506 Chem. Res. Toxicol. 1993,6, 506-510

Carbamate Derivatives of 2-Arylimidazo[ 1,Z-alpyridinium Salts as Acetylcholinesterase Inhibitors and Protective

Agents against Organophosphorus Compounds Richard J. Sundberg,**t Deepak Dalvie,? Joehassin Cordero,? and H. A. Musallami Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, and Walter Reed

Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20307

Received November 6, 1992

A series of 2-arylimidazo[l,2-alpyridinium salts with (N,N-dimethylcarbamoy1)oxy or (N- methylcarbamoy1)oxy groups a t the 3'- or 4'-position on the phenyl substituent and various substituents on the imidazo[l,2-alpyridine ring have been synthesized. The compounds show in vitro inhibitory activity against electric eel acetylcholinesterase (AChE), type 111, and several of the compounds show protective effects toward the organophosphorus AChE inhibitor soman in mice. The possible structural relationship of these compounds to physostigmine and pyr- idostigmine is considered.

Introduction The alkaloid physostigmine is well-known to be an

acetylcholinesterase (AChE) inhibitor and has found therapeutic application in treatment of glaucoma and myasthenia gravis (1). The compound functions as a reversible inhibitor of AChEl by carbamoylation of the nucleophilic serine at the enzyme active site. More recently, physostigmine derivatives have become of interest as potential therapeutic agents for Alzheimer's disease (2, 3). While physostigmine has prophylactic activity against organophosphorus compounds which irreversibly inhibit AChE (4, 5 ) , the occurrence of central nervous system side effects has been considered to be a drawback of this potential application (6). A number of physostigmine analogs, most notably pyridostigmine, have been examined for potential prophylactic activity against AChE inhibitors (7). We undertook the synthesis of anumber of quaternary imidazo[ 1,2-a]pyridinium carbamates of structures 1-4 which have a general structural resemblance to both physostigmine and pyridostigmine and have evaluated the in vitro activity of these compounds as AChE inhibitors and their in vivo prophylactic and antidotal activity toward soman.2

CH,HNC02 mN, ~ 0 2 c N ( c H 3 ) 2

CH3 N ' r ? I

1 C"3 6H3

PHYSOSTIGMINE PYRlDOSTlGMINE

Experimental Section General. Chemical reagenta were obtained from commercial

sources, mainly Aldrich Chemical Co. Electric eel acetylcho- linesterase, type 111, was obtained from Sigma Chemical Co. and used without further purification. All compounds which were tested for biological activity were characterized by IR and 'H- NMR spectra and by elemental analyses. A table giving chemical

* To whom correspondence should be addressed. f University of Virginia. t Walter Reed Army Medical Center. 1 Abbreviations: AChE, acetylcholinesterase; DNTB, 5,5'-dithiobis-

(2-nitrobenzoic acid). 2 Soman is methyl (1,2,2-trimethylpropyl)phosphonofluoridate.

shifts and peak multiplicity is included in the supplementary material. Elemental analyses were obtained for all elements except oxygen and fluorine and agreed with the calculated value within h0.4 % . Several of the salts analyze as hydrates.

Procedure for the Synthesis of 6-Substituted-2-phen- ylimidazor l&a]pyridines. A mixture of the appropriate 5-substituted-2-aminopyridine (10 mmol) and an appropriately substituted phenacyl bromide (10 mmol) was maintained a t reflux in acetonitrile for 24 h. The solid obtained was then filtered, washed with acetone (2 X 50 mL), and dried. The hydrobromide salt of the imidazopyridine was then suspended in methanol/ water (1:2, 100 mL) and neutralized with saturated sodium bicarbonate solution. The solid obtained was filtered, washed with water (2 X 50 mL), and dried. The substituted imidazo- [1,2-aIpyridine thus obtained was used for the next reaction without further purification.

Procedures for Carbamoylation of (Hydroxyphenyl)- imidazopyridines. Method A. A suspension of an appropriate (hydroxypheny1)imidazopyridine (10 mmol) in N,N-dimethyl- carbamoyl chloride (30 mL) was heated a t 120 O C for 8-24 h. The reaction mixture was allowed to cool to room temperature and quenched with ice water. After carbon dioxide evolution had ceased, the mixture was neutralized with saturated sodium bicarbonate solution, and the precipitate was filtered and dried to give the desired carbamoylated product. The product was then subjected to quaternization without further purification.

Method B. A suspension or solution of an appropriate (hydroxypheny1)imidazopyridine (10 mmol) in pyridine (20 mL) was treated with N,N-dimethylcarbamoyl chloride (30 mmol). The reaction mixture was refluxed for 1-2 h and then poured into ice water, and the precipitate was filtered and dried to give the desired carbamoylated product. The product obtained was quaternized without further purification.

Procedure for the Preparation of N-Methylcarbamates. A suspension of the substituted (hydroxypheny1)imidapyridine (1 mmol) in dry acetonitrile (50-100 mL) was treated with methyl isocyanate (10 equiv) and a catalytic amount of dibutyltin diacetate (2-3 drops). The reaction mixture was stirred for 24 h and filtered. The solid obtained was washed with ether and dried. The product was then subjected to quaternization without further purification.

Procedure for Preparation of Quaternary Imidazo[ I f - alpyridinium Iodides. To a suspension or solution of an appropriate imidazopyridine in tetrahydrofuran (150 mL) was added iodomethane (large excess), and the mixture was main- tained at reflux for 60 h. The precipitate was filtered, washed with tetrahydrofuran and ether, and dried to give the iodide salt. The producta were recrystallized from acetonitrile/ether or ethyl

0893-228x/93/2706-0506$04.0010 0 1993 American Chemical Society

Carbamates of Imidazo[l1,2-a]pyridinium Salts

acetate. In some cases the iodide sal& were dissolved in methanol/ acetonitrile and passed through an ion-exchange column (Am- berlite IRA 400 C1-). The solvent was evaporated, and the product obtained was recrystallized from acetonitrile/ethyl acetate or ether to give the chloride salt.

Procedures for Compounds 5. B-Nitroimidazo[ 1,2-a]- pyridine. A mixture of 2-amino-5-nitropyridine (4.6 g, 33.0 mmol), a-bromoacetaldehyde diethyl acetal (6.55 g, 33.0 mmol), and pyridinium p-toluenesulfonate (4.0 g, 15 mmole) in aceto- nitrile (50 mL) was heated for 24 h. The mixture was worked up as described in the general procedure to give 4.4 g (83%) of the desired imidazopyridine. The product obtained was carried to the next step.

6-Acetamidoimidazo[ l,2-a]pyridine. 6-Nitroimidazopyri- dine (5.0 g, 30 mmol) was suspended in methanol (250 mL) and hydrogenated at 40 psi for 6 h. The solution obtained was filtered through Celite and evaporated to dryness, and the product obtained (3.3 g, 82%) was warmed with acetic anhydride (40 mL). The solid obtained was filtered and dissolved in water and neutralized with sodium bicarbonate solution. The precipitate obtainedwasthenfilteredanddried (4.lg, 95%). Thecompound was subjected to quaternization without further purification. l-Methyl-6-acetamidoimidazo[ 1,2-a]pyridinium Iodide.

The 6-acetamidoimidazopyridine (2.4 g, 13.6 mmol) was qua- ternized and recrystallized from ethanol to give 3.1 g (72%) of the iodide salt: mp 294-296 "C.

6 4 (Methoxycarbonyl)amino]-2-phenylimidazo[ 14-alpyr- idine. A suspension of 6-amino-2-phenylimidazo[1,2-a]pyridine (3.5 g) and sodium carbonate (10.0 g, 94.3 mmol) in watedacetone (1:1,100 mL) was cooled to 0 "C, and methyl chloroformate (7.5 mL, 97.0 mmol) was added to it in a dropwise manner over a period of 10-20 min. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 1.5 h. The precipitate obtained was filtered, washed thoroughly with water, and dried to give the desired carbamate (3.6 g) in 80% yield.

l-Methyl-2-phenyl-6-[ (methoxycarbonyl)amino]imidazo- [ lJ-aIpyridinium Chloride. A solution of 2-phenyl-6-[(meth- oxycarbonyl)amino]imidazo[ l,a-a]pyridine (3.5 g 13.0 mmol) was quaternized and converted to the chloride in 74% yield; mp 225- 227 "C. 6-Hydroxy-2-phenylimidazo[ 1,2-a]pyridine. A solution of

6-(benzoyloxy)-2-aminopyridine (5.8 g, 27 mmol) and phenacyl bromide (8.5 g, 42 mmol) in acetonitrile (100 mL) was heated as described in the general procedure. The precipitate obtained was warmed in 10% sodium hydroxide solution, and the solution was cooled and neutralized with 10% hydrochloric acid. The precipitate was filtered and dried to give the desired product (4.6 g, 82%).

6 4 [ (NJV-Dimethylamino)carbonyl]oxy]-2-phenylimida- zo[ 1,2-a]pyridine. 6-Hydroxy-2-phenylimidazopyridine was converted to the desired carbamate by using Method A.

l-Methyl-6-[[ (N,N-dimethylamino)carbonyl]oxy]-2- phenylimidazo[ l,2-alpyridinium Chloride. A solution of 6-hydroxy-2-phenylimidazo [ 1,2-a] pyridine N,N-dimethylcar- bamate was quaternized as described in the general procedure, and the product was recrystallized from tert-butyl alcohol/ether. The product was obtained in 54% yield; mp 128-130 OC.

Procedure for Determination of Acetylcholinesterase Inhibition. In a typical run a predetermined amount of test compound to give a final concentration of 1W-1V M was placed in a photometric cuvette with 100 pL of 7.5 mM DNTB and 50 pL of 1 mg/mL enzyme solution (acetylcholinesterase, eel type 111). A final volume of 3 mL was obtained by adding the appropriate amount of buffer (52 mM NaH2POd) to the mixture. After incubation for 15 min, 100 pL of 0.1 M acetylthiocholine iodide was added, stirred for about 30 a, and placed in the spectrometer. The absorption of the solution was measured at 412 nm every 30 s for 6 min, and the rate of the enzymatic reaction was determined from the adsorbance change. The concentration a t which the enzyme is inhibited by 50% was obtained from a plot of enzymatic activity versus the logarithm of the inhibitor

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 507

X IR=CH3 y- 2 R = H y- 3R=CH3 y' 4R=H y- a H BMOZOZO o ~ ~ - EM02637 1- BM06475 a- BM05987 1' b 6-CH3 EM05978 a- BM05%9 I - B M W a- BM06699 I - c 7-CHg d 6 - C l e 6-CF3 I 6 - OCH3 8 6. HCONH- b 6 - CHaCONH- I 6. CH3CON(CH3t J 6 . C€I3m$W- k 6-PhCONH- I 6 . CHjNHCOhM- m 6. CH3qCNH

6 - (CH~~NCOZ-

BMMZO EM07641 B M W BM076% EM04364 BL55142 BM05M7 BM04917 EM04926 BM04935 BM04346 BM04337

a-

a-

a- BMM7M [ - a- BM06453 a-

a- a' a- a- a- a- a- )

a- BM03198 [ '

BMM11 a- BM07659 a- BM07678 1. EM07687 a- BM077M 1. BM08639 a- EM03689 TG. EM03670 [.

concentration. The value quoted is the average of 2-3 runs. The apparatus used to follow the reaction was a Hewlett Packard 8452A diode array spectrometer.

Procedure for in Vivo Evaluation of Antidotal and Prophylactic Activity. The in vivo biological evaluation was carried out by the Division of Drug Assessment, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, according to USAMRICD Protocol No. 105-87-000-A-447. In the pretreatment (prophylactic) assay, the test compound was administered a t the stated dose 15 or 60 min prior to challenge by a 2LDw dose of soman. Ten seconds after challenge atropine sulfate (11.2 mg/kg body wt) and pralidoxine (25 mg/kg body wt) were administered intramuscularly. The 24-h survival rate was compared to controls (saline instead of test compound). In the antidotal assay, the test compound was given simultaneously with atropine sulfate (25 mg/kg body wt) 10 s after challenge with 2LDw soman. The 24-h survival rate was compared with a negative control (pralidoxime sulfate, 25 mg/kg body wt, instead of the test compound).

Results

Most of the compounds are p - and m-(carbamoy1oxy)- phenyl derivatives represented by structures 1-4. Several related compounds were studied in order to determine the minimal structural requirements for AChE inhibition. The imidazo[l,2-a]pyridine 5a has only the acetamido substituent, while 5b also retains the phenyl ring. In Sc the carbamate function is moved to the heterocyclic ring, and in 6d it is replaced by a urethane group. The compounds synthesized are shown in Figure 1 and are designated by a sample number assigned a t the Walter Reed Army Institute of Research.

The most general synthesis of imidazo[l,2-a]pyridines involves cyclization condensation of an halomethyl ketone with a 2-aminopyridine (8-11). Using this method, a variety of substituents can be introduced on the hetero- cyclic ring. The pyridine substituent could also be modified if necessary. We used the nitro group to introduce a number of amido substituents. The hydrox- yphenyl substituent was converted to the N-methyl- or N,N-dimethylcarbamate after the cyclization. The final synthetic step was quaternization. The compounds were purified by crystallization. NMR, IR spectra, and ele- mental analyses for each compound confirmed the ex- pected structures.

508 Chem. Res. Toxicol., Vol. 6, No. 4, 1993 Sundberg et al.

Table 11. Prophylactic and Antidotal Activity toward 2LDm Soman

% survival % survival t - t - t + t - t - t - 60 15 10 60 15 10

compd dose min min s compd dose min min s

Table I. Inhibition of Electric Eel Acetylcholinesterase compd ICW (NM) compd ICm (rM) compd ICs0 (pM)

la lb Id le 1f It3 lh li lk

17.1 11 1.35 1.3 lm 1.1

12.6 In 1.6 10.0 2a 19.5 8.1 2b 4.0 1.2 2h 1.1 0.7 3a 7.1

24.5 3b 24.0 0.2 3d 12.6

Scheme I

3e 33.6 3f 20.5 3h 2.5 4a 0.35 4h 4.5 5a 27.0 5b 9.5 5c 50.0

R:CH, H

nil" whm R = CHI

' CH3

Most of the compounds were assayed as AChE inhibitors using the electric eel type I11 enzyme. The results are expressed as ICso, the concentration required for 50% inhibition of enzyme activity by the method of Ellman (12). Some of the compounds were also tested using fetal bovine serum AChE (data not shown). Due to compli- cations from facile hydrolysis, the monomethylcarbamates of the 2 and 4 series were not as extensively examined although an IC60 value of 0.35 pM was determined for 4a. Table I presents the results of the in vitro data.

The compounds were tested in vivo, using mice, as prophylactics and/or antidotes against the lethal toxicity of soman. For the prophylaxis screen, the test compound was administered subcutaneously 15 or 60 min prior to challenge with 2LDwsoman. Pralidoxime (25 mg/kg body wt) and atropine sulfate (11.2 mg/kg body wt) were administered 10 s after challenge. Under these conditions untreated negative controls show 0-20 9% survival while positive controls treated with pyridostigmine (0.1 mg/kg body wt) 15 min prior to challenge show about 50% survival. Survival of 40 % or more is considered to indicate potential for prophylactic activity. In the antidotal test, the compound is administered simultaneously with at- ropine (11.2mg/kgbodywt) lOsafterchallengewith2LDw of soman. As a negative reference, pralidoxime chloride (25mg/kgbodywt) andatropine (11.2mg/kgbodywt) are administered (<20% survival). As a positive control, HI- 63 (13 mg/kg body wt) and atropine (11.2 mg/kg body wt) are used (70-8096 survival). A survival rate of at least 40 % is considered to indicate the potential for antidotal activity. The animal survival data are given in Table 11. The prophylactic treatments are at t - 60 min and t - 15 min and the antidotal treatment a t t + 10 s, where t is the time of challenge with soman. Some of the compounds

* HI-6 is 1- [[ [4-(aminocarbonyl)pyridinio]methoxylmethyll-2- [(hy- droxyimino)methyll pyridinium dichloride.

la

lb

I C

Id

le

If

L3

lh

li

li

lk

11

lm

In

2a

2b

2d

5.0 20.0 80.0 2.2 8.9

35.4 2.6

10.2 40.8 6.2

25.0 100.0

6.2 25.0

100.0 4.0

15.9 63.4 0.8 3.1

12.4 1.5 5.8

23.0 3.1

12.5 50.0 3.1

12.5 50.0 3.1

12.5 50.0 2.1 8.4

33.5 2.1 8.4

33.5 6.25

25.0 100.0

0.9 3.6

14.5 1.5 6.0

24.0 3.1

12.5

90 80 30 100 80 20 60 10 0 60 50 10 30 40 40 80 70 10 50 50 40 20 50 60 90 80 70 40 70 10 40 60 0 40 30 60 50 10 80 50 40 20 20 56 30 90 90 40,70 70 70 60,90 80 20 20,lO 90 100 60 100 70 50 70 60 60,60

100 60 30,70 80 50 70,60 50 60 0,20 20 30 10,50 60 20 0,20 50 50 10,30 50 10 10,20 10 10 10,o 80 60 80,20 80 80 40,40 50 10 70,40 40 10 20,20 50 40 50,60 30 20 60,50 30 30 20,30 40 40 40,60 60 20 50,50 50 80 0,lO 60 60 10,lO 40 30 30,70 40 100 30 40 70 40

100 60 70 80 60 80 100

50.0 90 80 20 3.1 20 70

12.5 20 50 50.0 20 60

2h 0.6 2.5

10.0 3a 0.1

0.5 1.9

3b 6.2 25.0

100.0 3c 0.6

2.5 10.0

3d 3.1 12.5 50.0

36 6.2 25.0

100.0 3f 1.4

5.6 22.0

3h 3.1 17.5 50.0

4a 1.0 3.9

15.8 4b 2.2

8.8 35.0

4d 1.2 4.9

10.5 4e 3.1

12.5 50.0

4h 2.2 9.0

36.0 Sa 0.6

2.5 10.0

5b 0.2 1.0 4.0

5c 2.2 8.9

35.6 5d 0.9

3.8

50 60 40 40 50 50 40 20 10 20 30 50 30 10 50 50 30 40 70 40 70 60 30 20 60 50 60 50 60 80 70 90 60 40 50 60 60 60 30 30 20 10 0 0 0

10 20 80 10 10

80 60 30 50 50 50 40 0 0

60 40 60 30 20 10 30 50 40 80 70 80 50 50 30 70 80 40 70 60 40 80 60 40 50 50 50 50 50 70 0 0 0 0 0

10 0

40 20 10 10

15.0 0 10

0, 10 10,10 20,70 0 0 0

30,40 20,40 30,10 20 20 20

20,10 30,30 40,40

were also tested for oral activity. The test is done as described for subcutaneous administration except that the compound is administered 30 and 120 min prior to challenge. The results are given in Table 111.

Discussion The broad understanding of the action of the organo-

phosphorus AChE inhibitors recognizes that inhibition of the enzyme at the synapse results in repeated nerve impulse and muscle contraction culminating in asphyxiation (1 3). More recently, it has also been suggested that elevated acetylcholine levels cause morphologically damage at the synapse (14, 15). In the classical view, the prophylactic effect of carbamates can be attributed to slowly reversible

Carbamates of Imidazo[l,2-a]pyridinium Salts

Table 111. Prophylactic Activity after Oral Administration % survival % survival

compd dose 30min 120min compd dose 30min 120min la 15.6 0

62.5 70 250 60

le 15.6 20 62.5 80

250 100 lg 15.6 80

62.5 80 250 70

li 15.6 80 62.5 100

250 90 11 15.6 70

62.5 90 250 100

lm 15.6 60 62.5 90

250 70 In 15.6 10

62.5 40 250 40

2a 15.6 100 62.5 100

250 80

40 90 90 30 80 90 90 90 80

100 90

100 80 80

100 70

100 80 20 40 80 80

100 90

2b 15.6 20 62.5 60

250 100 2d 15.6 0

62.5 50 250 40

2h 13.1 0 52.5 20

250 80 3a 15.6 40

62.5 10 250 0

3b 15.6 40 62.5 0

250 10 3d 15.6 30

62.5 40 250 90

4h 15.6 0 62.5 20

250 30

80 70

100 20 70 70 50 80

100 20 70 80 20 10 0 70 40 70 50 30 50

carbamoylation of the active site serine which, while partially inhibiting AChE, spares the lethal effects. Some of the more recent results suggest that physostigmine may also have a protective effect against damage caused by elevated acetylcholine levels (14,15).

The properties of the imidazo[l,2-alpyridinium car- bamates we have synthesized suggest they are related to carbamates such as physostigmine and pyridostigmine. The data for inhibition of electric eel AChE shows that the carbamates are active with ICs0 ranging from 0.2 to 33 pM. The most active compounds were lk and 4a which have ICs0 C 0.5 pM. An activity level of about 1 pM is approached with compounds such as lb, lg, lh, 11, and lm. The acetamido derivative lh was among the most potent AChE inhibitors in both the 1 and 3 series. Even the related structure 5b, which lacks the carbamate function, has quite significant in vitro AChE inhibition activity. With the exception of lb (X = CH3), substituents such as chloro, trifluoromethyl, and methoxy appear to have little effect in the 1 series and are deleterious in the 3 series.

Several of the compounds show significant prophylactic activity. The most active appear to be la, Id, lg, and lh judging from survival at the lowest dose rate. Other compounds which exhibit consistent prophylactic activity include li, 2d, 4a, 4b, and 4d. Some of the compounds, notably l g and li, also have some antidotal activity. The carbamate functionality appears to be essential for in vivo activity. Compounds 5a, Sb, and 5d which lack the carbamate function are inactive in the prophylactic assay. Compound 5c in which the carbamate group is placed on the imidazo[l,2-alpyridinium ring does have modest activity as an in vivo AChE inhibitor but little in vivo protective effect. The carbamate group in 5c is structurally analogous to that in pyridostigmine. Compound 5b, while showing significant in vitro AChE inhibition, is inactive in vivo.

Assuming that the imidazo[l,2-alpyridinium carbam- ates act by mechanisms similar to pyridostigmine, they must interact with AChE and perhaps also with the acetylcholine receptor a t the synapse. Recently, the AChE

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 509

of Torpedo californica has been subjected to X-ray crystallographic structure determination (16). The active site is described as a “gorge” lined by a number of aromatic residues leading to a serine-histidine-glutamic acid cat- alytic triad. It is suggested that these aromatic structures might guide the substrate toward the active site by interaction between polarizable ?r-electrons and the qua- ternary ammonium group. It has often been assumed that the separation of the cationic charge and carbonyl group in acetylcholineof 5.OA (1 7) might represent the optimum for analogs. In an accompanying paper, it was shown that a number of compounds with larger apparent separation are active AChE inhibitors (17). For that group of compounds, however, there is substantial conformational flexibility, and it is conceivable the molecules adopt a conformation which is appropriate for AChE. The current series of compounds is much less flexible. Taking the N-1 ring nitrogen as the center for positive charge, the minimum separation of the carbonyl carbon is 5.1 A in the meta compounds and 7.2 A in the para compounds. Thus, the 5-A separation must not be an absolute requirement for activity. This observation is consistent with the conclusion from the crystallographic work that the “anionic site” is not a specific location but rather a diffuse charge gradient on the surface of the enzyme and perhaps within the active site gorge.

Acknowledgment. This research was carried out un- der the auspices of Walter Reed Army Institute of Re- search, Medical Research and Development Command, Contract DAMD-17-89-(2-9014. The authors acknowledge and thank the Division of Drug Assessment, US. Army Medical Research Institute of Chemical Defense, for biological testing and results.

Supplementary Material Available: Table IV giving ‘H- NMR peak positions and multiplicity for compounds la-d, lf- n, 2a,b, 2d, 2e, 2h, 3a-f, 3h, 4a, 4b, 4d, 40, 4h, and Sa-d (3 pages). Ordering information is given on any current masthead page.

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