Application of the Ugi Multicomponent Reaction in ... - ACS Publications

Article Cite This: J. Med. Chem. 2017, 60, 9376-9392

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Application of the Ugi Multicomponent Reaction in the Synthesis of Reactivators of Nerve Agent Inhibited Acetylcholinesterase Martijn C. de Koning,*,† Marloes J. A. Joosen,† Franz Worek,‡ Florian Nachon,§ Marco van Grol,† Steven D. Klaassen,† Duurt P. W. Alkema,† Timo Wille,‡ and Hans M. de Bruijn†,∥ †

TNO, Lange Kleiweg 137, 2288 GJ Rijswijk, The Netherlands Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany § Département de Toxicologie et Risques Chimiques, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France ‡

S Supporting Information *

ABSTRACT: Recently, a new class of reactivators of chemical warfare agent inhibited acetylcholinesterase (AChE) with promising in vitro potential was developed by the covalent linkage of an oxime nucleophile and a peripheral site ligand. However, the complexity of these molecular structures thwarts their accessibility. We report the compatibility of various oxime-based compounds with the use of the Ugi multicomponent reaction in which four readily accessible building blocks are mixed together to form a product that links a reactivating unit and a potential peripheral site ligand. A small library of imidazole and imidazolium reactivators was successfully synthesized using this method. Some of these compounds showed a promising ability to reactivate AChE inhibited by various types of CWA in vitro. Molecular modeling was used to understand differences in reactivation potential between these compounds. Four compounds were evaluated in vivo using sarin-exposed rats. One of the reactivators showed improved in vivo efficacy compared to the current antidote pralidoxime (2-PAM).

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INTRODUCTION Acetylcholinesterase (AChE) is a serine hydrolase that has an active site (or A-site) at the bottom of a long narrow gorge and a second substrate binding site at the entrance of the gorge, called peripheral anionic site (PAS or P-site).1−3 The A-site catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) at cholinergic synapses. The activity of AChE can be arrested by covalent phosphylation of the key serine residue in the active site with organophosphate (OP) compounds, such as insecticides (e.g., chlorpyrifos oxon) and nerve agents (e.g., sarin, soman, tabun, VX, etc.).4 The resulting accumulating levels of ACh lead to overstimulation of the cholinergic receptors and, as a consequence, neuromuscular failure, seizures, and ultimately death. Current treatment of OP poisoning comprises administration of atropine as an antimuscarinic agent, an anticonvulsant drug such as diazepam and an AChE reactivator. AChE reactivators such as the standard pyridinium oximes pralidoxime (2-PAM),5 obidoxime,6 and asoximchloride7 (HI-6) restore the activity of OPinhibited AChE by oxime-mediated nucleophilic displacement of the phosphyl adduct (1a−c, Figure 1). However, none of these oximes show sufficient broad-spectrum efficacy for the treatment of poisoning by the various OPs. A second drawback is that while OPs readily reach the brain the current oximes © 2017 American Chemical Society

provide little protection of the central nervous system (CNS) because their permanent charges thwart their penetration of the blood−brain barrier. Over the past decades much effort has been directed in the development of nonionic reactivators, but in general, the absence of charge reduces the affinity of reactivators for the active site resulting in low reactivation efficacy. Several alternative strategies for the development of lipophilic (or neutral) reactivators have been reported in recent years,8,9 such as the development of reactivators possessing a basic site so that the reactivator is in equilibrium between a neutral state, enhancing brain penetration, and a charged state facilitating reactivation.10,11 Recently, several compounds were reported that lack the oxime functionality and exert their actions through a yet unclear mechanism of reactivation.12−15 Finally, several research groups used a promising strategy comprising the covalent linkage of a P-site ligand to a nucleophilic entity, via a spacer.16−28 The P-site ligand increases the affinity of the whole construct to the inhibited enzyme resulting in an improved overall reactivation efficacy. Figure 2 shows examples of structures reported by different research groups that were Received: September 16, 2017 Published: November 1, 2017 9376

DOI: 10.1021/acs.jmedchem.7b01083 J. Med. Chem. 2017, 60, 9376−9392

Journal of Medicinal Chemistry

Article

Figure 1. Currently employed oximes in the treatment of patients intoxicated by nerve agents or OP pesticides.

Figure 2. Examples from recent papers on PSL-reactivator conjugates.

Figure 3. Synthesis of a possible Ugi product as an example of the envisaged design and synthetic strategy.

prepared following this strategy. The successful use of a variety of P-site targeting structures in these hybrid compounds illustrates that the P-site of the protein is capable of accommodating structurally versatile ligands. It is further evident that the covalent connection of two molecules with different tasks (affinity/nucleophilicity) via a spacer leads to compounds of increased structural complexity, at least compared to the current antidotes, which is in many cases reflected in longer synthesis routes, less flexibility, and low overall yields. Thus, there is a need for synthetic strategies that allow the rapid access to these types of structurally complex molecules. In this contribution, we have explored the Ugi multicomponent reaction29 as a synthetic tool to rapidly generate a library of potential reactivators of OP-inhibited human AChE (hAChE) from readily available or commercial building blocks. The Ugi reaction entails the condensation of (near) equimolar amounts of an aldehyde or ketone, an amine, a carboxylic acid, and an isocyanide to give the corresponding Ugi product in a

single chemical transformation with high atom efficiency (water is the only byproduct). The envisaged synthetic strategy is exemplified with the synthesis of a possible Ugi product in Figure 3. Thus, an oxime nucleophile connected to a carboxylic acid via a spacer serves as the starting compound in the Ugi multicomponent reaction and is reacted with a commercially available amine, formaldehyde, and an isocyanide to give the target compound in a single synthetic step. In this paper we demonstrate that this strategy provides rapid, flexible, and efficient access to hybrid compounds in which an oxime nucleophile is connected via a spacer to a peptide-like, branched structure (the Ugi scaffold structure) carrying molecular entities originating from the amine, isocyanide, and the aldehyde. It was anticipated that the variability of the latter three moieties could lead to the discovery of structures that favor interaction with the peripheral site residues of AChE. We demonstrate that some of these structurally novel reactivators possess promising in vitro reactivation efficacy toward nerve agent inhibited AChE. Finally, preliminary in vivo efficacy 9377



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Figure 4. A set of different nucleophiles connected to a carboxylic acid via spacers of varying lengths were used to probe the feasibility of the Ugi reaction in the presence of the reactive oxime functionality. The overall yields and the number of steps required for the synthesis of the starting compounds are provided in parentheses after the compound numbers. Yields (%) of isolated Ugi products are provided in parentheses.

gradient of methanol in water on a reversed phase (C18) column). In all cases, the desired Ugi products were successfully obtained except for the nitrile containing ketoxime (2c), which was converted under Ugi conditions into a single major (but unidentified) product having a molecular weight 10 Da lower than expected. All Ugi products were isolated in moderate to good yields except the Ugi product from the CF3containing ketoxime (2a) which could not be satisfactorily purified because of the formation of many byproducts. The losses in yields of the isolated Ugi compounds were predominantly due to the purification process rather than an incomplete conversion of starting materials. Inspection of 1H and 13C NMR spectra revealed double signals for all resonances, presumably incurred by the presence of rotamers. Further characterization was accomplished by HRMS and LC− MS, which corroborated the identity and purity of all compounds. To our best knowledge, there are no previous reports of Ugi reactions carried out in the presence of unprotected oxime groups. Actually, several reports demonstrate that unprotected oximes can serve as the electrophile in one of the key transformations in the Ugi reaction mechanism30,31 to furnish N-alkylated hydroxamic acids, albeit that these reactions required long reaction times or activation by a Lewis acid. Inspection of the LC−MS traces of the reaction mixtures of the Ugi reactions described in this paper did not reveal the formation of any N-alkylated hydroxamic acids. Apparently, the reaction with the imine species is favored over the reaction with the oxime functionality while having both imine and oxime functionalities present at the same time. An explanation is that the electrophilicity of the carbon atom in oximes is generally considered to be moderate compared to those of regular imines as a result of the low pKa of the nitrogen atom in oximes.32 This may also explain the need for long reaction times or the use of a Lewis acid to activate the oximes in the examples mentioned above.

evaluation was conducted using sarin-poisoned rats with a selection of the best performing compounds so far.

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RESULTS AND DISCUSSION

In order to probe the feasibility and scope of the synthetic strategy, a number of oxime nucleophiles of various nature were functionalized with a carboxylic acid linker of varying lengths to give compounds 2−5 in Figure 4. Thus, these compounds included charged pyridinium oximes, an uncharged imidazole oxime,8,25 several substituted ketoximes,27 and hydroximinoacetamides.8,9 These compounds were accessible in one to three simple steps from commercially available building blocks (the amount of synthetic steps required and the overall yields are provided in parentheses in Figure 4 for each of these compounds). Each of these building blocks (0.15−0.75 mmol) was reacted with a slight excess (1.05−1.5 equiv) of 4methoxybenzylamine (6a), isopropyl isocyanide (7a), and formaldehyde (8) in MeOH (or MeOH/water), using an end concentration of about 0.25 M of the starting oxime acid. At this stage, the choice of the amine (6a) was based on the notion that P-site ligands are often composed of aromatic groups and the aliphatic isocyanide was chosen to facilitate structural elucidation with NMR of the expected Ugi products. Formaldehyde (8) was chosen to prevent the formation of stereoisomers which was expected with the use of monosubstituted aldehydes or unsymmetrical ketones. Stereoisomer formation could also be avoided by using a symmetrical ketone (e.g., acetone), but those reactions were sluggish and gave much lower yields in a number of pilot experiments. TLC and HPLC analysis showed that conversion times varied between 1

0.18

>1

>1

>1

0.1 82 1.5 0.1 89 1.4 0.01 48 0.2 >1

0.2 213 0.7 0.1 167 0.7 0.02 121 0.2 >1

0.1 29 5.2 0.2 179 1.0 0.01 17 0.6 >1

0.3 352 0.9 0.06 122 0.5 0.04 80 0.5 ∼0.05

nr = negligible reactivation; kr in min−1; KD in μM; kr2 in mM−1 min−1; IC50 in mM.

several aliphatic and aromatic amines (6a−c) and isocyanides (7a and 7b) to give a range of Ugi products in which the Ugi scaffold structure was varied with different combinations of aliphatic and aromatic substituents of different polarity and positioned at varying distances from the imidazole nucleophile. All reactions successfully led to the isolation of the thirteen corresponding Ugi products (12, 13, and 14 series) in 17−76% yield. All compounds were characterized by HRMS, 1H NMR, and 13 C NMR. Additional characterization encompassed the determination of the pKa of the oxime functionalities in a couple of representative Ugi compounds. The pKa values were measured by using a 1H NMR method in which the chemical shifts of one of the two resonances of the CHNOH protons in 2 mM solutions of the oximes in various aqueous buffers (pH ranging from 4 to 13) were plotted against the respective pH (see Supporting Information). Two nonionic compounds from the 12 series both had a pKa of about 10.2. Similar pKa values were assumed for the other nonionic compounds (12, 13, and 14 series). For comparison, the pKa’s of the oxime groups in the pyridinium Ugi product 11a (pKa = 8.5) and 4-PAM (pKa = 8.6) were also determined. The pKa’s of the regular oximes (e.g., 1a) are also close to 8. It is generally accepted that the presence of a higher fraction of reactive oximate anions during reactivation (i.e., a lower pKa) would result in more effective reactivation. Thus, with the aim to lower the pKa of the imidazole oxime functionality all nonionic Ugi products were subjected to reaction with iodomethane to furnish the corresponding imidazolium oxime compounds (15, 16, and 17 series; see Figure 6). These alkylations proceeded uneventfully and in good yields for all compounds. The pKa’s of a couple of imidazolium compounds were measured and determined to be 8.3−8.4, much closer to the pKa value of about 8 of the current oximes. Although the alkylation step resulted in improvement of the pKa value, a down-side to that approach is the introduction of a (permanent) charge. On the other hand, the resulting compounds were expected to be much more lipophilic than the current oximes and might therefore have more likelihood to cross the brain barrier than current pyridinium oximes.35−38 Indeed, in silico calculations39 of the log D7.4 values (log distribution in octanol−water at pH 7.4) predicted a higher lipophilicity for the imidazolium compounds (log D7.4 ranges between −2.1 and −0.9) compared to 1a (−4.3), 1b (−7.0), and 1c (−8.8). Moreover, their relative lipophilicity was also practically illustrated by their ready purification, despite the

show an ability to reactivate GB-hAChE (Figure 5). While the hydroximinoacetamide products (10a and 10d) only showed marginal reactivation potency, both pyridinium oxime compounds (11a and 11b) effectively reactivated GB-hAChE. The former compound clearly displayed a biphasic reactivation curve in which the initial reactivation rate was attenuated at later time points, indicative of reinhibition of the enzyme by the accumulating in situ formed phosphonyloxime.34 Reactivation by compound 11b proceeded considerably more slowly than by compound 11a, showing that the distance between the reactivating pyridinium oxime part (five methylene groups in 11a and one methylene unit in 11b) and the alleged P-site binding part (originating from the amine and isocyanide) had a significant impact on the reactivation potency. The latter finding indicates that their ability to reactivate was not only facilitated by the presence of a pyridinium oxime functionality but also influenced by the positioning of the additional chemical entities attached to it. Moreover, it was found that the corresponding Ugi starting materials (i.e., compounds 4a and 4b) possessed very limited reactivation potency toward GB-inhibited hAChE (data not shown), indicating that the additional structural features installed by means of the Ugi reaction were beneficial for the reactivation rate. Similarly, we found that the nonionic imidazole oxime reactivator (12a) showed appreciable reactivation of GB-hAChE, while its oxime precursor (compound 3a) had no reactivation potency at all when tested at the same concentrations (data not shown). Although the overall reactivation rates of these Ugi oximes were still much lower than those of the current oximes, these initial results were considered very encouraging as they indicated that the reactivation potency of a suitable nucleophile can be improved by its attachment to an Ugi scaffold structure. It was further envisaged that optimization of the Ugi scaffold structure (i.e., structure and distance from the nucleophile) would be greatly facilitated by the ability to rapidly access new molecules by virtue of the one-step Ugi reaction from easily accessible or commercial building blocks. The imidazole oxime based Ugi compound (12a) was selected as a lead compound for further development as it was the best nonionic reactivator from the set tested. In order to explore whether its in vitro reactivation potency could be improved, additional imidazole oxime based starting materials 3b and 3c (Figure 6), having respectively three and one methylene groups between the imidazole and carboxylic acid moieties, were synthesized. The thus obtained starting compounds were now reacted with formaldehyde (8) and 9380



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Figure 7. Selected compounds for detailed kinetic analysis and preliminary in vivo evaluation.

Table 2. Summary of Reactivation Kinetics Parameters (Human Erythrocyte AChE) for a Selection of Ugi Oximes and Reference Oximes33,40,a compound GA

GB

GF

VX

a

kr KD kr2 kr KD kr2 kr KD kr2 kr KD kr2

1a

1b

0.01 706 0.014 0.250 27.6 9.06 0.182 3159.0 0.06 0.215 28.1 7.67

0.04 97.3 0.41 0.937 31.3 29.98 0.395 945.6 0.42 0.893 27.4 32.63

1c

12c

nr

nr

0.677 50.1 13.50 1.300 47.2 27.55 0.242 11.5 20.98

0.048 175.3 0.271 0.096 950.0 0.102 0.084 140.3 0.602

12d

15a

15c

0.108 ± 0.006 1199.5 ± 109.5 0.090 ± 0.003 0.073 ± 0.005 228.9 ± 31.7 0.322 ± 0.024 0.174 ± 0.006 815.8 ± 41.9 0.213 ± 0.004

0.012 ± 0.000 74.3 ± 0.3 0.168 ± 0.002 0.139 ± 0.004 98.0 ± 2.1 1.413 ± 0.013 0.106 ± 0.012 134.5 ± 24.7 0.800 ± 0.054 0.168 ± 0.012 115.9 ± 12.6 1.458 ± 0.055

0.010 ± 0.000 19.4 ± 0.4 0.536 ± 0.025 0.167 ± 0.021 66.1 ± 11.9 2.551 ± 0.141 0.182 ± 0.005 35.4 ± 2.3 5.156 ± 0.180 0.138 ± 0.001 52.0 ± 3.9 2.674 ± 0.172

nr

± ± ± ± ± ± ± ± ±

0.001 2.3 0.001 0.004 58.0 0.002 0.002 0.7 0.011

nr = negligible reactivation; kr in min−1; KD in μM; kr2 in mM−1 min−1.

extend (kr2 ≈ 0.03 mM−1·min−1), all other compounds from the 12 series demonstrated an equal or improved ability to reactivate GB-hAChE (kr2 varied between approximately 0.03 and 0.2 mM−1·min−1). The best compound of this series (12c) was about 7-fold more efficient in reactivating GB-hAChE than the lead compound 12a. Moreover, this compound also showed reasonable reactivation of GF-hAChE (kr2 ≈ 0.1 mM−1·min−1). The only other compound from this series that showed a comparable GF-hAChE reactivation rate was 12d. As judged from the obtained values for kr and KD (Table 1), the main determinant for the observed reactivation rates of the 12 series was the affinity of the reactivator for the inhibited enzyme (KD), which is quite evident for GF-hAChE and slightly less pronounced for GB-hAChE. Faster reactivation was observed by the corresponding imidazolium compounds (i.e., the 15 series). While the imidazolium Ugi products from the 16 and 17 series, having respectively three and one methylene units spacer length, showed no or only very slow reactivation, some of the 15 series compounds showed promising reactivation efficacies. All of the compounds from this series had the ability to reactivate GBhAChE as well as GF-hAChE in rates varying at 0.7−5.2 mM−1· min−1 (GB-hAChE) and 0.5−1.4 mM−1·min−1 (GF-hAChE). For instance, compound 15c reactivated GB-hAChE about 175fold faster than the aforementioned nonionic lead compound. It was further very gratifying to find that the 15 series of oximes were also able to reactivate tabun-inhibited hAChE, which is notoriously difficult to reactivate. The reactivation rates of GAhAChE were mainly attributable to high affinities (KD’s of ∼20−100 μM), while the reactivation rate constants kr were still quite low (∼0.01−0.04 min−1). From the compounds

presence of a permanent charge, using regular straight phase silica gel column chromatography using an eluent of methanol in dichloromethane (8−12%, v/v). All 23 compounds in Figure 6 were evaluated for their ability to reactivate nerve agent inhibited hAChE. To this end GB, GA, and GF adducts of hAChE (ghosts) were incubated with three different concentrations (ranging from 10 to 1000 μM) of reactivator and the fraction of reactivated hAChE was determined at multiple time points up to 60 min. This way, the screening of reactivation efficacy of multiple OP-hAChE adducts by the 23 Ugi reactivators allowed the efficient generation of rough estimates of the kinetic parameters kr (reactivation rate constant), KD (affinity constant), and the overall bimolecular reactivation rate constant kr2 (=kr/KD). The IC50 values were also determined. These numbers provided an idea of the underlying mechanism of reactivation and were used for down-selection of candidates for more detailed kinetic analysis. The compounds from the 14 series (one methylene group) did not show any reactivation up to 0.25 mM concentrations (Table S1). The compounds from the 13 series (three methylene units) only showed marginal reactivation of GB- and GF-inhibited hAChE and no reactivation of GAhAChE at 1 mM concentration (Table S2). In contrast, the compounds from the 12 series (five methylene units) all showed the ability to reactivate GB-hAChE as well as GFhAChE but not GA-hAChE (Table 1). First of all, these results confirmed the dependency of the reactivation efficacy on the number of methylene units between the nucleophile and the Ugi scaffold structure for all compounds tested. For instance, compared to the lead compound 12a, which only reactivated GB-hAChE to some 9381



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Figure 8. Molecular docking of selected compounds in the active site of GA-inhibited human acetylcholinesterase (model based on PDB entries 4ey4 and 3dl4; see Supporting Information). The binding energy determined by the scoring function of Autodock Vina and the distance between the phosphorus atom of GA and the oxime oxygen atom are indicated in the top left. The reactivator (C atoms in green) and the catalytic serine GA conjugate (C atoms in light gray) are represented in ball and stick. Key residues of the active site are represented in sticks.

tested 15c showed the best overall reactivation efficacy followed by 15a. The use of aromatic amines and/or isocyanides (i.e., 6a and 7b) seemed beneficial for reactivation, presumably by improving the affinity of the compounds. The improved affinity was also reflected in the IC50 value (0.18 mM) for 12c. On the other hand, 15c did not exhibit a low IC50 value. All other compounds had IC50 values of >0.25 mM. When the reactivation rates of individual imidazolium compounds from the 15 series (i.e., 15c) were compared with the corresponding nonionic imidazole compounds from the 12 series (i.e., compounds that only differ in the nucleophilic entity), it can be concluded that the reactivation rate constant kr increased by 3- to 5-fold and that the affinity constants KD improved by 4- to 9-fold with the use of an imidazolium nucleophile. The improved reactivation rate constants may be attributed to the more favorable pKa, while the observed higher affinity may be incurred by the presence of charge. For comparison, the oxime acids 3a−c as well as the Nalkylated version of 3a (i.e., compound 18) were also tested for reactivation efficacy (data not shown). Compound 18 was separately synthesized for this purpose. None of these oxime acid starting compounds showed notable reactivation up to 1 mM concentration except for 18, which gave some reactivation of GB-hAChE and GF-hAChE at 1 mM concentration but was still much slower than the corresponding Ugi products. The GA-hAChE adduct was not reactivated at all by 18 (up to 1 mM for 60 min). The slow or absence of reactivation by these compounds further demonstrates that the presence of the Ugi configuration contributed to the reactivation efficacy of the whole construct. The two best compounds from both the 12 series (i.e., 12c and 12d) and the 15 series (i.e., 15a and 15c) were selected (Figure 7) for a more elaborate kinetic analysis, in which the reactivation kinetics of GB-, GF-, GA-, and VX-inhibited hAChE were determined using additional oxime concentrations. The results of these experiments are provided in Table 2. For comparison, the kinetic parameters of the current oximes (i.e., 1a−c) were also added.33,40

The new Ugi oximes all performed worse than 1c for all OPs except for tabun and worse than 1a and 1b in the cases of GBand VX-inhibited hAChE. On the other hand, 15c reactivates GA-hAChE with a comparable rate as 1b and about 40 times faster than 1a. For the reactivation of GF-hAChE all four (ionic as well as nonionic) oximes outperformed 1a (2−85 times faster). Moreover, the two charged oximes (15 compounds) performed either comparable to or better (about 12 times) than 1b in the reactivation of GF-hAChE. Molecular Docking. In order to get structural insights on the reactivation of selected compounds 12d, 12c, 15a, and 15c, we performed molecular docking on hAChE phosphylated by GA, GB, GF, and VX. The primary aim of this modeling study was to determine if the molecule can bind in a productive way, i.e., with the oxime function in close proximity to the catalytic serine-OP adduct. Flexible docking experiments were undertaken in which the side chains of active site gorge residues Tyr337 and Trp286, both known to be highly flexible and key to the binding of the reactivators, and the alkoxy chain of the nerve agents were allowed to wander from their native position (see Supporting Information). The oxime was protonated as well as the abnormally basic Glu202, whereas the catalytic His447 was unprotonated, in agreement with a recent QM/ MM study on the protonation state of active site residues that are compatible with the reactivation reaction.41 One additional difficulty with docking of Ugi compounds is their high number of rotatable bonds (>10) yielding a large conformational space that is difficult to sample with reasonable computation times, especially when it is combined with flexibility of active site residues. Therefore, we optimized the search for a reactivator conformation relevant to reactivation by developing a specific algorithm based on the evaluation of the phosphorus−oxime distance and performing docking trials until a distance threshold is met (see Supporting Information). This strategy allowed us to rapidly verify if a particular molecule candidate can adopt a productive conformation. Analysis of the docking results shows that each of the four molecules can adopt this productive conformation for GA, GB, and VX inhibited with the oxime at 4−5 Å distance from the P 9382



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Figure 9. Percentage of animals showing convulsions (A) and seizures (B) over time. Development of convulsions and seizures was significantly different between groups over the 4 h observational period (Gehan−Breslow−Wilcoxon test). Oxime 15c significantly diminished the development of convulsions and seizures over time compared to 1a (p < 0.025, Gehan−Breslow−Wilcoxon test, corrected for multiple comparisons).

Figure 10. Total EEG power of rats following 1.8 LD50 sarin (130 μg/kg), treated with 3 mg/kg atropine im and ∼16 μmol/kg of each oxime iv (15c and 15a in panel A and 12c and 12d in panel B compared to naive controls and 1a). The 4 h AUC is shown in panel C. All data are shown as average ± standard error of the mean for n = 5/6 animals per group. Only 15c performed significantly better than 1a (p < 0.05, one-way ANOVA, followed by Bonferoni’s post hoc test, compared to animals treated with 1a).

Neither a significant difference in terms of binding energy (approximately −9/−10 kcal/mol) nor a significant difference in terms of distance (∼4 Å) is evidenced, suggesting that binding in a productive way is not discriminatory for the four molecules. Therefore, the absence of significant reactivation for 12d/12c compared to 15a/15c must be principally related to the higher pKa of the imidazole aldoxime function compared to the imidazolium aldoxime. There seems to be no impact of the removal of one aromatic on binding as 12c/12d on the one side and 15a/15c on the other side all have similar energy scores. Finally, comparison of 12c and 15c which differ only by the imidazolium exemplifies the diversity of binding modes of the aromatic part due to the multiple rotatable bonds that largely compensate for the slight rigidity of the two peptide

atom and the scoring function yielding binding energies of about −9 kcal/mol (see Supporting Information). In these cases, the oxime group makes a hydrogen bond with Glu202 or His447, and the imidazole ring tends to make π−π parallel stacking with Trp86 at the bottom of the gorge, with additional aromatic−aromatic interactions at peripheral aromatic site (Trp286, Tyr72, Tyr124). For GF, none of the selected molecules can approach the P atom at less than 6.9 Å due to the steric hindrance of the cyclohexyl group (see Supporting Information). Yet reactivation kinetic data are similar for GF, GB, and VX, suggesting that a large conformational change of the active site, not simulated by the docking software, must occur to yield a productive binding mode. The biggest difference in kinetics data was with tabun, so we focused on the docking results for GA-hAChE (Figure 8). 9383



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bonds in the Ugi structure which are not allowed to rotate during docking. In Vivo Efficacy. The compounds depicted in Figure 7 were also used to conduct limited in vivo efficacy testing. Male Wistar rats (±300 g) were chosen for this purpose because rat AChE reactivatability was reported to be more comparable with the human enzyme than that of guinea pig AChE.40 All experiments received approval from the animal ethical committee. Briefly, rats were surgically equipped with a jugular vein catheter and EEG electrodes according to previously described procedures.42,43 After 4 days of recovery, the rats were sc exposed to sarin (130 μg/kg, ∼1.8 LD50) followed after 1 min by an intramuscular treatment of 3 mg/kg atropine sulfate and an iv administration (1 mL/kg) with 16 μmol/kg of the new oximes (8−10 mg/kg) or the reference oxime 1a (2.8 mg/kg). This molar dose was based on solubility and tolerability of the new oximes. A higher molar dose of oxime 15c (32 μmol/kg (iv) without nerve agent led to labored breathing and lethality, urging to lower the dose. At the lower dose of 16 μmol/kg, sensitivity to light was observed. For the other compounds, no signs of toxicity were observed at this equimolar dose. No extensive toxicological assessment was conducted in view of the aim of the study at this time. A relatively low challenge dose was chosen to allow for either improved or impaired efficacy compared to 1a. After exposure to the nerve agent, cholinergic signs (chewing, shivering, salivation, tremor, convulsions, respiratory distress, and death) were monitored visually and the onset of these signs was scored according to standard procedures.44 Total EEG power was telemetrically recorded throughout the experiment with a minimum baseline of 30 min prior to sarin exposure. Blood samples were drawn at 30 and 240 min after exposure for measurement of cholinesterase activity. Brain cholinesterase activity was measured from brain homogenates after the 240 min end point. None of the oxime treatments could prevent all clinical signs such as chewing, shivering, or tremors. More severe signs, such as convulsions (Figure 9A) and seizures (Figure 9B) occurred in part or in all of the animals despite treatment with 1a or oximes 12c, 12d, and 15a. In contrast, oxime 15c prevented convulsive activity as well as seizures in all animals. Although the numbers between groups varies, none of the oximes performed significantly better compared to 1a with regard to the development of the clinical signs, except oxime 15c, which suppressed convulsions significantly better than 1a. These results were complemented by the EEG measurements (Figure 10). Figure 10A shows the EEG power measured over time of the animals treated with oximes 15c and 15a. For comparison, the graphs of the control group and the 1a treated group are displayed as well. Figure 10B shows the results for the oximes 12c and 12d. Analysis of the 4 h AUC of the EEG total power (Figure 10C) confirmed that only 15c performed significantly better than 1a. Blood cholinesterase activity at 30 min and 4 h after exposure is shown in Figure 11. None of the oximes induced a significant reactivation in blood 30 min after sarin injection, and no significant differences between oximes were observed. At 4 h after sarin injection 15c and 12d performed significantly worse than 1a. Except for 15c and 12d, the oximes showed significantly higher reactivation in blood at 4 h compared to the 30 min time point. The brain cholinesterase activity at 4 h after sarin challenge and iv treatment with the various oximes is shown in Figure 12,

Figure 11. Residual cholinesterase activity in blood of animals exposed to sarin (130 μg/kg sc) and treated with atropine sulfate (3 mg/kg im) and one of the new oximes iv (16 μmol/kg at 1 min), at 30 min, and at 4 h after sarin exposure. Cholinesterase activity is expressed as % of pre-exposure blood cholinesterase activity of the individual animals. Significant differences compared to 1a (∗) and between 30 min and 4 h after exposure (#) are shown (two-way ANOVA followed by Dunnett’s post hoc test; results were considered significantly different for p < 0.05).

panel A. In this case, only minimal brain cholinesterase reactivation was achieved by 1a and 12c. In contrast, 15c reached significantly higher brain cholinesterase reactivation than 1a. In Figure 12B the results of the animals are split based on the presence or absence of seizures. Animals not displaying seizures showed significantly higher levels of brain cholinesterase activity over the various groups. This shows that, in contrast to 1a, most of the newly designed oximes were able to induce higher levels of brain reactivation in some animals. Assuming similar pharmacokinetic profiles of the new oximes, these results might indicate that the oximes show potential for increased efficacy as a result of increased brain penetration.

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CONCLUSION We report that the Ugi four-component reaction is compatible with the presence of unprotected oxime functionalities and enabled fast access to relatively complex AChE reactivators in a single condensation step from readily available and commercial building blocks. It was further shown that this flexible route gave rapid access to a small library of reactivators, the majority of which comprised imidazole oxime and imidazolium oxime nucleophiles connected via a spacer to a peptide-like scaffold structure. It was further demonstrated that this type of compound was capable of reactivating various OP-inhibited hAChEs in vitro. The presence of the substituted scaffold enhanced the reactivation potency of the whole construct compared to the nucleophile alone, predominantly by improvement of the affinity for the inhibited enzyme. The substituents on the scaffold could be easily varied by simply choosing different amine/isocyanide combinations in the Ugi reaction, thus allowing ready optimization. The in vitro reactivation potency of the uncharged imidazole reactivators was lower than that of the imidazolium compounds, caused by the more reactive nucleophile (lowered pKa) as well as improved affinity (as result of the charge). The imidazolium oximes were overall less potent in vitro than the current oximes, although the new oximes sometimes outperformed current oximes in specific OP/oxime combinations. The in vivo efficacy of a selection of four new compounds was compared with 1a and administrated in equimolar doses to 9384



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Figure 12. Cholinesterase activity in the brain of rats exposed to sarin (130 μg/kg sc) at 4 h after exposure. Animals were treated at 1 min with an oxime (16 μmol/kg iv) and atropine sulfate (3 mg/kg, im). The left panel shows average + standard error of the mean of all animals per group. Panel B shows results split based on the presence or absence of EEG seizure activity; numbers indicate the number of animals in each group. Significant reactivation was achieved compared to 1a for 15c (∗, p < 0.05). # indicates a statistically significant difference in reactivation activity between seizing and nonseizing animals for 12d (one-way ANOVA followed by Dunnet’s post hoc test; results were considered significant for p < 0.05). collision energy between 11 and 25 eV, with argon as the collision gas (at an indicated pressure of 10−4 mbar). HRMS spectra were recorded on a Bruker maXis Impact Q-TOF. Other mass spectrometric analyses were carried out on a TSQ Quantum Ultra mass spectrometer (Finnigan, Thermo Electron Corporations, San Jose, CA, USA) equipped with an Acquity Sample Manager and Binary Solvent Manager (Waters, Milford, USA). For LC−MS experiments, the liquid chromatograph was connected to the mass spectrometer source via the sample manager equipped with a 10 μL loop and an Acquity BEH C18 column (1.7 μm particles, 1 mm × 100 mm; Waters, Milford, USA). The liquid chromatography system was run with a 25 min linear gradient from 100% A to A/B 55.5/45.5 v/v (A, 0.2% formic acid in water; B, 0.2% formic acid in acetonitrile) at a flow rate of 0.09 mL/ min. The TSQ Quantum Ultra mass spectrometer was operated with a spray voltage of 3 kV, a sheath gas pressure of 41 A.U., auxiliary gas pressure of 2 A.U., and a capillary temperature of 350 °C. Positive electrospray product ion spectra were recorded at an indicated collision energy of 15−20 eV, using argon as the collision gas at a pressure of 1.5 mTorr. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance III spectrometer operating at 400 and 100 MHz, respectively. Chemical shifts (δ) are given in ppm relative to tetramethylsilane (δ 0 ppm) or to residual solvent signals. Identification and purities (≥95%) of compounds were determined using 1H NMR, 13C NMR, and HRMS. Pan Assay Interference Compounds. All relevant compounds were screened for PAINS using tier 0 calculations at the Cheminformatics Web site of the ADME Center of Excellence at USAMRICD, USA. http://cheminfo.bhsai.org. None of the compounds showed positive. Results are displayed in the Supporting Information (molecular formula strings file). Synthesis Procedures. 2-(Hydroxyimino)-3,3,3-trifluoropropanoic Acid (2a). 35 mg (0.5 mmol) of hydroxylamine hydrochloride was dissolved in 0.5 mL of water. The mixture was cooled to 0 °C, and then 78 mg (0.49 mmol) trifluoropyruvic acid was added. The mixture was stirred and allowed to warm to room temperature. After overnight reaction the mixture was neutralized with HCl, extracted with EtOAc (3×), dried, and concentrated. Yield: 0.24 mmol (48%). 1H NMR (400 MHz, MeOD): no peaks. 13C NMR (101 MHz, DMSO): δ 166.91; 159.75; 125.79; 123.5; 121.04; 118.34. 2-(Hydroxyimino)acetic Acid (2b). 6.95 mL (8.80 g, 100 mmol) of 2-oxoacetic acid (glyoxalic acid) was dissolved in 18 mL of water. Added was 6.95 g (100 mmol, 1 equiv) hydroxylamine hydrochloride. After overnight stirring the crystals were filtered and washed with icecold water. Yield: 93%. 1H NMR (400 MHz; CD3OD): δ1.99 (s, 3H, CH3). 13C NMR (100 MHz; CD3OD): δ9.07 (CH3); δ148.68 (C N); δ165.76 (COOH). ESI-MS: calcd, 104.0348; found, 104.0450 (MH+).

sarin-exposed rats. It was shown that, in contrast to 1a and the other oximes, 15c was able to fully suppress development of convulsions and seizures in all rats. Because of toxicity observed in an initial study with 15c, the doses for all compounds were lowered by a factor of 2. It is not known whether the other compounds would have shown higher tolerable doses and thus higher efficacy. We choose to adhere to equimolar administration of compounds to keep the study comparative. In contrast to 1a, two of the four tested oximes were able to induce higher levels of brain reactivation in some animals. Overall, the results showed that imidazole oxime or imidazolium oxime having the general and readily accessible Ugi structure constitute a novel and promising class of new reactivators. The disadvantage of the imidazolium oxime is that a permanent charge was again introduced. Therefore, current work aims at using nonionic nucleophiles with a sufficiently low pKa.

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EXPERIMENTAL SECTION

General Information. All compounds and solvents were used as received from the suppliers (Aldrich). Purifications were conducted by automated flash chromatography using a Biotage Isolera 4. Biotage columns were used for this purpose. Analytical LC was conducted on an AKTA system using an Alltima C18 analytical column (5 μm particle size, flow of 1.0 mL/min). Absorbance was measured at 214 and 254 nm. Solvent system: A, 5% ACN, 0.1% TFA; B, 80% ACN, 0.1% TFA. Gradients of B were applied over 20 min unless otherwise stated. HPLC purifications were conducted on the AKTA system supplied with a semipreparative Alltima C18 column (5 μm particle size, running at 5 mL/min). LC/electrospray tandem mass spectrometric analyses for obtaining structural information were conducted on a Q-TOF hybrid instrument equipped with a standard Z-spray electrospray interface (Micromass, Altrincham, U.K.) and an Alliance type 2690 liquid chromatograph (Waters, Milford, MA, USA). The chromatographic hardware consisted of a precolumn splitter (type Acurate; LC Packings, Amsterdam, The Netherlands), a six-port valve (Valco, Schenkon, Switzerland) with a 10 or 50 μL injection loop mounted, and a PepMap C18 (LC Packings) column (15 cm × 1 mm i.d., 3 μm particles). A gradient of eluents A (H2O with 0.2% (v/v) formic acid) and B (acetonitrile with 0.2% (v/v) formic acid) was used to achieve separation. The following gradient was applied: 0−5 min, 100% solution A, flow 0.1 mL/min; 5−60 min, 100% A to 30% A, flow 0.6 mL/min. The flow delivered by the liquid chromatograph was split precolumn to allow a flow of approximately 6 μL/min through the column and into the electrospray MS interface. MS/MS product ion spectra were recorded using a cone voltage between 15 and 30 V and a 9385



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2-(Hydroxyimino)-3-cyanopropanoic Acid (2c). 910 mg (23 mmol) NaOH was dissolved in water (5 mL). 2.855 g (20 mmol) of ethyl (hydroxyimino)cyanoacetate was dissolved in water (30 mL) and added dropwise to the NaOH solution. After overnight heating at 50 °C the mixture was neutralized with HCl and extracted 3× with EtOAc. After evaporation the obtained crystals were recrystallized using a mixture of EtOAc/hexane. Yield: 10.56 mmol (53%). 1H NMR (400 MHz, MeOD): no peaks. 13C NMR (101 MHz, MeOD): δ 160.36; 126.45; 108.07. 2-(Hydroxyimino)propanoic Acid (2d). 9.00 g (100 mmol) of 2oxopropanoic acid was dissolved in water (18 mL). Hydroxylammonium chloride (6.95 g, 100 mmol, 1 equiv) was added. After overnight stirring at room temperature the mixture was extracted thrice with diethyl ether (20 mL). After drying, filtration, and concentration the crude product was recrystallized from hexane/EtOAc (5:1). Yield: 60%. 1H NMR (400 MHz; CD3OD): δ7.46 (s, 1H, CH). ESI-MS (M + H+): calcd, 104.2; found, 104.1. 6-(2-Hydroxyiminomethyl-1H-imidazol-1-yl)hexanoic Acid (3a). A heterogeneous solution was made with 4.92 g (44.5 mmol, 1 equiv) of 1H-imidazole-2-carbaldehyde, ethyl bromohexanoate (16.99 g, 66.75 mmol), potassium carbonate (14.03 g, 89 mmol), and KI (8.43 g, 44.5 mmol) in acetonitrile (100 mL). The mixture was stirred for 64 h at 52 °C under argon. After the mixture was cooled to room temperature, it was filtered and the solvent of the filtrate was evaporated under reduced pressure. Flash chromatography (2 CV 100% DCM, 5 CV 100% EtOAc) was used to purify the product. Yield: 8.734 g, 36.7 mmol (82%). Rf = 0.6 (MeOH/DCM, 1/19, v/v); Rf = 0.46 (EtOAc). 1H NMR (400 MHz, CDCl3) δ 9.8 (s, 1H, CHO), 7.25 (d, J = 0.9 Hz, 1H, CHCH-arom), 7.14 (s, J = 0.9 Hz, 1H, CHCH-arom), 4.35 (t, J = 7.3 Hz, 2H, CH2), 4.05 (q, J = 7.1 Hz, 2H, CH2), 2.25 (t, J = 7.3 Hz, 2H, CH2), 1.75 (m, J = 7.4 Hz, 7.7 Hz, 2H, CH2), 1.6 (m, J = 7.4 Hz, 7.7 Hz, 2H, CH2), 1.3 (m, J = 8.3 Hz, 7.1 Hz, 2H, CH2), 1.2 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3) δ 181.0, 172.4, 142.6, 130.8, 125.8, 59.4, 46.7, 33.1, 29.9, 25.0, 23.6, 13.5. [M + H+] calcd (found): 239.1390 (239.1392). To a stirring solution of ethyl 2-(2-formyl-1H-imidazol-1-yl)hexanoate (3.83 g, 16 mmol) in MeOH (5 mL) were added 24 mL of an aqueous solution of Na2CO3 (1.9 g, 18 mmol) and hydroxylamine hydrochloride (1.68 g, 24 mmol). The emulsion was left to stir for another hour. Water (50 mL) and EtOAc (50 mL) were added. After separation of the layers, the organic layer was washed with 10% NaHCO3 (50 mL), water/brine (1/1, 50 mL), and brine (50 mL). After drying (MgSO4), filtration, and evaporation of the solvent the crude product (4 g) was purified using silica gel column chromatography using 68% EtOAc in hexane to give 3.23 g, 13 mmol (80%) of the product. Rf = 0.5 (MeOH/DCM, 1/19, v/v); Rf = 0.35 (EtOAc). 1H NMR (400 MHz, CDCl3) δ 12.6 (bs, 1H, N-OH), 8.3 (s, 1H, CHN), 7.3 (s, 1H, CH imid), 6.9 (s, 1H, CH imid), 4.3 (t, 2H, CH2), 4.1 (q, 2H, CH2), 2.2 (t, 2H, CH2), 1.8 (m, 2H, CH2), 1.6 (m, 2H, CH2), 1.3 (m, 2H, CH2), 1.2 (t, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 173.8, 171.2, 141.5, 140.4, 128.2, 122.8, 60.3, 47.6, 33.9, 29.9, 25.7, 24.2, 14.1. [M + H+] calcd (found): 254.1499 (254.1504). To a solution of ethyl 2-(2-hydroxyiminomethyl-1Himidazol-1-yl)hexanoate (260 mg, 1.02 mmol) in EtOH (2 mL) was added a solution of NaOH (200 mg, 5 mmol) in water (2 mL). The homogeneous mixture was stirred for 30 min. DOWEX 50X-400 (H+form) was added until the pH was about 6.5. The solution was filtered, and the DOWEX was washed with MeOH. The filtrate was concentrated and coevaporated with ACN (2×) and MeOH (2×) to give 193 mg (0.85 mmol) of the title compound as a foam (83%). HPLC (0−100% B in 20 min): tR = 8.4 min. 1H NMR (400 MHz, methanol-d4) δ 8.1 (s, 1H, CHN), 7.2 (s, 1H, CH imid), 7.05 (s, 1H, CH imid), 5.5−5.0 (bs, 2H, COOH/NOH ↔ NH exchanged), 4.3 (t, 2H, CH2), 2.25 (t, 2H, CH2), 1.8 (m, 2H, CH2), 1.65 (m, 2H, CH2), 1.35 (m, 2H, CH2). A single peak at 3.4 ppm (0.5H) was observed as an impurity originating from the Dowex resin; 13C NMR (101 MHz, methanol-d4) δ 179.7, 141.42, 141.39, 128.9, 124.7, 48.47, 36.4, 31.4, 27.0, 26.0. [M + H+] calcd (found): 226.1186 (226.1187). 4-(2-Hydroxyiminomethyl-1H-imidazol-1-yl)butanoic Acid (3b). A heterogeneous solution was made with 1.44 g (15.0 mmol, 1 equiv)

of 1H-imidazole-2-carbaldehyde, ethyl bromobutanoate (4.39 g, 22.5 mmol), potassium carbonate (4.15 g, 30 mmol), and KI (2.49 g, 15 mmol) in acetonitrile (35 mL). The mixture was stirred for 64 h at 52 °C under argon. After the mixture was cooled to room temperature, it was filtered and the solvent of the filtrate was evaporated under reduced pressure. Flash chromatography (2 CV 34% EtOAc/hexane, 3.6 CV 58% EtOAc/hexane) was used to purify the product. Yield: 2.46 g (11.7 mmol, 78%). Rf = 0.35 (EtOAc/hexane, 1/1, v/v); Rf = 0.55 (EtOAc). 1H NMR (400 MHz, CDCl3) δ 9.8 (s, 1H, CHO), 7.3 (s, 1H, CH-imid), 7.2 (s, 1H, CH-imid), 4.5 (t, 2H, CH2), 4.1 (m, 2H, CH2), 2.3 (t, 2H, CH2), 2.1 (m, 2H, CH2), 1.3 (t, 3H, CH3). MS [M + H]+: found, 211.2; calcd, 211.2. To a stirring solution of ethyl 2(2-formyl-1H-imidazol-1-yl)butanoate (2.46g, 11.7 mmol) in MeOH (4 mL) was added 18 mL of an aqueous solution of Na2CO3 (1.39g, 13.1 mmol) and hydroxylamine hydrochloride (1.67 g, 17.6 mmol). The emulsion was left to stir for another hour. Water (37 mL) and EtOAc (37 mL) were added. After separation of the layers, the organic layer was washed with 10% NaHCO3 (37 mL), water/brine (1/1, 50 mL), and brine (37 mL). After drying (MgSO4), filtration, and evaporation of the solvent the crude product was purified using silica gel column chromatography using 73% EtOAc in hexane to give 1.69 g (7.5 mmol, 64%) of the intermediate. Rf = 0.23 (70% EtOAc/hexane); Rf = 0.63 (EtOAc). 1H NMR (400 MHz, CDCl3) δ 11.4 (s, 1H, NOH), 8.3 (s, 1H, CHN), 7.1 (s, 1H, CH imid), 7,0 (s, 1H, CH imid), 4.4 (t, 2H, CH2), 4.1 (q, 2H, CH2), 2.3 (t, 2H, CH2), 2.0 (m, 2H, CH2), 1.2 (t, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 172.9, 140.4, 140.0, 128.8, 123.0, 60.7, 46.8, 30.7, 25.7, 14.2. [M + H] found: 226.24. To ethyl 2-(2-hydroxyiminomethyl-1H-imidazol-1-yl)butanoate (1.69 g, 7.5 mmol) in THF (15 mL) was added a solution of NaOH (1.5 g, 37.5 mmol) in water (15 mL). The homogeneous mixture was stirred for 30 min. DOWEX 50X-400 (H+-form) was added until the pH was about 6.5. The solution was filtered, and the DOWEX was washed with MeOH. The filtrate was concentrated and coevaporated with MeOH (4×) to give 1.41 g (7.2 mmol, 96%) of the title compound as a foam. 1H NMR (400 MHz, MeOD) δ 8.1 (s, 1H, CHN), 7.2 (s, 1H, CH-imid), 7.1 (s, 1H, CH-imid), 4.4 (t, 2H, CH2), 2.3 (t, 2H, CH2), 2.1 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 177.1, 140.0, 140.1, 127.7, 123.4, 46.8, 31.8, 26.4. [M + H] found: 198.19. (2-Hydroxyiminomethyl-1H-imidazol-1-yl)acetic Acid (3c). A heterogeneous solution was made with 1.64 g (17 mmol, 1 equiv) of 1H-imidazole-2-carbaldehyde, 2.01 mL (3.22 g, 20.4 mmol, 1.2 equiv) of methyl bromoacetate, 4.72 g (34 mmol, 2 equiv) of potassium carbonate, and 2.84 g (17 mmol, 1 equiv) of potassium iodide in 30 mL of acetonitrile. The mixture was stirred for 72 h at 50 °C. After the mixture was cooled to room temperature, it was filtered and the solvent of the filtrate was evaporated under reduced pressure. Flash chromatography (DCM) was used to purify the product, 930 mg (5.53 mmol, 33% yield). Methyl 2-(2-formyl-1H-imidazol-1-yl)acetate was obtained. Rf = 0.6 (MeOH/DCM, 1/19, v/v); 1H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H, COH), 7.34 (d, J = 0.9 Hz, 1H, CHCHarom), 7.14 (s, J = 0.9 Hz, 1H, CHCH-arom), 5.16 (s, 2H, NCH2CO), 3.79 (s, 3H, COOCH3); 13C NMR (101 MHz, CDCl3) δ 182.3, 167.4, 131.7, 126.9, 52.9, 48.5. [M + H+] calcd (found): 169.0608 (169.0607). To a stirring solution of methyl 2-(2-formyl-1Himidazol-1-yl)acetate (750 mg, 4.5 mmol) in MeOH (3 mL) was added, in 6 portions of 1 mL, an aqueous solution (total 6 mL) of hydroxylamine hydrochloride (400 mg, 6 mmol) and Na2CO3 (482 mg, 4.5 mmol). After the last addition the product crystallized immediately. The emulsion was left to stirr for another hour. The solids were filtered, washed with water, and dried to give 500 mg, 2.7 mmol (60%) of the intermediate. 1H NMR (400 MHz, DMSO) δ 11.5 (s, 1H, N-OH), 8.0 (s, 1H, CHN), 7.3 (d, 1H, J = 0.9 Hz, CH imid), 7.0 (d, 1H, J = 0.9 Hz, CH imid), 5.1 (s, 2H, CH2), 3.7 (s, 3H, CH3). 13C NMR (101 MHz, DMSO) δ 168.6, 141.3, 140.3, 128.6, 124.7, 52.2, 48.6. [M + H+] calcd (found): 184.0717 (184.0718). To a suspension of methyl 2-(2-hydroxyiminomethyl-1H-imidazol-1-yl)acetate (175 mg, 0.95 mmol) in MeOH (2 mL) was added a solution of NaOH (215 mg, 5.4 mmol) in water (2 mL). After a short time (seconds to minutes) the solution became clear and the mixture was 9386



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(400 MHz, DMSO): δ 12 (s, 1H, NOH), δ 10.30 (s, 1H, OH), δ 2.2 (dt, 2H, CH2 E + 2H CH2 Z), δ 2.10 (dt, 2H, CH2 E + 2H, CH2 Z), δ 1.75 (s, 3H, CH3 Z), δ 1.71 (s, 3H, CH3 E), δ 1.68 (m, 2H, CH2 E + Z). 13C NMR (400 MHz, DMSO): δ 174.68; 174.60; 155.50; 155.11; 34.91; 33.92; 33.41; 27.77; 21.66; 20.96; 19.83; 13.67. MS: M + 1 calcd 146, found 146. 4-N-(2-Hydroxyimino-1-oxoethyl)aminobutanoic Acid (5d). Procedure was identical as for 16 using 4-aminobutanoic acid. 2.39 g yield (6%) as light yellow solid. 1H NMR (400 MHz, MeOH-d4) δ 7.5 (s, 1H, CH), 3.3 (t, 2H, CH2), 2.4 (t, 2H, CH2), 1.8 (q, 2H, CH2). 13C NMR (101 MHz, MeOH-d4) δ 175.6, 163.7, 142.7, 38.2, 30.8, 24.3. General Protocol for the Ugi Reactions. 0.5 mmol of the oxime was dissolved in 3 mL of MeOH. To this solution were added successively 4-methoxybenzylamine (98 μL, 0.75 mmol, 1.5 equiv), 38% formaldehyde (58 μL, 0.75 mmol, 1.5 equiv), and isopropyl isocyanide (73 μL, 0.75 mmol, 1.5 equiv). The mixture was stirred overnight, concentrated, and purified. N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(2hydroxyimino)acetamide (9a). HPLC indicated 100% conversion. Purification: MeOH/DCM (3 → 10%). Yield: 100 mg (0.34 mmol, 68%). Crystalline white solid. 1H NMR (400 MHz, MeOD); all signals are doubled. δ 7.95 and 7.8 (s, 1H, CHN), 7.2 + 7.25 (d, 2H, CHBn), 6.9 and 6.85 (d, 2H, CHBn), 4.75 and 4.6 (s, 2H, CH2), 4.1 and 3.95 (s, 2H, CH2), 3.9 (m, 1H, CH iPr), 3.75 (s, 3H, OMe), 1.15 and 1.1 (d, 6H, CH3 iPr). 13C NMR (101 MHz, CDCl3) δ 169.20/169.16, 166.07/165.80, 160.92/160.82, 144.04/143.53, 130.97/130.79, 129.36/129.21, 115.24/115.09, 55.71, 53.23, 50.87, 42.72/42.64, 22.50. [M + H+] calcd (found): 308.1605 (308.1603). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(2(hydroxyimino))propanamide (9b). HPLC indicated 100% conversion. Purification: 70% EtOAc in hexane. Yield: 110 mg (0.34 mmol, 68%). Crystalline white solid. 1H NMR (400 MHz, CDCl3 + few drops of MeOD); all signals are doubled. δ 10.8 and 10.3 (2 × bs, 0.2H OH), 7.25 and 7.2 (2H, 2 × CHBn), 6.8 (d, 2H, 2 × CHBn), 6.35 and 6.25 (bs, 1H, NH); 4.65, 4.6 (s, 2H, CH2), 3.9 and 3.8 (s, 2H, CH2), 3.85 (m, 1H, CH iPr), 3.75 (s, 3H, OMe), 2.05 (s, 3H, Me), 1.1 and 1.0 (d, 6H, CH3 iPr). 13C NMR (101 MHz, CDCl3) δ 168.66, 167.93, 167.55, 167.12, 15931, 151.5, 130.11, 129.30, 128.22, 127.76, 114.14, 60.41, 55.18, 52.90, 50.36, 49.50, 49.29, 49.08, 48.90, 41.41, 41.30, 22.22, 22.07, 20.89, 14.01, 12.09, 11.72. [M + H+] calcd (found): 322.1761 (322.1762). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-N-(2hydroxyimino-1-oxoethyl))hexanamide (10a). Purification: 6% MeOH/DCM. Yield: 86%; 1H NMR (400 MHz, chloroform-d3) δ 11.2 (bs, 1H), 7.5 (s, 1H), 7.2−6.8 (m, 5H); 6.3/5.9 (2 × bs, 1H), 4.6 (s, 2H), 4.0 (m, 1H), 3.9 (m, 2H), 3.8 (2 × s, 3H), 3.4 (m, 1H), 3.3 (m, 2H), 2.4 (m, 1.5H), 2.3 (m, 1H), 1.7 (m, 2H), 1.5 (m, 2H), 1.3 (m, 2H), 1.1 (m, 4H), 1.0 (m, 2H). 13C NMR (101 MHz, chloroforml-d3) δ 174.5, 168.6/167.7, 162.9, 159.4, 143.7, 129,9, 129.0, 128.1, 127.6, 114.5/114.4, 55.5, 52.2, 50.0, 41.6, 41.5, 36.9, 36.8, 33.1, 32.8, 29.0, 26.4, 26.3, 24.6, 22.5, 22.3. [M + H+] calcd (found): 421.2445 (421.2448). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(3-N-(2hydroxyimino-1-oxopropyl))aminoethanamide (10b). Purification: 20% EtOAc/n-hexane → 100% EtOAc. Yield: 61%. 1H NMR (400 MHz, CDCl3): δ 7.20−7.18 (dd, 2H, arom), δ 6.88 (dd, 2H, arom), δ 6.11 + 5.71 (d + d, 1H, NH), δ 4.63 + 4.57 (ds, 2H, CH2), δ 4.05− 4.01 (m, 1H, CH), δ 3.94 (s, 2H, CH2), δ 3.82 (s, 3H, CH3), δ 2.64− 2.23 (m, 4H, 2 × CH2), δ 1.92 (ds, 3H, CH3), δ1.13−1.01 (dd, 6H, 2 × CH3). 13C NMR (101 MHz, CDCl3): δ 173.32; 157.47; 130.11; 129.12; 128.14; 114.33, 55.33; 52.05; 50.57; 41.38; 31.29; 29.,70; 28.61; 22.45; 14.32. [M + H+] calcd (found): 393.2093 (393.2096). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(4-N-(2hydroxyimino-1-oxopropyl))aminopropanamide (10c). Purification: 20% EtOAc/n-hexane → 100% EtOAc. Yield 56%. 1H NMR: δ 7.21− 7.05 (dd, 2H, arom), δ 6.85 (dd, 2H, arom), δ 6.20 + 5.66 (d + d, 1H, NH), δ 4.57 + 4.54 (ds, 2H, CH2), δ 4.03−4.00 (m, 1H, CH), δ 3.90 (s, 2H, CH2), δ 3.78 (s, 3H, CH3), δ 2.45−2.13 (m, 6H, 3 × CH2), δ 1.89 (ds, 3H, CH3), δ1.14−1.08 (dd, 6H, 2 × CH3). 13C NMR: δ 173.77; 168.23; 159.23 ; 157.50; 129.22; 128.09; 114.34; 58.21; 52.03;

allowed to stirr for another 15 min. DOWEX 50X-400 (H+-form) was added until the pH was about 6.5. The solution was filtered, and the DOWEX was washed with MeOH. The combined filtrates were concentrated and the residue was coevaporated with ACN (2×) and MeOH (2×) to give 129 mg, 0.76 mmol (80%) of the title compound. HPLC (0−25% B in 20 min): tR = 8.4 min. 1H NMR (400 MHz, DMSO-d6) δ 11.7 (bs, 1H, N-OH), 7.9 (s, 1H, CHN), 7.1 (s, 1H, CH imid), 6.8 (s, 1H, CH imid), 4.65 (s, 2H, CH2). A single peak at 3.4 ppm (0.5H) was observed as an impurity originating from the Dowex resin. 13C NMR (101 MHz, methanol-d4) δ [M + H+] calcd (found): 170.0560 (170.0563). 1-(Carboxymethyl)-4-((hydroxyimino)methyl)pyridinium Bromide (4a). 1.536 g (12.5 mmol) of 4-pyridinealdoxime was dissolved in 50 mL of acetone. 4.538 g (31.25 mmol, 2.5 equiv) of bromoacetic acid was added and stirred overnight at reflux temperature. After cooling, the crystals were filtered and washed with cold acetone. Yield: 91%. 1H NMR (400 MHz; D2O): δ 5.45 (s, 2H, CH2); δ 8.27 (d, 2H, J = 6 Hz, CH pyridine); δ 8.44 (s, 1H, CH oxime); δ 8.82 (d,2H, J = 6 Hz, CH pyridine). 13C NMR (100 MHz; D2O): δ 60.93 (CH2); δ 124.47 (CN); δ 145.73 (CH pyridine); δ 146.04 (CH pyridine); δ 149.48 (C pyridine); δ 168.93 (COOH). ESI-MS: calcd, 181; found, 181. 1-(Carboxypentyl)-4-((hydroxyimino)methyl)pyridinium Bromide (4b). 1.22 g (10 mmol) of 4-pyridinealdoxime was dissolved in 40 mL of acetone. 4.878 g (25 mmol, 2.5 equiv) of 6-bromohexanoic acid was added and stirred overnight at reflux temperature. After cooling the crystals were filtered and washed with cold acetone. Yield: 70%. 1H NMR (400 MHz; D2O): δ 1.42 (d, 2H, J = 7 Hz, CH2); δ 1.67 (t, 2H, J = 7 Hz, CH2); δ 2.04 (t, 2H, J = 7 Hz, CH2); δ 2.34 (t, 2H, J = 7 Hz, CH2); δ 4.59 (t, 2H, J = 7 Hz, CH2); δ 8.22 (d, 2H, J = 6 Hz, CH pyridine); δ 8.33 (s, 1H, CH CN); δ 8.86 (d, 2H, J = 6 Hz, CH pyridine). 13C NMR (100 MHz; CDCl3): δ 23.55 (CH2); δ 24.66 (CH2); δ 30.07 (CH2); δ 33.41 (CH2). ESI-MS: calcd, 237.3; found, 237.3. 6-N-(2-Hydroxyimino-1-oxoethyl)hexanoic Acid (5a). To a stirred suspension of 23.07 g of 6-aminohexanoic acid (175.5 mmol) in 40 mL of MQ is added 7.02 g of NaOH (175.5 mmol). To the resulting solution is added 130 mL of EtOH and 13.73 g of ethyl oximinoglyoxylate (117 mmol), and the mixture is heated overnight at 50 °C. The mixture is acidified (pH 2) with concentrated HCl and extracted with EtOAc (5 × 100 mL). The combined EtOAc was dried with MgSO4, filtered, and concentrated. The residue was purified by automated flash chromatography (20% → 80% to 80% → 100% EtOAc/hexane) to give the title compound (4.45g, 22 mmol, 19%) as a white solid after coevaporation with MeOH and ACN. 1H NMR (400 MHz, MeOH-d4) δ 8.2 (bs, 1H, NH), 7.4 (s, 1H, CH), 3.3(t, 2H, CH2), 2.3 (t, 2H, CH2), 1.7−1.5 (2xq, 4H, 2x CH2), 1.5 (q, 2H,CH2). 13 C NMR (101 MHz, MeOH-d4) δ 176.1, 163.5, 142.7, 38.6, 33.4, 25.7, 26.1, 24.3. ESI-MS: calcd (M + H+), 203.2; found, 203.2. 3-N-(2-Hydroxyimino-1-oxopropyl)aminopropanoic Acid (5b). 2.084 g (30 mmol) of hydroxylamine hydrochloride and 1.800 g (45 mmol) of NaOH were dissolved in 25 mL of water. The mixture was cooled to 0 °C, and then 1.515 mL (15 mmol) of levulinic acid was added. The mixture was stirred and allowed to warm to room temperature. After 4 h the mixture was neutralized with HCl, extracted with EtOAc (3×), dried, and concentrated. Crystals were recrystallized using a mixture of EtOAc/hexane. Yield 9.1 mmol (61%). 1H NMR (400 MHz, DMSO): δ 12.00 (s, 1H, NOH), δ 10.30 (s, 1H, OH), δ 2.32−2.45 (m, 4H, CH2-CH2 E + 4H CH2-CH2 Z), δ 1.78 (s, 3H, CH3 Z), δ 1.73 (s, 3H, CH3 E). 13C NMR (400 MHz, DMSO): δ 174.44; 174.33; 155.03; 154.31; 30.97; 30.60; 24.24; 19.92; 14.19. MS: M + 1 calcd 132, found 132. 4-N-(2-Hydroxyimino-1-oxopropyl)aminobutanoic Acid (5c). 2.084 g (30 mmol) of hydroxylamine hydrochloride and 1.890 g (47 mmol) of NaOH were dissolved in 10 mL of water. The mixture was cooled to 0 °C, and then 1.79 mL (15 mmol) of 5-oxohexanoic acid was added. The mixture was stirred and allowed to warm to room temperature. After 4 h the mixture was neutralized with HCl, extracted with EtOAc (3×), dried, and concentrated. Crystals were recrystallized using a mixture of EtOAc/hexane. Yield: 6.85 mmol (46%). 1H NMR 9387



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41.38; 21.65; 21.02; 13.60. [M + H+] calcd (found): 407.2250 (407.2254). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(4-N-(2hydroxyimino-1-oxoethyl))aminobutanamide (10d). 1H NMR (400 MHz, chloroform-d3) δ 11.6 (s, 1H), 7.6−6.3 (m, 6H), 4.40 (s, 2H), 4.2−3.9 (m, 3H), 3.8 (s, 2H), 2.6−2.2 (2x t, 2H), 1.9 (bt, 2H), 1.2− 0.9 (m, 6H). 13C NMR (101 MHz, chloroform-d3) δ 173.9, 168.5/ 167.6, 163.38/163.34, 159.3, 143.4, 129.8, 128.7, 128.2, 127.6, 114.4/ 114.2, 55.3/55.25, 52.2, 50.3, 49.9, 49.8, 41.8/41.6, 38.8/38.0, 30.6/ 29.5, 25.1/24.6, 22.3. [M + H+] calcd (found): 393.2132 (393.2138). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-(4pyridiniumaldoxime)hexanamide) Bromide (11a). HPLC indicated 80% conversion. Purification: automated flash chromatography (10 → 20% MeOH/DCM). Product comes between 6 and 7 CV. The product is isolated as a light yellow foam. Yield: 115 mg (0.21 mmol, MW = 535; 43%). 1H NMR (400 MHz, MeOD); all signals are doubled. δ 9.05 (d, 2H, CHpyr), 8.20 (s, 1H, CHN), 8.15 (d, 2H, CHpyr), 7.15 and 7.1 (d, 2H, CHBn), 6.85 and 6.8 (d, 2H, CHBn), 4.65 (t, 2H, CH2), 4.55 (s, 2H, CH2), 3.9 (m, 3H, CH2 and CH iPr), 3.8 (s, 3H, OMe), 2.5 and 2.35 (t, 2H, CH2), 2.0 (m, 2H, CH2), 1.7 (m, 2H, CH2), 1.4 (m, 2H, CH2), 1.1 (d, 6H, CH3 iPr). 13C NMR (101 MHz, MeOD) δ 176.0, 168.8/169.39, 160.69/160.54, 150.93, 145.98/ 145.61, 161.64, 130.6, 130.09, 129.57/129.47, 126.23, 125.44/ 125.42, 115.44/115.32, 62.22, 55.85/55.76, 53.14, 50.76/50.45, 43.88, 42.72/42.56, 33.53/33.46, 31.94/31.90, 26.55/26.50, 25.34/ 25.25, 22.55. [M+] calcd (found): 455.2653 (455.2650). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(4pyridiniumaldoxime)acetamide Bromide (11b). HPLC indicated 85% conversion. Purification: MeOH/H2O (10 → 50%) on C18-silica. Yield: 74 mg (0.15 mmol, MW = 478; 31%). 1H NMR (400 MHz, MeOD); all signals are doubled. δ 8.9 (d, 2H, 2xCHpyr); 8.45 (s, 1H, CHN); 8.25 (d, 2H, 2 × CHpyr), 7.4 and 7.25 (d, 2H, 2 × CHBn), 7.0 and 6.9 (d, 2H, 2 × CHBn), 5.9 and 5.8 (2H, s, CH2), 4.7 and 4.6 (s, 2H, CH2), 4.15 and 4.0 (s, 2H, CH2), 3.9 (m, 1H, CH iPr), 3.8 (2 × s, 3H, OMe); 1.1 (2 × d, 6H, 2 × CH3 iPr). 13C NMR (101 MHz, CDCl3) δ 169.04/168.57, 167.46/166.97, 161.23/160.95, 151.82, 147.97/147.66, 145.66, 131.28/130.66, 128.97/127.88, 124.82/124.71, 115.51/115.06, 62.44/62.39, 55.81/55.73, 52.43/51.48, 50.19/49.28, 42.97, 22.48. [M+] calcd (found): 399.2027 (399.2030). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-(2hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12a). Rf = 0.33 (MeOH/DCM, 1/19, v/v). Yield 50 mg, 0.12 mmol, 39%). HPLC (0−100% B in 20 min): tR = 13.1 min. 1H NMR (400 MHz, CDCl3) δ 11.5 (bs, 1H, NOH), 8.2 (s, 1H, CHN), 7.4−6.8 (multiple resonances, 6H, 4 × CH arom, 2 × CH imid), 6.15 (ma, 0.6H, NH), 5.6 (mi, 0.4H, NH), 4.6,4.55 (s, ma/mi, 2H, CH2), 4.25 (m, 2H, CH2), 4.0 (m, 1H, CH iPr), 3.9,3.85 (s, mi/ma, CH2), 3.8 (s, 3H, OMe), 2.5,2.3 (t, ma/mi, 3H, CH2), 1.75 (m, 4H, 2 × CH2), 1.4 (m, 2H, CH2), 1.15,0.95 (d, ma/mi, 6H, CH3 iPr); 13C NMR (101 MHz, CDCl3) δ 175.02/174.67, 168.09/167.57, 159.54/159.33, 142.12/141.98, 140.38/140.32, 130.06, 128.99, 128.12/127.69, 122.89/122.85, 114.48/114.37, 55.33, 52.33/51.77, 50.39/49.58, 47.27/47.20, 41.63/41.59, 32.80/32.57, 29.45/29.12, 25.47/25.38, 24.21/23.67, 22.57/22.32. [M + H+] calcd (found): 444.2605 (444.2605). N-Butyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-(2-hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12b). Purification: 6% MeOH/DCM. Yield: 90 mg (0.24 mmol, 33%). 1H NMR (400 MHz, CDCl3), all signals are doubled. 1H NMR (400 MHz, chloroform-d) δ 11.90 (s, 1H), 8.22 (s, 1H), 7.06 (t, J = 2.1 Hz, 1H), 6.96−6.90 (m, 1H), 6.47 (d, J = 7.9 Hz, 1H), 4.29−4.18 (m, 2H), 4.13−3.93 (m, 1H), 3.91 (s, 2H), 3.34 (t, J = 7.7 Hz, 2H), 2.37 (dd, J = 9.1, 6.4 Hz, 2H), 1.83−1.62 (m, 3H), 1.60−1.20 (m, 6H), 1.12 (dd, J = 10.9, 6.5 Hz, 6H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 174.43, 174.21, 168.41, 167.46, 141.58, 140.29, 128.67, 122.71, 77.31, 77.20, 77.00, 76.68, 51.56, 50.62, 49.72, 47.24, 47.13, 41.65, 41.36, 32.78, 32.28, 30.70, 29.42, 29.39, 29.20, 25.46, 25.42, 24.16, 23.74, 22.44, 22.42, 19.96, 19.80, 13.69, 13.63. [M + H+] calcd (found): 380.2656 (380.2664).

N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-(6-(2hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12c). Purification: 6% MeOH/DCM. Yield: 79 mg (0.16 mmol, 23%). 1H NMR (400 MHz, CDCl3), all signals are doubled. 1H NMR (400 MHz, chloroform-d) δ 12.10 (s, 1H), 8.22 (s, 1H), 7.32−7.00 (m, 9H), 6.93−6.79 (m, 2H), 4.57 (s, 1H), 4.51 (s, 1H), 4.33 (dd, J = 15.1, 5.8 Hz, 2H), 4.28−4.14 (m, 2H), 3.95 (s, 1H), 3.90 (s, 1H), 3.74 (d, J = 15.9 Hz, 3H), 2.45−2.37 (m, 1H), 2.25 (dt, J = 10.3, 7.5 Hz, 1H), 1.69 (ddt, J = 21.9, 14.9, 6.5 Hz, 4H), 1.32 (p, J = 8.4, 7.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 174.82, 174.36, 174.21, 168.89, 168.24, 159.14, 159.09, 141.69, 141.63, 141.57, 140.20, 140.17, 137.79, 137.43, 129.66, 128.86, 128.60, 128.51, 128.46, 127.95, 127.81, 127.57, 127.47, 127.45, 127.40, 127.23, 126.48, 122.71, 122.68, 122.65, 114.23, 114.06, 77.30, 77.18, 76.98, 76.67, 55.16, 55.08, 52.15, 51.46, 50.60, 50.20, 49.66, 49.26, 47.26, 47.16, 47.08, 43.41, 43.23, 33.58, 32.77, 32.48, 29.79, 29.38, 29.27, 25.54, 25.44, 25.36, 24.05, 24.03, 23.72, 18.20. [M + H+] calcd (found): 492.2605 (492.2611). N-Butyl-N-[2-(benzylamino)-2-oxoethyl]-(6-(2-hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12d). 70 mg (0.17 mmol; 36%). Rf = 0.72 (5% MeOH/DCM). 1H NMR (400 MHz, methanol-d4) δ 8.08 (s, 1H), 7.38−7.16 (m, 6H), 7.04 (d, J = 1.2 Hz, 1H), 4.40 (s, 2H), 4.35−4.25 (m, 2H), 4.07 (d, J = 16.6 Hz, 2H), 3.38 (q, J = 7.5 Hz, 2H), 2.45 (t, J = 7.5 Hz, 1H), 1.86−1.21 (m, 10H), 0.94 (dt, J = 13.1, 7.3 Hz, 3H). 13C NMR (101 MHz, methanol-d4) δ 174.52, 140.17 (d, J = 12.5 Hz), 128.18 (d, J = 9.7 Hz), 127.79, 127.43−126.70 (m), 123.33 (d, J = 1.8 Hz), 50.48, 49.20, 48.95, 47.08 (d, J = 5.4 Hz), 42.77 (d, J = 22.8 Hz), 32.02, 30.45, 30.13 (d, J = 8.5 Hz), 25.62, 24.26 (d, J = 9.2 Hz), 19.68 (d, J = 14.6 Hz), 12.85 (d, J = 4.6 Hz). [M + H+] calcd (found): 428.2656 (428.2659). N-(2-Methoxyethyl)-N-[2-(benzylamino)-2-oxoethyl]-(6-(2hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12e). 40 mg (0.09 mmol; 17%). 1H NMR (400 MHz, methanol-d4) δ 8.08 (s, 1H), 7.37−7.25 (m, 6H), 7.21 (d, J = 1.2 Hz, 1H), 7.04 (d, J = 1.3 Hz, 1H), 4.41 (s, 2H), 4.35−4.25 (m, 2H), 4.18 (s, 1H), 4.10 (s, 1H), 3.66− 3.45 (m, 5H), 3.26 (d, J = 4.9 Hz, 3H), 2.50 (t, J = 7.5 Hz, 1H), 2.30 (t, J = 7.5 Hz, 1H), 1.76 (tq, J = 13.7, 6.8, 6.2 Hz, 2H), 1.70−1.56 (m, 2H), 1.44−1.26 (m, 2H). 13C NMR (101 MHz, methanol-d4) δ 174.52, 140.16 (d, J = 12.3 Hz), 128.19 (d, J = 9.7 Hz), 127.78, 127.63−126.66 (m), 123.35, 49.21, 48.94, 47.08 (d, J = 4.8 Hz), 42.77 (d, J = 23.0 Hz), 32.02, 30.45, 30.13 (d, J = 8.6 Hz), 25.62, 24.26 (d, J = 9.1 Hz), 19.69 (d, J = 14.6 Hz), 12.86 (d, J = 4.7 Hz). [M + H+] calcd (found): 430.2449 (430.2451). N-(2-Methoxyethyl)-N-[2-(isopropylamino)-2-oxoethyl]-(6-(2hydroxyiminomethyl-1H-imidazol-1-yl))hexanamide (12f). Purification: 6% MeOH/DCM. Yield: 90 mg (0.24 mmol, 33%). 1H NMR (400 MHz, CDCl3), all signals are doubled. 1H NMR (400 MHz, chloroform-d) δ 11.79 (s, 1H), 8.21 (d, J = 1.7 Hz, 1H), 7.06 (d, J = 1.4 Hz, 1H), 6.93 (s, 1H), 4.29−4.16 (m, 2H), 4.14−3.92 (m, 3H), 3.67−3.53 (m, 3H), 3.49 (t, J = 5.0 Hz, 1H), 3.33 (d, J = 19.0 Hz, 3H), 2.47−2.38 (m, 1H), 2.30−2.21 (m, 1H), 1.83−1.62 (m, 4H), 1.37 (pd, J = 7.1, 2.6 Hz, 2H), 1.11 (dd, J = 7.5, 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 175.01, 174.90, 168.57, 168.39, 141.86, 141.66, 140.26, 140.19, 128.72, 128.69, 122.76, 122.72, 77.31, 76.99, 76.67, 69.93, 69.88, 58.86, 58.32, 54.41, 51.08, 49.55, 49.13, 47.28, 47.17, 41.38, 41.30, 32.60, 32.34, 29.44, 29.12, 25.40, 25.32, 23.93, 23.38, 22.42, 22.37. [M + H+] calcd (found): 382.2449 (382.2451). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(4-(2hydroxyiminomethyl-1H-imidazol-1-yl))butanamide (13a). Purification: 1% MeOH/DCM 4 CV, 3% MeOH/DCM 3 CV, 8% MeOH/ DCM 4 CV. Yield: 169 mg (0.41 mmol, 41%). Rf = 0.52 (1/19 MeOH/DCM). 1H NMR (400 MHz, MeOD) δ 8.1 (d, 1H, CHN), 7.2 (d, 1H CH-imid), 7.2−7.1 (dd, 2H, CH-arom), 7.0 (d, 1H, CHimid), 6.9 (dd, 2H, 2 × CH-arom), 4.5 (s, 2H, CH2), 4.4 (t, 2H, CH2), 4.0 (s, 2H, CH2), 3.9 (s, 1H, CH), 3.8 (d, 3H, CH3), 2.5−2.4 (triple t, 2H, CH2), 2.1 (m, 2H, CH2), 1.1 (dd, 6H, 2 × CH3). 13C NMR (101 MHz, MeOD) δ 173.8, 168.5, 167.8, 159.4, 140.2, 129.4, 128.6, 128.1, 123.4, 113.9, 54.3, 51.7, 49.1, 46.4, 36.4, 29.3, 25.9, 21.2. [M + H+] calcd (found): 416.2292 (416.2297). N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-(4-(2hydroxyiminomethyl-1H-imidazol-1-yl))butanamide (13b). Purifica9388



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tion: 6% MeOH/DCM. Yield: 180 mg (0.39 mmol, 39%). 1H NMR (400 MHz, MeOD), δ 8.1 (s, 1H, CH-oxime), 7.3−7.2 (multiple resonances, 6H, 1 × CH-imid, 5 × CH-arom), 7.2 (dd, 2H, 2 × CHarom), 7.0 (s, 1H, CH-imid), 6.9 (dd, 2H, 2 × CH-arom) 4.5 (d, 2H, CH2), 4.2−4.4 (multiple resonances, 4H, 2 × CH2), 4.0 (ds, 2H, CH2), 3.8 (d, 3H, CH3), 2.5−2.4 (dt, 2H, CH2), 2.1 (m, 2H, CH2). 13 C NMR (101 MHz, MeOD) δ 173.8, 169.7, 168.9, 159.4, 140.2, 138.4, 129.4, 128.6, 128.0, 127.2, 126.9, 123.4, 114.1, 54.4, 51.8, 49.2, 49.1, 46.6, 42.7, 29.1, 25.9. [M + H+] calcd (found): 474.2292 (464.2292). N-Butyl-N-[2-(benzylamino)-2-oxoethyl]-(4-(2-hydroxyiminomethyl-1H-imidazol-1-yl))butanamide (13c). Purification: 6% MeOH/DCM. Yield: 129 mg (0.33 mmol 33%). 1H NMR (400 MHz, MeOD) δ 8.1 (s, 1H, CHN), 7.3−7.1 (multiple resonances, 6H, 5 × CH-arom, 1 × CH-imid), 7.0 (d, 1H, CH-imid), 4.4 (dt, 4H, 2 × CH2), 4.1 (s, 2H, CH2), 3.3 (m, 2H, CH2), 2.4 (dt, 2H, CH2), 2.1 (m, 2H, CH2), 1.5 (m, 2H, CH2), 1.5 (m, 2H, CH2), 1.3 (m, 3H, CH3) 0.9 (m, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 173.5, 169.9, 169.2, 140.3, 138.5, 128.3, 127.4, 126.8, 123.4, 50.5, 48.9, 46.6, 42.7, 30.2, 29.3, 28.7, 25.9,19.7, 12.8. [M + H+] calcd (found): 400.2343 (400.2344). N-Butyl-N-[2-(isopropylamino)-2-oxoethyl]-(2-hydroxyiminomethyl-1H-imidazol-1-yl)acetamide (14a). Yield: 56%. 1H NMR (400 MHz, methanol-d4) δ 8.06 (d, J = 5.6 Hz, 1H), 7.17 (dd, J = 6.6, 1.3 Hz, 1H), 7.07 (s, 1H), 5.36 (s, 1H), 5.26 (s, 1H), 4.11−3.91 (m, 3H), 3.47−3.33 (m, 2H), 1.69 (p, J = 7.6 Hz, 1H), 1.58−1.25 (m, 4H), 1.24−1.11 (m, 7H), 1.08−0.89 (m, 4H). 13C NMR (101 MHz, MeOD) δ 168.41, 168.38, 168.07, 168.04, 167.88, 141.04, 141.00, 140.63, 140.35, 127.89, 127.58, 127.56, 124.82, 124.70, 124.49, 51.21, 49.89, 49.81, 49.00, 48.83, 48.79, 48.70, 48.38, 48.29, 48.15, 48.08, 47.94, 47.86, 47.72, 47.65, 47.63, 47.44, 47.23, 47.01, 41.56, 41.33, 41.02, 38.94, 31.22, 31.10, 29.95, 29.18, 21.28, 21.20, 21.17, 19.97, 19.68, 19.65, 12.90, 12.84, 12.76, 12.72. [M + H+] calcd (found): 324.2030 (324.2034). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(2hydroxyiminomethyl-1H-imidazol-1-yl))acetamide (14b). Yield: 42%. 1H NMR (400 MHz, methanol-d4) δ 8.08 (d, J = 7.7 Hz, 1H), 7.32−7.23 (m, 1H), 7.23−7.13 (m, 2H), 7.07 (dd, J = 4.0, 1.3 Hz, 1H), 7.02−6.94 (m, 1H), 6.90−6.83 (m, 1H), 5.40 (s, 1H), 5.34 (s, 1H), 4.64 (s, 1H), 4.53 (s, 1H), 4.02−3.86 (m, 3H), 3.79 (d, J = 16.1 Hz, 3H), 1.11 (dd, J = 6.6, 3.1 Hz, 6H). 13C NMR (101 MHz, MeOD) δ 168.77, 168.37, 168.08, 167.62, 159.64, 159.38, 141.09, 141.03, 140.72, 140.52, 140.43, 129.49, 128.83, 128.74, 128.50, 128.00, 127.92, 127.64, 127.27, 124.79, 124.76, 114.14, 113.70, 113.56, 54.44, 54.37, 51.03, 49.74, 49.06, 48.94, 48.72, 48.31, 48.10, 48.02, 47.88, 47.67, 47.46, 47.24, 47.03, 44.17, 41.49, 41.30, 21.19, 21.17. [M + H+] calcd (found): 388.1979 (388.1985). N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-(2-hydroxyiminomethyl-1H-imidazol-1-yl)acetamide (14c). Purification: 4% MeOH/DCM. Yield 28 mg (30%). 1H NMR (400 MHz, chloroform-d) δ 8.20 (s, 1H), 8.13 (d, J = 16.9 Hz), 7.84 (t, J = 5.9 Hz, 1H), 7.29 (s), 7.16 (p, J = 2.9, 2.5 Hz, 4H), 7.03−6.86 (m, 5H), 6.86−6.70 (m, 4H), 6.62 (s, 1H), 5.09 (s, 2H), 4.91 (s), 4.68 (s), 4.28 (d, J = 5.4 Hz), 4.18 (s, 2H), 4.09 (d, J = 5.6 Hz, 2H), 3.99 (s), 3.81 (s, 1H), 3.77 (s, 3H), 3.74 (s, 1H), 3.53 (s, 2H). [M + H+] calcd (found): 436.1979 (436.1982). N-Butyl-N-[2-(benzylamino)-2-oxoethyl]-(2-hydroxyiminomethyl1H-imidazol-1-yl)acetamide (14d). Yield: 76%. 1H NMR (400 MHz, methanol-d4) δ 8.03 (d, J = 6.1 Hz, 1H), 7.40−7.21 (m, 5H), 7.25− 7.10 (m, 1H), 7.07 (dd, J = 5.4, 1.3 Hz, 1H), 5.36 (s, 1H), 5.24 (s, 1H), 4.43 (d, J = 21.9 Hz, 2H), 4.19 (s, 1H), 4.09 (s, 1H), 3.50−3.33 (m, 2H), 1.77−1.64 (m, 1H), 1.57−1.34 (m, 2H), 1.30 (h, J = 7.4 Hz, 1H), 1.06−0.93 (m, 2H), 0.91 (t, J = 7.3 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 169.50, 169.05, 168.38, 168.18, 141.02, 140.98, 140.71, 140.36, 138.28, 138.23, 128.27, 128.22, 128.11, 127.60, 127.54, 127.38, 127.30, 127.07, 126.79, 124.80, 124.67, 49.83, 49.21, 48.80, 48.50, 48.26, 48.05, 47.91, 47.84, 47.70, 47.62, 47.41, 47.20, 46.99, 43.04, 42.69, 29.99, 29.11, 19.66, 19.65, 12.79, 12.73. [M + H+] calcd (found): 372.2030 (372.2024).

General Protocol for N-Methylation of Imidazole-Based Ugi Products. Ugi products were dissolved in acetone to a concentration of 0.2 M. To this solution was added MeI (10 molar equiv), and the resulting mixture was allowed to stir overnight at room temperature. TLC (10% MeOH/DCM) was used to assess the progress of the reaction (usually near complete conversion of the starting material after overnight reaction). The mixture was concentrated, coevaporated with acetone, and purified using silica gel chromatography. N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-N-(1Me-2-hydroxyiminomethyl-1H-imidazolium))hexanamide Iodide (15a). Purification: 12% MeOH/DCM. Yield: 102 mg (0.17 mmol, 77%). 1H NMR (400 MHz, MeOD), all signals are doubled. 1H NMR (400 MHz, methanol-d4) δ 8.37 (s, 1H), 7.66 (d, J = 13.4 Hz, 1H), 7.54 (s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.2 Hz, 2H), 6.82 (d, J = 8.5 Hz, 1H), 4.36 (t, J = 8.6 Hz, 2H), 4.03−3.91 (m, 4H), 3.82−3.72 (m, 6H), 3.67 (s, 1H), 3.56 (s, 1H), 2.19 (t, J = 7.7 Hz, 2H), 1.89 (h, J = 15.5, 7.9 Hz, 2H), 1.66 (h, J = 17.9, 9.5 Hz, 2H), 1.46−1.32 (m, 2H), 1.13 (dd, J = 8.1, 7.6 Hz, 3H). 13 C NMR (101 MHz, MeOD) δ 176.14, 169.87, 169.79, 169.43, 169.35, 160.66, 160.53, 138.60, 138.57, 135.63, 130.63, 130.03, 129.90, 129.54, 129.48, 125.69, 124.53, 124.52, 115.31, 114.98, 114.83, 55.87, 55.79, 53.17, 50.83, 50.79, 50.65, 50.41, 49.84, 49.78, 49.73, 49.63, 49.49, 49.42, 49.28, 49.20, 49.06, 48.99, 48.78, 48.57, 48.35, 42.84, 42.73, 42.67, 42.56, 37.51, 37.50, 33.73, 33.58, 30.72, 30.69, 26.73, 26.64, 25.46, 25.38, 22.61, 22.58. [M + H+] calcd (found): 458.2762 (458.2765). N-Butyl-N-[2-(isopropylamino)-2-oxoethyl]-(6-N-(1-Me-2hydroxyiminomethyl-1H-imidazolium))hexanamide Iodide (15b). Purification: 12% MeOH/DCM. Yield: 60 mg (0.12 mmol, 58%). 1 H NMR (400 MHz, MeOD), all signals are doubled. 1H NMR (400 MHz, methanol-d4) δ 8.51 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 4.42 (td, J = 7.5, 5.2 Hz, 2H), 4.08 (s, 1H), 4.01 (d, J = 15.1 Hz, 5H), 3.44−3.32 (m, 2H), 2.50 (t, J = 7.3 Hz, 1H), 2.38 (t, J = 7.4 Hz, 1H), 1.97−1.81 (m, 2H), 1.76−1.25 (m, 7H), 1.18 (dd, J = 7.7, 6.6 Hz, 6H), 0.96 (dt, J = 14.3, 7.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 175.95, 175.71, 170.19, 170.11, 169.85, 169.76, 138.67, 135.61, 125.71, 124.56, 51.86, 50.68, 50.50, 50.13, 50.09, 49.64, 49.50, 49.43, 49.29, 49.22, 49.08, 49.01, 48.79, 48.58, 48.37, 48.34, 42.93, 42.82, 42.70, 42.59, 37.46, 37.45, 33.71, 33.27, 31.77, 31.46, 30.82, 30.72, 30.60, 26.84, 26.74, 25.55, 25.39, 22.64, 22.61, 21.10, 20.98, 19.37, 14.24, 14.21. [M + H+] calcd (found): 394.2813 (394.2813). N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-(6-N-(1-Me2-hydroxyiminomethyl-1H-imidazolium))hexanamide iIodide (15c). Purification: 12% MeOH/DCM. Yield: 154 mg (0.12 mmol, 55%). 1H NMR (400 MHz, MeOD), all signals are doubled. 1H NMR (400 MHz, methanol-d4) δ 8.49 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 6.5, 2.0 Hz, 1H), 7.66 (dd, J = 6.9, 2.0 Hz, 1H), 7.39−7.19 (m, 5H), 7.18 (dd, J = 8.7, 3.1 Hz, 2H), 6.96−6.82 (m, 2H), 4.65 (s, 1H), 4.55 (s, 1H), 4.37 (t, J = 9.8 Hz, 4H), 4.06 (d, J = 17.3 Hz, 2H), 3.99 (d, J = 2.5 Hz, 3H), 3.78 (d, J = 6.9 Hz, 3H), 3.37 (s, 2H), 2.58 (t, J = 7.3 Hz, 1H), 2.43 (t, J = 7.4 Hz, 1H), 1.85 (q, J = 7.6 Hz, 2H), 1.69 (t, J = 7.6 Hz, 2H), 1.48−1.37 (m, 2H). 13C NMR (101 MHz, MeOD) δ 176.21, 176.08, 175.58, 170.89, 170.36, 160.60, 160.45, 139.75, 139.62, 138.47, 138.44, 135.63, 130.59, 129.95, 129.92, 129.61, 129.51, 129.48, 129.45, 129.42, 129.03, 128.56, 128.36, 128.23, 128.06, 127.92, 125.72, 125.66, 124.48, 124.43, 115.30, 114.97, 55.89, 55.81, 53.29, 52.08, 50.81, 50.60, 50.43, 50.06, 49.84, 49.62, 49.48, 49.41, 49.27, 49.20, 49.06, 48.99, 48.77, 48.56, 48.35, 44.06, 43.82, 37.55, 37.52, 34.36, 33.72, 33.59, 30.66, 30.64, 30.60, 26.64, 26.60, 26.54, 25.36, 25.30, 25.20. [M + H+] calcd (found): 506.2762 (506.2757). N-Butyl-N-[2-(benzylamino)-2-oxoethyl]-(6-N-(1-Me-2-hydroxyiminomethyl-1H-imidazolium))hexanamide Iodide (15d). Yield: 68%. 1H NMR (400 MHz, MeOD), all signals are doubled. 1H NMR (400 MHz, methanol-d4) δ 8.49 (s, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.65 (dd, J = 8.9, 2.0 Hz, 1H), 7.39−7.20 (m, 5H), 4.45−4.33 (m, 4H), 4.15 (s, 1H), 4.09−3.97 (m, 4H), 3.40 (dt, J = 18.2, 7.7 Hz, 2H), 2.51 (t, J = 7.2 Hz, 1H), 2.36 (t, J = 7.3 Hz, 1H), 1.88 (dp, J = 15.1, 7.6 Hz, 2H), 1.77−1.24 (m, 7H), 0.96 (dt, J = 16.3, 7.3 Hz, 3H). 13 C NMR (101 MHz, MeOD) δ 174.51, 174.41, 169.86, 169.41, 9389



Article

H NMR (400 MHz, methanol-d4) δ 8.45 (d, J = 3.4 Hz, 1H), 7.73− 7.63 (m, 2H), 7.43−7.20 (m, 5H), 5.58 (s, 1H), 5.49 (s, 1H), 4.48 (s, 1H), 4.42 (s, 1H), 4.27 (s, 1H), 4.14 (s, 1H), 4.05 (d, J = 6.2 Hz, 3H), 3.51−3.35 (m, 2H), 1.83−1.67 (m, 1H), 1.56−1.39 (m, 2H), 1.44− 1.24 (m, 1H), 1.03 (t, J = 7.4 Hz, 2H), 0.91 (t, J = 7.3 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 169.15, 168.73, 166.18, 165.78, 138.80, 138.75, 138.32, 138.11, 134.46, 134.42, 128.28, 128.11, 127.43, 127.16, 127.07, 126.82, 124.64, 124.33, 124.09, 123.89, 51.04, 51.00, 49.89, 49.12, 48.62, 48.27, 48.13, 48.05, 47.91, 47.84, 47.70, 47.63, 47.42, 47.20, 46.99, 43.11, 42.69, 36.26, 35.95, 29.97, 29.11, 19.69, 19.61, 12.76, 12.74. [M + H+] calcd (found): 386.2187 (386.2180). 6-(3-Methyl-2-hydroxyiminomethyl-1H-imidazolium-1-yl)hexanoic Acid Iodide (18). To a stirring solution of ethyl 2-(2hydroxyiminomethyl-N-methylimidazol-1-yl)hexanoate (55 mg, 0.22 mmol) in acetone (1 mL) was added 319 mg of iodomethane, and the mixture was stirred overnight. The mixture was concentrated to give 87 mg, 0.22 mmol (quantative) of product. Rf = 0.05−0.25 (MeOH/ DCM, 1/9, v/v); 1H NMR (400 MHz, MeOH-d4) δ 8.5 (s, 1H, CH N), 7.8 (s, 1H, CH imid), 7.7 (s, 1H, CH imid), 4.4 (t, 2H, CH2), 4.1 (q, 2H, OCH2), 4.0 (s, 3H, N+CH3), 2.4 (t, 2H, CH2), 1.9 (m, 2H, CH2), 1.7 (m, 2H, CH2), 1.4 (m, 2H, CH2), 1.3 (t, 3H, CH3). 13C NMR (101 MHz, MeOH-d4) δ 174.0, 137.3, 134.5, 124.5, 123.2, 60.2, 49.3, 36.4, 33.4, 29.4, 25.3, 24.0, 13.3. [M + H+] calcd (found): 268.1656 (268.1662). Next step was identical to the final step of the synthesis of 2-(2-hydroxyiminomethyl-1H-imidazol-1-yl)hexanoic acid (3a). Yield 60%. 1H NMR (400 MHz, MeOH-d4) δ 8.5 (s, 1H, CH N), 7.8 (s, 1H, CH imid), 7.7 (s, 1H, CH imid), 4.4 (t, 2H, CH2), 4.0 (s, 3H, CH3), 2.2 (t, 2H, CH2), 1.9 (m, 2H, CH2), 1.7 (m, 2H, CH2), 1.4 (m, 2H, CH2). 13C NMR (101 MHz, MeOH-d4) δ 179.8, 137.4, 134.1, 124.3, 123.3, 49.4, 36.0, 29.2, 25.5, 24.9. [M + H+] calcd (found): 240.1343 (240.1346). 1

138.50, 138.41, 137.35, 134.20, 134.18, 128.21, 128.11, 127.30, 127.06, 126.96, 126.76, 124.32, 123.16, 50.52, 49.27, 48.95, 48.26, 48.12, 48.04, 47.90, 47.83, 47.69, 47.62, 47.41, 47.19, 47.08, 46.98, 42.84, 42.61, 35.98, 35.95, 32.28, 31.86, 30.45, 29.42, 29.29, 29.21, 25.36, 25.31, 24.12, 23.92, 19.74, 19.61, 12.82, 12.80. [M + H+] calcd (found): 442.2813 (442.2817). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-(4-N-(1Me-2-hydroxyiminomethyl-1H-imidazolium))butanamide Iodide (16a). Purification: 17% MeOH/DCM. Yield: 74 mg (0.17 mmol, 44%). 1H NMR (400 MHz, MeOD) δ 8.5 (s, 1H, CHN), 7.8 (d, 1H, CH-imid), 7.7 (d, 1H, CH-imid), 7.2 (d, 2H, 2 × CH-arom), 6.9 (dd, 2H, 2 × CH-arom), 4.6 (dd, 2H, CH2), 4.5 (m, 2H, CH2), 3.9− 4.1 (multiple resonances, 5H, 1 × CH2, 1 × CH3), 3.8 (d, 3H, CH3), 2.6 (dt, 2H, CH2), 2.2 (m, 2H, CH2), 1.1 (dd, 6H, 2 × CH3). 13C NMR (101 MHz, MeOD) δ 173.3, 168.5, 159.5, 137.6, 134.3, 129.4, 128.3, 124.5, 123.2, 114.0, 54.4, 51.7, 49.2, 48.8, 41.4, 36.2, 29.1, 25.2, 21.2. [M + H+] calcd (found): 430.2449 (430.2453). N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-(4-N-(1-Me2-hydroxyiminomethyl-1H-imidazolium))butanamide Iodide (16b). Purification: 17% MeOH/DCM. Yield: 38 mg (0.08 mmol, 67%). 1H NMR (400 MHz, MeOD) δ 8.5 (s, 1H, CHN), 7.6 (s, 1H, CHarom), 7.6 (d, 1H, CH-arom), 7.2−7.6 (multiple resonances, 5H, 3 × Ch-arom, 2 × CH-imid), 7.2 (d, 2H, 2 × CH-arom), 6.9 (dd, 2H, 2 × CH-arom) 4.5−4.6 (ds, 2H, CH2), 4.4−4.5 (multiple resonances, 4H, 2 × CH2), 4.1(s, 2H, CH2), 4.0 (s, 3H, CH3), 3.8 (d, 3H, CH3), 2.6 (dt, 2H, CH2), 2.2 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 173.3, 169.6, 168.9, 159.4, 138.5, 137.5, 134.3, 129.4, 128.3, 127.3, 124.5, 123.0, 114.0, 54.5, 53.5, 51.9, 49.3, 48.7, 42.8, 36,3, 29.1, 25.1. [M + H+] calcd (found): 478.2449 (478.2448). N-Butyl-N-[2-(isopropylamino)-2-oxoethyl]-N-(1-Me-2-hydroxyiminomethyl-1H-imidazolium)acetamide Iodide (17a). Yield: 74%. 1 H NMR (400 MHz, methanol-d4) δ 8.46 (d, J = 1.7 Hz, 1H), 7.71 (s, 1H), 7.74−7.65 (m, 1H), 5.57 (s, 1H), 5.51 (s, 1H), 4.19 (s, 1H), 4.13−3.89 (m, 5H), 3.49−3.34 (m, 2H), 1.79−1.66 (m, 1H), 1.59− 1.40 (m, 2H), 1.43−1.29 (m, 1H), 1.20 (dd, J = 25.1, 6.6 Hz, 6H), 1.03 (t, J = 7.3 Hz, 1H), 0.94 (t, J = 7.3 Hz, 2H). 13C NMR (101 MHz, MeOD) δ 168.09, 167.77, 166.20, 165.68, 138.79, 138.74, 134.47, 134.44, 124.65, 124.34, 124.11, 123.91, 51.05, 49.91, 48.95, 48.52, 48.28, 48.06, 47.92, 47.85, 47.74, 47.64, 47.43, 47.21, 47.00, 41.72, 41.40, 36.31, 36.02, 29.94, 29.16, 21.20, 21.17, 19.70, 19.62, 12.77. [M + H+] calcd (found): 338.2187 (338.2189). N-p-Methoxybenzyl-N-[2-(isopropylamino)-2-oxoethyl]-N-(1-Me2-hydroxyiminomethyl-1H-imidazolium))acetamide Iodide (17b). Yield: 87%. 1H NMR (400 MHz, methanol-d4) δ 8.48 (d, J = 19.9 Hz, 1H), 7.72 (dt, J = 5.1, 2.1 Hz, 3H), 7.38−7.29 (m, 1H), 7.25−7.18 (m, 2H), 7.04−6.96 (m, 1H), 6.92−6.85 (m, 2H), 5.60 (d, J = 17.6 Hz, 3H), 4.67 (s, 1H), 4.55 (s, 2H), 4.12−4.02 (m, 6H), 3.97 (s, 1H), 3.95−3.76 (m, 5H), 3.37 (s, 1H), 1.12 (dd, J = 6.6, 5.1 Hz, 9H). 13C NMR (101 MHz, MeOD) δ 167.78, 167.28, 166.54, 166.09, 159.80, 159.52, 138.84, 138.76, 134.51, 134.45, 129.74, 129.09, 127.58, 126.70, 124.63, 124.39, 124.12, 123.98, 114.17, 113.70, 54.47, 54.38, 51.20, 51.11, 50.03, 48.88, 48.48, 48.27, 48.13, 48.05, 47.98, 47.91, 47.84, 47.70, 47.63, 47.42, 47.20, 46.99, 41.58, 41.35, 36.27, 36.05, 21.14. [M + H+] calcd (found): 402.2136 (402.2129). N-p-Methoxybenzyl-N-[2-(benzylamino)-2-oxoethyl]-N-(1-Me-2hydroxyiminomethyl-1H-imidazolium))acetamide Iodide (17c). Yield: 26%. 1H NMR (400 MHz, methanol-d4) δ 8.50 (s, 1H), 8.45 (s, 1H), 7.76−7.66 (m, 2H), 7.43−7.20 (m, 7H), 7.24−7.16 (m, 1H), 7.01−6.94 (m, 1H), 6.92−6.83 (m, 1H), 5.64 (s, 1H), 5.57 (s, 1H), 4.70 (s, 1H), 4.56 (s, 1H), 4.37 (d, J = 5.0 Hz, 2H), 4.18 (s, 1H), 4.11−4.02 (m, 4H), 3.81 (d, J = 12.1 Hz, 3H), 3.62 (s). 13C NMR (101 MHz, MeOD) δ 168.84, 168.31, 166.57, 166.19, 159.80, 159.50, 138.81, 138.73, 138.23, 137.94, 134.51, 134.45, 129.70, 129.09, 128.23, 128.12, 127.50, 127.35, 127.19, 127.01, 126.84, 126.61, 124.63, 124.40, 124.13, 123.99, 114.18, 113.72, 62.89, 54.45, 54.38, 51.18, 51.08, 50.05, 48.78, 48.26, 48.12, 48.05, 47.90, 47.83, 47.62, 47.47, 47.41, 47.19, 46.98, 43.05, 42.68, 36.24, 36.03. [M + H+] calcd (found): 450.2136 (450.2144). N-Butyl-N-[2-(benzylamino)-2-oxoethyl]-N-(1-Me-2-hydroxyiminomethyl-1H-imidazolium)acetamide Iodide (17d). Yield: 81%.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01083. pKa measurement method, further details on in vitro reactivation kinetics, additional reactivation data, further details on molecular modeling, and further details on in vivo experiments (PDF) Molecular formula strings and some data (CSV)

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Corresponding Author

* [email protected]. ORCID

Martijn C. de Koning: 0000-0001-6482-9502 Florian Nachon: 0000-0003-0293-2429 Hans M. de Bruijn: 0000-0002-4026-337X Present Address ∥

H.M.d.B.: Gorlaeus Laboratories, Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the support from Dr. Peter Falk and funding from the Defense Threat Reduction Agency (DTRA, U.S.) via Contract HDTRA1-13-C-0025. Dr. Ben Capacio from 9390



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the U.S. Army Medical Research Institute of Chemical Defense (USMRICD, U.S.) is acknowledged for conducting ADME studies and the use of the online chemoinformatics system (BHSAI). F.W. and T.W. acknowledge funding from the Ministry of Defence, Bonn, Germany. F.N. acknowledges funding from the French Ministry of Armed Forces (DGA and SSA).

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ABBREVIATIONS USED

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REFERENCES

2-PAM, 2-pyridinealdoxime methiodide; 4-PAM, 4-pyridinealdoxime methiodide; ACN, acetonitrile; ANOVA, analysis of variance; A-site, active site; chlorpyrifos oxon, O,O-diethyl O3,5,6-trichloropyridin-2-ylphosphorothioate; CWA, chemical warfare agent; cyclosarin, O-cylohexyl methylphosphonofluoridate; Da, dalton; GA, ethyl N,N-dimethylphosphoramidocyanidate; GB, O-isopropyl methylphosphonofluoridate; GD, O-pinacolyl methylphosphonofluoridate; GF, O-cylohexyl methylphosphonofluoridate; HI-6, 4-carbamoyl-1-[[[2[(hydroxyimino)methyl]pyridinium-1-yl]methoxy]methyl]pyridinium dichloride; IC50, concentration at which 50% of the enzyme activity is inhibited; MeOH, methanol; MM, molecular modeling; obidoxim, 1,1′-[oxybis(methylen)]bis{4-[(E)(hydroxyimino)methyl]pyridinium} dichloride; OP, organophosphate; PAINS, pan assay interference compounds; PAS, peripheral anionic site; pralidoxim, 2-pyridinealdoxime methiodide; P-site, peripheral anionic site; PSL, peripheral site ligand; QM, quantum mechanics; sarin, O-isopropyl methylphosphonofluoridate; soman, O-pinacolyl methylphosphonofluoridate; tabun, ethyl N,N-dimethylphosphoramidocyanidate; VX, O-ethyl S-(2(diisopropylamino)ethyl)methylphosphonothiolate

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