CN108976147B

CN108976147B - Amino n-hexanoyl amido methyl-n-hexanoyl basic amino acid, synthesis, activity and application thereof

Info

Publication number: CN108976147B
Application number: CN201710400008.0A
Authority: CN
Inventors: 赵明; 彭师奇; 王玉记; 吴建辉; 黄凌燕
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2021-02-12
Anticipated expiration: 2037-05-31
Also published as: CN108976147A

Abstract

The invention discloses an amino N-caproyl acylamino-N-caproyl basic amino acid with the following formula (N-amino N-caproyl acylamino) -amino N-caproyl-AA (wherein AA is an L-Lys residue and an L-Arg residue), and synthesis, activity and application thereof. Discloses a preparation method thereof, discloses the antitumor activity thereof, discloses the anti-tumor metastasis activity thereof and discloses the anti-inflammatory activity thereof, so that the invention discloses the application thereof in preparing antitumor drugs, anti-tumor metastasis drugs and anti-inflammatory drugs.

Description

Amino n-hexanoyl amido methyl-n-hexanoyl basic amino acid, synthesis, activity and application thereof

Technical Field

The invention relates to (N-amino-N-caproyl carbamoyl) -amino-N-caproyl-AA, a preparation method thereof, antitumor activity thereof, antitumor metastasis activity thereof and anti-inflammatory activity thereof, and thus relates to application thereof in preparing antitumor drugs, antitumor metastasis drugs and anti-inflammatory drugs. The invention belongs to the field of biological medicine.

Background

Invasion and metastasis processes are the basic biological features of malignant tumors and are one of the difficulties in tumor research. Cancer metastasis is the leading cause of morbidity and mortality in cancer patients, accounting for approximately 90% of cancer deaths. The present study on tumor infiltration and metastasis has been extensively discussed in different aspects. Among them, the role of urokinase-type plasminogen activator (uPA) series in tumor invasion and metastasis has become one of the hot spots in current research. The urokinase-type plasminogen activator (uPA) system, a family of serine proteases, plays a crucial role in tumor infiltration and metastasis. The system includes urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), Plasminogen Activator Inhibitor (PAI), which is involved in a variety of physiological and pathological processes including cell migration, angiogenesis, inflammation, embryonic development, tumor growth and metastasis.

The amino n-hexanoic acid can generate competitive inhibition with plasminogen activator, so that the plasminogen can not be activated into plasmin, and the amino n-hexanoic acid is a medicament for clinically treating fibrinolytic hemorrhage. Tranexamic acid binds to plasminogen and also exerts an antifibrinolytic effect. The two can respectively achieve the relevant effect of inhibiting the uPA system by preventing the uPA/uPAR interaction and inhibiting the plasmin activation. The lowest effective dose of amino-n-hexanoic acid and tranexamic acid for inhibiting the uPA system is 3.8mmol/kg and 3.2mmol/kg respectively. The inventors believe that their toxic side effects are directly related to their minimum effective dose. The inventor realizes that the novel u-PA inhibitor constructed by reasonably combining two known inhibitors of the uPA system and connecting amino acids has triple effects of resisting tumor, tumor metastasis and inflammation at low dose. Based on this knowledge, it was found that amino-n-hexanoyl formylaminocyclohexylamido-n-hexanoic acid derivatives modified with basic amino acids (L-Lys and L-Arg) have not only antitumor activity but also antitumor and anti-inflammatory activities at a dose of 0.5. mu. mol/kg, after 3 years of exploration. Because the toxic and side effects of the medicine can disappear along with the reduction of the dosage, the effective dosage is reduced by at least 6400 times compared with the amino n-hexanoic acid and the tranexamic acid, and the structure modification has outstanding technical effect. Based on these findings, the inventors have proposed the present invention.

Disclosure of Invention

In a first aspect of the invention there is provided (N-amino-N-hexanoylcycloacyl) -amino-N-hexanoyl-AA of the formula wherein AA is an L-Lys residue and an L-Arg residue.

The second aspect of the present invention provides a method for synthesizing (N-amino-N-hexanoylcyclohexanoyl) -amino-N-hexanoyl-AA (AA is an L-Lys residue and an L-Arg residue), which comprises:

(1) condensing Boc-tranexamic acid and amino methyl hexanoate to obtain Boc-tranylcarbonylamino methyl hexanoate (1);

(2) removing Boc from Boc-cycloacyl-amino-n-hexanoic acid methyl ester in ethyl acetate solution of hydrogen chloride to obtain cycloacyl amino-n-hexanoic acid methyl ester (2);

(3) condensing Boc-amino N-hexanoic acid and methyl carbamoylamino N-hexanoate (2) to obtain (N-Boc-amino N-hexanoyl carbamoylamino) -amino N-hexanoate methyl ester (3);

(4) saponifying and demethylating the compound 3 to obtain (N-Boc-amino-N-caproyl carbamoylamino) -amino-N-hexanoic acid (4);

(5) removing Boc from the compound 4 in ethyl acetate solution of hydrogen chloride to obtain (N-amino-N-hexanoyl formyl) -amino-N-hexanoic acid (7);

(6) condensing the compound 4 with AA-OBzl (AA is L-Lys residue and L-Arg residue) to obtain (N-Boc-amino-N-hexanoyl carbamoylamino) -amino-N-hexanoyl amino acid benzyl ester (5a, b).

(7) The compound 5a, b is subjected to hydrogenolysis to remove benzyloxycarbonyl and then to remove Boc in an ethyl acetate solution of hydrogen chloride to obtain (N-amino-N-hexanoyl carbamyl) -amino-N-hexanoyl-AA (6a, b) (AA is an L-Lys residue and an L-Arg residue).

The third aspect of the present invention is to evaluate the anti-lung cancer metastasis activity of (N-amino-N-hexanoyl-carbamoyl) -amino-N-hexanoyl-AA (AA is L-Lys residue and L-Arg residue) inhibition C57BL/6 mice.

A fourth aspect of the present invention is the evaluation of the use of (N-amino-N-hexanoylcarboxamide) -amino-N-hexanoyl-AA (AA is the L-Lys residue and the L-Arg residue) for inhibiting tumor growth in S180 mice.

The fifth aspect of the present invention is to evaluate the inhibition of inflammation in ICR mice by (N-amino-N-hexanoylcyclo-yl) -amino-N-hexanoyl-AA (AA is L-Lys residue and L-Arg residue).

Drawings

FIG. 1 (N-amino-N-hexanoylaminocyclyl) -amino-N-hexanoyl-AA the AA in FIGS. 5a and 6a is the L-Lys residue; AA in 5b and 6b is an L-Arg residue. i) Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), dry Tetrahydrofuran (THF); ii) a solution of hydrogen chloride in ethyl acetate (4M); iii) CH₃OH,2MNaOH；iv)Pd/C,H₂,CH₃OH; hydrogen chloride in ethyl acetate (4M).

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of (N-Boc-carbamoylamino-N-hexanoic acid methyl ester (1)

0.69g (2.68mmol) of Boc-tranexamic acid was suspended in 50mL of dry tetrahydrofuran, and 0.66g (3.20mmol) of Dicyclohexylcarbodiimide (DCC) and 0.44g (3.26mmol) of 1-hydroxybenzotriazole (HOBt) were added in this order at 0 ℃ and stirred for 30 min. Then 0.49g (2.70mmol) methyl aminohexanoate was added and the solution pH was adjusted to 9 with N-methylmorpholine (NMM), stirred at rt for 6h and TLC (dichloromethane/methanol-30/1) showed completion. The solvent was removed by concentration under reduced pressure, and the residue was dissolved in 50mL of ethyl acetate and filtered to obtain a filtrate. The filtrate was successively diluted with 20mL of saturated NaHCO₃The solution was washed 3 times, 20mL of saturated NaCl solution was washed 3 times, and 20mL of saturated KHSO was added₄The solution was washed 3 times, 20mL of saturated NaCl solution 3 times, 20mL of saturated NaHCO₃The solution was washed 3 times, 20mL of a saturated NaCl solution was washed 3 times, and the ethyl acetate layer was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate to dryness under reduced pressure gave 0.83g (80%) of the title compound as a colorless solid. ESI-MS (m/e): 385[ M + H ]]⁺。

EXAMPLE 2 preparation of N-carbamoylamino-N-hexanoic acid methyl ester hydrochloride (2)

6.00g (15.62mmol) of Compound 1 are slowly mixed with 60mL of a solution of hydrogen chloride in ethyl acetate (4M) at-10 ℃ with stirring and kept at-10 ℃ for 5h with stirring. TLC (dichloromethane/methanol-30/1) showed the reaction was complete. Concentrating under reduced pressure, dissolving the residue with anhydrous ethyl acetateThe resulting solution was concentrated under reduced pressure. This operation was repeated 3 times. The solid was then suspended thoroughly with anhydrous ether and the ether was removed to give a colorless solid which was used directly in the next reaction. ESI-MS (M/e):322[ M + H]⁺。

EXAMPLE 3 preparation of (N-Boc-amino-N-caproylcarboxamide-amino-N-hexanoic acid methyl ester (3)

Using the procedure of example 1, from 3.36g (14.55mmol) Boc-amino-n-hexanoic acid and 4.67g (14.57mmol) Compound 2, a pale yellow solid was obtained. The solid was thoroughly triturated with ethyl acetate to give 6.20g (85%) of the title compound as a colorless solid. ESI-MS (m/e): 498[ M + H]⁺。

EXAMPLE 4 preparation of (N-Boc-amino-N-hexanoylaminocyclic-acyl) -amino-N-hexanoic acid (4)

6.20g (12.47mmol) of Compound 3 are dissolved in 20mL of methanol and the pH is adjusted to 12 at 0 ℃ with aqueous NaOH (2M). Stirring was carried out for 4h at 0 ℃ and pH 12, and TLC (dichloromethane/methanol-25/1) indicated completion of the reaction. The reaction mixture was saturated with KHSO₄The solution was adjusted to pH 7 and concentrated under reduced pressure. The residue was further saturated with KHSO₄The solution was adjusted to pH 2, extracted with ethyl acetate, and the ethyl acetate layers were combined and washed to neutrality with saturated NaCl solution. The ethyl acetate phase was dried over anhydrous sodium sulfate for 12 h. Filtration and concentration of the filtrate to dryness under reduced pressure gave 4.60g (76%) of the title compound as a colorless solid. ESI-MS (m/e): 484[ M + H]⁺。

EXAMPLE 5 preparation of (N-amino-N-hexanoylaminocyclic-acyl) -amino-N-hexanoic acid (7)

Using the method of example 2, 0.87g (84%) of the title compound was obtained as a colorless solid from 1.30g (2.69mmol) of Compound 4. Mp 236-239 ℃;

(c＝0.1，H₂O).ESI-MS(m/e):384[M+H]⁺.IR(cm^-1):3265,3081,2927,2857,1633,1557,1470,1416,1396,1362,1327,1235,1210,726,697.¹H-NMR(300MHz,D₂O):δ/ppm＝3.11(t,J＝6.6Hz,2H),2.98(d,J＝6.6Hz,2H),2.93(t,J＝7.8Hz,2H),2.20(t,J＝7.2Hz,2H),2.12(t,J＝7.2Hz,2H),2.09(m,1H),1.76(m,4H),1.67～1.55(m,4H),1.52～1.40(m,5H),1.37～1.24(m,6H),0.93(m,2H)。

EXAMPLE 6 preparation of (N-Boc-amino-N-hexanoylaminocyclyl) -amino-N-hexanoyl lysine benzyl ester (5a)

Using the method of example 1, 2.49g (41%) of the title compound were obtained as a colorless solid from 4.00g (8.28mmol) of Compound 4 and 2.80g (7.52mmol) of HCl.Lys (Boc) -OBzl. ESI-MS (m/e): 802[ M + H ]]⁺。

EXAMPLE 7 preparation of (N-Boc-amino-N-hexanoylaminocycloyl) -amino-N-hexanoylarginine benzyl ester (5b)

Using the method of example 1, from 4.00g (8.28mmol) of Compound 4 and 3.33g (6.92mmol) of tos.Arg (NO)₂) OBzl gave 1.03g (19%) of the title compound as a colorless solid. ESI-MS (m/e): 775[ M + H]⁺。

EXAMPLE 8 preparation of (N-amino-N-caproylcyclo-acyl) -amino-N-caproyl lysine (6a)

1.10g (1.37mmol) of 5a was dissolved in 20mL of methanol, 110mg of Pd/C was added thereto, and the mixture was evacuated under stirring at room temperature for 10 hours. TLC (dichloromethane/methanol-4/1) showed the reaction was complete. Pd/C was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was added to 5mL of anhydrous ethyl acetate and stirred well, -10 ℃ 10mL of a solution of hydrogen chloride in ethyl acetate (4M) was slowly added and stirred until TLC (ethyl acetate/water/glacial acetic acid ═ 3/1/1) showed completion of the reaction. Concentrating under reduced pressure, dissolving the residue with anhydrous ethyl acetate, concentrating under reduced pressure, and dissolving the residue with anhydrous ethyl acetate. This operation was repeated 3 times. Mixing the obtained solid with anhydrous diethyl ether, standing, removing diethyl ether to obtain colorless solid, and adding saturated NaHCO at 0 deg.C₃The solution was adjusted to pH 7 and purified by C18 column chromatography to give 480mg (68%) of the title compound as a colorless solid. Mp 208-.

(c＝0.1，H₂O).ESI-MS(m/e):512[M+H]⁺.IR(cm^-1):3292,2927,2857,1634,1548,1437,1392,1257,1232,1207,1168,696.¹H-NMR(300MHz,D₂O):δ/ppm＝4.07(dd,J₁＝7.8Hz,J₂＝5.1Hz,1H),3.07(t,J＝6.3Hz,2H),2.95(d,J＝6.3Hz,2H),2.88(t,J＝7.2Hz,4H),2.18(m,4H),2.08(m,1H),1.75(m,5H),1.63～1.46(m,9H),1.44～1.35(m,3H),1.33～1.22(m,8H),0.89(m,2H)。

EXAMPLE 9 preparation of (N-amino-N-hexanoylaminocyclohexyl) -amino-N-hexanoylarginine (6b)

Using the method of example 8, 120mg (19%) of the title compound are obtained from 0.92g (1.19mmol) of 5b as a colorless solid. Mp 219-.

(c＝0.1，H₂O).ESI-MS(m/e):540[M+H]⁺.IR(cm^-1):3282,3084,2927,2858,1634,1549,1446,1396,1235,1208,692.¹H-NMR(300MHz,D₂O):δ/ppm＝4.06(m,1H),3.08(m,4H),2.93(d,J＝6.3Hz,2H),2.80(t,J＝6.6Hz,2H),2.18(m,4H),2.06(m,1H),1.71(m,5H),1.62～1.50(m,8H),1.40～1.35(m,4H),1.29～1.20(m,6H),0.87(m,2H)。

EXAMPLE 10 determination of the anti-tumor metastasis Activity of Compounds 6a, b

The assay model was inoculated with Lewis mouse lung carcinoma cells (LLC, purchased from ATCC) in DMEM medium (containing 10% inactivated fetal bovine serum, 1X 10)⁵U/L penicillin and 100mg/L streptomycin), and the cells are enriched by passage every two days according to an adherent cell culture method. Digesting the cells when the cells are in good growth state and in logarithmic growth phase, and adjusting the cell density to 1 × 10 with physiological saline⁷one/mL. Staining with placental blue to count viable cells>95 percent. Inbred C57BL/6 male mice (SPF grade, body weight 20. + -.2 g) were taken and left-handed mice fixed. The right anterior limb axillary skin of the mouse was disinfected with 75% ethanol. The LLC tumor cell suspension is injected subcutaneously into the axilla of a mouse with a 1mL sterile syringe held in the right hand, and 0.2mL is injected into each mouse. After the mice are inoculated for 10 days, tumors with the diameter of about 4-5mm grow out, namely the tumor source. The Lewis lung cancer tumor-bearing mice are inoculated for 10 days and anesthetized by ether, and then the cervical vertebrae are removed for killing. Soaking in 75% ethanol for 10min, sterilizing, and removing tumor on clean bench. Well-grown tumor tissue was selected, minced in a sterile plate, and placed in a tissue homogenizer made of glass. Adding 4 deg.C physiological saline at a ratio of tumor mass to physiological saline volume of 1: 3 (g: mL), and lightly grinding to obtain cell suspension. The cell suspension is screened by 200-mesh cells to prepare single cell suspension. Adjusting the cell density of the single cell suspension to 1.5X 10 with physiological saline⁷one/mL. Staining with placental blue to count viable cells>95 percent. Left-handed inbred C57BL/6 male mice were fixed and their right anterior limb axillary skin was disinfected with 75% ethanol. The tumor cell suspension was injected subcutaneously into the mouse axilla with a 1mL sterile syringe in the right hand, 0.2mL each. 10 days after inoculation, the mice developed tumors of 4-5mm in diameter, and the inoculated mice were randomly grouped by the measured tumor volume. Each group had 12 mice. On day 11 of tumor inoculation mice were orally administered either a recognized physiological saline solution of the anti-tumor metastasis peptide RGDS (dose of 20. mu. mol/kg/day) or compound 6a, b (dose of 0.5. mu. mol/kg/day) or compound 7 (dose of 5. mu. mol/kg/day) or compound 7 (dose of 10 mL/kg/day) 1 dose per day for 12 consecutive days, and tumor volumes were measured and recorded every two days. The next day of the last administration, tumor volume was measured, cervical spine was removed by ether anesthesia and sacrificed, tumor of the mice was weighed, lung of the mice was taken and tumor nodules transferred from the lung of the tumor were counted. Statistical analysis of the data was performed using the t-test. The results are shown in Table 1. Compounds 6a, b were not only effective in inhibiting tumor lung metastasis at 0.5 μmol/kg dose, but also had no significant difference in activity from RGDS at doses 400-fold higher and compound 7 at doses 10-fold higher than them. These data indicate that the present invention has significant technical effects.

TABLE 1 antitumor metastatic Activity of Compounds 6a, b

a) P <0.01 to saline, p >0.05 to RGDS and compound 7; n is 12.

EXAMPLE 11 determination of the anti-tumor growth Activity of Compounds 6a, b

Doxorubicin, compound 7 and compounds 6a, b were all dissolved in saline prior to assay for administration to S180 mice. Inoculating 10-day-vigorous S180 ascites fluid from male ICR mice in sterile environment, diluting with normal saline to obtain (1:2) fluidAnd (3) fully mixing, staining the tumor cell suspension with freshly prepared 0.2% trypan blue, uniformly mixing, and counting according to a white cell counting method, wherein the cells stained with blue are dead cells, and the cells not stained with blue are live cells. The cell concentration is 4-large-grid viable cell number/4 × 10⁴The cell density was calculated as x dilution factor ═ cell number/mL, and the cell survival rate was calculated as live cell number/(live cell number + dead cell number) × 100%. Homogenizing tumor solution with survival rate of more than 90% to density of 2.0 × 10⁷Cell suspension per mL. This cell suspension was inoculated subcutaneously (0.2 mL/mouse) in the right axilla of a mouse to prepare S180 tumor-bearing mice. 24h after inoculation, S180 tumor-bearing mice were intraperitoneally injected daily with a saline solution of doxorubicin (dose 2. mu. mol/kg/day g) or daily orally administered with a saline solution of Compound 7 (dose 5. mu. mol/kg/day) or daily orally administered with a saline solution of Compounds 6a, b (dose 0.5. mu. mol/kg/day). The administration is once daily for 12 days. The day after the last dose, tumor volume was measured, cervical spine was removed under ether anesthesia and sacrificed, then the right axillary tumor growth site of the mouse was fixed with forceps, and the skin was excised and the tumor was blunt-stripped and weighed. Efficacy was expressed as tumor weight (mean ± SD g), and data were analyzed by t-test and variance. The results are shown in Table 2. Not only can compounds 6a, b effectively inhibit tumor growth at a dose of 0.5 μmol/kg, but there was no significant difference in activity from compound 7 at a dose 10-fold higher than them. These data indicate that the present invention has significant technical effects.

TABLE 2 Effect of Compounds 6a-b on tumor growth in S180 mice

a) P <0.01 to saline, p >0.05 to compound 7; n is 12.

EXAMPLE 12 determination of the anti-inflammatory Activity of Compounds 6a, b

Since xylene-induced ear swelling in mice is recognized as an acute inflammation model, the present invention measures the therapeutic effect of compounds 6a, b on a xylene-induced ear swelling model in mice. Because aspirin is a positive drug for treating acute inflammation, aspirin is selected as a positive control in the present invention. ICR male mice (body weight 42 + -3 g) were allowed to rest for 2 days at 22 deg.C, with free access to water and food. Thereafter, the mice were randomly divided into a saline group (dose of 0.2 mL/mouse), an aspirin group (dose of 1.11mmol/kg), a compound 7 group (dose of 5. mu. mol/kg) and compound 6a, b groups (dose of 0.5. mu. mol/kg), and 12 mice per group. Mice were tested either orally in normal saline, orally in aspirin, orally in compound 7, or orally in compound 6a, b, as indicated. After 30min of administration, the left auricle of the mouse was evenly smeared with 30 μ L of xylene, and after 2h, the mouse was subjected to ether anesthesia, the neck was cut off, the left and right ears were cut off, round ears were taken at the same positions of the two ears by a 7mm punch, and the difference in swelling between the two ears was weighed and found to be the swelling degree. Namely the swelling degree is equal to the weight of the left ear disk to the weight of the right ear disk. The results are shown in Table 3. Not only can compounds 6a, b effectively inhibit xylene-induced ear swelling in mice at a dose of 0.5 μmol/kg, but there was no significant difference in activity from compound 7, which was 10-fold higher than them at the dose. These data indicate that the present invention has significant technical effects.

TABLE 3 Effect of Compounds 6a, b on xylene swelling of mouse ears

a) P <0.01 to saline, p >0.05 to compound 7; n is 12.

Claims

1. (N-amino-N-hexanoylaminocyclyl) -amino-N-hexanoyl-AA of the formula,

wherein AA is an L-Lys residue or an L-Arg residue.

2. A process for preparing (N-amino-N-hexanoylcycloacyl) -amino-N-hexanoyl-AA according to claim 1, comprising:

(6) condensing the compound 4 with AA-OBzl to obtain (N-Boc-amino-N-hexanoyl formamide cycloacyl) -amino-N-hexanoyl amino acid benzyl ester (5a, b);

(7) the compound 5a, b is subjected to hydrogenolysis to remove benzyloxycarbonyl and then to remove Boc in an ethyl acetate solution of hydrogen chloride to obtain (N-amino-N-hexanoyl carbamyl) -amino-N-hexanoyl-AA.

3. Use of (N-amino-N-hexanoylcycloacyl) -amino-N-hexanoyl-AA according to claim 1 for the preparation of a medicament against tumor metastases.

4. The use of (N-amino-N-hexanoylcycloacyl) -amino-N-hexanoyl-AA according to claim 1 for the preparation of an antitumor medicament.

5. Use of (N-amino-N-hexanoylcycloacyl) -amino-N-hexanoyl-AA according to claim 1 for the preparation of an anti-inflammatory medicament.