Microwave-Promoted Total Synthesis of Puniceloid D for Modulating the Liver X Receptor

A growing global health concern is metabolic syndrome, which is defined by low HDL, diabetes, hypertension, and abdominal obesity. Nuclear receptors are attractive targets for treatment of diseases associated with metabolic syndrome. Liver X receptors (LXRs) have become one of the most significant pharmacological targets among nuclear receptors. Multiple research studies emphasize the essential function of the liver X receptor (LXR) in the pathophysiology of metabolic syndrome. Puniceloid D, among natural products, demonstrated promising effects on LXRα. However, attempts at the total synthesis of natural products were faced with challenges, including long synthetic steps and low yields, requiring a more efficient approach. In this study, for the first time, we successfully synthesized puniceloid D through a seven-step process and conducted docking studies to gain a comprehensive understanding of the interactions involved in the binding of puniceloid D to LXR within different heterodimeric contexts. Our understanding of the pathophysiology of metabolic syndrome could be improved by these findings, which might assist with the development of novel treatment strategies.


Introduction
One of the major obstacles for health systems worldwide is the diseases associated with metabolic syndrome.The condition consists of several risk factors, particularly linked to diabetes and cardiovascular disease.The cluster of metabolic factors includes low HDL cholesterol, high triglyceride levels, impaired fasting glucose, high blood pressure, and abdominal obesity [1].Therefore, the management and treatment of metabolic syndrome are of highest priority.Recent study data indicate that nuclear receptors play a critical role in the pathophysiology of this condition [2].
Target gene expression in a wide range of physiological pathways, including metabolic processes, is regulated by nuclear receptors, which are ligand-activated transcription factors [3][4][5].Nuclear receptors associated with metabolism are categorized as type 2 nuclear receptors, which are found inside the nucleus regardless of ligand binding [6].Liver X receptors (LXRα (NR1H3) and LXRβ (NR1H2)), farnesoid X receptor (FXR, NR1H4), peroxisome proliferator-activated receptors (PPARs, NR1C1/NR1C2/NR1C3), and RXRs are examples of type 2 nuclear receptors for the regulation of metabolic processes.Fatty acids, oxysterols, bile acids, and rexinoids are ligands for these receptors, highlighting their importance in the control of metabolic pathways [7,8].
An essential role for liver X receptors is to regulate innate immunity, inflammatory responses, and lipid and cholesterol metabolism [9][10][11].There are two isoforms of the liver X receptor: LXRα and LXRβ [12].While LXRβ (NR1H2) is expressed everywhere, LXRα (NR1H3) is expressed in metabolically active tissues such as the liver, adipose, kidney, macrophages, and intestines [13,14].LXR inhibits gluconeogenesis, promotes bile secretion, raises cholesterol clearance in the liver, suppresses the macrophage inflammatory response, increases clearance of cholesterol in the small intestine, and facilitates glucose reabsorption into cells by promoting the expression of GLUT4 (glucose transporter type 4) in adipose tissue [15].In addition to their role in regulating lipid metabolism, LXRs play a crucial role in controlling blood sugar levels by modulating glucose transport proteins such as GLUT4/5, and regulatory proteins like ChREBP (carbohydrate responsive element-binding protein), which are involved in glucose homeostasis [16].Therefore, the majority of the research performed to identify LXR modulators with therapeutic potential has focused on the development of LXR agonists [17,18].
The search for LXR modulators for the treatment of metabolic diseases is ongoing.Utilizing natural products that potentially offer decreased potency and fewer side effects or demonstrate tissue-or subtype-selective activity represents a viable approach [1]. Figure 1 displays some discovered LXRα agonists derived from natural products (marine-derived fungi) [19].Based on a study by Liang et al., [19] puniceloid C and D were identified as the most potent LXRα agonists.
X receptor: LXRα and LXRβ [12].While LXRβ (NR1H2) is expressed everywher (NR1H3) is expressed in metabolically active tissues such as the liver, adipose, macrophages, and intestines [13,14].LXR inhibits gluconeogenesis, promotes bi tion, raises cholesterol clearance in the liver, suppresses the macrophage inflam response, increases clearance of cholesterol in the small intestine, and facilitates reabsorption into cells by promoting the expression of GLUT4 (glucose transpor 4) in adipose tissue [15].In addition to their role in regulating lipid metabolism, LX a crucial role in controlling blood sugar levels by modulating glucose transport such as GLUT4/5, and regulatory proteins like ChREBP (carbohydrate respon ment-binding protein), which are involved in glucose homeostasis [16].Therefore jority of the research performed to identify LXR modulators with therapeutic p has focused on the development of LXR agonists [17,18].
The search for LXR modulators for the treatment of metabolic diseases is o Utilizing natural products that potentially offer decreased potency and fewer sid or demonstrate tissue-or subtype-selective activity represents a viable approach ure 1 displays some discovered LXRα agonists derived from natural products ( derived fungi) [19].Based on a study by Liang et al., [19] puniceloid C and D wer fied as the most potent LXRα agonists.Quinazolinones are known for their diverse and significant biological prope cluding cholinesterase inhibition and antitumor, antiviral, anti-inflammatory, an and protein kinase inhibitory effects [20].The presence of quinazolinone and its tives is noted in over 100 naturally occurring alkaloids, comprising a crucial class heterocycles [21].Several families of alkaloids are represented by fungal quinaz metabolites that contain the core pyrazino[2,1-b]quinazoline-3,6-dione (A) scaffol Scaffold A is a structural motif found in several natural products known notable biological activities, such as glyantripine (B), fiscalin B (C), fumiquinaz (D) and G (E), as well as puniceloids A−D (Figure 2).
Derivatives of this system have been synthesized using three distinct meth Quinazolinones are known for their diverse and significant biological properties, including cholinesterase inhibition and antitumor, antiviral, anti-inflammatory, anticancer, and protein kinase inhibitory effects [20].The presence of quinazolinone and its derivatives is noted in over 100 naturally occurring alkaloids, comprising a crucial class of fused heterocycles [21].Several families of alkaloids are represented by fungal quinazolinone metabolites that contain the core pyrazino[2,1-b]quinazoline-3,6-dione (A) scaffold [22].
Scaffold A is a structural motif found in several natural products known for their notable biological activities, such as glyantripine (B), fiscalin B (C), fumiquinazolines F (D) and G (E), as well as puniceloids A−D (Figure 2).
X receptor: LXRα and LXRβ [12].While LXRβ (NR1H2) is expressed everywher (NR1H3) is expressed in metabolically active tissues such as the liver, adipose, macrophages, and intestines [13,14].LXR inhibits gluconeogenesis, promotes bi tion, raises cholesterol clearance in the liver, suppresses the macrophage inflam response, increases clearance of cholesterol in the small intestine, and facilitates reabsorption into cells by promoting the expression of GLUT4 (glucose transpor 4) in adipose tissue [15].In addition to their role in regulating lipid metabolism, LX a crucial role in controlling blood sugar levels by modulating glucose transport such as GLUT4/5, and regulatory proteins like ChREBP (carbohydrate respon ment-binding protein), which are involved in glucose homeostasis [16].Therefore jority of the research performed to identify LXR modulators with therapeutic p has focused on the development of LXR agonists [17,18].
The search for LXR modulators for the treatment of metabolic diseases is o Utilizing natural products that potentially offer decreased potency and fewer sid or demonstrate tissue-or subtype-selective activity represents a viable approach ure 1 displays some discovered LXRα agonists derived from natural products ( derived fungi) [19].Based on a study by Liang et al., [19] puniceloid C and D wer fied as the most potent LXRα agonists.Quinazolinones are known for their diverse and significant biological prope cluding cholinesterase inhibition and antitumor, antiviral, anti-inflammatory, an and protein kinase inhibitory effects [20].The presence of quinazolinone and its tives is noted in over 100 naturally occurring alkaloids, comprising a crucial class heterocycles [21].Several families of alkaloids are represented by fungal quinaz metabolites that contain the core pyrazino[2,1-b]quinazoline-3,6-dione (A) scaffol Scaffold A is a structural motif found in several natural products known notable biological activities, such as glyantripine (B), fiscalin B (C), fumiquinaz (D) and G (E), as well as puniceloids A−D (Figure 2).
Derivatives of this system have been synthesized using three distinct meth cyclization of 4(3H)-quinazolinones (a), the cyclocondensation of 2,5-piperazin  Considering the increasing interest in natural products as potential sources of novel drugs [24] or as lead structures for drug discovery in medicinal and organic chemistry [25,26], and drawing inspiration from existing data [19] highlighting puniceloid D as the most potent LXRα agonist, our study presents the total synthesis of puniceloid D for the first time.Furthermore, significant results from molecular docking studies were obtained with puniceloid D.

Total Synthesis of Puniceloid D
The first total synthesis of puniceloid D (1) involved seven steps.In our study, we chose route (b) (Figure 3) based on literature findings, as protecting N-2 was reported to enhance reactivity and prevent racemization [27].In the first step, the reaction of (tertbutoxycarbonyl)-D-alanine with glycine ethyl ester hydrochloride as a nucleophile produced ethyl (tert-butoxycarbonyl)-D-alanylglycinate (7) at a 73.4% yield.This reaction was facilitated by using EDC and HOBt, which convert amine to amide [28].According to the literature, the stereochemistry is preserved [29,30].In the second step, intramolecular cyclization occurred under microwave conditions, involving the removal of the Boc group.Subsequently, intramolecular nucleophilic addition of the amin in compound 7 to the carbonyl group, followed by the elimination of the ethoxy group, produced compound 6 [31].The literature states that compound 6 was obtained with complete retention of the original stereochemical configuration of 7 through the use of HPLC analysis on the chiral phase [31].Next, compound 6 was heated under reflux conditions in acetic anhydride to yield compound 5. Classically, an excess of acetic anhydride is used in solvent-free acetylation reactions and it preserves the stereochemistry [32].To facilitate base-catalyzed condensation with aldehydes, compound 5 was diacetylated in the previous step.At room temperature, an aldol reaction is carried out between compound 5 and benzaldehyde, in the presence of potassium t-butoxide and DMF as a solvent, to form compound 4 [27].The acetyl group reduces the pKa of the alpha carbonyl, making it easily deprotonated.Based on existing research, compound 4 maintains its stereochemical integrity [27,33].Challenges in E/Z isomer selectivity could arise depending on the location of the phenyl ring.However, in the literature, the absence of NOE enhancement in vinyl protons upon N-H signal irradiation confirmed the Z configuration [27,34].Compound 4 was deacetylated through treatment with hydrazine monohydrate, resulting in the formation of compound 3 [27].The removal of the acetyl group increased the polarity of the compound, making it less soluble in commonly used solvents such as ethyl acetate and dichloromethane.The Considering the increasing interest in natural products as potential sources of novel drugs [24] or as lead structures for drug discovery in medicinal and organic chemistry [25,26], and drawing inspiration from existing data [19] highlighting puniceloid D as the most potent LXRα agonist, our study presents the total synthesis of puniceloid D for the first time.Furthermore, significant results from molecular docking studies were obtained with puniceloid D.

Total Synthesis of Puniceloid D
The first total synthesis of puniceloid D (1) involved seven steps.In our study, we chose route (b) (Figure 3) based on literature findings, as protecting N-2 was reported to enhance reactivity and prevent racemization [27].In the first step, the reaction of (tert-butoxycarbonyl)-D-alanine with glycine ethyl ester hydrochloride as a nucleophile produced ethyl (tert-butoxycarbonyl)-D-alanylglycinate (7) at a 73.4% yield.This reaction was facilitated by using EDC and HOBt, which convert amine to amide [28].According to the literature, the stereochemistry is preserved [29,30].In the second step, intramolecular cyclization occurred under microwave conditions, involving the removal of the Boc group.Subsequently, intramolecular nucleophilic addition of the amin in compound 7 to the carbonyl group, followed by the elimination of the ethoxy group, produced compound 6 [31].The literature states that compound 6 was obtained with complete retention of the original stereochemical configuration of 7 through the use of HPLC analysis on the chiral phase [31].Next, compound 6 was heated under reflux conditions in acetic anhydride to yield compound 5. Classically, an excess of acetic anhydride is used in solvent-free acetylation reactions and it preserves the stereochemistry [32].To facilitate base-catalyzed condensation with aldehydes, compound 5 was diacetylated in the previous step.At room temperature, an aldol reaction is carried out between compound 5 and benzaldehyde, in the presence of potassium t-butoxide and DMF as a solvent, to form compound 4 [27].The acetyl group reduces the pKa of the alpha carbonyl, making it easily deprotonated.Based on existing research, compound 4 maintains its stereochemical integrity [27,33].Challenges in E/Z isomer selectivity could arise depending on the location of the phenyl ring.However, in the literature, the absence of NOE enhancement in vinyl protons upon N-H signal irradiation confirmed the Z configuration [27,34].Compound 4 was deacetylated through treatment with hydrazine monohydrate, resulting in the formation of compound 3 [27].The removal of the acetyl group increased the polarity of the compound, making it less soluble in commonly used solvents such as ethyl acetate and dichloromethane.The reaction of compound 3 with Meerwein's salt (triethyloxonium tetrafluoroborate) in the presence of sodium carbonate resulted in the formation of the monoiminoether compound (2) [28,31].Meerwein's salt can act as an alkylating agent by serving as a source of ethyl groups (C 2 H 5 + ) [35].In accordance with published research [36], thermal treatment of compound 3 does not cause any changes to the stereocenter, leading to compound 2. Despite the reaction running for 24 h, the yield was low because the starting material for this reaction was diketopiperazine, which has low reactivity.The number of equivalents of the Meerwein's salt was increased to enhance the yield, but when 2.2 equivalents were used, it was discovered that ethylation occurred on both carbonyl oxygens in the diketopiperazine ring, highlighting the importance of controlling the number of equivalents.Finally, compound 2 interacts with 3-hydroxyanthranilic acid under microwave conditions at 200 • C, followed by cyclization and elimination of hydroxy, resulting in the formation of puniceloid D (1) [37] (Scheme 1).In the literature, certain research groups have focused on producing 1-aryl methylene pyrazino[2,1-b]quinazoline-3,6-diones, which share similarities with puniceloid D. Cledera et al. detailed a seven-step synthesis of (R,Z)-1-(4-methoxybenzylidene)-4methyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione under heating conditions at 120 • C for 2 h, achieving a 15% yield [27].The same group also employed a 9-min microwave irradiation reaction, obtaining the same compound at a 26% yield [36]. Taking inspiration from this, we successfully synthesized puniceloid D (1) for the first time with a 9.45% yield, utilizing 3-hydroxyanthranilic acid under microwave irradiation for 2 h.reaction of compound 3 with Meerwein's salt (triethyloxonium tetrafluoroborat presence of sodium carbonate resulted in the formation of the monoiminoether com (2) [28,31].Meerwein's salt can act as an alkylating agent by serving as a source groups (C2H5 + ) [35].In accordance with published research [36], thermal treatment pound 3 does not cause any changes to the stereocenter, leading to compound 2. the reaction running for 24 h, the yield was low because the starting material for action was diketopiperazine, which has low reactivity.The number of equivalen Meerwein's salt was increased to enhance the yield, but when 2.2 equivalents we it was discovered that ethylation occurred on both carbonyl oxygens in the diket zine ring, highlighting the importance of controlling the number of equivalents.compound 2 interacts with 3-hydroxyanthranilic acid under microwave condition °C, followed by cyclization and elimination of hydroxy, resulting in the form puniceloid D (1) [37] (Scheme 1).In the literature, certain research groups have on producing 1-aryl methylene pyrazino[2,1-b]quinazoline-3,6-diones, which sha larities with puniceloid D. Cledera et al. detailed a seven-step synthesis of (R,Z)-1oxybenzylidene)-4-methyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-di der heating conditions at 120 °C for 2 h, achieving a 15% yield [27].The same gr employed a 9-min microwave irradiation reaction, obtaining the same compound yield [36]. Taking inspiration from this, we successfully synthesized puniceloid the first time with a 9.45% yield, utilizing 3-hydroxyanthranilic acid under mi irradiation for 2 h.
The 1 H NMR and 13 C NMR spectra of the final synthesized compound (1) e a complete match with the reported ones [19] (Table 1) (Figure S15).The 1 H NMR and 13 C NMR spectra of the final synthesized compound (1) exhibited a complete match with the reported ones [19] (Table 1) (Figure S15).

Discussion
According to the literature [19], the transactivation effects of puniceloid D and oxepinamide K on LXRα were investigated.Puniceloid D exhibited significant transcriptional activation on LXRα, demonstrating an EC 50 value of 1.7 µM.In contrast, oxepinamide K, which contains an oxepin unit, showed 29 times less active transcriptional activation on LXRα, with EC 50 values of 50 µM compared to puniceloid D. These findings indicate that the quinazolinone skeleton found in puniceloid D is essential in determining the observed bioactivity in the context of LXRα transactivation [38].To enhance our understanding of the binding modes of puniceloid D and oxepinamide K with LXRs, we conducted molecular docking studies (in silico) using Glide in Extra Precision (XP), as implemented in Schrödinger (version 2023-4).The docking studies were performed with LXRα in the context of two different LXRα-RXRβ and LXRα-RXRα LBD heterodimers, represented by PDB IDs 1UHL and 2ACL, respectively.
In our in silico docking study, we investigated the interactions of puniceloid D and oxepinamide K with two LXRα LBD (PDB ID 1UHL and PDB ID 2ACL).Puniceloid D notably exhibited π-π stacking interactions with Tyr32 and Phe257 when docked with LXRα from the LXRα-RXRβ heterodimer (Figure 4).Similarly, in the docking study with LXRα from the LXRα-RXRα heterodimer, puniceloid D displayed π-π stacking interactions with Phe313 and hydrogen bonding interactions with Phe255 (Figure 5).This revealed different binding modes and potential molecular mechanisms underlying its interactions with LXRα in various heterodimeric contexts.Furthermore, we used the "dock" mode to calculate the binding affinity of compounds based on minimum energy values (kcal/mol) (Table 2).In both PDB IDs (1UHL and 2ACL), puniceloid D exhibited the lowest docking scores (−8.535 and −9.783 kcal/mol, respectively) and G scores (−8.841 and −10.098 kcal/mol, respectively) compared to oxepinamide K, indicating more stable binding conformations.This work provides more evidence that the quinazolinone skeleton is a useful model for the synthesis of LXR agonists.

Molecular Docking Experiment
The docking studies were conducted using Glide in Extra Precision (XP) employed by Schrödinger (version 2023-4).LXRα structures were acquired from the Protein Data Bank (PDB) with codes 1UHL and 2ACL [42].The 2D structures of the compounds were obtained in .sdffile format using CS ChemDraw (version 20) and were subsequently converted into 3D structures.The ligands were independently docked into the energy-minimized structures of the ligand-binding domains (LBD) of LXRα.Docking scores and G scores were calculated to assess the binding of each tested compound with LXRα.

Conclusions
Diseases related to metabolic syndrome, such as obesity and diabetes, exhibit a concerning global prevalence, making them significant health priorities.The LXRs play a major role in regulating lipid and cholesterol metabolism, in addition to their anti-inflammatory activities.Natural products can act as both independent sources for novel drugs and foundational structures for drug discovery.In the present study, we pursued a seven-step synthetic pathway, starting from readily available and inexpensive starting materials, to accomplish the synthesis of the natural product puniceloid D-a novel and potent liver X receptor agonist.Furthermore, the molecular docking study revealed that puniceloid D exhibited favorable binding scores with two LXRα LBD structures (PDB ID: 1UHL and 2ACL) in comparison to oxepinamide K. Puniceloid D demonstrated a favorable conformational state, engaging in hydrogen bond and π-π stacking interactions within the active sites of LXRα.Promising natural products can be chemically modified to overcome their low yield and complicated synthetic pathways, thereby increasing their commercial potential.Our future research will explore the chemical modification of puniceloid D and investigating the mechanisms of action of its compounds would also be of interest.

Table 2 .
Glide XP docking scores for puniceloid D and oxepinamide K with two LXRα LBD (PDB ID: 1UHL and 2ACL).

Table 2 .
Glide XP docking scores for puniceloid D and oxepinamide K with two LXRα LBD (PDB ID: 1UHL and 2ACL).

Table 2 .
Glide XP docking scores for puniceloid D and oxepinamide K with two LXRα LBD (PDB ID: 1UHL and 2ACL).