阅读说明：本技术 一类具有联合治疗作用的cddo/川芎嗪醇杂合物及其制备方法和用途 (CDDO/ligustrazine heterozygote with combined treatment effect and preparation method and application thereof ) 是由 凌勇 钱建强 许中原 孟迟 吴红梅 谢旭东 陶维志 丁倩 张雨婷 于 2021-08-06 设计创作，主要内容包括：本发明涉及生物医药技术领域,尤其涉及一类具有联合治疗作用的CDDO/川芎嗪醇杂合物及其制备方法和用途,具有通式Ⅰ所示结构：本发明采用双前药策略,利用LAP响应片段亮氨酸酰基对CDDO类药物的CUK进行前药保护,并与川芎嗪醇活性片段进行偶联,形成CDDO/川芎嗪醇杂合物。本发明利用前药在LAP过表达的肝脏炎症组织中,选择性地裂解亮氨酸酰胺键片段,释放出CDDO类药物和川芎嗪醇,从而提高两种药物在肝损伤炎症组织的浓度,增加了炎症治疗作用,降低了药物剂量,改善了CDDO类药物的毒副作用,提高了药物安全性。(The invention relates to the technical field of biomedicine, in particular to a CDDO/ligustrazine heterocomplex with combined treatment effect and a preparation method and application thereof, wherein the CDDO/ligustrazine heterocomplex has a structure shown in a general formula I:)

1. A CDDO/ligustrazine heterocomplex with combined treatment effect is characterized in that the CDDO/ligustrazine heterocomplex has a structure shown in a general formula I:

wherein, R represents one of H and tert-butyloxycarbonyl; r' represents OMe, NHMe,one of (1); n is 0 or 1.

2. The class of CDDO/ligustrazine hybrids according to claim 1, wherein said formula I has a structure wherein R, R' and n are selected from the group consisting of:

R＝H,R’＝NHMe,n＝0；

or R ═ H, R ═ OMe, n ═ 1;

or R-t-butoxycarbonyl,n＝1。

3. the method of claim 1, wherein the CDDO/ligustrazine hybrid is prepared by the steps of:

A. condensing L-leucine 2 protected by N-Boc with p-aminobenzyl alcohol 1 in the presence of HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate) and DIPEA (N, N-diisopropylethylamine) to generate an amide compound 3, and obtaining a bromide 5 under the action of phosphorus tribromide of the compound 3;

B. on the one hand, CDDO technical material 4 and ligustrazine alcohol are processed by DMF/K₂CO₃Processing to obtain enol intermediate 8, and performing etherification reaction on the enol intermediate 8 and the compound 5 to obtain enol ether compound 6 or I of CDDO-Me and ligustrazine_a(ii) a On the other hand, the enol intermediate 8 and N-tert-butyloxycarbonyl-L-leucine 2 are subjected to EDCI and DMAP condensation acylation reaction to obtain a compound I_a；

C. Finally, removal of Boc by MeOH/HCl from intermediate 6 resulted in the formation of compound I in high yield_b；

I_aAnd I_bAll belong to the general formula I;

the synthetic route is as follows:

wherein, R represents one of H and tert-butyloxycarbonyl; r' represents OMe, NHMe,one of (1); n is 0 or 1.

4. Use of a class of CDDO/ligustrazine hybrids with combination therapeutic effect according to claim 1 or 2 for the treatment of various inflammatory diseases of liver injury.

5. Use of a class of CDDO/ligustrazine hybrids with combination therapeutic effect according to claim 1 or 2 for liver function protection against various liver injury inflammatory diseases.

6. The use of a class of CDDO/ligustrazine hybrids with combination therapy according to claims 1 or 2 to achieve the combination therapy of CDDO and ligustrazine in vivo in liver injury inflammatory diseases.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a CDDO/ligustrazine heterocomplex with a combined treatment effect and a preparation method and application thereof.

Background

Hepatitis is a general term for liver inflammation, and is mainly caused by drinking, drugs and virus infection. If the hepatitis can not be reasonably treated in time, the hepatitis can even become hepatic fibrosis, liver cirrhosis and even liver cancer, so that the search of a medicament capable of effectively treating the hepatitis becomes one of the hot spots of research.

The pentacyclic triterpenoid has wide bioactivity, especially in the aspects of tumor resistance, inflammation resistance, body immunity regulation and the like. Research shows that Oleanolic Acid (OA) has a protective effect on the liver and has anti-inflammatory activity. In a series of OA derivatives, 2-nitrile-3, 12-dioxooleanolic acid-1, 9(11) -diene-28-carboxylic acid methyl ester (CDDO-Me) is most concerned, clinical tests for treating viral hepatitis and non-alcoholic fat are carried out by Reata company, and CDDO drugs in the same series show good anti-hepatitis activity, but clinical results show that the drugs have serious cardiotoxicity, which severely limits the clinical application of the drugs, and subsequent molecular mechanism researches find that alpha-cyano-alpha, beta-unsaturated ketone (CUK) of the CDDO drugs can activate Nrf2 signal passage to generate anti-inflammatory effect by combining with Keap, but the CUK activity is too high and can be combined with nucleophilic macromolecules except for the Keap to generate larger toxic and side effects. If the prodrug mode can be utilized, the CUK with the active structure is protected, and the CDDO drugs are selectively released at the disease position, so that the side effect of the CDDO drugs on normal tissues is avoided, and the toxicity of the drugs is reduced.

Tetramethylpyrazine (TMP) is an alkaloid, also known as tetramethylpyrazine, widely found in the traditional Chinese medicine, Chuan Xiong. TMP has wide pharmacological actions, such as anti-inflammatory, anti-oxidation, myocardial protection, liver protection and the like, and has good clinical application prospect. Mechanistic studies indicate that the anti-inflammatory and antioxidant functions of TMP are also related to its activation of Nrf2 signaling pathway. 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (ligustrazine) is a metabolite in vivo of TMP, and has anti-inflammatory, antioxidant and other activities similar to that of TMP.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides a CDDO/ligustrazine heterocomplex with a combined treatment effect, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a CDDO/ligustrazine heterocomplex with combined treatment effect has a structure shown in a general formula I:

wherein, R represents one of H and tert-butyloxycarbonyl; r' represents OMe, NHMe,one of (1); n is 0 or 1.

Preferably, in the structure of formula I, R, R' and n are selected from the following combinations:

R＝H,R’＝NHMe,n＝0；

or R ═ H, R ═ OMe, n ═ 1;

or R-t-butoxycarbonyl,n＝1。

the preferred structure is shown in table 1:

TABLE 1 partial compound code of formula I and corresponding structure

Wherein, I₁:(4aS,6aR,6bS,8aS,12aS,14aR,14bS) -10- (2-amino-4-methylpentanamide) -11-cyano-N, 2,2,6a,6b,9,9,12 a-octamethyl-14-oxo-12- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14 b-octadecylhydropyran-4 a-carbamate;

I₂(4aS,6aR,6bS,8aR,12aS,14aR,14bS) -methyl-10- ((4- (2-amino-4-methylpentanamide) benzyl) oxy) -11-cyano-2, 2,6a,6b,9,9,12 a-heptamethyl-14-oxo-12- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14 b-octadecylhydropyran-4 a-carboxylate;

I₃tert-butyl (1- ((4- (((4aR,6aS,6bR,8aS,12aS,12bR,14bS) -2-cyano-8 a- (1H-imidazole-1-carbonyl) -4,4,6a,6b,11,11,14 b-heptamethyl-13-oxo-1- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14 b-octadecylhydropyran-3-yl) oxo) methyl) phenyl) amino) -4-methyl-1-oxopentanoate-2-yl) carbamate.

The invention also provides a preparation method of the CDDO/ligustrazine heterozygote with the combined treatment effect, which comprises the following steps:

A. condensing L-leucine 2 protected by N-Boc with p-aminobenzyl alcohol 1 in the presence of HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate) and DIPEA (N, N-diisopropylethylamine) to generate an amide compound 3, and obtaining a bromide 5 under the action of phosphorus tribromide of the compound 3;

B. on the one hand, CDDO technical material 4 and ligustrazine alcohol are processed by DMF/K₂CO₃Processing to obtain enol intermediate 8, and performing etherification reaction on the enol intermediate 8 and the compound 5 to obtain enol ether compound 6 or I of CDDO-Me and ligustrazine_a(ii) a On the other hand, the enol intermediate 8 and N-tert-butyloxycarbonyl-L-leucine 2 are subjected to EDCI and DMAP condensation acylation reaction to obtain a compound I_a；

C. Finally, removal of Boc by MeOH/HCl from intermediate 6 resulted in the formation of compound I in high yield_b。

I_aAnd I_bAll belong to the general formula I.

The synthetic route is as follows:

wherein, R represents one of H and tert-butyloxycarbonyl; r' represents OMe, NHMe,one of (1); n is 0 or 1.

The invention also provides application of the CDDO/ligustrazine heterocomplex with the combined treatment effect in selective targeted treatment of liver injury inflammatory tissues.

Wherein, the CDDO technical material and the ligustrazine alcohol can be selectively released in the liver injury inflammation tissue in vivo; can realize the combined treatment effect of CDDO technical material and ligustrazine in the tissues of liver injury inflammation in vivo.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention adopts a double-prodrug strategy, utilizes LAP response fragment leucine acyl to perform prodrug protection on CUK of CDDO drugs, and couples with ligustrazine alcohol active fragments to form CDDO/ligustrazine alcohol heterozygotes.

2. According to the invention, the prodrug is used for selectively cracking leucine amido bond fragments in liver inflammation tissues with LAP over-expression, so that CDDO drugs and ligustrazine alcohol are released, the concentration of the two drugs in liver injury inflammation tissues is increased, the inflammation treatment effect is increased, the drug dosage is reduced, the toxic and side effects of the CDDO drugs are improved, and the drug safety is improved.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.

Example 1(4aS,6aR,6bS,8aS,12aS,14aR,14bS) -10- (2-amino-4-methylpentanamide) -11-cyano-N, 2,2,6a,6b,9,9,12 a-octamethyl-14-oxo-12- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14 b-octadecylhydropyran-4 a-carbamate (I)₁)

CDDO-NHMe (0.198mmol,110mg) was dissolved in anhydrous DMF (4ml) and K was added₂CO₃(0.396mmol,54.7mg)，N₂Protection, ice-bath, reaction for 12h, then (3,5, 6-trimethylpyrazin-2-yl) methanol (0.198mmol,30.1mg) was dissolved with DMF (1ml), and then Boc-leucyl chloride (0.30mmol,74.7mg) was added dropwise to the mixture under vigorous stirring for 2 h. After the reaction is finished, using CH₂Cl₂The aqueous solution was extracted three times (60mL) and the organic layer was washed with Na₂SO₄Drying and concentration, column separation gave intermediate 6a in 60% yield.

Intermediate 6a (0.980mmol,992mg) was dissolved in methanolic hydrochloric acid and stirred at room temperature for 12 h. After the reaction is finished, concentrating and carrying out column separation to obtain a compound I₁The yield was 60%.

¹H NMR(400MHz,CDCl₃)δ8.50(s,1H,NH),7.23(s,1H,NH),5.78(s,1H,CH＝C),5.45(m,2H,CH₂),4.97(d,J＝8.0Hz,1H,CH),4.31(t,J＝6.7Hz,2H,CH₂),4.01(s,1H,CH),3.47(s,3H,CH₃),3.30(s,3H,OCH₃),3.07(s,3H,NCH₃),3.06(m,1H,CH),2.92(d,J＝4.7Hz,1H,CH),1.99(dd,J＝10.0,4.4Hz,1H,CH),1.89(dd,J＝9.5,4.3Hz,1H,CH),1.79(m,4H,2CH₂),1.26(m,12H,4CH₂),1.13(d,J＝10.2Hz,6H,2CH₃),1.03(m,8H,4CH₂),0.97(t,J＝6.5Hz,6H,2CH₃),0.91(s,3H,CH₃).

Example 2(4aS,6aR,6bS,8aR,12aS,14aR,14bS) -methyl-10- ((4- (2-amino-4-methylpentanamide) benzyl) oxy) -11-cyano-2, 2,6a,6b,9,9,12 a-heptamethyl-14-oxo-12- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,14,14a,14 b-octadecaneHydropyrane-4 a-carboxylates (I)₂)

4-aminobenzol (0.739g, 6mmol) was dissolved in anhydrous DMF (15mL), N₂After protection, the mixture was cooled to 0 ℃ and, while stirring, DIPEA (1.55g, 12mmol) and HATU (1.62g, 12mmol) were added in that order. After stirring for a further 10min, N- (tert-butoxycarbonyl) -L-leucine 2(1.388g, 6mmol) was added. Then the reaction system is returned to room temperature for 2 h. After completion of the reaction, the reaction solution was washed with saturated NaCl solution (3X 200mL), extracted with ethyl acetate (3X 150mL), and the organic layer was extracted with Na₂SO₄Drying and concentration, column chromatography purification gave compound 3 in 85% yield. Intermediate 3(1.01g, 3mmol) was reacted with CH₂Cl₂(30mL), stirred in ice bath for 10min, slowly added phosphorus tribromide (0.81g, 3mmol) dropwise, and reacted for 1 h. After the reaction is finished, the pH is adjusted to 7 by using 2mol/L sodium bicarbonate solution, and then CH is used₂Cl₂Extraction, drying of the organic layer and concentration under reduced pressure gave compound 5 in 80% yield.

CDDO-Me (0.198mmol,100mg) was dissolved in anhydrous DMF (4ml) and K was added₂CO₃(0.396mmol,54.7mg)，N₂Protection, ice-bath, reaction for 12h, then (3,5, 6-trimethylpyrazin-2-yl) methanol (0.198mmol,30.1mg) was dissolved with DMF (1ml), and then intermediate 5(0.30mmol,119.4mg) was added dropwise to the mixture under vigorous stirring for 2 h. After the reaction is finished, using CH₂Cl₂The aqueous solution was extracted three times (60mL) and the organic layer was washed with Na₂SO₄Drying and concentration, column separation gave target compound 6b in 60% yield.¹H NMR(400MHz,CDCl₃)δ8.63(s,1H,NH),7.49(d,J＝8.2Hz,2H,2ArH),7.25(d,J＝8.0Hz,2H,2ArH),5.79(s,1H,CH＝C),5.37(m,2H,CH₂),4.93(d,J＝9.9Hz,1H,CH),4.53(d,J＝10.0Hz,1H,CH),4.28(s,1H,CH),4.22(s,1H,CH),3.60(s,3H,CH₃),2.96(dt,J＝13.9,4.1Hz,1H,CH),2.77(d,J＝4.7Hz,1H,CH),2.39(m,9H,3CH₃),2.02(d,J＝18.5Hz,2H,CH₂),1.72(m,4H,2CH₂),1.49(m,10H,5CH₂),1.22(m,6H,2CH₃),1.08(m,12H,4CH₃),0.96(d,J＝9.1Hz,6H,2CH₃),0.85(m,15H,5CH₃).

Intermediate 6b (0.11mmol,100mg) was dissolved in methanolic hydrochloric acid and stirred at room temperature for 12 h. After the reaction is finished, concentrating and carrying out column separation to obtain a compound I₂The yield was 60%.¹H NMR(400MHz,CDCl₃)δ9.64(s,1H,NH),7.65(d,J＝8.5Hz,2H,NH₂),7.37(d,J＝8.4Hz,2H,2ArH),5.87(s,1H,ArH)),5.52(d,J＝11.1Hz,1H,ArH),5.42(d,J＝11.1Hz,1H,CH＝C),5.01(d,J＝9.9Hz,1H,OCH),4.60(d,J＝9.9Hz,1H,CH),4.39(d,J＝23.4Hz,2H,OCH₂),3.67(s,3H,OCH₃),3.54(dd,J＝9.9,3.7Hz,1H,CH),3.36(s,1H,CH),3.02(dd,J＝13.7,4.2Hz,1H,CH),2.85(d,J＝4.7Hz,1H,CH),2.46(d,J＝5.8Hz,9H,3CH₃),2.00(dd,J＝8.8,5.7Hz,4H,2CH₂),1.81(m,8H,4CH₂),1.49(m,6H,3CH₂),1.26(s,3H,CH₃),1.20(s,3H,CH₃),1.16(s,3H,CH₃),1.11(s,3H,CH₃),1.04(s,3H,CH₃),1.01(d,J＝3.3Hz,6H,2CH₃),0.97(d,J＝3.4Hz,3H,CH₃),0.93(s,3H,CH₃).

Example 3 tert-butyl (1- ((4- (((4aR,6aS,6bR,8aS,12 bR,14bS) -2-cyano-8 a- (1H-imidazole-1-carbonyl) -4,4,6a,6b,11,11,14 b-heptamethyl-13-oxo-1- ((3,5, 6-trimethylpyrazin-2-yl) methoxy) -1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14 b-octadecylhydropyran-3-yl) oxo) methyl) phenyl) amino) -4-methyl-1-oxopentanoic acid-2-yl) carbamate (I).₃)

4-aminobenzol (0.739g, 6mmol) was dissolved in anhydrous DMF (15mL), N₂After protection, the mixture was cooled to 0 ℃ and, while stirring, DIPEA (1.551g, 12mmol) and HATU (1.62g, 12mmol) were added in that order. After stirring for a further 10min, N- (tert-butoxycarbonyl) -L-leucine (1.388g, 6mmol) was added. Then the reaction system is returned to room temperature for 2 h. After completion of the reaction, the reaction solution was washed with saturated NaCl solution (3X 200mL), extracted with ethyl acetate (3X 150mL), and the organic layer was extracted with Na₂SO₄Drying and concentration, column chromatography purification gave compound 3 in 85% yield. Intermediate 3(1.01g, 3mmol) was reacted with CH₂Cl₂(30mL), stirred in ice bath for 10min, and slowly added phosphorus tribromide (0.812g, 3mmol) dropwise) And reacting for 1 h. After the reaction is finished, the pH is adjusted to 7 by using 2mol/L sodium bicarbonate solution, and then CH is used₂Cl₂Extraction, drying of the organic layer and concentration under reduced pressure gave compound 5 in 80% yield.

CDDO-Im (0.198mmol,107mg) was dissolved in anhydrous DMF (4ml) and K was added₂CO₃(0.396mmol,54.7mg)，N₂Protection, ice-bath, reaction for 12h, then (3,5, 6-trimethylpyrazin-2-yl) methanol (0.198mmol,30.1mg) was dissolved with DMF (1ml), and then intermediate 5(0.30mmol,119.4mg) was added dropwise to the mixture under vigorous stirring for 2 h. After the reaction is finished, using CH₂Cl₂The aqueous solution was extracted three times (60mL) and the organic layer was washed with Na₂SO₄Drying, concentrating, and separating with column to obtain target compound I₃The yield was 62%.¹H NMR(400MHz,CDCl₃)δ8.63(s,1H,NH),8.15(m,1H,ArH),7.49(d,J＝8.2Hz,2H,2ArH),7.46(m,1H,ArH),7.26(m,1H,ArH),7.25(d,J＝8.0Hz,2H,2ArH),5.79(s,1H,CH＝C),5.37(m,2H,CH₂),4.93(d,J＝9.9Hz,1H,CH),4.53(d,J＝10.0Hz,1H,CH),4.28(s,1H,CH),4.22(s,1H,CH),2.96(dt,J＝13.9,4.1Hz,1H,CH),2.77(d,J＝4.7Hz,1H,CH),2.39(m,9H,3CH₃),2.02(d,J＝18.5Hz,2H,CH₂),1.72(m,4H,2CH₂),1.49(m,10H,5CH₂),1.22(m,6H,2CH₃),1.08(m,12H,4CH₃),0.96(d,J＝9.1Hz,6H,2CH₃),0.85(m,15H,5CH₃).

EXAMPLE 4 protection of liver cells from inflammatory injury by Compounds of the invention

The protection effect of the compound on human liver cell LO2 cell strain is evaluated by adopting a tetramethylazole blue colorimetric Method (MTT) in vitro toxicity test. Firstly, a bottle of LO2 cells in exponential growth phase is taken, 0.25% trypsin is added for digestion to make adherent cells fall off, and the product is prepared with 2X 10 per ml⁴～4×10⁴A suspension of individual cells. Inoculating the cell suspension on a 96-well plate, placing 180 μ L of the cell suspension in each well, and placing in a constant temperature CO₂Culturing in an incubator for 24 h. Changing the solution, adding 0.1% carbon tetrachloride to perform hepatocyte injury for 24h, dissolving the compound with DMSO, diluting with PBS, respectively testing the compound concentration at 10 nM and 40nM, and culturing at 20 μ L per wellAnd (5) cultivating for 24 hours. MTT was added to a 96-well plate at 20. mu.L per well and reacted in an incubator for 4 hours. The supernatant was aspirated, 150. mu.L of DMSO was added to each well, and shaken on a flat shaker for 5 minutes. The absorbance of each well was measured at a wavelength of 570nm using an enzyme linked immunosorbent assay to calculate the cell viability. The cell survival rate is determined as OD value of the test group/OD value of the negative control group × 100%.

Experimental results show that the compound can effectively reverse damage of liver cells under lower dose (10 nM and 40nM), inhibit death of liver cells, and has a cell survival rate remarkably higher than that of a model group and higher than that of a positive drug CDDO + ligustrazine combined administration group. Therefore, the compound has stronger treatment effect on injured liver cells.

TABLE II cellular Activity of the Compounds of the invention against liver cell injury

Example 5 in vivo test of the Compounds of the present invention for liver injury in mice

Male ICR mice were fasted overnight and injected intraperitoneally with CCl the following day₄Solution (CCl)₄: olive oil 1: 4 dilutions, 2mL/kg), injected every three days, twice in total. Liver function indexes of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in blood are detected on the seventh day. Dividing the liver injury mice into four groups, namely a model group, positive medicine CDDO + ligustrazine alcohol group (34 mu mol/kg), I₂(17μmol/kg)，I₂(34. mu. mol/kg). A blank control group (PBS injected at the same frequency and volume) was also set. The medicine is administered three times within 5 days, and is administered by intraperitoneal injection, and liver function indexes ALT and AST in blood are measured the next day after administration.

The experimental results show that the compound I of the invention₂Has stronger treatment effect on a mouse model with liver injury, and discovers the compound I of the invention by detecting liver function indexes ALT and AST in blood of a mouse₂The (17 and 34 mu mol/kg) groups have obvious liver injury treatment effect, the AST index of the liver function is reduced by 6-7 times compared with the model group, and the ALT index is reduced by nearly 7 times compared with the model group and is obviously superior to the model groupThe ALT and AST indexes of the positive drug CDDO-Me and ligustrazine alcohol (34 mu mol/kg) combined treatment group are 2-3 times lower than those of the positive drug CDDO-Me and ligustrazine alcohol combined treatment group, so that the compound has better treatment effect, and the liver function index is basically recovered to the normal level. Therefore, the compound has the function of remarkably treating in-vivo liver injury inflammation.

The description and practice of the disclosure herein will be readily apparent to those skilled in the art from consideration of the specification and understanding, and may be modified and modified without departing from the principles of the disclosure. Therefore, modifications or improvements made without departing from the spirit of the invention should also be considered as the protection scope of the invention.