Design and syntheses of some new ... - NOPR

Indian Journal of Chemistry Vol. 47B, October 2008, pp. 1559-1567

Design and syntheses of some new diphenylaminoisoxazolines as potent anti-inflammatory agent B R Dravyakar1, D P Kawade1, P B Khedekar2 & K P Bhusari*2 1

J L Chaturvedi College of Pharmacy, New Nandanvan, Nagpur 440 009 (MS)

2

Sharad Pawar College of Pharmacy, Wanadongri, Hingna Road, Nagpur 441 110 (MS) E-mail: [email protected] , [email protected] , [email protected] , [email protected]. Received 17 March 2008; accepted(revised) 5 August 2008

In the present investigation, a QSAR study is performed on twenty diphenylamino-2-isoxazoline derivatives using modified version of Allinger MM2 force field in Chem3D ultra structural descriptors. The relationship between antiinflammatory activity and various descriptors is established by stepwise multiple regression analysis. The analyses have produced well predictive and statistically significant QSAR models which are further cross validated. This study helps to design some expectedly potent compounds and synthesized, accordingly. These compounds are auxiliary screened spectroscopically and tested for anti-inflammatory activity. All compounds have showed better activity when compared with ibuprofen as standard. These compounds are further subjected to ulceration studies showing good results with almost no ulcerogenic activity. Keywords: Quantitative structure–activity relationships, nonsteroidal anti-inflammatory drugs, isoxazoline derivatives

Nonsteroidal anti-inflammatory drugs are mainly used in the treatment of pain and inflammation related to variety of pathologies1. Their anti-inflammatory effects are exerted by blocking the biosynthesis of prostaglandins2. Nonsteroidal anti-inflammatory drugs (especially, COX-inhibitors) under current clinical usage are highly acidic in nature and having common drawback of gastrointestinal toxicity, nephrotoxicity, etc3. This indicates the urge to develop newer agents. Several isoxazolines and its Mannich’s bases (i.e., aminomethyl derivatives) exhibited anti-inflammatory activity4-10. Furthermore, diphenylamine derivatives were also reported to possess good anti-inflammatory activity11. This increases our interest as it is having very low ulcerogenic toxicity to carryout the QSAR study. Several aminomethylisoxazoline diphenylamine derivatives were selected for the same study with the hope to obtain better anti-inflammatory agents with low ulcerogenic index and thus chosen for synthesis. (Figure 1 and Table I).

subjected to stepwise, multiple and sequential regression analysis with respect to biological activity (Table II). Correlation of each parameter was generated with biological activity. The number of developed models was high, so further analysis was based on statistically significant parameters, namely correlation coefficient (R), its square (R2), variance ratio (F), cross-validation method (Q2), standard deviation based on predicted residual sum of squares (SPRESS) and standard deviation of error of prediction (SDEP). The descriptors of diphenylaminoisoxazoline derivatives found to have good correlation with biological activity for designing anti-inflammatory agents which are summarized in Table III. Herein the results of the QSAR study for anti-inflammatory activity of mentioned series 4 are reported. R CH 2 NH

N N

Results and Discussion QSAR study In QSAR studies, all physicochemical parameters of each compound from the series were calculated and

O

R

Figure 1

1

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INDIAN J. CHEM., SEC B, OCTOBER 2008

Table I __ Anti-inflammatory activity data for diphenylamino-2isoxazoline derivatives used in this study Compd

R

R1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

3-OCH3, 4-OH H 4-N(CH3)2 4-OCH3 3-OCH3, 4-OH 3-OCH3, 4-OH 3-OCH3, 4-OH 3-OCH3, 4-OH H H H H 4-N(CH3)2 4-N(CH3)2 4-N(CH3)2 4-N(CH3)2 4-OCH3 4-OCH3 4-OCH3 4-OCH3

----H 2-Cl 3-Cl 2-OCH3 H 2-Cl 3-Cl 2-OCH3 H 2-Cl 3-Cl 2-OCH3 H 2-Cl 3-Cl 2-OCH3

a

IC50(µM) 0.7332 0.5799 0.9542 0.6686 0.5296 0.6512 1.0048 0.8451 0.8310 0.8992 1.3442 0.8952 0.4520 0.1224 0.9031 0.7011 0.4395 0.3234 0.5678 0.1880

b

pIC

6.13 6.24 6.02 6.17 6.28 6.19 6.00 6.07 6.08 6.05 5.87 6.05 6.34 6.91 6.04 6.15 6.36 6.49 6.25 6.73

IC50 values were determined by Carragennan induced rat paw oedema method b Negative logarithmic value of IC50 (in molees)[pIC = –log10IC50]

Table II __ Descriptors calculated for the QSAR study Sr. No.

Model II pIC = [-2.11486(± 0.811146)] +MR [0.0134564 (±0.00644908)]+PMY[0.00016579±0.000144508)] +DMZ [-0.0556885( ± 0.0677747)] N = 19, R = 0.9023, R2 = 0.8142, Variance = 0.0337, SD = 0.1837, F = 21.9244, Q2= 0.7076, SPRESS= 0.2305, SDEP=0.2048.

Type

1

Heat of Formation (HF)

Thermodynamic

2

Boiling Point (BP)

Thermodynamic

3

Critical Pressure (CP)

Thermodynamic

4

Critical Temperature (CT)

Thermodynamic

5

Critical Volume (CV)

Thermodynamic

7

Henry's Law Constant (HLC)

Thermodynamic

8

Ideal Gas Thermal Capacity (IGTC)

Thermodynamic

9

Log P

Thermodynamic

10

Melting Point (MP)

Thermodynamic

11

Molar Refractivity (MR)

Thermodynamic

12

Standard Gibbs Free Energy (SGFE)

Thermodynamic

13

Connolly Accessible Area (CAA)

Steric

14

Connolly Moleecular Area (CMA)

Steric

15

Connolly Solvent–Excluded Volume (CSEV)

Steric

16

Ovality (OVA)

Steric

17

Principal Moment of Inertia – X (PMI–X)

Steric

18

Principal Moment of Inertia – Y (PMI–Y)

Steric

19

Principal Moment of Inertia – Z (PMI–Z)

Steric

20

Dipole Moment (D)

Electronic

21

Dipole Moment –X Axis (DX)

Electronic

22

Dipole Moment –Y Axis (DY)

Electronic

23

Dipole Moment –Y Axis (DZ)

Electronic

24

Electronic Energy (EE)

Electronic

25

HOMO Energy (HOMO)

Electronic

26

LUMO Energy (LUMO)

Electronic

27

Repulsion Energy (RE)

Electronic

28

Bend Energy (Eb)

Thermodynamic

29

Charge–Charge Energy (CCE)

Thermodynamic

30

Charge–Dipole Energy (CDE)

Thermodynamic

31

Dipole–Dipole Energy (DDE)

Thermodynamic

32

Non–1, 4 VDW Energy (Ev)

Thermodynamic

33

Stretch Energy (SE)

Thermodynamic

34

Stretch–Bend Energy (SBE)

Thermodynamic

35

Torsion Energy (Et)

Thermodynamic

36

Total Energy (E)

Thermodynamic

37

VDW 1,4 Energy (VDWE)

Thermodynamic

38

Partition coefficient

Thermodynamic

a

Model I pIC=[-1.94512 (±1.00503)] +MR[0.0124763 (±0.00802789)] +PMY[0.000144693 (±0.000179957)]+DMZ[-0.0805902 (±0.0822254)] N = 20, R = 0.8451, R2 = 0.7142, Variance = 0.0535, SD = 0.2313, F=13.3331, Q2= 0.5787, SPRESS= 0.2809, SDEP=0.2512. This model has an outlier (Compound 14) because their residual values exceeded twice the standard error of estimate. When this outlier was removed from the dataset, a highly significant model II has been found which is able to explain 0.0337 of variance of antiinflammation. This model has a high internal predictivity as shown by the good Q2 value of 0.7076.

Descriptor

DRAVYAKAR et al.: SYNTHESES OF DIPHENYLAMINOISOXAZOLINES Table III __ Descriptors, observed, calculated and predicted anti-inflammatory activity data of compounds of training set Compd

MR

Descriptors PMY DMZ

Obsa

pIC Cal.b

LOO*

5.36 137.33 5412.13 0.96 6.13 5.56 1 5.36 142.13 5167.29 2.00 6.24 6.32 2 142.13 6850.19 0.11 6.02 5.43 4.96 3 5.21 143.79 5519.40 1.71 6.17 6.19 4 5.53 129.17 4437.73 1.82 6.28 5.27 5 133.98 4795.26 2.93 6.19 5.42 5.48 6 133.98 5414.67 0.53 6.00 5.05 5.30 7 5.41 135.64 4782.84 0.93 6.07 5.33 8 143.60 5430.06 1.02 6.08 5.47 4.99 9 148.41 5550.12 1.98 6.05 5.35 5.04 10 4.81 148.41 6528.12 -0.19 5.87 5.00 11 150.06 5565.55 2.15 6.05 5.15 4.93 12 135.64 5425.65 -0.57 6.34 5.17 5.31 13 140.44 5724.22 -0.49 6.91 * * 14 140.44 6544.99 -2.25 6.04 4.66 5.09 15 142.10 5178.33 2.79 6.15 5.10 5.31 16 102.99 4866.01 2.03 6.36 5.55 5.94 17 94.83 3678.13 4.53 6.49 5.88 6.13 18 109.26 6139.06 3.75 6.25 5.10 5.57 19 101.30 4912.69 1.28 6.73 5.30 5.70 20 a Observed value. b Calculated (Cal.) and predicted (LOO) values of pIC from Model II. * Compound removed as outlier Table IV __ Pearson correlation matrix for descriptors influencing inhibition of inflammation MR PMY DMZ

MR 1.0000 0.5206 0.2930

PMY

DMZ

1.0000 0.3022

1.0000

Figure 2

The parameters used in the model are almost independent, which can be seen from the Pearson correlation matrix (Tale IV, Figure 2).

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Two best models are selected from series, out of which model II was selected as the best. Because, this model has better statistically significant value, minimum standard deviation, low variance and statistically significant F- value. Molar refractivity (related to the volume of molecules and London dispersive forces), principle moment of inertia (steric parameter) and dipole moments (indicate the strength and orientation behaviour in electrostatic field) have better correlation with biological activity and have low value of standard deviation. The data showed (Model II) overall significant level greater than a 99.9 as it exceeded the tabulated F value. The above equation was validated by leave one out cross validation method and bootstrapping method as an internal validation, which gives statistically significant value. Whereas, Q2 was found to be greater than 0.5. Inflammatory mediators are membrane based and increasing the lipophilic nature of anti-inflammatory molecule may improve its pharmacokinetic and pharmacodynamic properties. Increase in lipophilicity and minimum steric interference increases antiinflammation suggested by positive value of molar refractivity (MR) and principle moment of inertia at Y axis, as both are moleecular properties describing electronic and steric parameters of the moiety. Normally both these properties are referred to certain group, thus substitution of lipophilic group will contribute to molar refractivity and principle moment of inertia. Furthermore, reduced electron density of amino group increases the electrostatic attraction for receptor. The dipole moment descriptor indicates the strength and orientation behaviour of molecule in an electrostatic field. It is also important in determining the behavior of the molecule in vicinity of the receptor. The Model II was tested for 19 compounds as a test set. The predicted activity shows linear relationship with observed activity in the test set (R = 0.90) showing the robustness of the model. Based on the above results, some new derivatives of diphenylaminoisoxazoline were designed and synthesized, accordingly. Chemistry The chemicals used were of Loba, Hi-media, E. Merck, SD Fine and National chemicals grade. The percentage yields are based upon the products obtained after purification through crystallization. The solvent used for crystallization has been mentioned in preceding text. The melting points of compounds

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O N

Diphenylamine

H +

Cl

C

O N

C

CH 3

CH 3

N, N-diphenylacetamide (1)

Acetyl chloride

CHO

_ OH

R O N

R

C CH

CH

N, N-Diphenylamino-3-substitutedphenyl2,3-propenamide (2a-c) NH 2OH. HCl R

N

O

N

3-Diphenylamino-5-substitutedphenyl2-isoxazoline(3a-c) HCHO,

Mannich reaction

R1 NH 2

H N H N N

H

O

R1

R

3-Diphenylamino-4-(substitutedphenyl)aminomethyl5-substitutedphenyl-2-isoxazoline derivatives(4a-x)

Scheme I

were determined in Celsius scale using Thermonik Precision apparatus (C-PMP-2, Mumbai, India). Silica gel G plates (activated at 1100, 30 min) were used for thin layer chromatography and were developed in iodine vapour chamber. Acetone:ethyl acetate (in equal ratio) was selected as solvent system for determining the Rf values, which is mentioned in the preceding text. The ultraviolet absorptions were measured in methanol (HPLC grade) on a Shimadzu 1601 spectrophotometer. IR spectra of compounds were recorded using KBr pellets on FTIR 8400s, Shimadzu, at Sharad Pawar College of Pharmacy, Wanadongri, Nagpur. 1H NMR spectra were recorded on a Varian EM 390 spectrophotometer (chemical shift in δ, ppm) at Pune University, Pune.

Diphenylamine taken as a starting material was subjected to nucleophilic substitution in the presence of acetyl chloride to form corresponding acetamide. The total reaction proceeded at low temperature due to low boiling point of acetyl chloride and gave better yield as compared to the reported4. Claisen-Schmidt condensation of corresponding acetamide with substituted benzaldehydes, gave the corresponding α,β-unsaturated carbonyl compounds, which on cyclisation with hydroxylamine hydrochloride resulted in isoxazoline formation. The isoxazolines were subjected to Mannich’s reaction using formaldehyde and corresponding substituted anilines to give the desired product (Scheme I).

DRAVYAKAR et al.: SYNTHESES OF DIPHENYLAMINOISOXAZOLINES

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Table V __ Physicochemical data of synthesized compounds 3a-c and 4a-x Compd 3a 3b 3c 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s 4t 4u 4v 4w 4x

R p-OH p-OCH3 o- OH, m-OCH3 p-OH p- OH p-OH p-OH p-OH p-OH p-OH p-OH o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 o- OH, m-OCH3 p-CH3 p-CH3 p-CH3 p-CH3 p-CH3 p-CH3 p-CH3 p-CH3

R1

Yield (%)

m. p. ( oC)

---p-Cl o-Cl m-Cl o, p-dichloro p-Br m- NO2 o- NO2 p-OCH3 p-Cl o-Cl m-Cl o, p-dichloro p-Br m- NO2 o- NO2 p-OCH3 p-Cl m-Cl o-Cl o, p-dichloro p-Br m- NO2 o- NO2 p-OCH3

80 74 59 67 64 42 49 42 93 65 42 57 37 33 39 43 78 81 58 59 45 68 34 62 78 75 48

108-10 112-14 102-04 140-42 154-56 120-22 106-08 164-66 212-14 154-56 118-20 128-30 80-82 116-18 94-96 120-22 182-84 128-30 88-90 146-48 94-96 110-32 106-08 146-46 212-14 164-66 108-10

All synthesized compounds were further characterized by melting point determination, Rf value calculation and UV spectra. The characterization data of synthesized compounds are given in Table V. The IR spectra of compounds could be obtained showing strong bands about 1153-1112 (C-O, stretching of five membered heterocyclic ring), 15981618 (C=N stretching of five membered heterocyclic ring), 842-632 (CH-Ar stretching), 3529-3359 (N-H stretching of 2o amine) and 1483-1433 cm-1 (CH2-N stretching) were in conformity of functional groups present in the structure assigned. In 1H NMR of the compounds, the protons present in isoxazoline appeared as doublet between δ 8.135-8.290 integrating for two protons. The signal due to aromatic proton appeared as multiplet at δ 7.015 integrating for six protons. The singlet was observed for secondary amine at δ 5.801 integrating for one

Rf value

λmax (nm)

0.68 0.42 0.50 0.64 0.58 0.42 0.38 0.59 0.61 0.49 0.42 0.36 0.72 0.64 0.45 0.75 0.61 0.45 0.42 0.54 0.51 0.42 0.38 0.68 0.81 0.69 0.42

280 278 274 287 282 285 289 275 268 267 278 247 247 249 254 249 268 270 272 267 232 255 251 281 288 277 248

proton. The methyl protons appeared as singlet at δ 2.518-2.978 integrating for two protons. Anti-inflammatory studies The test compounds 3a-c and 4a-x were evaluated in vivo for their anti-inflammatory using carrageenan induced rat paw edema method. The test compounds were administered by oral and intraperitoneal route at a dose level of 50 mg/Kg of body weight 1 hr before carrageenan injection. The paw volume was measured after 2 hr and 3.5 hr of carrageenan injection. The antiedematous effects of the test compounds were estimated and compared in terms of percent inhibition with ibuprofen as a standard. All the test compounds were also assessed for their gastric ulcer inducing action. The results of anti-inflammatory and ulcerogenic activity are shown in Table VI.

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INDIAN J. CHEM., SEC B, OCTOBER 2008 Table VI __ Anti-inflammatory activity and gastric ulceration of compounds 3a-c and 4a-x Compd

Ulcer index

3a 3b 3c 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s 4t 4u 4v 4w 4x Ibuprofen Control

0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 1/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 0/6 0/6 5/6 0/6

Swelling in thickness[x 10-2mm]a(% inhibition) Per oral (p.o.) Intraperitonial(i.p.) 2 hr 3.5 hr 2 hr 3.5 hr 12.4±1.02(40.39) 15.3±1.54(49.84) 13.6±2.12(44.30) 16.1±1.8 (52.44) 9.40±0.1 (30.62) 15.6± 1.2 (50.81) 13.9±2.4 (45.28) 11.9±1.2 (38.76) 20.2±1.4 (65.80) 12.7±1.8 (41.37) 13.6±2.8 (44.30) 11.5±2.1 (37.46) 10.2±1.5 (33.22) 13.3±1.5 (43.32) 11.7±0.7 (38.11) 12.5±0.2 (40.72) 20.5±3.5 (66.78) 8.6±1.7 (28.01) 10.1±0.5 (32.90) 13.4±2.4 (43..65) 12.5±2.3 (40.72) 20.4±2.5 (66.45) 19.6±3.4 (63.84) 11.3±0.9 (36.81) 12.4±1.1 (40.39) 15.8±2.3 (51.47) 18.7±3.6 (60.91) 16.2± 1.2 (52.77) 30.7 ± 0.96

13.0±1.24(40.88) 16.4±1.68(51.57) 14.1±1.47(44.34) 20.7± 2.1 (63.11) 13.5±1.4 (41.16) 19.8± 1.8 (60.37) 16.7±1.2 (50.91) 12.6±0.7 (38.41) 22.6±1.5 (68.90) 14.3±2.1 (43.60) 17.1±2.1 (52.13) 16.2±1.4 (49.39) 13.1± 2.0 (39.94) 12.4±1.6 (37.80) 14.2±4.5 (43.29) 12.9±2.4 (39.33) 21.1±2.0 (64.33) 12.1±2.3 (36.89) 13.1±1.4 (39.94) 13.7±4.0 (41.77) 13.4±2.1 (40.85) 22.1±2.6 (67.38) 21.2±2.7 (64.63) 14.1±2.1 (42.99) 1.45±0.12(44.21) 16.0±3.7 (48.78) 21.1±2.1 (64.33) 21.4±1.4 (65.24) 31.8 ± 1.1

15.1±0.98(48.40) 16.7±1.32(53.53) 16.2±1.26(51.92) 16.6±0.9 (53.21) 11.0±1.0 (35.26) 16.1± 2.0 (51.60) 14.3±1.3 (45.83) 12.8±2.2 (41.03) 21.3±1.4 (68.27) 12.8±2.4 (41.03) 13.9±2.8 (44.55) 12.0±0.3 (38.46) 10.5±1.5 (33.65) 13.8±2.5 (44.23) 11.8±3.1 (37.2) 13.4±1.1 (42.95) 22.1±3.4 (70.83) 11.2±1.5 (35.90) 10.5±0.7 (33.65) 13.5±1.9 (43.27) 13.1±3.8 (41.99) 20.9±3.2 (66.99) 20.5±2.1 (65.71) 12.4±0.8 (39.74) 13.2±2.4 (42.31) 16.7±3.4 (53.63) 19.9±1.2 (63.78) 17.6±1.7 (56.41) 31.2 ± 1.6

16.2±1.17(49.85) 18.4±0.87(56.62) 17.8±1.20(54.77) 21.5±1.5 (68.91) 145± 1.4 (46.47) 21.9±1.1 (70.19) 17.8±0.5 (57.05) 13.1±2.3 (41.99) 23.3±2.0 ( 74.68) 15.7±3.1 (50.32) 19.2±1.4 (61.54) 15.9±1.2 (50.96) 14.4±1.8 (46.15) 14.2±1.2 (45.51) 13.8±2.4 (44.23) 13.1±1.9 (41.99) 21.0±4.1 (67.31) 11.8±1.7 (37.82) 12.4±2.6 (39.74) 13.8±2.8 (44.23) 14.1±1.3 (45.19) 21.8±2.9 (69.87) 21.6±4.5 (69.23) 14.6±3.7 (46.79) 154±10 (49.36) 16.8±1.5 (53.85) 23.5±2.5 (75.32) 22.6±1.5 (72.44) 32.5 ± 1.2

Note: Number of animals used, n=6, Dose 50 mg/Kg body weight, inhibition %=[1–(Vt/Vc) × 100] where Vt is mean relative change in paw volume in test animals ND Vc is mean relative change in control group. All the test compounds are significant at P < 0.001 from the control. (Two way ANOVA followed by Bonferroni post test) a mean±SD.

The in vivo anti-inflammatory activity results indicate that all the test compounds possess statistically significant activity. Amongst these compounds 3b, 4a, c, f, l, q, r, v and x are found to be more potent when compared with standard. Furthermore, all the test compounds were generally found to be safer from the viewpoint of ulcer induction compared to the reference standard. Almost all the compounds showed gastric tolerance at 50 mg/ kg body wt. Thus, it can be concluded that study of

quantitative structure activity relationship helps to deduce good correlation between chemical structure and anti-inflammatory activity. The values in the results showed that compounds 4a-x were found to be more potent than compounds 3a-c. The most likely reason appears to be the probable good absorption of test compounds when administered by oral route or intraperitoneal route. The presumably reason was the test compounds are having bulkier groups at R and R1. This increases the lipophilicity which in turn enhances

DRAVYAKAR et al.: SYNTHESES OF DIPHENYLAMINOISOXAZOLINES

the permeability across the biological membrane to act directly upon either prostaglandin or lipoxygenase biosynthesis by inhibiting cyclooxygenase or lipoxygenase accordingly, ensuing good antiinflammatory activity. Experimental Section QSAR study The Dataset and parameters The anti-inflammation data of isoxazolines have been reported in terms of inhibitory concentration 50% of paw edema (IC50 in micromolees)4. The inhibition data were converted to negative logarithmic values (concentration in moles). These values were used for subsequent QSAR analyses as response variable. The models for inflammation inhibition were constructed based on the training set and the generated models were then validated: internally (using the leave one out technique) and externally (predicting the activities of the test set) 13-16. Moleecular structures were generated with ChemDraw Ultra 6.0 and optimized in CS Chem3D Ultra (Cambridge soft)17, first by molecular mechanics (MM2) and re-optimized by MOPAC– AM1 until the root mean square (RMS) gradient value becomes smaller than 0.0001 kcal/mole Å. Statistical computation The relationship between response variable (as a dependent variable) and various physicochemical as well as structural descriptors (as independent variables), were established by step–wise linear multiple regression analysis using SYSTAT 10.2 (ref. 18) and VALSTAT19 running on a Pentium 4 processor (CPU 3.00 GHz HT). Significant descriptors were chosen on the basis of statistical data of analysis. Synthetical study General procedure Synthesis of N, N-diphenylacetamide 1 Acetyl chloride (0.02 molee) was added drop wise to a cooled (0-5 oC) and constantly stirred solution of diphenylamine (0.1 mole) in chloroform (100 mL). The reaction-mixture was stirred on magnetic stirrer for 3 hr maintaining the temp (0-5oC). The solvent was distilled off. The residue was recrystallised from methanol. Yield: 15.3 g (70.83 %); m.p.: 98-100 oC; Rf : 0.65; λmax: 285 nm; Lit.Yield4: 68%; m.p.:103 o C; Rf : 0.59; λmax: 285 nm.

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Synthesis of N, N-diphenyl-3-(substitutedphenyl)2, 3-propenamides 2a-c To a solution of compound 1 (0.05 mole) in absolute ethanol (50 mL), the solution of sodium hydroxide (5 mL, 8%) and substituted benzaldehyde (0.05 mole) were added with constant stirring at temperature between 0-5 oC. The reaction-mixture was stirred for next 10-12 hr at room temperature and then poured into the cold water. The precipitated solid was filtered, washed with water, dried and recrystallised from ethanol. Yield: 10.8 g (65.93 %); m.p.: 78-80 oC; Rf : 0.7; λmax: 287 nm. Synthesis of 3–diphenylamino–5-(substituted) phenyl-2-isoxazolines 3a-c To a solution of compound 2a-c (0.03 mole) in ethanol (50 mL), hydroxylamine hydrochloride (0.03 mole) and sodium hydroxide (0.4 g) were added. The reaction- mixture was refluxed for 8 hr and poured into ice water. The precipitated solid was filtered, dried and recrystallised from ethanol. Yield: 4.5 g (73.28 %); m.p.: 132-34 oC; Rf : 0.57; λmax: 243 nm. Synthesis of 3-diphenylamino-4-(substitutedphenyl)-aminomethyl-5-substituted- phenyl- 2isoxazolines 4a-n To a solution of compound (3a-c) (0.01 mole) in methanol (50 mL), formaldehyde (0.02mole) and corresponding substituted aniline (0.02 mole) were added. The reaction-mixture was refluxed for 6 hr. The solvent was distilled off and the residue was poured into ice water. The precipitated solid was filtered off, dried and recrystallised from ethanol. Following the same procedure, all derivatives were synthesized. Some representative spectral data for compound 4 a-x is as follows. IR spectra for compound 4a: 806-644 (Ar-CH streching), 3579-3726 (O-H), 3379 (N-H stretching, secondary amine), 2893-2788 (C-H stretching), 1112 (C-O stretching of five membered heterocyclic ring), 1618 (C=N stretching of five membered heterocyclic ring), 1483-1450 (CH2-N stretching), 1400-1296 (C-O stretching), 3429-3379 ( aromatic N-H) cm-1. 1HNMR: δ 2.518 (d, 2H, CH2NHR), 3.404 (s, 1H, CHCH2) 5.801 (s, 1H, Ar-NH), 6.608 (d, 2H, CH2CH) 7.044-7.577 (m, 6H, C6H5), 8.135-8.290 (d, 2H, isoxazole). IR spectra for compound 4j: 824-632 (Ar-CH streching), 3728-3568 (O-H), 3438 (N-H stretching, secondary amine), 2873-2765 (C-H stretching), 1112

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(C-O streching of five membered heterocyclic ring), 1631 (C=N stretching (five membered heterocyclic ring)), 1481-1444 (CH2-N stretching), 1326 (C-O stretching), 3568-3438 (aromatic N-H) cm-1. 1H NMR for compound 4j: δ 2.978 (d, 2H, CH2NHR), 3.671 (s, 1H, CHCH2), 6.743 (d, 2H, CH2CH) 7.015 (m, 6H, C6H5), 8.561-8.410 (d, 2H, isoxazole). IR spectra for compound 4m: 811-686 (Ar-CH stretching), 3526-3579 (O-H), 3415-3359 (N-H stretching, secondary amine), 2738-2725 (C-H stretching), 1153 (C-O stretching, five membered heterocyclic ring), 1618-1599 (C=N stretching, five membered heterocyclic ring), 1433 (CH2-NH stretching), 1411 (C-O stretching), 3440-3359 (aromatic N-H) cm-1. 1H NMR: δ 2.624 (d, 2H, CH2NHR), 3.548 (s, 1H, CHCH2), 6.715 (d, 2H, CH2CH) 7.315 (m, 6H, C6H5), 8.162-8.201(d, 2H, isoxazole). Pharmacological studies Local bread albino mice of either sex weighing between 20 to 25 g, obtained from Biological E. limited, Hyderabad ( India ) were used in the present study. Animals were housed in wired mesh cages under the laboratory conditions (23 ± 2oC, 12 hr light) and maintained on a standard pellet diet (Hindustan Lever Ltd., Mumbai, India ) and water ad libitum before the day of the experiment. On the last day, food was withdrawn and they were given water only. During the course of experiment, the general behavior of animals was normal. All the experimental protocols were approved by the institutional animal ethical committee. The experiments were conducted in accordance with the standard guidelines. The animals were divided into three groups (control, standard and test) and each experimental group consisted of six animals.

solution was injected into the left hind paw as an internal control. The difference in footpad thickness between the right and left foot were measured with a pair of dial thickness gauge calipers in a different pattern of internals. The control groups received appropriate volumes of the vehicle only. Ibuprofen (50 mg/Kg body weight) was used a reference standard. The anti-edematous effects can be showed by the following equation. % anti-inflammatory activity = (1–V/Vc) × 100 Where, Vt = average difference in thickness between the left hind paw of test group Vc = control group of animals Gastrointestinal ulceration studies24 Mice were fasted 24 hr (with water available ad libitum) the compounds were suspended in a carboxymethyl cellulose dosage at 100 mg/Kg/day dose for five days in a volume of 0.5 mL/100g of body weight. The animals were sacrificed with diethyl ether inhalation, their stomachs removed by cutting along the greater curvature, washed under running water and fixed in formalin solution (5%). The stomachs were then examined for lesions under a dissecting microscope. Conclusion In the present study, an attempt has been made to design, synthesize and characterize some active antiinflammatory agents with minimal ulcerogenic side effect. From the results, it has been also pointed out that the bulkier substituents increase lipophilicity that may give better anti-inflammatory activity. The designed compounds 4a-x were synthesized according to the synthetic scheme.

Anti-inflammatory activity (in vivo)

Acknowledgement

The carrgeenan induced rat paw edema model according to the method reported by Winter et al.22,23, was employed for anti-inflammatory activity testing with some modifications. All the test compounds were suspended in carboxymethyl cellulose (0.5%) and administered either orally or intraperitoneally (50 mg/ Kg body weight) 60 min. prior the injection of 0.1 mL of freshly prepared solution of carrageenan (0.5 mg/25 mL) in physiological saline (154 mmole/ 1N NaCl )into the sub-planar tissue of the right hind paw of each mouse. The same volume of saline

The author wishes to thank Dr. K. P. Bhusari, Principal, Sharad Pawar College of Pharmacy, Nagpur and Dr. P. Trivedi, S.G.S.I.T.S., Indore, for providing facilities for this work. References 1 Hantoon M A, Minnesota Medicine, 84, 2001, 102. 2 Ross N, Encyclopedia of Medicine: Non Steroidal Antiinflammatory Drugs, http://www.enotes.com. 3 Mullican M D, Wilson M W, Connor D T, Kostlan C R, Schrier D J & Dyer, R D, J Med Chem, 36, 1993, 1090.

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17 CS Chem Office, version 6.0, Cambridge Soft Corporation, software publisher Association, 1730 M Street, NW, Suite 700, WashingtonDC, 20036 (202), 452-1600, USA. 18 SYSTAT 10.2 version supplied by SYSTAT SOFTWARE INC. 19 Gupta A K, Babu M A & S G Kaskhedekar, Indian J Pharm Sci, 66, 2004, 396. 20 Perun T J & Propst C L, Computer Aided Drug Design Methods and Applications, (Marcel Decker Inc, New York), 1989, 2. 21 Gerhard K & Abrahum U J, Computer Aided Moleecular Designs, 22, 1999, 473. 22 Winter C A, Edevin A R & Nuss G W, Proc Soc Exp Biol Res, 3, 1962, 544. 23 Vineger R, Schreiber W & Hugo J R, J Pharmacol Expt Therap, 166 1969, 96. 24 Turner R A, Screening Methods in Pharmacology, 1 (Academic Press Inc. London), 1971, 152.