Field

The present invention relates to a synthetic route to bis-quaternary pyridinium oximes which can be utilized to restore activity of acetylcholinesterase inhibited by combination with organopho sphates .

Background

Stimulating signals are typically carried by acetylcholine within a nervous system synapse. Such signals may be discontinued by a specific type of cholinesterase enzymes, acetylcholinesterase, which breaks down acetylcholine. If cholinesterase inhibiting chemicals are present, they may then prevent the breakdown of acetylcholine thereby disrupting normal nervous system activity. For example, certain chemical classes of pesticides, such as

organophosphates and carbamates, may result in toxic cholinesterase inhibition. Accordingly, if an individual is regularly exposed to such inhibitors, there remains a need to prophylactically or therapeutically treat such toxicity. Among other things, individuals or animals who may have been exposed to a carbamate type cholinesterase inhibitor may currently be treated with atropine, and those exposed to organophosphates may beneficially be treated with a pralidoxime antidote.

Organophosphorous nerve agents (OPNA) have been used as chemical weapons, and as noted, in pesticides, have reportedly cause an estimated 300,000 deaths per year worldwide. See, e.g., Eyer, P. et al, Toxicol. Rev. 2003, 22, 165-90. Currently, the bis-pyridinium oximes known as: (1) HLo-7 dimethylsulfate (DMS), otherwise known as l-[[[4- (aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis[(hydroxyimi no)methyl]pyridinium dimethane sulfonate); (2) HI-6 DMS, otherwise known as (l-[[[4-

(aminocarbonyl)pyridinio]methoxy] methyl] -2- [(hydroxyimino)methyl]pyridinium dimethane sulfonate); and (3) obidoxime DMS, otherwise known as oxo-[[l-[[4-

(oxoazaniumylmethylidene)pyridin-l-yl]methoxymethyl]pyrid in-4-ylidene]methyl]azanium dimethane sulfonate, are reportedly among the most effective reactivators of OPNA inhibited acetylcholinesterase (AChE).

However, current methods to synthesize the above referenced antidotes require the use of chemical compounds which are extremely toxic and which lead to relatively large amounts of side products that are difficult to remove from the reaction media. Accordingly, a continuing need exists for more efficient pathways to produced desired reactivators of OPNA inhibited AChE.

Summary

A method of forming bis-quaternary pyridinium oximes comprising:

supplying benzoic anhydride having the following structure:

reacting said benzoic anhydride with trioxane to form a dibenzoyloxymethyl ether having the following structure

supplying a substituted pyridine having the following structure: wherein Ri and R ₂ may be independently selected from the group consisting of hydrogen, alkyl, -CH=NOH or -CONH ₂ ; combining said dibenzyl acetoxymethyl ether with said substituted pyridine and forming a salt having the following structure:

reacting said salt with a substituted pyridine having the following structure:

wherein Ri and R ₂ may be independently selected from the group consisting of hydrogen, alkyl group, -CH=NOH or -CONH _¾ and forming a bis-pyridinium aldoxime salt of the following structure:

wherein R ₃ and R ₄ may be independently selected from the group consisting of consisting of hydrogen, alkyl, -CH=NOH or -CONH ₂ and X comprises C1-, Br-, I- or OSO ₂ CH ₃ .

The present invention also relates to a therapeutic method of treating a person or animal for intoxication with a phosphorous containing cholinesterase inhibitor, comprising administering to a person or animal a bis-pyridinium aldoxime salt formed by the above method.

Detailed Description

As noted above, current methods to synthesize HLo-7 DMS, HI-6 DMS and obidoxime DMS require use of chemical linkers such as bis(2-chloromethyl) ether (BCME) (2) or bis(2- methylsulfonoxymethyl) ether (BMME) (3), which are identified as extremely carcinogenic, with an exposure limit of 0.0003 ppm. In addition, BMME is relatively difficult to prepare and is unstable. Below is a summary of the relevant chemical reactions utilized employing such toxic reagents:

In the above scheme, Ri, R ₂ , R ₃ and R ₄ may be independently selected from the group consisting of hydrogen, alkyl, -CH=NOH or -CONH ₂ . Reference to -OMS is reference to - OSO ₂ CH ₃ . Reference to X is therefore reference to the anionic form, either C1-, Br-, I- or OSO ₂ CH ₃ . Accordingly, in compound 6, when Ri is hydrogen, R ₂ is -CH=NOH, and R ₃ and R ₄ are both -CH=NOH, and X is OSO ₂ CH _3, the compound is HLo-7. When Ri= -CH=NOH, R ₂ is hydrogen, R3 is hydrogen and R ₄ is -CH=NOH, and X is OSO ₂ CH _3, the compound is HI-6 DMS. When Ri and R ₃ are hydrogen, and R ₂ and R ₄ are -CH=NOH, and X is OSO ₂ CH _3, the compound is obidoxime DMS. As can be seen from the above, aside from the use of relatively toxic reagents (BCME or BMME), these methods produce relatively large amounts of symmetrical side products 7 and 8, which are relatively difficult to remove from the desired oximes.

The present invention is directed at the formation of a dibenzoyloxymethyl ether 10 which can be formed by reacting compound 9, a benzoic anhydride, optionally containing one or more electron withdrawing groups (EWG) on each of the two aromatic rings, preferably in the ortho and/or para position with respect to the carbonyl functionality, with trioxane and H2SO4 as an acid catalyst. Preferably, each aromatic ring on compound 9 contains one or two electron withdrawing groups. Accordingly, the reaction occurs preferably in an organic solvent, such as dicholorethane (DCE), where the optional and preferred one or more EWG is illustrated on the reaction scheme below:

In the above, the optional electron withdrawing groups (EWG), if present, may be preferably and independently selected from an organic trihalide (-CF3, -CCI3), sulfonate (-SO3H), nitro group (-NO2), ammonium (-NH3+), aldehyde (-CHO), ketones (-COR), carboxylic acid (- COOH), acyl chloride (-COC1), benzoate esters (-COOPh), amide (-CONH2) or halides (-F, -Cl, -Br, -I). Preferably, the EWG is the same on each of the two aromatic rings.

The dibenzoyloxymethyl ether 10 can then provide a reactive species which can alkylate substituted pyridines such as compound 1 to form a salt 11:

As illustrated below, when an additional Lewis acid and a second substituted pyridine such as compound 5 is added and reacted with compound 11 the desired bis-pyridinium OPNA antidotes 6 are formed at a yield in the range of 30-90%. Preferred Lewis acids include trimethylsilyl iodide (TMSI), Trimethylsilyloxytrifluoromethanesulfonate (TMSOTf), BFi»OEt2 or Trimethylsilyloxymethanesulfonate (TMSOMs). It should be noted that in this synthetic scheme, Ri, R2, R3 and R4 may be independently selected from the group consisting of hydrogen, alkyl, -CH=NOH or -CONH2 _. In addition, to X is again reference to the anionic form, either C1-, Br-, I- or OSO ₂ CH _3. Reference to alkyl groups preferably include, e.g., methyl (-CH ₃ ), ethyl (-

CH ₂ CH ₃ ), propyl (-CH ₂ CH ₂ CH ₃ ) or butyl (-CH ₂ CH ₂ CH ₂ CH ₃ ) groups.

Accordingly, the present invention relates to a synthetic procedure for the formation of bis-quatemary pyridinium oximes which can be utilized to restore activity of

acetylcholinesterase by combination with organophosphates. The procedure avoids the use of bis(2-chloromethyl) ether (BCME) (2) or bis(2-methylsulfonoxymethyl) ether (BMME) (3) as well as the formation of compounds 7 and 8 (symmetrical alkylation side products). Moreover, once prepared, the bis-quaternary pyridinium oximes may be readily incorporated into a pharmaceutically accepted carrier and administered in an antidotal amount to therapeutically treat exposure to a phosphorous containing cholinesterase inhibitor. A pharmaceutical accepted carrier may be understood herein as an aqueous formulation containing the bis-quaternary pyridinium oximes in the form of an aqueous solution, suspension or emulsion. The pharmaceutically acceptable carrier herein may also include other diluents suitable for preparing oral pharmaceutical suspensions. For example, pharmaceutically acceptable additives including stabilizing agents, suspending agents, surface tension modifiers, viscosity modifiers, colorants, preservatives, flavoring agents.

Working Examples Unless otherwise noted, solvents and reagents were used without purification. 1,2- dichloroethylene (DCE) was dried over 4 A molecular sieves for 48 h prior to use. Volatile solvents were removed under reduced pressure using a Buchi rotary evaporator. Infrared (IR) spectra were obtained using a Nicolet iS550 FT IR spectrophotometer using a diamond crystal attenuated total reflection (ATR) accessory and reported as wave numbers. Melting points were determined using differential scanning calorimetry (DSC) on a TA Instruments Differential Scanning Calorimeter Model Q100. Thin layer chromatography (TLC) was performed on glass- backed precoated silica gel plates (0.25 mm thick with 60 F254) and were visualized using one or both of the following manners: UV light (254 nm) and staining with h impregnated silica. Flash chromatography was performed using the Biotage Isolera One using pre-loaded Silicycle 25g high performance (14-40 mM) columns. 'H nuclear magnetic resonance (NMR) spectra were obtained at 400 MHz as indicated as solutions in CDC13 with 0.05% v/v tetramethylsilane (TMS) unless indicated otherwise. ¹³ C-NMR were obtained at 100 MHz as shown in the indicated deuterated solvent. Chemical shifts are reported in parts per million (ppm, d), and referenced to TMS, and coupling constants are reported in Hertz (Hz). Spectral splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; quint, quintuplet; sex, sextet; sept, septuplet; m, multiplet; comp, overlapping multiplets of magnetically nonequivalent protons; br, broad; and app, apparent.

General Procedure for Anhydride Formation

Pyridine (1 eq) was added to a stirring solution of the benzoic acid (1 eq) in CH2CI2 (2 mF/mmol) at room temperature. Thionyl chloride (0.55 eq) was added to the solution and was allowed to stir at room temperature. The reaction was monitored by TFC (1:1:8

CH ₂ Cl ₂ /Et ₂ 0/hexanes) and stopped after 2 hours. Diethyl ether (2 mF/mmol) was added to the mixture and vortexed. The slurry was filtered through a silica plug and washed with ether and the filtrate was concentrated under reduced pressure to afford the crude anhydride.

General Procedure for Zh ^' s-benzoyloxymethyl Ether Formation

Sulfuric acid (-0.03 eq) was added to a stirring solution of the benzoic anhydride (1 eq) and trioxane (0.55 eq) in DCE (1.22 mL/mmol). The mixture was heated to 60 °C and monitored by TLC (3:3:4 CHiCh/EtiO/hcxancs) unless otherwise indicated. After 3 hours the reaction was removed from heat and a mixture of 1:1 CH2CI2/H2O (1.5 mL/mmol) was added to the reaction. Ammonium hydroxide (2 drops) was added to the mixture and was vortexed. The organic phase was collected and concentrated under reduced pressure. The residue was preloaded onto silica and purified by flash chromatography.

s-benzoyloxymethyl ether (12). Utilized the following TLC conditions: (1:1:8 CH ₂ Cl ₂ /Et ₂ 0/hexanes). Isolated 1.31 g (41%) of pure 21a as a white solid. MP: 50 °C. ^-NMR (400 MHz) d 8.02 (app d, / = 9.6 Hz, 4 H), 7.56 (app t, / = 7.2 Hz, 2 H), 7.39 (t, / = 7.6 Hz, 4 H), 5.73 (s, 4 H); ¹³ C-NMR (100 MHz) d 165.8, 133.5, 130.0, 129.4, 128.5, 87.9; IR (pellet) 1722 (C=0), 1600, 1452, 1426, 1316, 1264, 1196, 1143, 1113, 1090, 1069, 1035, 1025, 1012, 968, 936, 846, 804, 703, 691, 683, 599, 495, 449 cm ¹ .

Z>zs-2-chlorobenzoyloxymethyl ether (13). Isolated 477 mg (8%) of pure 13 as a clear oil. ^ NMR (400 MHz) d 7.86 (m, 2 H), 7.47-7.40 (comp, 4 H), 7.26 (m, 2 H), 5.74 (s, 4 H); ¹³ C-NMR (100 MHz) d 164.5, 134.2, 133.1, 131.8, 131.2, 128.8, 126.6, 87.9; IR (film) 1731 (C=0), 1591, 1472, 1436, 1291, 1243, 1161, 1108, 1072, 1011, 920, 742, 722, 689, 650, 599, 473 cm ¹ .

s-2-flourobenzoyloxymethyl ether (14). Isolated 162 mg (3%) of pure 14 as a clear oil. ^ NMR (400 MHz) d 7.94 (td, J = 1.3, 6.1 Hz, 2 H), 7.52 (m, 2 H), 7.16 (t, 7 = 5.8 Hz, 2 H), 7.10 (dd, 7 = 7.4, 8.6 Hz, 2 H), 5.73 (s, 4 H); ¹³ C-NMR (100 MHz) d 163.3 (d, 7 = 3.8 Hz), 162.2 (d, 7 = 259.7 Hz), 135.1 (d, 7 = 9.1 Hz), 132.3, 124.0 (d, 7 = 4.0 Hz), 117.8 (d, 7 = 9.2 Hz),

117.0 (d, 7 = 22.2 Hz), 87.6; IR (film) 1724 (C=0), 1612, 1488, 1456, 1293, 1246, 1230, 1154, 1116, 1018, 923, 835, 790, 751, 691, 600, 541, 521 cm ¹ . s -2,6-diflourobenzoyloxymethyl ether (15). Isolated 442 mg (7%) of pure 15 as a white solid. MP: 59 °C. ^ NMR (400 MHz) d 7.45 (m, 2 H), 6.97 (t, / = 8.4 Hz, 4 H), 5.72 (s, 4 H); ¹³ C-NMR (100 MHZ) d 161.0 (dd, / = 5.8, 256.6 Hz), 160.6, 133.5 (t, / = 10.6 Hz), 112.0 (dd, / = 3.0, 18.6 Hz), 110.1 (t, / = 16.9 Hz), 87.1; IR (pellet) 1743 (C=0), 1623, 1465, 1284, 1260, 1236, 1150, 1121, 1033, 1008, 962, 923, 884, 829, 799, 772, 690, 580, 525, 512, 489, 405 cm ¹ .

s-2,6-dichlorobenzoyloxymethyl ether (16). Isolated 264 mg (5%) of pure 16 as a white solid. MP: 61 °C. ^-NMR (400 MHz) d 7.36-7.29 (comp, 6 H), 5.75 (s, 4 H); ¹³ C-NMR (500

MHz) d 163.9, 132.7, 131.9, 131.3, 127.9, 87.4; IR (pellet) 1750 (C=0), 1564, 1432, 1289, 1252, 1195, 1158, 1126, 1096, 1069, 1012, 965, 945, 911, 814, 800, 774, 751, 724, 605 cm ¹ .

Z>zs-4-chlorobenzoyloxymethyl ether (17). Utilized the following TLC conditions: (2:2:6 CH ₂ Cl ₂ /Et ₂ 0/hexanes). Isolated 78 mg (1%) of pure 17 as a white solid. MP: 97 °C. ^-NMR (400 MHz) d 7.92 (d, 7 = 8.4 Hz, 4 H), 7.36 (d, 7 = 8.4 Hz, 4 H), 5.71 (s, 4 H); ¹³ C-NMR (100 MHz) d 164.8, 140.1, 131.2, 128.8, 127.6, 88.1; IR (pellet) 1727 (C=0), 1713, 1593, 1487, 1457, 1402, 1288, 1267, 1181, 1165, 1090, 1045, 1010, 960, 848, 812, 758, 734, 685, 630, 598, 521, 475, 451, cm ¹ .

6w-4-flourobenzoyloxymethyl ether (18). Utilized the following TLC conditions: (1:1:8 CH ₂ Cl ₂ /Et ₂ 0/hexanes). Isolated 698 mg (11%) of pure 18 as a white solid. MP: 77 °C. ^-NMR (400 MHz) d 8.02 (m, 4 H), 7.06 (t, J = 8.8 Hz, 4 H), 5.71 (s, 4 H); ¹³ C-NMR (100 MHz) d 166.0 (d, 7 = 253.5 Hz), 164.7, 132.4 (d, 7 = 9.4 Hz), 125.5 (d, 7 = 2.9 Hz), 115.6 (d, 7 = 21.9 Hz), 88.0; IR (pellet) 1713 (C=0), 1598, 1505, 1460, 1411, 1267, 1234, 1217, 1196, 1152, 1110, 1095, 1080, 1031, 1011, 954, 923, 860, 852, 821, 792, 763, 688, 585, 542, 499, 485, 465, 423 cm ¹ .

Z>zs-2,4-dichlorobenzoyloxymethyl ether (19). Utilized the following TLC conditions: (1:1:8 C^Ch/EUO/hexanes). Isolated 3.14 g (28%) of pure 19 as a white solid. MP: 80 °C. ¾- NMR (400 MHz) d 7.82 (d, / = 8.4 Hz, 2 H), 7.46 (d, / = 2 Hz, 2 H), 7.25 (dd, J = 2, 8.4 Hz, 2 H)l, 5.51 (s, 4 H); ¹³ C-NMR (100 MHz) d 163.5, 139.1, 135.5, 132.8, 131.2, 127.0, 126.9, 88.2; IR (pellet) 1710 (C=0), 1583, 1469, 1396, 1375, 1292, 1273, 1251, 1161, 1130, 1110, 1074, 1024, 972, 933, 895, 841, 826, 781, 762, 675, 611, 563, 545, 487, 441. 404 cm ¹ .

s-3-chlorobenzoyloxymethyl ether (20). Utilized the following TLC conditions: (1:1:8

CH ₂ Cl ₂ /Et ₂ 0/hexanes). Isolated 680 mg (12%) of pure 20 as a white solid. MP: 65 °C. ^-NMR

(400 MHz) d 7.96 (t, / = 1.6 Hz, 2 H), 7.89 (dt, / = 1.2, 8.0 Hz, 2 H), 7.52 (ddd, / = 1.2, 2.0, 8.0 Hz, 2 H), 7.33 (t, / = 7.6 Hz, 2 H), 5.72 (s, 4 H); ¹³ C-NMR (100 MHz) d 164.5, 134.6, 133.5, 130.9, 129.8, 129.7, 127.9, 88.3; IR (pellet) 1713 (C=0), 1574, 1469, 1420, 1395, 1295, 1255, 1169, 1118, 1101, 1079, 1059, 1014, 945, 897, 890, 809, 749, 737, 672, 658, 575, 497, 464, 424 cm ¹ .

HI-6 bistriflate (21). Trimethylsilyl trifluoromethanesulfonate (0.92 g, 0.75 mL, 4.13 mmol) was added to a 1 dram vial containing linker 10 (250 mg, 0.59 mmol), pyridine-2-aldoxime (72 mg, 0.59 mmol), 2,6-di-tert-butylpyridine (0.53 g, 0.60 mL, 2.65 mmol), and nitromethane (0.25 mL) and the mixture was stirred at 40 °C for 1.5 h, whereupon isonicotinamide (72 mg, 0.59 mmol) was added and the reaction temperature raised to 60 °C and stirred for 16 h. The reaction mixture was then diluted in a mixture (1: 1) of MeCN and H2O (2000 mL) and a sample was run on the HPLC according to the HI-6 calibration curve method. The area response of the sample was determined to be 32.73 mAU, which corresponds to a 101% yield of HI-6 bistriflate (21) by the

Dx(A+N)

following relationship: % yield = where D is the dilution factor of the sample, A is

IOOOXEXLXM

the absorbance (in mAU), N is the Y intercept, e is the molar absorptivity coefficient, L is the moles of linker 10 used in the reaction, and M is the molecular weight of HI-6 DMS (478.50 g/mol).

22

Hlo-7 bistriflate (22). Trifluoromethanesulfonate (0.92 g, 0.75 mL, 4.13 mmol) was added to a 1 dram vial containing linker 10 (250 mg, 0.59 mmol), pyridine-2, 4-dialdoxime (97 mg, 0.59 mmol), 2,6-di-tert-butylpyridine (0.53 g, 0.60 mL, 2.65 mmol), and nitromethane (0.25 mL). The mixture was stirred at 40 °C for 1.5 h. Isonicotinamide (72 mg, 0.59 mmol) was added to the mixture and stirred at 60 °C for 16 h. The reaction mixture was then diluted in a mixture (1:1) of MeCN and H2O (800 mL) and a sample was run on the HPLC according to the Hlo-7 calibration curve method. The area response of the sample was determined to be 20.78 mAU, which corresponds to a 29% yield of Hlo-7 bistriflate (22) by the following relationship: % yield =

Dx(A+N )

1000 xexLxM where D is the dilution factor of the sample, A is the absorbance (in mAU), N is the Y intercept, e is the molar absorptivity coefficient, L is the moles of linker 10 used in the reaction, and M is the molecular weight of Hlo-7 DMS (521.52 g/mol).