One-pot synthesis of tetrazole-1,2,5,6-tetrahydronicotinonitriles and cholinesterase inhibition: Probing the plausible reaction mechanism via computational studies

Accepted Manuscript

One-Pot Synthesis of Tetrazole-1,2,5,6-Tetrahydronicotinonitriles and Choli-

nesterase Inhibition: Probing the Plausible Reaction Mechanism via Computa-

tional Studies

Abdul Hameed, Syeda Tazeen Zehra, Saba Abbas, Riffat Un Nisa, Tariq

Mahmood, Khurshid Ayub, Mariya al-Rashida, Jürgen Bajorath, Khalid

Mohammed Khan, Jamshed Iqbal

PII:

S0045-2068(16)30004-9

DOI:

http://dx.doi.org/10.1016/j.bioorg.2016.01.004

YBIOO 1872

Reference:

Bioorganic Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

26 December 2015

18 January 2016

Please cite this article as: A. Hameed, S.T. Zehra, S. Abbas, R.U. Nisa, T. Mahmood, K. Ayub, M. al-Rashida, J.

Bajorath, K.M. Khan, J. Iqbal, One-Pot Synthesis of Tetrazole-1,2,5,6-Tetrahydronicotinonitriles and

Cholinesterase Inhibition: Probing the Plausible Reaction Mechanism via Computational Studies, Bioorganic

Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.01.004

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One-Pot Synthesis of Tetrazole-1,2,5,6-Tetrahydronicotinonitriles and

Cholinesterase Inhibition: Probing the Plausible Reaction Mechanism via

Computational Studies

Abdul Hameed^a, Syeda Tazeen Zehra^a, Saba Abbas^b, Riffat Un Nisa^c, Tariq Mahmood^c,

Khurshid Ayub^c, Mariya al-Rashida^d, Jürgen Bajorath^e, Khalid Mohammed Khan^a, and Jamshed

Iqbal^*b

^aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological

Sciences, University of Karachi, Karachi-75270, Pakistan.

^bCenter for Advanced Drug Research, COMSATS Institute of Information Technology,

Abbottabad 22060, Pakistan.

^cDepartment of Chemistry, COMSATS, Institute of Information Technology, Abbottabad 22060,

Pakistan.

^dDepartment of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road-

54600, Lahore, Pakistan

^eDepartment of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and

Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmann str. 2, D-53113

Bonn, Germany

Abstract

In the present study, one-pot synthesis of 1H-tetrazole linked 1,2,5,6-tetrahydronicotinonitriles

under solvent-free conditions have been carried out in the presence of tetra-n-butyl ammonium

fluoride trihydrated (TBAF) as catalyst and solvent. Computational studies have been conducted

to elaborate two plausible mechanistic pathways of this one-pot reaction. Moreover, the

synthesized compounds were screened for cholinesterases (acetylcholinesterase and

butyrylcholinesterase) inhibition which are consider to be major malefactors of Alzheimer’s

disease (AD) to find lead compounds for further research in AD therapy.

Keywords: Ionic liquids, Click chemistry, Tetrazole, 1,2,5,6-Tetrahydronicotinonitrile, Density

Functional Theory (DFT), Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE)

^Corresponding Authors: A.H. Tel.: 0092-21-99261701-2; Fax: 0092-21-3481901; abdul.hameed@iccs.edu

J.I., Tel.: +92-992-383591-96; fax: +92-992-383441; e-mail: drjamshed@ciit.net.pk

1

1. Introduction

Solvent-free chemistry holds a unique place in organic synthesis to reduce environmental

hazards. The reaction avoids the use of toxic and volatile conventional organic solvents. For

solvent-free reactions ionic liquids provide excellent media for organic syntheses. Ionic liquids

serve as catalysts in many organic reactions [1]. Tetra-n-butylammonium fluoride (TBAF) is a

type of an ionic liquid that has been diversely used in organic syntheses. A range of different

solvent-free reactions have also been performed in the presence of TBAF as catalyst. Fluoride (F^-

) counter anion present in TBAF serves as mild base to promote different chemical reactions [2-

4]. In our solvent-free chemistry, a one-pot reaction between acetophenone, malononitrile and

trimethylsilyl azide (TMSN₃) was explored in the presence of neat TBAF to afford tetrazole

linked 1,2,5,6-tetrahydronicotinonitriles 2(a-h). The reaction involved 1) fluoride (F^-) mediated

Knoevenagel condensation 2) followed by multisteps ring closure to afford corresponding

1,2,5,6-tetrahydronicotinonitriles (5). Consequently 3) the click reaction furnished tetrazole

linked 1,2,5,6-tetrahydronicotinonitriles (2) (Scheme 1) [5]. Plausible mechanism of such

interesting one-pot reaction upto intermediate (5) has been suggested by two different logical

pathways (I) and (II) (Scheme 2) [5, 6]. The intermediate 1,2,5,6-tetrahydronicotinonitrile (5)

also served as excellent precursor for the synthesis of [1,6]-naphthyridine ring system [5] found

in many biological important molecules [7, 8].

Computational study using density field theory (DFT) calculations was carried out to elaborate

the most feasible pathway of this one-pot reaction. The scope of reaction was explored with

different phenyl substituted acetophenones. A series of tetrazole linked 1,2,5,6-

tetrahydronicotinonitriles 2(a-h) was synthesized under solvent-free conditions in moderate to

excellent yield. The structures of compounds 2(a-h) were confirmed using different

spectroscopic techniques.

Medicinal chemistry has witnessed an expansion in the use of biologically active nicotine based

molecules. Recently, Carreiras et. al. [9] synthesized nicotine containing pyridonepezils (1) and

evaluated their inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase

(BChE). Based on close structural similarities of our synthetic tetrazole-1,2,5,6-

tetrahydronicotinonitrile (2) with pyridonepezils (1) (Figure 1), we assessed them for their

cholinesterases (AChE and BChE) inhibitory activities. According to so-called cholinergic

hypothesis, [10, 11] cholinesterases are considered to be major responsible enzymes for the

2

pathogenesis of Alzheimer’s disease (AD) complications. AD is a progressive neurodegenerative

disorder that leads to gradual memory loss, decline in language skills and other effects on

cognitive functions. A report from Alzheimer's disease international describes it as the most

common type of dementia which now affects around 36 million people worldwide with 6% of

the population being over the age of 65 [12-14]. The major factors contributing to Alzheimer’s

disease include 1) deposition of -amyloid [15], 2) oxidative stress [16], 3) aggregation of tau

protein [17], and 4) low level of acetylcholine due to severe loss of cholinergic cells in the

brain.[10] Thus, the strategy to inhibit the activities of these enzymes is thought to provide

opportunities for treatment of AD and its symptoms. It is now well-known that the AChE level is

low in the presence of AD, while BChE shows an increased level. This suggests that BChE

might be the more attractive target for selective AD therapy.

Figure-1: Structures of pyridonepezil (1) and tetrazole linked1,2,5,6-tetrahydronicotinonitrile (2)

2. Results and discussion

2.1 Chemistry

One-pot solvent-free synthesis of tetrazole linked 1,2,5,6-tetrahydronicotinonitrile derivatives

2(a-h) was carried out using neat TBAF trihydrated as catalyst Scheme 1. Reaction between

corresponding acetophenones (1 equiv.) and malononitrile (1.5 equiv.) first underwent

Knoevenagel condensation to an in situ arylidene intermediate which via multisteps ring closure

afforded 1,2,5,6-tetrahydronicotinonitrile (5). Consequently, addition of azide furnished the

tetrazole-linked 1,2,5,6-tetrahydronicotinonitrile 2(a-h) in moderate to excellent yield. Two

plausible mechanisms of this one-pot reaction have been shown in Scheme-2. The structures of

1

final compounds 2(a-h) have been elucidated with spectroscopic techniques including H, C¹³

NMR spectroscopy etc. X-ray crystal structure of intermediate 2-dicyanomethylene-6-methyl-

4,6-diphenyl-1,2,5,6-tetrahydronicotinonitrile (5a) has been reported in our previous publication

3

[18]. Tetrazole linked 1,2,5,6-tetrahydronicotinonitriles 2(a-h) were further assessed for

biological potential against cholinesterase, acetylcholinesterase (AChE) and

butyrylcholinesterase (BChE).

Scheme-1: One-pot synthesis of tetrazole-1,2,5,6-tetrahydronicotinonitrile 2(a-h)

4

2.2 Investigating a plausible reaction mechanism

Scheme-2: Plausible pathways (I) Michael addition of (7a) to (6), or (II) Dimerization reaction

between (7b) and (6) to yield 1,2,5,6-tetrahydronicotinonitrile (5).

Plausible mechanism of this reaction to form 1,2,5,6-tetrahydronicotinonitrile derivative (5) via

two pathways (I) and (II), emanating from intermediate (7a) or its tautomer (7b), has been

depicted in Scheme-2. The reaction involves a Knoevenagel condensation, followed by a

dimerization to afford 1,2,5,6-tetrahydronicotinonitrile (5) and click chemistry to form final

product (2). Pathway (I) suggests Michael addition [5, 19] of carbanion (7a) to its neutral species

(6) followed by ring closure to form cyclohexene (9) which could tautomerized to

cyclohexadiene (10). It is evident that such type of cyclohexadiene (10) is thermally unstable and

expeditiously undergoes ring opening to acyclic hexatriene type intermediate 12 [20, 21].

Further, the base (F^‾) mediated multistep ring closure generated the dimer (5) [19, 22]. In

pathway (II), the nucleophilic nitrogen of tautomer (7b) attacks arylidenemalononitrile (6) to

5

form intermediate (11) which then affords dimer (5) via intermediate (14) (Scheme 2) [2, 23, 24].

Computational studies were preformed to understand plausible mechanism pathway (I) or (II)

for the formation of 1,2,5,6-tetrahydronicotinonitrile (5) (see Section-2.3).

The structures of dimer (5a) and tetrazole linked 2-dicyanomethylene-6-methyl-4,6-diphenyl-

1,2,5,6-tetrahydronicotinonitrile (2a), were fully characterized with different NMR techniques.

The compound (5a) showed three characteristic signals; a) a broad singlet (brs) for NH¹proton at

δ 9.71 ppm, b) two doublets for each methylene protons H^5aand H^5bat δ 3.77 and δ 3.30^ppm,

respectively, and c) a singlet for C-12 methyl protons at δ 1.71 ppm. The NH¹proton showed

COSY correlation of with H^5aand HMBC correlations with C², C³, C⁵, C⁶, C⁷, C⁸, C⁹which

confirm its position in the structure. In NOSEY spectrum, correlations with H⁷and aromatic H^2′,

H^6′were also observed. In COSY spectrum, only H^5amethylene proton showed COSY

correlation with NH¹. The C⁷methyl proton showed HMBC correlation with C^1′, C², C⁴, C⁵, C⁶

1

and NOSEY correlation with H¹, H^2′, H^5b, H^6′. A detail summary of H- and C¹³-NMR and

correlation data of compound (5a) has been presented in Table-1. While in the case of compound

(2a) characteristic signals for NH¹group appears at δ 10.33 ppm in d-DMSO, may be

deshielding effects due to tetrazole ring. The possible position of tetrazole formation to one of

the three nitrile groups (CN-9, CN-10, or C-11) was illustrated by using 2D NMR spectra. In

HMBC spectrum, the correlations of C⁹(114.2 ppm) nitrile group with H^5band NH¹protons

confirmed its position. However, the positions of C¹⁰and C¹¹nitrile groups in both compounds

(2a and 5a) were difficult to assign from NMR data. Therefore, the tetrazole formation at C¹¹

position was assigned on the basis of reactivity and availability of CN-11 nitrile group in

compound (5a). Some literature showed the addition of nucleophile to such type of synthetic

precursor (5a) [5, 23]. Moreover, NH¹group showed HMBC correlations with C³, C⁵, C⁶, C⁷,

and C⁸carbon atoms. Each proton, H^5aand H^5b, from methylene group (CH₂) group showed

doublet at δ 3.77 and 3.30 ppm, respectively. Corresponding correlation data include HMBC,

COSY, and NOESY has mentioned in the Table -2. Singlet of C⁷methyl group appears almost at

the same position (δ 1.78 ppm) as in the case of (5a). Attempts have also been made to obtain the

X-ray crystal structure of compounds in 2(a-h) series, even with solvent system which has been

^= obscured by H₂O signal in DMSO

6

used to develop the crystals of (5a) [18], were unsuccessful. Therefore, different NMR spectra

including 2D data has been discussed and provided in Table-2 to adapt the most favourable

structure of compounds in series 2(a-h).

7

Table-1: Characteristic ¹H, C¹³NMR correlation data of (5a)

Atoms No

1

2

C

-

3

C

-

4

C

-

5

6

C

-

7

8

C

-

9

10

11

1'

C

-

1''

C

-

2'/2''/3'/3''/4'/4''/5'/5''/6'/6''

CH x 10 (Aromatic H)

7.58-7.28 (m)

NH

CH^a

CH^b

CH₃

CN

9.71

(brs)

3.77

(d)

3.30

1.71

(s)

δ_H

δ_C

132.1/128.9/128.6/128.2

/127.5/124.9

-

157.8 101.1 168.9

44.03

56.9

28.2

49.5 113.7 115.5 114.9 142.8 135.5

C^1′

C^1′′

C²

C³

C⁴

C⁶

C⁷

C⁹

C²

C³

C⁵

C⁶

C⁷

C⁸

C⁹

C^1′

C²

C⁴

C⁵

C⁶

C^1′′

C³

C⁴

C⁶

C⁷

C^1′C²C^2'C^3′C⁴C^4′′C^5′C^6′

HMBC

-

H^2′H^2′′H^3′H^3′′H^4′H^4′′H^5′

H^5′′H^6′H^6′′

H^5a

-

H¹H^5b

H^5a

-

COSY

H^2′H^6′

H⁷

H¹H^2′

H^2′H^2′'H^3′H^3′'H^4′H^4′'

H^5′H^5′′H^5aH^6′H^6′'H⁷

H^2′H^5b

NOESY

H^5bH^6′

= obscured by H₂O signal in DMSO

8

Table-2: Characteristic ¹H, C¹³NMR correlation data of (2a)

Atoms No

1

2

C

-

3

C

-

4

C

-

5

6

7

8

9

10

11

1'

1''

2'/2''/3'/3''/4'/4''/5'/5''/6'/6''

CH x 10 (Aromatic H)

7.55-7.29 (m)

NH

CH^a

3.77

CH^b

3.30

C

CH₃

C

CN

C=N

C

10.33

(brs)

-

1.71 (s)

-

δ_H

δ_C

(d)

131.6/128.85/128.82/128.1

/127.6/124.9

-

150 102 167

43.9

56.1 29.2

64.1 114.2 116.4 143.4 143.4 136.3

C^1′

C³

C⁵

C⁶

C⁷

C⁸

C⁹

C^1′′

C³

C⁴

C⁶

C^1′′

C³

C⁴

C⁶

C⁷

C⁹

C^1′

C⁵

C⁶

-

C^1′C²C^2'C^3′C⁴C^4′′C^5′C^6′

HMBC

-

H^2′H^2′′H^3′H^3′′H^4′H^4′′H^5′H^5′′

H^6′H^6′′

H^5a

-

H¹H^5bH^5a

-

COSY

H¹H^2′

H^5aH^5b

H^6′

H^2′H^6′

H⁷

H^2′H^5bH^2′H^5a

H⁶H^{7 ′}H⁶H^{7 ′}

H^2′H^2′'H^3′H^3′'H^4′H^4′'H^5′H

^5′′H^5aH^6′H^6′'H⁷

NOESY

= obscured by H₂O signal in DMSO

9

2.2 Computational Studies

All calculations were performed by using GAUSSIAN 09 [25]. Geometries of the structures

were optimized without any symmetry constraints at hybrid B3LYP (Becke 3-Parameter, Lee,

Yang and Parr) method using 6-31G(d) [26-28]. B3LYP is a cost effective method for accurate

prediction of geometries of a number of synthetic [29-31] and natural products [32, 33]. Each

optimized structure was confirmed by frequency analysis at the same level to confirm the

stationary point either as a true minimum (no imaginary frequency) or a transition state (with one

imaginary frequency). Intrinsic reaction coordinates (IRC) calculations were performed to

confirm that the transition states connect to the right starting materials and products. IRC was

performed until the stationary point was reached with RMS gradient less than 1 x 10^-4. The

stationary points located through IRC were then completely optimized at the above mentioned

method. The reported energies for all structures are in kcal mol^-1and include unscaled zero point

energy corrections.

Density Functional Theory (DFT) calculations have been performed to explore possible reaction

mechanism for the formation of (5) from (6) and (7). The first step involves Knoevenagel

condensation in which active hydrogen of (4) undergoes nucleophilic addition to a carbonyl

group resulting in dehydration reaction to produce intermediate (6). The intermediate (6) has an

acidic proton which is abstracted by a mild base (TBAF) to generate the intermediate (7) (exists

in two tautomeric forms (7a and 7b). Condensation of (7a) or (7b) with (6) leads to two different

mechanistic pathways for the synthesis of 1,2,5,6-tetrahydronicotinonitrile; 1) Michael addition

(Path I) and 2) Dimerization reaction (Path II, Scheme-2).

In the Michael addition reaction, active methylene carbon (CH₂) of intermediate (7a) adds to

electrophilic carbon of intermediate (6) in a 1,4-addition manner (Figure-2a). The Michael

addition is kinetically and thermodynamically highly favorable. The reaction takes place without

any activation barrier. A transition state is located at a lower energy compared to the reactants (-

0.06 kcal mol^-1). The bond distance among both joining carbons is 2.14 Å (Figure-2). The small

negative activation barrier is within the error limits of the DFT method applied here. Therefore,

it can be concluded safely that the process is virtually barrier less. The reaction is

thermodynamically favorable by 4 kcal mol^-1; the products are more stable than the reactants.

10

Figure-2: Energy profile of Michael addition and Dimerization reaction of (7a) and (6),

calculated at B3LYP/6-31G(d). All energies are relative to (7a), hydrogen atoms have been

removed for clarity. Bond lengths and bond angles are shown in Angstroms and degrees,

respectively.

An alternative mechanism proposed for the reaction of (7b) with (6) is the dimerization reaction,

where two monomers (7b) and (6), join together to form covalently bonded intermediate (11).

The nucleophilic nitrogen of intermediate (7b) attacks the electrophilic carbon of intermediate

(6) (Scheme-2). Transition state (TS6-11), for the dimerization reaction, is located at a higher

energy (2.82 kcal mol^-1) from the reactants. The dimerization reaction is also a kinetically and

thermodynamically favorable process. The activation barrier for (TS6-11), although higher than

the competitive (TS6-8), is still negligible, and easily accessible under the reaction conditions.

The joining nitrogen and carbon atoms are at a distance of 1.80 Å in the transition state.

11

Since, the activation barriers for both reactions (Michael addition and dimerization reactions) are

easily accessible at room temperature; therefore, these initial steps don’t deliver any concluding

information regarding the selective operation of any mechanism. We believe that the subsequent

steps may play decisive role therefore, activation barrier for the subsequent steps are also

analyzed and discussed below.

Path I

In path I, The intermediate (8) undergoes ring closure through transition state (TS8-9) resulting

in the generation of intermediate (9) (Figure-3). The nucleophilic attack of electron rich carbon

of (7) on the electrophilic nitrile group is again a kinetically favorable process. The activation

barrier is 14.72 kcal mol^-1, which is easily accessible under the reaction conditions. This

activation energy is higher as compared to the first step (TS6-8), mainly due to the bulky groups

attached to both carbons that undergo reaction. The bond distance between the joining atoms is

2.07 Å which confirms the bond formation among two carbons to form a cyclohexene structure

(9).

Figure-3: Energy profile of generation of intermediate (9) from (8), calculated at B3LYP/6-

31G(d). The energies are relative to (8) at 0.0 kcal mol^-1, hydrogen atoms have been removed for

clarity.

The intermediate (9) undergoes tautomerization to deliver cyclohexadiene (10) which is

thermally unstable and undergoes ring opening to acyclic hexatriene type intermediate (12)

through transition state (TS10-12) (Figure-4). Activation energy for the reaction is calculated to

12

be 19.93 kcal mol^-1that show reaction feasibility. The bond distance, in the transition state,

among the two carbons is 2.39 Å which shows bond breakage of the cyclic structure.

Figure-4: Energy profile for the conversion of (10) into (13a) through a cascade of

electrocyclization and proton transfer, calculated at B3LYP/6-31G(d), hydrogen atoms have been

removed for clarity. All energy values are relative to (10) at 0.0 kcal mol^-1.

In the next step recyclization reaction takes place in which electrocyclization involves NH₂

moiety incorporated in the hexatriene moiety undergoing electrocyclization. The C-N bond

distance is 2.07 Å in the transition state (TS 12-13) (Figure-5) and the activation energy for the

reaction is 24.69 kcal mol^-1. The intermediate (13) undergoes sigmatropic 1,3-shift of hydrogen

between the nitrogen and carbon of the closed ring followed by formation of ionic product (13a)

13

which on protonation will convert to final 1,2,5,6-tetrahydronicotinonitrile (5) (Figure-4). The

reaction proceeds with an activation energy 27.15 kcal^-1. The activation barrier is relatively high

º

but still accessible under reaction conditions (100 C). The proton shift can alternatively take

place by a low energy pathway involving intermolecular proton shift where the activation barrier

is generally about 10-12 kcal mol^-1.

In pathway II, the arylidenemalononitrile (6), underwent dimerization reactions via tautomer

(7b) to generate intermediate (11). The intermediate (11) can undergo 1,3-sigmatropic shift of

CH(CN)₂group to deliver the intermediate (14); however, all attempts to locate the transition

state for such transformation met with failure. This sigmatropic shift is not believed to be a low

energy process because such sigmatropic shifts are kinetically highly unfavorable (activation

barriers are generally more than 40 kcal mol^-1). Therefore an alternative mechanism is proposed

where intermediate (11) first cyclizes to produce intermediate (15) (with a cyclobutane moiety)

which opens up at another position to deliver intermediate (14). The carbanion of intermediate

(11) undergoes intramolecular nucleophilic attack on the electrophilic carbon to deliver cyclized

product (15). The (TS11-15) shows the formation of a four membered ring which is responsible

for the formation of intermediate (15) (Figure-5).

Figure-5: Energy profile of generation of (15) from (11), calculated at B3LYP/6-31G(d),

hydrogen atoms have been removed for clarity. All energies are relative to (11) at 0.0 kcal mol^-1.

14

Opening of cyclobutane ring of intermediate (15) occurs in the next step in which transition state

(TS15-14) (Figure-6) shows the loosening of bond between two carbons. The C-C bond length is

2.22 Å in the transition state which was 1.58 Å in Intermediate (15). The reaction proceeds with

an activation energy of 11.01 kcal mol^-1. Both cyclization (to produce a cyclobutane ring in

intermediate 15) and then opening of the ring (to produce intermediate 14) are kinetically

favorable processes, and the activation barriers are easily accessible under the reaction

conditions. Finally, Intermediate (14) undergoes cyclization reaction with an activation barrier of

11.43 kcal mol^-1to deliver the final product (5) after the protonation of intermediate (13a). The

calculated kinetic barriers and thermodynamics associated with reactions for Path (I) and (II) are

easily accessible under the reaction conditions; therefore, it can be concluded that both

mechanisms are operational under the reaction conditions.

Figure-6: structure of transition state (TS15-14), calculated at B3LYP/6-31G(d), hydrogen

atoms have been removed for clarity. Bond length is in Angstroms.

15

Figure-7: Energy profile for generation of ionic product (13a) from (14) through

elelctrocyclization, calculated at B3LYP/6-31G(d), hydrogen atoms have been removed for

clarity.

2.3 Bioactivity and Structure Activity Relationship (SAR)

All successfully synthesized tetrazole linked 1,2,5,6-tetrahydronicotinonitriles 2(a-h) were

evaluated for their anticholinesterase activities against AChE and BChE, which were obtained

from electric eel and equine serum, respectively, by using Ellman’s assay [34]. The previously

reported 1,2,5,6-tetrahydronicotinonitriles 5(a-h) [18] were also evaluated for cholinesterase

inhibition. The bioactivity results of the both series tetrazole linked 1,2,5,6-

tetrahydronicotinonitriles 2(a-h) and its precursors 5(a-h) demonstrated that mostly compounds

were active against both AChE and BChE compared to standard inhibitors i.e. neostigmine and

donepezil (Table-3). From the bioactivities data, 4'/4''-fluoro (2d) derivatives was found to

exhibited highest inhibitory activity for AChE with IC₅₀values of and 2.01 ± 0.027 M while the

2'/2''-methoxy derivative (2g) revealed highest inhibitory activity in submicromolar level for

BChE with IC₅₀value of 0.290 ± 0.130 M. Limited SAR suggested that the activity of these

compounds depend on the nature of substitution and its position on the phenyl rings belong to

acetophenone.

16

In 1,2,5,6-tetrahydronicotinonitriles 5(a-h) series, all compounds displayed better AChE

inhibitory activity as compared to BChE inhibitory activity (Table 1). The 3'/3''-methoxy

derivative (5g) exhibited highest activity with IC₅₀value of 1.62 ± 0.041 M against AChE,

which indicated the importance of substituent pattern on phenyl ring during enzyme inhibition.

On the other hand, among the tetrazole linked 1,2,5,6-tetrahydronicotinonitriles 2(a-h), the

inhibitory activity was also varies due to different substituent pattern on the phenyl ring. The

derivative (2a) without any substituents was found moderately active only against AChE with

IC₅₀values of 7.02 ± 0.19 M. The 4'/4''-methyl substituted derivative (2b) showed inhibition

potential against both AChE and BChE with IC₅₀value 4.45 ± 0.180 M and 5.87 ± 0.128 M,

respectively, while the 3'/3''-methyl derivative (2c) which was found poorly or almost inactive

against both enzymes (Table-1). In halogen compounds, the derivative (2d) was found more

active against AChE than BChE while the 3'/3''-chloro analogue (2e) acted as potent dual

inhibitor for AChE with IC₅₀value of 2.32 ± 0.091 M and as well as for BChE with IC₅₀values

of 2.78 ± 0.216 M. The 4'/4''-hydroxy derivative (2h) with IC₅₀value of 7.42 ± 0.489 M

showed moderate activity against AChE, whereas it displayed significant activity against BChE

with an IC₅₀value of 2.45 ± 0.30 M. In the case of methoxy derivatives, the 3'/3''-methoxy

derivative (2f) showed selectively moderate inhibitory activity with IC₅₀values of 5.11 ± 0.12

M for only AChE, while the 2'/2''-methoxy derivative (2g) displayed excellent inhibitory

activity with IC₅₀values of 0.290 ± 0.130 M (Table 1). The above described results strongly

suggested that substituent’s electronic effect and position on phenyl ring influenced the

inhibitory activity of compounds.

17

Table-3: In Vitro AChE and BChE inhibition activities of 1,2,5,6-tetrahydronicotinonitrile 5(a-

h) and tetrazole linked derivatives 2(a-h).

Compounds

R

AChE

BChE

Selectivity^b

IC₅₀^a(µM)

H

20.3 ± 0.481

16.9 ± 0.95

5.21 ± 0.092

11.9 ± 0.420

11.2 ± 0.401

8.01 ± 0.201

1.56 ± 0.041

48.1 ± 0.691

36.5 ± 1.19

35.9 ± 1.54

55.2 ± 0.89

45.3 ± 2.581

68.8 ± 1.98

48.2 ± 1.96

2.37

2.16

6.89

4.64

4.04

8.59

30.9

5a

5b

5c

5d

5e

5f

4′/4′′-Methyl

3′/3′′-Methyl

4′/4′′-Fluoro

4′/4′′-Iodo

3′/3′′-Fluoro

3′/3′′-Methoxy

5g

4′/4′′-Hydroxy

5.38 ± 0.125

42.2 ± 0.070

7.84

5h

H

7.02 ± 0.19

4.45 ± 0.180

20.8 ± 0.582

2.01 ± 0.027

2.32 ± 0.091

5.11 ± 0.12

20.3 ± 0.861

7.42 ± 0.489

22.1 ± 1.40

5.87 ± 0.128

62.2 ± 0.80

38.8 ± 0.98

2.78 ± 0.216

39.2 ± 1.58

0.290 ± 0.130

2.45 ± 0.30

3.14

1.31

2.30

19.3

1.19

7.67

0.014

0.33

2a

2b

2c

2d

2e

2f

4′/4′′-Methyl

3′/3′′-Methyl

4′/4′′-Fluoro

3′/3′′-Chloro

3′/3′′-Methoxy

2′/2′′-Methoxy

4′/4′′-hydroxy

2g

2h

18

2.23

442

Neostigmine^c

Donepezil^c

19.3 ± 3.21

43.2 ± 4.21

0.019 ± 0.002

8.41 ± 0.381

^aInhibitory concentration; ^bSelectivity ratio = IC₅₀ratio (BChE/AChE); ^cStandard

inhibitors for cholinesterase activities

3. Conclusion

In conclusion, one-pot solvent-free synthesis of tetrazole linked 1,2,5,6-tetrahydronicotinonitrile

derivatives 5(a-h) has been carried out in the presence of TBAF. Two plausible reaction

mechanisms (I) and (II) (Scheme 2) were elaborated by using DFT calculations which

demonstrated pathway (I) to be the most feasible one. Moreover, bioactivity studies of 1,2,5,6-

tetrahydronicotinonitriles 5(a-h) and tetrazole linked derivatives 2(a-h) were performed against

cholineresatease, AChE and BChE. In most of the cases, tetrazole linked 1,2,5,6-

tetrahydronicotinonitriles 2(a-h) were found more active than intermediates 5(a-h). The tetrazole

analogue (2g) was found a potent selective inhibitor with IC₅₀values of 0.290 ± 0.130 M

against BChE, which has elevated activity in AD brain, as compared to reference inhibitors,

neostigmine and donepezil. The compound (2g) could be potentially served as lead for further

structural modifications in order to enhance potency and selectivity against cholinesterase to

affect AD symptoms.

4. Material and Methods (Chemistry)

The starting materials which include the different derivatives of acetophenone (≥99%),

malononitrile (≥99%), and tetrabutylammonium fluoride trihydrated (TBAF) (98%) were

purchased from Sigma-Aldrich and used without any purification unless otherwise stated. The

reagent grade ethyl acetate, diethyl ether, hexane, HCl laboratory grade and distilled water were

used as solvents during reaction workup and purification. All the reactions were performed in

screw capped Micro Reaction Vessels (5 mL), purchased from Sigma-Aldrich. Thin layer

chromatography (TLC) was used to monitor reaction using silica gel 60 aluminium-backed plates

0.063-0.200 mm as the stationary phase with analytical grade solvents such as ethyl acetate

(EtOAc), diethyl ether, n-hexane etc. Chromatograms were visualized with UV light (254 nm) or

using different staining mixtures such as basic potassium permanganate, vanillin, ninhydrin and

bromocresol green. Infrared spectra IR (KBr discs) were recorded between 4000-500 cm^-1on a

Bruker Vector-22 spectrometer. TGA and DSC analyses were carried out on a TA instrument,

model number SDT Q600. Aluminum heating/sample pans were used. The sample weight used

19

was 3-6 mg. Decomposition profiles were obtained at four different constant heating rates, that

º

ᵒ

is, 5 C/min, 10 C/min, 15 C/min and 20 C/min. All analyses were carried out under nitrogen

atmosphere (flow rate was 100 ml/min). The data were processed by use of Universal Analysis

2000 software, version 4.2E (TA Instruments, USA), and MS Excel 2013. Bruker spectrometers

1

13

were used to record H NMR spectra at 300, 400 or 500 MHz and C NMR at 75, 100 or 125

MHz as dilute solutions in the appropriate deuterated solvent at 25 ^ºC.

All the chemical shifts were recorded on the δ-scale (ppm) using residual solvents as an internal

1

13

1

13

standard (DMSO; H 2.50, C 39.43 and CHCl₃; H 7.26, C 77.16). Coupling constant were

calculated in Hertz (Hz) and multiplicities were labelled s (singlet), d (doublet), t (triplet), q

(quartet), quint (quintet) and the prefixes br (broad) or app (apparent) were used. Mass spectra

(ESI⁺) were recorded at Finnigan MAT-321A, Germany by using electrospray (ES+), electron

impact (EI+ EI+) or FAB (Fast atom bombardment) techniques. Melting points of solids were

determined using a Stuart™ melting point apparatus SMP3 apparatus and are uncorrected.

Butyrylcholinesterase (BuChE) (EC 3.1.1.8, from horse serum), acetylcholinesterase (AChE)

(EC 3.1.1.7, type VI-S from electric eel), S-butyrylthiocholine chloride (BTCCl),

acetylthiocholine iodide (ATCI), 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), neostigmine

methylsulphate and dimethylsulphoxide (DMSO) and donepezil hydrochloride were purchased

from Sigma-Aldrich (St. Louis, MO, USA).

5.1 General procedure for the synthesis of 1,2,5,6-Tetrahydronicotinonitrile Derivatives

2(a-h)

In a general procedure, an oven dried sealed vessel was charged with the corresponding

acetophenone (1.0 mmol), malononitrile (1.5 mmol) and TBAF (1.0 mmol) at room temperature.

º

The reactions were then heated at 100-103 C with constant stirring until the complete

consumption of the starting material, monitored by TLC analysis. At this reaction stage,

trimethylsilyl azide (1.0 mmol) was added into the same sealed vessel and left the resulting

º

mixture for further stirring at 100-103 C for 3-4 hr. The reaction mixture was cooled to room

temperature, poured in to 10% HCl to remove TBAF and extracted with EtOAc. The organic

layer was dried with MgSO₄, filtered and evaporator on rotary evaporator. The crude product

was purified by silica gel column chromatography with 80% EtOAc in hexane to afford the

desired pure compounds 2(a-h) in different yields (Figure 2). The structures of each compound

20

2(a-h) were confirmed with NMR, IR spectroscopy and mass spectrometry. The structures of

each compound 2(a-h) were confirmed with NMR, IR spectroscopy and mass spectrometry. The

spectral data of all compounds 5(a-h) was reported previously reported [18].

5.2 Spectral data compounds

2-Dicyanomethylene-6-methyl-4,6-bis(phenyl)-1,2,5,6-tetrahydronicotinonitrile (5a) [18]

¹H and C¹³NMR (Table-1); EI-HRMS calculated for C₂₂H₁₆N₄(M): 336.1375, Found: 336.1355;

Anal. calcd for C₂₄H₂₀N₄O₂; C, 78.55; H, 4.79; N, 16.66 found: C = 78.24, H = 4.99, N =

16.99%.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(phenyl)-1,2,5,6-

tetrahydronicotinonitrile (2a)

º

1

Brown solid (334 mg, 84.8%), mp 166 C, H and C¹³NMR (Table-2); HRMS (ESI) calculated

for C₂₂H₁₈N₇: 380.1623 (M+H)⁺, Found: 380.1596.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(4′/4′′-methylphenyl)-1,2,5,6-

tetrahydronicotinonitrile (2b)

Following the general procedure, the corresponding 1,2,5,6-tetrahydronicotinonitrile derivative

º

(2b) was isolated as a yellow solid (224 mg, 53.8%). mp 201 C; υ_max/cm^-1(KBr solid) 3429,

1

3138, 2923, 2203 (CN), 2138, 1733, 1606, 1542, 1446, 1408, 1380, 1190; H NMR (300 MHz,

DMSO) _H10.30 (1H, brs, NH), 7.41 (2H, d, J 8.1 Hz, ArH), 7.33 (4H, d, J 8.4 Hz, ArH), 7.18

(2H, d, J 8.1 Hz, ArH), 3.74 (1H, d, J 18.6 Hz, CH^5aH^5b), 3.32 (1H, d, CH^5aH^5b, obscured by

13

H₂O in DMSO), 2.36 (3H, s, ArCH₃), 2.26 (3H, s, ArCH₃), 1.74 (3H, s, CH₃); C NMR (75

MHz, DMSO) _C166.9, 150.4, 142.1, 140.4, 136.7, 133.2, 129.4, 129.3, 128.3, 124.7, 116.4,

114.5, 101.1, 64.1, 55.9, 43.7 (CH₂), 29.2 (CH₃), 20.9 (CH₃), 20.4 (CH₃); HRMS (ESI)

calculated for C₂₄H₂₂N₇: 408.1936 (M+H)⁺, Found: 408.1903.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(3′/3′′-methylphenyl)-1,2,5,6-

tetrahydronicotinonitrile (2c)

Following the general procedure, the corresponding 1,2,5,6-tetrahydronicotinonitrile derivative

º

(2c) was isolated as a brown solid (360 mg, 86.7%). mp 202 C; υ_max/cm^-1(KBr solid) 3456,

1

3130, 3045, 2922, 2206 (CN), 1606, 1558, 1450, 1380, 1274, 1043; H NMR (300 MHz,

DMSO) _H10.25 (1H, brs, NH), 7.43-7.33 (3H, m, ArH), 7.30-7.24 (3H, m, ArH), 7.20 (1H, app

21

d, J 8.0 Hz, ArH), 7.11 (1H, d, J 7.2 Hz, ArH), 3.73 (1H, d, J 18.4 Hz, CH^5aH^5b), 3.32 (1H, d,

CH^5aH^5b, obscured by H₂O in DMSO), 2.35 (3H, s, ArCH₃), 2.31 (3H, s, ArCH₃), 1.77 (3H, s,

CH₃); ¹³C NMR (75 MHz, DMSO) _C167.4, 150.5, 143.2, 138.2, 138, 136.4, 132.2, 128.7,

128.6, 128.5, 128.2, 125.6, 126.3, 121.9, 116.6, 114.2, 101.9, 64, 56.1, 44 (CH₂), 29.1 (CH₃),

21.1 (CH₃), 20.8 (CH₃); HRMS (ESI) calculated for C₂₄H₂₂N₇: 408.1936 (M+H)⁺, Found:

408.1919.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(4′/4′′-fluorophenyl)-1,2,5,6-

tetrahydronicotinonitrile (2d)

Following the general procedure, the corresponding 1,2,5,6-tetrahydronicotinonitrile derivative

º

(2d) was isolated as a brown solid (360 mg, 47.8%). mp 170 C; υ_max/cm^-1(KBr solid) 3371,

1

3236, 2924, 2858, 2205 (CN), 1606, 1556, 1479, 1387, 1276, 1068, 1002; H NMR (300 MHz,

DMSO) _H10.29 (1H, each brs, NH), 7.91 (2H, d, J 8.4 Hz, ArH), 7.73 (2H, d, J 8.4 Hz, ArH),

7.24 (4H, t, J 8.2 Hz ArH), 3.76 (1H, d, J 18 Hz, CH^5aH^5b), 3.36 (1H, d, CH^5aH^5b, obscured by

H₂O in DMSO), 1.78/1.71 (3H, each s, CH₃); ¹³C NMR (75 MHz, DMSO) _C165.8, 165, 164.4,

162.57/162.54, 160.2, 154.2, 149.8, 139.4, 130.9/130.8, 127.2/127.1, 116.2, 116.1/115.9,

115.6/115.4, 114, 102, 64.2, 55.8, 43.9 (CH₂), 29.1 (CH₃); HRMS (ESI) calculated for

C₂₂H₁₆F₂N₇: 416.1435 (M+H)⁺, Found: 416.1438.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(3′/3′′-chlorophenyl)-1,2,5,6-

tetrahydronicotinonitrile (2e)

Following the general procedure, the corresponding 1,2,5,6-tetrahydronicotinonitrile derivative

2e was isolated as a brown solid (224 mg, 49.8%). mp 152 ^ºC; υ_max/cm^-1(KBr solid) 3400, 2924,

1

2206 (CN), 1609, 1568, 1514, 1415, 1269, 1080, 1029; H NMR (400 MHz, DMSO) _H10.23

(1H, brs, NH), 7.63 (2H, app d, J 8.0 Hz, ArH), 7.57 (1H, t, J 7.8 Hz, ArH), 7.48 (1H, brs, ArH),

7.43 - 7.33 (4H, m, ArH), 3.78 (1H, d, J 18.4 Hz, CH^aH^b), 3.38 (1H, d, CH^aH^b, obscured by H₂O

in DMSO), 1.79 (3H, s, CH₃); ¹³C NMR (100 MHz, DMSO) _C165.2, 149.4, 147.5, 145.9,

138.1, 133.7, 133.5, 131.2, 130.8, 130.7, 127.8, 127.7, 127.6, 125.2, 123.8, 116, 113.6, 103 1,

64.1, 56, 43.5 (CH₂), 28.5 (CH₃); HRMS (ESI) calculated for C₂₂H₁₅Cl₂N₇: 448.0844 (M+H)⁺,

Found: 448.0849.

22

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(3′/3′′-methoxyphenyl)-1,2,5,6-

tetrahydronicotinonitrile (2f)

Following the general procedure, the corresponding 1,2,5,6-tetrahydronicotinonitrile derivative

º

(2f) was isolated as a brown solid (141 mg, 56.8%). mp 173 C; υ_max/cm^-1(KBr solid) 3414,

3233, 2836, 2205 (CN), 1642, 1603, 1542, 1488, 1457, 1430, 1380, 1290, 1257, 1174, 1041; ¹H

NMR (300 MHz, DMSO) _H10.28 (1H, brs, NH), 7.43 (1H, t, J 7.8 Hz, ArH), 7.32 (1H, t, J 8

Hz, ArH), 7.11 (1H, dd, J 2, 8.4 Hz, ArH), 7.04 (1H, d, J 9.2 Hz, ArH), 7.03 (1H, brs, ArH), 6.99

(1H, d, J 8 Hz, ArH), 6.93 (1H, brs, ArH), 6.88 (1H, dd, J 2, 8.4 Hz, ArH), 3.79 (3H, s,

ArOCH₃), 3.73 (1H, d, J 18.4 Hz, CH^5aH^5b), 3.73 (3H, s, ArOCH₃), 3.30 (1H, d, CH^5aH^5b,

13

obscured by H₂O in DMSO), 1.78 (3H, s, CH₃). C NMR (125 MHz, DMSO) _C167.1, 159.9,

158.8, 147.8, 142.7, 140.6, 133.2, 130.7, 129.7, 121.8, 116.5, 116.2, 116, 115.1, 113.8, 114.2,

110.5, 102.1, 65.2, 56.2, 55.2 (OCH₃), 55 (OCH₃), 43.8 (CH₂), 28.9 (CH₃); HRMS (ESI)

calculated for C₂₄H₂₂N₇O₂: 440.1834 (M+H)⁺, Found: 440.1807.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(2′/2′′-methoxyphenyl)-1,2,5,6-

tetrahydronicotinonitrile (2g)

Following the general procedure, the corresponding naphthyridine derivative (2g) was isolated as

a brown solid (295 mg, 65.8%). mp 171 ^ºC; υ_max/cm^-1(KBr solid) 3474, 3414, 3234, 2936, 2839,

1

2202 (CN), 1612, 1556, 1488, 1461, 1435, 1291, 1254, 1164, 1023; H NMR (300 MHz,

DMSO) _H10.46 (1H, brs, NH), 7.48 (1H, t, J 7.6 Hz, ArH), 7.33 (1H, t, J 7.6 Hz, ArH), 7.14

(2H, dd, J 8.8, 11.6 Hz, ArH), 7.06-6.99 (3H, m, ArH), 6.92 (1H, t, J 7.2 Hz ArH), 3.83 (3H, s,

ArOCH₃), 3.74 (3H, s, ArOCH₃), 3.78 (1H, d, J 25.5 Hz, CH^5aH^5b), 3.32 (1H, d, CH^5aH^5b,

13

obscured by H₂O in DMSO), 1.80 (3H, s, CH₃); C NMR (75 MHz, DMSO) _C166.3, 156.4,

156.1, 149.2, 132.3, 129.44, 129.38, 128.6, 126.07, 125.96, 120.54, 120.50, 116.3, 113.6, 112.4,

112.1, 103.7, 65.2, 55.8, 55.6 (OCH₃), 55.5 (OCH₃), 41.2 (CH₂), 26.3 (CH₃); HRMS (ESI)

calculated for C₂₄H₂₂N₇O₂: 440.1834 (M+H)⁺, Found: 440.1805.

2-(Cyano(1H-tetrazol-5-yl)methylene)-6-methyl-4,6-bis(4′/4′′-hydroxyphenyl)-1,2,5,6-

tetrahydronicotinonitrile (2h)

Following the general procedure, the corresponding naphthyridine derivative (2h) was isolated as

a brown solid (209 mg, 70.8%). mp 130 ^ºC; υ_max/cm^-1(KBr solid) 3357, 2924, 2208 (CN), 1639,

1

1569, 1512, 1440, 1381, 1267, 1232, 1172, 1109, 1034; H NMR (400 MHz, DMSO) _H10.71

23

(1H, s, ArOH) 10.15 (1H, brs, NH), 9.33 (1H, s, ArOH), 7.37 (2H, d, J 8.8 Hz, ArH), 7.17 (2H,

d, J 8.4 Hz, ArH), 6.83 (2H, d, J 8.4 Hz, ArH), 6.69 (2H, d, J 8.4 Hz ArH), 3.53 (1H, d, J 17.6

13

Hz, CH^5aH^5b), 3.32 (1H, d, J 18 Hz, CH^5aH^5b), 1.64 (3H, s, CH₃); C NMR (75 MHz, DMSO)

_C160.8, 156.4, 134, 130.7, 130.4, 126.7, 126.1, 125.9, 117.5, 115.5, 115.4, 115.2, 99.4, 69.2,

55.2, 43.9 (CH₂), 29.6 (CH₃); HRMS (ESI) calculated for C₂₂H₁₈N₇O₂: 412.1521 (M+H)⁺,

Found: 412.1500.

5.3 Determination of AChE and BChE inhibitory activity

Inhibitory activities of AChE and BChE were evaluated by using the Ellman’s method.[34]

Herein compounds 2(a-h) and 5(a-h) were evaluated as inhibitors of AChE and BChE. For

initial screening each compound was dissolved in DMSO (end concentration of DMSO was less

than 1% in assay) and tested at a final concentration of 0.5 mM. The compounds with

considerable inhibition (more than 50%) were subjected to further analysis by making their 8 to

10 serial dilutions in assay buffer (50 mM Tris-HCl, 0.1 M NaCl and 0.02 M MgCl_2,pH 8.0).

Reaction mixture comprised of 20 μL assay buffer, 10 μL of test compound, 10 µL of 0.031

U/mL of enzyme (0.5 and 3.4 U/mg of AChE or BuChE respectively). This mixture was

º

incubated at 25 C for 10 min. After this pre-incubation, enzymatic reaction was started by

addition of 10 µL of 1 mM acetylethiocholine iodide or butyrylthiocholine chloride (depending

on the enzyme) and the mixture was incubated again for 15 min. The amount of enzymatic

product was estimated by measuring change in absorbance at 405 nm using a microplate reader

(Bio-Tek ELx 800TM, Instruments, Inc. USA). Neostigmine and donepezil were used as

standard inhibitors. Enzyme dilution buffer consisted of 50 µM Tris HCl buffer containing 0.1%

(w/v) bovine serum albumin (BSA) and pH 8. The effect of DMSO on activity of enzyme was

subtracted by a negative control containing DMSO, instead of inhibitor. The IC₅₀values were

calculated using non-linear curve fitting program PRISM 5.0 (GraphPad, San Diego, California,

USA).

Conflict of interest

The authors have declared no conflict of interest.

Supplementary Information

24

The supplementary information includes the NMR spectra of tetrazole-1,2,5,6-

Tetrahydronicotinonitriles 2(a-h).

Acknowledgement

We are thankful to Higher Education Commission (HEC), Pakistan and H. E. J. Research

Institute of Chemistry, International Center for Chemical and Biological Sciences, University of

Karachi, Karachi-75270, Pakistan for providing financial support of this project. Prof. Dr.

Jamshed Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons

(OPCW), The Hague, The Netherlands and Higher Education Commission of Pakistan for the

financial support through Project No. 20-3733/NRPU/R&D/ 14/520 for the financial support. Dr.

Mariya al-Rashida is grateful to Dr. M. Saeed Iqbal and Rashid Masih for helpful discussions

regarding thermal analysis.

6. References

[1]

[2]

[3]

J. P. Hallett and T. Welton, Chem. Rev. 111 (2011) 3508-3576.

J. H. Clark, Chem. Rev. 80 (1980) 429-452.

A. R. Gangloff, J. Litvak, E. J. Shelton, D. Sperandio, V. R. Wang and K. D. Rice,

Tetrahedron Lett. 42 (2001) 1441-1443.

[4]

G. V. M. Sharma, V. G. Reddy, A. S. Chander and K. R. Reddy, Tetrahedron:

Asymmetry 13 (2002) 21-24.

[5]

[6]

C. Mukhopadhyay, P. Das and R. J. Butcher, Org. Lett. 13 (2011) 4664-4667.

H. Karlsen, P. H. Songe, L. K. Sunsby, L. C. Hagen, P. Kolsaker and C. Romming, J.

Chem. Soc., Perkin Trans. 1 (2001) 497-507.

[7]

O. Di Pietro, E. Viayna, E. Vicente-García, M. Bartolini, R. Ramón, J. Juárez-Jiménez,

M. V. Clos, B. Pérez, V. Andrisano, F. J. Luque, R. Lavilla and D. Muñoz-Torrero, Eur.

J. Med. Chem. 73 (2014) 141-152.

[8]

[9]

Desmond J. Brown, Jonathan A. Ellman and E. C. Taylor, The Chemistry of Heterocyclic

Compounds, The Naphthyridines, John Wiley and Sons, Inc.Hoboken, New Jersey, 2008.

D. Silva, M. Chioua, A. Samadi, P. Agostinho, P. Garção, R. Lajarín-Cuesta, C. de los

Ríos, I. Iriepa, I. Moraleda, L. Gonzalez-Lafuente, E. Mendes, C. Pérez, M. I. Rodríguez-

25

Franco, J. Marco-Contelles and M. Carmo Carreiras, ACS Chem. Neurosci. 4 (2013)

547-565.

[10] P. Davies and A. J. F. Maloney, Lancet 308 (1976) 1403.

[11] P. J. Whitehouse, D. L. Price, A. W. Clark, J. T. Coyle and M. R. DeLong, Ann. Neurol.

10 (1981) 122-126.

[12] N. L. Batsch and M. S. Mittleman, World Alzheimer’s Report 2012, 2012.

[13] K. Maslow, Alzheimers Dement. 6 (2010) 158−194.

[14] F. H. Darras, S. Pockes, G. Huang, S. Wehle, A. Strasser, H.-J. Wittmann, M. Nimczick,

C. A. Sotriffer and M. Decker, ACS Chem. Neurosci. 5 (2014) 225-242 and reference

therein.

[15] A. Castro and A. Martinez, Curr. Pharm. Des. 12 (2006) 4377-4387.

[16] D. Pratico, Ann. N.Y. Acad. Sci. 1147 (2008) 70-78.

[17] C. X. Gong and K. Iqbal, Curr. Med. Chem. 15 (2008) 2321-2328.

[18] A. Hameed, A. Anwar, S. Yousaf, K. M. Khan and F. Z. Basha, Eur. J. Chem. 3 (2012)

179-185.

[19] H. Karlsen, P. H. Songe, L. K. Sunsby, L. C. Hagen, P. Kolsaker and C. Rømming, J.

Chem. Soc., Perkin Trans. 1 (2001) 497-507.

[20] Y. T. Abramenko, A. Ivashchenko, K. Nogaeva and Y. A. Sharanin, Chem. Heterocycl.

Compd. 22 (1986) 508-512.

[21] Y. T. Abramenko, A. V. Ivashchenko, K. A. Nogaeva, N. A. Andronova and E. B.

Putsykina, Zh. Org. Khim. 22 (1986) 264-269.

[22] Y. U. Abramenko, A. V. Ivaschchenko, K. A. Nogaeva, N. A. Andronova and E. B.

Putsykina, J. Org. Chem. USSR (Engl. Transl.) 22 (1986) 230.

[23] F. M. Abdelrazek, N. H. Metwally, N. A. Kassab, N. A. Sobhy, P. Metz and A. Jaeger, J.

Heterocyclic Chem. 47 (2010) 384-388.

[24] S. Dunkel, U. He and G. Reck, J. prakt. Chem. 339 (1997) 414-419.

[25] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman,

G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li,

H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M.

Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,

H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J.

26

Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K.

Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega,

N. J. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.

Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.

Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.

Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J.

Cioslowski and D. J. Fox, Gaussian 09, 2009.

[26] P. C. Hariharan and J. A. Pople, Theor. Chim. Acta. 28 (1973) 213-222.

[27] A. D. Becke, J. Chem. Phys. 98 (1993) 5648-5652.

[28] C. Lee, W. Yang and R. Parr, J. Phys. Chem. 37 (1998) 785–789.

[29] T. Q. Hung, D. H. Hoang, N. N. Thang, T. T. Dang, K. Ayub, A. Villinger, A. Friedrich,

S. Lochbrunner, G.-U. Flechsig and P. Langer, Org. Biomol. Chem. 12 (2014) 6151-

6166.

[30] I. Javed, A. Khurshid, M. N. Arshad and Y. Wang, New J. Chem. 38 (2014) 752-761.

[31] H. Ullah, A.-u.-H. A. Shah, K. Ayub and S. Bilal, J. Phys. Chem. C. 117 (2013) 4069-

4078.

[32] H. Ullah, A. Rauf, Z. Ullah, S. Fazl i, M. Anwar, A.-u.-H. A. Shah, G. Uddin and K.

Ayub, Acta. A. Mol. Biomol. Spectrosc. 118 (2014) 210-214.

[33] M. A. Hashmi, A. Khan, K. Ayub and U. Farooq, Acta. A. Mol. Biomol. Spectrosc. 128

(2014) 225-230.

[34] G. L. Ellman, K. D. Courtney, V. Andres jr and R. M. Featherstone, Biochem.

Pharmacol. 7 (1961) 88-95.

27

Graphical Abstract

One-Pot

Tetrahydronicotinonitriles

Inhibition: Probing the

Mechanism via Computational Studies

Synthesis

of

Tetrazole-1,2,5,6-

Leave this area blank for abstract info.

and

Cholinesterase

Plausible

Reaction

Abdul Hameed, Syeda Tazeen Zehra, Saba Abbas, Riffat Un Nisa, Tariq Mahmood, Khurshid Ayub, Mariya al-Rashida,

Norbert Furtmann, Jürgen Bajorath, Khalid M. Khan, and Jamshed Iqbal

28

Research highlights

 A series of tetrazole-1,2,5,6-tetrahydronicotinonitriles was synthesized.

 Tetrazole-1,2,5,6-tetrahydronicotinonitriles as cholinesterase inhibitors.

 Plausible reaction mechanism was studied via computational methods.

29

Article Doi

One-pot synthesis of tetrazole-1,2,5,6-tetrahydronicotinonitriles and cholinesterase inhibition: Probing the plausible reaction mechanism via computational studies

DOI: 10.1016/j.bioorg.2016.01.004

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2016.01.004

Full text of DOI:10.1016/j.bioorg.2016.01.004

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