Chem. Res. Toxicol. 1990, 3, 413-422
413
Abiotic Transformations and Decomposition Kinetics of 4-Carbamoyl-2’-[ (hydroxyimino)methyl]-1 ,l’-(0xydimethylene)bis(pyridinium chloride) in Aqueous Phosphate Buffers Raymond E. Mdachi,? William D. Marshall,*$?Donald J. Ecobichon,’ Fouad M. Fouad,? and Celso E. Connolley-Mendozas Department of Food Science and Agricultural Chemistry, Macdonald College, Ste. Anne de Bellevue, Quibec, Canada H9X 1C0, Department of Pharmacology and Therapeutics, McGill University, Montreal, Qubbec, Canada H3G 1 Y6, and Defense Research Establishment Suffield, Ralston, Alberta, Canada TOJ 2NO Received November 18, 1988 T h e rate of disappearance of 4-carbamoyl-2’- [ (hydroxyimino)methyl]-1,l’-(oxydimethylene)bis(pyridinium chloride) (HI-6) from aqueous phosphate buffers (pH 3.0-9.1) was both p H and temperature sensitive. In midrange buffers (pH 6.0-9.1, p = 0.2 M)a t 37,25,or 4 “C the decomposition followed first-order kinetics consistent with hydroxide-promoted decomposition of the un-ionized drug or with hydrolysis of the ionized oxime anion t o result in 4-carbamoyl-2’-hydroxy-l,l’-(oxydimethylene)bis(pyridinium)cation (intermediate 1). The subsequent conversion of intermediate 1to 4-carboxy-2’-hydroxy-l,1’-(oxydimethylene)bis(pyridinium) cation (intermediate 2) followed higher order kinetics which were consistent with either acid- or base-promoted hydrolysis of the B-ring amide functionality. After approximately 138 days in the dark, the sum of the residual HI-6, intermediate 1, and intermediate 2 in the crude decomposition mixture accounted for 89.9 f 10.0% of the initial substrate. Minor byproducts included 4-carbamoyl-2’-carboxyoxy-1,1’-(oxydimethylene) bis(pyridinium) cation, 2-pyridinealdoxime, 2-pyridinecarboxaldehyde,2-hydroxypyridine, isonicotinamide, isonicotinic acid, and traces of cyanide. In addition, 2-cyanopyridine appeared to be a transient intermediate in more alkaline media. In total, this drug resembles other mono- and bis(pyridinium) aldoximes in terms of the decomposition routes in aqueous solutions a t intermediate pHs.
Introduction There is a continuing interest in bis(pyridinium) aldoximes in terms of their ability to reactivate the enzyme acetylcholinesterase (AChE) which has been inhibited by organophosphorus esters or carbamates (1-4). Pyridinium aldoximes, with an oxamino group ortho or para to a quaternary N, have been demonstrated to react with organophosphorus compounds (5,6) and carbamates in vitro to yield rapidly decomposing products. Provided that the methine hydrogen of the oximino group is sufficiently acidic, decomposition of the phosphorylated or carbamylated oxime is rapid and the adduct shows little tendency to act as a transfer agent and rephosphorylate (carbamylate) the liberated enzyme (7, 8). Equally well, if the phosphorylated (carbamylated) oxime is sufficiently stable, the toxic effects of certain OP or carbamate pesticides (8-1 0) may be exacerbated in the presence of the drug. The combination of the reactivating component, a pyridinium aldoxime, with various nicotinic acid analogues has resulted in compounds I-IV with increased lipoid solubility and decreased toxicity relative to obidoxime chloride (V) (see Chart I). Several comparative studies, both in vivo and in vitro, of various reactivators have indicated that 4-carbamoyl-2’-[(hydroxyimino)methyl]1,l’- (oxydimethylene)bis(pyridinium chloride) (HI-6; I)
* Correspondence should be addressed to this author at the Department of Food Science and Agricultural Chemistry, Macdonald College of McGill, 21 111 Lakeshore Rd., Ste. Anne de Bellevue, QuBbec, Canada H9X 1CO. Macdonald College. McGill University. *Defense Research Establishment Suffield.
*
0893-228x/90/2703-0413$02.50/0
possesses a relatively lower acute toxicity and appreciable reactivating properties for OP intoxicated rats ( l l ) ,mice (11, 12), dogs (13), and monkeys (14). In addition to reactivating AChE in the peripheral nervous system, it would appear that HI-6 does gain access to the central nervous system and that this drug possesses ganglionic activity (15) and antimuscarinic activity (16)and inhibits benzylate to the binding of [3H]-N-methyl-4-piperidy1 brain muscarinic receptors (17). The chemical stability and abiotic fate of HI-6 have not been fully studied. This product was demonstrated (18) to decompose to isonicotinic acid and to isonicotinamide in aqueous buffers (pH 2-9). Two additional products were not identified. The apparent activation energy for the decomposition was 113 kJ mol-’, and the rate was unaffected by oxime concentration, buffer composition, light, or oxygen (18). Brown et al. (19) demonstrated the formation of isonicotinamide, isonicotinic acid, pyridinepyridine4-carboxaldehyde, pyridine-2-carboxaldehyde, 2-aldoxime, and 4-cyanopyridine if HI-6 was stored in acetate buffer (pH 4.0) at 40 “C for six weeks. Given the preponderance of monopyridine ring products it was suggested that HI-6 may be decomposed via routes which are appreciably different from those of other bis(pyridinium) aldoximes as reported by Christenson (20-23). An earlier study (23) indicated that the aldoxime group of HS-6 (11)was decomposed to the corresponding aldehyde in acidic solutions (pH 2-3), and to the corresponding amide or hydroxy derivative in less acidic buffers (pH 4-9). In highly alkaline media hydrolysis of the oxydimethylene bridge was favored; products included pyridine-Zaldoxime, nicotinamide, and formaldehyde. Recently, Eyer et al. (24) tentatively identified two transformation products of HI-6 0 1990 American Chemical Society
414 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
Mdachi et al.
Q
+ t b
N i
VI
1
ak,
VI11
X
IX
Figure 1. Proposed decomposition route for HI-6 in neutral and moderately alkaline media. Chart I A-ring
33. R:*h(6 B
z3
kDA;&yR4 113 iy--
I
e5 ( 7 )
B-r,ng
compd no. I
I1 111
IV V
compd HI-6 HS-6 2,2-LuH6 2,4-LuH6 obidoxime
R1 CH=NOH CH=NOH CH=NOH CH=NOH H
R2
H H H H CH=NOH
in aqueous phosphate buffer (pH 7.4) as the 2-pyridone analogue of HI-6, 4-carbamoyl-2’-hydroxy-l,l’-(oxydimethylene)bis(pyridinium) cation (VI), and a deamination product of the 2-pyridone analogue, 4-carboxy-2‘hydroxy-1,l’-(oxydimethylene)bis(pyridinium)cation (VII). This group has also reported (25) that cyanide was detected in the serum of dogs which had received a single intravenous dose of HI-6. The objectives of the current study were to further define the routes, to determine the kinetics, and to corroborate the identity of the products of abiotic decomposition on HI-6 in aqueous phosphate buffers. Emphasis was to be placed on the pH range 6-9 where competing degradative routes were anticipated (functional group modification vs bridge hydrolysis). By analogy to the products observed for the abiotic decomposition of HS-6, anticipated products for the degradation of HI-6 are outlined in Figure 1. The nitrile VIII is considered to be a key (but reactive) intermediate in the reaction sequence at intermediate pHs. Hydrolysis of VIII would result sequentially in diamide IX and in amide-acid X. Alternately, hydroxide displacement of the nitrile (via cyanohydrin formation) would result in VI and subsequently in VI1 via hydrolysis of the
R3
H H CH=NOH H H
R4 H CONH2 H CH=NOH H
R5 CONHP H H H CH=NOH
B-ring amide functionality. In this reaction sequence, hydrolysis of the oxydimethylene bridge to result in monopyridine ring products has been deliberately deemphasized although this route may be competitive with functional group modification.
Materials and Methods Melting points were determined on an electrothermal melting point apparatus and have not been corrected. Nuclear magnetic resonance spectra were recorded on a 200-MHz XL 300 spectrometer in D20unless specified otherwise. HI-6 and analytical samples of authentic VI1 and XVI were kindly provided by P. Lockwood, Defense Research Establishment Suffield, Ralston, Alberta. Infrared spectra were recorded in aqueous solutions using a redesigned (26)Spectromaster-1double-beam spectrophotometer. HPLC analysis was performed with a Beckman Model lOOA pump controlled with a Beckman Model 420 programmer and either a 5-pm octyl-bonded,’S5C8”, 15 X 0.46 cm column or a phndapak C18,30 x 0.46 cm column. The mobile phase, delivered at 1 mL min-’, consisted of mixtures of approximately 85% phosphate buffer (pH 4.18, ionic strength 0.2 M) containing 0.03 M sodium octanesulfonate and approximately 15% acetonitrile. Samples (20 pL) were injected with a Beckman Model 120
Abiotic Transformations of a Bisbyridinium) Aldoxime fixed-loop injector. Column eluate was monitored with a Gilson Model 1 l l B fixed-wavelength ultraviolet (280 nm) detector, and chromatograms were recorded with a Hewlett-Packard Model 3390A recording integrator. Stability Trials of HI-6 in Aqueous Phosphate Buffers. A series of stock solutions of HI-6 (1.0 or 5.0 mg mL-') in aqueous phosphate buffers (pH 4-9 in unit increments, 0.2 M ionic strength) were prepared. Aliquots, 0.5 mL, of the standard solutions were added to 4.5 mL of the same phosphate buffer, and the vials were capped, thoroughly mixed, and incubated at 37 "C, at room temperature, or at 4 OC in the dark. At convenient intervals the crude decomposition mixture was assayed by HPLC. The ultraviolet detector was calibrated, by the method of external standards, using eight aqueous HI-6 standards ranging in concentration from 0.1 to 1.0 mg mL-'. Linear regression analysis of peak area (the average of three replicate determinations) on concentration of the corresponding standard resulted in a correlation coefficient, r, of 0.9989. HI-6 Anion (XI). A solution of 0.359 g (1mmol) of HI-6 in 2 mL of distilled water was added dropwise (with stirring) to 0.5 mL of 1.0 M aqueous sodium carbonate. 2-Propanol (1mL) was then added, and the resulting solution was cooled to -4 "C until yellow crystals formed. The crystals were recovered by filtration, washed sparingly with 2-propanol, dried in a vacuum, and stored at 0 "C: mp 160-162 "C dec; 'H NMR (D20) 6 6.32 (s, 2 H, methylene), 6.37 (s, 2 H, methylene), 7.92 (t, 1 H, aryl proton), 8.14 (d, 1 H, aryl proton), 8.43 (d, 2 H, aryl protons), 8.48 (t, 1 H, aryl proton), 8.53 (s, 1H, CH=NO), 8.78 (d, 1 H, aryl proton), 9.20 ppm (d, 2 H, aryl protons); 13CNMR (DzO)6 88.2,89.9 ppm (methylene carbons); 129.05, 129.05, 144.87, 147.37, 153.62 ppm (A-ring carbons); 129.56 (2 C), 149.96 (2 C), 152.06 ppm (B-ring carbons); 147.07 ppm (aldoxime carbon); 169.13 ppm (carbonyl carbon, B-ring). Chloride Ion Content of XI. HI-6 anion, 0.1002 g (0.278 mmol), was dissolved in 50 mL of distilled deionized water in a 100-mL beaker equipped with a magnetic stirrer and stirring bar. Barium nitrate, 0.5 g, and six drops of 6 M HN03 were added, and the resulting solution was titrated with 0.0190 M silver nitrate. The course of the titration was monitored potentiometrically with a silver indicating electrode and a saturated calomel electrode dipping in a solution of 2.5 M KN03. The change in potential with each incremental addition of titrant was recorded. Intermediate 1 (VI). HI-6 anion, XI, 0.1 g (0.278 mmol), was dissolved in 4 mL of CH30H in a 25-mL round-bottomed flask which was sealed with a rubber septum. The air in the flask was replaced with N2, and 60 pL of (C2H5)3Nwas added. Dimethyl phosphorochloridothioate, 100 pL, was added to the stirred reaction mixture, and the course of the reaction was monitored by HPLC. After 2 h, the reaction mixture was concentrated under reduced pressure and the oily residue, dissolved in a minimum of methanol, was added to a short column of silica gel (8.0 x 0.6 cm) and eluted with CH30H. The portion of column eluate corresponding to intermediate 1was concentrated to dryness and triturated with CHC13, and the residues were resolubilized in 1:1 acetone/methanol. White needle-like crystals formed on standing: mp 139-140 "C; 'H NMR 6 5.58 (s, 2 H, methylene), 6.01 (s, 2 H, methylene), 6.28 (d, J = 8.3 Hz, 1 H, A-ring proton), 6.38 (t, J = 8 Hz, 1 H, A-ring proton), 7.39 (t, J = 2 Hz, 1 H, A-ring proton), 7.53 (d, J = 2 Hz, A-ring proton), 8.20 (d, J = 7 Hz, 2 H, B-ring protons), 8.96 ppm (d, J = 7 Hz, 2 H, B-ring protons); 13C NMR 6 112.21, 122.34, 140.92, 146.53, 166.69 ppm (A-ring carbons); 128.92 (2 C), 146.69 (2 C), 152.98 ppm (B-ring carbons); 169.13 ppm (amide carbon, B-ring); 196.47 ppm (carbonyl carbon). Isolation of Intermediate 2 (VII). Intermediate 2 was prepared by the intentional decomposition of intermediate 1 (50 mg) in 0.02 M sodium bicarbonate solution (2.5 mL) at 37 "C. The course of the decomposition was monitored by HPLC. When approximately 95% decomposition had been achieved, the solvent was removed under reduced pressure (room temperature) and the residue was resolubilized in a minimum of methanol, applied to a short column of silica gel (8 X 0.6 cm), and eluted with CHC13/MeOH (3:l). The product crystallized slowly on evaporating the solvent: mp 191-193 "C; 'H NMR 6 5.59 (s, 2 H, methylene), 6.07 (s, 2 H, methylene), 6.30 (d, 1H, A-ring proton), 6.40 (m, 1 H, A-ring proton), 7.44 (m, 1 H, A-ring proton), 7.60 (d, 1 H, A-ring proton), 8.27 (d, 2 H, J = 6.5 Hz,B-ring protons),
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 415 8.97 (d, 2 H, J = 6.5 Hz, B-ring protons; 13C NMR 6 81.45,90.45 (methylene carbons), 112.24, 122.43, 140.83, 146.48, 166.64 ppm (A-ring carbons); 129.41 (2 C, B-ring carbons), 146.17 (2 C, B-ring carbons), 157.32 (B-ring carbon), 171.30 (carbonyl C, B-ring), 196.41 ppm (pyridone C, A-ring). 4-Carbamoyl-N-(methoxymet hyl) pyridinium Chloride (XIV). Chloromethyl methyl ether, 100 pL, dissolved in 3 mL of acetone was added dropwise to an ice-cold stirred solution of isonicotinamide, 0.1 g (0.819 mmol), dissolved in 12 mL of acetone. The precipitate, which formed immediately, was removed by filtration, and the filtrate was allowed to stand for 2 h. The white crystals that formed were recovered by filtration and allowed to air-dry: mp 178-180 "C; 'H NMR 6 3.53 (s, 3 H, methoxy group), 5.96 (s, 2 H, methylene group), 8.30 (d, J = 6 Hz, 2 H, aryl protons), 8.97 ppm (d, J = 6 Hz, 2 H, aryl protons). 2-Hydroxy-N-(methoxymethyl)pyridinium Chloride (XIII). This product was prepared according to the general method above. The white crystals that formed were recovered by filtration and air-dried: mp 147-149 "C; 'H NMR (D20 NaZCO3)6 3.50 (s, 3 H, methoxy group), 5.51 (s, 2 H, methylene), 6.24 (d, 1H), 6.33 (d of d, 1H), 7.37 (d of d, 1 H), 7.49 (d, 1 H). ['4C]HI-6. [carbonyl-14C]Isonicotinamide (17.5 mCi/mmol, nominal total activity 250 pCi, ICN Laboratories) was reacted with N-(chloromethoxymethyl)-2-[(hydroxyimino)methy1]pyridinium chloride (2-2) according to the general method of Schoene (27). The [14C]isonicotinamide,in 1mL of CH3CN, was diluted with 10 mg of radioinactive isonicotinamide and reacted with 48 mg of 2-2 in an inert atmosphere at 45 "C for 8 h. The solvent was reduced under N2, and the crude product was separated from the reaction mixture by centrifugation, washed sparingly with CH,CN, and purified by preparative thin-layer chromatography on silica gel using 2% of glacial acetic acid in methanol containing 0.1 M NaBr as eluent. A total of 106 pCi (42% yield) cochromatographed with authentic HI-6 in this system. The recovered product also cochromatographed with authentic HI-6 on the pBondapak HPLC column. Decomposition Trial of ['%]HI-6. Aqueous ['Y!]HI-6,1 mL of a 0.1 mg mL-' solution (78 pCi mmol-l), was added to 4 mL of saturated aqueous sodium bicarbonate solution and incubated in the dark at 37 "C. The course of the decomposition was assayed periodically by separating 20 pL of the crude decomposition mixture on the S5C8 HPLC column. Portions of column eluate corresponding to HI-6, to intermediate 1,and to monopyridinium products were trapped in separate liquid scintillation vials and concentrated under a gentle stream of NP. Decomposition of [ 14C]Intermediate 1. Radiolabeled HI-6 which had completely decomposed to [14C]intermediate1during storage at 4 "C, 0.5 mL, was added to 2.0 mL of saturated aqueous sodium bicarbonate and incubated at 37 "C in the dark. HPLC analysis indicated that there was no residual HI-6 nor any other decomposition products in this solution. The course of the decomposition wm monitored by HPLC. Fractions of column eluate corresponding to intermediate 1, to intermediate 2, and to monopyridinium ring products were trapped in separate scintillation vials and concentrated with a gentle stream of NP. Liquid Scintillation Counting. Samples were diluted with universal cocktail and assayed for activity by using a Model-1219 Rackbeta Spectral (LKB Wallac) liquid scintillation counter. Counts were automatically corrected for background, quenching (if any), and counting efficiency.
+
Results and Discussion A preliminary investigation of the effects of pH and temperature on t h e stability of HI-6 (1.39 X M) in phosphate buffers (ionic strength, 0.2 M; pH 3.0-9.1) indicated that t h e stability of this substrate was sensitive t o both factors (Figure 2). Additionally, HPLC analyses of t h e crude incubation mixtures indicated that HI-6 was transformed to several products (Figure 3), one of which appeared rapidly in t h e crude mixture b u t which subsequently decomposed over t h e course of t h e trials. T h i s product, intermediate 1, had a retention time (relative to HI-6 = 1.0) of 0.48 which was different from any of t h e anticipated monopyridine ring products. A subsequent
Mdachi et al.
416 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
Figure 2. Stability, with time, of HI-6 in aqueous phosphate buffers at (A) 37 "C, (B) room temperature, or (C) 4 "C. Scheme I
Table 1. Chemical Shifts of the Protons of HI-6 and Transformation Products product B-ring.~ protons I 9.23 (d, 2 H, J = 7 Hz) 8.49 (d, 2 H, J = 7 Hz) VI 8.96 (d, 2 H, J = 7 Hz) 8.20 (d, 2 H, J = 7 Hz) 8.97 (d, 2 H, J = 6.5 Hz) VI1 8.27 (d, 2 H, J = 6.5 Hz) XI 9.20 (d, 2 H, J = 7 Hz) 8.43 (d, 2 H, J = 7 Hz) XI11
XIV
methylene 6.44 ( 8 , 2 H) 6.31 (s, 2 H) 6.01 (s, 2 H) 5.58 (s, 2 H) 6.07 (s, 2 H) 5.59 (s, 2 H) 6.37 (e, 2 H) 6.32 (s, 2 H) 5.51 ( 8 , 2 H) 3.50 (s, methoxy)
oxime A-ring- protons carbon proton _ 9.08 (d, 1 H, J = 6 Hz),8.63 ( 8 , 1 H) 8.69 (8, 1 H) 8.48 (d. 1 H. J = 6 Hz),8.14 (d of d. 1 H) 7.53 (d, 1 H, J = 6 Hz),7.39 (m, 1 H) 6.38 (d of d, 1 H), 6.28 (d, 1 H, J = 6 Hz) 7.60 (d of d, 1 H), 7.44 (d of d, 1 H) 6.40 d, 1 H, J = 6 Hz),6.30 (d, 1 H, J = 6 Hz) 8.53 (s, 1 H) 8.78 (d, 1 H, J = 6 Hz),8.48 (d of d, 1 H) 8.14 (d, 1 H, J = 6 Hz),7.92 (d of d, 1 H) 7.49 (d, 1 H, J = 6 Hz),7.37 (d of d, 1 H) 6.33 (d of d, 1 H, J = 6 Hz),6.24 (d, 1 H, J = 6 Hz)
8.97 (d, 2 H, J = 6.4 Hz),5.96 (s, 2 H) 8.30 (d, 2 H, J = 6.5 Hz),3.53 (s, methoxy group)
reinvestigation of the crude decomposition profiles using the NBondapak column provided a superior resolution of products with shorter retention times and revealed the presence of a second decomposition intermediate (intermediate 2, RRT = 0.13, Figure 4) in addition to intermediate 1 (RRT = 0.38, Figure 4). Qualitative tests for CN- among the terminal products were positive; however, levels of this analyte were appreciably lower (
