Synthesis and biological evaluation of novel 2,3-pyrazole ring-substituted-4,4-dimethyl lithocholic acid derivatives as selective protein tyrosine phosphatase 1B (PTP1B) inhibitors with cellular efficacy

Abstract

In our continued efforts to develop lithocholic acid (LCA) analogues as selective PTP1B inhibitors, 14 novel 2,3-pyrazole ring-substituted-4,4-dimethyl derivatives were synthesized and evaluated against PTP1B, as well as homologous protein tyrosine phosphatases (PTPs). All compounds were shown to be more potent and selective PTP1B inhibitors than LCA (IC₅₀ = 12.74 μM) with IC₅₀ values ranging between 0.42 to 4.49 μM. Moreover, treatment of CHO/hIR cells with 4,4-dimethyl-2′-(p-fluoro phenyl)-2′H-chola-2,5-dieno[3,2-c]pyrazol-24-oic acid (30) or 4,4-dimethyl-2′-(o-chloro phenyl)-2′H-chola-2,5-dieno[3,2-c]pyrazol-24-oic acid (34) increased the phosphorylation levels of IR and Akt in a dose dependent manner. The promising findings in this study suggest that further investigation of these compounds for the treatment of metabolic disorders is warranted.

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Introduction

Type 2 diabetes and obesity are metabolic disorders that are characterized by insulin and leptin resistance.¹ Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that belongs to the protein tyrosine phosphatase (PTPs) family, which is involved in the regulation of several important physiological pathways. PTP1B dephosphorylates the insulin receptor (IR), IR substrate 1 (IRS1), and Janus kinase 2, resulting in a negative regulation of insulin and leptin signal transduction.^2,3 Moreover, PTP1B activity and expression are increased in insulin resistant humans and rodents,^4,5 whereas PTP1B deficient mice were found to resistant to weight gain and remain the insulin sensitivity as compared to the wild type mice.^6,7 Therefore, PTP1B has been proposed as novel target for the treatment of type 2 diabetes and obesity. It is worth noting that PTPs share a high degree of structural conservation in the active site.⁸ T-cell protein tyrosine phosphatase (TCPTP) has a sequence identity of about 74% in the catalytic domains with PTP1B.⁹ Although it is unknown whether the combined inhibition of PTP1B and TCPTP will lead to severe side effects, pancreatic-TCPTP knockout mice exhibited impaired glucose tolerance and attenuated glucose-stimulated insulin secretion when challenged with prolonged high fat feeding.¹⁰ Another drawback limited the application of most inhibitors for further development is poor intracellular PTP1B inhibitory potency. Because of the highly cationic character of the active site of PTP1B, most potent phosphotyrosine (pTyr) mimetics are highly negative charged providing these compounds with poor membrane permeability.¹¹

Natural products have been widely regarded as privileged structures in drug discovery efforts, and scaffolds derived from natural sources have led to the nearly half of the approved drugs in the past 2 decades. Many natural products and their derivatives were found possessing PTP1B inhibitory activities with moderate to good selectivities.^12,13 Trodusquemine (MSI-1436, 1, Fig. 1) is a noncompetitive and selective PTP1B inhibitors with moderate potency (IC₅₀ = 1 μM) which induces weight loss without significant associated toxicities in animal models.^14,15 Recently, several pentacyclic acid triterpenoids (ursolic, oleanolic, moronic and morolic acids, 2–5, Fig. 1) were reported to present significant antidiabetic activities in non-insulin dependent diabetic rat models.¹⁶ In our previous study,¹⁷ natural steroid compound lithocholic acid (LCA, 6, Fig. 1) was identified as a PTP1B inhibitor with an IC₅₀ value of 12.74 μM. Embedding of fused heterocycles (substituted pyrazole, oxazole, substituted pyrimidine, aminothiazole and pyrazine) on ring A resulted in an improvement in potency and selectivity over TCPTP as exemplified by compound 7 (IC₅₀ = 1.62 μM; TCPTP/PTP1B = 14.1-fold, Fig. 1). Herein, we reported our continued efforts in the structure–activity relationship (SAR) studies and biological evaluations of 2,3-pyrazole ring-substituted-4,4-dimethyl lithocholic acid derivatives.

Fig. 1 Structures of selected pentacyclic triterpenoids and steroids as PTP1B inhibitors.

Results and discussion

Chemistry

As shown in Scheme 1, compound 10 with two methyls at C-4 position was obtained from lithocholic acid in 5 steps.¹⁷ A typical formylation at C-2 of 10 was carried out by using sodium hydride and ethyl formate to yield 11, which was then reacted with various substituted hydrazine hydrochlorides in ethanol to afford substituted pyrazoles over two steps in 36–62% overall yields. The substitution position of phenyl at the pyrazole ring was determined via X-ray crystallography. Single crystal of compound 33 was obtained by slow evaporation method using mixed solvent of chloroform and methanol (4/1 v/v). The crystal structure¹⁸ (Fig. 2) shows that the phenyl group was at the N-1 position, and this is well agreement with previous report.¹⁹ When it comes to the methyl substituted pyrazoles, both of the isomers (12a and 12b, structures determined by NOE, see ESI†) were obtained. 12a, 12b and 13–24 were hydrolyzed in the presence of lithium hydroxide to give the desired carboxylic acids 25a, 25b and 26–37. All the final compounds were characterized by ¹³C NMR, ¹H NMR and HRMS.

Scheme 1 Reagents and conditions: (a) (i) MeOH, SOCl₂, 99%, (ii) PCC, DCM, 93%; (b) (i) HOAc, DCM, Br₂, (ii) Li₂CO₃, LiBr·H₂O, DMF, 48%; (c) t-BuOK, t-BuOH, MeI, 50%; (d) NaH, HCOOEt, 98%; (e) R-NHNH₂·HCl, EtOH, 36–62%; (f) MeOH/H₂O, LiOH·H₂O, 62–81%.

Fig. 2 X-ray structure of compound 33.

Structure–activity relationship

Compounds 25a, 25b and 26–37 were evaluated in the enzyme inhibition assay (p-NPP assay) against PTP1B. Oleanolic acid (OA) was used as reference compound (Table 1). The inhibitory activities on the homologous enzyme TCPTP were investigated. We also tested the inhibitory activities on other homologous PTPs (LAR, SHP1 and SHP2).

Table 1 Inhibitory activities against PTP1B and TCPTP

Compounds	IC50 (μM)		TCPTP/PTP1B^a
	PTP1B	TCPTP
a TCPTP/PTP1B, the ratio of IC50 of TCPTP and PTP1B.b IC50 > 40 μM, the inhibition (%) on TCPTP was between 5% and 50% at 40 μM.c The inhibition (%) on TCPTP was below 5% at 40 μM.d Oleanolic acid as positive control.
25a	2.82 ± 0.57	>40^b	>14.2
25b	4.49 ± 0.68	NA^c	>8.9
26	2.66 ± 0.51	>40	>15
27	0.81 ± 0.06	>40	>49.4
28	1.12 ± 0.11	NA	>35.7
29	2.35 ± 0.27	>40	>17
30	0.42 ± 0.07	4.53 ± 0.19	10.8
31	0.93 ± 0.19	NA	>43
32	1.17 ± 0.27	>40	>34.2
33	1.24 ± 0.22	NA	>32.3
34	0.86 ± 0.03	3.99 ± 0.18	4.6
35	1.34 ± 0.25	28.99 ± 1.61	21.6
36	0.73 ± 0.09	23.41 ± 0.53	32.1
37	1.03 ± 0.05	5.70 ± 0.36	5.5
6	12.74 ± 1.41	20.50 ± 2.10	1.6
7	1.62 ± 0.08	22.78 ± 4.36	14.1
OA^d	2.78 ± 0.19	6.00 ± 0.15	2.1

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It was intriguing that all of the 14 compounds were found to be more potent PTP1B inhibitors than that of LCA (6, IC₅₀ = 12.74 μM) suggesting that substitutions on 2,3-pyrazole-substituted-4,4-dimethyl lithocholic acid are well tolerated. 10 derivatives (27, 28, 30–37) with substituted phenyl groups presented improved PTP1B inhibitory activities than compound 7 (IC₅₀ = 1.62 μM). 30 (IC₅₀ = 0.42 μM) emerged as the most potent PTP1B inhibitor with almost 4-fold improved inhibitory potency compared to compound 7. Replacement of phenyl by methyl group (25a) reduced the inhibitory activity with IC₅₀ value of 2.82 μM, which suggested that the phenyl group is favorable for the activity. Among the para-position substituted compounds (26, 27, 28, 29 and 30), the para-methyl substituted compound 27 (IC₅₀ = 0.81 μM) and the para-chloro substituted compound 28 (IC₅₀ = 1.12 μM) showed improved PTP1B inhibitory activities compared to compound 7 (IC₅₀ = 1.62 μM). The para-fluoro substituted compound 30 was most active towards PTP1B with an IC₅₀ value of 0.42 μM. However, the para-methoxy substituent 26 and the para-bromo substituent 29 reduced the activity to 2.66 μM and 2.35 μM, respectively. Compared to the para-methyl substituted compound 27, ortho-methyl compound (32) and meta-methyl compound (31) exhibited similar PTP1B inhibitory activities, with IC₅₀ values of 1.17 μM and 0.93 μM, respectively. The 3,4-dimethyl-substituted compound 36, in the combination of para-methyl substitution and meta-methyl substitution, showed an increased inhibitory activity (IC₅₀ = 0.73 μM). All the chloro-substituted compounds (28, 33, 34 and 37) exhibited similar inhibitory activities at around 1 μM.

Selectivity over other homologous PTPs

The methyl substituted compounds (25a and 25b) did not show noticeable inhibitory activities against TCPTP. When it comes to the phenyl substituted compounds, the para-fluoro substituted compound 30, which emerged as the most potent PTP1B inhibitor, remained about 11-fold selectivity over TCPTP. All the methyl substituted compounds (27, 31, 32, 35 and 36) had good selectivity over TCPTP, 27, 31 and 32 did not exhibit noticeable inhibition towards TCPTP under 40 μM. The inhibition (%) of the chloro-substituted compounds 28 and 33 against TCPTP at 40 μM was below 5%, while the dichloro-substituted compound (37) showed decreased selectivity over TCPTP. It seemed that the electron density of the phenyl ring had influence on the selectivity. In detail, fluoro and dichloro substituted compounds (30 and 37) with lower electron density on the phenyl showed more potent inhibition to TCPTP, while remaining similar inhibition to PTP1B when compared with methyl, bromo or monochloro substituted compounds with one exception (34). Encouragingly, all the tested six compounds (27, 28, 30, 31, 32 and 33) exhibited no obvious inhibition on LAR, SHP1 and SHP2 at 40 μM (Table 2).

Table 2 Inhibition (%) of compounds 27, 28, 30, 31 32 and 33 against other homologous PTPs at 40 μM

Compounds	Inhibition^a (%)
	LAR	SHP1	SHP2
a Inhibition at 40 μM.
27	20.62	23.79	12.31
28	6.25	5.29	2.15
30	24.56	19.82	22.25
31	24.75	6.26	4.12
32	1.02	3.90	5.59
33	4.09	8.30	3.90

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Cellular efficacy

PTP1B is a negative regulator in the insulin signaling pathway by dephosphorylating specific phosphotyrosine residues of IR. Inhibition of PTP1B activity could enhance the phosphorylation of IR and downstream protein Akt, and thus activate the downstream signaling pathway. Four compounds (30, 31, 34 and 36) with good potency and/or selectivity were selected to evaluate the cellular efficacy in CHO/hIR cells. As shown in Fig. 3, two compounds (30 and 34) were found to remarkably increase the phosphorylation levels of IR and its downstream protein Akt in a concentration-dependent manner (3, 10 μM), suggesting that 30 and 34 may effectively stimulate the insulin signaling by inhibiting the activity of PTP1B.

Fig. 3 Effect of 30 and 34 on IR phosphorylation and Akt phosphorylation in CHO/hIR cells. CHO/hIR cells were incubated with 200 μM sodium orthovanadate, 0.2% DMSO, or compound 30 (or 34) for 3 h, and then treated with 10 nM insulin for 10 min. V: 200 μM sodium orthovanadate used as a positive control (PC).

Enzyme kinetic study and molecular docking analysis

In our previous study, the binding modality of compound 7 with PTP1B was investigated,¹⁷ since the substituent on the phenyl group had a crucial influence on potency and selectivity, it is necessary to find out whether the binding modality was changed after substituent introduction.

Firstly, the most active compound 30 was selected for PTP1B enzyme kinetic study. As shown in Fig. 4, the V_max value retained constant while K_m value increased with the mounting compound concentration, indicating that 30 was a competitive PTP1B inhibitor (K_i = 0.26 μM). Afterwards, a molecular docking analysis of 30 was carried out using LibDock available with Discovery Studio 2.1.²⁰ The preferred coordination mode of 30 with PTP1B was presented in Fig. 5, Fig. 5(b) shows the binding interactions of 30 with PTP1B. The carboxylic acid group of 30 is bound into the active site. Similar to that of 7, the carbonyl of the –COOH may interact with Arg221 via a salt bridge and the hydroxyl of the –COOH shows H-bond interaction with Glu115. However, the para-fluoro substituted phenyl group of 30 binds in the second phosphotyrosine (pTyr) binding site of PTP1B by H-bond interaction with guanidine group of Arg254, which was different from the ion–π interaction between 7 and PTP1B. The key H-bond interaction with Arg254 might be crucial for tightening the complex of PTP1B and compound 30. It indicated that the substituted phenyl group of these compound series had a diverse binding modality with PTP1B enzyme.

Fig. 4 Characterization of 30 to PTP1B. (a)Time-independent inhibition of PTP1B by 30. (b) Typical competitive inhibition of 30 shown by Lineweaver–Burk plot. (c) At various fixed concentrations of 30 the initial velocity was determined with various concentrations of pNPP.

Fig. 5 (a) Binding pose of 30 in the protein surface of PTP1B; (b) key residues of PTP1B binding site surrounding 30.

Conclusion

14 novel pyrazole-fused 4,4-dimethyl LCA derivatives were synthesized, the potency against PTP1B and selectivity over homologous PTPs were explored. Intriguingly, all of the 14 compounds exhibited improved activities (IC₅₀ = 0.42–4.49 μM) toward PTP1B compared to that of LCA (6, IC₅₀ = 12.74 μM). In particular, 4-fluoro phenyl substituted compound 30 (IC₅₀ = 0.42 μM) was the most potent PTP1B inhibitor, which also possessed good selectivity towards TCPTP, SHP1, SHP2 and LAR. Moreover, both 30 and 34 (3, 10, 30 μM) significantly increased the insulin-induced phosphorylation of IR and Akt in cell-based assays. In summary, we identified a series of 2,3-pyrazole-4,4-dimethyl lithocholic acid derivatives as novel PTP1B inhibitors with good potency, selectivity as well as cell permeability. Further investigation of these compounds may lead to the development of novel antidiabetic agents derived from natural steroids.

Experimental section

General methods

Starting materials, reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. The progress of reactions was monitored by silica gel thin layer chromatography (TLC) plates, visualized under UV or charred using concentrated H₂SO₄ in the solution of EtOH followed by heating. Flash column chromatography was performed using Qingdao Haiyang silica gel (200–300) with distilled solvents. At 26.5 °C, ¹H NMR (400 MHz) spectra were recorded on Bruker Avance 400 spectrometers in CDCl₃ or DMSO-d6 [using TMS as internal standard] and ¹³C NMR (100 MHz) spectra on Bruker Avance 400 spectrometers in CDCl₃/CD₃OD = 2/1 v/v [the residual peak of CDCl₃ (¹H NMR δ 7.26, ¹³C NMR δ 77.16); the residual peak of DMSO-d6 (¹H NMR δ 2.50); the residual peak of CD₃OD (¹³C NMR δ 49.00)]. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, coupling constant (in Hz) and integration. High resolution mass spectra were performed using a Bruker ESI-TOF high-resolution mass spectrometer. Melting points were uncorrected and were recorded on a Buchi B-54 melting point apparatus.

Synthesis of intermediate compound 10

A solution of lithocholic acid (6) (3.76 g, 10.0 mmol) in methanol (150 ml) at 0 °C was treated with SOCl₂ (2.92 ml, 40.0 mmol) over 30 min, then the mixture was stirred for 4 h at room temperature. The solvent was concentrated in vacuo, then the residue was dissolved in H₂O (30 ml) and EtOAc (30 ml). The EtOAc phase was separated, and the aqueous phase was extracted with EtOAc (2 × 20 ml). The combined organic extract was washed with saturated NaHCO₃, brine, dried over anhydrous Na₂SO₄, concentrated to give white solid and used for the next step without further purification.

To a solution of the white solid obtained above in CH₂Cl₂ (60 ml) was added PCC (4.32 g, 20.0 mmol), after stirring for 12 h at room temperature, the mixture was filtered and the filtrate was washed with saturated aqueous NaHSO₃, brine, dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc 7/1 v/v) to give compound 8 (3.56 g, 93%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 3.67 (s, 3H), 2.70 (t, J = 14.2 Hz, 1H), 2.40–2.29 (m, 2H), 2.26–2.20 (m, 1H), 2.20–2.13 (m, 1H), 2.06–1.99 (m, 3H), 1.92–1.19 (m, 16H), 1.16–1.05 (m, 4H), 1.02 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.68 (s, 3H); HRMS (ESI): calcd for C₂₅H₄₀NaO₃ [M + Na]⁺ 411.2870, found [M + Na]⁺ 411.2898.

Compound 8 (3.56 g, 9.2 mmol) was dissolved in CH₂Cl₂ (120 ml) and a solution of Br₂ (0.52 ml, 10.1 mmol) in HOAc (20 ml) was added at 0 °C over 30 min, the stirring was continued for 1 h. The mixture was washed with water, saturated aqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, concentrated to give a yellow oil mixture which was used for the next step without further purification.

To a solution of the yellow oil mixture in DMF (60 ml) was added Li₂CO₃ (2.72 g, 36.8 mmol) and LiBr·H₂O (1.93 g, 18.4 mmol) under N₂, the reaction mixture was stirred for 6 h at 90 °C. Then the mixture was cooled to room temperature, and added H₂O (50 ml) and EtOAc (50 ml). The EtOAc phase was separated, and the aqueous phase was extracted with EtOAc (3 × 20 ml). The organic phase were combined and washed with brine, dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc 10/1 v/v) to give compound 9 (1.83 g, 48%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 5.72 (s, 1H), 3.67 (s, 3H), 2.46–2.30 (m, 4H), 2.30–2.18 (m, 2H), 2.06–1.98 (m, 2H), 1.93–1.75 (m, 3H), 1.73–1.37 (m, 9H), 1.18 (s, 3H), 1.17–0.97 (m, 4H), 0.92 (d, J = 6.5 Hz, 3H), 0.71 (s, 3H); HRMS (ESI): calcd for C₂₅H₃₈NaO₃ [M + Na]⁺ 409.2713, found [M + Na]⁺ 409.2755.

A solution of compound 9 (1.83 g, 4.7 mmol) in dry t-BuOH (30 ml) was treated with t-BuOK (2.13 g, 19.0 mmol) under N₂, the reaction mixture was stirred at room temperature for 1 h, and then CH₃I was slowly introduced. After another 24 h at room temperature the reaction mixture was poured into ice water (50 ml), and the pH value was adjusted to 5 by 1 M HCl aqueous solution. The mixture was extracted with EtOAc (3 × 20 ml). The organic phase was washed with brine, dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc 20/1 v/v) to give compound 10 (0.98 g, 50%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 5.56 (dd, J = 2.0, 4.8 Hz, 1H), 3.67 (s, 3H), 2.63–2.18 (m, 4H), 2.15–1.75 (m, 5H), 1.72–1.29 (m, 9H), 1.23 (s, 6H), 1.20–1.00 (m, 5H), 0.93 (d, J = 6.4 Hz, 3H), 0.85 (s, 3H), 0.69 (s, 3H); HRMS (ESI): calcd for C₂₇H₄₂NaO₃ [M + Na]⁺ 437.3015, found [M + Na]⁺ 437.3005.

General procedure for the synthesis of 2,3-pyrazole ring-substituted-4,4-dimethyl lithocholic acid ethyl esters 12a, 12b, and 13–24

To a solution of compound 10 (300 mg, 0.7 mmol) in dry toluene (20 ml) was added ethyl formate (3.0 ml, 36.0 mmol) and NaH (579 mg, 60%, 14.1 mmol) under N₂, the reaction mixture was stirred for 6 h at room temperature. The mixture was poured into ice water and then the pH value was adjusted to 7 by 1 M HCl aqueous solution. The aqueous phase was extracted with EtOAc (3 × 20 ml). The combined organic phase was washed with brine, dried over anhydrous Na₂SO₄ and concentrated to give a light yellow solid (compound 11) which was used for the next step without further purification.

Compound 11 (320 mg, 0.7 mmol) was dissolved in ethanol (15 ml) and substituted hydrazine hydrochloride (3.5 mmol) was added under N₂, the reaction mixture was heated to reflux for 2 h. After cooling to room temperature, the solvent was removed in vacuo, then H₂O (10 ml) was added and the mixture was extracted with EtOAc (2 × 10 ml). The organic phase was separated and washed with brine, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by a flash chromatography (petroleum ether/EtOAc 10/1 v/v) to afford the corresponding pure 2,3-pyrazole ring-substituted-4,4-dimethly lithocholic acid ethyl esters 12a, 12b and 13–24.

Characterization data

Compound 12a. Isolated in 32% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.11 (s, 1H), 5.71 (dd, J = 5.4, 2.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 2.60 (d, J = 14.6 Hz, 1H), 2.32–2.24 (m, 1H), 2.19–1.95 (m, 4H), 1.85–1.70 (m, 2H), 1.66–1.42 [m, 8H, containing 1.48 (s, 3H)], 1.33 (s, 3H), 1.31–1.21 (m, 3H), 1.19 (t, J = 7.1 Hz, 3H), 1.14 (d, J = 4.6 Hz, 1H), 1.10–0.94 (m, 4H), 0.88 (d, J = 6.5 Hz, 3H), 0.84 (s, 3H), 0.63 (s, 3H).

Compound 12b. Isolated in 29% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 6.91 (s, 1H), 5.67 (dd, J = 5.2, 2.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.61 (d, J = 14.4 Hz, 1H), 2.32–2.24 (m, 1H), 2.18–1.95 (m, 4H), 1.87–1.69 (m, 3H), 1.65–1.47 (m, 4H), 1.43 (s, 3H), 1.38 (m, 1H), 1.29 (s, 3H), 1.26–1.24 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.15–0.93 (m, 5H), 0.87 (d, J = 6.4 Hz, 3H), 0.78 (s, 3H), 0.62 (s, 3H).

Compound 13. Isolated in 43% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.28 (s, 1H), 7.22 (m, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.61 (d, J = 3.3 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.68 (d, J = 14.6 Hz, 1H), 2.32–1.97 (m, 5H), 1.87–1.72 (m, 2H), 1.64–1.35 (m, 6H), 1.30–1.17 [m, 9H, containing 1.22 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)], 1.11–0.94 [m, 7H, containing 1.12 (s, 3H)], 0.88 (d, J = 8.5 Hz, 3H), 0.87 (s, 3H), 0.63 (s, 3H).

Compound 14. Isolated in 43% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.29 (s, 1H), 7.20–7.13 (m, 4H), 5.60 (dd, J = 5.3, 2.1 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.6 Hz, 1H), 2.34 (s, 3H), 2.32–2.24 (m, 1H), 2.19–1.97 (m, 4H), 1.87–1.34 (m, 10H), 1.31–1.24 (m, 2H), 1.22 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.15 (m, 1H), 1.08 (s, 3H), 1.04–0.94 (m, 2H), 0.88 (d, J = 6.5 Hz, 3H), 0.87 (s, 3H), 0.63 (s, 3H).

Compound 15. Isolated in 47% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.33 (m, 2H), 7.30 (s, 1H), 7.28–7.24 (m, 2H), 5.62 (dd, J = 5.4, 2.3 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.6 Hz, 1H), 2.32–2.24 (m, 1H), 2.19–1.97 (m, 4H), 1.87–1.70 (m, 2H), 1.64–1.34 (m, 7H), 1.31–1.23 (m, 2H), 1.21 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.16 (m, 1H), 1.09 (s, 3H), 1.07–0.94 (m, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H).

Compound 16. Isolated in 47% yield as a brown solid; ¹H NMR (400 MHz, CDCl₃) δ 7.49 (d, J = 8.4 Hz, 2H), 7.30 (s, 1H), 7.20 (d, J = 8.4 Hz, 2H), 5.61 (d, J = 3.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.67 (d, J = 14.6 Hz, 1H), 2.32–1.97 (m, 5H), 1.87–1.70 (m, 2H), 1.64–1.34 (m, 6H), 1.31–1.24 (m, 2H), 1.21 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.16 (m, 1H), 1.13–0.94 [m, 7H, containing 1.09 (s, 3H)], 0.89 (d, J = 8.4 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H).

Compound 17. Isolated in 46% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.27 (m, 3H), 7.05 (t, J = 8.5 Hz, 2H), 5.62 (d, J = 3.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.6 Hz, 1H), 2.28 (m, 1H), 2.19–1.98 (m, 4H), 1.87–1.70 (m, 2H), 1.65–1.34 (m, 7H), 1.31–1.24 (m, 2H), 1.21 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.16–1.11 (m, 2H), 1.08 (s, 3H), 1.05–0.96 (m, 2H), 0.88 (d, J = 6.7 Hz, 3H), 0.87 (s, 3H), 0.63 (s, 3H).

Compound 18. Isolated in 43% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.29 (s, 1H), 7.25–7.12 (m, 2H), 7.11 (m, 2H), 5.60 (dd, J = 5.2, 2.0 Hz, 1H), 4.04 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.6 Hz, 1H), 2.31 (s, 3H), 2.29–2.23 (m, 1H), 2.18–1.96 (m, 4H), 1.86–1.34 (m, 9H), 1.31–1.25 (m, 2H), 1.22 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.17 (m, 1H), 1.08 (s, 3H), 1.06–0.95 (m, 3H), 0.88 (d, J = 8.4 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H).

Compound 19. Isolated in 45% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.33 (m, 1H), 7.31–7.25 (m, 2H), 7.21–7.14 (m, 2H), 5.60 (dd, J = 5.2, 2.1 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.69 (d, J = 14.6 Hz, 1H), 2.32–2.24 (m, 1H), 2.19–1.97 (m, 4H), 1.89 (s, 3H), 1.85–1.70 (m, 2H), 1.64–1.35 (m, 6H), 1.31–1.16 [m, 9H, containing 1.30 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)], 1.14–1.02 [m, 4H, containing 1.05 (s, 3H)], 1.00–0.86 [m, 9H, containing 0.88 (d, J = 6.5 Hz, 3H), 0.86 (s, 3H)], 0.63 (s, 3H).

Compound 20. Isolated in 46% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.34 (m, 2H), 7.31–7.27 (m, 2H), 7.24–7.20 (m, 1H), 5.61 (dd, J = 5.2, 1.9 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.6 Hz, 1H), 2.31–2.24 (m, 1H), 2.18–1.97 (m, 4H), 1.87–1.70 (m, 2H), 1.64–1.34 (m, 6H), 1.31–1.25 (m, 2H), 1.23 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.16 (m, 1H), 1.11 (s, 3H), 1.08–0.94 (m, 4H), 0.88 (d, J = 6.5 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H).

Compound 21. Isolated in 47% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.47–7.40 (m, 2H), 7.38 (s, 1H), 7.35–7.23 (m, 2H), 5.61 (dd, J = 5.4, 2.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.69 (d, J = 14.6 Hz, 1H), 2.32–1.98 (m, 5H), 1.87–1.70 (m, 2H), 1.64–1.35 (m, 6H), 1.31–1.27 (m, 1H), 1.24 (s, 3H), 1.20–1.11 [m, 6H, containing 1.18 (t, J = 7.1 Hz, 3H)], 1.10–1.02 (m, 3H), 1.00 (s, 3H), 0.89 (d, J = 6.2 Hz, 3H), 0.88 (s, 3H), 0.63 (s, 3H).

Compound 22. Isolated in 49% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.31 (d, J = 4.9 Hz, 1H), 7.20–7.11 (m, 1H), 7.02–6.94 (m, 2H), 5.60 (d, J = 3.5 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 14.5 Hz, 1H), 2.29 (s, 3H), 2.26 (m, 1H), 2.19–1.97 (m, 4H), 1.84 (s, 3H), 1.82–1.70 (m, 2H), 1.64–1.35 (m, 6H), 1.30–1.16 [m, 9H, containing 1.27 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)], 1.14–0.99 (m, 4H), 0.93 (s, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.85 (s, 3H), 0.63 (s, 3H).

Compound 23. Isolated in 50% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.27 (s, 1H), 7.10–7.03 (m, 3H), 5.60 (d, J = 3.2 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 2.67 (d, J = 14.5 Hz, 1H), 2.32–2.10 [m, 9H, containing 2.24 and 2.21, both (s, 3H)], 2.07–1.98 (m, 2H), 1.87–1.71 (m, 2H), 1.64–1.35 (m, 6H), 1.31–1.16 [m, 9H, containing 1.23 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)], 1.13–0.95 [m, 7H, containing 1.09 (s, 3H)], 0.88 (d, J = 8.8 Hz, 3H), 0.87 (s, 3H), 0.63 (s, 3H).

Compound 24. Isolated in 47% yield as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.44 (m, 2H), 7.30 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 5.63 (d, J = 3.1 Hz, 1H), 4.05 (q, J = 7.0 Hz, 2H), 2.67 (d, J = 14.6 Hz, 1H), 2.32–1.97 (m, 5H), 1.87–1.72 (m, 2H), 1.64–1.35 (m, 6H), 1.28–1.16 [m, 9H, containing 1.22 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)], 1.12 (s, 3H), 1.09–0.96 (m, 4H), 0.88 (d, J = 6.4 Hz, 3H), 0.86 (s, 3H), 0.63 (s, 3H).

General procedure for the synthesis of 2,3-pyrazole ring-substituted-4,4-dimethyl lithocholic acid derivatives 25a, 25b and 26–37

To a solution of the 2,3-pyrazole ring-substituted-4,4-dimethly lithocholic acid ethyl esters (0.5 mmol) in MeOH/H₂O (20 ml/2 ml v/v) was added LiOH·H₂O (420 mg, 10.0 mmol), the reaction mixture was stirred at room temperature for 24 h. Aqueous HCl (1 M) was introduced to adjust the pH = 5. After solvent removal, the residue was extracted by EtOAc (3 × 10 ml). The combined organic phase was dried over anhydrous Na₂SO₄ and concentrated. The crude product was flash-chromatographed on a silica gel column (CH₂Cl₂/MeOH 30/1 v/v) to give the corresponding pure 2,3-pyrazole ring-substituted-4,4-dimethly lithocholic acid derivatives 25a, 25b and 26–37.

Characterization data

Compound 25a. Isolated in 76% yield as a white solid; mp: 141.5–142.4 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.93 (br, 1H), 7.09 (s, 1H), 5.82 (d, J = 2.8 Hz, 1H), 3.87 (s, 3H), 2.63 (d, J = 14.5 Hz, 1H), 2.24 (m, 1H), 2.15–1.97 (m, 4H), 1.83 (m, 1H), 1.73–1.63 (m, 2H), 1.62–1.54 (m, 2H), 1.51 (s, 3H), 1.49–1.38 (m, 2H), 1.36 (s, 3H), 1.30–1.16 (m, 4H), 1.13–1.00 (m, 4H), 0.90 (d, J = 6.2 Hz, 3H), 0.85 (s, 3H), 0.66 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.49, 151.58, 144.65, 136.20, 121.44, 114.91, 57.19, 56.07, 49.93, 42.56, 40.03, 38.89, 38.65, 36.32, 35.69, 33.58, 32.52, 31.72, 31.61, 31.40, 31.34, 29.54, 28.39, 24.46, 21.39, 21.25, 18.46, 12.03; HRMS (ESI): calcd for C₂₈H₄₃N₂O₂ [M + H]⁺ 439.3319, found [M + H]⁺ 439.3313.

Compound 25b. Isolated in 80% yield as a white solid; mp: 142.6–143.7 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.27 (s, 1H), 5.73 (d, J = 3.1 Hz, 1H), 3.74 (s, 3H), 2.66 (d, J = 14.4 Hz, 1H), 2.24 (m, 1H), 2.15–1.97 (m, 4H), 1.83 (m, 1H), 1.70–1.44 (m, 6H), 1.43–1.35 [m, 4H, containing 1.39 (s, 3H)], 1.29–1.16 [m, 6H, containing 1.26 (s, 3H)], 1.14–1.00 (m, 4H), 0.90 (d, J = 6.5 Hz, 3H), 0.77 (s, 3H), 0.66 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.50, 156.30, 149.65, 129.67, 121.70, 114.83, 57.27, 56.18, 49.71, 42.64, 40.12, 38.88, 38.81, 36.17, 35.77, 33.61, 32.77, 32.61, 32.36, 31.96, 31.47, 31.43, 28.45, 24.51, 21.53, 21.44, 18.50, 12.10; HRMS (ESI): calcd for C₂₈H₄₃N₂O₂ [M + H]⁺ 439.3319, found [M + H]⁺ 439.3330.

Compound 26. Isolated in 74% yield as a white solid; mp: 233.1–234.2 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.31 (s, 1H), 7.29 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 5.71 (d, J = 3.1 Hz, 1H), 3.82 (s, 3H), 2.72 (d, J = 14.7 Hz, 1H), 2.23 (m, 1H), 2.14–1.98 (m, 4H), 1.81 (m, 1H), 1.71–1.36 (m, 8H), 1.31–1.17 [m, 6H, containing 1.24 (s, 3H)], 1.14–1.08 [m, 4H, containing 1.09 (s, 3H)], 1.06–1.00 (m, 2H), 0.91 (d, J = 6.7 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.80, 160.32, 151.30, 146.69, 137.46, 134.74, 130.27 (2C), 121.25, 114.85, 113.98 (2C), 57.20, 56.10, 55.73, 49.90, 42.61, 40.06, 38.75, 37.23, 35.73, 33.59, 32.47, 32.39, 31.74, 31.56, 31.48, 31.40, 28.41, 24.47, 21.45, 21.32, 18.50, 12.09; HRMS (ESI): calcd for C₃₄H₄₇N₂O₃ [M + H]⁺ 531.3581, found [M + H]⁺ 531.3561.

Compound 27. Isolated in 64% yield as a white solid; mp: 235.8–236.9 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.94 (br, 1H), 7.32 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 5.70 (d, J = 3.1 Hz, 1H), 2.73 (d, J = 14.6 Hz, 1H), 2.39 (s, 3H), 2.24 (m, 1H), 2.16–2.00 (m, 4H), 1.80 (m, 1H), 1.72–1.35 (m, 8H), 1.28–1.18 [m, 5H, containing 1.24 (s, 3H)], 1.15–1.01 [m, 7H, containing 1.08 (s, 3H)], 0.91 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.49, 151.33, 146.80, 139.85, 139.44, 137.50, 129.57 (2C), 128.94 (2C), 121.35, 115.01, 57.27, 56.17, 49.99, 42.67, 40.13, 38.81, 37.31, 35.77, 33.62, 32.52, 32.42, 31.81, 31.58, 31.46, 31.41, 28.47, 24.52, 21.49, 21.39, 21.35, 18.54, 12.14; HRMS (ESI): calcd for C₃₄H₄₇N₂O₂ [M + H]⁺ 515.3632, found [M + H]⁺ 515.3652.

Compound 28. Isolated in 63% yield as a white solid; mp: 228.5–229.7 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.93 (br, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 7.37 (s, 1H), 5.72 (d, J = 3.1 Hz, 1H), 2.73 (d, J = 14.7 Hz, 1H), 2.24 (m, 1H), 2.16–1.97 (m, 4H), 1.82 (m, 1H), 1.73–1.34 (m, 8H), 1.29–1.18 [m, 5H, containing 1.24 (s, 3H)], 1.14–1.01 [m, 7H, containing 1.10 (s, 3H)], 0.91 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.73, 151.10, 147.02, 140.71, 138.19, 135.74, 130.61 (2C), 129.30 (2C), 121.60, 115.45, 57.28, 56.19, 49.99, 42.68, 40.13, 38.81, 37.29, 35.79, 33.57, 32.52 (2C), 31.82, 31.71, 31.46, 31.44, 28.47, 24.53, 21.49, 21.40, 18.53, 12.13; HRMS (ESI): calcd for C₃₃H₄₄ClN₂O₂ [M + H]⁺ 535.3086, found [M + H]⁺ 535.3095.

Compound 29. Isolated in 71% yield as a brown solid; mp: 236.1–237.4 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.94 (br, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.37 (s, 1H), 7.36 (d, J = 8.2 Hz, 2H), 5.72 (d, J = 3.0 Hz, 1H), 2.73 (d, J = 14.4 Hz, 1H), 2.24 (m, 1H), 2.17–1.98 (m, 4H), 1.83 (m, 1H), 1.72–1.35 (m, 8H), 1.29–1.19 [m, 5H, containing 1.24 (s, 3H)], 1.16–0.99 [m, 7H, containing 1.10 (s, 3H)], 0.91 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.66, 151.18, 147.27, 141.28, 138.27, 132.51 (2C), 131.09 (2C), 123.98, 121.81, 115.68, 57.45, 56.36, 50.16, 42.83, 40.29, 38.95, 37.44, 35.96, 33.69, 32.66, 32.62, 31.98, 31.81, 31.60, 28.62, 24.66, 21.58, 21.55, 18.62, 12.22; HRMS (ESI): calcd for C₃₃H₄₄BrN₂O₂ [M + H]⁺ 579.2581, found [M + H]⁺ 579.2599.

Compound 30. Isolated in 75% yield as a white solid; mp: 231.8–232.6 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.93 (br, 1H), 7.48–7.43 (m, 2H), 7.37–7.30 (m, 3H), 5.72 (dd, J = 5.0, 1.9 Hz, 1H), 2.73 (d, J = 14.7 Hz, 1H), 2.24 (m, 1H), 2.16–1.99 (m, 4H), 1.82 (m, 1H), 1.73–1.33 (m, 8H), 1.28–1.18 [m, 5H, containing 1.24 (s, 3H)], 1.15–1.00 [m, 7H, containing 1.09 (s, 3H)], 0.91 (d, J = 6.6 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.78, 163.55 (d, J_C,F = 249.5 Hz), 151.38, 147.28, 138.42, 138.17, 131.46, 131.37, 121.82, 116.28, 116.06, 115.58, 57.55, 56.47, 50.29, 42.91, 40.39, 39.04, 37.51, 36.05, 33.79, 32.75, 32.58, 32.08, 31.78, 31.70 (2C), 28.69, 24.73, 21.62 (2C), 18.65, 12.25; HRMS (ESI): calcd for C₃₃H₄₄FN₂O₂ [M + H]⁺ 519.3381, found [M + H]⁺ 519.3360.

Compound 31. Isolated in 80% yield as a white solid; mp: 236.5–237.5 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.41–7.29 (m, 3H), 7.22–7.14 (m, 2H), 5.71 (d, J = 3.0 Hz, 1H), 2.73 (d, J = 14.7 Hz, 1H), 2.36 (s, 3H), 2.22 (m, 1H), 2.16–1.99 (m, 4H), 1.83 (m, 1H), 1.71–1.56 (m, 4H), 1.51–1.36 (m, 3H), 1.29–1.19 [m, 6H, containing 1.25 (s, 3H)], 1.14–1.01 [m, 7H, containing 1.09 (s, 3H)], 0.91 (d, J = 6.6 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.86, 151.32, 146.60, 142.00, 139.08, 137.54, 130.33, 129.72, 128.71, 126.16, 121.31, 114.97, 57.24, 56.14, 49.95, 42.63, 40.09, 38.78, 37.29, 35.77, 33.57, 32.49, 32.36, 31.78, 31.60 (2C), 31.44, 28.44, 24.49, 21.46, 21.35, 21.26, 18.51, 12.10; HRMS (ESI): calcd for C₃₄H₄₇N₂O₂ [M + H]⁺ 515.3632, found [M + H]⁺ 515.3647; C₃₄H₄₆NaN₂O₂ [M + Na]⁺ 537.3457, found [M + Na]⁺ 537.3444.

Compound 32. Isolated in 73% yield as a white solid; mp: 236.2–239.0 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.97 (br, 1H), 7.51–6.99 (m, 5H), 5.71 (d, J = 3.2 Hz, 1H), 2.74 (d, J = 14.5 Hz, 1H), 2.24 (m, 1H), 2.15–1.99 (m, 4H), 1.86 (s, 3H), 1.80 (m, 1H), 1.72–1.54 (m, 4H), 1.51–1.36 (m, 3H), 1.31 (s, 3H), 1.27–1.15 (m, 4H), 1.13–1.01 [m, 5H, containing 1.05 (s, 3H)], 0.97 (m, 1H), 0.91 (s, 3H), 0.89 (d, J = 8.5 Hz, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.26, 150.91, 146.02, 140.85, 137.66, 130.85, 129.66, 128.67, 126.14, 121.15, 120.82, 114.70, 57.01, 55.90, 49.89, 42.42, 39.86, 38.61, 36.88, 35.50, 33.30, 32.23, 32.13, 31.55, 31.19, 31.13, 29.29, 28.22, 24.29, 21.20, 21.14, 18.36, 17.14, 11.95; HRMS (ESI): calcd for C₃₄H₄₇N₂O₂ [M + H]⁺ 515.3632, found [M + H]⁺ 515.3655.

Compound 33. Isolated in 72% yield as a white solid; mp: 232.9–233.8 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.62 (d, J = 8.1 Hz, 1H), 7.57–7.50 (m, 2H), 7.41 (d, J = 8.5 Hz, 1H), 7.38 (s, 1H), 5.72 (d, J = 3.2 Hz, 1H), 2.73 (d, J = 14.7 Hz, 1H), 2.21 (m, 1H), 2.14–1.98 (m, 4H), 1.81 (m, 1H), 1.71–1.36 (m, 8H), 1.29–1.19 [m, 5H, containing 1.25 (s, 3H)], 1.15–1.00 [m, 7H, containing 1.11 (s, 3H)], 0.91 (d, J = 6.7 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.80, 151.09, 146.99, 143.29, 138.32, 134.61, 130.14, 130.01, 129.55, 127.61, 121.63, 115.52, 57.30, 56.21, 50.00, 42.69, 40.14, 38.82, 37.33, 35.83, 33.56, 32.51, 31.83, 31.80, 31.78, 31.66, 31.52, 28.49, 24.54, 21.51, 21.41, 18.55, 12.14; HRMS (ESI): calcd for C₃₃H₄₄ClN₂O₂ [M + H]⁺ 535.3086, found [M + H]⁺ 535.3087; C₃₃H₄₃NaClN₂O₂ [M + Na]⁺ 557.2911, found [M + Na]⁺ 557.2897.

Compound 34. Isolated in 68% yield as a white solid; mp: 232.8–234.4 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.95 (br, 1H), 7.68–7.62 (m, 2H), 7.61–7.47 (m, 2H), 7.42 (s, 1H), 5.72 (d, J = 2.9 Hz, 1H), 2.74 (d, J = 14.7 Hz, 1H), 2.22 (m, 1H), 2.16–1.99 (m, 4H), 1.82 (m, 1H), 1.71–1.36 (m, 7H), 1.28 (s, 3H), 1.25–1.17 (m, 3H), 1.16–1.04 (m, 4H), 0.99 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.54, 151.08, 147.25, 139.84, 138.73, 135.07, 132.01, 131.39, 130.63, 127.61, 121.51, 115.41, 57.32, 56.23, 49.99, 42.73, 40.18, 39.04, 37.32, 37.16, 35.83, 33.58, 32.52, 31.88, 31.50, 31.47, 29.38, 28.51, 24.57, 21.46, 21.20, 18.57, 12.16; HRMS (ESI): calcd for C₃₃H₄₄ClN₂O₂ [M + H]⁺ 535.3086, found [M + H]⁺ 535.3112; C₃₃H₄₃NaClN₂O₂ [M + Na]⁺ 557.2911, found [M + Na]⁺ 557.2917.

Compound 35. Isolated in 69% yield as a white solid; mp: 228.4–229.4 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.94 (br, 1H), 7.36 (s, 1H), 7.23 (m, 1H), 7.18–7.06 (m, 2H), 5.70 (d, J = 3.0 Hz, 1H), 2.73 (d, J = 14.6 Hz, 1H), 2.35 (s, 3H), 2.23 (m, 1H), 2.15–1.98 (m, 4H), 1.82 (m, 1H), 1.80 (s, 3H), 1.71–1.55 (m, 4H), 1.51–1.37 (m, 3H), 1.30 (s, 3H), 1.25–1.18 (m, 3H), 1.14–1.04 (m, 4H), 0.92 (s, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.87 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.44, 151.09, 146.43, 139.96, 138.39, 137.46, 131.69, 129.72, 128.57, 126.96, 121.38, 114.93, 57.24, 56.14, 49.89, 42.62, 40.09, 38.80, 38.70, 37.27, 37.11, 35.73, 33.80, 33.50, 32.58, 32.43, 32.22, 31.79, 31.37, 29.39, 28.41, 24.47, 21.35, 18.54, 12.11; HRMS (ESI): calcd for C₃₅H₄₉N₂O₂ [M + H]⁺ 529.3832, found [M + H]⁺ 529.3813; C₃₅H₄₈NaN₂O₂ [M + Na]⁺ 551.3613, found [M + Na]⁺ 551.3615.

Compound 36. Isolated in 70% yield as a white solid; mp: 229.4–230.5 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.31 (s, 1H), 7.24 (d, J = 7.9 Hz, 1H), 7.14 (s, 1H), 7.08 (dd, J = 7.7, 1.8 Hz, 1H), 5.70 (d, J = 3.1 Hz, 1H), 2.72 (d, J = 14.6 Hz, 1H), 2.29 (s, 3H), 2.26 (s, 3H), 2.22 (m, 1H), 2.15–1.99 (m, 4H), 1.82 (m, 1H), 1.71–1.35 (m, 8H), 1.28–1.20 [m, 5H, containing 1.25 (s, 3H)], 1.14–1.01 [m, 7H, containing 1.09 (s, 3H)], 0.91 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.92, 151.73, 146.93, 140.06, 138.71, 137.79, 137.65, 130.35, 130.24, 126.72, 121.63, 115.21, 57.65, 56.57, 50.39, 42.98, 40.49, 39.11, 37.62, 36.13, 33.92, 32.83, 32.56, 32.17, 31.81 (2C), 31.75, 28.75, 24.80, 21.69, 21.65, 19.84, 19.70, 18.69, 12.29; HRMS (ESI): calcd for C₃₅H₄₉N₂O₂ [M + H]⁺ 529.3832, found [M + H]⁺ 529.3849.

Compound 37. Isolated in 71% yield as a white solid; mp: 237.8–238.8 °C; ¹H NMR (400 MHz, DMSO-d6) δ 11.91 (br, 1H), 7.82–7.74 (m, 2H), 7.46 (dd, J = 8.5, 2.3 Hz, 1H), 7.40 (s, 1H), 5.73 (d, J = 3.1 Hz, 1H), 2.74 (d, J = 14.7 Hz, 1H), 2.23 (m, 1H), 2.16–1.99 (m, 4H), 1.82 (m, 1H), 1.71–1.55 (m, 4H), 1.52–1.37 (m, 3H), 1.34–1.18 [m, 6H, containing 1.25 (s, 3H)], 1.15–1.00 [m, 7H, containing 1.13 (s, 3H)], 0.91 (d, J = 6.5 Hz, 3H), 0.89 (s, 3H), 0.67 (s, 3H); ¹³C NMR (101 MHz, CDCl₃/CD₃OD) δ 177.50, 150.88, 147.05, 141.48, 138.62, 134.11, 132.92, 131.16, 130.75, 128.64, 121.69, 115.68, 57.21, 56.12, 49.91, 42.63, 40.06, 38.74, 37.25, 35.74, 33.48, 32.60, 32.46, 31.79, 31.74, 31.43, 31.38, 28.43, 24.48, 21.47, 21.34, 18.50, 12.10; HRMS (ESI): calcd for C₃₃H₄₃Cl₂N₂O₂ [M + H]⁺ 569.2696, found [M + H]⁺ 569.2698; C₃₃H₄₂NaCl₂N₂O₂ [M + Na]⁺ 591.2521, found [M + Na]⁺ 591.2520.

Biological assays

Enzyme-based assay of PTP1B and related homologous PTPs. PTP1B hydrolyzes pNPP to pNP which can be detected at 405 nm. Briefly, the tested compounds were solubilized in DMSO and serially diluted into series of concentrations for the inhibitory test. The assays which were performed in 96-well plates were carried out in a final volume of 100 μL containing 50 mmol L⁻¹ MOPS, pH 6.5, 2 mmol L⁻¹ pNPP, 30 nmol L⁻¹ GST-PTP1B, and 2% DMSO, and the catalysis of pNPP was continuously monitored on a SpectraMax 340 microplate reader at 405 nm for 2 min at 30 °C. The IC₅₀ value was calculated from the nonlinear curve fitting of the percent inhibition [inhibition (%)] vs. the inhibitor concentration [I] using the following equation: % inhibition = 100/{1 + (IC₅₀/[I])k}, where k is the Hill coefficient. The inhibition assay of TCPTP was conducted by the same procedure.

To study the inhibition on the other homologous PTPs, SHP1, SHP2 and LAR assays were performed according to procedures described previously.²¹

Effect of PTP1B inhibitors on the phosphorylation level of IR and Akt in CHO-hIR cells. CHO-hIR cells were cultured in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) FBS, 100 units per mL penicillin and 100 mg ml⁻¹ streptomycin at 37 °C in 5% CO₂. The cells were starved in serum-free medium for 2 h and then treated with PTP1B inhibitors for 3 h, followed by stimulation with 10 nM insulin for 10 min. Then cells were washed three times with ice cold PBS and lysed with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM Na₃VO₄, 1 mM PMSF, 1 mM DTT, 1 mM EDTA, 1 mM EGTA) containing complete protease inhibitors (Roche). The immunoblots were visualized by chemiluminescence using the enhanced chemiluminescence Western blotting system.

Characterization of the inhibitor on enzyme kinetics²². In the time-independent inhibition experiment, PTP1B were preincubated with compounds (2% DMSO) on the ice for different times, and then add 10 μL mixture of enzyme and compounds to 90 μL assay system. To characterize the inhibitor of PTP1B, the assay was carried out in a 100 μL system containing 50 mmol L⁻¹ MOPS, pH 6.5, 30 nmol L⁻¹ PTP1B, pNPP in 2-fold dilution from 80 mmol L⁻¹, and different concentrations of the inhibitor. In the presence of the competitive inhibitor, the Michaelis–Menten equation is described as 1/v = (K_m/[V_max[S]])(1 + [I]/K_i) + 1/V_max, where K_m is the Michaelis constant, v is the initial rate, V_max is the maximum rate, and [S] is the substrate concentration. The K_i value was obtained by the linear replot of apparent K_m/V_max (slope) from the primary reciprocal plot versus the inhibitor concentration [I] according to the equation K_m/V_max = 1 + [I]/K_i.

Acknowledgements

This research was financially supported by Shanghai Science and Technology Council (No. 13142200900, 14DZ0511800, 14142201200, 13ZR1410600), National Science and Technology Major Projects for Major New Drugs Innovation and Development (2012ZX09304011 and 2013ZX09507002), National science Fund for Distinguished Young Scholars (81125023). We also thank the Laboratory of Organic Functional Molecules, Sino-French Institute of ECNU for support.

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 1401731. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra20238h

‡ These authors contributed equally to this work.

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