Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library—Reactivators of Acetylcholinesterase

A Journal of

Accepted Article

Title: Reversal of tabun toxicity, enabled by a triazole annulated oxime

library-reactivators of acetylcholinesterase

Authors: Zrinka Kovarik, Jarosław Kalisiak, Nikolina Maček Hrvat, Maja

Katalinić, Tamara Zorbaz, Suzana Žunec, Carol Green, Zoran

Radić, Valery V. Fokin, K. Barry Sharpless, and Palmer Taylor

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To be cited as: Chem. Eur. J. 10.1002/chem.201805051

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Reversal of tabun toxicity enabled by a triazole-annulated oxime

library – reactivators of acetylcholinesterase

Zrinka Kovarik,^[b]Jarosław Kalisiak,^[c]Nikolina Maček Hrvat,^[b]Maja Katalinić,^[b]Tamara Zorbaz,^[b]

Suzana Žunec,^[b]Carol Green,^[d]Zoran Radić,^[a]Valery V. Fokin,^[c,e]K. Barry Sharpless^[c]and Palmer

Taylor*^[a]

Abstract: Acetylcholinesterase (AChE), an enzyme that degrades the

neurotransmitter acetylcholine, when covalently inhibited by

organophosphorus compounds (OPs), such as nerve agents and

pesticides, can be reactivated by oximes. However, tabun remains

among the most dangerous nerve agents due to the low reactivation

efficacy of standard pyridinium aldoxime antidotes. Therefore, finding

an optimal reactivator for prophylaxis against tabun toxicity and for

post-exposure treatment is a continued challenge. In this study, we

analyzed the reactivation potency of 111 novel nucleophilic oximes

mostly synthesized using the CuAAC triazole ligation between alkyne

and azide building blocks. We identified several oximes with

significantly improved in vitro reactivating potential for tabun-inhibited

human AChE, and in vivo antidotal efficacies in tabun-exposed mice.

Our findings offer a significantly improved platform for further

development of antidotes and scavengers directed against tabun and

related phosphoramidate exposures, such as the Novichok

compounds.

limitations, one of which is the inability of currently applied

standard oximes (TMB-4, obidoxime, HI-6 and 2-PAM) to

efficiently

reactivate

AChE

inhibited

by

certain

organophosphates.^[2]Current therapy is particularly ineffective in

case of tabun. Tabun is a highly toxic organophosphorus

compound (OP) and nerve agent of warfare and terrorism

originally developed as a pesticide in Germany in 1936. An

electron pair located on the phosphoramido group of tabun

prevents efficient nucleophilic attack by oximates (Figure 1).^[3]In

addition, the active center gorge of the tabun-AChE conjugate is

sterically congested, thus reducing access of the oxime to the

phosphorus center.^[4]Accordingly, phosphoramide conjugates

show resistance to the classical reactivators. Among standard

oximes, TMB-4 shows the highest reactivation potency in test

cases of tabun inhibition.^[2d]However, it is also the most toxic

among the four, prompting further search for efficient tabun

5]

reactivators.^[1b,

Our previous studies on pyridinium and

imidazolium oximes show that only pyridinium oximes with para-

positioned oxime group possess sufficient potency to reactivate

tabun-inhibited AChE. Particularly effective were K074, K075 and

K203, with overall reactivation rate constants about seven times

higher than that of TMB-4 and about four times higher than with

K048, their analogue sharing a similar butylene linker.^{[2d, 4c, 6]}

Click chemistry enables rapid synthesis of molecular libraries

from readily available building blocks^[7]for in vitro and in vivo

development of a landscape of agents to counteract OP exposure.

Herein, we report design and synthesis of over 100 oximes,

mainly those encompassing a triazole ring synthesized using the

copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to form

a linking 1,4-triazole (Figure 1). The library was screened for

oxime-dependent reactivation kinetics of tabun-, VX-, sarin-,

cyclosarin-, and paraoxon- inhibited human recombinant AChE.

Several novel oximes were particularly efficient reactivators in

Introduction

Toxicity and mortality associated with nerve agents arise primarily

from irreversible inhibition of acetylcholinesterase (AChE; EC

3.1.1.7), an enzyme whose role is essential in cholinergic

neurotransmission. Inhibition of AChE activity results in

accumulation of the neurotransmitter acetylcholine at vital

cholinergic sites, which in turn leads to life-threatening toxic

manifestations. Currently used therapies rely on anticholinergic

(atropine) agents and oximes as reactivators of inhibited AChE^[1]

along with anti-seizure agents. However, this approach has

[a] Dr. Z. Radić, Prof. P. Taylor*

Skaggs School of Pharmacy and Pharmaceutical Sciences, University

of California at San Diego, La Jolla, CA 92093-0650, USA

E-mail: pwtaylor@ucsd.edu

tabun

inhibition.

The

lead

compounds

[b] Dr. Z. Kovarik, Dr. N. Maček Hrvat, Dr. M. Katalinić, T. Zorbaz, Dr. S.

Žunec

Institute for Medical Research and Occupational Health, HR-10000

Zagreb, Croatia

[c] Dr. J. Kalisiak, Prof. K. B. Sharpless

Skaggs Institute for Chemical Biology, The Scripps Research Institute,

La Jolla, CA 92037, USA

[d] Dr. C. Green

SRI International, Menlo Park, CA 94025-3493, USA

[e] Dr. V. V. Fokin

The Bridge@USC, University of Southern California, Los Angeles, CA

90089, USA

Z. K. and J. K. contributed equally to this study

Supporting Information for this article is given via a link at the end of

the document.

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IR, MS, elemental analysis). Supporting Information details

synthetic and characterization data.

Figure 1. Structure of racemic nerve agent tabun, (R/S)-ethyl N,N-

dimethylphosphoramido cyanidate, and general scheme of CuAAC employed

to synthesize oxime library tested for the antidotal activity against tabun.

Reactivation of human recombinant AChE inhibited by tabun.

The novel oximes, partially presented in Cochran et al.^[7c], were

tested as reactivators of human recombinant AChE inhibited by

tabun (Figure 1), phosphoramide compounds with cyanide

leaving group that show resistance to the classical reactivators.

The oxime library was screened initially at a single oxime

concentration for reactivation of tabun-conjugated AChE. Since

some of the oximes exhibited a high binding affinity for AChE

resulting in inhibition of its activity, their reactivation rates were

were investigated in vivo. Since these oximes present an

incompletely studied structural scaffold, we also tested their

cytotoxicity on neuroblastoma cell line, SH-SY5Y. Our findings

offer a platform for further development of more efficacious

antidotes not only for counteracting tabun, but also exposure to

related nerve agents.

assayed at lower concentrations. We employed

a high

Results

percentage of reactivation achieved within a short time as the

main criterion for selection among the oximes. Out of 111 novel

oximes, 40 were deemed more efficient reactivators of AChE than

2-PAM and were then ranked in terms of the observed

reactivation rate (k_obs), as shown in Figure 2 and Table 1

(Appendix). Although reactivation was monitored up to 24h,

restoration of enzyme activity approaching 100% was evident in

only 9 of these oximes, while maximal reactivation of greater than

50% was found for 59 oximes. Six novel triazole compounds were

more efficient in the screening than obidoxime.^[8]Under our

experimental conditions, which employed a 10-fold excess of OP

over AChE for its inhibition, it is likely that one enantiomer of the

racemic tabun reacted preferentially with AChE (Figure 1),

because of steric differences between R_Pand S_Pconjugates

fitting into the spatially constrained acyl pocket of AChE.^[9a,b]We

expected dominant (> 90%) formation of a single tabun-AChE

conjugate. Restoration of the full 100% of the AChE activity may

have required reactivation of both diasteromeric conjugates of the

serine-linked phosphoester, including the one of lower prevalence

formed with the less reactive enantiomer. Although this

explanation is plausible, inherent experimental variability in

Chemistry. Our search for efficient reactivators of phosphylated

AChE was facilitated by rapid synthesis of a large number of

candidate compounds using CuAAC reaction (Figure 1). For this

study, in addition to 1,4-triazole-oximes (1A−62A) obtained by

CuAAC, a series of N-alkyl-2 pyridinium compounds (1B−5B),

some of which contain more complex N-alkyl substituents

(1C−17C) and a series of miscellaneous oximes (1D−27D) were

synthesized (chemical structure of all oximes are given in Table

1, Appendix). Owing to the nearly quantitative yields in all of the

triazole linkage steps, libraries of candidate compounds were

directly employed in the initial reactivation screens. Most

structurally characterized compounds were then made in 10–30

mg quantities; those used for in vivo efficacy and

pharmacokinetics experiments were synthesized in quantities

from 100 mg to several grams. Some of triazole aldoxime

compounds of little or no activity were screened directly as

reaction mixtures as described previously.^[7c]All of the

compounds were prepared in good yields and were characterized

by standard analytical spectroscopy methods (1D and 2D NMR,

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Figure 2. Reactivation screening of tabun-inhibited AChE by 40 out of 111 oximes with higher reactivation efficacy (k_obs) than 2-PAM. Oximes were 1 mM in the

reactivation buffer, except 0.2 mM 3A and 0.1 mM 9A, 22A, 23A and 25A due to reversible high affinity for binding. Presented values are means of at least three

experiments (standard errors were less than 10%). The rate constants for reactivation, k_obs, are shown in purple, maximal estimated reactivation percentages in

green, and the approximate times to achieve maximum plateau values at the given oxime concentration in orange.

our assay precludes us from emphasizing this detail in order to

avoid over-interpretation of our data.

reactivation efficiency depends not only on the rate of

displacement of conjugated tabun regenerating the original

activity, but also on the apparent affinity (low dissociation

constant) of oxime for the phosphorylated enzyme. On the other

hand, the highest reactivation rate (k₂) that was evaluated for

oxime 1B, was coupled with a low binding affinity (1/K_OX) for the

phosphoramidate conjugated AChE, resulting in a compromised

overall reactivation rate (k_r). Nevertheless, five of the most

efficient reactivators of tabun-phosphorylated AChE are triazole

oximes with an aldoxime-positioned ortho- to the quaternary

pyridinium nitrogen or at the 2-position of the imidazolium ring.

As detailed in Table 1 (Appendix), maximal reactivation was

equal or less than 20% for 41 of the oximes, and the observed

reactivation rate constant was not calculated. Compounds with

low reactivation rates and a lower percentage of reactivation, in

our view, do not provide data for precise quantitation. In addition,

we determined the degree of reversible inhibition by oximes for

AChE by assessing enzyme activity when not exposed to tabun.

Of the 111 oximes, 99 exhibited some inhibitory potency at 0.01

mM: 37 of which inhibited AChE less than 30%, 31 oximes

inhibited AChE between 30 and 50%, 26 oximes between 50 and

80%, and 17 oximes more than 80% (Table 1, Appendix). The

additional 12 oximes, identified as strong reversible inhibitors of

AChE, inhibiting more than 95% at 0.01 mM concentration, were

re-examined at 5–50-fold lower oxime concentrations, where they

displayed inhibitory potencies between 10 and 85%.

For the most efficient oximes that reactivated AChE approaching

completion within short time, we ran reactivation kinetics over a

concentration range of the oximes enabling us to deconstruct

reactivation constants. Reactivation constants involved in the two-

step reactivation mechanism (equilibrium binding followed by the

irreversible reactivation) are shown in Figure 3. In Table 2, results

are arranged by the second-order rate constants since the

Out of 8 bis-quaternary reactivators,

quaternary nitrogen atoms in an extended distance of 8.4 Å,

equivalent to methylenes, two oximes have distances

corresponding to methylenes and one has distance

6 oximes have two

8

7

a

corresponding to 6 methylenes. A distance of ~8 Å or more

between the two quaternary nitrogens enables optimal positioning

of the oxime molecule in the phosphoramidated AChE gorge.

Moreover, the highest k_rwas achieved by oximes 3A and 9A, that

have two quaternary nitrogens separated by ~8.4 Å primarily

resulting from their low K_OX(Table 2) – a value at least 10-fold

lower than that of TMB-4 or bis-quaternary pyridinium

4c, 6a,b]

aldoximes.^[2d,

Oxime 2A stands out from several

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Figure 3. General scheme of oxime-assisted reactivation of phosphylated AChE and kinetics of reactivation by selected oximes. Mean values from at least three

experiments are presented. Reactivation constants were determined by nonlinear regression of experimental data and are presented in Table 2.

Table 2. Reactivation kinetic analysis of tabun-inhibited AChE with the most efficient oximes.^[a]

React_max

(%)

Oxime

Oxime Structure

Chemical Name

k₂(min^-1)

K_OX(mM)

k_r(M^-1min^-1)

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(2-

((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

3A

0.015 ± 0.001 0.0056 ± 0.0016

0.010 ± 0.001 0.0043 ± 0.0016

3000 ± 630

90

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(2-

(hydroxyimino)methyl)-1-methyl-1H-imidazol-

3-ium-3-yl)ethyl)-1H-1,2,3-triazol-1-

9A

2A

2500 ± 1275

355 ± 180

80

yl)propyl)pyridinium hexafluorophosphate

1-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)butyl)-2-

((hydroxyimino)methyl)pyridinium

hexafluorophosphate

0.026 ± 0.003

0.073 ± 0.036

100

4-((Hydroxyimino)methyl)-1-(2-(4-(2-(2-

((hydroxyimino)methyl)-1-methyl-1H-imidazol-

3-ium-3-yl)ethyl)-1H-1,2,3-triazol-1-

yl)ethyl)pyridine-1-ium dichloride

4A

1A

0.018 ± 0.001

0.028 ± 0.002

0.070 ± 0.016

0.126 ± 0.035

250 ± 57

220 ± 65

80

3-(2-(1-(3-((2-

(Hydroxyimino)ethyl)dimethylammonio)propyl)-

1H-1,2,3-triazol-4-yl)ethyl)-2-

100

((hydroxyimino)methyl)-1-methyl-1H-imidazol-

3-ium hexafluorophosphate

1-(2-(1-(3-(4-Carbamoylpyridinium-1-

yl)propyl)-1H-1,2,3-triazol-4-yl)ethyl)-2-

((hydroxyimino)methyl)pyridinium

hexafluorophosphate

20A

0.006 ± 0.0006

0.103 ± 0.041

60 ± 24

60

1-(2-(1-(3-((2-

(Hydroxyimino)ethyl)dimethylammonio)propyl)-

1H-1,2,3-triazol-4-yl)ethyl)-2-

((hydroxyimino)methyl)pyridinium

hexafluorophosphate

5A

0.014 ± 0.002

0.011 ± 0.002

0.29 ± 0.25

0.24 ± 0.11

47 ± 42

45 ± 21

80

90

2-((Hydroxyimino)methyl)-1-((1-(2-(4-

((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-4-yl)methyl)pyridinium

hexafluorophosphate

14A

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1-(2-(1-(2-(4-Carbamoylpyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-4-yl)ethyl)-2-

((hydroxyimino)methyl)pyridinium

hexafluorophosphate

15A

0.013 ± 0.003

0.39 ± 0.33

33 ± 30

70

1-Butyl-2-((hydroxyimino)methyl)pyridinium

chloride

2B

1B

0.020 ± 0.002

0.033 ± 0.008

0.82 ± 0.19

3.9 ± 1.7

25 ± 6.1

90

80

2-((Hydroxyimino)methyl)-1-propylpyridinium

chloride (1B)

8.5 ± 4.3

[a] Kinetic parameters (± S.E.): the maximal first-order reactivation rate constant (k₂), the apparent dissociation constant of the conjugated enzyme-oxime reversible

complex (K_OX), the overall second-order reactivation rate constant (k_r) and maximal percentage of reactivation (React_max) were determined from at least three

experiments.

selected mono-pyridinium oximes with high reactivation rate and

moderate affinity. Its overall reactivation rate is comparable to the

most efficient reactivators of tabun-inhibited AChE – bis-

pyridinium K048, K074, K075 and K203^{[4c, 6a,c, 10]}and uncharged

reactivators^[11]. Although prior computational analyses of bis-

pyridinium oximes for optimal tabun reactivation indicate the

oxime group should be in the para-position on the pyridinium

ring^[6b], our results show that it is not the case for this group of

oximes, since out of the oximes analyzed herein, only 14A and

4A have one oxime group in para-position on the pyridinium ring

(Table 2).

The absorption spectral analyses and estimation of pK_avalues

given in Figure 4 show that nucleophilicity of three lead oximes is

similar to the pK_avalue of the ortho-pyridinium oximes such as HI-

6.^[12]Therefore, for reactivation of tabun-inhibited AChE_,an

altered orientation and an angle of the oxime group with an anti-

regioisomer orientation promote more efficient reactivation

kinetics.

Figure 4. Absorption spectra at 25 °C and pH-dependence of normalized absorbance wherefrom the pK_avalues of the oxime group for oximes 2A, 3A and 14A

were evaluated (goodness of the fit was R²> 0.99).

Screening of oximes as broad spectrum reactivators of VX-,

sarin-, cyclosarin-, and paraoxon-inhibited AChE. While no

currently used oxime is sufficiently effective against all nerve

agents and pesticides, they show substantially different rate

constants depending on the conjugated organophosphate. Since

oximes selected from this study exhibited the potential to

reactivate tabun-inhibited AChE, a conjugate that is particularly

resistant to reactivation, we examined the 19 most potent

reactivating oximes for tabun as reactivators of VX-, sarin-,

cyclosarin- and paraoxon-inhibited AChE. As presented in Figure

5, several of the selected oximes exhibit reasonable reactivation

rates and maximal activities for the tested organophosphates

revealing an activity spectrum of universal reactivation. For

example, the bis-pyridinium aldoxime 14A displays a high

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potency to reactivate both phosphonate and phosphorate AChE

conjugates. Moreover, several of these oximes reactivate

conjugated AChE more efficiently than the standard reference

oxime, 2-PAM, but may lack the wide spectrum of potency of HI-

6^[11c]on phosphorates and methylphosphonates.

Pharmacokinetics of lead reactivators of phosphorylated

AChE. Oximes 2A, 3A and 14A, with the high reactivation rates

and moderate binding affinities, emerged as the lead reactivators

of tabun-inhibited AChE, and were chosen for further

pharmacokinetic and antidotal testing in mice. Since these oximes

are quaternary cations, rapid glomerular filtration and renal

tubular secretion are expected, as it was previously shown for

similar pyridinium aldoximes.^[13]The clearance profile (Figure 7),

shows that at 5 mg kg^-1injected intramuscularly, most of the initial

concentration was eliminated from mice plasma within 60 min. A

low volume of distribution, where the antidote is confined to

extracellular compartments, seems contributory. In any event,

clearance is rapid and multiphasic, but the rapid clearance (short

half-life) persists for close to an hour, where the plasma

concentration is 10% or less of the original value. This short

resident time in the plasma and rapid clearance would require

multiple dosing for a sustained therapeutic action of a long-acting

organophosphate.^[14]It is noteworthy to mention that typically

upon i.m. administration of a quaternary oxime, there is a short

redistribution phase followed by the kinetics of elimination from

the plasma. That redistribution phase probably takes place within

15 min, a time when the first

Cytotoxicity. Since the CNS, peripheral autonomic synapses,

and the neuromuscular junction are the primary sites of

organophosphate inhibition and oxime reactivation, we have

chosen a neuroblastoma cell line SH-SY5Y for initial cytotoxicity

screens. For testing, we have selected oximes 2A, 3A, and 14A

that display different reactivation potencies, as demonstrated by

the kinetic constants presented in Table 2 and Figure 3. Oximes

were assayed in concentrations up to 800 µM, and the results are

presented in Figure 6.

As shown, the maximum percentage inhibition of cell-growth and

differentiation was about 20% and occurred only for the highest

oxime concentration tested, a concentration level exceeding

those employed for in vitro reactivation assays. Practically, a

dose-response relationship was not evident in the studied

concentration range, so the lead oximes appeared not to show

significant cytotoxicity. Therefore, despite these oximes

presenting a new structural scaffold, the absence of toxicity in the

neuronal cell assay is comparable to standard oximes as well as

to the bis-pyridinium oximes: TMB-4, ICD-585 or K048.^[6b]

Figure 5. Reactivation screening of AChE inhibited by VX, sarin, cyclosarin and paraoxon by selected 0.1 mM oximes. Observed first order reactivation rates (k_obs

)

are compared to 2-PAM. Presented values are means of at least three experiments (standard errors were less than 10%).

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either dose (5% or 25% of oxime’s LD₅₀), giving a satisfactory in

vivo Protective Index after exposure to tabun.^[6a]When combined

with atropine, 3A ensured survival of all mice against 7.9  LD₅₀

of tabun or 2.4 mg kg^-1. The administration of a single atropine

dose of 10 mg kg^-1one minute after tabun resulted in the

Protective Index of 1.3. For other nerve agent poisoning, therapy

with atropine alone resulted in a Protective Index below 2.^[1b]

Despite their toxicity, tested oximes provides significant protection

against the lethal effects of tabun, presumably due to their

enhanced reactivation capacity for tabun-phosphorylated AChE

and to concomitant inhibition of unconjugated AChE that may

protect residual active AChE from irreversible phosphorylation. It

is worthwhile to note that these Protective Indices were obtained

by an up to 20-fold lower dose of triazole oximes than for TMB-4,

selected on the basis of their acute toxicity and LD₅₀(Table 3).

Out of three tested oximes, 14A provided the greatest antidotal

potential in treatment upon exposure to the other OPs and

ensured survival of all mice against 10 x LD₅₀of VX and paraoxon.

The antidotal action of tested compounds appears related to the

reactivation kinetics of phosphylated AChE, since their

reactivating efficacy shown in vitro (Figure 5) corresponds to their

therapeutic efficacy in vivo (Table 3). Further examination of the

Protective Indices, administering a lead oxime antidote against

the phosphoramidate nerve agents by intramuscular (i.m.)

administration at various times after OP exposure, should now be

considered.

Figure 6. Cytotoxicity of oximes on SH-SY5Y cells plotted as a logarithmic

function of concentration. The results were expressed as a percentage of

inhibition of cell growth compared with control (non-treated) cells.

point was taken. An extrapolation of concentration to zero time

yields values of ~9,000 ng mL^-1or 9 mg L^-1for 3A and 14A; this

yields a Volume of Distribution of about 0.55 L kg^-1, which is

reasonable for a quaternary agent. For oxime 2A the extrapolation

value might be closer to 2,000 ng mL^-1or 2 mg L^-1. This finding

may implicate sequestration of some of the 2A compound or a

more rapid and complete redistribution phase. Based on these

initial findings, a more complete pharmacokinetic analysis,

involving plasma and tissue levels with

a single lead,

phosphoramide-effective oxime seems warranted.

Antidotal activity of these oximes in OP-exposed mice. We

evaluated in vivo antidotal activity of the oximes in therapy against

tabun and four other OPs. The antidotal action of oximes in mice

was tested in these early studies by intraperitoneal (i.p.)

administration of oxime admixed with atropine one min after

subcutaneous (s.c.) OP exposure (Table 3). The oximes showed

significant acute toxicity. However, all three selected oximes (2A,

3A and 14A) showed better antidotal efficacy than TMB-4 with

Figure 7. Oxime clearance profile upon i.m. administration of 5 mg kg^-1of three

oximes in mouse plasma (mean of 3 mice per group and time point). Limits of

detection were ~2 ng mL^-1, 50 ng mL^-1and 100 ng mL^-1for 2A, 3A and 14A,

respectively.

Table 3. Antidotal efficacy of oximes applied at 5 or 25 % of their LD₅₀after OP exposure of mice by administering the oxime (i.p.) with

atropine (10 mg kg^-1) 1 min after OP (s.c.) ^[a]

Oxime Dose

Oxime LD₅₀

Oxime

Protective Index (MD)

(mg kg^-1)

(mol kg^-1)

Tabun

VX

Sarin

Paraoxon

Soman

1.4

7.1

2.2 (1.6)

8.9 (4.0)

6.4 (5.0)

9.4 (6.3)

1.6 (1.3)

2.1 (1.6)

11.6 (7.9)

11.2 (7.9)

2.5 (1.6)

3.8 (2.5)

2A

26.7

8.4

0.63

3.1

1.6 (1.0)

8.9 (7.9)

5.8 (5.0)

6.8 (6.3)

1.4 (1.3)

4.0 (2.0)

14.7 (6.3)

11.6 (10.0)

1.8 (1.3)

1.8 (1.6)

3A

3.6

19

1.5 (1.3)

5.0 (3.2)

8.9 (7.9)

10.3 (10.0)

2.0 (1.6)

5.4 (2.0)

13.6 (10.0)

14.7 (10.0)

2.2 (1.6)

2.5 (2.0)

14A

18.5

13

3.4 (2.5)

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TMB-4

89.8

63

4.0 (2.0)

[a] LD₅₀- acute toxicity (i.p.) was based upon 24h mortality rates. Protective Index was the ratio of LD₅₀between OP with antidote and

OP given alone. Maximal dose of OP, MD, is the highest multiple of the LD₅₀of OPs, which was fully counteracted by the antidote.

We have synthesized and pharmacologically characterized a

substantial number of 1,2,3-triazole-linked N-methylpyridinium

Discussion

and 2-methylimidazolium oximes with significantly improved in

vitro reactivating efficacies for tabun-inhibited AChE, when

compared to standard bis-pyridinium oximes. This underscores

the importance of an efficient cyclization azide-alkyne CuAAC

process in forming congeneric libraries of triazole test compounds.

Pharmacokinetic properties of the lead compounds were

monitored in initial studies in mice, and in vivo antidotal efficacy

for three leads was established in tabun-exposed mice. To assess

the spectrum of activity, we selected nineteen of the most efficient

tabun reactivators to test their efficacy in reactivating AChE

inhibited with other OPs. Although several of the tabun antidote

leads showed reasonable potency in reactivating other tested

AChE-OP conjugates, a universally superior antidote against all

organophosphates was not identified. Multidimensional and

hydrogen bonding constraints in the AChE active center gorge

conjugated with methylphosphonates, phosphorates and

phosphoramidates, mitigate against the likelihood of finding a

single reactivator most efficient for tabun, sarin, VX, cyclosarin,

paraoxon and their congeners. Nevertheless, our findings offer a

valuable and comprehensive platform for further development of

antidotes and scavengers against tabun and related

phosphoramidate exposures, such as the Novichok series of

compounds used in poisoning in Salisbury, England in the Spring

of 2018¹⁸.

Our study has identified a series of oximes with a high propensity

for reactivation of human recombinant AChE inhibited by

organophosphates. The organophosphate conjugates resemble

transition state analogues of acyl ester catalysis, forming

relatively stable conjugates of cholinesterases and other serine

hydrolases.^[15]As found initially by Irwin Wilson and colleagues,

nucleophilic oximes, that are site-directed, show a preferential

activity as reactivating agents.^[16]While most of the

alkylphosphate inhibitors of AChE are either phosphorates or

methylphosphonates, tabun is unique in representing the

phosphoramidates. Hence, the differing chemistry of the

phosphoramidates, while forming

a tetrahedral conjugate,

presents a free electron pair on the phosphoramide nitrogen. Also,

the substantial steric hindrance of the dimethylamido group may

influence the resistance of tabun to reactivation by certain oximes.

Moreover, the phosphoramides have two potential modes of

ageing in contrast to the alkyl methylphosphonates and

phosphorates.^[17]

Recently,

a new set of highly toxic phosphoramidates,

synthesized in Russia, were brought to light through an attempted

homicide, with a compound in the Novichok series, possessing a

fluoride leaving group.^[18]The triazole compounds synthesized

here and screened in terms of reactivation of the tabun conjugate,

in relation to other methylphosphonate and alkyl phosphorate

conjugates, suggest that they could serve as lead

countermeasures for tabun and other phosphoramidate exposure.

The lead reactivators contain an aldoxime substituted ortho- to

the methylpyridinium nitrogen or a 2-methylimidazole aldoxime

moiety. Our study has included reactivation measurements of

AChE inhibited by common nerve agents, VX, sarin and

cyclosarin, and pesticides forming a toxicant analogous to

paraoxon, along with initial assessments of their toxicity in cell

culture and mice. Novel reactivators, or recently developed non-

oxime reactivators, while showing generally broad activity

towards the phosphorates and phosphonates good activity, and

potential to penetrate CNS exhibit limitations toward reactivation

of the tabun conjugate.^[19]As pyridinium and imidazolium

aldoximes, they are not likely to cross the blood-brain barrier

Experimental Section

Chemistry. The majority of oximes were synthesized by Cu(I)-catalyzed

azide-alkyne cycloaddition to form the anti-triazole using a library of small

molecule building blocks, as shown in Figure 1. 1H and 13C NMR spectra

were recorded on a Varian INOVA-400 spectrometer in CDCl3 (7.26 ppm

for 1H, 77.00 ppm 13C), d6-DMSO (2.50 ppm for 1H, 39.43 ppm for 13C)

as standards. Reagents and solvents were purchased from Aldrich, Acros

or GFS and used without further purification.

General method for the synthesis of 1,4-triazole-oximes. The alkyne

(1.66 mmol) and the azide (1.66 mmol) were dissolved in the mixture of t-

BuOH:H₂O, 2:1 (10 mL), then sodium ascorbate (65.7 mg, 0.33 mmol, 20%

mol.) and CuSO₄(13.2 mg, 0.083 mmol, 5% mol.) were added. Reactions

were carried out for 16-24h at room temperature or 60 °C until LC/MS

analysis shows no starting materials. Subsequently the mixture was

evaporated, re-dissolved in H₂O (10 mL) and filtered if necessary. The

aquatic layer was washed with CHCl₃and a solution of NaPF₆(1.11 g, 6.64

mmol, 4 equiv.) in H₂O (5 mL) was added. The mixture was cooled to 0 °C

and the hexafluorophosphate salt was separated, washed with H₂O and

dried on high-vacuum. Products were further purified by dissolving the

hexafluorophosphate salt in small amount of acetone, filtrated and adding

a solution of Bu₄N⁺Clˉ (1 equiv.) in small amount of acetone. The chloride

salt is then separated (hygroscopic) and washed with acetone.

Subsequent an anion exchange from Clˉ to PF₆ˉ was conducted using the

method described above and gives the product as non-hygroscopic

hexafluorophosphate salt.^[23]Other attempts to purify triazole-oximes on

silica-gel or using RP-C18 columns were unsuccessful.

rapidly, conferring their antidotal activity only to the peripheral

13e, 20]

nervous system and not the CNS.^[6c,

As quaternary

ammonium compounds, they are likely to be rapidly cleared by

the kidney, similar to other pyridinium aldoximes. Also, their

antidotal activity towards inhaled sarin in a primate system has

yet to be examined as has been the case for sarin exposure

followed by the antidotal actions of zwitterionic, non-quaternary

oximes, such as RS194B.^[21]

Synthesis

of

1,4-triazole-oximes.

3-(2-(1-(3-((2-

Conclusions

(Hydroxyimino)ethyl)dimethylammonio)propyl)-1H-1,2,3-triazol-4-

10.1002/chem.201805051

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yl)ethyl)-2-((hydroxyimino)methyl)-1-methyl-1H-imidazol-3-ium

3.20-3.13 (m, 8H, A+B), 2.37-2.30 (m, 2H, A+B). ESI HR (MeOH) calcd

hexafluorophosphate (1A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈ 2:8 (84%). ¹H NMR (400

MHz, DMSO)  = 13.01 (s, 1H, A+B), 12.06 (s, 0.2H, A), 11.89 (s, 0.8H,

B), 8.48 (s, 1H, A+B), 7.95 (s, 1H, A+B), 7.81-7.79 (m, 2H, A+B), 7.61 (t,

J = 6.2Hz, 0.8H, B), 7.28 (t, J = 4.6Hz, 0.2H, A), 4.61 (t, J = 7.2Hz, 2H,

A+B), 4.39 (t, J = 6.8Hz, 2H, A+B), 4.27 (d, J = 4.8Hz, 0.4H, A), 4.10 (d,

J = 6.4Hz, 1.6H, B), 3.91 (s, 3H, A+B), 3.39-3.29 (m, 2H, A+B, the signal

overlaps with residual H₂O), 3.16 (t, J = 7.0 Hz, 2H, A+B), 3.11 (s, 1.2H,

A), 3.04 (s, 4.8H, B), 2.34-2.24 (m, 2H, A+B). ESI HR (MeOH) calcd for

C₁₆H₂₈N₈O₂0.5[M]²⁺: 182.1163 found: 182.1163.

for C₁₆H₂₇N₈O₂[M-H]⁺: 363.2251 found: 363.2257.

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(2-((hydroxyimino)methyl)-1-methyl-

1H-imidazol-3-ium-3-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate (9A). The title compound was obtained using

general method (88%). ¹H NMR (400 MHz, DMSO)  = 13.20 (s, 1H), 12.98

(s, 1H), 8.94 (d, J = 6.0Hz, 1H), 8.75 (s, 1H), 8.56-8.52 (m, 1H), 8.47 (s,

1H), 8.43-8.41 (m, 1H), 8.10-8.07 (m, 1H), 7.91 (s, 1H), 7.78 (s, 2H), 4.75

(t, J = 7.6Hz, 2H), 4.61 (t, J = 7.0Hz, 2H), 4.48 (t, J = 7.0Hz, 2H), 3.90 (s,

3H), 3.15 (t, J = 7.0Hz, 2H), 2.44-2.36 (m, 2H). ESI HR (MeOH) calcd for

C₁₈H₂₃N₈O₂[M-H]⁺: 383.1938 found: 383.1945.

1-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)butyl)-2-

2-((Hydroxyimino)methyl)-1-(2-(1-(2-(2-((hydroxyimino)methyl)pyridinium-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (2A). The title

compound was obtained using general method (67%). ¹³C NMR for the Cl

salt (100 MHz, DMSO)  = 146.8, 145.9, 145.0, 141.2, 136.1, 128.7, 128.6,

127.9, 127.7, 127.3, 125.7, 122.2, 57.5, 52.6, 29.8, 25.2, 24.2. ESI (MeOH)

for [M]⁺= 3 36.0.

1-yl)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)pyridinium

hexafluorophosphate

(10A). The title compound was obtained using general method (78%). ¹H

NMR (400 MHz, DMSO)  = 13.18 (s, 1H), 13.14 (s, 1H), 8.82 (d, J = 6.0Hz,

1H), 8.70 (s, 1H), 8.61-8.60 (m, 2H), 8.53 (q, J = 7.6Hz, 2H), 8.40-8.37 (m,

2H), 8.03-7.96 (m, 3H), 5.21 (brt, J = 5.2Hz, 2H), 4.97 (brt, J = 6.8Hz, 2H),

4.90 (brt, J = 5.2Hz, 2H), 3.23 (brt, J = 6.8Hz, 2H). ¹³C NMR (100 MHz,

DMSO)  = 147.5, 147.0, 146.0, 145.9, 145.4, 141.7, 141.2, 141.0, 127.2,

127.1, 125.7, 124.5, 118.0, 57.0, 56.9, 48.8, 25.9. ESI HR (MeOH) calcd

for C₁₈H₂₀N₇O₂[M-H]⁺: 366.1673 found: 366.1672.

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(2-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

(3A). The title compound was obtained using general method (86%). ¹H

NMR (400 MHz, DMSO)  = 13.11 (brs, 2H), 8.94 (d, J = 6.0Hz, 1H), 8.88

(d, J = 6.0Hz, 1H), 8.75 (s, 1H), 8.68 (s, 1H), 8.55-8.49 (m, 2H), 8.42-8.36

(m, 2H), 8.09-8.00 (m, 2H), 7.94 (s, 1H), 5.02 (t, J = 6.8Hz, 2H), 4.74 (t, J

= 7.4Hz, 2H), 4.48 (t, J = 7.0Hz, 2H), 3.27 (t, J = 6.8Hz, 2H), 2.43-2.36 (m,

2H). ¹³C NMR (100 MHz, DMSO)  = 147.1, 147.1, 146.1, 145.9, 145.3,

145.2, 141.5, 141.3, 141.2, 127.4, 127.2, 125.8, 125.7, 123.6, 57.0, 55.4,

46.2, 30.4, 26.2. ESI HR (MeOH) calcd for C₁₉H₂₂N₇O₂[M-H]⁺: 380.1829

found: 380.1831.

2-((Hydroxyimino)methyl)-1-((1-(4-(2-((hydroxyimino)methyl)pyridinium-1-

yl)butyl)-1H-1,2,3-triazol-4-yl)methyl)pyridinium

hexafluorophosphate

(11A). The title compound was obtained using general method (36%). ¹H

NMR (400 MHz, DMSO)  = 13.17 (s, 1H), 13.15 (s, 1H), 9.14 (d, J = 5.6Hz,

1H), 8.96 (d, J = 5.6Hz, 1H), 8.88 (s, 1H), 8.78 (s, 1H), 8.61-8.52 (m, 2H),

8.45-8.40 (m, 2H), 8.27 (s, 1H), 8.17-8.12 (m, 1H), 8.10-8.05 (m, 1H), 6.11

(s, 2H), 4.73 (t, J = 6.8Hz, 2H), 4.43 (t, J = 6.8Hz, 2H), 1.90-1.79 (m, 4H).

ESI HR (MeOH) calcd for C₁₉H₂₂N₇O₂[M-H]⁺: 380.1829 found: 380.1821.

1-(2-(1-(3-((2-(Hydroxyimino)ethyl)dimethylammonio)propyl)-1H-1,2,3-

triazol-4-yl)ethyl)-2-((hydroxyimino)methyl)pyridinium

2-((Hydroxyimino)methyl)-1-(2-(1-(3-(pyridinium-1-yl)propyl)-1H-1,2,3-

triazol-4-yl)ethyl)pyridinium hexafluorophosphate (12A). The title

compound was obtained using general method (86%). ¹H NMR (400 MHz,

DMSO)  = 13.06 (s, 1H), 8.98 (d, J = 5.6Hz, 2H), 8.84 (d, J = 5.6Hz, 1H),

8.64 (s, 1H), 8.57 (t, J = 7.8Hz, 1H), 8.49 (t, J = 7.8Hz, 1H), 8.34 (dd, J¹=

8.0Hz, J²= 1.2Hz, 1H), 8.14-8.10 (m, 2H), 8.02-7.98 (m, 1H), 7.89 (s, 1H),

4.97 (t, J = 7.0Hz, 2H), 4.57 (t, J = 7.2Hz, 2H), 4.38 (t, J = 6.8Hz, 2H), 3.21

(t, J = 6.8Hz, 2H), 2.48-2.41 (m, 2H). ¹³C NMR (100 MHz, DMSO)  =

147.1, 146.0, 145.6, 145.3, 144.8, 141.5, 141.2, 128.1, 127.2, 125.7, 123.6,

58.3, 57.0, 46.2, 30.8, 26.1. ESI HR (MeOH) calcd for C₁₈H₂₁N₆O [M-H]⁺:

337.1771 found: 337.1771.

hexafluorophosphate (5A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈ 2:8 (75%). ¹H NMR (400

MHz, DMSO)  = 13.12 (s, 1H, A+B), 12.06 (s, 0.2H, A), 11.88 (s, 0.8H,

B), 8.89 (d, J = 5.6Hz, 1H, A+B), 8.70 (s, 1H, A+B), 8.54 (t, J = 8.0Hz, 1H,

A+B), 8.38 (d, J = 8.0Hz, 1H, A+B), 8.06-8.02 (m, 1H, A+B), 7.96 (s, 1H,

A+B), 7.61 (t, J = 6.0Hz, 0.8H, B), 7.28 (t, J = 4.4Hz, 0.2H, A), 5.02 (t, J =

6.6Hz, 2H, A+B), 4.38 (t, J = 6.6Hz, 2H, A+B), 4.26 (d, J = 4.4Hz, 0.4H,

A), 4.09 (d, J = 6.0Hz, 1.6H, B), 3.40-3.25 (m, 4H, A+B, the signal overlaps

with residual H₂O), 3.11 (s, 1.2H, A), 3.04 (s, 4.8H, B), 2.32-2,23 (m, 2H,

A+B). ESI HR (MeOH) calcd for C₁₇H₂₇N₇O₂0.5[M]²⁺: 180.6108 found:

180.6115.

2-((Hydroxyimino)methyl)-1-(2-(1-(3-(4-((hydroxyimino)methyl)pyridinium-

2-((Hydroxyimino)methyl)-1-(((4-(2-(2-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)methoxy)methyl)pyridinium

1-yl)propyl)-1H-1,2,3-triazol-4-yl)ethyl)pyridinium

hexafluorophosphate

(13A). The title compound was obtained using general method (84%). ¹H

NMR (400 MHz, DMSO)  = 13.09 (s, 1H), 12.86 (s, 1H), 8.96 (d, J = 6.8Hz,

2H), 8.88 (d, J = 6.0Hz, 1H), 8.68 (s, 1H), 8.55-8.51 (m, 1H), 8.43 (s, 1H),

8.39-8.37 (m, 1H), 8.23 (d, J = 6.8Hz, 2H), 8.06-8.02 (m, 1H), 7.93 (s, 1H),

5.01 (t, J = 6.8Hz, 2H), 4.57 (t, J = 7.2Hz, 2H), 4.42 (t, J = 6.8Hz, 2H), 3.26

(t, J = 7.2Hz, 2H), 2.47-2.43 (m, 2H, the signal overlaps with residual

DMSO). ESI (MeOH) for [M-H]⁺= 380.2.

hexafluorophosphate (6A). The title compound was obtained using

general method (60%). ¹H NMR (400 MHz, DMSO)  = 13.22 (s, 1H), 13.14

(s, 1H), 9.03 (d, J = 5.6Hz, 1H), 8.86 (d, J = 5.6Hz, 1H), 8.73 (s, 1H), 8.62-

8.59 (m, 2H), 8.56-8.52 (m, 1H), 8.43 (dd, J¹= 8.4Hz, J²= 1.2Hz, 1H), 8.39

(dd, J¹= 8.2Hz, J²= 1.4Hz, 1H), 8.14-8.09 (m, 2H), 8.05-8.02 (m, 1H), 6.27

(s, 2H), 6.02 (s, 2H), 4.99 (t, J = 7.2Hz, 2H), 3.23 (t, J = 7.2Hz, 2H). ¹³C

NMR (100 MHz, DMSO)  = 147.1, 147.1, 146.8, 146.2, 145.5, 145.3,

142.3, 141.4, 141.2, 127.3, 127.2, 126.0, 125.5, 124.5, 85.3, 77.1, 57.0,

26.0. ESI HR (MeOH) calcd for C₁₈H₂₀N₇O₃[M-H]⁺: 382.1622 found:

382.1625.

2-((Hydroxyimino)methyl)-1-((1-(2-(4-((hydroxyimino)methyl)pyridinium-1-

yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)pyridinium

hexafluorophosphate

(14A). The title compound was obtained using general method (64%). ¹H

NMR (400 MHz, DMSO)  = 13.21 (brs, 1H), 12.93 (brs, 1H), 9.11 (d, J =

6.4Hz, 1H), 8.85 (s, 1H), 8.79 (d, J = 6.4Hz, 2H), 8.60-8.56 (m, 1H), 8.44

(d, J = 8.8Hz, 1H), 8.40 (s, 1H), 8.24 (s, 1H), 8.16-8.12 (m, 3H), 6.10 (s,

2H), 5.07 (s, 4H). ESI (MeOH) for [M-H]⁺= 352.3.

1-(3-(4-(2-((2-(Hydroxyimino)ethyl)dimethylammonio)ethyl)-1H-1,2,3-

triazol-1-yl)propyl)-2-((hydroxyimino)methyl)pyridinium

hexafluorophosphate (7A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈ 2:8 (91%). ¹H NMR (400

MHz, DMSO)  = 13.19 (s, 1H, A+B), 12.05 (s, 0.2H, A), 11.89 (s, 0.8H,

B), 8.95 (d, J = 6.0Hz, 1H, A+B), 8.75 (s, 1H, A+B), 8.55-8.51 (m, 1H,

A+B), 8.41 (d, J = 8.4Hz, 1H, A+B), 8.09-8.05 (m, 1H, A+B), 7.98-7.95 (m,

1H, A+B), 7.67 (t, J = 6.0Hz, 0.8H, B), 7.35 (t, J = 3.8Hz, 0.2H, A), 4.78 (t,

J = 7.0Hz, 2H, A+B), 4.52 (t, J = 7.0Hz, 2H, A+B), 3.35 (d, J = 4.4Hz, 0.4H,

A), 4.18 (d, J = 6.0Hz, 1.6H, B), 3.70-3.57 (m, 2H, A+B), 3.20-3.13 (m, 8H,

A+B), 2.46-2.39 (m, 2H, A+B). ESI HR (MeOH) calcd for C₁₇H₂₆N₇O₂[M-

H]⁺: 360.2142 found: 360.2144.

1-(2-(1-(2-(4-Carbamoylpyridinium-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)-

2-((hydroxyimino)methyl)pyridinium hexafluorophosphate (15A). The title

compound was obtained using general method (70%). ¹H NMR (400 MHz,

DMSO)  = 13.13 (s, 1H), 9.03 (d, J = 6.4Hz, 2H), 8.83-8.80 (m, 1H), 8.71

(s, 1H), 8.66 (brs, 1H), 8.56-8.50 (m, 1H), 8.42-8.36 (m, 3H), 8.31 (brs,

1H), 8.05-8.00 (m, 1H), 7.95 (s, 1H), 5.14-5.10 (m, 2H), 5.05-4.96 (m, 4H),

3.23 (t, J = 6.8Hz, 2H). ESI (MeOH) for [M-H]⁺= 366.4.

2-((Hydroxyimino)methyl)-1-(3-(4-((2-((hydroxyimino)methyl)pyridinium-1-

3-(3-(4-(2-((2-(Hydroxyimino)ethyl)dimethylammonio)ethyl)-1H-1,2,3-

triazol-1-yl)propyl)-2-((hydroxyimino)methyl)-1-methyl-1H-imidazol-3-ium

hexafluorophosphate (8A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈ 2:8 (95%). ¹H NMR (400

MHz, DMSO)  = 13.03 (s, 1H, A+B), 12.05 (s, 0.2H, A), 11.89 (s, 0.8H,

B), 8.55 (s, 1H, A+B), 7.96-7.94 (m, 1H, A+B), 7.80 (s, 2H, A+B), 7.66 (t,

J = 6.2Hz, 0.8H, B), 7.34 (t, J = 4.6Hz, 0.2H, A), 4.44-4.35 (m, 4H, A+B),

4.18 (d, J = 6.0Hz, 1.6H, B), 3.91 (s, 3H, A+B), 3.71-3.59 (m, 2H, A+B),

yl)methyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

(16A). The title compound was obtained using general method (47%). ¹H

NMR (400 MHz, DMSO)  = 13.18 (s, 1H), 13.17 (s, 1H), 9.15 (d, J = 5.6Hz,

1H), 8.94 (d, J = 5.6Hz, 1H), 8.91 (s, 1H), 8.75 (s, 1H), 8.59 (t, J = 8.0Hz,

1H), 8.52 (t, J = 8.0Hz, 1H), 8.44 (dd, J¹= 8.4Hz, J²= 1.2Hz, 1H), 8.40 (dd,

J¹= 8.4Hz, J²= 1.2Hz, 1H), 8.31 (s, 1H), 8.17-8.13 (m, 1H), 8.08-8.04 (m,

1H), 6.12 (s, 2H), 4.78 (t, J = 7.6Hz, 2H), 4.55 (t, J = 7.0Hz, 2H), 2.41 (p,

J = 7.4Hz, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.3, 147.1, 146.1, 145.9,

10.1002/chem.201805051

Chemistry - A European Journal

FULL PAPER

145.9, 145.2, 141.6, 141.3, 139.8, 127.7, 127.4, 125.8, 125.6, 124.8, 55.4,

52.5, 46.6, 30.3. ESI HR (MeOH) calcd for C₁₈H₂₀N₇O₂[M-H]⁺: 366.1673

found: 366.1680.

hexafluorophosphate (25A). The title compound was obtained using

general method (80%). ¹H NMR (400 MHz, DMSO)  = 13.21 (s, 1H), 12.86

(s, 1H), 9.04-9.03 (m, 1H), 8.95 (d, J = 6.8Hz, 2H), 8.62-8.58 (m, 2H), 8.43-

8.41 (m, 2H), 8.20 (d, J=6.8Hz, 2H), 8.13-8.09 (m, 1H), 8.06 (s, 1H), 6.28

(s, 2H), 6.02 (s, 2H), 4.81 (t, J = 7.0Hz, 2H), 3.33-3.31 (m, 2H, the signal

overlaps with residual H₂O). ESI (MeOH) for [M-H]⁺= 382.1.

2-((Hydroxyimino)methyl)-1-((1-(((2-((hydroxyimino)methyl)pyridinium-1-

yl)methoxy)methyl)-1H-1,2,3-triazol-4-yl)methyl)pyridinium

hexafluorophosphate (17A). The title compound was obtained using

general method (45%). ¹H NMR (400 MHz, DMSO)  = 13.22 (s, 1H), 13.19

(s, 1H), 9.11 (d, J = 5.6Hz, 1H), 9.02 (d, J = 5.6Hz, 1H), 8.88 (s, 1H), 8.62-

8.53 (m, 3H), 8.47-8.45 (m, 2H), 8.36 (dd, J¹= 8.2Hz, J²= 1.0Hz, 1H), 8.18-

8.15 (m, 1H), 8.08-8.05 (m, 1H), 6.30 (s, 2H), 6.103 (s, 2H), 6.098 (s, 2H).

¹³C NMR (100 MHz, DMSO)  = 147.4, 147.0, 146.8, 146.2, 146.0, 145.2,

141.5, 141.2, 140.5, 127.8, 127.1, 125.7, 125.5, 125.4, 85.4, 77.4, 52.2.

ESI HR (MeOH) calcd for C₁₇H₁₈N₇O₃[M-H]⁺: 368.1466 found: 368.1457.

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(pyridinium-1-yl)ethyl)-1H-1,2,3-

triazol-1-yl)propyl)pyridinium hexafluorophosphate (26A). The title

compound was obtained using general method (93%). ¹H NMR (400 MHz,

DMSO)  = 13.19 (s, 1H), 9.02 (d, J = 5.6Hz, 2H), 8.94 (d, J = 6.4Hz, 1H),

8.74 (s, 1H), 8.61-8.52 (m, 2H), 8.41 (dd, J¹= 8.2Hz, J²= 1.0Hz, 1H), 8.14-

8.06 (m, 3H), 7.91 (s, 1H), 4.90 (t, J = 7.2Hz, 2H), 4.74 (t, J = 7.6Hz, 2H),

4.48 (t, J = 7.0Hz, 2H), 3.37 (t, J = 7.0Hz, 2H), 2.42-2.35 (m, 2H). ¹³C NMR

(100 MHz, DMSO)  = 146.9, 145.8, 145.6, 145.2, 144.8, 142.1, 141.2,

127.8, 127.4, 125.7, 123.5, 59.9, 55.4, 46.1, 30.3, 26.5. ESI HR (MeOH)

calcd for C₁₈H₂₁N₆O [M-H]⁺: 337.1771 found: 337.1773.

1-(2-(1-Benzyl-1H-1,2,3-triazol-4-yl)ethyl)-2-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (18A). The title

compound was obtained using general method (98%). ¹³C NMR (100 MHz,

DMSO)  = 147.0, 146.0, 145.3, 141.6, 141.3, 135.9, 128.7, 128.1, 127.7,

127.1, 125.6, 123.6, 56.9, 52.7, 26.1. ESI (MeOH) for [M]⁺= 308.0.

4-Carbamoyl-1-(3-(4-(2-(4-((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-1-yl)propyl)pyridinium hexafluorophosphate (27A). The

title compound was obtained using general method (78%). ¹H NMR (400

MHz, DMSO)  = 12.85 (s, 1H), 9.18 (d, J = 6.8Hz, 2H), 8.95 (d, J = 6.4Hz,

2H), 8.66 (brs, 1H), 8.45-8.40 (m ,3H), 8.29 (brs, 1H), 8.19 (d, J = 6.4Hz,

2H), 7.90 (s, 1H), 4.85 (t, J = 6.8Hz, 2H), 4.65 (t, J = 7.2Hz, 2H), 4.44 (t, J

= 6.8Hz, 2H), 3.37-3.31 (m, 2H, the signal overlaps with residual H₂O),

2.55-2.47 (m, 2H, the signal overlaps with residual DMSO). ESI (MeOH)

for [M-H]⁺= 380.4.

2-((Hydroxyimino)methyl)-1-(2-(1-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)pyridinium

hexafluorophosphate

(19A). The title compound was obtained using general method (84%). ¹H

NMR (400 MHz, DMSO)  = 13.13 (brs, 1H), 12.92 (brs, 1H), 8.82 (d, J =

6.0Hz, 1H), 8.78 (d, J = 6.4Hz, 2H), 8.71 (s, 1H), 8.52 (t, J = 8.0Hz, 1H),

8.43-8.37 (m, 2H), 8.19 (d, J = 6.4Hz, 2H), 8.03-8.00 (m, 1H), 7.94 (s, 1H),

5.04-4.96 (m, 6H), 3.24 (t, J = 6.8Hz, 2H). ESI (MeOH) for [M-H]⁺= 366.2.

2-((Hydroxyimino)methyl)-1-(2-(4-(2-(4-((hydroxyimino)methyl)pyridinium-

1-(2-(1-(3-(4-Carbamoylpyridinium-1-yl)propyl)-1H-1,2,3-triazol-4-

yl)ethyl)-2-((hydroxyimino)methyl)pyridinium hexafluorophosphate (20A).

The title compound was obtained using general method (72%). ¹H NMR

(400 MHz, DMSO)  = 13.09 (s, 1H), 9.18 (d, J = 6.4Hz, 2H), 8.90-8.87 (m,

1H), 8.69 (s, 1H), 8.66 (brs, 1H), 8.56-8.50 (m, 1H), 8.40 (d, J = 6.4Hz, 2H),

8.40-8.35 (m, 1H), 8.29 (brs, 1H), 8.07-8.01 (m, 1H), 7.93 (s, 1H), 5.01 (t,

J = 6.8Hz, 2H), 4.65 (t, J = 7.0Hz, 2H), 4.42 (t, J = 6.8Hz, 2H), 3.25 (t, J =

7.0Hz, 2H), 2.54-2.46 (m, 2H, the signal overlaps with residual DMSO).

ESI (MeOH) for [M-H]⁺= 380.4.

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)pyridinium

hexafluorophosphate

(28A). The title compound was obtained using general method (88%). ¹H

NMR (400 MHz, DMSO)  = 13.18 (s, 1H), 12.86 (s, 1H), 8.92 (d, J = 6.8Hz,

2H), 8.62-8.60 (m, 2H), 8.54 (t, J = 8.2Hz, 1H), 8.41-8.38 (m, 2H), 8.19 (d,

J = 6.8Hz, 2H), 8.01-7.98 (m, 1H), 7.93 (s, 1H), 5.22 (t, J = 5.2Hz, 2H),

4.91 (t, J = 5.2Hz, 2H), 4.81 (t, J = 7.2Hz, 2H), 3.32 (t, J = 7.2Hz, 2H, the

signal overlaps with residual H₂O). ESI (MeOH) for [M-H]⁺= 366.3.

1-(3-(1-Benzyl-1H-1,2,3-triazol-4-yl)propyl)-2-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (29A). The title

compound was obtained using general method (90%). ¹H NMR (300 MHz,

DMSO)  = 13.15 (s, 1H), 8.99 (d, J = 6.0Hz, 1H), 8.79 (s, 1H), 8.53-8.45

(m, 1H), 8.40-8.33 (m, 1H), 8.08-7.99 (m, 1H), 7.91 (s, 1H), 7.39-7.23 (m,

5H), 5.52 (s, 2H), 4.78 (t, J = 7.3Hz, 2H), 2.73 (t, J = 7.3Hz, 2H), 2.20-2.10

(m, 2H). ¹³C NMR for the Cl salt (100 MHz, DMSO)  = 146.9, 145.9, 145.4,

145.0, 141.1, 136.0, 128.6, 128.0, 127.8, 127.2, 125.5, 122.3, 57.3, 52.6,

29.6, 21.5. ESI (MeOH) for [M]⁺= 322.0.

1-((1-(2-(4-Carbamoylpyridinium-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-

2-((hydroxyimino)methyl)pyridinium hexafluorophosphate (21A). The title

compound was obtained using general method (65%). ¹H NMR (400 MHz,

DMSO)  = 13.21 (s, 1H), 9.13-9.09 (m, 1H), 9.05 (d, J = 6.4Hz, 2H), 8.86

(s, 1H), 8.66 (brs, 1H), 8.62-8.56 (m, 1H), 8.47-8.43 (m, 1H), 8.38 (d, J =

6.4Hz, 2H), 8.30 (brs, 1H), 8.26 (s, 1H), 8.15-8.12 (m, 1H), 6.10 (s, 2H),

5.17-5.08 (m, 4H). ESI (MeOH) for [M-H]⁺= 352.4.

2-((Hydroxyimino)methyl)-1-(3-(1-(2-(2-((hydroxyimino)methyl)pyridinium-

1-(2-(4-(2-(4-Carbamoylpyridinium-1-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)-

2 ((hydroxyimino)methyl)pyridinium hexafluorophosphate (30A). The title

compound was obtained using general method (76%). ¹H NMR (400 MHz,

DMSO)  = 13.17 (s, 1H), 9.16 (d, J = 6.8Hz, 2H), 8.66 (brs, 1H), 8.62-8.59

(m, 2H), 8.57-8.52 (m, 1H), 8.42-8.37 (m, 3H), 8.29 (brs, 1H), 8.02-7.97

(m, 1H), 7.94 (s, 1H), 5.24-5.19 (m, 2H), 4.93-4.86 (m, 4H), 3.38-3.32 (m,

2H, the signal overlaps with residual H₂O). ESI (MeOH) for [M-H]⁺= 366.4.

1-yl)ethyl)-1H-1,2,3-triazol-4-yl)propyl)pyridinium

hexafluorophosphate

(22A). The title compound was obtained using general method (64%). ¹H

NMR (400 MHz, DMSO)  = 13.19 (s, 1H), 13.14 (s, 1H), 8.99 (d, J = 6.0Hz,

1H), 8.79 (s, 1H), 8.67 (d, J = 6.0Hz, 1H), 8.55-8.51 (m, 3H), 8.43-8.37 (m,

2H), 8.10-8.07 (m, 1H), 8.02-7.98 (m, 1H), 7.90 (s, 1H), 5.24-5.22 (m, 2H),

4.92-4.90 (m, 2H), 4.77 (t, J = 7.6Hz, 2H), 2.72 (t, J = 7.6Hz, 2H), 2.18-

2.11 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.6, 146.9, 146.0, 145.8,

145.6, 145.1, 141.3, 140.9, 127.4, 127.2, 125.8, 125.6, 123.2, 57.4, 57.0,

48.8, 29.7, 21.4. ESI HR (MeOH) calcd for C₁₉H₂₂N₇O₂[M-H]⁺: 380.1829

found: 380.1834.

4-((Hydroxyimino)methyl)-1-(3-(4-(2-(2-((hydroxyimino)methyl)-1-methyl-

1H-imidazol-3-ium-3-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate (31A). The title compound was obtained using

general method (82%). ¹H NMR (400 MHz, DMSO)  = 12.98 (s, 1H), 12.86

(s, 1H), 8.96 (d, J = 6.4Hz, 2H), 8.48 (s, 1H), 8.43 (s, 1H), 8.23 (d, J =

6.4Hz, 2H), 7.90 (s, 1H), 7.784-7.779 (m, 2H), 4.59 (q, J = 7.6Hz, 4H), 4.43

(t, J = 6.8Hz, 2H), 3.90 (s, 3H), 3.16-3.13 (m, 2H), 2.52-2.46 (m, 2H, the

signal overlaps with residual DMSO). ESI (MeOH) for [M-H]⁺= 383.5.

2-((Hydroxyimino)methyl)-1-(2-(1-(3-(2-((hydroxyimino)methyl)-1-methyl-

1H-imidazol-3-ium-3-yl)propyl)-1H-1,2,3-triazol-4-yl)ethyl)pyridinium

hexafluorophosphate (23A). The title compound was obtained using

general method (78%). ¹H NMR (400 MHz, DMSO)  = 13.06 (brs, 2H),

8.87 (d, J = 6.0Hz, 1H), 8.69 (s, 1H), 8.55 (s, 1H), 8.54-8.50 (m, 1H), 8.39-

8.37 (m, 1H), 8.05-8.01 (m, 1H), 7.92 (s, 1H), 7.83 (d, J = 2.0Hz, 1H), 7.80

(d, J = 2.0Hz, 1H), 5.02 (t, J = 6.8Hz, 2H), 4.37 (t, J = 7.2Hz, 2H), 4.32 (t,

J = 7.2Hz, 2H), 3.92 (s, 3H), 3.26 (t, J = 6.8Hz, 2H), 2.32-2.25 (m, 2H). ESI

HR (MeOH) calcd for C₁₈H₂₃N₈O₂[M-H]⁺: 383.1938 found: 383.1948.

2-((Hydroxyimino)methyl)-1-(2-(4-(3-(4-((hydroxyimino)methyl)pyridinium-

1-yl)propyl)-1H-1,2,3-triazol-1-yl)ethyl)pyridinium

hexafluorophosphate

(32A). The title compound was obtained using general method (69%). ¹H

NMR (400 MHz, DMSO)  = 13.13 (s, 1H), 12.85 (s, 1H), 8.99 (d, J = 6.8Hz,

2H), 8.67 (d, J = 5.6Hz, 1H), 8.56-8.52 (m, 2H), 8.43 (s, 1H), 8.38 (d, J =

8.0Hz, 1H), 8.23 (d, J = 6.8Hz, 2H), 8.03-8.00 (m, 1H), 7.89 (s, 1H), 5.22

(t, J = 5.2Hz, 2H), 4.91 (t, J = 5.2Hz, 2H), 4.59 (t, J = 7.2Hz, 2H), 2.64 (t, J

= 7.2Hz, 2H), 2.25-2.17 (m, 2H). ESI (MeOH) for [M-H]⁺= 380.2.

1-((1-(3-(4-Carbamoylpyridinium-1-yl)propyl)-1H-1,2,3-triazol-4-

yl)methyl)-2-((hydroxyimino)methyl)pyridinium

hexafluorophosphate

(24A). The title compound was obtained using general method (41%). ¹H

NMR (400 MHz, DMSO)  = 13.18 (s, 1H), 9.18 (d, J = 6.8Hz, 2H), 9.16-

9.12 (m, 1H), 8.90 (s, 1H), 8.66 (brs, 1H), 8.62-8.56 (m, 1H), 8.47-8.40 (m,

3H), 8.29 (s, 1H), 8.28 (brs, 1H), 8.18-8.13 (m, 1H), 6.13 (s, 2H), 4.68 (t, J

= 7.2Hz, 2H), 4.50 (t, J = 7.2Hz, 2H), 2.56-2.48 (m, 2H, the signal overlaps

with residual DMSO). ESI (MeOH) for [M-H]⁺= 366.4.

4-carbamoyl-1-(2-(1-(2-(4-((Hydroxyimino)methyl)pyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-4-yl)ethyl)pyridinium hexafluorophosphate (33A). The title

compound was obtained using general method (89%). ¹H NMR (400 MHz,

DMSO)  = 12.91 (s, 1H), 9.15 (d, J = 6.4Hz, 2H), 8.79 (d, J = 6.4Hz, 2H),

8.66 (brs, 1H), 8.42-8.38 (m, 3H), 8.28 (brs, 1H), 8.20 (d, J = 6.4Hz, 2H),

7.92 (s, 1H), 5.06-4.97 (m, 4H), 4.90 (t, J = 7.0Hz, 2H), 3.37 (t, J = 6.8Hz,

2H, the signal overlaps with residual H₂O). ESI (MeOH) for [M-H]⁺= 366.4.

2-((Hydroxyimino)methyl)-1-(((4-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)methoxy)methyl)pyridinium

10.1002/chem.201805051

Chemistry - A European Journal

FULL PAPER

1-(3-(4-(2-(4-Carbamoylpyridinium-1-yl)ethyl)-1H-1,2,3-triazol-1-

yl)propyl)-2-((hydroxyimino)methyl)pyridinium hexafluorophosphate (35A).

The title compound was obtained using general method (99%). ¹H NMR

(400 MHz, DMSO)  = 13.19 (s, 1H), 9.19 (d, J = 6.8Hz, 2H), 8.94 (d, J =

6.0Hz, 1H), 8.74 (s, 1H), 8.65 (brs, 1H), 8.57-8.51 (m, 1H), 8.43-8.38 (m,

3H), 8.27 (brs, 1H), 8.11-8.05 (m, 1H), 7.93 (s, 1H), 4.93 (t, J = 6.8Hz, 2H),

4.74 (t, J = 7.6Hz, 2H), 4.48 (t, J = 7.0Hz, 2H), 3.39 (t, J = 7.0Hz, 2H), 2.42-

2.35 (m, 2H). ESI (MeOH) for [M-H]⁺= 380.4.

4-((Hydroxyimino)methyl)-1-(3-(4-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

(45A). The title compound was obtained using general method (87%). ¹H

NMR (400 MHz, DMSO)  = 12.85 (s, 2H), 8.97-8.94 (m, 4H), 8.42 (d, J =

6.0Hz, 2H), 8.22 (d, J = 6.8Hz, 2H), 8.19 (d, J = 6.8Hz, 2H), 7.90 (s, 1H),

4.85 (t, J = 6.8Hz, 2H), 4.57 (t, J = 7.2Hz, 2H), 4.43 (t, J = 6.8Hz, 2H), 3.37-

3.33 (m, 2H, the signal overlaps with residual H₂O), 2.50-2.46 (m, 2H, the

signal overlaps with residual DMSO). ESI (MeOH) for [M-H]⁺= 380.2.

4-Carbamoyl-1-(2-(4-(2-(4-((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-

1H-1,2,3-triazol-1-yl)ethyl)pyridinium hexafluorophosphate (36A). The title

compound was obtained using general method (86%). ¹H NMR (400 MHz,

DMSO)  = 12.85 (s, 1H), 9.03 (d, J = 6.8Hz, 2H), 8.93 (d, J = 6.8Hz, 2H),

8.67 (brs, 1H), 8.42-8.38 (m, 3H), 8.31 (brs, 1H), 8.19 (d, J = 6.8Hz, 2H),

7.92 (s, 1H), 5.15-5.10 (m, 2H), 5.06-5.00 (m, 2H), 4.81 (t, J = 7.0Hz, 2H),

3.36-3.30 (m, 2H, the signal overlaps with residual H₂O). ESI (MeOH) for

[M-H]⁺= 366.4.

N-(2-(Hydroxyimino)ethyl)-3-(4-(2-((2-

(hydroxyimino)ethyl)dimethylammonio)ethyl)-1H-1,2,3-triazol-1-yl)-N,N-

dimethylpropan-1-aminium hexafluorophosphate (46A). The title

compound was obtained using general method as a mixture of isomers;

A+B; ≈ 1:3 (75%). ¹H NMR (400 MHz, DMSO)  = 12.06 (s, 0.5H, A), 11.89

(s, 1.5H, B), 8.01-7.99 (m, 1H, A+B), 7.67 (t, J = 6.2Hz, 0.75H, B), 7.61 (t,

J = 6.2Hz, 0.75H, B), 7.35 (t, J = 4.8Hz, 0.25H, A), 7.28 (t, J = 4.8Hz,

0.25H, A), 4.43 (t, J = 6.8Hz, 2H, A+B), 4.36 (d, J = 4.8Hz, 0.5H, A), 4.28

(d, J = 4.8Hz, 0.5H, A), 4.19 (d, J = 6.4Hz, 1.5H, B), 4.10 (d, J = 6.4Hz,

1.5H, B), 3.72-3.59 (m, 2H, A+B), 3.45-3.31 (m, 2H, A+B, the signal

overlaps with residual H₂O), 3.21 (s, 1.5H, A), 3.19-3.11 (m, 8H, A+B),

3.05 (s, 4.5H, B), 2.36-2.27 (m, 2H, A+B). ESI HR (MeOH) calcd for

C₁₅H₃₁N₇O₂0.5[M]²⁺: 170.6264 found: 170.6259.

4-((Hydroxyimino)methyl)-1-(2-(4-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)ethyl)pyridinium

hexafluorophosphate

(37A). The title compound was obtained using general method (91%). ¹H

NMR (400 MHz, DMSO)  = 12.89 (brs, 2H), 8.92 (d, J = 6.4Hz, 2H), 8.78

(d, J = 6.8Hz, 2H), 8.41 (s, 2H), 8.20-8.18 (m, 4H), 7.91 (s, 1H), 5.06-4.96

(m, 4H), 4.82 (t, J = 7.0Hz, 2H), 3.45-3.31 (m, 2H, the signal overlaps with

residual H₂O). ESI (MeOH) for [M-H]⁺= 366.3.

1-(3-(1-Benzyl-1H-1,2,3-triazol-4-yl)propyl)-4-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (47A). The title

compound was obtained using general method (74%). ¹³C NMR for the Cl

salt (100 MHz, DMSO)  = 148.4, 145.4, 145.1, 144.8, 136.1, 128.6, 128.0,

127.8, 123.8, 122.3, 59.7, 52.6, 30.0, 21.6. ESI (MeOH) for [M]⁺= 322.0.

2-((Hydroxyimino)methyl)-1-(3-(4-(4-(2-((hydroxyimino)methyl)pyridinium-

1-yl)butyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

(38A). The title compound was obtained using general method (74%). ¹H

NMR (400 MHz, DMSO)  = 13.18 (s, 1H), 13.16 (s, 1H), 9.00 (d, J = 6.0Hz,

1H), 8.94 (d, J = 6.0Hz, 1H), 8.79 (s, 1H), 8.73 (s, 1H), 8.55-8.50 (m, 2H),

8.41-8.39 (m, 2H), 8.09-8.04 (m, 2H), 7.84 (s, 1H), 4.78-4.74 (m, 4H), 4.48

(t, J = 6.8Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.47-2.39 (m, 2H), 1.91-1.84 (m,

2H), 1.71-1.64 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.0, 146.8,

145.91, 145.87, 145.8, 145.2, 145.1, 141.4, 141.3, 127.4, 127.4, 125.83,

125.76, 122.0, 57.7, 55.5, 46.0, 30.3, 29.8, 25.3, 24.3. ESI HR (MeOH)

calcd for C₂₁H₂₆N₇O₂[M-H]⁺: 408.2142 found: 408.2145.

1-(2-(1-(3-((2-(Hydroxyimino)ethyl)dimethylammonio)propyl)-1H-1,2,3-

triazol-4-yl)ethyl)-4-((hydroxyimino)methyl)pyridinium

hexafluorophosphate (48A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈ 2:8 (85%). ¹H NMR (400

MHz, DMSO)  = 12.85 (s, 1H, A+B), 12.05 (s, 0.2H, A), 11.88 (s, 0.8H,

B), 9.02 (d, J = 5.6Hz, 0.4H, A), 8.95 (d, J = 5.6Hz, 1.6H, B), 8.40 (s, 1H,

A+B), 8.26 (d, J = 5.6Hz, 0.4H, A),8.19 (d, J = 5.6Hz, 1.6H, B), 7.92 (s,

1H, A+B), 7.63-7.58 (m, 0.8H, B), 7.28-7.24 (m, 0.2H, A), 4.89-4.83 (m,

2H, A+B), 4.41-4.36 (m, 2H, A+B), 4.28-4.25 (m, 0.4H, A), 4.11-4.06 (m,

1.6H, B), 3.38-3.26 (m, 4H, A+B, the signal overlaps with residual H₂O),

3.10 (s, 1.2H, A), 3.03 (s, 4.8H, B), 2.33-2.23 (m, 2H, A+B). ESI (MeOH)

for 0.5[M]²⁺= 180.9.

2-((Hydroxyimino)methyl)-1-(3-(4-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate

(40A). The title compound was obtained using general method (85%). ¹H

NMR (400 MHz, DMSO)  = 13.19 (s, 1H), 12.85 (s, 1H), 8.96-8.92 (m,

3H), 8.74 (s, 1H), 8.54 (t, J = 8.0Hz, 1H), 8.42-8.40 (m, 2H), 8.19 (d, J =

6.8Hz, 2H), 8.09-8.06 (m, 1H), 7.92 (s, 1H), 4.85 (t, J = 7.0Hz, 2H), 4.74

(t, J = 7.4Hz, 2H), 4.49 (t, J = 7.0Hz, 2H), 3.39-3.35 (m, 2H, the signal

overlaps with residual H₂O), 2.43-2.35 (m, 2H). ESI (MeOH) for [M-H]⁺=

380.2.

2-((Hydroxyimino)methyl)-1-((1-(2-(2-((hydroxyimino)methyl)pyridinium-1-

yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)pyridinium

hexafluorophosphate

(50A). The title compound was obtained using general method (66%). ¹H

NMR (400 MHz, DMSO)  = 13.22 (s, 1H), 13.11 (s, 1H), 9.10 (d, J = 5.6Hz,

1H), 8.85 (s, 1H), 8.60-8.52 (m, 4H), 8.45 (dd, J¹= 8.0Hz, J²= 1.2Hz, 1H),

8.38 (dd, J¹= 8.4Hz, J²= 1.2Hz, 1H), 8.27 (s, 1H), 8.17-8.14 (m, 1H), 7.94-

7.90 (m, 1H), 6.09 (s, 2H), 5.23 (brt, J = 5.4Hz, 2H), 4.99 (brt, J = 5.4Hz,

2H). ¹³C NMR (100 MHz, DMSO)  = 147.6, 147.3, 146.1, 146.0, 145.9,

145.9, 141.4, 141.0, 140.0, 127.7, 127.2, 125.8, 125.7, 125.6, 57.0, 52.3,

49.1. ESI HR (MeOH) calcd for C₁₇H₁₈N₇O₂[M-H]⁺: 352.1516 found:

352.1516.

3-Fluoro-4-((hydroxyimino)methyl)-1-(2-(4-(2-(2-

((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-1H-1,2,3-triazol-1-

yl)ethyl)pyridinium hexafluorophosphate (41A). The title compound was

obtained using general method (74%). ESI (MeOH) for [M-H]⁺= 384.4.

1-(3-(4-(2-((2-(Hydroxyimino)ethyl)dimethylammonio)ethyl)-1H-1,2,3-

triazol-1-yl)propyl)-4-((hydroxyimino)methyl)pyridinium

2-((Hydroxyimino)methyl)-1-(3-(4-(5-(2-((hydroxyimino)methyl)pyridinium-

1-yl)pent-2-ynyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium

hexafluorophosphate (42A). The title compound was obtained using

general method as a mixture of isomers; A+B; ≈1:3 (93%). ¹H NMR (400

MHz, DMSO)  = 12.86 (s, 1H, A+B), 12.05 (s, 0.25H, A), 11.89 (s, 0.75H,

B), 8.97 (d, J = 6.6Hz, 2H, A+B), 8.42 (s, 1H, A+B), 8.21 (d, J = 6.6Hz,

2H, A+B), 7.97-7.96 (m, 1H, A+B), 7.66 (t, J = 6.2Hz, 0.75H, B), 7.34 (t, J

= 4.8Hz, 0.25H, A), 4.62 (t, J = 6.8Hz, 2H, A+B), 4.47 (t, J = 6.8Hz, 2H,

A+B), 4.35 (d, J = 4.8Hz, 0.5H, A), 4.18 (d, J = 6.4Hz, 1.5H, B), 3.70-3.57

(m, 2H, A+B), 3.20-3.13 (m, 8H, A+B), 2.55-2.47 (m, 2H, A+B, the signal

overlaps with residual DMSO). ESI (MeOH) for 0.5[M]²⁺= 180.9.

hexafluorophosphate (51A). The title compound was obtained using

general method (75%). ¹H NMR (400 MHz, DMSO)  = 13.16 (brs, 2H),

8.99 (d, J = 6.0Hz, 1H), 8.95 (d, J = 6.0Hz, 1H), 8.82 (s, 1H), 8.76 (s, 1H),

8.56-8.51 (m, 2H), 8.42-8.40 (m, 2H), 8.09-8.03 (m, 2H), 7.88 (s, 1H), 4.90

(t, J = 6.2Hz, 2H), 4.78 (t, J = 7.4Hz, 2H), 4.50 (t, J = 7.0Hz, 2H), 3.55 (s,

2H), 2.88-2.85 (m, 2H), 2.47-2.40 (m, 2H). ¹³C NMR (100 MHz, DMSO) 

= 147.1, 147.0, 146.3, 145.9, 145.4, 145.2, 141.6, 141.3, 127.4, 127.0,

125.8, 125.7, 122.5, 81.4, 75.8, 55.8, 55.5, 46.2, 30.3, 20.3, 15.4. ESI HR

(MeOH) calcd for C₂₂H₂₄N₇O₂[M-H]⁺: 418.1986 found: 418.1984.

3-Fluoro-4-((hydroxyimino)methyl)-1-(2-(1-(2-(2-

((hydroxyimino)methyl)pyridinium-1-yl)ethyl)-1H-1,2,3-triazol-4-

yl)ethyl)pyridinium hexafluorophosphate (43A). The title compound was

obtained using general method (60%). ESI (MeOH) for [M-H]⁺= 384.4.

1-(2-(1-Benzyl-1H-1,2,3-triazol-4-yl)ethyl)-4-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (52A). The title

compound was obtained using general method (95%). ¹H NMR (400 MHz,

DMSO)  = 13.0 (brs, 1H), 8.98 (d, J = 6.8Hz, 2H), 8.36 (s, 1H), 8.12 (d, J

= 6.8Hz, 2H), 7.95 (s, 1H), 7.32-7.24 (m, 3H), 7.18-7.14 (m, 2H), 5.51 (s,

2H), 4.85 (t, J = 7.0Hz, 2H), 3.32 (t, J = 7.0Hz, 2H). ¹³C NMR (100 MHz,

DMSO)  = 148.5, 145.1, 144.8, 141.8, 136.0, 128.6, 128.0, 127.6, 123.6,

123.4, 59.2, 52.6, 26.5. ESI (MeOH) for [M]⁺= 308.0.

2-((Hydroxyimino)methyl)-3-(3-(4-(2-(2-((hydroxyimino)methyl)-1-methyl-

1H-imidazol-3-ium-3-yl)ethyl)-1H-1,2,3-triazol-1-yl)propyl)-1-methyl-1H-

imidazol-3-ium hexafluorophosphate (44A). The title compound was

obtained using general method (87%). ¹H NMR (400 MHz, DMSO)  =

13.02 (s, 1H), 12.98 (s, 1H), 8.56 (s, 1H), 8.47 (s, 1H), 7.90 (s, 1H), 7.84-

7.81 (m, 2H), 7.79-7.77 (m ,2H), 4.61 (t, J = 7.2Hz, 2H), 4.38 (t, J = 7.0Hz,

2H), 4.33 (t, J = 7.0Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.15 (t, J = 7.2Hz,

2H), 2.34-2.26 (m, 2H). ESI HR (MeOH) calcd for C₁₇H₂₄N₉O₂[M-H]⁺:

386.2047 found: 386.2054.

2-((Hydroxyimino)methyl)-1-(3-(1-(2-(4-((hydroxyimino)methyl)pyridinium-

1-yl)ethyl)-1H-1,2,3-triazol-4-yl)propyl)pyridinium

hexafluorophosphate

(54A). The title compound was obtained using general method (99%). ¹H

NMR (400 MHz, DMSO)  = 13.17 (s, 1H), 12.90 (s, 1H), 8.98 (d, J = 6.4Hz,

1H), 8.80 (d, J = 6.4Hz, 2H), 8.78 (s, 1H), 8.54 (t, J = 7.8Hz, 1H), 8.42-

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8.40 (m, 1H), 8.39 (s, 1H), 8.19 (d, J = 6.8Hz, 2H), 8.10-8.07 (m, 1H), 7.88

(s, 1H), 5.06-4.98 (m, 4H), 4.77 (t, J = 7.6Hz, 2H), 2.73 (t, J = 7.6Hz, 2H),

2.17-2.10 (m, 2H). ESI (MeOH) for [M-H]⁺= 380.2.

MHz, DMSO+CD₃OD)  = 147.7, 146.1, 145.6, 141.6, 128.1, 126.6, 54.6,

16.0. ESI (MeOH) for [M]⁺= 151.3.

2-((Hydroxyimino)methyl)-1-pentylpyridinium chloride (4B). The title

compound was obtained using procedure for 3B and n-pentyl triflate

(85%).¹H NMR (400 MHz, DMSO)  = 13.43 (s, 1H), 9.27-9.25 (m, 1H),

8.79 (s, 1H), 8.59-8.55 (m, 1H), 8.38-8.36 (m, 1H), 8.13-8.09 (m, 1H), 4.81

(t, J = 7.6Hz, 2H), 1.81-1.74 (m, 2H), 1.32-1.24 (m, 4H), 0.83 (t, J = 7.2Hz,

3H). ¹³C NMR (100 MHz, DMSO)  = 146.8, 146.0, 145.1, 141.0, 127.3,

125.7, 57.6, 30.0, 27.4, 21.5, 13.6. ESI (MeOH) for [M]⁺= 193.3.

2-((Hydroxyimino)methyl)-1-(3-(4-(3-(2-((hydroxyimino)methyl)pyridinium-

1-yl)propyl)-1H-1,2,3-triazol-1-yl)propyl)pyridinium hexafluorophosphate

(56A). The title compound was obtained using general method (74%). ¹H

NMR (400 MHz, DMSO)  = 13.16 (brs, 2H), 9.00 (d, J = 6.0Hz, 1H), 8.94

(d, J = 6.0Hz, 1H), 8.79 (s, 1H), 8.74 (s, 1H), 8.54-8.51 (m, 2H), 8.42-8.40

(m, 2H), 8.09-8.05 (m, 2H), 7.90 (s, 1H), 4.83-4.75 (m, 4H), 4.50 (t, J =

6.8Hz, 2H), 2.76 (t, J = 7.4Hz, 2H), 2.47-2.39 (m, 2H), 2.23-2.15 (m, 2H).

¹³C NMR (100 MHz, DMSO)  = 147.0, 146.9, 145.9, 145.8, 145.40,

145.39, 145.2, 145.1, 141.4, 141.3, 127.4, 125.8, 125.7, 122.3, 57.43,

55.47, 46.2, 30.3, 29.6, 21.5. ESI HR (MeOH) calcd for C₂₀H₂₄N₇O₂[M-

H]⁺: 394.1986 found: 394.1985.

1-Hexyl-2-((hydroxyimino)methyl)pyridinium chloride (5B). The title

compound was obtained using procedure for 3B and n-hexyl triflate (86%).

¹H NMR (400 MHz, DMSO)  = 13.44 (s, 1H), 9.27-9.26 (m, 1H), 8.79 (s,

1H), 8.59-8.55 (m, 1H), 8.38-8.36 (m, 1H), 8.13-8.09 (m, 1H), 4.81 (t, J =

7.6Hz, 2H), 1.80-1.73 (m, 2H), 1.32-1.22 (m, 6H), 0.82 (t, J = 7.2Hz, 3H).

¹³C NMR (100 MHz, DMSO)  = 146.8, 146.0, 145.0, 141.0, 127.3, 125.7,

57.6, 30.4, 30.2, 24.9, 21.7, 13.7. ESI (MeOH) for [M]⁺= 207.3.

4-Carbamoyl-1-(2-(1-(3-(4-((hydroxyimino)methyl)pyridinium-1-yl)propyl)-

1H-1,2,3-triazol-4-yl)ethyl)pyridinium hexafluorophosphate (57A). The title

compound was obtained using general method (89%). ¹H NMR (400 MHz,

DMSO)  = 12.86 (s, 1H), 9.17 (d, J = 6.8Hz, 2H), 8.96 (d, J = 6.8Hz, 2H),

8.65 (brs, 1H), 8.43 (s, 1H), 8.40 (d, J = 6.4Hz, 2H), 8.28 (brs, 1H), 8.23

(d, J = 6.4Hz, 2H), 7.91 (s, 1H), 4.93 (t, J = 7.0Hz, 2H), 4.57 (t, J = 7.0Hz,

2H), 4.43 (t, J = 6.8Hz, 2H), 3.38 (t, J = 6.8Hz, 2H), 2.52-2.44 (m, 2H, the

signal overlaps with residual DMSO). ESI (MeOH) for [M-H]⁺= 380.4.

1-(3-Azidopropyl)-2-((hydroxyimino)methyl)pyridinium chloride (1C). The

title compound was synthesized analogously as for C2 using 3-

azidopropanol triflate to give greenish solid (76%). ¹H NMR (400 MHz,

DMSO)  = 13.35 (s, 1H), 9.14 (d, J = 6.0Hz, 1H), 8.79 (s, 1H), 8.56 (t, J =

7.6Hz, 1H), 8.40 (dd, J¹= 8.4Hz, J²= 1.2Hz, 1H), 8.12-8.09 (m, 1H), 4.84-

4.80 (m, 2H), 3.53 (t, J = 6.6Hz, 2H), 2.12-2.05 (m, 2H). ¹³C NMR (100

MHz, DMSO)  = 147.0, 146.1, 145.2, 141.0, 127.3, 125.7, 55.4, 47.4, 29.3.

ESI HR (MeOH) calcd for C₉H₁₂N₅O [M]⁺: 206.1036 found: 206.1032.

1-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)butyl)-4-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (58A). The title

compound was obtained using general method (64%). ¹³C NMR for the Cl

salt (100 MHz, DMSO)  = 148.3, 146.4, 144.9, 144.8, 136.2, 128.6, 127.9,

127.8, 123.9, 122.2, 59.8, 52.6, 30.2, 25.2, 24.3. ESI (MeOH) for [M]⁺=

336.0.

1-(2-Azidoethyl)-2-((hydroxyimino)methyl)pyridinium chloride (2C). To the

cooled (-30 °C) mixture of trifluoromethanesulfonic anhydride (28.0 g, 16.5

mL, 99.3 mmol, 1.1 equiv.) in CH₂Cl₂(250 mL) a solution of pyridine (7.9

g, 8.1 mL, 99.3 mmol, 1.1 equiv.) and 2-azidoethanol (7.8 g, 89.6 mmol) in

CH₂Cl₂(100 mL) was added dropwise maintaining the temperature at -

30 °C. The reaction was carried out for 30 min. at -30 °C, then H₂O (150

mL) was added. The organic layer was separated, dried over MgSO₄,

filtered and evaporated to give yellowish oil (12.3 g, 63%, stable for few

months at -30°C) used in the next step without purification. The triflate,

from the previous step, (12.3 g, 56.2 mmol) was diluted with CH₃NO₂

(200mL) then pyridine-2-carboxaldoxime (6.8 g, 56.2 mmol) was added in

one portion. The reaction was carried out for 3h at room temperature then

a solution of Bu₄N⁺Clˉ (31.2 g, 112.4 mmol, 2 equiv.) in CH₂Cl₂(150 mL)

was added. The product was separated and washed with EtOAc to give

greenish solid (10.1 g, 80%). ¹H NMR (400 MHz, DMSO) = 13.34 (s, 1H),

9.08 (dd, J¹= 6.2Hz, J²= 1.0Hz, 1H), 8.83 (s, 1H), 8.62-8.58 (m, 1H), 8.43

(dd, J¹= 8.2Hz, J²= 1.4Hz, 1H), 8.15-8.11 (m, 1H), 4.98 (t, J = 5.6Hz, 2H),

3.97 (t, J = 5.6Hz, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.5, 146.5, 145.6,

141.3, 127.0, 125.5, 56.2, 49.9. ESI HR (MeOH) calcd for C₈H₁₀N₅O [M]⁺:

192.0880 found: 192.0879. CAUTION: 2-azidoethanol is potentially

explosive and must be always synthesized and kept in solution.

1-(5-(1-Benzyl-1H-1,2,3-triazol-4-yl)pent-3-ynyl)-2-

((hydroxyimino)methyl)pyridinium hexafluorophosphate (61A). The title

compound was obtained using general method (99%). ¹³C NMR for the Cl

salt (100 MHz, DMSO)  = 147.1, 146.3, 145.2, 142.5, 141.2, 136.0, 128.6,

128.0, 127.9, 126.7, 125.4, 122.5, 81.4, 75.7, 55.6, 52.6, 20.2, 15.3. ESI

(MeOH) for [M]⁺= 346.3.

4-((Hydroxyimino)methyl)-1-(2-(1-(3-(2-((hydroxyimino)methyl)-1-methyl-

1H-imidazol-3-ium-3-yl)propyl)-1H-1,2,3-triazol-4-yl)ethyl)pyridinium

hexafluorophosphate (62A). The title compound was obtained using

general method (85%). ¹H NMR (400 MHz, DMSO)  = 1 3.02 (s, 1H),

12.84 (s, 1H), 8.95 (d, J = 6.4Hz, 2H), 8.54 (s, 1H), 8.40 (s, 1H), 8.18 (d, J

= 6.4Hz, 2H), 7.90 (s, 1H), 7.82-7.80 (m, 2H), 4.86 (t, J = 6.8Hz, 2H), 4.38

(t, J = 7.0Hz, 2H), 4.32 (t, J = 7.0Hz, 2H), 3.91 (s, 3H), 3.38-3.34 (m, 2H,

the signal overlaps with residual H₂O), 2.33-2.26 (m, 2H). ESI (MeOH) for

[M-H]⁺= 383.2.

Synthesis of oximes 4A, 34A, 39A, 49A, 53A, 55A, 59A and 60A was

reported earlier in Cochran et al.^[7c]

4-((Hydroxyimino)methyl)-1-(pent-4-ynyl)pyridinium chloride (3C). The title

compound was synthesized analogously as for 6C using 4-pentyn-1-ol

triflate and isonicotinaldehyde oxime (yellow solid, 89%). ¹H NMR (400

MHz, DMSO+1drop of D₂O)  = 8.89 (d, J = 7.2Hz, 2H), 8.33 (s, 1H), 8.13

(d, J = 7.2Hz, 2H), 4.59 (t, J = 7.2Hz, 2H), 2.62 (t, J = 2.4Hz, 1H), 2.25-

2.21 (m, 2H), 2.13-2.06 (m, 2H). ¹³C NMR (100 MHz, DMSO+1drop of

D₂O)  = 149.2, 145.9, 145.6, 124.9, 83.2, 72.9, 60.5, 29.6, 15.4. ESI

(MeOH) for [M]⁺= 189.1.

Synthesis of N-alkyl-2 pyridinium compounds, compounds with more

complex N-alkyl substitutions and of miscellaneous oximes (1B-5B and

1C-17C). 2-((Hydroxyimino)methyl)-1-propylpyridinium chloride (1B). The

title compound was obtained using procedure for 3B and n-propyl triflate

(57%). ¹H NMR (400 MHz, DMSO+CD₃OD)  = 9.00-8.98 (m, 1H), 8.70 (s,

1H), 8.52-8.48 (m, 1H), 8.38-8.36 (m, 1H), 8.03-7.99 (m, 1H), 4.70-4.66

(m, 2H), 1.88-1.79 (m, 2H), 0.91 (t, J = 7.2Hz, 3H). ¹³C NMR (100 MHz,

DMSO+CD₃OD)  = 147.7, 146.5, 145.7, 141.6, 127.9, 126.6, 60.0, 24.4,

10.3. ESI (MeOH) for [M]⁺= 165.3.

1-((Azidomethoxy)methyl)-2-((hydroxyimino)methyl)pyridinium

chloride

(4C). To the suspension of 12E (5.6 g, 23.7 mmol) in DMF (50 mL), NaN₃

(1.7 g, 26.1 mmol, 1.1 equiv.) was added. The reaction was carried out for

2h at room temperature, then diluted with CH₃CN (250 mL) and filtered.

The solid residue (NaCl) was disposed and the filtrate was subsequently

diluted with Et₂O (500 mL). The product was filtered as grey solid (2.4 g,

42%). ¹H NMR (400 MHz, DMSO)  = 13.39 (brs, 1H), 9.35-9.32 (m, 1H),

8.72 (s, 1H), 8.67 (t, J = 8.0Hz, 1H), 8.50 (dd, J¹= 8.2Hz, J²= 1.0Hz, 1H),

8.19-8.16 (m, 1H), 6.39 (d, J=2.0Hz, 2H), 5.12 (d, J = 1.0Hz, 2H). ¹³C NMR

(100 MHz, DMSO)  = 147.1, 147.0, 145.4, 141.0, 127.1, 125.2, 84.9, 81.9.

ESI HR (MeOH) calcd for C₈H₁₀N₅O₂[M]⁺: 208.0829 found: 201.0825. The

title compound decomposes within 1 month at room temperature, and few

months at -20 °C. Any other purification method (chromatography,

crystallization) has not been successful.

1-Butyl-2-((hydroxyimino)methyl)pyridinium chloride (2B). The title

compound was obtained using procedure for 3B and n-butyl triflate

(65%).¹H NMR (400 MHz, DMSO)  = 13.46 (s, 1H), 9.29-9.27 (m, 1H),

8.79 (s, 1H), 8.60-8.55 (m, 1H), 8.38-8.36 (m, 1H), 8.13-8.10 (m, 1H), 4.82

(t, J = 7.2Hz, 2H), 1.79-1.72 (m, 2H), 1.37-1.28 (m, 2H), 0.87 (t, J = 7.2Hz,

3H). ¹³C NMR (100 MHz, DMSO)  = 146.8, 146.0, 145.0, 141.0, 127.2,

125.7, 57.5, 32.3, 18.6, 13.3. ESI (MeOH) for [M]⁺= 179.3.

1-Ethyl-2-((hydroxyimino)methyl)pyridinium chloride (3B). The ethyl triflate

(2.1 g, 11.8 mmol) was diluted with CH₃NO₂(50 mL) then pyridine-2-

carboxaldoxime (1.44 g, 11.8 mmol) was added in one portion. Reaction

was carried out for overnight at room temperature then evaporated. Solid

residue was dissolved in H₂O (20 mL) and the solution was passed through

the ion exchange resin (IRA-400). Aqueous layer was washed with CHCl₃

(2 x 50 mL) then evaporated. Crude product was crystallized from

MeOH/CH₃CN (1.3 g, 60%). ¹H NMR (400 MHz, DMSO+CD₃OD)  = 8.97-

8.96 (m, 1H), 8.69 (s, 1H), 8.50-8.46 (m, 1H), 8.37-8.35 (m, 1H), 8.02-7.98

(m, 1H), 4.73 (q, J = 7.2Hz, 2H), 1.46 (t, J = 7.2Hz, 3H). ¹³C NMR (100

1-(4-Azidobutyl)-2-((hydroxyimino)methyl)pyridinium chloride (5C). The

mixture of 11E (10 g, 27.6 mmol) and NaN₃(2.1 g, 32.3 mmol, 1.2 equiv.)

in DMSO (25 mL) was stirred for 1 day at 50 °C then diluted with H₂O (50

mL) and passed through IRA-400(Clˉ) column. Aquatic layer was

evaporated and the residue was diluted with MeOH. NaCl was filtered off

10.1002/chem.201805051

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and the solution was evaporated again. The crude product was diluted with

CH₃CN and Et₂O and yellow crystals crashed out (4.4 g, 62%). ¹H NMR

(400 MHz, DMSO)  = 13.46 (s, 1H), 9.30 (d, J = 6.0Hz, 1H), 8.81 (s, 1H),

8.58 (t, J = 7.8Hz, 1H), 8.38 (d, J = 8.0Hz, 1H), 8.12 (t, J = 6.8Hz, 1H),

4.86 (t, J = 7.5Hz, 2H), 3.39 (t, J = 6.8Hz, 2H), 1.88-1.80 (m, 2H), 1.64-

1.57 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 146.9, 145.9, 145.1, 141.0,

127.3, 125.6, 57.0, 49.9, 27.6, 24.8. ESI HR (MeOH) calcd for C₁₀H₁₄N₅O

[M]⁺: 220.1193 found: 220.1197. The title compound cannot be

synthesized directly because 4-azidobutyl triflate quickly decomposes

during the synthesis.

3-azidopropano triflate and isonicotinaldehyde oxime to give yellow solid

which can be further purified by washing with CH₃CN (98%). ¹H NMR (400

MHz, DMSO)  = 13.04 (s, 1H), 9.19 (d, J = 6.8Hz, 2H), 8.44 (s, 1H), 8.24

(d, J = 5.6Hz, 2H), 4.71 (t, J = 6.8Hz, 2H), 3.49 (t, J = 6.4Hz, 2H), 2.23-

2.16 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 148.5, 145.1, 144.8, 123.8,

57.8, 47.5, 29.6. ESI (MeOH) for [M]⁺= 206.2.

1-(2-Azidoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (13C). The

title compound was synthesized analogously as for 2C using

isonicotinaldehyde oxime (yellowish solid, 83%). ¹H NMR (400 MHz,

DMSO)  = 13.09 (s, 1H), 9.17 (d, J = 6.8Hz, 2H), 8.45 (s, 1H), 8.28 (d, J

= 6.8Hz, 2H), 4.84 (t, J = 5.6Hz, 2H), 4.05 (t, J = 5.6Hz, 2H). ¹³C NMR (100

MHz, DMSO)  = 148.9, 145.4, 144.8, 123.7, 58.8, 50.2. ESI (MeOH) for

[M]⁺= 192.2.

2-((Hydroxyimino)methyl)-1-(pent-4-ynyl)pyridinium chloride (6C). The title

compound was synthesized using 4-pentyn-1-ol triflate obtained

analogously as before. The triflate (20.4 g, 94.4 mmol) was diluted with

CH₃NO₂(300 mL) then pyridine-2-carboxaldoxime (11.5 g, 94.3 mmol)

was added in one portion. The reaction was carried out for overnight at

room temperature then after evaporation the crude product was dissolved

in H₂O and passed through IRA-400(Clˉ) column. Aquatic layer was

washed with CHCl₃(3x) and evaporated. Grey solid was dissolved in

MeOH (25 mL) then acetone (200 mL) was added and the product was

filtered (16.2 g, 77%). ¹H NMR (400 MHz, DMSO)  = 13.43 (s, 1H), 9.23-

9.21 (m, 1H), 8.78 (s, 1H), 8.59-8.55 (m, 1H), 8.39-8.36 (m, 1H), 8.12-8.08

(m, 1H), 4.86-4.82 (m, 2H), 2.86 (t, J = 2.6Hz, 1H), 2.35 (dt, J¹= 7.0Hz, J²

= 2.6Hz, 2H), 2.05-1.97 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.0,

146.1, 145.2, 141.0, 127.3, 125.6, 82.5, 72.2, 56.8, 28.9, 14.6. ESI HR

(MeOH) calcd for C₁₁H₁₃N₂O [M]⁺: 189.1022 found: 189.1026.

1-(But-3-ynyl)-4-((hydroxyimino)methyl)pyridinium chloride (15C). The title

compound was synthesized analogously as for 6C using

isonicotinaldehyde oxime (yellow solid, 99%). ¹H NMR (400 MHz, DMSO)

 = 13.06 (s, 1H), 9.18 (d, J = 6.8Hz, 2H), 8.45 (s, 1H), 8.28 (d, J = 6.8Hz,

2H), 4.77 (t, J = 6.8Hz, 2H), 3.08 (t, J = 2.4Hz, 1H), 2.98 (dt, J¹= 6.8Hz,

J²=2.4Hz, 2H). ¹³C NMR (100 MHz, DMSO)  = 148.8, 145.2, 144.8, 123.6,

79.0, 75.0, 57.9, 20.2.

2-((Hydroxyimino)methyl)-1-(prop-2-ynyl)pyridinium bromide (17C). To the

cooled (-30 °C) mixture of trifluoromethanesulfonic anhydride (33.0 g, 19.4

mL, 116.9 mmol, 1.1 equiv.) in CH₂Cl₂(250 ml) a solution of pyridine (9.2

g, 9.4 mL, 116.9 mmol, 1.1 equiv.) and propargyl alcohol (6 g, 6.2 mL,

107.0 mmol) in CH₂Cl₂(100 mL) was added dropwise maintaining the

temperature at -30 °C. The reaction was carried out for 30 min. at -30 °C,

then cold H₂O (200 mL) was added. The organic layer was quickly

separated, dried over MgSO₄, filtered and carefully evaporated to the final

volume ca. 100 mL (DO NOT evaporate organic mixture completely). The

reaction yield (10.7 g, 53%) has been assumed based on the literature

results.^[22]The organic fraction from the previous step was immediately

diluted with CH₃NO₂(150 mL) then pyridine-2-carboxaldoxime (6.9 g, 56.5

mmol) was added in one portion. Reaction was carried out for 2h at room

temperature then solution of the Bu₄N⁺Brˉ (36.4 g, 113.0 mmol, 2 equiv.)

in CH₂Cl₂(150 mL) was added. The product as the bromide salt was

separated and washed with CH₂Cl₂and EtOAc to give white solid (12.2 g,

89%). ¹H NMR (400 MHz, DMSO)  = 13.22 (s, 1H), 9.26-9.25 (m, 1H),

8.78 (s, 1H), 8.66-8.62 (m, 1H), 8.47-8.45 (m, 1H), 8.20-8.16 (m, 1H), 5.87

(d, J = 2.4Hz, 2H), 4.08 (t, J = 2.4Hz, 1H). ¹³C NMR (100 MHz, DMSO) 

= 146.8, 146.2, 145.3, 141.2, 127.8, 125.6, 81.8, 75.3, 47.9. ESI HR

(MeOH) calcd for C₉H₉N₂O [M]⁺: 161.0709 found: 161.0710. The title

compound can be stored at room temperature for months without

decomposition, however in solution it quickly changes color to dark brown.

Any attempts to purify and/or anion exchange lead to the decomposition

of the product in solution. Black impurities are formed when this alkyne is

used in the triazole synthesis.

1-(Hex-5-ynyl)-2-((hydroxyimino)methyl)pyridinium chloride (7C). The title

compound was synthesized using 5-hexyn-1-ol triflate obtained

analogously as for 6C to give greenish solid (14.3 g, 75%). ¹H NMR (400

MHz, DMSO)  = 13.45 (s, 1H), 9.26 (d, J = 5.6Hz, 1H), 8.80 (s, 1H), 8.57

(t, J = 7.6Hz, 1H), 8.38 (dd, J¹= 8.0Hz, J²= 1.2Hz, 1H), 8.13-8.09 (m, 1H),

4.84 (t, J = 7.6Hz, 2H), 2.82 (t, J = 2.8Hz, 1H), 2.21 (dt, J¹= 7.2Hz, J²=

2.8Hz, 2H), 1.90-1.82 (m, 2H), 1.54-1.47 (m, 2H). ¹³C NMR (100 MHz,

DMSO)  = 146.9, 145.9, 145.1, 141.0, 127.3, 125.6, 83.7, 71.7, 57.1, 29.5,

24.3, 17.2. ESI HR (MeOH) calcd for C₁₂H₁₅N₂O [M]⁺: 203.1179 found:

203.1179.

1-(But-3-ynyl)-2-((hydroxyimino)methyl)pyridinium chloride (8C). To the

cooled (-30 °C) mixture of trifluoromethanesulfonic anhydride (40.0 g, 23.5

mL, 141.8 mmol, 1.1 equiv.) in CH₂Cl₂(300 mL) a solution of pyridine (11.2

g, 11.4 mL, 141.8 mmol, 1.1 equiv.) and 3-butyn-1-ol (9 g, 9.7 mL, 128.6

mmol) in CH₂Cl₂(300 mL) was added dropwise maintaining the

temperature at -30 °C. The reaction was carried out for 30 min. at -30 °C,

then H₂O (300 mL) was added. The organic layer was separated, dried

over MgSO₄, filtered and evaporated to give yellowish oil (24.6 g, 95%,

stable for few months at -30 °C) used in the next step without purification.

The triflate, from the previous step, (10 g, 49.5 mmol) was diluted with

CH₃NO₂(200 mL) then pyridine-2-carboxaldoxime (6 g, 49.5 mmol) was

added in one portion. Reaction was carried out for 4h at room temperature

then solid Bu₄N⁺Clˉ (27.2 g, 98.0 mmol, 2 equiv.) was added. After 5 min

white product was filtered (8.6 g, 83%). ¹H NMR (400 MHz, DMSO)  =

13.43 (s, 1H), 9.21-9.20 (m, 1H), 8.82 (s, 1H), 8.63-8.59 (m, 1H), 8.42-8.40

(m, 1H), 8.17-8.13 (m, 1H), 4.98 (t, J = 6.4Hz, 2H), 3.08 (t, J = 2.5Hz, 1H),

2.87 (dt, J¹= 6.4Hz, J²= 2.5Hz, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.2,

146.4, 145.6, 141.2, 127.0, 125.5, 78.7, 75.4, 55.2, 19.8. ESI HR (MeOH)

calcd for C₁₀H₁₁N₂O [M]⁺: 175.0866 found: 175.0868

3-(But-3-ynyl)-2-((hydroxyimino)methyl)-1-methyl-1H-imidazol-3-ium

chloride (3D). The title compound was synthesized analogously as for 6C

using 1-methyl-1H-imidazole-2-carbaldehyde oxime (yellow solid 95%).

The product can be further purified by washing with small amount of

CH₃CN. ¹H NMR (400 MHz, DMSO)  = 13.26 (s, 1H), 8.59 (s, 1H), 8.00-

7.97 (m, 2H), 4.50 (t, J = 6.8Hz, 2H), 3.95 (s, 3H), 3.02 (t, J = 2.4Hz, 1H),

2.77-2.73 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 136.9, 135.2, 124.5,

123.5, 79.6, 74.2, 46.9, 36.5 19.3. ESI HR (MeOH) calcd for C₉H₁₂N₃O

[M]⁺: 178.0975 found: 178.0979.

1-(Hepta-3,6-diynyl)-2-((hydroxyimino)methyl)pyridinium chloride (9C).

The title compound was synthesized using hepta-3,6-diyn-1-ol triflate

obtained analogously as for 6C to give greenish solid (5.3 g, 76%). ¹H NMR

(400 MHz, DMSO)  = 13.40 (s, 1H), 9.18-9.17 (m, 1H), 8.82 (s, 1H), 8.62-

8.58 (m, 1H), 8.40 (dd, J¹= 8.0Hz, J²= 1.2Hz, 1H), 8.15-8.11 (m, 1H), 4.96

(t, J = 6.4Hz, 2H), 3.18 (q, J = 2.3Hz, 2H), 3.01 (t, J = 2.8Hz, 1H), 2.86-

2.83 (m, 2H). ¹³C NMR (100 MHz, DMSO)  = 147.2, 146.4, 145.5, 141.2,

127.0, 125.5, 78.4, 78.1, 75.3, 71.7, 55.5, 20.1, 8.6. ESI HR (MeOH) calcd

for C₁₃H₁₃N₂O [M]⁺: 213.1022 found: 213.1025.

3-(3-Azidopropyl)-2-((hydroxyimino)methyl)-1-methyl-1H-imidazol-3-ium

chloride (4D). The title compound was synthesized analogously as for 2C

using 3-azidopropanol triflate and 1-methyl-1H-imidazole-2-carbaldehyde

oxime to give yellow solid which can be further purified by washing with

CH₃CN (91%). ¹H NMR (400 MHz, DMSO)  = 13.24 (s, 1H), 8.55 (s, 1H),

7.97 (dd, J¹= 12.8Hz, J²= 2.0Hz, 2H), 4.40 (t, J = 7.2Hz, 2H), 3.93 (s, 3H),

3.43 (t, J = 6.4Hz, 2H), 2.04-1.97 (m, 2H). ¹³C NMR (100 MHz, DMSO) 

= 136.9, 135.0, 124.6, 123.2, 47.4, 46.3, 36.49, 36.47, 28.6. ESI HR

(MeOH) calcd for C₈H₁₃N₆O [M]⁺: 209.1145 found: 209.1143.

1-(Hex-5-ynyl)-4-((hydroxyimino)methyl)pyridinium chloride (10C). The

title compound was synthesized analogously as for 6C using 5-hexyn-1-ol

triflate and isonicotinaldehyde oxime (light yellow solid, 82%). ¹H NMR

(400 MHz, DMSO)  = 13.01 (s, 1H), 9.17 (d, J = 6.8Hz, 2H), 8.44 (s, 1H),

8.24 (d, J = 6.8Hz, 2H), 4.66 (t, J = 7.2Hz, 2H), 2.82 (t, J = 2.4Hz, 1H),

2.23-2.19 (m, 2H), 2.02-1.95 (m, 2H), 1.49-1.42 (m, 2H). ¹³C NMR (100

MHz, DMSO)  = 148.3, 144.9, 144.8, 123.9, 83.7, 71.7, 59.5, 29.7, 24.3,

17.1. ESI (MeOH) for [M]⁺= 203.1.

3-Azido-N-(2-(hydroxyimino)ethyl)-N,N-dimethylpropan-1-aminium

chloride (5D). The title compound was synthesized analogously as for D13

using E5 to give the product as a mixture of isomers, A+B; ≈ 7:3 as light-

purple oil (97% overall). ¹H NMR (400 MHz, DMSO)  = 7.61 (t, J = 6.4Hz,

1H, A), 7.37 (t, J = 4.8Hz, 0.4H, B), 4.32 (d, J = 4.8Hz, 0.8H, B), 4.18 (d,

J = 6.4Hz, 2H, A), 3.52-3.37 (m, 4H, A+B), 3.19 (s, 2.4H, B), 3.11 (s, 6H,

A), 2.02-1.93 (m, 2H, A+B). ¹³C NMR (100 MHz, DMSO)  = 139.8, 138.9,

61.5, 61.2, 60.9, 56.9, 50.6, 50.2, 47.8, 31.2, 22.0, 21.8. ESI HR (MeOH)

calcd for C₇H₁₆N₅O [M]⁺: 186.1349 found: 186.1351.

1-(3-Azidopropyl)-4-((hydroxyimino)methyl)pyridinium chloride (4-PZ3)

(12C). The title compound was synthesized analogously as for 2C using

10.1002/chem.201805051

Chemistry - A European Journal

FULL PAPER

2-(Hydroxyimino)-N,N,N-trimethylethanaminium iodide (6D). The mixture

of 3E (1 g, 3.3 mmol), concentrated HCl (5 mL) and H₂O (40 mL) was

stirred under reflux for 2h then evaporated. The residue was dissolved in

H₂O (5 mL) and treated with 50% NH₂OH (5 ml). Reaction mixture was

stirred at rt for overnight then evaporated to give a product as a mixture of

two, unseparable isomers (A+B; ≈ 4:6; 655mg, 81%). ¹H NMR (400 MHz,

DMSO)  = 11.94 (s, 0.7H, A), 11.84 (s, 1H, B), 7.63 (t, J = 6.4Hz, 1H, B),

7.36 (t, J = 4.8Hz, 0.7H, A), 4.32 (d, J = 4.8Hz, 1.4H, A), 4.14 (d, J = 6.4Hz,

2H, B), 3.19 (s, 6.3H, A), 3.12 (s, 9H, B). ¹³C NMR (100 MHz, DMSO)  =

140.0, 139.2, 63.1, 58.74, 58.72, 52.7, 52.2. ESI HR (MeOH) calcd for

C₅H₁₃N₂O [M]⁺: 117.1022 found: 117.1024.

1-(But-3-ynyl)pyridinium chloride (6E). But-3-ynyl triflate (from 8C) (3 g,

14.8 mmol) was diluted with CH₃NO₂(60 mL) then pyridine (1.17 g, 1.19

mL, 14.8 mmol) was added. Reaction was carried out for 4h at room

temperature then solid Bu₄N⁺Clˉ (8.2 g, 29.6 mmol, 2 equiv.) was added.

The mixture was evaporated and the residue was dissolved in CH₃CN (15

mL) then Et₂O (60 mL) was added. Approximately 5 min later the product

crystallized (2.1 g, 85%). An analytical sample can be further purified by

crystallization from CH₃CN. ¹H NMR (400 MHz, DMSO)  = 9.28 (d, J =

6.0Hz, 1H), 8.67 (t, J = 7.8Hz, 1H), 8.22 (t, J = 7.2Hz, 1H), 4.83 (t, J =

6.6Hz, 2H), 3.08 (t, J = 2.4Hz, 1H), 3.00 (dt, J¹= 6.4Hz, J²= 2.4Hz, 2H).

¹³C NMR (100 MHz, DMSO)  = 145.9, 145.0, 127.7, 79.0, 75.0, 58.3, 20.3.

ESI HR (MeOH) calcd for C₉H₁₀N [M]⁺: 132.0808 found: 132.0806.

N-(2-(Hydroxyimino)ethyl)-N,N-dimethylbut-3-yn-1-aminium

chloride

(13D). The mixture of 4E (7 g, 28.1 mmol) and HCl_conc.(5 mL) in H₂O (100

mL) was stirred under reflux with Dean-Stark apparatus for 2h, then

evaporated and used in the next step without purification. The residue was

dissolved in 50% NH₂OH in H₂O (17.1 mL) and the reaction was carried

out at room temperature for overnight and then evaporated. Crude product

was washed with CH₃CN (10 mL) to give crystalline product as a mixture

of isomers, A+B; ≈ 1:1 (4.7 g, 88% overall). An analytical sample can be

further purified by crystallization from MeOH. ¹H NMR (400 MHz, DMSO)

 = 12.11 (brs, 1H), 7.61 (t, J = 6.4Hz, 1H), 7.38 (t, J = 4.8Hz, 1H), 4.35 (d,

J = 4.8Hz, 2H), 4.21 (d, J = 6.4Hz, 2H), 3.64 (t, J = 8.0Hz, 2H), 3.55 (t, J =

8.0Hz, 2H), 3.21 (s, 6H), 3.13 (s, 7H), 2.82-2.76 (m, 4H). ¹³C NMR (100

MHz, DMSO)  = 139.7, 138.7, 79.3, 79.2, 73.9, 73.9, 61.4, 61.3, 60.9,

56.9, 50.4, 50.0, 12.9, 12.8. ESI HR (MeOH) calcd for C₈H₁₅N₂O [M]⁺:

155.1179 found: 155.1178.

1-(3-Azidopropyl)pyridinium chloride (7E). The title compound was

synthesized analogously as for 2C using 3-azidopropanol triflate. For the

anion exchange IRA-400(Clˉ) column has been used to give yellow oil

(93%). ¹H NMR (400 MHz, DMSO)  = 9.32 (d, J = 5.6Hz, 2H), 8.64 (t, J =

7.8Hz, 1H), 8.21-8.17 (m, 2H), 4.79 (t, J = 7.2Hz, 2H), 3.50 (t, J = 6.6Hz,

2H), 2.21 (p, J = 6.8Hz, 2H). ¹³C NMR (100 MHz, DMSO)  = 145.5, 145.0,

127.9, 58.2, 47.5, 29.7. ESI HR (MeOH) calcd for C₈H₁₁N₄[M]⁺: 163.0978

found: 163.0976.

1-(But-3-ynyl)-4-carbamoylpyridinium chloride (8E). But-3-ynyl triflate

(from 8C) (7.8 g, 38.5 mmol) was diluted with CH₃NO₂(30 mL) then solid

isonicotinamide (4.7 g, 38.5 mmol) was added in one portion. Reaction

was carried out for overnight at room temperature then a solution of

Bu₄N⁺Clˉ (10.7 g, 38.5 mmol) in CH₃CN (100 mL) was added. The product

was filtered and crystallized from CH₃CN:EtOH (100 mL:300 mL) to give

white solid (5.1 g, 63%). ¹H NMR (400 MHz, DMSO)  = 9.31 (d, J = 6.8Hz,

2H), 8.88 (s, 1H), 8.54 (d, J = 6.8Hz, 2H), 8.30 (s, 1H), 4.81 (t, J = 6.4Hz,

2H), 3.08 (t, J = 2.4Hz, 1H), 3.01-2.97 (m, 2H). ¹³C NMR (100 MHz,

DMSO:D₂O; 1:1)  = 166.0, 149.8, 146.8, 127.0, 79.7, 76.2, 60.5, 21.6.

ESI (MeOH) for [M]⁺= 175.4.

Synthesis of oximes 11C, 14C, 16C, 1D, 2D, 7D, 8D−12D, 14D−27D was

reported earlier in Cochran et al.^[7c]

Synthesis of the building blocks (1E-12E). 1-(But-3-ynyl)-3-fluoro-4-

((hydroxyimino)methyl)pyridinium chloride (1E). The title compound was

synthesized analogously as for 6C using 3-fluoroisonicotinaldehyde oxime

(89%). ¹H NMR (400 MHz, DMSO)  = 13.35 (s, 1H), 9.81 (d, J = 5.2Hz,

1H), 9.11 (d, J = 6.4Hz, 1H), 8.44-8.38 (m, 2H), 4.83 (t, J = 6.6Hz, 2H),

3.12-3.09 (m, 1H), 3.04-2.98 (m, 2H). ¹³C NMR (100 MHz, DMSO)  =

156.0 (d, J = 255.3Hz), 141.83 (d, J = 3.7Hz), 139.6, 136.97 (d, J = 9.0Hz),

135.8 (d, J = 38.5Hz), 123.92 (d, J = 3.5Hz), 78.9, 75.1, 58.4, 20.2.

1-(2-Azidoethyl)-4-carbamoylpyridinium chloride (9E). The title compound

was synthesized analogously as for 8E using 2-azidoethanol triflate (from

2C) and isonicotinamide. The product was crystallized from CH₃CN:MeOH

(1:1) to give yellow solid (70%). ¹H NMR (400 MHz, DMSO)  = 9.36 (d, J

= 6.8Hz, 2H), 9.10 (s, 1H), 8.61 (d, J = 6.8Hz, 2H), 8.32 (s, 1H), 4.91 (t, J

= 5.6Hz, 2H), 4.07 (t, J = 5.6Hz, 2H). ¹³C NMR (100 MHz, DMSO)  =

163.1, 148.5, 146.2, 125.8, 59.3, 50.1. ESI (MeOH) for [M]⁺= 192.1.

1-(2-Azidoethyl)-3-fluoro-4-((hydroxyimino)methyl)pyridinium

chloride

(2E). The title compound was synthesized analogously as for 2C using 3-

fluoroisonicotinaldehyde oxime (61%). ¹H NMR (400 MHz, DMSO)  =

13.42-13.40 (m, 1H), 9.83 (d, J = 5.2Hz, 1H), 9.08 (d, J = 6.4Hz, 1H), 8.44-

8.37 (m ,2H), 4.90 (t, J = 5.4Hz, 2H), 4.08 (t, J = 5.4Hz, 2H). ¹³C NMR (100

MHz, DMSO)  = 156.1 (d, J = 255.3Hz), 142.03 (d, J = 4.0Hz), 139.5,

137.02 (d, J = 8.8Hz), 136.1 (d, J = 38.1Hz), 123.92 (d, J = 3.7Hz), 59.1,

50.0.

1-(3-Azidopropyl)-4-carbamoylpyridinium chloride (10E). The title

compound was synthesized analogously as for 8E using 3-azidopropanol

triflate and isonicotinamide. The product was crystallized from

CH₃CN:EtOH (1:1) to give yellow solid (69%). ¹H NMR (400 MHz, DMSO)

 = 9.38 (d, J = 6.8Hz, 2H), 9.06 (s, 1H), 8.58 (d, J = 6.8Hz, 2H), 8.30 (s,

1H), 4.80 (t, J = 6.8Hz, 2H), 3.49 (t, J = 6.8Hz, 2H), 2.26-2.19 (m, 2H). ¹³

C

NMR (100 MHz, DMSO)  = 163.1, 148.0, 145.9, 125.9, 58.3, 47.5, 29.6.

2,2-Diethoxy-N,N,N-trimethylethanaminium

iodide

(3E).

ESI (MeOH) for [M]⁺= 206.4.

(Dimethylamino)acetaldehyde diethyl acetal (5.78 mL, 5 g, 31.0 mmol)

was dissolved in Et₂O (50 mL) then MeI (2.9 mL, 6.6 g, 46.5 mmol, 1.5

equiv.) was added. Reaction was stirred at rt for 1 day then white solid was

1-(4-Chlorobutyl)-2-((hydroxyimino)methyl)pyridinium

trifluoromethanesulfonate (11E). To the cooled (-30 °C) mixture of

trifluoromethanesulfonic anhydride (26.2 g, 15.4 mL, 92.9 mmol, 1.1

equiv.) in CH₂Cl₂(250 mL) a solution of pyridine (7.4 g, 7.6 mL, 92.9 mmol,

1.1 equiv.) and 4-chlorobutan-1-ol (10 g, 9.2 mL of 90%, 82.9 mmol) in

CH₂Cl₂(100 mL) was added dropwise maintaining the temperature at -

30 °C. The reaction was carried out for 30 min. at -30 °C, then H₂O (150

mL) was added. The organic layer was separated, dried over MgSO₄,

filtered and evaporated to gave yellowish oil (21.5 g, 97%) used in the next

step without purification. The triflate, from the previous step, (21.0 g, 87.3

mmol) was diluted with CH₃NO₂(300 mL) then pyridine-2-carboxaldoxime

(10.6 g, 86.9 mmol) was added in one portion. The reaction was carried

out for 2h at room temperature then evaporated. The residue was

dissolved in H₂O (200 mL), washed with CHCl₃(2 x 150 mL) and

evaporated again to give green wax (27 g, 85%). An analytical sample can

be further purified by washing with EtOAc. ¹H NMR (400 MHz, DMSO)  =

13.17 (s, 1H), 9.03-9.01 (m, 1H), 8.80 (s, 1H), 8.56-8.52 (m, 1H), 8.41 (dd,

J¹= 8.2Hz, J²= 1.4Hz, 1H), 8.11-8.07 (m, 1H), 4.77 (t, J = 7.4Hz, 2H), 3.68

(t, J = 6.4Hz, 2H), 1.99-1.90 (m, 2H), 1.84-1.77 (m,2H). ¹³C NMR (100 MHz,

DMSO)  = 146.9, 145.8, 145.1, 141.3, 127.4, 125.8, 122.2, 119.0, 57.1,

44.5, 28.4, 27.8. ESI HR (MeOH) calcd for C₁₀H₁₄ClN₂O [M]⁺: 213.0789

found: 213.0793.

1

separated, washed with Et₂O and dried (8.9 g, 94%). H NMR (400 MHz,

DMSO)  = 4.95 (t, J = 4.8Hz, 1H), 3.58 (4.95 (d, J = 4.8Hz, 2H), 3.35 (s,

6H), 3.19 (s, 9H). ¹³C NMR (100 MHz, DMSO)  = 98.2, 64.6, 53.7, 53.2.

ESI HR (MeOH) calcd for C₉H₂₂NO₂[M]⁺: 176.1645 found: 176.1646.

N-(2,2-Diethoxyethyl)-N,N-dimethylbut-3-yn-1-aminium chloride (4E). To

the mixture of 2,2-diethoxy-N,N-dimethylethanamine (6.3 g, 7.2 mL, 39.1

mmol) in CH₃NO₂(50 mL), but-3-ynyl triflate (from 6C, 8 g, 39.1 mmol) was

added. The reaction mixture became immediately warm and after 1h a

solution of Bu₄N⁺Clˉ (21.7 g, 78.2 mmol, 2 equiv.) in CH₃CN:Et₂O (20

mL:100 mL) was added. White crystals of the product were separated (8.2

g, 84%). An analytical sample can be further purified by crystallization from

CH₃CN. ¹H NMR (400 MHz, DMSO)  = 5.03 (t, J = 4.8Hz, 1H), 3.62-3.54

(m, 8H), 3.17 (s, 6H), 3.08 (t, J = 2.4Hz, 1H), 2.75-2.71 (m, 2H), 1.09 (t, J

= 7.2Hz, 6H). ¹³C NMR (100 MHz, DMSO)  = 96.4, 79.3, 73.7, 64.8, 63.5,

62.1, 61.7, 51.0, 14.9, 12.9. ESI HR (MeOH) calcd for C₁₂H₂₄NO₂[M]⁺:

214.1801 found: 214.1802.

3-Azido-N-(2,2-diethoxyethyl)-N,N-dimethylpropan-1-aminium

chloride

(5E). The title compound was synthesized analogously as for 4E using 3-

azidopropanol triflate. For the anion exchange IRA-400(Clˉ) column has

been used to give colorless oil (87%). ¹H NMR (400 MHz, DMSO)  = 5.06

(t, J = 5.2Hz, 1H), 3.69-3.59 (m, 4H), 3.53 (d, J = 5.2Hz, 2H), 3.50-3.44 (m,

4H), 3.16 (s, 6H), 2.01-1.94 (m, 2H), 1.15 (t, J = 7.2Hz, 6H). ¹³C NMR (100

MHz, DMSO)  = 96.4, 63.5, 62.2, 61.8, 51.2, 47.8, 21.9, 14.9. ESI HR

(MeOH) calcd for C₁₁H₂₅N₄O₂[M]⁺: 245.1972 found: 245.1976.

1-((Chloromethoxy)methyl)-2-((hydroxyimino)methyl)pyridinium chloride

(12E). To the suspension of the pyridine-2-carboxaldoxime (4.4 g, 36.0

mmol) in CH₃NO₂(50 mL) bis(chloromethyl) ether (12.4 g, 108.0 mmol, 3

equiv.) was added. Reaction was carried out for overnight at room

temperature, then brown product was filtered and washed with CH₃CN and

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Et₂O (7.2 g, 84%). ¹H NMR (400 MHz, CD₃OD)  = 9.20-9.19 (m, 1H),

8.69-8.65 (m, 2H), 8.59 (dd, J¹= 8.2Hz, J²= 1.4Hz, 1H), 8.13-8.09 (m, 1H),

6.38 (s, 2H), 5.78 (s, 2H). ¹³C NMR (100 MHz, CD₃OD)  = 149.7, 148.7,

146.8, 141.8, 128.4, 127.2, 86.9, 81.3. ESI HR (MeOH) calcd for

Absorbances were read at 490 nm on a plate reader spectrophotometer

(InfiniteM200 Pro, Tecan, USA), and results were expressed as the

percentage of inhibition compared to control cells. Total percentage of

DMSO in cytotoxicity assay was 0.8% and did not appear to influence the

test. Data were evaluated using predefined IC₅₀equation from a GraphPad

Prism program (San Diego, USA).

C₈H₁₀ClN₂O₂

[M]⁺:

201.0425

found:

201.0431.

CAUTION:

Bis(chloromethyl) ether is very toxic and carcinogenic. The title compound

decomposes within 1 month at room temperature, and few months at -

20 °C. Any additional purification method has not been successful.

Oxime pharmacokinetics in mice. Female CD-1 mice, 4−8 weeks old

(22−34 g body weight), were purchased from Harlan (Livermore, CA). Mice

were fed Purina Certified Rodent Chow #5002 food and purified water was

provided ad libitum. Mice were kept in hanging polycarbonate cages at

21−23 °C, exchanging light and dark cycles every 12h. General

procedures for animal care and housing were in accordance with the

National Research Council (NRC) Guide for the Care and Use of

Laboratory Animals (1996) and the Animal Welfare Standards

incorporated in 9 CFR Part 3, 1991. For pharmacokinetic experiments

mice were divided into groups of three, and 5 mg kg^-1of the oximes was

administered i.m. in a single dose. Three animals were injected for each

time point analyzed. Blood (~300 µL) was collected from the retro-orbital

sinus of mice under isoflurane anesthesia into tubes containing EDTA,

processed to plasma within 30 minutes of collection, and then stored

frozen at ≤ −80 °C (± 10 °C). Concentrations of the oxime in body

compartments were determined by LC-MS using multiple reaction

monitoring (MRM) electrospray ionization detection in positive ion mode

as described previously.^[26]

Chemicals. Tabun, VX, sarin, cyclosarin, and soman were purchased

from NC Laboratory, Spiez, Switzerland. Nerve agent stock solutions were

prepared in isopropyl alcohol, while further dilutions were made in water

immediately prior to use. Paraoxon ethyl (Sigma-Aldrich, St. Louis, MO,

USA) was diluted in ethanol just before use. Acetylthiocholine iodide

(ATCh), standard oximes and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)

were purchased from Sigma Chemical Co., St. Louis, MO, USA. Stock

solutions of oximes were prepared in water or dimethyl sulfoxide (DMSO;

Kemika, Zagreb, Croatia) and diluted in sodium phosphate buffer (0.1 M,

pH 7.4) just before use. Atropine sulfate was purchased from Kemika,

Zagreb, Croatia.

Cholinesterase activity measurements. Recombinant human AChE

(AChE), wild type, was prepared as described earlier.^[7c]For reactivation

experiments, the enzyme was incubated with a ten-fold excess of OP until

inhibition was greater than 95%. Such inhibited enzyme was passed

through a Sephadex G-50 spin column (Roche Diagnostic GmbH,

Mannheim, Germany) to remove an excess of unconjugated OP. After gel

filtration by centrifugation, enzyme was diluted 10-fold in 0.1 M sodium

phosphate buffer, pH 7.4, containing 0.01% BSA and incubated with oxime

at 25 °C. At specified time intervals, an aliquot was diluted 100-fold in

phosphate buffer containing DTNB; upon addition of ATCh, residual

enzyme activity was measured. Final concentrations of ATCh and DTNB

in the assay were 1.0 and 0.3 mM, respectively. An equivalent sample of

uninhibited enzyme was passed through a parallel column, diluted to the

same extent as the inhibition mixture, and control activity was measured

in the presence of oxime at concentrations used for reactivation. Both

activities of control and reactivation mixture were corrected for oxime-

induced hydrolysis of ATCh. Reactivation of the enzyme was measured

immediately upon addition of ATCh at nine time points until reaching

maximal percentage of reactivation (React_max). Reactivation screening

was conducted at a given oxime concentration (0.1 or 1 mM) up to 24 h,

wherefrom the observed first-order reactivation rate constant (k_obs) and

Antidotal tests in OP exposed mice. Male NIH/OlaHsd mice (20−25 g

body weight; purchased from Imunološki zavod, Zagreb, Croatia) were fed

a standard diet, had free access to water and were kept in Macrolone

cages at 21 ^oC, exchanging light and dark cycles every 12h. For the

experiments mice were divided into groups of four. Acute toxicity (LD₅₀

)

was based upon 24h mortality rates and 95% confidence limits were

estimated from data tables, as described elsewhere.^[27]Antidotal activity of

oximes in OP-exposed mice was tested by administration (i.p.) of selected

oxime (in doses of 5% or 25% of its LD₅₀) dissolved in the solution of

atropine sulphate (10.0 mg kg^-1b.w.), one minute after OP exposure (s.c.).

Antidotal efficacy was expressed as a Protective Index (PI) with 95%

confidence limits and maximal dose of OP (MD). The PI is the ratio of LD₅₀

between OP with treatment and OP given alone, without treatment. The

MD is the highest multiple of the OP’s LD₅₀, that was fully counteracted by

treatments. The mice described in these experiments were treated in

accord with the approval of the Ethics Committee of the Institute for

Medical Research and Occupational Health in Zagreb, Croatia.

maximal percentage of reactivation (React_max

) were determined as

previously described.^[23]More detailed reactivation kinetics using a wider

oxime concentration range enables the determination of reactivation

constants: k₂(maximal first-order reactivation rate constant), K_ox(apparent

phosphylated enzyme-oxime dissociation constant) and k_r(second-order

reactivation rate constant) as described previously.^[9b]

Acknowledgements

This work was supported by the CounterACT Program, the

National Institute of Neurological Disorders and Stroke (NINDS),

National Institute of Health (NIH, Grant Number U01 NS058046)

and by the Croatian Science Foundation (HrZZ-IP-2013-11-4307).

All cholinesterase activity measurements were performed in 0.1 M sodium

phosphate buffer containing 0.01% BSA, pH 7.4 at 25 °C using the Ellman

spectrophotometric method^[24]at 412 nm on

a

CARY 300

spectrophotometer (Varian Inc., Australia) with temperature controller.

pK_adetermination of selected oximes. Protonation of ionizable groups

in the oximes was monitored by UV-VIS spectrometry (CARY 300

spectrophotometer, Varian Inc., Australia) and proton dissociation

constants were determined from pH-absorbance profiles at 25 °C using

Conflict of interest

equation 1 as described earlier.^[12]

The authors declare no conflict of interest.

+

A

∙ [H ] + A ∙ K

a

1

2

A_tot

=

(1)

+

[H ] + K

a

The observed absorbance at maximum absorption wavelength A_totis the

sum of the absorption fractions of all aldoxime ionization states present.

A₁and A₂are absorbances of the species with protonated (HL⁺) and

deprotonated (L) oxime group, respectively.

Keywords:

nerve

agents

•

acetylcholinesterase

•

organophosphate antidotes • 2-PAM • triazole-linked oximes

Supporting Information. Content: S1. 1H and 13C NMR spectra

for compounds 1A-5A, 9A, 14A, 15A, 20A, 1B and 2B. This

material is available free of charge via the internet at http://...

Cytotoxicity assay. SH-SY5Y cells were obtained from ATCC LGC

Standards GmbH, Germany, and cultured according to the protocol in

DMEM F12 medium (2 mM glutamine + 1% non-essential amino acids +

15% fetal bovine serum, Sigma-Aldrich, Germany). Two days before the

toxicity study, cells were seeded on 96 F-bottom clear well plates in 1x10⁵

/ well. On the day of the experiment, medium was removed and fresh

medium containing oximes in a designated concentration was added (120

μL volume per well). Oximes were tested at eight concentrations; in a 1:2

dilution sequence, starting from 800 µM. Plates were incubated for 24h at

37 °C. After incubation, cells were washed twice with PBS and the MTS

reagent was added to determine cytotoxic activity according to the

manufacturer’s protocol (CellTiter 96^®AQueous One Solution Cell

Proliferation Assay, Promega, USA). The MTS detection reagent assays

succinate dehydrogenase mitochondrial activity of living cells.^[25]

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Chemistry - A European Journal

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10.1002/chem.201805051

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Appendix

Table 1. Reactivation screening of the tabun-inhibited AChE by A) sixty-two triazole compounds, B) five N-alkyl-2 pyridinium compounds,

C) seventeen compounds with more complex N-alkyl substitutions, D) twenty-seven miscellaneous oximes, 2-PAM, and obidoxime. The

reactivation rate at the given oxime concentration, k_obs(1 mM; except for 12 oximes as marked in the table), maximal percentage of

reactivation, React_maxobtained in time (t), and reversible inhibition by the oxime (in concentration 100-fold lower than employed in

reactivation) were evaluated. Reactivation was monitored up to 24h.

A) triazole compounds

k_obs/ min^-1

React_max/%

t / h

Inhibition [OX]

1A

0.0270

95

1.5

40% (0.01 mM)

2A

0.0260

0.0170

95

80

2.5

65% (0.01 mM)

55% (0.002 mM)

3A (0.2 mM)

3

4A

0.0160

0.0153

0.0150

85

95

90

85

4

60% (0.01 mM)

20% (0.01 mM)

85% (0.01 mM)

11% (0.01 mM)

5A

6A

Obidoxime ^[8]

7A

0.0140

80

6

40% (0.01 mM)

8A

0.0136

0.0099

85

75

5

4

70% (0.01 mM)

55% (0.001 mM)

9A (0.1 mM)

10A

0.0093

100

7

40% (0.01 mM)

11A

12A

13A

14A

15A

0.0091

0.0090

0.0088

0.0085

95

80

90

60

7

7.5

6

85% (0.01 mM)

63% (0.01 mM)

70% (0.01 mM)

45% (0.01 mM)

20% (0.01 mM)

6

4

16A

0.0083

90

7

75% (0.01 mM)

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17A

0.0083

90

7

80% (0.01 mM)

18A

19A

0.0078

0.0068

90

80

7

8

80% (0.01 mM)

30% (0.01 mM)

20A

0.0061

0.0050

45

65

50

4

7

8

70% (0.01 mM)

30% (0.01 mM)

30% (0.001 mM)

21A

22A (0.1 mM)

23A (0.1 mM)

0.0043

0.0036

80

65

10

12

70% (0.001 mM)

70% (0.01 mM)

24A

25A (0.1 mM)

0.0035

0.0029

80

55

11

12

75% (0.001 mM)

80% (0.01 mM)

26A

27A

0.0027

55

12

30% (0.01 mM)

28A

0.0027

0.0026

0.0024

50

80

12

24

40% (0.01 mM)

10% (0.01 mM)

60% (0.01 mM)

2-PAM

29A

30A

31A

0.0023

0.0022

50

90

24

30% 0.01 mM)

85% (0.01 mM)

32A

33A

0.0022

75

60

24

80% (0.01 mM)

15% (0.01 mM)

34A

0.0019

90

24

30% (0.01 mM)

35A

36A

0.0018

0.0017

40

55

24

85% (0.01 mM)

25% (0.01 mM)

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37A

0.0017

0.0016

35

45

24

40% (0.01 mM)

10% (8 nM)

38A (0.8 µM)

39A

0.0014

80

24

30% (0.01 mM)

40A (0.1 mM)

0.0013

40

24

85% (0.001 mM)

20% (0.01 mM)

41A

42A

43A

0.0012

0.0011

50

35

24

55% (0.01 mM)

30% (0.01 mM)

44A (20 µM)

0.0010

40

24

45% (0.2 µM)

45A

0.0009

0.0008

0.0006

-

65

55

24

60% (0.01 mM)

15% (0.01 mM)

40% (0.01 mM)

20% (0.01 mM)

60% (0.01 mM)

10% (8 nM)

46A

47A

55

48A

50

49A

45

50A

50

51A (0.8 µM)

52A

≤20

-

20% (0.01 mM)

53A

-

≤20

24

30% (0.01 mM)

54A (0.1 mM)

55A

-

≤20

24

40% (0.001 mM)

40% (0.01 mM)

40% (8 nM)

56A (0.8 µM)

57A

45% (0.01 mM)

58A

-

≤20

24

45% (0.01 mM)

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59A

-

≤20

24

t / h

6

70% (0.01 mM)

80% (0.01 mM)

85% (0.001 mM)

Inhibition [OX]

15% (0.01 mM)

40% (0.01 mM)

5% (0.01 mM)

45% (0.01 mM)

20% (0.01 mM)

10% (0.01 mM)

Inhibition [OX]

20% (0.01 mM)

40% (0.01 mM)

25% (0.01 mM)

30% (0.01 mM)

20% (0.01 mM)

65% (0.01 mM)

10% (0.01 mM)

55% (0.01 mM)

20% (0.01 mM)

15% (0.01 mM)

50% (0.01 mM)

60A

≤20

61A

-

≤20

62A (0.1 mM)

-

≤20

B) N-alkyl-2 pyridinium compounds

k_obs/ min^-1

0.0123

0.0111

0.0089

0.0045

0.0030

0.0026

k_obs/ min^-1

0.0145

0.0129

0.0058

0.0052

0.0035

0.0031

0.0026

0.0020

0.0014

0.0009

React_max/%

1B

90

2B

85

4

3B

100

8

4B

65

8

5B

70

12

24

t / h

6

2-PAM

80

C) compounds with more complex N-alkyl substitutions

React_max/%

1C

95

85

90

80

75

70

80

70

60

50

55

2C

5

3C

10

11

12

24

4C

5C

6C

2-PAM

7C

8C

9C

10C

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11C

0.0006

50

≤20

24

40% (0.01 mM)

5% (0.01 mM)

10% (0.01 mM)

60% (0.01 mM)

85% (0.01 mM)

Inhibition [OX]

12C

13C

14C

15C

16C

17C

-

≤20

24

-

≤20

24

-

≤20

24

-

≤20

24

-

≤20

24

D) miscellaneous oximes

k_obs/ min^-1

React_max/%

t / h

1D

0.0092

100

7

60% (0.01 mM)

2-PAM

0.0026

80

24

10% (0.01 mM)

2D

0.0014

90

24

40% (0.01 mM)

3D

0.0009

60

24

50% (0.01 mM)

60% (0.01 mM)

5% (0.01 mM)

10% (0.01 mM)

15% (0.01 mM)

20% (0.01 mM)

4D

0.0007

40

5D

-

≤20

6D

7D

8D

9D

10D

11D

12D

13D

14D

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15D

-

≤20

25

24

20% (0.01 mM)

30% (0.01 mM)

35% (0.01 mM)

40% (0.01 mM)

16D

17D

18D

19D

20D

21D

22D

23D

24D

25D

26D

27D

-

≤20

24

40% (0.01 mM)

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Entry for the Table of Contents

FULL PAPER

We have synthesized and characterized

pharmacologically a substantial number

of N-methylpyridinium and

2-methylimidazolium oximes with

significantly improved in vitro

Zrinka Kovarik, Jarosław Kalisiak,

Nikolina Maček Hrvat, Maja Katalinić,

Tamara Zorbaz, Suzana Žunec, Carol

Green, Zoran Radić, Valery V. Fokin, K.

Barry Sharpless and Palmer Taylor*

reactivating efficacies for tabun-inhibited

AChE. Our findings offer a valuable and

comprehensive platform for further

development of antidotes and

scavengers against tabun and related

phosphoramidate exposures.

Page No. – Page No.

Reversal of tabun toxicity enabled

by a triazole-annulated oxime library

– reactivators of

acetylcholinesterase

Article Doi

Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library—Reactivators of Acetylcholinesterase

DOI: 10.1002/chem.201805051

Source and publish data:

Authors:

Article abstract of DOI:10.1002/chem.201805051

Full text of DOI:10.1002/chem.201805051

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