Intermediate 1: 4-N, N-Dimethylamino cyclohexanone

S1

Supporting Information

Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-

methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-

502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6)

Receptor Antagonist for Potential Treatment of Alzheimer’s Disease

Ramakrishna Nirogi*, Anil Shinde, Rama Sastry Kambhampati, Abdul Rasheed Mohammed,

Sangram Keshri Saraf, Rajesh Kumar Badange, Thrinath Reddy Bandyala, Venugopalarao

Bhatta, Kumar Bojja, Veena Reballi, Ramkumar Subramanian, Vijay Benade, Raghava

Choudary Palacharla, Gopinadh Bhyrapuneni, Pradeep Jayarajan, Vinod Goyal and Venkat Jasti

Discovery research, Suven Life Sciences Ltd, Serene Chambers, Road - 5, Avenue - 7, Banjara

Hills, Hyderabad 500 034, India

*Corresponding Author Tel.: +91-40-23556038; Fax: +91-40-23541152; e-mail:

[email protected]

S2

Table of Contents Page

Determination of 5-HT6 receptor Ki by Radioligand binding assay S3

Determination of Kb values for 5-HT6 Receptor S3

5-HT1A Binding Experimental Procedures S4

5-HT2A Binding Experimental Procedures S5

5-HT2C Binding Experimental Procedures S6

5-HT4B Binding Experimental Procedures S6

Determination of IC50 values for rat 5-HT7 Receptor S7

Pharmacokinetic Study in Rats S9

Rodent Brain Penetration Study S10

Protocol for Object Recognition Test S11

Selectivity Panel for compound 5al S12

Determination of Adrenergic Alpha 2A receptor Ki by Radioligand binding assay S15

Determination of Kb values for Adrenergic alpha 2C Receptor S16

Determination of Dopamine D3 receptor Ki by Radioligand binding assay S17

Permiability study S17

Protocol for protein binding S18

Pharmacokinetic Study in Dogs S19

CYP inhibition S20

CYP induction S20

Protocol for Fear Conditioning Model S21

Procedure for aqueous solubility S22

Analytical Characeterization Data S24

Supplementary Figures S39

References S67

S3

Determination of 5-HT6 receptor Ki by Radioligand Binding Assay:

The procedure used was previously described.1 In brief, receptor source and radioligand

used were human recombinant receptor expressed in HEK-293 cells and [3H] Lysergic Acid

Diethylamide (LSD), respectively. The final ligand concentration was 1.5 nM and non-specific

determinant was Methiothepin mesylate – [0.1 µM]. Methiothepin mesylate was used as a

positive control. Reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 10 mM

MgCl2, 0.5 mM EDTA for 60 min at 37 °C. The reaction was terminated by rapid vacuum

filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and

compared to control values in order to ascertain any interactions of test compound(s) with the

cloned serotonin - 5-HT6 binding site and reported as Ki values. These studies were conducted

and the data were analyzed at Novascreen Biosciences Corporation, Hanover, Maryland, U.S.A.

using standard radioligand binding techniques as described above. Under these tested assay

conditions the Ki value obtained for methiothepin mesylate (reference compound) is 0.5 ± 0.04

nM.

Determination of Kb for 5-HT6 Receptor:

The procedure used was previously described.2, 3

A stable CHO cell line expressing

recombinant human 5-HT6 receptor and pCRE-Luc reporter system was used for cell-based

assay. The assay offers a non-radioactive based approach to determine binding of a compound to

GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by

activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase

reporter gene under the control of cAMP response element.

The above cells were plated in 96 well clear bottom white plates at a density of 5 x 104

cells/well using Hams F12 medium containing 10% fetal bovine serum (FBS) and incubated

S4

overnight at 37 oC and 5% CO2 followed by serum starvation for 18-20 h. Increasing

concentrations of test compounds were added along with 10 µM serotonin in OptiMEM to the

cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4 h cells were lysed

using lysis buffer and luciferase assay buffer was added to each well and counts per second were

recorded using luminescence counter. From counts per second (CPS) obtained, percent binding

was calculated for each well by taking 10 µM 5-HT as 100% bound and vehicle as 0% bound.

The percent bound values were plotted against compound concentrations and data were analyzed

using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software. The

Kb and IC50 values were calculated using concentration of the agonist used in the assay and its

EC50 values in the same software. Under these tested assay conditions the Kb value obtained for

methiothepin mesylate (reference compound) is 0.7 ± 0.05 nM.

5-HT1A Binding Experimental Procedures:

The procedure used was previously described.4 Membrane preparation from recombinant

human 5-HT1A cell line (Cat no.RBHS1AM400UA) and radioligand 8-Hydroxy-DPAT, [Propyl-

2,3-ring-1,2,3-3H] (Cat no. NET929250UC) were purchased from Perkin Elmer. All other

reagents used in buffer preparation were purchased from Sigma. The final ligand concentration

was 1.75 nM; non-specific determinant was 5-HT [10 µM] and 5-HT1A membrane protein (16

µg/ well). Serotonin was used as a positive control. Reactions were carried out in 50 mM Tris pH

7.4 containing 0.5 mM EDTA, 10 mM MgSO4 and 0.1% ascorbic acid buffer for 120 min at 25

ºC. Reaction was stopped by rapid filtration followed by six washes of the binding mixture using

96 well harvest plate (Millipore Cat no. MSFCNXB50) pre coated with 0.33%

polyethyleneimine. The plate was dried and the bound radioactivity collected on the filters was

determined by scintillation counting using MicroBeta TriLux. Radioligand binding in the

S5

presence of non labeled compound was expressed as a percent of the total binding and plotted

against the log concentration of the compound. Ki values were determined using a nonlinear,

iterative curve-fitting computer program of Graph pad Prism 4 software (Table 1). Under these

tested assay conditions the Ki value obtained for serotonin (reference compound) is 0.2 ± 0.03

nM.

5-HT2A Binding Experimental Procedures:

The procedure used was previously described.5 Membrane preparation from recombinant

human 5-HT2A cell line (Cat no.ES-313-M400UA) and radioligand Ketanserin hydrochloride,

[Ethylene-3H]-(R-41468) (Cat no. NET791250UC) were purchased from Perkin Elmer. All other

reagents used in buffer preparation were purchased from Sigma. The final ligand concentration

was 1.75 nM; non-specific determinant was 1-NP [10 µM] and 5-HT2A membrane protein (5µg/

well). 1-NP was used as a positive control. Reactions were carried out in 67 mM Tris pH 7.6

containing 0.5 mM EDTA buffer for 60 min at 25 ºC. Reaction was stopped by rapid filtration

followed by six washes of the binding mixture using 96 well harvest plate (Millipore Cat no.

MSFBNXB50) pre coated with 0.33% polyethyleneimine. The plate was dried and the bound

radioactivity collected on the filters was determined by scintillation counting using MicroBeta

TriLux. Radioligand binding in the presence of non labeled compound was expressed as a

percent of the total binding and plotted against the log concentration of the compound. Ki values

were determined using a nonlinear, iterative curve-fitting computer program of Graph pad Prism

4 software (Table 1). Under these tested assay conditions the Ki value obtained for 1-(1-

naphthyl)piperazine hydrochloride (reference compound) is 15.4 ± 0.7 nM.

5-HT2C Binding Experimental Procedures:

S6

The procedure used was previously described.6 In brief, membrane preparation from

recombinant human 5-HT2C cell line (Cat no. 6110548400UA), radioligand Mesulergine, [N-

Methyl-3H] (Cat no. NET1148250UC) were purchased from Perkin Elmer. All other reagents

used in buffer preparation were purchased from Sigma. The final ligand concentration was 1.25

nM; non-specific determinant was Mianserine [10 µM], 5-HT2C membrane protein (30 µg/well)

and Ysi Polylysine SPA Beads (Perkin Elmer), 1.0 mg/ well. Mianserine was used as a positive

control. Reactions were carried out in 54 mM Tris (pH 7.4) containing 10.8 mM MgCl 2, 0.54

mM EDTA, 10.8 µM Pargyline, 0.108% Ascorbic acid, pH 7.4 buffer for 180 min at 25 °C.

The plate was read in a MicroBeta TriLux. Radioligand binding in the presence of non labeled

compound was expressed as a percent of the total binding and plotted against the log

concentration of the compound. Ki values were determined using a nonlinear, iterative curve-

fitting computer program of Graph pad Prism 4 software (Table 1). Under these tested assay

conditions the Ki value obtained for mianserine (reference compound) is 2.8 ± 0.3 nM.

5-HT4B Binding Experimental Procedures:

The procedure used was previously described.7 In brief, membrane preparation from

recombinant human 5-HT4B cell line was purchased from Millipore (Cat no. HTS110M),

Radioligand GR113808, [N-methyl-3H] was purchased from Perkin Elmer (Cat no.

NET1152010UC). All other reagents used in buffer preparation were purchased from Sigma. The

final ligand concentration was 0.5 nM; non-specific determinant was GR113808 [10 µM] and 5-

HT4B membrane protein (5 µg/well). GR113808 was used as a positive control. Reactions were

carried out in 25 mM Tris-HCl (pH 7.4) buffer for 120 min at 25 °C. Reaction was stopped by

rapid filtration followed by six washes of the binding mixture using 96 well harvest plate

(Millipore Cat no. MSFCNXB50) pre coated with 0.33% polyethyleneimine. The plate was dried

S7

and the bound radioactivity collected on the filters was determined by scintillation counting

using MicroBeta TriLux. Radioligand binding in the presence of non labeled compound was

expressed as a percent of the total binding and plotted against the log concentration of the

compound. Ki values were determined using a nonlinear, iterative curve-fitting computer

program of Graph pad Prism 4 software (Table 1). Under these tested assay conditions the Ki

value obtained for GR113808 (reference compound) is 16.2 ± 0.8 nM.

Determination of IC50 Values for Rat 5-HT7 Receptor:

The procedure used was previously described.8


recombinant rat 5-HT7 receptor and pCRE-Luc reporter system was used for cell-based assay.

The assay offers a non-radioactive based approach to determine binding of a compound to

GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by

activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase

reporter gene under the control of cAMP response element.




concentrations of test compounds were added along with 10 µM serotonin in OptiMEM to the

cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4 h cells were lysed

using lysis buffer and luciferase assay buffer was added to each well and counts per second were

recorded using luminescence counter. From CPS obtained, percent binding was calculated for

each well by taking 10 µM serotonin as 100% bound and vehicle as 0% bound. The percent

bound values were plotted against compound concentrations and data were analyzed using a

nonlinear, iterative curve-fitting computer program of Graph pad Prism 4 software. The IC50

S8

value was calculated using the concentration of the agonist used in the assay and its EC 50 values

in the same software (Table 1). Under these tested assay conditions the Kb value obtained for

methiothepin mesylate (reference compound) is 0.6 ± 0.04 nM (IC50 = 117 ± 6.6 nM).

Table 1: Serotonin Receptor Subtypes Selectivity Data

aDisplacement of [

3H]-LSD binding to cloned human 5-HT6 receptors stably expressed in HEK293 cells. K i values

were determined in duplicate and expressed as K i ± SEM. b, c, d, e

Recombinant membrane protein and radioligands used in the

binding assays were as follows: 5-HT1A (human cloned receptors in HEK293-EBNA cells, [3H]-8-OH-DPAT), 5-HT2A (human

cloned receptors in CHO-K1 cells, [3H]-Ketanserine), 5-HT2C (human cloned receptors in HEK293 cells, [

3H]-Mesulergine), 5-

HT4B (human cloned receptors in Chem-1 cells, [3H]-GR113808) and values are expressed as Ki ± SEM.

fIC50 values were

determined using non-radioactive cell based assay using recombinant 5-HT7 receptor and luciferase reporter expressed in CHO-

K1 cells and values are mean of duplicate experiments.

Pharmacokinetic Study in Rats:

Compound 5-HT6

Ki (nM)a

5-HT1A

Ki

(nM)b

5-HT2A

Ki (nM)c

5-HT2C

Ki

(nM)d

5-HT4B

Ki (nM)e

5-HT7

IC50

(nM)f

5a 6.05 ±

0.3

5230 ±

302

2016 ±

93 >1000 >10000 >10000

5h 8.76 ±

0.8

4782 ±

285

3574 ±

198 >1000 >10000 >1000

5ad 3.56 ±

0.3

3531 ±

245

248.8 ±

18 >1000

2133 ±

129 >10000

5ai 11.6 ±

1.6

2045 ±

121

199.3 ±

12 >1000

440.4 ±

28 >10000

5al 2.04 ±

0.2

7020 ±

358

2514 ±

377 >1000

4166 ±

285 >10000

5am 1.63 ±

0.2

4352 ±

268

926 ±

317 >1000

5465 ±

328 >10000

5an 4.96 ±

0.6

4428 ±

285 >10000 >1000

4695 ±

302 >10000

5bg 1.98 ±

0.1

1928 ±

83

179.4 ±

9 >1000

1843 ±

98 >10000

S9

Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were

housed in each cage. Two days prior to dosing day, male Wistar rats (225 - 250 g) were

anesthetized with isoflurane for surgical placement of jugular vein catheter. Animals were fasted

overnight before oral dosing (p.o.) and food pellets were allowed 2 h post dosing, whereas during

intravenous dosing food and water were provided ad libitum. Three rats each were dosed with

test compound orally (10 mg/kg) and intravenously (10 mg/kg). 10 mL/kg for oral and 2 mL/kg

for intravenous was used as dosing volume and water as a vehicle for preparation of dose

formulation.

At each time point blood was collected through jugular vein and immediately replenished

with an equivalent volume of normal saline into freely moving rats. Collected blood was

transferred into a labeled vial containing 10 µL of heparin as anticoagulant. Blood samples were

collected at following time points: pre dose, 0.08 (only i.v.), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post

dose (n=3). Blood was centrifuged at 4000 rpm for 10 min. Plasma was prepared and stored at -

70 °C until analysis. The concentrations of the compounds were quantified in plasma by

qualified LC-MS/MS method using suitable extraction technique. The compounds were

quantified in the calibration range around 2-2000 ng/mL in plasma. Study samples were analyzed

using calibration samples in the batch and quality control samples spread across the batch.

Pharmacokinetic parameters Cmax, AUC0-t, t1/2 and bioavailability were calculated by non

compartmental model using standard non-compartmental model by using WinNonLin 5.0.1

version Software package.

We have evaluated the pharmacokinetic profiling of a reported reference 5-HT6 antagonist

(SAM-531) in rats under similar experimental conditions to that of lead compounds (Table 2).

The reported values are in agreement with the published report.9

S10

Table 2. Pharmacokinetic Profile of SAM-531 in Wistar ratsa

aFasted male Wistar rats, 0.05 % HEC and 10% PEG-400 in Water was used as a vehicle for both oral and i.v. routes. Dosing

Volumes: 10 mL/kg for p.o. and 2 mL/kg for i.v.

Rodent Brain Penetration Study:

Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were

housed in each cage. Animals were given water and food ad libitum throughout the experiment

and maintained on a 12 h light/dark cycle. Male Wistar rats were fasted overnight and compound

was administered per orally (p.o.; dosing volume 2.5 mL/kg) at 10 mg/kg (n = 3). Blood was

collected by cardiac puncture at pre dose 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 32 h after

dosing (n=3/ time point), immediately brain was isolated and carcasses were discarded. The

brain was homogenized with 4 volumes of ice cooled water. Plasma and brain homogenates

(20%) were stored frozen at -20 °C until analysis. The plasma and brain levels of compound was

determined by validated LC-MS/MS method using solid phase extraction technique, quantified

in the calibration range of 2-2000 ng/mL in plasma and brain homogenates. Pharmacokinetic

evaluation was performed using the validated software WinNonlin version 4.1 (Pharsight

Compound SAM-531

10 mg/kg, p.o.

Cmax (ng/mL) 127 ± 18

AUC (ng.h/mL) 376 ± 19

F (%) 35 ± 1.5

5 mg/kg, i.v.

t1/2 (h) 1.24 ± 0.06

Vz (L/kg) 18 ± 2

CL (mL/min/kg) 162 ± 37

S11

Corporation, Mountain View, California 94040/USA). Extent of brain-plasma ratio was

calculated (Cb/Cp) at Tmax.

Protocol for Object Recognition Test:

For object recognition test, Male Wistar rats 10-12 weeks old were used. Arena was 50 x

50 x 50 cm. open field was made up of acrylic. On day 1, rats were habituated to individual test

arenas for 20 min in the absence of any objects. On day 2 (24 h after the habituation), rats were

subjected to familiarization phase (T1). The rats were placed individually in the open field

containing two identical objects (a1 and a2) for 3 min. The recognition trial (T2) trial was carried

out after 24 h after the T1 trial. Rats were allowed to explore the open field in presence of one

familiar object (a3) and one novel object (b) for 3 min. The exploratory behavior towards the

objects during the familiarization and recognition phases were recorded.

We have evaluated a reported reference 5-HT6 antagonist (SAM-531) in object recognition

task model under similar experimental conditions, except the route of administration. SAM-531

showed efficacy at doses of 1 and 3 mg/kg as shown below (Figure 1).

Data represents Mean SEM of Exploration Time (Students paired ‘t’ test) N=8-12

Figure 1. Effect of compound SAM-531 in time induced object recognition task.

5

1 0

1 5

2 0

2 5

***

V e h ic le

2 m L /k g , s.c.

S A M -5 3 1

1 m g /k g , s.c.

S A M -5 3 1

1 0 m g /k g , s.c.

S A M -5 3 1

3 m g /k g , s.c.

Ex

plo

ra

tio

n t

ime

(S

)

O p e n C o lu m n - F a m il ia r O b je c t

F i l le d C o lu m n - N o v e l O b je c t

S12

Selectivity Panel for Compound 5al:

S13

S14

S15

Determination of Adrenergic Alpha 2A Receptor Ki by Radioligand Binding Assay:


In brief; receptor source and

radioligand used were HT29 cells and [3H] MK-912, respectively. The final ligand concentration

was 0.75 nM and non-specific determinant was L (-)-Norepinephrine - [100 µM]. Oxymetazoline

HCl was used as a positive control. Reactions were carried out in 33 mM Tris-HCl (pH 7.5)

containing 1 mM EDTA, 140 mM NaCl and 100 µM Gpp(NH)p (guanyl-5'-yl-imido-

diphosphate) for 60 min at 25 °C. The reaction was terminated by rapid vacuum filtration onto

glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control

values in order to ascertain any interactions of test compound(s) with the human adrenergic alpha

2A binding site and reported as Ki values. These studies were conducted and the data were

analyzed at Novascreen Biosciences Corporation, Hanover, Maryland, U.S.A. using standard

radioligand binding techniques as described above. Under these tested assay conditions the Ki

value obtained for oxymetazoline HCl (reference compound) is 1.3 ± 0.06 nM.

S16

Determination of Kb Values for Adrenergic Alpha 2C Receptor:



recombinant human adrenergic alpha 2C receptor and pCRE-Luc reporter system was used for

cell-based assay. The assay offers a non-radioactive based approach to determine binding of a

compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is

modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor

luciferase reporter gene under the control of cAMP response element.




concentrations of test compounds were added along with 1 µM epinephrine and 1 µM forskolin

in OptiMEM to the cells. The incubation was continued at 37 oC in CO2 incubator for 4 h. After 4

h cells were lysed using lysis buffer and luciferase assay buffer was added to each well and

counts per second were recorded using luminescence counter. From CPS obtained, percent

binding was calculated for each well by taking 10 µM epinephrine as 100% bound and vehicle as

0% bound. The percent bound values were plotted against compound concentrations and data

were analyzed using a nonlinear, iterative curve-fitting computer program of Graph pad Prism 4

software. The Kb and IC50 values were calculated using concentration of the agonist used in the

assay and its EC50 values in the same software. Under these tested assay conditions the Kb value

obtained for spiroxatrine (reference compound) is 3.6 ± 0.3 nM.

S17

Determination of Dopamine D3 receptor Ki by Radioligand Binding Assay:


In brief; receptor source and

radioligand used were rat recombinant receptor expressed in CHO cells and [3H] 7-OH DPAT,

respectively. The final ligand concentration was 0.8 nM and non-specific determinant was

Dopamine – [1 µM]. (±)-7-OH-DPAT HBr was used as a positive control. Reactions were

carried out in 50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl for 60 min at 25 °C. The

reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped

onto the filters was determined and compared to control values in order to ascertain any

interactions of test compound(s) with the cloned Dopamine D3 binding site and reported as Ki

values. These studies were conducted and the data were analyzed at Novascreen Biosciences

Corporation, Hanover, Maryland, U.S.A. using standard radioligand binding techniques as

described above. Under these tested assay conditions the Ki value obtained for (±)-7-OH-DPAT

HBr (reference compound) is 0.4 ± 0.02 nM.

Permeability study:

Trans epithelial transport in Caco-2 cells was conducted at RCC Gen biotec GmbH (RCC-

GBT) Switzerland as per their standard operating procedure. The cells were maintained in

DMEM medium (PAA GmbH) supplemented with 10% fetal calf serum (FCS, Gibco), 1% non-

essential amino acids (NEAA, PAA GmbH) and 1% Gentarnicin (PAA GmbH). The cells were

seeded in 12 well Polyester (PET) transwell dishes (Corning) at a density of about 300,000 cells/

cm2 using Caco-2 medium. The cells were cultivated at 37 °C/ 5% CO2 in a humidified

incubator. The medium was renewed every 2-3 days. After cultivation of about 10 days the

TEER was determined every three days until it reached a plateau. Transwell cavities with a

TEER of >280Ω cm2 were used in the assay. For transepithelial transport study TEER was

S18

measured prior to the start of the assay, medium was removed and cells were washed twice with

pre-warmed (37 ºC) Hank's buffer to remove traces of medium. The assay was started by adding

test compound dissolved in to either the apical or basolateral cavity to a final concentration of 10

µM. Samples of 100 µl were taken at different time points (20, 40, 60, 80, 100 and 120 min).

The removed volume was replaced by adding 100 µL Hank's buffer and the reduction in

concentration was taken into account in the evaluation of the results. The samples were analyzed

using LCMS/MS analysis.

The apparent permeability coefficient Papp [cm/s] is a factor for the transport velocity of a

certain substance across the cell monolayer and it was determined using the following

Equation: 16

Papp = dQ/dt x 1/mo x 1/A x VDonor

Papp: apparent permeability coefficient [cm/s]

dQ : amount of substance transported, e.g. [nmol] or [µg]

dt : time, normally the endpoint 120 min will be used

mo: applied amount of substrate in donor compartment, e.g. [nmol] or [µg]

A : surface area of transwell membrane [cm2]

VDonor: Volume of the donor compartment [cm3]

Protocol for Protein Binding:

Unbound fractions of 5al in plasma were determined using Rapid Equilibrium dialysis. The

dialysate chambers were loaded with 750 µL of 100 mM phosphate buffer (pH 7.4) in triplicates.

The matrix chambers were loaded with 500 µL of the plasma spiked with 5al at a final

concentration of 1 µM. 50 µL of the sample was removed from both the chambers at 0 h. The

S19

plate was sealed and incubated at 37 °C for 6 h at 100 rpm. After 6 h, 50 µL of the sample was

removed from both the chambers. Equal volumes of buffer or plasma were added to the plasma /

microsomal and buffer samples respectively to create identical sample matrices for analysis. The

samples were precipitated with 150 µL of acetonitrile containing internal standard. All the

samples were centrifuged at 10000 rpm for 10 min at 4 °C. The supernatants were transferred to

vials and were analyzed by LC-MS/MS.

Pharmacokinetic Study in Dogs:

Compound 5al was administered to fasted Beagle dogs (n = 3/dose/ group) at 10 mg/kg,

orally on Day 8 and non-fasted dogs were treated with intravenous route at a dose of at 3.0

mg/kg on Day 1. For oral administration, compound was weighed directly into a gelatin capsule.

Water for injection was used as a vehicle for intravenous administration at a dosing volume of 1

mL/kg.

On Day 1 and Day 8 of the study, blood samples were obtained for plasma drug

concentrations following oral (capsule) and intravenous (bolus) administration. Samples were

collected at 15 min, 30 min, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h after the oral

dose. Whereas, after the intravenous dose, blood samples were collected at 5 min, 10 min, 20

min, 30 min and 1, 2, 4, 6, 8, 12, 24, 48 and 72 h. On each occasion, approximately 3 ml of blood

was drawn from the jugular vein and collected into lithium heparin blood collection tubes. Blood

samples were centrifuged at 4000 rpm for 10 min at 4 °C. Plasma was transferred to plastic

(polypropylene) tubes and placed on dry ice. The samples were stored at -80 ± 10 °C until

analysis.

S20

The compounds were quantified in the calibration range around 2-2000 ng/mL in plasma.

Study samples were analyzed using calibration samples in the batch and quality control samples

spread across the batch.

Pharmacokinetic parameters Cmax, AUC0-t, t1/2 and bioavailability were calculated by Non-

compartmental analysis was performed with the WinNonlin software version 4.1 (Pharsight

Corporation, Mountain View, California 94040/USA).

CYP Inhibition:

The inhibition profiles of test compounds were determined using the marker probe substrate

reactions of CYP enzymes in human liver microsomes as described elsewhere.17

The final

incubation mixture is comprised of phosphate buffer (100 mM, pH 7.4), marker probe substrate

specific to each enzyme, and human liver microsomes. Reactions were initiated by adding

NADPH (1 mM) to a final volume of 200 µL. After specified incubation times for each CYP

isoform, 120 µL of the incubation mixture was terminated with 240 µL of acetonitrile containing

internal standard. Samples were centrifuged at 2500xg for 10 min at 4 °C. The supernatants were

mixed with equal volumes of 0.1% formic acid in water. The analysis of samples was performed

by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

CYP Induction:

Human cryopreserved hepatocytes from three donors were thawed and recovered in universal

cryopreserved recovery medium and centrifuged at 100 g for 10 min. Viability was assessed via

trypan blue dye exclusion. The hepatocytes were plated in collagen coated 96-well plates with

approximately 65,000 cells per well and were cultures in an incubator kept at 37 °C in a

humidified atmosphere of 5% carbon dioxide and 95% air. The hepatocytes were cultured for 1

S21

day before they were treated with test compounds and positive control inducers (1A2: 50 μM

omeprazole; 2B6: 1000 μM phenobarbital; 3A4: 20 μM rifampin). Human hepatocytes from 3

different donors were used in the study. The treatment was performed for an additional 3 days.

After the 3 day treatment period, medium was changed to HIM containing a specified P450

substrate and the cells were incubated for 30 min. At the end of the incubation period, the

medium was harvested in to 96 well plates and were stored at -80 °C until analysis. Cell viability

of the hepatocytes was determined with the WST cell viability assay using the tetrazolim salt,

WST-1. The hepatocytes were incubated with the HIM containing WST-1 reagent at 1: 10

dilution for 2 h, followed by quantification of absorbance at 450 nm using a UV plate reader.

Total RNA was isolated from hepatocytes using the RNeasy 96 kit according to instructions

provided by the manufacturer for isolation. Reverse transcription was performed with 100 ng of

isolated RNA using the High capacity cDNA reverse transcription kit. Gene expression was

measured using 7500 Fast Real-Time PCR. RT Reactions were quantitated using Taqman

universal mix and specific primer sets. The relative quantity of the target gene was compared

with that of the endogenous control housekeeping gene expression (GAPDH) as determined by

the ΔΔCT method.

Protocol for Fear Conditioning Model:

Experiment was carried out over a period of two days. On day 1, rats were placed in the

operant behavior chamber and allowed to acclimatize for 2 min. Rats received a conditioned

stimulus (CS) (tone for 10 sec) followed by an unavoidable foot shock (unconditioned stimulus

(US): electric shock of 0.5 - 0.7 mA for 3 sec). Following a 1 min interval between each

administration, tone and shock were repeated to deliver a total of three CS-US pairings. Rats

were administered 5al (SUVN-502) (1 h). Scopolamine (0.3 mg/kg, s.c.) was administered 120

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min after training. On day 2, rats were placed in the operant behavior chamber and total freezing

time scored for a period of 5 min.

Procedure for Aqueous Solubility:

a) Sample Preparartion:

Weigh accurately about 2.0 g of sample and transfer it into a 2 mL eppendorf tube and

add 1 mL HPLC grade water and vortex for 2-5 min, place the same tube on a rugged rotator and

keep for rotation at 30 % speed by adjusting the knob. Keep rotation for 2 h and remove the

eppendorf tubes from rotator.

Centrifuge for 2 min at 2000 rpm and filter through 0.45 µm syringe filer into sample

vial.

Transfer 0.1 mL from the above solution in to a 100 mL volumetric flask and make up to

mark with water.

Transfer 5.0 mL from the above solution in to a 25 mL volumetric flask and make up to

mark with water.

b) Standard Prepartion:

Weigh accurately about 5 mg of same sample and transfer to 10 mL volumetric flask add

2 mL of water and vortex to dissolve finally make up to mark with water.

Transfer 2 mL from the above solution in to a 10 mL volumetric flask and make up to

mark with water (0.1 mg /mL).

Separately inject one blank as water, sample and standard in duplication and note the

areas of standard and sample.

NOTE: Keep the sample solution for rotation in duplicate / triplicate.

HPLC analysis

Chromatography conditions:

The liquid chromatograph equipped with Agilent 1100 series.

Mobile Phase: Buffer : Acetonitrile (Gradient)

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Buffer: 0.05% TEA in HPLC grade water, adjust the pH to 2.5 with TFA.

Gradient Programme:

S. No. Time (min) Buffer (%) ACN (%) Flow rate

1 0 80 0 1.00 mL/min

2 2 80 0 1.00 mL/min

3 10 20 100 1.00 mL/min

4 15 20 100 1.00 mL/min

5 16 80 0 1.00 mL/min

6 25 80 0 1.00 mL/min

Column: C18; 250 x 4.6 mm, 5 µm

Detection: 220 nm

Flow rate: 1.0 mL/min

Inj. Vol.: 10 µL

Calculation Area of sample

Sample Conc. (in µg/mL) = ------------------------- x Standard Concentration

Area of standard

S24

Analytical Characeterization Data:

1-[(2-Bromophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5b): IR (KBr,

cm-1

): 1123, 1179, 1373, 1447; 1H NMR (CDCl3, ppm): 8.11 – 8.14 (1H, dd, J = 1.6 , 7.9 Hz),

7.64 – 7.69 (4H, m), 7.39 – 7.47 (2H, m), 7.22 – 7.24 (2H, m), 3.66 (2H, s), 2.45 (8H, bs), 2.28

(3H, s); ESI mass (m/z): 448, 450 [M+H]+.

1-[(3-Bromophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5c): 1H NMR

(DMSO-d6, ppm): 8.08 (1H, s), 7.87 – 7.96 (3H, m), 7.73 (1H, s), 7.69 – 7.70 (1H, d, J = 7.7

Hz), 7.49 – 7.53 (1H, m), 7.34 – 7.38 (1H, m), 7.25 – 7.29 (1H, m), 3.57 (2H, s), 2.29 – 2.33

(8H, m), 2.12 (3H, s); ESI mass (m/z): 448.1, 450.0 [M+H]+.

1-[(4-Bromophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5d): IR (KBr,

cm-1

): 1145, 1178, 1372, 1380; 1H NMR (CDCl3, ppm): 7.92 – 7.96 (1H, d, J = 7.4 Hz), 7.70 –

7.74 (1H, m), 7.66 – 7.68 (3H, m), 7.52 – 7.57 (1H, dd, J = 1.8, 7.0 Hz), 7.42 (1H, s), 7.21 –

7.33 (2H, m), 3.59 (2H, s), 2.45 (8H, bs), 2.27 (3H, s); ESI mass (m/z): 448, 450 [M+H]+;

Elemental analysis calculated (%) for C20H22BrN3O2S: C 53.57, H 4.95, N 9.37. Found: C 53.69,

H 4.96, N 9.34.

1-[(3-Chlorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5e): 1H NMR

(CDCl3, ppm): 7.94 – 7.96 (1H, d, J = 8.2 Hz), 7.83 (1H, m), 7.73 – 7.74 (1H, m), 7.68 – 7.70

(1H, d, J = 7.7 Hz), 7.47 – 7.50 (1H, m), 7.44 (1H, s), 7.27 – 7.38 (3H, m), 3.61 (2H, s), 2.45

(8H, bs), 2.28 (3H, s); ESI mass (m/z): 403.3, 405.2 [M+H]+; HPLC = 98.5%.

1-[(2-Fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5f): 1H NMR

(DMSO-d6, ppm): 8.09 – 8.13 (1H, m), 7.72 – 7.79 (3H, m), 7.62 (1H, s), 7.40 – 7.47 (2H, m),

7.24 – 7.32 (2H, m), 3.61 (2H, s), 2.30 – 2.38 (8H, m), 2.13 (3H, s); ESI mass (m/z): 388.2

[M+H]+.

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1-[(3-Fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5g): 1H

NMR (DMSO-d6, ppm):7.92 – 7.94 (1H, d, J = 8.2 Hz), 7.85 – 7.87 (1H, d, J = 8.1 Hz), 7.78

– 7.80 (1H, d, J = 7.8 Hz), 7.68 – 7.76 (2H, m), 7.60 – 7.64 (1H, m), 7.53 – 7.58 (1H, m), 7.33

– 7.37 (1H, m), 7.25 – 7.29 (1H, m), 3.57 (2H, s), 2.29 – 2.33 (8H, m), 2.12 (3H, s); ESI mass

(m/z): 388.3 [M+H]+.

1-[(4-Isopropylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5i): IR

(KBr, cm-1

): 1121, 1144, 1190, 1371; 1H NMR (CDCl3, ppm): 7.97 – 7.97 (1H, d, J = 8.2 Hz),

7.66 – 7.81 (3H, m), 7.48 (1H, m), 7.21 – 7.33 (4H, m), 3.61 (2H, s), 2.85 – 2.92 (1H, m), 2.43

(8H, bs), 2.27 (3H, s), 1.10 – 1.19 (6H, d); ESI mass (m/z): 411 [M+H]+.

1-[(4-Methylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5j): 1H NMR

(CDCl3, ppm): 7.94 – 7.96 (1H, d, J = 8.2 Hz), 7.73 – 7.76 (2H, d, J = 8.1 Hz), 7.65 – 7.67

(1H, d, J = 7.7 Hz), 7.46 (1H, s), 7.28 – 7.32 (1H, m), 7.19 – 7.24 (3H, m), 3.60 (3H, m), 3.60

(2H, s), 2.43 – 2.47 (8H, bs), 2.33 (3H, s), 2.28 (3H, s); ESI mass (m/z): 384.2 [M+H]+; HPLC =

95.4%.

1-[(3-Methoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5k): 1H

NMR (CDCl3, ppm):7.90 – 7.93 (1H, d, J = 8.2 Hz), 7.68 – 7.70 (2H, m), 7.47 –7.48 (2H, m)

7.32 – 7.36 (2H, m), 7.21 – 7.25 (2H, m), 3.76 (3H, s), 3.58 (2H, s), 2.31 – 2.33 (8H, bs), 2.15

(3H, s); ESI mass (m/z): 400.1 [M+H]+.

1-[(4-Methoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5l): IR

(KBr, cm-1

): 2943, 2799, 1593, 1451, 1358, 1171, 744; 1H NMR (CDCl3, ppm): 7.93 – 7.97

(1H, d, J = 7.6 Hz), 7.77 – 7.84 (2H, m), 7.64 – 7.72 (1H, m), 7.46 (1H, s), 7.19 – 7.35 (2H, m),

6.82 – 6.91 (2H, m), 3.80 (3H, s), 3.60 (2H, s), 2.45 – 2.55 (8H, bs), 2.23 (3H, s); ESI mass

(m/z): 400.2 [M+H]+; HPLC = 99.4%.

S26

1-[(4-Trifluoromethoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5m): IR (KBr, cm-1

): 2937, 2797, 1447, 1377, 1259, 1181, 1012, 746; 1H NMR (CDCl3, ppm):

7.95 – 7.97 (1H, d, J = 8.2 Hz), 7.90 – 7.93 (2H, m), 7.69 – 7.71 (1H, d, J = 7.7 Hz), 7.44 (1H,

s), 7.32 – 7.36 (1H, m), 7.23 – 7.28 (3H, m), 3.60 (2H, s), 2.44 – 2.46 (8H, bs), 2.28 (3H, s); ESI

mass (m/z): 454.1 [M+H]+; HPLC = 96.7%.

1-[(4-Cyanophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5n): 1H

NMR (CDCl3, ppm): 7.93 – 7.98 (3H, m), 7.71 – 7.73 (2H, d, J = 8.3 Hz), 7.66 – 7.68 (1H, d, J

= 7.7 Hz), 7.42 (1H, s), 7.33 – 7.36 (1H, m), 7.27 – 7.29 (1H, m), 3.62 (2H, s), 2.55 – 2.59 (8H,

bs), 2.05 (3H, s); ESI mass (m/z): 394.9 [M+H]+; HPLC = 95.5%.

1-[(2,4-Difluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5o): 1H

NMR (CDCl3, ppm): 8.20 – 8.26 (1H, m), 7.73 – 7.75 (2H, d, J = 7.9 Hz), 7.54 – 7.65 (2H,

m), 7.28 – 7.39 (3H, m), 3.64 (2H, s), 2.32 – 2.34 (8H, bs), 2.37 (3H, s); ESI mass (m/z): 406.0

[M+H]+.

1-[(3,4-Difluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5p): 1H

NMR (CDCl3, ppm): 7.92 – 7.94 (1H, d, J = 8.1 Hz), 7.67 – 7.70 (2H, m), 7.41 (1H, s), 7.20 –

7.34 (3H, m), 3.61 (2H, s), 2.46 – 2.48 (8H, bs), 2.29 (3H, s); ESI mass (m/z): 406.5 [M+H]+;

HPLC = 95.7%.

1-[(2,6-Difluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5q): 1H

NMR (DMSO-d6, ppm): 7.71 – 7.81 (3H, m), 7.60 (1H, s), 7.27 – 7.36 (4H, m), 3.62 (2H, s),

2.30 – 2.38 (8H, m), 2.13 (3H, s); ESI mass (m/z): 406.3 [M+H]+.

1-[(2,4-Dichlorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5r): 1H

NMR (CDCl3, ppm): 8.25 – 8.27 (1H, d, J = 8.6 Hz), 7.91 (1H, d, J = 1.6 Hz), 7.72 – 7.76

S27

(3H, m), 7.59 – 7.61 (1H, m), 7.27 – 7.31 (2H, m), 3.65 (2H, s), 2.32 – 2.34 (8H, bs), 2.32 (3H,

s); ESI mass (m/z): 438.0, 439.9 [M+H]+.

1-[(2,6-Dichlorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5s): 1H

NMR (DMSO-d6, ppm): 7.72 – 7.78 (4H, m), 7.66 – 7.68 (1H, m), 7.47 – 7.49 (1H, d, J = 8.1

Hz), 7.27 – 7.29 (2H, m), 3.63 (2H, s), 2.40 – 2.45 (4H, m), 2.29 – 2.32 (4H, m), 2.13 (3H, s);

ESI mass (m/z): 438.3, 440.1 [M+H]+.

1-[(2-Chloro-4-fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5t):

1H NMR (CDCl3, ppm): 8.22 – 8.25 (1H, m), 7.62 – 7.73 (3H, m), 7.16 – 7.23 (3H, m), 6.89

(1H, m), 3.65 (2H, s), 2.51 – 2.54 (8H, bs), 2.27 (3H, s); ESI mass (m/z): 421.5, 423.8 [M+H]+;

HPLC = 96.3%.

1-[(2,5-Dimethoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5u): 1H

NMR (DMSO-d6, ppm): 7.65 – 7.71 (2H, m), 7.60 (1H, s), 7.50 – 7.51 (1H, d, J = 3.0 Hz),

7.21 – 7.27 (3H, m), 7.08 – 7.11 (1H, m), 3.79 (3H, s), 3.62 (5H, s), 2.30 – 2.40 (8H, m), 2.13

(3H, s); ESI mass (m/z): 430.3 [M+H]+.

1-[(2,5-Dimethylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5v): 1H

NMR (DMSO-d6, ppm): 7.72 – 7.76 (3H, m), 7.63 – 7.65 (1H, d, J = 7.6 Hz), 7.39 – 7.41 (1H,

d, J = 7.5 Hz), 7.23 – 7.29 (3H, m), 3.63 (2H, s), 2.35 (6H, s), 2.17 – 2.32 (8H, m), 2.14 (3H, s);

ESI mass (m/z): 398.4 [M+H]+.

3-(4-Methylpiperazin-1-ylmethyl)-1-(naphthalene-1-sulfonyl)-1H-indole (5w): 1H NMR

(CDCl3, ppm): 8.68 – 8.70 (1H, d, J = 8.5 Hz), 8.18 – 8.20 (1H, d, J = 7.3 Hz), 8.03 – 8.05

(1H, d, J = 8.1 Hz), 7.86 – 7.88 (1H, d, J = 8.1 Hz), 7.77 – 7.79 (1H, d, J = 8.1 Hz), 7.70 (1H,

s), 7.59 – 7.64 (2H, m), 7.49 – 7.56 (2H, m), 7.16 – 7.24 (2H, m), 3.65 (2H, s), 2.56 – 2.59 (8H,

bs), 2.41 (3H, s); ESI mass (m/z): 420.0 [M+H]+; HPLC = 95.6%.

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4-Chloro-1-[(4-isopropylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5x): IR (KBr, cm-1

): 2941, 2797, 1592, 1455, 1373, 1179, 774; 1H NMR (CDCl3, ppm): 7.88 –

7.93 (1H, m), 7.75 – 7.77 (2H, d, J = 8.4 Hz), 7.52 (1H, s), 7.26 – 7.28 (2H, d, J = 8.5 Hz), 7.17

– 7.21 (2H, m), 3.81 (2H, s), 2.86 – 2.93 (1H, m), 2.46 – 2.57 (8H, bs), 2.29 (3H, s), 1.18 – 1.20

(6H, d, J = 6.9 Hz); ESI mass (m/z): 446.4, 448.3 [M+H]+; HPLC = 98.7%; Elemental analysis

calculated (%) for C23H28ClN3O2S: C 61.94, H 6.33, N 9.42. Found: C 61.89, H 6.34, N 9.44.

4-Chloro-1-[(4-fluorophenyl)sulfonyl]- 3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5y): IR (KBr, cm-1

): 2938, 1572, 1443, 1372, 1175, 789; 1

H NMR (CDCl3, ppm): 7.86 – 7.90

(3H, m), 7.49 (1H. s), 7.19 – 7.21 (2H, m), 7.09 – 7.14 (2H, m), 3.62 (2H, s), 2.45 – 2.47 (8H,

bs), 2.26 (3H, s); ESI mass (m/z): 421.3, 423.5 [M+H]+; HPLC = 98.9%.

1-[(2-Bromophenyl)sulfonyl]-4-chloro-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5z): IR (KBr, cm-1

): 2936, 2796, 1574, 1449, 1375, 1178, 787; 1

H NMR (CDCl3, ppm): 8.12 –

8.14 (1H, dd, J = 1.6, 7.9 Hz), 7.74 (1H, s), 7.66 – 7.68 (1H, m), 7.55 – 7.57 (1H, d, J = 7.8 Hz),

7.49 (1H, d, J = 1.1 Hz), 7.41 – 7.42 (1H, d, J = 1.6 Hz), 7.18 – 7.20 (1H, d, J = 7.4 Hz), 7.11 –

7.13 (1H, d, J = 8.1 Hz), 3.86 (2H, s), 2.46 – 2.67 (8H, bs), 2.30 (3H, s); ESI mass (m/z): 482.2,

484.1 [M+H]+; HPLC = 95.6%.

5-Chloro-1-[(4-fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5aa): IR (KBr, cm-1

): 2936, 2796, 1591, 1493, 1376, 1181, 674, 587; 1H NMR (DMSO-d6,

ppm): 8.03 – 8.07 (2H, m), 7.92 – 7.94 (1H, d, J = 8.8 Hz), 7.77 (1H, s), 7.75 (1H, d, J = 1.8

Hz), 7.42 – 7.46 (2H, t, J = 8.7 Hz), 7.36 – 7.39 (1H, dd, J = 1.9, 8.7 Hz), 3.55 (2H, s), 2.30 –

2.34 (8H, bs), 2.15 (2H, s); ESI mass (m/z): 421.9, 423.8 [M+H]+; Elemental analysis calculated

(%) for C20H21ClFN3O2S: C 56.93, H 5.02, N 9.96. Found: C 56.90, H 5.03, N 9.94.

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6-Chloro-1-(phenylsulfonyl)-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5ab): IR

(KBr, cm-1

): 2937, 2796, 1557, 1447, 1372, 1176, 753; 1H NMR (CDCl3, ppm): 7.99 (1H, d, J

= 1.7 Hz), 7.85 – 7.88 (2H, m), 7.60 – 7.62 (2H, d, J = 8.4 Hz), 7.54 – 7.56 (2H, m), 7.43 – 7.48

(3H, m), 7.19 – 7.21 (1H, dd, J = 1.8, 8.4 Hz), 3.56 (2H, s), 2.36 – 2.48 (8H, bs), 2.10 (3H, s);

13C NMR (CDCl3, ppm): 137.8, 135.6, 133.9, 130.7, 129.2, 129.2, 126.6, 124.8, 123.7, 121.4,

119.6, 113.6, 54.9, 53.2, 52.9, 45.8; ESI mass (m/z): 404.4, [M+H]+; HPLC = 99.2%, Melting

range (°C): 130 – 133.3; Elemental analysis calculated (%) for C20H22ClN3O2S: C 59.47, H 5.49,

N 10.40. Found: C 59.52, H 5.50, N 10.42.

1-[(4-Bromophenyl)sulfonyl]-6-chloro-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ac): IR (KBr, cm-1

): 2938, 2801, 1574, 1377, 1137, 755; 1H NMR (CDCl3, ppm): 7.96 (1H,

d, J = 1.7 Hz), 7.71 – 7.73 (2H, m), 7.58 – 7.62 (3H, m), 7.39 (1H, s), 7.21 – 7.23 (1H, dd, J =

1.8, 8.4 Hz), 3.56 (2H, s), 2.43 – 2.45 (8H, bs), 2.27 (3H, s); ESI mass (m/z): 482.2, 484.3

[M+H]+; HPLC = 98.5%.

1-[(2-Bromophenyl)sulfonyl]-5-Fluoro-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ae): IR (KBr, cm-1

): 3122, 2937, 2838, 2794, 1447, 1373, 1175, 1145, 897, 593; 1H NMR

(CDCl3, ppm): 8.10 – 8.12 (1H, dd, J = 1.6, 7.9 Hz), 7.69 (1H, s), 7.66 – 7.68 (1H, dd, J = 1.3,

7.8 Hz), 7.56 – 7.58 (1H, m), 7.40 – 7.48 (3H, m), 6.93 – 6.95 (1H, m), 3.61 (2H, s), 2.45 (8H,

bs), 2.28 (3H, s); 13

C NMR (DMSO-d6, ppm): 160.2, 157.9, 136.8, 136.5, 131.9, 131.8, 131.0,

129.2, 128.6, 119.8, 118.1, 114.3, 112.8, 106.9; ESI mass (m/z): 466.4, 468.3 [M+H]+; HPLC =

98.8%.

1-[(4-Bromophenyl)sulfonyl]-5-Fluoro-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5af): IR (KBr, cm-1

): 2940, 2795, 1445, 1368, 1183, 741, 587; 1H NMR (CDCl3, ppm): 7.86 –

7.90 (1H, m), 7.68 – 7.70 (2H, m), 7.55 – 7.58 (2H, m), 7.44 (1H, s), 7.36 – 7.39 (1H, dd, J =

S30

2.5, 8.8 Hz), 7.02 – 7.07 (1H, m), 3.52 (2H, s), 2.44 (8H, bs), 2.28 (3H,s); ESI mass (m/z):

466.4, 468.3 [M+H]+; HPLC = 98.6%.

5-Bromo-1-[(4-fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ag): IR (KBr, cm-1

): 2940, 2803, 1590, 1444, 1376, 1182, 682, 584; 1H NMR (CDCl3, ppm):

7.81 – 7.88 (4H, m), 7.40 – 7.44 (2H, m), 7.09 – 7.13 (2H, m), 3.54 (3H, s), 2.44 (8H, bs), 2.28

(3H, s); ESI mass (m/z): 468.3, 466.2 [M+H]+; HPLC = 98.7%.

5-Bromo-1-[(4-isopropylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ah): IR (KBr, cm-1

): 2966, 2795, 2768, 1438, 1371, 1179, 809, 653; 1H NMR (CDCl3, ppm):

7.73 – 7.87 (3H, m), 7.37 – 7.45 (2H, m), 7.25 – 7.29 (3H, m), 3.55 (2H, s), 2.82 – 2.96 (1H, m),

2.41 – 2.49 (8H, bs), 2.28 (3H, s), 1.19 – 1.22 (6H, d, J = 6.8 Hz); ESI mass (m/z): 490.2, 492.1

[M+H]+; HPLC = 97.6%.

1-[(2-Bromophenyl)sulfonyl]-4-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

Dihydrochloride (5aj): IR (KBr, cm-1

): 3422, 2949, 2848, 1497, 1368, 1095, 768, 610, 589; 1H

NMR (D2O, ppm): 8.28 – 8.26 (1H, d, J = 7.9 Hz), 8.00 (1H, s), 7.63 – 7.65 (1H, d, J = 7.8

Hz), 7.52 – 7.56 (1H, m), 7.45 – 7.47 (1H, m), 7.13 – 7.17 (1H, m), 7.05 – 7.07 (1H, d, J = 8.2

Hz), 6.74 – 6.76 (1H, d, J = 8.0 Hz), 4.62 (2H, s), 3.84 (3H, s), 3.58 (8H, bs), 2.90 (3H, s); 13

C

NMR (D2O, ppm): 153.5, 136.2, 135.9, 135.1, 134.9, 132.3, 130.1, 128.3, 127.1, 119.7, 118.0,

107.1, 105.5, 105.0, 55.4, 52.2, 50.0, 48.0, 42.8. ESI mass (m/z): 478.3, 480.3 [M+H]+; HPLC =

97.6%.

1-[(4-Fluorophenyl)sulfonyl]-4-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ak): IR (KBr, cm-1

): 2938, 2796, 1591, 1373, 1182, 789, 675; 1H NMR (CDCl3, ppm): 7.85 –

7.89 (2H, m), 7.54 – 7.56 (1H, d, J = 8.2 Hz), 7.35 (1H, s), 7.18 – 7.22 (1H, t, J = 8.1 Hz), 7.06

– 7.10 (2H, m), 6.62 – 6.64 (1H, d, J = 8.4 Hz), 3.85 (3H, s), 3.79 (2H, s), 2.35 – 2.48 (8H, bs),

S31

2.28 (3H, s); 13

C NMR (CDCl3, ppm): 166.7, 164.2, 154.4, 136.6, 134.0, 129.5, 125.6, 122.8,

120.3, 116.4, 106.3, 104.0, 55.1, 55.1, 53.9, 52.9, 45.9; ESI mass (m/z): 418.5 [M+H]+; HPLC =

95.7%; Melting range (°C): 111.8 – 113.9.

5-Methoxy-1-(phenylsulfonyl)-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5ao): IR

(KBr, cm-1

): 1145, 1162, 1366, 1344; 1H NMR (CDCl3, ppm): 7.79 – 7.85 (3H, m), 7.36 – 7.50

(4H, m), 7.07 – 7.08(1H, d, J = 2.2 Hz), 6.85 – 6.90 (1H, dd, J = 2.6, 9.0 Hz), 3.80 (3H, s), 3.53

(2H, s), 2.41 (8H, bs), 2.25 (3H, s); ESI mass (m/z): 400 [M+H]+; Melting range (°C): 120 – 123.

1-[(4-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ap): IR (KBr, cm-1

): 1147, 1162, 1365, 1451; 1H NMR (CDCl3, ppm): 7.82 – 7.84 (1H, d, J =

8.9 Hz), 7.66 – 7.69 (2H, m), 7.52 – 7.55 (2H, m), 7.37 (1H, s), 7.13 – 7.15 (1H, d, J = 2.6 Hz),

6.91 – 6.94 (1H, dd, J = 2.5, 7.8 Hz), 3.82 (3H, s), 3.55 (2H, s), 2.44 (8H, bs), 2.28 (3H, s); ESI

mass (m/z): 479, 481 [M+H]+.

5-Methoxy-1-(naphthalene-1-sulfonyl)-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5aq): IR (KBr, cm-1

): 2936, 2797, 1475, 1365, 1171, 769, 596; 1H NMR (CDCl3, ppm): 8.69 –

8.71 (1H, d, J = 8.6 Hz), 8.07 – 8.09 (1H, dd, J = 1.0, 7.4 Hz), 8.01 – 8.03 (1H, d, J = 8.2 Hz),

7.85 – 7.87 (1H, d, J = 7.8 Hz), 7.66 – 7.68 (1H, d, J = 9.0 Hz), 7.64 (1H, s), 7.48 – 7.60 (3H,

m), 7.10 – 7.11 (1H, d, J = 2.4 Hz), 6.83 – 6.85 (1H, dd, J = 2.5, 9.0 Hz), 3.79 (3H, s), 3.55 (2H,

s), 2.42 – 2.43 (8H, bs), 2.27 (3H, s); 13

C NMR (CDCl3, ppm): 156.1, 136.3, 134.1, 132.5,

131.7, 130.5, 129.7, 129.1, 127.6, 127.3, 126.7, 124.9, 123.6, 119.0, 113.9, 113.5, 103.8, 55.6,

55.0, 52.7, 52.4, 46.0; ESI mass (m/z): 450.2 [M+H]+; HPLC = 95.8%.

5-Methoxy-1-[(4-methylphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ar): IR (KBr, cm-1

):2927, 1598, 1383, 1227, 1172, 871; 1H NMR (DMSO-d6, ppm):7.76 –

7.79 (3H, m), 7.59 (1H, s), 7.34 – 7.36 (2H, d, J = 8.0 Hz), 7.16 – 7.17 (1H, d, J = 1.6 Hz), 6.92

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– 6.95 (1H, dd, J = 2.0, 8.9 Hz), 3.74 (3H, s), 3.56 (2H, s), 2.36 – 2.40 (8H, bs), 2.30 (3H, s),

2.26 (3H, s); ESI mass (m/z): 414.0 [M+H]+; HPLC = 98.6%.

5-Methoxy-1-[(4-methoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5as): IR (KBr, cm-1

): 2926, 1595, 1475, 1369, 1165, 678; 1H NMR (DMSO-d6, ppm): 7.77 –

7.84 (3H, m), 7.58 (1H, s), 7.16 – 7.17 (1H, d, J = 1.2 Hz), 7.04 – 7.06 (2H, d, J = 8.7 Hz), 6.92

– 6.95 (1H, dd, J = 2.0, 9.0 Hz), 3.77 (3H, s), 3.75 (3H, s), 3.56 (2H, s), 2.41 – 2.49 (8H, bs),

2.25 (3H, s); ESI mass (m/z): 43.0 [M+H]+; HPLC = 99.7%.

5-Methoxy-1-[(4-trifluoromethoxyphenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-

1H-indole (5at): IR (KBr, cm-1

): 2973, 2723, 1411, 1377, 1145, 761, 616; 1H NMR (CDCl3,

ppm): 7.84 – 7.89 (3H, m), 7.38 (1H, s), 7.21 – 7.23 (2H, m), 7.14 – 7.15 (1H, d, J = 2.5 Hz),

6.92 – 6.95 (1H, dd, J = 2.5, 9.0 Hz), 3.83 (3H, s), 3.56 (2H, s), 2.43 – 2.45 (8H, bs), 2.28 (3H,

s); 13

C NMR (DMSO-d6, ppm): 156.4, 152.1, 135.8, 132.1, 129.7, 129.6, 126.0, 122.0, 121.0,

114.5, 113.9, 103.9, 55.7, 55.0, 52.7, 52.6, 46.0. ESI mass (m/z): 484.1 [M+H]+; HPLC =

97.7%; Melting range (°C): 99 – 100.6.

1-[(4-Bromophenyl)sulfonyl]-5-isopropoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5au): IR (KBr, cm-1

): 2972, 2928, 1463, 1387, 1102, 737; 1H NMR (CDCl3, ppm): 8.02 – 8.07

(1H, dd, J = 1.8, 7.8 Hz), 7.63 – 7.68 (2H, m), 7.49 – 7.54 (1H, d, J = 8.8 Hz), 7.37 – 7.48 (2H,

m), 7.19 – 7.20 (1H, d, J = 2.6 Hz), 6.78 – 6.84 (1H, dd, J = 2.6, 8.8 Hz), 4.49 – 4.55 (1H, m),

3.60 (2H, s), 2.39 – 2.45 (8H, bs), 2.28 (3H, s), 1.35 – 1.32 (6H, d, J = 6.2 Hz); ESI mass (m/z):

506.4, 508.3 [M+H]+; HPLC = 96.7%.

1-[(4-Bromophenyl)sulfonyl]-5-trifluoroethoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-

indole (5av): IR (KBr, cm-1

): 2937, 2796, 1573, 1370, 1174, 797, 675; 1H NMR (CDCl3, ppm):

7.86 – 7.88 (1H, d, J = 9.0 Hz), 7.66 – 7.69 (2H, m), 7.54 – 7.56 (2H, m), 7.40 (1H, s), 7.21 (1H,

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d, J = 2.5 Hz), 6.97 – 6.99 (1H, dd, J = 2.5, 9.0 Hz), 4.33 – 4.39 (2H, m), 3.55 (2H, s), 2.32 –

2.51 (8H, bs), 2.28 (3H, s); ESI mass (m/z): 546.4 [M+H]+; HPLC = 95.3%.

1-[(4-Isopropylphenyl)sulfonyl]-6-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5aw): 1H NMR (CDCl3, ppm): 7.75 – 7.78 (2H, m), 7.51 – 7.56 (1H, d, J = 8.6 Hz), 7.51 –

7.52 (1H, d, J = 2.2 Hz), 7.34 (1H, s), 7.25 – 7.27 (2H, m), 6.84 – 6.87 (1H, dd, J = 2.2, 8.6 Hz),

3.87 (3H, s), 3.55 (2H, s), 2.86 – 2.93 ( 1H, m), 2.43 (8H, bs), 2.27 (3H, s), 1.18 – 1.20 (6H, d, J

= 6.6 Hz); ESI mass (m/z): 442.5 [M+H]+; HPLC = 99.0%; Elemental analysis calculated (%)

for C24H31N3O3S: C 65.28, H 7.08, N 9.52. Found: C 65.47, H 7.06, N 9.51.

1-[(4-Fluorophenyl)sulfonyl]-6-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5ax): 1H NMR (CDCl3, ppm): 7.85 – 7.88 (2H, m), 7.55 – 7.57 (1H, d, J = 8.68 Hz), 7.49 –

7.49 (1H, d, J = 2.2 Hz), 7.31 (1H, s), 7.07 – 7.12 (2H, m), 6.86 – 6.88 (1H, dd, J = 2.2, 8.7 Hz),

3.87 (3H, s), 3.55 (2H, s), 2.44 (8H, bs), 2.27 (3H, s); 13

C NMR (DMSO – d6, ppm): 166.8,

164.3, 158.0, 136.1, 133.4, 130.3, 124.6, 123.8, 121.8, 120.6, 117.4, 112.4, 97.9, 55.8, 55.0, 52.8,

46.0. ESI mass (m/z): 418.5 [M+H]+; HPLC = 98.9%.

1-[(4-Isopropylphenyl)sulfonyl]-5-hydroxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

Dihydrochloride (5az): IR (KBr, cm-1

): 3402, 3244, 2961, 1595, 1461, 1373, 1174, 596; 1H

NMR (D2O, ppm): 7.83 (1H, s), 7.49 – 7.51 (2H, d, J = 8.2 Hz), 7.26 – 7.28 (1H, d, J = 8.9

Hz), 6.91 (1H, d, J = 1.7 Hz), 6.81 – 6.83 (2H, d, J = 8.3 Hz), 6.36 – 6.38 (1H, d, J = 9.9 Hz),

4.29 (2H, s), 3.41 – 3.45 (8H, bs), 2.87 (3H, s), 2.25 – 2.30 (1H, m), 0.56 – 0.57 (6H, d, J = 6.8

Hz); ESI mass (m/z): 428.4 [M+H]+; HPLC = 99.1%.

1-[(2-Fluorophenyl)sulfonyl]-5-hydroxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

Dihydrochloride (5ba): IR (KBr, cm-1

): 3420, 3193, 2987, 1589, 1445, 1366, 1173, 587; 1H

NMR (DMSO-d6, ppm): 11.56 – 11.86 (2H, bs), 9.54 (1H, bs), 8.04 – 8.07 (3H, m), 7.68 –

S34

7.70 (1H, d, J = 8.8 Hz), 7.40 – 7.44 (2H, t, J = 8.7 Hz), 7.13 (1H, s), 6.86 – 6.88 (1H, d, J = 7.7

Hz), 4.34 (2H, s), 3.30 – 3.34 (4H, bs), 3.54 – 3.58 (4H, bs), 2.77 (3H, s); ESI mass (m/z): 404.3

[M+H]+; DSC: 234.88 °C; HPLC = 99.6%.

1-[(2-Bromophenyl)sulfonyl]-5-nitro-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (5bb):

IR (KBr, cm-1

): 1121, 1175, 1286, 1330; 1370, 1508; 1H NMR ( ppm): 8.68 – 8.70 (1H, d, J =

2.6 Hz), 8.28 – 8.32 (1H, dd, J = 1.8, 9.6 Hz), 8.10 – 8.16 (1H, dd, J = 2.2, 9.1 Hz), 7.80 (1H, s),

7.23 – 7.75 (4H, m), 3.70 (2H, s), 2.53 (8H, bs), 2.33 (3H, s); ESI mass (m/z): 461, 463 [M+H]+;

Melting Range (°C): 224.7 – 226.1.

1-[(2-Bromophenyl)sulfonyl]-5-methylsulfanyl-3-[(4-methyl-1-piperazinyl)methyl]-1H-

indole (5bc): 1H NMR (CDCl3, ppm): 8.09 – 8.12 (1H, dd, J = 1.6, 7.9 Hz), 7.65 – 7.67 (3H,

m), 7.54 – 7.56 (1H, d, J = 8.6 Hz), 7.45 – 7.51 (1H, m), 7.37 – 7.43 (1H, m), 7.16 – 7.19 (1H,

dd, J = 1.8, 8.6 Hz), 3.63 (2H, s), 2.50 (8H, bs), 2.47 (3H, s), 2.30 (3H, s); ESI mass (m/z):

494.3, 496.7 [M+H]+; HPLC = 95.2%.

1-[(4-Fluorophenyl)sulfonyl]-2-methyl-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5bd): 1H NMR (CDCl3, ppm): 8.14 – 8.16 (2H, d, J = 8.0 Hz), 7.73 – 7.76 (2H, m), 7.60 –

7.62 (1H, d, J = 7.1 Hz), 7.22 – 7.29 (2H, m), 7.04 – 7.08 (2H, t, J = 8.3 Hz), 3.5 (2H, s), 2.56

(3H, s), 2.35 – 2.39 (8H, bs), 2.28 (3H, s); 13

C NMR (CDCl3, ppm): 166.7, 164.2, 136.2, 134.8,

130.9, 129.1, 129.0, 124.1, 123.6, 124.1, 123.6, 119.5, 117.5, 116.6, 116.4, 114.4, 55.0, 52.9,

52.1, 45.9, 13.0; ESI mass (m/z): 402.3 [M+H]+.

2-Chloro-1-(4-isopropyl benzenesulfonyl)-5-methoxy-3-(4-methyl piperazin-1-ylmethyl)-

1H-indole (5be): 1H NMR (CDCl3, ppm): 7.85 – 7.87 (2H, d, J = 7.9 Hz), 7.41 – 7.43 (2H, d, J

= 7.8 Hz), 7.13 – 7.15 (1H, d, J = 7.2 Hz), 6.72 – 6.74 (1H, dd, J = 2.1, 7.1 Hz), 6.65 (1H, d, J =

S35

2.1 Hz), 3.73 (3H, s), 3.50 (2H, s), 2.82 (1H, m), 2.42 (8H, bs), 2.27 (3H, s), 1.12 – 1.14 (6H, d,

J = 6.9 Hz); ESI mass (m/z): 475.1, 477.4 [M+H]+.

1-(2-Bromo benzenesulfonyl)-5-methoxy-3-(4-methyl-[1,4]diazepane-1-yl methyl)-1H-

indole (5bf): 1H NMR (CDCl3, ppm): 8.03 – 8.05 (1H, dd, J = 1.6, 7.8 Hz), 7.64 – 7.67 (1H,

dd, J = 1.2, 7.8 Hz), 7.62 (1H, s), 7.52 – 7.54 (1H, d, J = 9.0 Hz), 7.38 – 7.45 (2H, m), 7.20 (1H,

d, J = 2.8 Hz), 6.85 (1H, d, J = 2.5 Hz), 3.83 (3H, s), 3.74 (2H, s), 2.67 – 2.79 (8H, bs), 2.41

(3H, s), 1.87 – 1.90 (2H, m); ESI mass (m/z): 492.4, 494.3 [M+H]+; HPLC = 97.1%.

1-(4-Fluoro benzenesulfonyl)-5-methoxy-3-(4-methyl-[1,4]diazepane-1-yl methyl)-1H-

indole Dihydrochloride (5bh): 1H NMR (D2O, ppm): 7.95 – 7.97 (2H, m), 7.47 – 7.49 (2H,

m), 7.34 – 7.36 (1H, d, J = 7.6 Hz), 7.15 (1H, s), 6.71 – 6.73 (2H, m); ESI mass (m/z) 432.5

[M+H]+.

1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-ethyl-1-piperazinyl)methyl]-1H-indole

(5bi): IR (KBr, cm-1

): 2934, 2810, 1373, 1178, 758; 1H NMR (CDCl3, ppm): 8.02 – 8.05 (1H,

dd, J = 1.7, 7.9 Hz), 7.64 – 7.66 (2H, dd, J = 1.4, 8.2 Hz), 7.52 – 7.55 (1H, d, J = 9.0 Hz), 7.37 –

7.45 (2H, m), 7.18 – 7.19 (1H, d, J = 2.4 Hz), 6.83 – 6.85 (1H, dd, J = 2.5, 6.4 Hz), 3.82 (3H, s),

3.62 (2H, s), 2.54 (8H, bs), 2.39 – 2.44 (2H, q, J = 7.2 Hz), 1.06 – 1.10 (3H, t, J = 7.2 Hz); ESI

mass (m/z): 492.1, 494.3 [M+H]+; HPLC = 98.1%.

1-[(4-Isopropylphenyl)sulfonyl]-5-methoxy-3-[(4-ethyl-1-piperazinyl)methyl]-1H-indole

(5bj): IR (KBr, cm-1

): 2964, 2809, 1474, 1371, 1173, 778; 1H NMR (CDCl3, ppm):7.85 – 7.87

(1H, d, J = 9.0 Hz), 7.73 – 7.75 (2H, d, J = 8.4 Hz), 7.42 (1H, s), 7.23 – 7.25 (2H, m), 7.13 (1H,

d, J = 2.5 Hz), 6.90 – 6.93 (1H, d, J = 2.5, 9.0 Hz), 3.82 (3H, s), 3.57 (2H, s), 2.85 – 2.91 (1H,

m), 2.41 – 2.47 (8H, bs), 2.37 – 2.39 (2H, m), 1.17 – 1.19 (6H, d, J = 6.9 Hz), 1.05 – 1.09 (3H, t,

J = 7.2 Hz); ESI mass (m/z): 456.2 [M+H]+; HPLC = 97.7%.

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1-[(4-Fluorophenyl)sulfonyl]-5-methoxy-3-[(4-ethyl-1-piperazinyl)methyl]-1H-indole

(5bk): IR (KBr, cm-1

): 2945, 1591, 1371, 1178, 772; 1H NMR (CDCl3, ppm): 7.82 – 7.85 (3H,

m), 7.36 (1H, s), 7.15 – 7.16 (1H, d, J = 2.4 Hz), 7.05 -7.09 (2H, m), 6.91 – 6.94 (1H, dd, J =

2.5, 9.0 Hz), 3.82 (3H, s), 3.66 (2H, s), 2.45 (8H, bs), 2.31 – 2.39 (2H, q, J = 7.0 Hz), 1.02 –

1.05 (3H, t, J = 7.1 Hz); ESI mass (m/z): 432.2 [M+H]+; HPLC = 95.9%.

5-Methoxy-1-(phenylsulfonyl)-3-[(4-ethyl-1-piperazinyl)methyl]-1H-indole (5bl): IR (KBr,

cm-1

): 2936, 2811, 1474, 1371, 1175, 724; 1H NMR (CDCl3, ppm): 7.82 – 7.87 (3H, m), 7.49 –

7.54 (1H, m), 7.39 – 7.43 (3H, m), 7.12 – 7.13 (1H, d, J = 2.5 Hz), 6.90 – 6.93 (1H, dd, J = 2.5,

9.0 Hz), 3.81 (3H, s), 3.56 (2H, s), 2.45 – 2.48 (8H, bs), 2.37 – 2.43 (2H, q, J = 7.1 Hz), 1.05 –

1.09 (3H, t, J = 7.1 Hz); ESI mass (m/z): 414.5 [M+H]+; HPLC = 98.5%.

1-(2-Bromo benzenesulfonyl)-5-methoxy-3-(4-methyl piperazin-1-ylmethyl)-1H-indazole

(5bp): 1H NMR (CDCl3, ppm): 8.27 – 8.29 (1H, dd, J = 1.5, 7.9 Hz), 8.04 – 8.06 (1H, d, J =

9.1 Hz), 7.65 – 7.66 (1H, d, J = 7.1 Hz), 7.49 – 7.53 (1H, t, J = 7.5 Hz), 7.40 – 7.44 (1H, m),

7.37 – 7.38 (1H, d, J = 2.3 Hz), 7.17 – 7.20 (1H, d, J = 2.4, 9.1 Hz), 3.88 (3H, s), 3.80 (2H, s),

2.50 (8H, bs), 2.30 (3H, s); ESI mass (m/z): 479.2, 481.2 [M+H]+; HPLC = 95.5%.

3-[(4-Methyl-1-piperazinyl)methyl]-1H-indole (8a): 1H NMR (DMSO-d6, ppm): 10.89

(1H, bs), 7.60 – 7.62 (1H, d, J = 7.8 Hz), 7.32 – 7.34 (1H, d, J = 8.0 Hz), 7.19 (1H, s), 7.03 –

7.07 (1H, t, J = 7.2 Hz), 6.94 – 6.98 (1H, t, J = 7.4 Hz), 3.58 (2H, s), 2.17 – 2.37 (8H, m), 2.12

(3H, s); ESI mass (m/z): 230.2 [M+H]+.

1-(Phenylsulfonyl)-3-[(1-piperazinyl)methyl]-1H-indole (11a): 1H NMR (DMSO-d6,

ppm): 7.90 – 7.95 (3H, m), 7.66 – 7.70 (3H, m), 7.55 – 7.59 (2H, m), 7.32 – 7.35 (1H, m), 7.23 –

7.27 (1H, m), 3.54 (2H, s), 3.29 (1H, bs), 2.62 – 2.66 (4H, m), 2.18 – 2.38 (4H, m); ESI mass

(m/z): 356.3 [M+H]+.

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1-[(2-Bromophenyl)sulfonyl]-3-[(1-piperazinyl)methyl]-1H-indole Dihydrochloride

(11b): 1H NMR (D2O, ppm): 7.82 – 7.84 (1H, m), 7.71 – 7.73 (1H, m), 7.49 – 7.52 (2H, m),

7.21 – 7.35 (4H, m), 7.12 (1H,s), 3.51 (2H, s), 2.68 (4H, bs), 2.48 (4H, bs); ESI mass (m/z):

434.1, 436.2 [M+H]+.

1-[(4-Isopropylphenyl)sulfonyl]-5-methoxy-3-[(1-piperazinyl)methyl]-1H-indole

Dihydrochloride (11d): IR (KBr, cm-1

): 3446, 2962, 1593, 1372, 1173, 962, 662, 597; 1H NMR

(D2O, ppm): 7.91 (1H, s), 7.53 – 7.59 (3H, m), 7.04 – 7.05 (1H, d, J = 1.8 Hz), 6.92 – 6.94

(2H, d, J = 8.3 Hz), 6.58 – 6.61 (1H, dd, J = 1.6, 8.8 Hz), 4.38 (2H, s), 3.57 (3H, s), 3.42 (8H,

bs), 2.42 – 2.45 (1H, m), 0.69 – 070 (6H, d, J = 6.8 Hz); 13

C NMR (D2O, ppm): 156.2, 133.7,

130.6, 130.4, 128.8, 127.4, 126.9, 114.1, 113.9, 110.3, 102.5, 55.6, 50.6, 47.9, 40.9, 33.4, 22.5;

ESI mass (m/z): 428.3 [M+H]+; DSC: 143.15 – 153.95 °C; HPLC = 98.5%.

1-[(4-Fluorophenyl)sulfonyl]-5-methoxy-3-[(1-piperazinyl)methyl]-1H-indole Dimesylate

(11e): 1H NMR (D2O, ppm): 7.86 – 7.89 (3H, m), 7.74 – 7.76 (1H, d, J = 9.0 Hz), 7.09 – 7.14

(3H, m), 6.93 – 6.96 (1H, dd, J = 2.2, 9.2 Hz), 4.45 (2H, s), 3.75 (3H, s), 3.45 – 3.49 (8H, bs),

2.68 (6H, s); 13

C NMR (D2O, ppm): 167.1, 164.5, 156.3, 131.9, 131.9, 130.5, 130.5, 129.8,

129.7, 128.9, 116.9, 116.7, 114.3, 114.2, 109.9, 102.3, 55.7, 50.6, 47.9, 40.7, 40.7, 38.4; ESI

mass (m/z): 404.2 [M+H]+.

1-(2-Bromo benzenesulfonyl)-3-(piperazin-1-ylmethyl)-1H-pyrrolo[3,2-b]pyridine

Dihydrochloride (11f): 1H NMR (DMSO-d6, ppm): 9.54 (2H, bs), 8.64 (1H, s), 8.61 – 8.62

(1H, d, J = 4.1 Hz), 8.38 – 8.40 (1H, d, J = 7.5 Hz), 8.01 – 8.03 (1H, d, J = 8.2 Hz), 7.86 – 7.87

(1H, d, J = 7.4 Hz), 7.65 – 7.74 (2H, m), 7.37 – 7.39 (1H, m), 4.6 (2H, s), 3.44 (8H, bs); ESI

mass (m/z): 435.1, 437.2 [M+H]+; HPLC = 99.6%.

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4-[1-(2-Bromobenzenesulfonyl)-1H-indol-3-ylmethyl]piperazin-1-ylphenylmethanone

Hydrochloride (12a): 1H NMR (DMSO-d6, ppm): 10.81 (1H, bs), 8.34 – 8.36 (1H, d, J = 7.3

Hz), 8.27 (1H, s), 7.93 – 7.95 (1H, d, J = 6.7 Hz), 7.63 – 7.73 (2H, m), 7.57 – 7.59 (1H, d, J =

7.7 Hz), 7.46 – 7.49 (5H, m), 7.31 – 7.38 (2H, m), 4.57 (2H, s), 3.34 – 3.44 (4H, m), 3.07 – 3.20

(4H, m); ESI mass (m/z): 538.1, 540.1 [M+H]+.

3-(4-Benzyl piperazin-1-ylmethyl)-1-(2-bromo benzenesulfonyl)-1H-indole (13a): 1H NMR

(DMSO-d6, ppm): 8.15 – 8.17 (1H, d, J = 7.5 Hz), 7.84 – 7.86 (1H, d, J = 7.5 Hz), 7.75 – 7.79

(1H, m), 7.57 – 7.66 (3H, m), 7.23 – 7.30 (8H, m), 3.64 (2H, s), 3.44 (2H, s), 2.36 – 2.42 (8H,

m); ESI mass (m/z): 524.1, 526.2 [M+H]+.

5-Bromo-1-(3-bromo benzenesulfonyl)-3-[2-(4-methyl piperazin-1-yl)ethyl]-1H-indole

Dihydrochloride (21b): 1H NMR (DMSO-d6, ppm): 11.44 – 11.54 (2H, bs), 8.16 (1H, s), 7.91

– 7.99 (4H, m), 7.85 (1H, s), 7.53 – 7.56 (2H, m), 3.46 – 3.68 (4H, m), 3.09 – 3.27 (8H, m), 2.82

(3H, s); ESI mass (m/z): 542.2, 544.1 [M+H]+.

S39

Supplementary Figures:

NH

O

NN

(8h)

S40

NH

O

NN

(8h)

S41

N

O

NN

SO2

Br

Free base of 5al

S42

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S43

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S44

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S45

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S46

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S47

S48

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S49

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S50

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S51

N

O

NN

SO2

Br

CH3SO3H

CH3SO3H

H2O

(5al)

S52

N

OCHO

SO2

(15d)

S53

N

O

SO2

(16d)

OH

S54

N

O

SO2

(17d)

Cl

S55

N

O

SO2

(5bg)

N

N

2HCl

S56

N

O

SO2

(5bg)

N

N

2HCl

S57

NH

N

OBr

N

(19)

S58

NH

N

Br

N

(20)

S59

N

N

Br

N

(21a)

SO2

Br

2HCl

S60

N

HO

SO2

Br

NN

2HCl

(5ay)

S61

N

HO

SO2

Br

NN

2HCl

(5ay)

S62

N

O

SO2

Br

NNH

CH3SO3H

(11c)

CH3SO3H

S63

N

O

SO2

Br

NNH

CH3SO3H

(11c)

CH3SO3H

S64

N

N N

SO O

Br

O

(12b)

S65

N

O

SO2

NN

F

Br

(13b)

S66

N N

SO2

NN

(5bo)

S67

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Intermediate 1: 4-N, N-Dimethylamino cyclohexanone