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Quaternary Salts of 3,3’-Bis-Pyridinium Monooximes Synthesis and Biological Activity
Quaternary Salts of 3,3'-Bis-Pyrid inium Monooximes: Synthesis and Biological Activity ARUNK. SIKDER, ASHIM K. GHOSH,AND DEVENDRAK.JAISWAL' Received June 11, 1990, from the Synthetic Chemistfy Division, Defence Research and Development Establishment, Gwalior-474002, India. Accepted for publication July 20, 1992. Abstract 0 Two new series of asymetrically substituted 3,3'-bispyridinium monooximes bridged by oxopropane and propane groups were synthesized and characterized by spectral data and acid dissociation constants (pKs). Both the in vitro reactivation potency, in experiments with lyophilized electric eel acetylcholinesterase (AChE) inhibited by diisopropylfluorophosphate, and in vivo protection efficacy against diisopropylfluorophosphate intoxication in mice of these compounds were evaluated and compared with those of trimedoxime and 2-pyridinealdoxime methiodide. The compounds were also evaluated for in vitro inhibition of AChE. The compounds with the oxopropane link were stronger inhibitors and weaker reactivators than the corresponding propane derivatives.No significantcorrelation was observed among pK,, oxime inhibition of AChE, reactivation of inhibited AChE, and protection index. Changing substituents in pyridine rings or altering linking groups between pyridine rings did not improve antidotal efficacy compared with trimedoxime and 2-pyridine-aldoximemethiodide.
The toxicity of organophosphorouscompounds arises from the inhibition of acetylcholinesterase (AChE, EC 3.1.1.7).1 The antidotal effect of quaternary pyridine aldoximes is due to their capacityto displace the phosphoryl residue from the active site of the inhibited enzyme and restore the enzymatic activity.2.3 Numerous attempts have been made to improve the antidotal eacaCy of mono- and bis-pyridinium oximes by changing the central chain and substituent position in pyridine rings.a Significant information is available on 2,4'-, 2,3'-, and 4,4'substituted derivatives of bis-pyridinium monooximes,7~8but there is a paucity of literature on systematic studies on 3,3'substituted derivatives. Therefore,it was considered worthwhile to investigate the biological activities of these compounds. The reactivating potency of 3,3'-bis-pyridinium monooximes towards AChE that had been inhibited by diisopropylfluorophosphate (DFP) was determined. Acid dissociation constants (pKJ with respect to the hydroxyiminomethyl group of all the compounds were also determined to look for an effective reactivator within the optimal pK, range.9 We also evaluated the protective efficacyof these oximes in mice against DFP intoxication measured in terms of protection index (P.I.);the P.I. values of the oximes were compared with those of trimedoxime (TMB-4)and 2-pyridine-aldoxime methiodide (2-PAM). For these purposes, 23 compounds were prepared, and elemental analysis, and IR and NMR spectral data were used to confirm their structures.
Experimental Section Melting points are uncorrected. IR and NMR spectra were recorded on Perkin-Elmer 577 and JEOL FX 9OQ spectrometers, respectively; chemical shifts (6 units) are reported in parts per million (ppm) down field from tetramethylsilane (TMS) or sodium 2,2-dimethyl-2silapentane-5-sulfonate (DSS). Mass spectra were recorded on a JEOL DX-300 spectrometer. The W spectra (A, i n nm) were measured in triple-distilled water on a Shimadzu W-vis spectrophotometer. The compounds synthesized were purified by flash chromatographylo on cellulose powder and their purity was checked by 258 I Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
thin-layer chromatography (TLC, cellulose, DS-0, Fluka), with 1-butanol:a acetic acidwater (3:l:l) as the solvent system, and also by high-performance liquid chromatography, with a Waters C18 kBP column, a mobile phase comprised of acetonitrile and water (20:80) brought to pH 3.4 with 0.01 M heptanesulfonic acid, a flow rate of 1.5 mL/min, and W detection at 254 nm. Chemicals-Electric eel AChE (EC 3.1.1.7), 5,5'-dithiobis-(2nitrobenzoicacid) (DTNB),acetylthimholine iodide,3-W-morpholinopropane)sulfonicacid (MOPS),and Tris were purchased from Sigma Chemi d s Company (St. Louis, MO). Nicotinamide, isonicotinamide,N-methylnicotinamide, and 3-acetylpyridine were obtained from Aldrich Chemical Company, Milwaukee, WI.The solvents used in the syntheses were puritied and dried by standard procedures.11 Syntheses-The intermediate compounds, N-substituted nicotinamides and esters of nicotinic acid, were prepared by a previously described procedure.12 1-Chloro-l'-[3-(hydroxyiminomethyl)-pyridiniuml-2-oxopropane Chloride (2a)-To a solution of 10.16 g (0.08 mol) of 1,3dichloroacetone in 20 mL of acetone was added to a solution of 2.5 g (0.02 mol) of 3-pyridine-carboxaldoximein acetone (150 ml) in a dropwise manner over a period of 2 h at room temperature with constant stirring. The reaction mixture was stirred for an additional 24 h at room temperature. The precipitate formed was filtered and washed thoroughly with acetone. Recrystallization from methano1:acetone (40:60), after decolorizing with charcoal gave 2.5 g (49%)of 2a, mp, 196 "C (decomp);IR (KBr):3200-2700,1750,1600,1400, and 980 em-'; 'HNMR (DMSO-d,): 6 12.5 (8,lH, OH), 9.34.1 (m, 4H, Py), 8.35 (s, l H , CH = NOH), and 6.0 (8,2H, CH,), 4.8 (s, 2H, CH,). A n a L C a l c d for C,H,,N,O,Cl,: C, 43.37; H, 4.02; N, 11.24. Found: C, 43.10; H, 4.05; N, 10.93. 1-[3-(Hydroxyiminomethyl)-pyridiniuml-l'-[3'-(carbcyclohexyloxy)-pyridiniuml-2-oxopropaneDichloride (3g)-A mixture of 1.25 g (5 mmol) of 2a and 3.07 g (15 mmol) of cyclohexylnicotinate i n methanol (15mL) was refluxed i n a water bath for 20 h. The progress of the reaction was monitored by TLC. After the disappearance of the intermediate 2a, the reaction mixture was allowed to stand overnight at room temperature. The product formed on trituration with acetone was thoroughly washed with hot acetone. Recrystallization from acetone:methanol (8:2) after decolorizing with charcoal gave 1.2 g (52.8%)of 3g, mp, 195 "C (decomp);IR (KBr):3400-2900,1760,1140, and 1020 cm-'; 'H NMR (DMSO-d,): 6 12.4 (s, lH, OH), 9.50-8.76 (m, 4H, Py), 8.35 (s, l H , CH = NOH), 8.32-8.10 (m, 4H, Py),6.2 (br s, 4H, ~ x C H ~3.7-3.1 ), (m, lH, CeH,,), 2.0-1.2 (m, lOH, C,H11)Anal.-Calcd for C,,H2,N,O4Cl,: C, 55.51; H, 5.51; N, 9.25. Found: C, 55.26; H, 5.43; N, 9.01. All other compounds listed i n Table I were similarly prepared and characterized. 1-Bromo-l'-~3-(hydroxyiminomethyl)-pyridiniuml-propane Bromide (2b)-The procedure used was essentially that of Hagedorn et al.,13 with pyridine-3-carboxaldoximeand 1,3-dibromopropane in the molar ratio 1:3. 2b was synthesized in a yield of 77.2%, mp, 146-147 "C (lit.13 141 "C);IR (KBr): 3180, 1630, and 980 cm-'; 'H NMR (D,O): 6 9.22-9.10 (m, 2H, Py), 8.38 (6, lH, CH = NOH), 8.3-8.15 (m, 2H, Py), 4.96 (t, 2H, J = 7 Hz, N*CH,), 3.65 (t, 2H, J = 7 Hz, CH,), and 2.78-2.41 (m, 2H, CH,). 1-~3-(hydroxyiminomethyl)-pyridiniuml-1 '-[33'-(N-methylcarboxamido)-pyridiniuml-propane Dibromide (3e')-A solution of 3.24 g (0.01 mol) of 2b and 4.08 g (0.03 mol) of N-methylnicotinamide in methanol (25 mL) was refluxed for 16 h. The progress of the reaction was monitored by TLC. After refluxing for 16 h, the solvent was 0022-3549/93/0300-0258$02.50/0 0 1993, American Pharmaceutical Association
Table I-Selected
Physlcochemlcal Data for 3,3'-Bls-pyrldlnlum Monooxlmes of 2-Oxopropane Bridge Derivatives
0
Compound
3a 3b 3c 3d 3e 3f 3g 3h
Substituent (Y)
mp, 'Ca
Yield, %
180 190 200 21 0 210 230 195 206
26.8 32.3 51.3 56.7 46.4 16.5 52.7 26.8
CONH, CONHCH CONH Bu' CONHC5H9 CONHC6Hll CONHC,H, C02C6H1
1
CH=NOH
Molecular Formulab C15H16N403C12 cl gH1 8N403C12
C19H24N403C12
C20H24N403C12 cP1 H26N,03C12
c Z l H20N403C12 c Z l H25N304C12 cl 5H1 6N403C12
UV Spectral Data
PKa
max, nm
E,,,~x 104c
387 226 226 224 226 250 383 228
0.41 1.94 2.05 3.15 2.05 2.25 0.42 3.10
9.03 9.06 9.08 9.0 9.08 9.03 9.24 9.05
a Decomposition mp. All compounds were analyzed for C, H, and N; analytical results were within +0.4%of the theoretical values except for 3d (N: calcd, 12.76;found, 12.23). Molar absorptivity at maximum absorption; M-l . cm-l.
evaporated and the residue was triturated with acetone. The product obtained was crystallized from ethanolacetone (8:2), which afforded 4.3 g (93.4%), mp, 211-212 "C (decomp); IR (KBr): 3600-3000, 1600, and 1000 cm-l; 'H NMR (DMSO-4): 6 12.3 ( 8 , lH, OH), 9.50-8.76 (m, 4H, Py), 8.31 (8, lH, CH = NOH), 8.30-8.16 (m, 4H, Py), 4.9-4.5 (m, 4H, N+CH2), 2.9-2.4 (m, 5H, CH,, CH,). Anal.-Cdcd for C1,H2,N,O,Br,: C, 41.74; H, 4.35: N, 12.17. Found C, 41.40; H, 4.43, N, 12.18. Following the above-mentioned procedure, other compounds (Table 11) were synthesized as dibromide salts. Some compounds were prepared as iodide salts by addition of sodium iodide (3.75 g, 0.025 moll to the reaction mixture prior to reflux. Determination of PK, Values-The PK, valuea of the hyhxyiminomethyl group were determined spectrophotometricallyby the method of Albert and Sargentl4 in phosphate buffer (pH 7.0-8.21, Tris buffer (pH 7.2-9.0) and glycine-NaOH buffer (pH 8.6-10.4). Aliquota (0.5 mL) of aqueous oxime solution (5 x M) were diluted to 10 mL in buffer at 10 individual pH valuea, and optical densities were determined at a n analytical wavelength with buffer blank at 25 f 0.1 "C. The average value of the 10 meamrementa was considered the PK, of the cumpound. Of all compoundsinvestijpted, the values of bis-pyridinum monooximea of propane and 2-oxopropane bridge groups were in the ranges 8.30-9.14 and 9.0-9.24, respectively (Tablea I and 11). Reactivation of DFP-Inhibited AChElS--AChE that was initially inhibited with DFP was treated with reactivators (inhibited enzyme M reactivator) and then the percentage incubated with 1.5 x reactivation was determined. Inhibition cocMail(2 mL) contained 40 pL
of stockAChE (1350unitslmg of protein dissolved in 1mL of 0.1 M MOPS buffer, pH 7.6)and 10 pL of DFP in0.l M MOPS buffer (pH 7.6).The final concentration of DFP in the medium was 1.25 x M; at this concentration, 99% enzyme activity was inhibited after 10 min of incubation at 25°C. The concentration of DFP in the medium was determined by titratingthe enzyme with different concentrations of DFP. A 1-mLaliquot of inhibition cocktailwas kept aside to study spontaneous reactivation (1mL of MOPS buffer was added to obtain a final volume of 2 mL). Another 1 mL was reserved to study reactivation and a final volume of 2 mL was made by addition of different reactivators (final concentration 1.5 x lo-' M)and MOPS buffer. Enzyme activity was determined in 5-pL aliquota taken from the incubation cocktail at 0 min (i.e., before addition of DF'P), at 10 min aRer incubation with DFP,and at 2,10,20, and 60 min after incubation with or without reactivator. No spontaneous reactivation was observed even after 1 h of incubation following 10 min of inhibition of enzyme with DFP. In another set of experiments, enzyme inhibition with reactivator was studied under similar conditions, keeping enzyme and reactivator concentration the same as above. All assays were d e d out in triplicate. Percentage reactivation was calculated with the following equation:
E, - Ei x 100 Eo - Ei In eq 1, E, is the control enzyme activity at 0 min, Ei is the inhibited
Table 11-Selected Physlcochemlcal Data for 3,3'-Bls-pyrldlnlum Monooxlmes of Propane Bridge Derivatives
Compound
3a' 3b' 3c' 3d' 3e' 31' 3g' 3h' 31' 31' 3k 31' 3m' 3n' 30Ie
(Y) CONH, CONH, CONHCH, CONHCSHg CONHC& 1 CONHC& COCH, C02CH3 COzCH3 COzC3H,' C0&6H1
1
COC,H, CH=NOH CH3 CH3
Position
'X
mp, "Ca
Yield, %
3 4 3 3 3 3 3 3 3 3 3 3 3 3 4
Br Br
21 9 239 211-212 199 237 240 180 196197 197 176179 175 205-208 230-231 218-220 200
72.6 90.0 53.8 70.0 75.8 63.0 65.0 65.0 80.2 52.9 70.0 80.0 75.0 68.9 61 .O
Br Br Br Br
I I Br I Br I Br I I
Molecular Formulab cl 5H, eN40ZBr2
C15H18N402Br2 C16H20N402Br2
CE9H26N402Br2 c Z l H28N402Br'2 cP1 H22N402Br2 C16H19N30212 C16H19N30312 C16H19N303Br2
C18H23N30312 c Z l H2i"3°3Br2
c,
H2lN,O,I,
C15H18N402Br2 C15H18N3012
C15H19N3012
UV Special Data PKa
8.65 9.14 8.78 8.71 8.55 8.42 9.13 8.84 9.14 8.30 8.65 8.31 8.74 8.43 8.25
max, nm
Em, x lo4
258 257 256 256 255 258 258 257 257 -d 255 261 253 259 200
1.21 3.84 4.06 1.38 1.97 1.97 1.15 1.19 3.84 1.38 1.84 1.63 1.22 1.95
a Decomposition mp. *All compounds were analyzed for C, H, and N; analytical results were within f0.4% of the theoretical values except for 3a' and 3h'; 3a': calcd for C, 40.36;found, 39.80,3hf calcd for C, 34.59;found, 35.16. See Table I,footnote c. '-, Not determined. Compound with oxime group at 4-position.
Journal of PharmaceuticalSciences I259 Voi. 82, No. 3,March 1993
enzyme activity, and E, is the activity of inhibited enzyme after incubation with the oxime test compounds. Reversible AChE Inhibition-A reaction cocktail for inhibition studiesconsisted of20 pL of AChE (1350unitdmg protein)and different concentrations of reactivators in a total volume of 2 mL made up with MOPS buffer (0.1 M, pH 7.6). Following 10 min of incubation at 25 "C, a suitableamount was taken out to assay AChE activity as discussed in the following section. Attempts were made to determine IC,, values (concentration in the incubation medium required to inhibit 50% enzyme activity by reactivators) of reactivators. Propane-bridgedcompounds did not significantly inhibit AChE activity even at a reactivator concentration of 2 x lo-' M in the incubation medium. The results are presented as percentage of inhibition of enzyme at a reactivator concentration 2 x lo-' M (Tables I11 and M. Assay of AChE-AChE was assayed by the method of Ellman et al.16 A typical incubation mixture contained 1.89 mL of phosphate buffer (0.1 M, pH 8.0), 0.06 mL of DTNB (0.01 M prepared in 0.1 M phosphate buffer, pH 7.0), and 0.005 mL of enzyme and was incubated at 25 "Cfor 30 s in a constant-temperaturecell holder of a Shimadzu UV-vis spedrophotometer. The enzyme reaction was started by addition of 0.04 mL of acetylthiocholine (7.5 x lo-' M, prepared in 0.1 M phosphate buffer, pH 8.01, and the rate of reaction was studied. Enzyme activity was calculated assuming the molar extinction coefficient of the product formed was 13 600 M-' cm-'. Protection Studies-The P.I. was determined in mice as already described.16
-
Results and Discussion The compounds were prepared by the general synthetic route shown in Scheme I. Tables I and I1 provide structures and selected physiocochemical data of 2-oxopropane and propane bridge compounds. All oximes were obtained configurationally pure as evidencedby 'H NMR spectra (taken in DMSO-4). They exhibited chemical shift values for the oxime hydroxyl proton between 6 12.25 and 12.60 at different dilutions, and the positionsof the oximemethine proton ranged from 6 8.18 to 8.41. The chemical shifts difference between them are in the range and its 3.85-3.99. This suggeststhat pyridine-3-carboxaldoxime quaternary salts possess an E configuration.17-20 This is also in agreement with the higher acidities observed for these compounds (pK, 8.30-9.24).21,22 The results given in Table I11show that some of the oxopropane compounds in combinationwith atropine are effective in protedTable Ill-lnhlbition of AChE by Reactivators: In Vitro Reactivation Effect and In Vlvo Protection Efficacy of 2-Oxopropane Bridge Series
Inhibition at
2 x 10-3 M
3a 3b
3c 3d
3e 3f
3g 3h
43.0 55.0 57.2 71.8 68.2 85.0 25.0 60.0
IC50,
10-4
~a
-C 2.92 3.38 2.50 2.29 0.93 -C
1.50 ~~~~
P.I. in Mice Percent Reactivation (DFP) 17.0' NSe NS
NS NS
-f NS -f
4.0 3.60 7.18 6.40 9.54 5.36 7.18 4.53 ~
For compoundswhose percentage of inhibition is >50% at 2 x M, IC,, values were calculated by plotting percentage of enzyme inhibition versus log concentration of oximes. Male albino mice were used. Subcutaneous LD,, of DFP was determined. Reactivator (0.1 mmol/kg) was injected intramuscularlyfollowing intraperitoneal injection of atropine (10mg/kg) 30 s after DFP treatment"? P.I. = LD,, of poison with oxime/LD,, of poison. 'Attempts were not made to determine IC,, values of compounds because still higher concentration was required. 'The in vitro percents reactivation after shorter incubation periods of 2 , 10, and 20 min were 6.1,6.1,and 8.3,respectively. NS,not significant (p 15%).'Not determined. a
260 / Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
ing against DFP intoxication in mice, but are ineffective as reactivators of inhibited AChE, as indicated by the 60-min, in vitro percent reactivation data; good reactivating ability however, is exhibited by the compounds with a propane chain (Table IV).Some selected compounds were further screened for percent reactivationafter shorter reactivation times of 2,10, and 20 min. The compounds reactivated DFP-inhibited AChE even after 2 min of incubation;the percent reactivation increasedwith longer incubation times, exceptfor 30' and TMB-4. In the case of TMB-4, the decrease in percent reactivation with increasing incubation period can be explained by the formation of a rather stable phosphorylated 0xime23.24 that can rephosphorylatethe enzyme. The phosphorylatedoxime results from the nucleophilicdisplacement reaction on the phosphorylated AChE by the pyridinium oxime during the process of reactivation, whereas such intermediates break down quickly in most other 0xirnes.a The results show (Table IV)that even though 3c', 3d', 3e', 3f', and 31' are good protective agents (indicated by high in vivo P.I. values), they do not exhibit significant in vitro reactivation activity. The AChE inhibition values of 2-0x0propane chain derivatives of oximes are in most instances higher than those of corresponding propane chain-bridged compounds, indicating that 2-oxopropane derivatives are stronger inhibitors. Again, the nature of substituents (3b versus 3g of the oxopropane series) and substituent position (3a' versus 3b' of the propane series) on the second pyridinium ring significantly influence in reversing AChE inhibition values of the reactivator. The structure and properties of the central chain have some significance for better reactivators and weak inhibitors of AChE. No clear relationship among anti-AChE activity, reactivating potency, and P.I. in the synthesized compounds studied can be found; to establish a clear relationship among chemical constitution, reactivating power, and therapeutic efficiency of oximes is very difficult as was also observed in other compounds.25-26 The dephosphorylation of phosphorylated enzyme is dependent on various factors, such as physicochemicalproperties of the reactivating agent and including steric and electronic factors, lipophilic-hydrophilic balance, pK,, etc.27 Readivation of DFP-inhibited AChE is a nucleophilic displacement reaction on the phosphorus atom by the anion of a reactivator. The pK, of a n oxime is a measure of both the supply and the basicity of the anion and is of prime importance for its antidotal efficacy in contributing sufficient amount of the ionized form of oximes at physiological pH.2S.29 In the series of pyridinium oximes, the optimum pK, values for a reactivator to be effective are in the range -7.5-8.0,7-9although the anion would be certainly available at a pK, near the optimum value but might be a weak nucleophile. The evaluated pK, values of the present series of compounds lie above the optimum value, but nevertheless show significant antidotal efficacy in the propane link series of compounds. Obviously, optimal pKa is a requirement for effective reactivation, but not an absolute necessity. This has also been observed in some excellent antidotes such as HI-6 (1-[2-(hydroxyiminomethyl)pyridinium] - 1'- [4 '- (carboxamid0)-pyridinium]-2-oxapropane dichloride; pK,, 7.2), HS-6 (1-[2-(hydroxyiminomethyl)pyridinium)-1'-[3'-(carboxamido)-pyridiniuml-2-oxapropane dichloride; pK,, 7.3), and phenyl hydroxyiminomethyl quinolinium compounds (pK, 10.4).27.3032 It can be concluded that AChE inhibition by oxime, reactivation of DFP-inhibited AChE, and protection studies of the present series of compounds could not be correlated with their pK, values as has been reported earlier with different classes of reactivators. The oxopropane derivatives are more toxic and less effective in reactivating the inhibited or phosphorylated enzyme activity compared with propane derivatives. Changing the substituent positions or groups on one of the pyridine rings and modifying the linking aliphatic three-
Table IV-inhibltlon Compounds
of AChE by Reactivators: In Vltro Reactivation Effect and in Vivo Protection Efficacy of Propane Bridge Series
-
Percent Reactivationb
InhihitinnI a at 3 v ,L .I ,I llYlll",
Compound
10-3 M"
3a' 3b' 3C' 3d' 36' 3f' 3g' 3h' 31' 3J' 3k 31' 3m' 3n' 30' 2-PAM TMB-4
15.0 36.7 33.2 19.0 36.5 33.0 27.8 25.0 27.2 39.0 32.8 28.1 15.8 21.9 13.6 27.1 30.5
P.I. in Mice (DFP)"
r2
rio
120
rm
18.8 19.3
21.9 26.0
27.5 30.6
41 .O 47.0
4
-
-
-
-
-
-
-
7.9 10.7 13.3 4.8
12.0 13.6 28.0 9.2
21.7 22.7 32.2 16.2
-
-
17.5 10.8 58.8 23.0 71.4
21.1 13.7 61.5 34.6 62.2
-
-
36.3 21.2 60.7 40.6 53.9
NS"
21 .o 19.0 18.5 34.7 33.8 46.0 35.0
,
NS
9.5 49.0 20.6 35.8 46.0 50.0
8.0 10.10 8.05 9.98 9.98 9.98 8.18 9.52 9.55 4.86 3.40 7.66 13.13 12.43 13.94 7.6 22.9
" Attempts were not made to determine IC, values of compounds because still higher concentration was required. * In vitro percent reactivation after incubation of DFP inhibited AChE with test compounds for 2 min (r2),10 rnin (r,o), 20 min (r2,,), and 60 min (re0)." See Table 111, footnote b. -, Not determined. NS, Not significant (p 55%).
20 : 2
.-*-, -L, x . x a CI
zb : 2 =
Reflux
flcH -0' N
X@
s
CH~ZCHZ
N *2X0
3
11. Vogel, I. In Text Book of Practical Organic Chemistry; Longman: London, U.K., 1951; p 161. 12. Budgett, C. 0.;Raymond, C.; Provostclyde, L. 0.; Woodward, C. F. J. Am. Chem. SOC. 1945,67, 1135. 13. Heedorn. I.: Stark. I.: Schoene. K.: Schenkel. H. Aneim.Fo&h.lDrug'Res. 1978,'28, 2055.' ' 14. Albert, A.; Sargent, E. P. In Ionization Constants of Acids and Bases; John Wiley: New York, NY, 1962; p 69. 15. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88. 16. Das Gupta, S.; Ghosh, A. K.; Chowdhri, B. L.; Asthma, S. N.; Batra, B. S. Drug Chem. Toxicol. 1991, 14, 283. 17. Kleinspehn, G. G.; Jung, J. A.; Studniarz, S. A. J. Org. Chem. 1967.32. , --,460. 18. Bedford, C. D.; Harris, R. N.; Howd, R. A.; Kinley, R. A.; Miller, A.; Noeln, H. W. J. Med. Chem. 1984,27, 1431. 19. Grifaniti, M.; Merklli, S.; Stein, M. L. J. Pharm. Sci. 1972,61,631. 20. Forman, S. E. J. Org. Chem. 1964,29, 3323. 1957, 79, 481. 21. Ginsburg, S.;Wilson, I. B. J. Am. Chem. SOC. 22. Poziomek, E. J.; Kramer, D. N.; Mosher, W. A.; Michel, H. 0. J. Am. Chem. Soc. 1961.83.3916. 23. Hackley, B. E., Jr.; Steinberg, G. M.; Lamb, J. C. Arch. Biochem. Biophys. 1959,80, 211. 24. Schoene, K. In Medical Protection Against Chemical Warfare Agents; Almqvist & Wikeell: Sweden, 1976; p 88. 25. Maskimovic, M.; Bregoven, I.; Deljac, V.; Binenfeld, Z. Arch. Toxicol. 1984,55, 199. 26. Binenfeld, Z.; Deljac, V.; Knezevic, M.; Pavlov, L. J.; Maksimovic, M.; Markov, V.; Radovic, L. J.; Rakin, D. Acta Pharm. Jugosl. 1981, 31, 5. 27. Gray, A. P. Drug Metab. Rev. 1984,15, 557. 28. Ashani, J.; Cohen, S. J. Med. Chem. 1970, 13, 471. 29. Grifantini, M.; Martelli, S.; Stein, M. L. J.P h r m . Sci. 1969,58,460. 30. Granov, A.; Maksimovic, M.; Binenfeld, Z. Nauchno-Tech. Pregled Nr. 2 1984.34. 34. 31. Binenfeld, Z.; Boskovic, B.; Rakin, D.; Cosic, M. Acta Pharm. Jugosl. 1971,21, 113. 32. Bregovec, I.; Maksimovic, M.; Deljac, A.; Binenfeld, Z. Acta Phann. Jugosl. 1986,36, 9. ~~
- -G, -*-, 0
13a-sl : 2 13d-3n'): 2
x=u
I
X = B ~ W I
Scheme I atoms chain also did not appreciably improve the antidotal properties compared with those of TMB-4 and 2-PAM.
References and Notes 1. Eto, M. In Organophosphorous Pesticides: Organic and Biological Chemistry; CRC: New York, NY, 1974; p 123. 2. Hobbiger, F. In Handbuck Der Experimentellen Pharmakologie; Koelle, G. B. Ed.; Springer-Verlag: Berlin, Germany, 1963; Vol. 15, p 921. 3. Wills, J. H. Int. Encycl. Pharmacol. Ther. 1970, I, 357. 4. Binenfeld, T.; Kamenar, B.; Vickovic, I. Acta. Pharm. Jugosl. 1984,34. 195. 5. Bev&dic, Z. D.; Deljac, A.; Maksimovic, M.; Boskovic, B.; Binenfeld, T. Acta. Pharm. Jugosl. 1985,35, 213. 6. Deljac, V.; Maksimovic, M.; Radovic, L.; Rakin, D.; Markov, V.; Bnegovee, I.; Binenfeld, 2.Arch. Toxtcol. 1982,49, 285. I.; Stark, I.; Lorenz, H. P. Angew Chem. Znt. Ed. 1972, 7. Hagdorn, "r." 1 -
11,
avi.
8. Schoene, K.; Strake, E. M. Biochem. Pharmacol. 1971,20,2527. 9. Engelhard, N.; Erdmann, N. Arzneim.-Forsch. 1964,14, 870. 10. Clark Still, W.; Khan, M.; Mitra, A. J. Org. Chem. 1978,43,2923.
Acknowledgments The authors thank Dr. R. V. Swam (Director Defence Research and Development Establishment, G w d o r ) for his keen interest in the work, Dr. P. K. Ramachandran (Emeritus Scientist) and Dr. B. K. Bhattacharya for their helpful discussions, Mr. R. P. Semwal, Mr. Basant Lal, Mr. L. R. Chauhan, Mr. C. D. Raghuveeran, and Mr. R. S. Dangi for their help in the experimental work, and Mr. Mitthan Lal for his secretarial assistance.
Journal of Pharmaceutical Sciences I 261 Vol. 82, No. 3, March 1993
The toxicity of organophosphorouscompounds arises from the inhibition of acetylcholinesterase (AChE, EC 3.1.1.7).1 The antidotal effect of quaternary pyridine aldoximes is due to their capacityto displace the phosphoryl residue from the active site of the inhibited enzyme and restore the enzymatic activity.2.3 Numerous attempts have been made to improve the antidotal eacaCy of mono- and bis-pyridinium oximes by changing the central chain and substituent position in pyridine rings.a Significant information is available on 2,4'-, 2,3'-, and 4,4'substituted derivatives of bis-pyridinium monooximes,7~8but there is a paucity of literature on systematic studies on 3,3'substituted derivatives. Therefore,it was considered worthwhile to investigate the biological activities of these compounds. The reactivating potency of 3,3'-bis-pyridinium monooximes towards AChE that had been inhibited by diisopropylfluorophosphate (DFP) was determined. Acid dissociation constants (pKJ with respect to the hydroxyiminomethyl group of all the compounds were also determined to look for an effective reactivator within the optimal pK, range.9 We also evaluated the protective efficacyof these oximes in mice against DFP intoxication measured in terms of protection index (P.I.);the P.I. values of the oximes were compared with those of trimedoxime (TMB-4)and 2-pyridine-aldoxime methiodide (2-PAM). For these purposes, 23 compounds were prepared, and elemental analysis, and IR and NMR spectral data were used to confirm their structures.
Experimental Section Melting points are uncorrected. IR and NMR spectra were recorded on Perkin-Elmer 577 and JEOL FX 9OQ spectrometers, respectively; chemical shifts (6 units) are reported in parts per million (ppm) down field from tetramethylsilane (TMS) or sodium 2,2-dimethyl-2silapentane-5-sulfonate (DSS). Mass spectra were recorded on a JEOL DX-300 spectrometer. The W spectra (A, i n nm) were measured in triple-distilled water on a Shimadzu W-vis spectrophotometer. The compounds synthesized were purified by flash chromatographylo on cellulose powder and their purity was checked by 258 I Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993
thin-layer chromatography (TLC, cellulose, DS-0, Fluka), with 1-butanol:a acetic acidwater (3:l:l) as the solvent system, and also by high-performance liquid chromatography, with a Waters C18 kBP column, a mobile phase comprised of acetonitrile and water (20:80) brought to pH 3.4 with 0.01 M heptanesulfonic acid, a flow rate of 1.5 mL/min, and W detection at 254 nm. Chemicals-Electric eel AChE (EC 3.1.1.7), 5,5'-dithiobis-(2nitrobenzoicacid) (DTNB),acetylthimholine iodide,3-W-morpholinopropane)sulfonicacid (MOPS),and Tris were purchased from Sigma Chemi d s Company (St. Louis, MO). Nicotinamide, isonicotinamide,N-methylnicotinamide, and 3-acetylpyridine were obtained from Aldrich Chemical Company, Milwaukee, WI.The solvents used in the syntheses were puritied and dried by standard procedures.11 Syntheses-The intermediate compounds, N-substituted nicotinamides and esters of nicotinic acid, were prepared by a previously described procedure.12 1-Chloro-l'-[3-(hydroxyiminomethyl)-pyridiniuml-2-oxopropane Chloride (2a)-To a solution of 10.16 g (0.08 mol) of 1,3dichloroacetone in 20 mL of acetone was added to a solution of 2.5 g (0.02 mol) of 3-pyridine-carboxaldoximein acetone (150 ml) in a dropwise manner over a period of 2 h at room temperature with constant stirring. The reaction mixture was stirred for an additional 24 h at room temperature. The precipitate formed was filtered and washed thoroughly with acetone. Recrystallization from methano1:acetone (40:60), after decolorizing with charcoal gave 2.5 g (49%)of 2a, mp, 196 "C (decomp);IR (KBr):3200-2700,1750,1600,1400, and 980 em-'; 'HNMR (DMSO-d,): 6 12.5 (8,lH, OH), 9.34.1 (m, 4H, Py), 8.35 (s, l H , CH = NOH), and 6.0 (8,2H, CH,), 4.8 (s, 2H, CH,). A n a L C a l c d for C,H,,N,O,Cl,: C, 43.37; H, 4.02; N, 11.24. Found: C, 43.10; H, 4.05; N, 10.93. 1-[3-(Hydroxyiminomethyl)-pyridiniuml-l'-[3'-(carbcyclohexyloxy)-pyridiniuml-2-oxopropaneDichloride (3g)-A mixture of 1.25 g (5 mmol) of 2a and 3.07 g (15 mmol) of cyclohexylnicotinate i n methanol (15mL) was refluxed i n a water bath for 20 h. The progress of the reaction was monitored by TLC. After the disappearance of the intermediate 2a, the reaction mixture was allowed to stand overnight at room temperature. The product formed on trituration with acetone was thoroughly washed with hot acetone. Recrystallization from acetone:methanol (8:2) after decolorizing with charcoal gave 1.2 g (52.8%)of 3g, mp, 195 "C (decomp);IR (KBr):3400-2900,1760,1140, and 1020 cm-'; 'H NMR (DMSO-d,): 6 12.4 (s, lH, OH), 9.50-8.76 (m, 4H, Py), 8.35 (s, l H , CH = NOH), 8.32-8.10 (m, 4H, Py),6.2 (br s, 4H, ~ x C H ~3.7-3.1 ), (m, lH, CeH,,), 2.0-1.2 (m, lOH, C,H11)Anal.-Calcd for C,,H2,N,O4Cl,: C, 55.51; H, 5.51; N, 9.25. Found: C, 55.26; H, 5.43; N, 9.01. All other compounds listed i n Table I were similarly prepared and characterized. 1-Bromo-l'-~3-(hydroxyiminomethyl)-pyridiniuml-propane Bromide (2b)-The procedure used was essentially that of Hagedorn et al.,13 with pyridine-3-carboxaldoximeand 1,3-dibromopropane in the molar ratio 1:3. 2b was synthesized in a yield of 77.2%, mp, 146-147 "C (lit.13 141 "C);IR (KBr): 3180, 1630, and 980 cm-'; 'H NMR (D,O): 6 9.22-9.10 (m, 2H, Py), 8.38 (6, lH, CH = NOH), 8.3-8.15 (m, 2H, Py), 4.96 (t, 2H, J = 7 Hz, N*CH,), 3.65 (t, 2H, J = 7 Hz, CH,), and 2.78-2.41 (m, 2H, CH,). 1-~3-(hydroxyiminomethyl)-pyridiniuml-1 '-[33'-(N-methylcarboxamido)-pyridiniuml-propane Dibromide (3e')-A solution of 3.24 g (0.01 mol) of 2b and 4.08 g (0.03 mol) of N-methylnicotinamide in methanol (25 mL) was refluxed for 16 h. The progress of the reaction was monitored by TLC. After refluxing for 16 h, the solvent was 0022-3549/93/0300-0258$02.50/0 0 1993, American Pharmaceutical Association
Table I-Selected
Physlcochemlcal Data for 3,3'-Bls-pyrldlnlum Monooxlmes of 2-Oxopropane Bridge Derivatives
0
Compound
3a 3b 3c 3d 3e 3f 3g 3h
Substituent (Y)
mp, 'Ca
Yield, %
180 190 200 21 0 210 230 195 206
26.8 32.3 51.3 56.7 46.4 16.5 52.7 26.8
CONH, CONHCH CONH Bu' CONHC5H9 CONHC6Hll CONHC,H, C02C6H1
1
CH=NOH
Molecular Formulab C15H16N403C12 cl gH1 8N403C12
C19H24N403C12
C20H24N403C12 cP1 H26N,03C12
c Z l H20N403C12 c Z l H25N304C12 cl 5H1 6N403C12
UV Spectral Data
PKa
max, nm
E,,,~x 104c
387 226 226 224 226 250 383 228
0.41 1.94 2.05 3.15 2.05 2.25 0.42 3.10
9.03 9.06 9.08 9.0 9.08 9.03 9.24 9.05
a Decomposition mp. All compounds were analyzed for C, H, and N; analytical results were within +0.4%of the theoretical values except for 3d (N: calcd, 12.76;found, 12.23). Molar absorptivity at maximum absorption; M-l . cm-l.
evaporated and the residue was triturated with acetone. The product obtained was crystallized from ethanolacetone (8:2), which afforded 4.3 g (93.4%), mp, 211-212 "C (decomp); IR (KBr): 3600-3000, 1600, and 1000 cm-l; 'H NMR (DMSO-4): 6 12.3 ( 8 , lH, OH), 9.50-8.76 (m, 4H, Py), 8.31 (8, lH, CH = NOH), 8.30-8.16 (m, 4H, Py), 4.9-4.5 (m, 4H, N+CH2), 2.9-2.4 (m, 5H, CH,, CH,). Anal.-Cdcd for C1,H2,N,O,Br,: C, 41.74; H, 4.35: N, 12.17. Found C, 41.40; H, 4.43, N, 12.18. Following the above-mentioned procedure, other compounds (Table 11) were synthesized as dibromide salts. Some compounds were prepared as iodide salts by addition of sodium iodide (3.75 g, 0.025 moll to the reaction mixture prior to reflux. Determination of PK, Values-The PK, valuea of the hyhxyiminomethyl group were determined spectrophotometricallyby the method of Albert and Sargentl4 in phosphate buffer (pH 7.0-8.21, Tris buffer (pH 7.2-9.0) and glycine-NaOH buffer (pH 8.6-10.4). Aliquota (0.5 mL) of aqueous oxime solution (5 x M) were diluted to 10 mL in buffer at 10 individual pH valuea, and optical densities were determined at a n analytical wavelength with buffer blank at 25 f 0.1 "C. The average value of the 10 meamrementa was considered the PK, of the cumpound. Of all compoundsinvestijpted, the values of bis-pyridinum monooximea of propane and 2-oxopropane bridge groups were in the ranges 8.30-9.14 and 9.0-9.24, respectively (Tablea I and 11). Reactivation of DFP-Inhibited AChElS--AChE that was initially inhibited with DFP was treated with reactivators (inhibited enzyme M reactivator) and then the percentage incubated with 1.5 x reactivation was determined. Inhibition cocMail(2 mL) contained 40 pL
of stockAChE (1350unitslmg of protein dissolved in 1mL of 0.1 M MOPS buffer, pH 7.6)and 10 pL of DFP in0.l M MOPS buffer (pH 7.6).The final concentration of DFP in the medium was 1.25 x M; at this concentration, 99% enzyme activity was inhibited after 10 min of incubation at 25°C. The concentration of DFP in the medium was determined by titratingthe enzyme with different concentrations of DFP. A 1-mLaliquot of inhibition cocktailwas kept aside to study spontaneous reactivation (1mL of MOPS buffer was added to obtain a final volume of 2 mL). Another 1 mL was reserved to study reactivation and a final volume of 2 mL was made by addition of different reactivators (final concentration 1.5 x lo-' M)and MOPS buffer. Enzyme activity was determined in 5-pL aliquota taken from the incubation cocktail at 0 min (i.e., before addition of DF'P), at 10 min aRer incubation with DFP,and at 2,10,20, and 60 min after incubation with or without reactivator. No spontaneous reactivation was observed even after 1 h of incubation following 10 min of inhibition of enzyme with DFP. In another set of experiments, enzyme inhibition with reactivator was studied under similar conditions, keeping enzyme and reactivator concentration the same as above. All assays were d e d out in triplicate. Percentage reactivation was calculated with the following equation:
E, - Ei x 100 Eo - Ei In eq 1, E, is the control enzyme activity at 0 min, Ei is the inhibited
Table 11-Selected Physlcochemlcal Data for 3,3'-Bls-pyrldlnlum Monooxlmes of Propane Bridge Derivatives
Compound
3a' 3b' 3c' 3d' 3e' 31' 3g' 3h' 31' 31' 3k 31' 3m' 3n' 30Ie
(Y) CONH, CONH, CONHCH, CONHCSHg CONHC& 1 CONHC& COCH, C02CH3 COzCH3 COzC3H,' C0&6H1
1
COC,H, CH=NOH CH3 CH3
Position
'X
mp, "Ca
Yield, %
3 4 3 3 3 3 3 3 3 3 3 3 3 3 4
Br Br
21 9 239 211-212 199 237 240 180 196197 197 176179 175 205-208 230-231 218-220 200
72.6 90.0 53.8 70.0 75.8 63.0 65.0 65.0 80.2 52.9 70.0 80.0 75.0 68.9 61 .O
Br Br Br Br
I I Br I Br I Br I I
Molecular Formulab cl 5H, eN40ZBr2
C15H18N402Br2 C16H20N402Br2
CE9H26N402Br2 c Z l H28N402Br'2 cP1 H22N402Br2 C16H19N30212 C16H19N30312 C16H19N303Br2
C18H23N30312 c Z l H2i"3°3Br2
c,
H2lN,O,I,
C15H18N402Br2 C15H18N3012
C15H19N3012
UV Special Data PKa
8.65 9.14 8.78 8.71 8.55 8.42 9.13 8.84 9.14 8.30 8.65 8.31 8.74 8.43 8.25
max, nm
Em, x lo4
258 257 256 256 255 258 258 257 257 -d 255 261 253 259 200
1.21 3.84 4.06 1.38 1.97 1.97 1.15 1.19 3.84 1.38 1.84 1.63 1.22 1.95
a Decomposition mp. *All compounds were analyzed for C, H, and N; analytical results were within f0.4% of the theoretical values except for 3a' and 3h'; 3a': calcd for C, 40.36;found, 39.80,3hf calcd for C, 34.59;found, 35.16. See Table I,footnote c. '-, Not determined. Compound with oxime group at 4-position.
Journal of PharmaceuticalSciences I259 Voi. 82, No. 3,March 1993
enzyme activity, and E, is the activity of inhibited enzyme after incubation with the oxime test compounds. Reversible AChE Inhibition-A reaction cocktail for inhibition studiesconsisted of20 pL of AChE (1350unitdmg protein)and different concentrations of reactivators in a total volume of 2 mL made up with MOPS buffer (0.1 M, pH 7.6). Following 10 min of incubation at 25 "C, a suitableamount was taken out to assay AChE activity as discussed in the following section. Attempts were made to determine IC,, values (concentration in the incubation medium required to inhibit 50% enzyme activity by reactivators) of reactivators. Propane-bridgedcompounds did not significantly inhibit AChE activity even at a reactivator concentration of 2 x lo-' M in the incubation medium. The results are presented as percentage of inhibition of enzyme at a reactivator concentration 2 x lo-' M (Tables I11 and M. Assay of AChE-AChE was assayed by the method of Ellman et al.16 A typical incubation mixture contained 1.89 mL of phosphate buffer (0.1 M, pH 8.0), 0.06 mL of DTNB (0.01 M prepared in 0.1 M phosphate buffer, pH 7.0), and 0.005 mL of enzyme and was incubated at 25 "Cfor 30 s in a constant-temperaturecell holder of a Shimadzu UV-vis spedrophotometer. The enzyme reaction was started by addition of 0.04 mL of acetylthiocholine (7.5 x lo-' M, prepared in 0.1 M phosphate buffer, pH 8.01, and the rate of reaction was studied. Enzyme activity was calculated assuming the molar extinction coefficient of the product formed was 13 600 M-' cm-'. Protection Studies-The P.I. was determined in mice as already described.16
-
Results and Discussion The compounds were prepared by the general synthetic route shown in Scheme I. Tables I and I1 provide structures and selected physiocochemical data of 2-oxopropane and propane bridge compounds. All oximes were obtained configurationally pure as evidencedby 'H NMR spectra (taken in DMSO-4). They exhibited chemical shift values for the oxime hydroxyl proton between 6 12.25 and 12.60 at different dilutions, and the positionsof the oximemethine proton ranged from 6 8.18 to 8.41. The chemical shifts difference between them are in the range and its 3.85-3.99. This suggeststhat pyridine-3-carboxaldoxime quaternary salts possess an E configuration.17-20 This is also in agreement with the higher acidities observed for these compounds (pK, 8.30-9.24).21,22 The results given in Table I11show that some of the oxopropane compounds in combinationwith atropine are effective in protedTable Ill-lnhlbition of AChE by Reactivators: In Vitro Reactivation Effect and In Vlvo Protection Efficacy of 2-Oxopropane Bridge Series
Inhibition at
2 x 10-3 M
3a 3b
3c 3d
3e 3f
3g 3h
43.0 55.0 57.2 71.8 68.2 85.0 25.0 60.0
IC50,
10-4
~a
-C 2.92 3.38 2.50 2.29 0.93 -C
1.50 ~~~~
P.I. in Mice Percent Reactivation (DFP) 17.0' NSe NS
NS NS
-f NS -f
4.0 3.60 7.18 6.40 9.54 5.36 7.18 4.53 ~
For compoundswhose percentage of inhibition is >50% at 2 x M, IC,, values were calculated by plotting percentage of enzyme inhibition versus log concentration of oximes. Male albino mice were used. Subcutaneous LD,, of DFP was determined. Reactivator (0.1 mmol/kg) was injected intramuscularlyfollowing intraperitoneal injection of atropine (10mg/kg) 30 s after DFP treatment"? P.I. = LD,, of poison with oxime/LD,, of poison. 'Attempts were not made to determine IC,, values of compounds because still higher concentration was required. 'The in vitro percents reactivation after shorter incubation periods of 2 , 10, and 20 min were 6.1,6.1,and 8.3,respectively. NS,not significant (p 15%).'Not determined. a
260 / Journal of Pharmaceutical Sciences Vol. 82, No. 3, March 1993
ing against DFP intoxication in mice, but are ineffective as reactivators of inhibited AChE, as indicated by the 60-min, in vitro percent reactivation data; good reactivating ability however, is exhibited by the compounds with a propane chain (Table IV).Some selected compounds were further screened for percent reactivationafter shorter reactivation times of 2,10, and 20 min. The compounds reactivated DFP-inhibited AChE even after 2 min of incubation;the percent reactivation increasedwith longer incubation times, exceptfor 30' and TMB-4. In the case of TMB-4, the decrease in percent reactivation with increasing incubation period can be explained by the formation of a rather stable phosphorylated 0xime23.24 that can rephosphorylatethe enzyme. The phosphorylatedoxime results from the nucleophilicdisplacement reaction on the phosphorylated AChE by the pyridinium oxime during the process of reactivation, whereas such intermediates break down quickly in most other 0xirnes.a The results show (Table IV)that even though 3c', 3d', 3e', 3f', and 31' are good protective agents (indicated by high in vivo P.I. values), they do not exhibit significant in vitro reactivation activity. The AChE inhibition values of 2-0x0propane chain derivatives of oximes are in most instances higher than those of corresponding propane chain-bridged compounds, indicating that 2-oxopropane derivatives are stronger inhibitors. Again, the nature of substituents (3b versus 3g of the oxopropane series) and substituent position (3a' versus 3b' of the propane series) on the second pyridinium ring significantly influence in reversing AChE inhibition values of the reactivator. The structure and properties of the central chain have some significance for better reactivators and weak inhibitors of AChE. No clear relationship among anti-AChE activity, reactivating potency, and P.I. in the synthesized compounds studied can be found; to establish a clear relationship among chemical constitution, reactivating power, and therapeutic efficiency of oximes is very difficult as was also observed in other compounds.25-26 The dephosphorylation of phosphorylated enzyme is dependent on various factors, such as physicochemicalproperties of the reactivating agent and including steric and electronic factors, lipophilic-hydrophilic balance, pK,, etc.27 Readivation of DFP-inhibited AChE is a nucleophilic displacement reaction on the phosphorus atom by the anion of a reactivator. The pK, of a n oxime is a measure of both the supply and the basicity of the anion and is of prime importance for its antidotal efficacy in contributing sufficient amount of the ionized form of oximes at physiological pH.2S.29 In the series of pyridinium oximes, the optimum pK, values for a reactivator to be effective are in the range -7.5-8.0,7-9although the anion would be certainly available at a pK, near the optimum value but might be a weak nucleophile. The evaluated pK, values of the present series of compounds lie above the optimum value, but nevertheless show significant antidotal efficacy in the propane link series of compounds. Obviously, optimal pKa is a requirement for effective reactivation, but not an absolute necessity. This has also been observed in some excellent antidotes such as HI-6 (1-[2-(hydroxyiminomethyl)pyridinium] - 1'- [4 '- (carboxamid0)-pyridinium]-2-oxapropane dichloride; pK,, 7.2), HS-6 (1-[2-(hydroxyiminomethyl)pyridinium)-1'-[3'-(carboxamido)-pyridiniuml-2-oxapropane dichloride; pK,, 7.3), and phenyl hydroxyiminomethyl quinolinium compounds (pK, 10.4).27.3032 It can be concluded that AChE inhibition by oxime, reactivation of DFP-inhibited AChE, and protection studies of the present series of compounds could not be correlated with their pK, values as has been reported earlier with different classes of reactivators. The oxopropane derivatives are more toxic and less effective in reactivating the inhibited or phosphorylated enzyme activity compared with propane derivatives. Changing the substituent positions or groups on one of the pyridine rings and modifying the linking aliphatic three-
Table IV-inhibltlon Compounds
of AChE by Reactivators: In Vltro Reactivation Effect and in Vivo Protection Efficacy of Propane Bridge Series
-
Percent Reactivationb
InhihitinnI a at 3 v ,L .I ,I llYlll",
Compound
10-3 M"
3a' 3b' 3C' 3d' 36' 3f' 3g' 3h' 31' 3J' 3k 31' 3m' 3n' 30' 2-PAM TMB-4
15.0 36.7 33.2 19.0 36.5 33.0 27.8 25.0 27.2 39.0 32.8 28.1 15.8 21.9 13.6 27.1 30.5
P.I. in Mice (DFP)"
r2
rio
120
rm
18.8 19.3
21.9 26.0
27.5 30.6
41 .O 47.0
4
-
-
-
-
-
-
-
7.9 10.7 13.3 4.8
12.0 13.6 28.0 9.2
21.7 22.7 32.2 16.2
-
-
17.5 10.8 58.8 23.0 71.4
21.1 13.7 61.5 34.6 62.2
-
-
36.3 21.2 60.7 40.6 53.9
NS"
21 .o 19.0 18.5 34.7 33.8 46.0 35.0
,
NS
9.5 49.0 20.6 35.8 46.0 50.0
8.0 10.10 8.05 9.98 9.98 9.98 8.18 9.52 9.55 4.86 3.40 7.66 13.13 12.43 13.94 7.6 22.9
" Attempts were not made to determine IC, values of compounds because still higher concentration was required. * In vitro percent reactivation after incubation of DFP inhibited AChE with test compounds for 2 min (r2),10 rnin (r,o), 20 min (r2,,), and 60 min (re0)." See Table 111, footnote b. -, Not determined. NS, Not significant (p 55%).
20 : 2
.-*-, -L, x . x a CI
zb : 2 =
Reflux
flcH -0' N
X@
s
CH~ZCHZ
N *2X0
3
11. Vogel, I. In Text Book of Practical Organic Chemistry; Longman: London, U.K., 1951; p 161. 12. Budgett, C. 0.;Raymond, C.; Provostclyde, L. 0.; Woodward, C. F. J. Am. Chem. SOC. 1945,67, 1135. 13. Heedorn. I.: Stark. I.: Schoene. K.: Schenkel. H. Aneim.Fo&h.lDrug'Res. 1978,'28, 2055.' ' 14. Albert, A.; Sargent, E. P. In Ionization Constants of Acids and Bases; John Wiley: New York, NY, 1962; p 69. 15. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88. 16. Das Gupta, S.; Ghosh, A. K.; Chowdhri, B. L.; Asthma, S. N.; Batra, B. S. Drug Chem. Toxicol. 1991, 14, 283. 17. Kleinspehn, G. G.; Jung, J. A.; Studniarz, S. A. J. Org. Chem. 1967.32. , --,460. 18. Bedford, C. D.; Harris, R. N.; Howd, R. A.; Kinley, R. A.; Miller, A.; Noeln, H. W. J. Med. Chem. 1984,27, 1431. 19. Grifaniti, M.; Merklli, S.; Stein, M. L. J. Pharm. Sci. 1972,61,631. 20. Forman, S. E. J. Org. Chem. 1964,29, 3323. 1957, 79, 481. 21. Ginsburg, S.;Wilson, I. B. J. Am. Chem. SOC. 22. Poziomek, E. J.; Kramer, D. N.; Mosher, W. A.; Michel, H. 0. J. Am. Chem. Soc. 1961.83.3916. 23. Hackley, B. E., Jr.; Steinberg, G. M.; Lamb, J. C. Arch. Biochem. Biophys. 1959,80, 211. 24. Schoene, K. In Medical Protection Against Chemical Warfare Agents; Almqvist & Wikeell: Sweden, 1976; p 88. 25. Maskimovic, M.; Bregoven, I.; Deljac, V.; Binenfeld, Z. Arch. Toxicol. 1984,55, 199. 26. Binenfeld, Z.; Deljac, V.; Knezevic, M.; Pavlov, L. J.; Maksimovic, M.; Markov, V.; Radovic, L. J.; Rakin, D. Acta Pharm. Jugosl. 1981, 31, 5. 27. Gray, A. P. Drug Metab. Rev. 1984,15, 557. 28. Ashani, J.; Cohen, S. J. Med. Chem. 1970, 13, 471. 29. Grifantini, M.; Martelli, S.; Stein, M. L. J.P h r m . Sci. 1969,58,460. 30. Granov, A.; Maksimovic, M.; Binenfeld, Z. Nauchno-Tech. Pregled Nr. 2 1984.34. 34. 31. Binenfeld, Z.; Boskovic, B.; Rakin, D.; Cosic, M. Acta Pharm. Jugosl. 1971,21, 113. 32. Bregovec, I.; Maksimovic, M.; Deljac, A.; Binenfeld, Z. Acta Phann. Jugosl. 1986,36, 9. ~~
- -G, -*-, 0
13a-sl : 2 13d-3n'): 2
x=u
I
X = B ~ W I
Scheme I atoms chain also did not appreciably improve the antidotal properties compared with those of TMB-4 and 2-PAM.
References and Notes 1. Eto, M. In Organophosphorous Pesticides: Organic and Biological Chemistry; CRC: New York, NY, 1974; p 123. 2. Hobbiger, F. In Handbuck Der Experimentellen Pharmakologie; Koelle, G. B. Ed.; Springer-Verlag: Berlin, Germany, 1963; Vol. 15, p 921. 3. Wills, J. H. Int. Encycl. Pharmacol. Ther. 1970, I, 357. 4. Binenfeld, T.; Kamenar, B.; Vickovic, I. Acta. Pharm. Jugosl. 1984,34. 195. 5. Bev&dic, Z. D.; Deljac, A.; Maksimovic, M.; Boskovic, B.; Binenfeld, T. Acta. Pharm. Jugosl. 1985,35, 213. 6. Deljac, V.; Maksimovic, M.; Radovic, L.; Rakin, D.; Markov, V.; Bnegovee, I.; Binenfeld, 2.Arch. Toxtcol. 1982,49, 285. I.; Stark, I.; Lorenz, H. P. Angew Chem. Znt. Ed. 1972, 7. Hagdorn, "r." 1 -
11,
avi.
8. Schoene, K.; Strake, E. M. Biochem. Pharmacol. 1971,20,2527. 9. Engelhard, N.; Erdmann, N. Arzneim.-Forsch. 1964,14, 870. 10. Clark Still, W.; Khan, M.; Mitra, A. J. Org. Chem. 1978,43,2923.
Acknowledgments The authors thank Dr. R. V. Swam (Director Defence Research and Development Establishment, G w d o r ) for his keen interest in the work, Dr. P. K. Ramachandran (Emeritus Scientist) and Dr. B. K. Bhattacharya for their helpful discussions, Mr. R. P. Semwal, Mr. Basant Lal, Mr. L. R. Chauhan, Mr. C. D. Raghuveeran, and Mr. R. S. Dangi for their help in the experimental work, and Mr. Mitthan Lal for his secretarial assistance.
Journal of Pharmaceutical Sciences I 261 Vol. 82, No. 3, March 1993