CN108948137A - Piperazine -2,5- diketone of 3S- indolylethyl -6S- polar amino acid modification, synthesis, activity and application - Google Patents

Abstract

The invention discloses (the 3S of following formula, 6S) -3- (the positive hexanoyl amino normal-butyl of AA- amino) -6- (indoles -3- ethyl)-piperazine-2,5-dione (AA is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residue in formula).It discloses their preparation method, disclose their anti-tumor activity, disclose their activity of resisting tumor metastasis, and their anti-inflammatory activity activity is disclosed, thus anti-tumor drug is being prepared the invention discloses them, the application in medicine for anti transfer of tumor and anti-inflammatory drug.

Description

3S-indoleethyl-6S-polar amino acid modified piperazine-2,5-dione, its synthesis, Activity and Application

technical field

The present invention relates to (3S,6S)-3-(AA-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione. Related to their preparation method, related to their anti-tumor activity, related to their anti-tumor metastasis activity, and related to their anti-inflammatory activity, thus the present invention relates to their preparation of anti-tumor drugs, anti-tumor metastasis drugs and anti-inflammatory drugs Applications. The invention belongs to the field of biomedicine.

Background technique

Tumors seriously threaten human health. In addition to the poor prognosis of cancer patients, the concurrent metastasis of tumors further worsens the prognosis of patients. For example, more than 90% of cancer patients died of metastasis. Because the existing anti-tumor drugs have no anti-tumor metastasis effect, the clinical curative effect of tumor chemotherapy is unsatisfactory. It is an urgent clinical need to invent drugs against tumor metastasis. Previously, the inventors had disclosed that diketopiperazines with four configurations of S, S-, R, R-, R, S- and S, R- could inhibit the migration of HCCLM3 (highly metastatic human liver cancer cells) at a concentration of 0.5 μM and invasion. Later, the inventors disclosed that diketopiperazine in the R,R-configuration can inhibit tumor metastasis to the lung in C57BL/6 mice at a dose of 5 μmol/kg. However, the lowest effective dose is 5 μmol/kg. In order to reduce the minimum effective dose, the inventors made various modifications to the butylamino group of diketopiperazine in S,S-configuration. After 3 years of exploration, it was found that amino n-hexyl acylated with polar side chain amino acids (L-Asp, L-Arg, L-Gln, L-Glu, L-Lys, L-Asn, L-Ser and L-Thr) Acid acylation of the butylamino group of diketopiperazine in S,S-configuration not only reduces the minimum effective dose of anti-tumor metastasis to 0.5 μmol/kg, but also reduces the minimum effective dose of anti-tumor and anti-inflammation to 0.5 μmol/kg. A 10-fold reduction in the effective dose indicates that this structural modification has an outstanding technical effect. Based on these findings, the inventors have made the present invention.

Contents of the invention

The first content of the present invention is to provide (3S,6S)-3-(AA-aminon-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5 - diketone (AA in the formula is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residues).

The second content of the present invention is to provide (3S,6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (AA in the formula is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residue residue), the method comprises:

(1) L-Boc-Lys(Cbz) is condensed with L-Trp-OBzl to obtain Boc-Lys(Cbz)-Trp-OBzl;

(2) Boc-Lys(Cbz)-Trp-OBzl de-Boc in hydrogen chloride ethyl acetate solution to obtain Lys(Cbz)-Trp-OBzl;

(3) Lys(Cbz)-Trp-OBzl is cyclized in 5% aqueous sodium bicarbonate saturated ethyl acetate solution to generate (3S,6S)-3-(benzyloxycarbonylamino-n-butyl)-6-(indole -3-Ethyl)-piperazine-2,5-dione (1);

(4) Hydrogenolysis of compound 1 to obtain (3S,6S)-3-(butylamino)-6-(indole-3-ethyl)-piperazine-2,5-dione (2);

(5) Compound 2 is condensed with Boc-amino n-caproic acid to obtain (3S,6S)-3-(Boc-amino n-n-caproylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2, 5-diketone (3);

(6) De-Boc of compound 3 in hydrogen chloride ethyl acetate solution to obtain (3S,6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2 ,5-diketone (4);

(7) Compound 4 and Boc-AA (AA is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L- Ser residues and L-Thr residues) were condensed to give (3S,6S)-3-(Boc-AA-aminon-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2 , 5-diketones (5a-h).

(8) Compounds 5a-h de-Boc in ethyl acetate solution of hydrogen chloride to obtain (3S,6S)-3-(AA-aminon-hexanoylamino-n-butyl)-6-(indole-3-ethyl)- Piperazine-2,5-dione (6a-h).

The third content of the present invention is to evaluate (3S,6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (AA in the formula is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residue residues) inhibit the anti-metastatic activity of lung cancer in C57BL/6 mice.

The fourth content of the present invention is to evaluate (3S,6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (AA in the formula is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residue residues) inhibited inflammation in ICR mice.

The fifth content of the present invention is to evaluate (3S,6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (AA in the formula is L-Asp residue, L-Arg residue, L-Gln residue, L-Glu residue, L-Lys residue, L-Asn residue, L-Ser residue and L-Thr residue residues) to inhibit tumor growth in S180 mice.

Description of drawings

Figure 1 Synthesis of (3S,6S)-3-(AA-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6a-c) Route. AA is L-Asp residue in 5a and 6a; AA is L-Arg(NO ₂ ) residue in 5b, AA is L-Arg residue in 6b; AA is L-Gln residue in 5c and 6c; AA is L-Glu (OBzl) residue in 5d, AA is L-Glu residue in 6d; AA is L-Lys (Cbz) residue in 5e, AA is L-Lys residue in 6e; 5f and 6f AA is L-Asn residue; AA is L-Ser residue in 5g and 6g; AA is L-Thr residue in 5h and 6h; i) dicyclohexylcarbodiimide (DCC), 1-hydroxybenzo Triazole (HOBt), N-methylmorpholine (NMM), tetrahydrofuran (THF); ii) hydrogen chloride in ethyl acetate; iii) ethyl acetate, 5% sodium bicarbonate; iv) dimethylformamide ( DMF), Pd/C, H ₂ .

Detailed ways

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Embodiment 1 prepares Boc-Lys(Cbz)-Trp-OBzl

Suspend 7.7g (20mmol) Boc-Lys (Cbz) in 100mL of anhydrous tetrahydrofuran (THF), and add 2.7g (20mmol) 1-hydroxybenzotriazole (HOBt) to the suspension successively under ice-cooling And 5.0g (25mmol) dicyclohexylcarbodiimide (DCC), then stirred for 30min. Afterwards, 8.0 g (25 mmol) Trp-OBzl was added. The reaction compound was added dropwise to N-methylmorpholine (NMM) to adjust the pH to 9. The reaction mixture was first stirred under ice bath for 1 h, and then stirred at room temperature for 12 h. The reaction compound was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 150 mL of ethyl acetate solution. The obtained ethyl acetate solution was washed successively with 5% KHSO ₄ aqueous solution 3 times, and saturated NaCl aqueous solution 3 times. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ for 12 h, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained yellow syrup was purified by silica gel column chromatography (CH ₂ Cl ₂ /CH ₃ OH, 100:1) to obtain 12.1 g (88%) of the title compound as a colorless solid. ESI-MS (m/e): 657 [M+H] ⁺ .

Embodiment 2 prepares Lys(Cbz)-Trp-OBzl

3.8 g (5 mmol) Boc-Lys(Cbz)-Trp-OBzl was slowly mixed with 52 mL of hydrogen chloride in ethyl acetate under stirring in an ice bath. The resulting solution was stirred for 5 h in an ice bath. After that, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 50 mL of anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. This operation was repeated three times. The residue was washed well with anhydrous ether to afford 3.45 g (92%) of the title compound as a yellow powder. ESI-MS (m/e): 557 [M+H] ⁺ .

Example 3 Preparation of (3S, 6S)-3-(benzyloxycarbonylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (1)

3.45 g (6.2 mmol) of Lys(Cbz)-Trp-OBzl were dissolved in 150 mL of ethyl acetate. After the obtained solution was washed three times with 5% aqueous sodium bicarbonate solution, the ethyl acetate solution was stirred at room temperature for 12 h to fully separate out the colorless solid. 1.9 g (55%) of the title compound were filtered off. ESI-MS (m/e): 449 [M+H] ⁺ .

Example 4 Preparation of (3S, 6S)-3-amino-n-butyl-6-(indole-3-ethyl)-piperazine-2,5-dione (2)

To 1.9g (4.2mmol) (3S,6S)-3-(benzyloxycarbonylbutylamino)-6-(indole-3-ethyl)-piperazine-2,5-dione (1) with 20mL Add 200 mg of Pd/C to the water N,N-dimethylformamide (DMF) solution, feed H ₂ , and stir at room temperature for 48 hours. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 1.1 g (83%) of the title compound as a white powder. ESI-MS (m/e): 315 [M+H] ⁺ .

Embodiment 5 prepares Boc-amino n-caproic acid

Add 5.23g (Boc) ₂ O and 30mL dioxane to a solution of 2.62g (20mmol) amino-n-caproic acid and 30mL distilled water, and adjust the pH of the resulting reaction solution to 9 with 2N NaOH aqueous solution. Stir at room temperature for 24h, during which the vacuum was continuously pumped. The reaction compound was adjusted to pH 7 with KHSO ₄ aqueous solution, and concentrated under reduced pressure to remove dioxane. The remaining solution was further adjusted to pH 2 with KHSO ₄ . The remaining solution was extracted with 100 mL of ethyl acetate and dried with anhydrous NaSO ₄ for 8 h. Filtration and concentration of the filtrate under reduced pressure afforded 4.36 g (94%) of the title compound. ESI-MS (m/e): 232 [M+H] ⁺ .

Example 6 Preparation of (3S,6S)-3-(Boc-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (3)

Using the method of Example 1, from 0.97g (4.2mmol) Boc-amino n-hexanoic acid and 1.9g (3.5mmol) (3S, 6S)-3-aminobutyl-6-(indole-3-ethyl)- Piperazine-2,5-dione (2) afforded 2.21 g (71%) of the title compound as a colorless solid. ESI-MS (m/e): 528[M+H] ⁺ . ¹ H-NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.865(s, 1H), 8.018(s, 1H), 7.918( s,3H),7.586(m,2H),7.313(d,J＝8.1Hz,1H),7.042(m,2H),6.952(m,1H),6.744(s,1H),4.109(m,1H ),3.640(m,1H),3.228(m,1H),3.031(m,1H),2.891(m,2H),2.745(m,2H),2.004(t,J＝7.2Hz,2H),1.575 (m,2H), 1.431(m,10H), 1.118(m,3H), 0.970(m,3H), 0.612(m,3H).

Example 7 Preparation of (3S,6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (4)

Using the method of Example 3, from 2.21g (4mmol) (3S,6S)-3-(Boc-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5 - Diketone (3) yielded 1.45 g (82%) of the title compound as a colorless powder. ESI-MS (m/z): 428[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.947(s, 1H), 8.056(s, 1H), 7.936(s ,3H),7.664(s,1H),7.598(d,J＝7.8Hz,1H),7.314(d,J＝8.1Hz,1H),7.045(m,2H),6.906(m,1H),4.116 (m,1H),3.507(m,1H),3.267(m,2H),3.059(m,1H),2.773(m,4H),2.067(t,J=7.2Hz,2H),1.574-1.463( m,4H), 1.290(m,2H), 0.986(m,3H), 0.601(m,3H).

Example 8 Preparation of (3S,6S)-3-[Boc-Asp(OBzl)-amino-n-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piperazine-2,5-dione (5a)

Using the method of Example 1, from 965mg (3.6mmol) Boc-Asp (OBzl) and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethane (4)-piperazine-2,5-dione (4) afforded 1034 mg (47%) of the title compound as a colorless solid. ESI-MS (m/z): 733[M+H] ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm=10.874(s, 1H), 8.034(m, 1H), 7.932(m ,1H),7.785(m,1H),7.591(m,2H),7.360(m,6H),7.076(m,3H),6.953(m,1H),6.869(m,1H),5.084(m, 2H), 4.342(m, 1H), 4.311(m, 2H), 3.554(m, 2H), 3.276(m, 3H), 3.009(m, 3H), 2.765(m, 3H), 2.624(m, 1H ),2.508(m,1H),2.026(m,2H),1.478(m,3H),1.455(m,12H),1.215(m,3H),1.039(m,4H),1.460(m,3H) .

Example 9 Preparation of (3S,6S)-3-[Boc-Arg(NO ₂ )-amino-n-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piperazine-2,5-di Ketone (5b)

Using the method of Example 1, from 876mg (3.6mmol) Boc-Arg (NO ₂ ) and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3- Ethyl)-piperazine-2,5-dione (4) afforded 985 mg (49%) of the title compound as a yellow solid. ESI-MS (m/z): 670[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.871(s, 1H), 8.034(m, 1H), 7.932(m ,3H),7.775(m,1H),7.590(m,1H),7.316(d,J＝7.8Hz,1H),7.048(m,1H),6.931(m,1H),6.805(m,1H) ,4.114(m,2H),3.848(m,1H),3.502(m,1H),3.282(m,5H),3.031(m,3H),2.770(m,2H),2.028(t,J=6.9 Hz, 2H), 1.581(m, 6H), 1.380(m, 12H), 1.256(m, 3H), 0.973(m, 4H), 0.609(m, 3H).

Example 10 Preparation of (3S,6S)-3-(Boc-Gln-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5c)

Using the method of Example 1, from 885mg (3.6mmol) Boc-Gln and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)- Piperazine-2,5-dione (4) afforded 1120 mg (64%) of the title compound as a colorless solid. ESI-MS (m/z): 656[M+H] ⁺ . ¹ H-NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.872(s, 1H), 8.033(s, 1H), 7.938( s,1H),7.743(s,1H),7.591(m,2H),7.317(m,2H),7.047(m,2H),6.931(m,1H),6.759(m,2H),4.112(m ,1H),3.927(m,1H),3.502(m,1H),3.268(m,2H),3.083(m,3H),2.769(m,2H),2.036(m,4H),1.729(m, 2H), 1.492(m, 2H), 1.444(m, 12H), 1.237(m, 3H), 0.952(m, 3H), 0.617(m, 3H).

Example 11 Preparation of (3S,6S)-3-[Boc-Glu(OBzl)-amino-n-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piperazine-2,5-dione (5d)

Using the method of Example 1, from 1007mg (3.6mmol) Boc-Glu (OBzl) and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethane (4)-piperazine-2,5-dione (4) afforded 1235 mg (55%) of the title compound as a colorless solid. ESI-MS (m/z): 747[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.872(s, 1H), 8.033(m, 1H), 7.932(m ,1H),7.785(m,1H),7.564(m,2H),7.400(m,4H),7.287(m,1H),7.039(m,2H),6.953(m,1H),6.869(m, 1H),5.084(m,2H),4.113(m,1H),3.907(m,1H),3.642(m,2H),3.281(m,1H),3.000(m,3H),2.768(m,2H ),2.387(m,2H),2.362(m,2H),1.904(m,1H),1.809(m,1H),1.508(m,3H),1.437(m,10H),1.460(m,3H) ,1.435(m,3H),1.184(m,1H),1.095(m,3H).

Example 12 Preparation of (3S,6S)-3-[Boc-Lys(Cbz)-amino-n-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piperazine-2,5-dione (5e)

Using the method of Example 1, from 1100mg (3.6mmol) Boc-Lys (Cbz) and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethane (4)-piperazine-2,5-dione (4) afforded 1265 mg (56%) of the title compound as a colorless solid. ESI-MS(m/z):641[M+H] ⁺ . ¹ H NMR(300MHz,DMSO-d ₆ ):δ/ppm＝10.880(s,1H),8.044(s,1H),7.939(s ,1H),7.743(s,1H),7.743(s,1H),7.587(m,2H),7.316(m,7H),7.027(m,2H),6.929(m,1H),6.727(m, 1H), 5.004(s, 1H), 4.113(m, 2H), 3.642(m, 2H), 3.231(m, 4H), 3.024(m, 5H), 2.953(m, 2H), 2.004(t, J =7.5Hz, 2H), 1.437(m, 18H), 1.392(m, 4H), 0.981(m, 4H), 0.621(m, 3H).

Example 13 Preparation of (3S,6S)-3-(Boc-Asn-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5f)

Using the method of Example 1 from 832mg (mmol) Boc-Asn and 1280mg (3mmol) (3S, 6S)-3-(amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piper Oxazine-2,5-dione (4) afforded 980 mg (49%) of the title compound as a colorless solid. ESI-MS (m/z): 642[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.882(s, 1H), 8.035(m, 1H), 7.934(m ,1H),7.770(s,1H),7.591(m,2H),7.318(m,2H),7.045(m,2H),6.955(m,3H),6.843(s,1H),4.114(m, 2H),3.506(m,1H),3.233(m,1H),3.034(m,4H),2.770(m,2H),2.425(m,1H),2.029(m,2H),1.591(m,2H ), 1.458(m,13H), 1.239(m,3H), 0.991(m,3H), 0.605(m,3H).

Example 14 Preparation of (3S,6S)-3-(Boc-Ser-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5g)

Using the method of Example 1, from 738mg (3.6mmol) Boc-Ser and 1280mg (3mmol) (3S, 6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)- Piperazine-2,5-dione (4) afforded 598 mg (32%) of the title compound as a colorless solid. ESI-MS(m/z):615[M+H] ⁺ . ¹ H NMR(300MHz,DMSO-d ₆ ):δ/ppm＝10.929(s,1H),8.035(s,1H),7.920(s ,1H),7.760(m,1H),7.590(m,2H),7.319(d,J＝8.1Hz,1H),7.045(m,2H),6.927(m,1H),6.562(m,1H) ,4.817(m,1H),4.118(m,1H),3.927(m,1H),3.642(m,1H),3.280-3.091(m,1H),3.005(m,3H),2.750(m,2H ), 2.009 (t, J=7.5Hz, 2H), 1.482 (m, 2H), 1.384 (m, 11H), 1.233 (m, 3H), 0.977 (m, 4H), 0.618 (m, 3H).

Example 15 Preparation of (3S,6S)-3-(Boc-Thr-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5h)

Adopt the method of embodiment 1 from 430mg (3.6mmol) Boc-Thr and 1280mg (3mmol) (3S, 6S)-3-(amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl) -Piperazine-2,5-dione (4) afforded 880 mg (46%) of the title compound as a colorless solid. ESI-MS (m/z): 629[M+H] ⁺ . ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.869(s, 1H), 8.027(m, 1H), 8.134(m ,1H),7.732(m,1H),7.592(m,2H),7.317(m,1H),7.048(m,2H),6.956(m,1H),6.266(m,1H),5.763(s, 1H),4.113(m,1H),3.867(m,1H),3.807(s,2H),3.504(m,1H),3.268(m,1H),3.120(m,2H),2.771(m,2H ), 2.008(t, J=7.2Hz, 2H), 1.490(m, 1H), 1.393(m, 9H), 1.242(m, 2H), 1.035(m, 5H), 0.620(m, 2H).

Example 16 Preparation of (3S,6S)-3-(Asp-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6a)

According to the method of Example 3, from 980mg (1.3mmol) (3S, 6S)-3-[Boc-Asp(OBzl)-amino n-hexanoylamino n-butyl]-6-(indole-3-ethyl)-piper Oxazine-2,5-dione (5a) yielded 698 mg (82%) of (3S,6S)-3-[Asp(OBzl)-aminon-hexanoylbutylamino]-6-(indole-3-ethyl)- Piperazine-2,5-dione, a colorless solid. ESI-MS (m/z): 646 [M+H] ⁺ . According to the method of Example 4, from 450 mg (0.7 mmol) (3S, 6S)-3-[Asp(OBzl)-aminon-hexanoylbutylamino]-6-(indole-3-ethyl)-piperazine-2, 5-Diketone afforded 304 mg (67%) of the title compound as a colorless solid. ESI-MS(m/z):543[M+H] ⁺ .Mp152-154℃; [α] _D ²⁵ ＝-19.8(c＝0.1, methanol); IR(KBr,cm ^-1 ):3207,3055 , 2930, 1660, 1556, 1455, 1385, 1331, 1231, 1096, 742; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm=10.952(s,1H),8.059(s,1H),7.627 (m,2H),7.319(d,J=7.8Hz,1H),7.027(m,2H),6.953(m,1H),4.114(m,1H),3.505(m,1H),3.279(m, 1H), 3.539-3.141(m, 3H), 3.079(m, 2H), 2.770(m, 1H), 2.030(m, 1H), 2.044(t, J＝7.2Hz, 2H), 1.507-1.383(m ,4H), 1.247(m,2H), 0.947(m,3H), 0.643(m,3H).

Example 17 Preparation of (3S,6S)-3-(Arg-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6b)

According to the method of Example 3, from 700 mg (1 mmol) (3S, 6S)-3-[Boc-Arg(NO ₂ )-aminon-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piper Oxazine-2,5-dione (5b) yielded 312 mg (60%) of (3S,6S)-3-[Arg(NO ₂ )-aminon-hexanoylamino-n-butyl]-6-(indole-3-ethane base)-piperazine-2,5-dione as a colorless solid. ESI-MS (m/z): 630 [M+H] ⁺ . According to the method of Example 4, from 410mg (3S,6S)-3-[Arg(NO ₂ )-amino n-hexanoylamino n-butyl]-6-(indole-3-ethyl)-piperazine-2,5 - The dione afforded 298 mg (73%) of the title compound as a colorless solid. ESI-MS (m/z): 584[M+H] ⁺ .Mp167-169℃; [α] _D ²⁵ =-29.9 (c = 0.1, methanol); IR (KBr, cm ^-1 ): 3226,2928 , 1651, 1539, 1454, 1328, 1259, 1095, 742; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm=10.952(s, 1H), 8.568(s, 1H), 8.235(s, 3H ),7.927(m,1H),7.319(d,J=7.8Hz,1H),7.319(d,J=8.1Hz,1H),7.027(m,2H),6.953(m,1H),4.114(m ,1H),3.730(m,1H),3.279(m,1H),3.539-3.141(m,6H),3.079(m,2H),2.770(m,1H),2.044(t,J＝7.2Hz, 2H), 1.712(m, 2H), 1.507-1.383(m, 6H), 1.247(m, 2H), 0.947(m, 3H), 0.643(m, 3H).

Example 18 Preparation of (3S,6S)-3-(Gln-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6c)

According to the method of Example 3, from 655 mg (1 mmol) (3S, 6S)-3-(Boc-Gln-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2, 5-Diketone (5c) afforded 425 mg (76%) of the title compound as a colorless solid. ESI-MS(m/z): 556[M+H] ⁺ .Mp: 161-163℃; [α] _D ²⁵ ＝-153.5(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3187 _. ^_ 1H), 8.289(s, 3H), 8.054(m, 1H), 7.948(m, 1H), 7.665(t, J=5.4Hz, 1H), 7.590(d, J=7.8Hz, 1H), 7.516( s,1H),7.321(d,J＝8.1Hz,1H),7.046-7.000(m,2H),6.996-6.901(m,2H),4.115(m,1H),3.927(m,1H),3.502 (m,1H),3.264-3.076(m,4H),2.796(m,2H),2.202(m,2H),2.037(m,2H),1.928(m,2H),1.508-1.389(m,4H ), 1.269-1.219 (m, 2H), 0.954 (m, 3H), 0.646-0.526 (m, 3H).

Example 19 Preparation of (3S,6S)-3-(Glu-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6d)

According to the method of Example 3, from 1000mg (1.3mmol) (3S, 6S)-3-[Boc-Glu(OBzl)-aminon-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piper Oxazine-2,5-dione (5d) yielded 786 mg (90%) of (3S,6S)-3-[Glu(OBzl)-aminon-hexanoylamino-n-butyl]-6-(indole-3-ethyl )-piperazine-2,5-dione as a colorless solid. ESI-MS (m/z): 647 [M+H] ⁺ . According to the method of Example 4, from 500mg (0.77mmol) (3S, 6S)-3-[Glu(OBzl)-aminon-hexanoylbutylamino)]-6-(indole-3-ethyl)-piperazine-2 ,5-Diketone afforded 380 mg (76%) of the title compound as a white solid. ESI-MS(m/z): 557[M+H] ⁺ .Mp: 144-146℃; [α] _D ²⁵ ＝-23.4(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3223 ,3079,2929,1660,1549,1455,1396,1259,1097,1010,742; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm=10.952(s,1H),8.559(s,1H) ,7.935(s,1H),7.607(m,2H),7.319(d,J=7.8Hz,1H),7.045(m,2H),6.953(m,1H),5.083(s,5H),4.114( m,1H),3.505(m,1H),3.279(m,2H),3.539-3.141(m,3H),3.079(m,2H),2.770(m,2H),2.030(m,2H),2.054 (m,2H), 1.507-1.383(m,4H), 1.247(m,2H), 0.947(m,3H), 0.643(m,3H).

Example 20 Preparation of (3S,6S)-3-(Lys-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6e)

According to the method of Example 3, from 755mg (1mmol) (3S, 6S)-3-[Boc-Lys(Cbz)-amino n-hexanoylamino n-butyl]-6-(indole-3-ethyl)-piperazine -2,5-Diketone (5e) yielded 598 mg (91%) of (3S,6S)-3-[Lys(Cbz)-amino-n-caproylamino-n-butyl]-6-(indole-3-ethyl) -Piperazine-2,5-dione, a colorless solid. ESI-MS (m/z): 655 [M+H] ⁺ . According to the method of Example 4, from 520mg (0.8mmol) (3S, 6S)-3-[Lys(Cbz)-amino n-hexanoylamino-n-butyl]-6-(indole-3-ethyl)-piperazine- 2,5-Diketone afforded 425 mg (84%) of the title compound as free solid. ESI-MS(m/z): 556[M+H] ⁺ .Mp 159-162℃; [α] _D ²⁵ ＝-36.7(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3214, 3054, 2929, 2861, 1651, 1556, 1455, 1328, 1254, 1099, 742; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm=10.952(s,1H), 8.668(s,1H), 8.310(s,3H),8.076(s,3H),7.960(s,1H),7.717(s,1H),7.319(d,J=7.8Hz,1H),7.319(d,J=7.8Hz,1H ),7.027(m,2H),6.953(m,1H),4.114(m,1H),3.730(m,1H),3.279(m,1H),3.265(m,1H),3.170-3.005(m, 3H), 2.766(m, 4H), 2.062(t, J=8.1Hz, 2H), 1.745(m, 2H), 1.593(m, 2H), 1.480(m, 6H), 1.247(m, 2H), 0.947(m,3H),0.643(m,3H).

Example 21 Preparation of (3S,6S)-3-(Asn-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6f)

According to the method of Example 3, from 710mg (1.1mmol) (3S,6S)-3-(Boc-Asn-aminon-hexanoylbutylamino)-6-(indole-3-ethyl)-piperazine-2,5 - Diketone (5f) afforded 412 mg (68%) of the title compound as a colorless solid. ESI-MS(m/z): 542[M+H] ⁺ .Mp 183-185℃; [α] _D ²⁵ ＝-23.9(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3232, 2931,2161,1650,1557,1456,1330,1096,744; ¹ H NMR (300MHz, DMSO-d6): δ/ppm=10.882(s,1H),8.045(s,1H),7.941(s,1H ),7.856(m,1H),7.590(m,2H),7.382(s,1H),7.314(d,J＝8.1Hz,1H),7.046(m,2H),6.955(m,1H),6.843 (s,1H),4.114(m,1H),3.506(m,1H),3.444(m,1H),3.280(m,1H),3.045(m,3H),2.768(m,2H),2.425- 2.363(m,1H),2.159(m,1H),2.014(m,3H),1.492-1.367(m,4H),1.239(m,2H),0.991(m,3H),0.605(m,3H) .

Example 22 Preparation of (3S,6S)-3-(Ser-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (6g)

According to the method of Example 3, from 620mg (1mmol) (3S, 6S)-3-(Boc-Ser-amino n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2, 5-Diketone (5 g) gave 390 mg (75%) of the title compound as a colorless solid. ESI-MS(m/z): 515[M+H] ⁺ .Mp 155-157℃; [α] _D ²⁵ ＝-15.3(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3227, 3080, 2929, 1651, 1328, 1261, 1095, 1062, 743; ¹ H NMR (300MHz, DMSO-d ₆ ): δ/ppm＝10.929(s, 1H), 8.514(t, J＝4.8Hz, 1H) ,8.155(s,3H),8.046(m,1H),7.940(m,1H),7.658(m,2H),7.648(d,J＝8.1Hz,1H),7.047-7.024(m,2H), 6.997-6.902(m,1H),5.482(s,1H),4.118(m,1H),3.927(m,3H),3.642(m,1H),3.280-3.091(m,1H),3.005(m, 3H), 2.750(m, 2H), 2.050(t, J=7.2Hz, 2H), 1.526-1.378(m, 4H), 1.154(m, 2H), 0.977(m, 3H), 0.645-0.529(m ,3H).

Example 23 Preparation of (3S,6S)-3-(Thr-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5h)

According to the method of Example 3, from 628mg (1mmol) (3S, 6S)-3-(Boc-Thr-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2, 5-Diketone (5h) afforded 405 mg (64%) of the title compound as a colorless solid. ESI-MS(m/z): 529[M+H] ⁺ .Mp 152-154℃; [α] _D ²⁵ ＝-17.2(c＝0.1, methanol); IR(KBr,cm ^-1 ): 3231, ₍ ^_ s,3H),8.046(m,1H),7.940(m,1H),7.661(m,1H),7.625(d,J=7.8Hz,1H),7.319(d,J=8.1Hz,1H), 7.047-7.000(m,2H),6.997-6.902(m,1H),5.561(s,1H),4.113(m,1H),3.867(m,1H),3.642(m,2H),3.228(m, 1H), 3.076(m, 3H), 3.005(m, 2H), 2.750(t, J=7.2Hz, 2H), 1.531-1.383(m, 4H), 1.297-1.179(m, 2H), 1.123(m ,3H), 1.001-0.931(m,3H), 0.647-0.529(m,3H).

Example 24 Determination of anti-tumor metastasis activity of compound 6a-h

The assay model was inoculated with Lewis mouse lung cancer cells (LLC, purchased from ATCC), and DMEM medium (containing 10% inactivated fetal bovine serum, 1×10 ⁵ U/L penicillin and 100 mg/L streptomycin) was used. , according to the adherent cell culture method, subculture once every two days to enrich the cells. When the cells were in good growth state and in the logarithmic growth phase, the cells were digested, and the cell density was adjusted to 1×10 ⁷ cells/mL with normal saline. Placental blue staining, so that the viable cell count > 95%. Take an inbred C57BL/6 male mouse (SPF grade, body weight 20±2g), and fix the mouse with the left hand. Disinfect the skin of the right forelimb axilla of the mouse with 75% ethanol. Hold a 1mL sterile syringe in the right hand and subcutaneously inject LLC tumor cell suspension into the axilla of mice, 0.2mL per mouse. Ten days after inoculation in the mice, a tumor with a diameter of about 4-5mm grows out, which is the source of the tumor. Lewis lung cancer tumor-bearing mice that were inoculated for 10 days were anesthetized with ether, and sacrificed by cervical dislocation. Soak in 75% ethanol for 10 minutes, disinfect, and peel off the tumor body on an ultra-clean workbench. Select well-grown tumor tissue, cut it into pieces in a sterile plate, and place it in a tissue homogenizer made of glass. Add physiological saline at a temperature of 4°C at a ratio of 1 to 3 (g to mL) of the tumor mass to the volume of normal saline, and gently grind to make a cell suspension. The cell suspension was passed through a 200-mesh cell sieve to prepare a single-cell suspension. Adjust the cell density of the single cell suspension to 1.5×10 ⁷ cells/mL with physiological saline. Placental blue staining, so that the viable cell count > 95%. The inbred C57BL/6 male mouse was fixed in the left hand, and the skin of the axilla of the right forelimb of the mouse was disinfected with 75% ethanol. Hold a 1mL sterile syringe in the right hand and subcutaneously inject tumor cell suspension into the axilla of mice, 0.2mL per mouse. Ten days after the inoculation, the mice developed tumors with a diameter of 4-5 mm, and the inoculated mice were randomly divided into groups according to the measured tumor volume. 12 mice per group. On the 11th day inoculated with tumors, the mice were orally administered with a normal saline solution of the recognized anti-tumor metastasis peptide RGDS (at a dose of 20 μmol/kg/day) or with a normal saline solution of compounds 6a-h (at a dose of 0.5 μmol/kg/day) Or oral administration of compound 4 in normal saline solution (dose of 5 μmol/kg/day) or oral administration of normal saline (dose of 10 mL/kg/day), given once a day, for 12 consecutive days, measured and recorded every two days tumor volume. The day after the last administration, the tumor volume was measured, ether anesthetized and killed by dislocation of the cervical spine, the tumors of the mice were weighed, and the lungs of the mice were taken to calculate the number of metastatic tumor nodes. Statistical analysis of data was performed with t test. The results are shown in Table 1. At the dose of 0.5 μmol/kg, compounds 6a-h not only effectively inhibited lung metastasis of tumors, but also had no significant difference in activity from RGDS whose dose was 40 times higher and compound 4 whose dose was 10 times higher than them. These data show that the present invention has remarkable technical effects.

Table 1 Anti-metastatic activity of compound 6a-h

b) Compared with normal saline, p<0.01, compared with RGDS and compound 4, p>0.05; n=12

Example 25 Determination of anti-tumor growth activity of compound 6a-h

Doxorubicin, compound 4 and compound 6a-h were all dissolved in saline before the measurement and administered to S180 mice. In a sterile environment, take the vigorously growing S180 ascites tumor fluid inoculated in male ICR mice for 10 days, dilute it with normal saline (1:2) and mix well, and the tumor cell suspension is freshly prepared with 0.2% trypan Stain with blue, and count according to the white blood cell count method after mixing. Those stained with blue are dead cells, and those without staining are live cells. Calculate cell density according to cell concentration = number of living cells in 4 squares/4×10 ⁴ ×dilution factor=number of cells/mL, and calculate cell density according to cell survival rate=number of living cells/(number of living cells+number of dead cells)×100% Calculate cell viability. The tumor fluid with a survival rate greater than 90% was made into a cell suspension with a density of 2.0×10 ⁷ cells/mL by homogenization. The cell suspension was inoculated subcutaneously in the right axilla of mice (0.2 mL/mouse) to produce S180 tumor-bearing mice. After 24 hours of inoculation, the S180 tumor-bearing mice were injected intraperitoneally with a normal saline solution of doxorubicin (a dose of 2 μmol/kg/day g) or a daily oral administration of a normal saline solution of Compound 4 (a dose of 5 μmol/kg/day) or every day. Daily oral administration of compound 6a-h in physiological saline solution (dose 0.5 μmol/kg/day). Dosing once a day for 12 consecutive days. The day after the last administration, the tumor volume was measured, ether anesthetized and sacrificed by cervical dislocation, and then the tumor growth site in the right axilla of the mouse was fixed with forceps, the skin was cut open and the tumor was bluntly peeled off and weighed. The curative effect was expressed by tumor weight (mean ± SD g), and the data were analyzed by t test and analysis of variance. The results are shown in Table 2. Compound 6a-h not only effectively inhibited tumor growth at a dose of 0.5 μmol/kg, but also had no significant difference in activity from compound 4 whose dose was 10 times higher. These data show that the present invention has remarkable technical effect.

The influence of table 2 compound 6a-h on tumor growth of S180 mice

a) p<0.05 compared with normal saline, p>0.05 compared with compound 4; b) p<0.01 compared with normal saline, p>0.05 compared with compound 4; c) p<0.01 compared with normal saline and normal saline; n =12.

Example 26 Determination of anti-inflammatory activity of compound 6a-h

Because xylene-induced mouse ear swelling is recognized as an acute inflammation model, the present invention measures the therapeutic effects of compounds 6a-h on the xylene-induced mouse ear swelling model. Because aspirin is a positive medicine for treating acute inflammation, so the present invention selects aspirin as a positive control medicine. ICR male mice (body weight 42±3g) rested for 2 days in an environment with a temperature of 22°C, and had free access to water and food. After that, they were randomly divided into normal saline group (0.2 mL/rat), aspirin group (1.11 mmol/kg), compound 4 group (5 μmol/kg) and compound 6a-h group (0.5 μmol/kg). kg), 12 mice in each group. During the measurement, mice were orally administered with normal saline, or aspirin, or with compound 4, or with compounds 6a-h according to the group they belonged to. After 30 minutes of drug administration, 30 μL of xylene was evenly applied to the left auricle of the mouse. After 2 hours, the mouse was anesthetized with ether, and the neck was cut off to kill the left and right ears. Shape the ears, weigh them, and calculate the swelling difference between the two ears as the degree of swelling. That is, the degree of swelling = the weight of the left ear disk – the weight of the right ear disk. Compounds 6a-h not only effectively inhibited xylene-induced mouse ear swelling at a dose of 0.5 μmol/kg, but also had no significant difference in activity from compound 4, which was 10 times higher than their doses. These data show that the present invention has remarkable technical effects.

The effect of table 3 compound 6a-h on the mouse ear swelling that xylene causes

a) p<0.05 compared with normal saline, p>0.05 compared with compound 4; b) p<0.01 compared with normal saline and compound 4; n=12.

Claims (5)

1. (3S,6S)-3-(AA-amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione of the following formula, where AA are L-Asp residues, L-Arg residues, L-Gln residues, L-Glu residues, L-Lys residues, L-Asn residues, L-Ser residues and L-Thr residues, 2. The preparation method of claim (3S, 6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione, the Methods include: (1) L-Boc-Lys(Cbz) is condensed with L-Trp-OBzl to obtain Boc-Lys(Cbz)-Trp-OBzl; (2) Boc-Lys(Cbz)-Trp-OBzl de-Boc in hydrogen chloride ethyl acetate solution to obtain Lys(Cbz)-Trp-OBzl; (3) Lys(Cbz)-Trp-OBzl is cyclized in 5% aqueous sodium bicarbonate saturated ethyl acetate solution to generate (3S,6S)-3-(benzyloxycarbonylbutylamino)-6-(indole-3 -Ethyl)-piperazine-2,5-dione (1); (4) Hydrogenolysis of compound 1 to obtain (3S,6S)-3-(amino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (2) ; (5) Compound 2 is condensed with Boc-amino n-caproic acid to obtain (3S,6S)-3-(Boc-amino n-n-caproylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2, 5-diketone (3); (6) De-Boc of compound 3 in hydrogen chloride ethyl acetate solution to obtain (3S,6S)-3-(amino-n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2 ,5-diketone (4); (7) Compound 4 is condensed with Boc-AA (AA is L-Phe residue, L-Tyr residue and L-Trp residue) to obtain (3S,6S)-3-(Boc-AA-amino n-hexanoylamino n Butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione (5a-h); (8) Compounds 5a-h de-Boc in ethyl acetate solution of hydrogen chloride to obtain (3S,6S)-3-(AA-aminon-hexanoylamino-n-butyl)-6-(indole-3-ethyl)- Piperazine-2,5-dione (6a-h). 3. (3S, 6S)-3-(AA-amino n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione of claim 1 is preparing anti- Applications in tumor metastasis drugs. 4. (3S, 6S)-3-(AA-amino n-hexanoylamino n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione of claim 1 is prepared in the preparation of anti- Application in tumor medicine. 5. (3S, 6S)-3-(AA-amino n-hexanoylamino-n-butyl)-6-(indole-3-ethyl)-piperazine-2,5-dione of claim 1 is preparing anti- Application in inflammatory drugs.