Synthesis of Diosgenin-Ibuprofen Derivatives and Their Activities against Insulin-Dependent Diabetes Mellitus

Abstract

The synthesis and anti-diabetes activities of diosgenin-ibuprofen derivatives were investigated. Ibuprofen (IBU) was chemically coupled with diosgenin either directly or through amino acid esters linkers. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation were assessed. The results showed spirost-5-en-3β-yl (2-(4-isobutyl-phenyl)-propionate) (4) was of better activity to suppress the production of NO in the supernatant of LPS-stimulated RAW264.7 cells. In vivo investigation on nonobese diabetic (NOD) mice indicated that compound 4 decreased the incidence of insulin-dependent diabetes mellitus (IDDM; type 1 diabetes) of NOD mice which suggested a potential activity of compound 4 against type 1 diabetes.

Diabetes mellitus (DM) is a common but complicated metabolic disease characterized by hyperglycemia. Insulin-dependent diabetes mellitus (IDDM; type 1 diabetes) results from the damage of the insulin-secreting β-cells in the pancreas through an autoimmune process, leading to permanent deficiency of endogenous insulin.^1–3) Although patients with this form of diabetes could depend on exogenous insulin to sustain life, serious complications such as cardiovascular diseases, blindness, kidney failure and stroke caused by hyperglycemia will lead to a high fatality rate.^4–6) Therefore, applying processes or compounds that can strictly control the level of blood glucose or protect islet β-cells against destruction may be an alternative way to treat type 1 diabetes.

Proinflammatory cytokines are cytotoxic to β-cells and have been implicated in the pathogenesis of type 1 diabetes.^7–9) Insulin which is the primary medication used to treat the diabetes and prevent complications in all DM patients suppresses the inflammatory process of type 1 diabetes through preventing hyperglycemia and modulating key inflammatory molecules.¹⁰⁾ On one hand, there were studies suggesting that some non-steroidal anti-inflammatory drugs (NSAIDs) are effective in the treatment of type 1 diabetes, such as lisofylline (LSF),¹¹⁾ pentoxifylline (PTX)¹²⁾ and aspirin.^13–17) Ibuprofen (IBU), also a NSAID, was reported to have the capacity of controlling blood glucose concentration and promoting glucose tolerance.¹³⁾ It becomes toxic only at very high doses and has a wide therapeutic window.¹⁸⁾ The anti-inflammatory activities of IBU were connected with the inhibition of cyclooxygenase enzymes, which reduce the synthesis of prostaglandins. It has also an inhibitory effect on the inducible nitric oxide synthase (iNOS) protein which diminishes the synthesis of the proinflammatory cytokines.^19–21) On the other hand, steroidal anti-inflammatory drugs (SAIDS) could induce apoptosis or cell death of inflammatory cells and inhibit the production of inflammatory cytokines which are involved in the regulation of glucose metabolism and β-cell secretion.²²⁾ Diosgenin, an aglycone of steroidal saponins, possesses a variety of bioactivities such as anticarcinogenic, immunostimulant, antioxidant activity, antidiabetic activity and anti-inflammatory properties.^23–30) Research about anti-inflammatory activity of diosgenin has shown that it inhibited the production of inflammatory mediators in macrophages, one islet cell type which participate in the β-cells damage during the development of autoimmune diabetes.^31,32)

It is well known that chemotherapeutic twin drugs improve the pharmaceutical properties of the individual drugs.^33,34) Some studies of primaquine-NSAIDs twin drugs have been reported.³⁵⁾ To our knowledge, there are few studies that focused on the combination of NSAIDs and SAIDS as potential therapeutic agents against type 1 diabetes. IBU and diosgenin were both effect on diabetes, we supposed that diosgenin-IBU twin drugs obtained though substitution at OH–C (3) of diosgenin with IBU would lead to better activity to the treatment of autoimmune insulitis. Based on this consideration, IBU was chemically combined with diosgenin either directly or through amino acid ester linkers, and these structures reflect a new diversity of chemical combinations. Their effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production of RAW264.7 macrophages and nonobese diabetic (NOD) mice which naturally developed IDDM with remarkable similarity to that of human IDDM patients were performed and reported herein.

Results and Discussion

Synthesis

Up to now, no efforts have been undertaken to combine IBU (2-(4-isobutyl-phenyl)-propionic acid) (1) with diosgenin (2). The synthetic strategies we adopted are the classical esterification as shown in Chart 2. The transformation of IBU to 2-(4-isobutyl-phenyl)-propionyl chloride (3) was achieved by using thionyl chloride (SOCl₂) in dichloromethane at 70–80 °C in good yield³⁶⁾ (Chart 1). Diosgenin (2) was then coupled with 3 directly by esterification to give spirost-5-en-3β-yl (2-(4-isobutyl-phenyl)-propionate (4) in an excellent yield of 93% (Chart 2). The esterification was operated using 4-N,N-dimethylaminopyridine (DMAP) as a base catalyst in anhydrous dichloromethane at r.t. for 1 h.

Chart 1. Synthesis of Compound 3

a) SOCl₂/DMF, CH₂Cl₂, 70°C.

Chart 2. Synthesis of Compound 4

b) DMAP, CH₂Cl₂, r.t., 1 h.

Chart 3 shows the synthetic procedure of IBU (1) connection to diosgenin through amino acids linkers. Glycine and 6-aminohexanoic acid were chosen for the reason that neither additional alkaline nor acidic groups are introduced when they are used to connect IBU and diosgenin. On the other hand, glycine and 6-aminohexanoic acid have similar structures, but the length of the carbon chain is different. The amino groups of glycine and 6-aminohexanoic acid were protected as Boc derivatives. Boc-l-glycine (5) and Boc-6-aminohexanoic acid (6) were used to react with diosgenin (2) by esterification in the presence of DMAP and DCC in dry dichloromethane to offer the esters spirost-5-en-3β-yl N-(tert-butoxycarboyl)-l-glycine (7) and spirost-5-en-3β-yl N-(tert-butoxycarbonyl)-6-aminohexanoic acid (8). By deprotection of the amino groups with trifluoroacetic acid, spirost-5-en-3β-yl l-glycine (9) was obtained in 83% yield and spirost-5-en-3β-yl-6-aminohexanoic acid (10) in 81% yield after column chromatography. The target compounds spirost-5-en-3β-yl N-(2-(4-isobutyl-phenyl)-propionyl)-l-glycine (11) and spirost-5-en-3β-yl N-(2-(4-iso-butyl-phenyl)-propionyl)-6-aminohexanoic acid (12) were achieved in good yields from compounds 9 and 10 upon reacting with compound 3.

Chart 3. Synthesis of Compounds 7–14

c), DCC/DMAP, CH₂Cl₂, r.t. d), CF₃COOH/CH₂Cl₂, r.t., 3 h. e), Et₃N, CH₂Cl₂, 0°C, 1 h. f), EDC/DMAP, CH₂Cl₂, r.t. 3 h. g), (Ac)₂O/C₅H₅N, 6 h, r.t.

In addition, two reference compounds, cyclohexyl ester of IBU (13) and acetylated diosgenin (14), were also synthesized from compounds 1 and 2 upon reacting with cyclohexanol and acetic anhydride in good yields^37,38) (Chart 3). The processes of the above reactions were monitored by TLC. All compounds were purified by silica gel column chromatography and recrystallization from dichloromethane, characterized by high resolution mass spectra and NMR.

In Vitro Studies

LPS-stimulated macrophages generate high levels of nitric oxide synthase (iNOS), following overproduction of NO which is a potential mediator of cytokine inducing β-cell dysfunction.^39–45) Overproduction of NO induces apoptosis of beta cells in rodent models of diabetes.^46,47) Controlling the overproduction of NO of macrophage may have the effect of treating autoimmune diabetes. Therefore, we analyzed the effects of compounds 4, 11, 12, 13, and 14 on LPS-induced NO production of RAW264.7 macrophages and compared with IBU and diosgenin. The results were shown in Fig. 1. The production of NO in the supernatant of LPS-stimulated RAW264.7 cells was examined by measuring the amount of nitrite, a stable metabolite of NO. RAW264.7 macrophages in the resting state produced 9.2±0.83 μm nitrite during the 18 h incubation. When LPS (1 µg/mL) was added, the concentration of nitrite increased to 14.9±1.2 μm (Fig. 1). In general, pre-treatment of RAW264.7 macrophages with compounds 1, 2, 4, 11, 12, 13, and 14 inhibited LPS-induced NO production in a concentration-dependent manner (Fig. 1). Our experimental data demonstrated that compounds 4, 11, 12, 13, and 14 exhibited inhibitory activity against LPS-induced generation of NO (Fig. 1). At the concentration of 1.25, 5, 10 μm, the inhibitory activities of compound 4 (the concentration of nitrite was 9.2±1.23, 9.0±1.16, 8.1±0.95 μm) were much stronger than both of diosgenin (2) (the concentration of nitrite was 11.4±1.23, 11.5±0.88, 9.9±1.85 μm) and IBU (1) (the concentration of nitrite was 13.4±1.30, 9.2±1.30, 9.1±1.00 μm). Therefore, compound 4 in which IBU is connected to diosgenin directly exhibited much higher inhibitory activities than diosgenin (2) and IBU (1) alone. At the concentration of 1.25, 5 μm, compound 11 (the concentration of nitrite was 9.2±1.27, 9.3±1.38 μm) exhibited better inhibitory activities than that of diosgenin (2) (the concentration of nitrite was 11.4±1.23, 11.5±0.88 μm). It was worth mentioning that at the level of 10 μm, compound 11 (the concentration of nitrite was 11.6±1.23 μm) exhibited moderate activities which was equal to that of diosgenin (2) (the concentration of nitrite was 11.5±1.85 μm). Seemingly, increasing the concentration of compound 11 may promote the generation of NO. Compound 12 (the concentration of nitrite was 13.1±1.19, 13.3±1.27, 11.4±1.03 μm) displayed slightly weaker activity than diosgenin (2) alone. These results indicated that different amino acid spacers have different effects on the NO production inhibitory activity. Compared with the normal cell (the concentration of nitrite was 9.2±0.83 μm), compound 4 controlled the generation level of NO of RAW264.7 macrophages in a concentration dependent manner. It is also worth mentioning that both compounds 14 (the concentration of nitrite was 11.0±1.03, 10.1±1.02, 9.6±1.01 μm) and compounds 13 (the concentration of nitrite was 13.4±1.05, 10.4±1.06, 10.2±1.1 μm,) displayed weaker activities than compound 4 and were almost equal with parent compounds 2 and 1. These phenomena indicated that the potential activity of compound 4 was really resulted from the combination of the whole IBU and diosgenin. On account of this, we chose compound 4 as a candidate to determine its anti-diabetic activity in vivo.

Fig. 1. Inhibitory Effect of Compounds 1, 2, 4, 11, 12, 13, and 14 against LPS-Induced NO Production in RAW264.7 Macrophages

In Vivo Studies

The activity of compound 4 against the autoimmune insulitis was evaluated with the NOD mice model. The NOD mice, which spontaneously develop type 1 diabetes with a pathogenesis similar to the human disease, represent a useful model for the study of autoimmune diabetes.⁴⁸⁾ Observations of the pathology showed that the autoimmune insulitis of NOD mice occurred at the age of 4–5 weeks. NOD mice with type 1 diabetes showed the physiological characteristics of diabetes: frequent urination, polydipsia and hyperglycemia. And within a few weeks, the blood glucose level rapidly increased, then gradually decreased, but remained at higher level than normal. The weight of mice plummeted, and finally they fell into coma and died. In our study, we use the NOD mice model to determine the anti-diabetes activity of compound 4. It was dissolved in ethanol and then diluted with miglyol 812. The NOD mice were dosed orally three times per week with vehicle (miglyol 812) containing compound 4 or miglyol 812 alone as blank control. The effect of compound 4 on the growth of mice, the glucose tolerance and development of type 1 diabetes were assayed. Glucose levels in the tail venous blood were quantified. A diagnosis of diabetes was made after two sequential glucose measurements higher than 11.1 mm.

Effect of compound 4 on growth of mice weight of normal mice will gain during feeding, but body weight of diabetes mice will significantly reduce. Figure 2A shows the changes of the body weight of mice for 20 weeks of experimentation period. Although all groups showed increased body weight, the body weights of the groups that were dosed with compound 4 were greater than the ones of the blank control group. High administration (100 mg/kg) group tended to induce a greater body weight than that of blank control and the low exposure (50 mg/kg). Remarkably, from Fig. 2A, we can see that the mice weight within 100 mg/kg dose group and of the 50 mg/kg dose group did not plummet, compared with the blank control group (from 26.53±0.13 g to 21.52±0.21 g) during a 17–18 week period.

Fig. 2. The Activity of Compound 4 against Type 1 Diabetes of NOD Mice

Glucose Tolerance Tests

Glucose tolerance tests were performed to determinate the effect of compound 4 on controlling the concentration of blood glucose and the results are shown in Fig. 2B. Two grams per kilograms glucose in 0.85% NaCl was given to mice by intraperitoneal injection (IPGTT) after fasting for 16 h. Blood glucose was measured at interval of 5, 10, 15, 20, 30, 45, 60, and 90 min. When glucose (2 g/kg) was injected to the mice of blank control group, their blood glucose level rapidly increased to 22.5±0.07 mm (Fig. 2B) and remained at over 11.1 mm for about 20 min. In contrast, the blood glucose level in the group treated with a 50 mg/kg dose of compound 4 was slightly increased to 15 mm and then fell immediately. Moreover, the blood glucose level of 100 mg/kg dose group only increased to 10±0.04 mm, which was lower than the 50 mg/kg dose group. In short, compound 4 displayed the effect to enhance blood glucose tolerance and the 100 mg/kg dose of compound 4 gave better control of blood homeostasis.

Inhibition of the Development of Type 1 Diabetes

To analyze the ability of compound 4 to inhibit type 1 diabetes development, the glucose levels of NOD mice were monitored up to 32 weeks of age. The mice which the glucose value of two consecutive measurements higher than 11.1 mm were diagnosed to have developed diabetes. The type 1 diabetes incidence of groups treated with compound 4 was significantly lower compared with blank control, and the 100 mg/kg dose treatment was of greatest effect (Fig. 2C). Of the blank control mice, 50% were diabetics by 20 weeks old, compared with only 25% of mice treated with 50 mg/kg dose compound 4 for 8 weeks. The high doses treatment provided even higher protection, with only 18% of NOD mice developed type 1 diabetes. So the activity of compound 4 against autoimmune insulitis was certified.

Conclusion

In the current work, a new type of diosgenin-ibuprofen derivatives 4, 11, and 12 were synthesized in excellent yields. All the target compounds were synthesized for the first time and characterized by NMR and high resolution mass spectrometry. Compound 4 was found to have good anti-inflammatory effect on RAW264.7 cells in vitro. Then it was proven to reduce the risk of NOD mice to develop type 1 diabetes significantly.

Experimental

General All chemicals were commercially available. The solvents and reagents were analytical grade. 1,2-Dichloroethane was purified by distilling from phosphorus pentoxide. Thin-layer chromatography (TLC) was performed on aluminium sheet covered with silica gel 60 F254 (0.2 mm, Merck, Germany). Flash column chromatography (FC): silica gel 60 (Haiyang Chemical Co., P. R. China) at 0.4 bar. All compounds were characterized by NMR, high-resolution and ESI Mass spectra. NMR spectra were recorded on a AV II (Bruker, Germany) spectrometer at 400 MHz. ESI mass spectra were performed on a mass spectrometer (Q-TOF-premier, Waters Co., U.S.A.).

Synthesis of 2-(4-Isobutyl-phenyl)propionyl Chloride (3)

Ibuprofen (1, 5.00 g, 24 mmol) was dissolved in thionyl chloride (SOCl₂, 3 mL, 36 mmol). And N,N-dimethylformamide (DMF, 100 µL) was added under stirring. Then, the solution was heated to 70°C slowly and stirred at 70–75°C until no more bubbles discharged. The excess thionyl chloride was removed under high vacuum to obtain the compound 3 as oily liquid, which was dissolved in anhydrous dichloromethane (24 mL) and kept in an airtight vessel as a stock solution for next step.

Spirost-5-en-3β-yl (2-(4-Isobutyl-phenyl)propionate) (4)

Diosgenin (2, 829 mg, 2 mmol) and 4-N,N-dimethylaminopyridine (DMAP, 2.4 mg, 0.02 mmol) were dissolved in anhydrous dichloromethane (20 mL). Then a stock solution of compound 3 (3 mL) was added dropwise. The reaction was monitored by TLC and quenched with addition of water after about 1h. The organic layer was washed with 5% sodium chloride solution, saturated NaHCO₃ solution, and water, dried with Na₂SO₄ and evaporated at 50°C. The crude product was purified by column chromatography on silica gel (AcOEt/PE 1 : 9) which afforded compound 4 (93%, 1.12g) as white powder. Rf (AcOEt/PE 1 : 9) 0.42. mp 160.2–160.9 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.20 (d, J=7.8 Hz, 2H), 7.08 (d, J=7.7 Hz, 2H), 5.34 (dd, J=15.8, 4.2 Hz, 1H), 4.63–4.55 (m, 1H), 4.40 (dd, J=14.8, 7.4 Hz, 1H), 3.67–3.62 (q, J=7.0 Hz, 1H), 3.48–3.45 (m, 1H), 3.37 (t, J=10.9 Hz, 1H), 2.44 (d, J=7.1 Hz, 2H).¹³C-NMR (100 MHz, CDCl₃) δ: 174.2; 140.4; 139.8; 139.7; 138.0; 129.3; 127.1; 122.3, 109.3, 80.8, 74.0, 66.9, 62.1, 56.4, 49.9, 45.3, 45.1, 41.6, 40.3, 39.7, 38.0, 37.8, 36.7, 32.1, 31.9, 31.4, 30.3, 30.2, 28.8, 27.7, 27.5, 22.4, 20.8, 19.4, 18.6, 17.2, 16.3, 14.5. HR-MS: 625.4233 (C₄₀H₅₈O₄ Na⁺; calc. 625.4257).

Spirost-5-en-3β-yl N-(tert-Butoxycarbonyl)-l-glycine (7)

Diosgenin (2, 415 mg, 1 mmol), N-(tert-butoxycarbonyl)-l-glycine (5, 210 mg, 1.2 mmol) and DMAP (2.4 mg, 0.02 mmol) were dissolved in anhydrous dichloromethane (15 mL). A solution of DCC (309 mg, 1.5 mmol) dissolved in anhydrous dichloromethane (5 mL) was added dropwise under stirring at room temperature. The reaction was monitored by TLC until diosgenin was consumed completely. The reaction mixture was filtered to get the light yellow solution. And the solution was washed with 5% NaCl solution, saturated NaHCO₃ solution and water. The organic layer was concentrated at 50°C in vacuum after drying over Na₂SO₄. The crude product was purified by column chromatography on silica gel (AcOEt/PE 1 : 5) to yield compound 7 (92%, 528 mg) as white powder. Rf (AcOEt/PE 1 : 9) 0.31. mp 137.2–137.9 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.27 (s, 1H), 5.38 (d, J =3.9 Hz, 1H), 5.01 (s, 1H), 4.71–4.63 (m, 1H), 4.41 (dd, J=14.9, 7.4 Hz, 1H), 3.88 (d, J=5.0 Hz, 2H), 3.49–3.46 (m, 1H), 3.37 (t, J=10.9 Hz, 1H), 2.34 (d, J=7.6 Hz, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ: 169.8, 155.7, 139.4, 122.7, 109.3, 80.8, 79.9, 75.1, 66.8, 62.1, 56.4, 49.9, 42.7, 41.6, 40.3, 39.7, 38.0, 36.9, 36.7, 32.0, 31.8, 31.4, 30.3, 28.8, 28.3, 27.7, 20.8, 19.3, 17.2, 16.3, 14.5. HR-MS: 594.3793 (C₃₄H₅₃NO₆ Na⁺; calc. 594.3771).

Spirost-5-en-3β-yl N-(tert-Butoxycarbonyl)-6-aminohexanoic Acid (8)

From diosgenin (2, 415 mg, 1 mmol) and N-(tert-butoxycarbonyl)-6-aminohexanoic acid (6, 277 mg, 1.2 mmol) compound 8 (82%, 514 mg) was accomplished by the same procedure as described for compound 7. Rf (AcOEt/PE 1 : 9) 0.40. mp 129.6–130.7 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.27 (s, 1H); 5.37 (d, J=4.3 Hz, 1H); 4.64–4.54 (m, 2H); 4.41 (dd, J=15.0, 7.4 Hz, 1H); 3.49–3.46 (m, 1H); 3.37 (t, J=10.9 Hz, 1H); 3.11 (d, J=6.3 Hz, 2H); 2.33–2.26 (m, 4H). ¹³C-NMR (100 MHz, CDCl₃) δ: 173.0; 155.9; 139.70; 122.34; 109.26; 80.80; 79.03; 73.70; 66.83; 62.08; 56.43; 49.93; 41.60; 40.25; 39.7; 38.1; 36.9; 36.7; 34.5; 33.9; 32.0; 31.8; 31.4; 30.3; 29.7; 28.8; 28.4; 27.8; 26.3; 24.7; 20.8; 19.3; 17.1; 16.3; 14.5. HR-MS: 650.4396 (C₃₉H₆₁NNa⁺O⁶; calc. 650.4397).

Spirost-5-en-3β-yl l-Glycine (9)

Spirost-5-en-3β-yl N-(tert-butoxycarbonyl)-l-glycine (7, 457 mg, 0.8 mmol) was dissolved in dichloromethane (10 mL). Trifluoroacetic acid (CF₃COOH, 0.6 mL, 8 mmol) was added dropwise under stirring at room temperature. The reaction was monitored by TLC and allowed to continue for 3 h. The reaction mixture was cooled to 0°C and triethylamine (Et₃N, 1 mL) was added dropwise to adjust the pH of the reaction solution to 7–8 to terminate the reaction. Solvent was removed by vacuum evaporation and the residue was dissolved in dichloromethane and washed with water, saturated NaHCO₃ solution. The organic layer was dried with Na₂SO₄ and evaporated at 50°C. The crude product was purified by column chromatography on silica gel (AcOEt/PE 1 : 5). Compound 9 (83%, 312 mg) was obtained as white powder. Rf (AcOEt/PE 1 : 9) 0.44. mp 165.6–166.7 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.27 (s, 1H), 5.38 (d, J=4.1 Hz, 1H), 4.67–4.62 (m, 1H), 4.41 (dd, J=14.9, 7.4 Hz, 1H), 3.48–3.46 (m, 1H), 3.40–3.30 (m, 3H), 2.33 (d, J=6.9 Hz, 2H), 2.02–1.92 (m, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ: 173.7, 139.5, 122.6, 109.3, 80.8, 74.5, 66.8, 62.1, 56.4, 49.9, 44.2, 41.6, 40.3, 39.7, 38.1, 36.9, 36.7, 32.0, 31.8, 31.4, 30.3, 28.8, 27.8, 20.8, 19.3, 17.2, 16.3, 14.5. HR-MS: 494.3248 (C₂₉H₄₅NO₄ Na⁺; calc. 494.3247).

Spirost-5-en-3β-yl 6-Aminohexanoic Acid (10)

From spirost-5-en-3β-yl N-(tert-butoxycarbonyl)-6-aminohexanoic acid (8, 520 mg, 0.8 mmol), compound 10 (81%, 343 mg) was obtained as white solid by the same procedure described for compound 9. Rf (AcOEt/PE 1 : 2) 0.32. mp 157.6–158.7 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.28 (s, 1H), 5.56 (s, 1H), 5.37 (d, J=3.5 Hz, 1H), 4.61–4.53 (m, 1H), 4.41 (dd, J=14.8, 7.3 Hz, 1H), 3.58–3.53 (q, J=7.2 Hz, 2H), 3.46–3.42 (m, 1H), 3.37 (t, J =10.9 Hz, 1H), 2.99–2.96 (m, 2H), 2.29 (t, J=7.8 Hz, 4H). ¹³C-NMR (100 MHz, CDCl₃) δ: 172.9, 122.3, 109.3, 80. 8, 73.8, 66.8, 62.1, 56.4, 52.9, 49.9, 41.6, 40.2, 39.7, 38.1, 36.9, 36.7, 34.3, 32.0, 31.8, 31.4, 30.3, 28.8, 27.7, 27.2, 26.1, 24.4, 20.8, 19.3, 17.1, 16.3, 14.5, 8.2. HR-MS: 528.4072 (C₃₃H₅₄NO₄; calc. 528.4054).

Spirost-5-en-3β-yl N-(2-(4-Isobutyl-phenyl)-propionyl) l-Glycine (11)

To a solution of spirost-5-en-3β-yl l-glycine (9, 471 mg, 1 mmol) and triethylamine (Et₃N, 500 µL) in dichloromethane (50 mL), the stock solution of compound 1 (3 mL) was added dropwise at 0°C. After 1 h, the reaction mixture was washed with 5% sodium chloride solution and water, neutralized with saturated NaHCO₃ solution. Then the organic layer was dried with Na₂SO₄ and evaporated at 50°C. The crude product was purified by column chromatography on silica gel (AcOEt/PE 1 : 3). Compound 11 (82%, 540 mg) was obtained as white powder. Rf (AcOEt/PE 1 : 2) 0.21. mp 97.8–98.7 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.26 (s, 1H), 7.22 (d, J=7.7 Hz, 2H), 7.12 (d, J=7.7 Hz, 2H), 5.88 (s, 1H), 5.36 (s, 1H), 4.66–4.58 (m, 1H), 4.41 (dd, J=14.8, 7.3 Hz, 1H), 4.02–3.87 (m, 2H), 3.62–3.57 (q, J=7.1 Hz, 1H), 3.48–3.45 (m, 1H), 3.37 (t, J=10.9 Hz, 1H), 2.45 (d, J=7.1 Hz, 2H), 2.29 (d, J=7.8 Hz, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ: 174.6, 169.3, 140.8, 139.3, 138.1, 129.7, 127.3, 122.7, 109.3, 80.8, 66.9, 62.08, 56.4, 49.9, 46.5, 45.0, 41.7, 40.3, 39.7, 37.9, 36.8, 31.9, 31.4, 31.7, 31.4, 30.2, 29.7, 29.4, 28.8, 27.6, 22.7, 22.4, 20.8, 19.3, 18.4, 17.1, 16.3, 14.5, 14.1. HR-MS: 682.4462 (C₄₂H₆₁NNa⁺O₅; calc. 682.4448).

Spirost-5-en-3β-yl N-(2-(4-Isobutyl-phenyl)-propionyl)-6-aminohexanoic Acid (12)

From spirost-5-en-3β-yl 6-aminohexanoic acid (10, 528 mg, 1 mmol) and the stock solution of compound 1 (3 mL). White powder 12 (84%, 589 mg) was obtained with the same procedure for compound 11. Rf (AcOEt/PE 1 : 2) 0.32. mp 119.6–120.7 (CH₂Cl₂); ¹H-NMR (400 MHz, CDCl₃) δ: 7.27 (s, 1H), 7.18 (d, J=7.9 Hz, 1H), 7.11 (d, J=7.9 Hz, 1H), 5.37 (d, J=4.2 Hz, 2H), 4.6–4.55 (m, 1H), 4.41 (dd, J=15.0, 7.4 Hz,1H), 3.54–3.46 (m, J=17.6, 5.2 Hz, 2H), 3.37 (t, J=10.9 Hz, 1H), 3.18 (dd, J=13.2, 6.7 Hz, 2H), 2.45 (d, J=7.2 Hz, 2H), 2.30 (d, J=6.7 Hz, 2H), 2.22 (t, J=7.4 Hz, 2H). ¹³C-NMR (100 MHz, CDCl₃) δ: 174.4, 173.0, 140.7, 139.7, 138.7, 129.6, 127.4, 122.4, 109.3, 80.8, 73.7, 66.9, 62.1, 56.4, 49.9, 46.8, 45.0, 41.6, 40.3, 39.7, 39.3, 38.1, 36.9, 36.7, 34.4, 32.0, 31.9, 31.4, 30.3, 30.2, 29.1, 28.8, 27.8, 26.2, 24.5, 22.4, 20.8, 19.4, 18.4, 17.2, 16.3, 14.5. HR-MS: 738.5085 (C₄₆H₆₉NNa⁺O₅; calc. 738.5074).

Cyclohexyl (2-(4-Isobutyl-phenyl)propionate) (13)

Ibuprofen (217 mg, 1 mmol), EDC (159 mg, 1 mmol) and DMAP (20 mg, 0.16 mmol) were dissolved in anhydrous dichloromethane (10 mL), cyclohexanol (50 µL, 0.48 mmol) was added dropwise under stirring at room temperature. The reaction was monitored by TLC and quenched with addition of water after about 4 h. Then, the reaction solution was washed with 5% NaCl, saturated NaHCO₃ and water. After drying over Na₂SO₄, the organic layer was concentrated in vacuum at 50°C. The crude product was purified by column chromatography on silica gel (CH₂Cl₂) to yield compound 13 (92%, 528 mg) as colorless syrup. Rf (CH₂Cl₂/MeOH 9 : 1) 0.7; ¹H-NMR (400 MHz, DMSO) δ: 7.18 (d, J=8.1 Hz, 2H), 7.09 (d, J=8.1 Hz, 2H), 4.71–4.59 (m, 1H), 3.70 (q, J=7.1 Hz, 1H), 2.41 (d, J=7.2 Hz, 2H), 1.81 (m, J=13.5, 6.8 Hz, 1H), 1.7–1.53 (m, 3H), 1.42 (m, J=12.2, 11.2, 4.7 Hz, 2H), 1.36 (t, J=5.6 Hz, 4H), 1.32–1.14 (m, 4H), 0.84 (d, J=6.6 Hz, 6H); ¹³C-NMR (101 MHz, DMSO) δ: 173.67, 140.09, 138.56, 129.45, 127.41, 72.11, 44.85, 44.66, 31.30, 31.06, 30.09, 25.29, 23.29, 23.14, 22.55, 18.80. HR-MS: 311.2453 (C₁₉H₂₈O₂ Na⁺; calc. 311.2451).

Cell Culture and Nitrite Assay

RAW264.7, a mice celia macrophage cell line, was obtained from the American Type Culture Collection (Cryosite, Lane Cove, NSW, Australia). These cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 units/mL penicillin, and 100 pg/mL streptomycin in humidified atmosphere of 95% air, 5% CO₂ at 37°C. RAW264.7 cells were plated into 96-well plates at 2×10⁴ cells/well, and then cultured in the culture medium for 24 h. After pre-treated with various concentrations of testing drugs (1.25, 5, 10 μm) for 1 h, the cells were incentive with LPS (1 µg/mL). The nitrite accumulation in the supernatant was assessed by the Griess reaction after 24 h of LPS stimulation.⁴⁹⁾ Each 50 µL of culture supernatant was mixed with an equal volume of Griess reagent (0.1% N-(1-naphthyl)ethylenediamine and 1% sulfanilamide in 5% phophoric acid) and incubated at room temperature for 10 min. The absorbance was measured at 540 nm with an automated microplate reader, and a series of known concentrations of sodium nitrite was used as a standard.

In Vivo Studies

NOD mice obtained from Western China Experimental Animal Center, Sichuan University were used in the studies. They were kept under specific pathogen-free conditions. Animals were treated with humane care according to National Institutes of Health Guidelines of China during the experimental period. Twenty-one mice were randomly divided into three equally groups. Vehicle was administered as a blank control. The low dose (50 mg/kg) and high dose (100 mg/kg) of test compound groups were administered, respectively. Test compound was dissolved in ethanol and then diluted in miglyol 812. Three groups of mice were dosed orally with vehicle (miglyol 812) alone, low dose (50 mg/kg) and high dose (100 mg/kg) of test compound were given 0.4–0.5 mL per time and three times per week for eight weeks. Mice were continued to be observed until 32 weeks. Fasting blood glucose was measured using a glucometer (Bayer Corp, Mishawaka, IN, U.S.A.) before and after administration weekly. Body weight was monitored before and after administration once a week. Data was obtained as the mean and standard deviation (S.D.).

Glucose Tolerance Tests

Blood glucose levels of the mice after fasting for 16 h were measured as the value at 0 min. Then mice were given 2 g/kg glucose in 0.85% NaCl by intraperitoneal injection (IPGTT) and blood glucose was measured at 5, 10, 15, 20, 30, 45, 60, and 90 min using a glucometer.

Statistical Analysis

Statistical analysis was performed with the SPSS software system (SPSS for Windows, version 13.0; SPSS Inc., Chicago, IL, U.S.A.). Parametric data were statistically analyzed by the Student’s t-test or one-way ANOVA followed by post hoc tests when appropriate. Differences in non-parametric data were evaluated by the Mann–Whitney U test. Incidence curves were statistically analyzed using Kaplan–Meier test. Data were expressed as means±S.D. A significant difference was defined as p<0.05.

Acknowledgment

We thank the National Natural Science Foundations of China (document No. 20772087 and 20972105), the 12th-5-Year Plan Project of China (2011BAJ07B04) and the Open Foundation (SKLODSCUKF2012-04) from State Key Laboratory of Oral Diseases, Sichuan University for the financial supports. We also grateful to the Analytical and Testing Center of Sichuan University and Ming-Hai Tang from National Key Laboratory of Biotherapy for providing us with high quality nuclear magnetic resonance and high resolution mass spectra.

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