Synthesis of new Schiff bases bearing 1,2,4-triazole, thiazolidine and chloroazetidine moieties and their pharmacological evaluation.

New compounds based on oxindole moiety were synthesized via the reaction of 5-substitued isatins 1a-e with different nucleophiles such as benzidine, 3,3'-dimethoxybenzidine 2a,b and 2,6-diaminopyridine 3 to afford three different classes of bis-Schiff bases 4a-e, 5a-e and 6a-e, respectively. The structures of the new compounds were elucidated on the basis of their FTIR, 1H NMR, 13C NMR, GC/MS spectral data and elemental analysis. The in vitro antimicrobial activity of the new compounds was evaluated using a broth dilution technique in terms of minimal inhibitory concentration (MIC) against four bacterial and two fungal pathogens and anticancer activities against HELA cervix. The revealed data showed that compound 9d has excellent activity against Gram + ve and Gram -ve bacteria, and compounds 11b presented promising anticancer activity against HELA cervix. [Formula: see text].

Introduction

The oxo-derivatives of n class="Chemical">indole particularly isatins and related compounds are important class of compounds due to their biological effects, including antifungal, antiviral, anticancer and antiproliferative activities[1,]. These compounds are of great interest in oncology, microbiology and immunology[2]. Hence a significant rising research interest in the design of different oxindoles and related compounds as drugs is currently observed in the field of medicinal chemistry[3]. Schiff-bases and spiro-thiadiazoline derivatives of isatins have shown remarkable biological activities. Also oxindoles were reported earlier due to their marked cytotoxicity.

The compounds carrying n class="Chemical">azomethine functional group –C=N– which are known as Schiff bases have gained importance in medicinal and pharmaceutical fields due to the most versatile organic synthetic intermediates and also showing a broad range of biological activities, such as antituberculosis[11,], anticancer[13], analgesic and anti-inflammatory[14], anticonvulsant[15,], antibacterial and antifungal activities[17,].

Schiff bases are good intermediates n class="Chemical">for the synthesis of many heterocyclic ring systems like thiazolidinones[19] and azetidinones[20] etc.

Schiff bases are used as substrates in the preparation on class="Chemical">f a number of industrial and biologically active compounds via ring closure, cycloaddition and replacement reactions[21]. Moreover, Schiff bases derived from various heterocycles have been reported to possess cytotoxic[22], anticonvulsant[23], antiproliferative[24], antimicrobial[25] and anticancer activities[26].

Schiff bases are reported to possess antimicrobial activities. Heterocycles bearing n class="Chemical">nitrogen, sulfur and thiazole moieties constitute the core structure of a number of biologically interesting compounds[27]. Schiff base complexes derived from heterocyclic compounds have found increased interest in the context of bioinorganic chemistry.

The chemistry of n class="Chemical">1,2,4-triazole and its fused heterocyclic derivatives has received considerable attention owing to their synthetic and effective biological importance. 1,2,4-triazole moieties have been incorporated into a variety of therapeutically interesting drug candidates including antiviral (ribavarin), anti-migraine (rizatriptan), antifungal (fluconazole) and antianxiety compounds (alprazolam).The pharmacological importance of heterocycles derived from 1,2,4-triazole paved the way towards active research in a triazole chemistry[32].

In view of these reports and in n class="Chemical">continuation of our research on synthesis of biologically active molecules, we hereby report the synthesis of some new Schiff bases bearing triazole, thiazolidine and chloroazetidine moieties and evaluation of their biological activities as antimicrobial and anticancer agents.

Materials and methods

Chemistry

General remarks

All melting points are uncorrected and were determined on a Gallenkamp Instrument (London, UK). IR and NMR ren class="Chemical">corded on Perkin-Elmer-1430 infrared spectrophotometer (Waltham, MA) using the potassium bromide wafer or the Nujol mull technique for metal complexes and 1H NMR, 13C NMR spectra were measured in DMSO-d6 measured on a Varian Genini-300, 500 MHz spectrophotometer (Varian, Palo Alto, CA) and chemical shifts δ are in ppm. The mass spectra were measured on a HP GC MS-QPL000EX (Shimadzu, Tokyo, Japan) mass spectrophotometer at 70 ev. Microanalyses were carried out using a Perkin Elmer 2400 CHN elemental analyzer (Waltham, MA). The metal percentage was estimated using inductively coupled argon plasma (ICP) technique on a 6500 Duo apparatus, Thermo Scientific (Mahwah, NJ).

A 1000 mg/L multi-element and certified standard solution (Merck, Darmstadt, Germany) was used as the stock solution n class="Chemical">for instrument standardization. A microwave Digestion Lab Station closed system, Ethos Pro; Milestone, Italy was used to digest the organic matter in aqua regia. UV-Vis spectra were measured on UV-1600 spectrophotometer. The solid reflectance spectra were measured on a Shimadzu 3101 pc spectrophotometer. Magnetic susceptibilities of the metal complexes were measured at room temperature using a magnetic susceptibility Sherwood Scientific apparatus (Cambridge, UK). The molar conductance values of the metal chelates were calculated using a conductivity meter ORION model 150 with a 0.6 cell constant.

In this study, Explorer Automated Microwave Synthesis Work station (CEM) was used for the synthesis on class="Chemical">f the compounds.

Four bacterial strains and two n class="Chemical">fungal strains from the Basic Science Department, Faculty of Applied Medical Science, October 6th University were employed for minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) microbial counts.

The primary evaluation of the in vitro n class="Disease">cytotoxicity of the compounds under investigation against human tumor cells was tested at the National Cancer Institute (NCI), Cairo University, Egypt.

General procedure for the synthesis of bis N-[(1,3-dihydro)-5-substituted-indol-2-one]benzidine derivatives 4a–e and bis N-[(1,3-dihydro)-5-substituted-indol-2-one] 3,3′-dimethoxybenzidine derivatives 5a–e

Using thermal conditions

5-Substituted isatins 1a–e (0.02 mol) and each on class="Chemical">f the benzidine/3,3′-dimethoxybenzidine 2a,b (0.01 mol) were dissolved in warm ethanol (20 ml) containing glacial acetic acid (0.45 ml). The reaction mixture was refluxed for 3 h. The major part of product precipitated while hot. The solid formed on cooling was filtered, washed with hot ethanol and dried under vacuum to give 4a–e and 5a–e, respectively.

Using microwave irradiation

A mixture (0.02 mol) of n class="Chemical">5-substituted isatins 1a–e and benzidine/3,3′-dimethoxybenzidine 2a,b (0.01 mol) in the minimum quantity of ethanol (5 ml required to form a slurry) was irradiated with microwave radiation under controlled conditions [power: 150 watt, temperature: 78 °C]. On cooling, pure crystals were separated (TLC).

Bis N-[(1,3-dihydro)-2H-indol-2-one]benzidine 4a

Orange crystals, yield 50%; mp >350 °C; IR: 3174 for NH(n class="Chemical">oxindole), 3116 3001 for CH(aromatic), 1743 for C = O(oxindole) and 1612 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 10.97 (s, 2H, 2NH(oxindole), D2Oexchangeable), 7.90–6.60 (m, 16H, 4Ar–H) ppm. Anal. C28H18N4O2 (442.47): Calcd: N, 12.66. Found: N, 12.36. MS: m/z 442 (M+).

Bis N-[(1,3-dihydro)-5-methylindol-2-one]benzidine 4b

Red crystals, yield 82%; mp >350 °C; IR: 3240 (broad) for NH(n class="Chemical">oxindole), 3111 3047 for CH(aromatic), 1751 for C = O(oxindole) and 1612 for C=N cm −1. 1H NMR (DMSO d6, 300 MHz) δ: 10.84 (s, 2H, NH(oxindole), D2Oexchangeable), 7.69–6.42 (m,14H, 4Ar–H), 2.50 (s, 6H, 2CH3 of CH3–Ar) ppm. Anal. C30H22N4O2 (470.52): Calcd: C, 76.58; H, 4.71; N, 11.91. Found: C, 76.28; H, 4.62; N, 12.16. MS: m/z 471 (M+).

Bis N-[(1,3-dihydro)-5-chloroindol-2-one]benzidine 4c

Red crystals, yield 79%; mp >350 °C; IR: 3236 (broad) for NH(n class="Chemical">oxindole), 3109, 2989 for CH(aromatic), 1747 for C = O(oxindole) and 1608 for C=N cm−1. Anal. C28H16Cl2N4O2 (511.36): Calcd: C, 65.77; H, 3.15; N, 10.96; Cl, 13.87. Found: C, 65.65; H, 3.45; N, 11.10; Cl, 13.96. MS: m/z 511 (M+).

Bis N-[(1,3-dihydro)-5-nitroindol-2-one]benzidine 4d

Violet crystals, yield 58%; mp 316 °C; IR: 3255 (broad) for NH(n class="Chemical">oxindole), 3039 for CH(aromatic), 1751 for C = O (oxindole) and 1620 for C=N cm −1. 1H NMR (DMSO d6, 300 MHz) δ: 11.67(s, 2H, NH (oxindole), D2Oexchangeable), 8.37–6.66 (m, 14H, 4Ar–H). Anal. C28H16N6O6 (532.46): Calcd: N, 15.78. Found: N, 15.76. MS: m/z 532 (M+).

Bis N-[(1,3-dihydro)-5-fluoroindol-2-one]benzidine 4e

Brick red crystals, yield 59%; mp >350 °C; IR: 3298 for NH (n class="Chemical">oxindole), 3035, 2997 for CH(aromatic), 1747 for C = O(oxindole) and 1620 for C=N cm−1. Anal. C28H16F2N4O2 (478.45): Calcd: C, 70.29; H, 3.37; N, 11.71. Found: C, 70.15; H, 3.60; N, 11.41. MS: m/z 479 (M+), 480 (M++1), 481 (M++2).

Bis N-[(1,3-dihydro)-2H-indol-2-one] 3,3′-dimethoxybenzidine 5a

Orange crystals, yield 54.90%; mp 342–344 °C; IR: 3174 for NH(n class="Chemical">oxindole), 3000 for CH(aromatic), 2935 for CH(aliphatic), 1747 for C = O(oxindole) and 1608 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 10.95 (s, 2H, 2NH(oxindole), D2Oexchangeable), 7.49–6.66 (m, 14H, 4Ar–H), 3.84–3.76 (s, 6H, 2OCH3) ppm. Anal. C30H22N4O4 (502.16): Calcd: C, 71.70; H, 4.41; N, 11.51. Found: C, 72.00; H, 4.32; N, 10.86. MS: m/z 502 (M+), 503 (M++1).

Bis N-[(1,3-dihydro)-5-methylindol-2-one] 3,3′-dimethoxybenzidine 5b

Red crystals, yield 50%; mp 335–336 °C; IR: 3186 (broad) for NH(n class="Chemical">oxindole), 3000 for CH(aromatic), 2923 2835 for CH(aliphatic), 1732 for C = O(oxindole) and 1616 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 10.84 (s, 2H, 2NH(oxindole), D2Oexchangeable), 7.52–6.52 (m, 12H, 4Ar–H), 3.88–3.84 (s, 6H, 2OCH3), 2.50 (s, 6H, 2CH3 of CH3–Ar) ppm. Anal. C32H26N4O4 (530.57): Calcd: C, 72.44; H, 4.94. Found: C, 71.47; H4.70. MS: m/z 531 (M+).

Bis N-[(1,3-dihydro)-5-chloroindol-2-one] 3, 3′-dimethoxybenzidine 5c

Brick red crystals, yield 37.50%; mp 260 °C; IR: 3182 (broad) for NH(n class="Chemical">oxindole), 3082 3008 for CH(aromatic), 2966 for CH(aliphatic), 1728 for C = O(oxindole) and 1608 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 11.10 (s, 2H, 2NH(oxindole), D2Oexchangeable), 7.54–6.60 (m, 12H, 4Ar–H), 3.90–3.84 (s, 6H, 2OCH3) ppm. Anal. C30H20Cl2N4O4 (571.41): Calcd: N, 9.81; Cl, 12.41. Found: N, 9.87; Cl, 12.40. MS: m/z 571 (M+), 572 (M++1), 573 (M++2).

Bis N-[(1,3-dihydro)-5-nitroindol-2-one] 3,3′-dimethoxybenzidine 5d

Brick red crystals, yield 82.35%; mp 172–174 °C; IR: 3183 (broad) for NH(n class="Chemical">oxindole), 3115, 3014 for CH(aromatic), 2925, 2865 for CH(aliphatic), 1725 for C = O(oxindole) and 1618 for C=N cm−1. Anal. C30H20N6O8 (592.52): Calcd: C, 60.81; H, 3.40; N, 14.18. Found: C, 60.80; H, 3.70; N, 13.87. MS: m/z 593 (M+).

Bis N-[(1,3-dihydro)-5-fluoroindol-2-one] 3,3′-dimethoxybenzidine 5e

Violet crystals, yield 63.70%; mp 338–340 °C; IR: 3182, 3136 for NH (n class="Chemical">oxindole), 3077, 3011 for CH(aromatic), 2940 for CH(aliphatic), 1725 for C = O(oxindole) and 1621 for C=N cm−1. Anal. C30H20FN4O4 (538.50): Calcd: C, 66.91; H, 3.74; N, 10.40. Found: C, 66.66; H, 4.00; N, 10.06. MS: m/z 538 (M+), 539 (M++1), 540 (M++2).

General procedure for synthesis of bis N-[(1,3-dihydro)-5-substituted-indol-2-one]pyridine-2,6-diamine derivatives 6a–e

Pyridine-2,6-diamine 3 (0.004 mol, 0.436 gm) was added to a stirring solution on class="Chemical">f 5-substituted isatins 1a–e (0.008 mol) in ethanol (20 ml). The resulting mixture was refluxed at 60 °C and stirred for 3 h. The reaction mixture was cooled to room temperature and the resulting precipitate was filtered and washed with cold methanol. The solid was purified by column chromatography (20% ethyl acetate/80% petroleum ether (60–80 °C) to give pure title product.

A mixture of n class="Chemical">pyridine-2,6-diamine 3 (0.004 mol, 0.436 g) and 5-substituted isatins 1a–e (0.008 mol) in the minimum quantity of ethanol (5 ml, required to form a slurry) was irradiated with microwave radiation under controlled condition. On cooling, pure crystals were separated (TLC).

Bis N-[(1,3-dihydro)-2H-indol-2-one]pyridine-2,6-diamine 6a

Orange crystals, yield 86.9%; mp >350 °C; IR: 3166 for NH(n class="Chemical">oxindole), 3022 for CH(aromatic), 1720 for C = O(oxindole) and 1620 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 11.00 (s, 2H, NH(oxindole), D2Oexchangeable), 7.54–6.5 (m, 11H, 3Ar–H) ppm. Anal. C21H13N5O2 (367.36): Calcd: C, 68.66; H, 3.57; N, 19.06. Found: C, 68.25; H, 3.32; N, 18.86. MS: m/z 368 (M++1).

Bis N-[(1,3-dihydro)-5-methylindol-2-one]pyridine-2,6-diamine 6b

Bufn class="Chemical">f crystals, yield 48.20%; mp 238–240 °C; IR: 3197 for NH(oxindole), 3039 for CH(aromatic), 1712 for C = O(oxindole) and 1627 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 10.07 (s, 2H, NH(oxindole), D2Oexchangeable), 6.92–5.84 (m, 9H, 3Ar–H), 2.50 (s, 6H, 2CH3 of CH3–Ar) ppm. Anal. C23H17N5O2 (395.41): Calcd: N, 17.71. Found: N, 17.46. MS: m/z 397 (M++2).

Bis N-[(1,3-dihydro)-5-chloroindol-2-one]pyridine-2,6-diamine 6c

Orange crystals, yield 46.23%; mp 260 °C; IR: 3190 for NH (n class="Chemical">oxindole), 3050 for CH(aromatic), 1724 for C = O(oxindole) and 1616 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 11.09 (s, 2H, NH(oxindole), D2Oexchangeable), 7.58–6.87 (m, 9H, 3Ar–H) ppm. Anal. C21H11Cl2N5O2 (436.25): Calcd: N, 16.05; Cl, 16.25. Found: N, 16.38; Cl, 16.55. MS: m/z 437 (M++1), 440 (M++4).

Bis N-[(1,3-dihydro)-5-nitroindol-2-one]pyridine-2,6-diamine 6d

Green crystals, yield 95.23%; mp 212 °C; IR: 3200 for NH(n class="Chemical">oxindole), 3097 for CH(aromatic), 1735 for C = O(oxindole) and 1620 for C=N cm−1. Anal. C21H11N7O6 (457.36): Calcd: C, 55.15; H, 2.42; N, 21.44. Found: C, 54.89; H, 2.70. N, 21.87. MS: m/z 459 (M++2).

Bis N-[(1,3-dihydro)-5-fluoroindol-2-one]pyridine-2,6-diamine 6e

Orange crystals, yield 56.70%; mp >350 °C; IR: 3201 for NH (n class="Chemical">oxindole), 3085 for CH(aromatic), 1720 for C = O(oxindole) and 1627 for C=N cm−1. Anal. C21H11FN5O2 (403.34): Calcd: C, 62.53; H, 2.75; N, 17.36. Found: C, 62.66; H, 3.00; N, 17.06. MS: m/z 404 (M++1).

General procedures for synthesis of bis spiro[(5-methylindoline-3,2-(4H)thiazolidine)-2,4′(1H)-dione]1,1′-biphenyl 7a and bis spiro[(indoline-3,2-(4H)thiazolidine)-2,4′(1H)-dione]3,3′-dimethoxybenzidine 7b

[Method A]

A solution of n class="Chemical">Schiff bases 4b, 5a (0.001 mol) and mercaptoacetic acid (0.002 mol) in dry dioxane (50 ml) in presence of anhydrous ZnCl2 was refluxed for 6–10 h. After completion of the reaction excess of solvent was removed through distillation, and sticky solid obtained was poured onto crushed ice, then filtered, dried and recrystallized from ethanol to give 7a,b.

[Method B]

A mixture of n class="Chemical">Schiff bases 4b, 5a (0.001 mol) and mercaptoacetic acid (0.002 mol) was taken in DMF in a round-bottom flask fitted with a Dean Stark apparatus. The mixture was refluxed for 6 h with removal of water azeotropically. A sticky solid was formed on evaporating of solvent and was treated with a solution of sodium bicarbonate to remove excess of acid. The solid formed was filtered, washed with water, dried and recrystallized from ethanol to give 7a,b.

Bis spiro[(5-methylindoline-3,2-(4H)thiazolidine)-2,4′(1H)-dione]1,1′-biphenyl 7a

Orange crystals, yield 53%; mp 187–179 °C; IR: 3291(broad) for NH(n class="Chemical">oxindole), 3071, 3030 for CH(aromatic), 2915, 2828 for CH(aliphatic), 1700 for C = O(oxindole), 1679 for C = O(thiazolidine), 1246 for C–N and 638 for C–S–C cm−1. 1H NMR (DMSO d6,300 MHz) δ: 10.44 (s, 2H, NH(oxindole)), 8.27–6.47 (m,12H, 4Ar-H), 5.04 (s, 4H, 2CH2 of thiazolidinone), 2.46 (s, 6H, 2CH3 of CH3–Ar) ppm, 13C NMR (DMSO, 500 MHz) δ: 167.53, 167.26, 138.52, 137.15, 135.21, 135.11, 127.79, 127.02, 126.57, 126.26, 125.87 125.73, 120.86, 120.31, 119.33, 119.11, 118.87, 118.69, 118.44, 117.84, 113.63, 48.05, 46.78, 28.96, 28.16. Anal. C34H26N4O4S2 (618.12): Calcd: C, 66.00; H, 4.24; N, 9.06; S, 10.36. Found: C, 66.29; H, 4.25; N, 9.31; S, 10.45. MS: m/z 618 (M+).

Bis spiro[(indoline-3,2-(4H)thiazolidine)-2,4′(1H)-dione]3,3′-dimethoxybenzidine 7b

Yellow crystals, yield 58%; mp 218–220 °C; IR: 3247 for NH(n class="Chemical">oxindole), 3069, 3000 for CH(aromatic), 2927, 2873 for CH(aliphatic), 1681 for C = O(oxindole), 1658 for C = O(thiazolidine), 1257 for C–N and 675 for C–S–C cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 9.65 (s, 2H, NH(oxindole), D2Oexchangeable), 8.29–7.19 (m, 12H, 4Ar–H), 4.28–3.96 (s, 4H, 2CH2 of thiazolidinone), 3.85–3.82 (s, 6H, 2OCH3) ppm. Anal. C34H26N4O6S2 (650.13): Calcd: S, 9.86. Found: S, 9.60. MS: m/z 650 651(M++1) and 652 (M++2).

General procedures for synthesis of bis spiro[(5-methylindoline-3,2-(2H)-3-chloro-azetidine)-2,4′(1H)-dione]1,1′-biphenyl 8a and bis spiro[(indoline-3,2-(2H)-3-chloro-azetidine)-2,4′(1H)-dione]-3,3′-dimethoxybenzidine 8b

To a solution of n class="Chemical">Schiff bases 4b, 5a (0.001 mol) in DMF (30 ml), chloroacetyl chloride (0.002 mol) and triethylamine (0.002 mol) were added at 0–5 °C with constant stirring. The reaction mixture was refluxed on water bath for 7 h, and then excess of solvent was distilled off. The sticky solid was cooled, poured on ice water then filtered, further recrystallized from ethanol to give the title products.

Bis spiro [(5-methylindoline-3,2-(2H)-3-chloroazetidine)-2,4′(1H)-dione]1,1′-biphenyl 8a

Brown crystals, yield 52%; mp 148–150 °C; IR: 3267(broad) for NH(n class="Chemical">oxindole), 3103, 3071 for CH(aromatic), 2885 for CH(aliphatic), 1777 for C = O(azetidine), 1739 for C = O(oxindole), 1204 for C–N and 756 for C–Cl cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 11.19 (s, 2H, 2NH(oxindole)), 7.91–7.04 (m,16H, 4Ar-H), 5.83 (s, 2H, 2CH-Cl) ppm, 13C NMR (DMSO, 500 MHz) δ: 171.71, 169.16, 165.318, 163.36 163.06, 162.26, 160.86, 142.36, 135.55, 128.02, 127.90, 127.27, 126.64, 126.47, 122.52, 121.07, 120.54, 119.64, 116.95, 112.30, 111.27, 66.94, 63.83, 62.35. Anal. C32H20N4O4Cl2 (594.09): Calcd: C, 64.55; H, 3.39; N, 9.41; Cl, 11.91. Found: C, 64.76; H, 5.22; N, 9.31; Cl, 12.01. MS: m/z 595 (M++1).

Bis spiro [(indoline-3,2-(2H)-3-chloroazetidine)-2,4′(1H)-dione]-3,3′-dimethoxybenzidine 8b

Olive crystals, yield 67%; mp 196–198 °C; IR: 3217(broad) n class="Chemical">for NH(oxindole), 3100, 3078 for CH(aromatic), 2939, 2839 for CH(aliphatic), 1778 for C = O(azetidine), 1739 for C = O(oxindole), 1207 for C–N and 756 for C–Cl cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 10.90,10.81 (s, 2H, 2NH(oxindole), D2Oexchangeable), 8.18–6.75 (m,16H, 4Ar–H), 5.71–5.59 (s, 2H, 2CH–Cl), 3.94–3.89 (s, 6H, 2OCH3) ppm, 13C NMR (DMSO, 500 MHz) δ: 173.35, 172.22, 166.99, 162.03, 161.57, 149.76, 148.92, 143.34, 142.76, 138.45, 138.25, 131.58, 130.53, 125.46, 123.91, 122.87, 122.53, 121.77, 120.45, 119.33, 118.79, 69.696, 64.14, 62.67, 55.85, 54.712. Anal. C34H24N4O6Cl2 (654.11): Calcd: N, 8.55; Cl, 10.82. Found: N, 8.26; Cl, 10.55. MS: m/z 655 (M++1).

General procedures for synthesis of bis spiro[(3H)indole-3,3′-(3H)1,2,4-triazole-2–(1H)-one] 1,1′-biphenyl 9a and bis spiro[(5-substituted)indole-3,3′-(3H)1,2,4-triazole-2-(1H)-one]-3,3′-dimethoxybenzidines 9b–e

A mixture of n class="Chemical">Schiff bases 5a–d (0.01 mol) and thiosemicarbazide (0.02 mol) in a little amount of ethanol made as slurry was irradiated under MW. The solid product obtained after cooling was filtered and recrystallized from ethanol to give spiro compounds 9a–e.

Bis spiro[(3H)indole-3,3′-(3H)-1,2,4-triazol-2-(1H)-one] 1,1′-biphenyl 9a

Yellow crystals, yield 57%; mp 278–280 °C; IR: 3399 for NH2, 3328 n class="Chemical">for NH(triazole), 3195 for NH(oxindole), 3030 for CH(aromatic), 1694 for C = O(oxindole), 1616 for C=N and 1263 for C–N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 12.44 (s,2H,NH(oxindole) D2Oexchangeable), 11.21 (s, 2H, NH(triazole), D2Oexchangeable), 9.00, 8.62 (s, 4H, 2NH2, D2Oexchangeable), 7.61–6.86 (m, 14H, 4Ar–H) ppm, 13C NMR (DMSO, 500 MHz) δ: 179.19 178.63, 163.15, 158.65, 142.88 142.83, 132.58, 132.17, 131.40, 123.47, 122.31, 121.48, 120.47, 112.06, 111.02, 100.01, 72.60. Anal. C30H24N10O2 (556.21): Calcd: N, 25.17. Found: N, 25.82. MS: m/z 556 (M+).

Bis spiro [(3H) indole-3, 3′-(3H)-1,2,4-triazol-2–(1H)-one]-3,3′-dimethoxybenzidine 9b

Yellow crystals, yield 58.27%; mp 260–2 °C; IR: 3340 for NH2, 3260 n class="Chemical">for NH(triazole), 3166 for NH(oxindole), 3062 3012 for CH(aromatic), 2885 for CH(aliphatic), 1674 for C = O(oxindole), 1624 for C=N and 1276 for C–N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 12.46 (s, 2H, NH(oxindole) D2Oexchangeable), 11.15 (s, 2H, NH(triazole), D2Oexchangeable), 9.00,8.63 (s, 4H, 2NH2, D2Oexchangeable), 7.61–6.87 (m, 14H, 4Ar–H), 3.79 (s, 6H, 2OCH3) ppm, 13C NMR (DMSO, 500 MHz) δ: 179.19, 163.15 142.89 142.84, 141.79, 132.58 132.17, 131.40, 126.98, 123.46, 122.33, 121.49, 120.49, 112.08, 111.04, 83.15 (spiro C), 32.26 . Anal. C32H28N10O4 (616.63): Calcd: C, 62.33; H, 4.58; N, 22.71. Found: C, 62.00; H, 4.68; N, 23.12. MS: m/z 616 (M+).

Bis spiro [5-methylindole-3,3′-(3H)-1,2,4-triazol-2-(1H)-one]-3,3′-dimethoxybenzidine 9c

Violet crystals, yield 66.60%; mp 286–8 °C; IR: 3417 for NH2, 3255 n class="Chemical">for NH(triazole), 3166 for NH(oxindole), 3066 for CH(aromatic), 2931 2888 for CH(aliphatic), 1689 for C = O(oxindole), 1612 for C=N and 1261 for C–N cm−1. Anal. C34H32N10O4 (644.26): Calcd: N, 21.73. Found: N, 21.90. MS: m/z 644 (M+), 645 (M++1), 647 (M++3).

Bis spiro [5-chloroindole-3,3′3H)-1,2,4-triazol-2–(1H)-one]-3,3′-dimethoxybenzidine 9d

Yellow crystals, yield 77.30%; mp 305–8 °C; IR: 3413 for NH2, 3267 n class="Chemical">for NH(triazole), 3166 for NH(oxindole), 3062 for CH(aromatic), 2875 for CH(aliphatic), 1681 for C = O(oxindole), 1612 for C=N and 1253 for C–N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 12.29 (s, 2H, 2NH(oxindole), D2Oexchangeable), 11.26 (s, 2H, 2NH (triazole), D2Oexchangeable), 9.08, 8.78 (s, 4H, 2NH2, D2Oexchangeable), 7.75–6.92 (m, 14H, 4Ar–H), 3.31 (s, 6H, 2OCH3) ppm. Anal. C32H26Cl2N10O4 (684.15): Calcd: N, 20.43; Cl, 10.34. Found: N, 20.23; Cl, 10.48. MS: m/z 685 (M++1).

Bis spiro [5-nitroindole-3,3′-(3H)-1, 2, 4-triazol-2-(1H)-one]-3,3′-dimethoxybenzidine 9e

Green crystals, yield 63.75%; mp 320–22 °C; IR: 3371 for NH2, 3247 n class="Chemical">for NH(triazole), 3193 for NH(oxindole), 3066, 3008 for CH(aromatic), 2812 for CH(aliphatic), 1697 for C = O(oxindole), 1616 for C=N and 1257 for C–N cm−1. Anal. C32H26N12O8 (706.62): Calcd: C, 54.39; H, 3.71; N, 23.79. Found: C, 54.66; H, 4.00; N, 23.99. MS: m/z 706(M+).

General procedures for synthesis of bis N-[(1-morpholinomethyl)indolin-2-one]pyridine 2,6-diamines 10a,c and bis N-[(1-piperidinomethyl)indolin-2-one]pyridine-2,6-diamines 10b,d

A slurry consisting on class="Chemical">f the Schiff bases 6a,b (0.001 mol), absolute ethanol (2 ml) and 37% formalin (0.3 ml). To this slurry secondary amine (0.002 mol) was added drop wise, with cooling and shaking. The reaction mixture was irradiated for an appropriate time until the completion of the reaction, as the reactants disappeared (TLC). On cooling, crystals separated out were recrystallized from ethanol to give 10a–d.

Bis N-[(1-morpholinomethyl) indolin-2-one] pyridine 2,6-diamine 10a

Orange crystals, yield 55%; mp 216–8 °C; IR: 3039 for CH (aromatic), 2947, 2893, 2854, 2831 n class="Chemical">for CH(aliphatic), 1735 for C = O(oxindole) and 1612 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 7.69–7.12 (m, 11H, 3Ar–H),4.39 (s, 4H, 2NCH2N), 3.56–3.54 (t, 8H, 2CH2–O–CH2), 2.57–2.50 (t, 8H, 2CH2–N–CH2) ppm, 13C NMR (DMSO, 500 MHz) δ: 183.66, 159.48, 151.91, 139.25, 138.58, 138.39, 125.55, 124.65, 124.37, 123.94, 123.19, 118.084, 112.04, 111.93, 67.63, 67.39, 66.54, 66.31, 62.32, 51.29, 50.96, 50.36, 50.00. Anal. C31H31N7O4 (565.62): Calcd: C, 65.83; H, 5.52; N, 17.33. Found: C, 66.03; H, 5.59; N, 17.54. MS: m/z 565 (M+).

Bis N-[(1-piperidinomethyl) indolin-2-one] pyridine-2,6-diamine 10b

Orange crystals, yield 53%; mp 300 °C; IR: 3055 for CH(aromatic), 2935, 2854, 2804 n class="Chemical">for CH(aliphatic), 1716 for C = O(oxindole) and 1612 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 7.13–6.68 (m, 11H, 3Ar–H),4.37 (s, 4H, 2NCH2N), 2.249 (t, 8H, 2CH2–N–CH2) and 1.369 (m, 12H, 6CH2) ppm. Anal. C33H35N7O2 (561.68): Calcd: C, 70.57; H, 6.28; N, 17.46. Found: C, 70.23; H, 5.98; N, 17.87. MS: m/z 562 (M+).

Bis N-[(1-morpholinomethyl)-5-methylindolin-2-one] pyridine-2,6-diamine 10c

Orange crystals, yield 53.5%; mp 118–120 °C; 3088 for CH(aromatic), 2923, 2839, 2804 n class="Chemical">for CH(aliphatic), 1712 for C = O(oxindole) and 1589 for C=N cm−1. 1H NMR (DMSO d6, 300 MHz) δ: 7.44–7.13 (m, 9H, 3Ar–H),4.33 (s, 4H, 2NCH2N), 3.51–3.49 (t, 8H, 2CH2–O–CH2), 2.51–2.49 (t, 8H, 2CH2–N–CH2), 2.25 (s, 6H, 2CH3 of CH3–Ar) ppm. Anal. C33H35N7O4 (593.68): Calcd: N, 16.52. Found: N, 16.89. MS: m/z 594(M+).

Bis N-[(1-piperidinomethyl)-5-methylindolin-2-one] pyridine-2,6-diamine 10d

Orange crystals, yield 51%; mp 150–151 °C; 3028 for CH(aromatic), 2931, 2854, 2804 n class="Chemical">for CH(aliphatic), 1720 for C = O(oxindole) and 1600 for C=N cm−1. Anal. C35H39N7O2 (589.73): Calcd: N, 16.63. Found: N, 16.23. MS: m/z 590 (M++1), 592 (M++2), 593 (M++3).

General procedures for the synthesis of mononuclear Cu(II), Co(II) and Ni(II) macrocyclic complexes of bis-N-[(1,3-dihydro)-5-substitutedindol-2-one] pyridine-2,6-diamine 11a–f

To a stirring methanolic solution on class="Chemical">f 2,6-diamine pyridine 3 (0.01 mol), metal chloride (0.005 mol) dissolved in a minimum quantity of methanol was added. The resulting solution was refluxed for 30 min. Then 5-substituted isatins 1b,c (0.01 mol) dissolved in methanol (20 ml) was added to the refluxing mixture and complete reflux for 10 h. The reaction mixture was concentrated, the crystals obtained were filtered, washed with methanol, ether and dried in vacuum.

Synthesis of mononuclear Cu (II) macrocyclic complex of bis N-[(1, 3-dihydro)-5-methyl-indol-2-one] pyridine-2,6-diamine 11a

Brown crystals, yield 57.80%; mp >300 °C; IR: 3201 for NH, 1612 n class="Chemical">for C=N and 505 for M–N cm−1. UV λmax: 767, 737, 657, 516, 480, 393, 242 nm. μ eff: 1.8 B.M. Anal. C28H20CuCl2N8 (601.05): Calcd: N, 18.58; Cu, 10.54; Cl, 11.76. Found: N, 18.90; Cu, 10.41; Cl, 11.72.

Synthesis of mononuclear Cu (II) macrocyclic complex of bis N-[(1, 3-dihydro)-5-chloro-indol-2-one] pyridine-2, 6-diamine 11b

Violet crystals yield 56.89%; mp >300 °C; IR: 3213 for NH, 1612 n class="Chemical">for C=N and 525 for M–N cm−1. UV λmax: 752, 736, 706, 675, 658, 515, 506, 472, 393, 370, 333, 314, 234 nm. μ eff: 2.1 B.M. Anal. C26H14CuCl4N8 (640.94): Calcd: N, 17.41; Cu, 9.87; Cl, 22.03. Found: N, 16.70; Cu, 9.45; Cl, 21.98.

Synthesis of mononuclear Co (II) macrocyclic complex of bis N-[(1,3-dihydro)-5-methyl-indol-2-one] pyridine-2, 6-diamine 11c

Brown crystals, yield 67.90%; mp 320–22 °C; 3186 for NH, 1620 n class="Chemical">for C=N and 516 for M–N cm−1. UV λmax: 769, 734, 701, 656, 616, 510, 472, 433, 400, 369, 313, 254 nm. μ eff: 3.9 B.M. Anal. C28H20CoCl2N8 (597.05): Calcd: Co, 9.85; Cl, 11.85. Found: Co, 9.73; Cl, 11.84.

Synthesis of mononuclear Co (II) macrocyclic complex of bis N-[(1, 3-dihydro)-5-chloro-indol-2-one] pyridine-2, 6-diamine 11d

Olive crystals, yield 60.10%; mp >300 °C; 3209 n class="Chemical">for NH, 1616 for C=N and 555 for M–N cm−1. UV λmax: 769, 735, 697, 664, 514, 473, 438, 396, 370, 314, 243 nm. μ eff: 3.5 B.M. Anal. C26H14CoCl4N8 (636.94): Calcd: N, 17.53; Co, 9.22; Cl, 22.19. Found: N, 17.27; Co, 9.00; Cl, 21.99.

Synthesis of mononuclear Ni (II) macrocyclic complex of bis N-[(1,3-dihydro)-5-methyl-indol-2-one] pyridine-2,6-diamine 11e

Olive crystals, yield 45.70%; mp >300 °C; 3136 n class="Chemical">for NH, 1620 for C=N and 505 for M–N cm−1. UV λmax: 768, 737, 698, 659, 519, 498, 439, 411, 381, 320, 219 nm. μ eff: 2.6 B.M. Anal. C28H20NiCl2N8 (596.05): Calcd: Ni, 9.81; Cl, 11.85. Found: Ni, 9.67; Cl, 11.97.

Synthesis of mononuclear Ni (II) macrocyclic complex of bis N-[(1,3-dihydro)-5-chloro-indol-2-one] pyridine-2, 6-diamine 11f

Brown crystals, yield 55.30%; mp 278–280 °C; 3213 for NH, 1616 n class="Chemical">for C=N and 520 for M–N cm−1. UV λmax: 767, 738, 696, 674, 616, 520, 472, 432, 384, 326, 266, 212 nm. μ eff: 2.74 B.M. Anal. C26H14NiCl4N8 (635.94): Calcd: N, 17.54; Ni, 9.19; Cl, 22.19. Found: N, 17.21; Ni, 10.00; Cl, 22.21.

Pharmacological evaluation

In vitro antimicrobial measurement

The compounds were tested for their in vitro antimicrobial activity by the broth-dilution technique in terms of minimum inhibitory concentrations (MIC). Experimentally for potent Drugs[42]. The antimicrobial activities of the compounds in this study were evaluated against six pathogenic microbial species: Gram + ve bacteria Staphylococcus aureus and Staphylococcus epidermidis, Gram –ve bacteria Escherichia coli and Klebsiella pneumonia and fungi Aspergillus fumigatu, and Candida albicans. Reference drugs used were sulfamethoxazole as an antibacterial standard and fluconazole as an antifungal standard.

In vitro cytotoxicity evaluation

The primary evaluation of in vitro n class="Disease">cytotoxicity of the selected new compounds against human tumor cells was carried out at the NCI (Cairo University, Cairo, Egypt) using the method of Skehan and Storeng[43]. The cytotoxicity evaluation also involved the use of vinblastine sulfate or doxorubicin[44] as antitumor drug reference standards. The procedure used was as follows:

Cells were plated in 96-multiwell plate (105 cells/well) for 24 h. ben class="Chemical">fore treatment with the compound to allow attachment of the cell to the wall of the plate.

Difn class="Chemical">ferent concentrations of the compound under test (0, 1, 2.5, 5 and 10 mg/mL) were added to the cell monolayer triplicate walls that were prepared for each individual dose.

Monolayer cells were incubated with the n class="Chemical">compound for 48 h. at 37 °C and in atmosphere of 5% CO2.

After 48 h, the cells were n class="Chemical">fixed, washed and stained with sulfo-Rhodamine-B.

Excess stain was washed away with acetic acid and attached stain was ren class="Chemical">covered with Tris–EDTA buffer.

Color intensity was measured with an ELISA reader.

The relationship between the surviving fraction and drug n class="Chemical">concentration is plotted to give the survival curve of cancer breast cell line.

The results of the in vitro n class="Disease">cytotoxicity activity on human tumor cell line HELA (cervix) were determined according to the dose values of the drug exposure required to reduce survival in the cell lines to 50%.

Results and discussion

This study involve the synthesis of three din class="Chemical">fferent classes of bis-Schiff bases derived from three different diamines namely benzidine (4,4′-diamino-1,1′-biphenyl), 3,3′-dimethoxy-benzidine and 2,6-diaminopyridine. Thus condensation of (2 mol) of 5-substituted isatins 1a–e with (1 mol) of diamines such as benzidine, 3,3′-dimethoxybenzidine 2a,b and 2,6-diamino-pyridine 3 in ethanol at ambient temperature gave the desired bis-Schiff bases named by bis N-[(1,3-dihydro)-2H-indol-2-one] 4,4′-diamino-1,1′-biphenyl derivatives 4a–e, bis N-[(1,3-dihydro)-2H-indol-2-one] 3,3′-dimethoxybenzidine derivatives 5a–e and bis N-[(1,3-dihydro)-2H-indol-2-one]pyridine 2,6-diamine derivatives 6a–e, respectively, as illustrated in Scheme 1. The structure of these compounds was confirmed by elemental analysis, FTIR, 1H NMR and MS. 1H NMR spectrum of compound 4a showed bands at δ: 10.97 (s, 2H, 2NH(oxindole), D2Oexchangeable), 7.90–6.60 (m, 16H, 4Ar–H) ppm.

Scheme 1.

Synthesis of the target compounds 4a–e, 5a–e and 6a–e.

Synthesis of the target n class="Chemical">compounds 4a–e, 5a–e and 6a–e.

This study was extended to prepare new heterocycles such as bis spirothiazolidin-4-one derivatives 7a,b, bis spiroazetidinone derivatives 8a,b and bis spiron class="Chemical">1,2,4-triazole derivatives 9a–e, respectively by the reaction of bis-Schiff bases 4b, 5a–d with mercaptoacetic acid in the presence of anhydrous ZnCl2, chloroacetyl chloride in presence of triethylamine at temperature 80 °C and thiosemicarbazide as illustrated in Scheme 2. The structure of these compounds was confirmed by FTIR, 1H NMR 13C NMR, and MS spectra and elemental analysis. The 1H NMR spectrum of bis spiro[(3H)indole-3,3′3H)-1,2,4-triazol-2-(1H)-one]-3,3′-dimethoxybenzidine 9b showed bands at δ: 12.46 (s, 2H, NH(oxindole), D2Oexchangeable), 11.15 (s, 2H, NH(triazole), D2Oexchangeable), 9.00,8.63 (s, 4H, 2NH2, D2Oexchangeable),7.61–6.87 (m, 14H, 4Ar–H),3.79 (s, 6H, 2OCH3) ppm. The 13C NMR spectrum of bis spiro[(3H)indole-3,3′-(3H)-1,2,4-triazol-2-(1H)-one]-3,3′-dimethoxybenzidine 9b showed bands at δ: 179.19, 163.15, 142.89 142.84, 141.79, 132.58 132.17, 131.40, 126.98, 123.46, 122.33, 121.49, 120.49, 112.08, 111.04, 83.15(spiro C), 32.26.

Scheme 2. Synthesis of the target compounds 7a,b, 8a,b and 9a–e.

Scheme 2. Synthesis of the target n class="Chemical">compounds 7a,b, 8a,b and 9a–e.

Mechanism for the synthesis on class="Chemical">f target compounds 9a–e.

The reaction of n class="Chemical">bis-Schiff bases 6a,b with formaldehyde in the presence of a secondary amines such as piperidine and morpholine yielded the N-Mannich bases 10a–d, respectively, as illustrated in Scheme 4. The structure of these compounds was established from FTIR, 1H NMR, 13C NMR and MS spectra and elemental analysis. The 1H NMR spectrum of bis N-[(1-morpholinomethyl) indolin-2-one] pyridine 2,6-diamine 10a showed bands at δ: 7.69–7.12 (m, 11H, 3Ar–H),4.39 (s, 4H, 2NCH2N), 3.56–3.54 (t, 8H, 2CH2–O–CH2), 2.57–2.50 (t, 8H, 2CH2–N–CH2) ppm. 13C NMR spectrum of bis N-[(1-morpholinomethyl)indolin-2-one]pyridine 2,6-diamine 10a showed bands at δ: 183.66, 159.48, 151.91, 139.25, 138.58, 138.39, 125.55, 124.65, 124.37, 123.94, 123.19, 118.084, 112.04, 111.93, 67.63, 67.39, 66.54, 66.31, 62.32, 51.29, 50.96, 50.36, 50.00.

Scheme 4.

Synthesis of the target compounds 10a–d and 11a–f.

Synthesis of the target n class="Chemical">compounds 10a–d and 11a–f.

The complexes 11a–n class="Chemical">f were prepared by condensation reaction of 2,6-diamine pyridine with 5-substituted isatins 1b,c in the presence of MCl2.nH2O salts (where M = Cu, Co or Ni).The structure of metal complexes 11a–f was confirmed by elemental analysis and spectral studies. The elemental analysis showed a ratio of 2:2:1 [isatins:DAP:MCl2] as shown in Scheme 4. The theoretical values were in a good agreement with the found values. The presence of chlorine confirmed from element analysis and the low molar conductance values (15–82.30 S cm2mol−1) for the complexes 11a–f supports the non-electrolytic nature of the metal complexes.

IR spectral studies and mode of coordination of complexes 11a–f

In the spectrum of n class="Chemical">2,6-diaminopyridine a pair of medium intensity bands present at 3375–3400 cm−1 corresponding to (NH2) but these are absent in the infrared spectra of all the complexes. Further, no strong absorption band was observed at 1750–1700 cm−1 indicating the absence of (C = O) group of 5-substituted isatins. This indicates that the condensation of carbonyl groups of 5-substituted isatins and amino groups of pyridine – 2, 6-diamino might have taken place. These results provide strong evidence for the formation of macrocyclic frame. A strong absorption band in the region 1620–1612 cm−1 may be attributed to the (C=N) group. The lower values of (C=N) may be explained on the basis of drift of lone pair density of azomethine nitrogen towards the metal atom. The presence of band in the region 3213–3136 cm−1 in the isatin complexes may be assigned due to (N–H) stretching. New bands in the 555–505 cm−1 regions are assigned to stretching frequencies of (M–N) bonds. The unchanged pyridine ring vibrations in the complexes indicate non-coordination of the pyridine nitrogen atom. Moreover, the coordination through pyridine nitrogen is also ruled out on the basis that it will result in the formation of four membered heterocyclic rings, which are sterically unstable. Thus, in the presence of metal salts, a quadridentate macrocycle is formed which coordinates through suitably placed azomethine nitrogen while pyridine nitrogens do not take part in the coordination.

Electronic spectra and magnetic moment studies 11a–f

The magnetic susceptibility measurement for the solid n class="Chemical">Cu (II) complexes (1.8–2.1 B.M) is indicative of octahedral environment. The diffuse reflectance spectrum of the copper complexes 11a,b showed two band groups at 393–314 and 242–234 nm, these bands can be attributed to π–π* and n–π* transition states within the hydrazone ligand. Bands at 767–472 nm can be attributed to d–d transition states and ligand to metal charge transfer.

The magnetic susceptibility measurement for the solid n class="Gene">Co (II) complexes (3.50–3.90 B.M) is indicative of three unpaired electrons per Co (II) ion suggesting consistency with their octahedral environment. The diffuse reflectance spectrum of the cobalt complexes 11c,d showed two band groups at 396–313 and 254–243 nm, these bands can be attributed to π–π* and n–π* transition states within the hydrazone ligand. Bands at 769–400 nm can be attributed to d–d transition states and ligand to metal charge transfer.

The magnetic susceptibility measurement for the solid n class="Chemical">Ni (II) complexe (2.60–2.74 B.M) is indicative of octahedral environment. The diffuse reflectance spectrum of the nickel complexes 11e,f showed two band groups at 411–320 and 266–212 nm, these bands can be attributed to π–π* and n–π* transition states within the hydrazone ligand. Bands at 768–432 nm can be attributed to d–d transition states and ligand to metal charge transfer.

Pharmacological evaluation

In vitro antimicrobial measurement

Most of the synthesized n class="Chemical">compounds were tested for their in vitro antimicrobial activity by the broth-dilution technique in terms of MIC, MBC and minimum fungicidal concentration (MFC) [Experimentally, for potent drugs the MBC and MFC is usually 2 to 4 x the MIC for the same isolate.] The antimicrobial activity of the compounds against six pathogenic microbial species are present in Tables 1–3. The study also included the activity of reference compounds Sulfamethoxazole as antibacterial agent and Fluconazole as antifungal agent. From the data obtained, the following conclusion can be drawn:

Inhibition zone diameter in (mm) as a criterion of antibacterial and antin class="Chemical">fungal activities of the new synthesized compounds.

MIC in μg/ml of the new synthesized n class="Chemical">compounds.

MBC and Mn class="Chemical">FC in μg/ml of the new synthesized compounds.

MBC: Minimum Bactericidal n class="Chemical">Concentration; MFC: Minimum Fungicidal Concentration.

Schiff bases and n class="Chemical">bis-Schiff bases based on oxindole moiety have low antimicrobial activity compared to synthesized heterocyclic compounds obtained by formation of thiazolodine, azetidinone, 1,2,4 triazole and Mannich base derivatives.

Bis spiro thiazolodine 7b with 3,3′-dimethoxyn class="Chemical">benzidine fragment according to MICs count gave the highest antimicrobial activity of the thiazolodine derivatives.

All the azetidinone derivatives nearly gave the same activities.

Bis spiro 1,2,4 triazole derivatives showed remarkable activity and gave relative values on class="Chemical">f the reference drugs and may serve as useful lead compounds in search for potent antimicrobial agent.

Mannich bases with n class="Chemical">morpholine moiety 10a showed more activity compared to those with piperidine moiety 10b.

In vitro cytotoxicity evaluation of mononuclear Cu(II), Co(II) and Ni(II) macrocyclic complexes 11a–f

The activities of three din class="Chemical">fferent macrocyclic metal complexes 11a–f were performed against the cervix human cancer cell line (HELA). Unexpected low values of activity were obtained with the three series of macrocyclic complexes although the copper complex 11b gave remarkable activity comparable to the reference drug, also the activity obtained with the copper complex 11a may be considered as moderate one Figure 1.

Figure 1.

The cytotoxicity data of the activity of compounds 11a–f against cervix (HELA) tumor cell line compared to Vinblastine sulfate IC50:10.9.

The cytotoxicity data on class="Chemical">f the activity of compounds 11a–f against cervix (HELA) tumor cell line compared to Vinblastine sulfate IC50:10.9.

Conclusion

In this study, we report a convenient route for the synthesis of some novel heterocycles incorporating oxindole moiety in order to investigate their antimicrobial, antifungal activity. The in vitro evaluation of their antimicrobial against several pathogenic bacterial and fungal strains revealed that compound 9d showed the highest activity against Gram + ve and Gram –ve bacteria. The activities of three different macrocyclic metal complexes 11a–f were performed against the cervix human cancer cell line (HELA) .Unexpected low values of activity were obtained with the three series of macrocyclic complexes although the copper complex 11b gave remarkable activity comparable to the reference drug, also the activity obtained with the copper complex 11a may be considered as moderate one.

Table 1.

Inhibition zone diameter in (mm) as a criterion of antibacterial and antifungal activities of the new synthesized compounds.

Microorganism inhibition zone diameter(mm)
	Gram + ve bacteria		Gram − ve bacteria		Fungi
Compounds	S. aureus	S. epidermidis	E. coli	K. pneumonia	A. fumigatu	C. albicans
4a	16	16	19	15	10	13
4b	16	15	18	14	11	12
5a	17	15	17	14	10	11
5c	17	14	16	14	12	13
6a	16	18	19	15	13	15
7a	26	26	25	25	14	20
7b	25	30	30	27	17	21
8a	26	26	27	25	14	16
8b	26	28	25	23	15	15
9a	32	33	34	30	21	25
9d	34	35	36	32	23	25
10a	30	27	31	27	14	18
10b	30	29	30	29	16	19
10c	31	28	29	30	14	18
Sulfamethoxazole	36	36	39	33	–	–
Fluconazole	–	–	–	–	26	28

Table 2.

MIC in μg/ml of the new synthesized compounds.

Microorganism minimum inhibitory concentration
	Gram + ve bacteria		Gram − ve bacteria		Fungi
Compounds	S. aureus	S. epidermidis	E. coli	K. pneumonia	A. flavus	C. albicans
4a	24	24	24	48	48	48
4b	24	48	48	24	48	48
5a	24	48	48	48	48	48
5c	48	48	48	48	48	48
6a	48	24	24	48	48	24
7a	12	12	12	12	12	12
7b	6	6	6	6	12	6
8a	12	12	12	24	24	24
8b	12	6	12	24	24	12
9a	3	3	6	6	6	6
9d	3	3	3	3	6	6
10a	6	6	6	12	12	12
10b	12	12	12	12	24	24
10c	6	6	6	12	12	12
Sulfamethoxazole	3	3	3	3	–	–
Fluconazole	–	–	–	–	3	3

MIC: Minimum Inhibitory Concentration.

Table 3.

MBC and MFC in μg/ml of the new synthesized compounds.

Microorganism minimum bactericidal and fungicidal concentration
	Gram + ve bacteria		Gram − ve bacteria		Fungi
Compounds	S. aureus	S. epidermidis	E. coli	K. pneumonia	A. flavus	C. albicans
7a	24	24	24	24	48	48
7b	24	24	24	24	24	12
8a	48	48	24	48	48	96
8b	48	48	24	48	96	48
9a	12	12	24	24	12	12
9d	6	6	6	12	12	12
10a	12	12	24	24	48	48
10b	24	24	24	24	96	96
10c	24	24	12	48	24	24
Sulfamethoxazole	6	6	6	6	–	–
Fluconazole	–	–	–	–	6	6

MBC: Minimum Bactericidal Concentration; MFC: Minimum Fungicidal Concentration.