Synthesis and biological evaluation of novel N₁-phenylsulphonyl indole derivatives as potent and selective 5-HT₆R ligands for the treatment of cognitive disorders

Abstract

A series of 1-[3-(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole and 1-[3-(4-ethyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole derivatives were designed and synthesized as 5-HT₆ receptor (5-HT₆R) ligands. The lead compound 1-[4-methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6b), in this series, has shown potent in vitro binding affinity, selectivity, good pharmacokinetics (PK) profile and activity in the animal models of cognition.

Keywords:

• 5-Hydroxytryptamine-6 receptor
• animal models
• central nervous system
• cognition
• novel object recognition test
• structure–activity relationship

Introduction

Alzheimer disease (AD), the most common cause of dementia in the elderly, is clinically characterized by progressive cognitive impairment associated with severe neuropsychiatric disturbances. These behavioral and psychological symptoms of dementia (BPSD) include hallucinations, delusions, aggressive behavior, over activity, anxiety and affective disturbances1^,2. Current options for treating the cognitive symptoms associated with Alzheimer's are inadequate, giving urgency to the search for novel therapeutic strategies3–5. The 5-HT₆ receptor has been identified as a potential target for the treatment of cognitive deficits in Alzheimer’s disease and schizophrenia.

The serotonin-6 receptor (5-HT₆) is a G-protein-coupled receptor (GPCR) predominantly expressed in the central nervous system (CNS). In particular, it is widely reported to be located in brain regions associated with learning and memory such as the cerebral cortex, the hippocampus and the striatum6. It has been demonstrated that antagonism of the 5-HT₆ receptor modulates the release of a wide variety of neurotransmitters including elevation of extracellular levels of both glutamate and acetylcholine in brain regions such as the medial prefrontal cortex and the hippocampal formation7^,8. This modulatory activity suggests potential utility for 5-HT₆ receptor antagonists in the treatment of cognitive impairments associated with Alzheimer’s disease and schizophrenia.

Research efforts in this area have led to the discovery of a number of potent and selective 5-HT₆ receptor antagonists over the past decade, some of which have successfully undergone Phase-I clinical studies and some have been evaluated in clinical Phase-II studies for the treatment of AD9. As part of our own research program, we at Suven have designed and developed selective 5-HT₆ receptor competitive antagonist, SUVN-502 which has shown positive results in both preclinical and Phase-I study for cognitive impairment in schizophrenia and Alzheimer's disease10. Currently, several 5-HT₆R antagonists, for example SYN-120 (Biotie therapies)11, SAM-760 (Pfizer)12, AVN-211 (Figure 1, Avineuro Pharmaceuticals)13, Lu AE58054 (Figure 1, Lundbeck)14, SB-742457 (Figure 1, GlaxoSmithKline, Brentford, England, UK)15 are the advanced stage clinical candidates for the treatment of different cognitive and mental disorders including Alzheimer’s disease and schizophrenia.

Figure 1. Structures of 5-HT₆R antagonists in clinical trials for Alzheimer’s disease.

During this decade or so, there was a plenty of chemistry developed around different nuclei including the indole-based ones or even much simpler biphenyl sulfones and sulfonamides16–33^,34 (Figure 2). Suven has reported several series of 1-sulfonylindoles bearing the amino group at 3 or 4 or 5 position of the central core31–33. As part of our continuing research efforts in the 5-HT₆ area to identify potent and selective 5-HT₆ receptor ligands as potential treatments for cognitive dysfunction, we have attempted structural modification of earlier reported piperazinyl methyl-N₁-phenylsulfonyl indole derivatives33. We wished to determine whether introduction of piperazinyl methyl group on N₁-phenylsulfonyl moiety at C-3′ position would have a desired effect on the binding to 5-HT₆R and aimed to investigate the structure–activity relationship (SAR). Researchers have developed a pharmacophore model (Figure 3) for 5-HT₆ receptor antagonists, based on reported chemically diverse 5-HT₆ receptor antagonists35–42^,34. In this pharmacophore model, four core features were taken into consideration which include the positive ionizable atom (PI, usually a secondary or tertiary amino group), a hydrogen bond acceptor group (HBA, usually a sulfone or sulfonamide group), a hydrophobic site (HYD) and π-electron donor aromatic or heterocyclic ring (AR). We have designed and synthesized a series of sulfonyl indoles bearing basic cyclic amines (i.e. N₁-CH₃ and N₁-C₂H₅ substituted piperazines) on phenyl ring at C-3′ position (Figure 4), this designed series of compounds has all the required pharmacophoric features for binding to 5-HT₆R, and hence, we expected these compounds to be potent 5-HT₆R ligands. Herein, we report a series of 1-[3–(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole and 1-[3-(4-ethyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole derivatives as potent and selective 5-HT₆R ligands (Compounds-6, Figure 4).

Figure 2. Structures of known 5-HT₆ receptor ligands.

Figure 3. 5-HT₆ receptor pharmacophore model.

Figure 4. General structure of designed 5-HT₆ receptor ligands.

The selected compound 6b from this series was active in animal models of cognition. The synthesis, in vitro activity, selectivity, SAR, CYP liabilities in human liver microsomes, pharmacokinetics and pharmacological activities of this series of compounds are discussed in this paper.

Chemistry: The general synthetic strategy used for the preparation of 1-[3-(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole and 1-[3-(4-ethyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole derivatives (Compounds 6) was summarized in Scheme 1.

Scheme I. Reagents and conditions: (a) NaH, tetrahydrofuran, RT, 20–24 h; (b) LiAlH₄, 0–5 °C, 4–5 h; (c) MnO₂, Ethylene Dichloride, reflux, 3–4 h; (d) N-methyl (or) N-ethyl piperazine, Ethylene Dichloride, Na(OAC)₃BH, RT, 6–7 h; (e) Methanolic HCl (16% w/v), Diethylether, RT, 1 h.

Commercially available substituted 3-chlorosulfonyl benzoic acid 2 was reacted with substituted indoles 1 in the presence of sodium hydride as base at room temperature to obtain 3-(indole-1-sulfonyl) benzoic acid derivatives 3. The latter were treated with lithium aluminum hydride at 0 to 5 °C to obtain 1-(3-hydroxymethyl phenylsulfonyl)-1H-indole derivatives 4 in an overall yield of 33–55%.

The intermediates 4 were treated with manganese dioxide under reflux to afford 1-(3-formyl phenylsulfonyl)-1H-indole derivatives 5 in 75–85% yield. Finally, the aromatic aldehydes 5 were reacted with appropriate N₁-alkyl piperazines (N₁-CH₃ and N₁-C₂H₅) in the presence of sodium triacetoxy borohydride as reducing agent, to obtain the targeted compounds 6. The latter compounds were further treated with methanolic hydrochloric acid (16% w/v) to obtained the desired compounds as HCl salts (compounds 6) in 55–65% yield.

5-HT₆R-binding data (in vitro)

Radioligand binding assay for human 5-HT₆ receptor

The in vitro 5-HT₆ receptor-binding assay was carried out on human recombinant receptor expressed in HEK293 cells; radioligand used was [³H] LSD (60–80 Ci/mmol). Final ligand concentration was 1.5 nM, nonspecific determinant was methiothepin mesylate – [0.1 µM]; Methiothepin mesylate was used as reference compound and positive control. The radioligand binding study was carried out at NovaScreen, Hanover, MD.

Materials and methods

General considerations

Infrared spectra were recorded in KBr disc and in solid state using Perkin-Elmer model 1600 FT-IR spectrophotometer (Perkin-Elmer, Norwalk, CT). Electrospray ionization mass spectra were recorded on API 4000 triple quadrupole instrument (MDSSCIEX, Concord, Ontario, Canada). ¹H-NMR spectra were obtained on a Bruker proton NMR spectrometer (Fallanden, Switzerland) at 400 MHz. Deuterated reagents were used as solvents and were commercially procured. Tetramethylsilane (TMS) was used as an internal standard. Chemical shift values are expressed in parts per million (δ) and coupling constants are expressed in Hertz (Hz). Chromatography refers to column chromatography performed using 100–200 mesh silica gel and executed under nitrogen pressure (flash chromatography) conditions. All the reagents and chemicals used were of “reagent grade”.

Determination of % inhibition at 100 nM concentration, k_i and IC₅₀ values for 5-HT₆ receptor

The in vitro 5-HT₆ receptor-binding assay was carried out on human recombinant expressed receptor in HEK293 cells; Radioligand used was [³H]LSD (60–80 Ci/mmol). Final ligand concentration was 1.5 nM, nonspecific determinant was methiothepin mesylate – [0.1 µM]; methiothepin mesylate was used as reference compound and positive control. K_i values were determined in duplicate and the average values are reported here. 5-HT₆ receptor-binding studies were carried out at NovaScreen Biosciences Corporation, (caliper life sciences), Hanover, MD, as per their standard protocol.

Determination of k_b and IC₅₀ values for 5-HT₆ receptor antagonists

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 nonradioactive-based approach to determine potency of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by the 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 × 10⁴ cells/well using Hams F12 medium containing 10% fetal bovine serum (FBS) and incubated overnight at 37 °C 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 °C in CO₂ incubator for 4 h. After 4 h, cells were lysed using lyses buffer and luciface assay buffer was added to each well and counts per second were recorded using luminescence counter. From CPS obtained, percentage inhibition was calculated for each well by normalizing CPS values obtained in the presence of the compounds to those with 10 µM 5-HT (0% inhibition) and with vehicle (100% inhibition). The percent inhibition was determined for all concentrations of the antagonists (0.1 nM–10 µM range, half-log increment) and plotted as a function of the antagonist concentration. The K_b values were calculated with Prism (GraphPad Software Inc., La Jolla, CA) using built-in one site competition equation, by entering EC₅₀ values for 5-HT obtained in the same experiments (80–100 nM) and the 5-HT concentration 10 µM, as it was used in all the experiments.

Rodent pharmacokinetic study

Male Wistar rats (225 ± 25 gm) were used as experimental animals. Three animals were housed in each cage. Two days prior to dosing day, male Wistar rats (225–250 gm) were anesthetized with isoflurane for the surgical placement of jugular vein catheter. Animals were fasted over night before oral dosing (p.o) and food pellets were allowed 2-h postdosing, whereas during intravenous dosing, food and water were provided as ad libitum. Three rats were dosed with compounds (10 mg/kg) orally and intravenously (10 mg/kg).

At each time point, blood was collected through jugular vein and immediately replenish with an equivalent volume of normal saline from freely moving rats. Collected blood was transferred into a labeled appendorf 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 postdose (n = 3). Blood was centrifuged at 4000 rpm for 10 min. Plasma was prepared and stored at −20 °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, C_max, AUC_0-t, t_1/2 and bioavailability, were calculated by noncompartmental model using standard noncompartmental model by using WinNonLin 5.0.1 version Software package (Pharsight corporation, Mountain view, CA).

Rodent brain penetration study

Male Wistar rats (225 ± 25 gm) 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.

Brain penetration was determined at steady state in rat. One day prior to dosing day, male wistar rats (225 ± 25 gm) were anesthetized with halothane for the surgical placement of jugular and femoral vein catheters. After surgery, the rats were housed in individual rat infusion cage connected with infusion components and allowed free access to food and water.

The test compound was dissolved in water and administered at a constant infusion rate (5 mL/kg/h) upto 6 h at a target dose rate 1.0 mg free base/kg/h. Blood samples were removed during the latter part of the infusion to confirm steady-state blood concentrations, brian and blood was collected and estimated. The animals were sacrificed to collect the plasma and brain tissue and thenhomogenized using milli-Q water (20%). Plasma and brain was stored frozen at −20 °C until analysis. The concentrations of the test compound in plasma and brain were determined using LC-MS/MS method.

The test compound was quantified in plasma and brain homogenate by validated LC-MS/MS method using solid-phase extraction technique. The compound was quantified in the calibration range of 1–500 ng/mL in plasma and brain homogenate. Study samples were analyzed using calibration samples in the batch and quality control samples spread across the batch. Extent of brain-plasma ratio was calculated (C_b/C_p).

Protocol for NORT

For object recognition test, male Wistar rats 10–12 weeks old were used. Arena was 50 × 50 × 50 cm. Open field was made up of acrylic. Twnety-four hours prior to testing, rats were habituated to individual test arenas for 20 min in the absence of any objects. Twnety-four hours after the habituation, during the familiarization phase (T1), rats were placed individually in the open field for 3 min, containing two identical objects (a1 and a2). T2 trial was carried out after 24 h after the T1 trial. Rats were allowed to explore the open field for 3 min in the presence of one familiar object (a3) and one novel object (b). Discriminative index was calculated.

Experimental

Experimental procedures and analytical characterization data of compounds 3a–3ab, 4a–4ab, 5a–5ab and 6a–6ac

General (representative) procedure for the synthesis of compound 6 g (Di HCl Salt)

Preparation of 2-bromo-5-(indole-1-sulfonyl) benzoic acid (3 g). A solution of indole (6.24 g, 53.33 mmol) in 25 mL tetrahydrofuran (THF) was slowly added to a cooled (20 °C) suspension of sodium hydride (6.59 g, 164.74 mmol) in 20 mL of THF, over a period of 10 min maintaining the mass temperature at 25 °C under nitrogen atmosphere. The mass was further diluted with tetrahydrofuran (20 mL). The reaction mixture was further stirred for 1 h at the same temperature. Then, added a solution of 2-bromo-5-chlorosulfonyl benzoic acid (20.01 g, 66.81 mmol) in THF (30 mL) slowly over a period of 10 min. The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction mixture was quenched onto 600 mL cold water. The reaction mass was acidified with concentrated hydrochloric acid to pH 2 and extracted the product with ethyl acetate (2 × 200 mL). The combined organic layer was washed with brine solution (2 × 50 mL), water (2 × 100 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 17.81 g technical product. The material was used as such in the next step, with out further purification.

Melting range: 188.7–191.4 °C (dec); IR spectra (cm⁻¹): 1710, 1683, 1579, 1440, 1371, 1255, 1180, 1124, 744; ESIMS (m/z): 378.1, 380.0 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.6–6.61 (d, 1H, J = 3.88 Hz, Ar-H), 7.13–7.24 (m, 2H, Ar-H), 7.43–7.45 (m, 2H, Ar-H), 7.61–7.64 (m, 2H, Ar-H), 7.83–7.85 (d, 1H, J = 8.24, Ar-H), 8.23 (d, 1H, J = 2.04 Hz, Ar-H).

Similarly, compounds 3a–3ab were prepared from respective starting materials 1 (commercially available substituted indoles) and 2 (commercially available substituted 3-chlorosulfonyl benzoic acids) by using the method described previously, for example 3 g with some noncritical variations. The analytical data of compounds 3a–3ab are presented below. The obtained technical product was used as such in the next step, with out further purification.

3-(Indole-1-sulfonyl) benzoic acid (3a, R¹= H, R²= H, R³= H): ESIMS (m/z): 300.1 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.69–6.70 (d, 1H, J = 3.08 Hz, Ar-H), 7.23–7.27 (m, 2H, Ar-H), 7.33–7.37 (m, 1H, Ar-H), 7.52–7.61 (m, 2H, Ar-H), 8.00–8.02 (m, 1H, Ar-H), 8.09–8.12 (m, 1H, Ar-H), 8.23–8.26 (m, 1H, Ar-H), 8.60 (d, 1H, J = 1.60 Hz, Ar-H).

5-(Indole-1-sulfonyl)-2-methyl benzoic acid (3b, R¹= H, R²= H, R³= 4′-CH₃): Melting range (°C): 173.60–174.7 (Clear); IR (cm⁻¹): 3126, 2924, 1699, 1440, 1375, 1261, 1172, 1120; ESIMS (m/z): 314.4 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 2.62 (s, 3H, Ar-CH₃), 6.67–6.68 (d, 1H, J = 3.6 Hz, Ar-H), 7.21–7.25 (m, 1H, Ar-H), 7.30–7.34 (m, 2H, Ar-H), 7.51–7.53 (d, 1H, J = 7.8 Hz, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.89–7.91 (dd, 1H, J = 8.16, 2.04 Hz, Ar-H), 7.98–8.00 (d, 1H, J = 8.32 Hz, Ar-H), 8.55–8.56 (d, 1H, J = 1.96 Hz, Ar-H).

2-Chloro-5-(indole-1-sulfonyl) benzoic acid (3d, R¹= H, R²= H, R³= Cl): Melting range (°C): 177.7–182.1 (dec); IR (cm⁻¹): 3138, 1695, 1442, 1375, 1259, 1178, 1124; ESIMS (m/z): 334.1, 336.1 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.71–6.72 (d, 1H, J = 3.64 Hz, Ar-H), 7.24–7.28 (m, 1H, Ar-H), 7.33–7.37 (m, 1H, Ar-H), 7.54–7.56 (m, 3H, Ar-H), 7.90–7.93 (dd, 1H, J = 8.52, 2.40 Hz, Ar-H), 7.97–7.99 (d, 1H, J = 8.16 Hz, Ar-H), 8.50–8.51 (d, 1H, J = 2.36 Hz, Ar-H).

3-(5-Methoxy indole-1-sulfonyl) benzoic acid (3 h, R¹= 5-OCH₃, R²= H, R³= H): ESIMS (m/z): 330.3 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 3.80 (s, 3H, Ar-OCH₃), 6.62–6.63 (d, 1H, J = 3.63 Hz, Ar-H), 6.93–6.94 (d, 1H, J = 2.50 Hz, Ar-H), 6.96–6.97 (m, 1H, Ar-H), 7.53–7.58 (m, 2H, Ar-H), 7.89–7.91 (d, 1H, J = 8.76 Hz, Ar-H), 8.05–8.08 (m, 1H, Ar-H), 8.22–8.25 (m, 1H, Ar-H), 8.56–8.57 (d, 1H, J = 1.72 Hz, Ar-H).

3-(5-Ethoxy indole-1-sulfonyl) benzoic acid (3i, R¹= 5-OCH₂CH₃, R²= H, R³= H): ESIMS (m/z): 344.3 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 1.38–1.42 (t, 3H, Ar-OCH₂CH₃), 3.98–4.04 (q, 2H, Ar-OCH₂CH₃), 6.60–6.61 (d, 1H, J = 3.60 Hz, Ar-H), 6.93–6.99 (m, 2H, Ar-H), 7.52–7.57 (m, 2H, Ar-H), 7.88–7.90 (d, 1H, J = 9.16 Hz, Ar-H), 8.05–8.09 (d, 1H, J = 7.88 Hz, Ar-H), 8.21–8.23 (d, 1H, J = 7.80 Hz, Ar-H), 8.56 (s, 1H, Ar-H).

3-(5-Isopropoxy indole-1-sulfonyl) benzoic acid (3j, R¹= 5-OCH(CH₃)₂, R²= H, R³= H): ESIMS (m/z): 358.2 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 1.31–1.32 (d, 6H, J = 6.04 Hz, Ar-OCH(CH₃)₂), 4.42–4.54 (sept, 1H, Ar-OCH(CH₃)₂), 6.60 (d, 1H, J = 3.56 Hz, Ar-H), 6.91–6.94 (dd, 1H, J = 8.96, 2.40 Hz, Ar-H), 6.97 (d, 1H, J = 2.36 Hz, Ar-H), 7.52–7.53 (d, 1H, J = 3.68 Hz, Ar-H), 7.54–7.58 (m, 1H, Ar-H), 7.87–7.89 (d, 1H, J = 8.92 Hz, Ar-H), 8.06–8.09 (m, 1H, Ar-H), 8.23–8.25 (m, 1H, Ar-H), 8.58 (d, 1H, J = 1.61 Hz, Ar-H).

3-(5-Fluoro indole-1-sulfonyl) benzoic acid (3k, R¹= F, R²= H, R³= H): ESIMS (m/z): 318.1 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.65–6.66 (d, 1H, J = 3.08 Hz, Ar-H), 7.05–7.11 (m, 1H, Ar-H), 7.17–7.20 (dd, 1H, J = 8.64, 2.52 Hz, Ar-H), 7.59–7.62 (m, 2H, Ar-H), 7.94–7.98 (m, 1H, Ar-H), 8.07–8.09 (m, 1H, Ar-H), 8.25–8.27 (m, 1H, Ar-H), 8.57 (d, 1H, J = 1.64 Hz, Ar-H).

5-(5-Methoxy indole-1-sulfonyl)-2-methyl benzoic acid, (3 l, R¹= 5-OCH₃, R²= H, R³= CH₃): Melting range (°C): 190.2–194.9 (dec); IR (cm⁻¹): 3151, 1728, 1440, 1369, 1219, 1145; ESIMS (m/z): 344.1 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 2.69 (s, 3H, Ar-CH₃), 3.81 (s, 3H, Ar-OCH₃), 6.60–6.61 (d, 1H, J = 3.08 Hz, Ar-H), 6.93 (d, 1H, J = 2.48 Hz, Ar-H), 6.95–6.97 (dd, 1H, J = 6.68, 2.20 Hz, Ar-H), 7.32–7.34 (d, 1H, J = 8.24, Ar-H), 7.52–7.53 (d, 1H, J = 3.60 Hz, Ar-H), 7.86–7.90 (m, 2H, Ar-H), 8.52–8.53 (d, 1H, J = 2.12 Hz, Ar-H).

5-(5-Fluoro indole-1-sulfonyl)-2-methyl benzoic acid (3m, R¹= 5-F, R²= H, R³= CH₃): Melting range (°C): 198.7–200.7 (dec); IR (cm⁻¹): 3500, 3111, 1693, 1452, 1369, 1267, 1128; ESIMS (m/z): 331.9 [M − H]⁻; ¹H-NMR (400 MHz, DMSO-d₆): δ 2.49 (s, 3H, Ar-CH₃), 6.83–6.84 (d, 1H, J = 3.24 Hz, Ar-H), 7.18–7.23 (m, 1H, Ar-H), 7.40–7.43 (dd, 1H, J = 9.08, 2.60 Hz, Ar-H), 7.51–7.53 (d, 1H, J = 8.24, Ar-H), 7.90–7.92 (m, 2H, Ar-H), 8.00–8.03 (dd, 1H, J = 8.16 & 2.24 Hz, Ar-H), 8.24–8.25 (d, 1H, J = 2.20 Hz, Ar-H), 13.46–13.49 (bs, 1H, Ar-COOH).

2-Chloro-5-(5-fluoro indole-1-sulfonyl) benzoic acid (3o, R¹= F, R²= H, R³= Cl): Melting range (°C): 189.6–191.5 (Clear); ESIMS (m/z): 352.5, 354.5 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.67–6.68 (d, 1H, J = 3.18 Hz, Ar-H), 7.06–7.11 (m, 1H, Ar-H), 7.18–7.20 (dd, 1H, J = 8.60, 2.52 Hz, Ar-H), 7.55–7.56 (d, 1H, J = 2.34 Hz, Ar-H), 7.58–7.60 (d, 1H, J = 8.48 Hz, Ar-H), 7.89–7.90 (dd, 1H, J = 8.50, 2.41 Hz, Ar-H), 7.91–7.94 (m, 1H, Ar-H), 8.48–8.49 (d, 1H, J = 2.36 Hz, Ar-H).

2-Bromo-5-(5-fluoro indole-1-sulfonyl) benzoic acid (3q, R¹= F, R²= H, R³= Br): Melting range (°C): 193.6–196.5 (Clear); IR (cm⁻¹): 3140, 2922, 1701, 1456, 1375, 1294, 1174, 1130; ESIMS (m/z): 397, 399 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 6.66 (d, 1H, J = 3.40 Hz, Ar-H), 6.66–7.09 (m, 1H, Ar-H), 7.17–7.20 (dd, 1H, J = 8.60, 2.52 Hz, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.76–7.77 (m, 2H, Ar-H), 7.90–7.93 (dd, 1H, J = 9.12, 2.41 Hz, Ar-H), 8.40–8.41 (d, 1H, J = 1.32 Hz, Ar-H).

2-Bromo-5-(5-bromo indole-1-sulfonyl) benzoic acid (3s, R¹= Br, R²= H, R³= Br):

ESIMS (m/z): 456.1, 458.2, 460.2 [M − H]⁻.

2-Chloro-5-(5-methoxy indole-1-sulfonyl) benzoic acid (3t, R¹= 5-OCH₃, R²= H, R³= Cl): Melting range (°C): 156.75–157.45 (Clear); IR (cm⁻¹): 3115, 1705, 1462, 1373, 1217, 1135; ESIMS (m/z): 364.2, 366.2 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 3.81 (s, 3H, Ar-OCH₃), 6.63–6.64 (d, 1H, J = 3.16 Hz, Ar-H), 6.94–6.98 (m, 2H, Ar-H), 7.49–7.54 (m, 2H, Ar-H), 7.86–7.89 (m, 2H, Ar-H), 8.47 (d, 1H, J = 2.40 Hz, Ar-H).

2-Bromo-5-(5-methoxy indole-1-sulfonyl) benzoic acid (3v, R¹= 5-OCH₃, R²= H, R³= Br): Melting range (°C): 177.50–177.80 (Clear); IR (cm⁻¹): 3130, 1714, 1467, 1373, 1222, 1147; ESIMS (m/z): 407.9, 409.8 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 3.81 (s, 3H, Ar-OCH₃), 6.63–6.64 (d, 1H, J = 3.12 Hz, Ar-H), 6.94–6.96 (dd, 1H, J = 8.88, 2.48 Hz, Ar-H), 6.97–6.98 (d, 1H, J = 2.2 Hz, Ar-H), 7.48–7.49 (d, 1H, J = 3.6 Hz, Ar-H), 7.74–7.79 (m, 2H, Ar-H), 7.85–7.87 (d, 1H, J = 8.84 Hz, Ar-H), 8.42 (d, 1H, J = 1.32 Hz, Ar-H).

2-Chloro-5-(3-methyl indole-1-sulfonyl) benzoic acid (3x, R¹= H, R²= CH₃, R³= Cl): Melting range (°C): 188.40–191.50 (Clear); IR (cm⁻¹): 2921, 1714, 1444, 1372, 1276, 1182, 1121; ESIMS (m/z): 348.0, 350.1 [M − H]⁻; ¹H-NMR (400 MHz, CDCl₃): δ 2.25 (d, 3H, Ar-CH₃), 7.28–7.30 (m, 2H, Ar-H), 7.33–7.38 (m, 1H, Ar-H), 7.46–7.48 (d, 1H, J = 7.84 Hz, Ar-H), 7.51–7.53 (d, 1H, J = 8.52 Hz, Ar-H), 7.88–7.91 (dd, 1H, J = 8.48, 2.40 Hz, Ar-H), 7.97–7.99 (d, 1H, J = 8.20, Ar-H), 8.48–8.49 (d, 1H, J = 2.32 Hz, Ar-H).

2-Bromo-5-(3-methyl indole-1-sulfonyl) benzoic acid (3z, R¹= H, R²= CH₃, R³= Br): ESIMS (m/z): 392.2, 394.2 [M − H]⁻.

2-Chloro-5-(5-methoxy-3-methyl indole-1-sulfonyl) benzoic acid (3ab, R¹= 5-OCH₃, R²= CH₃, R³= Cl): ESIMS (m/z): 378.2, 380.2 [M − H]⁻.

Preparation of 1-[4-bromo-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4g)

A solution of 2-bromo-5-(indole-1-sulfonyl) benzoic acid (17.64 g, 46.42 mmol) in 30 mL tetrahydrofuran was slowly added to a cooled (0–5 °C) suspension of lithium aluminum hydride (2.3 g, 60.5 mmol) in 20 mL of THF. The reaction mass was stirred at 0–5 °C for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was cooled to −10 °C. Water (20 mL) was added slowly under nitrogen atmosphere. The reaction mass was diluted further with water (300 mL) and filtered through hyflow bed. The clear filtrate was extracted with ethyl acetate (4 × 100 mL). The combined organic layer was washed with brine solution (2 × 50 mL), water (2 × 50 mL) and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residual mass was purified by column chromatography, the eluent system being ethyl acetate and hexane (2:8) to obtain 6.025 g of the title product, Yield: 33% (over 2 steps).

Melting range: 102.6–103.4 °C (dec); I.R (cm⁻¹): 3311, 1444, 1371, 1257, 1128, 879; ESIMS (m/z): 366 [M + H]⁺, 368 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 4.69 (s, 2H, Ar-CH₂OH), 6.67–6.68 (dd, 1H, J = 3.64, 0.68 Hz, Ar-H), 7.23–7.25 (m, 1H, Ar-H), 7.32–7.33 (m, 1H, Ar-H), 7.55–7.64 (m, 4H, Ar-H), 7.97–7.99 (dd, 1H, J = 4.28, 0.8 Hz, Ar-H), 8.04–8.05 (d, 1H, Ar-H).

Similarly compounds 4a–4ab were prepared from respective starting materials 3a–3ab by using the method described earlier for the example 4 g with some noncritical variations. The analytical data of compounds 4a–4ab are presented below.

[3-(Indole-1-sulfonyl)-phenyl]-methanol (4a, R¹= H, R²= H, R³= H): Yield: 55% (over 2 steps); ESIMS (m/z): 288.2 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.80 (bs, 1H, Ar-CH₂OH), 4.69 (s, 2H, Ar-CH₂OH), 6.66–6.67 (d, 1H, J = 3.60 Hz, Ar-H), 7.21–7.24 (m, 1H, Ar-H), 7.30–7.34 (m, 1H, Ar-H), 7.40–7.44 (m, 1H, Ar-H), 7.51–7.53 (m, 2H, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.79–7.81 (d, 1H, J = 8.28 Hz, Ar-H), 7.88 (s, 1H, Ar-H), 7.78–8.00 (d, 1H, J = 8.32 Hz, Ar-H).

1-[4-Methyl-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4b, R¹= H, R²= H, R³= CH₃): Yield: 48% (over 2 steps); Melting range (°C): 101.0–102.8 (Clear); IR (cm⁻¹): 3280, 1444, 1365, 1163, 1126; ESIMS (m/z): 302.7 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.72 (bs, 1H, Ar-CH₂OH), 2.27 (s, 3H, Ar-CH₃), 4.64–4.65 (d, 2H, Ar-CH₂OH), 6.64–6.65 (d, 1H, J = 3.6 Hz, Ar-H), 7.19–7.30 (m, 3H, Ar-H), 7.51–7.52 (d, 1H, J = 6.56 Hz, Ar-H), 7.57–7.58 (d, 1H, J = 3.64 Hz, Ar-H), 7.92–7.93 (d, 1H, J = 1.92 Hz, Ar-H), 7.98 (d, 1H, J = 0.72 Hz, Ar-H), 8.05–8.07 (d, 1H, J = 0.68 Hz, Ar-H).

1-[4-Chloro-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4d, R¹= H, R²= H, R³= Cl): Yield: 39% (over 2 steps); Melting range (°C): 104.5–107.4 °C (dec); IR (cm⁻¹): 3429, 1446, 1371, 1261, 1166, 1126; ESIMS (m/z): 322.1 [M + H]⁺, 324.0 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 1.85 (bs, 1H, Ar-CH₂OH), 4.72 (s, 2H, Ar-CH₂OH), 6.67–6.68 (d, 1H, J = 3.64 Hz, Ar-H), 7.21–7.25 (m, 1H, Ar-H), 7.32–7.37 (m, 1H, Ar-H), 7.39–7.41 (m, 1H, Ar-H), 7.52–7.56 (m, 2H, Ar-H), 7.72–7.74 (dd, 1H, J = 8.36, 2.28 Hz, Ar-H), 7.97–7.99 (d, 1H, J = 8.28 Hz, Ar-H), 8.06–8.07 (d, 1H, J = 2.0 Hz, Ar-H).

5-Methoxy-1-[3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4 h, R¹= 5-OCH₃, R²= H, R³= H): Yield: 48% (over 2 steps); ESIMS (m/z): 318.2 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.85–1.87 (bs, 1H, Ar-CH₂OH), 3.80 (s, 3H, Ar-OCH₃), 4.69 (s, 2H, Ar-CH₂OH), 6.58–6.59 (d, 1H, J = 3.68 Hz, Ar-H), 6.91–6.93 (dd, 1H, J = 8.8, 2.52 Hz, Ar-H), 6.96 (d, 1H, J = 2.36 Hz, Ar-H), 7.38–7.42 (m, 1H, Ar-H), 7.51–7.52 (m, 2H, Ar-H), 7.75–7.77 (d, 1H, J = 7.84 Hz, Ar-H), 7.85–7.89 (m, 2H, Ar-H).

5-Ethoxy-1-[3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4i, R¹= 5-OCH₂CH₃, R²= H, R³= H): Yield: 44% (over 2 steps); ESIMS (m/z): 332.3 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.80 (bs, 1H, Ar-CH₂OH), 1.38 – 1.42 (t, 3H, Ar- OCH₂CH₃), 3.98–4.04 (q, 2H, Ar-OCH₂CH₃), 4.69 (s, 2H, Ar-CH₂OH), 6.57–6.58 (d, 1H, J = 3.16 Hz, Ar-H), 6.90–6.95 (m, 2H, Ar-H), 7.40–7.42 (m, 1H, Ar-H), 7.50–7.52 (m, 2H, Ar-H), 7.75–7.77 (d, 1H, J = 7.84 Hz, Ar-H), 7.85–7.88 (m, 2H, Ar-H).

5-Isopropoxy-1-[3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4j, R¹= 5-OCH(CH₃)₂, R²= H, R³= H): Yield: 43% (over 2 steps); ESIMS (m/z): 346.0 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.98 (bs, 1H, Ar-CH₂OH), 1.29–1.30 (d, 6H, J = 5.84 Hz, Ar-OCH(CH₃)₂), 4.46–4.52 (sept, 1H, Ar-OCH(CH₃)₂), 4.67 (s, 2H, Ar-CH₂OH), 6.55–6.56 (d, 1H, J = 3.0 Hz, Ar-H), 6.88–6.91 (dd, 1H, J = 9.0, 2.44 Hz, Ar-H), 6.95–6.96 (d, 1H, J = 2.40 Hz, Ar-H), 7.36–7.40 (m, 1H, Ar-H), 7.48–7.50 (m, 2H, Ar-H), 7.74–7.76 (d, 1H, J = 7.84 Hz, Ar-H), 7.84–7.86 (m, 2H, Ar-H).

5-Fluoro-1-[3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4k, R¹= F, R²= H, R³= H): Yield: 51% (over 2 steps); ESIMS (m/z): 306.2 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.0 (bs, 1H, Ar-CH₂OH), 4.68 (s, 2H, Ar-CH₂OH), 6.61 (d, 1H, J = 3.08 Hz, Ar-H), 7.03–7.06 (m, 1H, Ar-H), 7.15–7.18 (m, 1H, Ar-H), 7.41–7.43 (m, 1H, Ar-H), 7.51–7.54 (m, 1H, Ar-H), 7.58–7.59 (d, 1H, J = 3.68 Hz, Ar-H), 7.74–7.78 (m, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.90–7.92 (m, 1H, Ar-H).

5-Methoxy-1-[4-methyl-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4 l, R¹= 5-OCH₃, R²= H, R³= CH₃): Yield: 47% (over 2 steps); Melting range (°C): 144.3–145.2 (Clear); IR (cm⁻¹): 3422, 1606, 1467, 1365, 1224, 1136; ESIMS (m/z): 331.7 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.75–1.78 (t, 1H, J = 5.75 Hz, Ar-CH₂OH), 2.27 (s, 3H, Ar-CH₃), 3.79 (s, 3H, Ar-OCH₃), 4.63–4.64 (d, 2H, J = 5.51 Hz, Ar-CH₂OH), 6.56–6.57 (d, 1H, J = 3.08 Hz, Ar-H), 6.90–6.92 (dd, 1H, J = 8.98, 2.50 Hz, Ar-H), 6.95 (d, 1H, J = 2.42 Hz, Ar-H), 7.18–7.20 (d, 1H, J = 8.02 Hz, Ar-H), 7.52–7.53 (d, 1H, J = 3.59 Hz, Ar-H), 7.66–7.68 (d, 1H, J = 8.0 Hz, Ar-H), 7.86–7.89 (m, 2H, Ar-H).

5-Fluoro-1-[4-methyl-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4m, R¹= 5-F, R²= H, R³= CH₃): Yield: 45% (over 2 steps); Melting range (°C): 129.0–131.1 (Clear); IR (cm⁻¹): 3441, 1593, 1462, 1371, 1215, 1136; ESIMS (m/z): 319.9 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.73 (bs, 1H, Ar-CH₂OH), 2.29 (s, 3H, Ar-CH₃), 4.66 (s, 2H, Ar-CH₂OH), 6.60–6.61 (d, 1H, J = 3.12 Hz, Ar-H), 7.03–7.06 (m, 1H, Ar-H), 7.15–7.18 (dd, 1H, J = 8.76, 2.52 Hz, Ar-H), 7.20–7.22 (d, 1H, J = 8.04 Hz, Ar-H), 7.60–7.61 (d, 1H, J = 3.68 Hz, Ar-H), 7.67–7.70 (dd, 1H, J = 8.04, 2.12 Hz, Ar-H), 7.91–7.92 (m, 2H, Ar-H).

5-Fluoro-1-[4-chloro-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4o, R¹= F, R²= H, R³= Cl): Yield: 42% (over 2 steps); ESIMS (m/z): 340.2 [M + H]⁺, 342.2 [M + H]⁺²; IR (cm⁻¹): 3431, 1587, 1460, 1373, 1213, 1168, 1138; ¹H-NMR (400 MHz, CDCl₃): δ 2.0 (bs, 1H, Ar-CH₂OH), 4.75 (s, 2H, Ar-CH₂OH), 6.63–6.64 (d, 1H, J = 3.08 Hz, Ar-H), 7.02–7.08 (m, 1H, Ar-H), 7.16–7.19 (dd, 1H, J = 8.68, 2.48 Hz, Ar-H), 7.39–7.41 (d, 1H, J = 8.04 Hz, Ar-H), 7.58–7.59 (d, 1H, J = 3.68 Hz, Ar-H), 7.70–7.72 (dd, 1H, J = 8.4, 2.36 Hz, Ar-H), 7.91–7.95 (m, 1H, Ar-H), 8.05–8.06 (d, 1H, J = 2.36 Hz, Ar-H).

5-Fluoro-1-[4-bromo-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4q, R¹= F, R²= H, R³= Br): Yield: 39% (over 2 steps); Melting range (°C): 129.9–131.2 (Clear); ESIMS (m/z): 384 [M + H]⁺, 386 [M + H]⁺²; IR (cm⁻¹): 3431, 1585, 1462, 1369, 1215, 1168, 1136; ¹H-NMR (400 MHz, CDCl₃): δ 2.0 (bs, 1H, Ar-CH₂OH), 4.71 (s, 2H, Ar-CH₂OH), 6.63–6.64 (d, 1H, J = 3.72 Hz, Ar-H), 7.05 (d, 1H, J = 2.40, Ar-H), 7.16–7.19 (dd, 1H, J = 8.68, 2.52 Hz, Ar-H), 7.58–7.62 (m, 3H, Ar-H), 7.91–7.94 (dd, 1H, J = 8.80, 2.42 Hz, Ar-H), 8.03 (d, 1H, J = 1.56 Hz, Ar-H).

5-Bromo-1-[4-bromo-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4s, R¹= Br, R²= H, R³= Br): Yield: 41% (over 2 steps); 444.3 [M + H]⁺, 446.2 [M + H]⁺², 448.2 [M + H]⁺⁴.

5-Methoxy-1-[4-chloro-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4t, R¹= 5-OCH₃, R²= H, R³= Cl): Yield: 43% (over 2 steps); ESIMS (m/z): 352.4 [M + H]⁺, 354.5 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 2.11 (bs, 1H, Ar-CH₂OH), 3.80 (s, 3H, Ar-OCH₃), 4.72 (s, 2H, Ar-CH₂OH), 6.58–6.59 (d, 1H, J = 3.2 Hz, Ar-H), 6.91–6.96 (m, 2H, Ar-H), 7.36–7.38 (d, 1H, J = 8.0 Hz, Ar-H), 7.49–7.50 (d, 1H, J = 3.64 Hz, Ar-H), 7.67–7.68 (dd, 1H, J = 8.36, 2.40 Hz, Ar-H), 7.85–7.87 (d, 1H, J = 8.96 Hz, Ar-H), 8.03–8.04 (d, 1H, J = 2.32 Hz, Ar-H).

5-Methoxy-1-[4-bromo-3-(hydroxymethyl) phenylsulfonyl]-1H-indole (4v, R¹= 5-OCH₃, R²= H, R³= Br): Yield: 38% (over 2 steps); Melting range (°C): 112.7–114.0 (Clear); IR (cm⁻¹): 3348, 1612, 1471, 1375, 1224, 1141; ESIMS (m/z): 396.4 [M + H]⁺, 398.6 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 1.99 (bs, 1H, Ar-CH₂OH), 3.80 (s, 3H, Ar-OCH₃), 4.68 (s, 2H, Ar-CH₂OH), 6.60–6.61 (d, 1H, J = 3.08 Hz, Ar-H), 6.91–6.94 (dd, 1H, J = 8.96, 2.52 Hz, Ar-H), 6.96–6.97 (d, 1H, J = 2.40 Hz, Ar-H), 7.50–7.51 (d, 1H, J = 3.68 Hz, Ar-H), 7.56–7.61 (m, 2H, Ar-H), 7.86–7.88 (d, 1H, J = 9.0 Hz, Ar-H), 8.01 (d, 1H, J = 1.2 Hz, Ar-H).

1-[4-Chloro-3-(hydroxymethyl) phenylsulfonyl]-3-methyl-1H-indole (4x, R¹= H, R²= CH₃, R³= Cl): Yield: 41% (over 2 steps); Melting range (°C): 106.6–109.3 (Clear); IR (cm⁻¹): 3546, 3339, 1567, 1447, 1372, 1273, 1196, 1117; ESIMS (m/z): 336.1 [M + H]⁺, 338.6 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 1.97 (bs, 1H, Ar-CH₂OH), 2.24 (s, 3H, Ar-CH₃), 4.72 (s, 2H, Ar-CH₂OH), 7.23–7.27 (m, 1H, Ar-H), 7.29–7.30 (m, 1H, Ar-H), 7.32–7.34 (m, 1H, Ar-H), 7.35–7.38 (d, 1H, J = 8.40 Hz, Ar-H), 7.44–7.46 (d, 1H, J = 7.73 Hz, Ar-H), 7.69–7.72 (dd, 1H, J = 8.40, 2.38 Hz, Ar-H), 7.96–7.98 (d, 1H, J = 8.21 Hz, Ar-H), 8.05 (d, 1H, J = 2.3 Hz, Ar-H).

1-[4-bromo-3-(hydroxymethyl) phenylsulfonyl]-3-methyl-1H-indole (4z, R¹= H, R²= CH₃, R³= Br): Yield: 38% (over 2 steps); Melting range (°C): 122.7–123.9 (Clear); I.R (cm⁻¹): 3541, 1148, 1373, 1166; ESIMS (m/z): 380.1 [M + H]⁺, 382.1 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 1.98 (bs, 1H, Ar-CH₂OH), 2.24 (s, 3H, Ar-CH₃), 4.69 (s, 2H, Ar-CH₂OH), 7.27–7.30 (m, 2H, Ar-H), 7.31–7.33 (d, 1H, J = 7.16 Hz, Ar-H), 7.45–7.46 (d, 1H, J = 7.72 Hz, Ar-H), 7.55–7.57 (d, 1H, J = 8.36 Hz, Ar-H), 7.61–7.67 (d, 1H, J = 2.32 Hz, Ar-H), 7.96–7.99 (d, 1H, J = 8.24 Hz, Ar-H), 8.02–8.03 (d, 1H, J = 2.2 Hz, Ar-H).

5-Methoxy-1-[4-Chloro-3-(hydroxymethyl) phenylsulfonyl]-3-methyl-1H-indole (4ab, R¹= 5-OCH₃, R²= CH₃, R³= Cl): Yield: 43% (over 2 steps); Melting range (°C): 130.9–133.4 (Clear); I.R (cm⁻¹): 3293, 1611, 1453, 1366, 1228, 1133; ESIMS (m/z): 366.4 [M + H]⁺, 368.6 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 2.07 (bs, 1H, Ar-CH₂OH), 2.19 (s, 3H, Ar-CH₃), 3.82 (s, 3H, Ar-OCH₃), 4.71 (s, 2H, Ar-CH₂OH), 6.85–6.86 (d, 1H, J = 2.44 Hz, Ar-H), 6.90–6.93 (dd, 1H, J = 8.92, 2.48 Hz, Ar-H), 7.24 (d, 1H, J = 1.2 Hz, Ar-H), 7.33–7.35 (d, 1H, J = 8.4 Hz, Ar-H), 7.64–7.67 (dd, 1H, J = 8.40, 2.40 Hz, Ar-H), 7.85–7.87 (d, 1H, J = 9.0 Hz, Ar-H), 8.01–8.02 (d, 1H, J = 2.32 Hz, Ar-H).

Preparation of 1–(4-bromo-3-formyl phenylsulfonyl)-1H-indole (5g)

To a solution of 1-[4-bromo-3-(hydroxymethyl) phenyl sulfonyl]-1H-indole (4.73 g, 12.92 mmol) in ethylene dichloride (75 mL) was added manganese dioxide (8.92 g, 102.5 mmol). The reaction mass was heated to reflux temperature and maintained under reflux for 3 h, while monitoring the progress of the reaction by TLC. After completion of the reaction, the reaction mass was filtered through hyflow bed and the bed was washed with ethylene dichloride (2 × 10 mL). The clear filtrate was concentrated under reduced pressure to obtain the technical product. The technical material was triturated with of n-hexane (2 × 15 mL), decanted the solvent and dried under reduced pressure to obtain 3.42 g of the title product, Yield: 73%.

Melting range: 106.4–107.9 °C; I.R (cm⁻¹): 3074, 1691, 1571, 1440, 1371, 1253, 748; ESIMS (m/z): 364.0 [M + H]⁺, 366 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 6.7–6.71 (d, 1H, J = 3.52 Hz, Ar-H), 7.23–7.28 (m, 1H, Ar-H), 7.33–7.38 (m, 1H, Ar-H), 7.52–7.54 (m, 2H, Ar-H), 7.71–7.73 (d, 1H, J = 8.4 Hz, Ar-H), 7.88–7.90 (dd, 1H, J = 8.44, 1.92 Hz, Ar-H), 7.96–7.98 (d, 1H, J = 8.28 Hz, Ar-H), 8.35–8.36 (d, 1H, J = 2.28 Hz, Ar-H), 10.27 (s, 1H, Ar-CHO).

Similarly, compounds 5a–5ab were prepared from respective starting materials 4a–4ab by using the method described previously for the example 5 g with some noncritical variations. The analytical data of compounds 5a–5ab are presented below.

1-(3-Formyl phenylsulfonyl)-1H-indole (5a, R¹= H, R²= H, R³= H): Yield: 82%; ESIMS (m/z): 286.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 6.69–6.70 (d, 1H, J = 3.04 Hz, Ar-H), 7.24–7.25 (m, 1H, Ar-H), 7.31–7.36 (m, 1H, Ar-H), 7.51–7.53 (d, 1H, J = 7.76 Hz, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.60–7.63 (m, 1H, Ar-H), 7.99–8.03 (m, 2H, Ar-H), 8.09–8.12 (m, 1H, Ar-H), 8.35 (d, 1H, J = 1.6 Hz, Ar-H), 9.98 (s, 1H, Ar-CHO).

1-(4-Methyl-3-formyl phenylsulfonyl)-1H-indole (5b, R¹= H, R²= H, R³= CH₃): Yield: 85%; Melting range (°C): 88.3–89.3 (Clear); IR spectra (cm⁻¹): 1708, 1440, 1373, 1122; ESIMS (m/z): 300.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.65 (s, 3H, Ar-CH₃), 6.68–6.69 (d, 1H, J = 3.6 Hz, Ar-H), 7.21–7.25 (m, 1H, Ar-H), 7.31–7.35 (m, 2H, Ar-H), 7.52–7.54 (d, 1H, J = 7.68 Hz, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.92–7.94 (dd, 1H, J = 8.12, 1.96 Hz, Ar-H), 7.98–8.01 (dd, 1H, J = 8.28, 0.68 Hz, Ar-H), 8.29 (d, 1H, J = 2.12 Hz, Ar-H), 10.21 (s, 1H, Ar-CHO).

1-(4-Chloro-3-formyl phenylsulfonyl)-1H-indole (5d, R¹= H, R²= H, R³= Cl): Yield: 78%; Melting range (°C): 85.8–87.7 (Clear); IR (cm⁻¹): 1699, 1448, 1379, 1259, 1166; ESIMS (m/z): 320.1 [M + H]⁺, 322.3 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 6.70 (d, 1H, J = 3.64 Hz, Ar-H), 7.23–7.27 (m, 1H, Ar-H), 7.32–7.34 (m, 1H, Ar-H), 7.51–7.54 (d, 3H, Ar-H), 7.96–7.99 (m, 2H, Ar-H), 8.38–8.39 (d, 1H, J = 2.4 Hz, Ar-H), 10.37 (s, 1H, Ar-CHO).

5-Methoxy-1-(3-formyl phenylsulfonyl)-1H-indole (5 h, R¹= 5-OCH₃, R²= H, R³= H): Yield: 82%; ESIMS (m/z): 316.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 3.80 (s, 3H, Ar-OCH₃), 6.62–6.63 (d, 1H, J = 3.60 Hz, Ar-H), 6.93 (d, 1H, J = 2.57 Hz, Ar-H), 6.95–6.97 (m, 1H, Ar-H), 7.52–7.53 (d, 1H, J = 3.66 Hz, Ar-H), 7.60–7.64 (m, 1H, Ar-H), 7.88–7.91 (d, 1H, J = 8.88 Hz, Ar-H), 8.02–8.09 (m, 2H, Ar-H), 8.32–8.33 (d, 1H, J = 1.64 Hz, Ar-H), 9.99 (s, 1H, Ar-CHO).

5-Ethoxy-1-(3-formyl phenylsulfonyl)-1H-indole (5i, R¹= 5-OCH₂CH₃, R²= H, R³= H): Yield: 79%; ESIMS (m/z): 330.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.38–1.42 (t, 3H, Ar- OCH₂CH₃), 3.98–4.04 (q, 2H, Ar-OCH₂CH₃), 6.60–6.61 (d, 1H, J = 3.62 Hz, Ar-H), 6.92–6.93 (d, 1H, J = 2.47 Hz, Ar-H), 6.95–6.96 (m, 1H, Ar-H), 7.51–7.52 (d, 1H, J = 3.64 Hz, Ar-H), 7.59–7.63 (m, 1H, Ar-H), 7.87–7.89 (d, 1H, J = 8.64 Hz, Ar-H), 8.02–8.08 (m, 2H, Ar-H), 8.32 (d, 1H, J = 1.68 Hz, Ar-H), 9.99 (s, 1H, Ar-CHO).

5-Isopropoxy-1-(3-formyl phenylsulfonyl)-1H-indole (5j, R¹= 5-OCH(CH₃)₂, R²= H, R³= H): Yield: 79%; ESIMS (m/z): 344.1 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.31–1.32 (d, 6H, J = 6.04 Hz, Ar-OCH(CH₃)₂), 4.47–4.53 (sept, 1H, Ar-OCH(CH₃)₂), 6.60 (d, 1H, J = 3.52 Hz, Ar-H), 6.91–6.93 (dd, 1H, J = 8.96, 2.40 Hz, Ar-H), 6.96–6.97 (d, 1H, J = 2.36 Hz, Ar-H), 7.51 (d, 1H, J = 3.60 Hz, Ar-H), 7.62–7.64 (m, 1H, Ar-H), 7.86–7.88 (d, 1H, J = 8.96 Hz, Ar-H), 8.02–8.09 (m, 2H, Ar-H), 8.33 (d, 1H, J = 1.6 Hz, Ar-H), 9.99 (s, 1H, Ar-CHO).

5-Fluoro-1-(3-formyl phenylsulfonyl)-1H-indole (5k, R¹= F, R²= H, R³= H): Yield: 81%; ESIMS (m/z): 304 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 6.66–6.67 (d, 1H, J = 3.68 Hz, Ar-H), 7.06–7.09 (m, 1H, Ar-H), 7.17–7.19 (dd, 1H, J = 8.64, 2.52 Hz, Ar-H), 7.60–7.61 (d, 1H, J = 3.64 Hz, Ar-H), 7.64–7.66 (m, 1H, Ar-H), 7.93–7.98 (m, 1H, Ar-H), 8.04–8.09 (m, 2H, Ar-H), 8.34 (d, 1H, J = 1.56 Hz, Ar-H), 10.0 (s, 1H, Ar-CHO).

5-Methoxy-1-(4-methyl-3-formyl phenylsulfonyl)-1H-indole (5 l, R¹= 5-OCH₃, R²= H, R³= CH₃): Yield: 83%; Melting range (°C): 134.2–135.3 (Clear); IR (cm⁻¹): 3124, 1705, 1446, 1367, 1222, 1128; ESIMS (m/z): 330 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.60 (s, 3H, Ar-CH₃), 3.72 (s, 3H, Ar-OCH₃), 6.76–6.77 (d, 1H, J = 3.20 Hz, Ar-H), 6.92–6.95 (dd, 1H, J = 9.04, 2.56 Hz, Ar-H), 7.09 (d, 1H, J = 2.52 Hz, Ar-H), 7.52–7.54 (d, 1H, J = 8.20 Hz, Ar-H), 7.76–7.77 (d, 1H, J = 3.64 Hz, Ar-H), 7.80–7.82 (d, 1H, J = 9.04 Hz, Ar-H), 8.03–8.06 (dd, 1H, J = 8.12, 2.24 Hz, Ar-H), 8.26–8.27 (d, 1H, J = 2.16 Hz, Ar-H), 10.19 (s, 1H, Ar-CHO).

5-Fluoro-1-(4-methyl-3-formyl phenylsulfonyl)-1H-indole (5m, R¹= 5-F, R²= H, R³= CH₃): Yield: 79%; Melting range (°C): 96.5–99.4 (Clear); IR (cm⁻¹): 3145, 1712, 1460, 1373, 1213, 1138; ESIMS (m/z): 318 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.67 (s, 3H, Ar-CH₃), 6.64–6.65 (d, 1H, J = 3.16 Hz, Ar-H), 7.03–7.09 (m, 1H, Ar-H), 7.16–7.19 (dd, 1H, J = 8.68, 2.52 Hz, Ar-H), 7.34–7.36 (d, 1H, J = 8.16 Hz, Ar-H), 7.60 (d, 1H, J = 3.68 Hz, Ar-H), 7.89–7.94 (m, 2H, Ar-H), 8.26–8.27 (d, 1H, J = 2.12 Hz, Ar-H), 10.22 (s, 1H, Ar-CHO).

5-Fluoro-1-(4-chloro-3-formyl phenylsulfonyl)-1H-indole (5o, R¹= F, R²= H, R³= Cl): Yield: 78%; Melting range (°C): 92.5–94.4 (Clear); IR (cm⁻¹): 3136, 1699, 1579, 1452, 1377, 1207, 1163, 1132; ESIMS (m/z): 338.2 [M + H]⁺, 340.4 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 6.66–6.67 (d, 1H, J = 3.20 Hz, Ar-H), 7.06–7.09 (m, 1H, Ar-H), 7.17–7.19 (dd, 1H, J = 8.60, 2.52 Hz, Ar-H), 7.53–7.55 (d, 1H, J = 8.52 Hz, Ar-H), 7.57–7.58 (d, 1H, J = 3.72 Hz, Ar-H), 7.90–7.94 (m, 1H, Ar-H), 7.95–7.97 (dd, 1H, 8.52, 2.44 Hz, Ar-H), 8.36–8.37 (d, 1H, J = 2.40 Hz, Ar-H), 10.39 (s, 1H, Ar-CHO).

5-Fluoro-1-(4-bromo-3-formyl phenylsulfonyl)-1H-indole (5q, R¹= F, R²= H, R³= Br): Yield: 76%; Melting range (°C): 96.5–98.6 (Clear); IR (cm⁻¹): 3143, 1699, 1583, 1454, 1381, 1215, 1170, 1138; ESIMS (m/z): 382.2 [M + H]⁺, 384.4 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 6.66–6.67 (d, 1H, J = 3.20 Hz, Ar-H), 7.07–7.10 (m, 1H, Ar-H), 7.18–7.20 (dd, 1H, J = 8.60, 2.50 Hz, Ar-H), 7.53–7.55 (d, 1H, J = 8.50 Hz, Ar-H), 7.57–7.58 (d, 1H, J = 3.72 Hz, Ar-H), 7.91–7.95 (m, 1H, Ar-H), 7.95–7.97 (dd, 1H, 8.50, 2.42 Hz, Ar-H), 8.36–8.37 (d, 1H, J = 2.40 Hz, Ar-H), 10.36 (s, 1H, Ar-CHO).

5-Bromo-1-(4-bromo-3-formyl phenylsulfonyl)-1H-indole (5s, R¹= Br, R²= H, R³= Br): Yield: 74%; 442.3 [M + H]⁺, 444.2 [M + H]⁺², 446.2 [M + H]⁺⁴.

5-Methoxy-1-(4-chloro-3-formyl phenylsulfonyl)-1H-indole (5t, R¹= 5-OCH₃, R²= H, R³= Cl): Yield: 80%; Melting range (°C): 141.6–142.5 (Clear); IR (cm⁻¹): 3116, 1701, 1462, 1375, 1224, 1136; ESIMS (m/z): 350.2 [M + H]⁺, 352.3 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 3.80 (s, 3H, Ar-OCH₃), 6.62–6.63 (d, 1H, J = 3.62 Hz, Ar-H), 6.93–6.97 (m, 2H, Ar-H), 7.49–7.52 (m, 2H, Ar-H), 7.85–7.87 (d, 1H, J = 8.80 Hz, Ar-H), 7.93–7.96 (dd, 1H, J = 8.84, 2.48 Hz, Ar-H), 8.35 (d, 1H, J = 2.4 Hz, Ar-H), 10.38 (s, 1H, Ar-CHO).

5-Methoxy-1-(4-bromo-3-formyl phenylsulfonyl)-1H-indole (5v, R¹= 5-OCH₃, R²= H, R³= Br): Yield: 78%; Melting range (°C): 145.5–148.6 (Clear); IR (cm⁻¹): 1699, 1465, 1377, 1222, 1145; ESIMS (m/z): 394.2 [M + H]⁺, 396.3 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 3.80 (s, 3H, Ar-OCH₃), 6.62 (d, 1H, J = 3.60 Hz, Ar-H), 6.93–6.96 (m, 2H, Ar-H), 7.49 (s, 1H, Ar-H), 7.70–7.72 (d, 1H, J = 8.32 Hz, Ar-H), 7.85–7.87 (m, 2H, Ar-H), 8.32 (s, 1H, Ar-H), 10.27 (s, 1H, Ar-CHO).

1-[4-Chloro-3-formyl phenylsulfonyl]-3-methyl-1H-indole (5x, R¹= H, R²= CH₃, R³= Cl): Yield: 81%; Melting range (°C): 142.8–144.9 (Clear); IR (cm⁻¹): 2870, 1701, 1583 1447, 1371, 1166, 1115; ESIMS (m/z): 334.3 [M + H]⁺, 336.5 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 2.24 (s, 3H, Ar-CH₃), 7.25–7.28 (m, 2H, Ar-H), 7.32–7.36 (m, 1H, Ar-H), 7.44–7.46 (d, 1H, J = 7.72 Hz, Ar-H), 7.49–7.51 (d, 1H, J = 8.48 Hz, Ar-H), 7.95–7.97 (m, 2H, Ar-H), 8.37 (d, 1H, J = 2.37 Hz, Ar-H), 10.37 (s, 1H, Ar-CHO).

1-[4-Bromo-3-formyl phenylsulfonyl]-3-methyl-1H-indole (5z, R¹= H, R²= CH₃, R³= Br): Yield: 76%; Melting range (°C): 156.5–159.6 (Clear); IR (cm⁻¹): 1700, 1578, 1371, 1167; ESIMS (m/z): 378.2 [M + H]⁺, 380.2 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 2.24 (s, 3H, Ar-CH₃), 7.26–7.28 (m, 2H, Ar-H), 7.32–7.36 (t, 1H, J = 7.44 Hz, Ar-H), 7.44–7.46 (d, 1H, J = 7.04 Hz, Ar-H), 7.69–7.71 (d, 1H, J = 8.36 Hz, Ar-H), 7.86–7.88 (d, 1H, J = 6.72 Hz, Ar-H), 7.95–7.97 (d, 1H, J = 8.2 Hz, Ar-H), 8.34 (d, 1H, J = 1.28 Hz, Ar-H), 10.27 (s, 1H, Ar-CHO).

5-Methoxy-1-[4-Chloro-3-formyl phenylsulfonyl]-3-methyl-1H-indole (5ab, R¹= 5-OCH₃, R²= CH₃, R³= Cl): Yield: 80%; ESIMS (m/z): 364.2 [M + H]⁺, 366.3 [M + H]⁺²; ¹H-NMR (400 MHz, CDCl₃): δ 2.19 (s, 3H, Ar-CH₃), 3.82 (s, 3H, Ar-OCH₃), 6.85–6.86 (d, 1H, J = 2.48 Hz, Ar-H), 6.92–6.95 (dd, 1H, J = 8.96, 2.52 Hz, Ar-H), 7.23 (d, 1H, J = 1.16 Hz, Ar-H), 7.48–7.50 (d, 1H, J = 8.48 Hz, Ar-H), 7.84–7.86 (d, 1H, J = 9.0 Hz, Ar-H), 7.91–7.93 (dd, 1H, J = 8.52, 2.40 Hz, Ar-H), 8.33–8.34 (d, 1H, J = 2.4 Hz, Ar-H), 10.37 (s, 1H, Ar-CHO).

Preparation of 1-[4-bromo-3–(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6 g)

To a solution of 1–(4-bromo-3-formyl phenylsulfonyl)-1H-indole (1.004 g, 2.758 mmol) in 20 mL ethylene dichloride was added N-ethyl piperazine (0.3757 g, 3.295 mmol). Reaction mass was stirred at 25 °C for 10 min. Sodium triacetoxy borohydride (0.8842 g, 4.171 mmol) was added over a period of 10 min. The reaction mass was stirred at 25 °C for 6 h while monitoring the progress of the reaction by TLC. After completion of the reaction, reaction mass was quenched onto 5% sodium bicarbonate solution (100 mL) and extracted the product with chloroform (3 × 50 mL). The combined organic layer was washed with brine solution (2 × 50 mL), water (2 × 50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The technical product was purified by column chromatography, eluent system being ethyl acetate and n-hexane (7:1) to obtain 0.9344 g of the title product.

Melting Range: 122.2–126.3 °C; ESIMS (m/z): 462 [M + H]⁺; HRMS: [M + H]⁺ C₂₁H₂₄BrN₃O₂S calc. 462.0773, found. 462.0775.

Example 6 g (Di HCl salt): Preparation of 1-[4-bromo-3–(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride.

To a clear solution of 1-[4-bromo-3–(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (252.4  mg, 0.5463 mmol) in 15 mL diethyl ether under nitrogen atmosphere, a solution of methanolic hydrochloric acid (16% w/v, 0.32 mL, 1.402 mmol) was added in 5 min under stirring. The above reaction mass was further stirred at 25 °C, for a period of 1 h and concentrated under vacuum to obtain the above title product 262 mg, Yield: 65%, after 2 steps.

Melting range: 208.4–210.4 °C; IR spectra (cm⁻¹): 3429, 2935, 2245, 1440, 1172, 765; ESIMS (m/z): 462 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.20–1.24 (t, 3H, N-CH₂CH₃), 2.83 (bs, 4H, Piperazine-H), 3.10–3.15 (q, 2H, N-CH₂CH₃), 3.18 (bs, 4H, Piperazine-H), 3.91 (s, 2H, Ar-CH₂-N), 6.75 (d, 1H, J = 3.70 Hz, Ar-H), 7.21 (m, 1H, Ar-H), 7.28–7.30 (m, 1H, Ar-H), 7.53–7.55 (d, 1H, J = 7.7 Hz, Ar-H), 7.58–7.59 (d, 1H, J = 3.72 Hz, Ar-H), 7.69 (m, 2H, Ar-H), 7.78 (d, 1H, J = 1.8 Hz, Ar-H), 7.9 (d, 1H, J = 8.2 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄BrN₃O₂S calc. 462.0773, found. 462.0775.

Examples 6a–6ac

The compounds 6a–6ac were prepared from respective starting materials by using the method described above for the example 6g (Di HCl) with some noncritical variations. The analytical data of compounds 6a–6ac are presented below. The compound 6a was prepared from starting materials 5a and N-methyl piperazine.

1-[3-(4-Methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6a)

Yield: 63%; I.R (cm⁻¹): 2936, 1445, 1168, 823; ESI-MS (m/z): 370.3 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.29 (s, 3H, N-CH₃), 2.36 (bs, 8H, Piperazine-H), 3.47 (s, 2H, Ar-CH₂-N), 6.65–6.66 (d, 1H, J = 3.68 Hz, Ar-H), 7.19–7.23 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.36–7.38 (m, 1H, Ar-H), 7.45–7.53 (m, 2H, Ar-H), 7.56–7.57 (d, 1H, J = 3.68 Hz, Ar-H), 7.73–7.75 (m, 1H, Ar-H), 7.87 (m, 1H, Ar-H), 7.98–8.00 (d, 1H, J = 8.32 Hz, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₃N₃O₂S calc. 370.1511, found. 370.1514.

The compound 6b was prepared from starting materials 5b and N-methyl piperazine

1-[4-Methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6b)

Yield: 65%, after 2 steps; Melting range: 180.4–183.0 °C; I.R (cm⁻¹): 3383, 2960, 1629, 1446, 1130, 948; ESIMS (m/z): 384.6 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.45 (s, 3H, Ar-CH₃), 2.97 (s, 3H, N-CH₃), 3.29–3.58 (bs, 8H, Piperazine-H), 4.25 (s, 2H, Ar-CH₂-N), 6.72–6.73 (dd, 1H, J = 3.69, 0.39 Hz, Ar-H), 7.20–7.24 (m, 1H, Ar-H), 7.31–7.35 (m, 1H, Ar-H), 7.41–7.43 (d, 1H, J = 8.18 Hz, Ar-H), 7.52–7.54 (d, 1H, J = 7.8 Hz, Ar-H), 7.67–7.68 (d, 1H, J = 3.68 Hz, Ar-H), 7.83–7.85 (dd, 1H, J = 8.14, 2.02 Hz, Ar-H), 8.02–8.04 (dd, 1H, J = 8.28, 0.52 Hz, Ar-H), 8.12 (d, 1H, J = 1.24 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₅N₃O₂S calc. 384.1667, found. 384.1663.

The compound 6c was prepared from starting materials 5b and N-ethyl piperazine

1-[4-Methyl-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6c)

Yield: 62%, after 2 steps; Melting point: 250.13 °C; I.R (cm⁻¹): 3300, 2997, 1643, 1168, 752; ESIMS (m/z): 398.1 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.35–1.39 (t, 3H, N-CH₂CH₃), 2.42 (s, 3H, Ar-CH₃), 3.12–3.14 (bs, 6H, Piperazine-H), 3.23–3.29 (q, 2H, N-CH₂CH₃), 3.6 (bs, 2H, Piperazine-H), 4.05 (s, 2H, Ar-CH₂-N), 6.72–6.73 (dd, 1H, J = 3.12, 0.58 Hz, Ar-H), 7.20–7.24 (m, 1H, Ar-H), 7.30–7.34 (m, 1H, Ar-H), 7.38–7.40 (d, 1H, J = 8.17 Hz, Ar-H), 7.52–7.54 (d, 1H, J = 7.82 Hz, Ar-H), 7.65–7.66 (d, 1H, J = 3.69 Hz, Ar-H), 7.80–7.82 (dd, 1H, J = 8.11, 1.97 Hz, Ar-H), 8.0–8.02 (m, 2H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₇N₃O₂S calc. 398.1824, found. 398.1821.

The compound 6d was prepared from starting materials 5d and N-methyl piperazine.

1-[4-Chloro-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6d)

Yield: 59%, after 2 steps; Melting range: 210.6–213.90 °C; I.R (cm⁻¹): 3415, 2987, 1591, 1444, 1373, 1174, 736; ESIMS (m/z): 403.9 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.84 (s, 3H, N-CH₃), 3.05 (bs, 4H, Piperazine-H), 3.28 (bs, 4H, Piperazine-H), 4.10 (s, 2H, Ar-CH₂-N), 6.72–6.78 (d, 1H, J = 3.7 Hz, Ar-H), 7.18–7.22 (d, 1H, J = 7.4 Hz, Ar-H), 7.26–7.30 (d, 1H, J = 7.4 Hz, Ar-H), 7.49–7.51 (d, 2H, J = 8.4 Hz, Ar-H), 7.55–7.56 (d, 1H, J = 3.72 Hz, Ar-H), 7.82–7.84 (dd, 1H, J = 8.52, 2.32 Hz, Ar-H), 7.86–7.88 (dd, 2H, J = 7.92, 2.06 Hz, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₂ClN₃O₂S calc. 404.1121, found. 404.1117.

The compound 6e was prepared from starting materials 5d and N-ethyl piperazine.

1-[4-Chloro-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6e)

Yield: 62%, after 2 steps; Melting range: 196.0–197.1 °C; I.R (cm⁻¹): 3446, 2985, 2451, 1469, 1444, 1172, 740; ESIMS (m/z): 418.0 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.17–1.21 (t, 3H, N-CH₂CH₃), 3.04 (bs, 8H, Piperazine-H), 3.10–3.15 (q, 2H, N-CH₂CH₃), 4.10 (s, 2H, Ar-CH₂-N), 6.69–6.70 (d, 1H, J = 3.61 Hz, Ar-H), 7.15–7.19 (d, 1H, J = 7.5 Hz, Ar-H), 7.23–7.27 (m, 1H, J = 7.3 Hz, Ar-H), 7.46–7.48 (d, 2H, J = 8.34 Hz, Ar-H), 7.52–7.53 (d, 1H, J = 3.71 Hz, Ar-H), 7.79–7.81 (dd, 1H, J = 8.5, 2.2 Hz, Ar-H), 7.84–7.86 (m, 2H, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄ClN₃O₂S calc. 418.1278, found. 418.1276.

The compound 6f was prepared from starting materials 5 g and N-methyl piperazine.

1-[4-Bromo-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6f)

Yield: 59%, after 2 steps; Melting range: 214.1–216.0 °C; I.R (cm⁻¹): 3429, 2372, 1440, 1170, 740; ESIMS (m/z): 447.9 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.26 (bs, 2H, Piperazine-H), 2.79 (s, 3H, N-CH₃ & 2H, Piperazine-H), 3.09–3.11 (bs, 2H, Piperazine-H), 3.37–3.40 (bs, 2H, Piperazine-H), 3.7 (s, 2H, Ar-CH₂-N), 6.86 (dd, 1H, J = 3.7, 0.57 Hz, Ar-H), 7.24–7.28 (m, 1H, Ar-H), 7.33–7.37 (m, 1H, Ar-H), 7.59–7.61 (d, 1H, J = 7.67 Hz, Ar-H), 7.76–7.78 (dd, 1H, J = 8.4, 2.4 Hz, Ar-H), 7.80–7.81 (d, 1H, J = 3.6 Hz, Ar-H), 7.847.86 (d, 1H, J = 8.44 Hz, Ar-H), 7.93–7.95 (dd, 1H, J = 8.0, 0.35 Hz, Ar-H), 8.09 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₂BrN₃O₂S calc. 448.0616, found. 448.0611.

The compound 6h was prepared from starting materials 5 h and N-methyl piperazine.

5-Methoxy-1-[3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6h)

Yield: 64%; I.R (cm⁻¹): 2936, 2790, 1615, 1465, 1224, 996; ESIMS (m/z): 400.3 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.28 (s, 3H, N-CH₃), 2.35 (bs, 8H, Piperazine-H), 3.46 (s, 2H, Ar-CH₂-N), 3.80 (s, 3H, Ar-OCH₃), 6.57–6.58 (d, 1H, J = 3.6 Hz, Ar-H), 6.89–6.92 (dd, 1H, J = 8.96, 2.52 Hz, Ar-H), 6.95 (d, 1H, J = 2.44 Hz, Ar-H), 7.33–7.36 (m, 1H, Ar-H), 7.45–7.47 (m, 1H, Ar-H), 7.51–7.52 (d, 1H, J = 3.6 Hz, Ar-H), 7.69–7.71 (m, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 7.86–7.89 (d, 1H, J = 9.0 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₅N₃O₃S calc. 400.1617, found. 400.1619.

The compound 6i was prepared from starting materials 5i and N-methyl piperazine.

5-Ethoxy-1-[3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6i)

Yield: 62%; I.R (cm⁻¹): 3743, 2935, 1615, 1456, 1222, 900; ESIMS (m/z): 414.4 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.38–1.42 (t, 3H, Ar-OCH₂CH₃), 2.28 (s, 3H, N-CH₃), 2.35 (bs, 8H, Piperazine-H), 3.48 (s, 2H, Ar-CH₂-N), 3.98–4.03 (q, 2H, Ar-OCH₂CH₃), 6.56–6.57 (d, 1H, J = 3.60 Hz, Ar-H), 6.89–6.92 (dd, 1H, J = 8.96, 2.45 Hz, Ar-H), 6.93–6.94 (d, 1H, J = 2.38 Hz, Ar-H), 7.32–7.36 (m, 1H, Ar-H), 7.44–7.46 (m, 1H, Ar-H), 7.50–7.51 (d, 1H, J = 3.64 Hz, Ar-H), 7.69–7.71 (m, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 7.86–7.88 (d, 1H, J = 8.98 Hz, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₇N₃O₃S calc. 414.1773, found. 414.1778.

The compound 6j was prepared from starting materials 5j and N-methyl piperazine.

5-Isopropoxy-1-[3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6j)

Yield: 63%; I.R (cm⁻¹): 2974, 1612, 1455, 1221, 923; ESIMS (m/z): 428.3 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.31–1.32 (d, 6H, J = 6.04 Hz, Ar-OCH(CH₃)₂), 2.30 (s, 3H, N-CH₃), 2.46 (bs, 8H, Piperazine-H), 3.47 (s, 2H, Ar-CH₂-N), 4.47 (sept, 1H, Ar-OCH(CH₃)₂), 6.55–6.56 (d, 1H, J = 3.56 Hz, Ar-H), 6.87–6.90 (dd, 1H, J = 8.96, 2.44 Hz, Ar-H), 6.95–6.96 (d, 1H, J = 2.44 Hz, Ar-H), 7.33–7.37 (m, 1H, Ar-H), 7.45–7.47 (m, 1H, Ar-H), 7.50–7.51 (d, 1H, J = 3.56 Hz, Ar-H), 7.7–7.72 (m, 1H, Ar-H), 7.84–7.87 (m, 2H, Ar-H); HRMS: [M + H]⁺ C₂₃H₂₉N₃O₃S calc. 428.1930, found. 428.1936.

The compound 6k was prepared from starting materials 5k and N-methyl piperazine.

5-Fluoro-1-[3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole (6k)

Yield: 62%; I.R (cm⁻¹): 2937, 1590, 1460, 1216, 998; ESIMS (m/z): 388.3 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.30 (s, 3H, N-CH₃), 2.36 (bs, 8H, Piperazine-H), 3.48 (s, 2H, Ar-CH₂-N), 6.61–6.62 (d, 1H, J = 3.64 Hz, Ar-H), 7.03 (m, 1H, Ar-H), 7.15–7.18 (m, 1H, Ar-H), 7.37–7.39 (m, 1H, Ar-H), 7.47 (m, 1H, Ar-H), 7.59–7.60 (d, 1H, J = 3.64 Hz, Ar-H), 7.71 (m, 1H, Ar-H), 7.84 (m, 1H, Ar-H), 7.92–7.94 (m, 1H, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₂FN₃O₂S calc. 388.1417, found. 388.1421.

The compound 6l was prepared from starting materials 5 l and N-methyl piperazine.

1-[4-Methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-1H-indole dihydrochloride (6l)

Yield: 63%, after 2 steps; Melting range: 198.1–199.3 °C; I.R (cm⁻¹): 3427, 2974, 2372, 1616, 1465, 1224, 950; ESIMS (m/z): 414.3 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.37 (s, 3H, Ar-CH₃), 2.78 (s, 3H, N-CH₃), 2.79–3.46 (bs, 8H, Piperazine-H), 3.73 (s, 3H, Ar-OCH₃), 3.83–3.90 (s, 2H, Ar-CH₂-N), 6.73–6.74 (d, 1H, J = 3.6 Hz, Ar-H), 6.91–6.93 (dd, 1H, J = 9.01, 2.5 Hz, Ar-H), 7.08 (d, 1H, J = 2.46 Hz, Ar-H), 7.39–7.41 (d, 1H, J = 8.13 Hz, Ar-H), 7.73–7.76 (m, 2H, Ar-H), 7.82–7.84 (d, 1H, J = 9.02 Hz, Ar-H), 8.11 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₇N₃O₃S calc. 414.1773, found. 414.1775.

The compound 6m was prepared from starting materials 5m and N-methyl piperazine.

1-[4-Methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole dihydrochloride (6m)

Yield: 57%, after 2 steps; Melting range: 220.3–223.4 °C; I.R (cm⁻¹): 3370, 2642, 1596, 1464, 1171, 949; ESIMS (m/z): 402.3 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.45 (s, 3H, Ar-CH₃), 2.97 (s, 3H, N-CH₃), 3.29–3.62 (bs, 8H, Piperazine-H), 4.19 (s, 2H, Ar-CH₂-N), 6.72–6.73 (d, 1H, J = 3.6 Hz, Ar-H), 7.07–7.13 (m, 1H, Ar-H), 7.23–7.25 (dd, 1H, J = 8.9, 2.52 Hz, Ar-H), 7.41 7.43 (d, 1H, J = 8.18 Hz, Ar-H), 7.75–7.76 (d, 1H, J = 3.67 Hz, Ar-H), 7.82–7.89 (dd, 1H, J = 8.14, 1.96 Hz, Ar-H), 8.01–8.02 (m, 1H, Ar-H), 8.12 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄FN₃O₂S calc. 402.1573, found. 402.1569.

The compound 6n was prepared from starting materials 5m and N-ethyl piperazine.

1-[4-Methyl-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole dihydrochloride (6n)

Yield: 59%, after 2 steps; Melting range: 203.4–206.2 °C; I.R (cm⁻¹): 3425, 2458, 1594, 1443, 1171, 799; ESIMS (m/z): 416.3 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.23–1.27 (t, 3H, N-CH₂CH₃), 2.38 (s, 3H, Ar-CH₃), 3.03–3.06 (bs, 8H, Piperazine-H), 3.35–3.37 (q, 2H, N-CH₂CH₃), 3.8 (s, 2H, Ar-CH₂-N), 6.81–6.82 (d, 1H, J = 3.56 Hz, Ar-H), 7.16–7.21 (dd, 1H, J = 9.2, 2.62 Hz, Ar-H), 7.397.41 (dd, 1H, J = 9.16, 2.58 Hz, Ar-H), 7.41–7.43 (d, 1H, J = 7.86 Hz, Ar-H), 7.79–7.81 (d, 1H, J = 6.98 Hz, Ar-H), 7.87–7.88 (d, 1H, J = 3.66 Hz, Ar-H), 7.94–7.98 (dd, 1H, J = 9.02, 4.42 Hz, Ar-H), 8.13 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆FN₃O₂S calc. 416.1730, found. 416.1727.

The compound 6o was prepared from starting materials 5o and N-methyl piperazine.

1-[4-Chloro-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole dihydrochloride (6o)

Yield: 58%, after 2 steps; Melting range: 220.2–221.3 °C; I.R (cm⁻¹): 3429, 2983, 1591, 1463, 1377, 1138, 950; ESIMS (m/z): 422.0 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.84 (s, 3H, N-CH₃), 3.05 (bs, 4H, Piperazine-H), 3.34–3.36 (bs, 4H, Piperazine-H), 3.98 (s, 2H, Ar-CH₂-N), 6.60–6.61 (d, 1H, J = 3.67, Ar-H), 6.91–6.96 (dd, 1H, J = 9.18, 2.4 Hz, Ar-H), 7.04–7.07 (dd, 1H, J = 8.99, 2.41 Hz, Ar-H), 7.26–7.28 (d, 1H, J = 8.52 Hz, Ar-H), 7.53–7.54 (d, 1H, J = 3.68 Hz, Ar-H), 7.63–7.66 (dd, 1H, J = 8.5, 1.9 Hz, Ar-H), 7.72–7.75 (m, 1H, J = 4.34 Hz, Ar-H), 7.85–7.86 (d, 1H, J = 2.18 Hz, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₁ClFN₃O₂S calc. 422.1027, found. 422.1023.

The compound 6p was prepared from starting materials 5o and N-ethyl piperazine.

1-[4-Chloro-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole dihydrochloride (6p)

Yield: 56%, after 2 steps; Melting range: 204.1–205.5 °C; I.R (cm⁻¹): 3414, 2976, 1622, 1458, 1139, 804; ESIMS (m/z): 436.0 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.20–1.23 (t, 3H, N-CH₂CH₃), 3.04 (bs, 4H, Piperazine-H), 3.13–3.18 (q, 2H, N-CH₂CH₃), 3.32–3.34 (bs, 4H, Piperazine-H), 4.05 (s, 2H, Ar-CH₂-N), 6.65–6.66 (d, 1H, J = 3.67 Hz, Ar-H), 6.99–7.03 (m, 1H, Ar-H), 7.13–7.16 (dd, 1H, J = 9.0, 2.5 Hz, Ar-H), 7.41–7.43 (d, 1H, J = 8.55 Hz, Ar-H), 7.56–7.57 (d, 1H, J = 3.7 Hz, Ar-H), 7.74–7.76 (dd, 1H, J = 8.55, 2.26 Hz, Ar-H), 7.78–7.81 (dd, 1H, J = 9.0, 4.1 Hz, Ar-H), 7.86 (d, 1H, J = 2.3 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₃ClFN₃O₂S calc. 436.1184, found. 436.1182.

The compound 6q was prepared from starting materials 5q and N-methyl piperazine.

1-[4-Bromo-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole dihydrochloride (6q)

Yield: 55%, after 2 steps; Melting range: 219.4–221.6 °C; I.R (cm⁻¹): 3435, 2924, 2366, 1633, 1452, 1134, 943; ESIMS (m/z): 465.7 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.83 (s, 3H, N-CH₃), 2.93 (bs, 4H, Piperazine-H), 3.2–3.26 (bs, 4H, Piperazine-H), 3.96 (s, 2H, Ar-CH₂-N), 6.70–6.71 (d, 1H, J = 3.6 Hz, Ar-H), 7.02–7.07 (m, 1H, Ar-H), 7.19–7.22 (dd, 1H, J = 9.02, 2.5 Hz, Ar-H), 7.6–7.62 (d, 1H, J = 3.72 Hz, Ar-H), 7.67–7.72 (bs, 2H, Ar-H), 7.8 (s, 1H, Ar-H), 7.82–7.86 (m, 1H, Ar-H); HRMS: [M + H]⁺ C₂₀H₂₁BrFN₃O₂S calc. 466.0522, found. 466.0519.

The compound 6r was prepared from starting materials 5q and N-ethyl piperazine.

1-[4-Bromo-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-fluoro-1H-indole (6r)

Yield: 58%; Melting range: 92.2–93.9 °C; IR (cm⁻¹): 2939, 1589, 1458, 1138, 808; ESIMS (m/z): 479.8 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.11 (t, 3H, N-CH₂CH₃), 2.43–2.48 (bs, 8H, Piperazine-H & 2H, N-CH₂CH₃), 3.51 (s, 2H, Ar-CH₂-N), 6.62–6.63 (dd, 1H, J = 3.59, 0.44 Hz, Ar-H), 7.00–7.05 (m, 1H, Ar-H), 7.16–7.19 (dd, 1H, J = 8.66, 2.5 Hz, Ar-H), 7.5–7.6 (m, 3H, Ar-H), 7.917.95 (m, 1H, Ar-H), 7.97 (d, 1H, J = 1.94 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₃BrFN₃O₂S calc. 480.0678, found. 480.0675.

The compound 6s was prepared from starting materials 5s and N-ethyl piperazine.

1-[4-Bromo-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-bromo-1H-indole dihydrochloride (6s)

Yield: 56%, after 2 steps; Melting range: 205.1–207.1 °C; I.R (cm⁻¹): 3435, 2346, 1439, 1172, 815; ESIMS (m/z): 540.2 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.07–1.22 (t, 3H, N-CH₂CH₃), 2.59–2.89 (bs, 6H, Piperazine-H), 3.04–3.07 (q, 2H, N-CH₂CH₃), 3.10–3.12 (bs, 2H, Piperazine-H), 3.34 (s, 2H, Ar-CH₂-N), 6.50–6.51 (d, 1H, J = 3.39 Hz, Ar-H), 7.15 (m, 2H, Ar-H), 7.32 (m, 2H, Ar-H), 7.49 (dd, 1H, J = 9.24, 3.49 Hz, Ar-H), 7.61 (d, 1H, J = 8.74 Hz, Ar-H), 7.70 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₃Br₂N₃O₂S calc. 539.9878, found. 539.9881.

The compound 6t was prepared from starting materials 5t and N-methyl piperazine.

1-[4-Chloro-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-1H-indole dihydrochloride (6t)

Yield: 62%, after 2 steps; Melting range: 208.8–210.2 °C; I.R (cm⁻¹): 3446, 2981, 2372, 1585, 1465, 1222, 952; ESIMS (m/z): 434.1 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.82 (s, 3H, N-CH₃), 3.05 (bs, 4H, Piperazine-H), 3.23–3.28 (bs, 4H, Piperazine-H), 3.60 (s, 3H, Ar-OCH₃), 3.9 (s, 2H, Ar-CH₂-N), 6.55–6.56 (d, 1H, J = 3.64 Hz, Ar-H), 6.67–6.79 (dd, 1H, J = 9.03, 2.44 Hz, Ar-H), 6.88–6.89 (d, 1H, J = 2.42 Hz, Ar-H), 7.20–7.22 (d, 1H, J = 8.53 Hz, Ar-H), 7.42–7.43 (d, 1H, J = 3.65 Hz, Ar-H), 7.58–7.61 (dd, 1H, J = 8.5, 2.1 Hz, Ar-H), 7.65–7.68 (d, 1H, J = 9.04 Hz, Ar-H), 7.79 (d, 1H, J = 2.21 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄ClN₃O₃S calc. 434.1227, found. 434.1225.

The compound 6u was prepared from starting materials 5t and N-ethyl piperazine.

1-[4-Chloro-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-1H-indole dihydrochloride (6u)

Yield: 61%, after 2 steps; Melting range: 209.5–211.0 °C; I.R (cm⁻¹): 3448, 2983, 1585, 1465, 1222, 761; ESIMS (m/z): 448.0 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.19–1.23 (t, 3H, N-CH₂CH₃), 2.97 (bs, 4H, Piperazine-H), 3.11–3.16 (q, 2H, N-CH₂CH₃), 3.23 (bs, 4H, Piperazine-H), 3.67 (s, 3H, Ar-OCH₃), 4.0 (s, 2H, Ar-CH₂-N), 6.61–6.62 (d, 1H, J = 3.66 Hz, Ar-H), 6.83–6.86 (dd, 1H, J = 9.0, 2.5 Hz, Ar-H), 6.98 (d, 1H, J = 2.47 Hz, Ar-H), 7.39–7.41 (d, 1H, J = 8.54 Hz, Ar-H), 7.46–7.47 (d, 1H, J = 3.68 Hz, Ar-H), 7.71–7.74 (m, 2H, Ar-H), 7.80 (d, 1H, J = 2.2 Hz, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆ClN₃O₃S calc. 448.1383, found. 448.1378.

The compound 6v was prepared from starting materials 5v and N-methyl piperazine.

1-[4-Bromo-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-1H-indole (6v)

Yield: 63%; Melting range: 115.2–117 °C; I.R (cm⁻¹): 2929, 1606, 1371, 1217, 817; ESIMS (m/z): 477.9 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 2.33 (s, 3H, N-CH₃), 2.35 (bs, 8H, Piperazine-H), 3.50 (s, 2H, Ar-CH₂-N), 3.80 (s, 3H, Ar-OCH₃), 6.58 (dd, 1H, J = 3.6, 0.48 Hz, Ar-H), 6.9 (dd, 1H, J = 8.97, 2.50 Hz, Ar-H), 6.95 (d, 1H, J = 2.43 Hz, Ar-H), 7.48 (d, 1H, J = 3.6 Hz, Ar-H), 7.56 (m, 2H, Ar-H), 7.85 (d, 1H, J = 8.9 Hz, Ar-H), 7.96 (d, 1H, J = 1.33 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄BrN₃O₃S calc. 478.0722, found. 478.0715.

The compound 6w was prepared from starting materials 5v and N-ethyl piperazine.

1-[4-Bromo-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-1H-indole (6w)

Yield: 59%; Melting range: 196.1–198.6 °C; I.R (cm⁻¹): 3444, 2430, 1614, 1438, 1224, 761; ESIMS (m/z): 491.9 [M + H]⁺; ¹H-NMR (400 MHz, CDCl₃): δ 1.22–1.27 (t, 3H, N-CH₂CH₃), 2.65–2.66 (bs, 4H, Piperazine-H), 2.99–3.00 (bs, 2H, Piperazine-H), 3.12–3.14 (bs, 2H, Piperazine-H), 3.22–3.34 (q, 2H, N-CH₂CH₃), 3.35–3.37 (s, 2H, Ar-CH₂-N), 3.79 (s, 3H, Ar-OCH₃), 6.67–6.79 (d, 1H, J = 3.69 Hz, Ar-H), 6.93–6.96 (dd, 1H, J = 9.01, 2.52 Hz, Ar-H), 7.10–7.11 (d, 1H, J = 2.4 Hz, Ar-H), 7.71–7.72 (dd, 1H, J = 8.45, 2.42 Hz, Ar-H), 7.75–7.76 (d, 1H, J = 3.65 Hz, Ar-H), 7.80–7.85 (m, 2H, Ar-H), 8.03 (bs, 1H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆BrN₃O₃S calc. 491.0878, found. 491.0882.

The compound 6x was prepared from starting materials 5x and N-methyl piperazine.

1-[4-Chloro-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-3-methyl-1H-indole dihydrochloride (6x)

Yield: 61%, after 2 steps; Melting range: 216.9–218.7 °C; I.R (cm⁻¹): 3493, 2673, 1627, 1450, 1174, 962; ESIMS (m/z): 418.4 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.01 (s, 3H, Ar-CH₃), 2.83 (s, 3H, N-CH₃), 3.00 (bs, 4H, Piperazine-H), 3.2–3.35 (bs, 4H, Piperazine-H), 3.98 (s, 2H, Ar-CH₂-N), 7.12–7.14 (m, 1H, Ar-H), 7.15–7.31 (m, 4H, Ar-H), 7.62–7.63 (dd, 1H, J = 8.54, 1.96 Hz, Ar-H), 7.65–7.84 (m, 2H, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄ClN₃O₂S calc. 418.1278, found. 418.1275.

The compound 6y was prepared from starting materials 5x and N-ethyl piperazine.

1-[4-Chloro-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-3-methyl-1H-indole dihydrochloride (6y)

Yield: 63%, after 2 steps; Melting range: 218.9–220.5 °C; I.R (cm⁻¹): 3417, 2436, 1631, 1442, 1166, 966; ESIMS (m/z): 432.3 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.19–1.22 (t, 3H, N-CH₂CH₃), 2.00 (s, 3H, Ar-CH₃), 2.93 (bs, 4H, Piperazine-H) 3.11–3.16 (q, 2H, N-CH₂CH₃), 3.33–3.35 (bs, 4H, Piperazine-H), 4.01 (s, 2H, Ar-CH₂-N), 7.10–7.14 (m, 1H, Ar-H), 7.23–7.30 (m, 4H, Ar-H), 7.61 (dd, 1H, J = 8.64, 1.74 Hz, Ar-H), 7.82–7.84 (m, 2H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆ClN₃O₂S calc. 432.1434, found. 432.1436.

The compound 6z was prepared from starting materials 5z and N-methyl piperazine.

1-[4-Bromo-3-(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-3-methyl-1H-indole dihydrochloride (6z)

Yield: 56%, after 2 steps; Melting range: 210.5–213 °C; I.R (cm⁻¹): 3429, 2370, 1631, 1174, 948; ESIMS (m/z): 462.2 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.97 (s, 3H, Ar-CH₃), 2.78 (s, 3H, N-CH₃), 2.79 (bs, 4H, Piperazine-H), 3.10–3.17 (bs, 4H, Piperazine-H), 3.72 (s, 2H, Ar-CH₂-N), 7.06–7.08 (m, 1H, Ar-H), 7.17–7.21 (m, 4H, Ar-H), 7.37 (m, 1H, Ar-H), 7.72 (bs, 1H, Ar-H), 7.79–7.82 (d, 1H, J = 8.20 Hz, Ar-H); HRMS: [M + H]⁺ C₂₁H₂₄BrN₃O₂S calc. 462.0773, found. 462.0778.

The compound 6aa was prepared from starting materials 5z and N-ethyl piperazine

1-[4-Bromo-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-3-methyl-1H-indole dihydrochloride (6aa)

Yield: 62%, after 2 steps; Melting range: 204.1–206.5 °C; I.R (cm⁻¹): 3439, 2434, 1566, 1442, 1174, 966; ESIMS (m/z): 476.2 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.16–1.19 (t, 3H, N-CH₂CH₃), 1.96 (s, 3H, Ar-CH₃), 2.77–2.94 (bs, 4H, Piperazine-H), 3.04 (q, 2H, N-CH₂CH₃), 3.08 (bs, 4H, Piperazine-H), 3.69 (s, 2H, Ar-CH₂-N), 7.04–7.20 (m, 4H, Ar-H), 7.22 (bs, 1H, Ar-H), 7.35–7.37 (d, 1H, J = 8.2 Hz, Ar-H), 7.72 (bs, 1H, Ar-H), 7.8–7.82 (d, 1H, J = 8.16 Hz, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆BrN₃O₂S calc. 476.0929, found. 476.0931.

The compound 6ab was prepared from starting materials 5ab and N-methyl piperazine.

1-[4-Chloro-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-3-methyl-1H-indole dihydrochloride (6ab)

Yield: 61%, after 2 steps; Melting range: 213.3–214.2 °C; I.R (cm⁻¹): 3479, 2963, 2441, 1608, 1454, 1172, 883; ESIMS (m/z): 448.3 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 2.16 (s, 3H, Ar-CH₃), 2.77 (s, 3H, N-CH₃), 2.95–2.99 (bs, 6H, Piperazine-H), 3.45 (bs, 2H, Piperazine-H), 3.94 (s, 3H, Ar-OCH₃), 4.29 (s, 2H, Ar-CH₂-N), 6.92–6.96 (dd, 1H, J = 8.96, 2.52 Hz, Ar-H), 7.01 (d, 1H, J = 2.44 Hz, Ar-H), 7.54 (d, 1H, J = 1.04 Hz, Ar-H), 7.66–7.68 (d, 1H, J = 8.52 Hz, Ar-H), 7.79–7.83 (m, 2H, Ar-H), 8.22 (s, 1H, Ar-H); HRMS: [M + H]⁺ C₂₂H₂₆ClN₃O₃S calc. 448.1383, found. 448.1387.

The compound 6ac was prepared from starting materials 5ab and N-ethyl piperazine.

1-[4-Chloro-3-(4-ethyl piperazin-1-yl methyl) phenylsulfonyl]-5-methoxy-3-methyl-1H-indole dihydrochloride (6ac)

Yield: 59%, after 2 steps; Melting range: 217.5–220.8 °C; I.R (cm⁻¹): 3456, 2983, 1612, 1448, 1172, 962; ESIMS (m/z): 462.2 [M + H]⁺; ¹H-NMR (400 MHz, D₂O): δ 1.10–1.14 (t, 3H, N-CH₂CH₃), 1.91 (s, 3H, Ar-CH₃), 3.07–3.12 (bs, 10H, Piperazine-H & 2H, N-CH₂CH₃), 3.60 (s, 3H, Ar-OCH₃), 4.18 (s, 2H, Ar-CH₂-N), 6.74–6.8 (m, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 7.37–7.39 (d, 1H, J = 8.76 Hz, Ar-H), 7.61–7.67 (m, 2H, Ar-H), 7.74 (s, 1H, Ar-H); HRMS: [M + H]⁺ C₂₃H₂₈ClN₃O₃S calc. 462.1540, found. 462.1543.

Results and discussion

Structure–activity relationship (SAR)

Several analogs of 1-[3–(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indoles and 1-[3–(4-ethyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indoles were synthesized and their radioligand binding affinities at the human 5-HT₆R at 100 nM concentration are given in Table 1.

Table 1. 5-HT₆R binding data† for compounds 6.

Comp. No	R^1	R^2	R^3	R^4	% Inhibition at 100 nM concentration*	Ki (nM)
6a	H	H	H	CH3	92.41	7.50 ± 0.42
6b	H	H	CH3	CH3	95.11	7.01 ± 0.26
6c	H	H	CH3	C2H5	95.48	3.96 ± 0.28
6d	H	H	Cl	CH3	92.36	18.4 ± 0.84
6e	H	H	Cl	C2H5	92.06	15.6 ± 0.70
6f	H	H	Br	CH3	88.79	–
6g	H	H	Br	C2H5	87.68	–
6h	5-OCH3	H	H	CH3	67.58	–
6i	5-OC2H5	H	H	CH3	63.73	–
6j	5-O^iPr	H	H	CH3	61.48	–
6k	5-F	H	H	CH3	79.20	–
6l	5-OCH3	H	CH3	CH3	62.18	–
6m	5-F	H	CH3	CH3	86.18	–
6n	5-F	H	CH3	C2H5	78.54	–
6o	5-F	H	Cl	CH3	72.97	–
6p	5-F	H	Cl	C2H5	64.82	–
6q	5-F	H	Br	CH3	70.45	–
6r	5-F	H	Br	C2H5	62.52	–
6s	5-Br	H	Br	C2H5	55.86	–
6t	5-OCH3	H	Cl	CH3	57.03	–
6u	5-OCH3	H	Cl	C2H5	55.68	–
6v	5-OCH3	H	Br	CH3	54.21	–
6w	5-OCH3	H	Br	C2H5	50.53	–
6x	H	CH3	Cl	CH3	96.63	5.45 ± 0.28
6y	H	CH3	Cl	C2H5	92.91	7.85 ± 0.35
6z	H	CH3	Br	CH3	93.23	9.11 ± 0.24
6aa	H	CH3	Br	C2H5	91.51	12.8 ± 0.63
6ab	5-OCH3	CH3	Cl	CH3	71.53	–
6ac	5-OCH3	CH3	Cl	C2H5	68.10	–

*The data represent average of two determinations.

†5-HT₆ receptor-binding studies were carried out at NovaScreen Biosciences Corporation, (caliper life sciences), Hanover, MD, USA. Human recombinant/HEK293 cells; Radioligand: [³H] LSD (60–80 Ci/mmol).

“^_” means not tested for K_i (nM) determination.

The binding data of these compounds at 100 nM concentration indicated that the compounds have high to moderate affinity to the 5-HT₆R. Among the compounds synthesized 6a, 6b, 6c, 6d, 6e, 6x, 6y, 6z and 6aa have shown good binding affinity at human 5-HT₆R, when compared to other compounds in the series. Unsubstituted indole derivatives 6a, 6b, 6c and 6d have shown higher 5-HT₆R-binding affinities than those of the corresponding 5-substituted compounds, suggesting that the substitution at 5th position of indole ring is not well tolerated. This can be seen by comparing the in vitro binding affinities of the compound 6a with 6 h, 6i, 6j and 6k, compound 6b with 6 l and 6m, compound 6c with 6n or compound 6d with 6o and 6t. Compounds with electron donating and/or bulky groups (viz. OCH₃, OC₂H₅, OCH(CH₃)₂) at C₅ of indole showed moderate potency toward 5-HT₆R (compounds 6 h, 6i and 6j). One-fourth of the activity was lost when electron-donating groups like methoxy group was introduced at C₅ of indole as can be seen by comparing the binding affinities of the compound 6a with 6 h, compound 6d with 6t, compound 6e with 6u, compound 6f with 6v, compound 6 g with 6w, compound 6x with 6ab or compound 6y with 6ac.

More or less a very similar trend was observed in the case of electron withdrawing and/or bulky halogen groups (viz. F, Br) as can be seen by comparing the binding affinities of the compound 6 g with 6r and 6s. The C₅ halo substituted indole derivatives exhibited moderate affinity toward 5-HT₆R as can be seen from 6k–6s. In terms of in vitro affinity, the preferred order of substitution on phenylsulfonyl ring at C-4′ position was found to be CH₃, H_, Cl over Br as can be seen by comparing the binding affinities of the compounds 6b with 6a, 6d and 6f, compound 6k with 6m, 6o and 6q. In terms of activity, almost similar trend was observed in compounds with N₁-methyl or N₁-ethyl substitution on piperazine ring, as can be seen from comparing the binding affinities of the compound 6b with 6c, compound 6d with 6e, compound 6f with 6 g, compound 6t with 6u, compound 6v with 6w, compound 6x with 6y or compound 6z with 6aa.

Generally, replacement of the hydrogen atom at the indole-C₃ with the small methyl group is well tolerated and produced good in vitro potency, as can be seen by comparing affinities of the methyl-substituted derivatives 6x, 6y, 6z and 6aa with those of the corresponding C₃-unsubstituted compounds 6d, 6e, 6f and 6g.

The compounds showing >90% inhibition at 100 nM concentration at 5-HT₆R were further assessed for their K_i (nM) values by performing complete dose response from 10 µM to 0.1 nM in semilogarithmic concentrations (Table 1).

The compound 6b was also evaluated in 5-HT₆ reporter gene cell-based functional assay43^,44. The tested compound 6b has shown antagonistic activity by inhibiting the 5-HT-stimulated cAMP accumulation, with a K_b value of 4.92 ± 0.13 nM and IC₅₀ value of 548.2 ± 3.6 nM.

Selected compounds that displayed satisfactory potency towards 5-HT₆R in in vitro binding assay were further profiled in selectivity assays, against a panel of related serotonin receptors like 5-HT_4b, 5-HT₃, 5-HT2A and 5-HT2C, histamine H₁, dopamine D₂, adrenergic α_1B and the transporters such as SERT, NET and DAT.

We were also pleased to find that the tested compounds (6b, 6c and 6x) demonstrated <30% inhibition at maximum tested concentration of 10 µM, indicating that the tested compounds have very less activity toward these off targets and are highly selective toward 5HT₆ receptor (data not shown).

In drug discovery, a major challenge of CNS therapy is the blood–brain barrier (BBB). 5-HT₆ receptor antagonists should cross the blood brain barrier for treating cognitive dysfunction. This greatly limits the potential of a compound to become a successful CNS agent.

We have profiled the selected compounds (6b, 6c and 6x) for their PK parameters (Table 2). Brain and plasma levels were calculated in male Wistar rats after 6 h i.v. continuous infusion of compounds 6b, 6c and 6x at 1 mg/kg/h dose. Among these compounds, 6b has shown adequate brain to plasma ratio of 2.03 ± 0.42 (brain and plasma concentrations are 510 ± 145 ng/g, 286 ± 80 ng/mL), whereas compounds 6c and 6x were having lower to moderate brain to plasma ratio of 0.53 ± 0.13 (brain and plasma concentrations are 312 ± 82 ng/g, 59 ± 37 ng/mL) and 1.61 ± 0.39 (brain and plasma concentrations are 486 ± 250 ng/g, 293 ± 102 ng/mL), respectively. The pharmacokinetic profile of 6b, 6c and 6x was assessed in male Wistar rats (Table 2). Following i.v. administration of 10 mg/kg, the compounds 6b, 6c and 6x have shown good mean half-life of 2.69 ± 1.51 h, 6.25 ± 0.35 h and 5.84 ± 2.21 h, respectively. Following oral administration at 10 mg/kg, the compounds 6b and 6c have shown good plasma concentrations of 1375 ± 427 ng/mL, 749 ± 836 ng/mL, respectively, whereas compound 6x has shown low plasma concentration of 106 ± 1 ng/mL.

Table 2. Pharmacokinetic profile of compounds 6b, 6c, and 6x in male wistar rats*.

Compound	6b	6c	6x
i.v. (n = 3)
Dose (mg/kg)	10	10	10
t1/2 (h)	2.69 ± 1.51	6.25 ± 0.35	5.84 ± 2.21
Vz (L/kg)	3.6 ± 1.2	6.9 ± 4.3	7.5 ± 1.0
Cl (mL/min/kg)	34 ± 12	40 ± 7	52 ± 7
p.o. (n = 3)
Dose (mg/kg)	10	10	10
t1/2 (h)	7.85 ± 2.62	3.18 ± 1.71	1.73 ± 0.56
Cmax (ng/mL)	1375 ± 427	749 ± 836	106 ± 1
Tmax (h)	0.38 ± 0.18	0.50 ± 0.43	0.38 ± 0.18
AUC (ng.h/mL)	3194 ± 1748	1171 ± 1046	342 ± 152
F (%)	30 ± 7	14 ± 10	10 ± 6
Brain (ng/g)	570 ± 145	312 ± 82	486 ± 250
Plasma (ng/mL)	286 ± 80	592 ± 37	293 ± 102
Cb/Cp	2.03 ± 0.42	0.53 ± 0.13	1.61 ± 0.39

*Fasted male wistar rats, vehicle used: water for injection for both oral and intravenous routes. Dosing volumes: 10 mL/kg p.o. and 2 mL/kg for i.v.; values are mean ± SD.

i.v. = intravenous, p.o. = per oral, t_1/2 = half-life, Vz = volume of distribution, Cl = clearance, C_max = The peak plasma concentration of a drug after administration, T_max = Time of maximum drug concentration, AUC = Area Under the Curve, F (%) = bioavailability, C_b/C_p = brain to plasma ratio.

The clearance was low (34 ± 12 mL/min/kg) for compound 6b as compared to those of 6c and 6x. The compounds 6b, 6c and 6x have shown the volume of distributions (Vz, L/kg), that is, 3.6 ± 1.2, 6.9 ± 4.3 and 7.5 ± 1.0, respectively, at 10 mg/kg, i.v. dose.

The mean oral bioavailability for compound 6b was better (30 ± 7%) as compared to that of 6c (14 ± 10%) and 6x (10 ± 6%). The moderate-to-low oral bioavailability of these compounds may be because of their poor metabolic stability in rat liver microsomes.

The in vitro metabolic stability of compound 6b in rat and human liver microsomes was carried out for 30 minutes. Additional screening in in vitro ADME assays demonstrated that the compound 6b has extensively metabolized (100% and 94%) in rat and human liver microsomes, respectively.

Based on above pharmacokinetic profile data, the compound 6b was further evaluated for its CYP liabilities in human liver microsomes, the IC₅₀ values for compound 6b was found to be 6.9 µM for CYP 3A4 enzymes and 4.3 µM for CYP 2D6 enzymes (Table 3).

Table 3. Human CYP45045 inhibitory data and microsomal metabolic stability for compound 6b*.

	IC50 (µM)		% Metabolism in liver microsomes
Compound	CYP 3A4	CYP 2D6	Human	Rat
6b	6.9	4.3	94	100

*The cytochrome P450 inhibitory potential was determined using isoform-selective assays and heterologously expressed human CYP 2D6 and CYP 3A4. These values are the mean of duplicate determinations. Microsomal metabolic stability in wistar rat and human at 0.5 h, conc. 2.5 µM.

Based on the overall pharmacokinetic and brain penetration data (Table 2), compound 6b was selected for further profiling in pharmacological models of cognition. Object recognition is an attractive task for testing compounds for their potential against cognitive deficits in Alzheimer’s disease and schizophrenia.

Oral administration of compound 6b (10 mg/kg) has significantly improved performance of rats in animal models of cognition like novel object recognition test46 (NORT, Figure 5). Discriminative index of vehicle-treated group between familiar and novel object is 0.56, where as in compound 6b treated group is 0.73 (Figure 5).

Figure 5. Novel object recognition test data for compound 6b in rats. Compound 6b versus vehicle (paired t-test), n = 9–10/group, p.o., dosing drug: 60 min prior to test (p.o.). Vehicle PEG 400 50% v/v; 1 mL/kg, p.o. *p < 0.05 Student’s t-test. D.I. = discriminative index.

Conclusion

We have identified a new series of 1-[3-(4-alkyl piperazin-1-ylmethyl)- phenylsulfonyl]-1H-indoles (Compounds 6) as potent, selective, orally available and brain penetrant 5-HT₆ receptor ligands. The lead compound, 1-[4-methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6b) from this series has shown desired pharmacokinetic properties and is active in animal models of cognition such as NORT in preclinical trials. Based on its overall profile, the compound 6b was selected for further development.

Declaration of interest

The study is supported by discovery analytical, DMPK, In vitro, In vivo departments.

Acknowledgements

Mr. Venkateswarlu Jasti, CEO, Suven Life Sciences Ltd., Hyderabad, India is gratefully acknowledged.