Cytotoxicity study of some indophenines and isatin derivatives

Abstract

Eight indophenines were synthesized for the interest of studying biological activity especially for cytotoxicity. The cytotoxicity of some indophenines and some isatin derivatives was studied by the brine shrimp lethality bioassay. It was observed that all the indophenines from thiophene, thiazol and isatin derivatives showed potential cytotoxicity against brine shrimp nauplii and the structure activity relationships (SAR) of these compounds have been reported.

Introduction

Isatin (1H-indole-2,3-dione) and its derivatives possessing an indole nucleus have arose great attention in recent years due to their wide variety of biological activities and pharmacological studies as insecticides (Bahmaria and Deliwala, 1968), fungicides (Chen and Rhodes, 1996), anti-cancer (Holla et al., 2000) and anti-inflammatory. Isatins are very important compounds due to their antifungal properties (Islam et al., 1998). Moreover, isatins are the synthetic precursors of some biologically important compounds such as quinoline 1, 2, 3-thiadiazoline (Ram et al., 1980). Besides that, the triazins [5, 6-b] indole-3-thione derivatives have attracted considerable attention in the field of medicine due to their antifungal, antimalarial and antiparasitic properties (Pal et al., 1991). Therefore, the research interest on isatins has expanded day by day. Because of the continuing interest about isatins and its derivatives (Saha et al., 1992), the present study aims at the synthesis of some indophenines from isatins, thiophene, and thiazol. This work has been carried out from the synthetic point of view along with their biological (Islam et al., 1990) property especially cytotoxicity by brine shrimp lethality bioassay (Meyer et al., 1982; Solis et al., 1993).

Materials and Methods

Physical and spectral data of the compounds were recorded on a Fischer Johns electrothermal melting point apparatus by thin disc method, on a DR-8001 Shimadzu FT-IR spectrophotometer, on a WP-400 NMR spectrophotometer using DMSO-d₆ as the solvent with tetramethylsilane as internal standard and on a Kratas MS-25 using DH-88 data system.

Isatin and substituted isatins 1a, 1b and 1c (Sandmeyer, 1919) were synthesized from the condensation of corresponding aniline with chloralhydrate and hydro xylaminehydrochloride followed by the cyclization with concentrated sulfuric acid. Again, some indophenines (Gregory et al., 1993) were synthesized from thiophene and isatin by the standard procedure. Indophenines 2a and 2b were obtained when isatin reacts with thiophene under refluxing condition in the molar ratio of 2:1 respectively in the presence of concentrated sulfuric acid. Indophenines 3a, 3b, 4a and 4b were prepared under identical conditions (as 2a and 2b) from 5-chloroisatin and 7-bromoisatin respectively. Again, indophenines 5a and 5b were prepared by the reaction between isatin and thiazol in the molar ratio of 2:1 in presence of triethylamine under intensive stirring at 80°C (Scheme 1). The cytotoxicity of the synthesized compounds was studied by brine shrimp lethality bioassay (Solis et al., 1993).

Brine shrimps (Artemia Salina) were used as test animal for the investigation of cytotoxicity.

The preferred condition for brine shrimp (temperature 28-30°C, salinity 30-35 ppt, pH 8-9, and strong aeration) was established by mixing sodium chloride salts in water. After obtaining the desired condition, about one teaspoon of brine shrimp eggs was added to the beaker. After 12 hours hatching aggregated brine shrimp nauplii were collected in another beaker and rinse with fresh water and applied for testing.

For the cytotoxicity study, dimethylsulphoxide (DMSO) was used as a solvent and the mortality of brine shrimp nauplii in this DMSO solution was almost zero. Different concentrations (150, 100 and 50 μg/mL) of each test samples were prepared with DMSO. Then 10-12 brine shrimp nauplii were transferred to each test tube using micropipette.

The numbers of survived nauplii in each test tube were counted. The percentage of mortality of brine shrimp was calculated for each sample that gives different mortality for different concentrations. An approximate linear correlation was observed when logarithm of concentration was plotted against percentage of mortality and the values of LC₅₀ were calculated for each sample. The LC₅₀ represents the concentration of a compound, which will kill, or inactive 50 percent of the test animal. LC₅₀ is inversely proportional to the toxicity of a compound, i.e. the lower the LC₅₀ the higher the toxicity.

Results

Reaction I: After recrystallization, the products 1a, 1b and 1c were obtained as brickred solids with the yield of 90% and m. p. ranging from 188°-195°C. The structures of the compounds are consistent with the physical constants and spectral data. The IR spectra of the compounds 1a-c show a sharp band at 1772 cm^-1 corresponding to the nC=O keto group and another sharp band at 1740 cm^-1 indicates the nC=O lactam. Again, the ¹H-NMR signal for >NH is also observed in the respective region as a singlet and a signal for aromatic ring as multiplet.

Reaction II: The major product from crude mass afforded 2a in 60% yield, (deep blue, λ_max 625 nm) after purification over silica gel column chromatography. IR: n_max nujul (cm^-1) 3450 cm^-1 (m, sh, nN-H, lactam), 3100 cm^-1 (sh, nC-H, aromatic), 1749 cm^-1 (sh, nC=O), 1684 cm^-1 (sh, nC=C) and 1605, 1508 cm^-1 (sh, nC=C, aromatic); ¹H-NMR: δ 10.5 (s, 1H, N-H), 7.3-7.5 (m, 4H, alkene) and 6.9-7.0 (m, 8H, aromatic); mass spectrum: m/z 426 (, 25), 213 (33), 185 (20), 153 (100), 132 (38), 89 (20) and 77 (66). And 2b was afforded as minor product (λ_max 620 nm) in 15% yields which was brown in color with the melting point 260°- 265°C and also more polar comparing to 2a. IR: 3400 cm^-1 (s, sh, nN-H), 3050 cm^-1 (sh, nC-H aromatic), 1749 cm^-1 (sh, nC=O) and 1670 cm^-1 (sh, nC=C); ¹H-NMR: δ 10.4 (s, 1H, N-H), 8.5 (d, 2H, alkene), 7.2-7.5 (m, 8H, aromatic); mass spectrum: m/z 344 (, 40), 213 (67), 185 (45), 153 (100), 132 (56), 89 (69), 77 (80).

Reaction III: The major product 3a was obtained as a blue solid having a yield of 45% (λ_max 638 nm) with m.p. >300°C after purification over silica gel column chromatography. The IR spectra of compound 3a show a band at 1670 cm^-1 corresponding to the C=C in the conjugated system and another strong band at 1749 cm^-1 is observed for C=O. ¹H-NMR: δ 10 (s, 1H, N-H), 6.7-6.9 (m, 6H, aromatic); mass spectrum: m/z 494 (, 20), 247 (39), 187 (100), 166 (40), 89 (20) and 77 (60). Again, the minor product 3b was obtained as a brownish solid having a yield of 20%. IR: n_max nujul (cm^-1) 3300 cm^-1 (s, sh, nN-H), 3100 cm^-1 (sh, nC-H, aromatic), 1747 cm^-1 (sh, nC=O), 1682 cm^-1 (sh, nC=C); ¹H-NMR: δ 10.3 (s, 1H, N-H), 7.2-7.4 (m, 6H, aromatic); mass spectrum: m/z 412 (, 25), 247 (30), 219 (35), 187 (100), 89 (59) and 77 (60).

Reaction IV: The major product 4a was obtained as a blue solid in 55% yield (λ_max 650 nm) after purification over silica gel column chromatography. IR: n_max nujul (cm^-1) 3312 cm^-1 (s, sh, nN-H), 3100 cm^-1 (sh, nC-H, aromatic), 1716 cm^-1 (sh, nC=O), 1653 cm^-1 (sh, nC=C); ¹H-NMR: δ 11.4 (s, 1H, N-H), 6.9-7.1 (m, 6H, aromatic); mass spectrum: m/z 582 (, 15), 263 (30), 231 (100), 89 (50) and 77 (70). Second product 4b was obtained as brown solid, λ_max 620 nm and m. p. 220°-225°C. IR: n_max nujul (cm^-1) 3450-3500 cm^-1 (m, sh, nN-H), 3100 cm^-1 (sh, nC-H, aromatic), 1716 cm^-1 (sh, nC=O), 1693 cm^-1 (sh, nC=C); ¹H-NMR: δ 9.3 (s, 1H, N-H), 8.1-8.2 (d, 2H, alkene), 7.2-7.4 (m, 6H, aromatic); mass spectrum: m/z 500(, 40), 291 (54), 263 (40), 231 (100) and 155 (55).

Reaction V: The crude was chromatographed on silica gel column to separate the mixture which gives the major product 5a as a blue solid in 50% yield, m. p. 290°-295°C. IR: n_max nujul (cm^-1) 3300 cm^-1 (m, sh, nN-H, lactam), 3080 cm^-1 (sh, nC-H, aromatic), 1716 cm^-1 (sh, nC=O), 1684 cm^-1 (sh, nC=N), 1645 (sh, nC=C); ¹H-NMR: δ 10.0 (s, 1H, N-H, lactam), 8.0 (s, 2H, alkene), 6.9-7.0 (m, aromatic); mass spectrum: m/z 428 (, 15), 214 (72), 186 (22), 154 (100), 132 (58), 89 (20) and 77 (21). Another product 5b was obtained as brownish solid having a yield of 20%, m. p. 175°-180°C. IR: n_max nujul (cm^-1) 3450 cm-¹ (m, sh, nN-H), 3063 cm-¹ (sh, nC-H aromatic), 1740 cm-¹ (sh, nC=O), 1682 cm-¹ (sh, nC=N), 1670 cm-¹ (sh, nC=C); ¹H-NMR: δ 10.5 (s, 1H, N-H), 9.0 (s, 1H, a, N=CH), 7.1-7.3 (m, 8H, aromatic); mass spectrum: m/z 345 (, 35), 214 (46), 186 (40), 154 (100), 89 (15) and 77 (35).

The cytotoxicity study of the synthesized compounds was investigated on brine shrimp as a test organism and the LC₅₀ values for the synthesized compounds are shown in Figure 1.

Structure activity relationship (SAR)

1. From the Figure 1, it is found that compound 1c shows the pronounced cytotoxicity property than that of 1b. On the other hand, 1b had higher cytotoxicity property than 1a, the non-substituted isatin. Thus the order of cytotoxicity activity was Br>Cl>H.

2.The indophenines 2b with the insertion of one thiophene moiety showed enhanced cytotoxicity than 1a. The compound 2a with two thiophene moieties had still higher activity than 2b.

3. The indophenines 4a and 4b with Br atom in C–7 showed very high cytotoxicity that was similar to above trend.

4. Again the indophenines 3a and 3b with Cl atom in C-5 showed different cytotoxicity against brine shrimp nauplii, 3a showed greater cytotoxicity than 3b again the similar trend was observed.

5. The indophenines 5a and 5b also showed very high cytotoxicity. The cytotoxicity was enhanced due to the insertion of hetero atom N in the thiazol ring.

6. It is to be mention here that compounds (2a-5b) with other heterocyclic systems like isatin-1,3,4-thiadiazoline (Hossain et al., 2007) it was found that indophenines with thiophene moiety enhanced the cytotoxicity 24 times than isatin-1,3,4-thiadiazoline.

Discussion

The indophenines (2a-b, 3a-b, 4a-b and 5a-b) have been synthesized from isatins, thiophene and thiazol in moderate yields. The products have been characterized with the help of spectral analysis from Japan (Tokushima University) and German (Ruhr University).

The SAR study of the synthesized compounds showed different cytotoxicity against brine shrimp. The chemical structure of a compound is very important as the relatively minor modification in the compound may results in major change in biological properties. The isatin 1a is almost inactive but the introduction of a chlorine atom in 5-position of the benzene ring (1b) enhanced the cytotoxicity. Again, insertion of a bromine atom in 7-position of the benzene ring (1c) further enhanced the cytotoxicity as 1c>1b>1a, although the position of bromine and chlorine atoms are not identical. Indophenines 2a and 2b show higher cytotoxicity than that of isatins 1a, 1b and 1c due to the presence of thiophene moieties. Again, the indophenine 2a with two thiophene moieties shows still higher cytotoxicity than that of 2b with one thiophene moiety. On the other hand, the indophenines 4a and 4b show still greater cytotoxicity against brine shrimp nauplii than that of 3a and 3b, due to the presence of bromine atom in the indophenines. Also the indophenines 5a and 5b show very high cytotoxicity property against brine shrimp nauplii. The cytotoxicity enhancement is due to the presence of hetero atom N and S in the thiazol ring. Thus the compounds 1c, 4a, 4b, 5a and 5b are very active and the compounds 2a, 2b, 3a and 3b are moderately active.

Conclusion

The cytotoxicity increases by the introduction of Br atom in the benzene ring than that of Cl atom and the greater is the numbers of heterocycles adjacent to the isatin ring, higher is the cytotoxicity. Especially the bulky molecules that contain two thiophene moieties have better cytotoxicity, which means, it hinders the replication of DNA molecule.

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