CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.10 No.6, pp 190-201, 2017
Abstract : The manuscript describes synthesis of some novel indole bearing azetidinone derivatives and their evaluation for antianxiety activity. The compounds were synthesized following three step reaction to yield twenty derivatives as 3{[3-chloro-2-substituted-4oxoazetidin-1-yl]imino}-1,3-dihydro-2H-indol-2-one (23-42). All the final structures were assigned on the basis of IR, 1H NMR, mass spectra and elemental analyses.The final derivatives 3{[3-Chloro-2-(4-hydroxy,3-methoxy phenyl) -4-oxoazetidin-1-yl]imino}-1,3dihydro-2H-indol-2-one (28) and 3{[3-Chloro-2-(4-(Dimethylamino)phenyl) -4-oxoazetidin1-yl]imino}-1,3-dihydro-2H-indol-2-one (29) were found to be promising molecule in the series. The dimethyl amino and hydroxy substitution on the para position of phenyl ring system provided with active compounds having percentage preferences of open arm with 69.44and 83.33 respectively at 50 mg/kg dose level when compared to the standard drug. 3DQSAR results revealed that addition of electropositive and bulky groups at the phenyl ring will contribute towards increase in the antianxiety activity of the molecules while electronegative groups decreases the activity. Keywords : Indole, Azetidinone, Antianxiety activity, 3D-QSAR.
Anxiety is an emotion characterized by an unpleasant state of inner turmoil, often accompanied by nervous behaviour, such as pacing back and forth, somatic complaints, and rumination 1. It is also a generalized mood or condition that occurs without an identifiable triggering stimulus. As such, it is distinguished from fear, which occurs in the presence of an observed threat. Additionally, fear is related to the specific behaviours of escape and avoidance, whereas, anxiety is the result of threats that are perceived to be uncontrollable or unavoidable2. Anxiety can be appropriate, but when experienced regularly the individual may suffer from an anxiety disorder. People facing anxiety may withdraw from situations which have provoked anxiety in the past 3-5. The physiological symptoms of anxiety may include, headache, vertigo, nausea, palpitations, itchy skin and frequent urination6,7. The physiological reason for anxiety have not been completely established, but some facts suggested that there is decrease in the level of GABA or relative deficiency in the GABA neurotransmission, which can be augmented by agents acting on different components of the GABA system8.
In an attempt to synthesize and evaluate new compounds as antianxiety agents, we report herein, synthesis, biologicalevaluation and 3D-QSAR studies of a number of indole bearing azetidinone derivatives. Indoles are considered as the most promising bicyclic heteroaromatic nucleus in the field of medicinal
become widely identified as a 9-13 . Literature survey also agents 14. Compounds containing the
15-18
. The indole moiety is
19-21
moiety in experimental drugs. , whichare likely to exhibit antianxiety-like The scheme of the
Table 1.
Figure 2: Scheme synthetic route of the titled compounds Table 1: List of substitutions
Compound No. | -Ar | Compound No. | -Ar | ||
---|---|---|---|---|---|
3 | 23 | C6H5 | 13 | 33 | 4-CH3 -C6H5 |
4 | 24 | 4-Cl-C6H5 | 14 | 34 | 2-OCH3 -C6H5 |
5 | 25 | 4-C6H5 -CH2O-C6H5 | 15 | 35 | 4-OCH3 -C6H5 |
6 | 26 | 2-NO2 -C6H5 | 16 | 36 | 2-Cl-C6H5 |
7 | 27 | 4-OH-C6H5 | 17 | 37 | 3-Br-C6H5 |
8 | 28 | 4-OH,3-OCH3 C6H5 | 18 | 38 | 2,4-(OCH3)2 C6H5 |
---|---|---|---|---|---|
9 | 29 | 4-(CH3)2 -N-C6H5 | 19 | 39 | 4-NO2 -C6H5 |
10 | 30 | 3-NO2 -C6H5 | 20 | 40 | 2,3-(OCH3)2 C6H5 |
11 | 31 | 3,4,5-(OCH3)3 C6H5 | 21 | 41 | 3-Cl-C6H5 |
12 | 32 | 2,4-(Cl)2 -C6H5 | 22 | 42 | C4H3O-(2-furyl) |
Chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), S D Fine-Chem (Mumbai, MH, India) and Merck (Darmstadt, Germany), unless specified. Melting points (m.p.) were detected with open capillaries using ThermoNik precision melting point cum boiling point apparatus (model C-PMB-2, Mumbai, MH, India) and are uncorrected. Infrared (IR) spectra (KBr) were recorded on FTIR-8400s spectrophotometer (Shimadzu, Tokyo, Japan) at the Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharai (RTM) Nagpur University. Proton (1H) and carbon 13 (13C) nuclear magnetic resonance (NMR) were obtained using a BrukerAvance II 400 MHz spectrometer (Billerica, MA, USA), using tetramethylsilane (TMS) as internal standard. All chemical shift values were recorded as d (ppm), coupling constant value J is measured in hertz, the peaks are presented as s (singlet), d (doublet), t (triplet), brs (broad singlet), dd (double doublet), m (multiplet). The purity of compounds was controlled by thin layer chromatography (silica gel HF254e361, type 60, 0.25 mm; Merck, Darmstadt, Germany). Electrospray ionization mass spectrometry (ESI-MS) was recorded at Waters Q-TOF spectrometer (Waters, Milford, MA, USA) at the Sophisticated Analytical Instrumentation Facility (SAIF), Punjab University (Chandigarh, PB, India).
A mixture of isatin (1) (1mmol) and hydrazine hydrate (99%, 0.055 g, 1.1 mmol) in absolute methanol (25 ml) was refluxed for 1 h and then cooled to room temperature. The precipitate of hydrazones was filtered and dried. The crude product was recrystallized from ethanol to give hydrazones (2).
Yield: 70 %; mp 248-250 ºC; Rf0.39 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3411-3319, (NH, NH2), 1687 (C=O), 1609 (C=N); 1H NMR (DMSO, ppm): δ 6.87-7.37 (m, 4H, Ar-H), 10.57 (s, 2H, NH2), 9.39 (s, 1H, NH); EI-MS: m/z [M+H]+ 162.24
To a solution of compound 2 (0.01 mol) in ethanol (60 mL), substituted aromatic aldehyde (0.01 mol) along with few drops of glacial acetic acid were added. Then resulting mixture was refluxed for 7-8 h. The excess of the ethanol was distilled off and the remaining mixture was cooled, poured onto crushed ice and filtered. The crude product was recrystallized from 70% ethanol.
A mixture of Schiff base (3-22)(0.01 mol) and triethyl amine (0.02 mol) was dissolved in 1, 4Dioxane (15 m1). To this, a solution of chloroacetyl chloride (0.02 mol) was added in portion wise with vigorous shaking at room temperature for 20 min. The reaction mixture was heated under reflux for 3 h and the content was kept at room temperature for 48 h and poured into ice-cold water. The resulting solid was filtered, washed several times with water and then recrystalised from 70% ethanol.
Yield: 63%; mp 172-174 ºC; Rf0.53 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3367 (NH), 1704 (C=O), 1542 (C=N); 1H NMR (DMSO, ppm): δ 7.03-8.37 (m, 9H, Ar-H), 8.66 (s, 1H, NH), 6.88 (d, 1H, CH-Ar); 5.48 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 326.74. Anal. Calcd for C17H12ClN3O2: C, 62.76; H, 3.69; N, 12.92. Found: C, 61.45; H, 2.79; N, 11.77.
Yield: 71%; mp 191-193ºC; Rf0.61 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3369 (NH), 1699 (C=O), 1521 (C=N); 1H NMR (DMSO, ppm): δ 7.10-7.95 (m, 8H, Ar-H), 8.65 (s, 1H, NH), 5.88 (d, 1H, CH-Ar); 5.04 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 361.94. Anal. Calcd for C17H11Cl2N3O2: C, 56.66; H, 3.05; N, 11.66. Found: C, 55.21; H, 2.79; N, 10.75.
Yield: 61%; mp 135-137 ºC; Rf0.55 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3267 (NH), 1698 (C=O), 1545 (C=N); 1H NMR (DMSO, ppm): δ 7.05-8.89 (m, 13H, Ar-H), 9.56 (s, 1H, NH), 4.78 (d, 1H, CH-Ar); 6.12 (d, 1H, CH-Cl); 5.20 (d, 2H, CH2); EI-MS: m/z [M+H]+ 432.87. Anal. Calcd for C24H18ClN3O3: C, 66.70; H, 4.16; N, 9.72. Found: C, 65.61; H, 3.89; N, 9.15.
Yield: 63%; mp 168-170ºC; Rf0.42 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3395 (NH), 1675 (C=O), 1586 (C=N); 1H NMR (DMSO, ppm): δ 6.84-8.26 (m, 8H, Ar-H), 8.97 (s, 1H, NH), 5.07 (d, 1H, CH-Ar); 5.49 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 371.74. Anal. Calcd for C17H11ClN4O4: C, 55.13; H, 2.97; N, 15.13. Found: C, 54.33; H, 1.97; N, 14.94.
Yield: 72%; mp 211-213ºC; Rf0.58 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3324 (NH), 1723 (C=O), 1576 (C=N); 1H NMR (DMSO, ppm): δ 7.66-7.92 (m, 8H, Ar-H), 8.45 (s, 1H, NH), 5.66 (d, 1H, CH-Ar); 6.71 (d, 1H, CH-Cl); 9.56 (s, 1H, OH); EI-MS: m/z [M+H]+ 342.74. Anal. Calcd for C17H12ClN3O3: C, 59.82; H, 3.51; N, 12.31. Found: C, 58.51; H, 2.98; N, 11.87.
Yield: 67%; mp 178-180ºC; Rf0.44 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3289 (NH), 1689 (C=O), 1557 (C=N); 1H NMR (DMSO, ppm): δ 7.72-8.02 (m, 7H, Ar-H), 8.75 (s, 1H, NH), 5.21 (d, 1H, CH-Ar); 6.55 (d, 1H, CH-Cl); 2.76 (s, 3H, OCH3); 9.87 (s, 1H, OH); EI-MS: m/z [M+H]+ 372.77. Anal. Calcd for C18H14ClN3O4: C, 58.22; H, 3.77; N, 11.32. Found: C, 57.45; H, 2.94; N, 10.92.
Yield: 76%; mp 182-184 ºC; Rf0.61 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3295 (NH), 1745 (C=O), 1577 (C=N); 1H NMR (DMSO, ppm): δ 6.76-8.70 (m, 8H, Ar-H), 10.72 (s, 1H, NH), 5.05 (d, 1H, CH-Ar); 5.45 (d, 1H, CH-Cl); 3.57 (s, 6H, CH3); EI-MS: m/z [M+H]+ 369.81. Anal. Calcd for C19H17ClN4O2: C, 61.95; H, 4.61; N, 15.21. Found: C, 60.12; H, 4.12; N, 14.63.
Yield: 58%; mp 204-206 ºC; Rf0.43 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3292 (NH), 1672 (C=O), 1543 (C=N); 1H NMR (DMSO, ppm): δ 7.78-8.09 (m, 8H, Ar-H), 9.22 (s, 1H, NH), 5.23 (d, 1H, CH-Ar); 5.98 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 371.74. Anal. Calcd for C17H11ClN4O4: C, 55.13; H, 2.97; N, 15.13. Found: C, 54.37; H, 2.56; N, 14.61.
Yield: 61%; mp 221-223 ºC; Rf0.52 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3342 (NH), 1626 (C=O), 1537 (C=N); 1H NMR (DMSO, ppm): δ 7.66-8.16 (m, 6H, Ar-H), 8.78 (s, 1H, NH), 5.43 (d, 1H, CH-Ar); 5.51 (d, 1H, CH-Cl); 3.93 (s, 9H, CH3); EI-MS: m/z [M+H]+ 416.82. Anal. Calcd for C20H18ClN3O5: C, 57.83; H, 4.33; N, 10.12. Found: C, 56.37; H, 4.12; N, 9.82.
Yield: 64%; mp 224-226 ºC; Rf0.62 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3286 (NH), 1723 (C=O), 1529 (C=N); 1H NMR (DMSO, ppm): δ 7.23-8.15 (m, 7H, Ar-H), 9.45 (s, 1H, NH), 5.23 (d, 1H, CH-Ar); 5.03 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 395.63. Anal. Calcd for C17H10Cl3N3O2: C, 51.77; H, 2.53; N, 10.65. Found: C, 50.45; H, 1.98; N, 9.72.
Yield: 61%; mp 173-175 ºC; Rf0.57 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3294 (NH), 1741 (C=O), 1521 (C=N); 1H NMR (DMSO, ppm): δ 7.34-8.51 (m, 8H, Ar-H), 9.23 (s, 1H, NH), 5.13 (d, 1H, CH-Ar); 5.43 (d, 1H, CH-Cl); 2.42 (s, 3H, CH3); EI-MS: m/z [M+H]+ 340.77. Anal. Calcd for C18H14ClN3O2: C, 63.71; H, 4.12; N, 12.38. Found: C, 63.53; H, 3.23; N, 11.43.
Yield: 69%; mp 166-168 ºC; Rf0.41 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3334 (NH), 1698 (C=O), 1543 (C=N); 1H NMR (DMSO, ppm): δ 6.89-7.81 (m, 8H, Ar-H), 8.99 (s, 1H, NH), 5.07 (d, 1H, CH-Ar); 5.65 (d, 1H, CH-Cl); 2.31 (s, 3H, OCH3); EI-MS: m/z [M+H]+ 356.77. Anal. Calcd for C18H14ClN3O3: C, 60.84; H, 3.94; N, 11.83. Found: C, 59.57; H, 2.53; N, 10.43.
Yield: 61%; mp 172-174 ºC; Rf0.54 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3296 (NH), 1679 (C=O), 1521 (C=N); 1H NMR (DMSO, ppm): δ 6.89-8.61 (m, 8H, Ar-H), 10.88 (s, 1H, NH), 5.03 (d, 1H, CH-Ar); 5.34 (d, 1H, CH-Cl); 3.61 (s, 3H, OCH3); EI-MS: m/z [M+H]+ 356.77. Anal. Calcd for C18H14ClN3O3: C, 60.84; H, 3.94; N, 11.83. Found: C, 59.77; H, 2.61; N, 10.52.
Yield: 81%; mp 158-160 ºC; Rf0.61 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3345 (NH), 1709 (C=O), 1582 (C=N); 1H NMR (DMSO, ppm): δ 6.90-8.98 (m, 8H, Ar-H), 10.97 (s, 1H, NH), 5.48 (d, 1H, CH-Ar); 6.91 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 361.19. Anal. Calcd for C17H11Cl2N3O2: C, 56.66; H, 3.05; N, 11.66. Found: C, 55.51; H, 2.61; N, 10.72.
Yield: 67%; mp 182-184 ºC; Rf0.63 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3297 (NH), 1739 (C=O), 1567 (C=N); 1H NMR (DMSO, ppm): δ 7.15-8.65 (m, 8H, Ar-H), 9.77 (s, 1H, NH), 5.16 (d, 1H, CH-Ar); 6.32 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 405.64. Anal. Calcd for C17H11BrClN3O2: C, 50.49; H, 2.72; N, 10.39. Found: C, 49.32; H, 1.75; N, 9.84.
Yield: 73%; mp 156-158 ºC; Rf0.43 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3345 (NH), 1716 (C=O), 1513 (C=N); 1H NMR (DMSO, ppm): δ 6.48-8.92 (m, 7H, Ar-H), 10.42 (s, 1H, NH), 6.13 (d, 1H, CH-Ar); 5.89 (d, 1H, CH-Cl); 3.87 (s, 6H, OCH3); EI-MS: m/z [M+H]+386.80. Anal. Calcd for C19H16ClN3O4: C, 59.22; H, 4.15; N, 10.90. Found: C, 58.59; H, 3.98; N, 9.55.
Yield: 59%; mp 165-167 ºC; Rf0.65 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3298 (NH), 1675 (C=O), 1572 (C=N); 1H NMR (DMSO, ppm): δ 7.07-8.87 (m, 8H, Ar-H), 9.33 (s, 1H, NH), 5.31 (d, 1H, CH-Ar); 6.04 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 371.74. Anal. Calcd for C17H11ClN4O4: C, 55.13; H, 2.97; N, 15.13. Found: C, 54.65; H, 1.87; N, 14.75.
Yield: 59%; mp 165-167 ºC; Rf0.65 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3298 (NH), 1675 (C=O), 1572 (C=N); 1H NMR (DMSO, ppm): δ 7.12-8.91 (m, 7H, Ar-H), 9.51 (s, 1H, NH), 4.98 (d, 1H, CH-Ar); 6.13 (d, 1H, CH-Cl); 2.41 (s, 6H, OCH3); EI-MS: m/z [M+H]+ 386.80. Anal. Calcd for C19H16ClN3O4: C, 59.09; H, 4.14; N, 10.88. Found: C, 58.68; H, 3.87; N, 10.11.
Yield: 71%; mp 172-174 ºC; Rf0.43 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3223 (NH), 1702 (C=O), 1573 (C=N); 1H NMR (DMSO, ppm): δ 7.09-8.78 (m, 8H, Ar-H), 9.23 (s, 1H, NH), 5.34 (d, 1H, CH-Ar); 6.13 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 361.19. Anal. Calcd for C17H11Cl2N3O2: C, 59.09; H, 3.09; N, 11.66. Found: C, 58.65; H, 2.87; N, 11.02.
Yield: 59%; mp 165-167 ºC; Rf0.65 (Methanol: Toulene 1:4); IR (KBr, cm-1): 3298 (NH), 1675 (C=O), 1572 (C=N); 1H NMR (DMSO, ppm): δ 7.12-8.82 (m, 7H, Ar-H), 9.13 (s, 1H, NH), 5.21 (d, 1H, CH-Ar); 5.88 (d, 1H, CH-Cl); EI-MS: m/z [M+H]+ 316.71. Anal. Calcd for C15H10ClN3O3: C, 57.09; H, 3.16; N, 13.30. Found: C, 56.87; H, 2.87; N, 12.85.
The experimental protocols for the pharmacological screening on mice were done with Institutional Animal Ethics Committee, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj (RTM) Nagpur University, Nagpur, India (Reg. no.: IAEC/UDPS/2016/21).
Antianxiety activity
Female mice weighing 25-30 g were housed in a cage with controlled room temperature at 22-25ºC. Food and water were available ad libitum. Tests were performed only after the micehad been acclimatized to the above environment for at least 7 days. Each mouse received a single i.p. injection of drug or vehicle and was tested once in the elevated plus-maze (EPM).
The apparatus comprised of two open arms (35 × 5 cm) and two closed arms (30× 5× 15cm)that extended from a common central platform (5 × 5 cm). The floor and the walls of each arm were wooden and painted black. The entire maze was elevated to a height of 50 cm above floor level as validated and described
22-23
by Lister. Testing was conducted in a quiet room that was illuminated only by a dim light. Mice were given a single i.p. dose of various test compounds(50 mg/kg) and diazepam (2 mg/kg) 30 min before their placement on the EPM. To begin a test session, mice were placed on the open arm facing the centre of the maze. An entry into an arm was defined as the animal placing all four paws over the line marking that area. The number of entries and the time spent in the open and closed arms were recorded during a 5-min test period. The percentage of open arm entries (100 × open/total entries) was calculated for each animal. Between each trial, the maze was wiped clean with a damp sponge and dried with paper towels. All the results of the antianxiety activity are given in Table 2.
Table 2: Effect of 3{[2-chloro-3-substituted-4-oxoazetidin-1-yl]imino}-1,3-dihydro-2H-indol-2-oneseries of compounds in EPM
Sr. No. | Compound | % preference of open arm | No. of entries in open arm | Average time spent in open arm (s) |
---|---|---|---|---|
1. | 23 | 58.33 | 5.25 ± 0.73 | 27.00± 1.14 |
2. | 24 | 29.54 | 3.25 ± 0.64 | 28.25± 0.65 |
3. | 25 | 62.50 | 6.25 ±0.54 | 33.50± 0.12 |
4. | 26 | 41.66 | 5.00 ±0.35 | 35.50± 1.11 |
5. | 27 | 55.00 | 5.50±0.54 | 38.50± 0.45 |
6. | 28 | 69.44 | 6.25 ±0.73 | 34.00± 0.67 |
7. | 29 | 83.33 | 7.50±0.53 | 42.25± 0.12 |
8. | 30 | 37.50 | 4.50±0.55 | 34.25± 0.85 |
9. | 31 | 67.50 | 6.75 ±0.41 | 39.25± 0.75 |
10. | 32 | 43.75 | 5.25 ±0.21 | 27.75± 0.45 |
11. | 33 | 52.50 | 5.65±0.41 | 28.50± 0.65 |
12. | 34 | 67.50 | 6.75 ±0.41 | 32.50 ± 1.23 |
13. | 35 | 11.36 | 1.25 ±0.21 | 41.50 ± 0.22 |
14. | 36 | 25.00 | 3.00 ±0.35 | 35.75 ± 0.76 |
15. | 37 | 22.91 | 2.75 ±0.73 | 29.25 ± 1.54 |
16. | 38 | 47.50 | 4.75 ±0.41 | 36.00 ± 0.94 |
17. | 39 | 13.63 | 1.50±0.25 | 32.50 ± 0.65 |
18. | 40 | 38.46 | 5.00±0.50 | 26.25 ± 0.87 |
19. | 41 | 50.00 | 4.50±1.03 | 15.25 ± 0.65 |
20. | 42 | 12.50 | 1.25 ±0.21 | 22.00 ± 0.22 |
21. | Vehicle/control | 0.0 | 1.75 ±0.41 | 09.75 ± 0.45 |
22. | Diazepam (2mg/kg) | 85.00 | 8.50±0.55 | 42.50 ± 0.54 |
Data analyzed by using one-way analysis of variance (ANOVA) with post hoc Tukey test. n = 6; dose = 50 mg/kg. Values are represented as mean ± S.E.M. Values are significant at ∗∗∗P< 0.05, compared with control group.
The 3D-QSAR was performed using the molecular modeling software package VLife Molecular Design Suite (VLifeMDS) version 4.3.1 on HP-PC (HPLV1911) with a Pentium IVprocessor and Windows 7 operating system.The results obtained from antianxiety activity were employed for carrying out the 3D QSAR studies, the percentage preference to open armvalues of twenty compoundsTable 2 employed to obtain the statistical analysis on basis of various descriptors such as steric and electrostatic which were calculated and used as independent variables.The dataset of twenty molecules in were divided into training and test set by random selection method for MLR, PCR, PLSR and kNN-MFA model. The seventy eight percentages of total data selected as training set (15 molecules) and remaining as test set (5 molecules).The Modules >>QSARPlus>> 3D-QSAR from the main menu of MDS was selected to launch the worksheet. By default all the molecules in a directory were considered for QSAR. QSAR tool was chosen from which molecules were opened, the subfolder containing set of molecules was selected. Activity data which was stored as ‘activity.txt’ was inserted by selecting File >> Insert Data. The field parameters electrostatic, hydrophobic and steric were computed by selecting QSAR Tools >> Compute Field window. The Gasteiger-Marsili charge was selected for the computation and invariable columns were removed. The data selection was done by choosing QSAR Tools >> Data Selection. Training data set selection method was applied to create training and test set for this random selection was done. The data was selected in the range of 65% to 85%. Finally, Variable Selection and Model Building Wizard tool was selected for the application of statistical methods like kNN, PLSR, MLR and PCR from Advanced Methods >> Method. The statistical data was generated which results in coefficient of
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determination (r), cross validated coefficient of determination (q), rfor external test set (pred_r) fitness plot and points of distribution. The best model was selected on basis of co-relation coefficient (r2) and various statistical parameters such as standard error of co-relation coefficient (r2se), Sequential Fischer test (F), predicted co-relation coefficient for external test set (pred_r2), and standard error of predicted external corelation coefficient (pred_r2se). The models were cross-validated by ‘leave one out’ scheme and cross-validation co-relation coefficient q2 was calculated. The model with high q2 value is said to have high predictability.
The reported investigation deals with synthesis and characterization of several indole bearing azetidinone nucleuses to form final twenty derivatives. To achieve these, three different steps were carried out. In the first step, isatin (1H-indole-2,3-dione) 1was reacted with hydrazine hydrate in presence of methanol under conditions of reflux to yield the 3-hydrazinylidene-1,3-dihydro-2H-indol-2-one 2. NMR spectra of this compound exhibited prominent signals at δ 9.39 ppm and 10.57 ppm corresponding to the secondary amide proton and primary amine protons respectively. The aromatic protons belonging to fused benzene ring was exhibited around d 6.87 to 7.37 ppm presenting four protons. The major spectral change was observed in the IR
spectrum which provides with an appearance of primary amine functional group at 3411. cm−1. In the next
step, Schiff bases (3-22) were formed by refluxing 2 with various substituted aromatic aldehydes in the presence of few drops of glacial acetic acid. These compound were confirmed on the basis of spectral studies;1HNMR spectra showed, in each case, the signals as multiplet at 6.11-8.55 ppm attributed to Ar-H in addition to the singlet at the N-H group in the region 8.77-10.89 ppm. The singlet also appeared at d 8.93–10.86 ppm attributed toone proton of N=CH. Thus, it confirmed the formation ofSchiff bases. The final derivatives of this series3{[2-chloro-3-substituted-4-oxoazetidin-1-yl]imino}-1,3-dihydro-2H-indol-2-one(23-42)were synthesized by carrying out cyclization of compounds (3-22)with chloroacetyl chloridein presence of triethylamine.These products were obtained in satisfactory yield and purity as studied on the thin layer chromatography and melting point studies. The structural confirmation was carried out on the basis of spectral studies,the IR spectra of these compounds exhibited absorbance for important functional groups, such as secondary amide at 3267-3395 cm-1; the carbonyl group is indicated at 1699-1745 cm-1and the C=N bond is reflected around 1521-1586 cm-1.These groups are common to all the molecules from final derivatives.The 1H NMR spectra of these compounds exhibited several characteristic NMR shifts.The 1H NMR spectra showed, in each case the signals as multiplet at δ 7.07-8.87 ppm attributed to Ar-H in addition to the singlet of the N-H group in the region δ 8.45-9.45 ppm. The singlet appeared for C-2 of the azetidinonering in the regions δ 5.07
6.88 ppm integrating for one proton. The singlet also appeared at δ 5.04-6.91 ppm attributed to one proton of CH-Cl. Thus, it confirmed the formation of indole ring containing azetidinonederivatives.EI-MS of all compounds displayed the [M + H]+ confirming their molecular weight. The elemental (CHN) analyses were found within the limit of theoretical values. (cm-1)
The compounds (23-42)were evaluated for antianxiety activity by elevated plus maze test (EPM) in mice at dose of (50 mg/kg) and compared with the standard drugdiazepam(2 mg/kg). There were no mortality and noticeablebehavioural changes in acute oral toxicity for all the groups tested. The synthesized compounds were found to be safeup to 2000 mg/kg body weight. Initially, dose-dependentstudy of compound 23at different doses (25, 50, 100, and200 mg/kg, i.p.) were performed to ensure the maximumeffective dose for new synthesized compounds as antianxiety in EPM. From this study, we found that 50 mg/kg is themaximum effective dose and therefore it was selected for further pilot study of antianxiety-like effects of compounds (2342)in EPM. Antianxiety activity was assessed as number of entries in open arm, and data has been presented asTable 2.
The standard diazepam had percentage preference to open arm 85.00% at a dose level of2mg/kg. In our research all the synthesized derivatives canproduce significant percentage preference to open arm when comparedto the standard drug.Compounds 28and 29were found to be the most potent derivatives fromthe series, showing preference to open arm 69.44 and 83.33 respectively. At the same time, compounds 23, 25, 27, 31and34 showed moderate activity while compounds 24, 35and 42showed poor activity.The preliminary SAR of indole bearing azetidinone ring suggested that substitution of the phenyl ring by electron releasing groups on ortho and para position lead to an increase of antianxiety activity (28, 29).Whereas compounds containing electron withdrawing groups at ortho and para position of the aromatic core showed good antianxiety activity (23, 25, 27, 31 and 34).Moreover,compounds having electron withdrawing group specially chloro at ortho and para position of the aromatic nucleus caused decrease in antianxiety activity (24, 42). Therefore,such compounds would represent a useful matrix for thedevelopment of a new class of clinically useful antidepressantagents and deserve further investigation and derivatization.
In the present study, Partial least square regressionmodel (PLSR-SW) is developed coupled withstepwise variable selection method to develop 3D-QSAR models ofindole bearing azetidinone derivatives as antianxiety agents based on steric and electrostatic fields. The total dataset was divided into training and test sets using the sphere exclusionalgorithm for diversity of the sampling procedure. This approachresulted in selection of compounds 23, 26, 27, 30 and 31as thetest set and the remaining 15 compounds as the training set Table 3.Selection of molecules in the training set and test is a key andimportant feature of any QSAR model. Therefore the care was takenin such a way that biological activities of all compounds in test liewithin the maximum and minimum value range of biologicalactivities of trainingset of compounds. The UniColumn statistics fortraining set and test set were generated to check correctness ofselection criteria for trainings and test set molecules and resultreflected the correct selection of test and training sets Table 4.Several statistically significant 3D-QSAR models using stepwisevariable selection method were generated, of which thecorresponding best model is reported herein. The best 3D-QSAR PLSR-SWmodel selected based on the value of statisticalparameters has a q2 = 0.8442 and pred_r2 = 0.0712 Table 5.
From Table 3, it is evident that predicted activities of all thecompounds are in good agreement with their correspondingexperimental activities. The plots of observed versus predictedactivity of both training & test sets molecules helped in crossvalidationof PLSR-SW QSAR model and are depicted in Figure 3.The contribution plot and the 3D-QSAR graphical interface provides with thepoints, points generated in the model are E_698, E_671 and S_394 accountingfor positive electrostatic, negative electrostatic, positivesteric fieldsrespectively at the lattice points on the grid as shown in the Figure 3,4,5,these points suggestthe significance and requirements or removal of the various substitutions on the structure tomaximise the antianxiety activity.It shows that the removal of E_671 at thephenyl ring i.e., the electropositive group(electrostatic descriptor) which is in the negative range and thus suggests that removalof these would help in increase in the activity.In a similar fashion the positive contributorswould be the electrostatic and steric descriptors E_698 and S_394respectively. Addition of electropositive and bulky group at thephenyl ring will contributetowards enhancing the antianxiety activity of the molecules.Theseresults are in close agreement with the experimental observationsthat compounds 28 and 29 with electropositive groups showed greater antianxiety activity while compounds 24, 35and 42 with electronegative groups showed poor activity.
Table 3: Observed and predicted activity by QSAR equation along with residuals
Compounds | Sets | Activity | Residual | ||
---|---|---|---|---|---|
Experimental | Predicted | ||||
23 | Test | 58.33 | 41.53 | 16.80 | |
24 | Training | 29.54 | 41.01 | -11.47 | |
25 | Training | 62.50 | 64.26 | -1.76 | |
26 | Test | 41.66 | 33.68 | 7.98 | |
27 | Test | 55.00 | 42.75 | 12.25 | |
28 | Training | 69.44 | 70.77 | -1.33 | |
29 | Training | 83.33 | 82.21 | 1.12 | |
30 | Test | 37.50 | 10.90 | 26.6 | |
31 | Test | 67.50 | 49.44 | 18.06 | |
32 | Training | 43.75 | 36.21 | 7.54 | |
33 | Training | 52.50 | 70.26 | -17.76 | |
34 | Training | 67.50 | 61.25 | 6.25 | |
35 | Training | 11.36 | 10.71 | 0.65 | |
36 | Training | 25.00 | 27.50 | -2.5 | |
37 | Training | 22.91 | 32.58 | -9.67 | |
38 | Training | 47.50 | 52.08 | -4.58 | |
39 | Training | 13.63 | 20.12 | -6.49 | |
40 | Training | 38.46 | 42.06 | -3.6 | |
41 | Training | 50.00 | 31.84 | 18.16 | |
42 | Training | 12.50 | 29.68 | -17.18 |
Table 4: Uni Column statistics of the training and test sets for QSAR model
Data Set | Average | Maximum | Minimum | Standard Deviation | Sum |
---|---|---|---|---|---|
Training set | 45.1076 | 83.3330 | 11.3630 | 22.0918 | 676.6140 |
Test set | 46.1666 | 67.5000 | 12.5000 | 21.7354 | 230.8330 |
199
Table 5: Statistical results of 3D-QSAR PLSR-SW model generated by stepwise variable selection method
Sr. No. | Parameters | Results |
---|---|---|
1. | n | 15 |
2. | Degree_of_freedom | 12 |
3. | r2 | 0.8825 |
4. | q2 | 0.8442 |
5. | F_test | 45.0539 |
6. | r2_se | 8.1802 |
7. | q2_se | 9.4201 |
8. | pred_r2 | 0.0712 |
9. | pred_r2se | 20.9780 |
Figure 3:Comparison of observed activity versus predictedactivity for training set & test set compounds according to 3D-QSARmodel by PLSR-SW method
Figure 4: Contribution Plot for Steric and Electrostatic Descriptors Selected in 3D-QSARmodel by PLSR-SW method
Figure 5: Stereo view of molecular rectangular field gridaround the superposed molecular units of indole bearing azetidinone derivatives using PLSR-SW method
In summary, we have described the synthesis of novel indole bearing azetidinone derivatives by conventional method with high purity and better yields of product. All the spectral studies were in good agreement with the final structures of the titled derivatives. All synthesized compounds were evaluated for antianxiety activity in EPM. Among all derivatives tested in the present study, compounds 28 and 29 exhibiting promising antianxiety activity comparable to that of the diazepam. The substitution of electron releasing groups on the para position of phenyl ring system provided with active compounds having percentage preference to open arm of 69.44 and 83.33 respectively. The 3D-QSAR study results revealed that addition of electropositive and bulky group at thephenyl ring will contributetowards enhancing the antianxiety activity of the molecules while electronegative groups showed poor activity and theseresults are in close agreement with the experimental observations. Hence, these studies are useful inunderstanding the structural requirements for design of novel and potent antianxiety agents.
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