Synthesis and Anticholinesterase Activity of New Bispyridinium Compounds Fu-LIANHsu*', WDHARAMAN W9,OFFIEE. C I A R ~ SHEKAR , MUNAVALLI*§, AND WILLIAM P. ASHMAN* Received June 28, 1991, from the *U.S. Army Chemical Research, Development, and Engineering Center, Aberdeen Proving Ground, MD 21010-5423, and the *US.Arm Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425. Accepted for publication April 14, 1992. s6resent address: Geo-Centers, Inc., Ft. Washington, MD 20744.

Abstract Synthesis of new bis(1-methylpyridinium) compounds containing a 1,4diacetylbenzene linkage between the pyridinium moieties from commercially avallable 2-, 3-, and 4-picoline precursors was accomplished via metallation, reaction of the picolyllithium with 1,4dicyanobenzene, and subsequent quatemization of the resulting bispyridyl compounds. Acetylcholinesterase inhibitory activitywas determined colotimetrically with purified electric eel enzyme. Examination of structure-activity relationships indicated that the $substituted pyridinium compound is the most potent isomer, followed by the 2-substituted isomer, and that the +substituted analogue is the least active.

3e: 2,2'-dipyrldyl

3b: 33dpvrldvl

Among the various known neurotransmitters, acetylcholine (ACh) is a uniquely simple molecule, performing one of the most vital functions common to many living species. Impairment of ACh-mediated neurotransmission has been directly associated with many diseases.1.2 Maintaining the level of ACh via the inhibition of acetylcholinesterase (AChE) is one of the approaches used to treat diseases such as myasthenia gravis and Alzheimer's disease and poisoning by organophosphorus compounds.1.2 Thus, the development of safe and potent anti-AChE compounds is of considerable current interest. Long and Schueler in 1954 prepared a series of bisquaternary ammonium compounds with the general structure of 1 (see etructures).3.4 These pyridinium compounds were not only -10 times more potent than neostigmine but also caused rapid inhibition of AChE in human red blood cells. The anti-AChE activity of 1 may be due to the charge interaction and/or the interaction of the acetophenone moiety with the enzyme. To understand the importance of the geometry of the acetophenone moiety to the quaternary center on the inhibition of the enzyme, a new series of symmetrical bis(1methylpyridinium) compounds, 2a-2c, were synthesized, and their anti-AChE activities were investigated. These com-

s:

4.4'dipyrldyl

Scheme I

4

5

Scheme Ii

pounds are structurally similar to 1 but have different substitution patterns to permit the study of structureactivity relationships. Compounds 2 8 - 2 ~have a l-methylpyridinium function, which is ubiquitous among anti-AChE agents. The 1,4-diacetylbenzene linkage between the pyridinium moiety is similar to that in 1. This paper describes the syntheses and anti-AChE activities of 2a-2c.

Results and Discussion The syntheses of the bispyridinium compounds 2a-2c depended on the successful formation of the respective picolyllithium and its subsequent reaction with l,4-dicyanobenzene Table I-Chemlcal Shlft (6)of the Yethylene Protons of Blspyrldlnes and Their Derivatives'

1 R-H.C&

Compound

3a 3b 3c 2a 2b 2c

Chemical Shift of Indicated Proton, ppm -CH&O 4.53 4.53 4.53 5.43 4.91 5.02

-CH = G O H

Keto:enol Ratiob

6.4016.50

25:75 92:8 85: 15

-

C

6.08 6.25

a Solvent, Me,SO-d,; Me,Si chemical shift, 0.00 ppm. Measured by digital integration of peak areas. '-En01 tautomer was not observed.

0022-3549/92/12001 181$02.50/0 0 1992,American Pharmaceutical Association

Journal of Pharmaceutical Sciences I 1181 Vol. 81, No. 12, December 1992

~4

I

ni

c9

0

c7

Pyridostigmine 0 1

I

c7

C4'

0

0 1'

2a-c

(Scheme I). Reaction of phenyllithium (PhLi) in cyclohexane:ether (70:30)with the most reactive 2-picoline in tetrahydrofuran (THF) at room temperature6 generated a deepred 2-picolyllithiumsolution, and the addition of 1,4-dicyanobenzene followed by acid hydrolysis resulted in a yellow product (3a). Although 4-picolyllithium has been prepared from PhLi,6.7 under these conditions, reaction of PhLi and 4-picoline followed by 1,4dicyanobenzene resulted in the isolation of two undesired products, 4 and 5, in 15 and 36% yields, respectively (Scheme 11).The formation of 4 resulted from the nucleophilic addition of PhLi to 4-picoline.6 Interestingly, 5 appears to have formed from the nucleophilic displacement of one of the cyan0 groups by 4-picolyllithium. Thus, the reaction of 2-thienyllithium and 4-picoline in THF--benzene,8+9 followed by the addition of 1,ldicyanobenzene and acid hydrolysis of the imine intermediate yielded only 9% of the desired product 3c. Contrary to a n earlier report,7 the reaction of 3-picoline in the presence of 2-thienyllithium and 1,4dicyanobenzene did not furnish the desired product. Lithium diisopropylamide was then used to prepare 3-picolyllithium.gJO Reaction of 3-picolyllithium with 1,4-dicyanobenzene and subsequent acid hydrolysis produced 3b in 42%yield. Because of the moderate yield of 3b, the reaction of 4-picoline with lithium diisopropylamide was carried out, and 3c was isolated in 18%yield. The synthesis of bis(1-methylpyridinium) salts (2a-2c) from the corresponding dipyridyl compounds (3a-3~)was accomplished by reaction with methyl iodide in dimethylformamide (DMF). The reaction was fast in the w e of 3- and 4-substituted isomers but slow in the case of the 2-substituted derivative; this result was undoubtedly due to steric hindrance of the pyridine nitrogen. The methylene groups of 3a-3c are relatively acidic, and this phenomenon can be examined by 'H NMR spectrometry

(Table I). For 3a, two singlets, one at S 4.53 (methylene protons from keto form) and the other at S 6.40 and 6.50 (vinylic protons from enol form), were observed in deuterated dimethyl sulfoxide. However, the enolization was not observed with 3b and 3c. For the bispyridinium compounds 2a-2c the 1-methylpyridinium group enhanced the acidity of the methylene group, as expected and confirmed by the downfield shift of these resonances. Computational Analysis-Structurc+activity studies have identified important geometric and functional groups relating to anti-AChE activity.11-18 A minimum-energy conformational analysis was performed to achieve optimized threedimensional geometries of the compounds by using Allinger's MM2 (Molecular Mechanics).IQ-21 Molecular Modeling Analysis and Display System (MMADS122was used to incorporate the structures and perform theoretical calculations. Table I1 summarizes interatomic distances between each of the pyridinium nitrogens and the adjacent carbonyl carbon and the carbonyl oxygen of pyridostigmine and 2a-2c (seestructure). Anti-AChE Activity-The anti-AChE activities of the synthesized compounds were determined colorimetrically with purified electric eel AChE. The relative anti-AChE potencies were determined by studying enzyme inhibition at different concentrations of the substrates to obtain the IC,, values (molar concentration required to inhibit eel AChE activity in vitro by 500/0). For comparison purposes, the bkpyridinium compound (1;R = H) was prepared.3.4 The representative plots of percent control AChE activity versus concentrations are shown in Figure 1. Table I1 gives the IC,, values of 1, 2a-2c, and pyridostigmine. The importance of the position of the substituent of 2a-2c on the pyridinium moiety for anti-AChE action (Table 11) follows trends for many carbamoyl quaternary compounds known to interact with AChE.18 Therefore, the interatomic distances of the bispyridinium compounds were calculated. On the basis of structure-activity relationship of carbamoyl quaternary ammonium compounds, maximum activity against AChE was observed when the N+-carbonyl carbon distance was between 4.7 and 5.3 A.18 For pyridostigmine (a rneta-substituted N, N-dimethylcarbamoylpyridinium compound and the most active AChE inhibitor of the series),the N+-carbonyl carbon distances were 4 . 3 4 4 3 6 A. The N+carbonyl oxygen distances were 4.5-5.74 A. Interestingly, the anti-AChE activity of these novel bispyridinium derivatives

Table Il-hteratomlc Distances and Relative Anti-AChE Potencies of Ouaternary Compounds o

Distance between Atoms, A Compound 1 2a 2b 2c Pyridostigmine

Nl-C9

Nl-Ol

-a 3 . 1 M. 8 6 4.314.92 5.03-5.11 4.34-4.36'

-

N1-N1'

-

4 . m . 7 3 7.83-10.59 4.2S5.74 8.82-12.02 5.6W5.84 9.86-12.43 4.505.13c -

a -, Not applicable. Distance between Nl-C8. N1 and 02.

1182 I Journal of Pharmaceutical Sciences Vol. 81, No. 12, December 1992

IC,

M

1.3 x 10-7 3.0 x 4.0 X lo-' 3.0 X 5.0 x 10-7

Distance between

10-9

10-8

10 7

10-6

10-s

10-4

10-3

CONCENTRATION (M)

Figure 1-Effects of different test compounds on eel AChE activity. AChE was assayed colonmetrically as described in the Experimental Section. Amount of enzyme activity at each concentration of the test compounds shown was expressed as percent of control activity without any compound included in the assay after 30 min of reaction. Key: (0) 3-substituted isomer, 2b; (0)1 (R = H); (m) 2-substituted isomer,28; (0) 4-substituted isomer, 2c. Each point is the mean of 4-8 assays. The assay results were highly reproducible with a variation of -5% or less from the mean.

definitely appears to be related to their geometric orientations. Thus, the 3-substituted isomer, 2b, waa the most active campound, the 2-substituted isomer, 2a, waa -0.01 timea aa active aa 2b, and the 4-substituted isomer, 2c, was the least active. The interatomic distances for 2b were 4.31-4.92 A for the N+-carbonyl carbon bond and 4.23-6.74 A for the N+carbonyl oxygen bond. This close similarity to pyridostigmine suggests that 2b might be acting at the same active site of AChE. However, W-carbonyl carbon and N+-carbonyl oxygen distances of 2a were 3.133.86 A and 4.234.73 A, respectively. The corresponding interatomic distances of 2c, the least active of the series, were 6.03-5.11 A and 5.69-5.84 A, respectively.

Of the three biapyridinium salts (2a-24, the 3-substituted isomer, 2b, is the most potent compound. The results of this study indicate that the interatomic distance between the carbonyl group and the quaternary nitrogen and its orientation in space play a mqjor role in the anti-AChE activity of these compounds.

Experimental Section Melting points were determined with a Thomas-Hoover apparatus and are corrected. Infrared spectra were recorded on a Perkin-Elmer 1420 inetrument. 'H NMR apectra were obtained with a Varian EM 390 spectrometer with Me,Si as the internal reference. Chemical ionization mass spectra were obtained with a Finnigan 1015D spectrometer equipped with a model 6000 data collection system and with ammonia gas as the carrier. Elemental analyses (C, H, and N) were performed by Schwarzkopf MimanalyticalLaboratories, Woodside, NY. Where analyses are indicated by the symbols of the elements, analytical results were within +0.4% of the calculated values. THJ? was freshly distilled from L U , before the reactions. Picolines and diisopropylamine (i-Pr,NH) were distilled from KOH before use. Silica gel GF plates for TLC were purchased from Analtech, Incorporated, Newark, DE. Silica gel 60 (230400 mesh) was purchased from EM Laboratories, Darmstadt, Germany. 1,4-Bis(2-pyridylacetyl)benzene (3ab-A solution of 2-picoline (16.5g,0.177mol)in450mLofTHJ?wasaddedinadrop~manner to a solution of PhLi (2.7 M, 66 mL, 0.18 moll in cyc1ohexane:ether (70:30) at 20-30 "C under N, over a period of 30 min. The resulting deep-red solution was stirred at room temperature for 16 h. 1,4Dicyanobenzene(11.4 g, 0.09 mol) was added in one lot to the solution of 2-picolyllithiumat room temperature. The mixture was stirred for 24 h, treated with 2 N H,SO, (355 mL) at 1615 "C, and washed with ether (2 x 500 mL). The aqueous solution was brought to pH 10 by addition of 10% NaOH solution, during which time a yellow solid precipitated out. The yellow solid was filtered, washed with H,O, and dried to yield the crude product. Recrystallization from acetone afforded3a (15.4g, 55%):mp, 179-180 "C; IR (KBr):1680cm-' (weak, C = 0);'H NMR (Mew-rg): 6 4.53 (8, lH, CH,CO), 6.40, 6.50 (28, 1.5H, CH = W H ) , 7.067.43 (m, 4H, pyridine-H), 7.63-8.13 (m, 6H, pyridin+H and ArH), and 8.42 (m, 2H, pyridine-H); MS: mh 317

(MH').

M.--Calcd for C,,,HlaN9O,: -_ -- - - C. 75.93; H, 5.10; N, 8.85. Found C, 75.W; H, 5.25; N, 8.79. 1.4-Bis(3-~~dvlacetvllbenzene ( 3 b b T h e solution of i-Pr,NH (13g, 0.13 I&) G T H F 732 mL) was cooled to -70 "C and t r e a a in a dropwise manner with n-BuLi (1.6 M in hexane, 80 mL, 0.128 mol) during a 1-h period under N,. The resulting mixture gave a cloudy solution at -70 "C and became clear at -10 "C. The solution was stirred at - 10 "C for 1h, then treated in a dropwise manner with a solution of 3-picoline (10 g, 0.11 mol) in THF (10 mL). The resulting orange mixture was stirred at -10 to -20°C for 30 min, and 1,edicyanobenzene (8.2 g, 0.064 mol) was then added in small portions. The resulting dark-brown mixture was stirred at room temperature overnight. Then, 2 N H,SO, (220 mL) was added to a c i w the mixture, which was washed with EGO to remove neutral species. The separated dark-brown aqueous solution was filtered through diatomaceous earth (Celite), and then the pH of the filtrate was adjusted to 10 with 10% NaOH. The dark-brown precipitate was filtered, washed with H,O, and dried to give the crude product.

Recrystallization from EtOH yielded 3b (7.1 g, 42%):mp, 151-153 "C (reported mp for 3b * 2HC1, >260 "C);TLC (silica gel, CHCl,:acetone, 1:l):retardation factor UZ,, = 0.25; IR (Nujol): 1680 and 1695 cm-' (C = 0);'H NMR (Me,SO&): 6 4.53 (s,4H, 2CH,), 7.27-7.43 (m, 2H, pyridine-H), 7.60-7.77 (m, 2H, pyridine-H), 8.21 (8, 4H, AH),and 8.42-8.63 (m, 4H, pyridine-H); MS [chemical ionization (CI)/NH,]: mh 317 (MH+). Anal.-Calcd for C,,Hl,N,0,.2HC1: C, 61.71; H, 4.14; N, 7.20. Found C, 61.71; H, 4.53; N, 7.28. 1,4-Bie(4-pyridylacetyl)be.nzene(3c)-Method 1-A solution of n-BuLi (1.6 M in hexane, 146 mL, 0.234 moll was slowly added over a 1-h period to a solution of thiophene (23.2 g, 0.276 mol), THJ? (19 mL), and benzene (43 mL) at 3 0 3 5 "C under N,. The mixture was stirred at room temperature for 16 h, and then, 4-picoline (29.6 g, 0.319 mol) was added. The resulting dark-green crystalline suspension was heated to 5 0 6 3 "C for 2 h. 1,4-Dicyanobenzene(14.9 g, 0.116 moll was added to the reaction mixture, and the mixture was refluxed for 1.5 h. During this period, a dark, gummy slurry formed.A solution of 2 N H$04 (405 mL)was added to this mixture at 10-15 "C, and the mixture was washed with ether (3 x 500 mL) and then basified with 10% NaOH to pH 10 to give a brown solid precipitate. The crude solid (10 g) was collected, the filtrate was extracted with CHCl,, and the extract concentrated to give an additional 1.5 g of the product. T w o recrystallizations of the combined crude product from EtOH gave 3c (3.4 g, 9.2%): mp, 225-226 "C;IR (KBr): 1680 cm-' (C = 0);'H NMR (CDCl$Me,SO-4): 6 4.49 (s,4H, 2CH,), 7.27 (d, 4H, pyridlne-HJ = 5.0 Hz), 8.17 (s,4H, ArH), and 8.53 (d, 4H, pyridin+HJ = 5.0 Hz); MS (CVNH,): m/z 317 (MH'). M . - C a l c d for C2&,,N,0,: C, 75.93; H, 5.10; N, 8.85. Found C, 76.05; H, 5.25; N, 8.90. Method 2-A solution of n-BuLi (1.6M in hexane, 80 mL, 0.128 moll was added in a dropwise manner at - 70 "C to a solution of i-Pr,NH (13g, 0.128 moll in 32 mL of THF under N,. Then, the temperature of this turbid yellow solution was raised to - 10 to -20 "C and stirred at this temperature for 1h. To this solution, a solution of 4-picoline (10 g, 0.107 moll in 10 mL of THJ? was added in a dropwise manner. The resulting heterogeneous, orange mixture was stirred at - 10 to 0 "C for 1h, and 1,4slicyanobenzene (8.2 g, 0.064 mol) was added in several portions. The color of the reaction mixture gradually changed to dark brown. The mixture was stirred for 16 h a t room temperature. Then, 2 N HaO, (220 mL) was added to the mixture at the ice-bath temperature, and the mixture was washed with ether. The aqueous solution was brought to pH 10 with 10%NaOH to yield a light-brown precipitate. This mixture was extracted with CHC1,:isopropyl alcohol (3:2), and the organic layer was washed with brine, dried over anhydrous Na.#O,, and evaporated to give the crude product and Cpicoline. Recrystallization from EtOH yielded 3c (2.7 g, 18%).which was identical to the compound prepared according to method 1. General Procedure To Prepare Bisquaternary Compounds-A solution of 3 (1g, 3.16 mmol) in 80 mL of DMF was treated with 10 mL of CH,I, and the resulting mixture was stirred at room temperature. Compounds 2b and 2c precipitated out in the reaction mixture; however, 2a precipitated out after ether was added. 1,4-Diacetylbenzene-cu,a'-bis[2-(l-methylpyridinium)l diiodide (2a)-Reaction time, 14 days; recrystallization h m DMF-EtOH; yield, 54%; mp, 214-216 "C; IR (CsI): 1684 cm-' (C = 0); 'H NMR (Me$3O-$): 6 4.28 (s,6H, 2NCH3),5.43/6.08 (2s,4H, 2CH2 and trace amount of CH = W H ) , 8.13-8.37 (m, 8H, ArH and pyridin+H), 8.67 (m, 2H, pyridine-H), and 9.16 (d, 2H, pyridin+HJ = 7.0 Hz). Anal.-Calcd for C,,HZ2I,N,O2 * 1.5H20 C, 42.21; H, 3.78; N, 4.47. Found C, 42.22; H, 3.67; N, 4.36. 1,4-Diacetylbenzene-a,cr'-bis[3-(l-methylpyridinium)l diiodide (2b)-Reaction time, 4 h; recrystallization from DMF-EtOH; yield, 95%; mp, 267-268 "C; IR (Nujol): 1685 and 1690 cm-' (C = 0); 'H N M R (Me,SO-4): S 4.39 (8, 6H, 2CH,), 4.91 (s,4H, 2CH2),8.18 (dd, 2H, pyridine-HJ = 7.0, 8.0 Hz), 8.30 (8, 4H, ArH), 8.57 (d, 2H, pyridine-HJ = 7.0 Hz), and 8.92-9.02 (m, 4H, pyridin+H). M . - C a l c d for C,,HmI,N,Oz: C, 44.02; H, 3.69; N, 4.67. Found: C, 44.16; H, 3.66; N, 5.03. 1,4-Diacetylbenzene-a,a'-bis[4-(l-methyl~ridinium)l diiodide (&)-Reaction time, 16 h; recrystallized from H,O; yield, 95%; mp, r250 "C; IR (CsI): 1683c m - l (C = 0); 'H NMR (Me,SO-$): S 4.37 (8, 6H, 2CH,), 5.02 (8, 4H, 2CH,), 8.03-8.30 (m, 8H, ArH), and 8.98 (d, 4H, pyridine-HJ = 7.0 Hz). M . - C a l c d for C,,H,,I,N,O,: C, 44.02; H, 3.69; N, 4.67. Found: C, 44.02; H, 3.65; N, 4.65. Journal of Pharmaceutical Sciences I 1183 Vol. 81, No. 12, December 1992

4-Methyl-2-phenylpydine (4) and 4-(4-Cyanophenyl)mthylpyridine (5)-To a stirred solution of PhLi in cyc1ohexane:ether (70:30;2 M, 89 mL, 0.178 mol), a solution of 4-picoline (16.5g, 0.177mol) in 450 mL of dry THF was slowly added at 20-32 "C over a period of 30 min under N,. The resulting deep-red solution was stirred at room temperature for 16 h. Then, 1,4-dicyanobenzene (11.4g. 0.089 mol) was added, and the resulting dark-brown mixture was stirred at room temperature for 24 h. Then, 2 N H,SO, (355 mL) was added at 10-15 "C, and the mixture was washed with ether (2x 500 mL). The aqueous layer was brought to pH 10 with 10% NaOH and extracted with 700 mL of CHC1,. The combined extracts were dried over anhydrous MgSO, and concentrated to give 22.5 g of oil. The oil was chromatographed over a silica gel column and eluted with benzene: acetone (4:l). The fast-moving fractions were concentrated and recrystallized from n - h e m e to give 4 (2.3g, 15%): mp, 46-48 "C (literature value,28 50.41 "C); IR (KBr): 700 and 750 cm-'; 'H NMR (CDCl,): 6 2.39 (8,3H, CH,), 7.04 (d, lH, ArH,J = 5.0 Hz), 7.41-7.58 (m, 4H,ArH), 7.95 (dd, 2H,ArH, J = 8.0and 1.5 Hz), 8.26 (d, lH,ArH, J = 5.0Hz);MS (CINH,); m/z 170 (MH+). Another fraction gave 10 g of an oily product, which was recrystallized from n - h e m e to yield 5 (6.2g, 36%): mp, 72-73.5"C;IR (KBr);2225 ax-'(CN); 'H NMR (CDCl,); 6 4.04(s,2H, CH,), 7.10 (d, 2H, ArH, J = 6.0Hz), 7.31(d, 2H,ArH,J = 8.0Hz), 7.62 (d, 2H,ArH, J = 8.0 Hz), and 8.54 (d, 2H, ArH, J = 6.0 Hz); MS (CVNH,); m/z 195 (MH+). Anul.--Calcd for C12HIIN: C, 80.39;H, 5.19;N, 14.42.Found: C, 80.36;H, 5.29;N, 14.49. Anti-AChE Assay-Affinity-purified electric eel AChE was prepared in-house according to the procedure described for guinea pig brain.24 Acetylthiocholine and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Company (St. Louis, MO). Buffers and other chemicals used were analyticalreagent grade. AChE activity was determined colorimetridy by a modificationm of the micro Ellman AChE assay.m.27 Acetylthiocholine was used as the substrate, and DTNB was used as the color reagent. All reactions were carried out in 100 mM potassium phosphate buffer (pH 7.4)at mom temperature. The reaction mixtures were introduced into individual wells of a 96-well microtiter plate. Each assay mixture of 0.3-mLtotal volume contained the following: 0.2 mL of phosphate buffer containing 0.5 mM each of acetylthiocholine and DTNB, 0.03-mL solutions of test compounds in phosphate buffer to give desired final concentrations of lo-' to lo-' M, and 0.06 mL of phosphate buffer. Reactions were initiated by adding 0.01 mL of purified AChE in phosphate buffer to all wells except the enzyme blanks, which received 0.01 mL of buffer. The time-course of acetylthiocholine hydrolysis by AChE was determined by monitoring the increase ofyellow color produced from the reaction of thiocholine with 5,5'-dithiobis(2-nitrobenzoate) ion at a wavelength of 414 nm. AChE activity at each concentration of the teat compounds was expressed as a percentage of control (without any test compound added) activity after 30 min of reaction.

2. Volicer, L. Clinical Management of Alzheimer's Disease; Volicer, L.; Fabiszewski, K. J.; Rheaume, Y. L.; Lasch, K. E., Eds.; Aspen: Rockville, MD, 1988,pp 185-200. 3. Long, J. P.; Schueler, F. W. J. Am. Phnrm. Assoc. 1954,43,79. 4. Benz, F. W.; Long, J . P. J. Pharmacol. Exp. Ther. 1969,166,225. 5. Biichi, von J.; Kracher, F.; Schmidt, G. Helv. Chim.Acta 1962,45, 729. 6. Wibaut, J. P.; Hey, J. W. Rec. Tmv. Chin. Pays-Bas. 1953, 72, 513. 7. Fryer, I.; Brust, B.; Earley, J. V.; Sternbach, L. H. J. Org.Chem. 1966,31,2415. 8. Screttas, C. G.; Estham, J. F.;Kamienski, C. W. Chimiu 1970, 24,109. 9. Edward, W. B., 111 J. Heterucycl. Chem. 1975,12,413. 10. Kaiser, E. M.; Petty, J. D. Synthesis 1975,705. 11. Wombacher, H.; Wolf, H. U. Mol. Phnrmacol. 1971, 7,554. 12. Roufogalis, B. D.;Quist, E. E. Mol. Pharmacol. 1972,8, 41. 13. Berman, H. A.; Decker, M. M.; Now&, M. W.; Leonard, K. J.; McCauley, M.;Baker, W. M.; Taylor, P. Mol. Pharmacol. 1987, 31,610. 14. Cannon, J. G. Burger's Medicinnl Chemistry, Fourth Edition, Part 111; Wolff, M. E., Ed.; John Wiley & Sons: New York, 1981; pp 339-360. 15. Kabachnik, M. I.; Brestkin, A. P.; Godovikov, N. N.; Michelson, M. J.; Rozengart, E. V.; Rozengart, V. I. Pharmacol. Rev. 1970, 22,355. 16. Pauling, P.; Petcher, T. J. Chem.-Bwl. Interact. 1973,6,351. 17. Goldblum, A. Mol. Pharmacol. 1983,24,436. 18. Foldes, F. F.; Van Hees, G.; Davis, D. L.; Shanor, S. P. J. Pharmacol. Exp. Ther. 1958,122,457. 19. Allinger, N. L. J. Am. Chem. Soc. 1977,99,8127. 20. Allinger, N. L.; Yuh, Y .H.Quant. Chem. Progm. Exchange 1980, 13,395. 21. Burkert, U.;Allinger, N. L. Molecular Mechanics; ACS Monograph Series 177;American Chemical Society: Washington, D.C., 1982. 22. Leonard, J. M.;Famini, G. R. Technical Report, CRDEC-TR86039;U.S.Army Chemical Research, Development and Engineering Center: Aberdeen Proving Ground, MD 21010-5423, 1986. 23. Butler, D. E.; Bass, P. A; Nordin, I. C.; Hauck, F. P., Jr.; L'Italien, Y. J. J. Med. Chem. 1971,14,575. 24. Yamamura. H. I.: Reichard. D. W.: Gardner. T. L.: Morrisett. J. D.; Bmomfield,'C. A. Biochim. Bibphys. Acia 1973,302,305.' 25. Ray, R.; Clark, 0. E. FASEB J. 1988,2,A1541. 26. Ellman, G.L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R. M. Biochem. Pharrnacol. 1961, 7,88. 27. Brogdon, W. G.;Dickinson, C. M. Anal. Biochem. 1983,131,499.

References and Notes

We thank N. Wittaker of the National Institutes of Health for the mass spectra and W. T. Beaudry and L. L. Szdraniec of the Chemical Research, Development, and Engineering Center for the NMR spectra. We also thank Drs. J. G. Cannon and A. Brossi for fruitful discussions.

1. Taylor, P. The Pharmncological Basis of Thempeutics; Gilman, A. G.; Rall, T. W.; Nies, A. S.; Taylor, P., Eds.; Pergamon: New York, 1990;pp 131-149.

1184 I Journal of Pharmaceutical Sciences Vol. 81, No. 12, December 1992

Acknowledgments