Pyrano[2,3-c]pyrazole-pyrazolone compounds and process for preparation thereof

Abstract

The present invention relates to a hybrid pyrano[2,3-c]pyrazole-pyrazolones and analogous compounds of Formula 3; and a process for the preparation thereof. The invention further relates to identification of bioactivities of synthesized novel hybrid pyrano[2,3-c]pyrazole- pyrazolones. The novel hybrid pyrano[2,3-c]pyrazole-pyrazolones of Formula 3 displayed useful neuroprotective property and inhibited acetylcholine esterase enzyme at the lowest concentration. These compounds were also found to cross the Blood-Brain Barrier.

Description

[0001] PYRANO[2,3-c]PYRAZOLE-PYRAZOLONE COMPOUNDS AND PROCESS

[0002] FOR PREPARATION THEREOF

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a hybrid pyrano[2,3-c]pyrazole-pyrazolone compound of

[0005] Formula 3

[0006] Formula 3 wherein each Ri and R2, is independently selected from the group consisting of aryl, phenyl, 4-fluoro phenyl, 4-methyl phenyl, 4-methoxy phenyl and 3-thienyl; and

[0007] R3 is selected from the group consisting of phenyl, 4-chloro phenyl, 4-fluoro phenyl, 4- trifluoromethyl phenyl, 4-trifluoromethoxy phenyl and 2,2,2-trifluoroethyl; and

[0008] R4 is selected from the group consisting of alkyl, methyl, n-propyl and cyclopropyl.

[0009] Particularly, present invention relates to a process for the preparation of hybrid pyrano[2,3- c]pyrazole-pyrazolone compound of Formula 3. More particularly, present invention relates to the hybrid pyrano[2,3-c]pyrazole-pyrazolone compound of Formula 3 as neuroprotective agent and acetylcholine esterase [AChE] inhibitors.

[0010] BACKGROUND OF THE INVENTION

[0011] Pyrazolones are potent bioactive fertile sources for biologically important molecules possessing a broad spectrum of pharmacological activities, such as antimicrobial, antitumor, CNS effect, anti-inflammatory, antioxidant, anti-tubercular, antiviral, lipid-lowering, antihyperglycemic agents and protein inhibitors. Reference may be made to the Journal “Eur. J. Med. Chem. 2020, 186, 111893” which discloses a systematic summarization of biological activities and SAR of pyrazolones that could be beneficial for researchers to design novel pyrazolone derivatives, in addition, docking analysis is used to explain interactions between some typical pyrazolone.

[0012] Reference may be made to the Journal “Synlett, 2018, 29, 1037-1042” which discloses a protocol for synthesis of dihydrospirofuro[2,3-c]pyrazolones in aqueous medium involving pyrazolones and aldehydes at ambient temperature.

[0013] Reference may be made to the patent document CN102827172A which discloses derivatives of pyrazolones, the pyrano[2,3-c]pyrazole compounds, and preparation and application. Reference may be made to the patent document CN114920753A which discloses 6-fluoro- dihydropyrano [2, 3-c] pyrazole derivative as well as a synthesis method and application of the 6-fluoro-dihydropyrano [2, 3-c] pyrazole derivative.

[0014] Among a myriad of derivatives of pyrazolones, the pyrano[2,3-c]pyrazole has garnered significant attention owing to their diverse structural significance and promising biological activities including antimicrobial.

[0015] Reference may be made to the Journal “Bioorg. Med. Chem. Lett., 2012, 22, 5272” which discloses the synthesis of substituted pyrano[2,3-c]pyrazoles and evaluation for their antibacterial, anti-inflammatory and cytotoxic activities.

[0016] Reference may be made to the Journal “Bioorg. Med. Chem. Let I., 2012, 22, 4458” which discloses new substituted 4H-chromenes as anti-cancer agents.

[0017] Reference may be made to the patent document US11617738B2 which discloses 6-amino- 2,4-dihydropyrano [2, 3-c] pyrazoles as a ubiquitin specific protease 7 (USP7) inhibitor useful for the treatment of diseases such as inflammation, cancer, and immunological disorders.

[0018] Reference may be made to the Journal “J. Med. Chem., 1984, 27, 539” which discloses the synthesis of a series of N- substituted 3,4-dimethylpyrano[ 2,3-c]pyrazol-6-one derivatives in order to evaluate their biological activities, antiviral properties. Reference may be made to the Journal “PLoS One, 2017, 12, e0162642” which discloses the synthesis and biological evaluation of a series of dihydropyrano[2,3-c]pyrazole derivatives as a novel family of PPARy partial agonists.

[0019] Reference may be made to the Journal “Bioorg. Med. Chem., 2006, 14, 4792” which discloses that pyrano[2,3-c]pyrazole derivatives also inhibit the activity of the human Chkl kinase enzyme.

[0020] Based on the known art, both pyrazolones and pyrano[2,3-c]pyrazoles independently are highly potent scaffolds for many therapeutic activities although they have their own limitations. To overcome such limitations molecular hybridization is one of the proven and valuable approaches to obtain novel and improved potential drugs for the treatment of a number of diseases.

[0021] Reference may be made to “Curr. Med. Chem. 2007, 14, 1829” which discloses several examples of different strategies for drug design, discovery and pharmacomodulation focused on new innovative hybrid compounds presenting analgesic, anti-inflammatory, platelet anti-aggregating, anti-infectious, anticancer, cardio- and neuroactive properties.

[0022] In general, hybrid molecules are made by combining two different drug molecules through a linker. Reference may be made to the journal “RSC Adv., 2022,12, 19470” which discloses the hybridization of molecules for development as treatments for a number of key diseases is then outlined, including the design of hybrids for Alzheimer's, cancer, and malaria.

[0023] From a synthetic point of view, it involves many linear synthetic steps and the overall yield of the final target will be much less. Hence, synthetic methods need to be developed for hybrid molecules considering cost, time, and step economy. The present protocol focuses on the synthesis of hybrid pyrano[2,3-c]pyrazole -pyrazolones from diynones and active methylene containing pyrazolones. The present invention discloses a one-pot preparation of novel hybrid pyrano[2,3-c]pyrazole-pyrazolones scaffold from diynones and pyrazolones.

[0024] OBJECTIVE OF THE INVENTION

[0025] Main objective of the present invention is to provide a hybrid pyrano[2,3-c]pyrazole- pyrazolones compound of Formula 3. Another objective of the present invention is to provide an efficient process for the preparation of hybrid pyrano[2,3-c]pyrazole-pyrazolones compound of Formula 3, through the one-pot method using diynones of Formula 1 and pyrazolones of Formula 2.

[0026] Yet another objective of the present invention is to provide hybrid pyrano[2,3-c]pyrazole- pyrazolone compound of Formula 3 as useful neuroprotective agents.

[0027] Yet another objective of the present invention is to provide hybrid pyrano[2,3-c]pyrazole- pyrazolone compound of Formula 3 as useful acetylcholine esterase [AChE] inhibitors.

[0028] ABBREVIATIONS USED

[0029] TLC = Thin layer chromatography

[0030] NMR = Nuclear Magnetic resonance ppm = parts per million

[0031] HRMS = High resolution mass spectroscopy

[0032] FTIR = Fourier transform infrared

[0033] DCM = Dichloromethane

[0034] THF: tetrahydrofuran

[0035] DBU: l,8-diazabicyclo[5.4.0]undec-7-ene

[0036] DIPEA: N,N-Diisopropylethylamine

[0037] DBN: l,5-Diazabicyclo(4.3.0)non-5-ene

[0038] DABCO: 1,4-Diazabicyclo [2.2.2] octane

[0039] DMAP: N,N-dimethylaminopyridine

[0040] DMF: N.N-dimethylformamide

[0041] DMSO: Dimethyl sulfoxide

[0042] TEA: Triethylamine SUMMARY OF THE INVENTION

[0043] Accordingly, present invention provides a hybrid pyrano[2,3-c]pyrazole -pyrazolone compound of Formula 3

[0044] Formula 3 wherein each Ri and R2, is independently selected from the group consisting of aryl, phenyl, 4-fluoro phenyl, 4-methyl phenyl, 4-methoxy phenyl and 3-thienyl;

[0045] R3 is selected from the group consisting of phenyl, 4-chloro phenyl, 4-fluoro phenyl, 4- trifluoromethyl phenyl, 4-trifluoromethoxy phenyl and 2,2,2-trifluoroethyl; R4 is selected from the group consisting of alkyl, Me, n-propyl and cyclopropyl. and its analogous and their pharmaceutically acceptable salts, hydrates, solvates, optical isomers and combinations.

[0046] In an embodiment of the present invention, the compound of formula 3 is selected from the group consisting of: i. (E)-5-methyl-4-((E)-2-(3-methyl-l,6-diphenyl-3a,7a-dihydropyrano[2,3-c]pyrazol- 4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3a); ii. (E)-4-((E)-2-(l,6-diphenyl-3-propyl-3a,7a-dihydropyrano[2,3-c]pyrazol-4(lH)- ylidene)-l-phenylethylidene)-2-phenyl-5-propyl-2,4-dihydro-3H-pyrazol-3-one (3b); iii. (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-l,6-diphenyl-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3- one (3c); iv. (E)-2-(4-chlorophenyl)-4-((E)-2-(l-(4-chlorophenyl)-3-methyl-6-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (3d); v. (E)-2-(4-fluorophenyl)-4-((E)-2-(l-(4-fluorophenyl)-3-methyl-6-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (3e); vi. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethyl)phenyl)-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(4-

[0047] (trifluoromethyl)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3f); vii. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethoxy)phenyl)-3a,7a- dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(4-

[0048] (trifluoromethoxy)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3g); viii. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(2,2,2-trifluoroethyl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(2,2,2- trifluoroethyl)-2,4-dihydro-3H-pyrazol-3-one (3h); ix. (E)-4-((E)-l-(4-fluorophenyl)-2-(6-(4-fluorophenyl)-3-methyl-l-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5-methyl-2-phenyl-2,4- dihydro-3H-pyrazol-3-one (3i); x. (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-6-(4-fluorophenyl)-l-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-(4-fluorophenyl)ethylidene)-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3j); xi. (E)-4-((E)-l-(4-fluorophenyl)-2-(6-(4-fluorophenyl)-l-phenyl-3-propyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-2-phenyl-5-propyl-2,4- dihydro-3 H-pyrazol-3 -one (3k)

[0049] xii. (E)-4-((E)-2-(l,6-bis(4-fluorophenyl)-3-methyl-3a,7a-dihydropyrano[2,3-c]pyrazol-

[0050] 4(lH)-ylidene)-l-(4-fluorophenyl)ethylidene)-2-(4-fluorophenyl)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (31); xiii. (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(p-tolyl)-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-(p-tolyl)ethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3- one (3m); xiv. (E)-4-((E)-l-(4-methoxyphenyl)-2-(6-(4-methoxyphenyl)-3-methyl-l-phenyl-3a,7a- dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5-methyl-2-phenyl-2,4- dihydro-3H-pyrazol-3-one (3n);

[0051] xv. (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(thiophen-3-yl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-(thiophen-3-yl)ethylidene)-2-phenyl- 2,4-dihydro-3H-pyrazol-3-one (3o).

[0052] In another embodiment, present invention provides a one -pot process for the preparation of the compound of Formula 3 comprising the step of: i. reacting diynones of Formula 1 with pyrazolones of Formula 2 in a solvent at temperature in the range of 25-35°C optionally in the presence of a base for a period in the range of 30 min to 24 h to obtain hybrid pyrano[2,3-c]pyrazole -pyrazolones compound of Formula 3.

[0053] Formula 1 Formula 2 wherein Ri, R2, R3 and R4 are as defined in claim 1. In yet another embodiment of the present invention, Formula 1 is selected from the group consisting of:

[0054] In yet another embodiment of the present invention, Formula 2 is selected from the group consisting of:

[0055] In yet another embodiment of the present invention, the base is selected from the group comprising of DBU, DBN, DABCO, DIPEA, DMAP, EtsN, potassium carbonate, sodium carbonate, cesium carbonate, potassium bicarbonate, sodium bicarbonate, potassium phosphate and ammonium carbonate.

[0056] In yet another embodiment of the present invention, the solvent used is selected from the group consisting of ethers, alcohols, esters, acetone, toluene, THF, ethyl acetate, dimethylformamide, dimethylsulfoxide and acetonitrile.

[0057] In yet another embodiment of the present invention, compound of Formula 31 and 3n are found to display neuritogenic properties displaying a significant increase in average neuronal length compared to the positive control (NGF) across different concentrations ranging from 77.56 to 133.75 pM at 1.0 pM, 85.71-115.89 pM at 0.1 pM and 85.10-117.92 pM at 0.01 pM concentration.

[0058] In yet another embodiment of the present invention, compounds 3a, 3c, 3f, 31, 3n, 3o are showing non-cytotoxicity with IC50 values of 0.0022 to 0.8909 pM.

[0059] In yet another embodiment of the present invention, the compound 3a, 3c, 3f, 31, 3n, 3o inhibits acetylcholine esterase enzyme with a IC50 values ranging from 0.0682 to 0.5337 pM. In yet another embodiment of the present invention, the compound 3a, 3c, 3f, 31, 3n, 3o were found to cross the Blood-Brain Barrier (BBB) permeability within a range of logBB - 0.993 to 0.07.

[0060] BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 illustrates SRB cell viability assay with the compounds of Formula 3 at 1, 0.1 and 0.01 pM concentrations.

[0062] FIG. 2 illustrates the average neurite outgrowth at 24 h post-treatment with the compounds of Formula 3 at three different concentrations.

[0063] FIG. 3 illustrates the representation of % acetyl choline esterase enzyme activity inhibition of the compounds of Formula 3 in comparison with positive control (Galantamine (G)).

[0064] FIG. 4 represents a process step for the synthesis of Formula 3 from Formula 1 and Formula 2; wherein each Ri and R2, is independently selected from the group consisting of aryl, phenyl, 4-fluoro phenyl, 4-methyl phenyl, 4-methoxy phenyl and 3-thienyl; and R3 is selected from the group consisting of phenyl, 4-chloro phenyl, 4-fluoro phenyl, 4- trifluoromethyl phenyl, 4-trifluoromethoxy phenyl and 2,2,2-trifluoroethyl; and R4 is selected from the group consisting of alkyl, Me, n-propyl and cyclopropyl.

[0065] DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention provides a hybrid pyrano[2,3-c]pyrazole-pyrazolones compound of Formula 3 and an efficient process for the preparation thereof. The hybrid pyrano[2,3- c]pyrazole-pyrazolone compounds thus obtained are tested for their efficiency towards neurotrophic, neurogenic and neuroprotective activity.

[0067] The compounds of Formula 3 could serve as effective neuroprotective, neurotrophic and neurogenic agents and might be useful for the treatment of neurological and psychiatric diseases where the neurotrophic and / or neurogenic functions are compromised.

[0068] The present invention provides a compound of Formula 3

[0069]

[0070] Formula 3 wherein each Ri and R2, is independently selected from the group consisting of aryl, phenyl, 4-fluoro phenyl, 4-methyl phenyl, 4-methoxy phenyl and 3 -thienyl; and

[0071] R3 is selected from the group consisting of phenyl, 4-chloro phenyl, 4-fluoro phenyl, 4- trifluoromethyl phenyl, 4-trifluoromethoxy phenyl and 2,2,2-trifluoroethyl; and

[0072] R4 is selected from the group consisting of alkyl, methyl, n-propyl and cyclopropyl.

[0073] The compound of Formula 3 is selected from the group consisting of: a) (E)-5-methyl-4-((E)-2-(3-methyl-l,6-diphenyl-3a,7a-dihydropyrano[2,3-c]pyrazol- 4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3a); b) (E)-4-((E)-2-(l,6-diphenyl-3-propyl-3a,7a-dihydropyrano[2,3-c]pyrazol-4(lH)- ylidene)-l-phenylethylidene)-2-phenyl-5-propyl-2,4-dihydro-3H-pyrazol-3-one (3b); c) (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-l,6-diphenyl-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3- one (3c); d) (E)-2-(4-chlorophenyl)-4-((E)-2-(l-(4-chlorophenyl)-3-methyl-6-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (3d); e) (E)-2-(4-fluorophenyl)-4-((E)-2-(l-(4-fluorophenyl)-3-methyl-6-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (3e); f) (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethyl)phenyl)-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(4-

[0074] (trifluoromethyl)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3f); g) (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethoxy)phenyl)-3a,7a- dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(4-

[0075] (trifluoromethoxy)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3g); h) (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(2,2,2-trifluoroethyl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(2,2,2- trifluoroethyl)-2,4-dihydro-3H-pyrazol-3-one (3h); i) (E)-4-((E)- 1 -(4-fluorophenyl)-2-(6-(4-fluorophenyl)-3 -methyl- 1 -phenyl-3a,7 a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5-methyl-2-phenyl-2,4- dihydro-3H-pyrazol-3-one (3i); j) (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-6-(4-fluorophenyl)-l-phenyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-(4-fluorophenyl)ethylidene)-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3j); k) (E)-4-((E)-l-(4-fluorophenyl)-2-(6-(4-fluorophenyl)-l-phenyl-3-propyl-3a,7a- dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-2-phenyl-5-propyl-2,4- dihydro-3H-pyrazol-3-one (3k); l) (E)-4-((E)-2-(l,6-bis(4-fluorophenyl)-3-methyl-3a,7a-dihydropyrano[2,3-c]pyrazol- 4(lH)-ylidene)-l-(4-fluorophenyl)ethylidene)-2-(4-fluorophenyl)-5-methyl-2,4- dihydro-3H-pyrazol-3-one (31); m) (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(p-tolyl)-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-(p-tolyl)ethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3- one (3m); n) (E)-4-((E)-l-(4-methoxyphenyl)-2-(6-(4-methoxyphenyl)-3-methyl-l-phenyl-3a,7a- dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5-methyl-2-phenyl-2,4- dihydro-3H-pyrazol-3-one (3n); o) (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(thiophen-3-yl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-(thiophen-3-yl)ethylidene)-2-phenyl- 2,4-dihydro-3H-pyrazol-3-one (3o).

[0076] The present invention provides a process for the preparation of hybrid pyrano[2,3- c]pyrazole-pyrazolone compound of Formula 3 by the one-pot method.

[0077] The present process could be operated by employing the protocol using base and pyrazolones of Formula 2 in a one-pot approach using diynones of Formula 1 in high yields and purity. The newly developed process is illustrated in FIG 4.

[0078] The present process can be performed very effectively with a wide range of substrates and is a highly viable strategy that could be most suitable for the industrial-scale production of hybrid pyrano[2,3-c]pyrazole -pyrazolones compound of Formula 3 and analogous compounds. Further, this process is most suitable for the generation of a large library of intermediates and related molecules containing hybrid pyrano[2,3-c]pyrazole-pyrazolone moieties. All the reactions / experiments involve purification and systematic characterization of the individual reaction product as represented in the general process.

[0079] The present process for the preparation of hybrid pyrano[2,3-c]pyrazole-pyrazolones compound of Formula 3 by the one-pot method is the most convenient and simple method involving a protocol using base or without base in solvent media along with other reaction parameters.

[0080] The reaction of diynones of Formula 1 and pyrazolones of Formula 2 in a solvent such as toluene, DMF, THF, DMSO, dioxane, methanol, ethanol, isopropyl alcohol or acetone in the presence of bases such as K2CO3, CS2CO3, Na2CO3, DBU, DBN, DABCO, DIPEA, DMAP, EtsN, potassium bicarbonate, sodium bicarbonate, potassium phosphate and ammonium carbonate or absence of base at room temperature [25 to 35°C] provides the hybrid pyrano[2,3-c]pyrazole-pyrazolone compound of Formula 3.

[0081] The present invention provides a process for the preparation of hybrid pyrano[2,3- c]pyrazole-pyrazolone of Formula 3 comprising: i. treatment of diynones of formula 1 with pyrazolones of formula 2

[0082] Formula 1 Formula 2 wherein Ri, R2, R3 and R4 are as defined above. in a solvent at temperature in the range of 25-35°C optionally in presence of a base for a period in the range of 30 min to 24 h to obtain hybrid pyrano[2,3-c]pyrazole- pyrazolones compound of Formula 3.

[0083] The base used is selected from the group consisting of K2CO3, CS2CO3, Na2CO3, DBU, DBN, DABCO, DIPEA, DMAP, EtsN, potassium bicarbonate, sodium bicarbonate, potassium phosphate and ammonium carbonate.

[0084] The solvent used is selected from the group consisting of ethers, alcohols, esters, acetone, toluene, THF, ethyl acetate, dimethylformamide, dimethylsulfoxide and acetonitrile.

[0085] EXAMPLES

[0086] Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

[0087] Material and Methods used in experiments

[0088] The reagents and chemicals used in this process are bought from AVRA or Spectrochem or TCI or Sigma-Aldrich and were used as such without any further purification. In this process, the work-up and purification procedures were carried out with reagent grade solvents. All the reactions / experiments steps were monitored by thin layer chromatography and the crude products obtained were subjected to purification using crystallization or chromatography or distillation or extraction or filtration to get the pure compounds in good yields. Further, all the resultant compounds / products were systematically characterized using various analytical and spectral methods.

[0089] Measurement Method

[0090] High-resolution mass spectra (HRMS) were obtained from a Xero-G2-XS-QTOF HRMS instrument and Thermo Fisher Scientific Exactive (ESI) Instrument. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 600 or 500 or 400 or 300 MHz in CDCE solvent. Chemical shifts for ’ H NMR are expressed in parts per million (ppm) relative to tetramethylsilane (5 0.00 ppm). Chemical shifts for13C NMR are expressed in ppm relative to CDCI3 (8 77.0 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, quin = quintet, sext = sextet, m = multiplet), coupling constant (Hz), and integration.

[0091] Example 1

[0092] Synthesis of (E)-5-methyl-4-((E)-2-(3-methyl-l,6-diphenyl-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3a)

[0093] To a solution of diynone la (0.2 mmol) of Formula 1 and pyrazolone 2a (0.4 mmol) of Formula 2 in THF (3 mL) at 25°C was added base DBU (0.1 mmol; 50mol%). The reaction mixture was stirred at temperature (25°C) under open air atmosphere and monitored by TLC until the complete consumption of pyrazolone 2a occurred. After the appropriate period (30 min), the reaction mixture was quenched with water (5 mL) and diluted with EtOAc (10 mL). The organic phase was separated and the aqueous layer was washed with EtOAc (10 mL). Concentration of the combined organic layer under reduced pressure afforded the crude product, which was purified by column chromatography (ethyl acetate: hexane 1: 10) to afford the corresponding product 3a. The obtained crude compound was purified by silica gel column chromatography to afford hybrid pyrano[2,3-c]pyrazole -pyrazolone 3a as a reddish brown solid; yield: 90 mg (80%); mp 142-144°C.

[0094] IR (neat): vmax2923, 1626, 1515, 1004 cm’1; ’ H NMR (400 MHz, CDCI3) 6 8.64 (s, 1H), 8.11 - 7.91 (m, 2H), 7.87 - 7.69 (m, 2H), 7.69 - 7.57 (m, 3H), 7.52 (t, J = 8.0 Hz, 2H), 7.49 - 7.45 (m, 2H), 7.44 - 7.30 (m, 6H), 7.23 - 7.06 (m, 3H), 5.92 (s, 1H), 2.90 (s, 3H), 1.50 (s, 3H);13C NMR (151 MHz, CDCI3) 6 164.3, 158.0, 152.7, 149.1, 148.9, 146.7, 141.9, 139.7, 139.0, 137.1, 131.2, 130.6, 129.5, 129.2, 129.0, 128.78, 128.76, 127.2, 125.4, 124.3, 121.0, 119.4, 118.7, 111.6, 105.2, 103.9, 17.2, 16.5;

[0095] HRMS (ESI) Calcd for C37H28N4O2 [M+H]+561.2291; found, 561.2297.

[0096] Example 35 Synthesis of (E)-5-methyl-4-((E)-2-(3-methyl-l,6-diphenyl-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-2,4- dihydro-3H-pyrazol-3-one (3a) in absence of base

[0097] To a solution of diynone la (0.2 mmol) of Formula 1 was added pyrazolone 2a (0.4 mmol) of Formula 2 in THF (3 mL) at 25 °C and the reaction mixture was stirred at temperature (25°C) under open air atmosphere and monitored by TLC until the complete consumption of pyrazolone 2a occurred. After the appropriate period (24 h), the reaction mixture was concentrated and purified by column chromatography (ethyl acetate: hexane 1: 10) to afford the corresponding product 3a. The obtained crude compound was purified by silica gel column chromatography to afford hybrid pyrano[2,3-c]pyrazole-pyrazolone 3a as a reddish brown solid; yield: 47 mg (42%); mp 142-144°C. Example 2-34

[0098] The compounds of Formula 3b-3o were synthesized involving corresponding reactants of diynones of Formula 1, pyrazoles of Formula 2, with suitable solvent and base following the general procedure as described above under example l(3a) and or without base following the general procedure as described above under example 35 (3a).

[0099]

[0100] Example 36-40: Preparation of diynones of Formula 1

[0101] For the preparation of diynones (la-le) of Formula 1 that are utilized for the synthesis of representative hybrid pyrano[2,3-c]pyrazole-pyrazolones of Formula 3 known procedures were used as reported in the prior art (J. Org. Chem. 2022, 87, 1, 884).

[0102] “BuLi (2.2 eq. of a 2.5 M solution in hexanes) was added dropwise to a solution of ethynylbenzene (2.2 eq.) in dry THF at -78 °C. After a 30-minute stir period, ethyl formate (1 eq.) was added dropwise and the reaction was left stirring for 3 hours at the same temperature and allowed to warm to room temperature. The reaction was monitored by TLC and quenched by the saturated aqueous solution of NH4CI. The reaction mixture was extracted with ethyl acetate (3x10 ml) and the combined organic layer was washed with water followed by brine solution, dried with NaiSCh, and concentrated in vacuo. The crude material was then placed in a 50 mL round bottom flask and dissolved in DMSO. To the stirred solution, was added IBX (1.1 eq.). The reaction was monitored by TLC, after consumption of alcohol the reaction mixture was poured into ice water and extracted with ethyl acetate (3x10 ml) and the combined organic layer was washed with water followed by brine solution, dried with NaiSCL. and concentrated in vacuo. The crude material was purified by column chromatography (EtOAc / hexane, 1:5) to furnish diynone of Formula 1.

[0103] By following the general procedure 2 and corresponding specific starting materials, the following compounds were prepared.

[0104] Example 41-48: Preparation of pyrazolones of Formula 2

[0105] For the preparation of pyrazolones (2a- 2h) of Formula 2 that are utilized for the synthesis of representative hybrid pyrano[2,3-c]pyrazole-pyrazolones of Formual 3: known procedures were used as reported in the prior art (Org. Lett. 2014, 16, 19, 5060).

[0106] To a solution of P -ketoester (1 eq.) in acetic acid was added substituted phenylhydrazine (1 eq.) and the reaction mixture was refluxed for 24-36 h, and then cooled to room temperature and acetic acid was removed in vacuo. To the resulting crude precipitate, ethyl acetate was added and the suspended product was then filtered. Thus obtained product was dried to yield substituted pyrazolone of Formula 2.

[0107] By following the general procedure 3 and corresponding starting materials, the following compounds were prepared.

[0108] Example 49

[0109] Biological experiments

[0110] After developing a new process for the molecules of Formula 3, and since they have pyrazolone moiety embedded which generally tends to display biological activities, particularly in the CNS arena, preliminary screening of the selected molecules was taken up in neurobiologic al attributes.

[0111] The SRB assay was performed as described by Vanicha Vichai & Kanyawim Kirtikara (Nature protocols, 1(3), 1112-1116) to assess cytotoxicity. Briefly cytotoxicity against the Neuro 2a (N2A) cell line was performed by using three different drug concentrations (IpM, O.lpM, O.OlpM). N2A cells were passaged and seeded in 96-well plates at a density of 3200 cells per well. Following a 24-hour incubation, cells were treated with ice-cold 50% TCA and incubated at 4°C for 1 hour. Subsequently, plates were washed with distilled water, dried, and subjected to incubation with 0.04% SRB dye at room temperature for 1 hour. After rinsing with 1% acetic acid and drying, lOmM Tris base was added to each well, and absorbance was measured at 510 nm using a microplate reader. The Neurite outgrowth assay and imaging commenced with seeding Neuro2A cells at a density of 96000 cells per well in 6-well plates. Following a 24-hour incubation period, cells were subjected to serumdeprivation (0.1% serum) for 6 hours before exposure to compounds at three distinct nontoxic concentrations (IpM, O.lpM, O.OlpM). Following the incubation period, cellular imaging was conducted using a Motic microscope (Moticam Pro 282A) at 20x magnification. Neurite outgrowth was quantified by utilizing ImageJ software to calculate the average length (Organic & Biomolecular Chemistry, 22(4), 714-719).

[0112] For AChE inhibitory property studies; In this assay, 60 pL of Tris-HCl and 10 pL of acetylcholine enzyme (0.01 units) were added to each well of a 96- well plate. This was followed by the addition of 1.5 pL of the test compound and 3.5 pL of Tris-HCl. The plate was then incubated for 20 minutes at room temperature on a shaker. After incubation, 10 pL of substrate (0.075 M) and 60 pL of DTNB (0.001 M) were added to the wells. Spectrophotometric measurements were taken at 412 nm, and the percentage of enzyme inhibition was calculated.

[0113] When cytotoxicity against mouse neuroblastoma cells (Neuro 2a) was assessed using the SRB assay at three different concentrations (0.01, 0.1, and 1 pM) of the selected compounds at 24 hours from the drug treatment (FIG. 1). The findings indicated that the tested compounds remained within acceptable safety levels compared to the untreated or vehicle control group and displayed neuroprotective potential. The IC50 values have been tabulated in Table 1.

[0114] Table 1: IC50 (mM) values upon SRB assay. The data shown as mean concentration(mM)+standard deviation (SD).

[0115] The axonal development in Neuro2a-cells post-treatment of these molecules was studied to check the neurogenic activity with NGF as control following known prior art. As expected NGF showed a significant increase in neurite length compared to the untreated group. The vehicle control DMSO had no change in neurite length displaying no significant effect on neurite outgrowth. The tested compound of Formula 3 and formula 2 revealed that 2a,2e,3n and 31 displayed a significant increase in neuronal length compared to a positive control (NGF) across different concentrations (0.01, 0.1 and 1 mM) as shown in FIG. 2.

[0116] Additionally, when the selected compounds of Formula 3 and Formula 2 were assessed for their Blood-Brain Barrier (BBB) permeability following prior art in silico online bloodbrain barrier prediction server [https: / / biosig.lab.uq.edu.au / pkcsm / prediction, J. Med. Chem. 2015, 58, 4066-4072], Remarkably, all of the compounds were determined to be BBB permeable based on these computational analyses. The logBB values have been tabulated in table 2. Table 2: logBB values for Blood brain permeability assessment. Further, when the compounds of Formula 3 and Formula 2 were tested for acetycholine esterase enzyme inhibitory property with galantamine as the positive control following the prior art method as provided by Ellman et al (Biochem. Pharmacol., 1961, 7, 88-95). All of these compounds demonstrated notable inhibitory activity against AChE at the lowest concentration of (0.01 pM) in comparison to galantamine at same concentration of 0.01 p M as illustrated in FIG. 3 and Table 3 for IC50 values.

[0117] Table 3: IC50 values represent the concentration (pM) needed to achieve 50% inhibition of enzyme activity as determined by the AChE assay. Data are expressed as mean concentration (pM) ± standard deviation (SD).

[0118] ADVANTAGES OF THE INVENTION

[0119] The various advantages of the present process are given below.

[0120] • The process serves as a highly efficient and scalable production method.

[0121] • In view of the importance of multitarget-directed therapeutic and diverse bioactivities of pyrano[2,3-c]pyrazole as well as pyrazolone, a new and efficient process for the preparation of hybrid pyrano[2,3-c]pyrazole -pyrazolone from diynones of Formula 1 with pyrazolones of Formula 2 in presence or absence of base including cost, time and step economy. The diynones and pyrazolones can be easily accessible from the readily available commercial materials.

[0122] • The process could be operated by one-pot employing readily available diynones and pyrazolones.

[0123] • Another advantage of the present invention is; it includes very highly feasible reaction parameters.

[0124] • Isolation and / or purification of the product / s is straightforward. • This is an attractive and economic method for the production of hybrid pyrano[2,3- c]pyrazole-pyrazolone and analogs.

[0125] • This process could be adopted to generate a large library of process intermediates and hybrid pyrano[2,3-c]pyrazole-pyrazolone analogues.

[0126] • The hybrid pyrano[2,3-c]pyrazole-pyrazolones compounds of Formula 3 are useful as potent neuritogenic agents which are also BBB permeable.

[0127] • The hybrid pyrano[2,3-c]pyrazole-pyrazolones compounds are useful as potent acetylcholine esterase enzyme inhibitors.

Claims

We claim1. A hybrid pyrano[2,3-c]pyrazole-pyrazolone compound of Formula 3, and its analogous and their pharmaceutically acceptable salts, hydrates, solvates, optical isomers and combinations,Formula 3 wherein each Ri and R2, is independently selected from the group consisting of aryl, phenyl, 4-fluoro phenyl, 4-methyl phenyl, 4-methoxy phenyl and 3-thienyl;R3 is selected from the group consisting of phenyl, 4-chloro phenyl, 4-fluoro phenyl, 4-trifluoromethyl phenyl, 4-trifluoromethoxy phenyl and 2,2,2-trifluoroethyl;R4 is selected from the group consisting of alkyl, Me, n-propyl and cyclopropyl.

2. The compound as claimed in claim 1, wherein the compound of formula 3 is selected from the group consisting of: i. (E)-5-methyl-4-((E)-2-(3-methyl-l,6-diphenyl-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3a);ii. (E)-4-((E)-2-(l,6-diphenyl-3-propyl-3a,7a-dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl-5-propyl-2,4-dihydro-3H- pyrazol-3-one (3b);iii. (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-l,6-diphenyl-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-phenyl- 2,4-dihydro-3H-pyrazol-3-one (3c);iv. (E)-2-(4-chlorophenyl)-4-((E)-2-(l-(4-chlorophenyl)-3-methyl-6-phenyl-3a,7a-dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5- methyl-2,4-dihydro-3H-pyrazol-3-one (3d);v. (E)-2-(4-fluorophenyl)-4-((E)-2-(l-(4-fluorophenyl)-3-methyl-6-phenyl-3a,7a-dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-5- methyl-2,4-dihydro-3H-pyrazol-3-one (3e);vi. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethyl)phenyl)-3a,7a-dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(4-(trifluoromethyl)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3f);vii. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(4-(trifluoromethoxy)phenyl)-3a,7a-dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2- (4-(trifluoromethoxy)phenyl)-2,4-dihydro-3H-pyrazol-3-one (3g);viii. (E)-5-methyl-4-((E)-2-(3-methyl-6-phenyl-l-(2,2,2-trifluoroethyl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-phenylethylidene)-2-(2,2,2- trifluoroethyl)-2,4-dihydro-3H-pyrazol-3-one (3h);ix. (E)-4-((E)-l-(4-fluorophenyl)-2-(6-(4-fluorophenyl)-3-methyl-l-phenyl-3a,7a-dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5-methyl-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3i);x. (E)-5-cyclopropyl-4-((E)-2-(3-cyclopropyl-6-(4-fluorophenyl)-l-phenyl-3 a, 7 a-dihydropyrano [2,3 -c]pyrazol-4( lH)-ylidene)- 1 -(4- fluorophenyl)ethylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3j);xi. (E)-4-((E)-l-(4-fluorophenyl)-2-(6-(4-fluorophenyl)-l-phenyl-3-propyl-3a,7a-dihydropyrano [2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-2-phenyl-5- propyl-2,4-dihydro-3H-pyrazol-3-one (3k);xii. (E)-4-((E)-2-(l,6-bis(4-fluorophenyl)-3-methyl-3a,7a-dihydropyrano[2,3- c]pyrazol-4(lH)-ylidene)-l-(4-fluorophenyl)ethylidene)-2-(4-fluorophenyl)- 5-methyl-2,4-dihydro-3H-pyrazol-3-one (31);xiii. (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(p-tolyl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-(p-tolyl)ethylidene)-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3m);xiv. (E)-4-((E)-l-(4-methoxyphenyl)-2-(6-(4-methoxyphenyl)-3-methyl-l- phenyl-3a,7a-dihydro pyrano[2,3-c]pyrazol-4(lH)-ylidene)ethylidene)-5- methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (3n);xv. (E)-5-methyl-4-((E)-2-(3-methyl-l-phenyl-6-(thiophen-3-yl)-3a,7a- dihydropyrano[2,3-c]pyrazol-4(lH)-ylidene)-l-(thiophen-3-yl)ethylidene)-2- phenyl-2,4-dihydro-3H-pyrazol-3-one (3o).

3. A one-pot process for the preparation of the compound of Formula 3 as claimed in claim 1, comprising the step of: i. reacting diynones of Formula 1 with pyrazolones of Formula 2 in a solvent at temperature in the range of 25-35°C optionally in the presence of a base for a time period in the range of 30 minutes to 24 hours to obtain hybrid pyrano[2,3-c]pyrazole-pyrazolones compound of Formula 3;Formula 1 Formula 2 wherein Ri, R2, R3 and R4 are as defined in claim 1.

4. The process as claimed in claim 3, wherein Formula 1 is selected from the group consisting of:

5. The process as claimed in claim 3, wherein Formula 2 is selected from the group consisting of:

6. The process as claimed in claim 3, wherein the base is selected from the group comprising of DBU, DBN, DABCO, DIPEA, DMAP, EtsN, potassium carbonate, sodium carbonate, cesium carbonate, potassium bicarbonate, sodium bicarbonate, potassium phosphate and ammonium carbonate.

7. The process as claimed in claim 3, wherein the solvent used is selected from the group consisting of ethers, alcohols, esters, acetone, toluene, THF, ethyl acetate, dimethylformamide, dimethylsulfoxide and acetonitrile.

8. The compounds as claimed in claim 1 and 2, wherein compound of Formula 31 and 3n exhibit neuritogenic properties displaying a significant increase in average neuronal length compared to the positive control (NGF) across different concentrations ranging from 77.56 to 133.75 pM at 1.0 pM, 85.71-115.89 pM at 0.1 pM and 85.10-117.92 pM at 0.01 pM concentration.

9. The compounds as claimed in claim 1 and 2, wherein compounds 3a, 3c, 3f, 31, 3n, 3o exhibit non-cytotoxicity towards mouse neuroblastoma cells with IC50 values of 0.0022 to 0.8909 pM.

10. The compounds as claimed in claim 1 and 2, wherein the compound 3a, 3c, 3f, 31,3n, 3o inhibits acetylcholine esterase enzyme with a IC50 values ranging from 0.0682 to 0.5337 pM.

11. The compounds as claimed in claim 1 and 2, wherein the compound 3a, 3c, 3f, 31, 3n, 3o cross the Blood-Brain Barrier (BBB) permeability within a range of logBB - 0.993 to 0.07.

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