Document Type: Research Paper
Author
School of Pharmacy, Faculty of Medicine and Health Sciences, AIMST University, Kedah Darul Aman, Malaysia
Abstract
Keywords
1. Introduction
Quinoxaline derivatives are an important class of benzoheterocycles which has received much attention in recent years owing to their both biological properties and pharmaceutical applications. These derivatives are particularly interesting since some of them showed anti-microbial [1-9], anticancer [10-25], antimalarial [26-31], antiinflammatory [32-33], antinociceptive [34-36], antitubercular [37-39], anthelmintic [40-41], antidiabetic [42] and antiepileptic [43-44] properties. There is no report on the synthesis of indeno[1, 2-b]quinoxalin-11-ylidenamines and hence our research has focused to synthesize new indeno[1, 2-b]quinoxalin-11-ones substituted by aromatic amine moieties in 11 position and to investigate their antinociceptive, anti-inflammatory and antiepileptic activities.
2. Materials and methods
2.1. Chemistry
Titled compounds were prepared as shown in Figure1. Melting points were determined by Veego melting point apparatus and are not corrected. Thin layer chromatographic analysis were performed on a Merck grade Aluminum foil GF254 plates of 0.25 mm thickness in chloroform: water system (9:1). Spots were visualized under UV light. Infrared spectra were obtained on a Perkin Elmer-1600 series FTIR spectrophotometer using potassium bromide discs. Nuclear magnetic resonance spectra were recorded on Brucker 400 MHz spectrophotometer. Chemical shifts are reported in parts per million (δ) units relative to internal standard tetramethylsi-lane. Mass spectra were recorded on Joel JMS-DX 303 mass spectrophotometer. Elemental analysis was performed on Heraceus Carlo Erba 1108 and the analysis indicated by the symbols of the elements was within ±0.4 % of theoretical values.
Figure 1. Synthetic scheme of Indeno[1, 2-b] quinox-11-ylideneamines.
Table 1. Effect of synthesized compounds on Rota rod test for mice.
|
Behaviour |
Effect after 30 min of administration (mean ±SEM) (i.p) |
||||
|
|
0.5% v/v Tween 80 suspension |
20 mg.kg-1 |
50 mg.kg-1 |
100 mg.kg-1 |
200 mg.kg-1 |
|
Grip test |
No effect |
No effect |
No effect |
No effect |
1 ±0.42* |
2.2. Preparation of indeno[1, 2-b] quinoxalin-11-one (1) [45-48]
Equimolar quantities of benzene 1,2-diamine and indan-1,2,3 trione were refluxed in AcOH for 2 h at 100 °C. After 2 h, the mixture was poured into crushed ice and indeno[1,2-b]quinoxalin-11-one (1) obtained was filtered, dried and recrystallized from DMF. The desired product 1 obtained as brown solid in 68% yield: m.p. 107-108 °C. IR: 1725(C=O), 1608, 1506(C=C), 1334, 1189 (C=N), 863, 771, 734 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 7.7-8.1(m, 8H, Ar-H). Anal. Calcd for C15H8N2O: C, 77.58; H, 3.47; N, 12.06; O, 6.89. Found: C, 77.31, H,3.14; N, 11.86; O, 6.52. EI-MS (m/z) 232.24.
2.3. Preparation of (indeno [1, 2-b] quinoxalin-11-ylideneamino) benzene sulphonamide (2)[49]
Equimolar quantities of compound 1 and 4-aminobenzene sulphonamide were refluxed on a water bath in acetic acid medium for 3 h with continuous stirring. After 3 h, the crude reaction products were poured into crushed ice and the product obtained was filtered, dried and recrystalized from DMF. The desired product 2 was obtained as a brownish black solid in 64% yield: m.p. 120- 121 °C. IR: 3475, 3380 (N-H), 1609, 1596 (C=C), 1392 (C=N), 1309, 1151 (SO2), 831, 742 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 2 (s, 2H, NH2),7.2-7.9 (m, 12 H, Ar-H). Anal. Calcd for C21H14N4O2S: C, 64.76; H, 3.62; N, 50.35; O, 8.22; S, 8.23 Found: C, 64.42, H, 3.29; N, 50.04; O. 7.99; S, 7.98. EI-MS (m/z) 389.47.
Table 2. Behavioral study data of the synthesized compounds using actophotometer method.
|
Treatment |
Dose (mg.kg-1) |
Score (Mean±SEM) (time in s) |
|
Control Phenobarbitone 2
3
4
5
6
7
8
9 |
- 5 20 50 100 20 50 100 20 50 100 20 50 100 20 50 100 20 50 100 20 50 100 20 50 100 |
275±31* 36±7* 111±32* 83±8* 67±4* 294±13* 278±17* 240±6* 217±10* 189±4* 120±2* 141±10* 120±2* 92±6* 115±12* 76±4* 56±2* 44±4* 38±2* 22±9* 207±10* 168±2* 140±1* 173±23* 149±3* 120±7*
|
*p < 0.05 represent significant difference when compared with control groups
2.4. Preparation of indeno[1, 2-b]quinoxaline-11-ylidene phenylamine (3)
Compound 3 was prepared using the same procedure adopted for compound 2 by reacting equimolar quantities of compound 1 and aniline. The desired product 3 was obtained in 62% yield as a blackish brown solid: m.p. 197-198 °C. IR: 1641, 1619 (C=C),1336, 1182 (C=N), 867 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 7.25-8.2 (m, 13 H,Ar-H). Anal. Calcd for C21H13N3: C, 82.07; H, 5.57; N, 13.67; Found: C, 81.88, H, 5.12; N, 13.24. EI-MS (m/z) 307.35.
2.5. Preparation of indeno[1,2-b]quinoxaline-11-ylidene-(4-nitro phenylamine) (4)
Compound 4 was prepared using the same procedure adopted for compound 2 by using equimolar quantities of compound as a brown solid: m.p 185-186 °C. IR: 3434 (NH), 1606, 1502 (C=C), 1334 (C=N), 767 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 7 (s, 1H, NH), 7.65-8.25 (m, 13 H, Ar-H). Anal.
Calcd for C21H14N4: C, 78.24; H, 3.77; N, 17.38. Found: C, 77.89, H, 3.96; N, 17.01. EI-MS (m/z) 322.37.
2.6. Preparation of (indeno[1, 2-b]quinoxaline -11-ylidene-amino)phenol (5)
Compound 5 was prepared using the same procedure adopted for compound 2 by using equimolar quantities of compound 1 and 4-amino phenol. The desired product 5 was obtained in 60% yield as a black solid: m.p.67-68 °C. IR: 3424 (O-H), 1604, 1506 (C=C), 1376, 1334 (C=N), 1230 (C-O), 771, 734 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 5.9 (s, 1H, OH), 6.8-8.3 (m, 12 H, Ar-H). Anal. Calcd for C21H13N3O: C, 77.96; H, 4.05; N, 12.99; O, 4.95. Found: C, 77.58, H, 3.68; N, 12.60; O, 4.56.71. EI-MS (m/z) 323.53.
2.7. Preparation of indeno [1, 2-b] quinoxaline-11-ylidene-N'-phenylhydrazine (6)
Compound 6 was prepared using the same procedure adopted for 2 by using equimolar quantities of 1 and phenylhydrazine. The desired product 6 was obtained in 61% yield Anal. Calcd for C27H18N4: C, 81.38; H, 4.55; N, 14.06. Found: C, 79.99, H, 4.17; N, 13.68. EI-MS (m/z) 398.49.
Table 3. Evaluation of anti-nociceptive activity of the synthesized compounds by acetic acid induced writhing method.
|
Compound Dose(mg.kg-1) Writhing episodes in Percentage 15 min (mean±SEM) Protection |
|
Control ---- 53.83 ---- Paracetamol 2.5 09.17 ± 0.83 85
2 20 18.83 ± 0.81* 67
3 20 27.50 ± 0.88* 67
4 20 35.17 ± 0.96* 72
5 20 20.67 ± 0.86* 63
6 20 34.33 ± 0.93* 37
7 20 14.17 ± 0.73* 72
8 20 16.17 ± 0.79* 70
9 20 28.17 ± 0.98* 48
|
*p < 0.001 represent significant difference when compared with control groups
2.8. Preparation of N-(2,4-dinitrophenyl)-N'-indeno[1,2-b]quinoxalin-11-ylidene hydrazine(7)
Compound 7 was prepared using the same procedure adopted for 2 by using equimolar quantities of 1 and 2,4-dinitrophenylhy-drazine. The desired product 7 was obtained in 65% yield as a brownish red solid: m.p 164-165 °C. IR: 3434 (N-H), 1606, 1502 (C=C), 1552 (NO2), 1247 (C=N), 757 (aromatic hydrogen) cm-1. 1H-NMR (DMSO-d4) ) δ: 7(s, 1H, N-H), 7.05-8.05 (m, 11H, Ar-H).
Anal. Calcd for C21H12N6O4: C, 61.17; H, 3.18; N, 20.38; O, 15.52. Found: C, 59.78, H,2.81; N, 19.99; O, 15.14. EI-MS (m/z) 412.36.
2.9. Preparation of Indeno[1, 2-b]quinoxalin-11-ylidene phenylamine (8)
Compound 8 was prepared using the same procedure adopted for 2 by using equimolar quantities of 1 and biphenyl-4,4'-diamine. The desired product 8 was obtained in 63 % yield as a black solid: m.p. 137-138 °C. IR: 3407, 3328 (N-H), 1612 (C=C), 1263 (C=N), 819 (aromatic) cm-1. 1H-NMR (DMSO-d4) ) δ: 3.5 (s, 2H, NH2), 6.55-8.1 (m, 16 H, Ar-H).
1 and 4-nitroaniline. The desired product 4 was obtained in 59% yield as black sticky mass. IR: 3438 (N-H), 1619, 1596 (C=C), 1336, 1508 (NO2), 1247 (C=N), 727 (aromatic) cm-1. 1H-NMR (DMSO-d4) ) δ:
7.3-8.2 (m, 12 H, Ar-H). Anal. Calcd for C21H12N4O2: C, 71.59; H, 3.43; N, 15.90; O, 9.08. Found: C, 71.12, H, 3.09; N, 15.56; O, 8.71. EI-MS (m/z) 325.35
2.10. Preparation of 4-(Hydroxy-3-Indeno [1, 2-b] quinoxalin-11-ylidene amino) naphthalene-1-sulphonic acid (9)
Compound 9 was prepared using the same procedure adopted for 2 by using equimolar quantities of 1 and 1-amino-2-naphthol-4-sulphonic acid. The desired product 9 was obtained in 64 % yield as a yellow solid: m.p. 184-185 °C. IR: 3432 (OH), 1506 (C=C), 1334 (C=N), 1189, 1116 (SO3H), 773, 736 (aromatic) cm-1. 1H-NMR (DMSO-d4) δ: 2.52 (s, 1H, OH of SO3H), 3.5 (s, 1H, OH of naphthol), 7.6-8.2 (m, 13 H, Ar-H). Anal.
Calcd for C25H15N3O4S: C, 65.50; H, 2.86; N, 9.17, O, 13.96; S, 6.99. Found: C, 65.12, H,2.49; N, 8.78; O, 13.58, S.6.58. EI-MS (m/z) 458.47.
2.11. Pharmacological evaluation
The animals used for the studies were in accordance with principles of laboratory animal care and were approved by Institutional animal ethical committee. The antinociceptive evaluation was carried out using acetic acid induced writhing method. Swiss strain albino mice of either sex weighing 25-30 g were used for this study. The antiinflammatory activity evaluation was carried out using carrageenan induced rat paw edema method. Albino rats of Wistar strain weighing 100-200 g of either sex were used for this study.
The antiepileptic evaluation was carried out using maximal electro shock induced seizure method. Swiss strain albino mice of either sex weighing 25-30 g were used for this study. The test compounds were suspended in 0.5 % v/v Tween 80 in water. LD50 of the newly synthesized compounds were determined by Miller and Tainter [50] method by administering the compounds intraperitoneally.
2.11.1. Acetic acid induced writhing method [51]
The animals were housed and acclimated under standard laboratory conditions and were supplied with food and water ad libitum. The animals were divided into ten groups of six mice each. The control group of animals was administered with 0.5% v/v Tween 80 (0.5 ml) suspension. The standard drug was administered with paracetamol (Micro Labs) i.p. in a dose of 2.5 mg kg-1. Tween 80 suspension (0.5 % v/v) of the test compounds were administered i.p. in a dose of 20 mg.kg-1. After 20 min. of the administration of the test compounds and standard, all the groups of mice were given the writhing agent 3 % v/v aqueous acetic acid in a dose of 2 ml kg-1 i.p. The total number of writhing produced in these animals were counted visually for 15 min. and the number of writhing produced in treated groups were compared with those in control group. The results recorded in Table 3 are expressed as percentage protection and are analyzed statistically by “student t test”.
2.11.2. Anti-inflammatory activity
The antiinflammatory activity was evaluated by carrageenan induced paw edema method [52]. Albino rats of Wistar strain weighing 100-200 g of either sex were divided into ten groups each of six animals. Tween 80 suspension (0.5 % v/v) of the test compounds were administered intraperitoneally in a dose of 20 mgkg-1. The control group was given only 0.5% v/v Tween 80 (0.5 ml) suspension. One group was administered with diclofenac sodium (Novartis Laboratories) as standard, i.p. in a dose of 2 mg kg-1. After 30 min. of the administration of test compounds paw edema was induced in albino rats by injecting 0.1 ml of carrageenan (1% v/v in normal saline) suspension, into subplantar region of the left hind paw of each rat. After 1, 2 and 3 h of carrageenan injection, the increase in paw volume was measured by a plethysmometer. The antiinflammatory activity was measured in terms of percentage inhibition of edema and is analyzed statistically by “students t test” and recorded in Table 4.
Table 4. Evaluation of anti-inflammatory activity of the synthesized compounds by carrageenan Induced paw edema method.
|
Compound |
Dose (mg kg-1) |
Paw Volume (mean ± SEM) |
Percentage inhibition |
||||
|
1 h |
2 h |
3 h |
1 h |
2 h |
3 h |
||
|
Control Diclofenac sodium 2 3 4 5 6 7 8 9 |
-- 2.5
20 20 20 20 20 20 20 20 |
0.85 ± 0.00 0.68 ± 0.02**
0.77 ± 0.03** 0.75 ± 0.03** 0.75 ± 0.02** 0.78 ± 0.03** 0.80 ± 0.03* 0.72 ± 0.01** 0.74 ± 0.03** 0.79 ± 0.02** |
0.87 ± 0.00 0.62 ± 0.03**
0.74 ± 0.02** 0.73 ± 0.00** 0.73 ± 0.03** 0.74 ± 0.02** 0.75 ± 0.02** 0.69 ± 0.01** 0.72 ± 0.00** 0.70 ± 0.01** |
0.87± 0.00** 0.51± 0.02**
0.72± 0.01** 0.72± 0.01** 0.71± 0.01** 0.73 ±0.01** 0.73± 0.01** 0.67± 0.01** 0.7 ± 0.01** 0.72± 0.01** |
- 20.0
09.4 11.7 11.8 08.2 05.8 15.2 12.9 07.5 |
- 28.7
14.9 16.1 16.1 14.1 13.8 20.0 17.2 12.6 |
- 41.3
17.2 17.2 18.3 16.0 16.0 22.2 18.3 17.2 |
**p < 0.001 and *p < 0.01 represent significant difference when compared with control groups
2.11.3. Anti-epileptic activity [53]
The antiepileptic activity was evaluated by maximal electroshock induced seizure (MES) method. Swiss albino mice weighing 25-30 g of either sex were divided into ten groups each of six animals. The control group of animals was administered with 0.5%v/v Tween 80 (0.5 ml) suspension. One group was administered with phenobarbitone (Nicholas Primal) as standard, i.p. in a dose of 20 mg.kg-1. Tween 80 suspensions (0.5 % v/v) of the test compounds were administered i.p. in a dose of 20 mg kg-1 for other group of animals. After 1 h of the administration of the test compounds and standard, the animals were given electroshock through ear electrodes of 150 mA for 0.2s by electroconvulsiometer. Onset times for tonic, flexion, extension and clonic phase were noted. The protective index was observed as reduction time of tonic extensor phase and was tabulated in Table 5.
Table 5. Evaluation of antiepileptic activity of the synthesized compounds by Maximal Electroshock Induced Seizure (MES) method.
|
Compound |
Dose (mg.kg-1) |
Duration (mean±SEM, s) |
Recovery/ Death |
||
|
Extensor |
Clonus |
Stupor |
|||
|
Control Phenobarbitone 2 3 4 5 6 7 8 9 |
-- 20 20 20 20 20 20 20 20 20 |
16.50 ± 0.43 05.67 ± 0.46** 10.17 ± 0.48** 15.50 ± 0.43 15.17 ± 0.37* 10.17 ± 0.49** 10.26 ± 0.39** 08.17 ± 0.80** 13.33 ± 0.56* 10.27 ± 0.49** |
16.50 ± 0.43 02.17 ± 0.31 10.33 ± 0.42 15.33 ± 0.42 10.23 ± 0.41 15.33 ± 0.42 30.83 ± 0.60 07.33 ± 0.42 10.17 ± 0.49 15.46 ± 0.43 |
16.50 ± 0.43 01.33 ± 0.21 10.33 ± 0.42 14.33 ± 0.42 05.48 ± 0.42 06.33 ± 0.42 10.17 ± 0.49 20.50 ± 0.43 10.26 ± 0.38 05.33 ± 0.42 |
Death Recovery Recovery Recovery Death Recovery Death Recovery Recovery Recovery |
**p < 0.001 and *p < 0.01 represent significant difference when compared with control groups
2.11.4. Neurotoxicity screen
Minimal motor impairment was measured in mice by the Rota rod (Techno, India) test. The mice were trained to stay on an accelerating Rota rod of diameter 3.2 cm that rotates at 10 revolutions per min. Previously trained Albino mice were given test compounds i.p. in doses of 20, 50, 100, and 200 mg.kg-1. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one min. in each of the three trials. The results are summarized in Table 1.
2.11.5. Behavioral test
The titled compounds (20 mg kg-1 in 0.5 % v/v Tween 80 suspension) were screened for their behavioral effects using actophotometer (Techno, India) by Boissier and Simon method [54]. Albino mice were placed inside the actophotometer after 30 min. of administration of test compounds i.p. The behavior of animals inside the photocell was recorded as a digital score. The control animals were administered with 0.5 ml of Tween 80 (0.5% v/v) suspension. The observations are summarized in Table 2.
3. Results and discussion
3.1. Chemistry
Ketones readily undergo condensation reaction with amines. Indane-1,2,3-trione being a ketone yielded a condensation product indeno[1, 2-b] quinoxalin-11-one with benzene-1,2-diamine. Indeno[1, 2-b] quinoxalin-11-one yielded indeno[1, 2-b] quinoxalin-11-ylidienamines on reaction with various substituted amines.
Infrared spectrum of all eight compounds showed strong absorption bands for C=N group at 1393 cm-1 and aromatic absorptions at 1609 cm-1 and below 900 cm-1. Compound 2 showed strong absorptions for SO2 group at 1151 cm-1 and N-H group at 3380 cm-1. Compound 4 showed strong absorptions for NO2 group at 1508 cm-1. Compound 5 showed strong absorptions for OH group at 3424 cm-1. Compound 6 showed absorption band for NH group at 3434 cm-1. Compound 7 showed absorption band for N-H at 3434 cm-1 and for NO2 group at 1552 cm-1. Compound 8 showed absorption band for N-H group at 3428 cm-1. Compound 5 showed strong absorptions for OH group at 3432 cm-1 and for sulphonic acid group at 1116 cm-1.
All the eight synthesized compounds showed strong multiplets from δ 7.2-7.9 for aromatic protons. Compounds 2, 6, 7 and 8 showed singlet signals ranging from δ 2 to 7 for NH and NH2 protons. Hydroxyl protons were detected around δ 5.9 and δ 2.5 for compounds 5 and 9 respectively. Mass spectral data showed expected m/z values for all the eight synthesized compounds.
3.2. Pharmacological evaluation
3.2.1. Neurotoxicity screen
Minimum motor impairment was measured in mouse by Rota rod test showed that all the synthesized compounds have no effect which was indicated by their ability to maintain the equilibrium on the Rota rod for more than 1 min.
3.2.2. Behavioral test
Compound 7 produced significant decrease in the spontaneous motor activity in mice. This effect was dose dependent and the effect was observed within 30 min. of drug administration. Other compounds except compound 3 also showed good behavioral activity.
3.2.3. Anti-nociceptive activity
3.2.3.1. Acetic acid induced writhing method
Antinociceptive activity was evaluated by acetic acid induced writhing method. All the compounds tested exhibited antinociceptive activity in a dose of 20 mg.kg-1. The antinoci-ceptive activity of compounds 4, 7 and 8 is found to be superior compared to other synthesized compounds. The compounds 2, 3 and 5 exhibited moderate antinociceptive activity. The compounds 6 and 9 exhibited negligible anti-nociceptive activity.
3.2.4. Anti-inflammatory activity
The compounds 2, 3, 4, 5, 6, 7, 8 and 9 afforded 16-22% protection against carageenan induced paw edema, where as the standard drug diclofenac sodium (Novartis Laboratories) under similar conditions showed 42% inhibition. Among the compounds tested, compounds 4, 7 and 8 exhibited significant anti-inflammatory activity and compounds 2, 3, 5, 6 and 9 exhibited moderate anti-inflammatory activity.
3.2.5. Anti-epileptic activity
Maximal electroshock induced seizure model was adopted for evaluation of antiepileptic activity. Compound 7 exhibited significant antiepileptic activity by reducing the duration of extensor phase. The compounds 1, 3, 4 and 8 exhibited negligible anti-epileptic activity.
4. Conclusions
A series of indeno[1,2-b]quinoxalin-11-ylidenamines was prepared and demonstrated that these compounds possess good antinoci-ceptive activity tested by acetic acid induced writhing method, antiinflammatory activity tested by carageenan induced paw edema method and antiepileptic activity tested by maximal electroshock method. The most promising compounds having antinocicep-tive activity were indeno[1,2-b]quinoxalin -11-ylidene-(4-nitrophenyl)amine 4, N-(2,4-dinitrophenyl)-N'-indeno[1,2-b]quinoxalin-11-ylidene hydrazine 7 and N4-indeno[1,2-b]quinoxalin-11-ylidene biphenyl-4, 4'-diamine 8, N-(2,4-dinitrophenyl)-N'-indeno[1,2-b]quinoxalin-11-ylidene hydrazine 7, N4-indeno[1,2-b]quinoxalin-11-ylidene-biphenyl-4,4'-diamine 8 and indeno[1,2-b] quinoxalin-11-ylidene-(4-nitrophenyl) amine 4 exhibited promising antiinflammatory activity.
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