CN104784161A - Compound with effect of treating diabetes, pharmaceutical composition containing compound and application of compound - Google Patents

Abstract

The invention discloses a compound with the effect of treating diabetes, a pharmaceutical composition containing the compound and application thereof. In addition, the invention also provides a novel method for fully synthesizing the android quinuclidine. Also provided are novel compounds produced in a process for preparing android quinuclidine.

Description

A compound with the effect of treating diabetes, a pharmaceutical composition containing the compound and its application

technical field

The present invention generally relates to a compound for treating diabetes and its pharmaceutical composition. Specifically, the present invention relates to a method and composition for treating diabetes using specific compounds from Antrodia camphorate.

Background technique

Type 2 diabetes mellitus (T2DM) is the most common chronic disease with a high prevalence worldwide. Recent large-scale long-term trials have pointed out that aggressive blood sugar control can reduce the exacerbation of insulin resistance and the risk of cardiovascular disease (CVD), especially in T2DM (Avogaro, "Treating diabetes today with gliclazide MR: a matter of numbers." Diabetes, obesity & metabolism 14 Suppl 1:14-19, 2012). Accordingly, it is more hoped to develop blood sugar control agents superior to insulin analogs. For example, sitagliptin, which has the effect of controlling blood sugar, has been developed as an agent for the treatment of T2DM, and it is a highly selective dipeptidyl peptidase-4 inhibitor (dipeptidyl peptidase-4 inhibitor) ( Goldstein et al., "Effect of initial combination therapy with sitagliptin, a dipeptidyl peptidase-4 inhibitor, and metformin on glycemic control in patients with type 2diabetes." Diabetes Care 30(8):1979-1987, 2007). In addition, it has also been reported that metformin can effectively control blood sugar by activating AMP-activated protein kinase (AMPK) (Riddle, "Oral pharmacologic management of type2diabetes." American Family Physician 60(9): 2613-2620,1999; Zhou et al., "Role of AMP-activated protein kinase in mechanism of metformin action." Journal of Clinical Investigation 108(8):1167-1174,2001; Fryer et al., "The Anti- diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways.” Journal of Biological Chemistry 277(28):25226-25232, 2002; Leverve et al., “Mitochondrial specific metabolism. Diabetes and Metabolism 29(4 Pt 2):6S88-94, 2003). Sitagliptin and metformin are the first-line drugs widely used in the treatment of T2DM, which can lower blood sugar levels through different mechanisms. In previous clinical trials, the efficacy of sitagliptin combined with metformin in the initial treatment has been confirmed in T2DM patients.

Certain Chinese herbal medicines have been reported to have the potential to lower blood sugar levels (Lee et al., "Berberine, natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states." Diabetes 55(8):2256- 2264, 2006; Shi et al., "Tiliroside-derivative sensitivity GLUT4 translocation via AMPK in muscle cells." Diabetes research and clinical practice 92(2):e41-46, 2011). Cinnamomum kanehirai is a species of Lauraceae endemic to Taiwan (Wu et al., “Antrodia camphorata (“niu-chang-chih”), new combination of a medicinal fungus in Taiwan.” BOTANICAL BULLETIN-ACADEMIA SINICA TAIPEI 38:273-276, 1997). Antrodia camphorata is a parasitic fungus that grows in the inner cavity of the woody heart of C. kanehirae (Geetangili et al., “Review of Pharmacological Effects of Antrodia camphorata and Its Bioactive Compounds.” Evidence-Based Complementary and Alternative Medicine 2011:212641, 2011).

At present, it is still hoped to develop a new drug for the treatment of T2DM and its novel chemical synthesis method.

Contents of the invention

The present invention unexpectedly found that certain compounds isolated from Antrodia camphorata can effectively treat diabetes, especially T2DM.

In one aspect, the present invention provides the purposes of the compound with general formula (I) in the preparation treatment individual diabetes medicine:

Wherein, X is independently oxygen or sulfur, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen atom, methyl group or (CH ₂ )m-CH ₃ , m is an integer from 1 to 12, and n is an integer from 1 to 12.

In a specific embodiment of the present invention, the compound with general formula (I) can be isolated from Antrodia camphorate. An embodiment of the invention is antroquinol, specifically (+) or (−)-antroquinonol.

In an embodiment of the invention, the diabetes is type 2 diabetes and the individual is diabetic with insulin resistance. The compound has the effect of effectively improving glucose intake and controlling blood sugar.

In a specific embodiment of the present invention, the compound is androquinol, which has the activity of effectively inhibiting dipeptidyl peptidase-4 (Dipeptidyl peptidase-4, DPP4) and enhancing AMP-activated protein kinase (AMPK) the activation effect.

In yet another specific embodiment of the present invention, the drug further includes insulin, and the combination of androquinol and insulin can provide a synergistic effect.

In yet another aspect, the present invention provides a novel method for the preparation of androquinol, comprising asymmetric addition of diethyl zinc, Claisen rearrangement, ring-closing displacement reaction (ring-closing metathesis), and lactonization (lactonization) and other steps.

In yet another aspect, the present invention provides a novel compound, i.e. (–)-androquinol with general formula (IV):

wherein Me is methyl.

In a specific embodiment of the invention, the (−)-androquinol is non-toxic.

In yet another aspect, the present invention provides novel compounds produced during the preparation of androquinol, including:

Wherein PMB is p-methoxybenzyl (p-methoxybenzyl);

wherein Me is methyl and TBS is tert-butyldimethylsilyl; and

wherein Me is methyl and MOM is methoxymethyl.

In yet another aspect, the present invention provides a pharmaceutical composition in combination with a compound of general formula (I). An example of the invention is a pharmaceutical composition in combination with androquinol, specifically (+) or (−)-androquinol.

In yet another specific embodiment of the present invention, the drug further includes insulin or insulin analogues.

It is believed that one of ordinary skill in the art can utilize the description herein to the broadest extent without further elaboration. Therefore, the following description should be understood as being for the purpose of illustration and not limiting the scope of the present invention in any way.

Description of drawings

The aforementioned content of the invention, as well as the following detailed description of the invention, can be understood more clearly with reference to the accompanying drawings. In order to illustrate the present invention, the specific embodiments in the drawings are preferred presentations. It should be understood, however, that the invention is not limited to the precise arrangements and methodology shown. In the schema:

Figure 1 shows the MTT test results of (+) and (–)-androquinol (indicated by “Ant(+)” and “Ant(–)”, respectively), and compared with the control group (indicated by “Con”); The LNCaP cells were cultured overnight at 37°C in 5% CO ₂ , treated with 10 μM (+)-androquinol or (–)-androquinol for 48 hours, and then cultured for another 4 hours after MTT assay , The OD value of each well was measured at 570nm, where the data were expressed as mean and standard deviation (mean ± SD), and different letters represented significant differences among the treatment groups (p<0.05).

Figure 2 shows the ability of androquinol to inhibit DPP4 activity; the comparison of the DPP4 activity values of 100 μM androquinol (represented by "DPP4+Ant") and sitagliptin (represented by "DPP4+Sit") was determined by ELISA respectively (DPP4/CD26 detection kit, BML-AK498); the data are represented by mean and standard deviation (mean ± SD), and different letters represent significant differences among treatment groups (p<0.05).

Figure 3 (A) and Figure 3 (B) show the effect of androquinol on the phosphorylation of AKT Thr308 and AMPK Thr172:

Figure 3(A) provides the results of AKTThr308 phosphorylation induced by insulin, metformin (Met) and android quinol (Ant); where differentiated C2C12 cells were cultured in 100 nM insulin, 16 mM metformin (Met), or 25 μM android Quinole (Ant), and stood at 37°C for 30 minutes, then the cell lysate was separated by SDS-PAGE, and analyzed by phosphorylation-AKT Thr308 Western blotting; the data were expressed as the mean and the standard deviation of the mean (mean Value ± SEM), different letters represent significant differences among treatment groups (p<0.05).

Figure 3(B) provides the results of AMPK Thr172 phosphorylation induced by insulin, metformin (Met) and androquinol (Ant); where differentiated C2C12 cells were cultured in 100 nM insulin, 16 mM metformin (Met), or 25 μM anthroquinol (Ant), and stood at 37°C for 30 minutes, then the cell lysate was separated by SDS-PAGE, and phosphorylated-AMPK Thr172 Western blotting analysis was performed; the data were expressed as the mean value and the standard deviation of the mean value (mean ± SEM), different letters represent significant differences among treatment groups (p<0.05).

Figure 4 shows the effect of androquinol (Ant) on the PKA protein content induced by glucagon-like peptide-1 in AR42J cells; wherein the AR42J cells were treated with 1nM glucagon-like peptide-1 1, Glp-1), 1 nM insulin secretagogue-4 (Ex-4) and different concentrations (5 μM and 20 μM) of androquinol (Ant) were treated for 48 hours; the protein expression was determined by Western blotting, and GAPDH as an internal control group.

Figure 5(A) and Figure 5(B) show the effect of androquinol on GLUT4 transposition:

Figure 5(A) shows the effects of insulin (insulin, Ins), metformin (Met) and androquinol (Ant) on GLUT4 translocation; where C2C12 cells have been differentiated, and treated with 185μM insulin (Ins), 16mM metformin (Met) and 50 μM, 100 μM and 150 μM Ant androquinol (Ant) were treated for 55 minutes (the number of samples in each group was n=5); the data were represented by the mean value and the standard deviation of the mean value (mean ± SEM), and different letters represented each treatment group Significant difference between (p<0.05).

Figure 5(B) shows the GLUT4 translocation rate compared with the control group; in which C2C12 cells were differentiated and treated with 185 μM insulin (Ins), 100 μM androquinol and insulin plus 100 μM androquinol for 40 minutes (number of samples in each group n=5); the data are represented by the mean and the standard deviation of the mean (mean ± SEM), and different letters represent significant differences among the treatment groups (p<0.05).

Figure 6 provides the test results of glucose uptake by insulin (Ins), metformin (Met) and androquinol (Ant); wherein L6 cells have been differentiated and treated with 1 μM insulin (Ins), 2 mM metformin (Met), and/or 10 nM Androquinol (Ant), and/or 400nM S961 (an insulin receptor antagonist, used in simulated diabetes (diabetes mellitus, DM)) was treated for 30 minutes (n = 5 samples in each group); the data were averaged The standard deviation (mean ± SEM) of the value and the mean indicates that different letters represent significant differences among the treatment groups (p<0.05).

Figure 7(A) and Figure 7(B) show the hypoglycemic effect of androquinol, obtained through an oral glucose tolerance test (OGTT) under insulin resistance conditions:

Figure 7(A) shows the AUC (compared to the ratio of the control group) of Androquinol (Ant) in the Oral Glucose Tolerance Test (OGTT); wherein the blood glucose change is represented by AUC (area under the curve, area under curve) indicated that the mice were treated with S961 (an insulin receptor antagonist used in simulated DM) at a dose of 40 nmol/kg body weight (Bwt), and then metformin (Met) at 100 mg/kg Bwt as a positive control group , 50mg/kg Bwt of androquinol (Ant), and 10mg/kg Bwt of sitagliptin (Sit); then all mice were treated with 2g/kg Bwt of D-glucose (n=5 for each group of samples) ; The data are represented by the mean and the standard deviation of the mean (mean ± SEM), and different letters represent significant differences among the treatment groups (p<0.05).

Figure 7(B) shows the efficacy of (+)-androquinol (Ant(+)) versus (–)-androquinol (Ant(–)) in insulin-resistant conditions via an oral glucose tolerance test. (OGTT); where the changes in blood glucose are represented by AUC (area under the curve), the mice were treated with 50nmol/kg Bwt of S961, and then orally administered (p.o.) 50mg/kg Bwt of (+)-androquinol (Ant(+)) or (–)-androquinol (Ant(–)) (dissolved in PEG and EtOH), then all mice were treated with 2g/kg Bwt of D-glucose and D-glucose (2g/kg kg Bwt) treatment, and the OGTT test was performed; the data were expressed as the mean and the standard deviation of the mean (mean ± SEM), and different letters represented significant differences among the treatment groups (p<0.05).

Figure 8 shows the hypoglycemic effect of its androquinol in short-term processed DIO mice; wherein the DIO mice are treated with 25mg/kg Bwt of androquinol (Ant) or 20mg/kg Bwt of sitagliptin (Sit) , and an oral glucose tolerance test was carried out to determine the blood glucose changes (expressed in AUC) of mice (n=5 samples in each group); the data were expressed in mean and standard deviation of the mean (mean ± SEM) , Different letters represent significant differences among treatment groups (p<0.05).

Figure 9 shows the hypoglycemic effect of its Anthroquinol in long-term processed DIO mice; wherein DIO mice are treated with 25mg/kg Bwt of Androquinol (Ant) or 10mg/kg Bwt of Sitagliptin (Sit) 4 Week (number of samples in each group n=5), and an oral glucose tolerance test was carried out to measure blood glucose changes (expressed in AUC) in mice (number of samples in each group n=5); The standard deviation (mean ± SEM) indicates that different letters represent significant differences among the treatment groups (p<0.05).

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the invention. In case of conflict, the present document, including definitions, will control.

As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents known to those skilled in the art.

The following abbreviations used herein represent compounds of the present invention: methyl (methyl, "Me"), p-methoxybenzyl (p-methoxybenzyl, "PMB"), tertiary-butyldimethylsilyl ( tert-butyldimethylsilyl, "TBS"), and methoxymethyl ("MOM").

The term "individual" as used herein refers to a human being or a mammal, such as a patient, a companion animal (such as a dog, cat, and the like), a farm animal (such as a cow, sheep, pig, horse, and the like) ) or a laboratory animal (such as rats, mice, rabbits and similar animals).

The present invention finds for the first time that androquinol has the effect of inhibiting the activity of dipeptidylpeptidase-4 (DPP4) and/or enhancing the activation of AMP-activated protein kinase (AMPK). The present invention demonstrates that androquinol can provide efficacy similar to or superior to drugs for the treatment of diabetes, especially type 2 diabetes (T2DM), by enhancing AMPK activation similar to that of the first-line drug metformin, and/or by enhancing AMPK activation by similar Effect of sitagliptin to inhibit DPP4 activity. Accordingly, the present invention provides a novel method/pharmaceutical composition for use in the treatment of diabetes, specifically type 2 diabetes mellitus (T2DM), improved glucose uptake, and glycemic control in hyperglycemic mice (DM mice) , specifically those with insulin resistance. In addition, it has also been confirmed that Androquinol can combine with insulin to produce a synergistic effect.

According to the present invention, a method for treating diabetes in an individual comprises administering to the individual a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula (I):

Wherein X is independently oxygen or sulfur, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen atom, methyl group or (CH ₂ )m-CH ₃ , m is an integer from 1 to 12, and n is An integer from 1 to 12.

The compound of general formula (I) can be isolated from Antrodia camphorata using generally known techniques or standard methodology known to those skilled in the art.

An example of a compound is androquinol, which has the following general formula (II):

Androquinol is a known compound found only in the fermented product of Antrodia camphorata (Kuo et al., Novel compounds from antrodia camphorata, US20100130584A1). In traditional methods, the cost of purifying androquinol from fermented products is quite high and the recovery rate is quite low. Anthroquinol includes (+) and (–)-androquinol, however, (–)-androquinol was not isolated prior to the present invention. The (+)-androquinol has the general formula (III):

On the other hand, the (–)-androquinol has the general formula (IV), which is synthesized for the first time in the present invention:

In an embodiment of the present invention, the cell viability of cells treated with (–)-androquinol was similar to that in the control group (Con), as shown in Figure 1, showing that (–)-androquinol is non-toxic.

The present invention also provides a novel total synthesis method of androquinol. The preparation method of androquinol includes the steps of asymmetric addition of diethylzinc, Claisen rearrangement, ring closure replacement reaction, and lactonization.

This method is an enantioselective synthesis involving an iridium-catalyzed olefin isomerization-Claisen rearrangement (CR), a lactone The reaction is followed by a Grubbs ring closure displacement reaction to create three stereocenters. The necessary α,β-unsaturation reaction is achieved using a selenization/oxidation scheme.

The total synthesis method of this (+)-Androquinol is carried out according to the following process:

In the above-mentioned process for preparing androquinol, some new compounds are found and provided in the present invention, including:

as well as

In addition, the present invention provides a new use of a compound of general formula (I) in the preparation of a composition or medicine for treating diabetes.

The present invention also provides a pharmaceutical composition for treating diabetes, comprising a therapeutically effective amount of the compound of general formula (I).

In addition, the present invention provides a pharmaceutical composition for treating diabetes, comprising a combination of insulin or an insulin analogue and a compound of general formula (I).

In one embodiment of the pharmaceutical composition according to the present invention, the compound of general formula (I) is androquinol, specifically (+) or (−)-androquinol.

As used herein, the term "therapeutically effective amount" refers to an amount of an agent effective to achieve the desired therapeutic purpose. The therapeutically effective amount of a particular agent will vary depending on factors such as the identity of the agent, the route of administration, the size and species of animal receiving the agent, and the purpose of administration. The therapeutically effective amount for each individual case can be determined empirically by a skilled artisan based on the disclosure herein and methods established in the art.

As used herein, the term "insulin analogue", also known as "insulin receptor ligand", refers to a modified form of insulin that is different from that which occurs in nature, but which still performs the same activity in the human body as human insulin. Blood sugar control effect. Insulin analogues can be obtained through DNA genetic engineering, and the modified amino acid sequence of insulin can change its absorption, distribution, metabolism and secretion characteristics. Examples of insulin analogs include, but are not limited to, those described by Hirsch in "Insulin analogues", New England J Med 2005; 352:174-183, 2005.

The pharmaceutical compositions of the present invention may be administered by any suitable route, including but not limited to parenteral or oral administration. Pharmaceutical compositions for parenteral administration include solutions, suspensions, emulsions, and solid injectable compositions that can be dissolved or suspended in a solvent immediately before use. The injection can be prepared by dissolving, suspending or emulsifying one or more active ingredients in a diluent. Examples of the diluent are distilled water for injection, physiological saline, vegetable oil, alcohol and combinations thereof. In addition, the injection may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives and the like. Injections are sterilized or prepared by aseptic procedures in the final preparation step.

According to the present invention, the composition can be administered orally, wherein the composition can be in solid or liquid form. Such solid compositions include tablets, pills, capsules, dispersible powders, granules and the like. The oral compositions also include mouthwashes and sublingual lozenges which can be placed in the oral cavity. The capsules include hard capsules and soft capsules. In solid compositions for oral administration, one or more active compounds may be mixed alone with diluents, binders, disintegrants, lubricants, stabilizers, solubilizers, and then formulated into preparations in the usual manner. The formulation may be coated with a coating agent, or may be coated with two or more coats, as desired. In another aspect, the liquid compositions for oral administration include pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, elixirs, and the like. In this composition, one or more active compounds can be dissolved, suspended or emulsified in commonly used diluents (such as pure water, ethanol or their mixtures, etc.). Besides such diluents, the composition can also contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, perfuming agents, preservatives and buffers, and the like.

The following specific examples should be understood to be illustrative only and not limiting of the remainder of this document in any way. Without further elaboration, it is generally believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest.

Example

The preparation of embodiment 1 androquinol

The total synthesis of androquinol was carried out by an enantioselective synthesis method, involving an iridium-catalyzed olefin isomerization-Claisen rearrangement (ICR), a lactonization reaction, and a Grabby ring-closure displacement reaction. Three stereocenters are established, where the necessary α,β-unsaturation is achieved by a selenization/oxidation process.

1.1(+)-Android Quinol's Process

The total synthesis of the (+)-androquinol is carried out according to the following process:

1.2(–)-Android Quinol's Process

The total synthesis of the (–)-androquinol was carried out according to the following scheme:

1.3 Features

Spectral data for the Androquinol is provided below:

[α] _D ²⁵ : +42.5° (c=1.20 in CHCl ₃ ). IR (film): 3435, 2926, 1659.3, 1622, 1451, 1358, 1240, 1141, 1017, 944, 832, 749 cm ^–1 . ¹ H-NMR (400MHz, CDCl ₃ ): δ5.16(m, 1H), 5.08(m, 2H), 4.34(d, J=3.24Hz, 1H), 4.06(s, 3H), 3.66(s, 3H),2.52(m,1H),2.23(dd,J=7.48Hz,J=7.44Hz,2H),1.97-2.09(m,9H),1.75(m,1H),1.67(s,3H), 1.66 (s, 3H), 1.60 (s, 6H), 1.17 (d, J=6.92Hz, 3H). ¹³ C-NMR (100.6MHz, CDCl ₃ ): δ197.12, 160.49, 138.03, 135.92, 135.34, 131.31, 124.31, 123.85, 120.99, 67.91, 60.58, 59.19, 43.40, 40.266, 39.81, 27.04, 27.04, 39.04 25.69, 17.67, 16.12, 16.01, 12.31. HRMS-EI (m _{/ z} ) [M] ⁺ calcd for _C26H42O5 is 390.2770, observed 390.2764.

1.4 Characteristics of novel compounds

During the preparation of androquinol, the following novel compounds were found:

This spectral data is provided below:

¹ H-NMR (400MHz, CDCl ₃ ): δ9.64(d, J=2.20Hz, 1H), 7.23(m, 3H), 6.87(d, J=8.60Hz, 2H), 5.49(m, 1H) ,5.18(m,1H),4.42(d,J=11.52Hz,1H),4.36(d,J=11.52Hz,1H),3.80(s,3H),3.40(m,2H),2.34(m, 1H), 2.30(m, 1H), 2.02(m, 2H), 1.70(m, 1H), 1.56(m, 1H), 1.05(d, J=5.72Hz, 3H), 0.96(t, J=4.08 Hz,3H);

This spectral data is provided below:

[α] _D ²⁵ : −13.3° (c=1.05 in CHCl ₃ ). IR (film): 2929, 1729, 1463, 1255, 1099, 835 ^{cm -1} . ¹ H-NMR (400MHz, CDCl ₃ ): δ4.52(t, J=5.52Hz, 1H), 3.72(dd, J=5.20Hz, J=1.81Hz, 1H), 3.53(dd, J=8.02Hz ,J=5.40Hz,1H),3.46(s,3H),3.37(s,3H),3.24(dd,J=5.28Hz,J=2.32Hz,1H),2.62(dd,J=17.48Hz,J =8.04,Hz 1H),2.30(m,2H),1.33(m,1H),0.98(d,J=6.60Hz,3H),0.86(s,9H),0.07(s,3H),0.02(s ,3H). ¹³ C-NMR (100.6MHz, CDCl ₃ ): 176.35, 82.57, 81.29, 77.72, 73.93, 58.85, 58.36, 38.76, 38.40, 34.47, 25.83, 18.07, 17.31, -4.15, -4.65. HRMS-EI(m/z)[M] ⁺ calculated for C ₁₇ H ₃₂ O ₅ Si is 344.2019 and observed is 344.2015; and

This spectral data is provided below:

[α] _D ²⁵ : −69.9° (c=1.15 in CHCl ₃ ). IR (thin film): 2926, 1725, 1651, 1633, 1458, 1449, 1119, 1031cm ^–1 . ¹ H-NMR (400MHz, CDCl ₃ ): δ5.09(m, 3H), 4.74(d, J=6.84 Hz, 1H), 4.69(d, J=6.84Hz, 1H), 4.25(d, J=2.64Hz, 1H), 4.10(t, J=3.48Hz, 1H), 3.87(t, J=3.08Hz, 1H),3.48(s,3H),3.46(s,3H),3.43(s,3H),2.19(m,1H),1.94–2.08(m,10H),1.36(s,3H),1.33(s ,3H), 1.32(s,6H), 1.25(m,1H), 1.07(d,J=6.52Hz,3H). ¹³ C-NMR (100.6MHz, CDCl ₃ ): δ207.87, 136.84, 135.23, 131.23, 124.28, 123.95, 122.07, 98.29, 83.96, 83.03, 76.25, 58.99, 58.53, 56.09, 44.46, 494.05, 37.02, 37.02 26.73, 26.57, 25.66, 17.64, 16.21, 15.97, 11.09. HRMS-EI (m/z) [M] ⁺ calculated for C ₂₆ H ₄₄ O ₅ is 436.3189, observed 436.3184.

Embodiment 2 (+)-Androquinol and (–)-The effect of Androquinol

2.1 MTT test

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Invitrogen, USA) colorimetric basis assay to analyze cell viability.

The LNCaP cells were seeded in 96-well culture dishes at a density of 7 × ¹⁰³ cells per well and cultured overnight at 37 °C in 5% _CO2 . The cells were treated with 10 μM (+)-androquinol or (–)-androquinol for 48 hours, then MTT solution was added to each well and cultured in the culture dish for 4 hours. The culture medium was removed, and 100 μL of DMSO was added to each well to dissolve formazan, and the OD value of each well was measured at 570 nm using a microplate analyzer (Thermo Labsystems, Opsys MR, Thermo fisher scientific, Waltham, MA, USA).

As shown in Figure 1, the cell survival rate of the cells treated with (–)-androquinol was similar to that of the control group (Con), showing that (–)-androquinol was non-toxic.

Embodiment 3 androquinol's inhibitory effect on DPP4 enzyme activity

Dipeptidyl peptidase-4 (DPP4), also known as adenosine deaminase complexing protein 2 (adenosine deaminase complexing protein 2) or CD26 (differentiation cluster 26, cluster of differentiation 26), is a treatment for DM drug. DPP4 enzyme activity was determined by DPPIV/CD26 detection kit (BML-AK498, Enzo, NY, USA). This test is based on p-nitroaniline (p-nitroaniline, pNA) which is broken off from the chromogenic substrate (H-Gly-Pro-pNA), which will increase the absorbance at 405nm. First, add 50 μL of test buffer (50 mM glycine (Glycine), pH 8.7, 1 mM EDTA) to each well of a 96-well transparent microculture plate, then add 20 μL of DPP4 enzyme (13 μU/μL), 20 μL of test inhibitor agent, 100 μM androquinol or sitagliptin, a known clinical drug, and finally add 10 μL of pNA substrate to each well in sequence. After the reaction mixture was incubated at room temperature (RT) for 30 minutes, the absorbance of each sample was read at 405 nm using an ELISA plate reader (Thermo Labsystems, Opsy MR, Thermo fisher scientific).

The DPP4 activity of 100 μM androquinol (Ant) was compared with the conventional clinical drug sitagliptin (Sit). As shown in Figure 2, androquinol (Ant) provided a DPP4 activity inhibitory effect similar to that of sitagliptin (Sit), wherein the inhibition ratio (compared to the untreated control group) reached 50%.

Example 4 Effect of Androquinol on Inducing AMPK Thr172 Phosphorylation and Insulin Signaling Pathway

4.1 Culture and maintenance of mouse muscle myoblasts (C2C12)

Mouse muscle myoblasts (C2C12) were obtained from the Food Industry Development Institute (FIRDI, Hsinchu, Taiwan). The cells were grown and cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing high concentration of glucose (DMEM-high glucose; GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin streptomycin (penicillinstreptomycin) (GIBCO), and cultured in a 37°C cell culture incubator containing 5% CO ₂ . Before the experiment, cells were seeded and cultured in a 96-well culture dish at a density of 8×10 ³ cells per well or in a 6-well culture dish at a density of 2.5×10 ⁵ cells per well. When the myoblasts reached 80%, the cells were cultured in DMEM-high glucose medium supplemented with 1% FBS and 1% horse serum for 4 days to induce differentiation into myotubes.

4.2 Culture and maintenance of mouse muscle myoblasts (L6)

Mouse muscle myoblasts (L6) were donated from the laboratory of Professor Hitoshi Ashida (Kobe University, Kobe, Japan). The cells were grown and cultured in α-minimal essential medium (α-MEM, 12000022, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin/streptomycin (GIBCO), and cultured in 37 °C cell culture incubator with 5% _CO2 . Before the experiment, the cells were seeded and cultured in a 96-well culture dish at a density of 8×10 ³ cells per well or in a 6-well culture dish at a density of 2.5×10 ⁵ cells per well. When myoblasts reached 80% confluence, the cells were cultured in α-MEM supplemented with 2% FBS for 5 days to induce differentiation into myotubes.

4.3 Culture and maintenance of rat pancreatic tumor cells

Rat pancreatic tumor cells (AR42J) were obtained from the Food Industry Development Institute (FIRDI, Hsinchu, Taiwan). The cells were grown and cultured in DMEM (GIBCO) medium supplemented with 20% fetal bovine serum (FBS, GIBCO), 1% penicillin/streptomycin (GIBCO) and 2 mM L-glutamic acid, and cultured in 5 37°C cell culture incubator with % CO ₂ . Before the experiment, the cells were seeded and cultured for 16-24 hours.

4.4 Western blotting

After treatment, the cells were harvested and washed twice with cold KRH buffer (containing 50 mM HEPES, 137 mM NaCl, 4.8 mM KCl, 1.85 mM CaCl ₂ , 1.3 mM MgSO ₄ ), followed by ice-cold RIPA buffer (containing 50 mM Tris -HCl, pH 8.0, 150mM NaCl, 5mM NaF, 1% NP40, 1mM sodium orthovanadate (sodium orthovanadate), 0.5% sodium deoxycholate (sodium deoxycholate), 0.1% sodium dodecyl sulfate (sodium dodecyl sulphate, SDS ), protease inhibitors and phosphatase inhibitors (DE-68305, Roche, Mannheim, Germany)) and incubated at 4°C for 60 minutes. After the cells were centrifuged at 12,000 g for 30 minutes at 4°C, the supernatant was removed and quantified by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulphatepolyacrylamide gel electrophoresis, SDS-PAGE), and then transferred to PVDF (Perkin Elmer Life Sciences, Boston, MA, USA) membrane. Block the blotted film with 5% non-fat milk in TBS/T (containing 20 mM Tris-Base, 137 mM NaCl, pH 7.4, and 0.05% Tween-20) for 1 hour at room temperature (RT), followed by incubation at 4 °C. Incubate overnight with appropriate primary antibody. After washing, the blotted film was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (General Electric, Little Chalfont, Buckinghamshire, UK) for 1 hour. The signal was detected by WesternLightning™ Plus-ECL (Perkin Elmer Life Sciences) and the PVDF film was exposed to a luminescence image analyzer (LAS)-3000 (Fujifilm, Minato, Tokyo, Japan). The data obtained were analyzed and differences between the treatment groups were compared.

4.5 The effect of androquinol on inducing the phosphorylation of AKT Thr308 and AMPK Thr172

It is known that GLUT4 translocation in skeletal muscle requires the insulin-dependent PI3K/AKT activation signaling pathway or the insulin-independent AMPK activation pathway. Therefore, the determination of the activation of AKT and AMPK is to understand the role of androquinol through which pathway (insulin-dependent pathway or insulin-independent pathway) to induce GLUT4 translocation. In this test, metformin and insulin were administered separately to control glucose uptake by activating AMPK, and the insulin signaling pathway was used as a positive control group.

The C2C12 cells were cultured in DMEM supplemented with 1% FBS and 1% horse serum for 4 days to fully differentiate. The cells were washed twice with PBS containing 0.1% BSA, then incubated in PBS containing 0.1% BSA, and treated with or without insulin, metformin (Met) and androquinol (Ant) for 30 minutes. Treated cells were collected, washed twice with KRH, and then lysed in ice-cold RIPA buffer for 60 minutes. After centrifugation at 12,000g for 30 minutes at 4°C, the supernatant was stored at -80°C until use.

The C2C12 cells were treated with 100 nM insulin, 16 mM metformin (Met) and 25 μM androquinol (Ant) at 37° C. for 30 minutes. Subsequently, the cell lysates were separated by SDS-PAGE and analyzed by Western blotting for phosphorylated-AKT (Thr308) and phosphorylated-AMPK (Thr172). The protein expression levels including phospho-AMPK (Thr172), AMPKα, phospho-AKT (Thr308) and AKT (Cell Signaling, Boston, MA, USA) were detected and evaluated by Western blotting and primary antibodies.

As shown in Figure 3(A), insulin phosphorylation at AKT Thr308 induced insulin signaling, but not metformin and androquinol.

As shown in Figure 3(B), metformin (Met) and androquinol (Ant) can provide an AMPK Thr172 phosphorylation in mouse myoblasts C2C12; in particular, AMPK provided by androquinol (Ant) Phosphorylation effect of Thr172 was significantly better than that of insulin and metformin. It could be demonstrated that treatment with androquinol alone has the ability to improve insulin-independent GLUT4 translocation via the AMPK pathway in vitro.

4.7 The effect of androquinol on inhibiting the amount of PKA protein induced by DPP4 and enhancing glucagon-like peptide-1

It is known that in pancreatic β cells, the inhibition of DPP4 facilitates the binding of incretin peptides to G protein-coupled receptors (GLP-1R and GIP-R), and the downstream pathway is mainly regulated by cAMP , when GLP-1 binds to GLP-1R to increase cAMP, which results in PKA activation affecting subsequent Ca ²⁺ stimulation of insulin secretion.

The AR42J cells were treated with 1nM glucagon-like peptide-1 (GLP-1 (glucagon-like peptide-1), prospecbio, NJ, USA), 1nM insulin secretagogue-4 (Ex-4, Byetta, Eli Lilly , In, USA) and Androquinol were treated for 48 hours, and GAPDH was used as the internal control group. The PKA protein expression level was determined by Western blotting.

As shown in Figure 4, androquinol can effectively inhibit the enzyme activity of DPP4 in the enzyme experiment, and then use these compounds in AR42J cells to detect whether it can enhance the amount of PKA protein induced by glucagon-like peptide-1. It treated the cells with two hormones, glucagon-like peptide-1 (GLP-1) and insulin secretagogue-4, as well as natural compounds to measure the expression of PKA (wherein the androquinol treatment group had PKA protein expression).

The glucose uptake ability of embodiment 5 androquinol

Glucose uptake is known to be through the recruitment of GLUT4 to the cell membrane (GLUT4 translocation) and these transporters can assist glucose uptake. The glucose uptake capacity is determined by the cellular levels, which control the amount of GLUT4 glucose transporter present on the cell membrane.

5.1 Determination of GLUT4 translocation

C2C12 cells were cultured in DMEM supplemented with 1% FBS and 1% horse serum for 4 days to fully differentiate. The cells were washed twice with PBS containing 0.1% BSA, and then cultured in PBS with or without 185 μM insulin (Ins), 16 mM metformin (Met) or 50, 100 and 150 μM androquinol (Ant) for 55 minutes. (The number of samples in each group is n=5), then placed on ice and immediately fixed with 1% glutaraldehyde in PBS for 10 minutes at room temperature. After a 10-minute stop reaction with 0.1M glycine in PBS, the cells were blocked with PBS containing 5% mouse serum for 30 minutes. To determine the GLUT4 content on the cell surface, the cells were immediately incubated with 1 μg/mL anti-GLUT4 antibody (Santa Cruz, CA, USA) diluted in PBS containing 3% mouse serum for 1 hour. In the next step, the cells were treated with horseradish peroxidase (HRP)-conjugated anti-goat IgG secondary antibody (Jackson ImmunoResearch, Suffolk, UK) diluted (1:300) in PBS containing 3% mouse serum for 1 hour . After the washing step with PBS, TMB substrate (BioLegend, CA, USA) was added and incubated at room temperature for 30 minutes, and then 2N H ₂ SO ₄ was added to stop the reaction. HRP activity was measured by a spectrophotometer with absorbance at 450 nm (EnSpire 2300 Multilabel Reader, Perkin Elmer, Waltham, MA, USA).

The results are shown in Figure 5(A), which shows that androquinol has a good effect on GLUT4 translocation similar to insulin (Ins), and even better than metformin (Met).

On the other hand, the differentiated C2C12 cells were treated with insulin, androquinol, and insulin plus androquinol for 40 minutes (the number of samples in each group was n=5). The results are shown in Figure 5(B), which showed that simultaneous treatment with androquinol and insulin provided a synergistic effect.

5.2 Glucose uptake test

The glucose uptake assay was in accordance with a previous study (Yamamoto et al., "An enzymatic fluorimetric assay to quantitate 2-deoxyglucose and2-deoxyglucose-6-phosphate for in vitro and in vivo use." Analytical Biochemistry 404(2):238-240, 2010) with minor modifications and technical assistance from the laboratory of Prof. Hitoshi Ashida (Kobe University, Kobe, Japan). The differentiated L6 myotube cells planted in a 96-well microplate were cultured in 100 μL α-MEM containing 0.25% BSA per well for 30 minutes, and then added insulin, S961 (insulin receptor antagonist, donated from Dr.Lauge Novo-Nordisk, Denmark), metformin and androquinol. After incubation, the cells were washed twice with KRH. The L6 myotube cells were then incubated at 37° C. for 20 minutes in 5% CO ₂ in 60 μL of KRH buffer containing 1 mM 2-deoxyglucose (2DG, Sigma–Aldrich, St. Louis, MO USA) and 0.1% BSA. After incubation, the cells were washed twice with KRH buffer followed by the addition of 50 μL 0.1N NaOH. The microplates were incubated at 85°C for 90 minutes to dry. Subsequently, 50 μL of 0.1 N HCl was added to each well to neutralize the components in the wells, followed by 50 μL of 50 mM triethanolamine hydrochloride (TEA) buffer (200 mM KCl, 200 mM TEA, pH 8.1). Cellular 2DG uptake was measured by enzyme fluorescence assay. The fluorescent assay buffer is composed of 50 mM TEA buffer, 0.1% BSA, 2.5 mM β-NADP (Wako Pure Chemical, Osaka, Japan), 0.05 units of Diaphorase (Wako), 150 units of Leuconostoc mesenteris (L. mesenteriodes) G6PDH (sigma) and 0.5mM Resazurin sodium salt (sigma). 10 μL of 2DG sample was reacted with 100 μL of fluorescent assay buffer at 37° C. for 30 minutes. At the end of the reaction, the 570nm fluorescence value (EnSpire 2300 MultilabelReader, Perkin Elmer, Waltham, MA, USA) generated under 540nm excitation light was measured with a spectrophotometer.

As shown in Figure 6, insulin (Ins), metformin (Met) and androquinol (Ant) had similar effects on glucose uptake.

Taken together, androquinol enhanced GLUT4 translocation in a dose-dependent manner. Metformin and insulin are administered as clinical drugs to control glucose intake. Furthermore, the results of 100 μM androquinol were equivalent to the administration of 16 mM metformin in this trial (see Figure 5(A)). The results showed a synergistic effect when Androquinol was treated with insulin simultaneously (see Figure 5(B)). The results confirmed that androquinol alone has the ability to improve insulin-independent GLUT4 translocation in vitro. Androquinol provided glucose uptake effects similar to insulin and metformin (see Figure 6).

Embodiment 6 The effect of androquinol on blood sugar control (Glycemic control) in vivo

6.1 Animal experiments

Animal experiments were approved by the Animal Ethics Committee of Donghua University and carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals" of Donghua University. C57BL/6 mice and Imprinting Control Region (ICR) mice were obtained from the National Laboratory Animal Center (National Laboratory Animal Center, Taipei, Taiwan) and cultured at room temperature (22±2°C) and humidity (50 ±10%) in controlled environmental conditions. A cycle of 12 hours of light (0600am-1800pm) and 12 hours of darkness was maintained during the study. Mice had free access to food and water and were maintained on a standard laboratory diet (carbohydrates; 60%, protein; 28%, lipids; 12%, vitamins; 3%).

6.2 Oral Glucose Tolerance Test (OGTT) of Androquinol

Mice were subjected to this test after fasting for 12 hours. Mice were treated with D-glucose (2g/kg, p.o.) by oral gavage. At approximately 0, 30, 60, 90 and 120 minutes, blood was collected from the tail vein by venipuncture to measure blood glucose. Blood glucose was measured immediately by the glucose oxidase method using a glucose analyzer (Accu-Chek, Roche).

6.3 Efficacy of androquinol on short-term glucose tolerance in insulin-resistant mice

Eight-week-old male C57BL/6 mice were used for this experiment after a 12-hour fast. To investigate the effect of natural compounds on S961 (insulin receptor antagonist, Novo-Nordisk, Denmark)-induced hyperglycemia, mice were orally gavaged (p.o.) androquinol (dissolved in PEG and EtOH, 50 mg/kg Bwt) and D-glucose (2 g/kg Bwt) were injected intraperitoneally (i.p.) 15 min before S961 (50 nmol/kg Bwt). At approximately 0, 30, 60, 90 and 120 minutes, blood was collected from the tail vein by venipuncture for the OGTT test.

As shown in Figure 7(A), androquinol had hypoglycemic efficacy in insulin-resistant conditions in the oral glucose tolerance test (OGTT), similar to metformin. Furthermore, as shown in Figure 7(B), both (+)-androquinol and (–)-androquinol had hypoglycemic efficacy in insulin-resistant conditions in the oral glucose tolerance test (OGTT).

Embodiment 7 Androquinol is effective for the glucose tolerance of diet-induced obesity (Diet-induced obesity, DIO) ICR mice

7.1 Diet-induced obesity (DIO) ICR mice

Eight-week-old ICR males were induced by high-fat diet and 60% fructose water for 10 weeks. The high-fat diet contained 1 kg of conventional feed plus 150 g of conventional lard (23% total saturated fatty acids and 77% total unsaturated fatty acids, Qingxiang oil, Wei Li Foods Co., Changhua, Taiwan). The experimental mice were divided into two groups: (1) group A was diet-induced glucose intolerance (DIG, n=30) and group B was normal diet (Con, n=5). After being fed a high-fat diet and 60% fructose water for 10 weeks, ICR male mice were orally gavaged (p.o.) D-glucose (2g/kg) after 12 hours of fasting. At about 0, 30, 60, 90 and 120 minutes, blood was collected from the tail vein by venipuncture to measure blood glucose. Blood glucose was immediately measured with a glucose analyzer (Accu-Chek, Roche) using the glucose oxidase method. Hyperglycemia was defined as a blood glucose level higher than 200 mg/dL after 120 minutes of oral administration of glucose.

7.2 Short-term efficacy of androquinol on glucose tolerance in DIO ICR mice

DIO mice were administered 25 mg/kg Bwt of androquinol or 20 mg/kg Bwt of sitagliptin 15 minutes before oral gavage (p.o.) of D-glucose (2 g/kg) for the OGTT glucose tolerance test. The results obtained are shown in Figure 8, which shows that androquinol produces efficacy similar to that of sitagliptin.

7.3 Long-term efficacy of androquinol on glucose tolerance in DIO ICR mice

DIO mice were treated with androquinol (25 mg/kg Bwt) and sitagliptin (10 mg/kg Bwt) every other day (q.o.d) for 4 weeks. At the end of the long-term treatment, mice were assayed for glucose tolerance by the OGTT test. The results obtained are shown in Figure 9, which shows that androquinol produces efficacy similar to that of sitagliptin.

Statistical analysis

All data in these examples are presented as mean ± SEM. Statistical comparison of the results was performed using one-way analysis of variance (ANOVA). If the long histograms of each average value are marked with different letters, it means there is a significant difference after Tukey's test (p<0.05).

In view of the above in vitro and in vivo research results, it was concluded that androquinol could improve glucose uptake by enhancing the translocation of glucose transporter 4 and thus has the potential to control blood sugar, therefore, it can be developed into a drug for the treatment of diabetes, especially for type 2 diabetes.

Claims (18)

1. there is the purposes of compound in the individual diabetes medicament of preparation treatment of general formula (I),

Wherein X is oxygen or sulfur independently, R ₁, R ₂, R ₃with R ₄independently be selected from hydrogen atom, methyl or (CH ₂) m-CH ₃, m is the integer of 1 to 12, and n is the integer of 1 to 12.

2. purposes as claimed in claim 1, wherein the compound of this tool general formula (I) is separated from Antrodia camphorate (Antrodia camphorate).

3. purposes as claimed in claim 1, wherein the compound of this tool general formula (I) is Android tonquinol (antroquinonol).

4. purposes as claimed in claim 1, wherein the compound of this tool general formula (I) is (+)-Android tonquinol.

5. purposes as claimed in claim 1, wherein the compound of this tool general formula (I) is (–)-Android tonquinol.

6. purposes as claimed in claim 5 should (–)-Android tonquinol be wherein non-toxic.

7. purposes as claimed in claim 1, wherein these diabetes are Second-Type diabetes.

8. purposes as claimed in claim 1, wherein this individuality is the diabetic patients suffering from tool insulin resistance.

9. purposes as claimed in claim 1, wherein this compound has the effect effectively improving glucose uptake.

10. purposes as claimed in claim 1, wherein this compound has the effect controlling blood glucose.

11. purposes as claimed in claim 1, wherein this compound is Android tonquinol, it has the activity the effect strengthening the activation of AMP-activated form protein kinase (AMPK) that effectively suppress two peptidyls victory peptidase-4 (Dipeptidyl peptidase-4, DPP4).

12. purposes as claimed in claim 1, wherein said medicine also comprises insulin, and the combination of Android tonquinol and insulin can provide cooperative effect.

13. 1 kinds of methods preparing Android tonquinol, comprise the asymmetric addition effect of diethyl zinc (diethyl zinc), Ke Laisen (Claisen) transformation, cyclization displacement reaction (ring-closing metathesis), and the step such as lactonization (lactonization).

14. 1 kinds of compounds, it is tool general formula (IV) (–)-Android tonquinol:

Wherein Me is methyl.

15. compounds as claimed in claim 14, it is non-toxic.

16. 1 kinds of compounds, its be selected from by:

Wherein PMB is p-Jia Yang Ji Benzyl base (p-methoxybenzyl),

Wherein Me be methyl and TBS be three-Butyldimethyl silica-based (tert-butyldimethylsilyl), and

Wherein Me is methyl and MOM is methoxy (methoxymethyl), the group of composition.

17. 1 kinds of medical compositions being used for the treatment of diabetes, comprise compound as claimed in claim 14.

18. 1 kinds of medical compositions being used for the treatment of diabetes, comprise the combination of insulin or insulin analog and compound as claimed in claim 14.