Novel tetrahydrocarbazole benzyl pyridine hybrids as

European Journal of Medicinal Chemistry 155 (2018) 49e60

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl cholinesterase inhibitors with neuroprotective and b-secretase inhibition activities Roshanak Ghobadian a, Mohammad Mahdavi b, Hamid Nadri c, Alireza Moradi c, Najmeh Edraki d, Tahmineh Akbarzadeh a, e, Mohammad Sharifzadeh f, Syed Nasir Abbas Bukhari g, Mohsen Amini a, * a

Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran c Pharmaceutical Science Research Center and Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd 8915173143, Iran d Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran e Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran f Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran g Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Al-jouf, Sakaka 2014, Saudi Arabia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 March 2018 Received in revised form 17 May 2018 Accepted 20 May 2018 Available online 23 May 2018

Butyrylcholinesterase (BuChE) inhibitors have become interesting target for treatment of Alzheimer's disease (AD). A series of dual binding site BuChE inhibitors were designed and synthesized based on 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties. In-vitro assay revealed that all of the designed compounds were selective and potent BuChE inhibitors. The most potent BuChE inhibitor was compound 6i (IC50 ¼ 0.088 ± 0.0009 mM) with the mixed-type inhibition. Docking study revealed that 6i is a dual binding site BuChE inhibitor. Also, Pharmacokinetic properties for 6i were accurate to Lipinski's rule. In addition, compound 6i demonstrated neuroprotective and b-secretase (BACE1) inhibition activities. This compound could also inhibit AChE-induced and self-induced Ab peptide aggregation at concentration of 100 mM and 10 mM respectively. Generally, the results are presented as new potent selective BuChE inhibitors with a therapeutic potential for the treatment of AD. © 2018 Elsevier Masson SAS. All rights reserved.

Keywords: Alzheimer's disease Butyrylcholinesterase Docking study 2,3,4,9-Tetrahydro-1H-carbazole In-vitro assay

1. Introduction Alzheimer's disease (AD) is the most common form of dementia leading to psychiatric and behavioral disturbances, cognitive dysfunction and difficulties in performing daily activities [1]. Correspondingly, it has shown surge in the overall mortality rate by over 66% [2]. Decreasing Acetylcholine (Ach) in the cortex and hippocampus regions of brain is the most simulated neurotransmitter deficiency in AD [3]. Thus, the most promising approach to alleviate the symptoms of AD is the inhibition of cholinestrase (ChE) [4]. Though in the normal healthy brain, AChE has the main role in the regulation of ACh, It seems that there is a significant

* Corresponding author. E-mail address: [email protected] (M. Amini). https://doi.org/10.1016/j.ejmech.2018.05.031 0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

increase in BuChE activity in the most affected areas of the brain during the progression of AD [5,6]. According to a recent study, selective BuChE inhibitors increase brain ACh and enhance learning in rodents [7]. Moreover, selective BuChE inhibitors might not have common side-effects as AChE inhibitors such as classical cholinergic toxicity [8]. Thus, in recent years, BuChE inhibitors have become more interesting targets for the treatment of AD [3,6,7,9,10]. Although both ChE have almost similar anionic site and oxyanion hole in their active sites, spatial capacity of the Acyl pocket is the most prominent difference between the structures of AChE and BuChE. The replacement of Phe288 and Phe290 residues (in AChE) with Leu286 and Val288 (in BuChE) enables BuChE to hydrolyze bulkier substrates [6,11]. Selective BuChE inhibitors usually contain tri- or polycyclic structures (N1-phenetylnorcymserine and ethopropazine)

50

R. Ghobadian et al. / European Journal of Medicinal Chemistry 155 (2018) 49e60

[5,7,11e13]. Especially, indole containing tricycle derivatives based on Latrepirdine have recently shown selective BuChE inhibition [12,14,15]. All in all, to obtain more information on BuChE's selectivity of indole containing tricycle derivatives, we have designed and synthesized a novel series of 2,3,4,9-tetrahydro-1H-carbazole containing derivatives. On the other hand, the design of dual binding site ChE inhibitors seems to be a promising strategy to increase inhibitory activity through the enhancement of many drug-targeted interactions [16e20]. Recently, benzyl pyridine derivatives have been designed and synthesized, according to the donepezil structure, to have the cholinesterase inhibitory effect [21e24]. Therefore, in this study, a series of dual binding site BuChE inhibitors were designed and synthesized according to 2,3,4,9tetrahydro-1H-carbazole attached benzyl pyridine moieties. Moreover, in-vitro and MTT assay, study of b-secretase inhibition activity, pharmacokinetic and docking studies were performed (see Scheme 1). 2. Results and discussion 2.1. Chemistry According to Scheme 2, compound 1 was prepared as the literature reported [25]. Synthesis of 3a/b was carried out in a two phasic condition using TBAB as phase transfer catalyst agent. Purification of the product was carried out using column chromatography method. Condensation of 3a (or 3 b) with different benzyl halides (4a-j) gave the final compounds 5 (or 6)-a-j in reflux condition using dry acetone as solvent. 2.2. AChE and BuChE inhibitory activity The in-vitro IC50 values of the all derivatives 5a-j and 6a-j were determined according to the Ellman's method and compared with Donepezil as the reference drug (Table 1) [24,27]. All derivatives have much more potent anti-BuChE activities than anti-AChE activities (BuChE IC50: 0.088e2.1 mM, AChE IC50: 4.8e77.1 mM). So, according to the BuChE selectivity indexes of derivatives (SIBuChE: IC50 of AChE/IC50 of BuChE), all the designed compounds were

Scheme. 1. A): The structure of some recently reported AChE inhibitors B): new designed compounds 5a-j and 6a-j [15,17,22].

selective for BuChE's inhibition. During AD's progression, the loss of AChE-activity is compensated by unaffected BuChE [28,29]. Thus, the above result might have a therapeutic advantage for the treatment of AD. Among all the derivatives, the most potent BuChE inhibitor was compound 6i (IC50 ¼ 0.088 ± 0.0009 mM) with great BuChE selectivity (SIBuChE ¼ 180.68). According to Table 1, antiBuChE activities of eight of the synthesized derivatives (5a, b, e and 6a-c, h, i) were more potent than Donepezil (BuChE IC50 of donepezil: 0.35 ± 0.02 mM). Particularly, BuChE IC50 of 6i was 3.97 times less than that of donepezil. It is understood that derivatives having 3-(Chloromethyl) pyridine moiety in comparison to those having 4-(Chloromethyl) pyridine, show lower anti-AChE activity and higher anti-BuChE activity (for AChE Ic50 6a-6j > Ic50 5a-5j and for BuChE Ic50 5a-5j > Ic50 6a6j). Thus, the previously mentioned replacement led to a great enhancement of SIBuChE. It seems that both anti-AChE and antiBuChE activities can be changed by varying substituents on the benzyl group connected to 3 or 4-(Chloromethyl) pyridine moieties (replacement of benzyl bromide with different chlorine, fluorine and methyl benzyl halides). The introduction of fluorine in benzyl moiety diminished both anti-BuChEand anti-AChE activities. So, the fluorine especially in metha position of the benzyl moiety couldn't be tolerated. However, the insertion of chlorine or a methyl group increases both anti-BuChE and anti-AChE activities in all derivatives (except for anti-BuChE activity of 5f). Furthermore, it seems that among derivatives with ortho substitutions, 2-Cl derivatives (5a, 6a) led to the best enhancement of ChE's inhibition (AChE and BuChE IC50 5 d, 5 g, 6 d, 6 g > 5a, 6a). Similarly, among derivatives with para substitutions, 4-methyl derivatives (6i, 5f) showed the most improvement of ChE's inhibition (AChE and BuChE IC50 5c, 5i, 6c, 6f > 6i, 5f). Overall, derivatives having 3-(Chloromethyl) pyridine moiety were much more selective inhibitors of BuChE than those having 4(Chloromethyl) pyridine moiety and also the methyl substituent at para position of benzyl moieties due to its bulkiness was the most favorable group for BuChE's inhibition in derivatives having 3(Chloromethyl) pyridine moiety (6i has the best BuChE IC50 ¼ 0.088 mM). During the last two decades, few AChEIs have been introduced to clinic for treatment of mild and moderate stage of AD, such as tacrine, donepezil, rivastigmine and galanthamine. Limitation of oral bioavailability and other pharmacokinetic parameters of rivastigmine, and hepatoxicity of tacrine led to decrease their application in AD. Important of donepezil has been referred to good potency and low side effects. Two major interaction binding sites with AChE have been suggested in the structure of donepezil, Nbenzyl piperidine and indanone moiety. Consequently the efforts for introducing new AChEIs have been focoused to preparation of the hybrid molecules with keeping the important pharmacophoric groups from two active molecules. A hybrid derivative of donepezil-rivastigmine and donepezil-tacrine showed remarkable activity in inhibition of AChE and BChE [34,35]. Furthermore, some derivatives related to those hybrids showed significant b-amyloid anti-aggregation property [36]. Development of donepezil derivatives conduct ed to introduce ligand AP 2238 in which indanone moiety subunit has been substituted by dimethoxy coumarin, with an IC50 ¼ 44.5 nM for AChE and also a potency better than donepezil in inhibition of beamyloid aggregation [35]. Recently, some potent AChE inhibitor were reported in which, N-benzylpyridinum salt group was introduced into a series of aromatic scaffold with capability interact with catalytic active site (CAS) and peripheral anionic site (PAS). The activities of most potent compounds in each series are presented in Table 2. Comparison between their activity in inhibition of AChE and BChE (Table 2) and the synthesized compounds in the current study


51

Scheme. 2. Synthesis of compounds 5a-j and 6a-j.

Table 1 The IC50 values of the compounds 5a-j and 6a-j against AChE and BuChEa.

Entry

compounds

x

y

z

z’

AChE inhibition [IC50 (mM)]

BChE inhibition [IC50 (mM)]

SI BChE (IC50 AChE/IC50 BuChE)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j Donepezil

Cl Br Cl Br Cl Cl Cl Br Cl Br Cl Cl Cl Cl Cl Cl Cl Cl Cl Br e

Cl H H Me H H F H H H Cl H H F H H Me H H H e

H Cl H H Me H H F H H H Cl H H F H H Me H H e

H H Cl H H Me H H F H H H Cl H H F H H Me H e

6.5 ± 0.10 4.8 ± 0.10 25.6 ± 1.40 8.4 ± 0.20 5.1 ± 0.20 14 ± 0.90 30 ± 0.40 34.30 ± 0.60 32.8 ± 0.20 30.05 ± 0.60 36.1 ± 0.40 30.04 ± 0.007 27.3 ± 0.80 67.69 ± 0.012 77.1 ± 1.50 71.75 ± 0.009 47 ± 1.20 16.8 ± 0.80 15.90 ± 0.10 48.1 ± 0.50 0.023 ± 0.01

0.29 ± 0.009 0.26 ± 0.015 2.1 ± 0.044 0.40 ± 0.004 0.24 ± 0.005 0.7 ± 0.08 0.63 ± 0.003 0.98 ± 0.010 0.73 ± 0.012 0.48 ± 0.006 0.20 ± 0.004 0.25 ± 0.001 0.22 ± 0.013 0.51 ± 0.4 0.56 ± 0.028 0.52 ± 0.015 0.38 ± 0.023 0.11 ± 0.001 0.088 ± 0.0009 0.38 ± 0.007 0.35 ± 0.02

22.41 18.46 12.19 21 21.25 20 47.61 35 44.93 62.60 180.50 120.16 124.09 132.72 137.68 137.98 123.68 152.73 180.68 126.31 0.065

a

Data are expressed as Mean ± SE (three independent experiments).

52


Table 2 The biological data for reported compounds, benzylpyridinum derivative.

Ar

R1

R2, R3

AChE (IC50, mM)

BChE (IC50, mM)

SI

Ref

e

R2 ¼ H, R3 ¼ 3-F

7.19

45.61

6.34

[37]

e

R2 ¼ H, R3 ¼ 2-F

0.77

8.71

11.31

[37]

e

R2 ¼ H, R3 ¼ 2-Cl

0.0004

1370

3113

[17]

e

R2 ¼ 3-Me, R3 ¼ 3-Me

0.004

e

e

[21]

6-Br

R2 ¼ H, R3 ¼ 2-F

0.00011

0.489

4445

[38]

e

R2 ¼ H, R3 ¼ 3-F

10.14

12.16

0.83

[39]

e

R2 ¼ H, R3 ¼ 4-F

0.008

2.07

232

[40]

e

R2 ¼ H, R3 ¼ 4-F

0.038

1.84

48.4

[19]

clearly shows that potency of compounds 5, 6 (a-j) in inhibition of AChE is reduced. A significant increasing in activity of our compound and SI could be observed in inhibition of BChE. Apparently, substitution of tetrahydrocarbazole group as an aryl nonpolar ring in N-benzylpyridinum analogous changes the activity in favor of BChE inhibition and increases the selectivity index. However, there are some reports that emphasize in the positive role of BChE inhibitors in AD [3,6,7,9,10], the usefulness of BChE inhibition in AD needs more studies. Our findings could be considered in design of new compounds and have been used for regulation of balance between AChE and BChE inhibition activity.

concentrations of the chosen derivative (0, 0.043, 0.087 and 0.174 mM) and butrylcholine iodide (0.13, 0.32 and 0.69 mM) were used as an inhibitor and a substrate respectively. According to Fig. 1, it was comprehended that a mixed-type inhibition could be attributed to the compound 6i. Binding of compound 6i to BuChE changed both Vmax and Km values, a trend that is generally attributed to a mixed-type inhibition. Thus, in accordance with the docking studies, a dual binding site interaction with the receptor is expected. Furthermore, the inhibition constant Ki for compound 6i was calculated (Ki ¼ 0.09 mM) using a plot of slope versus the inhibitor's concentration (Fig. 2).

2.3. Kinetic studies of AChE and BuChE inhibition 2.4. Docking studies To investigate the mechanism of the BuChE's inhibition, the most potent derivative (6i, BuChE IC50 ¼ 0.088 mM) was chosen and Lineweaver-Burk curves were outlined (Fig. 1) [21]. Three different

Due to dynamic movement of some resides in the active site of cholinesterase that is crucial for accommodation of various ligands


53

Fig. 1. Kinetic study of compound 6i on the inhibition mechanism of BuChE by Lineweaver-Burk plot (1/v) sec. DA1 vs. (1/[s]) mM1. Fig. 3. The change of total potential energy of the complex system during 10 ns simulation.

Fig. 4. The calculated RMSD of the complex system during 10 ns simulation. Fig. 2. The plot of the slope versus the concentration of inhibitor 6i (mM) for calculating Ki.

in the active site; the molecular dynamics study has been done. To estimate stability of the trajectories, their total energy was calculated. An illustration of total system energy changes during the evolution of trajectories (Fig. 3) clearly displays the stability of the system. The molecular plasticity of the ligand-enzyme complex was estimated based on RMSD calculations. The RMSD of the complex system was shown in Fig. 4. According to the results the complex system was equilibrated around 3 ns. The RMSD remained constant until 10 ns. Finally the best frame of the trajectories was selected and further analyzed to finding the key interactions. The ligandenzyme schematic interactions were shown in Fig. 5. The plain structure of carbazole moiety contributes for making a p-p staking with Tyr332. The central pyridinium ring was attracted by the negative charge (made by Trp82 and Glu197) that deposited at the bottom of the active site. Moreover the extra charge transfer binding with Trp82 may be beneficial for better interaction. The rest of the molecule was fitted in the hydrophobic cavity made by Ile62, Pro84, Thr120 and Gly 121.

2.5. Screening of physicochemical properties Some of physicochemical properties of compound 6i including the polar surface area (tPSA), octanole/water partition coefficients (Clog P), a number of H-bond acceptors (HBA), a number of H-bond donors (HBD) and a number of rotatable bonds (RBC) were calculated and shown in Table 3. It is obvious that mentioned properties of 6i were confirmed by Lipinski rules of 5 [30]. Thus, 6i tends to have lower attrition rates during clinical trials and hence has an increased chance of reaching the market. 2.6. Neuroprotective effect against H2O2-induced cell death in PC12 neurons The MTT assay is a useful model for assessing neuronal differentiation and has been widely adopted. Hence, neuroprotective activity for the most potent compound (6i) against BuChE was determined by in-vitro MTT assay. The data is expressed as followed ± SEM (n ¼ 8) and the one-way analysis of the variance (ANOVA) continued by Newman-Keuls multiple comparisons test were carried out to determine the level of significance. According to Fig. 4, compound 6i demonstrated good neuroprotective activity at 10 mM (cell viability ¼ 65 and P < 0.01 vs. H2O2) (see Fig. 6).

54


Fig. 5. The binding pattern of the ligand (6i) in the active site of the enzyme. For simplification only the key residues were shown.

experiments were repeated for three times and compared with OM99-2 as the reference compound. According to Table 4 compound 6i possesses moderate inhibition against BACE1 at the concentration of 50 mM. The non-peptide structure of compound 6i is desired and acts as dual inhibitor of BuChE and BACE1. 2.8. Inhibition of AChE-induced and self-induced Ab aggregation

Fig. 6. Neuroprotective activity of compound 6i based on cell viability of PC12 cells in H2O2-induced damage. Data is expressed as followed ± SEM (n ¼ 8) and ANOVA proceeded by Newman-Keuls multiple comparisons test was carried out to determine the level of significance. ****: P < 0.0001 and **: P < 0.01 versus H2O2.

Compound 6i as a potent AChE inhibitor was selected to assess its ability to inhibit Ab1-42 peptide self-aggregation employing the thioflavin T (ThT) fluorescence method and compared with donepezil as reference compound. The obtained inhibition for compound 8f at 10 mM concentration was 55.7 ± 2.9% being higher than that of donepezil (16.4 ± 1.7%) and Tacrine (7.7 ± 1.5%). To further explore the dual action of this compound, the capacity to inhibit the AChE-induced Ab1-40 peptide aggregation was examined employing the same ThT-based fluorometric assay. Compound 6i exhibited a significant AChE-induced Ab aggregation inhibitory effect with percentage of inhibition 51.8 ± 1.5 more effective than Tacrine and donepezil (7.2 ± 1.2% and 25.2 ± 1.1, respectively), indicating that compound 8i binds to PAS of AChE. 3. Conclusion

Table 3 Molecular descriptorsa of compound 6i. Entry

Compound

HBD

HBA

ClogP

tPSA [Å2]

MW

RBC

19

6i

0

2

3.024

6.25

402.97

4

HBA: H-bond acceptors. Clog P: Calculated octanol-water partition coefficient. tPSA: topological polar surface area. RBC: Rotatable bond count. a HBD: H-bond donors.

2.7. BACE1 enzyme inhibitory activity of compound 6i BACE-1 inhibitory activity of the most potent BuChE inhibitor (6i) was evaluated via a fluorescence resonance energy transfer (FRET) based BACE-1 kit including BACE-1 enzyme and specific APP based peptide substrate (Rh-EVNLDAEFK-quencher). Also

In summary, a new series of potent selective BuChE inhibitors were designed. All of the compounds have shown selective inhibition of BuChE. They might have a therapeutic advantage for the treatment of AD as during AD's progression the loss of AChEactivity is compensated by unaffected BuChE. Also, eight of them had lower BuChE IC50 values than donepezil as the reference drug. In-vitro assay revealed that compound 6i indicated the most potent

Table 4 BACE1 inhibitory activitya of compound 6i. Compound

Inhibitionb at 50 mM (%)

Inhibitionb at 10 mM (%)

6i

30 ± 0.1

10 ± 0.2

a

The IC50 of standard drug OM99-2 was 3 nM. Values represent means ± standard error (S.E.) experiments. b

of

three

independent


anti-BuChE activity (IC50 ¼ 0.088 ± 0.0009 mM). Moreover, compound 6i was found to be an inhibitor of AChE-induced Ab aggregation, which can also interfere with Ab self-aggregation. It was understood from molecular modeling studies that compound 6i had important interactions with choline's binding site, peripheral binding site and catalytic site of BuChE. Furthermore, it was long enough to interact with the acyl pocket. Also, compound 6i demonstrated neuroprotective and BACE1 inhibition activities. Additionally, the pharmacokinetic properties of 6i were confirmed by Lipinski rules of 5. Generally, our study has presented new potent selective inhibitors of BuChE with a therapeutic potential for the treatment of AD. 4. Experimental 4.1. General chemistry All reagents were purchased from Sigma Aldrich, Merck and Fluka and were used as provided. For all the derivatives, 1H and 13C nuclear magnetic resonance (NMR) spectra's were recorded on a Bruker FT-500, using TMS as an internal standard and the IR spectra's were recorded with KBr disks using the Nicolet Magna FTIR 550 spectrophotometer. Furthermore, all of melting points were taken on a Kofler hot stage apparatus.

55

4.1.5. 9-(Pyridin-4-ylmethyl)-2,3,4,9-tetrahydro-1H-carbazole (2a) Yellow solid; yield: 58%, m. p. ¼ 215e217 C. IR (KBr): 2996, 2842, 1675, 1608, 1465 cm1, 1H NMR (CDCl3, 500 MHz): d ¼ 1.86e1.93 (m, 4H, tetrahydrocarbazole-CH2), 2.59 (t, J ¼ 6.5 Hz, 2H, H4), 2.76 (t, J ¼ 6 Hz, 2H, H1), 5.23 (s, 2H, N-CH2), 6.86 (d, J ¼ 5.5 Hz 2H, H3՛,5՛), 7.11 (m, 3H, H6,7,8), 7.24 (d, J ¼ 7.0 Hz, 1H, H5), 8.48 (d, J ¼ 5.5 Hz, 2H, H2՛,6՛) ppm. 4.1.6. 9-(Pyridin-3-ylmethyl)-2,3,4,9-tetrahydro-1H-carbazole (2b) Yellow solid; yield: 60%, m. p. ¼ 208e210 C. IR (KBr): 2980, 2845, 1678, 1600, 1475 cm1. 1H NMR (CDCl3, 500 MHz): d ¼ 1.77e1.83 (m, 4H, tetrahydrocarbazole-CH2). 2.64 (m, 2H, H4), 3.35 (m, 2H, H1), 5.34 (s, 2H, N- CH2), 6.99 (t, J ¼ 7.0 Hz, 1H, H5), 7.04 (t, J ¼ 7.0 Hz, 1H, H8), 7.26 (d, J ¼ 7.0 Hz, 1H, H6), 7.35 (d, J ¼ 8.0 Hz, 1H, H7), 7.39e7.41 (m, 2H, H4՛, 5՛), 8.3 (s, 1H, H2՛), 8.43 (d, J ¼ 6.0 Hz, 1H, H6՛) ppm. 13C- NMR (CDCl3, 500 MHz): d ¼ 20.6, 21.6 (C4), 22.6 (C1), 22.7 (C2), 43.1(C3), 109.1 (NCH2), 109.3 (NCH), 117.4 (CH), 118.6 (C8), 120.6 (C6՛), 123.6 (C5), 127.0 (C6), 134.2(C7), 135.3 (C4՛), 136.0 (tetrahydrocarbazole-C), 147.9 (C5՛), 148.0 (tetrahydrocarbazole -CN), 148.3(C3՛) ppm.

4.1.1. Synthesis of 2,3,4,9-tetrahydro-1H-carbazole (1) Synthesis of 2,3,4,9-tetrahydro-1H-carbazol (1) was prepared according to the published procedure [25]. To a mixture of cyclohexanone (10 mmol), acetic acid (60 mmol) and phenyl hydrazine (10 mmol) were added and reflux for 1 h. The progress of reaction was check by TLC. The mixture was kept in room temperature overnight and then white solid crude was filtered and washed with water and ethanol (75%) respectively. The solid material was crystalized in methanol to yield compound 1.

4.1.7. 1-(2-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (5a) Yellow solid; yield: 75%, m. p. ¼ 225e226 C. IR (KBr): 2986, 2852, 1678, 1464, 750 cm1 .1H NMR (DMSO, 500 MHz): d ¼ 1.81e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.60e2.61 (m, 2H, H4),2.66e2.68 (m, 2H, H1), 5.73 (s, 2H, N- CH2), 5.93 (s, 2H, NþCH2), 7.03e7.04 (m, 2H, H6,7), 7.31 (d, J ¼ 7 Hz, 1H, H8), 7.45e7.50 (m, 4H, H5, 4}, 5}, 6}), 7.56e7.59 (m, 3H, H3՛,5՛, 3}), 8.97 (d, J ¼ 5 Hz, 2H, H2՛,6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (C4), 21.2 (C2), 22.5 (C3), 44.8 (CH2-N), 60.6 (CH2-Nþ), 109.1 (tetrahydrocarbazole-C), 109.9 (C8), 117.7 (C5), 119.2 (C6), 120.9 (C7), 125.3 (C5}), 127.2 (C4}), 128.1 (C3}), 130.0 (C4‫׳‬, C5131.2 ,(‫( ׳‬C2}), 131.5 (C-N), 131.7 (C-N), 144.9 (C1}), 145.1 (C2‫׳‬, C6159.3 ,(‫( ׳‬C4‫ )׳‬ppm.

4.1.2. Synthesis of 9-(2-(pyridin-4-yl) ethyl)-2,3,4,9-tetrahydro-1Hcarbazole and 9-(2-(pyridin-3-yl)ethyl)-2,3,4,9-tetrahydro-1Hcarbazole (3a or 3 b) To solution of 2.73 mmol tetrabuthyl ammonium bromide (TBAB) in 15 mL of sodium hydroxide solution (50%) was added toluene (15 mL) and stirred for 15 min. Five mmol of compound 1 in toluene (15 mL) and 16.3 mmol of compound 2 (a or b) were added and stirred at room temperature for 12 h The solvent was removed under reduced pressure. To the residue 25 mL of water and chloroform (50 mL) were added and mixed. The organic layer was separated, dried over anhydrous sodium sulphate and filtered. The solvent was removed under reduced pressure and the residue was purified on a silica short column with using a mixture of petroleum ether: ETOAc (9:1) to give a yellow solid of 3a (or 3 b) [26].

4.1.8. 1-(3-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium bromide (5b) Cream solid; yield: 76%, m. p. ¼ 225e227 C. IR (KBr): 2925, 2852, 1678, 1642, 1464 cm1.1H NMR (DMSO), 500 MHz): d ¼ 180e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.59 (t, J ¼ 6.0 Hz, 2H, H4), 2.67 (t, J ¼ 6.0 Hz, 2H, H1), 5.73 (s, 2H, N- CH2), 5.90 (s, 2H, Nþ- CH2), 7.02e7.03 (m, 2H, H6,7), 7.30 (d, J ¼ 7.5 Hz, 1H, H8), 7.43e7.47 (m, 3H, H5, 5՛՛,6՛՛), 7.54 (d, J ¼ 7 Hz, 1H, H4՛՛), 7.59 (d, J ¼ 6.5 Hz, 2H, H3՛,5՛), 7.71 (s, 1H, H2՛՛), 9.18 (d, J ¼ 7 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): 20.5 (C4), 21.2 (C2), 22.5 (C3), 44.8 (CH2-N), 61.5 (CH2-Nþ), 109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.6 (C5), 119.1 (C6), 120.8 (C7), 125.3 (C4}), 127.2 (C6}), 127.7 (tetrahydrocarbazole-C), 128.9 (C2}), 129.3 (C3‫׳‬, C5131.0 ,(‫( ׳‬C3}), 133.5 (C-N), 135.3C1}), 136.2(C-N), 144.7(C2}, C6}), 159.0 (C4‫ )׳‬ppm.

4.1.3. General procedure for the synthesis of compounds 5a-j and 6a-j To a solution 1 mmol of 3a (or 3 b) in dry acetone (12 mL) was added 1.5 mmol of different benzyl halides (4a-j) and reflux for 1e6 h, checked by TLC. The solid material was filtered and washed with a mixture of petroleum ether/ETOAc (4:1) to give 5a-j and 6a-j. 4.1.4. 2,3,4,9-Tetrahydro-1H-carbazole (1) White solid; yield: 70% m. p. ¼ 132 C. IR (KBr): 3400, 3025, 2852, 1670, 1603, 1475,.1H NMR (CDCl3, 500 MHz): d ¼ 1.85e1.92 (m, 4H, tetrahydro-1Hcarbazole-CH2), 2.69e2.72 (m, 4H, tetrahydrocarbazole-CH2), 7.05e7.09 (t, 2H, H6, 7), 7.11 (d, J ¼ 7 Hz, 1H, H8), 7.24 (d, J ¼ 8 Hz, 1H, H5), 7.44 (s, 1H, NH) ppm.

4.1.9. 1-(4-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (5c) Cream solid; yield: 77%, m. p. ¼ 225e227 C. IR (KBr): 2928, 2852, 1678, 1643, 1463, 747 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.76e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.50e2.59 (m, 2H, H4), 2.66e2.68 (m, 2H, H1), 5.71 (s, 2H, N- CH2), 5.81 (s, 2H, NþCH2), 7.01e7.03 (m, 2H, H6,7), 7.28e7.30 (m, 1H, H8), 7.43e7.45 (m, 1H, H5), 7.51e7.55 (m, 4H, H 2՛՛,3՛՛,5՛՛,6՛՛), 7.56 (d, J ¼ 6.5 Hz, 2H, H3՛,5՛), 9.05 (d, J ¼ 7 Hz, 2H, H2՛,6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (C4), 21.2 (C1), 22.5 (C2), 44.7 (C3), 61.7 (CH2-N), 109.1 (CH2Nþ), 109.8 (tetrahydrocarbazole-C), 117.7 (C8), 119.2(C5), 120.9 (C6), 125.3 (C7), 127.2 (tetrahydrocarbazole-C), 129.1 (C3‫׳‬, C5130.9 ,(‫( ׳‬C3}, C5}), 132.9 (C2}, C6}), 134.2(C4}), 135.2 (C1}), 135.9 (C-N), 144.6 (C-N), 144.8(C2‫׳‬, C6159.0 ,(‫( ׳‬C 4‫ )׳‬ppm.

56


4.1.10. 1-(2-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium bromide (5d) Yellow solid; yield: 75%, m. p. ¼ 205e206 C. IR (KBr): 3035, 2925, 2852, 1638, 1463, 1424, 1373, 744 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.81 (t, J ¼ 6.0 Hz, 4H, tetrahydrocarbazole-CH2), 2.27 (s, 3H, CH3), 2.62 (t, J ¼ 6.0 Hz, 2H, H4), 2.68 (t, J ¼ 6.0 Hz, 2H, H1), 5.75 (2H, s, N- CH2), 5.91 (s, 2H, Nþ- CH2), 7.01e7.06 (m, 2H, H6,7), 7.19 (d, J ¼ 7.5 Hz, 1H, H3՛՛), 7.25 (t, J ¼ 7 Hz, H5՛՛), 7.27 (d, J ¼ 7 Hz, H6՛՛), 7.31e7.32 (m, 2H, H8,4՛՛),7.44 (d, J ¼ 7 Hz, 1H, H5), 7.56e7.59 (d, J ¼ 6 Hz, 2H, H3՛,5՛), 8.95 (d, J ¼ 5 Hz, 2H, H2՛,6՛) ppm.13CNMR (DMSO, 500 MHz): d ¼ 18.7 (CH3), 20.6 (C4), 21.2 (C1), 22.5 (C2), 44.8 (C3), 60.8 (CH2-N), 109.1 (CH2-Nþ), 109.8 (tetrahydrocarbazole-C), 117.6 (C8), 119.1 (C5), 120.9 (C6), 125.3 (C7), 126.6 (C4}, C5}), 127.2 (tetrahydrocarbazole-C), 129.4 (C3‫׳‬, C5130.8 ,(‫( ׳‬C6}), 132.0 (C3}), 135.3 (C-N), 135.9 (C2}), 137.0 (C-N), 144.6 (C1}), 144.8(C2‫׳‬, C6158.9 ,(‫( ׳‬C4‫ )׳‬ppm. 4.1.11. 1-(3-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium chloride (5e) Yellow solid; yield: 77%, m. p. ¼ 199e200 C. IR (KBr): 2925, 2855, 1643, 1463, 1371 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.80e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.29 (s, 3H, CH3), 2.58e2.60 (m, 2H, H4), 2.65e2.68 (m, 2H, H1), 5.70 (2H, s, N- CH2), 5.75 (2H, s, Nþ- CH2), 7.01e7.03 (m, 2H, H6,7), 7.22 (d, J ¼ 7 Hz, 1H, H4՛՛), 7.28e7.29 (m, 2H, H8,2՛՛), 7.32e7.33 (m, 2H, H5՛՛,6՛՛), 7.44 (d, J ¼ 7 Hz, 1H, H5), 7.55 (d, J ¼ 5.5 Hz, 2H, H3՛,5՛), 9.05 (d, J ¼ 5.5 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (C4), 20.8 (CH3), 21.2 (C1), 22.6 (C2), 44.8 (C3), 61.5 (N- CH2), 109.1 (Nþ- CH2), 109.9 (tetrahydrocarbazole-C), 117.6 (C8), 119.1(C5), 120.8 (C6), 125.4 (C7), 127.2 (C4}, C6}), 127.7, 128.9 (C3՛, C5՛), 129.4(C2}), 131.0 (C1}), 134.0 (C-N), 135.4 (C-N), 136.2(C3}), 144.8 (C2՛,C6՛), 158.9 (C4՛), ppm. 4.1.12. 1-(4-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium chloride (5f) Yellow solid; yield: 78%, m. p. ¼ 195e196 C. IR (KBr): 3027, 2928, 2850, 1638, 1615, 1464, 749 cm-1 1H NMR (DMSO, 500 MHz): d ¼ 1.81e1.84 (m, 4H, tetracarbazole-CH2), 2.29 (s, 3H, CH3), 258e2.59 (m, 2H, H4), 2.66e2.68 (m, 2H, H1), 5.70 (s, 2H, N- CH2), 5.75 (s, 2H, Nþ- CH2), 7.03 (t, J ¼ 7.5 Hz, 2H, H6,7), 7.23 (d, J ¼ 8 Hz, 2H, H3՛՛,5՛՛), 7.29 (d, J ¼ 8 Hz, 1H, H8), 7.40 (d, J ¼ 8 Hz, 2H, H2՛՛,6՛՛), 7.45 (d, J ¼ 7 Hz, 1H, H5), 7.55 (d, J ¼ 6 Hz, 2H, H3՛,5՛), 9.06 (d, J ¼ 6 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (C4), 20.7(C1), 21.2 (C2), 22.5 (C3), 44.7(N- CH2), 62.5 (Nþ- CH2), 109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.7 (C5), 119.2 (C6), 120.9 (C7), 125.3 (tetrahydrocarbazole-C), 128.9 (C2}, C3}, C5}, C6}), 129.7 (C3‫׳‬, C5131.1 ,(‫׳‬ (C1}), 135.2 (C4}), 138.9 (C-CH3), 144.4 (C-N), 144.6 (C2‫׳‬, C6158.8 ,(‫( ׳‬C4‫)׳‬ ppm. 4.1.13. 1-(2-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (5g) Yellow solid; yield: 73%, m. p. ¼ 231e232 C. IR (KBr): 3039, 2925, 2854, 1641, 1463, 759 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.81e1.84 (m, 4H, tetracarbazole-CH2), 2.59e2.61 (m, 2H, H4), 2.66e2.68 (m, 2H, CH1), 5.72 (s, 2H, N- CH2), 5.90 (s, 2H, Nþ- CH2), 7.01e7.03 (m, 2H, H6,7), 7.29e7.31 (m, 3H, H8, 4՛՛,6՛՛), 7.44e7.45 (m, 1H, H5), 7.50e7.51 (m, 1H, H5՛՛), 7.58e7.59 (m, 3H, H3՛,5՛,3՛՛), 8.97 (d, J ¼ 5 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole CH2), 21.2 (tetrahydrocarbazole CH2), 22.5 (tetrahydrocarbazole CH2, tetrahydrocarbazole CH2), 44.8 (N- CH2), 57.3 (Nþ- CH2)), 109.1 (tetrahydrocarbazole-C), 109.9 (C8), 116.0 (JCF ¼ 7.5 Hz, C3}), 117.7 (C5), 119.2 (C6), 120.9 (C7), 125.23 (JC-F ¼ 15.5 Hz, C5}), 125.3 (C1}), 127.3 (C4}), 127.6 (tetrahydrocarbazole-C), 128.9 (C3՛, C5՛), 131.54 (C6}), 132.13 (C-N), 137.2 (C-N), 145.0 (C2՛, C6՛), 159.2 (C4՛), 162.1 (JC-F ¼ 223.75 Hz, C2}) ppm.

4.1.14. 1-(3-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium bromide (5h) Yellow solid; yield: 74%, m. p. ¼ 231e232 C. IR (KBr): 2924, 2854, 1740, 1643, 1463, 1151, 838, 750 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.81e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.60e2.62 (m, 2H, H4), 2.67e2.68 (m, 2H, H1), 5.71 (s, 2H, N- CH2), 5.82 (s, 2H, Nþ- CH2), 7.01e7.03 (m, 2H, H6,7), 7.28e7.32 (m, 3H, H8, 5՛՛,6՛՛), 7.37 (d, J ¼ 7.5 Hz, 1H, H5) 7.45e7.49 (m, 3H, H2՛՛,5), 7.50e7.51 (m, 1H, H4՛՛), 7.57 (d, J ¼ 6.0 Hz, 2H, H3՛,5՛), 9.09 (d, J ¼ 6.0 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.7 (tetrahydrocarbazole-CH2), 20.8 (tetrahydrocarbazole-CH2), 21.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 44.8 (N- CH2), 57.5 (Nþ- CH2), 109.2 (tetrahydrocarbazole-C), 110.0 (C8), 116.1 (C4՛՛), 117.7 (C2՛՛), 119.2 (C5), 120.9 (C6), 125.2 (C7), 125.3 (C6՛՛), 127.6 (tetrahydrocarbazole-C), 128.9 (C3‫׳‬, C5130.2 ,(‫( ׳‬C5՛՛),135. 4 (C-N), 136.0 (C1՛՛), 137.2 (C-N), 146.5 (C2‫׳‬, C6159.6 ,(‫( ׳‬C4162.8 ,(‫( ׳‬JC-F ¼ 257.5 Hz) ppm. 4.1.15. 1-(4-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (5i) Pink solid; yield: 75%, m. p. ¼ 231e232 C. IR (KBr): 2925, 2855, 1642, 1464, 839 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.81e1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.59e2.60 (m, 2H, CH4), 2.66e2.67 (m, 2H, CH1), 5.70 (s, 2H, N- CH2), 5.79 (s, 2H, Nþ- CH2), 7.03 (t, J ¼ 7.5 Hz, 2H, H6,7), 7.26e7.30 (m, 3H, H8, 2՛՛,6՛՛), 7.44 (d, J ¼ 8.5 Hz,1H, H5), 7.56 (d, J ¼ 6.5 Hz,2H, H3՛,5՛), 7.61 (dd, J ¼ 5.5 Hz, J ¼ 3.0 Hz, 2H, H3՛՛,5՛՛), 9.07 (d, J ¼ 5 Hz, 2H, H2՛,6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.2 (tetrahydrocarbazoleCH2), 22.5 (tetrahydrocarbazole-CH2, tetrahydrocarbazole-CH2), 44.7 (N- CH2), 61.8 (Nþ- CH2), 109.1 (tetrahydrocarbazole-C), 116.0 (C8), 116.1 (C3}, C5}), 117.7 (C5), 119.3 (C6), 121.0 (C7), 125.3 (tetrahydrocarbazole-C), 130.2 (C3՛, C5՛), 131.6 (C1}), 135.3 (C2}, C6}), 135.9 (C-N), 144.9 (C-N), 148.0 (C2՛, C6՛), 159.2 (C3՛), 161.0 (JC-F ¼ 223.75 Hz) ppm. 4.1.16. 1-Benzyl-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl) pyridin-1-ium bromide (5j) Orange solid; yield: 77%, m. p. ¼ 231e232 C. IR (KBr): 3027, 2925, 2850, 1637, 1462 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.80e1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.60e2.61 (m, 2H, H4), 2.66e2.67 (m, 2H, H1), 5.77 (s, 2H, N- CH2), 5.92 (s, 2H, NþCH2), 7.03 (t, J ¼ 7.0 Hz, 2H, H6,7), 7.34 (d, J ¼ 8.0 Hz, 1H, H8), 7.42e7.45 (m, 4H, H5,2՛՛,4՛՛,6՛՛), 7.57e7.60 (m, 4H, H3՛՛,5՛՛, H3՛,5՛), 9.25 (d, J ¼ 5 Hz, 2H, H2՛,6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.5 (tetrahydrocarbazole-CH2), 21.2 (tetrahydrocarbazole-CH2), 22.3 (tetrahydrocarbazole-CH2, tetrahydrocarbazole-CH2), 44.8 (N-CH2), 62.2 (NþeCH2), 109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.6 (C5), 119.1(C6), 120.8 (C7), 125.2 (C4}), 127.1 (tetrahydrocarbazole-C), 128.9(C2}, C6}), 129.1 (C3}, C5}), 134.1(C1}), 135.3 (C-N), 135.9 (C-N), 144.5(C3‫׳‬, C5144.6 ,(‫( ׳‬C2‫׳‬, C6158.9 ,(‫( ׳‬C4‫ )׳‬ppm. 4.1.17. 1-(2-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6a) Yellow solid; yield: 76%, m. p. ¼ 209e210 C. IR (KBr): 2924, 2853, 1632, 1462, 751 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.82 (m, 4H, tetrahydrocarbazole-CH2), 2.50e2.63 (m, 4H, tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.97 (s, 2H, NþCH2), 7.01e7.02 (m, 2H, H6,7), 7.35e7.52 (m, 6H, H5,8,2՛՛,4՛՛,5՛՛,6՛՛), 8.13 (m, 1H, H5՛), 8.20 (m, 1H, H4՛), 8.62 (s, 1H, H2՛), 9.07 (d, J ¼ 5 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 61.3 (Nþ- CH2), 109.0 (tetrahydrocarbazole-C), 109.8 (C8), 117.5 (C5), 117.8 (C6), 127.2 (C7), 128.2 (C5129.9 ,(‫( ׳‬C5}), 130.9 (C4}), 131.7 (tetrahydrocarbazole-C), 131.9 (C3}), 135.1 (C6}), 135.7 (C3139.9 ,(‫( ׳‬C6‫)׳‬, 140 (C2}), 142.5 (C-N), 144.1 (C-N), 145 (C2}), 150 (C2153 ,(‫( ׳‬C4‫ )׳‬ppm.


4.1.18. 1-(3-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6b) Yellow solid; yield: 77%, m. p. ¼ 209e210 C. IR (KBr): 2925, 2854, 1630, 1465, 751, 713, 676 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.86 (m, 4H,tetrahydrocarbazole-CH2), 2.08 (t, J ¼ 5.5 Hz, 2H, tetrahydrocarbazole-CH2), 2.64 (t, J ¼ 5.5 Hz, 2H, carbazoleCH2), 5.58 (s, 2H, N- CH2), 5.87 (s, 2H, Nþ- CH2), 7.00e7.05 (m, 2H, H6,7), 7.37 (d, J ¼ 7.5 Hz, 1H, H8), 7.41 (d, J ¼ 7.5 Hz, 1H, H6՛՛), 7.44e7.48 (m, 2H, H4՛՛,5՛՛), 7.50 (d, J ¼ 7.5 Hz, 1H, H5), 7.66 (s, 1H, H2՛՛), 8.03 (d, J ¼ 7.5 Hz, 1H, H5՛), 8.08 (t, J ¼ 8.0 Hz, 1H, H4՛), 9.02 (s, 1H, H2՛), 9.17 (d, J ¼ 5 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.8 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazoleCH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazoleCH2), 42.6 (N- CH2), 63.3 (Nþ- CH2), 109.0 (tetrahydrocarbazole-C), 109.3 (C8), 109.8 (C5), 117.5 (C6), 117.75(C7), 119.0 (tetrahydrocarbazole-C),127.2 (C6}), 128.5 (tetrahydrocarbazole-C), 129.1 (C5129.32 ,(‫( ׳‬C5}), 129.8 (C2}), 133.9 (C4}), 135.2 (C-N), 135.8 (C-N), 138.5 (C3139.8 ,(‫( ׳‬C3}), 140.1 (C4142.3 ,(‫(׳‬C6143.57 ,(‫( ׳‬C2‫ )׳‬ppm. 4.1.19. 1-(4-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6c) Yellow solid; yield: 78%, m. p. ¼ 200e201 C. IR (KBr): 3021, 2921, 2851, 1498, 1400, 803 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.84 (m, 4H, tettahydrocarbazole-CH2), 2.61e2.65 (m, 4H, tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.58 (s, 2H, N- CH2), 5.87 (s, 2H, Nþ- CH2), 7.00e7.02 (m, 2H, H6,7), 7.34 (d, J ¼ 7.5 Hz, 1H, H8), 7.41 (d, J ¼ 7 Hz, 1H, H5), 7.49 (d, J ¼ 7.5 Hz, 2H, H2՛՛,6՛՛), 7.52 (d, J ¼ 7.5 Hz, 2H, H3՛՛,5՛՛), 8.03 (d, J ¼ 8 Hz, 1H, H5՛), 8.08 (t, J ¼ 6.5 Hz, 1H, H4՛), 8.92 (s, 1H, H2՛), 9.07 (d, J ¼ 6 Hz, 1H, H6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tettahydrocarbazole-CH2), 21.4 (tettahydrocarbazole-CH2), 22.5 (tettahydrocarbazole-CH2), 22.6 (tettahydrocarbazole-CH2), 42.6 (N-CH2), 62.4 (N-CHþ 2 ), 109.0 (tetrahydrocarbazole-C), 109.3 (tettahydrocarbazole-CH), 109.8 (tettahydrocarbazole-CH), 117.5 (tettahydrocarbazole-CH), 117.8 (tettahydrocarbazole-CH), 119.2 (tetrahydrocarbazole-C), 127.2 (C5՛), 128.3 (C3՛՛,5՛՛), 128.9 (H2՛՛,6՛՛), 129.2 (C4՛՛), 130.8 (C1՛՛), 131.0 (C-N), 132.8 (C-N), 134.2 (C3՛), 135.2 (C4՛), 135.7 (C2՛), 139.9 (C6՛) ppm. 4.1.20. 1-(2-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6d) Yellow solid; yield: 75%, m. p. ¼ 200e201 C. IR (KBr): 2926, 2858, 1645, 1460, 1376 cm1.1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.85 (m, 4H, tetrahydrocarbazole-CH2), 2.60e2.64 (m, 4H, tetrahydrocarbazole-CH2), 5.59 (s, 2H, N- CH2), 5.94 (s, 2H, NþCH2), 7.00e7.03 (m, 2H, H6,7), 7.27e7.30 (m, 2H, H4՛՛,6՛՛), 7.34 (d, J ¼ 8.0 Hz, 1H, H8), 7.41 (d, J ¼ 7.5 Hz, 2H, H5), 7.53e7.56 (m, 2H, H3՛՛,5՛՛), 8.02e8.09 (m, 2H, H5՛,4՛), 8.80 (s, 1H, H2՛), 9.10 (d, J ¼ 6 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 61.3 (Nþ- CH2), 109.0 (tetrahydrocarbazole-C), 109.8 (tetrahydrocarbazole-CH), 117.5 (C3}), 117.8 (tetrahydrocarbazole-CH), 121.0 (C1}), 127.2 (C5}), 128.2 (tetrahydrocarbazole-C), 130.9 (C4}), 131.7 (C5‫)׳‬, 131.9 (C6}), 135.1 (C-N), 135.7 (C-N), 140.0 (C3142.5 ,(‫( ׳‬C4144.1 ,(‫( ׳‬C2‫)׳‬, 145.12 (C6162.5 ,(‫( ׳‬JC-F ¼ 235 Hz, C2}) ppm. 4.1.21. 1-(3-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6e) Yellow solid; yield: 75%, m. p. ¼ 200e201 C. IR (KBr): 2925, 2867, 2837, 2724, 1639, 1457 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.79e1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.64e2.65 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.89 (s, 2H, NþCH2), 7.03 (m, 2H, H6,7), 7.28 (t, J ¼ 8.0 Hz, 2H, H5՛՛), 7.35 (m, 2H, H8, 6՛՛), 7.41 (m, 2H, H5, 2՛՛), 7.48 (d, J ¼ 7.0 Hz, 1H, H4՛՛), 8.02 (d, J ¼ 8 Hz, 1H, H5՛), 8.08 (t, J ¼ 6.5 Hz, 1H, H4՛), 9.04 (s, 1H, H2՛), 9.18 (d, J ¼ 6 Hz,

57

1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 30.6 (N- CH2), 42.6 (Nþ- CH2), 62.4 (tetrahydrocarbazole-C), 109.3 (tetrahydrocarbazole-CH), 110.0 (C4}), 116.0 (C2}), 117.5 (tetrahydrocarbazoleCH), 117.9 (tetrahydrocarbazole-CH), 119.2 (tetrahydrocarbazoleCH), 127.2 (C6}), 128.3 (tetrahydrocarbazole-C), 130.1 (C5131.4 ,(‫( ׳‬C5}), 131.6 (C-N), 135.2 (C1}), 135.8 (C-N), 140.0 (C3142.4 ,(‫( ׳‬C4143.6 ,(‫( ׳‬C2‫)׳‬, 144.0 (C6162.1 ,(‫( ׳‬JC-F ¼ 240 Hz, C3}) ppm. 4.1.22. 1-(4-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl) methyl)pyridin-1-ium chloride (6f) Yellow solid; yield: 77%, m. p. ¼ 200e201 C. IR (KBr): 2930, 2860, 1638, 1456 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.62e2.66 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.87 (s, 2H, Nþ- CH2), 7.02e7.03 (m, 2H, H6,7), 7.27 (t, J ¼ 8.0 Hz, 2H, H2՛՛,6՛՛), 7.35 (d, J ¼ 7.0 Hz, 1H, H8), 7.41 (d, J ¼ 7.5 Hz, 1H, H5), 7.59 (t, J ¼ 8.0 Hz, 1H, H3՛՛,5՛՛), 8.02 (d, J ¼ 8 Hz, 1H, H5՛), 8.08 (t, J ¼ 6.5 Hz, 1H, H4՛), 8.95 (s, 1H, H2՛), 9.17 (d, J ¼ 6 Hz, 1H, H6՛) ppm.13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.5 (tetrahydrocarbazoleCH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazoleCH2), 42.6 (N- CH2), 62.4 (Nþ- CH2), 109.2 (tetrahydrocarbazole-C), 109.8 (tetrahydrocarbazole-CH), 116.0 (tetrahydrocarbazole-CH), 117.5 (tetrahydrocarbazole-CH), 117.8 (JC-F ¼ 25.0 Hz, C3},5}), 119.2 (tetrahydrocarbazole-CH), 127.2 (C5128.4 ,(‫( ׳‬tetrahydrocarbazoleC), 130.1 (C1}), 131.5 (JC-F ¼ 8.75 Hz, C2},6}), 135.2 (C-N), 135.7 (C-N), 139.9 (C3142.4 ,(‫( ׳‬C4143.5 ,(‫( ׳‬C2143.9 ,(‫( ׳‬C6162.5 ,(‫( ׳‬JC-F ¼ 245 Hz, C4}) ppm. 4.1.23. 1-(2-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium chloride (6g) Yellow solid; yield: 75%, m. p. ¼ 200e201 C. IR (KBr): 3067, 3011, 2928, 2848, 1629, 1467, 741 cm-1 1H NMR (DMSO, 500 MHz): d ¼ 1.76e1.81 (m, 4H, tetrahydrocarbazole-CH2), 2.14 (s, 3H, CH3), 2.59e2.63 (m, 4H, tetrahydrocarbazole-CH2), 5.61 (s, 2H, N- CH2), 5.93 (s, 2H, Nþ- CH2), 7.00e7.04 (m, 2H, H6,7), 7.13 (d, J ¼ 7.0 Hz, 1H, H3՛՛), 7.21e7.24 (m, 2H, H5՛՛,6՛՛), 7.32e7.35 (m, 2H, H4՛՛,8), 7.40 (d, J ¼ 7.0 Hz, 2H, H5), 7.13 (t, J ¼ 6.5 Hz, 1H, H5՛), 8.18 (d, J ¼ 7.0 Hz, 1H, H4՛), 8.50 (s, 1H, H2՛), 9.05 (d, J ¼ 6 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 18.6 (CH3), 20.5 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 61.4 (Nþ- CH2), 109.0 (tetrahydrocarbazole-C), 109.3 (tetrahydrocarbazole-CH), 109.8 (tetrahydrocarbazole-CH), 117.5 (tetrahydrocarbazole-CH), 117.8 (tetrahydrocarbazole-CH), 118.9 (C5119.2 ,(‫( ׳‬C4}), 120.7 (tetrahydrocarbazole-C), 126.5 (C5}), 127.2 (C6}), 128.2 (C3}), 129.4 (C3130.8 ,(‫׳‬ (C4131.5 ,(‫( ׳‬C-N), 135.1 (C2}), 135.6 (C-N), 137.0 (C1}), 144.1 (C6143.2 ,(‫׳‬ (C2‫ )׳‬ppm. 4.1.24. 1-(3-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium chloride (6h) Yellow solid; yield: 74%, m. p. ¼ 200e201 C. IR (KBr): 3049, 3006, 2933, 2831, 1629, 1499, 1466, 1375 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.79e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.29 (s, 3H, CH3), 2.63e2.65 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.82 (s, 2H, Nþ- CH2), 7.01e7.04 (m, 2H, H6,7), 7.24 (t, J ¼ 7.0 Hz, 1H, H4՛՛), 7.27e7.29 (m, 2H, H2՛՛,5՛՛), 7.31 (d, J ¼ 7.5 Hz, 1H, H8), 7.37 (d, J ¼ 7.0 Hz, 1H, H6՛՛), 7.41 (d, J ¼ 7.0 Hz, 2H, H5), 8.04e8.09 (m, 1H, H5՛,4՛), 8.99 (s, 1H, H2՛), 9.16 (d, J ¼ 6 Hz, 1H, H6՛) ppm. 13CNMR (DMSO, 500 MHz): d ¼ 20.6 (CH3), 20.8 (tetrahydrocarbazoleCH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazoleCH2), 22.6 (tetrahydrocarbazole-CH2), 42.6 (N- CH2), 63.3 (NþCH2), 109.0 (tetrahydrocarbazole-C), 109.3 (C8), 109.8 (C5), 117.5 (C6), 117.75(C7), 119.0 (tetrahydrocarbazole-C),127.2 (C6}), 128.5

58


(tetrahydrocarbazole-C), 129.1 (C5129.32 ,(‫( ׳‬C5}), 129.8 (C2}), 133.9 (C4}), 135.2 (C-N), 135.8 (C-N), 138.5 (C3139.8 ,(‫( ׳‬C3}), 140.1 (C4‫)׳‬, 142.3(C6143.6 ,(‫( ׳‬C2‫ )׳‬ppm.

anti-AChE activity of the derivatives.

4.1.25. 1-(4-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9yl)methyl)pyridin-1-ium chloride (6i) Yellow solid; yield: 76%, m. p. ¼ 200e202 C. IR (KBr): 3033, 2932, 2851, 1618, 1499, 1462, 1377 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.77e1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.31 (s, 3H, CH3), 2.60e2.64 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.82 (s, 2H, Nþ- CH2), 7.01e7.03 (m, 2H, H6,7), 7.22 (d, J ¼ 7.0 Hz, 2H, H3՛՛,5՛՛), 7.35 (d, J ¼ 7.0 Hz, 1H, H8), 7.37 (t, J ¼ 7.0 Hz, 1H, H2՛՛,6՛՛), 7.41 (d, J ¼ 7.0 Hz, 1H, H5), 8.02 (d, J ¼ 8 Hz, 1H, H5՛), 8.07 (t, J ¼ 6.5 Hz, 1H, H4՛), 8.95 (s, 1H, H2՛), 9.15 (d, J ¼ 6 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (CH3), 20.7 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 63.1 (Nþ- CH2), 109.0 (tetrahydrocarbazole-C), 109.3 (C8), 109.8 (C5), 117.5 (C6), 117.7 (C7), 119.0 (tetrahydrocarbazole-C), 127.2 (C5128.3 ,(‫( ׳‬C2},C3}, C5}, C6}), 128.7(C1}), 129.0 (C-N), 129.6 (C4}), 130.9 (C-N), 135.2 (C3135.7 ,(‫( ׳‬C4‫)׳‬, 139.0 (C2139.8 ,(‫( ׳‬C6‫ )׳‬ppm.

Derivative (6i) with the best BuChE IC50 value was selected to investigate the mechanism of BuChE's inhibition. Double reciprocal plots (1/v versus 1/s) were outlined [21] for three different concentrations of 6i as the inhibitor (0.043, 0.087 and 0.174 mM) and butyrylthiocholine iodide as the substrate (0.13, 0.32 and 0.69 mM). Correspondingly, the inhibition constant (Ki) was calculated with the interception of the slope versus the concentration plot. All analyses were carried out using Microsoft Excel 2010.

4.1.26. 1-Benzyl-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl) pyridin-1-ium bromide (6j) Yellow solid; yield: 75%, m. p. ¼ 198e200 C. IR (KBr): 2924, 2850, 1560, 1460, 1371 cm1. 1H NMR (DMSO, 500 MHz): d ¼ 1.78e1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.62e2.66 (m, 4H, tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.90 (s, 2H, NþCH2), 6.99e7.05 (m, 2H, H6,7), 7.38 (d, J ¼ 7.5 Hz, 1H, H8), 7.40 (d, J ¼ 7.5 Hz, 1H, H5), 7.40e7.43 (m, 3H, H2՛՛,4՛՛,6՛՛), 7.49 (m, 2H, H3՛՛,5՛՛), 8.02 (d, J ¼ 8.0 Hz, 1H, H5՛), 8.08 (t, J ¼ 6.5 Hz, 1H, H4՛), 9.08 (s, 1H, H2՛), 9.19 (d, J ¼ 6.0 Hz, 1H, H6՛) ppm. 13C- NMR (DMSO, 500 MHz): d ¼ 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazoleCH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazoleCH2), 42.6 (N- CH2), 63.2 (Nþ- CH2), 109.1 (tetrahydrocarbazole-C), 109.3 (C8), 109.8 (C5), 117.5 (C6), 117.7 (C7), 119.2 (C4՛՛), 120.9 (tetrahydrocarbazole-C), 127.2 (C5128.4 ,(‫( ׳‬C3՛՛, C5՛՛), 128.6 (C2՛՛, C6՛՛), 128.9 (C1՛՛), 129.2 (C-N), 129.5 (C-N), 134.0 (C3135.2 ,(‫( ׳‬C4135.7 ,(‫( ׳‬C2‫)׳‬, 139.8 (C6‫ )׳‬ppm. 4.2. Cholinesterase inhibition assay Anti-AChE and anti-BuChE activity of derivatives were obtained using the Ellman's method [24,27]. Sodium hydrogen carbonate, potassium hydroxide, dipotassium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Fluka. Electric eel (Torpedo californica) AChE (type VI-S), BuChE (E.C.3.1.1.8, from equine serum), acetylthiocholine iodide, butyrylthiocholine iodide, 5,5‫׳‬-dithiobis [2-nitrobenzoic acid] (DTNB) and Donepezile hydrochloride was obtained from SigmaeAldrich (Steinheim, Germany). It is necessary to note that the last-mentioned drug was used as the reference drug. The stock solutions of the derivatives were prepared by dissolving them in the dimethyl sulfoxide (DMSO). Afterwards, they were diluted in the absolute ethanol to obtain three different assay concentrations and, for each concentration, done in triplicate. The assay solution contained 2 mL of phosphate buffer (0.1 M, pH ¼ 8), 60 mL DTNB, 30 mL of inhibitor and 20 mL of 5 IU/mL butryl cholinesterase solution. Then, the previous mixture was preincubated for 10 min at 25 C. The reaction was then initiated by adding 20 mL of butyrylthiocholine iodide as the substrate to each of the 24 wells. The absorbance changes were scored at 412 nm for 5 min using a Synergy HTX multimode plate reader. The IC50 values were calculated graphically from a concentration inhibition curve for each derivative. All plots were obtained with Microsoft Excel 2010. Also, the same assay was performed with AChE to obtain the

4.3. Kinetic studies of BuChE inhibition

4.4. Molecular dynamic simulation studies Molecular dynamic simulation study was performed using Gromacs 5.1 (University of Groningen, Netherlands) on an 8 core PC running Linux Ubuntu 14.0.1. According to tutorial of Gromacs (http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin/gmxtutorials/complex/index.html) GROMOS96-43a1 force field was used for preparation of topology file. The required format of ligand was prepared using PRODRG 2 online server [28,29]. To obtain 3D coordinates of ligand for simulation purpose, ligand docking was performed and the coordinates of the best pose (considering the best docking energy) was retrieved. The proper Gromacs input format (gro) was constructed by appending ligand coordinates to the end of protein gro file (Human butyrylcholinesterase, PDBID: 4BDS). The above complex was surrounded by a cubic box filled with simple point charge (SPC) water molecules and the minimum distance of 1.0 nm. After solvation the system was neutralized by adding 6 chloride ions (Cl-). Afterwards, the minimization step was performed using the following parameters: maximum steps ¼ 50000, maximum force threshold