Design, Synthesis and Therapeutic investigation of Tryptamine derivatives as Potential Antioxidant and Amyloid inhibitor/disaggregator

 

Saira Asghar^1,2, Nousheen Mushtaq¹, Ahsaan Ahmad³, Rabya Munawwar⁴,

Sumaira Ansari¹, Syeda Abiha Rizvi⁵

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences,

University of Karachi, Pakistan.

²Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Karachi, Pakistan.

³Institute of Pharmaceutical Sciences, Jinnah Sindh Medical University, Karachi, Pakistan.

⁴Dow College of Pharmacy, Dow University of Health Science, Karachi, Pakistan.

⁵Jinnah College of Pharmacy, Sohail University, Karachi, Pakistan.

 

ABSTRACT:

Oxidative stress is one of the main causative factors for pathogenesis of numerous disorders including psychological and neurodegenerative diseases (Alzheimer’s disease and Parkinsonism). Amyloid aggregates induced oxidative stress is involved in disfunction and degeneration of brain cells in Alzheimer’s disease. Antioxidants are important natural or synthetic molecules having tendency to curb free radicals and discontinuing their chain reactions prior to the damage of essential biomolecules. Drugs possessing antioxidant activity are being extensively anticipated for developing novel therapeutic agents in numerous pathological conditions accompanying oxidative stress like AD. In this study twelve tryptamine derivatives were synthesized and investigated for antioxidant, fibril inhibition and disaggregation potential. In vitro antioxidant potential of the derivatives was examined by using DPPH (2,2‑diphenyl‑1‑picrylhydrazyl) assay and revealed that SR10, SR14, SR23 and SR42 displayed better antioxidant activity (IC₅₀ in the range of 0.75±0.05µM-14.43±0.77µM) than the standard ascorbic acid (IC₅₀ =15.83±0.88µM). In vitro amyloid fibril inhibition and disaggregation assay was carried out by using HEWL (Hen egg white lysozyme) presenting moderate to high inhibition/disaggregation activities. Molecular dockingassessment recognized themode of bindingswithin active site of human antioxidant enzyme peroxiredoxin (PDB ID: 3MNG). These tryptamine derivatives with potential antioxidant and inhibition/disaggregation activitieswill be beneficialto develop and designdrug molecules forneurodegenerative disorders.

 

 

 

INTRODUCTION: 

Atoms or molecules having lone pair of electrons which are highly reactive are known to be as free radicals. They impart a very significant role in numerous chemical processes including human metabolism. Oxygen containing free radicals (hydroxyl, superoxide, peroxyl and hydroperoxyl) are named as reactive oxygen species (ROS)^1-3.

 

 

These ROS are generated inside the human body and can turn out to be noxious when engendered in excess quantity. Therefor equilibrium between the antioxidant systems of the body and rate of ROS generation is required for the homeostasis. The upsurge in degree of ROS generation and obstruction in antioxidant pathways result in oxidative stress^4,5. Oxidative stress is responsible to damage several biomolecules and tissues such as proteins, lipids and DNA, along with reducing their antioxidant defense control. Furthermore, Oxidative stressis one of the main causative factors for pathogenesis of numerous disorders including psychological and neurodegenerative diseases (Alzheimer’s disease and Parkinsonism). Though, ROS injury is central process responsible for neuronal demisein neurodegenerative diseases which has been supported by various studies with surrogate markers of oxidative stress^6-8. In vitro and in vivo studies revealed that different molecules with antioxidant capability decreased the oxidative stress initiated by β-amyloid protein fibrils⁹. The brains of patients with Alzheimer’s disease (AD) display ROS mediated molecular decline and is chiefly protuberant in surroundings of neurofibrillary tanglesand in senile plaques bearing neurons, signifyingthe roles of ROS for neuronal damage in pathogenesis of AD. Amyloid aggregates induced oxidative stress is involved in disfunction and degeneration of brain cells in AD because exposure to Amyloid-β proteinharms the working of glutamate transporters and membrane ion thereby oxidative stress mediated reactions compromises the mitochondrial functions¹⁰. Antioxidants are important natural or synthetic molecules having tendency to curb free radicals and discontinuing their chain reactions prior to the damage of essential biomolecules.Drugs possessing antioxidant activity are being extensively anticipated for developing novel therapeutic agents in numerous pathological conditions accompanying oxidative stress like AD^11-13.

 

Tryptamine belongs to the class of monoamine alkaloid containing indole ring and derived from tryptophan by decarboxylation. Tryptamine and its analogues show different biological activities such as antibacterial, anti-inflammatory, antioxidant, antifungal activities and cytoprotective properties. They are also found to be beneficial for the treatment of various psychological and neurodegenerative diseases like AD. Recent investigation showed that melatonin (natural tryptamine derivative) has strong peroxyl and hydroxyl radical scavenging potential. Tryptamine derivatives are capable to scavenge cations radicals and then oxidatively metabolized into indolic products by means of indolyl radicals consequently reducing the oxidative stress^14-16.

 

All above mentioned facts encouraged us to design, synthesize several tryptamine derivatives having antioxidant activity, amyloid fibrils inhibition and disaggregation potential.Compounds with better in vitro antioxidant activity than standard (ascorbic acid) were subjected to molecular docking study for investigating the mode of interaction with the human antioxidant enzyme peroxiredoxin (PDB ID: 3MNG).

 

Tryptamine derivatives were synthesized using various substituted phenacyl and benzoylhalides. DPPH (2,2‑diphenyl‑1‑picrylhydrazyl) assay was used to investigate the percent scavenging ability of the derivatives.The synthesized derivatives were assessed for inhibition of amyloid fibril formation triggered by hen egg white lysozyme (HEWL). Congo Red binding assay was used to check the tendency of derivatives to inhibit or disaggregate the formed fibril.

 

MATERIAL AND METHODS:

Chemistry:

Materials:

All the solvents and the chemicals utilized for the synthesis of tryptamine derivatives purchased from Sigma-Aldrich, USA and E. Merck, Germany. To confirm the synthesis of the products and their purity TLC plates coated with silica gel 60 F₂₅₄ purchased from Merck, Germany. TLC spots were visualized by HPUVIS Desaga, Heidelberg, Germany and iodine chamber. Analogue model SMP3 from STUART, Bibby Sterlin Ltd, UK was used to check the melting points of the compounds. Ultra-violet¹⁷ spectra were determined by Shimadzu UV-1601 spectrophotometer. Infra-red (IR) spectra were recorded using FT-IR spectrophotometer (NICOLET Avatar300). Mass spectrometer of JEOL JMS-HX110, Japan was used to obtain mass spectra.Bruker Advance AV-300 and AV-400 Nuclear magnetic resonance (H¹-NMR) spectrophotometer recorded the H¹-NMR spectra using 300 and 400MHz frequencies. Tetra methyl silane (TMS) was internal standard, solvents were d-D₂O and d6-DMSO for H¹-NMR spectra detection.

 

Scheme 1: General reaction scheme for the synthesis of tryptamine derivatives

 

List of synthesized tryptamine derivatives with different substituents on the structures

S. No.	Derivatives codes	Y	R	X
1	SR08			Br
2	SR10			Br
3	SR13			Cl
4	SR14			Br
5	SR17			Br
6	SR20			Cl
7	SR21			Br
8	SR22			Br
9	SR23			Br
10	SR24			Br
11	SR25			Br
12	SR42			Cl

 

General procedure for Chemical synthesis

Substituted phenacyl and benzoyl halides (0.0025M) added intoequimolar solution of tryptamine in 20-25ml THF. The reaction was stirred in basic medium at room temperature for about 4-5hrs then refluxed at 50°C for 8-10hrs till the formation of products. The progress of each reaction was monitored via TLC and visualized under UV-VIS lamp and iodine chamber. Ethanol (5): Ethyl acetate (5) was the solvent system for TLC. The solid precipitates were filtered by vacuum filtration after completion of reaction and thoroughly washed with THF. Single solvent recrystallization method was performed to get the pure products by means of THF solvent. The synthesized compounds were dried in vacuum desiccator with silica beads to remove moisture. Spectral studies and melting points were performed to confirm the synthesis of the target compounds.

 

N-[2-(1H-indol-3-yl) ethyl]-2-(naphthalen-2-yl)-2-oxoethanaminium bromide (SR08):

Off-White powder, yield: 19%, solubility: Ethanol, methanol, DMSO m.p:229-231°C, UV λmax (MeOH) nm: 218.8, IR νmax (cm^-1): 3305.33 (NH, stretch),2937.57 (=CH, aromatic, stretch), 2930.72 (CH, aliphatic, stretch),1593.19 (C=O, stretch), 1499.21 (NH, bend), 1454.26 (C=C, aromatic ,stretch), 1393.21 (CH, aliphatic, bend), 1233.60 (C=O, bend), 820.89(=CH, aromatic, bend), H¹-NMR (d₆-DMSO, 400MHz) δ (ppm): 10.94 (s, 1H, H-1’), 7.786 (s, 1H, H-7’’’), 7.551-7.537 (d, 1H, J=5.6 Hz, H-1’’) 7.365-7.349 (d, 5H, J = 6.4 Hz, H-2’’’, H-3’’’, H-4’’’, H-5’’’, H-H-6’’’), 7.082 (s, 2H, H-1’’), 7.00 (s, 1H, H-2’), 4.59 (s, 2H, H-2’’), 3.064-3.032 (t, 2H, J=12.8 Hz, H-2),  2.985-2.956(t, 2H, J=11.6 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 410.32

 

2-(4-fluorophenyl)-N-[2-(1H-indol-3-yl) ethyl]-2-oxoethanaminium bromide (SR10):

White powder, yield: 10%, solubility: Ethanol, methanol, water, DMSO m.p:241.2-242.7°C, UV λmax (H₂O) nm: 278.2, IR νmax (cm^-1): 3301.25 (NH, stretch),3007.04 (=CH, aromatic, stretch), 2929.40 (CH, aliphatic, stretch),1642.22 (C=O, stretch), 1586.02 (NH, bend), 1495.12 (C=C, aromatic ,stretch),1417.48 (CH, aliphatic, bend), 1233.60 (C=O, bend), 1094.67(aryl fluoride), 839.16 (=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.597-7.577 (d, 2H, J =8 Hz, H-1’’’, H-4’’’), 7.443-7.423 (d, 2H, J=8.0 Hz, H-2’’’,H-3’’’) 7.208-7.100 (m, 4H, H-3’, H-4’, H-5’, H-4’, H-6’), 7.107-7.069 (t, 1H, J = 15.2Hz, H-2’, H-1’’), 4.670 (s, 2H, H-2’’), 3.239-3.209 (t, 2H, J=12Hz, H-2) 3.083-3.050 (t, 2H, J=12Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 378.26

 

2-(2,4-difluorophenyl)-N-[2-(1H-indol-3-yl) ethyl]-2-oxoethanaminium chloride (SR13):

White powder, yield: 16%, solubility: Ethanol, methanol, water, DMSO m.p:259.2-260.7°C, UV λmax (H₂O) nm: 285, IR νmax (cm^-1): 3346.20 (NH, stretch), 3015.21 (=CH, aromatic, stretch), 2974.35 (CH, aliphatic, stretch),1642.22 (C=O, stretch), 1576.84 (NH, bend), 1548.24 (C=C, aromatic ,stretch),1499.21 (CH, aliphatic, bend), 1233.60 (C=O, bend), 1094.67 (arylfluoride), 837.23(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.599-7.579 (d, 1H, J =8 Hz, H-1’’’), 7.210-7.071 m, 4H, H-3’, H-4’, H-5’, H-6’), 7.109-7.090 (d, 2H, J = 7.6Hz, H-2’’’, H-3’’’), 7.071 (s, 1H, H-1’’), 6.80 (s, 2H, H-2’), 4.670 (s, 2H, H-2’’), 3.242-3.212 (t, 2H, J=14Hz, H-2) 3.087-3.053 (t, 2H, J=13.6 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 351.79

 

N-[2-(1H-indol-3-yl) ethyl]-2-(4-methylphenyl)-2-oxoethanaminium bromide (SR14):

White powder, yield: 18%, solubility: Ethanol, methanol, water, DMSO m.p:232.9-234.2°C, UV λmax (H₂O) nm: 286.6, IR νmax (cm^-1): 3313.51  (NH, stretch), 3051.99 (=CH, aromatic, stretch), 2925.31 (CH, aliphatic, stretch),1613.62 (C=O, stretch), 1580.95 (NH, bend), 1495.12 (C=C, aromatic ,stretch),1450.17 (CH, aliphatic, bend), 1241.77 (C=O, bend), 824.97(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.586-7.566 (d, 2H, J =8 Hz, H-1’’’, H-4’’’), 7.431-7.411 (d, 2H, J=8.0Hz, H-2’’’,H-3’’’) 7.197-7.145 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.095-7.076 (d, 2H, J = 7.6Hz, H-2’), 7.058 (s, 2H, H-1’’), 4.657 (s, 2H, H-2’’), 3.232-3.197(t, 2H, J=14Hz, H-2) 3.073-3.038 (t, 2H, J=14Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 374.29

 

N-[2-(1H-indol-3-yl) ethyl]-2-(4-methoxyphenyl)-2-oxoethanaminium bromide (SR17):

Light brown powder, yield: 22%, solubility: Ethanol, methanol, water, DMSO m.p:224.5-227.3°C, UV λmax (H₂O) nm: 285.2, IR νmax (cm^-1): 3313.51  (NH, stretch),3027.47 (=CH, aromatic, stretch), 2994.78 (CH, aliphatic, stretch),1658.57 (C=O, stretch), 1564.59 (NH, bend), 1491.03 (C=C, aromatic ,stretch),1348.01 (CH, aliphatic, bend), 1224.63 (C=O, bend), 861.75(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.598-7.578 (d, 2H, J =8 Hz, H-1’’’, H-4’’’), 7.443-7.423 (d, 2H, J=8.4 Hz, H-2’’’,H-3’’’) 7.209-7.158 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.108-7.089 (d, 2H, J =7.6 Hz, H-2’), 7-070 (s, 2H, H-2’’), 4.669 (s, 2H, H-2’’), 3.73 (s,3H, H-5’’’), 3.243-3.209 (t, 2H, J=13.6 Hz, H-2) 3.085-3.050 (t, 2H, J=14Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 390.29

 

2-(3,4-dihydroxyphenyl)-N-[2-(1H-indol-3-yl) ethyl]-2-oxoethanaminium chloride (SR20):

Grey powder, yield: 12%, solubility: Ethanol, methanol, water, DMSO m.p:260-262.9°C, UV λmax (H₂O) nm: 218.2, IR νmax (cm^-1): 3313.51  3566.86 (OH, stretch), 3293.08 (NH, stretch), 2970.26 (=CH, aromatic, stretch), 2888.54 (CH, aliphatic, stretch),1605.45 (C=O, stretch), 1499.22 (NH, bend), 1450.17 (C=C, aromatic ,stretch),1343.93 (CH, aliphatic, bend), 1233.63 (C=O, bend), 808.63(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.581-7.562 (d, 1H, J =7.6Hz, H-2’’’), 7.428-7.408 (d, 2H, J=8.0 Hz, H-1’’’,H-3’’’) 7.192-7.142 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.092-7.073(d,1H, J = 7.6Hz, H-2’), 7.055 (s, 2H, H-1’’), 5.0 (s, 2H, H-4’’, H-5’’), 4.668 (s, 2H, H-2’’), 3.224-3.190 (t, 2H, J=13.6 Hz, H-2) 3.066-3.032 (t, 2H, J=13.6 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 392.26

 

N-[2-(1H-indol-3-yl)ethyl]-2-(2-nitrophenyl)-2-oxoethanaminiumbromide (SR21):

White powder, yield: 35.09%, solubility: Ethanol, methanol, water, DMSO m.p:226.5-228.5°C, UV λmax (H₂O) nm: 278.4, IR νmax (cm^-1): 3313.52  (NH, stretch),3015.21 (=CH, aromatic, stretch), 2930.72 (CH, aliphatic, stretch),1601.36 (C=O, stretch), 1491.03 (NH, bend), 1450.17 (C=C, aromatic ,stretch),1335.75 (CH, aliphatic, bend), 1229.51 (C=O, bend), 882.18(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.593-7.574 (d, 2H, J =13.6Hz, H-1’’), 7.440-7.419 (d, 2H, J=8.4Hz, H-4’’’), 7.204(s, 2H, H-2’’’, H-3’’’), 7.173-7.154(d,4H, J = 7.6Hz, H-3’, H-4’, H-5’, H-6’), 7.103-7.084(d, 1H, J = 7.6Hz, H-2’), 7.066 (s, 2H, H-1’’), 4.669 (s, 2H, H-2’’), 3.236-3.204 (t, 2H, J=12.8 Hz, H-2) 3.079-3.045 (t, 2H, J=13.6 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 405.26

 

N-[2-(1H-indol-3-yl) ethyl]-2-(3-nitrophenyl)-2-oxoethanaminium bromide (SR22):

Creamy powder, yield: 22.47%, solubility: Ethanol, methanol, water, DMSO m.p:232.6-233.7°C, UV λmax (H₂O) nm: 217.8, IR νmax (cm^-1): 3317.59  (NH, stretch),3019.30 (=CH, aromatic, stretch), 2933.48 (CH, aliphatic, stretch),1654.48 (C=O, stretch), 1572.26 (NH, bend), 1491.03 (C=C, aromatic ,stretch),1446.08 (CH, aliphatic, bend), 1237.68 (C=O, bend), 820.89 (=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.598-7.578 (d, 1H, J =8 Hz, H-1’’’), 7.444-7.424 (d, 1H, J=8.0 Hz, H-2’’’), 7.209-7.196 (d, 2H, H-3’’’, H-4’’’), 7.178-7.158 (d,4H, J =8Hz, H-3’, H-4’, H-5’, H-6’), 7.108-7.089 (d, 1H, J = 7.6Hz, H-2’), 7.071(s, 2H, H-1’’), 4.670 (s, 2H, H-2’’), 3.244-3.210 (t, 2H, J=13.6Hz, H-2) 3.083-3.050 (t, 2H, J=14Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 405.27

 

N-[2-(1H-indol-3-yl) ethyl]-2-(4-nitrophenyl)-2-oxoethanaminiumbromide (SR23):

Light yellow powder, yield: 26%, solubility: Ethanol, methanol, DMSO m.p:225-230°C, UV λmax (MeOH) nm: 218.8, IR νmax (cm^-1): 3317.59 (NH, stretch), 3027.47 (=CH, aromatic, stretch), 2827.24 (CH, aliphatic, stretch),1646.31 (C=O, stretch), 1572.76 (NH, bend), 1491.03 (C=C, aromatic ,stretch),1433.83 (CH, aliphatic, bend), 1229.51 (C=O, bend), 808.63(=CH, aromatic, bend), H¹-NMR (d₆-DMSO, 400MHz) δ (ppm): 10.965 (s, 1H, H-1’), 7.84(s, 2H, H-2’’’, H-3’’’), 7.563-7.537 (d, 2H, J =10.4Hz, H-1’’’, H-4’’’) 7.374-7.082 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.020 (s, 2H, H-1’’), 6.996-6.971 (d, 1H, J = 10Hz, H-2’), 3.353(s, 2H, H-2), 3.062-3.014 (t, 2H, J=19.2Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 405.25

 

2-(biphenyl-4-yl)-N-[2-(1H-indol-3-yl) ethyl]-2-oxoethanaminium bromide (SR24):

Off-white powder, yield: 19%, solubility: Ethanol, methanol, DMSO m.p:212-214.5°C, UV λmax (MeOH) nm: 280.8, IR νmax (cm^-1): 3313.51 (NH, stretch), 2970.26(=CH, aromatic, stretch), 2921.23(CH, aliphatic, stretch),1683.09(C=O, stretch), 1499.21(NH, bend), 1339.84(C=C, aromatic ,stretch), 1237.68 (C=O, bend), 890.35(=CH, aromatic, bend), H¹-NMR (d₆-DMSO, 400MHz) δ (ppm): 10.952 (s, 1H, H-1’), 8.114-8.094 (d, 2H, J =8Hz, H-1’’’, H-4’’’), 7.932-7.912 (d, 2H, J = 8Hz, H-2’’’, H-3’’’), 7.621-7.601 (d, 2H, J =8Hz, H-5’’’, H-9’’’), 7.556-7.537 (d, 2H, J=7.6Hz, H-6’’’, H-8’’’), 7.369-7.349 (d, 1H, J=8 Hz, H-7’’’),7.102-7.019 (m, 4H, H-3’, H-4’, H-5’, H-6’), 6.982(s, 2H, H-2’), 4.931 (s, 2H, H-2’’), 3.185-3.164 (t, 2H, J=8 Hz, H-2) 3.002-2.985 (t, 2H, J =6.8 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 436.32

 

N-[2-(1H-indol-3-yl) ethyl]-3-oxo-3-phenylpropan-1-aminium bromide (SR25):

Light brown solid flakes, yield: 25%, solubility: Ethanol, methanol, DMSO m.p:215-217°C, UV λmax (MeOH) nm: 219, IR νmax (cm^-1): 3305.33 (NH, stretch), 2998.86(=CH, aromatic, stretch), 2904.88 (CH, aliphatic, stretch),1649.81 (C=O, stretch), 1572.76 (NH, bend), 1499.2(C=C, aromatic ,stretch), 1433.88(CH, aliphatic, bend), 1233.60 (C=O, bend), 890.35(=CH, aromatic, bend), H¹-NMR (d₆-DMSO, 400MHz) δ (ppm): 10.952(s, 1H, H-1’), 7.804 (s, 2H, H-1’’’,H-5’’’), 7.539 (s, 3H, H-2’’’, H-3’’’, H-4’’’), 7.350 (s, 4H, H-3’, H-4’, H-5’ H-6’), 7.081(s, 2H, H-1’’), 6.999(s, 1H, H-2’), 3.322(s, 2H, H-2’’), 3.29-3.250 (d, 2H, J =16Hz, H-3’’), 3.242-3.208 (t, 2H, J=13.6 Hz, H-2) 3.083-3.049 (t, 2H, J=13.6 Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 374.30

 

N- [2-(1H-indol-3-yl) ethyl]-3,5-dinitrobenzamide (SR42):

Crystalline white powder, yield: 37%, solubility: Ethanol, methanol, water, DMSO m.p:250.2-262.5°C, UV λmax (H₂O) nm: 218, IR νmax (cm^-1): 3301.25(NH, stretch),3035.64(=CH, aromatic, stretch), 2978.43 (CH, aliphatic, stretch),1687.17(C=O, stretch), 1540.07 (NH, bend), 1495.12 (C=C, aromatic ,stretch),1425.65 (CH, aliphatic, bend), 1241.77 (C=O, bend), 820.89(=CH, aromatic, bend), H¹-NMR (D₂O-d, 400MHz) δ (ppm): 7.595-7.577 (d, 2H, J =7.2 Hz, H-1’’’, H-3’’’), 7.442-7.422 (d, 1H, J=8.0 Hz, H-2’’’) 7.201-7.157 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.106-7.088(d, 1H,  J= 7.2Hz, H-2’), 7.069 (s, 2H, H-1’’), 3.244-3.210 (t, 2H, J=13.6 Hz, H-2) 3.085-3.057 (t, 2H, J=11.2Hz, H-1), MS (FAB) [M+1] ⁺ m/z: 355.73

 

Pharmacological evaluation:

In vitro antioxidant assay:

2,2-diphenyl-2-picrylhydrazyl (DPPH) procured from Sigma-Aldrich and methanol was obtained from Merk, Germany. The DPPH free radical used to evaluate the antioxidant potential of the synthesized compounds. 1mg of each synthesized derivativedissolved in methanol to prepare stock solution, serial dilutions (1-200µM) of test compounds were prepared, the respective dilutions of the test compounds and freshly prepared 0.1mM methanolic DPPH solution reacted together in equal ratio (1:1) and incubated for about 30min in the dark ^{18, 19}. The absorbance of each prepared test reaction recorded at 517nm through UV-VIS spectrophotometer (SHIMADZU UV-1800). All experiments executed in triplicates. DPPH used as negative control and the absorbance of the test compounds compared with ascorbic acid (positive control). Lower absorbance of the test solutions indicated higher antioxidant activity (free radical scavenging potential) of the test samples and all the results are displayed as 50 percent inhibitory concentration (IC₅₀ ±SEM). IC₅₀ valuesdetermined by graphical representation between concentration of test samples and percent inhibition by means of GraphPad PRISM software.

 

The percent free radical scavenging ability (antioxidant activity) using DDPH measured by following formula⁸,

Free radical scavenging potential (%) = ­(A_control− A_test)/ A_control × 100

 

In vitro amyloid fibril inhibition and disaggregation assay:

HEWL as lyophilized powder (HEW lysozyme≥ 40,000units/mg) purchased from Sigma-Aldrich (St. Louis, United States). 70µM Protein solution prepared freshly in 100mM glycine buffer having pH 2 holding 100mM NaCl. The protein concentrationdetermined spectrophotometrically at 280nm with an extinction coefficient (ε₂₈₀) of 2.65Lg^-1 cm ^-1. Stock solution conatining DMSO-1% and dilutions with desired concentrations (0-500µM) prepared in glycine buffer.

 

In vitro HEWL fibril inhibition assay:

Calculated volume of lysozyme solution, the test compounds or control and glycine buffer were agitated using water bath shaker (SHZ08, China) with 500rpm at 70 ̊C for 50-55hrs. 20µM Congo red solution made freshly in 10mM sodium phosphate buffer (pH 7.4) and filtered before use. Congo red (CR) binding assay used to determine the tendency of test samples to inhibit amyloid fibril in which the 0.5ml solution of test compounds or control mixed with 5ml of Congo red dye. Then scanrecorded in the range of 400-700nm by the UV-visible spectrophotometer (SHIMADZU UV-1800) of each test sample and control after incubation of 30minutes^20,21.

 

In vitro HEWL fibril disaggregation assay:

Final calculated protein solution and glycine buffer incubated with continuous shaking with speed of 500rpm on water bath (SHZ08, China)at 70°C for 50-55hrs till the aggregates formed. The formation of amyloid fibrils confirmed by measuring absorbance with freshly prepared 20µM CR in sodium phosphate buffer 100mM (pH 7.4). The absorption spectrum in the 400-700nm range recorded by UV-visible spectrophotometer. Presence of fibrils characterized by bathochromic shift from 495nm to 540nm. Solution containing the fibril (0.5ml) and test sample (0.5ml) incubated for 24hrs. Then 5ml CR solution added in 0.5ml of the solution (fibril and test samples mixture) and incubated for 30min at room temperature. The solution without test sample also run as control. UV absorption scan from 400-700nm recorded. Disaggregation potential of the test compounds assured by hypsochromic shift to 500-530nm from 540-550nm ²².

 

Docking studies:

The compounds depicted better antioxidant results as compared to the ascorbic acid selected to perform docking studies for exploring their mode of interactions with the target protein. 3D (three-dimensional structure of human peroxiredoxin (PDBID: 3MNG) obtained from the online data base protein data bank (https://www.rcsb.org/structure/3mng).All the heteroatoms and water molecules removed, and the protein was prepared by dock prep tool of the UCSF chimera 1.10.2. The structures of the compounds drawn by Chem Draw 2D Ultra from Chem office, energy minimization of each compound carried out by Open Babel and saved in pdb format. Docking was executed using PyRx virtual screening tool version 0.9.2 (AutoDock Vina) standard protocol. The grid box set to surrounds the active pocket site of the protein. Maximum eight conformation of every compound generated after the docking with AutoDock Vina, one with the best docking score (lowest binding energy) and root mean square deviation (RMSD) values between 1-2.0 Å selected to study the mode of binding between the ligand and target by UCSF Chimera^23-25. DTD (the bounded native co-crystalized ligand) redocked within the enzyme active pocket to validate the docking protocol. The ligands are presented in green ball and stick model, hydrogen bonds distances are indicated by red dotted line while all the residues are presented in orange wire frame model.

 

RESULTS AND DISCUSION:

Chemistry:

Twelve different derivatives of tryptamine scaffold were synthesized efficiently through targeting side chain amino group of alkylamine in tryptamine using nucleophilic substitution reaction (Scheme 1). Different substituted phenacyl, benzyl, benzoyl and naphthyl halides used for derivatization of tryptamine. Except SR08, all the compounds havesubstituted single aromatic ring with methyl, methoxy, hydroxyl, nitro, halogen and phenyl substituent at various positions.

 

The proposed derivatives divided into three main sections (chart 1).

1.   Tryptamine moiety (Blue)

2.   The linker (acyl, carbonyl, and ethyl carbonyl moieties) connecting tryptamine amino with terminal aromatic ring (Purple).  

3.   Single or two fused aromatic rings with and without substituents (flouro, nitro, methyl, phenyl, hydroxyl and methoxy groups attached at various positions of the ring) (Red).

 

These three segments in the synthesized compounds are very important to get obligatory conformation and better chemical interactions to bind with active site of the target.

 

Solid (white, off-white, grey, light yellow, light brown crystalline or powder state products) with 10-37% yield was obtained having solubility in methanol, ethanol and DMSO while some compounds demonstrated solubility also in water. The products were obtained by refluxing at 50-55^oC. Melting point and TLC techniques were utilized to indicate the successful completion of the respective reaction products. TLC card showed single and round spot of every compound at different distance from the parent (tryptamine) and the reactants. THF is used as a medium for single solvent recrystallization to get pure compounds.

 

Structure elucidation of the synthesized compounds done by different spectrophotometric techniques. UV- visible spectrophotometry determined the λ_max of the synthesize derivatives in the range of 193.4-286.6 nm. IR- spectroscopy indicated the presence of several vital functional groups by protuberant peaks as a part of the synthesized compounds. FAB positive technique revealed the molecular weight of each product in M+1 form. Furthermore, ¹HNMR (Proton nuclear magnetic resonance) established the number of protons by Bruker Advance at the frequency of AV-400 and AV-500 MHz in D₂O (deuterated water) and d6-DMSO (dimethyl sulfoxide) solvents.  Chemical shift reported as ppm unit. Peaks were appeared in ¹HNMR spectrum as singlet (s), doublet(d), triplet(t) and multiplet(m). J value (coupling constant) of the doublet and triplet was calculated in form of Hz.

 

Chart 1. Rationale to design tryptamine derivatives

Pharmacological evaluation:

 

In vitro antioxidant activity :

The synthesized compounds evaluated for their antioxidant potential using DPPH reagent. DPPHhas free electrons in its structure therefore present in the form of free radical and usually beneficial to estimate percent radical scavenging activity in in vitro assay. Oxidized (free electron) form of DPPH (2,2-diphenyl-1-picrylhydrazyl) absorbs at 516nm gives purple color and converted into reduced form yellow color DPPH.H (2,2-diphenyl-1-picrylhydrazin) after 30 mins of incubation by the effect of antioxidant molecules. This procedure is designated as rapid, simple, and useful technique for screening antioxidant potential of many samples. All these mentioned benefits madethe DPPH method valuable for assessing the potential of several synthesized agents to scavenge radicals and to discoverpotent antioxidant drug molecules^26-28.

 

Theabsorbance values of test samples at different concentrations (1-500 µM) used to determine the percent antioxidant activity and constructed graphically against respective concentration to get the linear equations for determining IC₅₀ values. The antioxidant activities of all the compounds by DPPH method are presented in Table 1.

 

Result revealedthat synthesized derivatives possessed strong to weak radical scavenging potential.SR23 (para-nitro phenacyl derivative),SR14 (para-methyl phenacyl tryptamine) showed the best antioxidant activity with IC₅₀ values of 0.75±0.05 µM and 1±1.33 µM respectively than standard ascorbic acid (IC₅₀ =15.83±0.88 µM). In SR42, presence of meta and ortho nitro groups in the terminal aromatic ring and replacing the linker with carbonyl reduced its scavenging potential (IC₅₀= 10.13±0.68 µM) more than ten folds in comparison with SR23 and SR14but still showing better activity than ascorbic acid. SR10with ortho, para-difluoro substituents demonstrated comparable activity (IC₅₀₌14.43±0.77 µM) with standard.  Percent scavenging ability of the synthesized compounds can be highly influenced by type of substituent and place of substitution. Presence of methylis linked with highly significant antioxidant activity of the compounds.Position of nitro group in SR21(IC₅₀₌57.6±0.87µM) and SR22 (IC₅₀₌22.3±0.47µM) highlighted the impact on scavenging potential. Shifting of nitro from ortho (SR21) to meta (SR22) improved the antioxidant potential more than two folds while the same nitro atpara position (SR23) enhanced the strength thirty to seventy times.  The antioxidant potential produced by nitro substituted derivatives represented as SR23>SR22>SR21.

 

Compounds having ortho,para-difluoro group (SR13, IC₅₀₌24.2±0.61µM) and para-methoxy group (SR17, IC₅₀₌26.13±1.16 µM) on aromatic ring exhibited moderate antioxidant potential. In the current series of tryptamine derivatives, SR08, SR20 andSR24 containing naphthyl, phenyl and hydroxyl moietiesdemonstrated poor activity as compared to ascorbic acid. In SR25 increase in linker and unsubstituted terminal aromatic rings made a drastic impact on the antioxidant potential by reducing the activity more than hundred times as compared to the most active compounds of series (SR14 and SR23). Interestingly SR42 having carbonyl linker with di nitro substituted phenyl ring appeared as the third best molecule in the series (IC₅₀10.13±0.68) presenting better result than standard. The potential of molecules for DPPH scavenging ability appeared as: SR23>SR14>SR42>SR10> SR22>SR13>SR17>SR21> SR20>SR24>SR08>SR25.

 

Table 1. IC₅₀ values for  Antioxidant acticity (DPPH radical scavanging potential) and amyloid inhibition/disaggregation activity of the synthesized ccompounds

S. No.	Test compounds	Antioxidant activity (IC50 ± SEM)	Amyloid inhibition and Disaggregation activity
			Test Concentration (µM)	Amyloid inhibition activity λ-max (nm)	Amyloid Disaggregation activity λ-max (nm)
1.	SR08	74.5±1.15	500	519.8	502.4
			10	531.6	532
2.	SR10	14.43±0.77	500	534	501.4
			10	541.2	543.2
3.	SR13	24.2±0.61	500	500.4	501
			10	544	504.4
4.	SR14	1±1.33	500	535.2	501
			10	539.4	541.2
5.	SR17	26.13±1.16	500	507.2	501.6
			10	538.4	538.4
6.	SR20	64.43±0.38	500	509.2	500.8
			10	549.8	535.8
7.	SR21	57.6±0.87	500	495.8	502.2
			10	548.6	537.8
8.	SR22	22.3±0.47	500	495.2	505.4
			10	500.2	535.8
9.	SR23	0.75±0.05	500	495.8	503.6
			10	557.2	535.8
10.	SR24	65.64±0.72	500	486	497.8
			10	544.6	543
11.	SR25	107.5±0.86	500	506.8	508.8
			10	550	541.2
12.	SR42	10.13±0.68	500	488	503.6
			10	552	543
13.	Tryptamine	29.02±0.57	500	549	503
			10	549	535.6
14.	Ascorbicacid	15.83±0.88	-----	-----	-----
15.	Congo Red	-----	----	493-495	494-495.6
16.	Control (Amyloid fibril)	------	------	540-550	544.6

 

In vitro amyloid fibril inhibition and disaggregation assay:

HEWL (Hen egg white lysozyme) is generally used to investigate the mechanism underlying the formation of amyloid fibril, inhibition and disaggregation through small molecules. Both the human lysozyme and HEWL are found to generate amyloid fibrils under particular environment^29,30. The HEWL protein was incubated at high temperature and lower pH to instigate the lysozyme amyloid fibrilization.  Several specific techniques are used to detect the amyloid fibril formation. Results are presented in table 2. In the current research study Congo Red (CR) binding assay is used to determine the inhibition and disaggregation of HEWL fibrils by the synthesized compounds. CR dye has tendency to complex with Aβ species (monomers to mature fibrils). CR binding assay executed to inquire β-sheet formed through amyloid fibrils and this persuades a characteristic increase in the absorption maximum red shift (490 to 540nm). This was observed in case of control (protein solution without test sample). Congo red alone showed a strong absorbance shoulder at 490-495nm^31,32. Compounds were tested at highest and lowest concentration (500 and 10µM). In the inhibition assay parent molecule tryptamine showed control pattern. Most of the compounds displayed moderate to high inhibition at 500µM. The CR and derivatives (SR08, SR13, SR17,SR20, SR22 and SR25) absorbance along with HEWL was intensely lower andobserved at 500-520nm, indicative of their higher efficacy. SR21, SR23, SR24 and SR42 showed absorbance in CR region 480-495nm. SR10, SR14 moderately inhibited HEWL fibril formation. At lower concentration 10µM all the compound showed poor efficacy except SR08, SR14, SR17 which demonstrated moderate efficacy. Only SR22 indicated excellent inhibition at 10µM.

 

In order to find out the HEWL amyloid disaggregation potential of the synthesized derivatives, first the fibrils were formed than incubated with test samples till 24hrs and then accessed by CR binding assay. It was observed thatall the derivatives at 500µM were effective HEWL amyloid fibril disaggregating agents because of bathochromic shift in 500-520nm while weak to excellent disaggregating potential was observed at 10µM. Absorbance at 503nm was the sign of excellent disaggregation ability of tryptamine at 500µM while 535nm in case of 10µM indicated moderate activity. The absorbance of CR with incubated HEWL andSR13 was distinctly lower thus indicating that this compound was more effective as HEWL fibril disaggregating agent. SR10, SR14, SR25 and SR42 displayed same absorbance pattern as observed in control spectrum (540-545nm) while rest of the compounds were proved to possess moderate disaggregation activity.

 

Molecular docking study for binding mode analysis:

In view of in vitro outcomes, docking investigation was done to analyze the probable binding interactions of most active compounds (SR23, SR42, SR10, and SR14) into the active site of human peroxiredoxin with bound ligand oxidized dithiothreitol (DTT) (PDB ID: 3MNG) by using Auto Dock Vina tool of PyRx Software. First of all, the interactions between native co-crystallized compound DTT were modeled to validate the Auto Dock Vina software.  Experimental co-crystallized ligand conformation of DTT and the one predicted by Auto Dock Vina were found to be superimposed indicating the successful reproducibility of experimental binding conformation of DTT in the enzyme active pocket. The binding score with hydrophobic and hydrogen bond interactions of ascorbic acid (reference compound), DTT and test compounds are displayed in Table 2.

 

SR23, SR42, SR10, SR14l and ed in the active site of 3MNG displayinggood docking scores and binding interactions. Their 3D-interactions in the active site of proteinshown in figure 1 and figure 2 respectively. Docking results of the compounds against the target protein revealed that all the mentioned compounds presented better docking scores (-6.0 and -7.7kcal/mol) as compared to standard (-4.9kcal/mol) and parent (-5 kcal/mol).

 

SR23 and SR42 involved in hydrophobic interactions with the help of sixteen residues including Glu16, Gly17, Glu18, Lys63, Val69, Val70, Thr81, Gly82, Glu83, Arg86, Glu91, Gly92, Val94, Arg95, Leu96 mimicking the docking mode the ascorbic acid. SR23 entered into the hot spot site horizontally but the indole ring and terminal aromatic ring flipped completely to face each other while flexible acyl linkerpart found to be completely twisted within the binding site presenting highest antioxidant activity. In SR23 amine of indole, carbonyl of linker and nitro of terminal phenyl ring are engaged with Val 94, Gly92 and Val70 via hydrogen bonding. Whereas in SR42 di-nitro substituted phenyl ring and rigid carbonyl as linker slightly changed the orientation of molecule though surrounded by same lipophilic residues but here indole linker and nitro groups are not able to make hydrogen bonding in the active pocket site of protein.

 

SR10 and SR14 showed same hydrophobic interaction. Moreover, the binding conformation analysis of the SR14 depicted that molecule is fixed in the hydrophobic region well and in compact form where both the indole ring and methyl phenyl ring positioned vertically over each other and completely twisted and made additional hydrophobic interaction with Lys17. SR10 was surrounded by maximum (three) hydrogen bonds with Glu16, Gly92, Leu96 while SR14 exhibited two hydrogen bonding with Glu16, Gly17. Whereas SR10 is extended well in the cavity surrounded by hydrophobic residues mentioned in Table2.  Size and nature of lipophilic substitution at para position is indicating the impact of substitution on in-vitro results. The standard compound ascorbic acid demonstrated hydrophobic interactions by interacting with hotspot residues including Glu16, Gly17, Thr81, Gly82, Gly85, Arg86, Lys89, Glu91, Gly92, Lys93, Val94, Arg95 and two hydrogen bond interaction with Gly92, Arg95were found in ascorbic acid. The parent compound, tryptamine was interacted with few residues for hydrophobic interactions and only one hydrogen bond via Val94. The binding interactions of selected test compounds showing good fit and stabilized position into the binding pocket of 3MNG protein validating the in vitro results.

 

Table 2: Docking scores and Ligands interactions (hydrogen bonding and hydrophobic interactions) with the protein (PDB ID:3MNG)

Compounds	Docking score (Kcal/mol)	Hydrogen bonding	Hydrophobic interactions
SR42	-7.7	Val70	Glu16, Gly17, Glu18, Lys63, Val69, Val70, Thr81, Gly82, Glu83, Arg86, Glu91, Gly92, Val94, Arg95, Leu96
SR23	-6.7	Val70, Gly92, Val94	Glu16, Gly17, Lys63, Val69, Val70, Thr81, Gly82, Glu83, Gly85, Arg86, Glu91, Gly92, Val94, Arg95, Leu96
SR10	-6.5	Glu16, Gly92, Leu96	Glu16, Gly17, Val69, Val70, Thr81, Gly82, Gly85, Arg86, Lys89, Glu91, Gly92, Lys93, Val94, Arg95, Leu96
SR14	-6	Glu16, Gly17	Glu16, Gly17, Glu18, Val69, Val70, Thr81, Gly82, Glu83, Gly85, Arg86, Glu91, Gly92, Lys93, Val94, Arg95, Leu96
Tryptamine	-5	Val94	Gly17, Thr81, Gly82, Glu83, Gly85, Arg86, Glu91, Gly92, Val94, Arg95, Leu96
Ascorbic acid	-4.9	Gly92, Arg95	Glu16, Gly17, Thr81, Gly82, Gly85, Arg86, Lys89, Glu91, Gly92, Lys93, Val94, Arg95, Leu96
DTT	-3.4	------	Gln68, Val69, Val70, Gly82, Glu83, Gly85, Arg86, Glu91, Gly92, Val94, Arg95, Leu96

 

CONCLUSION:

Among all the synthesized tryptamine derivatives SR23, SR42, SR10 and SR14 demonstrated promising antioxidant activity as compared to standard ascorbic acid and parent molecule tryptamine in DPPH radical scavenging assay. Moreover, the docking results also complemented to the results achieved through the in vitro studies of the compounds. SR23, SR42, SR10 and SR14 presented better binding scores and interactions as compared to reference drug (ascorbic acid).  Furthermore, docking analysis represented that these four compounds interacted with key amino acid residues within the active pocket of 3MNG such as Glu16, Gly17, Lys63, Val69, Val70, Thr81, Gly82, Glu83, Gly85, Arg86, Glu91, Gly92, Val94, Arg95, Leu96. All the compounds represented poor to good amyloid inhibition and disaggregation properties in hen egg white lysozyme inhibition and disaggregation assay. Present study demonstrated that the mentioned synthesized compounds might be the promising hit to lead molecules, which can efficiently improve as new drug molecules used in the treatment of various neurodegenerative diseaseslike Alzheimer’s and Parkinson’s diseases.

 

Figure 1. Binding poses of a) SR23 b) SR42 in green ball and stick model with the 3MNG protein. All residues are presented in orange wire frame while hydrogen bond interaction distances are displayed by red dotted lines.    

 

Figure2: Binding poses of a) SR10 b) SR14 in green ball and stick model with the 3MNG protein. All residues are presented in orange wire frame while hydrogen bond interaction distances are displayed by red dotted lines.

 

CONFLICT OF INTEREST:

The authors have declared no conflicts of interest concerning this study.

 

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Accepted on 29.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(8):3622-3632.