JPR_17_442 paper -3 to journal of pharmacy research_RA

1 1
2 Research Article 2
3 3
4 4
5 Synthesis and antitumor evaluation of indole-substituted 5
6
7
indole-fused keto hydrazide–hydrazones 6
7
8 Durgesh Rudavath,1 Reddymasu Sreenivasulu2, Rudraraju Ramesh Raju1* 8
9 9
10 10
AQ1
11 ABSTRACT 11
12 A new type of indole and substituted indole-linked keto hydrazide–hydrazones were designed, synthesized, and evaluated 12
13 for their cytotoxicity against eight human cancer cell lines HeLa, A549, MCF-7, K562, HEK293, HT29, SF295, and HL60. 13
14 These two synthesized hydrazide–hydrazone derivatives show potent antitumor activities on the above eight human cancer 14
15 cell lines. In this, compound 10 exhibited potent antitumor activity on four cancer cell lines MCF-7, A549, K562, and HL60 15
16 with 50% inhibitory concentration (IC50) value of 8.81 µM, 14.11 µM, 14.75 µM, and 15.631 µM, whereas compound 13 16
shows on five cancer cell lines A549, MCF-7, K562, SF295, and HL60 with IC50 value of 16.319 µM, 9.44 µM, 18.38 µM,
17 17
13.513 µM, and 19.31 µM than the standard drug cisplatin, respectively. These two compounds may be identified as promising
18 drug lead compounds. 18
19 19
20 KEY WORDS: 5-bromo indole, Cytotoxicity, Human cell lines, Indole, MTT assay 20
21 21
22 22
23 INTRODUCTION MATERIALS AND METHODS 23
24 Hydrazide–hydrazone exhibit a great interest All the chemicals, reagents, and solvents were 24
25 due to their importance in synthesis, a variety of purchased from AVRA Pvt. Ltd. and Sigma Aldrich. 25
26 valuable heterocyclic compounds which have The purity of the compounds was checked by thin- 26
27 pharmaceutical activity, potent antitumor agents layer chromatography on silica gel plates, and spots 27
28 in cancer chemotherapy.[1-9] These hydrazide– were visualized by exposure to ultraviolet light. 28
29 hydrazone fragments play a very important role due Microanalysis was performed on Perkin–Elmer 29
30 to their potentially high on antibacterial, antifungal, model 240 analyzer, and the values were found 30
31 antimicrobial, and anticonvulsant.[10-17] Moreover, many within ±0.4% of the theoretical values. 1H nuclear 31
32 of them showed analgesic and antiplatelet properties.[18-22] 32
magnetic resonance (NMR) spectra were recorded on
33 Therapeutic prominence of the hydrazide–hydrazone 33
BRUKER-400 Ultra ShieldTM spectrometer. Chemical
34 derivatives has been well established. In addition, these 34
shifts (δ) are expressed in ppm using dimethyl
35 hydrazide–hydrazone moiety compounds were reported 35
sulfoxide (DMSO)-d6 solvent and tetramethyl saline as
36 to elicit anticancer[22-29] and anti-HIV properties,[30] and 36
hence, they have gained an important place in medicinal internal standard. The physical constants and spectral
37 data of the synthesized compounds are presented. 37
38 chemistry. Recently, hydrazide–hydrazones have 38
39 gained great importance due to their diverse biological 39
Experimental Section
activity, including anti-inflammatory, antimalarial,
40 Synthesis of N’-((5-bromo-1H-indol-3-yl)methylene)-2-(1-
40
and antituberculosis activities.[19, 31-35] With the aim of
41 methyl-1H-indol-3-yl)-2-oxoacetohydrazide[9] 41
obtaining novel keto hydrazide–hydrazones, we report
42 42
here in the synthesis of a series of keto hydrazide- 500 mg (2.5 mmol, 1.0 eq) of 2-(1-methyl-1H-indol-
43 hydrazones and followed by antitumor evaluations on
43
3-yl)-2-oxoacetohydrazide 8 was dissolved in 3ml of
44 human cancer cell lines.[36,37] 44
acetic acid, to this 557mg (2.5mmol, 1 eq.) of 5-bromo-
45 45
1H-indole-3-carbaldehyde 9 was added and stir for 6 h
46 46
Access this article online at 90°C. Later, the reaction mass was neutralized with
47 47
a cold NaHCO3 solution, filtered, and recrystallized
48 Website: jprsolutions.info ISSN: 0974-6943 48
from ethanol then afforded 10.
49 49
50 50
1
Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar – 522 510, Andhra Pradesh, India, 2Department
51 of Chemistry, University College of Engineering (Autonomous), Jawaharlal Nehru Technological University, Kakinada – 51
52 533 003, Andhra Pradesh, India 52
53 53
54 *Corresponding author: Rudraraju Ramesh Raju, Department of Chemistry, Acharya Nagarjuna University, Nagarjuna 54
Nagar – 522 510, Andhra Pradesh, India. Phone: +91-9866190692. E-mail: rrraju1@gmail.com
55 55
Received on: 20-09-2017; Revised on: 23-10-2017; Accepted on: 27-11-2017

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 Durgesh Rudavath et al.

1 m.p.=328–330°C; mol.Wt=423; IR (KBrCm−1) υmax=319 of these hydrazide hydrazone compounds (10,13) 1

2 5,1666,1527,1344,1197,980,790; 1H-NMR (DMSO-d6, indicated by 50% inhibitory concentration (IC50) 2
3 400 MHz, δ ppm)=11.6(brs,1H NH); 8.01–8.47(m, 4H values was calculated by comparing to the standard 3
4 Ar-H); 8.17(s, 1H CH) 7.23–7.39(m, 4H Ar-H);3.3(s, drug cisplatin from the plotted absorbance data for 4
5 3H CH3); ESI-MS: 423 (M+H)+. the dose-response curves. Statistical analysis was 5
6 performed using SPSS software version  16.0. IC50 6
Synthesis of N’-((1H-indol-3-yl)methylene)-2-(5-bromo-1- values (in µM) are expressed as the mean ± standard
7 7
methyl-1H-indol-3-yl)-2-oxoacetohydrazide [12] deviation of five independent experiments.
8 8
9 500  mg (1.7 mmol, 1.0 eq.) of 2-(5-bromo-1- 9
methyl-1H-indol-3-yl)-2-oxoacetohydrazide 11 Human cancer cell lines were used HeLa (cervical
10 10
was dissolved in 3 ml of acetic acid, to this 244 mg cancer cell line), A549 (alveolar adenocarcinoma
11 cell line), SF295 (glioblastoma-multiforme cell line),
11
12 (1.7 mmol, 1 eq.) of 1H-indole-3-carbaldehyde 12 was 12
added and stir for 6 h at 90°C. Later, the reaction mass HT-29 (colorectal adenocarcinoma), MCF-7 (breast
13 cancer), K562 (chronic myelogenous leukemia cell 13
was neutralized with a cold NaHCO3 solution, filtered,
14 line), HEK 293 (normal embryonic kidney cell line), 14
and recrystallized from ethanol then afforded 13.
15 and HL60 (acute myelogenous leukemia) obtained 15
16 m.p.=326–328°C; mol. Wt=423; IR (KBrCm−1) from the Cell Bank of the Nexcelom Sciences (NCI 60). 16
17 υmax=3205,1696,1558,1327,1201,981,798; 1H-NMR All lines were maintained in Eagle’s minimal essential 17
18 (DMSO-d6, 400 MHz, δ ppm)=8.17(s,1H CH) medium with 5% of fetal bovine serum and gentamicin 18
19 11.8(brs,1H NH); 8.20-8.53(m, 4H Ar-H); 7.16- 50 µg/ml. Cultures were maintained in 75 cm2 culture 19
20 7.55(m, 4H Ar-H);3.3(s, 3H CH3); ESI-MS: 423 flasks at 37°C, 5% CO2, and 100% relative humidity, 20
21 (M+H)+. and media were changed at least twice a week. 21
22 22
23 Biological Evaluation - In Vitro Cytotoxicity Anticancer Activity 23
24 Cytotoxicity activities against human cancer cell lines The synthesized two derivatives were screened for 24
25 of these hydrazide–hydrazones 10,13 synthesized their anticancer activity against the selected human 25
26 compounds were tested by the method of MTT assay. cancer cell lines such as cervical (HeLa), alveolar 26
27 Compounds were dissolved in DMSO and diluted (A549), breast (MCF-7), chronic myelogenous 27
28 in culture media before the application. Different leukemia (K562), colorectal (HT29), glioblastoma- 28
29 dilutions of each compound were tested (0.1–100 µM) multiforme (SF295), and acute myelogenous leukemia 29
30 with an incubation period of not more than 48 h. To (HL60) as per reported protocol, and the values are 30
31 account for the toxicity of DMSO, the values obtained shown by IC50 in µM. The anticancer activities of 31
32 for the DMSO control were subtracted from those tested compounds ranged between 9.75 and 36 µM 32
33 of the test compounds. Dose-response curves were [Table  1]. Compound 10 shows potent cytotoxicity 33
34 plotted for the test compounds and controls after against four cell lines with IC50 values 14.11 µM 34
35 correction by subtracting the background absorbance (A549), 8.81 µM (MCF-7), 14.75 µM (K562), and 35
36 from that of the blanks. The anticancer potency 15.63 µM (HL60), whereas compound 13 shows 36
37 37
38 R R O 38
39 DMF/DMC
K2CO3 + Cl-C-C-Cl
Di ethyl ether 39
40 N
H
Reflux 130 0C (4 hrs) N
O
00C (4hrs)
40
41 1 2 CH3 41
42 O
O
O 42
43
O

NHNH2
43
44 44
Cl R
R
NH2NH2

45 N
Ethanol
N 45
46 3
CH3
4
CH3 46
47 Where R=Br/H
47
48 O O
R2
48
O
49 49
O

NHNH2 OHC N
50 R1
+
R2
Ethanol
R1
N
H 50
51 N
N
CH3COOH N
H
51
52 52
CH3 H N

5 6 7
53 53
CH3
Where R1= Br/H
R2= Br/H
54 54
55 Scheme 1: General schematic representation for the synthesis of novel keto hydrazide–hydrazones 55

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 Durgesh Rudavath et al.

1 potent anticancer activity against five cell lines with 1

HL60 cells IC50

IC50 values 16.319 µM (A549), 9.44 µM (MCF-7),

15.631±1.381

27.931±0.182
2 2

19.31±2.051
value (µM)
3 18.38 µM (K562), 13.513 µM (SF295), and 19.31 µM 3
4 (HL60). These two compounds may be identified as 4
5 promising drug lead compounds than the standard 5
6 drug Cisplatin. Almost, these two derivatives showed 6
7 potent anticancer activities against particular human 7
IC50 value (µM)
cancer cell lines.
34.605 ±0.481
8 8

13.513±1.02
SF295 cells

14.83±0.61
9 9
10 RESULTS AND DISCUSSION 10
11 Chemistry 11
12 12
The synthesis of novel indole-substituted indole-
13 13
HT29 cells IC50

fused keto hydrazide–hydrazone analogs was shown

18.521±0.511

14 14
value (µM)

9.75±0.904
11.1±1.61

in Scheme 1. Indole 1 on methylation with DMC and

15 K2CO3 at 130°C in DMF as solvent over 4  h gave 15
16 N-methyl indole 2. This compound 2 treated with 16
17 oxalyl chloride at 0°C in diethyl ether as a solvent 17
18 for 6 h then affords N-methyl indolyl mono oxalyl 18
19 chloride 3. This mono oxalyl chloride 3 was refluxed 19
IC50 value (µM)
HEK293 cells

21.73±0.338
20.13±0.718
19.59±0.528

20 with hydrazine hydrate with ethanol over 6 h gave 20

21 N-methyl indolyl keto-3-carbohydrazide 4. Finally, 21
22 this keto carbohydrazide 4 refluxed with substituted 22
23 23
Table 1: Biological activity of hydrazine–hydrazone derivatives with the standard anticancer drug cisplatin

indole aldehyde in ethanol and glacial acetic acid

24 over 6 h afforded new indole-substituted indole-fused 24
25 keto hydrazide–hydrazone 10 forms [Scheme 2]. The 25
K562 cells IC50

14.75±0.381
18.38±1.071
28.172±0.61
value (µM)

26 above reaction is repeat by interchange the places 26

27 of indole and substituted indole in the scheme then 27
28 forms substituted indole indole-fused keto hydrazide– 28
29 hydrazone 13 [Scheme 3] with good yields. 29
30 1. HeLa - Human cervical cancer cell line. 30
31 2. A549 - Human alveolar adenocarcinoma cell line. 31
IC50 value (µM)
MCF‑7 cells

3. MCF-7 - Human breast adenocarcinoma cell line.

8.81±0.594

32 32
9.44±1.88
15±0.294

33 4. K562 - Human chronic myelogenous leukemia cell 33

34 line. 34
35 5. HEK 293 - Human normal embryonic kidney cell 35
36 line. 36
37 6. HT29- Human colorectal adenocarcinoma. 37
A549 cells IC50

7. SF295-Human glioblastoma multiforme.

16.319±0.83
14.11±0.418
value (µM)

38 38
36±0.414

8. HL60-Human acute myelogenous leukemia.

39 39
40 Bar graphs of 50% inhibitory concentration values 40
41 (in µM) for hydrazide–hydrazones derivatives. 41
42 42
43 43
HeLa cells IC50

16.99±0.937
value (µM)

13.93±0.57

44 44
13±0.83

45 45
46 46
47 47
48 48
IC50: 50% Inhibitory concentration

49 49
Compound

50 50
10
13

51 ACKNOWLEDGMENT 51
AQ2
52 52
53 The authors will thankful to University Grants 53
Cisplatin

54 Commission, New  Delhi, India, for financial 54

S. No

55 assistance in the form of major research project 55

F.No.42-285/2013 (SR).
1
2

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 Durgesh Rudavath et al.

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O
O
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Br
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