Chem. Res. Toxicol. 1993,6, 500-505
500
Carbamates of (Hydroxyphen0xy)methylHeteroaromatic Salts as Acetylcholinesterase Inhibitors and Protective Agents against Organophosphorus Compounds Richard J. Sundberg,**tDeepak Dalvie,?Joehassin Cordero,? Michal Sabat,? 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 N,N-dimethylcarbamates of 2- [ (2'-, 3'-, and 4'-hydroxyphenoxy)methyl] heteroaromatic salts has been prepared. Pyridine, imidazole, quinoline, benzimidazole, and imidazo[ 1,2-a]pyridine derivatives were included. Most of the compounds are inhibitors of electric eel acetylcholinesterase and also show prophylactic activity toward a 2LDm dose of soman in mice. Introduction The toxicology of inhibitors of acetylcholinesterase (AChE)' has long been of interest in connection with chemical warfare agents (I, 2 ) and insecticides (3, 4). Therapeutic applications exist for AChE inhibitors in management of myasthenia gravis (5). More recently, the postulate that a cholinergic deficiency is associated with Alzheimer's disease has led to investigation of AChE inhibitors in this therapeutic context (6-9). The broadest classes of AChE inhibitors include the organophosphorus (0Pl1compoundsand aryl Carbamates. The former group are irreversible inhibitors which act by covalent modification of the nucleophilic serine of AChE (10). Partial hydrolysis at phosphorus, known as aging, prevents dephosphorylation (11,12). The carbamates act by carbamoylation of the same nucleophilic serine but are classified as reversible since the enzyme can be slowly regenerated by hydrolyticrelease of the carbamoyl moiety. Some carbamates (e.g., pyridostigmine and physostigmine) exhibit prophylaxis against the OP compounds (13-22). The mechanism presumably involves reversible carbamoylation which prevents irreversible phosphorylation. While carbamoylation causes an AChE deficit, it permits eventual recovery especially if used in conjunction with an anticholinergicagent such as atropine. Other protective mechanisms have also been suggested (23-25). We report here on a series of heteroaromatic carbamates encompassed by general structure 1-5. The synthesis, in vitro AChE inhibitory activity, and in vivo prophylactic and antidotal activity against OP AChE inhibitors are described. The structural requirements for activity and possible mechanism of action are discussed. The compounds evaluated are shown in Chart I. Sample numbers assigned by the Walter Reed Army Institute for Research are given. For comparisonwith the carbamates a series of l-methyl2-(phenoxymethy1)pyridinium salts with other substituents in the phenoxy group was also examined (compounds 6a-f), along with two related imidazolium salts (7a,f).
* To whom correspondence should be addressed.
+ University of
Virginia. Walter Reed Amy Medical Center. 1 Abbreviations: Ache, acetylcholinesterase; OP, organophosphorus; DNTB,5,5'-dithiobis(2-nitrobenzoic acid). t
Chart I
HETEROCYCLE
'0 I
BM02628 T s O
6 a
X H b C(CH3)3 e OCH, d CHO e CH=NOH f COhH,
BM08657 CI'
7
EM00197 BMOOZM BMOOlB8 B.VOO571 BM00593 BM00580
BM01238 BMOl247
Experimental Section General. Chemical reagents were obtained from commercial sources, mainly Aldrich Chemical Co. Electric eel acetylcholinesterase, 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 1HNMR spectra and by elementalanalyses. A table giving chemical shifts and peak multiplicity is included in the supplementary
0893-228~/93/2706-0500$04.00/0 0 1993 American Chemical Society
Carbamates of Heteroaromatic Salts material. Elemental analyses were obtained for all elements except oxygen and fluorine and agreed with the calculated value within f0.4%, Several of the salts analyze as hydrates. 2 4 (4’-Hydroxyphenoxy)methyl]pyridine. A solution of hydroquinone (8.1 g, 73.5 mmol) in ethanol (30 mL) was added to a mixture of 2-pyridylmethyl chloride hydrochloride (4.92 g, 29.9 mmol) and potassium hydroxide (1.68 g, 29.9 mmol) with stirring at room temperature. Potassium hydroxide (4.2 g, 74.8 mmol) was then added, and the reaction mixture was maintained at reflux under an atmosphere of nitrogen for 24 h. The mixture was cooled and filtered, the solid was washed with methanol (2 x 25 mL), and the combined filtrate and washings were evaporated in uacuo. The gummy residue was triturated with 10% sodium hydroxide (50 mL) and extracted with ether (2 X 50 mL). The aqueous layer was neutralized with concentrated hydrochloricacid and the precipitate filtered, washed with water, and dried to give the desired phenoxymethyl ether (4.4 g, 73%). This was used in the next step without further purification. 2-[[4’4[(N,N-Dimet hylamino)carbonyl]oxy]phenoxy 1methyllpyridine. 2- [(4’-Hydroxyphenoxy)methyllpyridine(4.1 g, 20.3 mmol) was suspended in (Nfl-dimethy1amino)carbamoyl chloride (30mL). The reaction mixture was maintained at reflux for 4 h, cooled to room temperature, and poured onto ice water (100mL). After the evolution of COz ceased,the reaction mixture was basified with saturated sodium bicarbonate solution to pH 8.8. The solid was filtered, washed with water (2 X 100 mL), and dried. The crude product was then subjected to quaternization. 4-hydroxy phenyl NJV-Dimet hylcarbamate. A suspension of hydroquinone (22 g, 0.2 mol) in xylene (600 mL) was heated to reflux, and to this was added Nfl-dimethylcarbamoyl chloride (24mL, 0.26 mol) dropwise over a period of 30 min. The reaction mixture was further maintained at reflux for 4-5 h after the addition was complete. The reaction mixture was then cooled to room temperature and treated with ice-coldsodium hydroxide solution (10%, 200 mL). The organic layer was separated and washed twice with 10% sodium hydroxide followed by water. All the aqueous extracts were combined and washed once with ether and then acidified with 10% HC1 to give a precipitate which was filtered and dried to give the desired monocarbamate (15.0 g, 41%). 3-Hydroxyphenyl N,N-Dimethylcarbamate. The compound was prepared from resorcinol by the same procedure as for the 4-isomer except that the acidified aqueous layer was extracted with dichloromethane. The organic layer was then evaporated to give the desired monocarbamate (16.7 g, 46%). 2-Hydroxyphenyl NJV-Dimethylcarbamate. The compound was prepared from catechol by the procedure and workup described for the 3-isomer and gave the desired product in 49% yield. General Procedure for Preparation of Heteroaromatic Carbamates. The appropriate hydroxyphenyl carbamate (1.2 equiv) was dissolved in MezSO and added dropwise to a suspension of potassium hydroxide (1.4 equiv) in MeZSO. The mixture was stirred for 30 min. To this reaction mixture was added potassium carbonate (1.0 equiv) followed by dropwise addition of a solution of the heteroarylmethyl chloride hydrochloride salt (1.0 equiv) in MezSO. The reaction mixture was stirred overnight at room temperature and poured into ice water. If a precipitate was obtained, it was filtered. Products which were oils were extracted with ethyl acetate, and the organic layer was washed with water, dried, and evaporated to give the desired heteroaromatic carbamate. This procedure was used for preparation of compounds 2, 3, and 5. [4-[[ (N,N-Dimet hylamino)carbonyl]oxy]phenoxy]acetonitrile. A suspension of potassium carbonate (7.74 g, 0.56 mol) in MezSO (100 mL) was treated with a solution of 4-hydroxyphenyl carbamate (7.64 g, 0.042 mol) in MezSO (100 mL). The mixture was stirred for 1.5 h at room temperature, and then a solution of chloroacetonitrile (3.94 mL, 0.626 mol) in MeZSO (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 3-4 h and then poured over ice water. The precipitate was filtered, washed with water, and dried
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 SO1 in air to give the desired product (6.9 g, 75%). The product was characterized by NMR and used for the next step. [3-[ [ (N,N-Dimet hylamino)carbonyl]oxy]phenoxy]acetonitrile. The compound was prepared from chloroacetonitrile and 3-hydroxyphenyl Nfl-dimethylcarbamate by the above procedure in 79 % yield. The product was characterized by NMR and used for the next step without further purification. 2 4 [4’-[[(N,N-Dimethylamino)carbonyl]oxy]phenoxy]methyl]benzimidazole. The method of Nabulsi and Gandour was followed (26).To a solution of [4-[[(NJV-dimethylamino)carbonyl]oxy]phenoxy]acetonitrile (Log,0.022 mol) inmethanol (80 mL) was added sodium (0.52 g, 0.022 mol). The solution was allowed to stir at room temperature for 2 h. Phenylenediamine dihydrochloride (4.1 g, 0.23mol) was then added, and the solution was stirred for 1.5 h. The reaction mixture was then quenched with ice water, and the turbid solution was extracted with ethyl acetate. The organic layer was washed with water, dried, and evaporated in UQCUO to give an oil (5.6 g, 70%). The product was characterized by NMFt and used for the next step without further purification. 2-[[Y-[ [(N,N-Dimethylamino)carbonyl]ox~]phenoxy]methyl]benzimidazole. The compound was prepared by the same procedure as for the 4-isomer. The desired product was obtained in 68% yield. The product was characterized by NMR and used for the next step without further purification. General Procedure for Quaternization and Conversion tochloride Salt. To a suspension or solution of an appropriate heteroaromatic carbamate in tetrahydrofuran (150mL) was added iodomethane (large excess), and the mixture was maintained at reflux for 60 h. The precipitate obtained was filtered and washed with tetrahydrofuran and ether and dried. The iodide salt was dissolved in methanoVacetonitrile and passed through an ionexchange column (Amberlite IRA 400 C1- supplied by Mallinckrodt Chemical Works or Aldrich Chemical (20.). The solvent was evaporated, and the product obtained was then recrystallized from acetonitrile/ethyl acetate or ether to give the chloride salt. General Procedure for Synthesis of Heteroaryl Ethers. A solution of appropriate phenol (1.1 mol) and potassium hydroxide (2.5 mol) in MezSO (30 mL) was stirred at room temperature for 1h. A solution of 2-pyridylmethyl chloride or 2-imidazolylmethyl chloride in MezSO (20 mL) was then added dropwise. The reaction mixture was stirred at room temperature and poured into water (300 mL). The water layer was extracted with ethyl acetate (3 X 100 mL) and washed with water (3 X 100 mL), 10% sodium hydroxide solution (3 X 50 mL), and brine (3 X 50mL). The organiclayer was dried (NazSO,) and concentrated in vacuo, and the residue was used for the next step without further purification. The following compounds were prepared by this method. 2-(Phenoxymethyl)pyridine from phenol in 70% yield. 2-[ (4’-Methoxyphenoxy)methyl]pyridinefrom 4-methoxyphenol in 90% yield. 2 4 (4-tert-Butylphenoxy)methyl]pyridinefrom 4-tert-butylphenol in 75% yield. 2-1(4’-Formylphenoxy)methyl]pyridine from 4-hydroxybenzaldehyde as described previously (38). 2 4 (4’-Carbamoylphenoxy)methyl]pyridinefrom 4-hydroxybenzamide in 87% yield. The reaction time was 24 h. 2 4 (4’-Formylphenoxy)methyl]imidazolein 54% yield from 4-hydroxybenzaldehyde. The reaction time was 2.5 h. 2-[(4’-Carbamoylphenoxy)methyl)imidazolefrom 4-hydroxybenzamide in 29% yield. The reaction time was 4 h. General Procedure for Preparation of Quaternary Salts 6 and 7. A solution of the appropriate ether and methyl p-toluenesulfonate (1.2-1.5 equiv) in anhydrous acetonitrile (4550 mL) was maintained at reflux for 60 h. The reaction mixture was cooled in an ice-water bath, and the solid precipitate was fiitered, washed with ether, dried, and recrystallizedwith absolute ethanol and anhydrous ether. Procedure for Determination of Acetylcholinesterase Inhibition. A predetermined amount of test compound to give a final concentration of 1O-g-1O-a M was placed in a photometric
502 Chem. Res. Toxicol., Vol. 6,No. 4, 1993
Sundberg et al. Table 1. Inhibition of Electric Eel AChE 01311
e
compd 01301
la Ib IC
2a 2b 2c 3a
C(211
Figure 1. ORTEP drawing of crystal structure of 2b. 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 thecorrespondingamout of buffer (52 mM NaHzPOd) to the mixture. After incubation for 15 min, 100 pL of 0.1 M acetylthiocholine iodide was added and stirred for about 30 s and placed in the spectrometer. The absorption of the solution was measured at 412 nm every 30 s for 6 min. From the slope of the curve obtained the rate of the enzymaticreaction was determined. The inhibitor concentration at which enzyme is inhibited by 50% was obtained from a plot of the percentage enzymatic activity ( % EA) versus the logarithm of the inhibitor concentration. The apparatus used to follow the reaction was a Hewlett Packard 8452A diode array spectrophotometer. 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 at the stated dose 15 and 60 min prior to challenge by a 2LDm dose of soman. Ten second 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 bodywt) 10s after challenge with 2LDm soman. The 24-h survival rate was compared with a negative control (pralidoxime sulfate, 25 mg/kg body wt, instead of the test compound). X-ray Crystal Structure Determination of 2b. All X-ray measurements were done on a Siemens/Nicolet P3m diffractometer at 25 "C using Cu Ka!radiation (A = 1.54178 A). The compound crystallizes in the triclinic space group Pi (No. 2) with a = 9.070(3) A, b = 10.827(3)A, c = 13.029(4) A; a! = 68.07(2)", 0 82.03(3)", 7 77.30(3)"; V = 1155.67(7)A, 2 = 2, D d d = 1.339 cma. A total of 2908 independent reflections were collected between 3" I29 I110" by the 8/29 scan method. The structure was solved by direct methods using SHELXTL PLUS: Version 4.0, and refiied by full-matrix least-squares refinement using 2494 reflections with Z > 2u(n to R = 0.063 and R, = 0.095. The goodness of fit was 1.86. Hydrogen atoms were located from a difference Fourier map and included as fixed contributions to the structure factors. The largest peak in the final difference map was 0.60 e/Aa. The ORTEP (50% probability ellipsoids) drawing is presented in Figure 1. Molecular Modeling. The MM2 force field as implemented on the CAChe Work Systema was used to investigate the energy of the alternate conformers. The strain energies were computed as the sum of bond length deformation, valenceangle deformation, torsional angle deformation,nonbonding interaction, electrostatic 2 SHELXTLPLUS Version 4.0,Siemens Analytical Instruments, Inc., Madison, WI, 1990. * CAChe Work Sptem, Version 3.0, CAChe Scientific Inc., 1992.
ICm(pM)
14.7 0.2
0.035 12.3 0.01 0.2
3.3
compd
ICm(pM)
3b 4a 4b Sa 5b 6a 6b
0.2 0.6 0.1 3.1 0.5 >75 80
compd
ICmoM)
60
>lo0 140 >lo0 >lo0
6d 60 6f
7e
45
7f
>lo0
interaction, and hydrogen bond interaction contributions at a dielectric constant of 1.5. The structure determined by crystallography was inputted and then minimized using the blockdiagonalNewton-Raphson technique. This structure minimized to a strain energy of -0.57 kcal/mol with a carbonyl carbon42 distance of 7.86 A. The molecule was then inputted and minimized in both (+I- and (-)-gauche conformations. Slightly lower strain energies of -1.13 and -2.27 kcal/mol were found for the (+)- and (-)-gauche conformations, respectively. The carbony142 distances were 5.12 and 4.57 A, for the minimized (+)and (-)-gauche conformers, respectively.
Results (A) Synthesis. A general synthetic route was used for most of the N,N-dimethylcarbamates. The appropriate benzenediol was monocarbamoylated and then alkylated with the appropriate chloromethyl heterocycle. The heterocyclic compound was then quaternized with methyl iodide or methyl tosylate. The iodides were converted to chlorides by ion exchange and purified by crystallization. All the compounds were characterized by elemental analysis and by IR and 'H-NMR spectra. An alternative synthetic route was initially used for the pyridinium compound, but it proved to be less general. In that procedure the benzenediol was first monoalkylated with 2-(chloromethyl)pyridine. The resulting phenol was then carbamoylated, quaternized, and purified by the general procedure. The benzimidazole compounds were prepared by cyclizationof [3- or [4- [(N,N-dimethylcarbamoy1)oxylphenoxylacetonitrile with phenylenediamine (26). The 4"Ibstituted (phenoxymethy1)pyridiniumsalts 6a-e were prepared by alkylation of the appropriate phenol with 2-(chloromethyl)pyridine. Compounds 7e and 7f were prepared similarly by using 2-(chloromethy1)imidazole. (B) Biological Evaluation. All the compounds were evaluated as AChE inhibitors by the method of Ellman using electric eel AChE, type I11 (27). The results are given in Table I. Representative compounds were also evaluated in vivo for prophylactic and antidotal activity against the organophosphorus AChE inhibitor soman. In the prophylaxis assays the test compound is administered to mice by subcutaneous injection 15 or 60 min prior to challenge by 2LDm of 50111811.~ Pralidoxime (25 mg/kg body wt) and atropine sulfate (11.2 mg/kg body wt) are administered 10s after challenge. Under these conditions untreated negative controls typically show 0-20 % survival. A positive control, pyridostigmine, at 0.1 mg/kg body wt results in -50% survival. Survival of 40% or more is considered to indicate potential for prophylactic activity. For the antidotal assay the compound is administered simultaneously with atropine (11.2 mg/kg body wt) 10 s after challenge with 2LDm of soman. As a negative reference pralidoxine chloride (25 mg/kg body wt) and atropine (11.2 mg/kg body wt) are administered. Usually ~~
~
Soman is methyl (1,2,2-trimethylpropyl)phoephonofluoridate.
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 503
Carbamates of Heteroaromatic Salts
Table 11. Prophylactic and Antidotal Activity against Soman compd
la
lb IC
2a
dose
0.9 3.5 6.2 14.0 15.6 25.0 62.5 100 250 0.4 1.6 6.2 0.2 0.9 3.5 1.25 5.0 20.0
% survival t 60 min 100
-
t - 15 min
100 90 80 90 90 90 80 30 70 50
50 60
tOX
tOX
80 50 30 60 50 60
50 90 50 40 70 20
40 50 60 30 20 10
2b
0.007 0.03 0.11
40 40 50
60 40 30
2c
0.11 0.44 1.78
60 70 60
70 50 40
6.25 25.0 100
40 20 10
60 60 50
3a
3b 4a 4b
0.06 0.24 0.96 0.9 3.7 14.6 0.020 0.075 0.30
t + 10 s 50 60
80 60 100 40 90 80 100 10 80
40 30 60 50 30 50 60 60 80
compd
dose
% survival t 60 min
Sa
3.1 12.2 49.0
50 50 70
t - 15 min 20 60 30
70 70 50
80 60 50
-
t + 10 s 10 30 40
40
40 50 50 30 40 10 40 50 60
there are
