Peripheral site acetylcholinesterase inhibitors targeting both inflammation and cholinergic dysfunction

Article abstract of DOI:10.1016/j.bmcl.2010.02.102

The design and study of two classes of noncompetitive acetylcholinesterase inhibitors (AChEIs) which also function as NSAID prodrugs are reported. The most potent AChEIs disclosed contain an aromatic alkyl-aryl linker between an NSAID and a lipophilic choline mimic and they inhibit acetylcholinesterase (AChE) in the submicromolar range. These agents have the therapeutic potential to dually target inflammation by releasing an NSAID in vivo and activating the cholinergic anti-inflammatory pathway via cholinergic up-regulation.

Full text of DOI:10.1016/j.bmcl.2010.02.102

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2987–2990
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peripheral site acetylcholinesterase inhibitors targeting both inflammation
and cholinergic dysfunction
Sherri Young a, Karine Fabio a, Christophe Guillon a, Pramod Mohanta a, , Timothy A. Halton a,à
,
Diane E. Heck b, Robert A. Flowers II a, Jeffrey D. Laskin c, Ned D. Heindel a,
*
a Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA
b Department of Environmental Health Science, New York Medical College School of Public Health, Valhalla, NY 10595, USA
c CounterACT Center of Excellence, Rutgers University/UMDNJ, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and study of two classes of noncompetitive acetylcholinesterase inhibitors (AChEIs) which
also function as NSAID prodrugs are reported. The most potent AChEIs disclosed contain an aromatic
alkyl-aryl linker between an NSAID and a lipophilic choline mimic and they inhibit acetylcholinesterase
(AChE) in the submicromolar range. These agents have the therapeutic potential to dually target inflam-
mation by releasing an NSAID in vivo and activating the cholinergic anti-inflammatory pathway via cho-
linergic up-regulation.
Received 7 January 2010
Accepted 26 February 2010
Available online 3 March 2010
Keywords:
NSAIDs
Acetylcholinesterase inhibitors
Prodrugs
Ó 2010 Elsevier Ltd. All rights reserved.
Noncompetitive inhibitor
Pro-inflammatory cytokine up-regulation plays a role in the
pathogenesis of a wide range of disorders including osteoarthritis,1
psoriasis,2 multiple sclerosis,3 and other autoimmune disorders.4
Despite decades of research, non-steroidal anti-inflammatory
drugs (NSAIDs) are still one of the most commonly used, highly
effective treatments for such disorders. However, since chronic
NSAID-use often leads to gastrointestinal (GI) side effects, NSAID
ester prodrugs have been explored to mask the acidic GI-irritating
portion of the NSAID.5 For conditions such as arthritis, topical
NSAID prodrugs are particularly useful as their therapeutic action
is localized, resulting in minimal systemic side effects.6
NSAID-AChEI conjugates have recently been explored in order
to target neuroinflammation16 and vesicant-induced inflamma-
tion.17 These novel agents contain the AChEI, pyridostimine, linked
Alternately, acetylcholinesterase inhibitors (AChEIs) have impli-
cations in the treatment of severe inflammation resulting from
sepsis,7 endotoxemia,8 and rheumatoid arthritis,9 as well as in
the treatment of neuroinflammation associated with Alzheimer’s
disease10–12 and Myasthenia Gravis.13 The administration of CNS-
active AChEIs such as galanthamine depletes systemic pro-inflam-
matory cytokines and ameliorates both central and peripheral
inflammation.8 AChEIs seem to suppress inflammation via the cho-
linergic anti-inflammatory pathway, a mechanism by which the
vagus nerve of the CNS regulates the production and release of tu-
mor necrosis factor and other cytokines.14,15
Class 1 (Ester-Carbonate Series)
Class 2 (Ester Series)
Compound
NSAID
Ibuprofen
X
C
Compound
NSAID
Ibuprofen
X
C
Si
N+
C
Si
N+
C
Si
N+
C
1
2
3
4
5
6
7
8
9
Ibuprofen
Naproxen
Si
C
10
11
12
13
14
15
16
17
18
19
Ibuprofen
Ibuprofen
Naproxen
Naproxen
Naproxen
Si
C
Si
C
Indomethacin
Indomethacin
Diclofenac
Diclofenac
Naproxen
Indomethacin
Indomethacin
Indomethacin
Diclofenac
Diclofenac
Si
* Corresponding author. Tel.: +1 610 758 3464; fax: +1 610 758 3461.
E-mail address: ndh0@lehigh.edu (N.D. Heindel).
Si
Present address: PTC Therapeutics, Inc., South Plainfield, NJ 07080-2449, USA.
Present address: Digestive Care, Inc., Bethlehem, PA 18017-7059, USA.
à
Figure 1. Generic structures of Class 1 (top) and Class 2 (bottom) agents.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.102

2988
S. Young et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2987–2990
Figure 2. Structure of 7, the most potent AChEI discussed herein (IC50 = 0.51 lM).
Class 1 compounds were obtained by coupling either 3,3-dim-
ethylbutyl carbonochloridate (Ia)20 or 2-(trimethylsilyl)ethyl carbo-
nochloridate (Ib)21 to the phenolic function of p-hydroxybenzyl
alcohol. Coupling of II to an NSAID activated with 4-dimethylamino-
pyridine (DMAP) completed the synthesis (Scheme 1). Overall yields
of this procedure ranged from 61% to 76%, including synthesis of the
chloroformate starting materials.
Scheme 1. Synthesis of Class 1 agents. Reagents and conditions: (a) Et3N, THF, À10
to 25 °C; (b) NSAID, EDC HCl, DMAP, CH2Cl2, 0–25 °C.
via a hydrocarbon spacer to ibuprofen. When screened against sul-
fur mustard, the bifunctional agents demonstrated longer and
more effective prevention of edema and inflammation than the
AChEI alone with a post-treatment anti-inflammatory effect com-
parable to that of the parent NSAID.17 Furthermore, the same
conjugates down-regulated nitric oxide and prostaglandin E2 pro-
duction and ameliorated autoimmune encephalomyelitis more
than either component alone.16 NSAID-AChEIs seem to prove
dually effective against inflammatory disorders by activating the
cholinergic anti-inflammatory pathway and inhibiting cyclooxy-
genase. Based on previous findings, two novel classes of noncom-
petitive AChEIs containing common NSAIDs linked via an ester or
an aromatic ester–carbonate backbone to choline bioisosteres have
been developed (Fig. 1).18 The synthesis and evaluation of these
compounds as anti-inflammatory and anticholinesterase agents
for topical or oral administration are presented herein.
rClass 1 molecules have recently been designed as both up-
regulators of choline and NSAID prodrugs.18 The cholinergic
functions (–CO–O–CH2CH2X(CH3)3; X = C, Si, N+) are known bio-
isosteres of choline which inhibit AChE in both a competitive
(as with the silicon derivative) and noncompetitive (as with
the carbon derivative) manner.19 It is proposed that AChE binds
to the choline mimic and activates the cholinergic anti-inflam-
matory pathway. Additionally, esterases in the plasma, skin
and lipid membranes release the NSAID, thus targeting dual
mechanisms of inflammation. Molecules containing NSAIDs di-
rectly linked to choline mimics via an ester bond (Fig. 1, Class
2) have been included both as a control class as well as an addi-
tional class for therapeutic exploration.
In the simple-ester series (Fig. 1, Class 2), standard coupling con-
ditions [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDCÁHCl)-DMAP] were employed for the carbon and
silicon derivatives in a one step, moderate- to high-yielding proce-
dure (55–97%). Synthesis of the nitrogen-based NSAID-choline es-
ters of Class 2 (11, 14, and 17) has been previously described by
Young and co-workers22 and will not be discussed further. Using
Young’s method, products were isolated in moderate yields over
three steps (30–40%). All Class 2 agents were purified using non-
chromatographic methods such as crystallization and extraction.
Following synthesis and characterization, all compounds were
evaluated for anticholinesterase activity using Ellman’s method23
with tacrine,24 one of the most potent commercial AChEIs, as a ref-
erence (Table 1). AChE contains a 20 Å deep gorge stretching from
the enzyme surface to the active-site serine residue.25 This struc-
tural moiety is often termed the aromatic or hydrophobic gorge
since it is lined with Trp, Tyr and Phe residues.25 A peripheral bind-
ing site termed the peripheral anionic site (PAS) is located at the
top of the gorge, near the surface of the enzyme. Keeping in mind
the nature of AChE, it was predicted that the highly lipophilic and
aromatic Class 1 and 2 agents would interact with the Trp, Tyr and
Phe residues at and near the PAS and inhibit enzyme activity.
Listed in Table 1 are IC50 values for each compound against
AChE. As a control, the parent NSAIDs were also screened for activ-
ity and were deemed inactive compared to most of the test com-
pounds (IC50 >1000 lM, data not shown). A direct comparison of
Class 1 and 2 activities reveals that Class 1 compounds are more
potent than Class 2 by at least a factor of 4, in all cases. The most
potent Class 1 inhibitor (7, Fig. 2) has an IC50 comparable to that
Table 1
Anticholinesterase activities and Clog P values of Class 1 and 2 agents
Class 1 (ester–carbonate series)
Class 2 (ester series)
a
a
Compound
IC50
(lM)
% AChE recoveryb
101
88 11
Clog Pc
Compound
IC50
(
lM)
% AChE recoveryb
Clog Pc
1
2
3
4
5
6
7
8
1.93 0.64
1.19 0.20
1.74 1.01
0.83 0.15
2.29 0.94
0.72 0.13
0.51 0.02
1.36 0.13
8
7.37
7.53
6.50
6.67
7.87
8.03
8.41
8.58
9
24.6 14.5
25.7 4.9
>2000
19.7 1.7
13.9 0.3
>1000
9.75 0.89
3.32 0.36
230 32
2.69 0.15
2.66 0.25
107 17
92 11
6.47
6.64
0.42
5.61
5.77
À0.45
6.97
7.14
0.92
7.52
7.68
10
11
12
13
14
15
16
17
18
19
99
98
78
86
4
3
2
1
99
117 13
100
93 13
5
1
87 11
104 23
80
73
101
99
2
4
6
4
Tacrine HCl
0.055 0.005
95
90 10
a
Source of AChE is electric eel (Electrophorus electricus). Each condition was run in duplicate.
b
EeAChE was incubated for 30 min with 55
lM inhibitor at 25 °C. Enzyme activity was assayed following gel filtration. Values reported are averages of at least two
experiments.
c
Values calculated using ChemBioDraw Ultra 11.0.

S. Young et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2987–2990
2989
Table 2
Hydrolysis in acetate buffer was also determined at the pH on
the surface of the skin (pH 5). No significant hydrolysis was ob-
served for up to seven days under acidic conditions, suggesting
that the ester and ester–carbonate linkages should be resistant to
cleavage on the surface of the skin. Preliminary hydrolysis data
suggest that Class 1 and 2 agents will act as NSAID prodrugs in
vivo. Class 1 compounds demonstrate a more gradual NSAID-re-
lease when compared to Class 2. Additionally, they are more lipo-
philic than their Class 2 counterparts, making them more suitable
as topical prodrugs.
Novel AChEIs have been developed with the potential to dually
target inflammation by activating the cholinergic anti-inflamma-
tory pathway and releasing an NSAID, in vivo. Lineweaver–Burk
plots indicate that all test compounds are reversible, noncompeti-
tive inhibitors of AChE. The most potent inhibitors (Class 1) contain
a lipophilic aromatic spacer and are four times more active than
those with solely an ester linkage between the anticholinergic
and anti-inflammatory functionalities (Class 2). Of the compounds
screened, diclofenac derivative 7 was most active likely due to its
high lipophilicity, aromaticity and the chlorine-substituted
ring.26,27 Hydrolysis data suggest that these agents should be effec-
tive as either oral or topical NSAID prodrugs.
Hydrolysis of Class 1 and 2 compounds in human plasmaa
Class 1 (ester–carbonate series)
Class 2 (ester series)
b
b
Compound
t1/2 (min)
Compound
t1/2 (min)
1
3
4
5
7
204
135
253
468
357
12
13
14
18
38
63
<5
111
a
Hydrolysis measured in 80% human plasma in PBS at 37 °C.
Half-lives determined by plotting the semi-log of prodrug disappearance.
b
of the AChEI, galanthamine, a natural alkaloid which has been ap-
proved for the treatment of Alzheimer’s disease.24 Since Class 2
compounds were found less active than Class 1, it is likely that
the lipophilic aromatic linker is needed to maximize potential
p–
p
stacking interactions26 with the Trp, Tyr and Phe residues of
the aromatic gorge.25 The ester–carbonates (Class 1) are also at
least ten times more lipophilic than the corresponding simple-es-
ters, which may also increase hydrophobic interactions with AChE
(refer to Table 1 for Clog P values). The more hydrophilic nitrogen
derivatives of Class 2 (11, 14, and 17) were at least 20 times less
potent than their carbon and silicon derivatives, further enhancing
the argument that hydrophobic interactions at and near the active-
site gorge may be responsible for the observed activities.
Acknowledgments
This work was funded in part by the National Institutes of
Health CounterACT Program through the National Institute of
Arthritis and Musculoskeletal and Skin Diseases (award
#U54AR055073). Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
federal government. The authors would like to thank Professors
Sam Niedbala and Jebrell Glover for use of their instrumentation.
Overall, the most active AChEIs in both classes (7, 18, and 19)
are all derivatives of the NSAID, diclofenac. This trend is likely
due to the high lipophilicity of diclofenac which strengthens inter-
actions with the hydrophobic residues of AChE. Furthermore, the
chlorine substituents on one of the aromatic rings of diclofenac
may enhance p–p stacking interactions with aromatic residues lin-
ing the gorge (likely Trp84, Phe330, or Trp279 in Torpedo californica
AChE), as suggested by previous SAR and computational studies
performed by Holzgrabe and co-workers26 and Sussman and co-
workers.27
Supplementary data
Supplementary data (synthetic procedures, physical character-
ization of compounds, in vitro assay methods and Lineweaver-Burk
plots) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2010.02.102.
The degree of reversibility of AChE-inhibitor interactions was
investigated for all inhibitors. Following an incubation period of
AChE with the test agents, gel filtration resulted in near-complete
restoration of enzyme activity (80–100%, see Table 1). It was
important to demonstrate the reversible nature of our compounds
because many of the known irreversible AChEIs are nerve agents,
toxins, or pesticides and are not considered for therapeutic use.28
The mechanism of AChE inhibition was further investigated by
generating Lineweaver-Burk plots.29 Briefly, the reciprocal of the
reaction velocity (mÀ1) was plotted against the reciprocal of the
substrate concentration ([acetylthiocholine, ATCh]À1) after assay-
ing enzyme activity for varying inhibitor and ATCh concentrations.
All compounds exhibited reversible, noncompetitive inhibition,
with quadrant III interceptions (for detailed assay method and
example Lineweaver–Burk plots, see Supplementary data). These
analyses support the reversibility studies previously discussed.
More in depth investigations which address whether compounds
1–19 are interacting with the PAS of AChE or an alternate site
are ongoing.
References and notes
1. Özgüney, I. Expert Opin. Pharmacother. 2008, 9, 1805.
2. Deeva, I.; Mariani, S.; De Luca, C.; Pacifico, V.; Leoni, L.; Raskovic, D.; Kharaeva,
Z.; Korkina, L.; Pastore, S. Wide-spectrum profile of inflammatory mediators in
the plasma and scales of patients with psoriatic disease. Cytokine 2009,
doi:10.1016/j.cyto.2009.09.014.
3. Centonze, D.; Muzio, L.; Rossi, S.; Furlan, R.; Bernardi, G.; Martino, G. Cell Death
Diff.. 2009, doi:10.1038/cdd.2009.179.
4. Kunz, M.; Ibrahim, S. Mediators Inflamm. 2009, 2009, 1.
5. Cannon, J. Concepts of Pharmacology. Pharmacology for Chemists, 2nd ed.; Oxford
University Press: New York, 2007; pp 13–17.
6. Trommer, H.; Neubert, R. Skin Pharmacol. Physiol. 2006, 19, 106.
7. Fodale, V.; Letterio, B. Crit. Care Med. 2008, 36, 622.
8. Pavlov, V.; Parrish, W.; Rosas-Ballina, M.; Ochani, M.; Puerta, M.; Ochani, K.;
Chavan, S.; Al-Abed, Y.; Tracey, K. Brain Behav. Immun. 2009, 23, 41.
9. van Maanen, M.; Vervoordeldonk, M.; Tak, P. Nat. Rev. Rheumatol. 2009, 5, 229.
10. Tsuno, N. Expert Rev. Neurother. 2009, 9, 591.
11. Guay, D. Consult. Pharm. 2008, 23, 598.
12. Marco, L.; do Carmo Carreiras, M. Recent Pat. CNS Drug Discovery 2006, 1, 105.
13. Brenner, T.; Nizri, E.; Irony-Tur-Sinai, M.; Hamra-Amitay, Y.; Wirguin, I. J.
Neuroimmunol. 2008, 201-202, 121.
14. Tracey, K. Nature 2002, 420, 853.
15. Tracey, K. J. Clin. Invest. 2007, 117, 289.
16. Nizri, E.; Adani, R.; Meshulam, H.; Amitai, G.; Brenner, T. Neurosci. Lett. 2005,
376, 46.
17. Amitai, G.; Adani, R.; Fishbein, E.; Meshulam, H.; Laish, I.; Dachir, S. J. Appl.
Toxicol. 2006, 26, 81.
18. Laskin, J. D.; Fabio, K.; Lacey, C. J.; Young, S.; Mohanta, P.; Guillon, C.; Heindel,
N. D.; Huang, M.-T.; Heck, D. E. Unique Dual-Action Therapeutics, U. S. Patent
Pending, PCT/US2009/005961, filed November 3, 2009.
19. Cohen, S.; Chishti, S.; Elkind, J.; Reese, H.; Cohen, J. J. Med. Chem. 1985, 28, 1309.
20. Hudson, H.; Koplick, A.; Poulton, D. J. Chem. Soc., Perkin Trans. 2 1979, 57.
To evaluate Class 1 and 2 agents as oral or topical prodrugs,
hydrolytic release of the NSAID was followed by HPLC under vari-
ous conditions. Gradual release of the parent NSAID was observed
for both Class 1 and 2 agents in human plasma (80% plasma in
PBS), with half-lives not exceeding 8 h (Table 2). Negligible hydro-
lysis was observed in the PBS control during the time of the half-
life determination. The ester–carbonate prodrugs released the
parent drug more slowly (t1/2 of 2–8 h) compared with the ester
prodrugs (t1/2 of 5–100 min). Compound 14 was completely hydro-
lyzed after 10 min, suggesting that the choline esters (11, 14, and
17) would not make suitable prodrugs.

2990
S. Young et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2987–2990
21. Berglund, A.; Akerblom, E.; Hedin, A. EU Patent 0,209,586, 1986.
22. Venuti, M.; Young, J.; Maloney, P.; Johnson, D.; McGreevy, K. Pharm. Res. 1989,
6, 867.
27. Dvir, H.; Wond, D.; Harel, M.; Barril, X.; Orozco, M.; Luque, F.; Muñoz-Torrero,
D.; Camps, P.; Rosenberry, T.; Silman, I.; Sussman, J. Biochemistry 2002, 41,
2970.
23. Ellman, G.; Courtney, K.; Andres, V.; Feather-Stone, R. Biochem. Phamacol. 1961, 7, 88.
24. Guillou, C.; Mary, A.; Zafiarisoa Renko, D.; Gras, E.; Thal, C. Bioorg. Med. Chem.
Lett. 2000, 10, 637.
28. Taylor, P. In The Pharmacological Basis of Therapeutics; Hardman, J., Limbird, L.,
Molinoff, P., Ruddon, R., Gilman, A., Eds.; McGraw-Hill: New York, 1996; pp
161–176.
25. Shen, T.; Tai, K.; Henchman, R.; McCammon, A. Acc. Chem. Res. 2002, 35, 332.
26. Kapkova, P.; Stiefl, N.; Sürig, U.; Engels, B.; Baumann, K.; Holzgrabe, U. Arch.
Pharm. Pharmacol. Med. Chem. 2003, 336, 523.
29. (a) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658; (b) Bergmeyer, H.;
Gawehn, K. ‘Principles of Enzymatic Analysis’; Verlag Chemie: NY, 1978. pp 36–
40.