intermediate 1: 4-n, n-dimethylamino cyclohexanone
TRANSCRIPT
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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:
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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
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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
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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:
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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
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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
A stable CHO cell line expressing
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.
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
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 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
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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
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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
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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
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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
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Selectivity Panel for Compound 5al:
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Determination of Adrenergic Alpha 2A Receptor Ki by Radioligand Binding Assay:
The procedure used was previously described.10, 11
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.
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Determination of Kb Values for Adrenergic Alpha 2C Receptor:
The procedure used was previously described.12, 13
A stable CHO cell line expressing
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.
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
overnight at 37 oC and 5% CO2 followed by serum starvation for 18-20 h. Increasing
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.
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Determination of Dopamine D3 receptor Ki by Radioligand Binding Assay:
The procedure used was previously described.14, 15
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
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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
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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.
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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
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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)
S23
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]+.
S25
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
S32
– 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,
S33
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%.
S36
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]+.
S37
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%.
S38
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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