New series of monoquaternary pyridinium oximes: synthesis and reactivation potency for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Abstract

The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE) are reported. Several newly synthesized compounds efficiently reactivated inhibited EeAChE, but were poor reactivators of inhibited rHuAChE. Compounds bearing a thiophene ring in the side chain (20, 23, 26 and 29) showed better reactivation (24–37% for EeAChE and 5–9% for rHuAChE) compared to compounds with furan and isoxazole heterocycles (0–8% for EeAChE and 2–3% for rHuAChE) at 10⁻⁵ M. The N-pyridyl-CH₂COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10⁻⁴ M with a k_r value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE.

Reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) are important therapeutic agents for the treatment of toxicity resulting from organophosphorus inhibitors (OPI) such as sarin, paraoxon, tabun, chlorpyrifos, cyclosarin, VX, and DFP. Some of these OPIs have been used as warfare agents (such as sarin, soman, tabun, VX) and are some of the most toxic man made compounds known.¹ OPIs inhibit AChE via formation of a covalent bond at a serine hydroxyl group within the enzyme’s active site. Once inhibited, AChE is not able to hydrolyze acetylcholine and accumulates in the synaptic cleft leading to cholinergic over-stimulation, compromised respiration and sometimes death.¹ AChE inhibited by OPI can be reactivated by 2-pyridine aldoxime methiodide (2-PAM, pralidoxime; 1)² that nucleophilically attacks the phosphoryl moiety displacing it from the active site serine (Fig. 1). In addition to 2-PAM^3–6, the bis-oximes trimedoxime (TMB-4, 2),^{7, 8} obidoxime (3)^{9, 10} and HI-6 (4)^{11, 12} show efficacy as reactivators in vivo and are widely used for the treatment of OPI poisoning.^{13, 14} Recently, several new pyridinium oxime reactivators were reported including bisquaternary pyridinium oximes connected with different linker chains viz. alkylene linker,^15–18 but-2-ene linker,^19–22 aromatic ring containing linkers^23–25 and oxygenated linker chain.^26–28 Monoquaternary pyridinium oximes include 2-PAM analogs with varying side chain^{29, 30} and 4-PAM analogs with modified oximes.³¹ Amongst these, but-2-ene linked bisquaternary 4,4′-bisoxime²² and 2-PAM analogs with a propyl side chain²⁹ showed reactivation of paraoxon-inhibited AChE comparable to clinically known reactivators.

Figure 1.

Equation representing inhibition and reactivation of AChE by OPI and oximes. Selected examples of oximes are shown.

Although bis-quaternary oximes are considered more effective AChE reactivators, many countries use 2-PAM as the therapeutic agent.^32,33 The efficacy of the oxime used is also dependent on the AChE type. Certain AChE types undergo rapid reactivation whereas others show little to no appreciable activity after oxime treatment. This and other limitations highlight that there has been no single broad-spectrum oxime reactivator identified to date.^1,34 Therefore, the development and selection of new, more effective AChE oxime reactivators is very important. The focus of this work was to determine the effect of different heterocyclic and aliphatic side chains (capable of hydrogen bonding) on the ability of monoquaternary pyridinium oximes to reactivate two forms of AChE inhibited by paraoxon.

Monoquaternary pyridinium oximes 5–35 were prepared in 40–92% yield via reaction of 2-, 3- or 4-pyridine aldoximes with different substituted alkyl halides in acetonitrile or acetone at 60–80 °C for 1–24 h.³⁵ Monoquaternary pyridinium oximes 5–16 were designed to contain an aliphatic side chain with a group capable of hydrogen bonding viz. carbamoyl, carboxyl and hydroxyl. Furthermore, highly electron rich heterocycles, viz. thiophene, furan and isoxazole were used as side chains 17–35 to explore interactions with cation-containing (cation-π bonding) or aromatic residues (π–π bonding) at or near the AChE active site. The oxime group was positioned at 2, 3 and 4 on the pyridinium ring to determine the best position for reactivation of AChE using the N-derivatized groups. All compounds were characterized by ¹H NMR, ¹³C NMR, ESI-MS and IR prior to assay.³⁵ Compounds 10, 11 and 13 have been reported as reactivators but not against EeAChE or rHuAChE.^7,36 All compounds were assayed for their capacity to reactivate EeAChE and rHuAChE using a modified Ellman assay.^37,38 2-PAM and paraoxon were chosen as a reference reactivator and as representative OPI.

Reactivation results are summarized in Table 1. Oximes with aliphatic side chains mostly showed concentration-dependent reactivation of AChE. Compounds containing carboxamide or carboxylic acid groups paired with the 2 or 4-oxime position (5, 7 and 8) gave 40–45% reactivation of EeAChE while compounds with hydroxyethyl side chain 11 and 13 showed 50 and 70% reactivation of EeAChE at 10⁻³ M comparable to 2-PAM 1. Compound 8 also showed promise as a reactivator of rHuAChE (15%) at 10⁻³ M. Compounds with aromatic side chains (thiophene, furan and isoxazole) displayed a reverse concentration-activity relationship. These compounds, with the exception of 19 and 35, showed ≤ 8% reactivation of EeAChE and no reactivation of rHuAChE at the highest concentration (10⁻³ M). Lower concentrations of oxime (10⁻⁴ to 10⁻⁵ M) 17–29 were better reactivators of both enzymes suggesting that these compounds may inhibit AChE at higher concentrations and explain, in part, the inability to reactivate inhibited AChE at 10⁻³ M.^19,23

Table 1.

Synthesis and in vitro reactivation potency of monoquaternary pyridinium oximes 5–35 for paraoxon-inhibited EeAChE and rHuAChE

Entry	R	Oxime position	X	Reaction time (h)	% Yield	% Reactivation = [(Ar-Ai)/(Ao-Ai)] × 100
						EeAChE (% R ± SEM)			rHuAChE (% R ± SEM)
						10−3 Ma	10−4 Ma	10−5 Ma	10−3 Ma	10−4 Ma	10−5 Ma
1	H	2	I	-	-	67 ± 1	67 ± 3	63 ± 3	42 ± 2	22 ± 2	7 ± 1
5	CONH2	2	I	24	62	41 ± 3	9 ± 0	1 ± 0	2 ± 1	2 ± 0	1 ± 0
6	CONH2	3	I	2	76	1 ± 1	0	0	0	1 ± 0	2 ± 0
7	CONH2	4	I	2	84	44 ± 2	6 ± 1	0	0	1 ± 0	1 ± 0
8	COOH	2	I	24	61	45 ± 3	36 ± 3	34 ± 2	15 ± 1	15 ± 2	5 ± 1
9	COOH	3	I	2	71	1 ± 0	0	0	0	1 ± 0	1 ± 0
10	COOH	4	I	2	78	4 ± 0	0	0	0	1 ± 0	2 ± 0
11	CH2OH	2	Br	24	58	50 ± 4	37 ± 3	13 ± 1	2 ± 2	5 ± 0	2 ± 0
12	CH2OH	3	Br	2	60	8 ± 1	0	0 ± 0	0	1 ± 0	1 ± 0
13	CH2OH	4	Br	2	68	70 ± 2	2 ± 2	3 ± 1	6 ± 1	3 ± 0	2 ± 0
14	CH2COOH	2	Br	24	48	10 ±1	0	0	0	1 ± 0	1 ± 0
15	CH2COOH	3	Br	2	56	5 ± 0	0	0	0	1 ± 0	1 ± 0
16	CH2COOH	4	Br	2	68	15 ±1	0	0	0	2 ± 0	1 ± 0
17	4-methoxybenzen-1-yl	2	Cl	12	70	8 ± 1	19 ± 2	18 ± 2	0	7 ± 1	3 ± 0
18	4-methoxybenzen-1-yl	3	Cl	1	84	1 ± 1	0	0	0	3 ± 0	2 ± 0
19	4-methoxybenzen-1-yl	4	Cl	1	92	19 ± 1	11 ± 1	3 ± 1	0	3 ± 1	2 ± 0
20	thiophen-2-yl	2	Cl	24	58	1 ± 0	24 ± 2	34 ± 2	0	9 ± 2	9 ± 1
21	thiophen-2-yl	3	Cl	2	65	0	3 ± 1	1 ± 0	0	4 ± 0	5 ± 0
22	thiophen-2-yl	4	Cl	2	78	3 ± 0	12 ± 2	12 ± 0	0	4 ± 1	5 ± 0
23	5-bromothiophen-2-yl	2	Cl	12	74	0	29 ± 2	37 ± 2	0	7 ± 0	8 ± 1
24	5-bromothiophen-2-yl	3	Cl	1	84	4 ± 1	7 ± 0	8 ± 1	0	5 ± 1	6 ± 0
25	5-bromothiophen-2-yl	4	Cl	1	90	3 ± 1	14 ± 1	10 ± 1	0	5 ± 1	4 ± 0
26	benzthiophen-3-yl	2	Cl	24	67	0	28 ± 2	24 ± 3	0	7 ± 1	8 ± 1
27	benzthiophen-3-yl	3	Cl	2	79	0	5 ± 1	8 ± 1	0	2 ± 0	5 ± 1
28	benzthiophen-3-yl	4	Cl	2	85	0	0	1 ± 1	0	2 ± 0	2 ± 0
29	thiophen-3-yl	2	Cl	24	79	0	8 ± 1	28 ± 2	0	2 ± 0	5 ± 1
30	furan-2-yl	3	Cl	1	62	0	0	0	0	2 ± 0	2 ± 0
31	furan-2-yl	4	Cl	1	60	8 ± 1	14 ± 2	1 ± 1	0	3 ± 0	2 ± 0
32	furan-3-yl	3	Cl	1	46	0	0	0	0	3 ± 0	2 ± 0
33	3-methyl-isoxazol-5-yl	2	Br	24	40	7 ± 1	25 ± 3	8 ± 1	0	7 ± 1	3 ± 0
34	3-methyl-isoxazol-5-yl	3	Br	2	64	0	0	0	0	3 ± 1	2 ± 0
35	3-methyl-isoxazol-5-yl	4	Br	2	67	20 ± 2	24 ± 2	6 ± 0	0	2 ± 1	2 ± 0

^*compounds 5–16 acetone, 60–70 °C, 2–24 h; 17–35 MeCN, 70–80 °C, 2–24 h;

^areactivator concentration

Each reactivator varied with respect to the optimal concentration used, and four trends were identified from the data (Table 1). First, a correlation was found between percent reactivation and increasing concentration of oxime (5, 7, 8, 11–16). A second trend was found that inversely correlates increasing reactivation with decreasing concentration of oxime (20, 22–24, 27–29). Third, compounds 17, 21, 25, 26, 31, 33, 35 showed the best percent reactivation at mid-range concentrations of oxime.¹⁹ Last, analogs bearing the oxime in the 2-position were better reactivators than those with oximes in the 3- or 4-position. Of the compounds with heteroaromatic side chains, thiophene analogs 20, 23, 26 and 29 with the 2-oxime group reactivated paraoxon-inhibited EeAChE 34%, 37%, 24% and 28% at 10⁻⁵ M, respectively. These thiophene analogs also reactivated rHuAChE but only ~5% at 10⁻⁵ M. Oximes with a thiophen-2-yl side chain (e.g., 20 and 23) were slightly better reactivators than the corresponding thiophen-3-yl structures (26 and 29). The furan analogs 30–32 were poor reactivators but the 3-methylisoxazole analogs 33 and 35 showed 24–25% reactivation of EeAChE at 10⁻⁴ M. The 4-methoxybenzyl analogs 17–19 were poor reactivators of paraoxon-inhibited EeAChE and rHuAChE although the 2- and 4-positional oximes 17 and 19 reactivated AChE up to 19% of its activity.

Owing to their greater activity, compounds 8, 13, 20, 23 and 26 were selected for analysis of the oxime-mediated reactivation rate constants (k_r; characterizes the dissociation of the enzyme and the phosphorylated-oxime). ³⁹ The k_r was calculated using the equation from the linear portion of activity curves (0–15 min).⁴⁰

As expected from the preliminary data (Table 1), these oximes showed 8 to 21-fold greater k_r values for reactivation of paraoxon-inhibited EeAChE than rHuAChE. Compound 8 showed the highest k_r values among the analogs tested against for both EeAChE and rHuAChE, and 8 was superior to 2-PAM for rHuAChE reactivation. Also interesting was the trend of k_r values 8 > 20 > 23 > 26 ≥ 13 that was the same for both EeAChE and rHuAChE (Table 2).

Table 2.

Reactivation rate constant (k_r) for 8, 13, 20, 23 and 26

Entry	kr [min−1]
	EeAChE	rHuAChE
2-PAM (1)	0.1766 ± 0.0134	0.0041 ± 0.0003
8	0.0406 ± 0.0021	0.0048 ± 0.0004
13	0.0108 ± 0.0002	0.0010 ± 0.0002
20	0.0339 ± 0.0026	0.0016 ± 0.0001
23	0.0189 ± 0.0004	0.0012 ± 0.0001
26	0.0138 ± 0.0008	0.0010 ± 0.0001

Paraoxon-inhibited EeAChE undergoes greater than 60% reactivation with 2-PAM from 10⁻³M to 10⁻⁵M (Table 1). Conversely, 2-PAM reactivates paraoxon-inhibited rHuAChE to a far lesser extent at 10⁻³M (42%) and is barely reactivatable at 10⁻⁵M (7%). This suggests that rHuAChE will eventually become unable to reactivate at all following inhibition by paraoxon. This may be due to one or more non-reactivation processes including denaturation or aging. The amino acid sequences of rHuAChE⁴¹ and EeAChE⁴² are 89% homologous and do not reveal any obvious differences to account for the 8 to 21 fold greater rate of reactivation observed for EeAChE. However, species-specific differences in reactivation rates following inhibition by a variety of OP agents are known^43,44 and any data comparisons should consider these differences.

In conclusion, a new series of monoquaternary pyridinium oximes with heteroaromatic side chains have been reported of which thiophene-containing analogs 20, 23 and 26 were the better reactivators. Compound 8, bearing an acetic acid side chain, was found to be the most effective reactivator of the 31 novel compounds tested with a higher k_r value than pralidoxime (1) for paraoxon-inhibited rHuAChE.