Description:Description of the Related Art
[0002] Natural substances obtained from plants, animals, and minerals have been used throughout human history for diagnosing, preventing, and treating medical conditions, as well as supporting various bodily functions. Many ancient societies utilized plants for their perceived medicinal properties. Early humans likely encountered bioactive substances through plant-based foods due to their diet. Over time, knowledge about the toxic and therapeutic characteristics of natural materials developed as humans interacted with their environment. Concerns about the toxicity and side effects of modern drugs have encouraged ongoing use of natural products in health care. Many views alternative remedies as safer and prefer herbal medicine for its natural origins and fewer side effects. As a result, herbal medicine has grown rapidly worldwide. The use of natural products in drug discovery has limited exploration of synthetic compounds. Secondary metabolites from natural sources are often considered more biologically active and suitable for drugs, as they evolve within living systems. Studies from 1981 to 2002 highlight the key role of natural products in developing and identifying new medications.
[0003] Alzheimer's disease is the most common form of dementia, a group of disorders caused by the loss or dysfunction of brain neurons. This neuronal decline affects memory, behavior, and thinking, and eventually impairs basic functions like walking and swallowing, ultimately leading to death. Curcumin, a main yellow pigment from turmeric rhizomes, exhibits diverse pharmacological and biological effects.
[0004] One natural medication that may be used to treat Alzheimer's disease is curcumin. When given to a mouse model of Alzheimer's disease lowers serum Aβ levels and lessens the burden of Aβ in the brain. This effect is primarily observed in the neocortex and hippocampus of the mouse model. Curcumin penetrates the blood-brain barrier, prevents Aβ plaques from forming, destroys preexisting Aβ fibrils, and stops them from extending. Curcumin therapy has a stronger therapeutic impact in Alzheimer's disease and can restore the distorted neurotic morphology that is seen close to plaques. It works better than ibuprofen or naproxen at preventing the development of Aβ plaques.4 Oxadiazoles are the five membered heterocyclic aromatic group containing two nitrogen and one oxygen having four different isomers. these isomers are having better pharmacological activities, such as antibacterial, anti-inflammatory, antimalarial, antiviral, anticancer, antidepressive, analgesic, neuroprotective.
SUMMARY
[0005] In view of the foregoing, an embodiment herein provides a method for synthesis and evaluation of oxadiazole-curcumin derivatives for anti-Alzheimer’s study. In some embodiments, wherein the study's chemicals and reagents were purchased from S.D. Fine Chem. Ltd., Merck, and LOBA Chemicals and were of analytical reagent (AR) and laboratory reagent (LR) grade. The melting points of the synthesized compounds were determined using a microcontroller-based Melting Point device, model CL 725/726, and were reported as uncorrected. Thin layer chromatography (TLC) was employed to confirm the purity of the compounds, using silica gel 60 F254 precoated plates (3 x 8 cm) as the stationary phase and various solvent combinations as the mobile phase. These TLC plates, sourced from E-Merck, Darmstadt, Germany, displayed spots under short-wavelength UV light.
[0006] Infrared spectra of the synthesized compounds were recorded using a Fourier transform infrared (FT-IR) spectrometer with KBr pellets, covering the 4000-400 cm⁻¹ range. The frequencies were noted in wave numbers. 1H-NMR (400 mhz) spectra were recorded in chloroform-d and in DMSO-d6 in Amx- 400 MHz liquid state NMR spectrometer BRUKER Chemical shifts (δ) are reported in parts per million downfield from internal reference Tetramethyl Silane (TMS) as well as Mass spectra (JEOL GC mate MASS spectrophotometer) also taken for identification of derivatives.
[0007] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The embodiments herein will be better understood from the following detailed description with reference to the drawings, in which:
[0009] FIG. 1 illustrates a method for synthesis and evaluation of oxadiazole-curcumin derivatives for anti-Alzheimer’s study according to an embodiment herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0010] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[0011] FIG. 1 illustrates a method for synthesis and evaluation of oxadiazole-curcumin derivatives for anti-Alzheimer’s study according to an embodiment herein. In some embodiments, the 10mg of purified curcumin was dissolved in 2ml of Methanol and 4mg of 4-Hydrazino benzoic acid along with 18µl of trimethylamine. A catalytic amount of acetic acid was added and incubated for 24 hours. The solution was evaporated in vaccum to get the dark orange powder of Hydrazinobenzoyl curcumin.
[0012] General procedure for the synthesis of 1-(4-(3,5-bis((E)-4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl) phenyl) butan-1-one
[0013] Preparation of intermediate was carried out by esterification, by reacting 4gm of Hydrazinobenzoyl curcumin with 40ml ethanol and catalytic amount of sulphuric acid by refluxing for 4hours. After cooling, ice-cooled water was added and the precipitate was collected, washed with dilute aqueous ammonia then with water, and recrystallized from aqueous ethanol, giving yellow crystals.7
[0014] General procedure for the synthesis of 1-(4-(3,5-bis((E)-4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl) phenyl) benzohydrazide
[0015] 0.01 mole of compound 3 and 0.07 moles of 80% hydrazine hydrate were taken in a round bottom flask and refluxed in presence of absolute ethanol for about 4-5 hours. Excess solvent was distilled off and solid obtained was filtered, dried and purified by crystallization using methanol.
[0016] General procedure for the synthesis of Curcumin-Oxadiazole derivatives [4a-e]: A mixture of an equimolar hydrazide (0.0082mole) and substituted benzoic acid derivatives (0.0082mole) in 10ml of phosphorous oxychloride was refluxed for 8hrs. The mass was then poured carefully into ice cold water. Neutralized with sodium bicarbonate. The resulting solid was filtered and recrystallized using methanol.8
[0017] Anti-Alzheimer’s Activity Hydrogen peroxide-induced oxidative stress in Saccharomyces cerevisiae is used to identify genetic factors involved in oxidative stress response. Gene deletion or overexpression assays help determine yeast cell adaptation under these conditions. Since oxidative stress can promote plaque formation linked to Alzheimer's disease, the survival of wild-type yeast cells is assessed using spot assays.
[0018] In this method yEG001 strain of yeast with the genotype MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 is used. Wild type cells were streaked onto YPD plates and incubated at 30°C for two days. Individual colonies were picked using sterile toothpicks, resuspended in 100 μl YPD, and adjusted to an OD600 of 0.0006 in 30 ml YPD using a UV-Visible spectrophotometer. The culture was diluted with 30 ml YPD and grown overnight at 220 rpm. The next day, the OD600 was measured, the culture volume was adjusted to 12 OD600, transferred to a 50 ml conical tube, centrifuged at 3,900 x g for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in 20 ml pre-warmed YPD. Cultures were mixed well by vertexing and added 10 ml of culture to two sterile flasks for each strain being tested. OD60, to one of the flasks and added H2O2 to 4 mM final concentration in 10 ml culture.
[0019] Commercially available H2O2 is usually 30% (w/w), density of 1.1 g/ml, and has a molecular weight of 34.01 g/mol. Therefore, the stock concentration was 9.79 M, and 4.08 μl of the stock gave a final concentration of 4 mM in a 10 ml culture volume. All flasks were placed in the shaker and continued incubating at 30 °C for 30 min. After the incubation, 1 ml from each culture was removed and placed in a 1.5 ml microfuge tube. Centrifuged at 12,200 x g for 2 min. Supernatant was removed and resuspended the cell pellet in 1 ml sterile, distilled water.
[0020] A 10-fold dilution series was prepared in a 96-well plate: 200 μl of undiluted cells were placed in the first well, and 180 μl sterile water was added to the next five wells. For each strain, 20 μl from one well was transferred to the next with 180 μl water using a multichannel pipette, repeating this process to complete the series. Then, 5 μl from each well was spotted onto YPD plates, starting from the most diluted sample. Plates were air-dried, incubated at 30 °C, and photographed daily. Untreated cultures showed visible growth by day 2, while H2O2-treated cultures became visible after 5 days.
[0021] In some embodiments, on Characterization of Hydrazinobenzoyl curcumin C28H24N2O6; M.W. 484.51g/mol; M.P. 218-220°C; Yield 75%; Recrystallized from ethanol; 1H NMR δ=3.58 (O-CH3), (OH)δ=7.428 (Ar), δ=9.95(OH); Orange yellow powder.
[0022] Characterization of 1-(4-(3,5-bis((E)-4-hydroxy-3-methoxystyryl)-1H-pyrazol-1-yl) phenyl) benzohydrazide
[0023] C28H26N4O5; M.W. 498.54g/mol; M.P. 248-250°C; Yield 70%; Recrystallized from ethanol; 1H NMR δ=3.59 (O-CH3), δ=7.2 (Ar), δ=9.59(NHNH2); Greenish yellow powder.
[0024] Characterization of 4,4’-((1E,1’E) -(1-(4-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1H-pyrazole-3,5-diyl) bis(2-methoxyphenol) [4a]
[0025] C35H27ClN4O5; M.W. 619.07g/mol; M.P. 187-189°C; Yield 60%; Recrystallized from methanol; IR: υ= 760.27 cm-1 (C-Cl), 3454.17 cm-1 (NH), 1681.13 cm-1 (C=C), 3543.96 cm-1 (OH); 1H NMR δ=3.58 (Cl), δ=9.6 (OH), δ=7.3-7.9(Ar); Light yellow powder.
[0026] Characterization of 4,4’-((1E,1’E) -(1-(4-(5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1H-pyrazole-3,5-diyl) bis(32thane-2,1-diyl) bis(2-methoxyphenol) [4b]
[0027] C35H27N5O7; M.W. 629.63g/mol; M.P. 175-178°C; Yield 75%; Recrystallized from methanol; IR: υ= 1528.37 cm-1 (C-H), 1641.02 cm-1 (C=C), 3452.27 cm-1(OH); 1H NMR δ=9.8 (OH), δ=7.2-8(Ar); yellowish orange powder.
[0028] Characterization of 4,4’-((1E,1’E) -(1-(4-(5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1H-pyrazole-3,5-diyl) bis(32thane-2,1-diyl) bis(2-methoxyphenol) [4c]
[0029] C35H26Cl2N4O5; M.W. 653.52g/mol; M.P. 168-170°C; Yield 75%; Recrystallized from methanol; IR: υ= 749.75 cm-1 (C-Cl), 1506.84 cm-1 (C=C), 3482.35 cm-1(OH) ; 1H NMR δ=9.58 (OH), δ=3.58 (Cl), δ=7.2-8.1(Ar); yellow powder.
[0030] Characterization of 4,4’-((1E,1’E)-(1-(4-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl)bis(2-methoxyphenol)[4d] C35H27ClN4O5; M.W. 619.07g/mol; M.P. 158-161°C; Yield 80%; Recrystallized from methanol; IR: υ= 659.03 cm-1 (C-Cl), 1500.97 cm-1 (C=C), 3456.74 cm-1(OH) ; 1H NMR δ=9.5 (OH), δ=3.598 (Cl), δ=7.2-8.1(Ar); Pale red powder.
[0031] Characterization of 4,4’-((1E,1’E) -(1-(4-(5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1H-pyrazole-3,5-diyl) bis(ethene-2,1-diyl) bis(2-methoxyphenol) [4e]
[0032] C35H29N5O5; M.W. 599.65g/mol; M.P. 235-238°C; Yield 80%; Recrystallized from methanol; IR: υ= 3397.58 cm-1 (N-H2), 1637.50 cm-1(C=C), 3514.55 cm-1(OH) ; 1H NMR δ=9.4(NH2), δ=9.5 (OH), δ=7.2-8(Ar); Red powder.
[0033] 3.2. Anti-Alzheimer Activity
[0034] All the synthesized curcumin derivatives were screened for anti-Alzheimer activity by assessing survival of yeast cells in absence of H2O2 and in the presence of H2O2 and samples, at the concentration of 10μg. Among all the synthesized derivatives, except 4b all the compounds showed excellent anti Alzheimer activity.
-H2O2 DF (105) +H2O2 DF (103) Colony Count Avg SD
Control 76 7600000 200 200000 2.631579 2.63 0.11
70 7000000 190 190000 2.714286
78 7800000 195 195000 2.5
4e 70 7000000 218 218000 3.114286 2.98 0.16
75 7500000 210 210000 2.8
68 6800000 205 205000 3.014706
4d 65 6500000 170 170000 2.615385 2.50 0.19
69 6900000 180 180000 2.608696
70 7000000 160 160000 2.285714
4c 65 6500000 160 160000 2.461538 2.37 0.11
71 7100000 170 170000 2.394366
80 8000000 180 180000 2.25
4b 81 8100000 140 140000 1.728395 1.70 0.09
62 6200000 110 110000 1.774194
68 6800000 109 109000 1.602941
4a 70 7000000 150 150000 2.142857 2.08 0.19
72 7200000 160 160000 2.222222
75 7500000 140 140000 1.866667
Sample Average Standard Deviation
Control 2.62 0.11
4e 2.98 0.16
4d 2.5 0.19
4c 2.37 0.11
4b 1.7 0.09
4a 2.08 0.19 , Claims:I/We Claim:
1. A method for synthesis and evaluation of oxadiazole-curcumin derivatives for anti-Alzheimer’s study, wherein the method comprising:
including oxadiazole-curcumin compounds, which are produced by conjugating an oxadiazole moiety with a curcumin scaffold. The compound's anti-Alzheimer's action is achieved by inhibiting the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE);
a tablets, capsules, nanoparticles, or injectable formulations appropriate for administration to the central nervous system (CNS) are the options from which the formulation is chosen.
a synaptic dysfunction is avoided and tau protein hyperphosphorylation is decreased by treatment.
2. The derivative of the chemical of claim 1 exhibits antioxidant activity by scavenging reactive oxygen species (ROS), which lowers the oxidative stress on neurons linked to Alzheimer's disease pathogenesis.