Synthesis, Drug Likeness and In-vitro Screening of Some Novel Quinazolinone Derivatives for Anti-Obesity Activity

Aim: A series of novel quinazolinone derivates was synthesized and assessed for their ability to inhibitory action on pancreatic lipase. The cyclization of quinazolinone-4(3H)-one derivatives was achieved, whereas carbon-carbon cross coupling reactions were carried out on cyclized quinazolinone-4(3H)-one. This synthesis method afforded corresponding 2, 3 and 6 substituted quinazolin-4(3H)-ones (3a to 3m) with moderate to high yields. Methods: Benzamide derivatives (1a-1b) were synthesized from anthranilic acid using acid-amine reaction, followed by cyclization using catalytic p-toluene sulfonic acid and oxidation using (diacetoxyiodo)benzene to give bromo substituted quinazolin-4(3H)-ones (2a-2b), which were cross coupled to suitable boronic acid using Suzuki-Miyaura condition to obtain desired compound (3a3m). All synthesized compounds were characterized by FTIR, proton NMR, LC-MS analysis, checked for their drug likeness, absorption and evaluated for in vitro pancreatic lipase inhibition activity. Results: Analytical interpretation of all compounds with infrared, proton NMR and LC-MS spectroscopy confirmed their correct structure. All compounds (3a-3m) show good absorption and Original Research Article Modh and Patel; JPRI, 33(28B): 81-92, 2021; Article no.JPRI.68512 82 have reasonably good molecular properties except 3c and 3m which violate two criteria for Lipinski’s rule. Whereas, Compounds 3l and 3m showed IC50 value of 13.13±0.84 μg/mL and 13.80±1.27 μg/mL respectively comparable to the Orlistat (12.72±0.97μg/mL), a US FDA approved drug for the treatment of obesity. Conclusion: Pancreatic lipase is an important lipolytic enzyme, synthesized and secreted through pancreas, plays an important role in dietary trigycerol absorption and metabolism. Therefore, reducing fat absorption through pancreatic lipase inhibition is a promising strategy to treat obesity. Based upon our findings, compounds 3l and 3m can be further developed as potent anti-obesity agents.

have reasonably good molecular properties except 3c and 3m which violate two criteria for Lipinski's rule. Whereas, Compounds 3l and 3m showed IC 50 value of 13.13±0.84 µg/mL and 13.80±1.27 µg/mL respectively comparable to the Orlistat (12.72±0.97µg/mL), a US FDA approved drug for the treatment of obesity. Conclusion: Pancreatic lipase is an important lipolytic enzyme, synthesized and secreted through pancreas, plays an important role in dietary trigycerol absorption and metabolism. Therefore, reducing fat absorption through pancreatic lipase inhibition is a promising strategy to treat obesity. Based upon our findings, compounds 3l and 3m can be further developed as potent anti-obesity agents.
In the present work, we synthesized novel quinazolin-4(3H)-one derivatives and evaluated for pancreatic lipase inhibition activity which support their future development as anti-obesity agent.

MATERIAL AND METHODS
The novel synthesis scheme for the title compounds is outlined in Fig. 2 and SQ detector. Samples were prepared in 2mM ammonium acetate and injected into the BEH C18 (502.1 mm) 1.7µm column for detection using 0.1% formic acid in water: acetonitrile as mobile phase with. 1H-NMR spectra were recorded on Brucker 400 MHz Avance III HD instrument with 5mm PABBO BB/19F-1H/D Z-GRD Z108618 probe using DMSO D6 as a solvent and data were processed using Topspin 3.2 software. TLC was performed on precoated alumina silica gel 60 F254 (Merck) using different polarity ratios of ethylacetate: nhexane as mobile phase and detection was done using UV light. The resulting compounds were purified by recrystallization using suitable solvent or by flash column chromatography.
General synthetic procedures used for the preparation of the target compounds are described below.

Synthesis of 2, 3 and 6 substituted quinazolin-4(3H)-one (3a to 3m)
To the mixture of 2a or 2b (1.0 mmol) and Boronic acid derivative (1.5 mmol) in 1, 4dioxane (20 time) was added cesium carbonate (3.0 mmol) in a glass sealed tube and the reaction mixture was degassed with nitrogen for 15 min, followed by the addition of tetrakis (triphenylphosphine) palladium (0) (Pd(PPh 3 ) 4 ) (0.05 mmol) and heated to 100 0 C for 12 h. The reaction was monitored with TLC using ethylacetate: n-hexane as mobile phase. The reaction mixture was poured to water, extracted with ethylacetate, washed with brine, dried over sodium sulfate, concentrated under reduced pressure to give the crude product which was purified by flash column chromatography using ethylacetate: n-hexane as mobile phase to give the desired product (3a-3m) with good yield.

Prediction of Drug Likeness and Absorption
The prediction of molecular properties like drug likeliness and absorption were carried out by Molinspiration Cheminformatics Software available online. All synthesized molecules were sketched in ChemDraw 15 and they were copied as SMILES and saved. The Molinspiration home page was opened online, where a link for "Calculation of Molecular Properties and Bioactivity Score" was opened. All saved SMILES for synthesized compounds were pasted and properties like Log P, molecular weight, number of hydrogen bond acceptors, number of hydrogen bond donors, number of rotatable bonds, molecular volume, total polar surface area were calculated and saved. Absorption (%abs) was calculated by %abs = 109-(0.345 X TPSA) [28].

Experimental method
In vitro pancreatic lipase inhibition activity was performed as described by Nakai et al. [29]. Pancreatic lipase activity was measured using 4methylumbelliferyl oleate (

RESULTS AND DISCUSSION
In the first step, benzamide derivatives (1a-1b) were synthesized from anthranilic acid using acid-amine coupling condition. In the second step, substituted benzamide derivatives were cyclized using catalytic p-toluene sulfonic acid, followed by oxidation using (diacetoxyiodo)benzene to give bromo substituted quinazolin-4(3H)-ones (2a-2b), which were cross coupled to suitable boronic acid using Suzuki-Miyaura condition in third step to obtain desired compound (3a-3m). All synthesized compounds were characterized and confirmed with physical parameters like melting point, IR, LC-MS and 1H-NMR spectroscopy.

Prediction of Drug Likeness and Absorption
Biological activity being the function of the complex influence of many molecular descriptors in a drug, highlighting the effect of some individual parameters makes it possible to estimate the drug-likeness of newly synthesized molecules. There are several strategies for defining drug-like properties, Lipinski's rule is most commonly preferred. It states that to be drug-like, a candidate should have less than five hydrogen bond donors (HBD), less than 10 hydrogen bond acceptors (HBA), a molecular weight of less than 500 Da, and a pa coefficient log P of less than 5. It aims to highlight possible bioavailability problems if two or more properties are violated [30, synthesized targets (3a to 3m), follow Lipinski's rule except 3c and violate two criteria of Log P and molecular  weight of less than 500 Da, and a partition coefficient log P of less than 5. It aims to highlight possible bioavailability problems if two 30,31]. Out of all molecules and 3m which criteria of Log P and molecular weight. However, they need be explored during formulation development and bioavailability study. Moreover, all synthesized targets 3m) show good percentage of absorption. Results of calculated molecular properties and absorption have been summarized in and Table 2.

Pancreatic Lipase Inhibitory Activity (Anti-Obesity Activity)
Compounds (3a to 3m) were tested for pancreatic lipase inhibition activity and results were compared with Orlistat (Positive control). The pancreatic lipase inhibitory effects of the test compounds were indicated by IC50 value in Table 3.
Orlistat prevents absorption of fat from human diet and thereby produces calorie intake. It works by inhibiting pancreatic lipase, an enzyme that breakdowns triglyceride in the intestine and in absence of this enzyme, triglycerides from the diet are prevented from being hydrolyzed into the absorbable free fatty acids and instead excreted unchanged and undigested [32].
Pancreatic lipase is an important lipolytic enzyme secreted into the duodenum via duct system of pancreas. It plays a significant role in dietary trigycerol absorption. Pancreatic lipase hydrolyses triglycerols to monoacyl glycerols and fatty acids and it accounts for the hydrolysis of 50-70% of total dietary fats. The synthesized compounds studied here may probably inhibit digestion and absorption of dietary lipids through an inhibitory action on pancreatic lipase and therefore they can be further developed as potent anti-obesity agents. IC 50 value of Orlistat (positive control) was found to be 12.72±0.97 µg/mL. From all the tested compounds, 3l and 3m exhibited IC 50 Value of 13.13±0.84 µg/mL and 13.80±1.27 µg/mL respectively, suggesting their potent pancreatic lipase inhibitory functions comparable to the standard Orlistat.

CONCLUSION
Quinazolin-4(3H)-one derivatives constitute an important class of heterocycles with diverse pharmacological activities. All the title compounds (3a-3m) were synthesized, characterized, and evaluated for their drug likeness, absorbance and pancreatic lipase inhibitory activity. Two most potent compounds 3l and 3m exhibited IC50 value of 13.13±0.84 µg/mL and 13.80±1.27 µg/mL respectively for pancreatic lipase inhibition which is analogous to the orlistat, a US FDA approved drug for the treatment of obesity. Two molecules 3l and 3m

CONSENT AND ETHICAL APPROVAL
It is not applicable.

AVAILABILITY OF DATA AND MATERIAL
All data and material are available upon request.

INTERESTS AND DISCLAIMER
Authors have declared that no competing interests exist. The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.