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United States Patent 0 " 3,093,541 Patented June 11, 1963 2 1 dioximes of the formula 3,093,541 BIS QUATERNARY OXIMES ‘ Brennie E. Hackley, Jr., and Edward J. Poziomek, Edge wood, and George M. Steinberg, Baltimore, Md, as i CH==NOH ‘ signors to the United States of America as represented (IVb) This invention further relates to unsymmetrical bis quaternary 4-formylpyridinium halide monoximes of the formula by the Secretary of the Army No Drawing. Original application Apr. 28, 1959, Ser. No. 809,578, now Patent No. 3,077,476, dated Mar. 12, 1963. Divided and this application Oct. 11, 1961, Ser. No. 151,126 1 Claim. (Cl. 167-65) (Granted under Title 35, US. Code (1952.), sec. 266) This invention described herein may be manufactured 0 and used by or for the Government of the United States of America for governmental purposes without the pay 15 CH=NOH ment to us of any royalty thereon. (V) wherein R" represents either three lower alkyl groups or This is a division of application Serial No. 809,578, ?led April 28, 1959, now Patent No. 3,077,476, granted March 12, 1963. the hydrocarbon portion of the pyridine ring. This invention also relates to the 1,1'-trimethylene bis ‘ This invention is directed to certain diquaternary 20 (3-amidooximopyridinium) halides pyridinium halide oximes which are useful as chemo therapeutic and prophylactic agents for mammals poisoned by anticholinesterases, particularly the “nerve gas” known as GB or sarin, i.e., isopropyl methylphosphono?uoridate. The invention relates to 1,1'-polymethylene bis(4 formylpyridinium) halide dioximes wherein the poly (VI) This invention also relates to the compounds methylene group contains from 2 to 6 carbon atoms; These compounds have the structural formula 30 .(vn) This invention also relates to the compounds (I) 35 where R is a polymethylene group having from two to six carbon atoms and Xi is chloride, bromide or iodide. This invention further includes compounds of Formula I, but in which R is a polymethylene group containing CH=NOH (VIII) While all these compounds are useful in varying degree 40 This invention further includes 1,l'-(2,5-dimethyl-p for the purposes set out above, the di?erent groups exhibit phenylenedimethylene) bis (4-formylpridinium) halide quite striking differences in effectiveness. In all the above formulas X— is chloride, bromide or iodide, which appear dioximes of the formula to be equivalents as to physiological action, except for from seven to ten carbon atoms. their effect on solubility. . Wilson et -al., in Patent No. 2,816,113, granted Decem ber 10, 1957, disclose a group of compounds which are ef fective as antidotes for treatment of mammals poisoned by compounds of high anticholinesterase activity, such 50 as The invention also relates to 1,1'-(2-'butenylene) bis (4-formylpyridinium) halide dioximes of the formula the “nerve gases” diisopropylphosphoro?uoridate (DFP), isopropyl methylphosphono?uoridate (GB), and O-ethyl, N,N dimethyl phosphoroamidocyanidate (GA), as well as other related organic phosphorus compounds, including many insecticides. The compound of the Wilson 55 et al. group which has received by far the most attention is 2-formyl-l-methyl pyridinium iodide oxime, commonly known as Z-pyridine aldoXime methiodide or 2-PAM. This compound is outstanding in its ability to reactivate, in vitro, oholinesterase which has been inhibited by, for ex 60 ample, GB. Thus, even as compared to the very closely The invention further relates to 1,1'-polymethylene bis related 4-PAM, disclosed in Example ll of the Wilson pat 0 H=N O H GH=N O E (III) (3-formylpyridinium) halide dioximes of the formula 4. rent, 2-PAM ‘shows much greater activity. With isopropyl methylphosphonylated acetylocholinesterase the rate con stant at pH 7.4 and 25° C. in the presence of 10-3 M 65 a'cetylocholine is 2x103 per mole per minute for Z-PAM and 1.4 X102 per mole per minute for 4-PA1M. The Wilson patent shows a' high rate of survival in mice which have been poisoned with paraoxon and then treated with 2-PAM. wherein R is a polymethylene group containing from 2 Nerve gas poisoning has been treated symptomatically 70 to 6 carbon atoms. with drugs which are pharamacologically antagonistic to Another class of compounds included are the 1,1’-(p acetyl choline. Such a compound is atropine and it is at phenylenedimethylene) bis (3-formy1pridinium) halide 3,093,541 .3 4 a) present the recommended remedy. Recently, 2-PAM has been reported to enhance considerably the activity of atropine in the chemotherapeusis of poisoning due to organophosphorus compounds. TMB-4 were ordinarily given intravenously. However, in the “therapeutic” tests on rabbits, dogs and cats, the GB was administered subcutaneously, since death vfrom 20 LD50 intravenous dose of GB occurs so quickly that it is » The compounds of Formula I above in which R contains virtually impossible to give timely administration of. the from 2 to 6 carbon atoms are appreciably more effective antidote. Atropine, when administered, was included in the fol than 2-PAM as reactivators of GB-inhibited acetylcholin esterase and also in enhancing the activity of atropine in both therapy and propylaxis. When If is bromide, the lowing amounts. variation of the rate constant for the in vitro reactivation 10 of GB inhibited eel acetylcholinesterase at pH 7.4 and 25° Rats ________________________________ -a ____ __ Rabbits C. was found to be as follows: 2 _ 0.5 The “prophylactic” doses were given within two minutes prior to the injection of the GB, the “therapeutic” doses 15 so soon as poisoning symptoms were visible. Rate constant Table 3 shows the results. (l/molcs/minutes) (cum (cum (OHM (CHM (cum 4 ___________________________________ _ _ Dogs and cats TABLE 1 R Mgjkg. TABLE 3 7><103 6x103 6x103 1x104 6x103 A. PROPHYLAOTIC 20 2—PA M TM B-4 Survival ratio ' ' Survival ratio When administered in combination with atropine to Animals Animals animals poisoned with GB the order of effectiveness was 25 DoscDose, nag/kg. With- With rug/kg. With- With somewhat diiferent. Under these conditions the com pound in which R=\(CH2)3, i.e., 1,1’-trin1ethy1ene bis (4 TMB-4, was most effective. atro- out atro pine atro- pine pine In rats challenged with a Miee_ _ _ _ 2 LD50 dose of GB administered intravenously, all of a 30 group of six animals survived if the atropine-TMB-4 com Rabbits bina’tion was administered intravenously immediately after poisoning. The atropine-2—PAM combination saved only Dogs____ B. THERAPEUTIC l\/Iice-_-_ treatments, which were given intravenously when symp~ toms appeared. However, the recovery time was much shorter ‘for the surviving animals which received the TMB-4, i.e., 2 hours, as against 24 hours for those receiv 40 ing the 2-PAM. A summary of the reactivation rates and survival ratios for these compounds when administered therapeutically to rats together with atropine is as follows: TABLE 2 ______ ._ Rats____ Rabbits Cats-___ Dogs____ 1/6 2/6 5/5 4/5 Miee____ Rats____ Rabbits Cats.___ D0gs____ The recovery periods, i.e., time for disappearance of sypmtorns of poisoning, among survivors in the above tests, with atropine, were as follows. TABLE 4 45 2~PAM Animals Reactivation Survival rate constant ratio (GB) _ Rats. _ __ Cats_ _ _ _ two of the group of animals. On the other hand, with dogs which were given a 20 LD50 dose of GB subcutane ously the survival ratios were the same (4/5) for the two (011m ____ ._ out atro- pine formylphyridinurn) bromide dioxime also known as TMB-4 Prophylactic Therapeutic Prophylactic Therapeutic 7X103 6X103 6X10a 1x104 6x103 These compounds constitute our presently preferred 55 group. our preferred group. For these compounds the reactiva tion rate constant and the survival ratio vfor rats (meas ured as given above) were as follows, X- being bromide. Our compounds may be employed prophylactically, i.e., injected before exposure to the anticholinesterase agent, e.g., GB, or therapeutically, i.e., injected subsequent to exposure. The following series of experiments compares the e?’ec— tiveness of our presently preferred compound, TMB-4, with 2-PAM applied to various animals by these two methods. The animals were poisoned by injections of GB as follows: Mice ___________________ __ 0.173 mg./lcg. (LD5O). Rats ___________________ __ 0.126 trig/kg. (2 LD5Q). Rabbits: Intravenous _________ __,_ 0.340 mg./kg. (20 LD50). Subcutaneous ________ __ 0.900 mg./kg. (20 LD50). Dogs and cats: Intravenous __________ __ 0.440 mg/kg. (20 LB“). Subcutaneous ________ __ 0.900 rug/‘kg. (20 LD50). To minimize absorption effects both the GB and The compounds of Formula I in which R contains from 7 to 10 carbon atoms are less effective than those of 60 TABLE 5 R Rate Survival ratio (GB) 2X10a 1. 2><10a 1. 4X103 0/4 0/4 0/4 0/6 While these compounds were ineffective in vivo against GB, they were, together with 2-PAM, very e?ective against certain other anticholinesterases, particularly that designated as VX by the U.S. Army Chemical Corps. All these compounds caused survival of all animals (sur~ vival rates of 4/ 4 and 6/6) when administered therapeuti cally to rats challenged by 2 LD50 doses of VX. 3,093,541 6 ing on a steam bath a neutralized aqueous solution of The compounds of Formula II exhibited properties 4-pyridinecarboxaldehyde and hydroxylamine hydrochlo intermediate those of the two subgroups of Formula I. When X- was chloride the compound had the following ride. The oxime had a melting point of 130-1305“ C. The 2- and 3-oximes were produced by similar methods. The quaternization to produce dioximes was carried properties. (In this and all following tables the survival ratios are those for rats challenged by 2 L135“ doses of . out by reacting the proper oxime with a 1, n dihaloalkane, (CH2)nX2 employing a 3 :1 molar ratio of oxime to halide. GB or (VX) and the oxime was employed therapeuti cally.) The unsymmetrical quaternary monoximes were obtained Compounds of Formula III showed reactivation rates by reacting the pyridine oxime with the appropriate very close to those of our preferred group. Thus when X'- in Formula III is bromide the reactivation rate con 10 omega-halopropyl quaternary salt in a 1.5:1 molar ratio. Two procedures were utilized. stant was 8><103 as compared to the value ‘for the Procedure A: A mixture of the pyridine oxime and R/=(CH2)4 member of our preferred group of 6x103. halide was dissolved in sufficient ethanol and re?uxed for For the unsaturated member (Formula III) the survival the period of time speci?ed in Table 9. ratio for rats challenged by GB was only 1/ 4 as compared to 6/6 for the saturated analogue (Formula I). Both 15 Procedure B: A mixture of the oxime and halide was dissolved in about 100 ml. of ethanol and heated in a 200 gave complete survival (ratios of 4/4 and 6/6) for animals ml. capped pressure bottle (carbonated beverage type) challenged by VX, however. for the length of time speci?ed. The reaction mixtures Compounds of Formula IVa showed anomalous were cooled to room temperature and the product re moved by ?ltration. In several instances it was necessary 20 They gave reactivation rates which were low, but sur vival ratios which were high as compared to Z-PAM, as ' to add absolute ether to eifect complete precipitation. The properties. products were recrystallized from ether. This procedure was usually employed because of its simplicity. shown by Table 6, X- being bromide. TABLE 6 Table 9 gives the procedure, yields and melting points 25 Reactivation Survival rate constant ratio (GB) (CH2): ------------------------------------ - (CHM ------------------------------------ - a. 5x101 4. 2x102 for representative compounds. TABLE 9 4/4 3/4 For- Substituents - Melting (n1) mula 30 The compounds of group IVb, which are closely related to those of IVa, were somewhat less effective. When X" was bromide the ‘compound had the following proper ties: Reactivation rate constant 2x102, survival ratio (GB)--2/4. 35 The compounds of group V were another group in which the results of therapeutic treatment against GB were better as compared to Z-PAM than the reactivation rate constants would suggest, as shown by the following table, X- being bromide. TABLE 7 40 Gondi- No. tions Halide R Yield or decom percent position (d) R” point, ° C. I ____ _- Br I ____ __ Br I ____ __ Br I ____ __ Br I ____ __ Br 35. 0 88. 2 81.0 95. 0 85. 0 >300 m. 238-241 (I. 239-241 (I. 208-210 (1. 219-223 6. II“... 01 IVa__ Br IVai. Br ___________________ __ B, as hr__ (011m _________ ._ B, 60 r__ (Slim _________ __ B, 60 hr__ 70 68 80 1%... Br ___________________ __ B, 20 111:. 83.5 >300 m. 208-211 m. 226-231 m. 248-251 in. V .... __ Br V ____ ._ Br ________ _- (021393“ ________ __ pyridine B, 69 11LB, 64 11L- 43 10 230-231 (1. 223-226 (1. VIII." Br rmg. ___________________ __ B, 90 hr__ 16 201-203 (1. Further details regarding the preparation and properties + R"EN-— Reactivation Survival rate constant ratio (GB) of certain of our compounds are given in the following 45 publications by us and our associates: 50 “Pyridine Alcloximes,” by Edward J. Poziomek, Bernnie E. Hackley, Jr., and George M. Steinberg, “Journal of Organic Chemistry,” vol. 23, pp. 714-717 (May 1958); and “Chemotherapeutic Effectiveness of Trimethylene Bis (4-Formy1 Pyridinium Bromide) Dioxime in Anticholin esterase Poisoning,” by Edmund Bay, S. Kropp and L. F. Yates, Proceedings of the Society for Experimental Biol + (0,115) iN1. 2x101 4/4 ogy and Medicine, vol. 98, pages 107-109 (May 1958). These articles are to be considered incorporated by refer The compounds of Formulas VI, VII and VIII, while 55 ence in this speci?cation. being of di?erent structure are alike in exhibiting reactiva While we have shown a number of speci?c examples of tion rate constants which are very low as compared to compounds and their use, it will be obvious that various Z-PAM but giving high survival ratios as shown by Table changes can be made without ‘departing from our inven 8, X- being bromide in each case. tion, which is de?ned by the following claim. 60 TABLE 8 We claim: A method of therapeutically treating a mammal which has been poisoned by a compound having high anticho Formula No. Reactivation Survival ratio (GB) rate linesterase activity which comprises injecting a composi 69 4/ 465 67 4/4 Negligible 4/4 tion comprising atropine and 1,1'-(2-butenylene) bis(4 formylpyridinium) halide dioxime. ' References Cited in the ?le of this patent Preparation of Compounds Poziomek: Am. Chem. Soc. Abst. of Papers, 132nd 4-pyridinecarboxaldehyde oxime was prepared by war-m 70 Meeting, 1957, pages 16-0.