Tricyclic phenothiazine and phenoselenazine derivatives as potential multi-targeting agents to treat Alzheimer's disease

Article abstract of DOI:10.1039/c5md00274e

A group of tricyclic phenothiazines (6a, 6b and 7a-l) and phenoselenazines (12a, 12b and 13a-l) was designed, synthesized and evaluated as multi-targeting ligands aimed at the cholinergic, amyloid and oxidative stress pathways of Alzheimer's disease. The phenothiazine derivative 7j (2-chloro-10H-phenothiazin-10-yl-(4-methoxyphenyl)methanone) was identified as the best dual, non-selective cholinesterase inhibitor (AChE IC₅₀ = 5.9 ± 0.6 μM; BuChE IC₅₀ = 5.3 ± 0.5 μM), whereas in the corresponding phenoselenazine series, 13j (2-chloro-10H-phenoselenazin-10-yl-(4-methoxyphenyl)methanone) exhibited good non-selective cholinesterase inhibition (AChE IC₅₀ = 5.8 ± 0.4 μM; BuChE IC₅₀ = 4.9 ± 0.5 μM). Interestingly, N-10 unsubstituted phenothiazine 6a (AChE IC₅₀ = 7.3 ± 0.6 μM; BuChE IC₅₀ = 5.8 ± 0.5 μM; Aβ_1-42 aggregation inhibition = 62%; DPPH scavenging = 92%), and the corresponding phenoselenazine bioisostere 12a (AChE IC₅₀ = 5.6 ± 0.4 μM; BuChE IC₅₀ = 3.0 ± 0.5 μM; Aβ_1-42 aggregation inhibition = 45.6%; DPPH scavenging = 84.4%) were able to exhibit multi-targeting ability by demonstrating cholinesterase inhibition, beta-amyloid aggregation and antioxidant properties. These results show that fused tricyclic ring systems based on either phenothiazine or phenoselenazine templates can be useful to develop hybrid small molecules to target multiple pathological routes associated with Alzheimer's disease.

Full text of DOI:10.1039/c5md00274e

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Tricyclic phenothiazine and phenoselenazine derivatives as potential
multiꢀtargeting agents to treat Alzheimer’s disease
Gary Tin, a Tarek Mohamed, a, b, Nyasha Gondora, a Michael A. Beazely, a and Praveen P. N.
Rao, a,*
Graphical Abstract
*Corresponding author:
Tel: +1 519 888 4567 ext: 21317; eꢀmail: praopera@uwaterloo.ca
aSchool of Pharmacy, Health Sciences Campus, University of Waterloo, 200 University Avenue West,
Waterloo, Ontario, Canada N2L 3G1
bDepartment of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario,
Canada N2L 3G1
1

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Tricyclic phenothiazine and phenoselenazines as potential multiꢀtargeting agents to treat
Alzheimer’s disease
a
a, b
a
a
Gary Tin, Tarek Mohamed,
Nyasha Gondora, Michael A. Beazely, and Praveen P. N.
Rao, a,*
a School of Pharmacy, Health Sciences Campus, University of Waterloo, 200 University Avenue West,
Waterloo, Ontario, Canada N2L 3G1
b Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario,
Canada N2L 3G1
______________________________________________________________________________
A group of tricyclic phenothiazine (6a, 6b, 7a–l) and phenoselenazines (12a, 12b, 13a–l) were
designed, synthesized and evaluated as multiꢀtargeting ligands aimed at the cholinergic, amyloid
and oxidative stress pathways of Alzheimer’s disease. The phenothiazine derivative 7j (2ꢀchloroꢀ
10Hꢀphenothiazinꢀ10ꢀylꢀ(4ꢀmethoxyphenyl)methanone), was identified as the best dual,
nonselective cholinesterase inhibitor (AChE IC50 = 5.9 ± 0.6 ꢁM; BuChE IC50 = 5.3 ± 0.5 ꢁM),
whereas in the corresponding phenoselenazine series, 13j (2ꢀchloroꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀ
(4ꢀmethoxyphenyl)methanone) exhibited good nonselective cholinesterase inhibition (AChE IC50
= 5.8 ± 0.4 ꢁM; BuChE IC50 = 4.9 ± 0.5 ꢁM). Interestingly, Nꢀ10 unsubstituted phenothiazine 6a
(AChE IC50 = 7.3 ± 0.6 ꢁM; BuChE IC50 = 5.8 ± 0.5 ꢁM; Aβ1ꢀ42 aggregation inhibition = 62%;
DPPH scavenging = 92%), and the corresponding phenoselenazine bioisotere 12a (AChE IC50 =
5.6 ± 0.4 ꢁM; BuChE IC50 = 3.0 ± 0.5 ꢁM; Aβ1ꢀ42 aggregation inhibition = 45.6%; DPPH
scavenging = 84.4%), were able to exhibit multiꢀtargeting ability by demonstrating
cholinesterase inhibition, betaꢀamyloid aggregation and antioxidant properties. These results
show that fused tricyclic ring systems based on either a phenothiazine or phenoselenazine
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templates can be useful to develop hybrid small molecules to target multiple pathological routes
associated with Alzheimer’s disease.
______________________________________________________________________________
1. Introduction
Alzheimer’s disease (AD) is a complex neurodegenerative disorder that is the leading cause of
dementia around the world.1–3 Recent years have seen a dramatic rise in its prevalence due to an
increase in the aging population, increasing life span and limited pharmacotherapy options. The
cholinesterase inhibitors such as donepezil, galantamine and rivastigmine which prevent the
degradation of the neurotransmitter acetylcholine (ACh), (by inhibiting the enzymes acetyl and
butyrylcholinesterase AChE and BuChE respectively), yet provide only symptomatic relief and
are ineffective as long term therapies for AD.4–6 The enzyme AChE is primarily responsible for
the degradation of the neurotransmitter ACh. However, as AD progresses, BuChE is known to
take over this function due to depletion of AChE. These observations highlight the benefits of
developing dual AChE and BuChE inhibitors. AD pathophysiology suggests the involvement of
few major pathways such as cholinergic dysfunction, amyloid aggregation, tau
hyperphosphorylation and oxidative stress, that can lead to neurodegeneration, loss of memory
and cognition.7–14 The involvement of multiple factors in the pathophysiology of AD supports
the development of hybrid small molecules as multiꢀtargeting agents to treat AD.15–17
Tacrine (Fig. 1, 1), the first cholinesterase inhibitor, is an example of a fused tricyclic ring. Its
chemical structure has been modified to design tacrine derivatives that exhibit multiꢀtargeting
ability as shown in Fig. 1 (2 and 3).18, 19 In this regard, we investigated the use of phenothiazine
(PTZ), and its bioisostere, phenoselenazine (PSZ, Fig. 1, 4 and 5) derivatives, as multiꢀtargeting
ligands to treat AD, since they also possess a fused tricyclic ring system similar to tacrine. PTZs
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represent an important class of bioactive molecules. For example, both chlorpromazine and
fluphenazine are used in clinical therapy as antipsychotics.20, 21 In addition, PTZ derivatives are
also known to exhibit cholinesterase inhibition and antioxidant properties.22ꢀ24
The trace element selenium (Se) is an essential micronutrient in the diet and is known to be
present in selenoproteins, such as glutathione peroxidase (GPx), which is part of body’s
antioxidant defense.25 Interestingly a recent study in AD patients shows lower levels of selenium
in the plasma, erythrocytes, and nails compared to the control group, suggesting that Se
deficiency is associated with AD.26 In this regard, some novel organoselenium compounds have
been developed as multiꢀtargeting compounds to treat AD, which further supports the
incorporation of selenium to design novel antiꢀAD molecules.27, 28 Based on this evidence, we
considered the organoselenium based PSZ derivatives in our study as a bioisostere of sulfur
containing PTZs.
Fused tricyclic ring systems based on either a PTZ or a PSZ have the potential to serve as
suitable ring templates to design novel small molecule multiꢀtargeting agents against AD
pathophysiology. In our study, we synthesized a library of PTZ and PSZ derivatives which were
evaluated for their potential to (i) inhibit AChE and BuChE enzymes; (ii) prevent amyloid
aggregation and (iii) possess antioxidant properties. The structure activity relationship (SAR)
data acquired demonstrates that PTZ and PSZ derivatives have the ability to inhibit
cholinesterases, exhibit antiꢀamyloid aggregation and antioxidant properties as well as represent
a novel class of fused tricyclic ring systems as multiꢀtargeting ligands to treat AD.
2. Results and discussion
2.1 Chemistry
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PTZ derivatives 7a–l were synthesized starting from either unsubstituted 10Hꢀphenothiazine (6a)
or 2ꢀchloroꢀ10Hꢀphenothiazine (6b), which were coupled to various acid chlorides (RCOCl,
where R = phenyl, benzyl, phenethyl, 3ꢀ or 4ꢀmethoxyphenyl or 3,4ꢀdimethoxyphenyl) under
reflux at 110 °C in toluene as shown in Scheme 1.29 These conditions afforded 7a–l in good
yields ranging from 70–95%. The synthesis of PSZ derivatives was accomplished in a stepwise
fashion. Initially, the starting precursor diphenylamine (11a or 11b) was synthesized by coupling
cyclohexꢀ2ꢀenone (8) with iodine in presence of dimethylaminopyridine (DMAP) and potassium
carbonate (K2CO3) to afford αꢀiodinated 2ꢀcyclohexꢀ2ꢀenone (9) in 70% yield (Scheme 2).30 In
the next step, 9 was coupled with substituted anilines (10) in a metalꢀfree approach by refluxing
overnight in presence of trace amounts of paraꢀtoluenesulfonic acid (pꢀTSOH) to afford 11a and
11b in 40ꢀ50% yields.31 The ring closing of 11a or 11b to obtain the PSZ tricyclic ring was
achieved by heating them at 150 °C in a pressure vial, in presence of selenium, selenium dioxide
(SeO2) and iodine, using sulfolane as solvent.32 It should be noted that the presence of SeO2
helps in regenerating selenium in situ and improves the PSZ yield. The final yield of PSZ
derivatives 12a and 12b, were 20–25% which was much better than our previous attempts to
cyclize diphenylamine (11a or 11b) in presence of selenium monochloride (Se2Cl2), under
reflux, which provided poor yields (5–10%) and difficulty in purification.33 In the final step, the
PSZ derivatives 12a and 12b, were subjected to nucleophilic addition/elimination reaction with
acid chlorides (RCOCl, where R = phenyl, benzyl, phenethyl, 3ꢀ or 4ꢀmethoxyphenyl or 3,4ꢀ
dimethoxyphenyl), to afford PSZ derivatives 13a–l in moderate to good yields (20–80%, Scheme
1).
2.2 Cholinesterase inhibition studies
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The cholinesterase inhibition activity profile of PTZ (6a, 6b, 7a–l) and PSZ derivatives (12a,
12b, 13a–l) toward both human AChE and BuChE enzymes, were evaluated using the Ellman
assay as per our previously reported protocol (Table 1).34 The unsubstituted PTZ derivative 6a,
exhibited an IC50 value of 7.4 ± 0.6 µM toward human AChE and was less potent compared to
reference agents tacrine (AChE IC50 = 0.16 ± 0.01 µM), donepezil (AChE IC50 = 0.04 ± 0.002
µM) and galantamine (AChE IC50 = 2.6 ± 0.6 µM, Table 1). Addition of an Nꢀ10 benzoyl
substituent in compound 7a (R = phenyl), retained AChE inhibition (IC50 = 8.0 ± 0.7 µM) and
the compound was less potent compared to 6a. The effect of Nꢀ10 aromatic acyl groups, with one
and two carbon spacers, was explored. These modifications provided AChE inhibition for
compounds 7b (R = benzyl; AChE IC50 = 7.4 ± 0.8 µM) and 7c (R = phenethyl; AChE IC50 = 7.1
± 0.8 µM), although they were less potent compared to reference agents. Our previous work, has
shown that the presence of a 3,4ꢀdimethoxyphenyl substituent provided good cholinesterase
inhibition.34 Accordingly, the effect of methoxyphenyl substituents was explored by evaluating
compounds 7d–f (Table 1). Interestingly, these compounds exhibited AChE inhibition ranging
from 5.8 to 6.3 µM (IC50s), and were more potent compared to compounds 6a and 7a–c. The 3,4ꢀ
dimethoxyphenyl compound (7f) was identified as the most potent AChE inhibitor (IC50 = 5.8 ±
0.4 µM). Furthermore, the addition of Cꢀ2 chlorine to the PTZ scaffold was investigated. It
appears that Cꢀ2 chlorine substituent was not a major factor in AChE inhibition and Cꢀ2 chloroꢀ
PTZ derivatives 6b and 7g–l, exhibited IC50 values in the range of 4.6 to ~10.0 µM (Table 1).
Similar to compounds from the nonꢀchlorinated PTZ series (6a and 7aꢀc), the presence of
methoxyphenyl substituents enhanced AChE inhibition (7j–l) with compound 7k (3ꢀ
methoxyphenyl) identified as the most potent compound (AChE IC50 = 4.6 ± 0.5 µM) among the
Cꢀ2 chloroꢀPTZ derivatives.
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The SAR studies of PTZs on BuChE inhibition indicates that, they were generally weak
inhibitors compared to their inhibition profile toward AChE (Table 1). Among the nonꢀ
chlorinated PTZs, compound 6a exhibited superior inhibitory potency (BuChE IC50 = 5.8 ± 0.5
µM) relative to AChE (IC50 = 7.4 ± 0.6 µM) and was the most potent. It was approximately 11ꢀ
fold more potent relative to the reference agent galantamine (BuChE IC50 = 66.5 ± 4.1 µM) and
was less potent compared to both tacrine (BuChE IC50 = 0.04 ± 0.001 µM) and donepezil
(BuChE IC50 = 3.6 ± 0.4 µM). Among the 2ꢀClꢀPTZ derivatives 7g–l, compound 7h (R =
benzyl), was identified as the most potent BuChE inhibitor (BuChE IC50 = 3.6 ± 0.4 µM). In
general, the presence of a 2ꢀCl substituent led to enhanced BuChE inhibition potency for Nꢀ10
acyl PTZ derivatives 7g–j. Interestingly, the presence of either a phenethyl (7i) or 4ꢀ
methoxyphenyl (7j) Nꢀ10 acyl substituents, provided similar BuChE inhibition (IC50 values of
5.0 ± 0.6 and 5.3 ± 0.5 µM respectively) whereas both 3ꢀmethoxy (7k) and 3,4ꢀdimethoxyphenyl
(7l) derivatives exhibited weak inhibition (IC50 values of ~30.8 and 32.6 µM respectively). The
cholinesterase SAR study for PTZ compound library demonstrates that they exhibit dual
inhibition of both AChE and BuChE enzymes. They showed nonselective inhibition of both the
cholinesterase enzymes and generally exhibited superior AChE inhibition potency.
The cholinesterase inhibition profile of PSZ derivative 12a toward AChE, shows an IC50 value
of 5.6 ± 0.4 µM and was less potent compared to reference agents tacrine (AChE IC50 = 0.16 ±
0.01 µM), donepezil (AChE IC50 = 0.04 ± 0.002 µM) and galantamine (AChE IC50 = 2.6 ± 0.6
µM, Table 2). However, it showed greater potency, than the organoselenium compound ebselen
(AChE IC50 = 6.2 ± 0.5 µM). Addition of an Nꢀ10 phenyl substituent in compound 13a, retained
AChE inhibition (IC50 = 5.4 ± 0.4 µM) compared to 12a. Compound 13b (R = benzyl; AChE
IC50 = 6.4 ± 0.6 µM) and 13c (R = phenethyl; AChE IC50 = 6.7 ± 0.7 µM) were less potent
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compared to 12a (AChE IC50 = 5.6 ± 0.4 µM). The effect of methoxyphenyl substituents, at Nꢀ10
was explored by evaluating compounds 13d–f (Table 2). Interestingly, these compounds
exhibited AChE inhibition ranging from ~4.6 to 6.2 µM (IC50). The 4ꢀmethoxyphenyl compound
(13d) was identified as the most potent AChE inhibitor (IC50 = 4.6 ± 0.3 µM). Furthermore, the
addition of a Cꢀ2 chlorine to the PSZ scaffold was investigated. Similar to the PTZ series, the Cꢀ
2 chlorine substituent was not a major factor in AChE inhibition. The Cꢀ2 chloroꢀPSZ
derivatives 12b and 13g–l, exhibited IC50 values in the range of ~5.8–6.5 µM (Table 2). Similar
to the compounds from the nonꢀchlorinated PSZ series (12a and 13a–c), the presence of
methoxyphenyl substituents, enhanced AChE inhibition (13j–l), with compound 13j (R = 4ꢀ
methoxyphenyl) identified as the most potent compound (AChE IC50 = 5.8 ± 0.4 µM), among the
Cꢀ2 chloroꢀPSZ derivatives. The SAR studies of PSZs on BuChE inhibition indicates that, they
were generally weak inhibitors compared to their inhibition profile toward AChE (Table 2).
Among the nonꢀchlorinated PSZs (12a and 13a–f), compound 12a exhibited superior inhibitory
potency (BuChE IC50 = 3.0 ± 0.5 µM) relative to AChE (IC50 = 5.6 ± 0.4 µM) and was the most
potent. It was approximately 22ꢀfold more potent relative to the reference agent galantamine
(BuChE IC50 = 66.5 ± 4.1 µM), and more potent than both donepezil (BuChE IC50 = 3.6 ± 0.04
µM) and ebselen (BuChE IC50 = 4.6 ± 0.6 µM). However, it was less potent compared to tacrine
(BuChE IC50 = 0.04 ± 0.001 µM). Among the 2ꢀClꢀPTZ derivatives 12b and 13g–l, compound
13g (R = phenyl), was identified as the most potent BuChE inhibitor (BuChE IC50 = 3.8 ± 0.1
µM). In general, the presence of a 2ꢀCl substituent, led to enhanced BuChE inhibition potency
for Nꢀ10 acyl PSZ derivatives 13g, 13h, 13j and 13l, relative to the corresponding nonꢀ
chlorinated Nꢀacyl PSZ derivatives (13a–f). Interestingly, the presence of either a 4ꢀ
methoxyphenyl (13j), or and 3,4ꢀdimethoxyphenyl (13l) Nꢀ10 acyl substituents, provided similar
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BuChE inhibition (IC50 values of ~4.9 and 5.7 µM respectively), whereas both 3ꢀmethoxyphenyl
(13i) and phenethyl (13k) derivatives, exhibited weak inhibition (IC50 values of ~40.0 and 19.3
µM respectively). The cholinesterase SAR study for PSZ compound library, demonstrates that
they exhibit dual inhibition of both AChE and BuChE enzymes. Their AChE inhibition ranged
from ~4.6ꢀ6.7 µM (IC50s), whereas their BuChE inhibition ranged from ~3.0ꢀ40.0 µM. Similar to
that of the PTZ series, the PSZ series exhibited nonselective inhibition of both cholinesterase
enzymes, and generally exhibited superior AChE inhibition potency. These studies show that
both PTZ and PSZ based compounds serve as useful ring scaffolds to design dual cholinesterase
inhibitors.
2.3. Molecular docking studies of PTZ (6a, 7j) and PSZ (12a, 13j) compounds with human
cholinesterases
The catalytic site of cholinesterases consists of His, Ser and Glu triad which are involved in
substrate hydrolysis, whereas an anionic subsite consists of a Trp residue. In addition, the active
site of AChE is made up of a peripheral anionic site (PAS) which is lined by amino acid residues
Trp72, Asp74, Tyr124, Trp286 and Tyr341, whereas BuChE lacks this PAS due to the presence
of smaller aliphatic amino acid residues closer to the entrance. As a consequence, BuChE active
site is more than 60% larger (501.91 Å3) compared to AChE (302.31 Å3). The cholinesterase
inhibitor tacrine is known to bind in the catalytic site and the anionic subsite.22, 35
Binding mode of 6a (AChE IC50 = 7.4 ± 0.6 ꢁM; BuChE IC50 = 5.8 ± 0.5 ꢁM), within the active
site of human AChE, shows that the tricyclic PTZ ring was oriented closer to catalytic and
anionic subsites (His447 and Trp86 respectively). The aromatic rings of 6a, underwent πꢀπ
stacking interactions with aromatic rings of Trp86, Tyr337 and Tyr341, whereas the central
thiazine ring was in van der Waal’s contact with Trp86 and Tyr337 (distance < 5 Å) as shown in
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Figure 2a. Interestingly, the sulfur atom of thiazine ring was in van der Waal’s contact with
His447 (sulfurꢀπ interaction, distance < 5 Å). Modeling of 6a in the human BuChE shows that
the PTZ ring was oriented in a flipped conformation compared to its binding mode in AChE
(Figure 2b). The aromatic rings of 6a underwent van der Waal’s interactions with Trp82 and
Ala328. These studies show that 6a was able to inhibit cholinesterases through hydrophobic
interactions.
The modeling study of the best PTZ derivative 7j (AChE IC50 = 5.9 ± 0.6 ꢁM; BuChE IC50 = 5.3
± 0.5 ꢁM, Figure 2c) shows that, in the AChE active site, the tricyclic PTZ ring, with a Cꢀ2
chloro substituent, underwent πꢀπ and πꢀhalogen interactions with Trp86, Tyr337 and Phe338
(Figure 2c). The central thiazine sulfur was closer to His447 (distance < 5 Å). The Nꢀ10 acyl
amide C=O formed a hydrogen bond with hydroxyl of Tyr337 (distance = 2.7 Å) and the 4ꢀ
methoxyphenyl substituent was in the vicinity of Asp74, Trp86, and Tyr124 (Figure 2c) closer to
the PAS. Compound 7j exhibits a different binding mode in BuChE, where the flipped PTZ ring
undergoes πꢀπ stacked and πꢀhalogen interactions with Trp82 (Figure 2d). The sulfur atom of
central thiazine ring was closer to Trp82 (distance < 5 Å). The 4ꢀmethoxyphenyl substituent
underwent nonpolar contacts with Gly117 and Phe329.
The binding mode of selenium containing tricyclic 12a (AChE IC50 = 5.6 ± 0.4 ꢁM; BuChE IC50
= 3.0 ± 0.5 ꢁM), in AChE active site, shows that the PSZ ring undergoes πꢀπ interactions with
Trp86, Tyr341 and Phe338 (distance < 5 Å, Figure 3a) and the selenium atom was in close
proximity to Trp86. In the BuChE active site, 12a exhibited a flipped conformation compared to
its binding mode in AChE (Figure 3b), with the selenium atom closer to His438 (distance < 5.5
Å) and the tricyclic PSZ ring underwent a number of nonpolar contacts with Trp82, Ala328,
Tyr338 and Tyr341 (distance < 5 Å). The binding mode of 4ꢀmethoxyphenyl compound 13j
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(AChE IC50 = 5.8 ± 0.4 ꢁM; BuChE IC50 = 4.9 ± 0.5 ꢁM, Figure 3c), in AChE, shows that the
PSZ ring was closer to the anionic subsite and catalytic site, where it undergoes πꢀπ and πꢀ
halogen interactions with Trp86 (distance < 5 Å). The selenium atom was closer to His447 and
Tyr337 (distance < 4.5 Å). Interestingly, the Nꢀ10 acyl amide C=O formed a hydrogen bond with
Tyr337 (distance = 2.7 Å) and the 4ꢀmethoxyphenyl moiety was oriented closer to the PAS
(Tyr124), as shown in Figure 3c. In the BuChE active site, the PSZ ring of 13j, was in van der
Waal’s contact with Trp82 and Ala328 (distance < 5 Å), whereas the 4ꢀmethoxyphenyl
substituent was oriented toward the mouth of BuChE and underwent Tꢀshaped πꢀπ stacking
interactions with Tyr332 (Figure 3d). These studies suggest that PTZ compound 7j and PSZ
compound 13j, exhibit bivalent inhibition of AChE where the tricyclic ring was oriented closer
to the catalytic and anionic subsite whereas the Nꢀ10 4ꢀmethoxyphenyl moiety was closer to the
PAS.
2.4. Selfꢀinduced amyloid aggregation (Aβ1ꢀ42) inhibition studies
The PTZ (6a, 6b, 7f and 7l) and PSZ (12a, 12b, 13f and 13l) compounds were evaluated for their
ability to inhibit selfꢀinduced Aβ1ꢀ42 aggregation, using the thioflavinꢀT (ThT) based fluorescence
measurements (Table 3). Their activity was compared to a known Aβꢀaggregation inhibitor,
orange G (Table 3). The unsubstituted PTZ (6a), exhibited superior antiꢀaggregation activity
compared to orange G at all concentrations tested (62% inhibition at 25 ꢁM, Table 3), whereas 7f
was inactive. A similar trend was observed for Cꢀ2 chloro PTZ compounds 6b and 7l (60.8% and
5.9% inhibition at 25 ꢁM, Table 3). Among the PSZ compounds tested, the Nꢀ10 unsubstituted
PSZs, 12a and 12b, exhibited good antiꢀaggregation activity (45.6 and 45.0% inhibition
respectively, at 25 ꢁM Table 3), whereas Nꢀ10 acyl substituted compounds 13f and 13l, exhibited
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weak antiꢀaggregation activity at the highest concentration tested (~11.0 and 5.9% inhibition
respectively, at 25 ꢁM, Table 3). The Aβꢀaggregation kinetics data for compounds 6a and 12a is
shown in Figure 4a and 4b. Both compounds were able to exhibit a concentration dependent
decline in ThT fluorescence intensity over a period of time. The chart shows that both 6a and
12a, were able to produce a rapid decline in the Aβꢀgrowth phase and reduce the formation of
Aβ fibrils.
2.5. 2,2ꢀDiphenylꢀ1ꢀpicrylhydrazyl (DPPH) radical scavenging activity
The antioxidant properties of PTZ (6a, 6b, 7f and 7l) and PSZ (12a, 12b, 13f and 13l)
compounds, were evaluated using the DPPH radical scavenging activity and was compared with
known radical scavenger trolox, and the organoselenium compound ebselen (Table 4).36 The
PTZ compound 6a, (92.1% inhibition at 50 ꢁM) exhibited excellent antioxidant activity. The Nꢀ
10 acyl substituted PTZ compound 7f, exhibited radical scavenging activity (44.3% inhibition at
50 ꢁM). However, it showed a 2ꢀfold decrease in antioxidant activity compared to 6a. Similarly,
the 2ꢀchloro PTZ compound 6b (76.4% inhibition at 50 ꢁM), and 7l (46.5% inhibition at 50 ꢁM)
exhibited similar trends, with the Nꢀ10 acyl substitution, proving to be detrimental to antioxidant
activity. All the PSZ compounds tested (12a, 12b, 13f and 13l), exhibited superior antioxidant
activity (38.3–84.4% inhibition at 50 ꢁM, Table 4), compared to the organoselenium reference
compound ebselen (34.7% inhibition at 50 ꢁM). In general, as observed with PTZ based
compounds, Nꢀ10 acylation led to a decline in their antioxidant properties (13f = 39%; 13l =
38.3% inhibition at 50 ꢁM, Table 4). This study shows that, the presence of a free amine at Nꢀ10,
of either PTZ or PSZ rings, provides excellent antioxidant properties, whereas Nꢀ10 acylation
reduces their ability to scavenge DPPH radicals. It is suggested that, this could be due to the
formation of a resonance stabilized radical in PTZ and PSZ’s, that possess a free NH group,
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while Nꢀacylation might compromise this stability and exhibit less efficient DPPH radical
scavenging activity (Figure 5).
2.6. Cell death assay studies of PTZ and PSZ compounds in SHꢀSY5Y neuroblastoma cells
The cell toxicity of PTZ (6a, 6b, 7f and 7l) and PSZ (12a, 12b, 13f and 13l) compounds, were
carried out in SHꢀSY5Y neuroblastoma cells, using the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium (MTT) bromide assay (Figure 6).34 In these assays, PTZ compounds 6a, 6b,
7f and 7l exhibited moderate to good cell viability (63.3–71.6% viability at 50 ꢁM). In contrast,
PSZ compounds 12a, 13f and 13l, exhibited good to excellent cell viability (76.0–100% viability
at 50 ꢁM) and were superior to the seleniumꢀcontaining reference compound ebselen (53.9%
viability at 50 ꢁM). The Cꢀ2 chloro substituted PSZ compound 12b, exhibited toxicity compared
to other compounds tested (43.0% viability at 50 ꢁM). These studies indicate that PTZ and PSZ
templates are viable scaffolds to design small molecules with multiꢀtargeting activity.
3. Conclusion
We have developed a novel class of fused tricyclic PTZ and PSZ based molecules as multiꢀ
targeting agents to treat AD. Among the PTZ and PSZ derivatives with Nꢀ10 acyl substitution, a
4ꢀmethoxyphenyl group provided good cholinesterase inhibition. However, Nꢀ10 acyl
substitution was detrimental to antiꢀAβ aggregation and antioxidant properties. In contrast, Nꢀ10
nonꢀacylated derivatives (6a, 6b, 12a and 12b), were able to (i) target and inhibit both AChE and
BuChE enzymes; (ii) prevent Aβꢀaggregation and (iii) scavenge DPPH radicals. It is anticipated
that our work will provide design strategies to develop novel diseaseꢀmodifying therapies for
AD.
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4. Experiments
4.1. Chemistry
All solvents, reagents and compounds 6a (10Hꢀphenothiazine) and 6b (2ꢀchloroꢀ10Hꢀ
phenothiazine) were purchased from various commercial vendors (Acros Organics, Sigma
Aldrich, and Alfa Aesar, USA) with minimum purity of 95% and were used without further
purification. Melting points were determined using a FisherꢀJohns apparatus and are uncorrected.
1H NMR and 13C NMR spectra were performed on a Bruker Avance (300 and 75 MHz
respectively) series spectrometer using CDCl3 or DMSOꢀd6 as the solvent. Coupling constants
(Jꢀvalues) were recorded in hertz (Hz) and the following abbreviations were used to represent
multiplets of NMR signals: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad. Carbon
multiplicities (C, CH, CH2, CH3) were assigned by DEPT 90/135 experiments. Highꢀresolution
mass spectrometry (HRMS) analysis was done through positive ion electrospray ionization (ESI)
using a Thermo Scientific QꢀExactiveTM mass spectrometer, Department of Chemistry,
University of Waterloo. The mass spectrometry data for PSZ and PSZ derivatives are reported
based on the most stable selenium isotope (80Se). Combustion analysis was carried out by
Analest, Department of Chemistry, University of Toronto and were within ± 0.4% of theoretical
values for C, H and N elements. Crude product purification was done using flash
chromatography with Merck 230ꢀ400 mesh silica gel. Thinꢀlayer chromatography (TLC) was
performed on a Merck 60F254 silica gel plates (0.2 mm) using three different solvent systems
(5:1 EtOAc:MeOH, DCM, 5:1 hexane:EtOAc) and spots were visualized at UV 254 nm. The
purity of final compounds were confirmed (> 95%) by running Agilent HPLC (1200 Infinity
series) using an analytical column (Agilent Zorbax Eclipse XDBꢀC8 column with 4.6 x 150 mm
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dimensions and 5 µm particle size) with 50:50 ACN/water in 0.1% v/v TFA as eluent and a flow
rate of 1.5 mL/min (detection at UV 254 nm).
4.2. General method for the preparation of PTZ derivatives (7aꢀl)
To a mixture of phenothiazine (6a) or 2ꢀCl phenothiazine (6b) (0.40 g, 2.01 mmol) in 7 mL of
anhydrous toluene at room temperature, the desired acyl chloride (R = phenyl, benzyl, phenethyl,
3ꢀmethoxyphenyl, 4ꢀmethoxyphenyl or 3,4ꢀdimethoxyphenyl) was added (1.5 eq). The reaction
mixture was refluxed overnight at 110 °C and was monitored by TLC. Upon completion, the
excess toluene was evaporated in vacuo. The crude product was purified via flash
chromatography using DCM as the solvent. Final compound yields ranged from 70–95%.
Analytical data for PTZ derivatives 7a–l is given below.
4.2.1. 10HꢀPhenothiazinꢀ10ꢀylꢀ(phenyl)methanone (7a): White solid (80%). mp: 175–
177°C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.63–7.55 (m, 2H), 7.49–7.41 (m, 2H), 7.40– 7.32 (m,
1H), 7.31–7.19 (m, 8H).
4.2.2. 1ꢀ(10HꢀPhenothiazinꢀ10ꢀyl)ꢀ2ꢀphenylethanone (7b): Yellow solid (94%). mp =
153–155 °C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.54 (d, J = 7.7 Hz, 2H), 7.41–7.31 (m, 5H),
7.24–7.18 (m, 4H), 7.11–7.06 (m, 2H), 3.83 (s, 2H).
4.2.3. 1ꢀ(10HꢀPhenothiazinꢀ10ꢀyl)ꢀ3ꢀphenylpropanꢀ1ꢀone (7c): Yellow solid (77%).
1
mp = 98–100 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.57 (d, J = 7.8 Hz, 2H), 7.52 (dd, J = 7.7
Hz, 1.3, 2H), 7.35 (td, J = 7.7, 1.5 Hz, 2H), 7.31–7.23 (m, 2H), 7.21–7.00 (m, 5H), 2.85–2.63 (br
s, 4H).
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4.2.4. (4ꢀMethoxyphenyl)ꢀ10Hꢀphenothiazinꢀ10ꢀylꢀmethanone (7d): White solid
1
(71%). mp = 170–175 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.58–7.50 (m, 2H), 7.46–7.39 (m,
2H), 7.25–7.18 (m, 6H), 6.78 (d, J = 8.9 Hz, 2H), 3.69 (s, 3H); HRMS (ESI) m/z calcd. for
C20H16NO2S [M + H]+ 334.0902, found 334.0895.
4.2.5. (3ꢀMethoxyphenyl)ꢀ10Hꢀphenothiazinꢀ10ꢀylꢀmethanone (7e): White solid
1
(83%). mp = 155–157 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.58–7.51 (m, 2H), 7.46–7.38 (m,
2H), 7.26–7.18 (m, 4H), 7.14 (t, J = 7.8 Hz, 1H), 6.88 (dd, J = 8.0, 2.2 Hz, 1H), 6.84–6.77 (m,
2H), 3.60 (s, 3H); HRMS (ESI) m/z calcd. for C20H16NO2S [M + H]+ 334.0902. Found
334.0895.
4.2.6. (3,4ꢀDimethoxyphenyl)ꢀ10Hꢀphenothiazinꢀ10ꢀylꢀmethanone (7f): White solid
(70%). mp = 176–178 °C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.58–7.51 (m, 2H), 7.46–7.39 (m,
13
2H), 7.26–7.19 (m, 4H), 6.91–6.86 (m, 1H), 6.84–6.76 (m, 2H), 3.69 (s, 3H), 3.50 (s, 3H); C
NMR (75 MHz, DMSOꢀd6): δ 167.4, 150.5, 147.7, 139.4, 131.3, 127.7, 127.2, 126.8, 126.6,
121.2, 112.0, 110.7, 55.5, 55.1; HRMS (ESI) m/z calcd. for C21H18NO3S [M + H]+ 364.1007,
found 364.1003.
4.2.7. 2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl (phenyl)methanone (7g): Yellow solid (80%)
mp: 157ꢀ160°C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.64 (d, J = 7.2 Hz, 2H), 7.48 (d, J = 7.7 Hz,
2H), 7.38 (td, J = 7.6 Hz, 3, 2H), 7.31–7.23 (m, 2H), 7.18–7.13 (m, 2H), 6.97– 6.90 (m, 2H).
4.2.8. 1ꢀ(2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl)ꢀ2ꢀphenylethanone (7h): Yellow solid
1
(94%). mp = 90–92 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.74 (s, 1H), 7.65 (d, J = 7.81, 1H),
7.52–7.45 (m, 2H), 7.44–7.22 (m, 3H), 7.21–7.12 (m, 3H), 6.98–6.90 (m, 2H), 3.82 (s, 2H);
HRMS (ESI) m/z calcd. for C20H15ClNOS ([M + H]+ 352.0562, found 352.0558.
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4.2.9. 1ꢀ(2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl)ꢀ3ꢀphenylpropanꢀ1ꢀone (7i): Yellow oil
1
(95%). H NMR (300 MHz, DMSOꢀd6): δ 7.67 (s, 1H), 7.61–7.49 (m, 3H), 7.41–7.33 (m, 2H),
7.31–7.25 (m, 1H), 7.22–7.14 (m, 2H), 7.11 (d, J = 7.0 Hz, 1H), 7.08–7.00 (m, 2H), 2.83–2.65
(br s, 4H); HRMS (ESI) m/z calcd. for C21H17ClNOS [M + H]+ 366.0719, found 366.0717.
4.2.10. 2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀylꢀ(4ꢀmethoxyphenyl)methanone (7j): Yellow
1
solid (94%). mp = 153–155 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.68 (s, 1H), 7.60–7.51 (m,
2H), 7.36–7.30 (m, 1H), 7.29–7.14 (m, 5H), 6.81 (d, J = 8.7 Hz, 2H), 3.70 (s, 3H); HRMS (ESI)
m/z calcd. for C20H15ClNO2S [M + H]+ 368.0512, found 368.0510.
4.2.11. 2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀylꢀ(3ꢀmethoxyphenyl)methanone (7k): White
1
solid (92%). mp = 140–142 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.64 (s, 1H), 7.56 (t, J = 8.6
Hz, 2H), 7.37–7.26 (m, 2H), 7.26–7.13 (m, 3H), 6.91 (d, J = 8.3 Hz, 1H), 6.86–6.77 (m, 2H),
3.62 (s, 3H); HRMS (ESI) m/z calcd. for C20H15ClNO2S [M + H]+ 368.0512, found 368.0509.
4.2.12. 2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀylꢀ(3,4ꢀdimethoxyphenyl)methanone (7l):
1
Yellow solid (73%). mp = 155–158 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.69 (s, 1H), 7.60–
7.52 (m, 2H), 7.37–7.31 (m, 1H), 7.29–7.15 (m, 3H), 6.92–6.77 (m, 3H), 3.70 (s, 3H), 3.51 (s,
3H); HRMS (ESI) m/z calcd. for C21H17ClNO3S [M + H]+ 398.0617, found 398.0613.
4.3. Synthesis of αꢀiodinated 2ꢀcyclohexꢀ2ꢀenone (9)
To a mixture of cyclohexꢀ2ꢀenone 8 (1 mL, 10.33 mmol) in 50 mL of 1:1 THF:H2O, iodine (1.5
eq), DMAP (1 eq) and potassium carbonate (1.2 eq) was added. The reaction mixture was
allowed to stir at room temperature for 30 min. Upon completion, the reaction mixture was
diluted with EtOAc. The organic layer was washed with saturated sodium thiosulfate and 10%
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HCl. The organic layer was separated and evaporated in vacuo and the crude product was
purified via flash chromatography using DCM as the solvent to afford 9.
1
4.3.1. 2ꢀIodocyclohexꢀ2ꢀenone (9): Yellow solid (70%). mp = 49–50 °C. H NMR (300
MHz, DMSOꢀd6): δ 7.78 (t, J = 4.4 Hz, 1H), 2.63–2.51 (m, 2H), 2.43–2.34 (m, 2H), 1.93 (quin, J
= 6.3 Hz, 2H).
4.4. General method for the preparation of diphenylamines (11a and 11b)
To a mixture of 10 (aniline or 3ꢀchloroaniline, 4.8 mmol) in 10 mL EtOH, 9 (1.2 eq) and pꢀTsOH
(0.2 eq) were added. The reaction mixture was refluxed overnight at 75 °C. Upon completion,
the reaction mixture was diluted with EtOAc. The organic layer was washed with 20% sodium
bicarbonate and saturated brine solution. The organic layer was separated and evaporated in
vacuo. The crude product was purified via flash chromatography using DCM as the solvent.
Final compound yield ranged between 40–50%. Analytical data for 11a and 11b is given below.
1
4.4.2. Diphenylamine (11a): Yellow solid (50%). mp = 50–52 °C H NMR (300 MHz,
DMSOꢀd6): δ 8.08 (s, 1H), 7.18 (t, J = 8.1 Hz, 4H), 7.02 (d, J = 8.1 Hz, 4H), 6.77 (t, J = 7.2 Hz,
2H). 13C NMR (75 MHz, DMSOꢀd6): δ 143.4, 129.1, 119.6, 116.7.
1
4.4.3. 3ꢀChloroꢀNꢀphenylaniline (11b): Brown oil (40%). H NMR (300 MHz, DMSOꢀ
d6): δ 8.36 (s, 1H), 7.30–7.22 (m, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.07 (d, J = 8.1 Hz, 2H), 7.01–
6.93 (m, 2H), 6.88 (t, J = 7.3 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H).
4.5. General method for the preparation of PSZ compounds 12a and 12b
To a mixture of selenium (1.00 g, 12.79 mmol) in 5 mL of sulfolane, the appropriate
diphenylamine (11a or 11b) (2 eq), selenium dioxide (1.20 eq), and iodine (0.1 eq) were added.
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The reaction mixture was sealed in a pressure vial with a Teflon bushing and placed in an oil
bath at 150 °C for 5 hours. Upon completion, the reaction mixture was cooled to room
temperature and was filtered through Celite plug using dichloromethane (DCM). The solvent
was evaporated in vacuo and recrystallized using EtOH and then subsequently purified twice or
thrice by flash chromatography using 5:1 hexanes:EtOAc as the eluent. Final compound yield
ranged from 20–25%. Analytical data for 12a and 12b are given below.
1
4.5.1. 10HꢀPhenoselenazine (12a): Yellow solid (20%). mp = 195–197 °C. H NMR
(300 MHz, DMSOꢀd6): δ 8.56 (s, 1H), 7.10–6.95 (m, 4H), 6.78–6.68 (m, 4H); 13C NMR (75
MHz, DMSOꢀd6): δ 142.1, 128.8, 127.8, 122.1, 115.1, 111.5; HRMS (ESI) m/z calcd. for
C12H10N80Se [M + H]+ 246.9900, found 246.9895. Anal. calcd. for C12H10NSe (246.16): C
58.55, H 3.69, N 5.69; found: C 58.61, H 3.73, N 5.64.
1
4.5.2. 2ꢀChloroꢀ10Hꢀphenoselenazine (12b): Purple solid (26%) mp = 199–200 °C. H
NMR (300 MHz, DMSOꢀd6): δ 8.74 (s, 1H), 7.14–6.97 (m, 3H), 6.83–6.66 (m, 4H); HRMS
(ESI) m/z calcd. for C12H9ClN80Se [M + H]+ 281.9588, found 281.9500.
4.6. General method for the preparation of PSZ derivatives 13a–l
To a mixture of 12a or 12b (0.356 mmol) in 7 mL of anhydrous toluene, the desired acyl
chloride (R = phenyl, benzyl, phenethyl, 3ꢀmethoxyphenyl, 4ꢀmethoxyphenyl or 3,4ꢀ
dimethoxyphenyl) was added (1.5 eq). The reaction mixture was refluxed overnight at 110 °C.
Upon completion, the excess toluene was evaporated in vacuo. The crude product was purified
via flash chromatography using DCM as the solvent. Some compounds required two columns to
purify (13f and 13l). Final compound yields ranged from 20–80%. Analytical data for
compounds 13a–l are given below.
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4.6.1. 10HꢀPhenoselenazinꢀ10ꢀylꢀ(phenyl)methanone (13a): Yellow solid (87%). mp =
1
157–159 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.73–7.65 (m, 2H), 7.45–7.39 (m, 2H), 7.26–
7.13 (m, 9H); HRMS (ESI) m/z calcd. for C19H14NO80Se [M + H]+ 352.0240, found 352.0235.
Anal. calcd. for C19H13NOSe (350.27): C 65.15, H 3.74, N 4.00; found: C 65.55, H 3.92, N 4.01.
4.6.2. 1ꢀ(10HꢀPhenoselenazinꢀ10ꢀyl)ꢀ2ꢀphenylethanone (13b): Yellow solid (91%). mp
1
= 111–113 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.66 (d, J = 7.6 Hz, 4H), 7.40 (t, J = 7.6 Hz,
2H), 7.30–7.22 (m, 2H), 7.21–7.13 (m, 3H), 6.99–6.90 (m, 2H), 3.72 (s, 2H); HRMS (ESI) m/z
calcd. for C20H16NO80Se [M + H]+ 366.0397, found 366.0393. Anal. calcd. for C20H15NOSe •0.1
CH2Cl2 (372.79): C 64.76, H 4.11, N 3.76; found: C 64.96, H 3.76, N 3.82.
4.6.3. 1ꢀ(10HꢀPhenoselenazinꢀ10ꢀyl)ꢀ3ꢀphenylpropanꢀ1ꢀone (13c): White solid (87%).
1
mp = 184–186 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.68 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.8
Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.28–7.06 (m, 5H), 7.02 (d, J = 7.2 Hz, 2H), 2.73 (s, 2H), 2.61
(br s, 2H); HRMS (ESI) m/z calcd. for C21H18NO80Se [M + H]+ 380.0553, found 380.0546.
Anal. calcd. for C21H17NOSe•0.1 H2O (378.32): C 66.29, H 4.57, N 3.68; found: C 65.90, H
4.17, N 3.68.
4.6.4. (4ꢀMethoxyphenyl)ꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀmethanone (13d): White solid
(79%). mp = 184–186 °C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.72–7.66 (m, 2H), 7.47–7.39 (d, J
= 7.3 Hz, 2H), 7.27–7.13 (m, 6H), 6.76 (d, J = 8.7 Hz, 2H), 3.68 (s, 3H); HRMS (ESI) m/z calcd.
80
for C20H16NO2 Se [M + H]+ 382.0346, found 382.0339. Anal. calcd. for C20H15NO2Se•0.5 H2O
(389.30): C 61.70, H 4.14, N 3.60; found: C 61.82, H 4.09, N 3.57.
4.6.5. (3ꢀMethoxyphenyl)ꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀmethanone (13e): White solid
(84%). mp = 157–159 °C. 1H NMR (300 MHz, DMSOꢀd6): δ 7.71 (dd, J = 7.1, 1.7 Hz, 2H), 7.44
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(d, J = 7.5 Hz, 2H), 7.29–7.09 (m, 5H), 6.92–6.77 (m, 3H), 3.60 (s, 3H); HRMS (ESI) m/z calcd.
80
for C20H16NO2 Se [M + H]+ 382.0346, found 382.0338. Anal. calcd. for C20H15NO2Se•0.2 H2O
(383.90): C 62.57, H 4.04, N 3.65; found: C 62.43, H 4.16, N 3.64.
4.6.6. (3,4ꢀDimethoxyphenyl)ꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀmethanone (13f): Yellow
1
solid (73%). mp = 185–187 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.71 (dd, J = 7.3, 1.7 Hz,
2H), 7.44 (dd, J = 7.7 Hz, 1.4, 2H), 7.14 (m, 4H), 6.93–6.87 (m, 1H), 6.84– 6.73 (m, 2H), 3.69
(s, 3H), 3.49 (s, 3H); 13C NMR (75 MHz, DMSOꢀd6): δ 167.3, 150.4, 147.6, 139.6, 130.3, 129.2,
127.8, 127.4, 126.8, 126.7, 122.1, 112.0, 110.7, 55.4, 55.2; HRMS (ESI) m/z calcd. for
80
C21H18NO3 Se [M + H]+ 412.0451. Found 412.0445. Anal. calcd. for C21H17NO3Se•0.5 H2O
(410.32): C 60.15, H 4.33, N 3.34; found: C 60.45, H 4.05, N 3.27.
4.6.7. 2ꢀChloroꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀ(phenyl)methanone (13g): Yellow solid
(79%). mp =155–158. 1H NMR (300 MHz, DMSOꢀd6): δ 7.78–7.68 (m, 3H), 7.37–7.13 (m, 9H);
HRMS (ESI) m/z calcd. for C19H13ClNO80Se [M + H]+ 385.9850, found 385.9841.
4.6.8. 1ꢀ(2ꢀChloroꢀ10Hꢀphenoselenazinꢀ10ꢀyl)ꢀ2ꢀphenylethanone (13h): Yellow oil
(73%). 1H NMR (300 MHz, DMSOꢀd6): δ 7.75 ( br s, 1H), 7.67 (d, J = 8.3 Hz, 3H), 7.42 (t, J =
7.9 Hz, 2H), 7.37–7.23 (m, 2H), 7.23–7.12 (m, 2H), 6.95–6.88 (m, 2H), 3.74 (s, 2H); HRMS
(ESI) m/z calcd. for C20H15ClNO80Se [M + H]+ 400.0007, found 399.9998.
4.6.9. 1ꢀ(2ꢀChloroꢀ10Hꢀphenoselenazinꢀ10ꢀyl)ꢀ3ꢀphenylpropanꢀ1ꢀone (13i): Yellow
1
solid (84%). mp = 90–92 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.74 (d, J = 8.5 Hz, 3H), 7.45–
7.10 (m, 9H), 2.87–2.69 (br s, 4H); HRMS (ESI) m/z calcd. for C21H17ClNO80Se [M + H]+
414.0163, found 414.0155.
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4.6.10. 2ꢀChloroꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀ(4ꢀmethoxyphenyl)methanone (13j):
1
White solid (47%). mp = 155–156 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.78–7.64 (m, 3H),
7.31 (d, J = 8.2 Hz, 2H), 7.26–7.16 (m, 4H), 6.80 (d, J = 8.4 Hz, 2H), 3.70 (s, 3H); HRMS (ESI)
80
m/z calcd. for C20H15ClNO2 Se [M + H]+); 415.9956, found 415.9947.
4.6.11. 2ꢀChloroꢀ10Hꢀphenoselenazinꢀ10ꢀylꢀ(3ꢀmethoxyphenyl)methanone (13k):
1
Yellow solid (52%). mp = 135–137 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.80–7.36 (m, 3H),
7.38–7.26 (m, 2H), 7.25–7.11 (m, 3H), 6.90 (d, J = 8.2 Hz, 1H), 6.86–6.78 (m, 2H), 3.61 (s, 3H);
HRMS (ESI) m/z calcd. for C20H15ClNO2 Se [M + H]+ 415.9956, found 415.9947.
80
4.6.12. 2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀylꢀ(3,4ꢀdimethoxyphenyl)methanone (13l):
1
Yellow solid (28%). mp = 138–140 °C. H NMR (300 MHz, DMSOꢀd6): δ 7.78–7.65 (m, 3H),
7.35–7.26 (m, 2H), 7.24–7.15 (m, 2H), 6.96–6.73 (m, 3H), 3.69 (s, 3H), 3.50 (s, 3H); HRMS
80
(ESI) m/z calcd. for C21H17ClNO3 Se [M + H]+ 446.0062, found 446.0054.
4.7. Cholinesterase inhibition studies
Human AChE and BuChE enzymes were obtained from SigmaꢀAldrich, St. Louis, MO, USA
(AChE product number C0663 and BuChE product number B4186 respectively). The inhibition
profile of PTZ (6a, 6b and 7a–l) and PSZ (12a, 12b and 13a–l) series was evaluated using
Ellman’s reagent.34 The cholinesterase inhibitors tacrine (item number 70240, Cayman Chemical
Company, Ann Arbor, MI), donepezil (product number D6821, SigmaꢀAldrich, St. Louis, MO)
and galantamine (product number G1660, SigmaꢀAldrich, St. Louis, MO) were used as reference
agents. Test compounds were prepared as stock solutions in DMSO (maximum 1% v/v in final
wells) and diluted in buffer solution (50 mM Tris.HCl, pH 8.0, 0.1 M NaCl, 0.02 M
MgCl2.6H2O). Then in 96ꢀwell plates, 160 µL of 5,5’ꢀdithiobis(2ꢀnitrobenzoic acid) (1.5 mm
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DTNB), 50 µL of hAChE (0.22 U/mL in 50 mm Tris.HCl, pH 8.0, 0.1% w/v bovine serum
albumin, BSA) or 50 µL of hBuChE (0.12 U/mL in 50 mM Tris.HCl, pH 8.0, 0.1% w/v BSA)
were incubated with 10 µL of test compounds (1, 5, 10, 25, 50 and 100 µM final concentrations)
at room temperature for 5 min followed by the addition of 30 µL of either acetylthiocholine
iodide (15 mM ATCl prepared in ultra pure water) or Sꢀbutyrylthiocholine iodide (15 mM BTCI
prepared in ultra pure water). Then the absorbance was measured at various time intervals (0, 60,
120, 180, 240 and 300 s) at 412 nm. The inhibitory concentration (IC50 values) were calculated
from the concentration–inhibition dose response curve on a logarithmic scale based on two
independent experiments (n = 3).
4.8. Molecular modeling studies of PTZ and PSZ compounds in cholinesterases
Molecular docking experiments were performed using Discovery Studio (DS) StructureꢀBasedꢀ
Design software program (version 4.0) from BIOVIA/Accelrys Inc. San Diego, USA. The
coordinates for the Xꢀray crystal structures of human AChE was obtained from the protein data
bank (pdb id – 4EY7). The protein was prepared using the macromolecule module in DS and
active site was defined by selecting a 12 Å sphere around the ligand donepezil after which it was
deleted. PTZ (6a and 7j) and PSZ (12a and 13j) compounds were built in 3D using the small
molecule module in DS. The ligands were docked to AChE active site using CDOCKER
algorithm using CHARMm force field. Docking steps include simulated annealing with 2000
heating steps, 700K heating target temperature and 5000 cooling steps, 300K cooling target
temperature to generate 10 docked ligand poses. The enzymeꢀligand complex obtained was
ranked based on CDOCKER energy and CDOCKER interaction energy in kcal/mol. Polar and
nonpolar contacts of ligands with cholinesterase enzymes were evaluated. A similar modeling
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experiments were carried out to investigate the binding of ligands with human BuChE enzyme
(pdb code = 2XQJ) after deleting the ligand VX 150.
4.9. Selfꢀinduced Aβ1ꢀ42 aggregation inhibition studies
The antiꢀAβ aggregation activity of test compounds (6a, 6b, 7f, 7l, 12a, 12b, 13f and 13l), were
evaluated using the ThTꢀbased fluorescence assay. The Aβ1ꢀ42 hexafluoroꢀ2ꢀpropanol (HFIP)
(rPeptide, Geogia, USA) stock solution was prepared by dissolving in 1% NH4OH solution, to a
1mg/mL stock solution, followed by dilution in phosphate buffer (pH 8.0) to 500 ꢁM. Stock
solutions of test compounds were prepared in DMSO solution, diluted in phosphate buffer (pH
8.0), and were sonicated for 30 min. The final DMSO concentration per each well was 1% v/v or
lower. The ThT fluorescent dye stock solution (15 ꢁM) was prepared in 50 mM glycine buffer
(pH 8.5). The aggregation kinetics assay was carried out using a Corning® 96ꢀwell flat, clear
bottom black plates. Each well contains 110 ꢁl of ThT, 50 ꢁl of phosphate buffer (pH 8.0), 20 ꢁl
of test compounds in different concentrations (1, 5, 10 and 25 ꢁM, final concentration) and 20 ꢁl
of Aβ1ꢀ42 (5 ꢁM final concentration). The plate was incubated at 37 °C with a plate cover under
shaking and fluorescence was measured every 5 min. using a SpetraMax M5 multimode plate
reader (excitation = 440 nm and emission = 490 nm) over a period of 16 h. Appropriate control
experiments that contain Aβ1ꢀ42 and test compound alone were evaluated as well. The known Aβ
aggregation inhibitor orange G (SigmaꢀAldrich, St. Louis, MO) was used as a reference
compound. The percentage inhibition was calculated using the equation 100% control – [(IFi –
IFo)] where 100% control indicates no inhibitor, IFi and IFo are the fluorescence intensities in
the presence and absence of ThT. The results were expressed as percentage inhibition of two
separate experiments of triplicate measurements.
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5.0. Antioxidant activity evaluation
The DPPH radical scavenging activity of test compounds (6a, 6b, 7f, 7l, 12a, 12b, 13f and 13l),
were tested in a 96ꢀwell plate format. The antioxidants, trolox and ebselen were used as
reference compounds. The test compound and DPPH (0.09 mM) stock solutions were prepared
in methanol. The final test compound concentration was 50 ꢁM. Each well contained 50 ꢁL of
test compound and 200 ꢁL of DPPH solution. Appropriate controls with no DPPH and no test
compounds were included. The 96ꢀwell plate was protected from light and incubated at room
temperature with shaking for 60 minutes and then absorbance was measured at 517 nm. The
percentage DPPH scavenging activity was calculated by subtracting the absorbance of the DPPH
control by the difference in absorbance value of the test compounds and blank which was
divided by the absorbance of the DPPH control. The value obtained was converted to percent
inhibition. The results were expressed as mean ± standard deviation (SD) of two separate
experiments (n = 3).
5.1. Cell death assay studies in SHꢀSY5Y neuroblastoma cells
The cell viability assay for test compounds (6a, 6b, 7f, 7l, 12a, 12b, 13f and 13l) were carried
out using SHꢀSY5Y neuroblastoma cells. They were plated at a density of 4 x105 per mL in 96ꢀ
well plates with complete growth media consisting of DMEM and Ham’s F12 in a 1:1 ratio,
supplemented with 2.5 nM glutamate and 10% fetal bovine serum at 37 °C in 5% CO2. The cells
were incubated overnight and treated with the test compounds and ebselen (50 ꢁM) for 24 h at 37
°C in triplicates (n = 3). The MTT reagent was added in 10% of the culture medium volume to
each well and the cells were cultured for an additional 3 h at 37 °C in 5% CO2. After incubation,
the resulting formazan crystals were solubilized with MTT reagent solution in each well and the
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absorbance was taken at 570 nm. All results were expressed as a relative percent of MTT to
untreated controls.
Acknowledgements
The authors would like to thank Dr. John Honek, Department of Chemistry, University of Waterloo, for
assistance with the fluorescence microplate reader, Ontario Mental Health Foundation for a PhD
studentship (TM), NSERCꢀDiscovery Grant to PR (RGPIN 03830ꢀ2014) and MB (RGPIN 371384ꢀ2013),
Canada Foundation for Innovation (CFIꢀJELF) Leaders Fund (for PR) and Ministry of Research and
Innovation, Government of Ontario, Canada for an Early Researcher Award (PR) for financial support.
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Fig. 1 Chemical structure of some fused tricyclic compounds 1, 2, 3, 4 and 5
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