CN110577518B - Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof - Google Patents

Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof Download PDF

Info

Publication number
CN110577518B
CN110577518B CN201810596333.3A CN201810596333A CN110577518B CN 110577518 B CN110577518 B CN 110577518B CN 201810596333 A CN201810596333 A CN 201810596333A CN 110577518 B CN110577518 B CN 110577518B
Authority
CN
China
Prior art keywords
lys
diketopiperazine
methyl
butyl
boc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201810596333.3A
Other languages
Chinese (zh)
Other versions
CN110577518A (en
Inventor
赵明
张筱宜
彭师奇
宋越
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Capital Medical University
Original Assignee
Capital Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Capital Medical University filed Critical Capital Medical University
Priority to CN201810596333.3A priority Critical patent/CN110577518B/en
Publication of CN110577518A publication Critical patent/CN110577518A/en
Application granted granted Critical
Publication of CN110577518B publication Critical patent/CN110577518B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention discloses 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl) of the formula]-2, 5-diketopiperazine (AA in the formula is L-Lys residue and L-Thr residue), discloses a preparation method thereof, discloses the anti-tumor activity thereof and discloses the anti-tumor metastasis activity thereof, so that the invention discloses the application thereof in preparing anti-tumor drugs and anti-tumor metastasis drugs.
Figure DDA0001692316830000011

Description

Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof
Technical Field
The invention relates to 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino n-butyl ] -2, 5-diketopiperazine, a preparation method thereof, antitumor activity thereof and antitumor metastasis activity thereof, and thus the invention relates to application thereof in preparing antitumor drugs and antitumor metastasis drugs. The invention belongs to the field of biological medicine.
Background
Tumors seriously threaten the health of human beings. In addition to the poor prognosis of patients with tumors by themselves, metastasis associated with tumors further worsens the prognosis of patients. For example, more than 90% of patients with tumors die from metastases. Because the existing antitumor drugs have no effect of resisting tumor metastasis, the clinical curative effect of tumor chemotherapy is not ideal. The invention relates to a medicament for resisting tumor metastasis, which is an urgent clinical need. The inventors have previously disclosed that diketopiperazines of the four S, S-, R, R-, R, S-and S, R-configurations inhibit migration and invasion of HCCLM3 (highly metastatic human liver cancer cells) at a concentration of 0.5. mu.M. Later, the inventors further disclosed that R, R-configured diketopiperazines inhibit tumor metastasis to the lung in C57BL/6 mice at a minimum effective dose of 5. mu. mol/kg/day. To reduce the minimum effective dose, the inventors have developed various modifications to the butylamino group of the diketopiperazine in the R, R-configuration. After 3 years of exploration, the acylation of the 4-amino-n-butyl group of diketopiperazines of R, R-configuration with L-Lys and L-Lys acylated side chain amino groups was found to reduce the minimum effective dose against tumor metastasis to 0.5. mu. mol/kg/day. The 10-fold reduction of the lowest effective dose indicates that the structural modification has outstanding technical effects. They were also effective in inhibiting tumor growth in S180 mice at a dose of 0.5. mu. mol/kg/day. This additional anti-tumor effect indicates a prominent technical effect of this structural modification. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide 3R- (indol-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine of the formula (wherein AA is a L-Lys residue and a L-Thr residue).
Figure GDA0001834603250000011
The second aspect of the present invention provides a process for synthesizing 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine (AA is L-Lys residue and L-Thr residue), which comprises:
(1) D-Boc-Lys (Cbz) is coupled with D-Trp-OBzl to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) D-Boc-Lys (Cbz) -D-Trp-OBzl removes Boc protecting group in 4N hydrogen chloride/ethyl acetate solution to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) cyclizing D-Lys (Cbz) -D-Trp-OBzl in ethyl acetate containing 5% sodium bicarbonate water solution to obtain 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine (1);
(4)1 catalyzing hydrogen to remove carbobenzoxy to obtain 3R- (indole-3-methyl) -6R- [ 4-amino-n-butyl ] -2, 5-diketopiperazine (2);
(5)2 is coupled with L-Boc-Lys (Cbz) to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino n-butyl ] -2, 5-diketopiperazine (3);
(6)3, removing benzyloxycarbonyl by catalytic hydrogenolysis to obtain 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino n-butyl) -2, 5-diketopiperazine (4);
(7)4 and Boc-AA (AA is L-Lys residue and L-Thr residue) to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino n-butyl ] -2, 5-diketopiperazine (5a, b);
(8)5a, b in 4N hydrogen chloride/ethyl acetate solution to remove Boc protecting group to obtain 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino N-butyl ] -2, 5-diketopiperazine (6a, b).
In the third aspect of the present invention, 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine (6a, b) was evaluated for its activity in inhibiting the metastasis of lung cancer in C57BL/6 mice.
The fourth aspect of the present invention is to evaluate the inhibition of tumor growth in S180 mice by 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine (6a, b).
Drawings
FIG. 13R- (indol-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl]-2, 5-diketopiperazines (6a, b) wherein AA is an L-Lys residue in 5a and 6 a; AA in 5b and 6b is an L-Thr residue; i) dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), Tetrahydrofuran (THF); ii)4N hydrogen chloride-ethyl acetate reagent; iii) ethyl acetate, 5% sodium bicarbonate; iv) Dimethylformamide (DMF), Pd/C, H2。
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 D-Boc-Lys (Cbz) -D-Trp-OBzl
7.7g (20mmol) of D-Boc-Lys (Cbz) was suspended in 100mL of anhydrous Tetrahydrofuran (THF), and 2.7g (20mmol) of 1-hydroxybenzotriazole (HOBt) and 5.0g (25mmol) of Dicyclohexylcarbodiimide (DCC) were added to the suspension in this order under ice bath, followed by stirring for 30 min. Then, 8.0g (25mmol) of D-Trp-OBzl was added. The reaction mixture was adjusted to pH 9 by dropwise addition of N-methylmorpholine (NMM). The reaction mixture was stirred first for 1h on ice and then for 12h at room temperature. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 150mL of ethyl acetate solution. The ethyl acetate solution obtained was successively treated with 5% KHSO4Washing with aqueous solution for 3 times to saturate NaCl aqueous solution, 3 times. Anhydrous Na for ethyl acetate layer2SO4Drying for 12h, filtering, and concentrating the filtrate under reduced pressure to dryness. The resulting yellow syrup was purified by silica gel column Chromatography (CH)2Cl2/CH3OH,100:1) 12.0g (88%) of the title compound are obtained as a colorless powder. ESI-MS (M/e):657[ M + H]+。
EXAMPLE 2 preparation of D-Lys (Cbz) -D-Trp-OBzl
3.8g (5mmol) D-Boc-Lys (Cbz) -D-Trp-OBzl were slowly mixed with 52mL of hydrogen chloride in ethyl acetate (4M) with stirring in an ice bath. The resulting solution was stirred in an ice bath for 5 h. After that, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 50mL of anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. This operation was repeated three times. The residue was washed thoroughly with anhydrous ether to give 3.41g (93%) of the title compound as a yellow powder. ESI-MS (M/e):557[ M + H ]]+。
Example 33R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino-n-butyl) -2, 5-diketopiperazine (1)
3.45g (6.2mmol) D-Lys (Cbz) -D-Trp-OBzl were dissolved in 150mL ethyl acetate. After the resulting solution was washed three times with a 5% aqueous solution of sodium hydrogencarbonate, the ethyl acetate solution was stirred at room temperature for 12 hours to sufficiently precipitate a colorless solid. 1.8g (51%) of the title compound are filtered off. ESI-MS (M/e):449[ M + H ]]+。
EXAMPLE 4 preparation of 3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine (2)
To a solution of 1.9g (4.2mmol) of 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino N-butyl) -2, 5-diketopiperazine (1) in 20mL of anhydrous N, N-Dimethylformamide (DMF) was added 200mg of Pd/C, and H was bubbled through2The reaction was stirred at room temperature for 12 h. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 1.2g (92%) of the title compound as a colorless powder. ESI-MS (M/e) 315[ M + H ]]+。
EXAMPLE 5 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl ] -2, 5-diketopiperazine (3)
Using the method of example 1, from 4.18g (11mmol) Boc-Lys (Cbz) and 3.14g (10.0mmol)3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine (2) 5.17g (76 g)%) the title compound as a colorless solid. ESI-MS (M/e):677[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.852(s,1H),8.045(d,J＝1.8Hz,1H),7.916(d,J＝1.8Hz,1H),7.581(m,2H),7.345(m,6H),7.223(t,J＝5.4Hz1H),7.025(m,2H),6.930(t,J＝6.9Hz,1H),6.714(d,J＝8.4Hz,1H),4.115(m,1H),3.812(m,1H),3.499(m,1H),3.206(dd,J1＝16.5Hz,J2＝3.9Hz,1H),3.172(d,J＝3.9Hz,1H),2.993(m,3H),2.764(m,2H),1.478(m,2H),1.381(m,11H),1.426(m,2H),0.971(m,3H),0.600(m,3H)。
EXAMPLE 6 preparation of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4)
From 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl using the procedure of example 4]1.65g (2.44mmol) of (E) -2, 5-diketopiperazine (3) give 1.49g (90%) of the title compound as a colorless powder. ESI-MS (M/e):543[ M + H]+。
EXAMPLE 7 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc) -amino-n-butyl ] -2, 5-diketopiperazine (5a)
From 1.14g (3.3mmol) Boc-Lys (Boc) and 1.49g (2.75mmol)3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) 1.57g (66%) of the title compound was obtained as colorless powder using the method of example 1. ESI-MS (M/e):771[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.847(s,1H),8.023(s,1H),7.915(s,1H),7.741(m,1H),7.580(m,2H),7.302(m,1H),7.048(s,1H),7.027(t,J＝8.1Hz 1H),6.930(t,J＝8.1Hz 1H),4.116(m,1H),3.814(m,2H),3.499(m,1H),3.256(dd,J1＝13.8Hz,J2＝3.0Hz,1H),3.033(m,3H),2.886(m,2H),2.738(m,2H),1.508(m,4H),1.375(m,22H),1.242(m,4H),0.975(m,3H),0.595(m,3H)。
EXAMPLE 8 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Thr) -amino-n-butyl ] -2, 5-diketopiperazine (5b)
From 1.05g (3.3mmol) Boc-Thr and 1.49g (2.75mmol)3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) 1.45g (71%) of the title compound were obtained as colorless powder using the method of example 1. ESI-MS (M/e):744[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.861(s,1H),8.052(s,1H),7.930(s,1H),7.769(m,1H),7.587(m,2H),7.297(d,J＝8.1Hz 1H),7.039(s,1H),7.020(t,J＝7.2Hz 1H),6.923(t,J＝7.2Hz 1H),6.729(d,J＝8.1Hz 1H),6.262(d,J＝8.1Hz 1H),4.722(d,J＝6.0Hz 1H),4.114(m,1H),3.873(m,1H),3.802(m,2H),3.489(m,1H),3.252(dd,J1＝14.4Hz,J2＝3.6Hz,1H),3.020(m,3H),2.758(m,2H),1.471(m,2H),1.384(m,20H),1.236(m,2H),1.018(d,J＝6.0Hz 1H),0.955(m,3H),0.565(m,3H)。
EXAMPLE 9 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Lys) -amino-n-butyl ] -2, 5-diketopiperazine (6a)
From 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc)) amino-n-butyl using the procedure of example 2]515mg (0.67mmol) of (E) -2, 5-diketopiperazine (5a) gave 20.0mg (5.2%) of the title compound as a pale yellow powder. ESI-MS (M/e):571[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝11.000(s,1H),8.579(m,1H),8.510(m,1H),8.082(s,1H),8.038(s,1H),7.584(d,J＝7.5Hz,1H),7.322(d,J＝7.5Hz,1H),7.041(s,1H),6.930(t,J＝6.9Hz 1H),6.839(t,J＝6.9Hz 1H),4.113(m,1H),3.865(m,1H),3.733(m,1H),3.512(m,1H),3.253(m,1H),3.125(m,1H),3.051(m,2H),2.824(m,2H),2.772(m,2H),1.379(m,4H),1.358(m,4H),1.302(m,2H),1.267(m,2H),0.955(m,3H),0.595(m,3H)。
EXAMPLE 10 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Thr) -amino-n-butyl ] -2, 5-diketopiperazine (6b)
From 515mg (0.67mmol) of 3R- (indol-3-methyl) -6R- [4-Boc-Lys (Boc-Thr) -amino n-butyl using the method of example 2]-2, 5-diketopiperazine (5b) gave 247mg (64%) of the title compound as a colorless powder. ESI-MS (M/e):571[ M + H]+；1H NMR(300MHz,DMSO-d6):1H NMR(300MHz,DMSO-d6):δ/ppm＝10.947(s,1H),8.052(m,1H),7.994(m,2H),7.936(m,1H),7.578(d,J＝7.8Hz,1H),7.214(d,J＝7.8Hz,1H),7.026(m,2H),6.928(m,1H),4.117(m,2H),3.796(m,1H),3.506(m,1H),3.346(m,1H),3.253(dd,J1＝14.4Hz,J2＝3.6Hz,1H),3.039(m,3H),2.783(m,2H),1.592(m,1H),1.561(m,1H),1.467(m,2H),1.289(m,2H),1.060(d,J＝6.3Hz,1H),0.998(m,3H),0.595(m,3H)。
EXAMPLE 11 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 physiological saline with the temperature of 4 ℃ according to the ratio of the tumor mass to the volume of the physiological saline of 1 to 3(g to mL), and lightly grinding to prepare the 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 either orally administered a recognized physiological saline solution of the anti-tumor metastasis peptide RGDS (at a dose of 20. mu. mol/kg/day) or orally administered a physiological saline solution of compounds 6a, b (at a dose of 0.5. mu. mol/kg/day) or orally administered a physiological saline solution (at a dose of 10 mL/kg/day), 1 dose per day, administered for 12 consecutive days, and tumor volumes were measured and recorded every other day. For the last administrationThe next day, tumor volume was measured, cervical spine was removed by ether anesthesia and sacrificed, mice tumors were weighed, mice lungs were taken and tumor nodules transferred from tumor lungs were counted. Statistical analysis of the data was performed using the t-test. The results are shown in Table 1. Not only was compound 6a, b effective in inhibiting tumor lung metastasis at the 0.5 μmol/kg dose, but the activity was not significantly different from RGDS at the 20 μmol/kg dose. These data indicate that the present invention has significant technical effects. Anti-tumor metastasis Activity of tables 16 a, b
Figure GDA0001834603250000051
Figure GDA0001834603250000061
a) P <0.01 to saline, p >0.05 to RGDS; n-10
Example 12 determination of dose dependence of anti-metastatic Activity of Compound 6a
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. The volume of the normal saline is 1 to 3(g to mL) according to the weight ratio of tumor mass) Adding physiological saline with the temperature of 4 ℃ into the mixture according to the proportion, and lightly grinding the mixture to prepare 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. Mice on day 11 of tumor inoculation were orally administered either a normal saline solution of the putative antitumor metastatic peptide RGDS (dose of 20. mu. mol/kg/day) or compound 6a (dose of 0.5. mu. mol/kg/day, 0.05. mu. mol/kg/day, 0.005. mu. mol/kg/day) or normal saline (dose of 10 mL/kg/day) 1 time daily for 12 consecutive days with tumor volumes 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 2. After the compound 6a can effectively inhibit the tumor lung metastasis at the dose of 0.5 mu mol/kg, the compound 6a can still effectively inhibit the tumor lung metastasis at the dose of 0.05 mu mol/kg, so that the effective dose is reduced by 400 times. These data indicate that the present invention has significant technical effects.
TABLE 2 determination of dose dependence of the anti-metastatic Activity of 6a
Figure GDA0001834603250000062
a) P <0.01 to saline, p >0.05 to RGDS, p >0.05 to 6a at a dose of 0.05. mu. mol/kg/day; b) p <0.05 to physiological saline, p <0.05 to 6a at a dose of 0.005 μmol/kg/day; c) p <0.05 to saline; n is 10.
EXAMPLE 13 determination of the anti-tumor growth Activity of Compounds 6a, b
Both doxorubicin and compound 6a, b were dissolved in physiological saline prior to assay for administration to S180 mice. Taking S180 ascites tumor liquid which is inoculated in a male ICR mouse and grows vigorously for 10 days in a sterile environment, diluting the S180 ascites tumor liquid into liquid (1:2) by using normal saline, fully mixing the liquid, dyeing the tumor cell suspension by using freshly prepared 0.2% trypan blue, uniformly mixing the liquid and the liquid, counting the liquid according to a white cell counting method, wherein the blue-dyed cell is a dead cell, and the non-dyed cell is a live cell. 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 physiological saline solution of doxorubicin (dose 2. mu. mol/kg/day g) or orally administered daily with a physiological 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 3. The data that compounds 6a, b were effective in inhibiting tumor growth at a dose of 0.5 μmol/kg indicate that the present invention has significant technical effects.
TABLE 3 Effect of Compounds 6a, b on tumor growth in S180 mice
Figure GDA0001834603250000071
a) P <0.01 to saline; n is 10.

Claims (4)

1. 3R- (indol-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine of the formula,
Figure FDA0002966198120000011
wherein AA is a L-Lys residue and a L-Thr residue.
2. A process for the preparation of 3R- (indole-3-methyl) -6R- [4-lys (aa) -amino-n-butyl ] -2, 5-diketopiperazine according to claim 1, which comprises:
(1) D-Boc-Lys (Cbz) is coupled with D-Trp-OBzl to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) D-Boc-Lys (Cbz) -D-Trp-OBzl removes Boc protecting group in 4N chlorine hydride-ethyl acetate reagent to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) cyclizing D-Lys (Cbz) -D-Trp-OBzl in ethyl acetate containing 5% sodium bicarbonate water solution to obtain 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine;
(4) the 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine catalyzes and hydrolyzes benzyloxycarbonyl to obtain 3R- (indole-3-methyl) -6R- (4-amino n-butyl) -2, 5-diketopiperazine;
(5)3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine is coupled with L-Boc-Lys (Cbz) to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl ] -2, 5-diketopiperazine;
(6)3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino n-butyl ] -2, 5-diketopiperazine is subjected to catalytic hydrogenolysis to remove benzyloxycarbonyl to obtain 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino n-butyl) -2, 5-diketopiperazine;
(7) coupling 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine with Boc-AA to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino-n-butyl ] -2, 5-diketopiperazine;
(8) and removing the Boc protecting group of the 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino N-butyl ] -2, 5-diketopiperazine in a 4N hydrogen chloride-ethyl acetate reagent to obtain the 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino N-butyl ] -2, 5-diketopiperazine.
3. The use of 3R- (indole-3-methyl) -6R- [4-lys (aa) -amino-n-butyl ] -2, 5-diketopiperazine according to claim 1 for the preparation of an anti-tumor medicament.
4. The use of 3R- (indole-3-methyl) -6R- [4-lys (aa) -amino-n-butyl ] -2, 5-diketopiperazine according to claim 1 for the preparation of a medicament for the treatment of tumor metastasis.
CN201810596333.3A 2018-06-11 2018-06-11 Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof Expired - Fee Related CN110577518B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810596333.3A CN110577518B (en) 2018-06-11 2018-06-11 Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810596333.3A CN110577518B (en) 2018-06-11 2018-06-11 Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof

Publications (2)

Publication Number Publication Date
CN110577518A CN110577518A (en) 2019-12-17
CN110577518B true CN110577518B (en) 2021-06-08

Family

ID=68809518

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810596333.3A Expired - Fee Related CN110577518B (en) 2018-06-11 2018-06-11 Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof

Country Status (1)

Country Link
CN (1) CN110577518B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105272980A (en) * 2014-06-10 2016-01-27 首都医科大学 (3R,12aS)-3-(4-aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione, and preparation and application thereof
CN105503833B (en) * 2014-10-20 2019-07-02 首都医科大学 Indoles piperazine-diketone, preparation and therapeutic effect
CN106349148B (en) * 2015-07-13 2019-07-05 首都医科大学 New indole class compound with anti-tumor metastasis and anti-inflammatory activity, synthesis and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Enantiomeric diketopiperazines:getting insight into the impact of the configuration on the conformation,nanoimage,u-PA inhibition and anti-metastatic activity;Feng Wang et al.;《Med.Chem.Commun.》;20150331;第6卷(第9期);第956-962页 *

Also Published As

Publication number Publication date
CN110577518A (en) 2019-12-17

Similar Documents

Publication Publication Date Title
CN110577518B (en) Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof
CN108948137B (en) 3S-indolylethyl-6S-polar amino acid modified piperazine-2, 5-diketone and synthesis, activity and application thereof
CN108929320B (en) 3R-indolylmethyl-6R-oxazolidinone modified piperazine-2, 5-dione, synthesis, activity and application thereof
CN110577568B (en) Methyl indole and amide side chain amino acid modified diketopiperazine, synthesis, activity and application thereof
CN110577517B (en) Methyl indole and aromatic amino acid modified 2, 5-diketopiperazine, synthesis, activity and application thereof
CN108976201B (en) 3R-indolylmethyl-6S-aromatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN110551108B (en) 3R-indolylmethyl-6R-amino acid modified 2, 5-diketopiperazines, synthesis, activity and use thereof
CN108947976B (en) 3S-indolylmethyl-6S-aromatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108929369B (en) 3R-indolylmethyl-6R-acidic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108947978B (en) 3R-indolylmethyl-6R-Tyr modified piperazine-2, 5-diketone and synthesis, activity and application thereof
CN109134595B (en) Theanyl amino acid benzyl ester modified curcumin, and synthesis, activity and application thereof
CN108947975B (en) 3S-indolylethyl-6S-fatty amino acid modified piperazine-2, 5-diketone and synthesis, activity and application thereof
CN108929313B (en) 3R-indolylmethyl-6R-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108976204B (en) 3S-indolylmethyl-6R-aspartic acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN109456311B (en) 3R-indolylmethyl-6R-aromatic amino acid modified piperazine-2, 5-diketone and synthesis, activity and application thereof
CN108947979B (en) 3R-indolylmethyl-6R-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108947977B (en) Piperazine-2, 5-diketone modified by 3R-indolylmethyl-6R-amide side chain amino acid, and synthesis, activity and application thereof
CN110551109B (en) Amino acid and tranexamic acid modified diketopiperazine, preparation and application thereof
CN108948141B (en) 3R-indolylmethyl-6S-Pro modified piperazine-2, 5-dione, synthesis, activity and application thereof
CN108976211B (en) 3S-indolylmethyl-6R-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108976277B (en) 3R-indolylmethyl-6S-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108976210B (en) 3S-indolylmethyl-6R-aromatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN108929314B (en) 3R-indolylmethyl-6S-acidic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof
CN110551175B (en) Amino acid and tranexamic acid modified diketopiperazine, preparation, activity and application thereof
CN108976203B (en) 3S-indolylmethyl-6R-Lys modified piperazine-2, 5-dione, and synthesis, activity and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20210608

CF01 Termination of patent right due to non-payment of annual fee