Synthesis and Cytotoxic Activity of New Indole Derivatives
Radhika C.1, Venkatesham Akena2, Venkateshwar Rao J.1, Sarangapani M.2*
1Talla Padmavathi College of Pharmacy, Orus, Warangal-506009, A.P, India
2University College of Pharmaceutical Sciences, Kakatiya University, Warangal-506009, A.P, India
*Corresponding Author E-mail: cradhika8@gmail.com
ABSTRACT:
Some new (Napthalen-1-yloxyl/ 2-yloxy) - acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (4 and 5) have been synthesized by condensing naphthyl-1-oxy/2-oxy acetic acid hydrazides with different isatins. Their chemical structures have been confirmed by IR, NMR, and Mass spectroscopy and by elemental analysis. Investigation of cytotoxic activity was done by MTT assay method using HeLa and MCF cell lines. The compounds with 6-Bromo substitution (4e, 5e) have shown the prominent cytotoxic activity among the synthesized compounds.
KEYWORDS: Synthesis/Indoles/cytotoxicity
A thorough survey of literature reveals indole derivatives have shown broad spectrum of biological activity like antibacterial, anti inflammatory, analgesic, antiviral, antifungal, anti tubercular, anti depressant, anti convulsant, anti cancer1-6. Keeping in view of these pharmacological activities of indole derivatives, we synthesized some new isatin Schiff bases by condensing Naphthalen-1-yloxy/2-yloxy acetic acid hydrazides with different isatins. All the synthesized compounds were screened for their cytotoxic activity by MTT Assay method by using HeLa and MCF cell lines.
Material and Methods
Synthesis
The desired products namely (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (4a-4f) and (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (5a-5f) were prepared by the reactions outlined in scheme-1. The reactions of α/β naphthol (1) with the ethylchloroacetate gave (Naphthalen-1-yloxy/-2-yloxy)-acetic acid ethyl ester (2) and converted to (Naphthalen-1-yloxy/-2-yloxy)-acetic acid hydrazides (3) with hydrazine hydrate. On condensing compound 3 with different isatins gave 4a-4f and 5a-5f.
EXPERIMENTAL:
Melting points were determined by open capillary using toshiwal melting point apparatus and are uncorrected. Purity of compounds was checked by TLC and silica gel coated plates. IR spectra were recorded in potassium bromide KBr on FTIR 8400 Shimadzu spectrophotometer. 1H NMR spectra were recorded on 300MHz Bruker DPX using CDCl3 and Mass spectra were recorded on LC-MS/MS. Elemental analysis was perform on Perkin-Elmer series 2400.
All the chemical analysis was performed at IICT, Hyderabad. All chemicals used in synthesis purchased from E.Merck and Aldrich. The purity of the compounds was controlled with Merck pre-coated TLC plates and spots were visualized with ultraviolet light.
A mixture of a or b-Naphthol (0.01 mol), ethylchloroacetate (0.01 mol) and anhydrous potassium carbonate (0.02 mol) in dry acetone (60 ml) was refluxed on water bath for 24 hr. The inorganic solid was filtered and the excess solvent was removed on a rota vapour. The product thus obtained was pure enough to prepare the corresponding hydrazide.
To a suspension of compound 2 (0.01 mol) in absolute ethanol (50 ml), hydrazine hydrate (99%, 0.015 mol) was added and the reaction mixture was refluxed for 15 hr. The solution was concentrated and allowed to cool overnight.
R=H, 5-CH3, 5-Cl, 7-CH3, 7-COOCH3, 6-Br
Scheme-1
1a or b-Naphthol, 2 (Naphthalen-1-yloxy/-2-yloxy)-acetic acid ethyl ester, 3 (Naphthalen-1-yloxy/-2-yloxy)-acetic acid hydrazides, 4(Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides, 5(Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides
a. Ethylchloroacetate (ClCH2COOC2H5) b. Hydrazine hydrate (99%) c. Indole-2, 3-done
Table 1: Physical data of (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3- ylidene)-hydrazides (4a - 4f)
|
S. No. |
Compound code |
R |
M.p. (°C) |
Yield (%) |
Molecular formula |
Mol. wt |
Elemental analysis found (calculated) (%) |
||
|
C |
H |
N |
|||||||
|
1 |
4a |
H |
268 |
75 |
C20H15O3N3 |
345 |
69.56 (69.52) |
4.34 (4.29) |
12.17 (12.13) |
|
2 |
4b |
5-CH3 |
242-243 |
78 |
C21H17O3N3 |
359 |
70.19 (70.16) |
4.73 (4.71) |
11.69 (11.65) |
|
3 |
4c |
5-Cl |
234-235 |
70 |
C20H14O3N3Cl |
379 |
63.32 (63.33) |
3.69 (3.66) |
11.08 (11.04) |
|
4 |
4d |
7-CH3 |
252-254 |
73 |
C21H17O3N3 |
359 |
70.19 (70.15) |
4.73 (4.75) |
11.69 (11.64) |
|
5 |
4e |
6-Br |
274 |
68 |
C20H14O3N3Br |
539 |
44.52 (44.49) |
2.59 (2.55) |
7.79 (7.76) |
|
6 |
4f |
7-COOCH3 |
210-211 |
70 |
C23H17O5N3 |
415 |
60.72 (60.74 |
4.09 (4.04) |
10.12 (10.14) |
The resulting solid obtained was filtered, washed with cold ethanol, dried, and recrystallised from ethanol8. The compound was separated as shining colorless needle shaped crystals. Adopting this procedure a) (Naphthalene-1-yloxy) acetic acid hydrazide yield 74%, MP 142-143°C.
b) (Naphthalene-2-yloxy) acetic acid hydrazide yield 83%, MP 122-124°C was prepared.
The IR (KBr) spectra of the compound (3a) exhibited absorption bands at 3295(NH2), 3033 (NH); 1661 (C=O, acid hydrazide), 1266 (C-O-C).
The IR (KBr) spectra of the compound (3b) exhibited absorption bands at 3307(NH2), 3031(NH); 1661 (C=O, acid hydrazide), 1260(C-O-C).
Table 2: Physical data of (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (5a – 5f)
|
S. No. |
Compound code |
R |
M.p. (°C) |
Yield (%) |
Molecular formula |
Mol. wt |
Elemental analysis found (calculated) (%) |
||
|
C |
H |
N |
|||||||
|
1 |
5a |
H |
270 |
85 |
C20H15O3N3 |
345 |
69.56 (69.52) |
4.34 (4.35) |
12.17 (12.13) |
|
2 |
5b |
5-CH3 |
254-256 |
80 |
C21H17O3N3 |
359 |
70.19 (70.16) |
4.73 (4.74) |
11.69 (11.65) |
|
3 |
5c |
5-Cl |
202-203 |
77 |
C20H14O3N3Cl |
379 |
63.32 (63.33) |
3.69 (3.71) |
11.08 (11.04) |
|
4 |
5d |
7-CH3 |
210-211 |
72 |
C21H17O3N3 |
359 |
70.19 (70.18) |
4.73 (4.75) |
11.69 (11.64) |
|
5 |
5e |
6-Br |
280-282 |
75 |
C20H14O3N3Br |
539 |
44.52 (44.49) |
2.59 (2.55) |
7.79 (7.76) |
|
6 |
5f |
7-COOCH3 |
226-227 |
70 |
C23H17O5N3 |
415 |
60.72 (60.70) |
4.09 (4.04) |
10.12 (10.13) |
Table 3: Cytotoxic activity of (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (4a – 4f)
|
S. No. |
Compound Code |
Substituent’s R |
HeLa cell lines IC50 value (µM) |
MCF cell lines IC50 value (µM) |
|
1 |
4a |
H |
126.66 |
137.24 |
|
2 |
4b |
5-CH3 |
154.35 |
149.54 |
|
3 |
4c |
5-Cl |
83.23 |
78.12 |
|
4 |
4d |
7-CH3 |
148.55 |
165.35 |
|
5 |
4e |
6-Br |
68.42 |
66.20 |
|
6 |
4f |
7-COOCH3 |
167.39 |
155.78 |
|
7 |
Cisplatin |
- |
30.12 |
32.62 |
Table 4: Cytotoxic activity of (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (5a – 5f))
|
S. No. |
Compound code |
Substituent’s R |
HeLa cell lines IC50 value (µM) |
MCF cell lines IC50 value (µM) |
|
1 |
5a |
H |
156.35 |
221.11 |
|
2 |
5b |
5-CH3 |
201.32 |
254.15 |
|
3 |
5c |
5-Cl |
112.21 |
151.31 |
|
4 |
5d |
7-CH3 |
148.55 |
212.57 |
|
5 |
5e |
6-Br |
87.33 |
91.22 |
|
6 |
5f |
7-COOCH3 |
NA |
265.97 |
|
7 |
Cisplatin |
- |
30.12 |
32.62 |
Synthesis of (Naphthalen-1-yloxy/2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (4and5) An appropriate indole-2, 3-dione (3, 0.01 mol) was heated under reflux in methanol (50 ml) containing two to three drops of glacial acetic acid with (Naphthalen-1-yloxy/2-yloxy)-acetic acid hydrazide (3, 0.01 mol) for half an hour. The product thus obtained was filtered and purified by recrystallization from suitable solvent(s).
Twelve compounds were prepared by adopting above described procedure and their physical and analytical data are presented in Table – 1and 2 and Figure-1 and 2. The IR spectrum of the compound (4a, R=H) showed absorption frequencies in (cm-1) at 3258 (NH), 3057 (NH); 1701 (C=O, lactam), 1680 (C=O, acid hydrazide), 1178 (C-O-C).
Figure 1. Cytotoxic activity of (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro- indole-3-ylidene)-hydrazides (4)
Figure 2. Cytotoxic activity of (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (5)
Its PMR spectrum (DMSO-d6) exhibited characteristic peaks (ind, ppm): 13.8 (s, 1H, lactam NH), 11.2 (s, 1H, NH-C=O), 4.99(s, 2H, CH2) 6.9 to 7.8 (m, 11H, ArH).
Mass spectrum of the compound (4a, R=H) recorded its molecular ion (M+1) at m/z 346.
The physical and analytical data were given in table 1and2.
Cytotoxic activity:
Cytotoxic activity was evaluated by MTT7-9 (Microculture tetrazolium) assay using HeLa and MCF cell lines. Microculture tetrazolium assay (MTT) is based on the metabolic reduction of 3-(4, 5-dimethylthiazol-2, 5-diphenyl) tetrazolium bromide (MTT) to water insoluble formazan crystals with mitochondrial dehydrogenase enzyme, which gives direct correlation of viable cells.
Cell suspension and the compound solution (Dissolved in 1% DMSO) were added to the 96 well plates and kept in carbon dioxide incubator with 5% CO2 at 37oC for 72 hours. Blank contains only cell suspension and control wells contain 1% DMSO and cell suspension. After 72 hours, MTT was added and kept in carbon dioxide incubator for 2 hours followed by of lysis buffer and 96 well plates was kept on rotary shaker for 8 hours. Then absorption was read on ELISA reader at 562 nm. The inhibitory activity of test compounds was compared with DMSO control.
RESULTS AND DISCUSSION:
Pharmacological activity of the compounds 4a-4f and 5a-5f were conducted at CDRI and were evaluated for the cytotoxic activity by MTT method against HeLa and MCF cell lines. The results are summarized in table 3 and 4.
The cytotoxic activity data of (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides 4 (table-3) indicates that all the compounds exhibited cytotoxic activity against HeLa cell lines and MCF cell lines. Most significant of them is the compound 4e (R=6-Br) with IC50 values of 68.42 mM and 66.20 mM against HeLa cell lines and MCF cell lines respectively. Compound 4c (R= 5-Cl) is next in the order of cytotoxic activity with IC50 values of 83.23 mM and 78.12 mM against HeLa cell lines and MCF cell lines respectively. This is followed by the compounds 4a, 4b and 4d exhibited moderate activity against HeLa cell lines and MCF cell lines. Among all the compounds, compound 4f (R=7-COOCH3) exhibited least activity against HeLa cell lines and MCF cell lines.
The data on IC50 values of cytotoxic activity of (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (5) is given in the Table 4. The compound 5e (R = 6-Br) showed highest cytotoxic activity among the series with IC50 values of 87.33 mM and 91.22 mM against HeLa cell lines and MCF cell lines respectively. Compound 5c (R= 5-Cl) is next in the order of cytotoxic activity with IC50 values of 112.21 mM and 151.31 mM against HeLa cell lines and MCF cell lines respectively. The compounds (5a, 5b, 5d) exhibited moderate activity against HeLa cell lines and MCF cell lines. Among all the compounds, compound (5f) did not shown activity against HeLa cell lines.
The cytotoxic study revealed that 6-Br substitution of isatin produced more active compounds in a series.
(Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (V) have shown better yields among two series of compounds.
Among the derivatives of the (Naphthalen-1-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (IV), compound (IVe, R=6-Br) is relatively more effective cytotoxic compound against both the cell lines employed.
Among the derivatives of the (Naphthalen-2-yloxy)-acetic acid (2-oxo-1, 2-dihydro-indole-3-ylidene)-hydrazides (V), the compound with 6-Br substitution (Ve) is relatively more effective cytotoxic compound against both the cell lines employed.
ACKNOWLEDGEMENTS:
This work was supported by TallaPadmavathi College of Pharmacy and Kakatiya University. IICT provided support for the acquisition of the NMR, Mass spectrometer and elemental analyser used in this work.
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Received on 13.05.2010 Modified on 19.05.2010
Accepted on 24.05.2010 © AJRC All right reserved
Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 965-968