Analgesic and Antiinflammatory Effects of Some New 6-Acyl-3-substituted-2(3H)-benzoxazolone Derivatives

J O U R N A L

O F

A publication of the American Pharmaceutical Association and the

American C hemicaI Society

March 1994 Volume 83. Number 3

RESEARCH ARTICLES

Analgesic and Antiinflammatory Effects of Some New 6-Acyl-3-substituted-2(3H)-benzoxazolone Derivatives DILEKDEMIREROL", RUMEYSADEMIRDAMAR (SUNAL)~, AND SUNADURU~ Received November 14, 1990, from the 'Pharmaceutical Chemistry Department, *the Pharmacology Department, and the §Toxicology Department, Faculty of Pharmacy, hkcettepe University, Ankara, Turkey. Accepted for publication June 19, 1993". Abstract 0 6-Acyl-2(3~benzoxazoionederivativeswere reacted with Csubstituted phenacyl bromide in ethanol to give the corresponding 69cyl-3-[(Csubstitutedbenzoyl)methyi]-2(3~benzoxazolones.The

compounds were screened for their analgesic, antiinflammatory activities. The eight compounds synthesized showed high analgesic activities and further Investigation revealed compounds 3, 6 and 7 to be potent inhibitors of carrageenan- and arachidonic acid-induced paw edema. The PGEl isolated from the carrageenan paw edema fluid was also significantly low in mice treated with compounds 3, 6 and 7.

Introduction 6-Acyl-3-substituted-2(3H)-benzoxazolonederivatives are reported to exhibit a broad spectrum of activity including antibacterial, analgesic, antiinflammatory, and anticonvulsant.14 Close et al.6 and Mercier et al.7 have reported that 3-acetyl2(3H)-benzoxazolonehas analgesic activity; Aries8 and Erol et al.9 and Erdogan et al.l0 observed that Mannich bases of 6-acyl2(3H)-benzoxazolonesare more potent analgesicswhen compared to aspirin. (Piperazinomethy1)benzoxazoloneswere also reported as analgesics with high antiinflammatory activities.11 We therefore aimed to investigate the effect of 2-acyl substitution on the analgesic and antiinflammatory activity of 2(3H)benzoxazolones.

Experimental Section (A)Chemistry-(a) 6-Acyl-2(3H)-benzoxazoZones-The title compounds were synthesized by heating 2(3H)-benzoxazolone with substituted benzoic acid in polyphosphoric acid for 7 h. 0

Abstract published in Advance ACS Abstracts, December 15,1993.

@ 1994, American Chemical Society and Amerlcen Phsrmeceirljcal Assocriatlon

After cooling,the solution was poured into ice-water. The crude product was filtered and crystallized from ethanol.12J3 (b) a-Bromo-4-substituted-acetophenones-Thesewere prepared by Immediate's method.14 (c)Sodium Salt of 6-AcyZ-2(3H)-benzoxazolones-The sodium salt of 6-acyl-2(3H)-benzoxazoloneswere made by dissolving 6-acyl-2(3H)-benzoxazolonesin a solition of sodium ethoxide. (d) 6-Acyl-3-[(4-substituted-benzoyZ)methyl]-2(3H)benzoxazolones-To the solution of sodium 6-acyl-2(3H)benzoxazolone in alcohol was added an equimolar amount of a-bromo-4-substituted-acetophenone. The solution was heated on a steam bath under reflux for 3 h, cooled, and filtered. The crude product was washed with cold water, dried, and crystallized from the acetone. (B) Pharmacology-(a) AnaZgesic Actiuity16-Locally bred albino mice of both sexes (23 f 2 g) were used. A modified Koster test for screening analgesic activity was employed. Compounds 1-8 were administered orally (100 mg/kg) as a suspension in 50% (w/w) simple syrup (T.F.). One hour later 3 % (w/w) acetic acid (300 mg/kg) was administered intraperitoneally. Ten minutes following ip acetic acid injection the number of stretches per animal was recorded for 10min. Control animals received an equal volume of vehicle, and aspirin was administered orally at a 100 mg/kg dose level as the standard drug. Analgesic activity was calculated using the formula (n-n') % Analgesic Activity = -x 100 n where n = the mean number of stretches of the control group and n' = the mean number of stretches of the test group. (b) Antiinf lamma tory Actiuity-(1) Carrageenan-Induced Mouse Paw Edema-Carrageenan-induced mouse paw edema (CPE) was measured using a Peacock dial thickness gauge (0.0110mm). Six mice per group were used. Sixty minutes after oral administration of the compound (100 mg/kg) 0.01 mL of 2% carrageenanwas injected subcutaneously into the plantar surface

00223549/94/ 120&273$04.50/0

Journal of Ph&vmaceutical Sciences / 273 VOI. a3, NO. 3, March 1994

Table 1-Physlcal

and Chemlcal Data of 6-Acyl-3-[(4-subslltuted-benzoyl)methyl]-2( BH)-benzoxazolones

Anal.c CompdNo.

R1

1

eCI

R2 H

Yielda (%)

mpb ("C)

75.1

201-3

2

&I

CI

77.2

205-6

3

eCI

Br

83.4

210-1

4

&I

NO2

81.2

21 1-1

5

mCI

H

78.1

200-1

6

mCI

GI

80.5

201-1

7

M I

Br

85.5

214-1

8

mCI

NO2

82.4

212-1

Empirical formula

Spectral Data

Calcd

Found

IRd (cm-l)

C: 67.44

67.46 3.52 3.48 61.32 3.35 3.18 56.36 2.97 2.84 60.38 3.40 6.74 68.65 3.83 3.70 62.11 3.22 3.49 56.33 2.87 3.10 60.6 1 3.05 6.44

1600' 1760" 1680"' 1610 1760 1680 1610 1765 1680 1610 1765 1690 1600 1750 1680 1610 1760 1680 1610 1760 1680 1610 1765 1690

H: 3.60 N: 3.57 C: 61.99 H: 3.07 N: 3.29 C: 56.14 H: 2.78 N: 2.98 C: 60.49 H: 3.00 N: 6.41 C: 67.44 H: 3.60 N: 3.57 C: 61.99 H: 3.07 N: 3.29 C: 56.14 H: 2.78 N: 2.98 C 60.49 H 3.00 N: 6.41

NMRe (ppm) 5.70(s) 8.20-7.29(m) 5.69(s) 8.19-7.48(m) 5.68(s) 8.11-7.47(m) 5.79(s) 8.40-7.44(m) 5.70(s) 8.16-7.29(m) 5.70(s) 8.19-7.48(m) 5.67(s) 8.11-7.47(m) 5.79(s) 8.42-7.44(m)

a Yield obtained after one crystallization from acetone. Melting points were determined on a Thomas-Hooverapparatusand were uncorrected. The C, H, N analyses were performed by Middle East Technical University, Department of Chemistry Microanalysis Laboratory, Ankara, Turkey. IR spectra were determined on a Perkin-Elmer Model 457 IR in KBr. *, aromatic ketone C=O; , lactam C=O; , methylene ketones C=O. NMR spectra were charted on a Brucker 80 MHz using tetramethylsilane as the internal standard and DMSO as the solvent. s = singlet, m = mukiplet.

of the right hind paw. Three hours later the edema volume was measured using a dial thickness gauge. (2) Arachidonic Acid-Induced Edema-Six mice per group was used. Sixty minutes after oral administration of the compound (100 mg/kg) 0.01 mL of arachidonic acid (100 pg/O.l mL) was injected subcutaneously into the plantar surface of the right hind paw and the volume of the resulting edema was measured with a dial thickness guage. (3)Isolation and Assay of Prostaglandin-like Activity-The source of prostaglandin-like activity was edema fluid from rat feet inflamed by carrageenan. A 1% suspension of carrageenan in saline was injected (0.01 mL) into the plantar surface of the right hind foot through a 26-gauge needle. To test animals, compounds 1-8 were administered orally (100 mg/kg) 1h prior to carragenen injection; 1.5 h after carragenan injection the animals were killed. The edema fluid was squeezed onto a blotting paper (0.5 mm diameter) and prostaglandin-likematerial extracted by the modified method described by Duru.lG The method used is a one-step method which minimizes the loss of material. Pasteur pipets were employed as colums and were filled with Amberlite XAD-2 resin, the blotting paper containing the edema fluid was placed on top, and the prostaglandin-like material was eluted with ethyl acetate-methanol (8.5:1.5). The eluate was evaporated to dryness at 40-45 "C and frozen at -20 "C until bioassayed on rat fundus strip. The rat fundus strip assay was performed according to the method of Vane.17 Results were statistically analyzed by Students t-test. 274 /Journal of Pharmaceutical Sciences Vol. 83, No. 3,March 1994

Results The physical properties of compounds 1-8 are given in Table 1. Compounds 1-8 all shoved high analgesic activity. Then they were all screened for their ability to inhibit CPE. Compounds 3,6,and 7 inhibited edemasignificantly when compared to indomethocin inhibition (p < 0.05) (Table 2). These three compounds were also found to be potent inhibitors of arachidonic acid-induced edema (Table 2). We then investigated the ability of compounds 1-8 to inhibit PGEz synthesis. In this assay compounds 3, 6,and 7 proved to inhibit PGEz synthesis too (Table 2).

Discussion The preparation of 6-acyl-2(3H)-benzoxaolone was previously reported.9 N-Alkylation on the 3-position was accomplished by refluxing a mixture of 4-substituted phenacyl bromide and the sodium salt of 6-acyl-2(3H)-benzoxaolone in alcohol. 2(3H)-Benzoxazolones have structural similarity to urethan; in fact, they are cyclic urethans. These compounds are used as anticonvulsants and have analgesic activities. We therefore and insynthesized 3-substituted-6-acyl-2(3H)-benzoxazolones vestigated their analgesic and antiinflammatory activities. The analgesic activity of the compounds were screened using aspirin as a reference analgesic. As can be seen in Table 2, the compounds 1-8 showed analgesic activities higher than aspirin. Then we screened the compounds for their inhibition of

Table 2-Pharmacologlcal

compd

Data of the Compounds.

Inhibn of Arachidonic Isolated PGEp inhibition of AcidC (ng/mL) from the Analgesic CPE (%)" Edema (%) Rat Paw Edema Activity

1 2(f2.1) 2 6(*1.9) 3 69'(f2.4) 4 36(f3.1) 5 17(*2.7) 6 59(f4.2) 7 77*(f1.6) 8 20(f3.7) Controls Indometd 36(f4) Aspirind

19.2(f1.4) 36.3(*3.2) 45.5(f2.7) 16.3(f1.9) 22.5(f3.1) 47.3*(*4.1) 44.5(f2.6) 16.2(f4.9)

5.9(f0.4) 5.l(f0.9) 1.3'(f0.2) 2.5(f0.6) 2.4(f0.1) 2.3(*0.7) 1.2'(f0.3) 5.1(*0.4)

80.57(f6.1) 90.48(*3.4) 90.48(f5.2) 85.38(*4.3) 84.76(f3.7) 85.05(f4.6) 92.52(*3.1) 75.45(f6.8)

37.0(f1.7)

l.g'(f0.3)

58.97(*4.1)

a Indomethacin 5 mg/kg ip injection. CPE = carrageenan paw edema. Arachidonic acid (100 pglO.1 mL) administered sc into the (100 mg/kg) orally plantar surface of the hind paw of mice. dA~pirin administered. 'Significantly different from the control (p < 0.05). Compounds 1-8 were orally administered at 100 mg/kg in a simple syrup. Carrageenan was injected 1 h after administration of the compounds. Edema was measured 1.5 h following carrageenan injection. Results are expressed as fSEM.

carrageenan-induced edema. The edema inhibition of compounds 1,2,5, and 8 were not significant when compared to the inhibition obtained by indomethacin;however,the inhibition of carrageenan-inducededema by compounds 3, 6, and 7 (69% , 59%,and 7776, respectively) was significantly high and compound 4 was equipotent to indomethacin (Table 2). The compoundswere then screened for their ability to inhibit arachidonic acid-inducededema. Compound 2 inhibited arachi-

donic acid-induced edema as well as indomethocin, but compounds 3, 6, and 7 inhibited arachidonic acid-induced edema significantly (Table 2). However, only compounds 3 and 7 significantlyinhibited PG& synthesis. From these results we concluded that compounds 3,6, and 7 (3-(4-bromobenzoyl)and 3-[4-(nitrobenzoyl)methylIderivatives of 6-acyl-2(3H)-benzoxazolones)are active analgesic, antiinflammatorycompounds and their activity is via arachidonicacid inhibition.

References and Notes 1. Erol, D. D.; Rosen, A.; Erdoian, H.; Yului, N. Arzneim. Forsch. 1989,39 (II851. ), 2. Erol, D. D.; Erdogan, H.; Yului, N. J. Pharm. Belg. 1989,44 (5), 334. 3. Ackrell, J.; Antonio, Y.; Franco, F.; et al. J. Med. Chem. 1978,21, 1035. 4. Dunn, J.; Muc owski, J. J . Med. Chem. 1981,24,1097. 5. Safak, C.; Erdokan, H.; Palaska, E.; Sunal,R.; Duru, S. J . Med. Chem. 1992,35,1296. 6. Close, J. W.; Tiffany, B. D.; Spielman, M. A. J. Am. Chem. SOC. 1949,71, 1265. 7. Mercier;J.; Lespagnol, C.; Sestier, M. R. Bull. SOC.Lille. 1953,35. 8. Aries, R. Fr. Pat. 1970, 1,593,066 03 Jul. 9. Erol, D.D.;Sunal, R.; Duru, R. Arzneim. Forsch. 1990,40(I), 478. 10. Erdoian, H.;Unlu, S.; Sunal,R. Arch. Pharm. 1989,322,75. 11. Sunal,R.;Erdo&n,H.;Duru,S.Proceedingsojthelstlnternationul Congress on Inflammation June Barcelona, 1990; pp 110. 12. Bonte, J. P.;Lesieur, D.; Lespagnol, C.; Cazin, J. C.; Cazin, M. Eur. J . Med. Chem.-Chim. Ther. 1974,9,491. 13. Renard, P.; Lesieur, D.; L e s p y l , C.; Lespagnol, D.; Cazin, J. C. Eur. J. Med. Chem.-Chim. her. 1980,15,453. 14. Immediate, T.;Day, A. R. J . Org. Chem. 1940,5,512. 15. Koster, S.;Anderson, M.; Debeer, E. J. Fed. R o c . 1959,18,412. 16. Duru, S.; Sahin, G. Bull. Hacettepe Uniu.Fac. Pharm. 1981,l (l), 18. 17. Vane, J. R. Br. J. Pharmacol. 1957,12,344.

Journal of Pharmaceutical Sciences / 275 Vol. 83,No. 3, March 1994