Differential binding of phenothiazine urea derivatives to wild-type human cholinesterases and butyrylcholinesterase mutants

Article abstract of DOI:10.1016/j.bmc.2010.01.066

A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic site.

Full text of DOI:10.1016/j.bmc.2010.01.066

Bioorganic & Medicinal Chemistry 18 (2010) 2232–2244
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Differential binding of phenothiazine urea derivatives to wild-type human
cholinesterases and butyrylcholinesterase mutants
d
d
d
b
Sultan Darvesh a,b,c,d, , Ian R. Pottie , Katherine V. Darvesh , Robert S. McDonald , Ryan Walsh ,
*
Sarah Conrad d, Andrea Penwell d, Diane Mataija d, Earl Martin d
a Department of Medicine (Neurology), Dalhousie University, Halifax, Nova Scotia, Canada
b Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada
c Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada
d Department of Chemistry, Mount Saint Vincent University, Halifax, Nova Scotia, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for
its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent
inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional
binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on
wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the
aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase
activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic
site.
Received 16 December 2009
Revised 27 January 2010
Accepted 29 January 2010
Available online 4 February 2010
Keywords:
Acetylcholinesterase
Butyrylcholinesterase
Enzyme kinetics
Ó 2010 Elsevier Ltd. All rights reserved.
Mutant butyrylcholinesterase
1. Introduction
The mechanism producing the positive effect of methylene blue
in AD is thought to relate, at least in part, to its interference with
Phenothiazines are heterocyclic molecules containing two ben-
zene rings linked in a tricyclic system through nitrogen and sulfur
atoms (Fig. 1). The first known member of this class of compounds,
the purple dye thionine, was synthesized by Lauth in 1876, fol-
lowed by the synthesis of another dye, methylene blue (Fig. 1),
by Caro in 1877. The evidence that both thionine and methylene
blue contained a phenothiazine tricycle was provided when the
parent compound, phenothiazine, was prepared and characterized
by Bernthsen in 1883.1,2 Since the late 19th century, many pheno-
thiazine derivatives have been synthesized and have proven to be
highly eclectic in their biological activities.1,2 Phenothiazines have
been used as dyes, as biological stains and as pharmaceutical
agents to treat a variety of diseases. Chlorpromazine (Fig. 1), for
example, was one of the first compounds used as a neuroleptic
to treat symptoms of psychosis.1 The earlier phenothiazine deriva-
tive, methylene blue, in addition to many other applications,3 has
been used as a treatment for parasitic, bacterial and viral infec-
tions, for cancer treatment and, most recently, for the treatment
of dementias, such as Alzheimer’s disease (AD).4
the aggregation of b-amyloid and tau,4 the neuropathological hall-
marks of this disease. Another prominent feature of AD is choliner-
gic dysfunction.5–7 In this regard, phenothiazine derivatives, in
general, are known to inhibit cholinesterases.8–13 Other drugs,
including amitriptyline and morphine (Fig. 1) also inhibit cholines-
terases,14–18 but certain phenothiazines tend to be more selective
inhibitors of butyrylcholinesterase (BuChE, EC 3.1.1.8) over acetyl-
cholinesterase (AChE, EC 3.1.1.7).8–13 In recent years, compounds
with the ability to selectively inhibit BuChE have gained interest
for the treatment of AD. It has been reported that selective BuChE
inhibitors raise acetylcholine levels in the brains of animals.19 Cho-
linesterase inhibition is deemed important because diminished
brain acetylcholine appears to give rise to the dominant cognitive
and behavioural symptoms of AD. Supporting this notion is the
observation that treatment with cholinesterase inhibitors allevi-
ates such symptoms.20 The drugs of this type that are in current
use for the treatment of AD, such as, donepezil, galantamine and
rivastigmine, produce the inhibition of both AChE and BuChE,
although to different degrees.21 Since both hydrolytic enzymes
are still present in AD brains, it is not clear whether the positive
symptomatic relief produced by these drugs results mainly from
AChE inhibition, BuChE inhibition or the combined inhibition of
both enzymes. A comparative study of the effect of a highly
selective inhibitor of BuChE, and one selective for AChE inhibition,
* Corresponding author. Address: Room 1308, Camp Hill Veterans’ Memorial
Building 1, 5955 Veterans’ Memorial Lane, Halifax, Nova Scotia, Canada B3H 2E1.
Tel.: +1 902 473 2490; fax: +1 902 473 7133.
E-mail address: sultan.darvesh@dal.ca (S. Darvesh).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.066

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2233
Phenothiazines
Thioridazine
Chlorpromazine
Methylene blue
N
N
N
N
S
Cl
N
S
N
N
S
S
Cl
Tricyclic antidepressants
Nortriptyline
Amitriptyline
Imipramine
N
H
N
N
N
Other Drugs
Procainamide
Morphine
Quinidine
HO
O
H
N
N
H
HO
O
N
H
H
N
H
H
N
O
HO
N
Figure 1. Structure of phenothiazines and other drugs that inhibit cholinesterases.
R1
with those that inhibit both, may help solve this dilemma by dem-
onstrating greater efficacy with one type of treatment. The obser-
vations that BuChE activity is elevated in AD, while AChE activity
is diminished22,23 and that BuChE activity predominates in regions
of the brain involved in cognition and behaviour,24,25 may favour
the use of BuChE-specific inhibitors in treating AD.26
We have previously reported the synthesis of phenothiazine N-
10-amide derivatives that are highly selective inhibitors of human
BuChE11,12 and a series of comparable phenothiazine carbamates27
some of which inhibit only AChE.
Here we report the synthesis of a series of phenothiazine-N-10-
urea derivatives which, for the most part, tend to be, like the cor-
responding amides, selective BuChE inhibitors. However, one
sub-class of this series, the substituted aminoureas, is found to
be able to produce potent inhibition of both BuChE and AChE.
We provide evidence, through the direct use of BuChE mutant spe-
cies and the effect of elevated substrate on wild-type human cho-
linesterases, that the aminoureas have a distinct mechanism for
binding to cholinesterases, not evident with the other urea deriva-
tives or the amide and carbamate derivatives of phenothiazine. In-
stead, these aminourea inhibitors have some features in common
with cationic phenothiazines like methylene blue.4,13,28
(a)
(b)
R1
N
R2
O
Cl
O
N
H
R2
N
S
N
S
CH2Cl2
R3
N
R3
H
R1
R2
R1
R2
O
Cl
O
CH2
N
N
CH2 N
X
X
N
N
S
S
CH2Cl2
Figure 2. Synthetic schemes: (a) synthesis of phenothiazine ureas; (b) synthesis of
phenothiazine aminoureas.
inhibit AChE or BuChE. That compound is reported here for struc-
tural comparison with the corresponding urea derivative (14),
which is a robust inhibitor of BuChE (Table 1). Urea products were
derived from alkyl, cycloalkyl and aryl amines, as well as certain
substituted diamines, giving compounds that represent two gen-
eral categories, ureas (Tables 1–3) and aminoureas (Table 4). Each
compound was then tested for its ability to inhibit AChE and BuChE
using a modification21,27 of Ellman’s spectrophotometric method.29
2. Results and discussion
2.1. Synthetic chemistry
A total of 37 urea derivatives of phenothiazine were prepared
from phenothiazine-10-carbonyl chloride and a variety of amines,
as depicted in the reaction schemes in Figure 2. Of these, 22 com-
pounds (see Section 4) have been synthesized previously. One
amide derivative, 1-adamantylamide (compound 38, see Section 4)
was prepared earlier11 but was not reported then, since it did not
All the reactions were carried out by mixing phenothiazine-10-
carbonyl chloride with an appropriate amine (Fig. 2a) or diamine
(Fig. 2b), in dichloromethane. The mixture was stirred at room
temperature and the reaction was monitored by thin layer chroma-
tography until all the starting material was consumed or no change

2234
S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
Table 1
7
6
5
4
3
Inhibition constants (Ki values), molecular volumes and Log P values for phenothi-
azine alkyl and cycloalkyl urea derivatives
14
R
N
8
13
12
O
5
4
H
11
9
7
6
N
2
3
10
S
R2 = 0.78
1
#
R
Ki (
l
M) BuChE
Ki (
lM) AChE
V (Å3)
Log P
1
2
H
CH3
90.8 11.8
18.3 2.2
89.3 2.6
9.36 3.51
233
253
2.96
3.20
200
250
300
Volume Å3
350
400
3
35.0 2.1
20.5 5.0
1450 321
None
272
290
3.54
4.02
Figure 3. A plot of extent of BuChE inhibition (pKi) versus molecular volume, of a
series of phenothiazine alkyl ureas, demonstrating a linear relationship between
potency of BuChE inhibition and molecular volume. Note that the best inhibitor of
this series has the largest volume.
4
5
6
3.41 0.83
24.7 5.4
691 39
None
290
308
3.86
4.44
(a). Amide (38)
(b). Urea (14)
O
H
7
8
10.6 1.6
None
None
308
326
3.97
3.97
N
O
N
N
S
S
2.66 0.64
9
29.1 7.5
42.5 1.6
None
None
300
283
3.04
3.49
O
10
11
12
13
10.0 1.7
2.99 0.89
2.81 0.33
None
None
None
300
315
332
3.91
4.33
4.75
14
0.35 0.07
None
379
5.03
Figure 4. Structural and conformational differences between urea (14) and amide
(38) phenothiazine adamantyl derivatives that determine the ability of these
compounds to inhibit butyrylcholinesterase. The urea derivative (14) exhibits
greater flexibility, permitting the phenothiazine tricycle to interact with the
butyrylcholinesterase active site gorge, thus inhibiting the enzyme catalysis. The
amide derivative (38) is more rigid and prevents the phenothiazine tricycle
interaction with the enzyme. The lower figures representing the most stable
conformers of these two compounds were generated using SPARTAN006.30
Log P indicates the relative solubility in n-octanol versus water, with the most
hydrophobic molecules having the highest Log P value.
terase inhibition at the highest inhibitor concentration, depending
on solubility limits (up to 0.167 mM in 50% aqueous acetonitrile),
and then through serial (1:10) dilutions in the same solvent to ob-
tain an inhibition–concentration profile that indicated a range of
inhibitor concentrations suitable for kinetic studies. Lineweaver–
Burk plots were then generated for each compound in the absence
of inhibitor and at two concentrations of inhibitor. A replot of
slopes of these lines against inhibitor concentration gave the
inhibition constant (Ki values, Tables 1–4) as the intercept on the
x-axis. Ki represents the equilibrium constant for the dissociation
of the enzyme–inhibitor complex (EI) into enzyme (E) and inhibi-
tor (I) {EI ? E + I, where Ki = [E] [I]/[EI]}. A smaller Ki value indi-
cates higher inhibitor potency. In certain of the data treatments,
where indicated, the inhibitor affinities, instead of being expressed
in exponential terms, are converted to the negative logarithm of
the affinity constant (ꢀlog Ki), or pKi (as in Fig. 3). In this treatment
a larger number represents a higher affinity. The inhibitor potency
(Ki) was found to vary greatly throughout the series of ureas and
in the reaction mixture could be discerned. After workup, the prod-
ucts were purified by crystallization from petroleum ether/dichlo-
romethane mixtures. All compounds were fully characterized by
IR, 1H and 13C NMR spectroscopy, as well as low- and high-resolu-
tion (accurate mass) mass spectrometry. Physical data were consis-
tent with the structure of each molecule synthesized. HPLC
analysis of all purified compounds showed them to be more than
98% pure (see Supplementary data).
2.2. Enzyme kinetic studies
Each phenothiazine derivative was evaluated for its ability to
inhibit purified human plasma BuChE and purified recombinant
human AChE. Initially, each derivative was examined for cholines-

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2235
Table 2
Table 3
Inhibition constants (Ki values), molecular volumes and Log P values for phenothi-
Inhibition constants (Ki values), molecular volumes and Log P values for phenothi-
azine aryl and alkylaryl urea derivatives
azine disubstituted alkyl urea derivatives
R
O
R
O
N
N
S
H
N
S
#
R
Ki (l
M) BuChE
Ki (l
M) AChE
V (Å3)
Log P
#
R
Ki (
l
M) BuChE
Ki (
l
M) AChE
V (Å3)
Log P
23
N
N
9.20 1.74
1.05 0.13
400 31
None
273
3.44
15
0.31 0.05
9.63 1.67
12.1 0.31
4.69 0.04
None
None
None
None
319
4.86
24
25
310
346
3.11
5.08
16
17
18
365
337
332
4.74
5.35
5.42
O
0.43 0.001
0.18 0.08
None
None
N
N
Cl
26
346
4.75
19
0.71 0.16
None
337
5.35
N
N
N
27
28
29
0.56 0.05
1.56 0.43
7.10 1.21
None
None
None
297
314
306
3.75
4.17
3.04
20
21
4.45 1.27
0.19 0.04
None
None
337
356
4.93
5.21
O
barrier by diffusion. Often the solubility of a molecule in water is too
low for the spectrophotometric analysis required in the experimen-
tal determination of log P values. In such cases, values can be ob-
tained indirectly via methods such as the ALOGPS v 2.0 system31
used herein. This method compares the structure of the molecule
with a large database of known molecular partition coefficients.
The larger the value of log P, the more hydrophobic the molecule
and, hence, the greater the facility with which it can cross the
blood–brain barrier. All the phenothiazine derivatives examined
had calculated log P values greater than 2.5 (Tables 1–4). These par-
tition coefficient values are all comparable to those calculated ear-
lier for other phenothiazine derivatives.12,27 In addition, although
factors other than hydrophobicity may be involved in crossing the
blood–brain barrier, the log P values in Tables 1–4 are comparable
to those calculated earlier by the same method for drugs known to
cross the blood–brain barrier.12
22
0.15 0.02
None
388
5.93
the factors contributing to this were explored by considering
molecular parameters related to the structure of each compound
and to that of the enzyme. The Ki values were independent of the
pH between 6.8 and 8.0, as has been observed previously for other
cholinesterase inhibitors.23
The inhibitory effect of compound 31 on wild-type BuChE and
several BuChE mutants (D70G, F329A and Y332A) was examined
at a single butyrylthiocholine concentration (1.6 ꢁ 10ꢀ4 M) using
the modified Ellman method. Michaelis–Menten plots to examine
aminourea inhibition were developed using fixed enzyme and
inhibitor concentrations and wide range of substrate concentra-
tions (3.5 ꢁ 10ꢀ5–4.0 ꢁ 10ꢀ2 M) also using the modified Ellman
method.
2.4. Butyrylcholinesterase inhibition by phenothiazine ureas
2.3. Molecular computational studies
The inhibition of human BuChE by urea and aminourea deriva-
tives of phenothiazine (Tables 1–4) was consistently of the revers-
ible type. The relative potency of this inhibition was found to be
directly related to the total molecular volume of the inhibitor.
For example, a plot of pKi (ꢀlog Ki) versus molecular volume for
the alkyl ureas shown in Table 1, produced a linear relationship,
as depicted in Figure 3. This type of BuChE inhibition and the obvi-
ous relationship between potency and inhibitor size, are compara-
ble to the inhibition of this enzyme by phenothiazine amides11,12
and carbamates.27 These similarities suggest a common mode for
the binding of such phenothiazine derivatives to BuChE and, as de-
Computational methods were used to determine certain molec-
ular characteristics that could govern specificity and potency for
cholinesterase inhibition.
The total molecular volume was calculated, and is reported in
Tables 1–4. Volumes were obtained by carrying out molecular
mechanics calculations, using SPARTAN006.30 The Merck molecular
force field (MMFF) was employed, selecting the most stable con-
former for the volume calculation.30
The log P values were calculated to predict the ability of the com-
pound to cross the blood–brain barrier.31 Experimentally, this is
done by determining the concentration of the compound in n-octa-
nol with that in water. The log of this partition value (P) provides an
estimate of how efficiently the compound will cross the blood–brain
scribed earlier,9,27 this entails a highly favoured
p–p association
between the aromatic rings of the phenothiazine tricycle and the
two aryl side chains of phenylalanine (F329) and tyrosine (Y332)
on the E-helix27 within the active site gorge of the enzyme. Thus,

2236
S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
Table 4
Inhibition constants (Ki values), molecular volumes, Log P values and pKa values (distal nitrogen) of phenothiazine aminourea derivatives
O
R
N
S
V (Å3)
Log P
pKa
a
#
Ki (lM) BuChE
Ki (
lM) AChE
H
N
30
0.51 0.06
16.3 7.2
359
3.87
9.83
N
H
N
31
32
0.009 0.0002
0.21 0.012
0.81 0.062
29.4 7.6
395
347
4.51
3.51
10
N
H
N
9.12
N
H
N
33
34
0.02 0.003
6.91 0.87
0.62 0.13
None
364
355
3.93
2.8
9
N
N
H
N
7.7
O
H
N
O
35
36
37
7.86 0.52
17.1 9.4
0.25 0.08
None
None
None
373
328
405
2.9
7.7
NCb
6.0
N
N
N
N
3.19
5.38
H
N
a
The pKa values were obtained from Scifinder Scholar, published by the American Chemical Society. This software uses Advanced Chemistry Development (ACD/Labs)
Software V8.19 for Solaris (Ó 1994–2009 ACD/Labs) calculate pKa value.
b
Program unable to calculate pKa value.
as the size of the urea substituent on the phenothiazine scaffold is
increased, the ability to block substrate access to the catalytic triad
exhibits a concomitant increase. However, in spite of similarities
between the inhibitory effects towards BuChE of phenothiazine
ureas and their corresponding amides and carbamates, there were
some notable exceptions. For example, the 1-adamantylcarbonyl
amide derivative of phenothiazine (38) showed no inhibition of
2.5. Acetylcholinesterase inhibition by phenothiazine ureas
As seen in Tables 1–3, urea derivatives of phenothiazine, like
their amide counterparts,11,12 showed little or no ability to inhibit
AChE. The small volume of the AChE active site gorge (302 Å3)9
undoubtedly contributes to the inability of many of the larger
ureas to gain access to this region of the enzyme for inhibition. An-
other factor thought to contribute to this absence of AChE inhibi-
tion is the presence of a tyrosine (Y337) residue9,32 on the E-
helix of human AChE.27 This amino acid side chain interferes with
BuChE up to its solubility limit of approximately 20 lM. This has
been attributed to the somewhat rigid nature of the amide bond
(Fig. 4a) that, having the bulky adamantyl group attached, cannot
be properly accommodated within the gorge to line up the pheno-
thiazine moiety for binding to F329 and Y332. A similar phenome-
non was earlier found to occur with the long 4-biphenylcarbonyl
amide derivative12 and carbamate derivative27 that, likewise,
showed no BuChE inhibition. In the case of 4-biphenylcarbonyl
amide, BuChE inhibition was attained by the addition of a flexible
methylene (CH2) group into the system (4-biphenylacetylamide,
Ki = 1.7 ꢁ 10ꢀ6 M).12 The extra tetrahedral carbon atom permits a
different 3-D geometry enabling the molecule to have increased
molecular flexibility within the active site region. Similarly, this
added flexibility, with concomitant BuChE inhibition, by the ada-
mantyl urea derivative (Table 1) can also be attributed to the addi-
tion of a tetrahedral nitrogen atom to the adamantyl moiety in
compound 14 (Fig. 4b), compared to the lack of BuChE inhibition
observed with the adamantyl amide derivative (38) (Fig. 4a).
the
p–p interaction between the phenothiazine tricycle and resi-
dues F338 and Y341, that are equivalent to F329 and Y332 in
BuChE, and that serve as the binding site for the phenothiazine
moiety in BuChE.9,11,12,27 The poor, but almost identical, AChE
inhibitor affinity constants for small (molecular volume <310 Å3)
amide phenothiazines11 appears also to operate with the alkyl
ureas (Tables 1 and 3) and may result from a weak interaction of
the carbonyl functionality with a hydrogen bonding group, such
as that of Y124 or Y341, within the AChE active site region.11
2.6. Cholinesterase inhibition by phenothiazine aminoureas
In sharp contrast to the ureas, a number of the substituted ami-
noureas, such as compound 31 (Table 4), were found to be potent
inhibitors of both AChE and BuChE (Fig. 5). There is little doubt that

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2237
(a). AChE
(b). BuChE
Inhibition Constant
Inhibition Constant
Plot
Plot
50
40
30
16
12
50
40
30
20
10
0
12
8
8
4
0
4
0
-1.E-06 5.E-07 2.E-06
[Inhibitor] M
-3.E-08 3.E-08 9.E-08
[Inhibitor] M
20
10
0
-5.0
-3.0
-1.0
1.0
3.0
-5.0
-3.0
-1.0
1.0
3.0
1/[s] (/10000)
1/[s] (/10000)
-10
-10
-20
-20
Figure 5. Lineweaver–Burk plots of; (a) acetylcholinesterase hydrolysis of acetylthiocholine in the absence (N) and presence of (d = 9.3 ꢁ 10ꢀ7 M and j = 1.9 ꢁ 10ꢀ6 M) of N-
[2-(N0,N0-diisopropylamino)ethyl]-10H-phenothiazine-10-carboxamide (31) and (b) butyrylcholinesterase hydrolysis of butyrylthiocholine in the absence (N) and presence
(d = 4.2 ꢁ 10ꢀ8 M and j = 8.3 ꢁ 10ꢀ8 M) of (31). Inhibition constants were derived from the plot in the inset, where the Ki for acetylcholinesterase is 8.1 ꢁ 10ꢀ7 M and the Ki
for butyrylcholinesterase is 9.0 ꢁ 10ꢀ9 M.
the BuChE inhibition involves the usual
p
–
p
interaction between
inhibition by aminoureas with the use of mutant BuChE species.
Thus, a comparative study was made of the inhibition of wild-type
BuChE and the BuChE mutants D70G, F329A and Y332A. The effect
of 2-(N,N-diisopropylamino ethyl) phenothiazine urea (31) on
these four BuChE enzyme species is summarized in Figure 6. As
can be seen in this representation, at an inhibitor concentration
of 50 nM, wild-type BuChE was inhibited by approximately 50%,
with no inhibitory effect on the D70G mutant enzyme at that con-
centration. A 10-fold increase in inhibitor concentration inhibited
wild-type BuChE almost completely, while D70G BuChE was still
at over 80% of its original activity with no inhibitor (Fig. 6). The
importance of Y332 in binding this inhibitor is also evident in
the retention of nearly 50% activity at 500 nM inhibitor with
Y332A BuChE. F329A BuChE also exhibits greater resistance to
F329 and Y332 of the enzyme and the phenothiazine moiety.11,12,27
However, the Ki value for compound 31, being in the nanomolar
range for BuChE, implies that other factors may be influencing
greater binding of this type of molecule to the enzyme. This addi-
tional binding site may also provide the unusually high AChE inhi-
bition (10ꢀ7 M range) by the same compound (Table 4). This robust
AChE inhibition occurs in spite of the large molecular volume
(395 Å3) of this inhibitor compared to the active site gorge volume
(302 Å3). This implies binding of the compound at the periphery of
the AChE gorge.
One factor that could contribute to the ability of a substituted
aminourea, such as compound 31, to provide robust inhibition of
both cholinesterases (Fig. 5) is the potential of the substituent dis-
tal amino group to be positively charged under the conditions (pH
8.0) of the Ellman essay. To test the importance of a potential po-
sitive charge in binding substituted aminoureas to cholinesterases,
calculated approximate pKa values of the distal substituent tertiary
amino groups (Table 4) were considered with respect to the ability
to produce potent AChE inhibition. As can be seen in that table,
only aminoureas that would be predominantly cationic under the
reaction conditions (pH 8.0 or below) (e.g., compounds 30–33,
but not 34–37) show the inhibition of AChE. That is, only those
substituents with pKa values well above the assay pH of 8.0 inhibit
AChE. The same reasoning could be invoked to explain why BuChE
showed unusually high sensitivity to inhibition by compounds
such as 31 (Table 4). That is, in addition to the novel cationic bind-
ing with the substituent nitrogen, BuChE should still be capable of
O
NH
N
S
100
75
50
25
0
forming p–p interaction with the phenothiazine moiety to provide
inhibition constant values in the nanomolar range (compound 31,
Ki = 9.0 ꢁ 10ꢀ9 M), comparable to that produced by the large 9-
anthrylcarbonyl amide.12
2.7. Inhibition of butyrylcholinesterase mutants
To further address the question of a common feature of both
AChE and BuChE that would facilitate inhibition through binding
a cationic nitrogen atom, the common anionic aspartic acid residue
(D74 in human AChE and D70 in human BuChE) was considered.
This common component of the peripheral anionic site (PAS) of
cholinesterases was tested as a major contributor to cholinesterase
Wild-type
F329A
Y332A
D70G
Figure 6. Comparison of residual enzymatic activity of wild-type BuChE and
mutant enzyme species, F329A, Y332A and D70G, in the absence (black bar) and
presence of two concentrations (grey bar = 5 ꢁ 10ꢀ8 M and white bar = 5 ꢁ 10ꢀ7 M)
of N-[2-(N0,N0-diisopropylamino)ethyl]-10H-phenothiazine-10-carboxamide (31).

2238
S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
inhibition by compound 31 than wild-type BuChE, but the effect is
less pronounced than for Y332A BuChE. Thus, robust inhibition of
BuChE by aminoureas, such as compound 31, can be attributed
to the additional salt linkage of this cationic urea with D70 of the
quence of conformational changes to the enzymes because of the
interaction between D74 in AChE and D70 in BuChE, with cationic
substrates such as acetylcholine.35 Thus, the interaction between
the cationic substrate and the anionic amino acid residue at the
PAS leads to conformational changes that produce the opposite ef-
fects in AChE and BuChE. Experiments with an aminourea (31) and
the BuChE D70G mutant suggest that the cationic amionoureas
form both a salt bridge between D70 and an electrostatic interac-
tion with aromatic amino acids F329 and Y332 of the enzyme. This
effect is lost when the D70 is mutated to the neutral amino acid
glycine. Based on these observations, AChE was assumed to inter-
act with aminoureas, like compound 31, solely through the ionic
linkage with D74.
peripheral anionic site, in addition to the usual p–p interaction be-
tween the phenothiazine tricycle and F329 and Y332 (Fig. 7b). It
may therefore be reasonable to assume that the corresponding an-
ionic group in human AChE (D74) is largely responsible for the
strong inhibition of that enzyme by compound 31 (Fig. 7a).
2.8. Effect of substrate concentration on aminourea inhibition
One of the most important differences between AChE and
BuChE is the effect on enzymatic activity at high substrate concen-
tration. At elevated substrate concentrations, AChE exhibits sub-
strate inhibition while BuChE exhibits substrate activation under
these conditions.33–35 This effect is known to result as a conse-
To further explore the involvement of the PAS of both cholines-
terases in binding cationic aminoureas, the known effect of sub-
strate binding to the anionic aspartate residues was exploited. As
can be seen in Figure 8, at substrate concentration above 1 mM
Figure 7. Acetylcholinesterase and butyrylcholineterase active site gorges showing ionic bonding between the cationic disubstituted aminourea, N-[2-(N0,N0–diisopropyl-
amino)ethyl]-10H-phenothiazine-10-carboxamide (31) and (a) D74 of the human acetylcholinesterase and (b) D70 human butyrylcholinesterase peripheral anionic sites.
With this enzyme p–p interaction between F329 and Y332 and the phenothiazine rings can also occur; in AChE this is prevented due to the presence of Y337. The figure was
generated using the SPARTAN006 program.30 First, the crystal structure of human acetylcholinesterase,32 (PDB ID: 1B41) and human butyrylcholinesterase (PDB ID: 1POI)52 was
obtained from the protein databank53 and PyMol was employed to delete all amino acids save for those selected residues found in the active site.54 Compound 31 was
optimized separately, using molecular mechanics and the MMFF force field, within SPARTAN006 to obtain the preferred conformer.30 Using SPARTAN006, the optimized compound
31 was manually fitted into the active site gorge to represent simulated binding.
(a) AChE
(b) BuChE
1.1
0.9
0.7
0.5
0.3
0.1
-0.1
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
30
35
40
45
0
10
20
30
40
Substrate Concentration (mM)
Substrate Concentration (mM)
Figure 8. Michaelis–Menten plots of the effect on cholinesterase hydrolysis of increasing concentration of acetylthiocholine (1.95 ꢁ 10ꢀ5–4.0 ꢁ 10ꢀ2 M) in the absence (d)
and presence (j) of N-[2-(N0,N0-diisopropylamino)ethyl]-10H-phenothiazine-10-carboxamide (31) on; (a) acetylcholinesterase, indicating inhibition of the enzyme at high
substrate concentration. Note that in the presence of 31 (1.8 ꢁ 10ꢀ6 M) there is no effect of this compound at substrate concentration greater that 20 mM and on (b)
butyrylcholinesterase, showing the activation of the enzyme at high substrate concentration. Note that in the presence of 31 (1.8 ꢁ 10ꢀ7 M) the inhibition by this compound
is not completely overcome even at 40 mM acetylthiocholine.

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2239
there is the expected diminished AChE activity (Fig. 8a) and ele-
vated BuChE activity (Fig. 8b). In the presence of the aminourea
(31), there is a marked inhibition of both enzymes at lower sub-
strate concentrations (up to 10 mM). However, for AChE, the inhi-
bition of the enzyme by 31 is eliminated at very high substrate
concentration (20 mM). This can be interpreted to be a conse-
quence of the constant occupation of D74 by the cationic acetylthi-
ocholine substrate at elevated concentration, preventing binding of
the cationic aminourea at this site to exert its inhibitory effect.
BuChE also shows a marked inhibition by 31 at lower substrate
concentration but this inhibition shows a trend, like AChE, to be
overcome at higher acetylthiocholine concentration (>10 mM). Un-
like the loss of aminourea inhibition at 20 mM for AChE, the inhi-
bition of BuChE by 31 is not completely lost, even at 40 mM, in
spite of the fact that the inhibitor concentration is one tenth that
used for AChE. This is attributed to the fact that, in addition to
being able to form a salt bridge with D70 in BuChE, the aminourea
5,50-dithiobis (2-nitrobenzoic acid) (DTNB) were obtained from
Sigma (St. Louis, MO). Phenothiazine-10-carbonyl chloride was
purchased from Acros Chemicals. Commercial reagents/solvents
were purchased from Fischer Scientific, Caledon Laboratories Inc.
or Aldrich Chemical Co. Inc., and were used as received, except
for the liquid amines, which were distilled prior to use.
4.2. Organic synthesis and purification
Of the total of 37 urea derivatives of phenothiazine prepared, 22
compounds (1,40 2,41 3,41 4,42 5,42 6,42 7,42 15,41,43,44 20,41,45 21,46
23,47 24,48 25,42 26,42 27,48 28,48 29,49 30,48 32,50 33,50 34,46
3651) have been synthesized previously.
For the procedure for synthesis of phenothiazine ureas, typi-
cally, a mixture of phenothiazine-10-carbonyl chloride (1.00 g,
3.82 mmol), and amine (9.55 mmol) in CH2Cl2 (40 mL) was stirred
at room temperature until analysis by thin layer chromatography
revealed no unreacted phenothiazine-10-carbonyl chloride re-
mained, or no further change was observed. All reactions were
monitored using thin layer chromatography (TLC) which was per-
formed on Macherey Nagel PolygramÒ Sil G/UV254 precoated silica
plates (0.20 mm thickness). The developing solvent was dichloro-
methane or dichoromethane/ethyl acetate mixtures. TLC plates
were visualized using short wave (254 nm) UV lamp, then an I2
chamber. Reaction periods were typically in the range of 1ꢀ3 h.
The reaction mixture was then washed with 0.1 M HCl
(3 ꢁ 30 mL), 0.1 M NaOH (2 ꢁ 30 mL), and water (1 ꢁ 30 mL) then
dried over MgSO4. The solvent was removed by reduced pressure
to afford the crude urea. This material was dissolved in the mini-
mum amount of petroleum ether, and precipitated by the addition
of CH2Cl2. All solids were recrystallized using a mixed solvent tech-
nique with petroleum ether/CH2Cl2 mixtures, except for compound
38 which was recrystallized from petroleum ether.
(31) is also able to bind (Fig. 7b), through p–p interaction, between
the phenothiazine tricycle and F329 and Y332 in BuChE.11,12,27 Be-
cause of the presence of Y337 in AChE,9 this additional
cannot occur with AChE (Fig. 7a).
p–p binding
3. Conclusions
Urea derivatives of phenothiazine inhibit human BuChE to an ex-
tent that can be directly related to the molecular volume of the com-
pound. These compounds show little or no inhibition of AChE
becauseof activesitevolume restrictions andbecauseof interference
with binding of the phenothiazine tricycle to the E-helix,12 due to the
presence of Y337 in AChE (seeFig. 7a). Substituted aminourea deriv-
ativesinhibittheactivityof bothcholinesterases throughionicbond-
ing between a cationic distal nitrogen on the substituent attached to
phenothiazine and the aspartate residue of the peripheral anionic
site. These aminoureas provide a unique class of phenothiazine
derivatives that can be potent inhibitors of both cholinesterases.
Such inhibitors could prove useful for comparison with phenothia-
zine derivatives that are selective inhibitors of AChE or BuChE in
the quest for the most effective treatment of cognitive impairment.
Currently, drugs used for the treatment of symptoms of AD are
thought to exert their effects by inhibiting the enzymatic activity
of cholinesterases, prolonging the action of acetylcholine in cholin-
ergic neurotransmission.20 In addition, long-term use of certain cho-
linesterase inhibitors has been shown to lead to overexpression of
cholinesterases, AChE in particular.36,37 Whether compounds de-
scribed herein are also capable of inducing changes in cholinesterase
expression will require further studies. It has also been shown that
the peripheral anionic site of AChE promotes amyloid fibril forma-
tion.38 Since phenothiazine aminoureas, such as 31, appear to affect
the peripheral anionic site of AChE, they should interfere with AChE
promoting amyloid fibril formation. It has also been reported that
the 40 amino acid residue segment at the carboxy terminal of BuChE
attenuates the process of amyloid fibril formation.39 Since aminou-
reas like 31 appear to bind to the peripheral anionic site of BuChE,
well away from the carboxy terminal, they should not interfere with
this enzyme’s ability to attenuate amyloid fibril formation.
4.3. Analysis of synthesized compounds
Melting points were measured using a Mel-Temp II apparatus
from Laboratory Services Inc. and are uncorrected. Infrared spectra
were recorded as Nujol mulls between sodium chloride plates on a
Nicolet model 205 or a Nicolet Avatar 330 FT-IR spectrometer. Peak
positions were reproducible within 1ꢀ2 cmꢀ1. Nuclear magnetic
resonance spectra were recorded at the Atlantic Region Magnetic
Resonance Centre, Dalhousie University, on a Bruker AC-250F oper-
ating at 250.1 MHz for proton and 62.9 MHz for carbon or a Bruker
AVANCE 500 operating at 500.13 MHz for proton and 125.76 MHz
for carbon-13. Chemical shifts are reported in ppm relative to TMS,
in CDCl3 solution. Reported multiplicities are apparent. Mass spec-
tra were recorded at Dalhousie University on a CEC 21-110B spec-
trometer using electron ionization at 70 V and an appropriate
source temperature with samples being introduced by means of
a heatable port probe. Accurate mass measurements were also
made on this machine operated at a mass resolution of 8000 by
computer controlled peak matching to appropriate PFK reference
ions. Mass measurements were routinely within 3 ppm of the cal-
culated value. The purity of the synthesized compounds was also
determined using an Agilent Technologies 1200 series HPLC sys-
tem with either a C18 reverse phase column with methanol as
the mobile phase or a silica gel column with 5% methanol in dichlo-
romethane as the mobile phase.
4. Experimental
4.1. Materials
4.4. Analytical data
Purified human plasma butyrylcholinesterase (BuChE, EC
3.1.1.8) and recombinant mutant BuChE species (D70G, F329A
and Y332A) were from Dr. Oksana Lockridge (University of Nebras-
ka Medical Center).35 Purified recombinant human acetylcholines-
terase (AChE, EC 3.1.1.7), acetylthiocholine, butyrylthiocholine and
4.4.1. Phenothiazine-10-carboxamide40 (1)
Compound 1 55%; colourless crystals, mp 191–194 °C. IR (Nu-
jol): 3451, 3286, 3183, 1655, 1591, 1566, 1308, 1268, 1240, 1093,

2240
S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
1033, 755 cmꢀ1
.
1H NMR: 5.19(s, 2H), 7.17–7.23(m, 2H), 7.28–
4.4.8. N-Neopentyl-10H-phenothiazine-10-carboxamide (8)
Compound 66%, colourless crystals, mp 80.4–82 °C. IR:
3467, 1688, 1512, 1315, 1279, 1250, 1224, 767, 760 cmꢀ1 1H
NMR: 0.87(s, 9H), 3.07(d, J = 6.2 Hz, 2H), 5.03–5.05(m, 1H),
7.19–7.24(m, 2H), 7.30–7.35(m, 2H), 7.41(dd, J = 7.7, 1.3 Hz,
2H), 7.59(dd, J = 7.9, 1.1 Hz, 2H). 13C NMR: 27.1, 32.1, 51.9,
126.5, 127.1, 127.2, 128.0, 133.5, 138.8, 154.8. EI-MS (m/z):
312(M+), 200, 199(base), 198, 197, 167, 166, 154, 114, 95, 71,
69. HRMS (EI) M+(found): 312.1294; calcd: 312.1296 (for
C18H20N2OS).
7.35(m, 2H), 7.40(dd, J = 7.6, 1.2 Hz, 2H), 7.59(dd, J = 7.9, 0.9 Hz,
2H). 13C NMR: 126.7, 126.9, 127.3, 128.1, 133.4, 138.7, 155.5. EI-
MS (m/z): 242(M+), 200, 199(base), 198, 197, 167, 166, 154. HRMS
(El) M+(found): 242.0505; calcd for C13H10N2OS: 242.0514.
8
.
4.4.2. N-Methyl-10H-phenothiazine-10-carboxamide41 (2)
Compound 2 68%; colourless crystals; mp 168–169 °C. IR: 3311,
1660, 1519, 1409, 1313, 1299, 1286, 1253, 1156, 760, 747,
734 cmꢀ1 1H NMR: 2.82(d, J = 4.6 Hz, 3H), 4.95(broad s, 1H),
.
7.16–7.22(m, 2H), 7.28–7.34(m, 2H), 7.39(dd, J = 7.6, 1.5 Hz, 2H),
7.57(dd, J = 7.9, 1.2 Hz, 2H). 13C NMR: 27.6, 126.5, 127.1, 127.2,
128.0, 133.4, 138.8, 155.3. EI-MS (m/z): 256(M+; base), 200, 199,
198, 167, 166, 154. HRMS (EI) M+(found): 256.0685; calcd:
256.0670 (for C14H12N2OS).
4.4.9. N-(2-Methoxyethyl)-10H-phenothiazine-10-carboxamide
(9)
Compound 9 81%; colourless crystals, mp 118–120 °C. IR: 3380,
1666, 1500, 1314, 1299, 1276, 1255, 1231, 1196, 1100, 1026, 998,
775, 750 cmꢀ1. 1H NMR: 3.31(s, 3H), 3.41–3.48 (m, 4H), 5.39(broad
s, 1H), 7.17–7.22(m, 2H), 7.28–7.33(m, 2H), 7.39(dd, J = 7.7, 1.3 Hz,
2H), 7.58(dd, J = 7.9, 1.1 Hz, 2H). 13C NMR: 40.7, 58.7, 71.3, 126.4,
126.9, 127.1, 1278.0, 133.3, 138.7, 154.6. EI-MS (m/z): 300(M+),
201, 200, 199(base), 198, 167, 166. HRMS (EI) M+(found):
300.0934; calcd: 300.0932 (for C16H16N2O2S).
4.4.3. N-Ethyl-10H-phenothiazine-10-carboxamide41 (3)
Compound 3 42%; colourless crystals; mp 114–116 °C. IR: 3439,
3310, 1645, 1589, 1518, 1320, 1300, 1281, 1249, 1236, 757 cmꢀ1
.
1H NMR: 1.10(t, J = 7.2 Hz, 3H), 3.22–3.32(m, 2H), 5.00(broad s,
1H), 7.13–7.20(m, 2H), 7.26–7.32(m, 2H), 7.32–7.38(m, 2H),
7.54–7.57(m, 2H). 13C NMR: 15.2, 35.8, 126.5, 127.1, 127.2, 128.1,
133.4, 138.8, 154.5. EI-MS (m/z): 270(M+), 201, 200, 199(base),
198, 167, 166, 154, 140, 127. HRMS (EI) M+(found): 270.0832;
calcd: 270.0827 (for C15H14N2OS).
4.4.10. N-Cyclopropyl-10H-phenothiazine-10-carboxamide (10)
Compound 10 58%; colourless crystals, mp 132–133.5 °C. IR:
3533, 3464, 3325, 1679, 1645, 15889, 1509, 1314, 1299, 1284,
1261, 1249, 778, 738 cmꢀ1 1H NMR: 0.45–0.51(m, 2H), 0.68–
.
4.4.4. N-Propyl-10H-phenothiazine-10-carboxamide42 (4)
Compound 4 71%; colourless crystals; mp 105–107 °C. IR: 3384,
3317, 1660, 1590, 1504, 1317, 1299, 1280, 1258, 1233, 1154, 762,
0.76(m, 2H), 2.61–2.70(m, 1H), 5.18(broad s, 1H), 7.15–7.21(m,
2H), 7.26–7.33(m, 2H), 7.38(dd, J = 7.6, 1.5 Hz, 2H), 7.54(dd,
J = 7.9, 1.2 Hz, 2H). 13C NMR: 7.1, 23.8, 126.8, 127.3, 127.5, 128.3,
133.6, 138.9, 155.7. EI-MS (m/z): 282(M+), 201, 200, 199(base),
198, 167, 166, 154. HRMS (EI) M+(found): 282.0846; calcd:
282.0827 (for C16H14N2OS).
750 cmꢀ1 1H NMR: 0.88(t, J = 7.5 Hz, 3H), 1.51(sextet, J = 7.3 Hz,
.
2H), 3.17–3.25(m, 2H), 5.00(broad s, 1H), 7.15–7.23(m, 2H),
7.27–7.35(m, 2H), 7.39(dd, J = 7.9, 1.8 Hz, 2H), 7.57(dd, J = 7.9,
1.2 Hz, 2H). 13C NMR: 11.3, 23.0, 42.6, 126.4, 127.0, 127.1, 128.0,
133.4, 138.8, 154.5. EI-MS (m/z): 284(M+), 201, 200, 199(base),
198, 167, 166, 154, 140, 84, 48. HRMS (EI) M+(found): 284.0992;
calcd: 284.0983 (for C16H16N2OS).
4.4.11. N-Cyclobutyl-10H-phenothiazine-10-carboxamide (11)
Compound 11 61%, colourless crystals, mp 133–135.5 °C. IR:
3319, 1673, 1655, 1589, 1567, 1514, 1315, 1300, 1284, 1253,
1206, 1032, 754 cmꢀ1 1H NMR: 1.57–1.85(m, 4H), 2.23–2.38(m,
.
4.4.5. N-Isopropyl-10H-phenothiazine-10-carboxamide42 (5)
Compound 5 68%; colourless crystals, mp 116–118 °C. IR: 3308,
1654, 1515, 1326, 1311, 1280, 1250, 1233, 1167, 760, 749 cmꢀ1. 1H
NMR: 1.12(d, J = 6.7 Hz, 6H), 3.94–4.10(m, 1H), 4.75–4.79(d,
J = 7.3 Hz, 1H), 7.14–7.21(m, 2H), 7.26–7.33(m, 2H), 7.38(dd,
J = 7.6, 1.5 Hz, 2H), 7.56(dd, J = 7.6, 1.5 Hz, 2H). 13C NMR: 23.0,
43.0, 126.4, 127.0, 127.1, 128.1, 133.4, 138.8, 153.8. EI-MS (m/z):
284(M+), 200, 199(base), 198, 197, 196, 171, 167, 166. HRMS (EI)
M+(found): 284.0984; calcd: 284.0983 (for C16H16N2OS).
2H), 4.22–4.38(m, 1H), 5.10(d, J = 7.3 Hz, 1H), 7.13–7.20(m, 2H),
7.25–7.33(m, 2H), 7.37(dd, J = 7.6, 1.5 Hz, 2H), 7.56(dd, J = 7.6,
1.2 Hz, 2H). 13C NMR: 15.0, 31.4, 46.2, 126.5, 127.0, 127.2, 128.1,
133.4, 138.7, 153.5. EI-MS (m/z): 296(M+), 201, 200, 199(base),
198, 167, 166, 154, 55. HRMS (EI) M+(found): 296.0986; calcd:
296.0983 (for C17H16N2OS).
4.4.12. N-Cyclopentyl-10H-phenothiazine-10-carboxamide (12)
Compound 12 52%; colourless crystals, mp 125–126 °C. IR:
3412, 1677, 1503, 1316, 1303, 1252, 1124, 1030, 763 cmꢀ1 1H
.
4.4.6. N-Butyl-10H-phenothiazine-10-carboxamide42 (6)
Compound 6 72%; colourless crystals, mp 111–114 °C. IR: 3334,
NMR: 1.25–1.38(m, 2H), 1.52–1.63(m, 4H), 1.91–2.03(m, 2H),
4.06–4.20(m, 1H), 4.89(d, J = 7.0 Hz, 1H), 7.15–7.21(m, 2H), 7.27–
7.33(m, 2H), 7.38(dd, J = 7.6, 1.5 Hz, 2H), 7.56(dd, J = 7.9, 1.2 Hz,
2H). 13C NMR: 23.6, 33.1, 52.7, 126.4, 126.9, 127.1, 128.0, 133.3,
138.8, 154.1. EI-MS (m/z): 310(M+), 200, 199(base), 198, 167, 166,
154, 127, 88, 86, 84. HRMS (EI) M+(found): 310.1140; calcd:
310.1140.0827 (for C18H18N20S).
1659, 1531, 1317, 1301, 1283, 1253, 1232, 1123, 756 cmꢀ1 1H
.
NMR: 0.91(t, J = 7.5 Hz, 3H), 1.26–1.36(m, 2H), 1.44–1.52(m, 2H),
3.22–3.27(m, 2H), 4.99(broad s, 1H), 7.17–7.22(m, 2H), 7.29–
7.34(m, 2H), 7.39(dd, J = 7.7, 1.5 Hz, 2H), 7.58(dd, J = 7.9, 1.3 Hz,
2H). 13C NMR: 13.7, 20.0, 31.9, 40.6, 126.4, 127.0, 127.1, 128.0,
133.4, 138.9, 154.5. EI-MS (m/z): 298(M+), 201, 200, 199(base),
198, 197, 171, 167, 166, 154. HRMS (EI) M+(found): 298.1136;
calcd: 298.1140 (for C17H18N2OS).
4.4.13. N-Cyclohexyl-10H-phenothiazine-10-carboxamide (13)
Compound 13 88%; colourless crystals, mp 159–160 °C. IR:
3416, 1676, 1507, 1321, 1256, 1215, 1030, 765 cmꢀ1
.
1H
4.4.7. N-tert-Butyl-10H-phenothiazine-10-carboxamide42 (7)
Compound 7 99%; colourless crystals, mp 115–118 °C. IR: 3436,
1679, 1505, 1264, 1315, 1283, 1255, 1203, 756, 746 cmꢀ1. 1H NMR:
1.34(s, 9H), 4.90(broad s, 1H), 7.16–7.21(m, 2H), 7.28–7.33(m, 2H),
7.40(dd, J = 7.8, 1.4 Hz, 2H), 7.57(dd, J = 7.8, 1.2 Hz, 2H). 13C NMR:
29.1, 51.2, 126.3, 127.0, 127.1, 128.0, 133.4, 139.0, 153.4. EI-MS
(m/z): 298(M+), 201, 200, 199(base), 198, 197, 167, 166, 57. HRMS
(EI) M+(found): 298.1140; calcd: 298.1140 (for C17H18N2OS).
NMR: 1.30–1.15(m, 3H), 1.30–1.38(M, 2H), 1.54–1.63(m, 3H),
1.90–1.93(m, 2H), 3.65–3.72(m, 1H), 4.85(d, J = 7.5 Hz, 1H),
7.15–7.18(m, 2H), 7.27–7.30(m, 2H), 7.37(dd, J = 7.7, 1.1 Hz,
2H), 7.56(dd, J = 7.9, 0.9 Hz, 2H). 13C NMR: 24.7, 25.5, 33.2,
49.7, 126.3, 126.9, 127.0, 128.0, 133.3, 138.8, 153.7. EI-MS
(m/z): 324(M+), 201, 200, 199(base), 198, 167, 166, 154, 128.
HRMS (EI) M+(found): 324.1290; calcd: 324.1296 (for
C19H20N2OS).

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2241
4.4.14. N-(Adamant-1-yl)-10H-phenothiazine-10-carboxamide
(14)
4.4.20. N-Benzyl-10H-phenothiazine-10-carboxamide41,45 (20)
Compound 20 94%; colourless crystals, mp 135–136.8 °C (lit.
Compound 14 75%, colourless crystals, mp 167–167.5 °C. IR:
3426, 1692, 1512, 1359, 1343, 1319, 1311, 1298, 1250, 1219,
mp 137–140 °C).44 IR (Nujol): 3355, 1663, 1508, 1313, 1283,
1259, 1230, 1028, 754, 699 cmꢀ1 1H NMR (CDCl3): 4.52 (d,
.
1085, 767, 7.38 cmꢀ1
.
1H NMR: 1.61–1.67(m, 6H), 1.87(d, 1H),
J = 5.7 Hz, 2H), 5.38–5.39 (m, 1H), 7.22–7.26 (m, 2H), 7.28–7.38
(m, 7H), 7.44 (dd, J = 7.8, 1.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H). 13C
NMR (CDCl3): 44.9, 126.7, 127.2, 127.3, 127.4, 127.5, 128.2,
128.7, 133.6, 38.7, 138.9, 154.7. EI-MS (m/z): 332 (M+), 201, 200,
199(base), 198, 167, 154, 91, 77. HRMS (EI) M+(found): 332.0982;
calcd: 332.0983 (for C20H16N2OS).
1.93–1.97(m, 6H), 2.02–2.10(m, 3H), 4.79(broad s, 1H), 7.16–
7.19(m, 2H), 7.25–7.35(m, 2H), 7.37(dd, J = 7.9, 1.2 Hz, 2H),
7.58(dd, J = 7.9, 1.2 Hz, 2H). 13C NMR (CDCl3): 29.6, 36.5, 42.0,
51.9, 126.4, 127.1, 127.2, 128.1, 133.4, 139.0, 153.1. EI-MS (m/z):
376(M+), 200, 199(base), 198, 167, 166, 154, 135, 120, 93, 79, 77.
HRMS (EI) M+(found): 376.1607; calcd: 376.1609 (for C23H24N2OS).
4.4.21. N-(2-Phenylethyl)-10H-phenothiazine-10-
carboxamide46 (21)
4.4.15. N-Phenyl-10H-phenothiazine-10-carboxamide41,43,44
(15)
Compound 21 18%, Colourless crystals, mp 111.5–113 °C. IR
(Nujol): 3419, 1677, 1515, 1306, 1272, 1251, 1031, 773, 757, 750,
Compound 15 68%; colourless crystals, mp 175.9–176.8 °C (lit.
mp 180 °C).43 IR: 3314, 3272, 1674, 1660, 1597, 1525, 1440,
706 cmꢀ1 1H NMR (CDCl3): 2.78(t, J = 6.7 Hz, 2H), 3.43–3.51(m,
.
1324, 1236, 757 cmꢀ1 1H NMR: 6.91(broad s, 1H), 7.02–7.09(m,
.
2H), 4.96–5.00(m, 1H), 7.09–7.43(m, 13H). 13C NMR (CDCl3):
35.7, 42.1, 126.4, 126.5, 127.0, 127.1, 127.9, 128.54, 128.8, 133.4,
138.6, 139.0, 154.5. EI-MS (m/z): 346(M+), 201, 200, 199(base),
198, 167, 166, 154. HRMS (EI) M+(found): 346.1129; calcd:
346.1140 (for C21H18N2OS).
1H), 7.22–7.41(m, 8H), 7.45(dd, J = 7.6, 1.5 Hz, 2H), 7.67(dd,
J = 7.9, 1.2 Hz, 2H). 13C NMR: 119.6, 123.6, 126.9, 127.0, 127.4,
128.3, 128.9, 133.7, 138.0, 138.3, 151.8. EI-MS (m/z): 318(M+),
201, 200, 199(base), 198, 167, 166, 154, 119, 84, 77. HRMS (EI)
M+(found): 318.0827; calcd: 318.0827 (for C19H14N2OS).
4.4.22. N-(Naphthalen-1-ylmethyl)-10H-phenothiazine-10-
carboxamide (22)
4.4.16. N-(4-Methoxyphenyl)-10H-phenothiazine-10-
carboxamide (16)
Compound 22 69%; colourless crystals, mp 182–184.5 °C. IR
Compound 16 77%; colourless crystals, mp 149–151.5 °C. IR
(Nujol): 3297, 1670, 1600, 1529, 1516, 1319, 1257, 1226, 1178,
(Nujol): 3338, 1650, 1527, 1255, 1230, 805, 776, 753 cmꢀ1 1H
.
1034, 829, 752 cmꢀ1 1H NMR (CDCl3): 3.77(s, 3H), 6.75–6.87(m,
.
NMR (CDCl3): 4.92(d, J = 5.2 Hz, 2H), 5.28–5.32(m, 1H), 7.12–
8.07(m, 15H). 13C NMR (CDCl3): 43.2, 123.5, 125.4, 126.0, 126.2,
126.55, 126.63, 127.0, 127.3, 128.1, 128.5, 128.8, 131.4, 133.5,
133.8, 138.6, 154.4. EI-MS (m/z): 382(M+), 200, 199(base), 198,
183, 167, 166, 154, 141, 139, 128, 127. HRMS (EI) M+(found):
382.1128; calcd: 382.1140 (for C24H18N2OS).
3H), 7.21–7.48(m, 8H), 7.66(d, J = 7.3 Hz, 2H). 13C NMR (CDCl3):
55.5, 114.1, 121.9, 126.8, 127.0, 127.4, 128.3, 131.1, 133.7, 138.4,
152.3, 156.2. EI-MS (m/z): 348(M+), 201, 200, 199(base), 198, 167,
166. HRMS (EI) M+(found): 348.0935; calcd: 348.0932 (for
C20H16N2O2S).
4.4.17. N-p-Tolyl-10H-phenothiazine-10-carboxamide (17)
Compound 17 62%; colourless crystals, mp 174.3–175 °C. IR
(Nujol): 3408, 1686, 1592, 1515, 1403, 1316, 1296, 1276, 1258,
4.4.23. N,N-Dimethyl-10H-phenothiazine-10-carboxamide (23)
Compound 23 50%; colourless crystals, mp 99–101 °C (lit. mp
94–96 °C).47 IR (Nujol): 1668, 1587, 1281, 1247, 1171, 1087,
1226, 1193, 813, 762, 755 cmꢀ1 1H NMR (CDCl3): 2.31(s, 3H),
.
1066, 1029, 1021, 945, 760, 750 cmꢀ1 1H NMR (CDCl3): 2.86(s,
.
6.85(broad s, 1H), 7.08–7.12(m, 2H), 7.24–7.30(m, 4H), 7.36–
7.41(m, 2H), 7.46(d, J = 7.6 Hz, 2H), 7.69(d, J = 7.9 Hz, 2H). 13C
NMR (CDCl3): 20.8, 119.8, 126.9, 127.1, 127.4, 128.3, 129.5,
133.2, 133.7, 135.5, 138.4, 152.0. EI-MS (m/z): 332(M+; base),
201, 200, 199, 198, 167, 166, 154, 133, 132. HRMS (EI) M+(found):
332.0984; calcd: 332.0983 (for C20H16N2OS).
6H), 7.05–7.10(m, 2H), 7.19–7.24(m, 2H), 7.28(dd, J = 7.6, 1.4 Hz,
2H), 7.61(dd, J = 8.1, 1.1 Hz, 2H). 13C NMR (CDCl3): 37.7, 121.1,
124.7, 127.5, 127.5, 128.0, 141.5, 157.9. EI-MS (m/z): 270(M+),
200, 199(base), 198, 197, 154, 72. HRMS (EI) M+(found):
270.0834; calcd: 270.0827 (for C15H14N2OS).
4.4.24. N,N-Diethyl-10H-phenothiazine-10-carboxamide (24)
Compound 24 84%; colourless crystals, mp 86.7–88 °C (lit. mp 91–
93 °C).48 IR (Nujol): 1676, 1415, 1311, 1289, 1269, 1237, 1215, 1157,
4.4.18. N-(4-Chlorophenyl)-10H-phenothiazine-10-
carboxamide (18)
Compound 18 85%; colourless crystals, mp 171.6–172.9 °C. IR
(Nujol): 3418, 1694, 1591, 1518, 1398, 1308, 1232, 1194, 1088,
1091, 1036, 847, 752 cmꢀ1 1H NMR (CDCl3): 1.03(t, J = 7.1 Hz, 6H),
.
3.37(q, J = 7.1 Hz, 4H), 6.99–7.03(m, 2H), 7.13–7.16(m, 2H),
7.21(dd, J = 7.7, 1.2 Hz, 2H), 7.43(d, J = 8.0 Hz, 2H). 13C NMR (CDCl3):
12.9, 42.4, 119.3, 124.3, 125.9, 127.3, 127.4, 141.6, 156.9. EI-MS (m/z):
298(M+), 199, 198(base), 197, 196, 167, 166, 101, 100, 72. HRMS (EI)
M+(found): 298.1142; calcd: 298.1140 (for C17H18N2OS).
1012, 824, 766, 758 cmꢀ1 1H NMR (CDCl3): 6.89(broad s, 1 H),
.
7.20–7.42(m, 8H), 7.46(dd, J = 7.6, 1.5 Hz, 2H), 7.65(dd, J = 7.9, 1.5
hz, 2H). 13C NMR (CDCl3): 120.9, 127.0, 127.1, 127.5, 128.4,
128.6, 128.9, 133.7, 136.8, 138.1, 151.7. EI-MS (m/z): 354(M+2),
352(M+), 200, 199(base), 198, 167, 166, 154, 153, 125. HRMS (EI)
M+(found): 352.0422; calcd: 352.0437 (for C19H13N2OSCl).
4.4.25. N,N-Dipropyl-10H-phenothiazine-10-carboxamide42
(25)
4.4.19. N-o-Tolyl-10H-phenothiazine-10-carboxamide (19)
Compound 19 69%; colourless crystals, mp 151–153.5 °C. IR
(Nujol): 3435, 3417, 1687, 1588, 1529, 1312, 1243, 1198,
Compound 25 51%; colourless crystals, mp 84.2–85.8 °C. IR (Nu-
jol): 1671, 1591, 1570, 1415, 1304, 1237, 1206, 1163, 1135, 1102,
1038, 931, 755, 741 cmꢀ1 1H NMR (CDCl3): 0.82(t, J = 7.6 Hz, 6H),
.
760 cmꢀ1 1H NMR (CDCl3): 2.02 (s, 3H), 6.81 (broad s, 1H), 6.95–
.
1.43–1.59(m, 4H), 3.21–3.25(m, 4H), 6.98–7.06(m, 2H), 7.11–
7.19(m, 2H), 7.22(dd, J = 7.7, 1.5 Hz, 2H), 7.41(dd, J = 7.9, 1.3 Hz,
2H). 13C NMR (CDCl3): 11.4, 21.2, 50.1, 119.1, 124.4, 125.8, 127.4,
127.5, 141.7, 157.2. EI-MS (m/z): 326(M+; base), 200, 199, 198,
197, 167, 166, 154, 129, 128, 86. HRMS (EI) M+(found):
326.1457; calcd: 326.1453 (for C19H22N2OS).
7.02 (m, 1H), 7.07–7.30 (m, 4H), 7.34–7.38 (m, 2H), 7.46
(dd, J = 7.6, 1.5 Hz, 2H), 7.68 (dd, J = 7.9, 1.2 Hz, 2H), 7.93 (d,
J = 7.9 Hz, 1H). 13C NMR (CDCl3): 17.7, 121.7, 124.1, 126.9, 127.0,
127.3, 127.4, 127.8, 128.3, 130.3, 133.8, 136.3, 138.4, 152.1.
EI-MS (m/z): 33(M+), 200, 199(base), 198, 167, 154, 133. HRMS
(EI) M+(found): 332.0975; calcd: 332.0983 (for C20H16N2OS).

2242
S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
4.4.26. N,N-Diisopropyl-10H-phenothiazine-10-carboxamide42
(26)
368, 354, 269, 199, 198, 172, 171(base), 167, 157, 129, 114, 72.
HRMS (EI) M+(found): 369.1875; calcd: 369.1875 (for
C21H27N3OS).
Compound 26 22%; colourless crystals, mp 163–165 °C. IR (Nu-
jol): 1675, 1592, 1570, 1432, 1314, 1285, 1243, 1225, 1205, 1155,
1089, 1041, 818, 748, 740 cmꢀ1. 1H NMR (CDCl3): 1.23(d, J = 6.7 Hz,
12H), 4.02 (m, 2H), 6.94–7.01(m, 2H), 7.08–7.19(m, 4H), 7.30(dd,
J = 7.9, 1.2 Hz, 2H). 13C NMR (CDCl3): 20.4, 48.3, 117.9, 124.0,
124.4, 127.3, 127.4, 141.7, 155.1. EI-MS (m/z): 326(M+), 200, 199,
198, 197, 167, 166, 154, 129, 128, 87, 86, 69. HRMS (EI) M+(found):
326.1462; calcd: 326.1453 (for C19H22N2OS).
4.4.32. N-[2-(Pyrrolidinyl)ethyl]-10H-phenothiazine-10-
carboxamide50 (32)
Compound 32 28%, colourless crystals, mp 94–95.5 °C. IR (Nu-
jol): 3373, 3331, 1673, 1536, 1494, 1309, 1277, 1247, 1153,
755 cmꢀ1 1H NMR: 1.65–1.71(m, 4H), 2.42–2.46(m, 4H), 2.57(t,
.
J = 6.3 Hz, 2H), 3.32–3.37(m, 2H), 5.71(t, J = 5.8 Hz, 1H), 7.15–
7.19(m, 2H), 7.26–7.30(m, 2H), 7.37(dd, J = 7.7, 1.3 Hz, 2H),
7.56(dd, J = 8.0, 1.0 Hz, 2H). 13C NMR: 23.7, 39.6, 53.7, 54.3,
126.5, 127.1, 127.2, 128.0, 133.4, 139.0, 154.8. EI-MS (m/z):
339(M), 269, 201, 200, 199(base), 198, 167, 166, 142, 141, 84.
HRMS (EI) M+ (found): 339.1400; calcd: 339.1405 (for
C19H21N3OS).
4.4.27. (10H-Phenothiazin-10-yl)(pyrrolidin-1-yl)methanone
(27)
Compound 27 74%; colourless crystals, mp 136.2–137.5 °C (lit.
mp 137–139 °C).48 IR (Nujol): 1653, 1588, 1567, 1308, 1244,
.
1233, 1033, 760 cmꢀ1 1H NMR (CDCl3): 1.72–1.80(m, 4H),
3.19(m, 4H), 7.05–7.08(m, 2H), 7.19–7.23(m, 2H), 7.27(dd, J = 7.7,
1.2 Hz, 2H), 7.69(d, J = 8.1 Hz, 2H). 13C NMR (CDCl3): 25.3, 47.7,
122.5, 124.9, 127.4, 127.5, 129.4, 141.4, 156.4. EI-MS (m/z):
296(M+), 199, 198, 197, 171, 154, 98. HRMS (EI) M+(found):
296.0997; calcd: 296.0983 (for C17H16N2OS).
4.4.33. N-[2-(Piperidinyl)ethyl]-10H-phenothiazine-10-
carboxamide50 (33)
Compound 33 63%, colourless crystals, mp 67–70 °C. IR (Nujol):
3427, 1685, 1505, 1316, 1298, 1282, 1250, 1219, 773, 752 cmꢀ1. 1H
NMR: 1.33–1.45(overlapping m, 6H), 2.23–2.34(m, 4H), 2.38(t,
J = 6.0 Hz, 2H), 3.28–3.32(m, 2H), 5.87(t, J = 5.6 Hz, 1H), 7.16–
7.19(m, 2H), 7.28–7.31(m, 2H), 7.37(dd, J = 7.7, 1.1 Hz, 2H),
7.56(dd, J = 7.9, 0.9 Hz, 2H). 13C NMR: 24.3, 26.0, 37.4, 53.9, 56.6,
126.3, 127.0, 127.1, 127.8, 133.2, 138.8, 154.5. EI-MS (m/z):
4.4.28. (10H-Phenothiazin-10-yl)(piperidin-1-yl)methanone
(28)
Compound 28 82%; colourless crystals, mp 118–121 °C (lit. mp
118–120 °C).48 IR (Nujol): 1651, 1589, 1567, 1481, 1404, 1307,
+
340(M+H+), 353(Mꢀ ), 269, 200, 199, 198, 167, 156, 155(base),
1277, 1246, 1219, 1133, 1028, 988, 939, 771, 762, 648 cmꢀ1 1H
.
99, 98. HRMS (EI) M+ (found): 353.1559; calcd: 353.1562 (for
C20H23N3OS).
NMR (CDCl3): 1.41–1.48(m, 4H), 1.51–1.56(m, 2H), 3.38–3.43(m,
4H), 7.08–7.12(m, 2H), 7.22–7.25(m, 2H), 7.29(d, J = 7.6 Hz, 2H),
7.63(d, J = 8.1 Hz, 2H). 13C NMR (CDCl3): 24.3, 25.3, 46.7, 121.1,
124.7, 127.38, 127.44, 127.7, 141.6, 156.9. EI-MS (m/z): 310 (M+),
199, 198, 113, 112, 69, 56. HRMS (EI) M+(found): 310.1128; calcd:
310.1140 (for C18H18N2OS).
4.4.34. N-(2-Morpholinoethyl)-10H-phenothiazine-10-
carboxamide46 (34)
Compound 34 67%, colourless crystals, mp 106.5–108 °C. IR
(Nujol): 3440, 1675, 1510, 1143, 1113, 936, 913, 850, 769 cmꢀ1
.
4.4.29. Morpholino(10H-phenothiazin-10-yl)methanone49 (29)
Compound 29 95%; colourless crystals, mp 135.2–137.2 °C. IR
(Nujol): 1653, 1409, 1357, 1315, 1301, 1283, 1267, 1249, 1226,
1H NMR: 2.31–2.39(m, 4H), 2.42(t, J = 6.4 Hz, 2H), 3.28–3.35(m,
2H), 3.48–3.60(m, 4H), 5.75(broad s, 1H), 7.14–7.22(m, 2H),
7.26–7.33(m, 2H), 7.38(dd, J = 7.6, 1.5 Hz, 2H), 7.54 (dd, J = 7.9,
1.2 Hz, 2H). 13C NMR: 37.0, 53.0, 56.5, 67.0, 126.5, 127.1, 127.2,
128.0, 133.4, 138.8, 154.6. EI-MS (m/z): 355(M+), 200, 199(base),
198, 167, 157, 100. HRMS (EI) M+ (found): 355.1367; calcd:
355.1354 (for C19H21N3OS).
1117, 998, 939, 757 cmꢀ1 1H NMR (CDCl3): 3.36–3.41(m, 4H),
.
3.55–3.59(m, 4H), 7.12–7.15(m, 2H), 7.26–7.30(m, 2H), 7.33–
7.35(m, 2H), 7.71(d, J = 8.1 Hz, 2H). 13C NMR (CDCl3): 46.1, 66.2,
122.1, 125.2, 127.5, 127.6, 128.9, 141.2, 157.1. EI-MS (m/z):
312(M+; base), 199, 198, 171, 154, 113, 112, 69. HRMS (EI)
M+(found): 312.0925; calcd: 312.0932 (for C17H16N2O2S).
4.4.35. N-(3-Morpholinopropyl)-10H-phenothiazine-10-
carboxamide (35)
4.4.30. N-[2-(N0,N0–Diethylamino)ethyl]-10H-phenothiazine-
10-carboxamide hydrochloride salt (30)
Compound 35 62%, colourless crystals, mp 112–114 °C. IR (Nu-
jol): 3348, 1656, 1516, 1319, 1278, 1254, 1119, 767, 759 cmꢀ1 1H
.
Compound 30 43%; colourless crystals, mp 184–186 °C(dec) (lit.
NMR: 1.60–1.70(m, 2H), 2.27–2.38 (m, 6H), 3.34–3.44 (m, 6H),
5.79(t, J = 5.0 Hz, 1H), 7.15–7.22(m, 2H), 7.27–7.34(m, 2H),
7.39(dd, J = 7.6 Hz, 2H), 7.61 (dd, J = 7.9, 1.3 Hz, 2H). 13C NMR:
25.4, 40.3, 53.7, 57.4, 66.6, 126.3, 126.9, 127.1, 128.0, 133.1,
139.0, 154.7. EI-MS (m/z): 369(M+), 201, 200, 199, 198, 172,
171(base), 167, 166, 100. HRMS (EI) M+ (found): 369.1506; calcd:
369.1511 (for C20H23N3O2S).
mp 184–186 °C(dec)).48 IR (Nujol): 3366, 1658, 1512, 1319, 1256,
.
771 cmꢀ1 1H NMR (DMSO-d6): 1.20(t, J = 7.2 Hz, 6H), 3.07–
3.11(m, 6H), 7.21–7.28(m, 2H), 7.32–7.39(m, 2H), 7.47(dd, J = 7.6,
1.5 Hz, 2H), 7.60(dd, J = 7.9, 0.9 Hz, 2H), 10.72(broad s, 1H). 13C
NMR (DMSO-d6): 8.5, 35.4, 46.9, 49.2, 126.6, 127.4, 127.6, 127.8,
132.3, 138.6, 154.3. EI-MS (m/z): 342(M++H+), 269, 201, 200, 199,
198, 167, 166, 154, 143, 100, 86. HRMS (EI) M+(found):
342.1418; calcd: 342.1640 (for C19H24N3OS+).
4.4.36. (4-Methylpiperazin-1-yl)(10H-phenothiazin-10-
yl)methanone (36)
4.4.31. N-[2-(N0,N0–Diisopropylamino)ethyl]-10H-
phenothiazine-10-carboxamide (31)
Compound 36 26%, colourless crystals, mp 237–238 °C(dec) (lit
mp: 228–230 °C).51 IR (Nujol): 1673, 1415, 1300, 1280, 1247,
975, 753, 742 cmꢀ1 1H NMR: 2.68–2.83(m, 5H), 3.27–3.38(m,
.
Compound 31 54%; colourless crystals, mp 109–111.5 °C. IR
(Nujol): 3336, 1657, 1511, 1284, 1177, 756 cmꢀ1
.
1H NMR
2H), 3.45–3.60(m, 2H), 3.80–3.91(m, 2H), 7.14–7.21(m, 2H),
7.26–7.34(m, 2H), 7.37 (dd, J = 7.6, 1.5 Hz, 2H), 7.69(dd, J = 7.9,
1.2 Hz, 2H), 12.97(broad s, 1H). 13C NMR: 42.6, 43.2, 52.5, 123.6,
126.0, 127.6, 127.8, 130.4, 140.1, 156.7. EI-MS (m/z): 325(M+, base
peak), 200, 199, 198, 167, 154, 128, 127, 85, 70. HRMS (EI) M+
(found): 325.1241; calcd: 325.1249 (for C18H19N3OS).
(DMSO-d6): 0.81(d, J = 6.6 Hz, 12H), 2.51(t, J = 6.0 Hz, 2H),
2.85(septet, J = 6.6 Hz, 2H), 3.22–3.26(m, 2H), 7.15–7.19(m, 2H),
7.26–7.30(m, 2H), 7.36(dd, J = 7.8, 1.2 Hz, 2H), 7.56(dd, J = 7.9,
1.0 Hz, 2H). 13C NMR (DMSO-d6): 20.6, 38.9, 42.6, 47.1, 126.4,
127.2, 127.5, 128.0, 133.6, 139.1, 154.9. EI-MS (m/z): 369 (M+),

S. Darvesh et al. / Bioorg. Med. Chem. 18 (2010) 2232–2244
2243
4.4.37. N-(3-(Methyl(phenyl)amino)propyl)-10H-
phenothiazine-10-carboxamide (37)
To examine the effect of high substrate concentration on the
inhibition of cholinetsrases by aminoureas, such as compound
31, Michaelis–Menten plots ([substrate] vs rate) were developed
over a wide range of substrate concentrations (0.039–40 mM of
acetylthiocholine) that would ensure substrate inhibition of AChE
and substrate activation of BuChE. These observations were made
in the absence and presence of an appropriate concentration of
compound 31.
Compound 37 26%, colourless crystals, mp 110–111.5 °C. IR
(Nujol): 3441, 1675, 1598, 1506, 1316, 1251, 1214, 1072, 990,
769, 756 cmꢀ1 1H NMR: 1.77–1.84(m, 2H), 2.82(s, 3H), 3.31–
.
3.38(m, 4H), 5.33(broad t, J = 5.3 Hz, 1H), 6.60–6.69(m, 2H),
6.75(t, J = 7.4 Hz, 1H), 7.18–7.25(m, 4H), 7.26–7.30(m, 2H),
7.40(dd, J = 7.7, 1.3 Hz, 2H), 7.55(dd, J = 7.9, 1.3 Hz, 2H). 13C NMR:
27.3, 38.4, 39.7, 51.5, 113.2, 117.1, 126.8, 127.4, 127.4, 128.3,
129.4, 133.7, 139.0, 149.8, 155.0. EI-MS on salt (m/z): 390(M+),
199(base), 191, 167, 166, 154, 104, 91, 77. HRMS on salt (EI) M+
(found): 390.1632; calcd: 390.1640 (for C23H24N3OS+).
4.6. Calculation of molecular parameters
Computational chemistry studies were carried out using molec-
ular mechanics methods, employing SPARTAN006.30 The Merck
molecular force field (MMFF) was used, selecting the most stable
conformer for the calculation of molecular volume.30 Figure 7
was generated using the SPARTAN006 program.30 First, the crystal
structure of human acetylcholinesterase,32(PDB ID: 1B41) and hu-
man butyrylcholinesterase (PDB ID: 1POI)52 was obtained from the
protein databank53 and PyMol was employed to delete all amino
acids save for those selected residues found in the active site.54
Compound 31 was optimized separately, using molecular mechan-
ics and the MMFF force field, within SPARTAN006 to obtain the pre-
ferred conformer.30 Using SPARTAN006, the optimized compound 31
was manually fitted into the active site gorge to represent simu-
lated binding.
4.4.38. (Adamant-1-yl)(10H-phenothiazin-10-yl)methanone (38)
Compound 38 10%, dark greenish-brown crystals, mp 177–
178.1 °C. IR (Nujol): 1655, 1585, 1318, 1284, 1257, 1234, 1174,
763, 740 cmꢀ1 1H NMR: 1.49–1.62(m, 6H), 1.82–1.88(m, 9H),
.
7.19(td, J = 7.6, 1.5 Hz, 2H), 7.29(td, J = 7.7, 1.5 Hz, 2H), 7.45(dd,
J = 7.6, 1.5 Hz, 2H), 7.62(dd, J = 7.6, 1.2 Hz, 2H). 13C NMR: 28.4,
36.3, 39.7, 44.9, 126.4, 126.7, 127.4, 128.1, 134.6, 141.0, 178.0.
EI-MS (m/z): 361(M+), 198, 136, 135(base), 107, 93, 79. HRMS (EI)
M+ (found): 361.1480; calcd: 361.1500 (for C23H23NOS).
4.5. Enzyme kinetic studies
The esterase activity of AChE and BuChE was determined by a
modification21 of the Ellman29 spectrophotometric method. Briefly,
1.35 mL of buffered DTNB solution (usually pH 8.0), 0.05 mL of hu-
man recombinant AChE (ꢂ0.03 units) or purified human serum
BuChE (ꢂ0.05 units) in 0.1% aqueous gelatin and 0.05 mL of 50%
aqueous acetonitrile or one of the phenothiazine derivatives dis-
solved in this solvent, in a glass, or quartz, stoppered cuvette of
1 cm path-length. Serial dilutions of (1:10) of each inhibitor com-
pound in 50% acetonitrile were tested for the ability to inhibit either
AChE or BuChE. The mixture was zeroed at 412 nm, and the reaction
was initiated by the addition of 0.05 mL acetylthiocholine (for
AChE) or butyrylthiocholine, except where acetylthiocholine is indi-
cated (for BuChE) in an aqueous solution at a final concentration of
1.6 ꢁ 10ꢀ4 M. Final volume was always 1.5 mL. The reactions were
4.7. Calculation of log P values
In order to assess the ability of the compound to cross the
hydrophobic blood–brain barrier, log P values were obtained using
the ALOGPS method.31
Acknowledgements
Canadian Institutes of Health Research, Canada Foundation for
Innovation, Vascular Health and Dementia Initiative (DOV-78344)
(through partnership of Canadian Institutes of Health Research,
Heart & Stroke Foundation of Canada, the Alzheimer Society of
Canada and Pfizer Canada Inc.), Natural Sciences and Engineering
Research Council of Canada, Multiple Sclerosis Society of Canada,
Capital District Health Authority Research Fund, Nova Scotia
Health Research Foundation, Brain Tumour Foundation of Canada,
the Committee on Research and Publications of Mount Saint Vin-
cent University. We would like to thank Jillian Soh and Dongbin
Zhang, for excellent technical support.
performed at 23 °C. The rate of change of absorbance (DA/min),
reflecting the rate of hydrolysis of acetylthiocholine or butyrylthi-
ocholine, was recorded every 5 s for 1 min, using a Milton-Roy
1201 UV–vis spectrophotometer (Milton-Roy, Ivyland, PA) set at
k = 412 nm. This provided a rate versus [I] profile for the selection
of suitable inhibitor concentrations for further analysis. These
experiments were generally done at least in triplicate and the values
averaged. The final concentration of acetonitrile (1.67% v/v), in the
Ellman assays, was found to have no measurable effect on the activ-
ity of AChE or BuChE. Lineweaver–Burk plots were generated by
using a fixed amount of cholinesterase and varying amounts of sub-
strate (3 ꢁ 10ꢀ5–1.6 ꢁ 10ꢀ4 M), in the presence or absence of the
inhibitors. Inhibitor concentrations to be used were based on the
previously generated inhibitor concentration/rate profile. The re-
plot of the slopes of the above double reciprocal plots against inhib-
itor concentration gave the inhibitor constant (Ki) as the intercept
on the x-axis. Kinetics were generally carried out at pH 8.0 because
of the faster rate of hydrolysis of choline esters,23 which allows for
more efficient kinetic analysis. Furthermore, it has been shown pre-
viously23 that Ki values are independent of pH between 6.8 and 8.0.
To confirm this in the present study, the Ki value for compound 31
with BuChE and butyrylthiocholine as substrate was 6.6 ꢁ 10ꢀ9 M
at pH 7.4 which was comparable to the value determined at pH
8.0 (9.0 ꢁ 10ꢀ9 M, Table 4). As a consequence, all the Ki values re-
ported in Tables 1–4 were determined at pH 8.0 for comparison of
relative inhibitor potency under the same conditions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.066.
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