Development Of New Potential GPCR171 Agonists As A Possible Solution For Metabolic Food Disorders

Abstract:

Synthesis of two novel compounds namely 5-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalic acid and 5-(3,4-diethoxybenzamido) isophthalic acid , as potential agonists of GPCR171 is achieved in two steps according to the standard amide coupling/deprotection sequence. The compounds are characterized by Nuclear Magnetic Resonance spectrometry. Structures of these compounds are envisaged by using common drug design approach, such as structure extension, starting from the MS0015203 (5-[(2-Methyl-1-oxo-2-propen-1-yl)amino]-1,3-benzenedicarboxylic acid) a potent synthetic agonist of GPCR171 as a lead compound. Among others, this receptor is found to modulate the appetite and food intake in mice. Based on this, the novel compounds presented here can be considered as candidates for pharmacological in vitro and in vivo tests of their activity towards the GPCR171, and possibly as a useful tool for further improvement of the treatment for these metabolic food disorders.

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Introduction:

G protein-coupled receptors (GPCR) are a well-known broad group of membrane receptors which are among the most significant therapeutic targets. Activation of GPCRs by extracellular stimuli, initiates the signal transduction that eventually leads to the response. Crystal structure of GPCRs by crystallization protocol is hard to obtain, because it is hard to remove and purify them from the membranes. One of the GPCRs that has crystal structure determined is P2Y12 (PDB: 4PXZ). P2Y12 is one of the main GPCR whose work is to stabilize the platelet aggregation in blood cloth forming, making this receptor as an essential target for current antiplatelet therapy.

In my proposed research, I have targeted G protein-coupled receptor 171 (GPR171), which is recently discovered as a receptor for modulation of appetite and metabolism in mice. The biological role of GPCR171 and gene that is coding for it (GPR171), is received a lot of attention lately, and it is found to involve feeding and metabolism in mice and myeloid differentiation. It was also found that GPR171 is overexpressed in the lung cancer tissue, suggesting him to be a target of promise for the development of antineoplastic drugs. Since GPR171 is expressed in the part of the brain that is responsible for psychological disorders, recently, it was found that it may be used as a novel target to develop anxiolytics. Up until recently, GPCR171 was thought to be an orphan receptor, because its natural agonist BigLen is discovered not long ago. BigLen is a 16-amino acid neuropeptide (LENSSPQAPARRLLPP) which acts as a natural agonist of GPCR171 in hypothalamus. Its binding affinity for the GPCR171 is high (low Kd= 0.5 Nm). The structural requirement of the BigLen that is essential for the binding to its target was found by the drug design approach named drug simplification i.e. by cutting the endopeptide piece by piece. Finally, after many tests, it was found that only four amino acids (LLPP) of the C-terminus are enough to trigger the receptor. These findings initiated the development of small chemical ligands which can selectively activate or deactivate the GPR171.

Since there are no crystal structure of the GPR171, homology modeling is an alternative option. For cooperation was used P2Y 12 receptor, because they are phylogenetically related as found by comparing their homologous sequences. The mechanism of action of the P2Y12 receptor demonstrates that the ADP binding in the extracellular domain of the receptor initiates the cascade of events inside the cell. The examination of the P2Y12 crystal structure revealed the binding pocket of this receptor has Histidine side chain as an essential for receptor activity. This was proven by the fact that when Histidine is replaced by Glutamine, the activity of the receptor is disturbed.

In ligand binding assays, it was found that ligands like 2MeSADP, ADP, and ADPβS which binds the P2Y12 receptor are not active towards the GPCR171 as they do not displace its natural ligand BigLen. These observations suggested that there is a significant structural difference in the binding site of GPR171 and P2Y12. However, the atomic coordinates from PDB file 4PXZ are all that is available to thread the GPR171 amino acid sequence.

Molsoft ICM-Pro Software was used to find out best fit of small chemical structure to the binding site of GPR171 by analyzing thousands of molecules from the database. After this rigorous screening process, MS0015203 was found as a promising candidate who can act as an agonist on GPR171. Molecule MS0015203 was found to be a partial agonist of GPR171 when checked through in vivo experiment. It was found that after the peripheral injection of MS0015203, food intake in mice is significantly increased. This effect is shown to be reversible through shRNA-mediated knockdown of hypothalamic GPR171. This result suggested that targeting the GPR171 may be of use for managing body weight and metabolism in mammals.

Experimental Procedures and Materials (Method and Materials is better)

Materials

3-ethyl-5-methylisoxazole-4-carboxylic acid, 3,4-Diethoxybenzoic acid, 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), N,N-dimethylformamide (DMF), Diisopropylamine, Diphenyl 5-amino-isophthalate, Liquid Nitrogen, Dichloromethane (DCM), Sodium bicarbonate, Hexane, Ethyl acetate, Sodium chloride(Brine), Sodium sulfate, Sodium hydroxide, Column chromatography, dimethyl sulfoxide-d6, and nuclear magnetic resonance (NMR) spectroscopy.

Experimental Procedures

There are some other small chemical molecules selected by the Molsoft ICM-Pro Software like MS0016574, but it showed quite lower ability to displace BigLen from GPR171 receptor compared to MS0015203. This finding prompted me for current research plan where I selected MS0015203 as a scaffold molecule and developed library of compounds based on it, which differ in group attached to the carbonyl carbon of amide group.

MS0015203 Synthetic Target

Synthesized Compounds

The target molecules are synthesized in two steps that involve liquid phase peptide bond synthesis followed by a Deprotection step in which benzyl diester is converted into a benzyl dicarboxylate.

Step 1: Peptide Bond Synthesis

Step 2: Deprotection to form the free acid

The reaction rate was increased by converting the carboxylic acid group into an ester. Three-mole equivalents of HCTU (1.77 g, 2.87mmol) at 0 °C were mixed with the same equivalents of the carboxylic acid derivative (R-COOH group). This mixture was dissolved in a minimal amount of DMF (6 mL). Then this solution was added into round bottom flask (RBF) and placed in an ice bath. The moist room air in the reaction mixture was replaced by anhydrous nitrogen through outfitted nitrogen balloon and bead syringe on the flask. Next, nine molar equivalents of N, N-Diisopropylethylamine (DIPEA) were added dropwise into the RBF. DIPEA is a strong base but weak nucleophile due to bulkier isopropyl groups which prevent the formation of side products. After around 15 minutes one molar equivalent of the starting amine precursor, dimethyl-5-aminoisophthalate was dissolved in minimal amount of DMF and added to the RBF in the reaction mixture. The reaction was allowed to proceed for completion for 1-2 days and monitored by aluminum-backed UV TLC for completion. After it was complete, the reaction mixture was diluted with 25 mL of DCM and transferred to a separatory funnel. DMF and polar impurities were removed by washing the organic layer twice with 50 mL of deionized water. An additional wash was made with 50 mL of saturated aqueous sodium bicarbonate solution to remove excess unreacted benzoic acid (R-group) by converting it to its sodium salt which is soluble in the aqueous layer. The organic layer which contains the desired product further washed twice with 50 mL of saturated aqueous sodium chloride solution, to remove excess DMF and residual water from the organic layer. Next, sodium sulfate was used for drying of the organic layer and then it was carefully transferred to a clean RBF. The rotary evaporator was used to remove solvents, and the product was dried under vacuum to remove residual solvents. UV-TLC (70% hexane to 30% ethyl acetate) and flash column chromatography was subsequently performed for purification. Proton NMR was performed to check product purity.

Deprotection to form Final Product and its Characterization

The purified intermediate (amide) was then transferred to RBF placed in an ice bath and dissolved in 15 mL of THF. Five molar equivalent of sodium hydroxide was dissolved in 5 mL of water in a test tube and added dropwise in the RBF. The reaction was left overnight and also monitored by TLC. This process resulted in the formation of a water-soluble sodium dicarboxylate salt. 50 mL of water was added to the RBF, and then the aqueous layer was washed with 10 mL of DCM and 10 mL of ethyl acetate respectively and then collected. The sodium dicarboxylate salt present in aqueous layer was later converted to free dicarboxylic acid group by adding dropwise acid into it for making pH around 1 to 1.5 of the solution so that dicarboxylic acid can precipitate out from the aqueous layer. Then the solution was filtered, and the precipitate was collected and washed with a saturated NaCl solution followed by sodium sulfate solution. Then the ethyl acetate was used to dissolve the precipitate and dried under a rotary evaporator followed by overnight vacuum to remove residual solvents. The compound was dissolved in dimethyl sulfoxide and characterized by 1H NMR and 13C NMR.

Results

1- Synthesis of 5-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalate acid (KM-1)

Three-mole equivalents of HCTU (1.77 g, 12.87mmol) were mixed with the same equivalents of the 3-ethyl-5-methylisoxazole-4-carboxylic acid (0.66g, 4.28mmol). This mixture was dissolved in DMF (6 mL). Then this solution was added into round bottom flask (RBF) and placed in an ice bath. The moist room air of the flask was replaced by anhydrous nitrogen through outfitted nitrogen balloon and bead syringe on the flask. Next, nine molar equivalents of N, N-Diisopropylethylamine (DIPEA) (2.20mL, 12.87mmol) were added dropwise into the RBF. After around 15 minutes one molar equivalent of the starting amine precursor, dimethyl-5-aminoisopthalate (0.300g, 1.43 mmol) was dissolved in 3ml DMF and added to the RBF in the reaction mixture. That was allowed to proceed for completion for 24 hours at 0 C0. After completion of the reaction, the reaction mixture was diluted with 25 mL of DCM and transferred to a separatory funnel. Then the organic layer was washed (2x) with 50 mL of deionized water to remove DMF and polar impurities. An additional wash was made with 50 mL of saturated aqueous sodium bicarbonate solution and 50 mL of saturated Sodium chloride(Brine). The organic layer dried with sodium sulfate, and it removed by the rotary evaporator. UV-TLC (70% hexane to 30% ethyl acetate) and flash column chromatography was subsequently performed for purification. Proton NMR was performed to check product purity. 15-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalate intermediates.

Synthesis of 5-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalic acid

The intermediate was then transferred to RBF placed in an ice bath and dissolved in 15 mL of THF. Five molar equivalent of sodium hydroxide (0.185g, 4.63 mmol) was dissolved in 5 mL of H2O in a test tube and added dropwise in the RBF, and the reaction was left overnight. This process resulted in the formation of a water-soluble sodium dicarboxylate salt. 50 mL of water was added to the RBF, and then the aqueous layer was washed with 10 mL of DCM and 10 mL of ethyl acetate respectively and then collected. The mixture was diluted with 50mL of water and washed with 10mL of DCM (dichloromethane). The organic layer acidified with HCl until a pH of ~1-1.5. Then the ethyl acetate was used to dissolve the precipitate and dried under a rotary evaporator followed by overnight vacuum to remove residual solvents. The compound was dissolved in dimethyl sulfoxide and characterized by 1H NMR and 13C NMR. 5-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalic acid was synthesized.

2- Synthesis of 5-(3,4-diethoxybenzamido) isophthalate:

Three-mole equivalents of HCTU (1.77 g, 12.87mmol) were mixed with the same equivalents of the 3,4-diethoxybenzoic acid (0.901g, 4.29mmol). This mixture was dissolved in DMF (6 mL). Then this solution was added into round bottom flask (RBF) and placed in an ice bath. The moist room air of the flask was replaced by anhydrous nitrogen through outfitted nitrogen balloon and bead syringe on the flask. Next, nine molar equivalents of N, N-Diisopropylethylamine (DIPEA) (2.20mL, 12.87mmol) were added dropwise into the RBF. After around 15 minutes one molar equivalent of the starting amine precursor, dimethyl-5-aminoisopthalate (0.300g, 1.43 mmol) was dissolved in 3ml DMF and added to the RBF in the reaction mixture. That was allowed to proceed for completion for 24 hours. After completion of the reaction, the reaction mixture was diluted with 25 mL of DCM and transferred to a separatory funnel. Then the organic layer was washed (2x) with 50 mL of deionized water to remove DMF and polar impurities. An additional wash was made with 50 mL of saturated aqueous sodium bicarbonate solution and 50 mL of saturated Sodium chloride(Brine). The organic layer dried with sodium sulfate, and it removed by the rotary evaporator. UV-TLC (70% hexane to 30% ethyl acetate) and flash column chromatography was subsequently performed for purification. Proton NMR was performed to check product purity. 5-(3,4-diethoxybenzamido) isophthalate was synthesized and characterized.

Synthesis of 5-(3,4-diethoxybenzamido) isophthalic acid

The intermediate was then transferred to RBF placed in an ice bath and dissolved in 15 mL of THF. Five molar equivalent of sodium hydroxide (0.185g, 4.63 mmol) was dissolved in 5 mL of H2O in a test tube and added dropwise in the RBF. The reaction was left overnight. This process resulted in the formation of a water-soluble sodium dicarboxylate salt. 50 mL of water was added to the RBF, and then the aqueous layer was washed with 10 mL of DCM and 10 mL of ethyl acetate respectively and then collected. The mixture was diluted with 50mL of water and washed with 10mL of DCM (dichloromethane). The organic layer acidified with HCl until a pH of ~1-1.5. Then the ethyl acetate was used to dissolve the precipitate and dried under a rotary evaporator followed by overnight vacuum to remove residual solvents. The compound was dissolved in dimethyl sulfoxide and characterized by 1H NMR and 13C NMR

5-(3,4-diethoxybenzamido) isophthalic acid is synthesized and characterized.

Discussion

In the present research work, I have synthesized two compounds based on MS0015203 scaffold namely 5-(3-ethyl-5-methylisoxazole-4-carboxamido) isophthalic acid and 5-(3,4-diethoxybenzamido) isophthalic acid. These novel analogs have different aromatic substituents at the carbonyl carbon of amide group of MS0015203.

The method for the synthesis, under the mild conditions, consisted of two steps: synthesis of phthalic acid esters, and the deprotection of acids. Phthalates were obtained under the condition of peptide coupling, using HCTU, aminium coupling reagent, which activates the carboxylic group of initial acid towards amidation. Once the amide bond was formed, the ester groups of the precursor amine were hydrolyzed by base treatment, affording the final products via two mild reaction procedures.

1H NMR and 13C NMR characterization of these compounds was done successfully.

Introduction of the aromatic and heterocyclic groups in the MS0015203 scaffold can influence on its pharmacological activity, in a manner that cannot be theoretically predicted. With their planar geometry, and p-p interaction that can form with other p bonds, and moderate basicity of isoxazole nitrogen, there are possibilities for emphasizing their pharmacological activity towards the GPCR171. One of the difficulties connected with the optimization of the potential ligand structures is that there is no crystal structure of GPRC171, which means that docking and other computational methods are approximate. However, these new analogs of MS0015203 should be further experimentally tested for their binding ability to GPR171 and may be a potential candidate for therapeutic modulations of food intake and metabolism. While inhibitory activity to GPR171 can be explored further for showing anti-obesity activity, but it is beyond the scope of the research presented here.

03 December 2019

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