Asymmetric Synthesis of Photophore-Containing Lactisole Derivatives to Elucidate Sweet Taste Receptors

Lactisole, which has a 2-phenoxy propionic acid skeleton, is well-known as an inhibitor of sweet taste receptors. We recently revealed some of the structure–activity relationships of the aromatic ring and chiral center of lactisole. Photoaffinity labeling is one of the common chemical biology methods to elucidate the interaction between bioactive compounds and biomolecules. In this paper, the novel asymmetric synthesis of lactisole derivatives with common photophores (benzophenone, azide and trifluoromethyldiazirine) for photoaffinity labeling is described. The synthetic compounds are subjected to cell-based sweet taste receptors, and the substitution with trifluoromethyldiazirinyl photophore shows the highest affinity to the receptor of the synthesized compounds.

Photoaffinity labeling [7][8][9][10] is a useful biochemical method for the analysis of biological interactions between low-molecular-weight bioactive compounds and biomolecules. The methodology may afford other information near the binding sites which cannot be obtained from structure-activity relationship studies. Three major photophores-phenylazide, benzophenone and (trifluoromethyl)phenyldiazirine-are used for photoaffinity labeling [11]. We reported several preparations of gustatory ligands to elucidate gustatory receptors [12][13][14][15][16][17][18][19][20]; however, there are few studies on the synthesis of photoreactive lactisole derivatives which have aimed to elucidate their biological activities. In this paper, we describe the comprehensive synthesis of photoreactive lactisole derivatives (Figure 1), which we then used in an inhibitory activity assay for cell-based human sweet taste receptors.

Synthesis
The key steps to construct a lactisole skeleton in an asymmetric manner are achieved by the Mitsunobu reaction for optically pure methyl lactate and phenol derivatives of photophore precursors. Details for each photophore are described below.

Benzophenone-Based Lactisole Derivatives
A few reports have described the preparations of racemic benzophenone-based lactisole derivatives and their separation by chromatography [21][22][23][24][25]. However, to date, the asymmetric synthesis of benzophenone-modified lactisole derivatives has not been reported. Hydroxybenzophenone derivatives (1)(2)(3), optically pure methyl lactate (4 and 5, 1.5 equiv.) and triphenylphosphine (1.2 equiv.) were preincubated at 0 °C for 10 min in dichloromethane and diethyl azodicarboxylate (1.5 equiv.) was slowly added to the solution. The reaction mixture was stirred at room temperature overnight and worked up with a general procedure. Purification with column chromatography afforded the optically pure methyl 2-benzoylphenoxypropionate derivatives 6-8 with a good yield. The methyl esters were subjected to chiral high-performance liquid chromatography (HPLC) with Chiralpak IG [2] to ensure the configuration of the chiral center. The methyl esters were hydrolyzed under reflux in the presence of K2CO3 to afford derivatives 9-13 in high yields ( Figure 2).

Azide-Based Lactisole Derivatives
An example of the synthesis of racemic azide-based lactisole ethyl ester has been reported previously [26], but to date, the asymmetric synthesis of azide modified on the aromatic ring of the lactisole skeleton has not been reported. Akazome et al. reported the asymmetric synthesis of (R)-2nitro (R)-23 [27] and (R)-3-nitro (R)-15 [28,29]-substituted lactisole methyl ester. Our retrosynthesis

Synthesis
The key steps to construct a lactisole skeleton in an asymmetric manner are achieved by the Mitsunobu reaction for optically pure methyl lactate and phenol derivatives of photophore precursors. Details for each photophore are described below.

Benzophenone-Based Lactisole Derivatives
A few reports have described the preparations of racemic benzophenone-based lactisole derivatives and their separation by chromatography [21][22][23][24][25]. However, to date, the asymmetric synthesis of benzophenone-modified lactisole derivatives has not been reported. Hydroxybenzophenone derivatives (1)(2)(3), optically pure methyl lactate (4 and 5, 1.5 equiv.) and triphenylphosphine (1.2 equiv.) were preincubated at 0 • C for 10 min in dichloromethane and diethyl azodicarboxylate (1.5 equiv.) was slowly added to the solution. The reaction mixture was stirred at room temperature overnight and worked up with a general procedure. Purification with column chromatography afforded the optically pure methyl 2-benzoylphenoxypropionate derivatives 6-8 with a good yield. The methyl esters were subjected to chiral high-performance liquid chromatography (HPLC) with Chiralpak IG [2] to ensure the configuration of the chiral center. The methyl esters were hydrolyzed under reflux in the presence of K 2 CO 3 to afford derivatives 9-13 in high yields ( Figure 2).

Synthesis
The key steps to construct a lactisole skeleton in an asymmetric manner are achieved by the Mitsunobu reaction for optically pure methyl lactate and phenol derivatives of photophore precursors. Details for each photophore are described below.

Benzophenone-Based Lactisole Derivatives
A few reports have described the preparations of racemic benzophenone-based lactisole derivatives and their separation by chromatography [21][22][23][24][25]. However, to date, the asymmetric synthesis of benzophenone-modified lactisole derivatives has not been reported. Hydroxybenzophenone derivatives (1)(2)(3), optically pure methyl lactate (4 and 5, 1.5 equiv.) and triphenylphosphine (1.2 equiv.) were preincubated at 0 °C for 10 min in dichloromethane and diethyl azodicarboxylate (1.5 equiv.) was slowly added to the solution. The reaction mixture was stirred at room temperature overnight and worked up with a general procedure. Purification with column chromatography afforded the optically pure methyl 2-benzoylphenoxypropionate derivatives 6-8 with a good yield. The methyl esters were subjected to chiral high-performance liquid chromatography (HPLC) with Chiralpak IG [2] to ensure the configuration of the chiral center. The methyl esters were hydrolyzed under reflux in the presence of K2CO3 to afford derivatives 9-13 in high yields ( Figure 2). Then, 2-Nitrophenol 22 was also subjected to the Mitsunobu reaction in an identical manner with another isomer to make nitro methyl lactate skeleton 23. The hydrogenation of the nitro group promoted subsequent intramolecular cyclization between the amino and methyl ester to afford 2methyl-2H-benzo[b] [1,4]oxazin-3(4H)-one as the sole product [27]. To prevent intramolecular cyclization, the methyl ester hydrolyzed 24, then converted to tert-butyl ester 25. The hydrogenation of nitro group of 25 proceeded smoothly to isolate aniline derivative 26 within an hour. Although tert-butyl ester was selected, intramolecular cyclization between the aniline and ester proceeded with a longer reaction time (>4 h). Compound 26 was subjected to diazotization followed by azidation to construct phenylazide moiety 27, which was checked by chiral center configuration.
The deprotection of tert-butyl group of 27 with trifluoroacetic acid in dichloromethane afforded a complex mixture because the azide group was not stable under this condition. Mild acidic deprotection with 4 M HCl in dioxane was acceptable, but the partial decomposition of starting material 27 was observed over an hour. The starting material 27 could be recovered by partition within 1 h, and the hydrolysis was repeated for the recovered 27 three times to afford compound 28 in a moderate yield ( Figure 4). The reaction with 2-nitrophenol 22 and optical pure tert-butyl lactate was also conducted to construct compound 25 directly, but unfortunately, the Mitsunobu reaction did not proceed for the tert-butyl lactate. Then, 2-Nitrophenol 22 was also subjected to the Mitsunobu reaction in an identical manner with another isomer to make nitro methyl lactate skeleton 23. The hydrogenation of the nitro group promoted subsequent intramolecular cyclization between the amino and methyl ester to afford 2-methyl-2H-benzo[b] [1,4]oxazin-3(4H)-one as the sole product [27]. To prevent intramolecular cyclization, the methyl ester hydrolyzed 24, then converted to tert-butyl ester 25. The hydrogenation of nitro group of 25 proceeded smoothly to isolate aniline derivative 26 within an hour. Although tert-butyl ester was selected, intramolecular cyclization between the aniline and ester proceeded with a longer reaction time (>4 h). Compound 26 was subjected to diazotization followed by azidation to construct phenylazide moiety 27, which was checked by chiral center configuration.
The deprotection of tert-butyl group of 27 with trifluoroacetic acid in dichloromethane afforded a complex mixture because the azide group was not stable under this condition. Mild acidic deprotection with 4 M HCl in dioxane was acceptable, but the partial decomposition of starting material 27 was observed over an hour. The starting material 27 could be recovered by partition within 1 h, and the hydrolysis was repeated for the recovered 27 three times to afford compound 28 in a moderate yield ( Figure 4). The reaction with 2-nitrophenol 22 and optical pure tert-butyl lactate was also conducted to construct compound 25 directly, but unfortunately, the Mitsunobu reaction did not proceed for the tert-butyl lactate.

Trifluotromethyldiazirine-Based Lactisole Derivatives
To date, trifluoromethyldiazirine-based lactisole derivatives have not been reported. The three-membered azi (N=N) partial structure of trifluoromethyldiazirine is not stable for the Mitsunobu condition because the reactant, diethyl azodicarboxylate DEAD also has an azo group in its structure. The reaction with diazirinyl phenol derivatives and 1 -hydroxy peracetylsucrose with DEAD or Tsuda reagents did not occur in our previous study [14]. The synthetic plan was based on the preparation of trifluoroacetyl modified on the aromatic ring of lactisole; then we constructed a diazirinyl three-membered ring on the trifluoroacetyl group. The reagent of liquid ammonia is essential to the construction of the Molecules 2020, 25, 2790 4 of 25 diazirine moiety, but the reagent also reacts with methyl ester to form amide. tert-Butyl ester was utilized for this purpose. p-Trifluoroacetyl anisole and m-trifluoroacetyl anisole 29 and 30 were selected as the starting material for 4 and 3-substituted lactisoles. Compound 29 was treated with lithium chloride under a reflux condition to obtain trifluoroacetyl phenol 31 [30,31]. m-Trifluoroacetyl phenol 32 was synthesized with a boron tribromide treatment of 30 at room temperature [32]. Mitsunobu reactions for these phenols with chiral methyl lactate were archived in an identical manner to construct lactisole methyl ester skeletons 33 and 34. After the hydrolysis of methyl esters with K 2 CO 3 under the reflux condition, corresponding trifluoroacetyl-substituted carboxylic acids 35 and 36 were subjected to diazirine formation. However, it was difficult to construct diazirine moiety for 35 and 36 directly. The carboxylic acids were converted to tert-butyl esters with tert-butyl bromide in the presence of K 2 CO 3 and tetrabutylammonium bromide (TBAB) under reflux conditions. Although the reactions were not completed within 5 h, the decompositions of the diazirinyl moiety of the starting material were observed over 5 h. tert-Butyl derivatives 37 and 38 were obtained with moderate yields and the starting materials could be recovered from the reaction mixture ( Figure 5). The tert-butyl thioester formations [33] were also conducted for the carboxylic acids to improve the chemical yields of the protection. The chemical yields were almost identical with tert-butyl ester, but the recovery of the starting material was very difficult from the reaction mixture. Mitsunobu reactions for trifluoroacetyl phenol 31 and 32 with chiral tert-butyl lactate were also conducted, but no reactions were observed. Then, 2-Nitrophenol 22 was also subjected to the Mitsunobu reaction in an identical manner with another isomer to make nitro methyl lactate skeleton 23. The hydrogenation of the nitro group promoted subsequent intramolecular cyclization between the amino and methyl ester to afford 2methyl-2H-benzo[b] [1,4]oxazin-3(4H)-one as the sole product [27]. To prevent intramolecular cyclization, the methyl ester hydrolyzed 24, then converted to tert-butyl ester 25. The hydrogenation of nitro group of 25 proceeded smoothly to isolate aniline derivative 26 within an hour. Although tert-butyl ester was selected, intramolecular cyclization between the aniline and ester proceeded with a longer reaction time (>4 h). Compound 26 was subjected to diazotization followed by azidation to construct phenylazide moiety 27, which was checked by chiral center configuration.
The deprotection of tert-butyl group of 27 with trifluoroacetic acid in dichloromethane afforded a complex mixture because the azide group was not stable under this condition. Mild acidic deprotection with 4 M HCl in dioxane was acceptable, but the partial decomposition of starting material 27 was observed over an hour. The starting material 27 could be recovered by partition within 1 h, and the hydrolysis was repeated for the recovered 27 three times to afford compound 28 in a moderate yield ( Figure 4). The reaction with 2-nitrophenol 22 and optical pure tert-butyl lactate was also conducted to construct compound 25 directly, but unfortunately, the Mitsunobu reaction did not proceed for the tert-butyl lactate.   Then, 2-trifluoroactyl phenol, which was prepared by the Fries rearrangement of phenyl 2,2,2-trifluoroacetate with AlCl 3 [34], was subjected to Mitsunobu reaction with chiral methyl lactate 4 and did not afford a 2-trifluoroacetyl lactisole skeleton. The Mitsunobu reaction of salicylaldehyde and methyl lactate 4 followed by the conversion of aldehyde to the trifluoroacetyl group also failed due to the instability of methyl ester for the condition of trifluoroacetyl construction. Protected salicylaldehyde was selected for trifluoroacetyl substitution at the 2-position of lactisole. The carbonyl group was protected by thioacetal 40 [35], then subjected to a Mitsunobu reaction with chiral methyl lactate 4 and 5 to construct lactisole methyl ester 41 in an identical manner to that described above. After the hydrolysis of methyl esters with methanolic NaOH under the reflux condition, carboxylic acid 42 was converted to lactisole tert-butyl ester 43, then thioacetal was deprotected with methyl iodide. Aldehyde 44 was treated with TMS-CF 3 followed by oxidation with Dess-Martin periodinane [20] to afford 2-trifluoroacetyl-substituted lactisole tert-butyl ester 45 ( Figure 6).   Each trifluoroacetyl-substituted lactisole tert-butyl esters (37, 38, 45) was subjected to diazirine constructions, oximation with hydroxylamine hydrochloride 46-48, tosylation for hydroxyl group of oxime 49-51, diaziridine formation with liquid ammonia 52-54, and oxidation with activated MnO2 to obtain 55-57, which were checked by chiral center configuration. tert-Butyl esters were hydrolyzed with trifluoroacetic acid to afford trifluoromethyldiazirine-based lactisole derivatives in moderate yields. The one-pot conversion of the tosyl oxime 49 to diazirine 55 [36] was also acceptable for the preparation with an identical yield ( Figure 7). Each trifluoroacetyl-substituted lactisole tert-butyl esters (37, 38, 45) was subjected to diazirine constructions, oximation with hydroxylamine hydrochloride 46-48, tosylation for hydroxyl group of oxime 49-51, diaziridine formation with liquid ammonia 52-54, and oxidation with activated MnO 2 to obtain 55-57, which were checked by chiral center configuration. tert-Butyl esters were hydrolyzed with trifluoroacetic acid to afford trifluoromethyldiazirine-based lactisole derivatives in moderate yields. The one-pot conversion of the tosyl oxime 49 to diazirine 55 [36] was also acceptable for the preparation with an identical yield ( Figure 7).

Cell-Based Sweet Taste Assay
The synthesized photoreactive lactisole derivatives were administrated to the Flp-In 293 cell line and were expressed as inhibitors to the human sweet taste receptors, with less than 3.2 mM comprising the final concentration. (S)-configurations of all photoreactive lactisole derivatives were preferred for sweet taste inhibition, and the results were consistent with our previous studies for lactisole and 2,4-dichlorophenoxy propanoic acid (2,4-DP) [5]. Benzophenone photophore (9-11) is too bulky to substitute for sweet taste receptors. Azide-substituted lactisoles (20, 21 and 28), which feature the linear linkage of three nitrogen atoms, had slightly higher affinity for the receptor than benzophenone derivatives. Trifluoromethyldiazirinyl substitution (58-60) had the highest affinity for sweet taste receptors among the photoreactive lactisole derivatives. The positions of photophores on aromatic rings are also influenced the activity, and the 4-position was preferred as a substitute. Fifty percent inhibitory concentration (IC 50 ) values (µM) for (S)-58 and (S)-59 are almost identical with that of (S)-lactisole ( constructions, oximation with hydroxylamine hydrochloride 46-48, tosylation for hydroxyl group of oxime 49-51, diaziridine formation with liquid ammonia 52-54, and oxidation with activated MnO2 to obtain 55-57, which were checked by chiral center configuration. tert-Butyl esters were hydrolyzed with trifluoroacetic acid to afford trifluoromethyldiazirine-based lactisole derivatives in moderate yields. The one-pot conversion of the tosyl oxime 49 to diazirine 55 [36] was also acceptable for the preparation with an identical yield ( Figure 7).

Discussion
The 2-phenoxypropanoic acid skeleton has a unique bioactivity and is known as a pesticide and sweetener inhibitor. It seems that the stereochemistry at the 2-position may play an important role in its biological activity. We reported a comparison of the biological activity of optically pure lactisole and 2,4-dichlorophenoxy propanoic acid, which were synthesized in an asymmetric manner. Photoaffinity labeling is one of the most reliable methods for functional analysis between small ligands and biomolecules. It is essential to prepare photophore-substituted ligand derivatives. In this paper, we achieved the asymmetric synthesis of benzophenone, azide and trifluoromethyldiazirine modified derivatives on the aromatic ring of lactisole. The Mitsunobu reaction was utilized to construct a lactisole skeleton in an asymmetric manner. Although benzophenone skeletons were acceptable for the Mitsunobu reaction conditions, azide and trifluoromethyldiazirine moieties are not suitable for the condition because of the nitrogen-nitrogen multiple bonds in their structures. The phenol derivatives of precursors for these photophores were utilized for the asymmetric synthesis of the lactisole skeleton, followed by the construction of photophores on these derivatives. Several instabilities of the intermediates to the reaction condition were found, which were overcome with improvements of the reaction condition or protecting groups. Human sweet receptor assays for the photophore-containing lactisole derivatives revealed that (S)-isomers at the 4-position of lactisole derivatives are important and that the trifluoromethyldiazirine-substituted derivatives have identical affinity to optically pure lactisole. These results indicated that photoaffinity labeling with synthetic lactisole derivatives will be useful for the functional analysis of sweet taste receptors.

(S)-2-(4-Azidophenoxy)propanoic acid ((S)-20). Methyl (S)-2-(4-azidophenoxy)propanoate (S)-18
(702 mg, 3.17 mmol) was dissolved in MeOH (15 mL) and 2M NaOH (3.2 mL). After the reaction mixture was stirred at reflux for 2 h, cooled to room temperature and then partitioned between ethyl acetate and water. The water layer was acidified by 1 M HCl aq and extracted by ethyl acetate. The organic layer was washed by H 2 O and brine, and dried over MgSO 4 , filtrated and concentrated to give (S)-20 (601 mg, 91%). 15 mmol) was dissolved in CH 2 Cl 2 (30 mL) and cooled to −78 • C. BBr 3 was added dropwise at −78 • C and the reaction mixture stirred at room temperature for 5 h. The mixture was cooled to 0 • C and 10% NaOH (30 mL) was added slowly, and then HCl was added to make pH 1 at same temperature. NH 4 OH was also added to make pH 7 at room temperature, then extracted by ethyl acetate. The organic layer was dried over MgSO 4 , filtrated and concentrated to give 31 (1.018 g, quant). 1