WO2016070712A1 - Amino acid derivatives and applications thereof - Google Patents

Abstract

Disclosed are amino acid derivatives and applications thereof. The amino acid derivatives contain the oxalyl group, and thus can interfere with energy metabolism of cells and induce apoptosis. Therefore, the amino acid derivatives can be widely applied.

Description

Amino acid derivatives and their applications Technical field

This invention relates to amino acid derivatives and their use.

Background technique

The incidence of cancer has been on the rise in the late 20th century. According to the World Health Organization (WHO), the number of new global cancer cases in 1990 was about 8.07 million, an increase of 37.4% over the 5.17 million in 1975. In 1997, the number of cancer deaths worldwide was about 6.2 million, and according to current trends, as the world population reaches 8 billion in 2020, there will be 20 million new cancer cases, of which 12 million will be killed. Most will happen in developing countries.

Tumors seriously endanger people's health and bring huge economic burden to patients and society. It is of great practical significance to effectively diagnose and treat tumors. The diagnosis and treatment of tumors is a hot spot in existing medical research.

The variety of tumors, the different characteristics, and the different reactions to drugs, which makes the treatment of tumors extremely difficult. At this stage, the treatment of tumors mainly includes surgery and medication. Surgical treatment is mainly applied to tumors that have not metastasized. The surgical results vary according to the tumor, and are more restricted, and the damage to normal humans is greater. Chemotherapy is mainly based on chemotherapy, and well-known, chemotherapy drugs have a strong killing effect on normal cells, and side effects are large, and their use is also limited.

Whether it is surgery or medical treatment, there is nothing to do with metastatic malignant tumors. It has been very practical to develop an anti-tumor drug that has a good killing effect on various tumors, especially on tumors that have metastasized.

Summary of the invention

It is an object of the present invention to provide a class of amino acid derivatives.

Another object of the present invention is to provide an application of the above amino acid derivative for the preparation of an antitumor drug.

Another object of the present invention is to provide an antitumor drug.

The technical solution adopted by the present invention is:

An amino acid derivative having the structural formula shown in Formulas I to VI.

In the formula:

X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

Y, Y ₁ , Y _{2 are} independently polypeptides;

The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

The amino acid derivative does not include

And other compounds which have been disclosed to meet the above formula.

As a further improvement of the above amino acid derivative, R, R' are independently selected from the group consisting of glutamic acid, glutamine, citrulline, ornithine, cystine, aspartic acid, asparagine, threonine, serine ,leucine,isoleucine,glycine,cysteine,methionine,tryptophan,tyrosine,phenylalanine,valine,lysine, Side chain substituents of hydroxylysine, hydroxyproline, valine, lysine, histidine, and simple modifications thereof.

As a further improvement of the above amino acid derivative, the structural formula of the amino acid derivative is

And its simple modifications.

As a further improvement of the above amino acid derivative, Y, Y ₁ and Y _{2 are} independently 2 to 12 peptides. In particular, Y, Y ₁ and Y _{2 are} independently 2 to 12 tumor targeting peptides.

As a further improvement of the above amino acid derivative, the pharmaceutically acceptable salt of the amino acid derivative is selected from the group consisting of sodium, potassium, calcium, zinc, lithium, iron, magnesium salt, sulfonate; the pharmaceutically acceptable ester of the amino acid derivative is selected from the group consisting of C2~ The ester between C4; the pharmaceutically acceptable ether of the amino acid derivative is selected from the group consisting of an ether between C2 and C4.

The use of amino acid derivatives in the preparation of antitumor drugs, the structural formula of amino acid derivatives is as shown in Formulas I to VI,

In the formula:

X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

Y, Y ₁ , Y _{2 are} independently polypeptides;

The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

Or an amino acid derivative as described above.

The tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, lymphocytic leukemia. , osteosarcoma, lymphosarcoma.

An antitumor drug whose active ingredient is at least one of the amino acid derivatives shown above.

Preferably, the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, Lymphocytic leukemia, osteosarcoma, lymphosarcoma.

The synthesis process of amino acid derivatives includes the following synthetic routes:

During the synthesis, the -OH, -NH ₂ or R groups are protected and deprotected as needed;

Wherein: X is selected from the group consisting of an oxalyl group and a simple modification thereof, or a residue obtained by coupling a compound containing an oxalyl group with an amino acid, and a simple modification thereof.

The beneficial effects of the invention are:

The amino acid derivative of the invention can target lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin. Lactate degeneration, citrate synthase, α-ketoglutarate dehydrogenase complex protein activity in malignant tumor tissues such as cancer, granulocyte leukemia, lymphocytic leukemia, osteosarcoma, lymphosarcoma, etc., specifically interfere with tumor cell production ATP destroys its energy metabolism, induces tumor cell death and apoptosis, enhances the inhibition of cancer cell proliferation, growth, metastasis, etc. It has high-efficiency and low-toxic anti-tumor pharmacodynamic activity, and has high energy consumption and high consumption. All kinds of malignant tumors have better killing or inhibition effects.

The amino acid derivative of the present invention has good water solubility and is easy to prepare into an injection preparation.

At the same time, some amino acid derivatives still have better or even better anti-tumor activity, which is not available in existing anti-tumor drugs, which greatly facilitates the use of patients, overcomes the existing anti-tumor drugs and relies on injection, safety. Low defects.

The amino acid derivative of the invention has simple synthesis process, low cost, easy purification and industrial production.

DRAWINGS

Figure 1 is a ¹ H NMR spectrum of Compound 2;

Figure 2 is a ¹³ C NMR spectrum of Compound 2;

Figure 3 is a ¹ H NMR spectrum of Compound 3;

Figure 4 is a ¹³ C NMR spectrum of Compound 3;

Figure 5 is a ¹ H NMR spectrum of Compound 4;

Figure 6 is a ¹³ C NMR spectrum of Compound 4;

Figure 7 is a ¹ H NMR spectrum of Compound 6;

Figure 8 is a ¹³ C NMR spectrum of Compound 6;

Figure 9 is a ¹ H NMR spectrum of Compound 7;

Figure 10 is a ¹³ C NMR spectrum of Compound 7;

Figure 11 is a ¹ H NMR spectrum of Compound 8;

Figure 12 is a ¹³ C NMR spectrum of Compound 8;

Figures 13 to 18 are in vitro antitumor profiles of different compounds;

Figure 19 is the effect of different compounds on the resting metabolic energy of animals;

Figure 20 is the effect of different compounds on ATP content in animal transplanted tumor tissues;

Figures 21 to 31 show the tumor inhibition rate of different compounds in different tumor models in animals.

detailed description

An amino acid derivative having the structural formula shown in Formulas I to VI.

In the formula:

X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

Y, Y ₁ , Y _{2 are} independently polypeptides;

The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

The amino acid derivative does not include

And other compounds which have been disclosed to meet the above formula.

In the present invention, a simple modification of a group refers to a simple substitution or addition of a group without changing the structure of the main body of the group, such as replacement of one or more of H by a hydroxyl group; or halogenation, Or esterification and etherification with a small molecule of C2 to C4; when the group has a carboxyl group or an amine group, the carboxyl group or the amine group is amidated.

The amino acid derivative of the present invention may be either L-form or D-form. The structure of L-form is closer to the structure of natural amino acids, but it is also easily metabolized in vivo, with shorter half-life and shorter onset time; D-amino acid derivatives can also be identified and better combined in vivo. It is more difficult to be metabolized, has a longer half-life, and has a longer onset of action. Different amino acid derivatives can be selected as antitumor active ingredients as needed.

As a further improvement of the above amino acid derivative, the structural formula of the amino acid derivative is

And its simple modifications. In particular, when the amino acid derivative is

Or a pharmaceutically acceptable salt, ester, ether or amide thereof, the amino acid derivative can be formulated into an oral preparation and has excellent antitumor activity.

Tumor cell targeting polypeptide (2-10 peptide) has a targeted distribution in tumor tissues, which can be better located in tumor cells and taken up by tumor cells, and is more prone to pharmaceutically active.

Preferably, Y, Y ₁ and Y ₂ in the above structural formula are independently 2 to 12 peptides, and further, Y, Y ₁ and Y _{2 are} independently 2 to 10 peptides, 2 to 8 peptides, 2 to 7 peptides, 2 to 4 peptides. More preferably, Y, Y ₁ and Y _{2 are} independently 2 to 12 tumor targeting peptides. Further, Y, Y ₁ and Y _{2 are} independently 2 to 10, 2 to 8, 2 to ₇ , ₂ to 4 tumor targeting peptides, especially those which have been confirmed to be more easily taken up by tumor cells and used for protein synthesis. Peptide.

As a further improvement of the above amino acid derivative, the pharmaceutically acceptable salt of the amino acid derivative is selected from the group consisting of sodium, potassium, calcium, zinc, lithium, iron, magnesium salt, sulfonate; the pharmaceutically acceptable ester of the amino acid derivative is selected from the group consisting of C2~ The ester between C4; the pharmaceutically acceptable ether of the amino acid derivative is selected from the group consisting of an ether between C2 and C4.

The use of amino acid derivatives in the preparation of antitumor drugs, the structural formula of amino acid derivatives is as shown in Formulas I to VI,

In the formula:

X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

Y, Y ₁ , Y _{2 are} independently polypeptides;

The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

Or an amino acid derivative as described above.

In particular, the amino acid derivative is

And its simple modifications.

The tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, lymphocytic leukemia. , osteosarcoma, lymphosarcoma.

An antitumor drug whose active ingredient is at least one of the amino acid derivatives shown above.

Preferably, the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, Lymphocytic leukemia, osteosarcoma, lymphosarcoma.

The synthesis process of amino acid derivatives includes the following synthetic routes:

During the synthesis, the -OH, -NH ₂ or R groups are protected and deprotected as needed;

Wherein: X is selected from the group consisting of an oxalyl group and a simple modification thereof, or a residue obtained by coupling a compound containing an oxalyl group with an amino acid, and a simple modification thereof.

For the amino acid derivative having the polypeptide structure, the corresponding solid phase synthesis can be carried out on the basis of the above synthesis steps.

Since the amino acid derivative of the present invention has a simple structure, it can also be directly entrusted to an existing compound synthesis company for synthesis.

The metabolism of tumor cells is vigorous, and a large amount of amino acids, small molecules for energy supply, etc. are required during the growth process. When the amino acid derivative of the present invention acts on tumor cells, a large amount of targeted accumulation in tumor tissues and tumor cells is observed. Aggregation, which can feedbackly inhibit the activity of citrate synthase and α-ketoglutarate dehydrogenase complex protein in tumor tissues through the mechanism of elevated substrate levels (the two enzymes are rate-limiting enzymes for the Krebs cycle) At the same time, it can specifically interfere with the tumor cell mitochondrial complex IV cytochrome c oxidase complex to produce ATP, thereby inducing tumor cell death and apoptosis, and enhancing the inhibition of cancer cell proliferation, growth, metastasis, etc.; The metabolic rate is significantly lower than that of tumor cells, and the amino acid derivative of the present invention is less ingested, and the energy metabolism and substance synthesis of the cells are also less affected, and there is no obvious killing effect. Therefore, the amino acid derivative of the present invention has high-efficiency and low-toxic antitumor pharmacodynamic activity.

The amino acid derivative of the invention has the effect of inhibiting and killing tumors from tumors, and can be applied to the treatment of tumors at various stages, and can also be combined with existing anti-tumor drugs to better anti-tumor. effect. Similarly, the amino acid derivatives of the present invention can also be used in combination to block the energy metabolism of the tumor as much as possible to achieve a better anti-tumor effect.

The synthesis process of the amino acid derivative of the present invention is further exemplified below in conjunction with specific synthetic examples.

Synthesis of amino acid derivatives

Synthetic route of oxalyl glutamic acid (compound 4)

The overall synthetic route is as follows,

The specific synthesis process is as follows:

Technical route of synthesis of compound 2

1.0 eq of Compound 1 was weighed and dissolved in an appropriate amount of CH ₂ Cl ₂ , and 2.2 eq of Et ₃ N was added to the above reaction solution under stirring. 1.1 eq of EtOOC-COCl was added dropwise to the above reaction solution under ice-cooling and stirring. After the completion of the dropwise addition, the ice bath was removed, stirred at room temperature, and sampled for HPLC monitoring until the disappearance of the starting material peak. The white solid insoluble solid was filtered off, and the organic phase was washed once with 5% aqueous NaHCO ₃ solution, saturated brine, 10% aqueous KHSO ₄ and brine, and the organic phase was collected, dried and concentrated to give a viscous compound 2 The yield was 90% and the HPLC purity was 95%.

The nuclear magnetic data of Compound 2 is:

¹ H NMR (400 Hz, CD ₃ OD) δ: 4.28-4.38 (m, 2H), 2.31-2.35 (t, 2H), 1.93-2.21 (m, 2H), 1.44-1.47 (d, 18H), 1. 1.37 (t, 3H), the hydrogen spectrum of which is shown in Figure 1;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 173.60, 171.38, 161.07, 159.27, 83.41, 81.92, 63.93, 54.22, 32.50, 28.33, 28.21, 27.30, 14.25, the carbon spectrum is shown in Figure 2;

MS (EI) m / z (100%): 358.1 (M ^- , 100), theory 359.3;

Technical route for the synthesis of compound 3

Compound 2 was dissolved in methanol, and pH 8-9 was adjusted by adding an appropriate amount of 1 mol/L NaOH aqueous solution, and the mixture was hydrolyzed at room temperature, and the reaction was monitored by HPLC. After the reaction is completed, the solution is adjusted to pH 2-3 with hydrochloric acid, and the mixture is evaporated to dryness to remove methanol. The mixture is extracted twice with ethyl acetate. The organic phase is combined, washed once with brine, concentrated to dryness and dried to give compound 3 85%, HPLC purity 96%.

The nuclear magnetic data of Compound 3 is:

¹ H NMR (400 Hz, CD ₃ OD) δ: 2.31-2.34 (t, 2H), 1.93-2.21 (m, 2H), 1.44-1.47 (d, 18H), the hydrogen spectrum is shown in Figure 3;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 173.62, 171.45, 162.20, 160.07, 83.40, 81.93, 54.21, 32.50, 28.33, 28.21, 27.37, the carbon spectrum of which is shown in FIG.

MS (EI) m / z (100%): 330.2 (M ^- , 100),

Technical route of synthesis of compound 4

Compound 3 was dissolved in 3 volumes of TFA and the reaction was monitored by HPLC. After the reaction was completed, the TFA was removed by rotary evaporation. Add appropriate amount of water to dissolve, wash once with CH ₂ Cl ₂ , and freeze the aqueous phase to obtain white powdery compound 4 in a yield of 90%, purity 96.8%.

The nuclear magnetic data of the compound 4 is: ¹ H NMR (400 Hz, CD ₃ OD) δ: 4.47-4.54 (m, 1H), 2.39-2.46 (m, 2H), 2.01-2.33 (m, 2H), the hydrogen spectrum is as Figure 5;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 176.31, 173.86, 162.22, 160.09, 53.45, 30.98, 27.37, the carbon spectrum of which is shown in FIG.

MS (EI) m / z (100%): 113 (M ^- -106, 100), theory 219;

Synthetic route of oxalyl glutamine (compound 8)

The overall synthetic route is as follows,

The specific synthesis process is as follows:

Technical route of synthesis of compound 6

1.0 eq of Compound 5 was weighed and dissolved in an appropriate amount of CH ₂ Cl ₂ , and 2.2 eq of Et ₃ N was added to the above reaction solution under stirring. 1.1 eq of EtOOC-COCl was added dropwise to the above reaction solution under ice-cooling and stirring. After the completion of the dropwise addition, the ice bath was removed, stirred at room temperature, and sampled for HPLC monitoring until the disappearance of the starting material peak. The white solid insoluble solid was filtered off, and the organic phase was washed once with 5% aqueous NaHCO ₃ solution, saturated brine, 10% aqueous KHSO ₄ and brine, and the organic phase was collected, dried and concentrated to give viscous compound 6 The yield was 88% and the HPLC purity was 96%.

The nuclear magnetic data of Compound 6 is:

¹ H NMR (400 Hz, CD ₃ OD) δ: 4.31-4.34 (m, 1H), 3.57-3.62 (m, 2H), 2.29-2.33 (t, 2H), 1.99-2.24 (m, 2H), 1.50 ( s, 9H), 1.15.19.19 (t, 3H), the hydrogen spectrum thereof is shown in Figure 7;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 177.45, 171.38, 160.12, 159.08, 83.47, 58.32, 54.66, 32.41, 28.18, 27.72, 18.37, the carbon spectrum of which is shown in FIG.

MS (EI) m / z (100%): 301.2 (M ^- , 82) 113.1 (100), theory, 302.2;

Technical route of synthesis of compound 7

Compound 6 was dissolved in methanol, and pH 8-9 was adjusted by adding an appropriate amount of 1 mol/L NaOH aqueous solution, and the mixture was hydrolyzed at room temperature, and the reaction was monitored by HPLC. After the reaction was completed, the solution was adjusted to pH 2-3 with hydrochloric acid, and concentrated to remove methanol by rotary evaporation. The white insoluble material was collected by filtration, and the filter cake was washed twice with cold water. The cake was collected and dried to give compound 7 in a yield of 85%. %

The nuclear magnetic data of Compound 7 is:

¹ H NMR (400 Hz, CD ₃ OD) δ: 4.31-4.35 (m, 1H), 2.27-2.33 (t, 2H), 1.97-2.24 (m, 2H), 1.47 (s, 9H), Figure 9 shows;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 177.47, 171.47, 162.35, 160.32, 83.49, 54.57, 32.38, 28.19, 27.83, the carbon spectrum of which is shown in FIG.

MS (EI) m / z (100%): 273.2 (M ^- , 100),

Technical route of synthesis of compound 8

Compound 7 was dissolved in 3 volumes of TFA and the reaction was monitored by HPLC. After the reaction was completed, the TFA was removed by rotary evaporation. Add appropriate amount of water to dissolve, wash once with CH ₂ Cl ₂ , and freeze the aqueous phase to obtain white powdery compound 8 in a yield of 90%, purity 90%.

The nuclear magnetic data of Compound 8 is:

¹ H NMR (400 Hz, CD ₃ OD) δ: 4.44 - 4.71 (s, 1H), 2.31-2.35 (m, 2H), 2.05-2.28 (m, 2H), the hydrogen spectrum is shown in Figure 11;

¹³ C NMR (400 Hz, CD ₃ OD) δ: 177.58, 173.79, 162.27, 160.13, 53.71, 32.50, 28.01, the carbon spectrum of which is shown in FIG.

MS (EI) m/z (100%): 1121. (M ^- -105, 100)

The compounds 2, 3, 4, 6, 7, and 8 synthesized by the above scheme were used for experiments to confirm their functions.

Safety experiment

1. Amino acid derivatives on L929 cytotoxicity experiment

Cell culture and cytotoxicity assessment

L-929 cells were supplemented with 10% fetal bovine serum and penicillin (100 units), streptomycin in a gaseous environment of 5% ± 1% carbon dioxide at 37 ± 1 ° C and in a humidified environment containing tissue culture flasks containing the recommended media. 100 μg) and other antibiotics (Invitrogen, USA) cell culture medium (Invitrogen, USA) were cultured to achieve the cell density required for the experiment. The monolayer cells were rinsed twice with PBS, the PBS was aspirated, and digested in a 0.25% trypsin/EDTA, 37 ± 1 °C tissue culture flask until the cells were separated and floated.

The in vitro cytotoxic effect of the synthesized compounds 2, 3, 4, 6, 7, 8 on L929 cells was performed and performed according to the morphological analysis of the cell strain of ISO 10993-part 5.

Table 1 Qualitative morphological grading of compound toxicity

For the test, the in vitro cytotoxic effects of compounds 2, 3, 4, 6, 7, and 8 on L929 cells were observed. All compounds were diluted with 5% DMEM, and diluted to 100 μM, and diluted to 50 μM, 25 μM, 12.5 μM, 6.25 μM 5% DMEM dilution, respectively, added 5% CO ₂ cultured L-929 mice. Fusion of fibroblasts into monolayer cells. In addition, three independent test solutions were positive control (cisplatin, positive control), negative control (normal saline) and vehicle control (5% DMEM). All test solutions were incubated for 24 hours at 5% ± 1% CO ₂ at 37 ± 1 °C. The monolayer cells were positive in the test, and the reagent control test solution was examined under the microscope for 24 ± 1 hour to determine any change in cell morphology.

result

Under the conditions of this study, compounds 2, 3, 4, 6, 7, 8 were observed at a concentration of 100 μM for 24 ± 1 hour and found to be less toxic or less cytotoxic to L-929 cells.

After 24 hours of incubation, morphological observation and biological reaction, the results are shown in Table 2.

Table 2 Qualitative morphological classification of the toxicity of the test compound

Test drug	Confluent single layer	percentage	Intracellular granule	Dissolve	level	reaction
100μM Compound 2	no	<50%	Have	no	2	mild
100μM compound 3	no	<50%	Have	no	2	mild
100μM Compound 4	Have	<20%	Have	no	1	slight
100μM Compound 6	Have	0%	Have	no	0	no response
100μM Compound 7	Have	0%	Have	no	0	no response
100μM Compound 8	Have	0%	Have	no	0	no response
Cisplatin	no	>70%	no	no	4	severe

Negative control (NS)	Have	0%	Have	no	0	no response
Blank control (solvent)	Have	0%	Have	no	0	no response

.

in conclusion

Toxicity of compounds 2, 3, 4, 6, 7, and 8 to L929 cells showed that 5% DMEM dilution at 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM was applied to L929 cells over 24 hours. No significant cytotoxicity was observed at a maximum concentration of 100 μM.

2. Killing experiments of in vitro cancer cells by amino acid derivatives (IC ₅₀ )

Experimental materials and instruments

Drugs 2, 3, 4, 6, 7, 8, control cisplatin, human lung cancer H460, H1650, SPC-A-1, A549 cell line, liver cancer HepG2 cell line, breast cancer MCF-7 cell line, colon cancer HT -29 cell line, prostate cancer PC-3, DU145 cell line, cervical cancer Hela cell line, gastric cancer MGC-803 cell line, DMEM medium, 10% FBS calf serum, MTT reagent, DMSO, 96-well plate, enzyme label Instrument and so on. Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory.

Experimental methods and steps

1) collecting log phase cells;

2) The number of cells is 4×10 ³ /L, and 100 uL per well;

3) Incubate for 12 hours;

4) Add the concentration of the test drug and the positive control drug to the concentration of 0.5, 2.5, 5.0, 10, 25, 50, 100 μM, and the positive control drug at the concentration of 0.5, 2.5, 5.0, 10, 25, 50, 100 μM. Platinum; another solvent control group

5) After culture 24, add 20 μl of MTT (5 mg / ml) solution per well;

6) Incubate for 4 hours, terminate the culture, and discard the supernatant;

7) Add DMSO 100 ul / well, shake for 10 min;

8) measured by a microplate reader, λ = 490 nm;

9) Record the results and draw a chart.

Calculate the cell growth inhibition rate and IC50 of the drug according to the following formula:

IR%=(1 - experimental group OD value / control group OD value) × 100%

RESULTS: Compounds 2, 3, 4, 6, 7, and 8 were diluted in 5% DMEM at 0.5, 2.5, 5.0, 10, 25, 50, and 100 μM for in vitro lung cancer H460, H1650, and SPC-A-1. , A549 cells, liver cancer HepG2 cells, breast cancer MCF-7 cells, colon cancer HT-29 cells, prostate cancer PC-3, DU145 cells, cervical cancer Hela cells, gastric cancer MGC-803 cells, observed after 24 hours, observed compound 2 The 50% inhibition rate (IC ₅₀ ) of the above cells at 3, 4, 6, 7, and 8.

Compounds 2, 3, 4, 6, 7, and 8 act on lung cancer H460, H1650, SPC-A-1, A549 cells, liver cancer HepG2 cells, breast cancer MCF-7 cells, colon cancer HT-29 cells, and prostate cancer PC, respectively. -3, DU145 cells, cervical cancer Hela cells, gastric cancer MGC-803 cells, after 24 hours incubation, MTT assay for anti-tumor activity found that compounds 2, 3, 4, 6, 7, 8 have certain anti-tumor effects (respectively See Figures 13 to 18), but the anti-tumor activity of the amino acid derivative in vitro is not high compared to the positive control group.

3. Acute toxicity test in mice

SPF grade KM mice were housed in an independent ventilation system (IVC) facility in an SPF laboratory animal chamber. Group raising, free drinking water, eating, laboratory temperature is 20 ~ 25 ° C, humidity is 40 ~ 70%. Animal room conditions are always stable to ensure the reliability of the test results. Feed and drinking water: The feed is SPF grade granular rat material purchased from Guangdong Medical Laboratory Animal Center. Nutrition and sanitation meet the requirements of SPF laboratory animals. Drinking water is free to ingest.

Clinically used route: oral administration.

Pre-test data: After a single oral administration of the compound 8 (20140304) synthesized by the applicant at the maximum dose, that is, the maximum concentration of 1800. mg/kg·bw, the mortality rate of the mice was 0%.

Grouping of animals: 140 animals with quarantine and weight requirements were randomly divided into 14 groups according to body weight and sex, 10 in each group, half male and half female. Preparation of test drug: Weighed compound 8 freeze-dried powder injection, dissolved in physiological saline. Route and number of administration: single oral administration. Dosing volume: 10 ml/kg. Fasting time of animals: fasting for 6 to 12 hours before administration and fasting for 4 hours after administration.

Observation and examination: General observation: Immediately after administration, observe the reaction of the animals, and continue to observe for 4 hours, and then observe once every morning for 14 days, record the performance and characteristics of the animal poisoning, the time of occurrence of toxicity, and the disappearance time. And the time of death of the animal.

Body weight measurement: before administration (d ₁ ), 24 hours after administration (d ₂ ), day 4 (d ₄ ), day 8 (d ₈ ), day 11 (d ₁₁ ), and day 15 (d ₁₅ ) Weigh the animal's body weight.

Pathological examination: poisoning death or sudden death of animals in time for necropsy, other animals at the end of the observation period after necropsy, when the tissue and organ changes in volume, color, texture, etc., the pathological organization of the changed tissue and organs Learn to check. Anatomical organs include thymus, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), lymph nodes, bladder, prostate, testis ( With epididymis), uterus, ovaries.

Observation indicators: detailed records of animal reactions, including appearance, behavioral activities, mental state, appetite, urine and color, fur, skin color, breathing, nose, eyes, oral cavity with abnormal secretions, weight changes and death.

Experimental results: Within 14 days, the mice did not die. After the gavage, the mice contracted and did not like the activity. The analysis was caused by the excessive concentration of the drug and the excessive volume of the gavage. It indicates that the toxicity of compound 8 is low, and the maximum tolerated dose of mice is about 1800 mg/kg, which is equivalent to 512 times of the clinically recommended dose.

Effect of Amino Acid Derivatives on Cell Energy Metabolism

1. Antitumor energy metabolism of amino acid derivatives

METHODS: The CCM metabolites produced by MEDGRAPHIC of the United States were used to allow the nude mice to rest without stimulation before the test. The measurements were taken after 30 minutes of calm (the measured indicators were O ₂ , CO ₂ and exhaled breath in inhalation and exhalation, according to The inspiratory and exhaled nitrogen concentrations are constant, and the inspiratory volume is calculated. The oxygen consumption (VO ₂ ) and carbon dioxide production (VCO ₂ ) are combined, and the total nitrogen output (24) is combined with the 24-hour urine. The ambient humidity during the test is 50-65%, the temperature is 18-24 ° C, the atmospheric pressure is 101 ~ 102.4 kPa (758-768 mmHg), and these data are input into the computer for instrument calibration.

The energy consumption is calculated according to the formula 3.94 × VO ₂ + 1.11 × VCO _{2 -} 2.17 × N. The respiratory quotient is calculated according to RQ = VCO ₂ / VO ₂ , and the non-protein respiratory quotient is calculated according to NRQ = (VCO _{2 -} 4.754 × N) = (VO _{2 -} 5.293 × N). The effect of amino acid derivatives on resting energy metabolism REE (Resting Energy Expenditure, total daily energy consumption: kJ/d) in nude mice bearing A549 animal model was determined. The results are shown in Figure 19:

Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory. As can be seen from Fig. 19, on the 10th day and the 14th day after administration, the model group and the cisplatin group and the compounds 2, 3, 4, 6, 7, and 8 groups can significantly reduce the energy metabolism of the tumor tissue.

2. ATP generation

The ATP (ATP Assay Kit) was used to detect ATP (adenosine 5'-triphosphate) levels in tumor-bearing nude mice. According to the firefly luciferase fluorescein, the fluorescence is required to be developed by ATP. When both firefly luciferase and fluorescein are excessive, the production of fluorescence is proportional to the concentration of ATP in a certain concentration range. This makes it possible to detect the ATP concentration in the solution with high sensitivity. The test kit used can detect ATP at concentrations as low as 5 nmol/L. The concentration of ATP in conventional cell or tissue lysates is only 0.1-1 μmol/L, and the intracellular ATP level of some common cells is about 10 nmol/mg protein. When the concentration of ATP in a cell or tissue is detected, the ATP concentration can be determined by lysing the lysate provided by the kit.

Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory. The effects of compounds 2, 3, 4, 6, 7, and 8 on ATP content in tumor-bearing nude mice A549 xenografts are shown in Figure 20.

As seen from Fig. 20, on the 14th day after administration, the model group and the cisplatin group and the compounds 2, 3, 4, 6, 7, and 8 all significantly reduced the ATP content of the tumor tissue, but compared with the cisplatin group. In contrast, Compounds 2, 4, and 6 have significant differences in the inhibition of ATP production.

Antitumor pharmacodynamics experiment of amino acid derivatives

Experimental animals:

Healthy Balb-c nude mice, male and female, about 4 weeks old, weighing 18-22g, were purchased from Guangdong Medical Laboratory Animal Center. Nude mice with a tumor diameter of about 0.5-1.0 cm were taken for 2-3 weeks after inoculation for the experiment.

Cell line:

Human malignant tumor cell lines H460, H1650, SPC-A-1, A549, HepG2, MCF-7, HT-29, PC-3, DU145, Hela, MGC-803.

Establishment of SPCA tumor-bearing nude mouse model:

70 healthy Balb-c nude mice, male, 4 to 5 weeks old, weighing 18-22 g. Human non-small cell lung cancer cell lines H460, H1650, SPC-A-1, A549, HepG2, MCF-7, HT-29, PC-3, DU145, Hela, MGC-803, adjusted cell density of about 1 × 10 ⁷ /ml, inoculated with 0.2ml/nose in the right iliac crest of the nude mice, and after 2-3 weeks, the tumor was grown to 1.0×1.0cm, and the tumor tissue was uniformly cut, and the cannula was inoculated to the tested nude mice. The anterior right axilla was subcutaneously and observed after 1-2 weeks of inoculation. The tumor formation rate was 98.6%, and nude mice with tumor growth of about 0.5×0.5 cm were selected for the experiment.

Antitumor effect of amino acid derivatives on H460 cell line-bearing nude mice

Grouping of animals:

Model nude mice bearing a tumor volume of approximately 0.5×0.5 cm were randomly assigned, Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) Synthesis from the inventor's laboratory. Therefore, the tumor-bearing animals were divided into eight groups, a model group, a positive control group, a compound 2 group, a compound 3 group, a compound 4 group, a compound 6 group, a compound 7 group, and a compound 8 group, and 8 animals in each group.

Dose setting:

Model group: equal volume of normal saline; positive control group: 10 μM/kg (cisplatin solution); compound 2 group: 50 μM/kg; compound 3 group: 50 μM/kg; compound 4 group: 50 μM/kg; compound 6 group: 50 μM /kg; Compound 7 group: 50 μM/kg; Compound 8 group: 50 μM/kg; each group was administered intraperitoneally (ip) according to 0.1 ml/10 g.

Method of administration:

Dosing daily for 14 consecutive days. All were administered by intraperitoneal injection (ip), weighed before daily administration, and the amount of each dose was determined according to the daily body weight. Weigh the next day and measure the tumor volume. On the 15th day, the animals were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor were taken out, weighed separately, and stored in 10% formaldehyde.

Detection Indicator:

(1) The formula for calculating the tumor volume (TV) is:

V = 1/2 × a × b ² (a, b respectively indicate the length and width of the tumor)

(a, b units are mm, tumor volume V is mm ³ )

(2) Tumor weight inhibition rate = (1 - average tumor weight of the administration group / average tumor weight of the control group) × 100%

(3) Tumor volume inhibition rate = (1 - (volume before administration - volume after administration) / (volume before administration - volume after administration)) × 100%.

Pharmacodynamic experiment results of amino acid derivatives

1. The inhibition rate of amino acid derivative amino acid oxalate on lung cancer H1650 model in nude mice is shown in Fig. 21.

As seen from Fig. 21, Compound 3 and Compound 8 were P>0.05 as compared with the positive control drug cisplatin, i.e., the efficacy of Compound 8 and Compound 2 against lung cancer H1650 proliferation was comparable to that of cisplatin.

2. The inhibition rate of amino acid derivative amino acid oxalate on lung cancer H460 model in nude mice is shown in Fig. 22.

As seen from Fig. 22, Compounds 2 and 6 were P>0.05 as compared with the positive control drug cisplatin, i.e., the potency of Compounds 2 and 6 against lung cancer H460 proliferation was comparable to that of cisplatin.

3. The inhibition rate of the amino acid derivative amino acid oxalate on the lung cancer SPC-A-1 model in nude mice is shown in FIG.

As seen from Fig. 23, compounds 2, 3, 4 and 7 were compared with the positive control drug cisplatin, P>0.05, ie the efficacy of compounds 2, 3, 4 and 7 against the proliferation of lung cancer SPC-A-1 and cisplatin quite.

4. The inhibition rate of amino acid derivative amino acid oxalate on lung cancer A549 model in nude mice is shown in Fig. 24.

As seen from Fig. 24, Compounds 2, 3 and 7 were P>0.05 compared with the positive control drug cisplatin, i.e., the effects of Compounds 2, 3 and 7 against lung cancer A549 proliferation were comparable to those of cisplatin.

5. Amino acid derivative amino acid oxalic acid amide inhibition rate in liver cancer HepG ₂ model in nude mice is shown in Fig. 25.

As seen from Fig. 25, Compounds 4 and 6 were comparable to cisplatin in the proliferation of anti-hepatocarcinoma HepG ₂ compared to the positive control drug cisplatin, but Compounds 2, 3 and 7 were more effective against liver cancer than cisplatin. Platinum (P < 0.05).

6. The inhibition rate of amino acid derivative amino acid oxalate on breast cancer MCF-7 model in nude mice is shown in Fig. 26.

As seen from Figure 26, Compounds 2, 3 and 7 were comparable to cisplatin in the proliferation of anti-breast cancer MCF-7 compared to the positive control cisplatin.

7. The inhibition rate of the amino acid derivative amino acid oxalate on the colon cancer HT-29 model in nude mice is shown in FIG.

As seen from Figure 27, Compounds 2, 3, 4 and 7 were comparable to cisplatin in anti-breast cancer MCF-7 proliferation compared to the positive control drug cisplatin.

8. The inhibition rate of the amino acid derivative amino acid oxalate on the prostate cancer PC-3 model in nude mice is shown in FIG.

As seen from Fig. 28, Compounds 2, 3 and 7 were comparable to cisplatin in anti-prostate cancer PC-3 proliferation compared to the positive control drug cisplatin.

9. The anti-tumor rate of the amino acid derivative amino acid oxalate on the prostate cancer DU145 model in nude mice is shown in FIG.

As seen from Figure 29, Compounds 2, 3, 4 and 7 were comparable to cisplatin in the proliferation of prostate cancer DU145 compared to the positive control cisplatin.

10. The inhibition rate of the amino acid derivative amino acid oxalic acid on the cervical cancer Hela model in nude mice is shown in FIG.

As seen from Fig. 30, the anti-Hela proliferation effects of Compounds 2, 3 and 4 were comparable to those of the cisplatin group, but the anti-Hela proliferation effect of Compound 7 compared with the cisplatin group. Higher than cisplatin.

11. The anti-tumor rate of amino acid derivative amino acid oxalate on gastric cancer MGC-803 model in nude mice is shown in FIG.

As seen from Fig. 31, Compounds 2, 3, 7 and 8 were comparable to cisplatin in the proliferation of anti-gastric cancer MGC-803 as compared with the positive control cisplatin group.

Claims (10)

1. An amino acid derivative having the structural formula shown in Formulas I to VI.

   

   In the formula:

   X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

   R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

   Y, Y ₁ , Y _{2 are} independently polypeptides;

   The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

   The amino acid derivative does not include

   
2. The amino acid derivative according to claim 1, wherein R and R' are independently selected from the group consisting of glutamic acid, glutamine, citrulline, ornithine, cystine, aspartic acid, asparagine , threonine, serine, leucine, isoleucine, glycine, cysteine, methionine, tryptophan, tyrosine, phenylalanine, valine, lysine, hydroxy Side chain substituents of lysine, hydroxyproline, valine, lysine, histidine, and simple modifications thereof.
3. The amino acid derivative according to claim 1, wherein the structural formula of the amino acid derivative is

   

   And its simple modifications.
4. The amino acid derivative according to any one of claims 1 to 3, wherein Y, Y ₁ and Y _{2 are} independently 2 to 12 peptides.
5. The amino acid derivative according to claim 4, wherein Y, Y ₁ and Y _{2 are} independently 2 to 12 tumor targeting peptides.
6. The amino acid derivative according to any one of claims 1 to 5, wherein the pharmaceutically acceptable salt of the amino acid derivative is selected from the group consisting of sodium, potassium, calcium, zinc, lithium, iron, magnesium salt, sulfonate; The pharmaceutically acceptable ester is selected from the group consisting of esters between C2 and C4; the pharmaceutically acceptable ether of the amino acid derivative is selected from the group consisting of ethers between C2 and C4.
7. The use of an amino acid derivative in the preparation of an antitumor drug, the structural formula of the amino acid derivative is as shown in Formulas I to VI,

   

   In the formula:

   X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;

   R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;

   Y, Y ₁ , Y _{2 are} independently polypeptides;

   The amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;

   Or an amino acid derivative according to any one of claims 2 to 6.
8. The use according to claim 7, wherein the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, Melanoma, skin cancer, granulocyte leukemia, lymphocytic leukemia, osteosarcoma, lymphosarcoma.
9. An antitumor drug, wherein the active ingredient is at least one of the amino acid derivatives according to any one of claims 1 to 6.
10. The synthesis process of amino acid derivatives includes the following synthetic routes:

    

    

    During the synthesis, the -OH, -NH ₂ or R groups are protected and deprotected as needed;

    Wherein: X is selected from the group consisting of an oxalyl group and a simple modification thereof, or a residue obtained by coupling a compound containing an oxalyl group with an amino acid, and a simple modification thereof.

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