Exploration of a library of triazolothiadiazole and triazolothiadiazine compounds as a highly potent and selective family of cholinesterase and monoamine oxidase inhibitors: Design, synthesis, X-ray diffraction analysis and molecular docking studies

Article abstract of DOI:10.1039/c5ra00906e

There is a high demand for the collection of small organic molecules (especially N-heterocycles) with diversity and complexity in the process of drug discovery. This need for privileged scaffolds in medicinal research gives an impetus for the development of nitrogen-containing compounds which are widely encountered in natural products, drugs and pharmaceutically active compounds. In this context, a diverse library of new triazolothiadiazole (4a-l) and triazolothiadiazine (5a-p) compounds was designed, synthesized and evaluated as potent and selective inhibitors of electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) by Ellman's method using neostigmine and donepezil as standard inhibitors. Among the screened triazolothiadiazoles, 4j emerged as a lead candidate showing the highest inhibition with an outstanding IC₅₀ value of 0.117 ± 0.007 μM against AChE, which is ～139-fold greater inhibitory efficacy as compared to neostigmine, whereas 4k displayed ～506-fold strong inhibition with IC₅₀ of 0.056 ± 0.001 μM against BChE. In the triazolothiadiazine series, 5j and 5e depicted a clear selectivity towards EeAChE with IC₅₀ values of 0.065 ± 0.005 and 0.075 ± 0.001 μM, respectively, which are ～250- and ～218-fold stronger inhibition as compared to neostigmine (IC₅₀ = 16.3 ± 1.12 μM). In addition, the synthesized compounds were also tested for their monoamine oxidase (MAO-A and MAO-B) inhibition, where 4a from the triazolothiadiazole series delivered the highest potency against MAO-A with an IC₅₀ value of 0.11 ± 0.005 μM which is ～33-fold higher inhibition as compared to the standard inhibitor, clorgyline (IC₅₀ = 3.64 ± 0.012 μM), whereas compound 5c from the triazolothiadiazine series turned out to be a lead inhibitor with an IC₅₀ value of 0.011 ± 0.001 μM which is ～330-fold stronger inhibition. Moreover, compounds 4b (triazolothiadiazole series) and 5o (triazolothiadiazine series) were identified as lead inhibitors against MAO-B. Molecular modelling studies were performed against human AChE and BChE to observe the binding site interactions of these compounds.

Full text of DOI:10.1039/c5ra00906e

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Exploration of a library of triazolothiadiazole and triazolothiadiazine compounds as a
highly potent and selective family of cholinesterase and monoamine oxidase inhibitors:
Design, synthesis, Xꢀray diffraction analysis and molecular docking studies
a†
b
a
b
a*
Imtiaz Khan , Syeda Mahwish Bakht , Aliya Ibrar , Saba Abbas , Shahid Hameed , Jonathan
c
d
b
e
b*
M. White , Usman Ali Rana , Sumera Zaib , Mohammad Shahid , Jamshed Iqbal
a
b
Department of Chemistry, QuaidꢀiꢀAzam University, Islamabadꢀ45320, Pakistan
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabadꢀ22060, Pakistan
c
School of Chemistry and Bioꢀ21 Institute, University of Melbourne, Parkvilleꢀ3052, Australia
d
Deanship of Scientific Research, College of Engineering, King Saud University, Riyadh 11421,
Saudi Arabia
e
German Center for Neurodegenerative Diseases (DZNE)
†
Present Address: School of Chemistry, University of Nottingham, University Park, Nottingham,
NG7 2RD (UK)
Graphical Abstract
___________________________________________________________________________
*Corresponding authors. Tel.: +92ꢀ51ꢀ90642133; Fax: +92ꢀ51ꢀ90642241; Eꢀmail:
shameed@qau.edu.pk
(S.
Hameed);
+92ꢀ992ꢀ383591/96;
Fax:
+92ꢀ992ꢀ383441.
drjamshed@ciit.net.pk (J. Iqbal)
1

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Abstract
There is a high demand for the collection of small organic molecules (especially Nꢀheterocycles)
with diversity and complexity in the process of drug discovery. This need for privileged
scaffolds in medicinal research gives an impetus for the development of nitrogenꢀcontaining
compounds which are widely encountered in natural products, drugs and pharmaceutically active
compounds. In this context, a diverse library of new triazolothiadiazole (4a–l) and
triazolothiadiazine (5a–p) compounds was designed, synthesized and evaluated as potent and
selective inhibitors of electric eel acetylcholinesterase (EeAChE) and horse serum
butyrylcholinesterase (hBChE) by Ellman’s method using neostigmine and donepezil as standard
inhibitors. Among the screened triazolothiadiazoles, 4j emerged as a lead candidate showing
highest inhibition with an outstanding IC value of 0.117 ± 0.007 µM against AChE, which is
5
0
~139–folds more inhibitory efficacy as compared to neostigmine, whereas 4k displayed ~506–
folds strong inhibition with IC of 0.056 ± 0.001 µM against BChE. In the triazolothiadiazine
5
0
series, 5j and 5e depicted a clear selectivity towards EeAChE with IC values of 0.065 ± 0.005
5
0
and 0.075 ± 0.001 µM, respectively, which is ~250– and ~218–folds stronger inhibition as
compared to neostigmine (IC = 16.3 ± 1.12 µM). In addition, the synthesized compounds were
5
0
also tested for their monoamine oxidases (MAOꢀA and MAOꢀB) inhibition, where 4a from
triazolothiadiazole series, delivered highest potency against MAOꢀA with IC50 value of 0.11 ±
0.005 µM which is ~33ꢀfolds higher inhibition as compared to standard inhibitor, clorgyline
(
IC = 3.64 ± 0.012 µM), whereas, compound 5c from triazolothiadiazine series turned to be a
5
0
lead inhibitor with IC50 value of 0.011 ± 0.001 µM which is ~330ꢀfolds strong inhibition.
Moreover, compounds 4b (triazolothiadiazole series) and 5o (triazolothiadiazine series) were
2

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identified as lead inhibitors against MAOꢀB. Molecular modelling studies were performed
against human AChE and BChE to observe binding site interactions of these compounds.
Key words: Acetylcholinesterase, Butyrylcholinesterase, Molecular modelling, Monoamine
oxidases, Triazolothiadiazole, Triazolothiadiazine.
Introduction
1
,2
Alzheimer’s disease (AD) is a chronic, progressive, neurodegenerative disease and it currently
1
–3
afflicts 24 million people worldwide. It is the most common cause of senile dementia and is
characterized by loss of both short term and long term memory due to neuronal loss,
disorientation, difficulty in speaking or writing, loss of reasoning skills, and delusions, among
4
other symptoms. A multitude of features, including metal ion dyshomeostasis and disruption of
both lipid production and membrane structure, have been implicated in AD onset and
3
progression. Recent research has suggested that some of these characteristics could be
2
,3
intertwined with other facets (i.e., protein aggregation) of the disease.
Two important enzymes from the group of serine hydrolases, acetylcholinesterase (AChE, EC
.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8; also known as pseudocholinesterase), are
3
usually defined as cholinesterases (ChEs). Structurally, these serine hydrolases belong to the
5
class of proteins known as the esterase/lipase family within the α/βꢀhydrolase fold superfamily.
6
They differ in kinetics and substrate selectivity and the major role of AChE is to catalyze the
hydrolysis of acetylcholine (ACh) in cholinergic synapses, whereas the function of BChE is less
7
,8
clearly defined because it can hydrolyze ACh as well as other esters. One of the major
therapeutic strategies adopted for primarily symptomatic AD is based on the cholinergic
9
hypothesis targeting cholinesterase enzymes (acetylꢀ and butyrylꢀcholinesterase). Inhibition of
3

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the hydrolysis of acetylcholine by blocking its metabolic enzyme acetylcholinesterase (AChE)
increases the ACh concentration in cholinergic synapses and can subsequently affect a number of
pathogenic processes and thereby provides a possible symptomatic treatment option for AD. On
the other hand, butyrylcholinesterase (BChE) has recently been considered as a potential target
1
0
because it also plays an important role in regulating ACh level. ChE inhibitors (ChEIs) are used
in the treatment of various neuromuscular disorders and have provided the first generation of
1
1
drugs for the treatment of Alzheimer’s disease. AChE inhibitors currently approved as drugs
for the treatment of Alzheimer’s disease are donepezil, rivastigmine, galanthamine and tacrine.
In view of the limited number of AChE inhibitors currently available on the market for the
treatment of AD, the search for new ChEIs is of great interest and ongoing worldwide.
Monoamine oxidase (MAO; EC 1.4.3.4) is an important target to be considered for the treatment
of AD, as catalyzes the oxidative deamination of a variety of biogenic and xenobiotic amines,
12
with the concomitant production of hydrogen peroxide. Monoamine oxidase is a flavin adenine
dinucleotide (FAD) containing enzyme, which is localized in the outer mitochondrial membranes
1
3
14
of neuronal, glial, and other cells particularly abundant in the liver and brain. MAO exists as
two isozymes: MAOꢀA and MAOꢀB, showing different substrate specificity, sensitivity to
inhibitors, and amino acid sequences. MAOꢀA preferentially oxidizes norepinephrine and
serotonine, and is selectively inhibited by clorgyline, while MAOꢀB preferentially deaminates βꢀ
1
5
phenylethylamine and is irreversibly inhibited by Lꢀdeprenyl. Selective MAOꢀA inhibitors are
used clinically as antidepressants and anxiolytics, while MAOꢀB inhibitors are used for reduction
of the progression of Parkinson’s disease and of symptoms associated with Alzheimer’s disease.
Earlier MAOꢀinhibitors introduced into clinical practice for the treatment of depression were
abandoned due to adverse side effects, such as ‘cheese effect’ characterized by hypertensive
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1
6
crises. For this reason, there is currently a need for compounds that are useful for enhancing
cognitive function and for treating cognitive deterioration in AD, as well as compounds that can
1
6
generally improve cognition in normal, diseased, and aging subjects, and presently the research
has been directed towards the synthesis of new potential agents with possible clinical practice.
Nitrogenꢀcontaining heterocyclic compounds are the most abundant and integral scaffolds that
occur ubiquitously in a variety of synthetic drugs, bioactive natural products, pharmaceuticals
and agrochemicals. Owing to their widespread applications, these skeletons have long been a
subject of immense interest, and substantial efforts have been made to the development of
synthetic strategies which could lead to the discovery of new bioactive compounds in medicinal
1
7
chemistry.
Among them, conjugated heterocycles like triazolothiadiazoles and
triazolothiadiazines are associated with a wide range of biological activities including
1
8
19
20
21
22
antimicrobial, antitumor, antiviral, anticancer, CNS depressant, antiꢀinflammatory,
2
3
24
25
analgesic, antioxidant, antiꢀHIV and antiꢀkinesin Eg5 activity. Numerous triazolothiadiazole
derivatives have also revealed potent urease, phosphodiesterase and cꢀMet protein inhibitory
2
6,27
potential.
We have recently demonstrated the utility of these hybrid structures incorporating
heteroꢀaromatic substituent (pyridine) at 3–position of the main skeleton displaying potential
28,29
cytotoxic, antiꢀcholinesterase, alkaline phosphatase and antiꢀleishmanial activities.
These Nꢀ
bridgehead cholinesterase inhibiting lead structures were found to exhibit moderate activity and
were used as a starting point for the development of highly potent and selective cholinesterase
inhibitors. The cholinesterase inhibitory activities of these classes of compounds and their
selectivity were dramatically improved by introducing a nonꢀheteroaromatic substituents at 3ꢀ
position. With this aim, we synthesized these hybrid fundamental templates with a variety of
structural modifications and investigated SARs to find structures with high cholinesterase
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inhibitory potential. Additionally, the synthesized hybrids were also screened for their
monoamine oxidase inhibition and were found to inhibit MAOꢀA selectively (Figure 1). The
results of this study are summarized in this paper.
Cholinesterase inhibitors
N
N
N
N
N
N
N
N
S
S
S
S
N
N
N
N
N
N
N
N
O
O
MeO
Br
Br
Br
4j
Me
4k
OMe
5j
5e
Cl
Monoamine oxidase inhibitors
N
N
N
N
N
N
S
S
S
N
N
N
N
N
N
OH
O
O
MeO
MeO
MeO
4
a
4b
OH
5c
F
Fig. 1 Synthesized heteroaromatic structures with remarkable cholinesterase and monoamine
oxidase inhibitory potential.
Results and Discussion
Chemistry
The target compounds were synthesized as outlined in Scheme 1. The key intermediate in the
preparation of triazolothiadiazoles (4a–l) and triazolothiadiazines (5a–p) was 4ꢀaminoꢀ1,2,4ꢀ
triazole (3a,b) which in turn was prepared by a oneꢀpot reaction of the corresponding benzoic
3
0
acids (1a,b) with thiocarbohydrazide (2) at 190–200 °C. While 4ꢀaminoꢀ3ꢀmercaptoꢀ1,2,4ꢀ
triazoles (3a,b) may exist in thione–thiol tautomeric forms, in the present study, the thiol
1
structure was dominated in the solid state. Routine spectroscopic methods including FTIR and H
NMR readily distinguished between these constitutional isomers.
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Scheme 1 Synthesis of triazolothiadiazoles (4a–l)and triazolothiadiazines (5a–p).
ꢀ1
The FTIR spectra of 3a and 3b showed absorption bands at 2554 and 2563 cm , respectively,
ꢀ1
indicative of –SH group and no absorption band in the range 1300–1200 cm attributable to the
ꢀ1
C=S group. The disappearance of the absorption bands for NꢀH in the range of 3400ꢀ3200 cm
1
also indicated the formation of thiol isomer. H NMR spectra of 4ꢀaminoꢀtriazoles (3a,b)
exhibited resonances for –NH protons as a singlet at 5.77 and 5.80 ppm (integrating two
2
protons) and the –SH proton as a singlet at 13.83 and 14.03 ppm, respectively.
The reaction of corresponding 4ꢀaminoꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol (3a,b) with aryloxy acids in the
presence of phosphorous oxychloride delivered 1,2,4ꢀtriazolo[3,4ꢀb][1,3,4]thiadiazoles (4a–
2
8,29
l).
Their proton NMR spectra showed new peaks in the range of 5.80–8.53 ppm (integration
for two protons) attributed to methylene protons, and lacked signals characteristic of –SH and –
NH protons, establishing that the –COOH group of the aryloxy acids reacted with –SH and –
2
NH groups of the intermediate 3a,b to afford the corresponding conjugated products in efficient
2
yields. Structural variations included electronꢀdonating (–OH, –OMe, –Me) as well as electronꢀ
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withdrawing (–F) substituents on the aryloxy ring attached to Cꢀ6 of triazolothiadiazole skeleton
in addition to those present on aryl ring of benzoic acid attached to Cꢀ3 of the conjugated core.
Moreover, 1,2,4ꢀtriazolo[3,4ꢀb][1,3,4]thiadiazines (5a–p) were prepared through the reaction of
corresponding 4ꢀaminoꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol (3a,b) with substituted phenacyl bromides in
2
8,29
1
absolute ethanol under reflux.
Their proton H NMR spectra showed that the signals due to –
SH and –NH protons had disappeared, and a new signal appeared at 4.58–4.40 ppm (integration
2
for two protons) attributed to the methylene protons. This confirmed that the intermediate (3a,b)
underwent ring closure to give the 1,2,4ꢀtriazolo[3,4ꢀb][1,3,4]thiadiazines in good yields. The
generality of this method is validated by using a diverse range of substituents (electron–rich and
electron–deficient) on the aryl ring affording corresponding hybrid products in high yields. The
cyclocondensation reaction also proceeds efficiently when αꢀbromo ketones with bulky biphenyl
and naphthyl groups were used as coupling partners affording desired products in good yields.
Furthermore, purity of the synthesized compounds was ascertained by elemental analysis.
Xꢀray Crystallography
For a full structural elucidation, compounds 4i and 5e were confirmed by Xꢀray diffraction
analysis. The crystal and instrumental parameters used in the unit cell determination, the data
collection, and structure refinement parameters are presented in Table 1.
Table 1 Crystal data and structure refinement for 4i and 5e
Structural parameters
Empirical formula
Formula weight
Compound 4i
Compound 5e
C H BrN OS
C H N OS
17
13
4
23 18
4
401.28
398.47
Temperature (K)
Wavelength (Å)
130(2)
0.7107
130(21)
0.7107
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Crystal system
Triclinic
Monoclinic
P 21/c
Space group
P ꢀ1
Unit cell dimensions (Å)
a = 7.5733(3), α = 105.441(5)°
a = 13.0300(3), α = 90°
b = 10.0581(7), β = 102.930(4)° b = 17.3539(4), β = 98.990(2)°
c = 11.2828(6), γ = 91.660(4)°
c = 8.6142(2), γ = 90°
3
Volume (Å )
803.82(8)
2
803.82(8)
4
Z
3
Density (calculated) (mg/m )
1.584
1.376
ꢀ1
Absorption coefficient (mm )
0.285
0.191
F(000)
404
832
3
Crystal size (mm )
0.59 × 0.39 × 0.18
0.35 × 0.2 × 0.15
Theta range for data collection (°)
Index ranges
2.973 to 29.121
2.903 to 29.188
ꢀ9<=h<=10, ꢀ13<=k<=13,
ꢀ15<=h<=17, ꢀ23<=k<=20,
ꢀ11<=l<=11
ꢀ14<=l<=12
Reflections collected
6521
11392
Independent reflections
3736 [R(int) = 0.0331]
4578 [R(int) = 0.0382]
Completeness to theta = 25.242° (%) 99.7
99.8
Absorption correction
Gaussian
Semiꢀempirical from equivalents
Max. and min. Transmission
0.639 and 0.328
1.00000 and 0.86346
2
2
Refinement method
Fullꢀmatrix leastꢀsquares on F
Fullꢀmatrix leastꢀsquares on F
Data / restraints / parameters
3736 / 0 / 218
4578 / 0 / 263
2
Goodnessꢀofꢀfit on F
1.028
1.053
Final R indices [I>2σ(I)]
R = 0.0349, wR = 0.0729
R = 0.0505, wR = 0.1156
1
2
1
2
R indices (all data)
R = 0.0448, wR = 0.0776
R = 0.0736, wR = 0.1343
1 2
1
2
ꢀ3
Largest diff. peak and hole (e Å )
0.472 and ꢀ0.512
0.316 and ꢀ0.400
Compound 4i: A thermal ellipsoid plot for the [1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole (4i) is
presented in Figure 2. The bond distances within the [1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole ring
system are in agreement with the mean values for the corresponding distances from 49 structures
3
1
in the Cambridge Crystallographic Database (CSD) containing this moiety. The bromophenyl
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substituent is essentially coplanar with the [1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole ring (C2ꢀC1ꢀ
C7ꢀN3 dihedral angle = ꢀ174.0(2)°) and the RMS deviation of atoms from the plane defined by
C1ꢀC9, N1ꢀN4, S1 = 0.077. The phenoxymethylene substituent is twisted from coplanarity with
the triazolothiadiazole ring (dihedral angle S1ꢀC9ꢀC10ꢀO1 = 55.8(2)°) while the conformation
about the C11ꢀO1 bond (dihedral angle C10ꢀO1ꢀC11ꢀC16 = ꢀ2.4(3)°) allows for conjugation of
the oxygen pꢀtype lone pair with the aromatic ring.
Fig. 2 Thermal ellipsoid plot for 4i, ellipsoids are at the 30% probability level.
Crystals of 4i are densely packed as a result a number of strong intermolecular interactions. The
most important interactions are summarized in Figures 3–5. The first of these is a strong
complementary interaction between a pair of triazolothiadiazole rings related by a centre of
inversion. The S1…N2 distance is 2.795(2) Å, which is 0.555 Å shorter than the sum of the van
der Waals radii for nitrogen and sulfur. The N2’…S1ꢀC9 angle which is 163.9(2)° suggests that
this may be an example of a ‘sigmaꢀhole’ type interaction between the nitrogen lone pair and the
sulfur atom. A search of the CSD for structures containing the triazolothiadiazole ring system,
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revealed no contacts between the S atom and N donors in the crystal which were significantly
shorter than 3.0 Å, therefore this structure provides the shortest example of a N…S interaction
3
2
yet observed in these types of compounds.
Fig. 3 Interaction between [1,2,4]triazolothiadiazole rings. Atoms labeled with a (‘) are related
by the symmetry operation (ꢀx, ꢀy, 1ꢀz).
The crystal packing of 4i is also stabilized by πꢀstacking interactions between pairs of the planar
aryl–substituted triazolothiadiazole moieties. The distance between the mean planes of aryl–
triazolothiadiazole is 3.342(2) Å while the centroid–centroid distance is 4.124 Å.
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Fig. 4 π–Stacking between centrosymmetrically related pair of the 4i (symmetry operation is (ꢀx,
ꢀy, 1ꢀz).
1
The third strong interaction evident in the packing of 4i is a complementary CꢀH…π interaction
between centrosymmetrically related pairs of phenoxymethylene moieties. The distance between
H10A and the mean plane defined by atoms C11ꢀC16 is 2.753(2) Å while the distance of H10A
to the centroid of this ring is 2.820 Å.
Fig. 5 CꢀH…π interaction between centrosymmetrically related pairs of 4i, symmetry operator
(1ꢀx, ꢀy,ꢀz).
A partial packing diagram indicating all these interactions is presented in Figure 6.
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Fig. 6 Partial packing diagram illustrating the N…S sigma–hole interaction and the π–π and Cꢀ
H…π interactions in crystals of 4i.
These three strong interactions, which extend in all 3ꢀdimensions in the crystal (Figures 7 and 8),
ꢀ3
result in a densely packed crystal which has a high crystal density of 1.658 g/cm .
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Fig. 7 Packing diagram of 4i in the xz plane stabilized by π…π interactions.
Fig. 8 Packing diagram of 4i in the yz plane stabilized by both N…S sigmaꢀhole interactions and
CꢀH…π interactions.
Compound 5e: A thermal ellipsoid plot for 7Hꢀ[1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazine (5e) is
presented in Figure 9. The bond distances within the 7Hꢀ[1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazine
ring system are closely similar to the mean bond distances obtained from 61 structures
containing this ring system in the CSD. The methoxyphenyl substituent is essentially coplanar to
the triazole ring( C2ꢀC1ꢀC7ꢀN1 dihedral angle = ꢀ5.0(2)°) while the first biphenyl ring is close to
coplanar with the C=N moiety in the thiadiazine ring (dihedral angles C16ꢀC11ꢀC10ꢀC9 = ꢀ
3.2(2)° and C16ꢀC11ꢀC10ꢀN4 = 172.5(2)°, this conformation, which allows for conjugation
between these two groups occurs at the cost of a close contact between hydrogen atoms attached
to C16 and C9 (H16…H9b 1.94 Å). The biphenyl rings are twisted from coplanarity (C18ꢀC17ꢀ
C14ꢀC13 dihedral angle = ꢀ26.5(2)°). The methoxyl substituent is near coplanar with the
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aromatic ring (dihedral angle C23ꢀO1ꢀC4ꢀC3 = 10.5(2)°) this conformation allows for extensive
delocalization of the oxygen loneꢀpair onto the ring through to the electronꢀdeficient
triazolothiadiazine moiety.
Fig. 9 Thermal ellipsoid plot for 5e. Ellipsoids are at the 30% probability level.
In contrast to compound 4i, there are only relatively weak intermolecular interactions between
molecules of 5e in the crystal. The shortest interactions are between hydrogen atoms H15 and
H16 and the nitrogen atoms N1 and N2 respectively (distances N1…h16 and N2…H15 are 2.68
and 2.46 Å, respectively (Figure 10). Consistent with the relatively weak intermolecular contacts
ꢀ3
in the structure of 5e, the crystal density 1.376 gcm is significantly less than 4i.
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Fig. 10 Close intermolecular contacts between molecules of 5e (symmetry code x, yꢀ0.5, 0.5+z).
Pharmacological studies
Cholinesterase inhibition
The newly synthesized triazolothiadiazoles (4aꢀl) and triazolothiadiazines (5aꢀp) were assayed
3
3
for inhibition against EeAChE and hBChE by Ellman’s method at micromolar level using
neostigmine and donepezil as standard inhibitors. Their IC values for AChE are 16.3 ± 1.12 µM
5
0
and 0.02 ± 0.003 µM, respectively. Whereas for BChE, neostigmine and donepezil showed IC50
value of 28.3 ± 2.06 and 7.23 ± 0.12 µM, respectively. The preliminary results revealed that the
synthesized hybrid compounds exhibited remarkable cholinesterase inhibition which is many
folds higher as compared to standard drugs. The results of this study are summarized in Tables 2
and 3.
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Table 2 AChE and BChE inhibition by newly synthesized triazolothiadiazoles (4a–l)
N
N
S
N
N
R1
O
R2
4a-l
1
2
Entry
R
R
AChE
BChE
IC ± SEM (µM)
5
0
4
a
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
3ꢀBr
2ꢀOH
4ꢀOH
2ꢀMe
4ꢀMe
0.282 ± 0.017
0.538 ± 0.046
3.302 ± 0.142
0.564 ± 0.016
0.245 ± 0.024
0.272 ± 0.018
0.213 ± 0.008
0.152 ± 0.004
0.311 ± 0.013
0.117 ± 0.007
0.159 ± 0.014
0.227 ± 0.013
16.3 ± 1.12
4.287 ± 0.023
0.478 ± 0.011
4.987 ± 0.028
0.184 ± 0.007
1.142 ± 0.013
0.175 ± 0.003
2.235 ± 0.014
2.354 ± 0.051
2.012 ± 0.018
1.971 ± 0.032
0.056 ± 0.001
0.168 ± 0.003
28.3 ± 2.06
4b
4
c
4d
4
e
4ꢀOMe
4ꢀF
4
f
4g
2ꢀOH
4ꢀOH
2ꢀMe
4ꢀMe
4ꢀOMe
4ꢀF
4h
3ꢀBr
4
i
3ꢀBr
4
j
3ꢀBr
4k
3ꢀBr
4
l
3ꢀBr
Neostigmine
Donepezil
—
—
—
—
0.02 ± 0.003
7.23 ± 0.12
IC = Concentration of inhibitor required for halfꢀmaximal enzyme inhibition and are reported as means
50
of two independent experiments, each performed in triplicate (SEM, standard error of the mean).
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Table 3 AChE and BChE inhibition by newly synthesized triazolothiadiazines (5a–p)
1
Entry
R
Ar
AChE
BChE
IC ± SEM (µM)
5
0
5
a
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
3ꢀBr
4ꢀOMeꢀPh
4ꢀClꢀPh
4ꢀFꢀPh
0.870 ± 0.051
1.09 ± 0.072
0.365 ± 0.013
0.220 ± 0.005
0.075 ± 0.001
0.496 ± 0.016
0.205 ± 0.013
0.301 ± 0.011
0.747 ± 0.042
0.065 ± 0.005
0.259 ± 0.031
0.950 ± 0.085
1.303 ± 0.12
1.066 ± 0.76
0.854 ± 0.93
0.248 ± 0.021
16.3 ± 1.12
0.176 ±0.013
0.362 ±0.015
3.422 ± 0.008
0.066 ± 0.021
3.936 ± 0.041
3.174 ± 0.007
2.382 ± 0.004
2.547 ± 0.016
0.748 ± 0.004
1.445 ± 0.0124
1.384 ± 0.016
3.981 ± 0.0015
0.480 ± 0.028
0.343 ± 0.012
0.780 ± 0.062
1.07 ± 0.084
28.3 ± 2.06
5b
5
c
5d
4ꢀMeꢀPh
biphenyl
naphthyl
5
e
5
f
5g
4ꢀNO
2
ꢀPh
ꢀPh
5h
3,4ꢀCl
2
5
i
4ꢀOMeꢀPh
4ꢀClꢀPh
4ꢀFꢀPh
5
j
3ꢀBr
5k
3ꢀBr
5
l
3ꢀBr
4ꢀMeꢀPh
biphenyl
naphthyl
5m
3ꢀBr
5n
3ꢀBr
5o
3ꢀBr
4ꢀNO
2
ꢀPh
ꢀPh
5p
3ꢀBr
3,4ꢀCl
2
Neostigmine
—
—
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Donepezil
—
—
0.02 ± 0.003
7.23 ± 0.12
IC = Concentration of inhibitor required for halfꢀmaximal enzyme inhibition and are reported as means
50
of two independent experiments, each performed in triplicate (SEM, standard error of the mean).
Structureꢀactivity relationship
On the basis of cholinesterase inhibitory activity results obtained for the prepared conjugated
hybrid structures, we have used a three–zone approach to analyze the results and to guide our
initial round of medicinal chemistry structureꢀactivity relationship studies (Figures 11 and 12). In
particular, zone 2, the core heteroaromatic scaffold has found to be nonꢀinfluential on the activity
results. The zone 3 possessed a broad variation of substituents (electron–rich and electron–
deficient) on the aryl ring and in certain cases aryl ring itself. The investigation of these
exclusive modifications combined with zone 1 least alterations indicated a profound effect on the
generation of lead structures with improved and selective inhibitory activity.
Fig. 11 The synthesized triazolothiadiazole structures (4a–l) investigated herein.
From the activity results presented in Table 2, it is evident that most of the hybrid compounds
were significantly active for both AChE and BChE enzymes with more selectivity towards
AChE. The variation of the substituents at aromatic rings (zone 1 and 3) significantly affected
cholinesterase inhibitory potency and the selectivity as well. Remarkably, the behavior of these
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compounds is more selective for AChE than BChE inhibition having electron–rich (–OMe)
group on phenyl ring attached at 3–position of conjugated skeleton. All the synthesized
compounds (4a–l) were found to be significantly more potent against AChE than the standard
drugs used. Among them, 4j was the lead candidate showing highest inhibition with IC value
5
0
of 0.117 ± 0.007 µM, which is ~139–fold stronger inhibition as compared to neostigmine. This
compound possessed a bromo substituent at the meta–position of the phenyl ring (zone 1)
attached at 3–position along with a 4–methyl substituent at the para–position of the aromatic ring
(zone 3) attached at 6–position of triazolothiadiazole core. The same compound showed nearly
50% inhibition against BChE demonstrating the selective inhibitory potential towards AChE.
Replacement of the zone 1 meta–bromo group with a para–methoxy substituent also afforded
good inhibition against AChE (compound 4d; IC50 = 0.564 ± 0.016 µM; ~29–fold strong
inhibition) in addition to the ~154–fold higher potency against BChE as compared to 4j. Among
the same set of derivatives, the substituent electronic effect at various positions in zone 3
modulated the ChE inhibition as shown by compounds 4g–i. In general, all the synthesized
triazolothiadiazole compounds were emerged as efficient inhibitors of AChE whereas
compounds 4b, 4e, 4f, 4k, and 4l were found as potent candidates for BChE inhibition. It was
also found that the combination of electron–rich and electron–deficient substituents in zone 1
and 3 resulted in stronger and selective inhibitors of AChE as compared to BChE where this
combination is underlined in selected examples.
The newly synthesized triazolothiadiazine hybrids were also evaluated in vitro as inhibitors of
electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE). The
inhibitory activities of novel compounds were compared to those of neostigmine and donepezil.
All of the novel target analogues proved to be very potent and selective inhibitors of
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cholinesterases displaying inhibition abilities in micromolar range. A careful insight and analysis
of the data listed in Table 3 led to several eventual structure–activity relationships (SAR) using a
similar three zone approach (Figure 12).
Fig. 12 The synthesized triazolothiadiazine structures (5a–p) investigated herein.
Some interesting considerations can be made by comparing enzyme inhibition data (Table 3) for
the two sets of products bearing the same substituent in the zone 3 but variable groups (electron–
rich and electron–deficient) on phenyl ring in zone 1. The majority of tested hybrids in the first
set of compounds bearing para–methoxy group in zone 1 depicted a clear selectivity towards
EeAChE highlighting 5e as the most potent AChE inhibitor in the series with IC value of 0.075
5
0
±
0.001 µM which is ~218–fold stronger inhibition as compared to neostigmine (IC = 16.3 ±
50
1.12 µM). This compound incorporates bulky biphenyl ring in zone 3 in addition to para–
methoxy group in zone 1. Similar results were also obtained when the biphenyl ring at C–6 is
replaced with another sterically demanding naphthyl group (compound 5f: IC = 0.496 ± 0.016
5
0
µM). The compounds incorporating electron–deficient substituents such as 5g (4–NO ) and 5h
2
(3,4–Cl ) also revealed notable selective inhibition towards AChE with IC of 0.205 ± 0.013 µM
2 50
(~79–fold stronger inhibition) and 0.301 ± 0.011 µM (~54–fold stronger inhibition), as compared
to neostigmine. Within the first set of derivatives, compound 5d was the lead candidate against
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BChE with IC50 value of 0.066 ± 0.021 µM (~427– and 109–fold stronger inhibition) as
compared to neostigmine and donepezil, respectively. Other potent inhibitors of BChE are
compounds 5a and 5b showing ~160– and 78–fold stronger inhibition (IC = 0.176 ± 0.013 and
5
0
0
.362 ± 0.015 µM, respectively) as compared to standard drug neostigmine.
In the second set of compounds where a metaꢀbromo substituent is present on the aromatic ring
zone 1), 5j emerged as a lead compound against AChE with IC50 value of 0.065 ± 0.005 µM
(
showing ~250–fold higher potency as compared to neostigmine in addition to ~20–fold stronger
inhibition against BChE (IC50 = 1.445 ± 0.0124 µM). This lead compound incorporates a
combination of electron–withdrawing substituents (Br and Cl) on aryl ring in zone 1 and 3 at
meta– and para–position, respectively. Similar results were acquired when another chloro
substituent is introduced at 3–position (compound 5p; IC = 0.248 ± 0.021 µM against AChE
5
0
and IC = 1.07 ± 0.084 µM against BChE). The replacement of electronically poor substituents
5
0
with sterically encumbered substituents like biphenyl and naphthyl in zone 3 (compounds 5m
and 5n; IC50 = 1.303 ± 0.12 and IC50 = 1.066 ± 0.76 µM, respectively) produced excellent
inhibitions of both AChE and BChE with more selectivity towards BChE. In general, most of the
compounds were found as potent and selective inhibitors of cholinesterases and the results
obtained clearly indicated the influence of electronic and steric properties of substituents on the
cholinesterase inhibition as well as selectivity.
Monoamine oxidase inhibition
The inhibitory activity of monoamine oxidases A and B was measured in vitro by using a
3
4
spectrophotometric method. The percentage of MAO inhibition was determined for all the
examined compounds and IC data were measured only for the evaluated compounds (Tables 4
5
0
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and 5). Clorgyline and deprenyl were used as standard inhibitors with IC values of 3.64 ± 0.012
5
0
and 0.007 ± 0.001 µM, respectively.
At a first glance, the inhibition data reported in Table 4 indicated that most of the examined
triazolothiadiazole compounds exhibited a very high and selective inhibition of MAOꢀA as
compared to MAOꢀB. Among the synthesized compounds, 4a delivered highest potency against
MAOꢀA with IC value of 0.11 ± 0.005 µM which is ~33ꢀfold higher inhibition as compared to
5
0
standard inhibitor, clorgyline (IC = 3.64 ± 0.012 µM). The previously mentioned SAR strategy
5
0
was applied to explain the activity results (Figure 11). According to that the most active analogue
a possesses electronꢀdonating groups at the paraꢀ and orthoꢀposition of both aryl rings in zone 1
4
and 3, respectively. The replacement of the 2ꢀhydroxy group with another electronꢀrich (ꢀMe)
group produced similar results (compound 4c; IC50 = 0.11 ± 0.007 µM, ~33ꢀfold strong
inhibition). The change of substitution position on the aryl ring in zone 3 had a positive influence
on the inhibitory activity. Compounds 4d and 4b were also found to exhibit significant
inhibitions with IC values of 0.24 ± 0.008 (15ꢀfold strong inhibition) and 0.25 ± 0.003 µM (14ꢀ
5
0
fold strong inhibition), respectively. Similarly, compounds obtained by replacing bromo group
with methoxy group (4i and 4j) also showed significant inhibition against MAOꢀA with IC50
values of 1.23 ± 0.06 and 1.23 ± 0.003 µM, respectively. The extent of this inhibition was found
to be higher (~3ꢀfold) as compared to the standard clorgyline. The introduction of electronꢀ
withdrawing (ꢀF) group at paraꢀposition of phenyl ring attached to Cꢀ6 of heteroaromatic core led
to reduced activity as compared to the clorgyline (compound 4f; IC = 4.77 ± 0.13 µM). The
5
0
combination of electronꢀdeficient (ꢀBr) group in zone 1 and electronꢀrich (ꢀOH) group in zone 3
abolished the activity (compound 4g; IC = 14.65 ± 0.09 µM).
5
0
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On the other hand, the synthesized triazolothiadiazoles showed reduced activity against MAOꢀB
as compared to the standard deprenyl (IC50 = 0.007 ± 0.001 µM). The most active MAOꢀB
inhibitor was compound 4b which showed an IC value of 0.03 ± 0.001 µM. This compound
50
incorporates electronꢀrich functional groups at paraꢀposition of both aryl rings in zone 1 and 3.
To sum up, MAOꢀA inhibitory potency of triazolothiadiazole compounds appeared to be mainly
modulated by electronic effect which is clearly evidenced by the results presented herein
whereas synthesized derivatives were inactive against MAOꢀB.
Similarly, all the synthesized triazolothiadiazine compounds were also evaluated for their ability
to inhibit MAOꢀA and MAOꢀB. The corresponding IC50 values are shown in Table 5. The
previously discussed SAR model (Figure 12) was used to construct correlation between
structural properties and biological activity results. Among the tested derivatives, compound 5c
turned to be a lead candidate with IC50 value of 0.011 ± 0.001 µM which is ~330ꢀfold strong
inhibition as compared to the standard inhibitor (clorgyline) used for comparison purpose. This
compound incorporates a paraꢀmethoxy group at aromatic ring in zone 1 along with an electronꢀ
deficient (ꢀF) functional group at aromatic ring in zone 3. The replacement of methoxy
substituent with bromo group and introduction of two electronꢀwithdrawing chloro substituents
also produced similar results (compound 5p; IC50 = 0.02 ± 0.0005 µM, ~182ꢀfold strong
inhibitory efficacy). Apart from electronic parameters, the steric effects were also found to
contribute positively towards the inhibition of MAOꢀA. In this regard, compound 5e exhibited
strong inhibition with an IC value of 0.09 ± 0.006 µM which is 40ꢀfold higher as compared to
5
0
the standard inhibitor (clorgyline), whereas, the introduction of bulky naphthyl group led to the
reduction in activity (IC = 1.23 ± 0.009 µM). But, still this inhibitory potential is 3ꢀfold higher
5
0
as compared to clorgyline. The synthesized structures with a combination of paraꢀmethoxy group
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and paraꢀnitro or –chloro were also emerged as potent inhibitors of MAOꢀA (compound 5g; IC50
0.82 ± 0.005 and 0.92 ± 0.007 µM; ~4.4ꢀ and ~4ꢀfold stronger inhibition, respectively). On the
=
other hand, prepared compounds were also tested for MAOꢀB inhibition but the activity profile
was diminished for the screened derivatives. In the evaluated series, compound 5o was found to
demonstrate a highest inhibitory potency towards MAOꢀB with an IC value of 0.08 ± 0.0003
5
0
µM as compared to the standard, deprenyl (IC50 = 0.007 ± 0.001 µM). This compound
incorporates a metaꢀbromo substituent on aromatic ring in zone 1 in addition to the highly
polarizable paraꢀnitro group on phenyl ring in zone 3. In general, both electronic and steric
factors were actively found to modulate the MAO inhibition and the presented derivatives
(triazolothiadiazoles and triazolothiadiazines) are proved to be better and selective MAOꢀA
inhibitors and these data are an important observation to follow up a rational design of more
potent and selective inhibitors of monoamine oxidases (MAO).
Table 4 Monoamine oxidase inhibition by triazolothiadiazoles (4aꢀl)
N
N
S
N
N
R1
O
R2
4a-l
1
2
Entry
R
R
MAOꢀA
MAOꢀB
IC ± SEM (µM)
5
0
4
a
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
2ꢀOH
4ꢀOH
2ꢀMe
4ꢀMe
0.11 ± 0.005
0.25 ± 0.003
0.11 ± 0.007
0.24 ± 0.008
14.24 ± 1.03
0.03 ± 0.001
0.33 ± 0.009
4.36 ± 0.21
4b
4
c
4d
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4
e
4ꢀOMe
4ꢀOMe
3ꢀBr
3ꢀBr
3ꢀBr
3ꢀBr
3ꢀBr
3ꢀBr
—
4ꢀOMe
1.85 ± 0.005
4.77 ± 0.13
14.65 ± 0.09
7.42 ± 0.2
1.23 ± 0.06
1.23 ± 0.003
2.89 ± 0.007
6.90 ± 0.34
3.64 ± 0.012
—
1.02 ± 0.003
0.606 ± 0.014
21.73 ± 0.027
25.31 ± 0.02
1.73 ± 0.09
1.42 ± 0.005
2.97 ± 0.009
0.87 ± 0.006
—
4
f
4ꢀF
2ꢀOH
4ꢀOH
2ꢀMe
4ꢀMe
4ꢀOMe
4ꢀF
4g
4h
4
i
4
j
4k
4
l
Clorgyline
Deprenyl
—
—
—
0.007 ± 0.001
IC = Concentration of inhibitor required for halfꢀmaximal enzyme inhibition and are reported as means
50
of two independent experiments, each performed in triplicate (SEM, standard error of the mean).
Table 5 Monoamine oxidase inhibition by triazolothiadiazines (5aꢀp)
1
Entry
R
Ar
MAOꢀA
MAOꢀB
IC ± SEM (µM)
5
0
5
a
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMeꢀPh
4ꢀClꢀPh
4ꢀFꢀPh
0.22 ± 0.004
0.92 ± 0.007
0.011 ± 0.001
5.64 ± 0.030
0.18 ± 0.002
3.66 ± 0.003
5.92 ± 0.005
15.21 ± 0.170
5b
5
c
5d
4ꢀMeꢀPh
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5
e
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
3ꢀBr
biphenyl
naphthyl
4ꢀNO ꢀPh
3,4ꢀCl ꢀPh
0.09 ± 0.006
1.23 ± 0.009
0.82 ± 0.005
16.5 ± 1.070
2.08 ± 0.010
1.69 ± 0.004
3.92 ± 0.620
2.17 ± 0.130
1.21 ± 0.004
3.51 ± 0.060
13.94 ± 0.900
0.02 ± 0.0005
3.64 ± 0.012
—
28.59 ± 2.070
6.03 ± 0.370
34.82 ± 0.041
42.78 ± 0.037
40.19 ± 0.025
32.09 ± 0.17
0.32 ± 0.017
3.68 ± 0.170
3.86 ± 0.050
18.7 ± 0.100
0.08 ± 0.0003
0.67 ± 0.009
—
5
f
5g
2
5h
2
5
i
4ꢀOMeꢀPh
4ꢀClꢀPh
4ꢀFꢀPh
5
j
3ꢀBr
5k
3ꢀBr
5
l
3ꢀBr
4ꢀMeꢀPh
biphenyl
naphthyl
5m
3ꢀBr
5n
3ꢀBr
5o
3ꢀBr
4ꢀNO
2
ꢀPh
ꢀPh
5p
3ꢀBr
3,4ꢀCl
2
Clorgyline
Deprenyl
—
—
—
—
0.007 ± 0.001
IC = Concentration of inhibitor required for halfꢀmaximal enzyme inhibition and are reported as means
50
of two independent experiments, each performed in triplicate (SEM, standard error of the mean).
Docking Studies
The Xꢀray structures of human AChE (PDB ID: 4BDT) and BChE (PDB ID: 4BDS) were
selected for the docking study because the available electric eel structures for AChE are of low
crystallographic resolutions (>4 Å) and structures of equine BChE are not available at the
moment. The active sites of AChE and BChE show a high degree of similarity despite the
replacement of residues Pro446, Tyr124 and Phe297 in the active site of AChE by residues
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Met437, Gln119 and Val288 in BChE. In addition, residue Tyr337 in AChE is replaced by the
smaller Ala328 in BChE.
Docking validation was carried out by reꢀdocking the ligand extracted from the experimentally
determined enzymeꢀligand adduct. The docking method was able to reproduce the
experimentally observed bound conformation of the coꢀcrystallized ligand with rmsd <2 Å. By
reꢀdocking the crystallographic ligand huprine W in the active site of AChE, similar interactions
were seen with the residues Tyr337, Trp86, Gly122, Ser203 as huprine W showed in the crystal
structure of AChE. The ring system of both compound series adapts a comparable position to
huprine W and it interacts with side chain atoms of several aromatic residues including Tyr337
and Trp86. Furthermore, residue Thr83 is found to be involved in hydrogen bonding with
triazolothiadiazole and triazolothiadiazine ring of compounds. The phenyl rings having different
substituents at both ends of the compounds are directed towards the hydrophobic pockets formed
by the residues Gly121, Phe297 and Gly122. πꢀπ stacking is also observed by most of the
compounds in the active site.
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Fig. 13 Binding site interactions of amino acids inside the active pockets of AChE for reference
ligand Huprine W (right) and the potent compound 4k (left)
Figure 13 shows the interactions of amino acids in the active site of AChE by coꢀcrystal ligand
Huprine W in the right side and potent compound 4k in the left side. The figure shows that
Trp86 and Tyr337 are involved in πꢀinteraction with the ligand as well as compound 4k. His447
is also showing πꢀinteraction in the active pocket. It is therefore predicted that the docking of the
compounds in the active site of enzyme showed the similar interactions as shown by coꢀcrystal
ligand.
29