ORIGINAL RESEARCH

In silico studies on 2,3-dihydro-1,5-benzothiazepinesas cholinesterase inhibitors

Farzana Latif Ansari • Saima Kalsoom •

Zaheer-ul-Haq • Zahra Ali • Farukh Jabeen

Received: 1 March 2011 / Accepted: 18 July 2011 / Published online: 3 August 2011

� Springer Science+Business Media, LLC 2011

Abstract In vitro studies on cholinesterase inhibitory

potential on the three sets of 2,3-dihydro-1,5-benzothiaze-

pines have been carried out. The compounds in Set 1 were

unsubstituted on ring A, while those in Sets 2 and 3 had a

20- and 30-hydoxy substituent, respectively, in ring A.

These studies revealed that they are mixed inhibitors of

both AChE and BChE as reflected from their IC50 values. It

was further observed that 30-hydroxy substituted ben-

zothiazepines (Set 3) were found to have stronger affinity

for both AChE and BChE compared with those of Sets 1

and 2. Moreover, all the compounds in Set 3 were found to

be stronger BChE inhibitors than AChE. These experi-

mental observations were rationalized by conducting in

silico studies using molecular docking tool of Molecular

Operating Environment (MOE) software, thereby, a good

correlation was observed between IC50 values and their

binding interactions within the enzyme active site. We

have observed that these interactions were electrostatic and

hydrophobic in nature besides hydrogen bonding. The high

BChE inhibitory potential of 30-hydroxy substituted ben-

zothiazepines was found to be cumulative effect of

hydrogen bonding and p–p interactions between the ligand

and BChE. These findings may serve as a guideline for

synthesizing more potent ChE inhibitors for the treatment

of Alzheimer’s disease and related dementias.

Keywords Cholinesterase inhibitors � MOE docking �1,5-benzothiazepines

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder

which is characterized by the loss of cholinergic activity in

patient’s brain (Terry and Buccafusco, 2003; Silman and

Sussman, 2005; Nachon et al., 2005); Acetylcholinesterase

(AChE) is one of nature’s most elegantly engineered pro-

teins. The physiological role of the enzyme is the accelera-

tion of the hydrolysis of neurotransmitter acetylcholine at

nerve–nerve and neuromuscular junctions. The enzyme is

ideally suited for this role, because it possesses one of the

fastest turnover rates known (Carreiras and Marco, 2004;

Shen et al., 2005). ACh is a neurotransmitter that is

hydrolyzed by acetylcholinesterase (EC 3.1.1.7) and butyr-

ylcholinesterase (EC 3.1.1.8) (Rodriguez et al., 2005; Zah-

eer-ul-Haq et al., 2003); Hence, the most promising

therapeutic strategy for activation of the central cholinergic

function has been the use of cholinomimetic agents. The

function of cholinesterase inhibitors is to boost up the

endogenous level of ACh in the brain of patients suffering

from AD, thereby, increasing cholinergic neurotrans-

mission.

Acetylcholinesterase (AChE) and butyrylcholinesterase

(BChE) are found to be quite similar, possessing 53%

sequence homology and both have their active sites at the

base of enzyme gorge of depth 20 A. Recent research has

revealed that in the brain of patients suffering from AD, the

level of AChE is considerably reduced whereas that of

BChE increases, thus, aggravating the toxicity of b-amy-

loid peptide. In such instances, it is possible that BChE

may be more suitable target than AChE (Harel et al.,

F. L. Ansari (&) � S. Kalsoom � Z. Ali � F. Jabeen

Department of Chemistry, Quaid-i-Azam University,

Islamabad 45320, Pakistan

e-mail: fla_qau@yahoo.com

Zaheer-ul-Haq

Dr. Panjwani Center for Molecular Medicine and Drug Research,

International Center for Chemical and Biological Sciences,

University of Karachi, Karachi 75270, Pakistan

123

Med Chem Res (2012) 21:2329–2339

DOI 10.1007/s00044-011-9754-6

MEDICINALCHEMISTRYRESEARCH

1993). It is not surprising that cholinesterase inhibitors

have shown better results in the treatment of AD than any

other strategy explored (Kryger et al., 1998); (Ansari et al.,

2005). Hence, the search for the discovery of novel cho-

linesterase inhibitors (ChE) is expected to continue in

future since the current ChEs inhibitors are reported to

have some side effects too (Ansari et al., 2008). The

availability of several crystal structures of both ChEs in

complexes with different inhibitors provides the possibility

to apply docking protocol to explore for protein inhibitor

complexes in terms of the nature of their interactions

(MOE 2005-06; Ellman et al., 1961).

We have reported earlier the synthesis of a variety of

2,3-dihydro-1,5-benzothiazepines by a [4?3] annulation of

a,b-unsaturated ketones with o-aminothiophenol (Esposito

et al., 2000; Kuntz, 1992). The elements of diversity were

introduced by substitution both on the rings A and B that

led to the following three sets of compounds.

It was interesting to note that all the compounds in Set 2

were inactive, while only one benzothiazepines (4) was

active in Set 1; however, quite a significant number of

compounds were found to possess strong BChE inhibitory

potential. Another striking observation was that ben-

zothiazepines having a 30-hydroxy substituent on ring A

were found to be more potent than either the unsubstituted-

or 20-hydroxy substituted analogs. These observations point

toward the necessity of having a deeper insight into their

mechanism of cholinesterase inhibition. The knowledge of

this mechanistic detail would help in developing new

potential drugs for treating AD and related dementias.

Herein, we report a comparative study of cholinesterase

inhibitory potential of the three sets of compounds syn-

thesized earlier by conducting molecular docking studies

using Molecular Operating Environment (MOE) software

(Morris et al., 1996). The main purpose of this study was to

provide a possible relationship between the experimentally

determined IC50 values and the docking interactions of

benzothiazepines 1–27 in the active site of ChEs. The

docking of these benzothiazepines with AChE and BChE

has provided valuable information about the nature of

binding interactions of these ligands with both these

enzymes. These results provide a rationale for the greater

inhibitory potential of some compounds than the others,

which was found to be primarily based on the presence and

interaction of the ligands with Trp84 and Arg515 in the

active sites of AChE and BChE, respectively.

Materials and methods

Solution phase synthesis of 2,3-dihydro-1,5-

benzothiazepines of Sets 1 and 2 (Ansari et al., 2005)

To a solution of chalcone (20 mmol) in dry acidic metha-

nol acidified by adding few drops of glacial acetic acid, o-

aminothiophenol (20 mmol) was added. The mixture was

refluxed until a crystalline solid separated out. After

cooling, the solid product was collected and washed with

diethyl ether and cold methanol. The crude solid was

recrystallized from ethanol.

Solid phase synthesis of 2,3-dihydro-1,5-

benzothiazepines of Set 3 (Ansari et al., 2008)

Solid phase synthesis of 2,3-dihydro-1,5-benzothiazepines

(12–27) has been carried out on Wang resin using chal-

cones as precursors which in turn were synthesized on the

same solid support through Claisen-Schmidt condensation

of 3-hydroxyacetophenone with different substituted

aldehydes.

Cholinesterase inhibition assay

Electric-eel acetylcholinesterase (EC 3.1.1.7), acetylthi-

ocholine iodide, butyrylthiocholine chloride, 5,5-dithio-

bis-[2-nitrobenzoic acid] (DTNB), galanthamine, and

eserine were purchased from Sigma. All the other chemi-

cals were of analytical grade. AChE and BChE inhibitory

activities were determined according to the reported pro-

tocol (Abdel-Hamid et al., 2007). Acetylthiocholine iodide

and butyrylthiocholine chloride were used as substrates to

evaluate AChE and BChE, respectively. Sodium phosphate

buffer (pH 8.0; 150 ll, 100 mM), DNTB, test-compound

solution (10 ll, in EtOH), and AChE (or BChE) solution

(20 ll) were mixed and incubated for 15 min at 25�C. The

reaction was then initiated by the addition of acetylthiocholine

SN

R

SN

R

OH SN

RHO

Set 1 Set 2 Set 3

2330 Med Chem Res (2012) 21:2329–2339

123

(or butyrylthiocholine) (10 ll), their hydrolysis was moni-

tored by the formation of a yellow 5-thio-2-nitrobenzoate

anion which was traced by UV/VIS at 412 nm. All the reac-

tions were performed in triplicate in a 96-well micro-titer

plate. The percentage inhibition was calculated as (E - S)/

E 9 100, where E is the activity of the enzyme without the test

compound, and S is the activity of enzyme with the test

compound.

Docking procedure

Docking programs are broadly classified into two classes

i.e., direct and unbiased; the former have the advantage of

having higher speed, but they have the disadvantage of

making assumptions about potential energy landscape to

save computational time (Stewart, 1989). The unbiased

methods such as Autodock and MOE Dock have abilities to

cover up disadvantages of direct docking methods.

Although both MOE and Autodock are quite similar,

however, the former has the advantage of graphical inter-

face as well as other modules such as analysis, molecular

mechanics, and molecular dynamics (Nawaz et al., 2008);

(Carolan et al., 2008). MOE-Dock by Chemical Computing

Group Inc was used in the current study. All the docking

studies were carried out on a Pentium 1.6 GHz worksta-

tion, 512 MB memory using the Windows Operating

system.

The crystal structure of protein complexes of AChE and

BChE were obtained from Protein Data Bank (PDB id

1ACl and 1POI, respectively), since they represent the

pharmacological target for the development of new drugs

to cure the Alzheimer disease. The edited crystal structure

after removing water molecules was imported into MOE

and all the hydrogen atoms were added to the structure with

their standard geometry followed by their energy minimi-

zation using MOPAC 7.0. The resulting model was sub-

jected to systematic conformational search at default

parameters with RMS gradient of 0.01 kcal/mol using Site

Finder. Enzymes were searched for their active sites and

dummy atoms were created from the resulting alpha

spheres. The backbone and residues were kept fixed, and

the energy minimization was performed. Root mean square

deviation values (RMSD) were used to compare the ligand

between the predicted and its corresponding crystal struc-

ture. The resulting docked poses with RMSD less than

1.5 A were clustered together. The lowest energy mini-

mized pose was used for further analysis.

All the benzothiazepines were docked following the

same procedure. Ten different conformations were selected

for each ligand. All the other parameters were maintained

at their default settings. The best conformation of each of

the ligand–enzyme complex was selected based on the

energetic grounds. The resulting ligand–enzyme complex

model was then used for calculating the energy parameters

using MMFF94x force field energy calculation and pre-

dicting the ligand–enzyme interactions at the active site.

Results and discussion

The solution phase synthesis of benzothiazepines of Sets 1

and 3 (1–11) was carried out by the reaction of a,b-

unsaturated ketones using o-aminothiophenol (Scheme 1).

Benzothiazepines of Set 3 (12–27) were synthesized by the

same synthetic strategy in the solid phase using Wang resin

as a solid support as shown in Scheme 2.

In vitro cholinesterase inhibition studies were carried

out on unsubstituted bezothiazepines (Set 1) and different

substituted 20-, and 30-hydroxybenzothiazepines (Sets 2 and

3) and the IC50 values of the screened compounds were

determined along with that of standard galanthamine as

shown in Table 1. It is evident that none of the ben-

zothiazepines in Set 2 was found to be active against any of

the ChEs. However, one benzothiazepines 4 in Set 1 was

found to be active against BChE with an IC50 value of

60 lM; the same compound was found to be only weakly

active against AChE (IC50 = 102 lM).

Parallel studies when carried out on 30-hydroxy-substi-

tuted benzothiazepines Set 3 revealed that quite a few

compounds were found to have potential as ChE inhibitors

with IC50 values varying from 5.9 to 102 lM against

AChE, and 3.97–142 lM against BChE. A comparison of

the inhibitory potential of these compounds against AChE

and BChE also revealed that the number of compounds

active against BChE was greater than that of those active

against AChE. Furthermore, compounds 17, 19, and 27

with IC50 values 4.70, 4.65, and 3.97 lM, respectively

were found to be even more active than the standard

galanthamine (IC50 = 8.0 lM). Therefore, it deemed nec-

essary to explain the possible ligand–enzyme interactions

in these three sets of benzothiazepines and to have a deeper

insight into the mechanism of their interaction to help in

the designing of new potent ChEs inhibitors.

More recently, we reported the cholinestrersase inhibi-

tory potential of 2,3,4,5-tetrahydrobenzothiazepines; the

reduced analogs of 2,3-dihydrobenzothiazepines with the

observation that the former set of compounds was much

more active than the latter. For getting further structural

insight into the inhibition mechanism of these compounds,

docking studies were carried out using SYBYL and the

results were reported. Herein, we report the theoretical and

computational studies on the mechanism of ChE inhibition

by the three sets of compounds through molecular docking

studies using MOE software.

Protein data bank contains several AChE and BChE

complexes with small molecules. The major difference in

Med Chem Res (2012) 21:2329–2339 2331

123

their complexes is the orientation of one or more amino

acids. In order to control the performance of our docking

approach in the case of AChE, we have selected the crystal

structure of AChE cocrystallized with decamethonium. The

active site is found to be located at 20 A from the protein

surface at the bottom of a deep and narrow gorge. The

CO

CH3

R1 +

HC

O

O

R1

R2

SH

NH2

SNR1

R2

H / MeOH

NaOH

Set 1 Set 2

No. R1 R2 No. R1 R2

1 H H 5 2-OH H 2 H 4-Me 6 2-OH 4-Cl 3 H 3,4-diOMe 7 2-OH 4-F 4 H 4-NO2 8 2-OH 3-OMe

9 2-OH 4-OMe 10 2-OH 4-Me 11 2-OH 3,4-OCH2O

Scheme 1 Solution phase

synthesis of 2,3-dihydro-1,5-

benzothiazepines (1–11)

CH3

O

HOOHODIAD, TPP,NMM

48 hrs.

CHO

KOH 6 equiv.

O

OO

CH3

O

OO

Ring B

Ring B

SH

NH2

i)

ii) 50% TFA

N S

HORing B2

34'3

'3

'3

'3

AA

AA

Set 3 Ring B Ring B 12 = Phenyl 20 = 3,4-OMe2C6H4

13 = 2-ClC6H4 21 = 4-OH-3-OMeC6H3

14 = 3-ClC6H4 22 = 3-NO2C6H4

15 = 4-ClC6H4 23 = 4-NO2C6H4

16 = 2-FC6H4 24 = 4-MeC6H4

17 = 3-OHC6H4 25 = 4-NMe2C6H4

18 = 4-OHC6H4 26 = Pyridin-3-yl 19 = 2-OCH3C6H4 27 = Thien-2-yl

Scheme 2 Solid phase

synthesis of 30-hydroxy-2,3-

dihydro-1,5-benzothiazepines

(Set 3)

2332 Med Chem Res (2012) 21:2329–2339

123

details of the docking procedure are given in the ‘‘Materials

and methods’’ section. Using docking protocol, the active

site of AChE enzyme was obtained, which contained

decamethonium as a ligand. Both the ligand and the side

chain of the binding site were relaxed, taking into account

the amino acids mobility within 5 A around the ligand,

whereby nine amino acids Tyr70, Ser81, Trp84, Tyr121,

Glu199, Phe330, Phe331, Tyr334, and His440 were found

in the active site of AChE. The docking of all the com-

pounds with BChE (PDB id = 1POI) was done following

the same docking protocol as for AChE using the crystal

structure of BChE cocrystallized with 2-(N-morpholino)-

ethanesulfonic acid (MES). The active site, in this case,

was also found to be located 20 A deep from the protein

surface at the bottom. The amino acids within 5 A in the

active sites were Lys323, Asp334, Tyr420, Val436,

Leu514, and Arg515. Most of the compounds were found

to bind with BChE between Lys323 and Arg515 making

direct contact through hydrogen bonding and p–p interac-

tions with these two residues.

All the synthesized benzothiazepines 1–27 were docked

in the active site of AChE and BChE and ten conformations

for each ligand were saved. Finally, the best conformation

was selected based on the lowest binding energy (Table 1).

The binding free energy data obtained after docking pro-

cedure did not show any apparent correlation between

docked binding energies and experimentally determined

IC50 values, which could be due to the neglect of aqueous

environment. Another reason could be that the experi-

mental data may not be precise enough to give consistent

values within the standard confidence limits.

Docking calculations also allow the prediction of the

structures of all the complexes between the enzymes and

ligands, thereby, suggesting different kinds of interactions.

These interactions were based on possible H-bonding and

p–p interactions between ligands and amino acids present

Table 1 In vitro cholinesterase inhibitory activities and binding energies of 2,3-dihydro-1,5-benzothiazepines (1–27)

Compound R1 R2 AChE BChE

IC50 (lM) Binding energy (KJ/mol) IC50 (lM) Binding energy (KJ/mol)

1 H H – -10.45 – -9.06

2 H 4-CH3 – -10.53 – -9.19

3 H 3,4-OCH2O – -11.13 – -10.56

4 H 4-NO2 102 -10.82 60.0 -9.72

5 2-OH H – -10.77 – -9.54

6 2-OH 4-Cl – -11.16 – -10.48

7 2-OH 4-F – -11.22 – -9.84

8 2-OH 3-OCH3 – -11.20 – -10.09

9 2-OH 4-OCH3 – -10.94 – -10.54

10 2-OH 4-CH3 – -11.08 – -9.87

11 2-OH 3,4-OCH2O – -11.71 – -10.63

12 3-OH H 40.0 -11.16 142.1 -10.37

13 3-OH 2-Cl – -11.00 – -10.54

14 3-OH 3-Cl – -11.23 29.4 -10.25

15 3-OH 4-Cl 18.8 -11.44 76.9 -11.01

16 3-OH 2-F 70.6 -11.47 – -10.41

17 3-OH 3-OH 71.1 -11.80 4.7 -10.69

18 3-OH 4-OH – -11.88 – -11.78

19 3-OH 2-OCH3 – -11.77 4.65 -10.56

20 3-OH 3,4-diCH3 – -10.85 19.7 -10.55

21 3-OH 3-OCH3-4-OH – -11.80 – -11.71

22 3-OH 3-NO2 – -11.63 – -11.27

23 3-OH 4-NO2 – -11.66 – -11.06

24 3-OH 4-CH3 49.8 -11.26 75.2 -10.64

25 3-OH 4-NMe2 – -11.31 34.4 -10.79

26 3-OH 3-Pyridyl – -11.46 – -10.09

27 3-OH 2-Thiophen 5.9 -11.49 3.97 -10.20

Galanthamine 0.5 -11.51 8.0 -10.04

Med Chem Res (2012) 21:2329–2339 2333

123

within 5 A. Besides, the activity of compounds was also

found to be dependent on the presence or absence of one or

more amino acids in the active site. Moreover, all the

compounds were found to bind in the same pocket of active

site as shown by their superimposition in Fig. 1. The binding

of all the compounds in the active site of AChE could be due

to the fact that they have aromatic rings and a nitrogen atom,

which is generally common to the other AChE inhibitors.

However, the difference in their binding affinity could be

due to the difference in their substitution pattern.

Geometrical features of ligand–enzyme complexes were

correlated on the basis of their morphology and amino acids

environment in the macromolecular cavity. It was observed

that p–p interactions played an important role in stabilizing

ligand–enzyme complexes. The hydrogen bonding and p–pinteractions were the main features of interaction of mostly

ligands with Trp84 of AChE as shown in Table 2. Figure 2

confirms the key contributing role of Trp84 in the ligand

pocket. It is evident from Table 2 that benzothiazepine 27

was most potent AChE inhibitor of the series (IC50 =

5.9 lM). This could be rationalized on the basis of strong

hydrogen bonding (64% with Glu199 with a distance of

2.9 A). The H-bonding was found between the 30-hydroxyl

group of compound 27 and the carbonyl oxygen of Glu199 at

the distance of 2.9 A (Fig. 3). The p–p interactions wereFig. 1 Superimposition of some benzothiazepines of Sets 1–3 in the

active site of AChE enzyme

Table 2 Binding interactions observed in 1,5-benzothiazepines (1–27) with AChE

Compounds IC50 (lM) H-bonding p–p interactions

Distance (A) Score (%) Amino acid

1 – – – – –

2 – – – – Trp84, Tyr 334

3 – – – – Trp84

4 102 – – – Trp84, Tyr 334

5 – – – – Trp84, Tyr 334

6 – – – – Phe331

7 – – – – Trp84

8 – – – – Trp84

9 – – – – Tyr334

10 – – – – Tyr334

11 – – – – –

12 40.0 2.07 45 His 440 Trp84

13 – – – – Phe330

14 – – – – Phe330

15 18.8 3.5 37 Tyr 130 Trp84

16 70.6 3.65 20 Ser 81 Trp84

17 71.1 2.9 25 Ser 81 Trp84, Phe330

18 – – – – Trp 84

19 – – – – Tyr334

20 – – – – Tyr334

21 – – – – –

22 – – – – –

23 – – – – Tyr334

24 49.8 2.52 21 Tyr 121 Trp84

2334 Med Chem Res (2012) 21:2329–2339

123

found with Trp84 and Phe330. The other active ben-

zothiazepines 15 (IC50 = 18.0 lM) also showed hydrogen

bonding (37%) with Tyr130 and p–p interaction with Trp84.

The equipotency of benzothiazepines 16 and 17 was evident

from the same type of H-bonding with Ser81 in both the

compounds. Table 3 showed the amino acid environment

around the ligand within 5 A. It is evident that Trp84, being

present in all the ligand–enzymes complexes could be con-

sidered as activity determinant amino acids as the same

amino acid was also found in galanthamine–enzyme

complex.

The fact that benzothiazepines were found to be stronger

inhibitor of BChE compared with AChE may be rational-

ized on the basis of the observation that numerous docked

poses of these compounds scored very high in terms of

H-bonding (Table 4). For example in benzothiazepines 27,

the extent of H-bonding was found to be 96% (Fig. 4)

besides having very strong p–p interaction with Arg515.

The equipotent benzothiazepines 17 and 19 bind to BChE

with the same degree of p–p interaction with Arg515 and

the same extent of H-bonding (81%) with Try500 and

Lys323, respectively.

Another remarkable observation was that none of the

benzothiazepines in Set 2 was found to be active against

Fig. 2 Key contribution of Trp84 in the active site of AChE enzyme with a 16, b 27

Fig. 3 3D docking pose of 27 (ball & stick) at AChE active site,

showing H-bonding (purple) with Glu199 (Color figure online)

Table 2 continued

Compounds IC50 (lM) H-bonding p–p interactions

Distance (A) Score (%) Amino acid

25 – – – – Tyr334

26 – – – – Phe330

27 5.9 1.49 46 Trp 84 Trp84

2.5 77 Tyr 121

Galanthamine 0.5 2.63 93 Tyr121 Trp84

2.84 65 Tyr121

2.0 55 Asn85

Med Chem Res (2012) 21:2329–2339 2335

123

Table 3 Hydrophobic interactions of 1,5-benzothiazepines (1–27) with different amino acids in the active site of AChE within 5 A radius

Compound Tyr70 Ser81 Trp84 Glu199 Phe330 Phe331 Tyr334 His440

1 ? ? ? - ? ? ? -

2 ? - ? ? ? ? - ?

3 ? - ? ? - - ? -

4 - - ? ? ? - ? -

5 ? ? ? - ? ? - -

6 ? - ? ? ? ? ? ?

7 ? ? ? ? ? ? ? ?

8 ? ? ? ? ? ? ?

9 ? - ? - ? ? - ?

10 ? - ? ? ? ? ? ?

11 ? - ? - ? ? - ?

12 - - ? - ? ? ? ?

13 ? ? ? - ? ? - ?

14 ? - ? ? ? ? ? -

15 - ? ? ? ? ? ? ?

16 - - ? - - - ? ?

17 - - ? - ? ? ? ?

18 ? - ? - - ? ? ?

19 ? ? ? ? ? ? - ?

20 ? ? ? - ? ? ? -

21 ? ? ? - ? ? ? ?

22 ? - ? - - ? ? -

23 ? ? ? ? ? ? ? -

24 - ? ? - ? ? - ?

25 ? ? ? ? ? ? ? -

26 ? - ? ? - - ? ?

27 - - ? ? ? ? ? ?

Galanthamine - ? ? - ? - - ?

Table 4 Binding interactions of 1,5-benzothiazepines (1–27) with BChE

Compound IC50 (lM) H-bonding p–p interactions

Distance (A) Score (%) Amino acid

1 – – – – Arg 515

2 – – – – Arg 515

3 – – – – Lys 323

4 60.0 2.78 65 Tyr 420 Arg 515

5 – – – – –

6 – – – – –

7 – – – – –

8 – – – – Arg 515

9 – – – – Arg 515

10 – – – – Arg 515

11 – – – –

12 142.1 – – – Arg 515

13 – – – –

2336 Med Chem Res (2012) 21:2329–2339

123

AChE and BChE. This could be explained on the basis of

the difference in the orientation and fitting in mechanism of

all the benzothiazepines in Set 2 compared with those in

Set 1 and 3. A careful glance at Fig. 5 reveals that the

orientation of ring A in these compounds does not allow

penetration of molecules deep into the gorge, thereby,

hindering their interactions with the amino acids in the

active site.

Based on these in silico studies, it was also possible to

predict that a benzothiazepine without a hydroxyl group on

ring A and with a thiophene moiety as ring B (shown in

Fig. 6 as white) could also be a strong candidate as BChE

Table 4 continued

Compound IC50 (lM) H-bonding p–p interactions

Distance (A) Score (%) Amino acid

14 29.4 2.23 93 Lys 323 –

2.5 12 Tyr 420

15 76.9 3 96 – Arg 515

16 – – – – –

17 4.7 2.6 81 Tyr 500 Arg 515

2.7 41 Lys 323

3.0 23 Lys 323

18 – – – – –

19 4.65 2.28 81 Lys 323 Arg 515

2.55 17 Tyr 420

20 19.7 2.7 96 Tyr 420 Arg 515

21 – – – – Arg 515

22 – – – – –

23 – – – –

24 75.2 3.04 25 Lys 323 Arg 515

25 34.4 2.47 49 Gln 518 Arg 515

26 – – – – Arg 515

27 3.97 2.4 96 Tyr 420 Arg 515

Galanthamine 8.0 2.1 76 Tyr240 Arg 515

Fig. 4 3D docking pose of 27 (ball & stick) at BChE active site,

showing H-bonding (purple) with Tyr500 (Color figure online)

Fig. 5 Comparison of orientation pattern of unsubstituted (blue), 20-hydroxy (white), and 30-hydroxy (green) substituted benzothiazepines

(Color figure online)

Med Chem Res (2012) 21:2329–2339 2337

123

inhibitor due to the similarity in fitting pattern to ben-

zothiazepine 27 (yellow) as shown in Fig. 6, where the

superimposition of all the three analogs have been shown.

On the basis of prediction, two analogs of compound 27

were synthesized using reported procedure. Theses analogs

were submitted for their cholinesterase inhibition assay. A

direct correlation was observed between the predicted

bioactivity and experimental results as shown in Table 5.

Conclusions

In vitro studies of 1,5-benzothiazepines have shown that

they are mixed inhibitors of both ChEs. The goal of our

docking studies was to explore the possible docking mode

of active compounds within the active site of AChE and

BChE. The docking results of these compounds show that

the active compounds bind to AChE and BChE with the

orientation and position very close to that resulting from

crystallographic analysis in both ChEs with their actual

ligands DME and MES, respectively. The stabilizing

interactions between the protein and ligands are p–p inter-

actions, H-bonding and of hydrophobic nature, and the high

affinity of these compounds are the results of cumulative

effect of all these interactions. In our docking experiments,

a good correlation of IC50 values with binding interaction

was found, evidencing the significant role of Trp84 and

Arg515 in AChE and BChE enzymes, respectively.

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