Synthesis of Some Substituted Quinazolinediones as Potential Inhibitors of Smooth Muscle Contraction

September 1988 Volume 77, Number 9

JOURNAL OF PHARMACEUTICAL SCIENCES

ARTICLES

Synthesis of Some Substituted Quinazolinediones as Potential Inhibitors of Smooth Muscle Contraction HULYAAKGUN*,ULRlCH HOLLSTEIN*’’, AND LEON HURWITZ* Received January 26, 1988, from the ‘Deparfment of Chemistry, and *Departmentof Pharmacology, School of Medicine, The University of New Mexico, Albuquerque, NM 87131. Accepted for publication April 26, 1988.

Dicyclohexylcarbodiimide (DCC) coupling with a carboxylic acid (e.g., p-methoxyphenylacetic acid) gave the corresponding amide 1 1 2 (Scheme I). Acid hydrolysis deprotected the other amine of the piperazine to give 2.1° The amide was reduced with lithium aluminum hydride (LAH) to yield 313 (this compound has recently been reported14). The piperazine fragment was combined with chloroacetonitrile to form 4, which was reduced to the primary amine 5 with Raney nicke115.16 (Scheme 11). The amine 5 reacted smoothly with isatoic anhydride to give a substituted anthranilamide 6, which upon treatment with ethyl chloroformate yielded 7. Ring closure of 7 in ethanolic KOH4J7produced the final substituted quinazolinedione 8. A number of reports in the literature indicate that com1,4-Dihydro-2,4-dioxo-3(2H)-quinazolineacetic acid and 1pounds containing the quinazolinedione unit in their strucacid ture possess pharmacological activity. 2,4(lH,3H)-Quinazo- methyl-1,4-dihydro-2,4-dioxo-3(~-quinazolineacetic (10) were synthesized by the procedure of Papadopoulosls linedione and 1,3-dimethyl-2,4(lH,3H)-q~inazolinedione~-~ [the former compound 10 (R= H)is described by Papadopouhave been shown to possess anticonvulsant3 activity against 10~181.Coupling of anthranilic acid or N-methylanthranilic electroshock- and pentylenetetrazole-induced seizures in mice. 3-(4-Aryl-1-piperazinylalkyl)-2,4(1H,3H)-quinazoline- acid with ethyl isocyanoacetate produced the substituted diones exhibit sedative and hypotensive activities.4 3-Alkyl- urea 9 which, upon treatment with conc. HC1, generated a substituted5 and 1,3-dialkyl-substituted 2,4(1H,3H)-quinazo- quinazolinedione fragment that is substituted with two carlinediones cause vasodilation in animals.s.7 Moreover, 3- bons at N-3 (10; Scheme 111). l,4-Dihydro-2,4-dioxo-3(2H)quinazolineacetic acid and the alkyl-substituted quinazolinediones are useful as antiinflammatory agents and 5-hydroxytryptamine (serotonin) corresponding piperazine fragments in the presence of DCC led to 3-(4-alkylaryl-l-piperazinylalkyl)-2,4-(1H,3H)-quinaantagonists.8.9 In review of these results, we synthesized a set of related structures containing, in addition to quinazo- zolinediones (Scheme IV). A number of the compounds synthesized were subjected to linedione, other structural elements that might possibly preliminary tests that would provide some indication of their confer useful biological action. The new structural determibiological activity. The tests consisted of exposing isolated nants a r e a two-carbon chain, connected to N-3 of quinazolinedione, carrying via a piperazine bridge a variable carbon chain and terminating in a substituted benzene nucleus. In view of previous reports indicating that derivatives of 1,3-disubstituted 2,4(lH,3H)-quinazolinediones exhibit a vasodilator action,5-6.7preliminary experiments were 1 performed to explore the inhibitory action of our quinazolinediones on the contractile function of smooth muscle.

Abstract 3-Substituted 2,4(1H,3H)-quinazolinedioneswere prepared from the corresponding Nsubstituted 2-arninobenzamides by treatment with ethyl chloroformate and KOH in ethanol. Also, a series of 3substituted and 1-methyl-3-substituted 2,4(1H,3H)-quinazolinediones were synthesized by the reaction of 1-methyl-1,Cdihydro- and 1,4dihydro-2,4-dioxo-3(2H)-quinazolineacetic acid with the corresponding Ksubstituted piperazines. The 13C NMR spectra and mass spectra of the compounds were measured and signals were assigned. Some of the compounds showed inhibitory action on contractile function of smooth muscle.

Results and Discussion The synthesis of the variable side chain started with piperazine blocked a t one side by benzyloxycarbonyl(Z-).10*11 aO22-3549/88/0900-0735$0 1 .OO/O 0 1988, American

Pharmaceutical Association

2 Scheme I

Journal of pharmaceutical Sciences / 735 Vol. 77, No. 9, September 1988

ti

H

1

s

Scheme II

@YH

~~%HCH2COOC2H5

I

OCN-CH2COOC2H5 >

R

R

9 0 conc. HC1, ~ J ; C H 2 - C O O H

i

R = CH3 or H

zs? Scheme 111

0

0

n

R

R'

11 --

12 --

H

3-p Cl 0

H

CH3

-O-;-CH3

-CH -CH - O - O C H 3

2

2

CH3 C1. 0

-O-!-CH3

Scheme IV 736 /Journal of Pharmaceutical Sciences Vol. 77, No. 9, September 1988

longitudinal muscles from the guinea pig ileum and/or isolated tracheal muscles from the guinea pig to these compounds and observing the effects that the compounds had on the contractile function of the smooth muscle tissues. When the isolated ileal muscle was used as the test preparation, it was suspended in a muscle bath that contained a standard physiological bathing medium (composition given below) and was subsequently transferred to a bathing medium to which a high potassium ion concentration, but no calcium ion, had been added. The muscle was then induced to contract isometrically by introducing a final concentration of 1.8 mM CaClz into the muscle bath. Each muscle was contracted both in the absence and presence of one of the synthetic compounds and the degree of inhibition of contraction produced by the compound was noted. When the isolated tracheal muscle was used as the test preparation, it was suspended in a muscle bath that contained a standard physiological bathing medium. An isometric contraction was then elicited by adding a final concentration of 1 x M acetylcholine to the muscle bath. As in the former case, each muscle was induced to contract both in the absence and presence of one of the synthetic compounds and the degree of inhibition of contraction produced by the compound was noted. Table I shows the preliminary results obtained with each compound tested. It would appear from these results that 8, at a concentration of 2 x M, had an appreciable inhibitory effect on the isolated tracheal muscle and a small inhibitory effect on the isolated ileal muscle. Compound 15, at a concentration somewhat less than 2 x M, had an appreciable inhibitory effect on the isolated ileal muscle, but not on the tracheal muscle. All the other data obtained indicate that the remaining compounds exert little or no inhibitory actions. The unique inhibitory effect of 15 is of some interest. Our observation that this compound can significantly reduce a potassium-activated contraction of ileal smooth muscle without producing any change in an acetylcholine-activated contraction of tracheal smooth muscle permits some speculation about its mechanism of action. The lack of any effect on the tracheal muscle would seem to rule out an anticholinergic action, a beta-adrenergic agonist action (and consequent increase in cyclic AMP), or a direct inhibitory action on the contractile apparatus as possible underlying mechanisms for its inhibitory effect. Thus, as one reasonable alternative, the inhibition of the potassium-induced contraction of the ileal muscle by 15 could have resulted from a blockade of voltagesensitive calcium channels in the cell membrane. Additional work will, of course, be required to confirm or negate this hypothesis.

Experimental Section Melting points were determined in capillaries using a ThomasHoover Unimelt apparatus and are uncorrected. The I3C NMR spectra were taken on a Varian FT-80 spectrometer using solutions in CDC13or DMSO-d,. Chemical shifts (6) are given in Tables I1 and 111. Mass spectra were recorded on a Finnigan 4600 automated gas chromatograph-mass spectrometer data system. All chemicals were from Aldrich Chemical Company. Microanalyses, indicated by the symbols of the elements, were within 2 0.3%of the calculated values and may be obtained from the author. Part A-I -Benzyloxycarbonyl-4-p-methoxybenzylcarbonylpiperazine (2)-A mixture of 1.5 g of l-benzyloxycarbonylpiperazine,lo,ll 10 mL of dry CH2Cl,, and 1.158 g ofp-methoxybenzylcarboxylic acid in 10 mL of dry tetrahydrofuran (THF) was stirred in a flask with a drying tube. The flask was placed in an ice bath and a solution of 1.42 g of dicyclohexylcarbodiimide (DCC) in 10 mL of dry CH2C12 was added. The mixture was stirred a t room temperature for 2 h, and cooled in the refrigerator for 1 h. 1,3-Dicyclohexylurea (DCU) was separated by filtration. The solvent was removed under reduced

Table Clnhibltlon of Smooth Muscle Contractlon by Qulnazolinediones Concentration Compound

of Compound, M

Ileal Smooth Muscle

Tracheal Smooth Muscle

No. of Expts.

% Inhibition

10 10

27.4 k 4.8 12.0 f 3.4

6 7 6 5

10.2 ? 4.2 45.9 f 3.5 10.3 k 1.3

2

SEM

No. of Expts.

% Inhibition

* SEM

~~~

~

8 11 14 12 15 13

2 2 2 2 2 2

x 10-5

x x x x x

10-58

10-5b 10-5a 10-5a 10-5*

7.0 k 3.3

64.7 f 6.3 0

0 0

a Compounds 11,12, and 15 were not readily soluble in water; the quantity of material necessary to make a final concentrationof 2 x 10-5 M could almost all be dissolved if the water was first acidified and then brought to a pH of 7.5 with Tris buffer; consequently, the concentrations of 11, 12, 15 used in the biological experiments were slightly less than 2 x 10 M. bCompounds 13 and 14, in the amounts required to make a final concentration of 2 x M, could not be entirely dissolved in H20.Therefore, the actual concentrations of the compounds used in the biological experiments are M close to, but not exceeding 2 x

pressure to give 2.56 g of 1: mp 95-97 "C (ethanol); yield 98%; MS: rnle (%) 368 (M+, 101,277 (27). 261 ll0),249(5), 233 (trace), 148 (101, 121 (731, 91 (100). Anal.-Calc. for CZ1Hz4NZO4: C,H,N. p-MethoxybenzylcarbonyLpipermineHBr (2)-To a solution of 0.400 g of 1in 5 mL of glacial acetic acid was added a mixture of 2 mL of acetic acid and 1mL of HBr (47%).The mixture was stirred for 2 h at 60 "C, cooled, and then filtered after the addition of 20 mL of ether, mp 208-210 "C (ethanol); yield 81%; MS: mle (%) 234 (M+, 17), 148 (33), 121 (loo), 113 (116), 91 (8), 56 (44). Anal.-Calc. for Cl3Hl9BrN2O2: C,H,N. p-Methoxyphenethylpiperazine@)-A solution of 76 mg of LiAlH, in 4 mL of dry ether was placed in a three-necked flask equipped with a condenser, a stirrer, and a dropping funnel. The solution was gently refluxed while 400 mg of 2 in 5 mL of dry ether was added over a period of 2 h. The mixture was stirred overnight. The reaction mixture was worked up by the addition of 0.5 mL of water. After filtration to remove the A1 and Li salts, ether was evaporated to give a yellow liquid, mp 260-262 "C (HC1 salt); yield 61%; MS: rnle (%) 220 (M+, trace), 135 (4), 121 (81, 99 (loo), 91 (4), 70 (12), 56 (21). Anal.-Calc. for C13H22ClzNz0:C,H,N. 1 -(~-MethoxyphenethyL)~-(cyunomethyl)piper~zne (4)-A mixture of 0.460 g of 3 and 0.100 g of chloroacetonitrile was stirred while heated at 80 "C for 3 h. First 5 mL of water, and then concentrated HC1 was added to the mixture until the pH reached 2. The acidic mixture was extracted three times with 5 mL of chloroform. The water layer was basified with 4 M NaOH until the pH reached 11, and then extracted three times with 5 mL of CHCl,. The chloroform was removed under reduced pressure, mp 105-108 "C (ethanol);yield 66%;MS: rnle (%) 233 (M+-CN,trace), 216 (trace), 189 (12), 157 (32), 135 (20), 121 (201, 113 (301, 99 (loo), 91 (141, 70 (44). Anal.-Calc. for C16H21N30 C,H,N. l-(p-MethoxyphenethyZ)4-(2-aminoethyl)pipermine (5)-A mixture of 0.400 g of 4,0.200 g of Raney nickel, and 18 mL of methanolic ammonia was placed in a hydrogen flask, treated with hydrogen for 2 h, and filtered. The methanol was removed under reduced pressure to give a yellow oil, mp 260-265 "C (dec; ethanol HCl salt); yield 80%; MS: rnle (Yo) 261 (M+-2)trace, 233 (24), 189 (ll),156 (ll),142 (1001, 135 (go), 121 (32), 97 (57), 91 (12), 70 (50), 55 (44), 42 (61). Anal.-Calc. for C15H&13N30: C,H,N. 2 -A mi no -N -[2 4 4 -met hoxyp he net hy 1-1 -piperaziny 1 ) ethyllbenzamid (6)-This product was obtained by the procedure of Hayao and Schut,16 mp 124-126 "C (ethanol); yield 48%; MS: rnle (%) 368 (M'-14, trace), 261 (77), 233 (15), 163 (15), 135 (54), 120 (1001, 97 (701, 83 (54), 69 (74). Anal.-Calc. for C22H31CIN40z:C,H,N. 2 -Carbethoxyamino-N-[244 -methoxyphenethyL)-1 -piperazinyZlethyllbenzamid (7)-A mixture of 0.5 g of 6 and 4.3 mL of ethyl chloroformate was heated over a steam bath for 3 h and evaporated under reduced pressure to a residue which was recrystallized from ethanol, mp 228-230 "C; yield 58%;MS: rnle (%) 545 (M+,trace), 409 (trace), 333 (82), 287 (25), 261 (trace), 233 (30), 189 (21), 146 (loo), 135 (44), 121 (40),98 (48). Anal.-Calc. for Cz5H3&lzN404.Hz0: C,H,N. 3 -[2 - [ 4 - ( 4 -Methoxyphenethyl) - 1 -piperazinyllethyll-2,4(lH,3H)quinmolinedione @)-A mixture of 0.4 g of 7 and 1g of KOH

in 45 mL of ethanol was refluxed over a steam bath for 4 h and evaporated to dryness. A solution of the residue in a minimum amount of water was adjusted to pH 7-8 with acetic acid. The precipitated product was recrystallized from ethanol, mp 207210 "C; yield 65%; MS: rnle (%) 408 (M+, trace), 287 (loo), 189 (15), 146 (15), 125 (48), 111 (12), 99 (20), 98 (20),97 (22). Anal.-Calc. for C23H28N403:C,H,N. Part B-241 -MethyL-3-ethoxycarbonylmethylureido)benzoicacid (9) and l-methyl-1,4-dihydro-2,4-dioxo3H~2H)-quinazolineacetic acid (10; adapted from PapadopouLous18)-A solution of 4 g of N methylanthranilic acid in 35 mL of saturated KHCO, was shaken vigorously with 3.3 mL of ethyl isocyanoacetate for 25 min. The solution was acidified with concentrated HCI and filtered. The final product was crystallized from ethanol (mp 135-137; yield 63%) to give 2-(l-methyl-3-ethoxycarbonylmethylureido~benzoic acid (9). Three grams of 9 and 30 mL of concentrated HCl was heated on a steam bath for 1.5 h to yield 10. The mixture was cooled and diluted with water. White crystalline needles were observed, mp 238240 "C; yield 80%;MS: mle (%) 234 (M+,trace), 189 (201, 142 (401, 121 (50), 111 (lo), 105 (25), 91 (73), 83 (50),69 (75),55 (100). Anal.-Calc. for CllH16N204:C,H,N. Procedure A-A mixture of 1mmol of 1,4-dihydro-2,4-dioxo-3(2H)quinazolineacetic acid in 5 mL of dry THF and 1 mmol of the corresponding piperazine chain in 5 mL of dry CH2C12was cooled in a flask with a drying tube. Then, 1 mmol of DCC in 5 mL of CH2C1, was added. The mixture was stirred for 15 min in an ice bath and for 2 h a t room temperature. The reaction mixture was cooled to 5 "C for 1 h, and DCU was removed by filtration. The solvent was removed under reduced pressure. Procedure B-A mixture of 1 mmol of 10 in 5 mL of dry THF and 1 mmol of the corresponding piperazine chain in 5 mL of dry CH,Cl, was cooled in a flask with a drying tube. Then, 1mmol of DCC in 5 mL of CHzClz was added. The mixture was stirred for 3 h in an ice bath, DCU was removed by filtration, and the solvent was evaporated. 3 -[2 -Ox0 -2-[4 4 4 -methoxyphenethyl)-1 -piperaziny LJethyL J 2,4(1H,3H)-quinazolinedione (11)-Procedure A-mp 230 "C (dec; ethanol); yield 53%; MS: rnle (%) 422 (M+, trace), 301 (71), 219 (trace), 203 (7), 175 (32), 146 (38), 135 (7), 121 (7), 99 (100). Anal.-Calc. for Cz3Hz6N,O4:C,H,N. 3 -[Z- 0 x o - 2 - [ 4 - ( 2 -chlorobenzyl) - 1 -piperazinyllethyll-2,4(lH,3H)-quinazolinedione (12)-Procedure A-mp 180-183 "C (ethanol); yield 55%;MS: mle (%) 412 (M+,trace), 287 (2),209 (9),203 (151, 180 (41),168 (441, 146 (601, 125 (loo), 111 (4). AmZ.--Calc. for Cz1HZ1N4O3Cl:C,H,N. 3 4 2 - 0 x o - 2 -[44 4 -acetylphenyl) - 1 -piperazinyl]ethyll-2,4(1H,3H)-quinazolinedione (13)-Procedure A-mp 225-227 "C (ethanol); yield 48%; MS: rnle (%) 406 (M+,trace), 203 (60), 175 (72), 146 (loo), 119 (48), 111 (10). 3 4 2 -Ox0 -2 - [ 4 4 4 -methoxyphenethyL) -1 -piperazinylJethylJ1 -methyl-2,4(3H)-quinazolinedione (14)-Procedure B-mp 210212 "C (ethanol); yield 5 0 4 ; MS: rnle (%) 325 (M'-107, trace), 315 (59). 301 (4). 217 (22). 189 (78). 146 (6). 135 (13). 121 (27). 105(12).99 (loo), 91 (14), 77 (15). Anal.-Calc. for C24H24N404.0.4H20: C,H,N. 3-[2 - 0 x o - 2 - [ 4 - ( 2- c h l o r o b e n z y l )- 1 - p i p e r a z i n y l l e t h y l l - 1 Journal of Pharmaceutical Sciences / 737 Vol. 77, No. 9, September 1988

Table II--'3C Chemical Shifts Assignments of Compounds 1-8

Carbon Number Compound

1

2

3 and 7

4 and 6

5

8

9

10 and13

11 and12

R Group

la

55.5

155

115

130

137

40

170

43

46

2a

56

158

115

131

128

36

170

41

43

H

3"

56

158.5

114

130.5

133

33

62

55

44

H

4b

55

158

115

130

133

33

60

51

52

5b

53

157

113

129.5

132

32

60

55

55

117

33

- 4 - CZN 55

32

- 4- 4- NH2 n

6b

57

157.5

114

129

133

32

55

53

127.5

53

0 7b

55

158

113.5

129.5

132

34

55.5

48

121.5

48 54

34

12G

18 6 4 153

55

158

114

129.5

132

32

60

methyl-2,4(3H)-quinazolinedione (15l-Procedure B-mp 164-167 "C (ethanol);yield 5 0 4 ; MS: mle ('%) 426 tM+,trace), 391 (M'-Cl, trace), 359 (trace), 315 (trace),301 (trace), 217 ( 2 4 , 209 (8), 189 (57), 180 (35), 168 (44), 125 (1001, 99 (191, 77 (14). 3 - 1 2 - 0 x 0 - 2- / 4 - ( 3- a c e t y l p h e n y l - 1 - p i p e r a z i n y l l e t h y l l - 1 methyL2,43HI -yuinazolinedione (16)-Procedure B-mp 205-207 "C (ethanol);yield 45%;MS: mle (9%)420 [M', 601,217 (481,203 (12), 189 (951, 174 (loo), 161 (41), 146 (17), 132 (48), 105 (15). Anal.-Calc. for C2:,H24N404.H20:C,H,N. Pharmacology-Segments of longitudinal smooth muscle were isolated from the guinea pig ileum by the method described by Weiss et a1.19The segments were suspended in muscle baths that contained a physiological bathing medium maintained a t 30 2 1 "C. The tracheal muscle was prepared by isolating the trachea from the guinea pig and cutting it into small rings. Each ring of tracheal muscle was then suspended in a muscle bath that contained the physiological bathing medium maintained a t 30 2 1 "C. The composition of the physiological bathing medium in millimoles per liter was as follows: NaCI, 125; KCI, 2.7; CaC12, 1.8; glucose, 11; Tris buffer, 23.8. The solution was adjusted to pH 7.5 with 6 M HCI and was saturated with 1009 oxygen. The high 738 /Journal of Pharmaceutical Sciences Vol. 77, No. 9, September 1988

53

53

potassium bathing medium used in experiments with the longitudinal muscle of the guinea pig ileum had the same composition as the physiological bathing medium except that all the NaCl was replaced by a n equal molar concentration of KCl and 1.8 M CaCl, was not added to the medium until it was necessary to elicit a mechanical response. Isometric contractions of the smooth muscles were measured by means of a Narco F50 microdisplacement myograph transducer and recorded on a Narco physiograph type DMP4A. Initial tension on the muscle preparations was set at 0.5 g.

References and Notes 1. Burckhalter, J. H.; Scarborough, H. C. J . Am. Phurm. Assoc. 1956.44, 545. 2. Das, B.; Mukherjee, R. J . Indian Chem. SOC.1963,40, 35. 3. Wenzel, D. G . J . Am. Pharm. Assoc. 1955,44,550. 4. Hayao, S.; Havera H. J.; Strycker, W. G.; Leipzig, T. J.; Kulp, R. A.; Hartzler, H. E. J . Med. Chem. 1965,8, 807. 5. Horacio, V. U.S. Patent 336 946, 1975; Chem. Abstr. 1975, 83, 37918~.

Table HC'3C Chemical Shifts Assignments of Compounds 10-16

Carbon Number Compound

1

2

3

4

5

6

7

8

9

10

11

10'

151 161

115 129

124

135 115

141

43

170 31

-

-

llb

151 163

115 128.5

123.5

136 116.5 141

35

167

-

35

52

150 162

114 126.5

123

136 115

42

165

-

45

56

12a

140

R' Group

12 13 and 14 and 15

-

58

30

c1

13'

151 161

114 128

124

136

116

140

42

169

-

43

51 112

60

14a

151 162.5

116 128.5

123.5

136 117

15'

151 164

114 127

123

136 114.1 140

140.5 32.5

43

173 35

48

53

166 30

33

50

165 13L.S

25

31

.54

c1

16'

152 164.5

112 129

121

134 111

140

43

166 30

42.5

47.5 160 25

~

112 a Solvent

3

13C

DMSO-d6. DMSO-ddCF3CO2H.'Spectrum was run at elevated temperature.

6. Havera, H. J.; Vidrio, H. J . Med. Chem. 1979,22, 1548. 7. Havera, H. J., Ger. Patent 2 428 858, 1979; Chem. Abstr. 1975, 82, 17102913. 8. Maillard, J.; Vincent, M.; Benard, M. Chim. Ther. 1968, 3(2), 100. 9. Hong, E. U.S.Patent 3 726 979, 1973; Chem. Abstr. 1973, 79, 27560a. Fedor, L. R. J . Am. Phurm. Assoc. 1959,48, 412. 10. Foye, W. 0.; 11. Goldman, L.; Williams, J. H. J . Org. Chem. 1953,18, 815. 12. Sheenan, J. C.;Hess, G. P. J . Am. Chem. SOC.1955, 77, 1067. 13. Brown, H. C.; Yoon, N. M. J . Am. Chem. Soc. 1966,88, 1464. 14. Jpn. Kokai Tokkyo Koho J.P. 60 197 660 [85 197 6601; Chern. Abstr. 1986,104, P16848Ow.

15. Pollard, C. B.; Rietz, E. G.; Robbins, R. J . Am. Chem. SOC.1953, 75, 2989. 16. Hayao, S.; Schut, R. N. J . Org. Chem. 1961,26, 3414. 17. Gadekar, M. S.; Kotsen, A.M. J. Chem. SOC.1964,4, 4666. 18. Papadopoulos, E. P. J . Heterocycl. Chem. 1981, 18, 515. 19. Weiss, G. B.; Coalson, R. E.; Hunvitz, L. Am. J . Pizysiol. 1961, 200, 789.

Acknowledgments We thank Kotobuki Seiyaku, Ltd., Japan, for financial support for this study.

Journal of Pharmaceutical Sciences / 739 Vol. 77, No. 9, September 1988