.49%; Found: C, 61.72, H, 5.51, N, 22.48%.

3.6

3.6 Procedure for in vitro cytotoxic activity of 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f) by Brine Shrimp Lethal Bioassay (BSLB)

Brine Shrimp Lethal Bioassay was used according to the method of Meyer et al., 1982. The lethality experiments were conducted in a 12 well microplate, each well consisting of 5 mL artificial seawater with required test concentration. The derivatives were dissolved in dimethylsulfoxide (DMSO) and diluted with artificial sea salt water to achieve the required test concentrations, and maintained final concentration of DMSO, which did not exceed 0.05%. The control experiments were also performed simultaneously with an addition of carrier solvent alone (without sample). Ten numbers of 48 h old nauplii were added into each well and exposed for 24 h to different test concentrations under illumination with a minimum of three replicates. A drop of dry east suspension (3 mg in 5 mL artificial seawater) was added as food to each well. The plates were then examined under magnification (10×) and the number of dead nauplii in each well was counted. Percent mortalities were corrected for natural mortality in controls using Abbots formula (Abbot, 1925), i.e., corrected percent mortality = (PI – C/100 – C) ∗ 100, where PI denotes the observed mortality rate, and C is natural mortality. The median lethal concentration (LC₅₀ for 24 h value) for each assay was calculated by taking average of three experiments using statistical software, BioStat-2008 based on the Finney Probit analysis (Finney, 1971). The results for test compounds were compared with the positive control Podophyllotoxin. All samples were done in triplicate. The LC₅₀ values for the test compounds (5a–f) obtained from BSLB were expressed in μM.

3.7

3.7 Procedure for 5-Lipoxygenase inhibition assay for 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f)

5-Lipoxygenase inhibition assay mixture contained 2.97 mL of 50 mmol phosphate buffer pH at 6.3. 5 μL of 80 mmol Linoleic acid and sufficient amount of potato 5-Lipoxygenase enzyme. The reaction was started by the addition of substrate (Linoleic acid) and the increase in UV absorption at 234 nm was followed at 25 °C. The activities were measured at 2 min. The reaction was linear during this time period. In the inhibition studies, the activities were measured in the presence of various concentrations of target molecules (5a–f). All the assays were performed in triplicate. The IC₅₀ values for the test compounds (5a–f) obtained from 5-LOX inhibition assay were expressed in μM.

4 Conclusion

In conclusion, we report a novel series of six N prenylated indole-3-carbazones, in which five compounds were interesting in cytotoxic and two compounds in 5-LOX inhibition assay.

Acknowledgments

We are grateful to DRDO, New Delhi, India for providing financial assistance through the Project ERIP/ER/1003916/M/01/1354 and UGC, New Delhi, India for the award of UGC-BSR JRF to the author P.C.

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Introduction

Results and discussion

Chemistry
Bioevaluation

Experimental

General
General procedure for the synthesis of substituted 1H-indole-3-carbaldehyde (2a–c)
General procedure for the synthesis of substituted 1-(3-methylbut-2-enyl)- 1H-indole-3-carbaldehyde (3a–)
General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) semicarbazide (5a, 5c, 5e)
General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) thiosemicarbazide (5b, 5d, 5f)
Procedure for in vitro cytotoxic activity of 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f) by Brine Shrimp Lethal Bioassay (BSLB)
Procedure for 5-Lipoxygenase inhibition assay for 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f)

Conclusion

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[2] Adam J., Cairns J., Caulfield W., Cowley P., Cumming I., Easson M., Edwards D., Ferguson M., Goodwin R., Jeremiah F., Kiyoi T., Mistry A., Moir E., Morphy R., Tierney J., York M., Baker J., Cottney J., Houghton A., Westwood P., Walker G., . 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] Balkan A., Berk B., . Novel approaches to anti-inflammatory agents as therapeutics for asthma. Curr. Med. Chem. Anti-Inflamm. Anti-Allergy Agents. 2003;2:9-18.
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Design, synthesis of novel N prenylated indole-3-carbazones and evaluation of in vitro cytotoxicity and 5-LOX inhibition activities

Abstract

Keywords

Brine shrimp lethality bioassay

Cytotoxicity

5-Lipoxygenase inhibition

N prenylated indole-3-carbazones

1 Introduction

2 Results and discussion

2.1 Chemistry

2.2 Bioevaluation

2.2.1 In vitro cytotoxic activity by Brine Shrimp Lethal Bioassay (BSLB)

2.2.2 5-Lipoxygenase inhibition assay

2.2.3 Structure–Activity Relationship Study (SARS)

3 Experimental

3.1 General

3.2 General procedure for the synthesis of substituted 1H-indole-3-carbaldehyde (2a–c)

3.2.1 1H-indole-3-carbaldehyde (2a): (James and Snyder, 1959)

3.2.2 5-bromo-1H-indole-3-carbaldehyde (2b)

3.2.3 3-formyl-1H-indole-5-carbonitrile (2c)

3.3 General procedure for the synthesis of substituted 1-(3-methylbut-2-enyl)- 1H-indole-3-carbaldehyde (3a–)

3.3.1 1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3a)

3.3.2 5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-carbaldehyde (3b)

3.3.3 3-formyl-1-(3-methylbut-2-enyl)-1H-indole-5-carbonitrile (3c)

3.4 General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) semicarbazide (5a, 5c, 5e)

3.4.1 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-yl)methylene) semicarbazide (5a)

3.4.2 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) semicarbazide (5c)

3.4.3 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) semicarbazide (5e)

3.5 General procedure for the synthesis of 1-(5-substituted-1-(3-methylbut-2-enyl)- 1H-indole-3 yl)methylene) thiosemicarbazide (5b, 5d, 5f)

3.5.1 1-((1-(3-methylbut-2-enyl)- 1H-indol-3-yl)methylene)thiosemicarbazide (5b)

3.5.2 1-(5-bromo-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) thiosemicarbazide (5d)

3.5.3 1-(5-cyano-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) thiosemicarbazide (5f)

3.6 Procedure for in vitro cytotoxic activity of 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f) by Brine Shrimp Lethal Bioassay (BSLB)

3.7 Procedure for 5-Lipoxygenase inhibition assay for 1-(5-substituted-1-(3-methylbut-2-enyl)-1H-indole-3-yl) methylene) carbazides (5a–f)

4 Conclusion

Acknowledgments

References

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