CF3-Substituted Mollugin 2-(4-Morpholinyl)-ethyl ester as a Potential Anti-inflammatory Agent with Improved Aqueous Solubility and Metabolic Stability

Although mollugin, the main ingredient of the oriental medicinal herb Rubia cordifolia, has considerable anti-inflammatory effects, it has poor aqueous solubility as well as poor metabolic and plasma stability. To overcome these shortfalls, various mollugin derivatives have been synthesized and evaluated for their ability to inhibit U937 monocyte cell adhesion to HT-29 colonic epithelial cells in TNF-α- or IL-6-induced models of colon inflammation. The 2-(4-morpholinyl)-ethyl ester of CF3-substituted mollugin (compound 15c) showed good water solubility, improved metabolic and plasma stability, and greater inhibitory activity than mesalazine in both the TNF-α- and IL-6-induced colonic epithelial cell adhesion assays, suggesting that 15c is a potential anti-inflammatory agent.


Introduction
Rubia cordifolia is a flowering plant in the coffee family (Rubiaceae), widely distributed from Africa to Asia and Australia [1]. Its roots have been used as a traditional medicine in India [2] and China [3] for their anti-inflammatory, astringent, tonic, antiseptic, deobstruent, and antidysenteric effects [4,5]. Mollugin (1), a methyl ester derivative of naphthoquinone (Figure 1), has been identified as a major active component of the roots of Rubia cordifolia [6]. Recent pharmacological data show that mollugin (1) manifests a wide range of biologically interesting properties, such as antibacterial, anti-inflammatory, antileukemia, and anti-allergic activities [7]. In particular, mollugin (1) displays efficacy against inflammatory bowel disease (IBD) in both a TNF-α-induced colon inflammation cell culture model and a DSS/TNBS-induced IBD animal model [8]. In the IBD study, mollugin inhibited NF-κB activation [9] and blocked the JAK-STAT signaling pathway [5]. As these results became well known, the naphthoquinone scaffold of mollugin was spotlighted. Diverse analogues [7,10] such as oxomollugin [11] and azamollugin [12] were synthesized to explore their anti-inflammatory, antioxidant, antibacterial, and anticancer activities. Regarding IBD medicines on the market, only a few synthetic anti-inflammatory drugs, such as mesalazine (2) [13] and sulfasalazine (3) [14] (Figure 1), are used in mild to moderate cases. However, those 4-aminosalicylic acid-type drugs have several drawbacks, such as the need for high dosages (3 g/day), low efficacy, and various adverse effects [13]. Because alternative medications and treatments such as corticosteroid therapy or the immunosuppressive agent infliximab [15] are also of limited use due to various serious adverse effects and treatment costs, an urgent and medically unmet need exists for the development of new oral drugs with improved efficacy and lower toxicity in comparison with the current drug mesalazine. Because mollugin had at least 1000-fold improved efficacy over mesalazine in the cell adhesion assay and in an IBD animal model [8], our previous efforts focused on development of mollugin as a therapeutic agent for patients with IBD. However, further study with mollugin was limited due to uneven assay results caused by its poor aqueous solubility as well as metabolic instability.
As a part of our ongoing research on the development of IBD treatments using mollugin, we have modified the methyl ester of mollugin to various aminoalkyl esters to increase its water solubility [16] via formation of an ammonium salt. We also added F, CH3, and CF3 groups to the naphthalene ring of mollugin to increase its metabolic stability. Herein, we report the synthesis of various mollugin esters and substituted mollugins and their water solubility, anti-IBD effects in the colonic epithelial cell adhesion assay, metabolic stability, and plasma stability.

Chemistry
The general procedure for the synthesis of mollugin derivatives 6a-o is depicted in Scheme 1. Scheme 1. Synthesis of various mollugin derivatives. Reagents and conditions: (a) Me2SO4 (1.05 eq.), NaHCO3 (2.0 eq.), DMF, r.t., 5 h, 96%; (b) 3-methylbut-2-enal (2.2 eq.), PhB(OH)2 (1.1 eq.), AcOH, toluene, reflux, 20 h, 67%; (c) alcohol or amine TsOH (5%), microwave, 160 °C, 3 h, 8-52%. Regarding IBD medicines on the market, only a few synthetic anti-inflammatory drugs, such as mesalazine (2) [13] and sulfasalazine (3) [14] (Figure 1), are used in mild to moderate cases. However, those 4-aminosalicylic acid-type drugs have several drawbacks, such as the need for high dosages (3 g/day), low efficacy, and various adverse effects [13]. Because alternative medications and treatments such as corticosteroid therapy or the immunosuppressive agent infliximab [15] are also of limited use due to various serious adverse effects and treatment costs, an urgent and medically unmet need exists for the development of new oral drugs with improved efficacy and lower toxicity in comparison with the current drug mesalazine. Because mollugin had at least 1000-fold improved efficacy over mesalazine in the cell adhesion assay and in an IBD animal model [8], our previous efforts focused on development of mollugin as a therapeutic agent for patients with IBD. However, further study with mollugin was limited due to uneven assay results caused by its poor aqueous solubility as well as metabolic instability.
As a part of our ongoing research on the development of IBD treatments using mollugin, we have modified the methyl ester of mollugin to various aminoalkyl esters to increase its water solubility [16] via formation of an ammonium salt. We also added F, CH 3 , and CF 3 groups to the naphthalene ring of mollugin to increase its metabolic stability. Herein, we report the synthesis of various mollugin esters and substituted mollugins and their water solubility, anti-IBD effects in the colonic epithelial cell adhesion assay, metabolic stability, and plasma stability.

Chemistry
The general procedure for the synthesis of mollugin derivatives 6a-o is depicted in Scheme 1. oxomollugin [11] and azamollugin [12] were synthesized to explore their anti-inflammatory, antioxidant, antibacterial, and anticancer activities. Regarding IBD medicines on the market, only a few synthetic anti-inflammatory drugs, such as mesalazine (2) [13] and sulfasalazine (3) [14] (Figure 1), are used in mild to moderate cases. However, those 4-aminosalicylic acid-type drugs have several drawbacks, such as the need for high dosages (3 g/day), low efficacy, and various adverse effects [13]. Because alternative medications and treatments such as corticosteroid therapy or the immunosuppressive agent infliximab [15] are also of limited use due to various serious adverse effects and treatment costs, an urgent and medically unmet need exists for the development of new oral drugs with improved efficacy and lower toxicity in comparison with the current drug mesalazine. Because mollugin had at least 1000-fold improved efficacy over mesalazine in the cell adhesion assay and in an IBD animal model [8], our previous efforts focused on development of mollugin as a therapeutic agent for patients with IBD. However, further study with mollugin was limited due to uneven assay results caused by its poor aqueous solubility as well as metabolic instability.
As a part of our ongoing research on the development of IBD treatments using mollugin, we have modified the methyl ester of mollugin to various aminoalkyl esters to increase its water solubility [16] via formation of an ammonium salt. We also added F, CH3, and CF3 groups to the naphthalene ring of mollugin to increase its metabolic stability. Herein, we report the synthesis of various mollugin esters and substituted mollugins and their water solubility, anti-IBD effects in the colonic epithelial cell adhesion assay, metabolic stability, and plasma stability.
Mollugin (1) was prepared from commercially available 1,4-dihydroxy-2-naphthoic acid (4) by known methodology [17][18][19][20]. Microwave-assisted transesterification of methyl ester of mollugin with various alcohols and catalytic p-toluenesulfonic acid gave target products 6a-o. Although the product yields at fixed reaction times in the microwave reactor were moderate to low, other side products were not formed and the mollugin starting material could be recovered after the reaction.
The synthetic procedure for mollugin derivatives 15a-c is outlined in Scheme 2. Intermediates 8a-c, obtained by the Wittig olefination between F-, CH 3 -, and CF 3 -substituted aldehydes and 4-methylstyrene, were treated with methyl 2-bromoacetate under zinc-mediated coupling conditions to yield alcohols 9a-c at 79-82% yields. Diketones 10a-c, which were prepared from DMP oxidation of secondary alcohol in 9a-c, were subjected to a Pd-catalyzed cyclization reaction to yield naphthols 11a-c at 90-95% yields. Hydrolysis and potassium persulfate-mediated oxidation and esterification yielded the mollugin core (1,4-dihydroxynaphthalene) 13a-c at 80-82% yields. The cyclized adducts 14a-c were obtained by a known method [17,18], and final direct esterification was performed by microwave irradiation to yield substituted mollugins 15a-c at 40-45% yields. Mollugin (1) was prepared from commercially available 1,4-dihydroxy-2-naphthoic acid (4) by known methodology [17][18][19][20]. Microwave-assisted transesterification of methyl ester of mollugin with various alcohols and catalytic p-toluenesulfonic acid gave target products 6a-o. Although the product yields at fixed reaction times in the microwave reactor were moderate to low, other side products were not formed and the mollugin starting material could be recovered after the reaction.
Alternatively, compounds 15a,d were prepared from 16a,b via Hauser annulation [21] (Scheme 3). The benzoic acids 16a-b were converted to diethylamide 17a-b via acyl halide formation, followed by reaction with diethylamine. Lithiation of 17a-b, which was followed by quenching with anhydrous DMF, provided aldehyde 18a-b in good yields. Subsequent treatment of formylbenzamide 18a-b with trimethylsilyl cyanide in the presence of KCN and 18-crown-6 gave the corresponding key intermediates 19a-b [22]. Hauser annulation of cyanophthalide 19a-b with methyl acrylate and LiOtBu yielded the fluorinated 1,4-naphthoquinol 13a,d, which further underwent cyclization and transesterification to yield 15a,d.

Biological Activity
Synthesized mollugin derivatives were subjected to the colonic epithelial cell adhesion assay [8], which mimics the initial stage of colon inflammation. The inhibition of TNF-α-induced or IL-6induced adhesion of U937 monocytic cells to HT-29 cells in 10 μM solution of synthesized molecules was measured, and the results are summarized in Table 1 and Table 2. Mesalazine (2), a commercially available medication for the treatment of IBD, and mollugin were chosen as assay positive controls. As shown in Table 1, mesalazine displayed ~30% inhibition of U937 adhesion to HT-29 cells at a 20 mM concentration. Surprisingly, at 2000-fold dilution (10 μM), monocyte cell adhesion to HT-29 was reduced to 40% when the cells were treated with mollugin or its various ester derivatives. In particular, 6a-b, 6d-g, 6i-k, and 6n significantly inhibited both TNF-α-and IL-6-induced monocyteepithelial adhesion. Although mollugin N-ethyl-pyrrolidine (6c), N-ethyl-piperazine esters (6h), and various amides (6l-m, 6o) showed good % inhibition in the TNF-α-induced adhesion model, they displayed reduced % inhibition in the IL-6-induced adhesion model.
Compounds 6a-j and 6n-o have a tertiary amine in their side chains; they showed good water solubility (>10 mM). Thus, the substitution of the methyl ester of mollugin with various solubilizing aminoalkyl esters enhances the aqueous solubility of the mollugin without significant reduction of inhibition capability in the TNF-α-or IL-6-induced monocyte-epithelial adhesion assay.
Substitution of the methyl ester of mollugin with various aminoalkyl esters also resulted in improved plasma stability [23]; although mollugin, a methyl ester, was unstable in rat plasma, aminoalkyl esters of mollugin 6d-h displayed good stabilities in rat and human plasma. Among the water-soluble mollugin derivatives (6a-i and 6n-o), compounds 6h and 6i, which have a piperazine moiety at the ester, were excluded because their IC50s for hERG channel inhibition [24] were 3.3 μM and 5.3 μM, respectively. They may therefore have cardiovascular adverse effects. However, it was shown that mollugin itself and its ester or amide derivatives (6a-b, 6d-h, 6k-l) were generally unstable, especially with dog, rat, and mouse liver microsomes in the metabolic stability assay [25]. On the basis of those initial in vitro ADME studies, the 2-(4-morpholinyl)-ethyl group of 6d was chosen as the alternative to methyl ester in mollugin for water solubility and plasma stability, but 6d still needs further modification to improve its metabolic stability, especially in small animals.

Biological Activity
Synthesized mollugin derivatives were subjected to the colonic epithelial cell adhesion assay [8], which mimics the initial stage of colon inflammation. The inhibition of TNF-α-induced or IL-6-induced adhesion of U937 monocytic cells to HT-29 cells in 10 µM solution of synthesized molecules was measured, and the results are summarized in Tables 1 and 2. Mesalazine (2), a commercially available medication for the treatment of IBD, and mollugin were chosen as assay positive controls. As shown in Table 1, mesalazine displayed~30% inhibition of U937 adhesion to HT-29 cells at a 20 mM concentration. Surprisingly, at 2000-fold dilution (10 µM), monocyte cell adhesion to HT-29 was reduced to 40% when the cells were treated with mollugin or its various ester derivatives. In particular, 6a-b, 6d-g, 6i-k, and 6n significantly inhibited both TNF-α-and IL-6-induced monocyte-epithelial adhesion. Although mollugin N-ethyl-pyrrolidine (6c), N-ethyl-piperazine esters (6h), and various amides (6l-m, 6o) showed good % inhibition in the TNF-α-induced adhesion model, they displayed reduced % inhibition in the IL-6-induced adhesion model.
Compounds 6a-j and 6n-o have a tertiary amine in their side chains; they showed good water solubility (>10 mM). Thus, the substitution of the methyl ester of mollugin with various solubilizing aminoalkyl esters enhances the aqueous solubility of the mollugin without significant reduction of inhibition capability in the TNF-α-or IL-6-induced monocyte-epithelial adhesion assay.
Substitution of the methyl ester of mollugin with various aminoalkyl esters also resulted in improved plasma stability [23]; although mollugin, a methyl ester, was unstable in rat plasma, aminoalkyl esters of mollugin 6d-h displayed good stabilities in rat and human plasma. Among the water-soluble mollugin derivatives (6a-i and 6n-o), compounds 6h and 6i, which have a piperazine moiety at the ester, were excluded because their IC 50 s for hERG channel inhibition [24] were 3.3 µM and 5.3 µM, respectively. They may therefore have cardiovascular adverse effects. However, it was shown that mollugin itself and its ester or amide derivatives (6a-b, 6d-h, 6k-l) were generally unstable, especially with dog, rat, and mouse liver microsomes in the metabolic stability assay [25]. On the basis of those initial in vitro ADME studies, the 2-(4-morpholinyl)-ethyl group of 6d was chosen as the alternative to methyl ester in mollugin for water solubility and plasma stability, but 6d still needs further modification to improve its metabolic stability, especially in small animals. To improve metabolic stability by blocking the metabolic hydroxylation of the aromatic ring, F-, CH 3 -, or CF 3 -moieties were added to the naphthalene ring of mollugin [26]. The bioactivity, metabolic stability, and plasma stability of the F-, CH 3 -, or CF 3 -substituted molecules 14a-d and their 2-(4-morpholinyl)-ethyl esters 15a-d are summarized in Table 2. The bioactivity levels of 14a-d in the TNF-α-or IL-6-induced monocyte-epithelial adhesion assay were similar or slightly better than mollugin. The metabolic stability of those molecules with rat or mouse liver microsomes improved over that of mollugin. As it is known that stability in human plasma is usually greater than that in rodent plasma [27], 15a-d are very stable in human plasma but displayed slightly reduced stability in rat plasma. It is noteworthy that the 2-(4-morpholinyl)-ethyl ester derivatives 15a-d had good inhibitory activity in the IL-6-induced monocyte-epithelial adhesion assay. They displayed over 50% inhibition of U937 adhesion to HT-29 cells at a 10 µM concentration. Based on the in vitro data, compound 15c was chosen as a lead, since it displayed better bioactivity than mollugin with favorable metabolic and plasma stability as well as good water solubility (>10 mM). As shown in Table 3, the results of the CYP inhibition assay suggest that 15c has no significant interaction (<50% inhibition at 10 µM drug concentration) with five major isozymes of CYPs (CYP 3A4, 2D6, 2C9, 1A2, 2C19). An MTT cell viability assay was performed, and the results suggested that neither 6d nor 15c affects the cell viability at 10 µM concentration. Serum amyloid A (SAA), one of the most well-known acute-phase proteins in mice [28], is up-regulated in the presence of DSS or DNBS stimulation of IBD and is measured for monitoring IBD [29]. We confirmed that 6d or 15c treatment reduces serum SAA level in a murine model of DSS-induced colitis ( Figure 2). As shown in Table 3, the results of the CYP inhibition assay suggest that 15c has no significant interaction (<50% inhibition at 10 μM drug concentration) with five major isozymes of CYPs (CYP 3A4, 2D6, 2C9, 1A2, 2C19). An MTT cell viability assay was performed, and the results suggested that neither 6d nor 15c affects the cell viability at 10 μM concentration. Serum amyloid A (SAA), one of the most well-known acute-phase proteins in mice [28], is up-regulated in the presence of DSS or DNBS stimulation of IBD and is measured for monitoring IBD [29]. We confirmed that 6d or 15c treatment reduces serum SAA level in a murine model of DSS-induced colitis (Figure 2).

Synthetic Methods and Molecular Characterization
All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA), Alfa-Aesar (Ward Hill, MA, USA), and TCI (Nihonbashi, Japan) and were used without further purification. Reactions were performed under an atmosphere of dry nitrogen. An Initiator microwave system (Biotage, Uppsala, Sweden) was used for microwave-assisted reaction. LC-MS was performed on a system consisting of an electrospray ionization (ESI) source in a LCMS-2020 liquid chromatography-

Synthetic Methods and Molecular Characterization
All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA), Alfa-Aesar (Ward Hill, MA, USA), and TCI (Nihonbashi, Japan) and were used without further purification. Reactions were performed under an atmosphere of dry nitrogen. An Initiator microwave system (Biotage, Uppsala, Sweden) was used for microwave-assisted reaction. LC-MS was performed on a system consisting of an electrospray ionization (ESI) source in a LCMS-2020 liquid chromatography-mass spectrometer system (Shimadzu, Kyoto, Japan; column: Shim-pack GIS, 100 × 3.0 mm, 3 µm ODS). A Teledyne ISCO flash purification system (Lincoln, NE, USA) with various prepacked silica gel cartridges was used for flash column chromatography. 1 H-and 13 C-NMR spectra were recorded in the indicated solvent on an AVANCE III HD (400 and 100 MHz for 1 H and 13 C, respectively) spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are reported as δ values in parts per million downfield from TMS (δ 0.0) as the internal standard in CD 3 OD, DMSO-d 6 or CDCl 3 . The purity of the compounds was evaluated on a Shimadzu reverse-phase analytical HPLC system (column: Ace C18, 150 × 4.6 mm, 3 µm). Purities of all compounds that were subjected to biological assay were >95%.

General Method A (6a-o)
The mixture of mollugin (0.35 mmol), alcohol (3.52 mmol), and catalytic p-TsOH (0.035 mmol) in 2 mL microwave vial was placed in the cavity of microwave reactor, and then stirred for 3 h at 160 • C. The produced brown mixture was dried under vacuum and subjected to purification (20 g silica gel cartridge, dichloromethane-MeOH) to give the title product.

General Method B (Compounds 8a-c)
Various 2-bormobenzaldehyde (25 mmol), 4-methylstyrene (1.5 eq.), palladium (II) acetate (0.1 eq.) and tris(o-tolyl)phosphine (0.2 eq.) were dissolved in triethylamine (0. 8 M) and added in a pressure tube. The pressure tube was tightly capped and heated for 15 h at 130 • C. The reaction mixture was quenched with saturated aqueous ammonium chloride and then organic materials were extracted with dichloromethane (two times). The combined organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The produced mixture was subjected to flash chromatography on silica gel using hexane/ethyl acetate (0 to 2% for 60 min) to give the wanted products. Yield 65-75%.

General Method C (Compounds 9a-c)
(4-Methylstyryl)benzaldehyde 8a-c (15 mmol) was dissolved in anhydrous benzene (50 mL). To this solution, Zinc powder (1.5 eq.) and methyl 2-bromoacetate (2.0 eq.) were added to the reaction mixture, which was then refluxed for 5 h. The reaction was quenched with saturated aqueous ammonium chloride and the organic materials were extracted with dichloromethane (two times). The combined organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The mixture was purified by flash chromatography on silica gel using hexane/ethyl acetate (5 to 20% for 60 min) to give the target products; Yield 79-82%.

General Method D (Compounds 10a-c)
Various methyl (4-methylstyryl) phenyl propanoate 9a-c (12 mmol) and Dess-Martin periodinane (1.3 equiv) were dissolved in anhydrous dichloromethane, and stirred at room temperature for 2 h. The reaction mixture was quenched with saturated sodium bicarbonate water solution and extracted with dichloromethane (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The product was purified by flash chromatography on silica gel using hexane/ethyl acetate (1 to 5% for 60 min). Product obtained as a pale yellow oil. Yield 86-90%.

General Method E (Compounds 11a-c)
Various methyl (4-methylstyryl) phenyl) oxopropanoate 10a-c (10 mmol) was dissolved in anhydrous dichloroethane (50 mL). Palladium (II) trifluoroacetate (0.2 equiv), copper (II) acetate (1.0 equiv) and methyl acrylate (3.0 equiv) were added in reaction mixture and stirred at 100 • C for 10 h. The reaction mixture was quenched with saturated ammonium chloride water solution and extracted with dichloromethane (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The product was purified by flash chromatography on silica gel using hexane/ethyl acetate (0 to 2% for 60 min). Product obtained as a white solid. Yield 90-95%.

General Method F (Compounds 12a-c)
Various methyl hydroxy-2-naphthoate 11a-c (9 mmol) was dissolved in tetrahydrofuran and stirred at 25 • C for 10 min. The excess amount of potassium hydroxide solution was added in the reaction mixture and stirred at 90 • C for 20 h. The reaction mixture was cooled, acidified with 6N hydrogen chloride solution and extracted with ethyl acetate (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The product was purified by flash chromatography on silica gel using dichloromethane/methanol (10 to 30% for 60 min). Product obtained as a yellow solid. Yield 95-98%.

General Method G (Compounds 13a-c)
Various 1-hydroxy-2-naphthoic acids 12a-c (6 mmol) was dissolved in a mixed solution of 10% sodium hydroxide and 1,4-dioxane and stirred at 0 • C for 1 h. A saturated aqueous solution of potassium persulfate (1.5 equiv) was slowly added to the reaction mixture during 4-5 h. The reaction mixture was continuously stirred at 20 • C for overnight. The reaction mixture was acidified with 6N HCl solution and extracted with ethyl acetate (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The dried mixture was dissolved in N,N-dimethyl-formamide, 3.0 equivalent of potassium hydrogen carbonate was added to reaction mixture. Which was stirred at 40 • C for 0.5 h 3.0 equivalent of Iodomethane was added in reaction mixture, which was stirred at 40 • C for overnight. The reaction mixture was quenched with saturated sodium bicarbonate water solution and extracted with dichloromethane (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The product was purified by flash chromatography on silica gel using hexane/ethyl acetate (5 to 10% for 60 min). Product obtained as a pale yellow solid. Yield 80-82%. LiOtBu (6 mL. 1 M in THF, 6.0 mmol) was added to stirred solution of the phthalide (0.35 g, 2.0 mmol) in dry THF (10 mL) at −60 • C under an inert atmosphere. The resulting solution was stirred at the same temperature for 30 min after which a solution of methyl acrylate (0.362 mL, 4.0 mmol) in dry THF (10 mL) was added. The reaction mixture was stirred for another 30 min at −60 • C followed by 8 h stirring at room temperature. The reaction was then quenched with saturated ammonium chloride solution (20 mL) and THF was evaporated under reduced pressure. The residue was then extracted with ethyl acetate (2 × 20 mL). The combined extracts were washed with brine, dried (Na 2 SO 4 ), filtered, and concentrated to afford the crude product which was purified by column chromatography to obtain the pure compound.

General Method H (Compounds 14a-d)
Various methyl 1,4-dihydroxy-2-naphthoate derivatives 13a-d (5 mmol), phenylboronic acid (2.0 equiv), glacial acetic acid (5.0 equiv), and 3-methylbut-2-enal (3.0 equiv) were dissolved in anhydrous toluene and refluxed for 6 h under nitrogen gas in an apparatus fitted with a Dean-Stark trap. The reaction mixture was cooled, quenched with saturated sodium bicarbonate water solution, and extracted with dichloromethane (two times). The combined organic layer was washed with brine and dried over magnesium sulfate, filtered and concentrated under vacuum. The product was purified by flash chromatography on silica gel using hexane/ethyl acetate (5 to 20% for 60 min). Product obtained as a pale yellow solid. Yield 63-66%;

General Method I (Compounds 15a-d)
Various methyl 6-hydroxy-2H-benzo[h]chromene-5-carboxylates 14a-c (3 mmol), sodium methoxide (2.0 equiv) and an alkyl alcohol (7.0 equiv) were dissolved in anhydrous toluene and added to a microwave vial, which was placed in the microwave cavity, and stirred at 150 • C for 1.5 h. After cooling and being neutralized via addition of 1.0 equivalent of the 6N hydrogen chloride solution, the mixture was concentrated under vacuum. The product was purified by flash chromatography on silica gel using hexane/ethyl acetate (30 to 100% 20 min). Product obtained as a pale yellow oil. Yield 40-59%.

Synthesis of 17a-b
To a benzoic acid (10 mmol) solution in CH 2 Cl 2 (20 mL), SOCl 2 (5 mL) was added via syringe. After addition of DMF (3 drops), the reaction mixture was stirred for 5 h at room temperature, and then concentrated to dryness under reduced pressure. The produced acid chloride was dissolved in CH 2 Cl 2 (30 mL) and cooled under ice bath. Diethylamine (25 mmol) was added dropwise and the resulting colorless suspension was stirred for 3 h at room temperature. After addition of brine (80 mL) and CH 2 Cl 2 (50 mL), the organic layer was partitioned, washed with brine, dried with MgSO 4 ,

Measurement of Plasma Stability
The plasma stability assay was performed by incubation of human or rat plasma with a test compound in a final concentration of 1 µM for 120 min at 37 • C. The incubation was terminated by addition of 40 µL of ice-cold acetonitrile and vortexing for 5 min. The precipitated proteins were removed by centrifugation at 14,000× g for 5 min at 4 • C. Aliquots of the supernatant were injected onto a Shimadzu Nexera XR LC system. Percentages of the parent compound remaining were calculated by comparing peak areas using the Xcalibur software (version 1.6.1, Waltham, MA, USA).

hERG Assay
hERG channel binding assay was performed using Invitrogen's hERG Fluorescence Polarization Assay kit (PV5365) with Synergy Neo plate reader (BioTek, Winooski, VT, USA). The binding assay was carried out according to the kit manufacturer's instructions.

CYP Inhibition Assay
All incubations were performed in duplicate, and the mean values were used for analysis. The assays of phenacetin O-deethylase, tolbutamide 4-hydroxylase, S-mephenytoin 4-hydroxylase, dextromethorphan O-demethylase and midazolam 1 -hydroxylase activities were determined as probe activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, using cocktail incubation and tandem mass spectrometry. Briefly, incubation reaction was performed with 0.25 mg/mL human liver microsomes in a final incubation volume of 100 µL. The incubation medium contained 100 mM phosphate buffer (pH 7.4) with probe substrates. The incubation mixture containing various inhibitors (10 µM) was pre-incubated for 5 min. After pre-incubation, an NADPH regenerating system was added. After incubation at 37 • C for 15 min, the reaction was stopped by placing the incubation tubes on ice and adding 40 µL of ice-cold acetonitrile. The incubation mixtures were then centrifuged at 10,000× g for 5 min at 4 • C. Aliquots of the supernatant were injected onto an LC-MS/MS system. The CYP-mediated activities in the presence of inhibitors were expressed as percentages of the corresponding control values.

Determination of Water Solubility at 10 mM
Water solubility (>10 mM) was determined using the following procedure at room temperature. The test compound was added to a vial containing sodium phosphate buffer, 0.1 M, pH 7.4 (1 mL) to make a 10 mM mixture, and the mixture was equilibrated during 10 min of sonication and then visually checked immediately and after 24 h for any undissolved parts or precipitation of the sample. If no precipitation was shown after 24 h, the aqueous solution was filtered (Syringe filter; PTFE; pore size: 0.45 µm; 25 mm DI) and subjected to LC-MS to confirm the reagent.

Conclusions
To improve metabolic stability, plasma stability, and water solubility, we synthesized various mollugin derivatives and assayed their inhibitory activity against U937 monocyte cell adhesion to HT-29 colonic epithelial cells in both TNF-α-and IL-6-induced models of colon inflammation at a 10 µM concentration. The water solubility and plasma stability were improved by replacing the methyl ester of mollugin with 2-(4-morpholinyl)-ethyl, and the metabolic stability was improved by substitution of F-or CF 3 -on the aromatic ring of mollugin. Compound 15c has good water solubility, plasma stability, and metabolic stability and has greater inhibitory activity than mesalazine or mollugin in the TNF-α-or the IL-6-induced colonic epithelial cell adhesion assay. The results suggest that 15c is a potential anti-inflammatory agent.