Total Synthesis and Metabolic Stability of Hispidulin and Its d-Labelled Derivative

Hispidulin is a naturally occurring flavone known to have various Central nervous system (CNS) activities. Proposed synthetic approaches to synthesizing hispidulin have proven unsatisfactory due to their low feasibility and poor overall yields. To solve these problems, this study developed a novel scheme for synthesizing hispidulin, which had an improved overall yield as well as more concise reaction steps compared to previous methods reported. Additionally, using the same synthetic strategy, d-labelled hispidulin was synthesized to investigate its metabolic stability against human liver microsome. This work may produce new chemical entities for enriching the library of hispidulin-derived compounds.

Our recent clinical case report described a patient with a refractory chronic motor tic disorder who dramatically improved after taking the leaf juice of a local herb, Clerodendrum inerme (L.) Gaertn (CI) [8]. The ethanol extract of CI leaf alleviated methamphetamine-induced hyperlocomotion (MIH) as a mouse model of motor tic [9]. Using a bioassay-guided purification procedure in this animal model, we further isolated the constituents from CI leaf extract to identify a flavone hispidulin (6-methoxy-4 ,5,7-trihydroxyflavone) to be the main active ingredient (Figure 1) [9]. Hispidulin significantly attenuated MIH and even in amounts up to 100 mg/kg was incapable of affecting spontaneous locomotor activity or performance in mice [9]. Importantly, hispidulin had no hit on human ether-à-go-go-related gene (hERG) channels, an undesirable target in drug development [10]. spontaneous locomotor activity or performance in mice [9]. Importantly, hispidulin had no hit on human ether-à-go-go-related gene (hERG) channels, an undesirable target in drug development [10]. Hispidulin is widely distributed in Asteraceae [11][12][13][14] and some of the Lamiaceae [15]. Further assays for screening towards 92 target proteins associated with Central nervous system (CNS) diseases indicated that hispidulin formed strong bonds with GABAA receptors (IC50 = 0.73−1.78 μM) and inhibited catecholamine-O-methyl-transferase (COMT) (IC50 1.32 μM) [10]. By acting as a positive allosteric modulator (PAM), it enhanced cerebellar α6GABAA receptor activity. Studies revealed some C6-substituted flavones are identified to be PAMs of GABAA receptors [16,17]. The results of a structure-activity relationship (SAR) study indicated the C6 substituent in flavones greatly contributes to activity [10].
Because of its interesting biological activity, several research groups have developed synthetic approaches to hispidulin [18][19][20][21][22][23]. For example, Shen and coworkers developed a strategy for semisynthesis of hispidulin in seven reaction steps by using a naturally occurring scutellarin (Scheme 1) [22]. Although this method is concise and has an overall yield as high as 10.7% [22], the researchers showed that, upon the large-scale synthesis of compound 1, the protection of the catechol moiety of scutellarein using dichlorodiphenylmethane at 175 °C failed [20,23]. A seven-step synthesis route developed by Lin and coworkers solved this problem but reduced the overall yield to 7.1% (Scheme 1) [20]. Zhang and coworkers then developed a scheme that only required four reaction steps (Scheme 1). Nonselective MOM (methoxymethyl) protection of scutellarein caused the overall yield of the synthesis of hispidulin to be decreased (6.3%) [23]. Despite the satisfactory overall yield of these strategies, the tedious purification procedure required to isolate scutellarin from plants limits their use for large-scale preparation of hispidulin.
Kavvadias and coworkers developed a method synthesizing hispidulin from 2,4,6-trihydroxyacetophenone compound 6 in nine reaction steps (Scheme 2). However, the overall yield of this method is very limited (1.1%) [18]. We recently developed a feasible and reproducible approach for synthesizing hispidulin (Scheme 3) [21]. This method slightly improved the overall yield due to the low yield of Friedel-Crafts acetylation of compound 8 as well as unsatisfactory regioselective MOM protection of compound 9. These facts motivated us to investigate an efficient and high-yield route to synthesize hispidulin. Hispidulin is widely distributed in Asteraceae [11][12][13][14] and some of the Lamiaceae [15]. Further assays for screening towards 92 target proteins associated with Central nervous system (CNS) diseases indicated that hispidulin formed strong bonds with GABA A receptors (IC 50 = 0.73−1.78 µM) and inhibited catecholamine-O-methyl-transferase (COMT) (IC 50 1.32 µM) [10]. By acting as a positive allosteric modulator (PAM), it enhanced cerebellar α 6 GABA A receptor activity. Studies revealed some C6-substituted flavones are identified to be PAMs of GABA A receptors [16,17]. The results of a structure-activity relationship (SAR) study indicated the C6 substituent in flavones greatly contributes to activity [10].
Because of its interesting biological activity, several research groups have developed synthetic approaches to hispidulin [18][19][20][21][22][23]. For example, Shen and coworkers developed a strategy for semisynthesis of hispidulin in seven reaction steps by using a naturally occurring scutellarin (Scheme 1) [22]. Although this method is concise and has an overall yield as high as 10.7% [22], the researchers showed that, upon the large-scale synthesis of compound 1, the protection of the catechol moiety of scutellarein using dichlorodiphenylmethane at 175 • C failed [20,23]. A seven-step synthesis route developed by Lin and coworkers solved this problem but reduced the overall yield to 7.1% (Scheme 1) [20]. Zhang and coworkers then developed a scheme that only required four reaction steps (Scheme 1). Nonselective MOM (methoxymethyl) protection of scutellarein caused the overall yield of the synthesis of hispidulin to be decreased (6.3%) [23]. Despite the satisfactory overall yield of these strategies, the tedious purification procedure required to isolate scutellarin from plants limits their use for large-scale preparation of hispidulin.
Kavvadias and coworkers developed a method synthesizing hispidulin from 2,4,6-trihydroxyacetophenone compound 6 in nine reaction steps (Scheme 2). However, the overall yield of this method is very limited (1.1%) [18]. We recently developed a feasible and reproducible approach for synthesizing hispidulin (Scheme 3) [21]. This method slightly improved the overall yield due to the low yield of Friedel-Crafts acetylation of compound 8 as well as unsatisfactory regioselective MOM protection of compound 9. These facts motivated us to investigate an efficient and high-yield route to synthesize hispidulin. Deuterium is a stable isotope of hydrogen. Because deuterium has a stronger chemical bond with carbon than hydrogen, deuterium-labelled compounds can affect the absorption, distribution, metabolism and toxicology of their counterpart compound [24,25]. The Food and Drug Administration (FDA) recently approved the first deuterated drug, deutetrabenazine, for treating involuntary writhing movements or chorea in Huntington's disease [26]. Deuterium incorporation of tetrabenazine markedly increased its half-life and area under the curve (AUC) in plasma [27]. Study showed that the pig caecal microflora metabolized hispidulin to scutellarein due to O-demethylation at the C6 position [28]. This result suggested that replacement of OCH3 of this position of hispidulin using OCD3 may reduce its metabolic liability. Therefore, this study replaced C6-OCH3 in hispidulin with C6-OD3 to investigate whether such modification allows enhancement of the metabolic stability. Notably, introducing deuterium into hispidulin leads to a new chemical entity (NCE) that meets the criteria for a 505(b) (2) patent application [27].
The new hispidulin synthesis scheme developed in this study is more feasible compared to all methods previously reported. In particular, it had the highest overall yield, which may help to synthesize C6-OCH3-containing hispidulin derivatives. The same strategy can also be used to synthesize d-labelled hispidulin. The microsome stability of hispidulin and its deuterium counterpart were compared. Figure 2 shows the retrosynthetic analysis to successfully synthesize hispidulin. Hispidulin can be produced from compound I via debenzylation and oxidative cyclization. Compound I is derived from compound II and commercially available compound III, which are used to conduct Claisen-Schmidt condensation and deprotection of MOM (methoxymethoxy). Compound II in turn can be prepared from compound IV via methylation. Compound IV is considered to be the key intermediate that possesses two different protecting groups as well as acetyl and hydroxy moieties. Selective Bayer-Villiger reaction of compound V provided compound IV. Compound V is prepared from compound VI via Stille coupling. Benzylation and regioselective iodination of compound VII gives compound VI. Starting from commercially available compound 7, Compound VII is synthesized via selective MOM protection. Deuterium is a stable isotope of hydrogen. Because deuterium has a stronger chemical bond with carbon than hydrogen, deuterium-labelled compounds can affect the absorption, distribution, metabolism and toxicology of their counterpart compound [24,25]. The Food and Drug Administration (FDA) recently approved the first deuterated drug, deutetrabenazine, for treating involuntary writhing movements or chorea in Huntington's disease [26]. Deuterium incorporation of tetrabenazine markedly increased its half-life and area under the curve (AUC) in plasma [27]. Study showed that the pig caecal microflora metabolized hispidulin to scutellarein due to O-demethylation at the C6 position [28]. This result suggested that replacement of OCH 3 of this position of hispidulin using OCD 3 may reduce its metabolic liability. Therefore, this study replaced C6-OCH 3 in hispidulin with C6-OD 3 to investigate whether such modification allows enhancement of the metabolic stability. Notably, introducing deuterium into hispidulin leads to a new chemical entity (NCE) that meets the criteria for a 505(b) (2) patent application [27].

Retrosynthetic Analysis of Hispidulin
The new hispidulin synthesis scheme developed in this study is more feasible compared to all methods previously reported. In particular, it had the highest overall yield, which may help to synthesize C6-OCH 3 -containing hispidulin derivatives. The same strategy can also be used to synthesize d-labelled hispidulin. The microsome stability of hispidulin and its deuterium counterpart were compared. Figure 2 shows the retrosynthetic analysis to successfully synthesize hispidulin. Hispidulin can be produced from compound I via debenzylation and oxidative cyclization. Compound I is derived from compound II and commercially available compound III, which are used to conduct Claisen-Schmidt condensation and deprotection of MOM (methoxymethoxy). Compound II in turn can be prepared from compound IV via methylation. Compound IV is considered to be the key intermediate that possesses two different protecting groups as well as acetyl and hydroxy moieties. Selective Bayer-Villiger reaction of compound V provided compound IV. Compound V is prepared from compound VI via Stille coupling. Benzylation and regioselective iodination of compound VII gives compound VI. Starting from commercially available compound 7, Compound VII is synthesized via selective MOM protection.

Synthesis of Hispidulin
Scheme 4 shows a synthetic approach originally developed for hispidulin. Using compound 6 as the starting material, selective MOM protection towards its C4-and C6-OH gave compound 11. First, N-bromosuccinimide (NBS) was used for bromination of C3 of compound 11; however, only undesirable C3 and C5 dibromination occurred. Alternatively, compound 11, when reacted with I2 in the presence of Lewis acid CF3CO2Ag, provided selectively iodinated product 12 in the high yield (90%). The experimental result resulted from the difference of steric hindrance between C3 and C5 upon using the more bulky reagent I2. Benzylation of compound 12 generated compound 13. Next, several reported oxidation methods were used [29][30][31] in attempts to convert the iodide group of compound 13 into phenol. These oxidation catalysts included CuI, Cu and Pd, as shown in Table 1. Reaction using Pd2dba3 coupled with the ligand 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (tBuXPhos) gave compound 14, but its yield was poor (27%). Despite the use of microwave irradiation, the yields were incapable of being improved. Instead, Claisen-Schmidt condensation of compound 13, reacted with 4-(benzyloxy)benzaldehyde prior to selective deprotection of MOM group in the presence of HCl, gave chalcone 15. The selective deprotection reaction can be manipulated by decreasing the reaction time to avoid further MOM deprotection of C4. Oxidative cyclization of compound 15 in the presence of catalytic I2 provided flavone 16. Notably, excess I2 caused an undesirable retro-aldol reaction that gave C4-and C6-deprotected compound 13. Treatment of catalytic Pd(PPh3)4 and tributyl(1-ethoxyvinyl)tin converted compound 16 to compound 17 through the Stille reaction. Baeyer-Villiger oxidation followed by basic hydrolysis of compound 17 by using H2O2 or m-chloroperoxybenzoic acid (MCPBA) [32] failed to give expected product 18.

Synthesis of Hispidulin
Scheme 4 shows a synthetic approach originally developed for hispidulin. Using compound 6 as the starting material, selective MOM protection towards its C4-and C6-OH gave compound 11. First, N-bromosuccinimide (NBS) was used for bromination of C3 of compound 11; however, only undesirable C3 and C5 dibromination occurred. Alternatively, compound 11, when reacted with I 2 in the presence of Lewis acid CF 3 CO 2 Ag, provided selectively iodinated product 12 in the high yield (90%). The experimental result resulted from the difference of steric hindrance between C3 and C5 upon using the more bulky reagent I 2 . Benzylation of compound 12 generated compound 13. Next, several reported oxidation methods were used [29][30][31] in attempts to convert the iodide group of compound 13 into phenol. These oxidation catalysts included CuI, Cu and Pd, as shown in Table 1. Reaction using Pd 2 dba 3 coupled with the ligand 2-di-tert-butylphosphino-2 ,4 ,6 -triisopropylbiphenyl (tBuXPhos) gave compound 14, but its yield was poor (27%). Despite the use of microwave irradiation, the yields were incapable of being improved. Instead, Claisen-Schmidt condensation of compound 13, reacted with 4-(benzyloxy)benzaldehyde prior to selective deprotection of MOM group in the presence of HCl, gave chalcone 15. The selective deprotection reaction can be manipulated by decreasing the reaction time to avoid further MOM deprotection of C4. Oxidative cyclization of compound 15 in the presence of catalytic I 2 provided flavone 16. Notably, excess I 2 caused an undesirable retro-aldol reaction that gave C4-and C6-deprotected compound 13. Treatment of catalytic Pd(PPh 3 ) 4 and tributyl(1-ethoxyvinyl)tin converted compound 16 to compound 17 through the Stille reaction. Baeyer-Villiger oxidation followed by basic hydrolysis of compound 17 by using H 2 O 2 or m-chloroperoxybenzoic acid (MCPBA) [32] failed to give expected product 18.     Thus, we developed a new synthesis (Scheme 5) for hispidulin using the retrosynthetic analysis as shown in Figure 2. First, 2,4,6-trihydroxybenzaldehyde 7 reacted with MOMCl gave the bis(methoxymethoxy)-protected compound 20. Regioselective iodination of compound 20 prior to reaction with BnBr produced compound 21. The chemical structure of compound 21 was confirmed by rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectrum. Figure 3 Table 2 shows how tributyl(1-ethoxyvinyl)tin was used to optimize Stille coupling. First, catalyst Pd(PPh 3 ) 4 was used in dioxane at 100 • C. The reaction had a satisfactory yield (68%), but the reaction time was up to 30 h. Replacement of the solvent by toluene led to the reaction time decreasing to 24 h. Further experiments using palladium catalysts such as PdCl 2 (PPh 3 ) 2 and Pd(dppf)Cl 2 in dioxane or toluene showed that PdCl 2 (PPh 3 ) 2 significantly improved the yield and decreased the reaction time. In particular, PdCl 2 (PPh 3 ) 2 coupled with toluene not only gave the highest yield, but also had the lowest reaction time. Baeyer-Villiger oxidation and basic hydrolysis of compound 22 afforded compound 14. Methylation of compound 14 using CH 3 I gave compound 23a. Claisen-Schmidt condensation of compound 23a with 4-(benzyloxy)benzaldehyde prior to MOM deprotection produced chalcone 24a. Cyclization of compound 24a in the presence of catalytic I 2 generated flavone 25a. Although debenzylation of compound 25a catalyzed by 10% Pd-C did not yield hispidulin, a reaction using BCl 3 at −80 • C successfully converted compound 25a into hispidulin. The chemical structure of hispidulin was identified as judged by 2D-NMR analyses. Figure 4 shows the key correlation in the ROESY spectrum of hispidulin that 5-OH (δ H 13.07) was correlated to 6-OMe (δ H 3.74) and H-8 (δ H 6.59) was correlated to H-2 and H-6 (δ H 7.92). Additionally, the HMBC spectrum showed that 5-OH (δ H 13.07) correlated to C-5 (δ C 152.8), C-6 (δ C 131.4), C-7 (δ C 157.3), C-9 (δ C 152.4) and C-10 (δ C 104.1); H-8 (δ H 13.07) correlated to C-6 (δ C 131.4), C-7 (δ C 157.3), C-9 (δ C 152.4) and C-10 (δ C 104.1); 6-OMe-H (δ H 3.74) correlated to C-6 (δ C 131.4) (Supplementary Materials). The 1 H-and 13 C-NMR data of synthesized hispidulin were similar to those of hispidulin previously isolated (Supplementary Materials) [33].

Synthesis of d-Hispidulin
The synthesis of d-hispidulin is described in Scheme 5. Methylation of compound 14 using CD3I gave compound 23b. The same method to hispidulin was used to synthesize d-labelled hispidulin starting from compound 23b. Due to the absence of a proton signal of the CD3O group in the 1 H-NMR spectra of the d-containing intermediate compounds 23b, 24b, 25b and d-hispidulin, these compound structures were identified depending on the 13 C-NMR spectra without 1 H decoupling and the mass technique. The 13 C-NMR spectra revealed a characteristic multiplet splitting pattern of the 13 C signal for the CD3O group in compounds 23b, 24b, 25b and d-hispidulin. The mass spectra also supported chemical structures of these d-labelled compounds. All synthesized compounds had an estimated purity of at least 98% as determined by HPLC analysis (Supplementary Materials).

Comparison of Hispidulin Synthesis Methods
Strategies for synthesizing hispidulin can be classified as semisynthesis and total synthesis strategies. The starting material used in most semisynthetic methods is scutellarin, which is a natural product. Table 3 shows that the semisynthesis routes had fewer reaction steps compared to the total synthesis methods; however, they need tedious isolation procedures for scutellarin, which limits the scale for further chemical modification. Furthermore, their overall yields are only 6.3-10.7% [20,22,23]. For total synthesis, Kavvadias and coworkers developed a nine-step synthesis approach. The starting material used in this method is commercially available 2,4,6-trihydroxyacetophenone. Although this method solves the issue of the source for starting material, its drawback is low overall yield [18]. We previously developed a feasible route of hispidulin synthesis that has an overall yield comparable to that of the method developed by Kavvadias and coworkers [21]. This present study

Synthesis of d-Hispidulin
The synthesis of d-hispidulin is described in Scheme 5. Methylation of compound 14 using CD3I gave compound 23b. The same method to hispidulin was used to synthesize d-labelled hispidulin starting from compound 23b. Due to the absence of a proton signal of the CD3O group in the 1 H-NMR spectra of the d-containing intermediate compounds 23b, 24b, 25b and d-hispidulin, these compound structures were identified depending on the 13 C-NMR spectra without 1 H decoupling and the mass technique. The 13 C-NMR spectra revealed a characteristic multiplet splitting pattern of the 13 C signal for the CD3O group in compounds 23b, 24b, 25b and d-hispidulin. The mass spectra also supported chemical structures of these d-labelled compounds. All synthesized compounds had an estimated purity of at least 98% as determined by HPLC analysis (Supplementary Materials).

Comparison of Hispidulin Synthesis Methods
Strategies for synthesizing hispidulin can be classified as semisynthesis and total synthesis strategies. The starting material used in most semisynthetic methods is scutellarin, which is a natural product. Table 3 shows that the semisynthesis routes had fewer reaction steps compared to the total synthesis methods; however, they need tedious isolation procedures for scutellarin, which limits the scale for further chemical modification. Furthermore, their overall yields are only 6.3-10.7% [20,22,23]. For total synthesis, Kavvadias and coworkers developed a nine-step synthesis approach. The starting material used in this method is commercially available 2,4,6-trihydroxyacetophenone. Although this method solves the issue of the source for starting material, its drawback is low overall yield [18]. We previously developed a feasible route of hispidulin synthesis that has an overall yield comparable to that of the method developed by Kavvadias and coworkers [21]. This present study

Synthesis of d-Hispidulin
The synthesis of d-hispidulin is described in Scheme 5. Methylation of compound 14 using CD 3 I gave compound 23b. The same method to hispidulin was used to synthesize d-labelled hispidulin starting from compound 23b. Due to the absence of a proton signal of the CD 3 O group in the 1 H-NMR spectra of the d-containing intermediate compounds 23b, 24b, 25b and d-hispidulin, these compound structures were identified depending on the 13 C-NMR spectra without 1 H decoupling and the mass technique. The 13 C-NMR spectra revealed a characteristic multiplet splitting pattern of the 13 C signal for the CD 3 O group in compounds 23b, 24b, 25b and d-hispidulin. The mass spectra also supported chemical structures of these d-labelled compounds. All synthesized compounds had an estimated purity of at least 98% as determined by HPLC analysis (Supplementary Materials).

Comparison of Hispidulin Synthesis Methods
Strategies for synthesizing hispidulin can be classified as semisynthesis and total synthesis strategies. The starting material used in most semisynthetic methods is scutellarin, which is a natural product. Table 3 shows that the semisynthesis routes had fewer reaction steps compared to the total synthesis methods; however, they need tedious isolation procedures for scutellarin, which limits the scale for further chemical modification. Furthermore, their overall yields are only 6.3-10.7% [20,22,23]. For total synthesis, Kavvadias and coworkers developed a nine-step synthesis approach. The starting material used in this method is commercially available 2,4,6-trihydroxyacetophenone. Although this method solves the issue of the source for starting material, its drawback is low overall yield [18]. We previously developed a feasible route of hispidulin synthesis that has an overall yield comparable to that of the method developed by Kavvadias and coworkers [21]. This present study further made the reaction steps more concise. In particular, the synthetic scheme showed the highest overall yield of all approaches to synthesize hispidulin.

Human Liver Microsome Stability
Metabolic stability is associated with susceptibility of compounds to biotransformation. Metabolic half-life (t 1/2 ) and intrinsic clearance (CL int ) was compared between hispidulin and d-hispidulin by testing these synthesized compounds in a human liver microsome stability assay. The study revealed that FDA-approved deuterated agent deutetrabenazine had a t 1/2 (8.6 h) superior to tetrabenazine (4.8 h). In addition to t 1/2 , the AUC of deutetrabenazine (542 ng·hr/mg) was also higher than that of its counterpart compound (261 ng·hr/mg) [27]. In contrast, the experimental results indicated hispidulin and d-hispidulin had no significant difference in t 1/2 and CL int (Table 4), which suggested that the C6-OMe of hispidulin is resistant to be modified by the human liver microsome. The metabolic site of hispidulin in the human liver microsome is worthy of further study. The experiments showed that this method of synthesizing hispidulin and its d-labelled derivative is highly feasible. Specifically, it increases overall yield compared to previous methods. The t 1/2 and intrinsic clearance of these two compounds were identified as well. Overall, this synthetic route can be applied to produce 6-OMe-containing hispidulin derivatives as new chemical entities for investigating their biological activities.
Supplementary Materials: The following are available online. 1 H-and 13 C-NMR spectra and HPLC chromatogram of all compounds synthesized; 1 H-and 13 C-NMR spectra comparison of experimental and reported hispidulin; ROESY spectra of compound 21; and ROESY, HMQC and HMBC spectra of final products hispidulin and d-hispidulin.