Design,synthesis,and evaluation of naphthalidinamide analogues as nonsteroidal mineralocorticoid receptor antagonists

Abstract

Inappropriate overactivation of the mineralocorticoid receptor by the steroid hormones aldosterone or cortisol contributes to the development of cardiovascular diseases. The new mineralocorticoid receptor antagonist can directly block mineralocorticoid receptor overactivation, inhibit inflammation and profibrotic gene expression, thereby reducing chronic kidney disease progression and cardiovascular risk, achieving dual benefits for both kidney and heart. Antergy of mineralocorticoid receptor is a proven therapeutic concept for the management of chronic kidney disease and type 2 diabetes associated diseases. Using finerenone (BAY 94-8862) as an inspiration, several novel naphthalidinamide analogues were designed, synthesized and their biological activities were determined as nonsteroidal mineralocorticoid receptor antagonists. In vitro results showed that A2 (IC₅₀ = 30.3 nM) and B (IC₅₀ = 5.6 nM) exhibited significant antagonistic activities against mineralocorticoid receptor. Among them, compound B showed the most potent antagonistic activity against mineralocorticoid receptor cells which was much superior to the control compound BAY-94-8862 (IC₅₀ = 28.1 nM). Besides, the two potent compounds showed promising (>170-fold) selectivity over the glucocorticoid receptor, androgen receptor and progesterone receptor. Furthermore, they displayed rapid metabolic profiles in human, dog, monkey, rat, and mouse liver microsomes than the control compound BAY-94-8862. These results highlighted the great potential compound B to become a promising mineralocorticoid receptor antagonist for cardiovascular disease.

Introduction

Cardiovascular diseases (CVDs) pose a significant threat to human health and life, ranking as one of the leading causes of mortality worldwide. ¹ With the advancements in living standards, the issues of obesity and aging population are becoming increasingly serious, and the CVDs have become the main killer as well as tumor.^2,3 The inappropriate overactivation of the mineralocorticoid receptor (MR) by steroid hormones such as aldosterone or cortisol is a critical contributor to the pathogenesis of CVDs. ⁴ Mineralocorticoid receptor antagonists (MRAs) have been shown to effectively reduce blood pressure in patients with primary aldosteronism and essential hypertension, particularly in individuals with resistant hypertension. ⁵ Moreover, combining MRAs with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) has demonstrated superior therapeutic benefits compared with conventional therapies alone, notably in reducing prote-inuria in patients with chronic kidney disease (CKD).^{6 –8} This combination has also been associated with reductions in markers of myocardial fibrosis. ⁹ Consequently, MR inhibition has emerged as a validated therapeutic strategy for managing CKD and diseases associated with type 2 diabetes (T2D).^10,11

The first-generation MRA, spironolactone (IC₅₀ = 24 nM), and the second-generation MRA, eplerenone (IC₅₀ = 990 nM) ¹² (Figure 1), have demonstrated significant efficacy in reducing morbidity and mortality in renal and cardiac diseases. Clinical studies further underscore the therapeutic potential of MRAs in treating CKD and diabetic nephropathy. However, the clinical utility of these steroidal spirolactones is limited by notable drawbacks. Spironolactone exhibits substantial off-target activity on androgen receptor (AR) and progesterone receptor (PR) due to its structural similarity to progesterone, resulting in poor selectivity and adverse effects such as gynecomastia, impotence, and menstrual irregularities. ¹³ Although eplerenone offers improved selectivity, it is less potent in vitro than spironolactone and has demonstrated lower efficacy in hypertensive clinical trials. ¹⁴

Figure 1.

Chemical structures of the representative mineralocorticoid receptor antagonist.

To address the limitations of spironolactone and eplerenone, novel, potent, and selective MRAs have been developed.^{15 –17} Finerenone (BAY 94-8862), ¹⁸ a third-generation nonsteroidal MRA with high potency was identified through high-throughput screening of cyano-1,4-dihydropyridines. Finerenone has been shown to reduce the risk of sustained eGFR decline, end-stage kidney disease, cardiovascular death, non-fatal myocardial infarction, and hospitalization for heart failure in adult patients with CKD associated with T2D.^{19 –22} In 2021, it received approval from the U.S. Food and Drug Administration, becoming the first nonsteroidal selective MRA to improve renal outcomes in adult patients with CKD and T2D. ²³

Encouraged by the successful discovery of the marked finerenone, for development of new nonsteroidal MRAs that are more potent, selective, and orally available, reducing the risk of hyperkalemia, we disclose an extended SAR (structure-activity relationship) exploration for dihydronapthyridine derivatives by a pharmacophore fusion strategy of finerenone (BAY 94-8862).

1,4-Dihydro-1,6-naphthyridine ring is a pharmacophore group that maintains MR inhibitory activity. Previous research has shown that the C3, C4, and C5 substituents on the naphthyridine have a strong impact on activity. ¹⁸ On the basis of retaining the pharmacophore of 1,4-dihydro-1,6-naphthoprim ring, C4 (A ring) was modified by introducing F and CF₃, C5 (B ring) was modified by introducing cyclopropane (Figure 2). Therefore, in this paper, three new naphthalidinamide analogues were designed and synthesized as novel MRAs, and the antagonistic activity and selectivity were evaluated based on MR cell-based transactivation assay.

Figure 2.

Design strategy of the novel naphthalidinamide analogues.

Results and discussion

Chemistry

The synthesis of targeted molecules was illustrated in Scheme 1. The protocols used for preparation of the intermediates and target compounds were described in detail in the “Experimental Section.” The classical Hantzsch reaction was successfully applied for the construction of dihydropyridines.^24,25 Condensation of 2-methoxy-4-(trifluoromethyl) benzaldehyde (1a), 4-amino-5-methylpyridin-2(1H)-one and benzyl 3-oxobutanoate which can be available through commercial sources by three-component in one-pot, the corresponding dihydropyri-dine benzyl 4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-5-oxo-1,4,5,6-tetrahydro-1,6-naphthopyridine-3-carboxylate（2a）was obtained with the yield of 84%. The subsequent alkylation of 2a using triethyl orthoformate and concentrated sulfuric acid at 130~135 °C provided benzyl 5-ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-1,4-dihydro-1,6-1,6-naphthopyridine-3-carboxylate (3a) with the yield of 66%. Removal of the protecting group was achieved under mild conditions using H₂ and Pd/C to provide carboxylic acid product 5-ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2, 8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylic acid （4a） with a good yield of 91%. Conversion into amide was carried out in DMF by acid activation using N, N, N′, N′-tetramethyl-O-(7- AzabenzoTriazol-1-yl) uranium hexafluorophosphate (HATU) and N,N-diiso-propylethylamine (DIPEA) following treatment with aqu-eous ammonia, and the aimed compound 5-ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2, 8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxamide（A1）was obtained in 76% yield. The same procedures were performed for the synthesis of the target products A2 started from the corresponding aldehyde 1b.

Scheme 1.

Synthesis of naphthalidinamide analogues.

Haloalkane is frequently employed as an alkylating agent for preparation of alkoxy pyridine from pyridine.^{26 –28} Compound 2b reacted with 1-(bromomethyl) cyclopropane in the presence of cesium carbonate at 45~50 °C for 6 h without purification, followed by reduction with H₂, the key intermediate 5-(cyclopropylmethoxy)-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylic acid (5) was generated with total yield of 71% for two steps, followed by amination with ammonia, the target compound B was obtained with 79% yield (Scheme 2).

Scheme 2.

Synthesis of compound B from 2b.

Cell-based transactivation assay

The cell-based transactivation assay of the targeted compounds was evaluated and finerenone (BAY-94-8862) was selected as the positive control. The MR activity assay results were listed in Table 1.

Table 1.

The inhibitory activities of target compounds against MR receptor. ^a

a

The values were means ± SD from three independent experiments.

b

Cell-based transactivation assay was performed with the luciferase assay. Each compound was performed at 10 doses from 10 mM to 0.51 nM with 3 fold concentration gradient, and each test in duplicate.

The development of novel nonsteroidal MRAs began with “A” ring modification using a pharmacophore fusion strategy of finerenone. Instead of CN in R1, CF₃ and F as new hydrogen bond acceptor, the novel 4-aryl-1,4-dihydro-1,6-naphthalidinamide derivatives A1 and A2 were synthesized for preliminary evaluation. MR activity assay results indicated that all the two compounds showed significant antagonistic activities (IC₅₀⩽161.6 nM). Improved activities against MR were found for the compound bearing F (R₁) group, and the promising candidate A2 (IC₅₀ = 30.3 nM) was identified. Furthermore, the “B” ring modification was also proceed based on the promising structure of A2, we decorated the naphthyridine scaffold with cyclopropane instead of CH₃, and the potent compound B (IC₅₀ = 5.6 nM) was discovered, which was more potent than the control compound BAY 94-8862 (IC₅₀ = 28.1 nM). It can be found that the rigid small ring as cyclopropane is beneficial to increase the potential. In summary, an active compound B with potent MR antagonistic activity was obtained for further development, and these results indicated that F at R₁ position was an important group for maintaining the activity.

CLogP represents the hydrophobicity of a compound, the larger CLogP means the greater hydrophobicity and the smaller hydrophilicity. TPSA refers to topological polar surface area, which is a physical parameter that describes the chemical properties of molecules. The higher tPSA value means the greater polarity and the higher solubility of the compound. The properties of the targeted compounds including molecular weight, CLogP and tPSA values is associated with activity and pharmacokinetic parameters. ²⁹ Regarding to these synthesized naphthalidinamide derivatives, they were found to be potential in terms of potency, among them, the promising compound B displayed the best potency which was superior to the marked BAY 94-8862. The properties comparison of compounds and BAY 94-8862 were summarized in Table 2.

Table 2.

Properties ^a of the target compounds and BAY 94-8862.

Compd.	Molecular weight （MW）	CLogP	tPSA
BAY-94-8862	378.43	4.11	109.71
A1	421.42	4.55	85.94
A2	371.41	4.63	85.94
B	399.46	5.08	85.94

a

CLogP as well as tPSA were predicted by ChemBioDraw 22. and the digitals were rounded to two digits.

The data indicated that the predicted CLogP values were less than 5.08 and the predicted tPSA values were less than 100.5 for all target compounds. According to the cell-based transactivation assay, compounds B displayed the most potent againstic activity against MR with larger CLogP than compounds A1 and A2. For MW, there was no consistency to the potencies. For the promising compound B, compared with BAY-94-8862 (MW = 378.43, CLogP = 4.11), B (MW = 399.46, CLogP = 5.08) displayed more potent antagonistic activity against MR with equivalent MW and higher CLogP value.

Selectivity against other nuclear hormone receptors

In order to investigate the selectivity to other nuclear hormone receptors (NHRs), such as the glucocorticoid receptor (GR), AR, and PR, the promising compounds A2 and B were selected for further test of againstic activity over GR, AR, and PR in stable steroid hormone receptor cell lines. In this program, spironolactone was selected as MR positive control, enzalutamide was selected as AR positive control, mifepristone was selected as PR and GR positive control, the cellular activity data were shown in Table 3.

Table 3.

Cell-based transactivation assay for MR, AR, PR, and GR. ^a

Compd.	Cell-based activity (IC50, nM) b
	MR	AR	PR	GR
Positive	28.5 (spironolactone)	319.2 (enzalutamide)	5.9 (mifepristone)	24.4 (mifepristone)
BAY-94-8862	28.1	>10 μmol	8532	4968
A2	30.3	3876	>10 μmol	6100
B	5.6	2841	>10 μmol	5310

a

MR: mineralocorticoid receptor, GR: glucocorticoid receptor, AR: androgen receptor, PR: progesterone receptor.

b

All experiments were repeated at least three times.

The results in Table 3 indicated that compound A2 and B displayed excellent (>300-fold) selectivity to progesterone receptor as well as the control compound BAY-94-8862 (>270-fold). Besides, the potent compounds A2 and B showed good (>200-fold) selectivity over the GR which is superior to BAY-94-8862 (>160-fold). For AR, compound B (>500-fold) showed higher selectivity than the control compound BAY-94-8862 (>320-fold), while B (>120-fold) displayed a weaker but satisfied selectivity.

Generally, the potent compounds A2 and B displayed good selectivity over the GR, AR, and PR, a breakthrough in terms of potency and selectivity of 4-aryl-1,4-dihydro-1,6-naphthalidinamide derivatives was achieved by introducing F to the “A” ring and cycloalkyl to the “B” ring.

Liver microsomes stability

The half-life time (t_1/2) and intrinsic clearance (CL_int) parameters were commonly used to evaluate their metabolic stabilities that could give good indications of the in vivo hepatic clearance.^30,31 BAY-94-8862 was selected as the control compound, the metabolic stability of the promising compound A2 and B was determined with various species of liver microsomes, such as human, monkey, dog, mouse, and rat liver microsomes (Table 4). The five types of liver microsomes used in this project are all commercial and purchased from BIOIVT, RILD, and Corning, all of which are transported to Hunan Hengxing Pharmaceutical Technology Co., Ltd. via cold chain transportation, with reliable quality. The reception data of liver microsomes from five species are all stored in the archives of Hunan Hengxing Pharmaceutical Technology Co., Ltd.

Table 4.

Stabilities of compounds A2 and B in liver microsomes of various species.

Liver microsomes	BAY-94-8862			A2			B
	t1/2 (min)	CL(mL/min/kg)	CLint (mL/min/kg)	t1/2 (min)	CL(mL/min/kg)	CLint (mL/min/kg)	t1/2 (min)	CL(mL/min/kg)	CLint (mL/min/kg)
Human	11.7	0.119	112	10.0	0.139	131	4.0	0.347	328
Monkey (Cynomolgus)	5.4	0.255	367	1.8	0.787	1134	0.7	2.110	3031
Dog (Beagle)	36.7	0.038	54	20.6	0.067	97	12.7	0.109	157
Mouse	9.9	0.140	252	9.6	0.144	260	6.5	0.214	385
Rat	11.8	0.117	285	6.5	0.215	522	5.6	0.246	597

Table 4 revealed that compound A2 and B were easily metabolized in human liver microsomes with half-life time of 10.0 and 4.0 min respectively, and the control BAY-94-8862 showed the similar result. For other species, significant discrepancy was generated among different species. The dog liver microsome metabolized compound A2 and B in 20.6 and 12.7 min, which was more slowly than other microsomes. Furthermore, compound A2 and B were metabolized in a shorter time than BAY-94-8862 in mouse, rat, and monkey liver microsomes. So, these compounds could be metabolized more rapidly than BAY-94-8862 after binding with the target and showed less toxicity, which was displayed during our in vivo experiments.

Theoretical docking analysis

To further explain the potent antagonistic activity against MR of compound B, the protein crystal (PDB ID: 2ABI) was selected as template, the binding mode of the molecule with the wild-type MR ligand in the active site was predicted by the AutoDock software. ³² The docking mode (Fig. 3) indicated that compound B bound to the outer edge of the ATP-binding pocket. As depicted in Fig 4, a traditional hydrogen bond was formed between the carbonyl oxygen atom and Lys785, besides, two additional hydrogen bonds were also observed between the hydrogen atoms of amide and Leu795. Furthermore, the π-alkyl interaction was formed between the benzene core and Leu795, the π-sigma interaction was formed between the pyridine core and Ile799. Interestingly, alkyl interactions were also observed between the exocyclic methyl of B with Ile802 and Val781. Briefly, the theoretical docking analysis was consistent with the potent antagonistic activities against MR of compound B.

Figure 3.

Interaction map of compound B with MR ligand.

Figure 4.

The 2D diagrams of protein–ligand interactions between compound B and 2ABI.

Conclusions

In summary, based on the structure of the third-generation MRA, Finerenone (BAY 94-8862), by modifying “A” and “B” ring using a structure-based design strategy, some novel naphthalidinamide derivatives were designed, synthesized, and biologically evaluated as nonsteroidal antagonist of the MR. The development of novel nonsteroidal MRAs began with “A” ring modification using a pharmacophore fusion strategy of finerenone. From this effort, the promising candidate A2 (IC₅₀ = 30.3 nM) was identified in vitro biological evaluation. Furthermore, “B” ring modification was also proceed based on the promising structure of A2, and the most potent compound B (IC₅₀ = 5.6 nM) was discovered, which was more potent than the control compound BAY 94-8862 (IC₅₀ = 28.1 nM). The results showed that a rigid small ring as cyclopropane is beneficial to increase the potent. Selectivity against other NHRs was also determined in stable steroid hormone receptor cell lines; the potent compounds A2 and B displayed good selectivity over the GR, AR, and PR. Besides, the microsomal stabilities in human, dog, monkey, rat, and mouse indicate that compounds A2 and B displayed shorter metabolism time than the related compound BAY-94-8862.

Experimental Section

Chemistry

Instruments

Unless additional specification, all reactions were performed in air or moisture site. Reaction processes were monitored by thin-layer chromatography (TLC) with silica gel aluminum plates (60F-254) and spots were visualized with UV light at 254 nm or iodine. Flash column chromatography was performed using silica gel (300~400 mesh). ¹ H NMR and ¹³C NMR spectra were recorded in CDCl₃ or DMSO-d₆ at room temperature on Bruker Avance spectrometers with Tetramethylsilane (TMS) as internal standard. Data of ¹H NMR are reported as follows: chemical shift, multiplicity (s = singlet, br = broad, d = doublet, t = triplet, m = multiple), coupling constants and integration. High-resolution mass spectra were analyzed by Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS.

Materials

Solvents for reaction, extraction and Flash column chromatography were reagent grade and used as received. Unless otherwise required, all the reagents were commercially obtained with analytical grade and used without further purification. Frequently dry solvents (CH₂Cl₂, Tetrahydrofuran (THF), DMF, etc.) were anhydrous packaged from suppliers with Sure/Seal bottles. Yields mean isolated yields unless otherwise stated.

Synthesis

General procedures for the synthesis of compounds A1 and A2

Benzyl 4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-5-oxo-1,4,5,6-tetrahydro-1, 6-naphthyridine -3-carboxylate (2a)

2-Methoxy-4-(trifluoromethyl) benzaldehyde 1a (2.04 g, 10.00 mmol) and 4-amino-5- methylpyridin-2(1H)-one (1.24 g, 10.00 mmol) was suspended in 50 mL isopropanol, benzyl 3-oxobutanoate (2.86 g, 12.00 mmol) was added drop wisely, then the mixture was sealed and reacted under vigorous stirring at 110 °C for 12 h. The reaction was cooled to 0~5 °C, filtrated by suction and washed with isopropanol, the crude product was obtained as a dark solid. Purification was achieved by column chromatography (acetate/petroleum = 1/3) to give the title compound 2a as an off-white solid (4.07 g, 84%). ¹H NMR (400 MHz, CDCl₃, ppm) δ 11.95 (s, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.29 (dd, J = 3.4, 1.6 Hz, 2H), 7.17-7.12 (m, 2H), 6.99 (m, 1H), 6.93 (d, J = 1.6 Hz, 1H), 6.76 (d, J = 1.3 Hz, 1H), 5.90 (s, 1H), 5.44 (s, 1H), 5.13-4.97 (dd, J = 12.0 Hz, 2H), 3.64 (s, 3H), 2.41 (s, 3H), 2.02 (s, 3H). MS (EI⁺): m/z = 485.27 [M + H]⁺.

Benzyl 5-ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-1,4-dihydro-1, 6-naphthyridine -3-carboxylate (3a)

To a mixture of triethyl orthoformate (25.0 g, 168.7 mmol) and compound 2a (2.50 g, 5.2 mmol), concentrated sulfuric acid (98%, 1 mL) was added at 90 °C, then, the mixture was sealed and stirred at 130~135 °C for 3 h and the reaction was clear, after cooling to room temperature, the excessive triethyl orthoformate was evaporated in vacuo, and the residue was purified by column chromatography (acetate/petroleum = 1/6), title compound 3a (1.75 g, 66%) was obtained as a light yellow solid. ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.65 (s, 1H), 7.33-7.29 (m, 3H), 7.20-7.07 (m, 2H), 7.01 (d, J = 7.9 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H), 5.93 (s, 1H), 5.54 (s, 1H), 5.22-4.94 (dd, J = 12.0 Hz, 2H), 4.14 (m, 2H), 3.58 (s, 3H), 2.47 (s, 3H), 2.16 (s, 3H), 1.19 (t, J = 7.0 Hz, 3H). MS (EI⁺): m/z = 513.30 [M + H]⁺.

5-Ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylic acid (4a)

Intermediate 3a (1.50 g, 2.93 mmol) was dissolved in 75 mL methanol, 10% Pd-C (0.075 g, 64% water) was added to the solution, the reaction was stirred under H₂ atmosphere at 40~45 °C for 6 h. Pd-C was filtered while the mixture was hot, the filtration was evaporated in vacuo, purified by column chromatography (acetate/petroleum = 1/2) title compound 4a (1.12 g, 91%) was obtained as a white solid. ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.68 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.16-7.03 (m, 2H), 5.47 (s, 1H), 4.17 (qd, J = 7.1, 4.5 Hz, 2H), 4.01 (s, 3H), 2.54 (s, 3H), 2.18 (d, J = 0.8 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H). MS (EI⁺): m/z = 423.23 [M + H]⁺.

5-Ethoxy-4-(2-methoxy-4-(trifluoromethyl)phenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxamide (A1)

Compound 4a (0.80 g, 1.89 mmol) was dissolved in 10 mL DMF, followed by introducing N, N, N′, N′-Tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (HATU) (1.437 g, 3.78 mmol) and N, N-Diisopropylethylamine (DIPEA) (0.488 g, 3.78 mmol), the mixture was stirred at room temperature for 1 h, then aqueous ammonia (28%, 5 mL) was added. The reaction mixture was sealed and stirred for 2 h at 100 ℃. After addition of H₂O (15 mL) and extraction with EtOAc (20 mL × 3), the combined organic phases were dried with anhydrous sodium sulfate, and the crude product was purified by column chromatography (DCM: MeOH = 0–10%) to give the title compound A1 as a pale-yellow solid (0.61 g, 76%). ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.68 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 7.9 Hz, 1H), 7.06 (s, 1H), 5.47 (s, 1H), 4.21–4.13 (m, 2H), 4.01 (s, 3H), 2.54 (s, 3H), 2.18 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃, ppm) δ 169.69, 160.08, 153.94, 144.71, 143.86, 142.66, 139.72, 131.15, 118.80, 110.39, 106.81, 105.21, 103.73, 61.41, 55.85, 30.55, 20.84, 14.54, 13.00. HRMS (ESI): 422.1692 calcd for C₂₁H₂₂F₃N₃O₃ [M + H]⁺, found: 422.1712.

Benzyl 4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-5-oxo-1,4,5,6-tetrahydro-1,6-naphthopyridine-3-carboxylate (2b)

Compound 1b as starting material, the title compound 2b was prepared according to the synthetic procedures for 2a as a white solid with the yield of 77%. ¹H NMR (400 MHz, CDCl₃, ppm) δ 11.55 (s, br, 1H), 7.45–7.38 (m, 2H), 7.32–7.29 (m, 2H), 7.17–7.14 (m, 2H), 6.86 (d, J = 1.2 Hz, 1H), 5.87 (d, J = 16.5 Hz, 1H), 5.57 (d, J = 2.3 Hz, 1H), 5.11 (d, J = 12.0 Hz, 1H), 5.01 (d, J = 12.0 Hz, 1H), 2.45 (s, 3H), 2.06 (s, 3H), 1.24 (s, 3H). MS (EI⁺): m/z = 435.29 [M + H]⁺.

Benzyl 5-ethoxy-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylate (3b)

Compound 2b as starting material, the title compound 3b was prepared according to the synthetic procedures for 3a as a pale solid with the yield of 59%. ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.64 (s, 1H), 7.32 (m, 3H), 7.19 (m, 2H), 7.13 (d, J = 7.7 Hz, 1H), 6.44 (m, 2H), 5.88 (s, 1H), 5.14 (d, J = 12.0 Hz, 1H), 5.04 (d, J = 12.0 Hz, 1H), 4.15 (qd, J = 7.0, 4.2 Hz, 2H), 3.53 (s, 3H), 2.46 (s, 3H), 2.15 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H). MS (EI⁺): m/z = 463.34 [M + H]⁺.

5-Ethoxy-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylic acid (4b)

Compound 3b as starting material, the title compound 4b was prepared according to the synthetic procedures for 4a as a white solid with the yield of 86%. ¹H NMR (400 MHz, CDCl₃, ppm) δ 8.11 (s, 1H), 7.28 (s, 2H), 7.10 (dd, J = 8.4, 2.5 Hz, 1H), 4.70 (s, 1H), 3.05–2.95 (m, 2H), 1.39 (s, 3H), 1.31 (s, 3H), 1.28 (s, 6H). MS (EI⁺): m/z = 373.26 [M + H]⁺.

5-Ethoxy-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxamide (A2)

Compound 4b as starting material, the title compound A2 was prepared according to the synthetic procedures for A1 as a pale solid with the yield of 71%. ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 7.67 (s, 1H), 7.56 (s, 1H), 6.95–6.99 (m, 1H), 6.78–6.81 (m, 1H), 6.59–6.64 (m, 1H), 5.22 (s, 1H), 4.51–4.55 (m, 2H), 4.19–4.25 (m, 3H), 4.04–4.09 (m, 1H), 3.77 (s, 3H), 3.08–3.15 (m, 1H), 2.03–2.19 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 170.27, 159.63, 156.99, 144.79, 144.16, 138.53, 132.56, 132.52, 131.20, 131.10, 112.13, 107.28, 107.08, 106.29, 104.34, 99.49, 99.24, 73.68, 73.60, 66.78, 56.38, 34.25, 32.10, 18.61, 14.20. HRMS (ESI): 372.1723 calcd for C₂₀H₂₂FN₃O₃ [M + H]⁺, found: 372.1741.

5-(Cyclopropylmethoxy)-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxylic acid (5)

To a suspension of 50 mL DMF, 2b (1.20 g, 2.76 mmol) and caesium carbonate (1.80 g, 5.53 mmol), a solution of 10 mL DMF and 1-(bromomethyl)cyclopropane (0.41 g, 3.04 mmol) were added drop wisely. Then, the mixture was stirred at 45~50 °C for 6 h. After completion of the reaction monitored by TLC, 100 mL water was added and the mixture was extracted with dichloromethane (60 mL × 3). Combined the organic phases, dried with anhydrous sodium sulfate, the solvent was evaporated in vacuo and the crude product was afforded. The residual was dissolved in 60 mL THF, 10% Pd-C (240 mg, 64% water) was added to the solution, the reaction was stirred under H₂ atmosphere at 55 °C for 8 h. Pd-C was filtered while the mixture hot, the filtration was evaporated in vacuo, purified by column chromatography (acetate/petroleum = 1/2) title compound 5 (780 mg, 71%) was obtained as a white solid. ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 7.56 (s, 1H), 7.28 (s, 1H), 7.00–6.96 (m, 1H), 6.78 (d, J = 11.5 Hz, 2H), 6.63–6.59 (m, 2H), 5.17 (s, 1H), 3.78 (s, 3H), 2.18 (s, 3H), 2.05 (s, 3H), 1.03–1.10 (m, 1H), 0.40–0.38 (m, 2H), 0.26 (d, J = 4.8 Hz, 2H). MS (EI⁺): m/z = 399.22 [M + H]⁺.

5-(Cyclopropylmethoxy)-4-(4-fluoro-2-methoxyphenyl)-2,8-dimethyl-1,4-dihydro-1,6-naphthopyridine-3-carboxamide (B)

Compound 5 (650 mg, 1.63 mmol) was dissolved in 10 mL DMF, followed by introducing N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) (932 mg, 2.45 mmol) and triethylamine (330 mg, 3.26 mmol), the mixture was stirred at room temperature for 1 h, then aqueous ammonia (28%, 5 mL) was added. The reaction mixture was sealed and stirred for 1 h at 80℃. After addition of H₂O (15 mL) and extraction with EtOAc (20 mL × 3), the combined organic phases were dried with anhydrous sodium sulfate, and the crude product was purified by column chromatography (DCM: MeOH = 0~10%) to give the title compound B as a white solid (513 mg, 79%). ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.65 (d, J = 1.0 Hz, 1H), 7.17 (dd, J = 8.4, 6.6 Hz, 1H), 6.69 (s, 1H), 6.62–6.55 (m, 2H), 5.77 (s, 1H), 5.38 (s, 1H), 4.02–3.97 (m, 1H), 3.94 (s, 3H), 3.92–3.88 (m, 1H), 2.52 (s, 3H), 2.17 (d, J = 0.9 Hz, 3H), 1.11–1.07 (m, 1H), 0.53–0.45 (m, 2H), 0.27–0.21 (m, 1H), 0.17 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 170.36, 159.91, 148.23, 145.07, 144.25, 136.30, 132.53, 120.95, 118.20, 112.12, 107.30, 103.14, 61.16, 38.04, 18.23, 14.81, 14.29. HRMS (ESI): 398.1880 calcd for C₂₂H₂₄FN₃O₃ [M + H]⁺, found: 398.1887.

Biological assay

Determination of IC₅₀ values in cell-based assays

IC₅₀ determinations for humanized oxosteroid receptors (MR: mineralocorticoid receptor, GR: glucocorticoid receptor, AR: androgen receptor, PR: progesterone receptor) were performed with Bright-Lite Luciferase Assay from Vazyme according to the manufacturer’s instructions. Luc2P-GAL4-HEK293 (transient MR HEK293 cell, E5800) was purchased from ATCC and taken as an experimental cell line. Using JetPRIME transfection reagent, pBIND-MR as plasmids, all cells were cultured in DMEM medium (Corning), supplemented with 10% FBS (Invitrogen) and 200 μg/mL Hygromycin B (Solarbio), cells were connected to 96-well plates at a density of 3000 cells/well using complete medium. A gradient-diluted compound was added to the cells to dilute 9 concentrations from a final concentration of 3 μM, 3 replicates per concentration. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂, and exposed to compounds treatment for 24 h, luciferase activity was determined with a luminescence-detecting video camera system. GraphPad Prism Software (version 5.0, GraphPad Software Inc., San Diego, CA, USA) was used for curve fitting and calculation of IC₅₀ values. The IC₅₀ values were determined from at least three independent experiments performed in duplicate.

Compound stability test in liver microsome

Compounds were incubated with human (BIOIVT), monkey (RILD), dog (Corning), rat (Corning), and mouse (Corning) liver microsomes. The liver microsomes (20 mg protein/mL) was incubated in 37 ℃ water bath for 3 min, then, 25 μL β-NADPH solution (4 mM) was added to 75 μL incubation solution without β-NADPH, vortexed for 30 s with 100 μL total volume, and reactions were initiated in 0.05 M Phosphate buffer (pH = 7.4) at 37 °C for 0, 5, 15, 30, 60 min. The termination agent (including internal standard) was added, followed by vortex and centrifuge, the reaction was quenched, and the amount of the remaining compound was analyzed using LC-MS/MS (ESI⁺). LC-MS conditions are as follows: mobile phase A: acetonitrile, B: 0.2% acetic acid with water; gradient, time, and flow rate (A%): initial (0.4, 15); 1 (0.4, 95); 2 (0.4, 95); 3 (0.4, 15). Chromatographic column: Waters ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm), injection volume: 2 µL.

Protein modeling and ligand docking

The X-ray structure of the wild-type MR ligand binding domain complexed with deoxycorticosterone (PDB ID: 2ABI) was selected as a model for docking study. Autodock was used to determine the molecules rotatable bonds and torsion angles, then saved with pdb.qt format after charge treatment. Structure of the compound B was constructed with GaussView 6.0 software, based on the density functional theory (DFT), and the geometry was optimized using the gasian16 program at the B3LYP/6-31+G (d) level. The docking simulation of small molecules and dominant conformation was performed with Autodock, the interaction analysis of the dominant conformation with the lowest energy was carried out using Discovery Studio (DS) software (https://www.3ds.com/products-services/biovia/).

Supplemental Material

sj-docx-1-chl-10.1177_17475198251351168 – Supplemental material for Design, synthesis, and evaluation of naphthalidinamide analogues as nonsteroidal mineralocorticoid receptor antagonists

Supplemental material, sj-docx-1-chl-10.1177_17475198251351168 for Design, synthesis, and evaluation of naphthalidinamide analogues as nonsteroidal mineralocorticoid receptor antagonists by Mingguang Zhang, Yan Pang, Yurui Zhao, Sibei Chen and Cheng Guo in Journal of Chemical Research