Long-acting anticholinesterases for myasthenia gravis: Synthesis and activities of quaternary phenylcarbamates of neostigmine, pyridostigmine and physostigmine

Article abstract of DOI:10.1016/j.bmc.2010.05.022

The N-monophenylcarbamate analogues of neostigmine methyl sulfate (6) and pyridostigmine bromide (8) together with their precursors (5), (7), and the N(1)-methylammonium analogues of (-)-phenserine (12), (-)-tolserine (14), (-)-cymserine (16) and (-)-phen

Full text of DOI:10.1016/j.bmc.2010.05.022

Bioorganic & Medicinal Chemistry 18 (2010) 4687–4693
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Long-acting anticholinesterases for myasthenia gravis: synthesis and
activities of quaternary phenylcarbamates of neostigmine,
pyridostigmine and physostigmine
Qian-sheng Yu a, Harold W. Holloway a, Weiming Luo b, Debomoy K. Lahiri c,
Arnold Brossi d, Nigel H. Greig a,
*
a Drug Design & Development Section, Laboratory of Neurosciences, Biomedical Research Center, National Institute on Aging, National Institutes of Health,
251 Bayview Blvd, Baltimore, MD 21224, USA
b MedStar Research Institute, Hyattsville, MD 20782, USA
c Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA
d School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The N-monophenylcarbamate analogues of neostigmine methyl sulfate (6) and pyridostigmine bromide
(8) together with their precursors (5), (7), and the N(1)-methylammonium analogues of (ꢀ)-phenserine
(12), (ꢀ)-tolserine (14), (ꢀ)-cymserine (16) and (ꢀ)-phenethylcymserine (18) were synthesized to pro-
duce long-acting peripheral inhibitors of acetylcholinesterase or butyrylcholinesterase. Evaluation of
their cholinesterase inhibition against human enzyme ex vivo demonstrated that, whereas compounds
5–8 possessed only marginal activity, 12, 14, 16 and 18 proved to be potent anticholinesterases. An
extended duration of cholinesterase inhibition was determined in rodent, making them of potential inter-
est as long-acting agents for myasthenia gravis.
Received 17 March 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 12 May 2010
Keywords:
Myasthenia gravis
Pyridostigmine
Published by Elsevier Ltd.
Neostigmine
N-Phenylcarbamate analogues of
(ꢀ)-physostigmine
(ꢀ)-Phenserine
(ꢀ)-Cymserine
Congenital myasthenic syndromes
1. Introduction
fects. A short duration of pharmacodynamic action, usually 30–
120 min,3 allied with rapid metabolism has made the routine clin-
The
compounds
3-[(dimethylamino)carboxyl]oxy]-N,N,N-
ical use of these compounds troublesome.3–5 This has resulted in
necessary multiple dosing (10 doses over 24 h) as well as the
development of slow-release formulations to achieve efficacy.
Recent studies have demonstrated that specific aromatic carba-
mate analogues of (ꢀ)-physostigmine (9), which include the Alz-
heimer’s disease experimental drug (ꢀ)-phenserine (11), whose
history, chemistry and pharmacology have been reviewed,6–9 as
well as (ꢀ)-tolserine (13), (ꢀ)-cymserine (15) and (ꢀ)-phenethyl-
cymserine (17), possess far longer durations of in vivo cholinester-
ase inhibitory action compared to not only their methyl carbamate
analogue, (ꢀ)-physostigmine (9),7–9 but also to neostigmine and
pyridostigmine.3–5,10–12 Phenyl carbamate analogues of neostig-
mine methyl sulfate (6) and pyridostigmine bromide (8) together
with their corresponding precursors (5) and (7), as well as methyl
quaternary analogues of (ꢀ)-phenserine (12), (ꢀ)-tolserine (14),
(ꢀ)-cymserine (16) and (ꢀ)-phenethylcymserine (18) were there-
fore synthesized to assess their potential pharmacological utility.
The assessment of their anticholinesterase activities is reported
trimethylammonium methyl sulfate, better known as neostigmine
methyl sulfate (3),1 and 3-[(dimethylcarbamoyl)oxy]-1-methylpy-
ridinium bromide, pyridostigmine bromide (4)2 (Fig. 1) are well
known peripheral cholinesterase inhibitors that have been exten-
sively used over the last five decades in the treatment of neuro-
muscular junction disorders, epitomized by the autoimmune
disorder myasthenia gravis and non-autoimmune congenital
myasthenic syndromes, and have additionally been used as pro-
phylactics for organophosphate poisoning.3–5 These quaternary
compounds are highly water-soluble, and consequently minimally
cross the blood–brain barrier. Restricted from entering the brain,
they exert potent systemic anticholinesterase activity at skeletal
muscles, in the absence of centrally mediated actions or side ef-
* Corresponding author. Tel.: +1 410 558 8278.
E-mail addresses: Greign@grc.nia.nih.gov, greign@mail.nih.gov, GreigN@vax.grc.
nia.nih.gov (N.H. Greig).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.05.022

4688
Q. Yu et al. / Bioorg. Med. Chem. 18 (2010) 4687–4693
HO
HO
N
N(Me)2
2
1
(Me)2N
O
(Me)2N
O
O
O
N
Me
Br
O
N(Me)3CH3SO4
3
4
H
H
N
N
O
O
O
R
R
7, R=N
8, R=NMeBr
5, R=N(Me3)2
6, R=N(Me)3CH3SO4
H
N
H
N
Me
Me
O
O
Me
R
R
O
O
N
N
Me
Me
11, R=NMe
9, R=NMe
10, R=N(Me)2Br
12, R=N(Me)2Br
H
N
Me
H
N
Me
Me
O
O
R
R
Me
O
O
N
N
Me
Me
Me
15, R=NMe
13, R=NMe
16, R=N(Me)2Br
14, R=N(Me)2Br
H
N
3
3a
2
Me
2'
4
1'
O
3'
4'
1
R
5
8a
Me
O
6'
N
6
8
5'
7
Me
Me
17, R=NCh2CH2Ph
18, R=N(Me)CH2CH2PhBr
Figure 1.
here as a first step in a search for better agents for treatment of
myasthenia gravis that are unencumbered by a short duration of
action.
and methylbromide, separately, to provide the corresponding qua-
ternary salts, 6 and 8, in yields of 80% and 70%. (ꢀ)-Phenserine
(11), (ꢀ)-tolserine (13), (ꢀ)-cymserine (15) and (ꢀ)-phenethyl-
cymserine (17) were obtained according to a known procedure13
and then, together with (ꢀ)-physostigmine (9), were transformed
to quaternary salts 10, 12, 14, 16 and 18 by reaction with meth-
ylbromide, in a manner similar to but different from our prior syn-
thesis of quaternary iodide salts (ꢀ)-phenserine (11).14 In this
manner our synthesis of a range of quaternary (ꢀ)-physostigmine
phenylcarbamate bromide salts could be directly compared to pyr-
idostigmine bromide (4).
2. Results and discussion
2.1. Chemistry
As reported previously,1,2 m-dimethylaminophenol (1) and 3-
hydroxypyridine (2) can be reacted with different N-alkyl or aryl
carbamoyl chlorides to give different N,N-disubstituted carba-
mates. Following the procedure for the synthesis of (ꢀ)-phenserine
(11),13 m-dimethylaminophenol (1) and 3-hydroxypyridine (2)
were reacted with phenylisocyanate to generate N-monophenyl-
substituted carbamates, 5 and 7, in yields of 86% and 70%, respec-
tively. Compounds 5 and 7 then were reacted with methylsulfate
2.2. Cholinesterase inhibition
The activity of compounds 3–18 to inhibit human acetyl-(AChE,
EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) was

Q. Yu et al. / Bioorg. Med. Chem. 18 (2010) 4687–4693
4689
assessed by a modification of the Ellman technique,15,16 in which
the concentration required to inhibit 50% enzymatic action (IC50
value) was determined in half-log steps over a 5 log concentration
range (0.3 nM to 30 lM), and is shown in Table 1. A lower IC50 va-
lue is associated with greater anticholinesterase potency.
The time-dependent cholinesterase inhibition of compound 16,
as a representative of a quaternary (ꢀ)-physostigmine phenylcar-
bamate, was assessed in the anesthetized rat following administra-
tion by the intrapertioneal (ip) route at a dose of 10 mg/kg. As
illustrated in Figure 2, onset of plasma cholinesterase inhibition oc-
curred rapidly, inducing peak inhibition of 66% at 60 min that grad-
ually declined to 33% by 24 h. No central (tremor) or peripheral
(lacrimation and salivation) cholinergically-mediated adverse ef-
fects were evident.
The introduction of a phenylcarbamoyl side chain into the pri-
mary pharmacophore of either neostigmine (3) or pyridostigmine
(4), to provide analogues 6 and 8, resulted in a substantial loss of
anticholinesterase activity. The IC50 value of neostigmine (3) de-
clined by 100- and 300-fold for AChE and BChE (from 18.8 to
1875 nM, and from 60 to 18,000 nM) for 6, respectively. This same
modification in pyridostigmine (4) resulted in entire loss of AChE
activity for 8 but, interestingly and unlike 6, the preservation of
BChE inhibitory action.
In contrast, the quaternization of (ꢀ)-physostigmine (9) and re-
lated phenylcarbamates to provide 10, 12, 14, 16 and 18 with
charge characteristics akin to neostigmine (3) and pyridostigmine
(4), retained or improved AChE inhibitory action. Specifically, for
the N(1)-methylamonium bromides (10, 12, 14) of (ꢀ)-physostig-
mine (9), (ꢀ)-phenserine (11) and (ꢀ)-tolserine (13), the high AChE
potency of the parent compounds was maintained, but the differ-
ential selectivity of 9 and 11 for AChE was lost consequent to im-
proved BChE inhibition versus the parent compounds. In the case
of the BChE-selective inhibitors, (ꢀ)-cymserine (15) and (ꢀ)-phen-
ethylcymserine (17), quaternization resulted in significantly im-
proved AChE inhibitory potency for 16 and 18 that, together with
a loss 10-fold loss of BChE activity for 18, resulted in the loss of
BChE selectivity of these quaternary compounds. In each case,
however, the resulting AChE IC50 values of these quaternary (ꢀ)-
physostigmine phenylcarbamates (10, 12, 14, 16 and 18) compared
favorably to neostigmine (3) and were more potent than pyrido-
stigmine (4). In addition the AChE and BChE IC50 values compared
favorably to those of prior synthesized quaternary (ꢀ)-physostig-
mine phenylcarbamate iodide salts.14
2.3. Discussion
Myasthenia gravis, the most common disorder affecting the
postsynaptic element of the neuromuscular junction, is an autoim-
mune disease. It afflicts up to 2 per 100,000 population, primarily
20–30 year old women and older (70–80 year) men.17 The majority
cases (85%) are associated with an IgG antibody raised against the
postsynaptic nicotinic acetylcholine receptor, reducing the number
of effective receptors to a third of normal levels.3–5 Impaired neu-
romuscular transmission together with diminished generation of
postsynaptic action potentials results in decreased muscle contrac-
tion, leading to the classical clinical features of fluctuating muscle
weakness and fatigability.18 Albeit an autoimmune disorder and
routinely treated by a wide array of immunosuppressive strate-
gies,4 cholinesterase inhibitors remain crucial to symptomatic
treatment, particularly for congenital myasthenic syndromes that
are a group of non-autoimmune disorders caused by mutations
in several proteins that compose the neuromuscular junction.5
Unfortunately, neostigmine methyl sulfate (3) and pyridostigmine
bromide (4), are plagued by the need for frequent dosing, side ef-
fects in the elderly and erratic absorption after oral
administration.19
The inhibitory interaction between carbamate-based cholines-
terase inhibitors, as epitomized by 3, 4 and 9, and the cholinester-
ase enzymes, AChE and BChE, occurs within a narrow gorge of
some 20 Å depth that contains several domains that bind and
cleave ACh,20–22 and provide a rationale for the described modifica-
tions to generate improved inhibitors.23–26 The three-dimensional
structure of the gorge is roughly equivalent to two hemispheres
that sandwich a catalytic center between two loops (the acyl-
and omega-loops) that form the sidewalls of the active site gorge,
with key amino acid differences between AChE and BChE in the
Computation of Log D values of compounds 3, 4, 6, 8, 10, 12, 14,
16 and 18 was undertaken utilizing Advanced Chemistry Develop-
ment, Inc. (ACD/Labs, Toronto, Ontario, Canada) (ACD/PhysChem
Suite version 12.01) and PrologD (CompuDrug, Pallas, Sedona,
AZ) software and is shown in Table 1. This log D value is a measure
of the aqueous versus lipid solubility of a compound, which relates
to its permeability at biological membranes, as determined from
the octanol–water distribution coefficient.
Table 1
50% Inhibitory concentration of compounds versus freshly prepared human erythrocyte AChE and plasma BChE, and computed log D value
No.
Compound
AChE
BChE
Selectivity A/B or B/Aa
log Dd
(IC50, nM, SEM)
3
4
5
6
7
Neostigmine methyl sulfate
Pyridostigmine bromide
18.8 3.0
360 30
2500 130
1875 110
>30,000b
>30,000b
27.9 2.4
26.1 6.2
24.0 6.0
25.4 2.8
10.3 1.6
14.6 1.2
760 21
145 14
>30,000b
300 22
60
2
3.2 AChE
2.5 AChE
7.2 AChE
9.6 AChE
None
BChE
1.7 BChE
5 AChE
65 AChE
8.3 AChE
189 AChE
9.6 AChE
14.9 BChE
3.4 BChE
>5000
ꢀ2.09
ꢀ2.84
3.06
900 100
18,000 3000
18,000 2200
>30,000b
550 200
16.0 2.9
130 15
1560 60.0
210 90
1950 245
140 60
N-Phenylcarbamate of 3-dimethylaminophenol
N-Phenylcarbamate of 3-phenol-1-trimethyl ammonium methylsulfate
N-Phenylcarbamate of 3-hydroxypyridine
N-Phenylcarbamate of 3-hydroxy-1-methyl pyridinium bromide
(ꢀ)-Physostigmine
ꢀ1.4
1.78
8
9
ꢀ2.21
0.42
10
11
12
13
14
15
16
17
18
(ꢀ)-N(1)-Methylammonium bromide of physostigmine
(ꢀ)-Phenserine
ꢀ1.04
2.22
(ꢀ)-N(1)-Methylammonium bromide of phenserine
(ꢀ)-Tolserine
ꢀ0.5
2.66
(ꢀ)-N(1)-Methylammonium bromide of tolserine
(ꢀ)-Cymserine
ꢀ0.17
51 1.0
43 23
6.0 1.0
51 2.6
3.51
(ꢀ)-N(1)-Methylammonium bromide of cymserine
(ꢀ)-Phenethylcymserine
1.0
5.72
2.43
(ꢀ)-N(1)-Methylammonium bromide of phenethylcymserine
5.9 BChE
a
b
c
A/B or B/A: selectivity for AChE or BChE from IC50 values.
None: insufficient activity in the range of 0.3 nM to 30 lM to calculate an IC50 value, and thus considered inactive.
The IC50 data of 9, 11, 13, 15 from Ref. (Brzostowska et al., 1992)13, and 17 from Ref. (Yu et al., 1999).23
d
ACD/PhysChem Suite version 12.01 and PrologD (CompuDrug, Pallas).

4690
Q. Yu et al. / Bioorg. Med. Chem. 18 (2010) 4687–4693
stigmine and (ꢀ)-phenserine (11), possess inhibition T1/2s of 10–
11 h.38,39
One of several factors that may further contribute to the inter-
action between the phenylcarbamate moiety of a carbamate-based
anticholinesterase and the acyl-binding site of AChE is the stability
provided by both hydrophobic and
p electron interactions due to
p–p
stacking of the phenylcarbamate between the flanking phenyl
moieties of two phenylalanines (Phe295 and Phe297) that are pres-
ent on the acyl-loop and form the base of the gorge of human
AChE.16,20,21,24 These key amino acids are not present within BChE,
where the gorge base is formed by the smaller aliphatic ones
Leu286 and Val288.
20,28 This allows the accommodation within BChE
of larger substrates and inhibitors, such as (ꢀ)-cymserine (15) and
(ꢀ)-phenethylcymserine (17) that bear a 40 isopropyl moiety on
their phenylcarbamate.23
Based on these factors, two approaches were taken to gener-
ate longer-acting anticholinesterases for myasthenia gravis: (i)
replacing the dimethyl carbamoyl side chain of the known
drugs neostigmine methyl sulfate (3) and pyridostigmine bro-
mide (4) with a phenyl carbamoyl side chain to form new qua-
ternary salts (6) and (8) and, (ii) modifying the known long-
acting anticholinesterases, (ꢀ)-phenserine (11) and (ꢀ)-tolserine
(13), by reacting them with iodomethane to form their corre-
sponding quaternary salts 12 and 14. In addition, following
our development of reversible, carbamate-based selective BChE
inhibitors, (ꢀ)-cymserine (15) and (ꢀ)-phenethylcymserine
(17), the same approach was applied to generate their corre-
sponding quaternary salts (16) and (18). Whereas the former
approach resulted in a dramatic loss of anticholinesterase activ-
ity, indicating that phenylcarbamates of neostigmine and pyri-
dostigmine (6, 8) cannot be readily accommodated within the
binding domains of AChE, the latter approach proved effective
in generating potent charged inhibitors (10, 12, 14, 16 and
18) that, albeit less cholinesterase subtype selective than their
parent compounds, were of similar or greater potency to neo-
stigmine (3) and pyridostigmine (4). As would be predicted
and shown in Table 1, the carbamoyl side chain replacement
as in 6 and 8 resulted in moderate decline in aqueous solubility
as reflected in Log D values, compared to 3 and 4. By contrast
and also expected, the quaternary salts 12, 14, 16 and 18 were
less lipophilic than their respective parent compounds. Although
these quaternary salts were not as water-soluble as either neo-
stigmine (3) or pyridostigmine (4) and thus may not have as
restricted a blood–brain barrier permeability, they would be
not expected to enter brain as freely as 11, 13, 15 and 17,
whose brain partition are high.7,8 As cognitive impairment has
been reported to occur in myasethenia gravis,40 but remains
controversial,41 some brain entry of drug could be of value.
As illustrated in Figure 2, the preliminary time-dependent cho-
linesterase inhibition of 16 in rat is relatively long-acting, and
in line with aryl carbamates of (ꢀ)-physostigmine in both ani-
mal models and humans.7,8,39
Figure 2. Time-dependent plasma cholinesterase inhibition following
intraperitoneal dose of compound 16 in rat.
a single
gorge region differentiating their respective shapes and catalytic
properties.20–22 Four different domains within the gorges of both
AChE and BChE have been defined and mapped. These include:
(i) a peripheral anionic site (PAS) at the mouth of the gorge that
is the first binding domain encountered by a positively charged
substrate or inhibitor as it enters, (ii) a slightly deeper cationic-
p
site, where the quaternary ammonium of choline interacts, (iii) a
deeper still acylation site that is the active center of the enzyme
and involves a catalytic triad and, (iv), an acyl-binding pocket at
the gorge base.20,23
The acylation site defines the active center involved in the
catalysis of ACh and is centered around an active serine residue
that, in human AChE, comprises a catalytic triad of Ser203, His447
and Glu334 (in human BChE: Ser198, His438, Glu325). The latter two
amino acids act as proton acceptors/donors to provide a ‘charge
relay’ system and the subsequent activation of the hydroxyl
group of Ser203. Substrates, exemplified by ACh, then bind cova-
lently to the active site serine consequent to the attack at the
carbonyl group of the substrate by the hydroxyl anion of the ser-
ine that leads to a tetrahedral alkoxy anion enzyme-substrate
intermediate. This tetrahedral intermediate then collapses, the
choline moiety of ACh leaves and the serine becomes acylated,
yielding a transient ‘acyl-enzyme’ complex. The presence of a
water molecule allows nucleophilic attack on the acyl-enzyme
intermediate that, via a tetrahedral intermediate, rapidly col-
lapses (in the order of milliseconds) to restore a free catalytic
site and carboxylate product.27–31 By contrast, in the presence
of a carbamate-based cholinesterase inhibitor, attack of the car-
bonyl function of the carbamate similarly through Ser203 gener-
ates a temporary (in the order of min to h) ‘carbamylated
drug-enzyme complex’ that is incapable of further substrate
catalysis.16,20,21,24
This complex, however, is considerably more stable than the
acetyl-enzyme one described for ACh, and its time-dependent
hydrolysis, or rate of decarbamylation, is in large part contingent
on the structure of the carbamate moiety.16,24 Whereas simple
short-chain alkyl carbamates (e.g., H and CH3) are subject to
relatively rapid decarbamylation32 to provide short-acting antich-
olinesterases (e.g., physostigmine: human pharmacodynamic
half-life (T1/2) 30 min,33 long-chain alkyl carbamates (e.g., (CH2)n
where n = 7 or 8)34,35 and simple aryl carbamates (e.g., phe-
nyl)7,8,36,37 provide more stable carbamylated drug-enzyme com-
plexes that undergo slower decarbamylation and, thereby, induce
a longer duration of cholinesterase inhibition. Consequently, the
alkyl heptyl and phenyl carbamate anticholinesterases, (ꢀ)-eptyl-
In summary, in the quest for longer-acting peripheral anticho-
linesterases modifying the structures of neostigmine methyl sul-
fate (3) and pyridostigmine bromide (4) led new quaternary salts
6 and 8 that lacked cholinesterase inhibitory action. In the case
of phenylcarbamate analogues of physostigmine, the quaternary
salts retained activity, compared to parent compounds 11, 13, 15
and 17, and appear long-acting.
3. Conclusion
To overcome the short duration of action of neostigmine methyl
sulfate (3) and pyridostigmine bromide (4) in the symptomatic
treatment of myasthenia gravis, the N-monophenylcarbamate ana-

Q. Yu et al. / Bioorg. Med. Chem. 18 (2010) 4687–4693
4691
logues of neostigmine (6) and pyridostigmine (8) were synthe-
sized. Evaluation of their cholinesterase inhibition demonstrated
that 6 and 8 possessed only marginal activity. As an alternative ap-
proach, the longer-acting cholinesterase inhibitory phenylcarba-
mate analogues of (ꢀ)-physostigmine 11, 13, 15 and 17 were
transferred into their corresponding N(1)-methylammonium bro-
mide analogues (12, 14, 16 and 18) and retained significant po-
tency that, for AChE, was equimolar to or great than their parent
compounds. These new quaternary salts (12, 14, 16 and 18) proved
more water-soluble than their parent compounds, and 16, as a rep-
resentative of the class, proved to be long-acting in rodent. To-
gether, these results suggest that such agents are of interest as
drug candidates for myasthenia gravis and congenital myasthenic
syndromes.
148–149 °C; 1H NMR (CD3OD): d 8.30–7.50 (m, 9H, Ar-H), 4.20 (s,
3H, N–CH3). Anal. (C13H13BrN2O2. HBr.H2O), C, H, N.
4.1.5. N(1)-Methylammonium bromide of (ꢀ)-physostigmine
(10)
(ꢀ)-Physostigmine 9 (78.8 mg, 0.286 mmol) was dissolved in
anhydrous CH3CN (1.5 ml), and 0.572 ml of bromomethane (2 M
in Et2O) was added. The solution was stirred at rt overnight. There-
after, evaporation of solvent and reagent left a residue that was
crystallized from H2O to provide crystalline product 10 (84.7 mg,
80%): mp 134–136 °C;
½
a 2D3
ꢁ
ꢀ124.6° (c 0.2 EtOH); 1H NMR
(DMSO-d6), d 7.65 (s, 1H, N–H), 7.13 (s, 1H, C4–H), 6.90 (d, 1H,
C6–H), 6.65 (d, 1H, C7–H), 5.10 (s, 1H, C8–H), 3.60 (m, 1H, C2a–
H), 3.15 (s, 6H, N1+–(CH3)2), 3.00 (m, 1H, C2b–H), 2.85 (s, 3H,
N8–CH3), 2.72 (d, 3H, N–CH3), 2.50–2.30 (m, 2H, C3–H2), 1.55 (s,
3H, C3a–CH3). Anal. (C16H24BrN3O2.H2O) C, H, N.
4. Experimental
4.1. Chemistry
4.1.6. N(1)-Methylammonium bromide of (ꢀ)-phenserine(12)
(ꢀ)-Phenserine 11 (168 mg, 0.5 mmol) was dissolved in anhy-
drous CH3CN (2 ml), and 1 ml of bromomethane (2 M in Et2O)
was added. The solution was stirred at rt overnight. Evaporation
of solvent and reagent provided a residue, which was crystallized
from H2O to give crystalline product 12 (184 mg, 85%): mp 185–
Melting points (uncorrected) were measured with a Fisher–
Johns apparatus; 1H NMR was recorded on a Bruker (Bellevica,
MA) AC-300 spectrometer; MS (m/z) were measured on an Agilent
5973 GC–MS (CI); elemental analyses were performed by Atlantic
Microlab, Inc. All reactions involving non-aqueous solutions were
performed under an inert atmosphere.
188 °C; ½a 2D3
ꢁ
ꢀ116.6° (c 0.1 EtOH); 1H NMR (DMSO-d6), d 10.18
(s, 1H, N–H), 7.70 (d, 1H, C40–H), 7.40 (m, 2H, C30–H and C50–H),
7.30 (d, 2H, C20–H and C60–H), 7.18 (m, 2H, C4–H and C6–H),
6.90 (d, 1H, C7–H), 5.18 (s, 1H, C8–H), 3.70 (m, 1H, C2a–H), 3.35
(s, 6H, N1+–(CH3)2), 3.15 (m, 1H, C2b–H), 2.95 (s, 3H, N8–CH3),
2.60–2.40 (m, 2H, C3–H2), 1.65 (s, 3H, C3a–CH3). Anal.
(C16H24BrN3O2ꢂ1/2H2O) C, H, N: calcd 9.52; Found 8.95.
4.1.1. N-Phenylcarbamate of 3-dimethylaminophenol (5)
3-Dimethylaminophenol (1) (500 mg, 3.65 mmol) was dis-
solved in anhydrous Et2O (50 ml) and a small piece of Na (about
1 mg) was added. After stirring for 2 min under nitrogen at rt,
phenylisocyanate (434 mg, 3.65 mmol) was added dropwise;
thereafter, the solvent was immediately evaporated under vacuum.
The residue was crystallized from CH2CL2 to provide crystalline
product 5 (650 mg, 70%): mp 150–152 °C; 1H NMR (CDCL3): d
9.00–7.70 (m, 9H, Ar-H), 4.20 (s, 6H, N–(CH3)2), 2.87 (br, H, NH);
CI-MS, m/e 257 (MH+). Anal. for (C15H16N2O2), C, H, N.
4.1.7. N(1)-Methylammonium bromide of (ꢀ)-tolserine (14)
(ꢀ)-Tolserine 13 (370 mg, 1.05 mmol) was dissolved in anhy-
drous CH3CN (4.2 ml), 2.1 ml of bromomethane (2 M in Et2O) was
added, and the solution stirred at rt overnight. Thereafter, evapora-
tion of solvent and reagent gave a residue that was crystallized
from H2O to provide crystalline product 14 (374 mg, 80%): mp
136–140 °C; ½a 2D3
ꢁ
ꢀ82.6° (c 0.2 EtOH); 1H NMR (DMSO-d6), d
10.01 (s, 1H, N–H), 7.40 (d, 2H, C30–H and C50–H), 7.20 (d, 2H,
C20–H and C60–H), 7.18 (s, 1H, C4–H), 7.00 (s, 1H, C6–H), 6.70 (d,
1H, C7–H), 5.18 (s, 1H, C8–H), 3.55 (m, 1H, C2a–H), 3.15 (s, 6H,
N1+–(CH3)2), 2.95 (m, 1H, C2b–H), 2.85 (s, 3H, N8–CH3), 2.50–
2.30 (m, 2H, C3–H2), 1.50 (s, 3H, C3a–CH3). Anal. (C24H32N3O2Br.-
H2O) C, H, N.
4.1.2. N-Phenylcarbamate of 3-phenol-1-trimethylammonium
methylsulfate (6)
Compound 5 (128 mg, 0.5 mmol) was dissolved in acetone
(2 ml), and (CH3)2SO4 (0.1 ml) was added. The mixture was kept
in a refrigerator overnight. A crystallized product was filtered
and recrystallized from EtOH to give compound 6 as white crys-
tals (153 mg, 80%): mp 125–127 °C; 1H NMR (CD3OD): d 7.90–
7.20 (m, 9H, Ar-H), 3.62 (m, 12H, 4CH3). Anal. (C17H22N2O6S),
C, H, N.
4.1.8. N(1)-Methylammonium bromide of (ꢀ)-cymserine (16)
(ꢀ)-Cymserine 15 (379 mg, 1.0 mmol) was dissolved in anhy-
drous CH3CN (4.5 ml) and 2 ml of bromomethane (2 M in Et2O)
was added. The solution was stirred at rt overnight, following
which evaporation of solvent and reagent provided a residue that
was crystallized from H2O to give crystalline product 14 (315 mg,
4.1.3. N-Phenylcarbamate of 3-hydroxypyridine (7)
3-Hydroxypyridine (500 mg, 5.26 mmol) was dissolved in anhy-
drous Et2O (50 ml) and a small piece of Na (about 1 mg) was added.
After stirring for 2 min under nitrogen at rt, phenylisocyanate
(626 mg, 5.26 mmol) was added dropwise. The solvent was then
immediately evaporated under vacuum to provide a crystalline
residue, which was recrystallized from CH2CL2 to give product 7
as white crystals (964 mg, 85%): mp 112–113 °C; 1H NMR (CDCL3):
d8.70–7.00 (m, 9H, Ar-H), 1.70 (br, 1H, NH). CI-MS, m/e: 215 (MH+).
Anal. for (C12H10N2O2), C, H, N.
80%): mp 160–162 °C; ½a D23
ꢁ
ꢀ92.2° (c 0.2 EtOH); 1H NMR (DMSO-
d6), d 10.02 (s, 1H, N–H), 7.55–7.30 (m, 5H, Ar-H and C4–H), 7.20
(d, 1H, C5–H), 6.90 (d, 1H, C7–H), 5.30 (s, 1H, C8–H), 3.70 (m, 1H,
C2a–H), 3.40 (s, 6H, N1+–(CH3)2), 3.20 (m, 1H, C2b–H), 3.05 (s,
3H, N8–CH3), 2.60–2.50 (m, 2H, C3–H2), 2.45 (s, 3H, Ar-CH3), 1.75
(s, 3H, C3a–CH3). Anal. (C24H32BrN3O2.H2O) C, H, N.
4.1.9. N(1)-Methylammonium bromide of (ꢀ)-phenethylcy-
mserine(18)
4.1.4. N-Phenylcarbamate of 3-hydroxy-1-methylpyridinium
bromide (8)
(ꢀ)-Phenethylcymserine 17 (469 mg, 1.0 mmol) was dissolved
in anhydrous CH3CN (5 ml), 2 ml of bromomethane (2 M in Et2O)
was added, and the solution was stirred at rt overnight. Evapora-
tion of solvent and reagent provided a residue that was crystalized
from H2O to give crystalline product 18 (462 mg, 82%): mp 150–
Compound 7 (107 mg, 0.5 mmol) was dissolved in anhydrous
Et2O (2 ml), and 1 ml of bromomethane (2 M in Et2O) was then
added into the ether solution. The mixture was maintained at rt
for 2 days to give crystalline product. Recrystallization of this prod-
uct from EtOH provided crystalline compound 8 (108 mg, 70%): mp
151 °C; ½a 2D3
ꢁ
ꢀ142.8° (c 0.2 EtOH); 1H NMR (DMSO-d6), d 10.02

4692
Q. Yu et al. / Bioorg. Med. Chem. 18 (2010) 4687–4693
(s, 1H, N–H), 7.45–6.70 (m, 12H, Ar-H) 5.22 (s, 1/2H, 1/2C8–H),
5.18 (s, 1/2H, 1/2C8–H), 3.70–2.80 (m, 10H, C2–H2, N–CH2CH2,
(Me)2CH– and N8–CH3), 3.40 (s, 6H, N1+–(CH3)2), 3.20 (m, 1H,
C2b–H), 3.05 (s, 3H, N8–CH3), 2.50–2.30 (m, 2H, C3–H2), 1.55 (s,
1.5H, 1.5C3a–CH3), 1.45 (s, 1.5H, 1.5C3a–CH3) Anal. (C31H38BrN3
O2ꢂ1/2H2O) C, H, N.
sample was taken to determine resting (no inhibition) cholinester-
ase activity. Compound 16 was administered a 10 mg/kg ip and
blood samples were taken at predefined times from 5 min to
24 h. These were centrifuged (8000g, 30 s) and the plasma was col-
lected and frozen (ꢀ80 °C) for determination of cholinesterase
inhibition. In rat plasma, unlike that in humans, both AChE and
BChE are present. Therefore a specific inhibitor of BChE, Iso-OMPA
(1 ꢃ 10ꢀ4 M), was used during the quantitative determination of
AChE inhibition. Throughout this procedure, the animal was as-
sessed for any acute adverse effects, particularly associated with
cholinergic overdrive (e.g., tremor, salivation).
4.2. Quantitation of anticholinesterase activity
The anticholinesterase activity of compounds 3–18 was as-
sessed by quantifying their capacity to inhibit the ability of freshly
prepared human AChE and BChE to enzymatically degrade their
respective specific substrates, acetyl-(b-methyl)thiocholine and s-
butyrylthiocholine (0.5 mmol/L) (Sigma Chemical Co., St. Lois,
MO).15,42 Samples of AChE and BChE were derived from freshly col-
lected human whole red blood cells and plasma, respectively. Com-
pounds were dissolved in Tween 80/EtOH 3:1 (v:v; <150 lL total
volume) and were diluted in 0.1 M Na3PO4 buffer (pH 8.0) in
half-log concentrations to provide a final concentration range that
spanned 0.3 nM to 30 lM. Tween 80/EtOH was diluted to in excess
of 1 in 5000. No inhibitory action of either AChE or BChE was de-
tected in separate experiments in which the anticholinesterase
activity of the known compound, 9, was quantified in excess and
without Tween 80/EtOH.
For the preparation of BChE, freshly collected blood was centri-
fuged (10,000g, 10 min, 4 °C) and plasma was removed and diluted
1:125 with 0.1 M Na3PO4 buffer (pH 7.4). Plasma was carefully
checked to insure an absence of hemolysis. For AChE preparation,
erythrocytes were washed five times in isotonic saline, lysed in 9
volumes of 0.1 M Na3PO4 buffer (pH 7.4) containing 0.5% Triton X
(Sigma) and then were diluted with an additional 19 volumes of
buffer to a final dilution of 1:200.
Plasma samples were thawed, and a 10
added immediately to 1390 l 0.1 mol/L sodium phosphate buffer,
pH 7.4, with and without iso-OMPA. Three minutes later, 100
ll sample was then
l
ll
5,50-dithiobis-2-nitrobenzoic acid (0.5 mmol/L; Sigma Chemical
Co.) plus S-butyrylthiocholine in 0.1 mol/L sodium phosphate
buffer, pH 7.4, was added. The production of a yellow 5-thio-2-ni-
tro-benzoate anion (produced by the reaction of 5,50-di-thiobis-2-
nitrobenzoic acid with thiocholine released by enzymatic
hydrolysis of butyrylthiocholine) was measured precisely 20 min
later by spectrophotometer (412 nmk). All reagents were main-
tained at rt and plasma samples were assayed together in dupli-
cate. Cholinesterase inhibition was calculated by subtracting the
absorption value of samples with iso-OMPA from those without
and expressing the difference (related to substrate hydrolysis by
cholinesterase alone) as a percent of the predrug level. The total as-
say time was 23 min, which minimized drug decomposition and
rejuvenation of enzyme in the plasma sample.
Acknowledgments
This work was supported in part by the Intramural Research
Program of the National Institute on Aging, National Institutes of
Health, and by National Institutes of health grants (AG18379 and
AG18884) to D.K.L. The authors are grateful for the aid and input
of Scott MacDonald, Advanced Chemistry Development, Inc., Tor-
onto, ON, Canada, for determination of Log D values of charged
compounds. Animal studies were undertaken on an approved pro-
tocol in accord with the Animal Care and Use Committees of the
Intramural Research Program, National Institute on Aging, and fol-
lowed National Institutes of Health guidelines. The authors declare
no conflicts of interest relating to the described research.
Analysis of anticholinesterase activity was undertaken by utiliz-
ing a 25 lL sample of each enzyme preparation, and was under-
taken at their optimal working pH 8.0,15 in 0.1 M Na3PO4 buffer
(0.75 mL total volume). Compounds were preincubated with en-
zymes (30 min, rt) and then were incubated with their respective
substrates and with 5,50-dithiobis-2-nitrobenzoic acid (25 min,
37 °C). The substrate/enzyme interaction was immediately halted
by the addition of excess enzyme inhibitor ((ꢀ)-physostigmine
(9) 1 ꢃ 10ꢀ5 M) and production of a yellow thionitrobenzoate an-
ion was then measured by spectrophotometer at 412 nmk. To cor-
rect for nonspecific substrate hydrolysis, aliquots were co-
incubated under conditions of absolute enzyme inhibition (by the
addition of 1 ꢃ 10ꢀ5 M (ꢀ)-physostigmine (9), and the associated
alteration in absorbance was subtracted from that observed
through the concentration range of each test compound. Each
agent was analyzed on four separate occasions and assayed
along-side (ꢀ)-physostigmine (9), as a control and external stan-
dard whose activity we have previously reported.42 The mean en-
zyme activity at each concentration of test compound was then
expressed as a percent of the activity in the absence of compound.
This was transformed into a logit format (where logit = ln(%activ-
ity/100 minus%activity)) and then was plotted as a function of its
log concentration. Inhibitory activity was calculated as an IC50, de-
fined as the concentration of compound (nM) required to inhibit
50% of enzymatic activity, which was determined from a correla-
tion between log concentration and logit activity. Only results ob-
tained from correlation coefficients of r2 P ꢀ0.98 were considered
acceptable. Studies that did not obtain this threshold were
repeated.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.022.
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