A novel class of apical sodium--dependent bile salt transporter inhibitors: 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides

Abstract

The apical sodium--dependent bile acid transporter (ASBT) is the main transporter to promote re-absorption of bile acids from the intestinal tract into the enterohepatic circulation. Inhibition of ASBT could increase the excretion of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. Therefore, ASBT is an attractive target for developing new cholesterol-lowering drugs. In this report, a series of 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides were designed as inhibitors of ASBT. Most of them demonstrated potency against ASBT transport of bile acids. In particular, compound 4a₁ was found to have the best activity, resulting in 80.1% inhibition of ASBT at 10 μmol/L.

Graphical abstract

The apical sodium--dependent bile acid transporter (ASBT) is the main transporter to promote re-absorption of bile acids from the intestinal tract into the enterohepatic circulation. Inhibition of ASBT could increase the excretion of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. Therefore, ASBT is an attractive target for developing new cholesterol-lowering drugs. A series of novel 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides were designed, synthesized and the inhibitory activities for ASBT were assessed.

1. Introduction

High serum cholesterol is a main cause of coronary artery disease (CAD), which greatly increases the risk of atherosclerosis¹. About 50% of cholesterol is eliminated from the body by conversion to bile acids. Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the intestine2, 3. More than 90% of the secreted bile acid is re-absorbed from the intestine and transported back to the liver via the enterohepatic circulation system, and re-secreted into the bile. The major mechanism for absorption of intestinal bile acids is active uptake by the apical sodium--dependent bile acid transporter (ASBT) located in the ileal brush-border membrane3, 4, 5. Inhibition of ASBT reduces re-absorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption6, 7. Because ASBT is localized on the apical membrane of the lumen in the ileum, inhibitors can block ASBT activity without entering the circulation system. Therefore, ASBT is an attractive target for developing new cholesterol-lowering drugs8, 9, 10, 11. At present, several ASBT inhibitors have been developed that are effective in animal models, which include S-1647, R-146224, 264W94 and SC-435 (Fig. 1)12, 13, 14, 15.

Figure 1.

Structures of apical sodium--dependent bile acid transporter (ASBT) inhibitors.

Our laboratory has been dedicated to the investigation and development of ASBT inhibitors and has obtained a series of compounds with good activity¹⁶. NC-1, a compound obtained by extensive screening in our lab, was found to have potent ASBT inhibitory activity. It showed 30.5% inhibition of ASBT at 10 μmol/L in an in vitro assay. NC-1 has a scaffold of 1-aryl-1,8-naphthyridine which is very similar to R-146224. To develop more potent ASBT inhibitors, we optimized the structure of NC-1 in this study. Using combinatorial principles, a 3-carboxamide was introduced to NC-1. At the same time, we observed that the 7 position of R-146224 was a quaternary ammonium salt with a long linker, while NC-1 held a chlorine in the corresponding position. It has been reported that a tertiary amine or a quaternary ammonium salt substituted in this position could exhibit the same potency as seen in the most commonly reported ASBT inhibitors17, 18, 19, 20. Thus, in order to simplify the structure, we replaced the chlorine atom with dimethylamine or diethylamine with reference to SC-435. Finally, twenty-three 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides were designed, synthesized through a three-step process and evaluated for their ASBT inhibitory activity by a radioactive binding assay (Fig. 2).

Figure 2.

Design of 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides.

2. Results and discussion

2.1. Chemistry

The synthetic pathways to this series of target compounds are shown in Scheme 1. Nucleophilic substitution of 1-(2,4-bifluorophenyl)-6-fluoro-7-chloro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-ethyl formate 1 with dimethylamine or diethylamine in the presence of triethylamine in tetrahydrofuran (THF) gave 7-aminonaphthyridine-3-ethyl formate 2a and 2b. Hydrolysis of the esters 2a and 2b in a NaOH-H₂O-EtOH system yielded the corresponding naphthyridine-3-carboxylic acid 3a and 3b. Coupling of the naphthyridine-3-carboxylic acid 3a and 3b with commercially available substituted anilines in the presence of isobutyl chloroformate and triethylamine in dry dichloromethane afforded the target compounds 4a₁--4a₁₃ and 4b₁--4b₁₀. All the target structures were confirmed by ¹H NMR, ¹³C NMR and mass spectrometry (MS).

Scheme 1.

The synthesis of 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides 4a₁--4a₁₃, 4b₁--4b₁₀. Reagents and conditions: (a) HN(R¹)₂, Et₃N, THF, r.t.; (b) NaOH, EtOH, reflux; (c) Et₃N, isobutylchloroformate, CH₂Cl₂, r.t.

2.2. ASBT inhibition assay

The inhibitory activity of the compounds was tested in vitro against ASBT by a radioisotope-based assay²¹. A human ASBT expression construct was prepared as previously described²². The inhibitory activity was expressed as inhibition (%) in 10 µmol/L (Table 1). The values are the average of three independent experiments with S-1647 as a positive control in each experiment.

Table 1.

The structures and ASBT inhibition of compounds 4a₁--4a₁₃ and 4b₁--4b₁₀.

Compd.	R1	R2	Inhibition (%)a
4a1	Methyl	3,5-Difluoro	80.1±1.5
4a2	Methyl	3,5-Dichloro	62.5±2.1
4a3	Methyl	2,4-Dichloro	51.7±2.6
4a4	Methyl	2,3-Dichloro	49.8±3.3
4a5	Methyl	2,5-Dichloro	47.6±1.9
4a6	Methyl	3-Chloro	45.4±4.1
4a7	Methyl	3-Chloro-4-fluoro	53.2±2.8
4a8	Methyl	3-Trifluoromethyl-4-methyl	52.6±1.2
4a9	Methyl	3-Difluoromethoxy	50.5±3.4
4a10	Methyl	2-Methyl-3-trifluoromethyl	48.3±4.2
4a11	Methyl	3,4-Dichloro	50.2±1.1
4a12	Methyl	3,5-Dimethoxy	19.6±3.1
4a13	Methyl	4-Methoxy	18.5±3.7
4b1	Ethyl	3,5-Difluoro	58.1±4.6
4b2	Ethyl	3,5-Dichloro	44.3±2.9
4b3	Ethyl	2,4-Dichloro	46.5±5.1
4b4	Ethyl	2,3-Dichloro	47.2±2.5
4b5	Ethyl	2,5-Dichloro	45.7±3.9
4b6	Ethyl	3-Chloro	42.9±2.8
4b7	Ethyl	3-Chloro-4-fluoro	44.3±4.1
4b8	Ethyl	2-Methyl-3-trifluoromethyl	27.1±2.3
4b9	Ethyl	3,4-Dichloro	35.6±3.4
4b10	Ethyl	4-Methoxy	13.7±2.6
NC-1			30.5±2.8
S-1647			77.9±3.3

^aValues represent the percent inhibition of ASBT at 10 µmol/L of the test compounds and are the average of three independent experiments. Each value represents the mean±SD.

As shown in Table 1, most of the newly synthesized derivatives exhibited ASBT inhibitory activity and most of them showed better activity than the lead compound NC-1. Particularly, 4a₁ exhibited an inhibition of 80.1% towards ASBT which was more potent than S-1647. Substitution at 7 positions of the 1,8-naphthyridine scaffold was considered an important factor in the activity. Indeed, compounds 4a₁, 4a₂ and 4a₃ with dimethylamino groups (R¹) showed better activity than the corresponding substituted diethylamino analogues 4b₁, 4b₂ and 4b₃, respectively. In addition, compounds 4a₁₂, 4a₁₃ and 4b₁₀ with an electron-donating group (R²) substituted on the 3-carboxamide phenyl ring showed observably lower potency than other corresponding electron-withdrawing group analogues. So R² with an electron-withdrawing group was of greatest benefit for ASBT inhibitory activity.

3. Conclusions

In conclusion, a series of novel 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides was designed, synthesized and the inhibitory activities for ASBT were assessed. Although the ASBT inhibitory activity for this group of compounds was not as high as expected, it is certain that 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides indeed possess ASBT inhibitory activity. Further structural modifications are necessary and in progress in our laboratory.

4. Experimental

4.1. General experimental procedures

All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. MS were taken in ESI mode or APCI mode on an Agilent 1100 LC--MS system (Agilent, Palo Alto, CA, USA). Nuclear magnetic resonance spectroscopy (¹H NMR and ¹³C NMR) were performed using a 400 MHz Bruker ARX-400 spectrometers (Bruker Bioscience, Billerica, MA, USA) with CDCl₃ as solvent and TMS as an internal standard. (J values are in Hz. Chemical shifts are expressed in ppm downfield from internal standard TMS.) All the starting materials were obtained from commercially available sources and used without further purification, unless otherwise specified. Yields were not optimized.

4.2. The synthesis of details of target compounds

4.2.1. 1-(2,4-Bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-ethyl formate (2a)

To a solution of 1-(2,4-bifluorophenyl)-6-fluoro-7-chloro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-ethyl formate (1, 10.0 g, 26.13 mmol) in THF (50 mL) was added dimethylamine hydrochloride (3.2 g, 39.20 mmol) and then triethylamine (7.92 g, 78.39 mmol). The reaction mixture was stirred for 10 h at room temperature and then filtered. The filtrate was concentrated and diethyl ether (20 mL) was added to the mixture. After stirred for 0.5 h, the resulting solid was filtered. The filter cake was dried to give 2a (81.6% yield) as a white solid; mp: 190.0–191.3  °C. ¹H NMR (CDCl₃) δ: 1.39–1.42 (3 H, m), 3.03 (6 H, s), 3.04 (3 H, s), 4.36–4.41 (2 H, m), 7.02–7.07 (2 H, m), 7.37—7.43 (1 H, m), 8.08 (1 H, d, J = 13.48 Hz), 8.38 (1 H, s). HR-MS m/z Calcd. C₁₉H₁₆O₃N₃F₃ [M+H]⁺ 392.1216, Found 392.1216.

4.2.2. 1-(2,4-Bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-ethyl formate (2b)

Compound 2b was obtained as a white solid (83.2% yield) from compound 1 as described for 2a; mp: 146.8–147.9 °C. ¹H NMR (CDCl₃) δ: 1.05 (6 H, t, J=6.8 Hz), 1.40 (3 H, t, J=7.2 Hz), 3.35—3.41 (4 H, m), 4.40 (2 H, dd, J₁=14.4 Hz, J₂=4.8 Hz), 7.01–7.08 (2 H, m), 7.45 (1 H, dd, J₁=13.6 Hz, J₂=8 Hz), 8.08 (1 H, d, J=13.6 Hz), 8.42 (1 H, s). HR-MS m/z Calcd. C₂₁H₂₀O₃N₃F₃ [M+H]⁺ 420.1529, Found 420.1530.

4.2.3. 1-(2,4-Bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid (3a)

To a solution of 2a (5.0 g, 12.78 mmol) in alcohol (30 mL) was added 10% NaOH (10 mL). The mixture was heated to 80 ^oC for 10 h and concentrated. The reaction mixture was diluted with water (12 mL), adjusted to pH 6 with 6 mol/L hydrochloric acid. After stirred for 0.5 h the precipitate was filtered and washed with water to pH 7. The filter cake was dried to give 3a (89.3% yield) as a greyish white solid; mp: 225.4–226.6 °C. ¹H NMR (CDCl₃) δ: 3.10 (6 H, s), 7.04–7.10 (2 H, m), 7.35–7.39 (1 H, m), 8.06 (1 H, d, J=12.8 Hz), 8.65 (1 H, s), 14.90 (1 H, s). HR-MS m/z Calcd. C₁₇H₁₂O₃N₃F₃ [M+H]⁺ 364.0903, Found 364.0901.

4.2.4. 1-(2,4-Bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid (3b)

Compound 3b was obtained as a greyish white solid (87.6% yield) from compound 2b as described for 3a; mp: 171.3–172.5 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.42 (4 H, t, J=6.8 Hz), 7.04–7.10 (2 H, m), 7.35–7.39 (1 H, m), 8.05 (1 H, d, J=13.6 Hz), 8.64 (1 H, s). HR-MS m/z Calcd. C₁₉H₁₆O₃N₃F₃ [M+H]⁺ 392.1216, Found 392.1218.

4.2.5. N-(3,5-bifluorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₁)

To a solution of 3a (0.5 g, 1.38 mmol) in dry dichloromethane (5 mL) was added triethylamine (0.42 g, 4.14 mmol) and then stirred for 0.5 h at room temperature. Isobutyl chloroformate (0.38 g, 2.76 mmol) was added at 0 ^oC and then stirred for 1 h. The mixture was upswing to room temperature and 3,5-difluorophenylamine (0.21 g, 1.66 mmol) was added. After stirred for 10 h the precipitate was filtered and washed with little diethyl ether. The filter cake was dried to give 4a₁ (71.5% yield) as a white solid; mp: 278.2–279.5 °C. ¹H NMR (CDCl₃) δ: 3.07 (6 H, s), 6.51–6.57 (1 H, m), 7.03–7.11 (2 H, m), 7.34–7.39 (2 H, m), 7.41–7.45 (1 H, m), 8.06 (1 H, d, J=13.2 Hz), 8.71 (1 H, s), 12.43 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.10, 164.44, 162.78, 162.01, 161.85, 159.19, 156.65, 150.63, 148.27, 146.39, 145.69, 145.53, 140.77, 129.99, 119.68, 113.01, 112.62, 112.22, 112.00, 105.04, 103.45, 103.16, 98.95, 39.72, 39.64. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₅ [M+H]⁺ 475.1188, Found 475.1188.

4.2.6. N-(3,5-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₂)

Obtained as a white solid (68.7% yield) from compound 3a as described for 4a₁; mp: 293.3–294.2 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 7.03–7.10 (3 H, m), 7.40–7.46 (1 H, m), 7.73 (2 H, s), 8.06 (1 H, d, J=13.6 Hz), 8.72 (1 H, s), 12.43 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.04, 162.78, 150.63, 150.53, 148.27, 146.38, 145.70, 145.52, 140.46, 135.03, 129.98, 129.88, 123.64, 119.66, 119.44, 118.56, 112.98, 112.57, 112.21, 112.02, 105.28, 105.02, 104.79, 39.74, 39.66. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₃Cl₂ [M+H]⁺ 507.0597, Found 507.0596.

4.2.7. N-(2,4-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₃)

Obtained as a white solid (65.3% yield) from compound 3a as described for 4a₁; mp: 281.3–282.6 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 7.03–7.10 (2 H, m), 7.23–7.24 (1 H, m), 7.39–7.44 (2 H, m), 8.16 (1 H, d, J=13.6 Hz), 8.58 (1 H, d, J=8.8 Hz), 8.74 (1 H, s), 12.61 (1 H, s). ¹³C NMR (CDCl₃) δ:176.13, 162.88, 150.62, 148.24, 146.48, 145.67, 145.53, 134.89, 130.00, 128.97, 128.58, 127.47, 124.40, 122.99, 119.99, 119.77, 113.11, 113.02, 112.19, 111.96, 105.28, 105.05, 104.78, 39.73, 39.65. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₃Cl₂ [M+H]⁺ 507.0597, Found 507.0597.

4.2.8. N-(2,3-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₄)

Obtained as a white solid (67.2% yield) from compound 3a as described for 4a₁; mp: 283.6–284 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 7.03–7.10 (2 H, m), 7.21 (2 H, d, J=4.8 Hz), 7.39–7.45 (1 H, m), 8.18 (1 H, d, J=13.6 Hz), 8.53–8.56 (1 H,m), 8.74 (1 H, s), 12.67 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.08, 163.00, 150.59, 148.23, 146.56, 145.66, 145.52, 137.80, 132.89, 129.95, 124.90, 122.50, 120.39, 120.00, 119.78, 113.11, 113.04, 112.14, 111.95, 105.26, 105.02, 104.76, 39.71, 39.64. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₃Cl₂ [M+H]⁺ 507.0597, Found 507.0597.

4.2.9. N-(2,5-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₅)

Obtained as a white solid (72.6% yield) from compound 3a as described for 4a₁; mp: 260.0–261.3 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 7.01–7.10 (3 H, m), 7.34 (1 H, d, J=8.4 Hz), 7.39–7.45 (1 H, m), 8.16 (1 H, d, J=13.2 Hz), 8.71 (1 H, d, J=2.4 Hz), 8.74 (1 H, s), 12.65 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.08, 162, 96, 150.61, 148.24, 146.59, 145.67, 145.52, 136.95, 132.99, 130.01, 129.88, 124.08, 122.11, 121.88, 119.98, 119.76, 113.11, 112.93, 112.21, 119.98, 105.28, 105.02, 104.79, 39.73, 39.65. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₃Cl₂ [M+H]⁺ 507.0597, Found 507.0598.

4.2.10. N-(3-chlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₆)

Obtained as a white solid (69.3% yield) from compound 3a as described for 4a₁; mp: 265.1–266.5 °C. ¹H NMR (CDCl₃) δ: 3.09 (6 H, s), 7.05–7.12 (2 H, m), 7.27–7.31 (2 H, m), 7.45 (1 H, d, J=6.4 Hz), 7.60 (1 H, d, J=8.4 Hz), 7.95 (1 H, s), 8.10 (1 H, d, J=13.6 Hz), 8.78 (1 H, s), 12.34 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.16, 162.59, 148.26, 147.10, 146.29, 145.69, 145.52, 139.82, 134.52, 129.98, 129.86, 123.84, 120.49, 119.73, 119.51, 118.38, 113.03, 112.96, 112.19, 111.97, 105.26, 105.00, 104.76, 39.73, 39.65. HR-MS m/z Calcd. C₂₃H₁₆O₂N₄F₃Cl [M+H]⁺ 473.0986, Found 473.0988.

4.2.11. N-(3-chloro-4-fluorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₇)

Obtained as a white solid (62.1% yield) from compound 3a as described for 4a₁; mp: 270.0–271.2 °C. ¹H NMR (CDCl3) δ: 3.08 (6 H, s), 7.04–7.13 (3 H, m), 7.41 (1 H, s), 7.53 (1 H, d, J=7.2 Hz), 7.98 (1 H, d, J=6 Hz), 8.08 (1 H, d, J=13.2 Hz), 8.74 (1 H, s), 12.30 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.14, 162.54, 155.69, 153.25, 150.62, 148.26, 146.24, 145.54, 135.37, 129.87, 122.37, 120.98, 119.90, 119.69, 119.47, 116.58, 116.36, 112.99, 112.80, 112.21, 111.95, 105.27, 105.04, 104.77, 39.73, 39.66. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₄Cl [M+H]⁺ 491.0892, Found 491.0892.

4.2.12. N-(3-trifluoro-4-methylphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₈)

Obtained as a white solid (68.7% yield) from compound 3a as described for 4a₁; mp: 227.4–228.9 °C. ¹H NMR (CDCl₃) δ: 2.45 (3 H, s), 3.08 (6 H, s), 7.04–7.09 (2 H, m), 7.24–7.26 (1 H, m), 7.39–7.44 (1 H, m), 7.85 (1 H, d, J=8.4 Hz), 8.02 (1 H, s), 8.08 (1 H, d, J=13.6 Hz), 8.76 (1 H, s), 12.32 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.18, 162.62, 150.53, 148.27, 146.26, 145.70, 145.53, 136.56, 132.41, 131.65, 129.96, 129.35, 129.05, 125.74, 123.23, 119.72, 119.50, 117.98, 113.04, 112.98, 112.18, 111.96, 105.26, 105.03, 104.77, 39.73, 39.65. HR-MS m/z Calcd. C₂₅H₁₈O₂N₄F₆ [M+H]⁺ 521.1406, Found 521.1407.

4.2.13. N-(3-difluoromethoxyphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₉)

Obtained as a white solid (73.6% yield) from compound 3a as described for 4a₁; mp: 204.7–205.5 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 6.55 (1 H, t, J=7 4 Hz), 6.87 (1 H, d, J=8.0 Hz), 7.04–7.10 (2 H, m), 7.32 (1 H, t, J=8.0 Hz), 7.42 (1 H, d, J=5.2 Hz), 7.49–7.52 (1 H, m), 7.77 (1 H, s), 8.08 (1 H, d, J=13.6 Hz), 8.77 (1 H, s), 12.35 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.17, 162.67, 151.80, 146.32, 145.53, 140.11, 129.97, 129.88, 119.73, 119.50, 118.67, 117.11, 116.10, 114.52, 113.53, 113.07, 112.95, 112.15, 111.93, 111.36, 105.26, 105.03, 104.77, 39.73, 39.65. HR-MS m/z Calcd. C₂₄H₁₇O₃N₄F₅ [M+H]⁺ 505.1293, Found 505.1290.

4.2.14. N-(3-trifluoromethyl-2-methylphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₁₀)

Obtained as a white solid (52.5% yield) from compound 3a as described for 4a₁; mp: 208.7–209.5 °C. ¹H NMR (CDCl₃) δ: 2.57 (3 H, s), 3.08 (6 H, s), 7.03–7.10 (2 H, m), 7.31 (1 H, t, J=8.0 Hz), 7.43(2 H, d, J=8.0 Hz), 8.13 (1 H, d, J=13.6 Hz), 8.42 (1 H, d, J=6.8 Hz), 8.77 (1 H, s), 12.22 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.34, 164.40, 162.81, 161.77, 159.10, 156.69, 150.62, 148.25, 146.40, 145.54, 138.49, 130.00, 127.57, 126.03, 125.92, 121.50, 119.85, 119.63, 113.20, 113.03, 112.19, 111.96, 105.26, 105.03, 104.77, 39.73, 39.65. HR-MS m/z Calcd. C₂₅H₁₈O₂N₄F₆ [M+H]⁺ 521.1407, Found 521.1406.

4.2.15. N-(3,4-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₁₁)

Obtained as a white solid (63.7% yield) from compound 3a as described for 4a₁; mp: 285.0–286.2 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 7.03–7.10 (2 H, m), 7.38 (1 H, d, J=8.8 Hz), 7.42–7.45 (1 H, m), 7.52–7.56 (1 H, m), 8.03–8.07 (2 H, m), 8.74 (1 H, s), 12.38 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.06, 162.65, 161.79, 159.06, 156.51, 150.45, 148.21, 146.30, 145.49, 138.18, 132.54, 130.32, 130.02, 126.65, 121.86, 119.51, 112.92, 112.63, 112.22, 112.00, 105.24, 105.01, 104.74, 39.73, 39.65. HR-MS m/z Calcd. C₂₃H₁₅O₂N₄F₃Cl₂ [M+H]⁺ 507.0597, Found 507.0599.

4.2.16. N-(3,5-bimethoxyphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₁₂)

Obtained as a white solid (62.6% yield) from compound 3a as described for 4a₁; mp: 237.2–238.2 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 3.81 (6 H, s), 6.26 (1 H, d, J=2.0 Hz), 7.03–7.09 (4 H, m), 7.41–7.44 (1 H, m), 8.08 (1 H, d, J=13.2 Hz), 8.77 (1 H, s), 12.19 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.17, 162.48, 160.39, 150.56, 150.47, 148.23, 146.22, 145.66, 145.49, 140.33, 130.01, 129.92, 119.70, 119.48, 113.17, 113.04, 112.13, 111.91, 105.20, 104.94, 104.71, 98.39, 97.13, 55.40, 39.70, 39.62. HR-MS m/z Calcd. C₂₅H₂₁O₄N₄F₃ [M+H]⁺ 499.1587, Found 499.1588.

4.2.17. N-(4-methoxyphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(dimethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4a₁₃)

Obtained as a white solid (71.7% yield) from compound 3a as described for 4a₁; mp: 261.5–262.7 °C. ¹H NMR (CDCl₃) δ: 3.08 (6 H, s), 3.81 (3 H, s), 6.90 (2 H, d, J=8.8 Hz), 7.02–7.09 (2 H, m), 7.42 (1 H, s), 7.68 (2 H, d, J=8.8 Hz), 8.08 (1 H, d, J=13.6 Hz), 8.78 (1 H, s), 12.07 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.17, 162.09, 156.08, 150.57, 148.23, 146.03, 145.66, 145.49, 131.95, 130.03, 121.89, 119.72, 119.50, 114.09, 113.34, 113.03, 112.14, 111.92, 105.19, 104.93, 104.70, 55.49, 39.71, 39.63. HR-MS m/z Calcd. C₂₄H₁₉O₃N₄F₃ [M+H]⁺ 469.1482, Found 469.1485.

4.2.18. N-(3,5-bifluorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₁)

Obtained as a white solid (62.9% yield) from compound 3b as described for 4a₁; mp: 212.0–213.4 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.37–3.44 (4 H, m), 6.53 (1 H, t, J=8.8 Hz), 7.03–7.10 (2 H, m), 7.34–7.45 (3 H, m), 8.05 (1 H, d, J=14.0 Hz), 8.70 (1 H, s), 12.47 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.11, 164.46, 164.31, 162.89, 162.01, 161.87, 149.31, 147.84, 146.24, 145.95, 145.27, 140.79, 129.98, 119.76, 119.54, 112.67, 112.58, 112.15, 111.93, 104.97, 103.52, 103.23, 99.00, 45.17, 45.10, 13.33. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₅ [M+H]⁺ 503.1501, Found 503.1500.

4.2.19. N-(3,5-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₂)

Obtained as a white solid (60.3% yield) from compound 3b as described for 4a₁; mp: 202.7–203.9 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.41 (4 H, t, J=6.8 Hz), 7.03–7.10 (3 H, m), 7.37–7.43 (1 H, m), 7.73 (2 H, d, J=1.2 Hz), 8.05 (1 H, d, J=14.0 Hz), 8.70 (1 H, s), 12.46 (1 H, s). ¹³C NMR (CDCl₃) δ:176.08, 162.88, 149.41, 149.31, 147.84, 146.22, 145.94, 145.27, 140.48, 135.04, 130.08, 129.98, 123.67, 119.75, 119.52, 118.62, 112.66, 112.54, 112.17, 111.94, 105.21, 104.98, 104.72, 45.17, 45.10, 13.34. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₃Cl₂ [M+H]⁺ 535.0910, Found 535.0909.

4.2.20. N-(2,4-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₃)

Obtained as a white solid (71.5% yield) from compound 3b as described for 4a₁; mp: 228.1–229.5 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.38–3.44 (4 H, m), 7.03–7.10 (2 H, m), 7.24 (1 H, d, J=2 Hz), 7.37–7.41 (1 H, m), 7.44 (1 H, d, J=2.4 Hz), 8.15 (1 H, d, J=13.6 Hz), 8.58 (1 H, d, J=8.8 Hz), 8.73 (1 H, s), 12.64 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.09, 162.96, 149.27, 147.78, 146.32, 145.92, 145.21, 134.92, 130.13, 128.96, 128.54, 127.46, 124.38, 122.97, 120.01, 119.78, 112.90, 112.69, 112.12, 111.86, 105.18, 104.95, 104.69, 45.12, 45.05, 13.33. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₃Cl₂ [M+H]⁺ 535.0910, Found 535.0912.

4.2.21. N-(2,3-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₄)

Obtained as a white solid (78.5% yield) from compound 3b as described for 4a₁; mp: 218.2–219.7 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.41 (4 H, t, J=6.8 Hz), 7.04–7.09 (2 H, m), 7.22 (2 H, d, J=5.2 Hz), 7.37–7.43 (1 H, m), 8.16 (1 H, d, J=13.6 Hz), 8.55 (1 H, t, J=4.8 Hz), 8.74 (1 H, s), 12.70 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.08, 163.10, 149.27, 147.79, 146.40, 145.92, 145.22, 137.82, 132.90, 130.03, 127.45, 124.91, 122.54, 120.41, 120.06, 119.83, 112.96, 112.11, 112.08, 111.89, 105.18, 104.95, 104.69, 45.12, 45.05, 13.34. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₃Cl₂ [M+H]⁺ 535.0910, Found 535.0910.

4.2.22. N-(2,5-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₅)

Obtained as a white solid (73.4% yield) from compound 3b as described for 4a₁; mp: 282.2–283.7 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.38–3.44 (4 H, m), 7.00–7.03 (1 H, m), 7.03–7.10 (2 H, m), 7.34 (1 H, d, J=8.8 Hz), 7.38–7.44 (1 H, m), 8.16 (1 H, d, J=13.6 Hz), 8.71 (1 H, t, J=2.4 Hz), 8.73 (1 H, s), 12.68 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.05, 163.04, 149.27, 147.78, 146.43, 145.92, 145.21, 136.98, 132.97, 130.13, 129.88, 124.04, 122.10, 121.87, 120.01, 119.78, 112.82, 112.69, 112.14, 111.91, 105.19, 104.96, 104.69, 45.12, 45.05, 13.33. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₃Cl₂ [M+H]⁺ 535.0910, Found 535.0909.

4.2.23. N-(3-chlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₆)

Obtained as a white solid (77.9% yield) from compound 3b as described for 4a₁; mp: 204.6–205.5 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.38–3.45 (4 H, m), 7.03–7.10 (3 H, m), 7.24–7.28 (1 H, m), 7.42 (1 H, d, J=5.6 Hz), 7.58 (1 H, d, J=8.0 Hz), 7.93 (1 H, s), 8.06 (1 H, d, J=13.6 Hz), 8.77 (1 H, s), 12.35 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.11, 162.67, 149.25, 147.79, 146.13, 145.91, 145.22, 139.84, 134.49, 130.14, 129.84, 123.78, 120.45, 119.74, 119.52, 118.36, 112.81, 112.68, 112.13, 111.90, 105.15, 104.92, 104.65, 45.13, 45.06, 13.34. HR-MS m/z Calcd. C₂₅H₂₀O₂N₄F₃Cl [M+H]⁺ 501.1299, Found 501.1296.

4.2.24. N-(3-chloro-4-fluorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₇)

Obtained as a white solid (78.1% yield) from compound 3b as described for 4a₁; mp: 183.1–184.5 °C. ¹H NMR (CDCl₃) δ: 1.08 (6 H, t, J=6.8 Hz), 3.40–3.45 (4 H, m), 7.03–7.13 (3 H, m), 7.42 (1 H, d, J=4.8 Hz), 7.56 (1 H, d, J=8.8 Hz), 7.99 (1 H, dd, J₁=6.8 Hz, J₂=2.0 Hz), 8.06 (1 H, d, J=14.0 Hz), 8.77 (1 H, s), 12.35 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.14, 162.63, 149.29, 147.83, 146.08, 145.93, 145.26, 135.36, 129.99, 122.43, 121.00, 119.94, 119.87, 119.75, 119.53, 116.60, 116.38, 112.73, 112.66, 112.14, 111.92, 105.20, 104.93, 104.70, 45.15, 45.09, 13.33. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₄Cl [M+H]⁺ 519.1205, Found 519.1208.

4.2.25. N-(3-trifluoro-2-methylphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₈)

Obtained as a white solid (55.7% yield) from compound 3b as described for 4a₁; mp: 240.8–241.9 °C. ¹H NMR (CDCl₃) δ: 1.08 (6 H, t, J=6.8 Hz), 2.57 (3 H, s), 3.40—3.44 (4 H, m), 7.04—7.10 (2 H, m), 7.31 (1 H, t, J=8.0 Hz), 7.41–7.45 (2 H, m), 8.12 (1 H, d, J=14.0 Hz), 8.43 (1 H, d, J=7.2 Hz), 8.77 (1 H, s), 12.25 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.29, 162.91, 149.38, 147.81, 146.25, 145.94, 145.24, 138.49, 130.03, 129.75, 129.46, 127.63, 126.03, 123.17, 121.51, 119.88, 119.66, 113.08, 112.68, 112.09, 111.90, 105.18, 104.91, 104.68, 45.14, 45.07, 14.08, 13.33. HR-MS m/z Calcd. C₂₇H₂₂O₂N₄F₆ [M+H]⁺ 549.1720, Found 549.1719.

4.2.26. N-(3,4-bichlorophenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₉)

Obtained as a white solid (56.2% yield) from compound 3b as described for 4a₁; mp: 163.1–164.6 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.40–3.43 (4 H, m), 7.03–7.10 (2 H, m), 7.38–7.43 (2 H, m), 7.57 (1 H, d, J=8.0 Hz), 8.06 (2 H, d, J=13.6 Hz), 8.76 (1 H, s), 12.42 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.08, 162.74, 149.39, 147.82, 146.15, 145.92, 145.25, 138.21, 132.56, 130.37, 130.01, 126.73, 124.87, 124.70, 121.98, 119.60, 112.62, 112.12, 111.93, 105.18, 104.92, 104.69, 45.16, 45.09, 13.33. HR-MS m/z Calcd. C₂₅H₁₉O₂N₄F₃Cl₂ [M+H]⁺ 535.0910, Found 535.0909.

4.2.27. N-(4-methoxyphenyl)-1-(2,4-bifluorophenyl)-6-fluoro-7-(diethylamino)-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxamide (4b₁₀)

Obtained as a white solid (69.5% yield) from compound 3b as described for 4a₁; mp: 217.3–218.5 °C. ¹H NMR (CDCl₃) δ: 1.07 (6 H, t, J=6.8 Hz), 3.37–3.44 (4 H, m), 3.81 (3 H, s), 6.81 (2 H, d, J=8.8 Hz), 7.01–7.08 (2 H, m), 7.38–7.42 (1 H, m), 7.68 (2 H, d, J=8.8 Hz), 8.06 (1 H, d, J=13.6 Hz), 8.75 (1 H, s), 12.09 (1 H, s). ¹³C NMR (CDCl₃) δ: 176.20, 162.18, 156.08, 149.20, 147.76, 145.85, 145.20, 131.98, 130.14, 121.91, 119.80, 119.58, 114.46, 114.10, 113.27, 112.67, 112.03, 111.84, 105.12, 104.88, 104.62, 55.49, 45.10, 45.03, 13.34. HR-MS m/z Calcd. C₂₆H₂₃O₃N₄F₃ [M+H]⁺ 497.1795, Found 497.1796.

4.3. ASBT inhibition assay

HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and grown in MEM supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin and 10% heat-inactivated fetal bovine serum. A human ASBT expression construct was prepared as previously described¹⁵. HEK293T cells were seeded in 12-well plates and transiently transfected with 0.5 μg/well pcDNA3.1/ASBT or the negative control plasmid pcDNA3.1 using lipofactamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer׳s instructions. Twenty-four hours after transfection, HEK293T cells were assayed for taurocholic acid uptake as previously described with minor modifications¹⁶. Briefly, cells were washed twice with warm wash and uptake buffer (116 mmol/L NaCl, 5.3 mmol/L KCl, 1.1 mmol/L KH₂PO₄, 0.8 mmol/L MgSO₄, 1.8 mmol/L CaCl₂, 11 mmol/L d-dextrose/d-glucose, and 10 mmol/L HEPES, pH 7.4), and then incubated with the same buffer containing the indicated concentrations of test compounds (dissolved in dimethyl sulfoxide) and 1 μCi/mL of [³H]-taurocholic acid (TCA, 0.2 μmol/L) (PerkinElmer Life Sciences) for 10 min. To terminate the transport process the plates were chilled on ice and the cells were immediately washed with ice-cold buffer three times. Cells were lysed with 0.3 mL lysis buffer (0.5% Triton X-100) and shaken vigorously for 20 min. The radioactivity of the cell lysate was counted using a MicroBeta² Liquid Scintillation and Luminescence Counter (PerkinElmer Life Sciences). Protein concentration of the lysate was used to normalize uptake activity.

The inhibition rate was calculated using the following formula in Eq. (1):

$$\mathrm{Inhibition}\mathrm{rate}(\%)=\left[(1-\left(\mathit{A}-\mathit{C}\right)/\left(\mathit{B}-\mathit{C}\right)\right]\times \text{100}$$ (1)

where A stands for [³H] uptake value of test compound in [³H]-TCA buffer added to pcDNA3.1/ASBT transfected cells; B stands for [³H] uptake value of DMSO (without inhibitor) in [³H]-TCA buffer added to pcDNA3.1/ASBT transfected cells; C stands for [³H] uptake value of blank sample (DMSO) in [³H]-TCA buffer added to empty vector (pcDNA3.1)-transfected cells.