Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit

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Pyridinium oximes with ortho-positioned chlorine moiety exhibit improved physicochemical properties and efficient reactivation of human acetylcholinesterase inhibited by several nerve agents Tamara Zorbaz, David Malinak, Nikola Marakovic, Nikolina Ma#ek Hrvat, Antonio Zandona, Michal Novotny, Adam Skarka, Rudolf Andrys, Marketa Benkova, Ondrej Soukup, Maja Katalinic, Kamil Kuca, Zrinka Kovarik, and Kamil Musilek J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01398 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Journal of Medicinal Chemistry

Pyridinium oximes with ortho-positioned chlorine moiety exhibit improved physicochemical properties and efficient reactivation of human acetylcholinesterase inhibited by several nerve agents

Tamara Zorbaz,1§ David Malinak,2-3§ Nikola Maraković,1 Nikolina Maček Hrvat,1 Antonio Zandona,1 Michal Novotny,2-3 Adam Skarka,2 Rudolf Andrys,2 Marketa Benkova,3 Ondrej Soukup,3 Maja Katalinić,1 Kamil Kuca,2 Zrinka Kovarik,1* and Kamil Musilek2-3*

1

Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000 Zagreb,

Croatia 2

University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62,

50003 Hradec Kralove, Czech Republic 3

University Hospital in Hradec Kralove, Biomedical Research Center, Sokolska 581, 50005 Hradec

Kralove, Czech Republic

§

These authors contributed equally.

*

Corresponding authors.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the pKa value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to nonchlorinated analogues. The nucleophilicity was examined and the pKa was found to be lower than that of analogous nonchlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the dichlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was respectively, three-, seven-, and eight-fold higher than by K027. Its universality, PAMPA permeability, favorable acid dissociation constant coupled with its negligible cytotoxic effect and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an organophosphorus antidote.

Key words: organophosphorus compound, cholinesterase, antidote, pralidoxime, asoxime, chlorinated pyridinium oxime

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INTRODUCTION Acute organophosphorus (OP; i.e. nerve agents and some pesticides) toxicity derives from inhibition of the pivotal enzyme acetylcholinesterase (AChE; EC 3.1.1.7) leading to cholinergic crises.1,2 However, the diverse chemical structures of OPs induce different physicochemical, toxicokinetic, and toxicodynamic properties which all influence the course of poisoning and treatment as well. Research into reactivators of acetylcholinesterase, undertaken since the 1950s,3 has produced an enormous number of reactivators. In the last couple of decades, K-oximes, especially K027, K048, and K203 (Figure 1), have been shown to be potent reactivators of AChE inhibited with various nerve agents and OP pesticides4–11, which reflects their universality in reactivation independent of the phosphoryl moiety attached to the active site serine, and according to both in vitro and in vivo results11–14 particularly significant improvement in reactivation of the tabun-AChE conjugate was achieved when compared to standard oximes used in practice (e.g. pralidoxime, trimedoxime or asoxime; Figure 1). Moreover, K027, K048 and K203 oximes showed low cytotoxic potential in different cell lines (fibroblasts, and hepatic, kidney, blood, and ovary cells),11,15 and K048 showed no significant cytotoxic or genotoxic potential.16 Additionally, their acute toxicity in mice was lower than for trimedoxime (LD50: K048>K027>K203).11,12 However, those oximes still lack the propensity for high penetration through the blood-brain barrier (BBB).17–19 Lipophilicity is an important property for centrally active drugs and fluorination of the oximes has been introduced in the past in an attempt to increase the lipophilic character of the compounds.20,21 Indeed, it was observed that the permeability of fluorinated oximes in parallel artificial membrane permeation assay (PAMPA) increased proportionately to the increase in the number of fluorine atoms in their structure.21 Moreover, the fluorinated analogue of oxime K203 showed slightly higher neuroprotection when compared to K203, but did not show higher reactivation of brain AChE, which suggests that it may have other pharmacological effects in the central nervous system (CNS).22

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O

O

NH2

NH2 N HON

N

N HON

O

2 Br K027 (1)

N

N HON

2 Br

NH2

N 2 Br K203 (3)

K048 (2) NOH

N

N

NOH

HON

X

N

N

NOH

2X

O

N O

2X

NH2 pralidoxime (2-PAM; 4, X = Cl, CH3SO3)

trimedoxime (TMB-4; 5, X = Cl)

asoxime (HI-6; 6, X = Cl, CH3SO3)

Figure 1. Structure of selected K-oximes and commercially available oxime reactivators.

To allow brain reactivation of OP intoxication, uncharged reactivators have been introduced and tested.23,24 The design of the potent uncharged molecules originated from AChE inhibitors that were coupled with the novel 3-hydroxy-2-pyridinealdoxime scaffold as an efficient nucleophile.25–27 Some uncharged amidine-oximes, 2-hydroxyiminoacetamides and 2-hydroxyiminoimidazoles were also investigated (Figure 2).28,29 Although efficient reactivation among uncharged molecules was confirmed for various OPs in vitro, in vivo data are rather limited30,31 or not available to date. The major advantage of an uncharged reactivator should be the enhanced penetrability through BBB and thus efficient reactivation of brain AChE.32 However, the increased bioavailability and barrier penetration could become a disadvantage considering the possible toxicity of oximes and/or oxime-OP conjugates (data are not available), various adverse effects on brain neurotransmitter cascades (off-target or pharmacodynamics data are not available), high plasma protein binding, or slow body elimination (pharmacokinetics data are not available).33 A substantial drawback might be limited brain bioavailability or limited solubility of published uncharged reactivators, as was recently confirmed for some lead candidates.31,34 HON HO HON

N N

R n

3-hydroxy-2-pyridinealdoximes

R

N

N NOH

N

NOH

R NOH

H N

R1

O 2-hydroximinoacetamides

amidine-oximes

Figure 2. Structure of selected uncharged oxime reactivators.

4


N

N R

2-hydroximinoimidazoles

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Herein are described the design, preparation, and evaluation of novel chlorinated double-charged monooxime reactivators (Chart 1). The novel reactivators were designed to keep the structural characteristics of their potent reference compounds, but with increased lipophilicity that would possibly increase their penetration through BBB and result in higher brain concentrations than achieved with the reference compounds. For biological characterization, we firstly determined the affinity of human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) for these chlorinated oximes. Additionally, their reactivation ability was evaluated for AChE inhibited by several nerve agents (sarin, cyclosarin, VX, and tabun). In order to determine the proportion of oxime molecules in oximate form at physiological pH, we determined the pKa values of the oxime moiety. Using molecular docking, the observed differences in reactivation potency between nonchlorinated and mono- and dichlorinated oxime analogues for two OP-enzyme conjugates, (cyclosarin- and tabun- inhibited mouse AChE) were rationalized. Further, the cytotoxic profile of nonchlorinated and chlorinated analogues was tested on the neuroblastoma-derived cell line SH-SY5Y, since chlorination of organic compounds could also contribute to their toxic effects in the cell.35 Finally, ex vivo experiments were carried out in order to test the ability of the selected most potent oxime for degradation of sarin, cyclosarin, and VX in whole human blood.

novel AChE reactivators

R1 H

HO

N

K027 (1) K048 (2) K203 (3)

N R2

R1

o-chlorinated oxime containing moiety (reactivation in catalytic site)

3´

N

Y

HON

N O

5´

R2

2 Br

carboxamide containing moiety (hydrogen bonding in peripheral site)

NH2

R1 Cl, R2 H, Y: 7 (CH2), 8 (CH2CH2), 9 ((E)-CH=CH) R1 Cl, R2 Cl, Y: 10 (CH2), 11 (CH2CH2), 12 ((E)-CH=CH)

Chart 1. Design of double-charged acetylcholinesterase reactivators with chlorine atoms.

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Molecular design The bisquaternary scaffold was chosen for its valuable properties that were confirmed both in vitro and in vivo, and the novel bisquaternary molecules were related to promising K-oximes (1-3). Previously the pKa of these mono-oxime molecules had been experimentally determined and their oximate forming properties quantified to be ~7.87-8.09, whereas the pKa of the very potent charged reactivator asoxime (HI-6) is about 7.42.36 Thus, one or two chlorine atoms were introduced as electron withdrawing group/s into the pyridinium ring carrying the oxime moiety (7-12) to decrease the pKa of the novel mono-oximes and increase the possibility of forming an oximate as the functional nucleophile. An ortho-positioned chlorine moiety was chosen as a relatively small electron-withdrawing group rather than the more bulky bromine that was assumed to be spatially inconvenient, or the previously-used fluorine derived reactivators which presented only limited reactivation ability.21,37 The linkages between oxime and amide pyridinium rings were kept the same as in the parent molecules 1-3.

RESULTS AND DISCUSSION Synthesis The reactivators were prepared via two step synthesis (Scheme 1). The mono- or dichlorinated pyridine aldoximes (13, 14) were prepared from the corresponding aldehydes using hydroxylamine hydrochloride in good to quantitative yields.38 The halogenated aldoximes were then treated with monoquaternary pyridinium amides (15-17), which were prepared as previously described39, resulting in the final mono- or dichlorinated pyridinium oximes (7-12). The final step took at least 12 hours for compounds with a double bond in the linker (9, 12) and several days for compounds with a fullysaturated linker (7-8, 10-11). The yields were found to be better for compounds with an unsaturated linker (9, 12), whereas compounds with a saturated linkage (7-8, 10-11) were obtained in generally lower yields, especially when two chlorine moieties were present. The low reactivity is related to the chlorine atoms which reduce the electron density on pyridine nitrogen and thus they decrease the yield during the SN2 reaction.

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Br R1

N

O H

R2

a)

N O

Br

R1 NH2OH.HCl

Y

N

HON

15-17

R1

NH2

b) or c) R

3

N

Y

N

HON

2

R

13 (R1 Cl, R2 H) 14 (R1 Cl, R2 Cl)

1

5 2

O 2 Br

NH2

2

R Cl, R H, Y: 7 (CH2), 8 (CH2CH2), 9 ((E)-CH=CH) R1 Cl, R2 Cl, Y: 10 (CH2,), 11 (CH2CH2), 12 ((E)-CH=CH)

Scheme 1. Synthesis of double-charged acetylcholinesterase reactivators (7-12) and their precursors (13-17). Conditions and reagents: (a) pyridine, ethanol, reflux, 5 h; (b) MeCN, reflux, 7 days; (c) DMF, 60°C, 12 h.

pKa determination Absorbance spectra of chlorinated oximes were monitored in solutions of pH from 4.6-11.1 and representatives are shown in Figure 3. Similar spectra were observed for all oximes tested (Figure S13). The acid dissociation constant, Ka, for the oxime group was determined from the pH-dependent change of the absorbance intensity at the specified wavelength that corresponds to the oximate form.40 It was observed that the wavelength of the absorption maximum of the oximate increased with the number of chlorine atoms (10-12 nm per Cl atom), where K027 without chlorine atoms had maximum at 344 nm (Figure S13). Chlorinated oximes generally showed a decrease of the oxime group’s pKa value (>0.7) when compared to nonchlorinated analogues. Therefore, it is expected that ~35-40% of the total concentration of chlorinated oximes are in the oximate form at the physiological pH. For comparison, reference compounds K027, K048, and K203 are expected to form ~10% of the oximate form at the physiological pH. Results obtained for K027 and asoxime are in good agreement with previously published pKa values determined by the same method (8.42 for K027 and 7.47 for asoxime).41

Table 1. Experimental pKa values of the oxime group and oximate percentage at pH 7.4. Oxime

pH range

pKa

Oximate (%)

K027 (1)

4.6-11.1

8.32

10

K048 (2)

6.0-9.5

8.44

10

K203a (3)

6.0-9.0

8.33

10

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asoxime (6)

4.6-9.2

7.46

50

7

4.6-11.1

7.57

40

8

4.6-10.3

7.68

35

9

4.6-9.2

7.60

40

10

4.6-10.3

7.53

45

11

4.6-10.3

7.66

35

12

4.6-9.2

7.55

40

afrom

ref.42

Figure 3. Absorption spectra of oximes 7 and 10 (100 µM) at different pH values (4.6-11.1).

Reversible inhibition of AChE and BChE Reversible inhibition of recombinant human AChE and purified human plasma BChE with oximes was described by the dissociation constants of the enzyme-oxime complex, Ki, obtained from measurements of enzyme activity in the presence of oximes (Table 2). Both enzymes exhibited a moderate affinity (i.e., 1/Ki) for binding of all oximes with a 4-12-fold higher affinity of AChE than BChE. The affinity of AChE for chlorinated oximes was comparable to the affinity for asoxime, and higher than for analogous oximes and pralidoxime. Additionally, both AChE and BChE showed a higher affinity for dichlorinated than for monochlorinated oximes. This confirms that the additional chlorine atom interacts with active site residues and it stabilizes the enzyme-oxime complex.

8


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Table 2. Reversible inhibition of human AChE and BChE with pyridinium oximes. Ki (μM) a

Oxime

Ki (BChE)/Ki (AChE)

AChE

BChE

K027 (1)

73 ± 10 b

660 ± 110 d

9.0

K048 (2)

110 ± 10 b

420 ± 60 d

3.8

K203 (3)

90 ± 12 c

910 ± 16 c

10.1

pralidoxime (4)

180 ± 10

390 ± 48

2.2

asoxime (6)

25 ± 1.4

215 ± 11

9.1

7

42 ± 4

273 ± 42

6.5

8

36 ± 5

265 ± 52

7.4

9

24 ± 3

294 ± 41

12.3

10

21 ± 2

195 ± 18

9.3

11

12 ± 2

140 ± 12

11.7

12

22 ± 2

187 ± 9

8.5

adissociation

inhibition constant; bfrom ref.11; cfrom ref.12; dfrom ref.43

Oxime-assisted reactivation of AChE inhibited with nerve agents The reactivation of phosphylated AChE by sarin, cyclosarin, VX, and tabun was evaluated over a wide oxime concentration range enabling determination of relevant reactivation constants. For several combinations of oxime and OP-AChE conjugate, reactivation curves showed a biphasic response with an initial burst (Table S1) as presented in Figures 4 and S15 implying a phenomenon reported elsewhere,44,45 but reactivation constants were calculated from one-phase fit for an easier comparison with other oximes. It is also interesting to note that an unusual biphasic dependence of kobs on oxime concentration (Figure 4B; different from usual Michaelis-Menten-type model shown in Figure 4D) was obtained only in the case of K027-assisted reactivation of sarin-inhibited AChE. The reactivation

9



appeared to be accelerated in a relatively narrow concentration range of K027, and this should be studied in the future. The sarin-inhibited AChE activity was promptly restored with all tested oximes, achieving 80-100% of reactivation. Reactivation parameters given in Table 1 showed that the sarin-inhibited AChE had more than 10 times lower affinity for non- and monochlorinated oximes than for their dichlorinated analogue. Thus, the most potent reactivator with the overall reactivation rate higher than that of asoxime was compound 10, due to having the lowest dissociation constant of inhibited enzyme-oxime complex KOX (i.e., high affinity of sarin-AChE conjugate for oxime 10), accompanied by high maximal reactivation rate k+2.

Figure 4. Reactivation of sarin-inhibited AChE with K027 and oxime 8 fitted with one phase (full line) and two-phase exponential association (dotted line) (A and C), and dependence of kobs and oxime concentration (B and D).

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Table 3. Reactivation of sarin-inhibited human AChE with pyridinium oximes at 25°C. a Oxime

k+2 (min-1)

KOX (μM)

kr (M-1 min-1)

Reactmax (%)

t (min)

K027 (1)

1.2 ± 0.06

320 ± 20

3640 ± 270

80

20

K048 (2)

1.2 ± 0.3

510 ± 250

2340 ± 640

100

300

K203 (3)

1.5 ± 0.3

650 ± 290

2250 ± 520

100

300

pralidoxime (4)

0.16 ± 0.01

190 ± 30

850 ± 110

85

40

asoxime (6)b

0.9 ± 0.1

170 ± 40

5420 ± 780

90

4

7

0.37 ± 0.14

745 ± 425

490 ± 100

80

90

8

0.38 ± 0.16

1150 ± 640

330 ± 50

80

90

9

0.35 ± 0.06

840 ± 195

420 ± 30

80

60

10

0.43 ± 0.04

35 ± 10

12000 ± 2500

80

10

11

0.20 ± 0.01

50 ± 10

3880 ± 430

80

30

12

0.17 ± 0.02

60 ± 10

2830 ± 390

80

30

ak , +2

maximal first-order reactivation rate; KOX, dissociation constant of conjugated enzyme-oxime

complex; kr, overall second-order reactivation rate; Reactmax, maximal percentage of reactivation achieved in time, t; bfrom ref.27

The cyclosarin-inhibited AChE was reactivated completely with all tested oximes and the evaluated reactivation parameters are given in Table 4. Although all tested oximes (except K048, K203 and pralidoxime) efficiently reactivated the cyclosarin-AChE conjugate, it is interesting to note that all monochlorinated oximes had four to tenfold lower reactivation rate than their dichlorinated analogues, while their dissociation constants were similar. The most potent reactivator of cyclosarin-inhibited AChE was the dichlorinated oxime 10 due to a supremely high k+2 which resulted in 100% reactivation within a minute. Moreover, achieved efficiency was similar to that of asoxime, known as potent reactivator in the case of cyclosarin.27

Table 4. Reactivation of cyclosarin-inhibited AChE with pyridinium oximes at 25°C. a

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Oxime

k+2 (min-1)

KOX (μM)

kr (M-1 min-1)

Reactmax (%)

t (min)

K027 (1)

1.2 ± 0.06

400 ± 40

2900 ± 180

100

5

K048 (2)

0.07 ± 0.02

1740 ±550

43 ± 4

100

240

K203 (3)

0.08 ± 0.02

740 ± 270

110 ± 20

100

240

pralidoxime (4)

0.05 ± 0.002

1178 ± 138

43 ± 3

100

120

asoxime (6)b

-

-

22100 ± 1300

100

Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit

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