KR102348728B1 - Pharmaceutical Composition for the Prevention or Treatment of Cancer - Google Patents

Abstract

The present invention relates to a novel oligopeptide AQTGTGKT analog compound, and more specifically, an analog compound of the oligopeptide AQTGTGKT that exhibits excellent anticancer effect and stably exists in human blood, a pharmaceutical composition comprising the same as an active ingredient, and a pharmaceutical composition thereof A manufacturing method is provided. The analog compound according to the present invention and a pharmaceutical composition comprising the same as an active ingredient have a small molecular weight compared to an antibody, which is an advantage of an oligopeptide preparation, so there is less concern about immune response, easy penetration into tissues, and cancer cells. It has a more excellent anti-proliferative effect and can be used as a useful anticancer agent to treat cancer because it exhibits an effect that can exist stably in human blood.

Description

Pharmaceutical composition for the prevention or treatment of cancer {Pharmaceutical Composition for the Prevention or Treatment of Cancer}

The present invention relates to a novel oligopeptide analog compound of AQTGTGKT, a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient, and a method for preparing the same.

Although the therapeutic effect of cancer is improving due to the development of early diagnosis methods for cancer and the continuous development of new anticancer therapies, cancer is still an important disease that competes as the first and second cause of death in Korea. Most of the currently used anticancer drugs are due to chemotherapy, and they have various pharmacological actions depending on the type of cancer and various side effects due to toxicity are pointed out as a problem in cancer treatment.

Existing anticancer drugs penetrate not only cancer cells but also normal tissues and damage the function and activity of normal cells. It shows major problems in cancer treatment, such as multidrug resistance. Therefore, research on the development of innovative anticancer drugs that can solve these serious problems of existing anticancer drugs is being actively conducted.

On the other hand, although the development of an antibody targeting a specific tumor antigen of a tumor cell is being made, in the case of the antibody, there are problems such as concerns about an immune response and low efficiency of penetration into the tissue. On the other hand, in the case of peptides, the molecular weight is small, so there is less concern about immune response, unlike antibodies, and it has the advantages of easy penetration into tissues. It is expected that there will be few side effects such as damage to cells. However, despite these advantages, it is only used for very limited carcinomas, and in particular, it is decomposed within a short time immediately after administration to humans and it is difficult to exert its effect.

As a method for solving the above problems, amidation, esterification, acylation, acetylation, PEGylation, cyclization, or alkylation of a peptide It is possible to improve the yield, increase in vivo activity, increase the affinity of the peptide for the receptor, delay the degradation of the protein, etc. by modifying it by There is a risk that it may lose its in vivo activity or cause unexpected side effects. Therefore, studies on the modification of peptides to minimize these negative effects and maximize the desired effects are being actively conducted.

KR 10-2073144 B1

The present invention has been devised to solve the problems of the prior art as described above, and the anticancer effect and half-life in the blood of the oligopeptide AQTGTGKT (alanine-glutamine-threonine-glycine-threonine-glycine-lysine-threonine) are further improved. As a result of earnest efforts to improve, the present invention was completed. In particular, it was confirmed that the amidation analog compound of the AQTGTGKT peptide had an excellent anticancer effect and an increased half-life.

Accordingly, it is an object of the present invention to provide a compound represented by the following general formula:

[general meal]

X-AQTGTGKT

In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

X is

It is at least one selected from the group consisting of.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising a compound represented by the above general formula as an active ingredient.

Another object of the present invention is to provide a method for preparing a compound represented by the above general formula.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention as described above, the present invention provides a compound represented by the following general formula:

[general meal]

X-AQTGTGKT

In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

X is

It is at least one selected from the group consisting of.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following general formula as an active ingredient:

[general meal]

X-AQTGTGKT

In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

X is

It is at least one selected from the group consisting of.

In addition, the present invention provides a method for preventing or treating cancer comprising administering to a subject a compound represented by the above general formula.

In addition, the present invention provides the use of the compound represented by the general formula for preventing, improving or treating cancer.

Furthermore, the present invention provides the use of a compound represented by the above general formula for producing a medicament for the treatment of cancer.

In one embodiment of the present invention, the cancer may be a cancer selected from the group consisting of lung cancer, breast cancer, blood cancer, colorectal cancer, pancreatic cancer, and combinations thereof, but is not limited thereto.

In another embodiment of the present invention, the lung cancer may be non-small cell lung cancer, but is not limited thereto.

In another embodiment of the present invention, the breast cancer may be triple negative breast cancer, but is not limited thereto.

In another embodiment of the present invention, the hematologic cancer may be selected from the group consisting of leukemia, lymphoma, multiple myeloma, and combinations thereof, but is not limited thereto.

In one embodiment of the present invention, X is

At least one selected from the group consisting of, the cancer may be lung cancer, but is not limited thereto.

이고, 상기 암은 유방암일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, X is

And, the cancer may be breast cancer, but is not limited thereto.

In another embodiment of the present invention, X is

At least one selected from the group consisting of, the cancer may be a blood cancer, but is not limited thereto.

,

및

로 이루어진 군으로부터 선택된 하나 이상이고, 상기 암은 췌장암일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, X is

,

and

At least one selected from the group consisting of, the cancer may be pancreatic cancer, but is not limited thereto.

In another embodiment of the present invention, X is

At least one selected from the group consisting of, the cancer may be colorectal cancer, but is not limited thereto.

인 경우, 인간 혈액 내에서의 반감기가 100분 내지 150분일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, X is

In the case of , the half-life in human blood may be 100 minutes to 150 minutes, but is not limited thereto.

In another embodiment of the present invention, X is

In the case of at least one selected from the group consisting of, the half-life in human blood may be 45 minutes to 70 minutes, but is not limited thereto.

인 경우, 인간 혈액 내에서의 반감기가 20분 내지 30분일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, X is

In the case of , the half-life in human blood may be 20 to 30 minutes, but is not limited thereto.

The present invention also provides a method for preparing an oligopeptide X-AQTGTGKT comprising the steps of:

(The A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

wherein X is

It is one selected from the group consisting of.)

(1) synthesizing TG and KT, respectively;

(2) synthesizing TGKT by combining the TG and KT;

(3) synthesizing TGTGKT by binding TG to the N-terminus of TGKT;

(4) synthesizing QTGTGKT by binding Q to the N-terminus of TGTGKT; and

(5) synthesizing X-AQTGTGKT by binding an alanine derivative (X-A) to the N-terminus of QTGTGKT.

The present invention relates to a novel analog compound of the oligopeptide AQTGTGKT, a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient, and a method for preparing the same, wherein the analog of the oligopeptide AQTGTGKT exhibits excellent anticancer effects, and It was confirmed that it was stably present in the blood. Therefore, the pharmaceutical composition according to the present invention has a smaller molecular weight compared to an antibody, which is an advantage of an oligopeptide, so there is less concern about an immune response, and in addition to being easy to penetrate into a tissue, the effect of inhibiting the proliferation of cancer cells is more excellent, and the human It is expected that it can be used as a useful anticancer agent to treat cancer because it exhibits the effect of being stably present in the blood.

^{1 to 7 are diagrams showing UPLC-MS (top) and 1} H NMR (bottom) results for identifying the compound according to the present invention and confirming the structure of the compound, FIG. 1 is 4-PhPh-AQTGTGKT, FIG. 2 is Ac-AQTGTGKT, Figure 3 is 3-PhPh-AQTGTGKT, Figure 4 is 4-MeOPh-AQTGTGKT, Figure 5 is 2-PhPh-AQTGTGKT, Figure 6 is Ph-AQTGTGKT, Figure 7 is the measurement result of Naphthyl-AQTGTGKT it has been shown
8 is a diagram showing the results of analyzing the cell activity inhibitory effect of the compound treatment of the present invention in lung cancer cell line H1299 by CTG assay ( ^* p <0.05, ^** p <0.01, ^*** p <0.001; 9 to 16 ).
9 is a diagram showing the results of analyzing the cell activity inhibitory effect of the compound of the present invention in the lung cancer (papillary adenocarcinoma) cell line H820 by MTT assay.
10 is a diagram showing the results of analyzing the cell activity inhibitory effect of the compound treatment of the present invention in the lung cancer cell line H1975 by MTT assay.
11 is a view showing the results of analysis by CTG assay on the cell activity inhibitory effect of the treatment with the compound of the present invention in the breast cancer cell line MDA-MB-231.
12 is a diagram showing the results of analyzing the cell activity inhibitory effect of the treatment with the compound of the present invention in the hematological cancer cell line Daudi by MTT assay.
13 is a diagram showing the results of analyzing the cell activity inhibitory effect of the treatment with the compound of the present invention in the pancreatic cancer cell line BxPC3 by MTT assay.
14 is a diagram showing the results of analyzing the cell activity inhibitory effect of the compound treatment of the present invention in colorectal cancer cell line HT29 by MTT assay.
15 is a diagram confirming the tumor growth inhibitory effect of treatment with the compound of the present invention by measuring the tumor volume over time after treatment with the compound of the present invention or PBS in mice implanted with the H1975 lung cancer cell line.
16 is a diagram confirming the tumor growth inhibitory effect of the compound of the present invention by measuring the tumor volume over time after treatment with the compound of the present invention or PBS in mice transplanted with the H820 lung cancer cell line.
17 to 23 are diagrams showing the results of measuring the residual amount of each compound according to the present invention over time in human blood.

The present inventors newly synthesized amidation analogs of the oligopeptide AQTGTGKT, confirmed that they exhibit excellent anticancer effects (see Example 2) and excellent stability in blood (see Example 3). The invention was completed. Accordingly, the present invention can provide a compound represented by the following general formula:

[general meal]

X-AQTGTGKT

In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

X is

It is at least one selected from the group consisting of.

As another aspect of the present invention, the present invention can provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the compound represented by the above general formula as an active ingredient.

As another aspect of the present invention, the present invention may provide a method for preventing or treating cancer comprising administering to an individual a compound represented by the above general formula.

As used herein, the term “prevention” refers to any action that blocks, suppresses, or delays symptoms caused by cancer by administering the composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms of cancer are improved or beneficially changed by administration of the composition according to the present invention.

As used herein, the term “subject” refers to a subject in need of prevention or treatment of a disease. For example, the subject can be a human or mammal, including non-human primates, mice, dogs, cats, horses, sheep and cattle.

As used herein, the term “oligopeptide” refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonds. The oligopeptide of the present invention can be prepared according to chemical synthesis methods known in the art (eg, solid-phase synthesis techniques) along with molecular and biological methods (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)).

The scope of the compound according to the present invention may also include a pharmaceutically acceptable salt thereof. As used herein, the term “pharmaceutically acceptable” means that the benefit/risk ratio is reasonable without undue toxicity, irritation, allergic reaction, or other problems or complications, so that it is not suitable for use in contact with the tissues of a subject (eg, a human). Suitable and means a compound within the scope of sound medical judgment. The pharmaceutically acceptable salts include, for example, acid addition salts formed with pharmaceutically acceptable free acids and pharmaceutically acceptable metal salts.

In addition, the scope of the compound according to the present invention may include a biological function equivalent having a mutation in the amino acid sequence that exerts a biological activity equivalent to that of the compound of the present invention. Such amino acid sequence variation can be made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge and size, and the like. According to the analysis of the size, shape and type of amino acid side chain substituents, alanine and glycine have similar sizes; Lysine is a positively charged residue; It can be seen that glutamine and threonine are not charged. Therefore, based on these considerations, alanine and glycine; And glutamine and threonine can be said to be biologically functional equivalents.

In introducing the mutation, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The hydrophobic amino acid index is very important in conferring an interactive biological function of a protein. It is a known fact that amino acids having a similar hydrophobicity index must be substituted to retain similar biological activity. When introducing a mutation with reference to the hydrophobicity index, the substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins having equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced with reference to the hydrophilicity value, the substitution is made between amino acids exhibiting a difference in the hydrophilicity value within preferably ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

Considering the mutation having the above-described biological equivalent activity, the protein of the amino acid sequence (AQTGTGKT) of the compound represented by the general formula of the present invention is interpreted as including a sequence showing substantial identity. The substantial identity is at least when the sequence of the present invention and any other sequences are aligned to correspond to the maximum, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence that exhibits 62.5% homology, more preferably 75% or more homology, and most preferably 87.5% or more homology. Alignment methods for sequence comparison are known in the art.

The pharmaceutical composition of the present invention is used for the prevention or treatment of cancer. Cancers for which the pharmaceutical composition of the present invention can be used may be cancers selected from the group consisting of lung cancer, breast cancer, blood cancer, colorectal cancer, pancreatic cancer, and combinations thereof, but is not limited thereto.

In the present invention, the lung cancer may be non-small cell lung cancer, but is not limited thereto.

In addition, the breast cancer may be triple negative breast cancer, but is not limited thereto.

In addition, the hematologic cancer may be a hematologic cancer selected from the group consisting of leukemia, lymphoma, multiple myeloma, and combinations thereof, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the pharmaceutical composition according to the present invention exhibits excellent anticancer activity against lung cancer, breast cancer, blood cancer, pancreatic cancer and colorectal cancer (see Example 2).

In the present invention, when the pharmaceutical composition is used for the prevention or treatment of lung cancer, it contains a compound represented by the general formula X-AQTGTGKT as an active ingredient, wherein X is

It may be one or more selected from the group consisting of, but is not limited thereto.

일 수 있으나, 이에 제한되는 것은 아니다.In addition, in the present invention, when the pharmaceutical composition is used for the prevention or treatment of breast cancer, it contains a compound represented by the general formula X-AQTGTGKT as an active ingredient, wherein X is

may be, but is not limited thereto.

In addition, in the present invention, when the pharmaceutical composition is used for the prevention or treatment of hematologic cancer, it contains a compound represented by the general formula X-AQTGTGKT as an active ingredient, wherein X is

It may be one or more selected from the group consisting of, but is not limited thereto.

및

로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In addition, in the present invention, when the pharmaceutical composition is used for the prevention or treatment of pancreatic cancer, it contains a compound represented by the general formula X-AQTGTGKT as an active ingredient, wherein X is

and

It may be one or more selected from the group consisting of, but is not limited thereto.

In addition, in the present invention, when the pharmaceutical composition is used for the prevention or treatment of colorectal cancer, it contains a compound represented by the general formula X-AQTGTGKT as an active ingredient, wherein X is

It may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, it was confirmed that the compounds represented by the general formula according to the present invention can exist stably in human blood and have an improved half-life compared to AQTGTGKT (see Example 3). Therefore, the results of the above examples show that the compounds according to the present invention have improved anticancer effect and stability.

인 경우, 인간 혈액 내에서의 반감기는 20분 이상일 수 있으나, 이에 제한되는 것은 아니다. 상기 반감기는 예를 들어 20분 내지 30분, 21분 내지 30분, 22분 내지 30분, 23분 내지 30분, 24분 내지 30분, 25분 내지 30분, 26분 내지 30분, 27분 내지 30분, 28분 내지 30분, 29분 내지 30분, 20분 내지 29분, 21분 내지 29분, 21분 내지 28분, 21분 내지 27분, 21분 내지 26분, 21분 내지 25분, 21분 내지 24분, 21분 내지 23분, 21분 내지 22분, 22분 내지 30분, 22분 내지 28분, 22분 내지 26분, 22분 내지 24분, 23분 내지 30분, 23분 내지 28분, 23분 내지 26분, 23분 내지 24분, 24분 내지 30분, 24분 내지 27분, 25분 내지 30분, 25분 내지 27분, 26분 내지 30분, 26분 내지 28분, 27분 내지 30분, 27분 내지 29분, 28분 내지 30분, 28분 내지 29분 또는 29분 내지 30분 등일 수 있다. 또한, 상기 반감기는 AQTGTGKT의 반감기와 비교하여 1900% 내지 2900% 증가한 것일 수 있으나, 이에 제한되는 것은 아니다. 상기 증가 비율은 예를 들어 1900% 내지 2700%, 2000% 내지 2700%, 2100% 내지 2700%, 2200% 내지 2700%, 2300% 내지 2700%, 2400% 내지 2700%, 2500% 내지 2700%, 2600% 내지 2700%, 1900% 내지 2600%, 2000% 내지 2600%, 2100% 내지 2600%, 2100% 내지 2500%, 2100% 내지 2400%, 2100% 내지 2300%, 2100% 내지 2200%, 2200% 내지 2700%, 2200% 내지 2600%, 2200% 내지 2500%, 2200% 내지 2400%, 2200% 내지 2300%, 2300% 내지 2700%, 2300% 내지 2500%, 2400% 내지 2700%, 2400% 내지 2600%, 또는 2500% 내지 2700% 등일 수 있다.In the compound represented by the general formula X-AQTGTGKT of the present invention, X is

In the case of , the half-life in human blood may be 20 minutes or more, but is not limited thereto. The half-life is, for example, 20 minutes to 30 minutes, 21 minutes to 30 minutes, 22 minutes to 30 minutes, 23 minutes to 30 minutes, 24 minutes to 30 minutes, 25 minutes to 30 minutes, 26 minutes to 30 minutes, 27 minutes. to 30 minutes, 28 to 30 minutes, 29 to 30 minutes, 20 to 29 minutes, 21 to 29 minutes, 21 to 28 minutes, 21 to 27 minutes, 21 to 26 minutes, 21 to 25 minutes, 21 minutes to 24 minutes, 21 minutes to 23 minutes, 21 minutes to 22 minutes, 22 minutes to 30 minutes, 22 minutes to 28 minutes, 22 minutes to 26 minutes, 22 minutes to 24 minutes, 23 minutes to 30 minutes, 23 minutes to 28 minutes, 23 minutes to 26 minutes, 23 minutes to 24 minutes, 24 minutes to 30 minutes, 24 minutes to 27 minutes, 25 minutes to 30 minutes, 25 minutes to 27 minutes, 26 minutes to 30 minutes, 26 minutes to 28 minutes, 27 to 30 minutes, 27 to 29 minutes, 28 to 30 minutes, 28 to 29 minutes, or 29 to 30 minutes, and the like. In addition, the half-life may be 1900% to 2900% increased compared to the half-life of AQTGTGKT, but is not limited thereto. The increase rate is for example 1900% to 2700%, 2000% to 2700%, 2100% to 2700%, 2200% to 2700%, 2300% to 2700%, 2400% to 2700%, 2500% to 2700%, 2600 % to 2700%, 1900% to 2600%, 2000% to 2600%, 2100% to 2600%, 2100% to 2500%, 2100% to 2400%, 2100% to 2300%, 2100% to 2200%, 2200% to 2700%, 2200% to 2600%, 2200% to 2500%, 2200% to 2400%, 2200% to 2300%, 2300% to 2700%, 2300% to 2500%, 2400% to 2700%, 2400% to 2600% , or 2500% to 2700%, and the like.

In addition, in the compound represented by the general formula X-AQTGTGKT of the present invention, X is

로 이루어진 군으로부터 선택된 하나 이상인 경우, 인간 혈액 내에서의 반감기는 45분 이상일 수 있으나, 이에 제한되는 것은 아니다. 상기 반감기는 예를 들어 45분 내지 70분, 46분 내지 70분, 47분 내지 70분, 48분 내지 70분, 49분 내지 70분, 50분 내지 70분, 51분 내지 70분, 53분 내지 70분, 55분 내지 70분, 57분 내지 70분, 59분 내지 70분, 65분 내지 70분, 45분 내지 65분, 50분 내지 65분, 55분 내지 65분, 60분 내지 65분, 45분 내지 60분, 50분 내지 60분, 55분 내지 60분, 45분 내지 55분, 50분 내지 55분, 또는 60분 내지 70분 등일 수 있다. 또한, 상기 반감기는 AQTGTGKT의 반감기와 비교하여 4400% 내지 6900% 증가한 것일 수 있으나, 이에 제한되는 것은 아니다. 상기 증가 비율은 예를 들어 4400% 내지 6900%, 4500% 내지 6900%, 4600% 내지 6900%, 4700% 내지 6900%, 4800% 내지 6900%, 4900% 내지 6900%, 5000% 내지 6900%, 5100% 내지 6900%, 5200% 내지 6900%, 5300% 내지 6900%, 5400% 내지 6900%, 5500% 내지 6900%, 5600% 내지 6900%, 5700% 내지 6900%, 5800% 내지 6900%, 5900% 내지 6900%, 6000% 내지 6900%, 6100% 내지 6900%, 6200% 내지 6900%, 6300% 내지 6900%, 6400% 내지 6900%, 6500% 내지 6900%, 6600% 내지 6900%, 6700% 내지 6900%, 6800% 내지 6900%, 4400% 내지 6500%, 4500% 내지 6500%, 4700% 내지 6500%, 5000% 내지 6500%, 5200% 내지 6500%, 5500% 내지 6500%, 5700% 내지 6500%, 6000% 내지 6500%, 6300% 내지 6500%, 4400% 내지 6000%, 4600% 내지 6000%, 4800% 내지 6000%, 5000% 내지 6000%, 5500% 내지 6000%, 4400% 내지 5500%, 4700% 내지 5500%, 5000% 내지 5500%, 5200% 내지 5500%, 4400% 내지 5000%, 4800% 내지 5000%, 4400% 내지 4700%, 또는 4500% 내지 4700% 등일 수 있다.

In the case of at least one selected from the group consisting of, the half-life in human blood may be 45 minutes or more, but is not limited thereto. The half-life is, for example, 45 minutes to 70 minutes, 46 minutes to 70 minutes, 47 minutes to 70 minutes, 48 minutes to 70 minutes, 49 minutes to 70 minutes, 50 minutes to 70 minutes, 51 minutes to 70 minutes, 53 minutes. to 70 minutes, 55 minutes to 70 minutes, 57 minutes to 70 minutes, 59 minutes to 70 minutes, 65 minutes to 70 minutes, 45 minutes to 65 minutes, 50 minutes to 65 minutes, 55 minutes to 65 minutes, 60 minutes to 65 minutes minutes, 45 minutes to 60 minutes, 50 minutes to 60 minutes, 55 minutes to 60 minutes, 45 minutes to 55 minutes, 50 minutes to 55 minutes, or 60 minutes to 70 minutes, and the like. In addition, the half-life may be increased by 4400% to 6900% compared to the half-life of AQTGTGKT, but is not limited thereto. The increase rate is for example 4400% to 6900%, 4500% to 6900%, 4600% to 6900%, 4700% to 6900%, 4800% to 6900%, 4900% to 6900%, 5000% to 6900%, 5100 % to 6900%, 5200% to 6900%, 5300% to 6900%, 5400% to 6900%, 5500% to 6900%, 5600% to 6900%, 5700% to 6900%, 5800% to 6900%, 5900% to 6900%, 6000% to 6900%, 6100% to 6900%, 6200% to 6900%, 6300% to 6900%, 6400% to 6900%, 6500% to 6900%, 6600% to 6900%, 6700% to 6900% , 6800% to 6900%, 4400% to 6500%, 4500% to 6500%, 4700% to 6500%, 5000% to 6500%, 5200% to 6500%, 5500% to 6500%, 5700% to 6500%, 6000 % to 6500%, 6300% to 6500%, 4400% to 6000%, 4600% to 6000%, 4800% to 6000%, 5000% to 6000%, 5500% to 6000%, 4400% to 5500%, 4700% to 5500%, 5000% to 5500%, 5200% to 5500%, 4400% to 5000%, 4800% to 5000%, 4400% to 4700%, or 4500% to 4700%, and the like.

인 경우, 인간 혈액 내에서의 반감기는 100분 이상일 수 있으나, 이에 제한되는 것은 아니다. 상기 반감기는 예를 들어 100분 내지 150분, 102분 내지 150분, 103분 내지 150분, 104분 내지 150분, 105분 내지 150분, 106분 내지 150분, 107분 내지 150분, 108분 내지 150분, 109분 내지 150분, 110분 내지 150분, 111분 내지 150분, 112분 내지 150분, 115분 내지 150분, 117분 내지 150분, 120분 내지 150분, 123분 내지 150분, 125분 내지 150분, 127분 내지 150분, 130분 내지 150분, 132분 내지 150분, 135분 내지 150분, 137분 내지 150분, 140분 내지 150분, 142분 내지 150분, 145분 내지 150분, 147분 내지 150분, 148분 내지 150분, 100분 내지 140분, 102분 내지 140분, 104분 내지 140분, 106분 내지 140분, 108분 내지 140분, 120분 내지 140분, 125분 내지 140분, 130분 내지 140분, 135분 내지 140분, 100분 내지 130분, 102분 내지 130분, 105 내지 130분, 110분 내지 130분, 115분 내지 130분, 120분 내지 130분, 125분 내지 130분, 100분 내지 120분, 105분 내지 120분, 110분 내지 120분, 115분 내지 120분, 100분 내지 110분, 105분 내지 110분, 또는 100분 내지 105분 등일 수 있다. 또한, 상기 반감기는 AQTGTGKT의 반감기와 비교하여 9900% 내지 14900% 증가한 것일 수 있으나, 이에 제한되는 것은 아니다. 상기 증가 비율은 예를 들어 9900% 내지 14900%, 10000% 내지 14900%, 10100% 내지 14900%, 10200% 내지 14900%, 10300% 내지 14900%, 10400% 내지 14900%, 10500% 내지 14900%, 10700% 내지 14900%, 11000% 내지 14900%, 11500% 내지 14900%, 12000% 내지 14900%, 12500% 내지 14900%, 13000% 내지 14900%, 13500% 내지 14900%, 14000% 내지 14900%, 14500% 내지 14900%, 9900% 내지 12000%, 10000% 내지 12000%, 10200% 내지 12000%, 10500% 내지 12000%, 10700% 내지 12000%, 11000% 내지 12000%, 11500% 내지 12000%, 11700% 내지 12000%, 9900% 내지 11000%, 10000% 내지 11000%, 또는 9900% 내지 10000% 등일 수 있다.In addition, in the compound represented by the general formula X-AQTGTGKT of the present invention, X is

In the case of , the half-life in human blood may be 100 minutes or more, but is not limited thereto. The half-life is, for example, 100 minutes to 150 minutes, 102 minutes to 150 minutes, 103 minutes to 150 minutes, 104 minutes to 150 minutes, 105 minutes to 150 minutes, 106 minutes to 150 minutes, 107 minutes to 150 minutes, 108 minutes. to 150 minutes, 109 to 150 minutes, 110 to 150 minutes, 111 to 150 minutes, 112 to 150 minutes, 115 to 150 minutes, 117 to 150 minutes, 120 to 150 minutes, 123 to 150 minutes minutes, 125 minutes to 150 minutes, 127 minutes to 150 minutes, 130 minutes to 150 minutes, 132 minutes to 150 minutes, 135 minutes to 150 minutes, 137 minutes to 150 minutes, 140 minutes to 150 minutes, 142 minutes to 150 minutes, 145 minutes to 150 minutes, 147 minutes to 150 minutes, 148 minutes to 150 minutes, 100 minutes to 140 minutes, 102 minutes to 140 minutes, 104 minutes to 140 minutes, 106 minutes to 140 minutes, 108 minutes to 140 minutes, 120 minutes to 140 minutes, 125 to 140 minutes, 130 to 140 minutes, 135 to 140 minutes, 100 to 130 minutes, 102 to 130 minutes, 105 to 130 minutes, 110 to 130 minutes, 115 to 130 minutes , 120 minutes to 130 minutes, 125 minutes to 130 minutes, 100 minutes to 120 minutes, 105 minutes to 120 minutes, 110 minutes to 120 minutes, 115 minutes to 120 minutes, 100 minutes to 110 minutes, 105 minutes to 110 minutes, or It may be 100 minutes to 105 minutes, and the like. In addition, the half-life may be increased by 9900% to 14900% compared to the half-life of AQTGTGKT, but is not limited thereto. The increase rate is for example 9900% to 14900%, 10000% to 14900%, 10100% to 14900%, 10200% to 14900%, 10300% to 14900%, 10400% to 14900%, 10500% to 14900%, 10700 % to 14900%, 11000% to 14900%, 11500% to 14900%, 12000% to 14900%, 12500% to 14900%, 13000% to 14900%, 13500% to 14900%, 14000% to 14900%, 14500% to 14900%, 9900% to 12000%, 10000% to 12000%, 10200% to 12000%, 10500% to 12000%, 10700% to 12000%, 11000% to 12000%, 11500% to 12000%, 11700% to 12000% , 9900% to 11000%, 10000% to 11000%, or 9900% to 10000%, and the like.

On the other hand, the pharmaceutical composition according to the present invention may further include an appropriate carrier, excipient and/or diluent commonly used to prepare a pharmaceutical composition in addition to the active ingredient. In addition, according to a conventional method, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, external preparations, suppositories, and sterile injection solutions.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. When formulating the composition, it can be prepared by using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. A preferred dosage may be selected according to the condition and weight of the subject, the degree of disease, the drug form, the route of administration, and the duration. As a specific example, the pharmaceutical composition may be administered in an amount of 0.001 to 1000 mg/kg, 0.01 to 100 mg/kg, 0.01 to 10 mg/kg, 0.1 to 10 mg/kg, or 0.1 to 1 mg/kg once a day to It can be administered in several divided doses.

In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be determined by a person skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, disease type, and drugs used in combination.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, it may be administered by oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The daily dose may be administered once to several times a day.

As another aspect of the present invention, the present invention may provide a method for preparing an oligopeptide X-AQTGTGKT, comprising the steps of:

(The A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,

wherein X is

It is one selected from the group consisting of.)

(1) synthesizing TG and KT, respectively;

(2) synthesizing TGKT by combining the TG and KT;

(3) synthesizing TGTGKT by binding TG to the N-terminus of TGKT;

(4) synthesizing QTGTGKT by binding Q to the N-terminus of TGTGKT; and

(5) synthesizing X-AQTGTGKT by binding an alanine derivative (X-A) to the N-terminus of QTGTGKT.

The terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor may properly define the concept of the term in order to best describe his invention. Based on the principle that there is, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

experimental method

1. CTG (CellTiter-Glo Luminescent) Analysis

After treatment with the AQTGTGKT analog compound according to the present invention to a cancer cell line, the degree of cell proliferation was measured through CTG analysis. Specifically, 5 × 10 ³ /100 μl of cells per well were seeded in a 96-well plate and cultured for 24 hours, followed by transfection with 7 types of AQTGTGKT analogs according to the present invention, respectively. After 72 hours, CellTiter-Glo® (Promega Co., USA) reagent was mixed in the same amount as the cell culture solution and reacted on an orbital shaker for 2 minutes. After reacting at room temperature for 10 minutes, the luminescence signal was measured using a luminometer (GloMax®, Promega).

2. MTT (tetrazolium) analysis

After treatment with the AQTGTGKT analog compound according to the present invention to a cancer cell line, the degree of cell proliferation was measured through MTT analysis. ^{Specifically, 5 × 10 3} /100 μl of cells were dispensed per well in a 96-well plate, and after culturing for 24 hours, 7 types of AQTGTGKT analogs according to the present invention were introduced respectively. After 72 hours, 10 μl of CellTiter-96® (Promega Co., USA) reagent was added to each well and _{reacted at 5% CO 2} , 37°C. After 3 hours, the absorbance was measured at 490 nm using a ^{spectrophotometer (SPECTROstar Nano, BMG).}

3. Lung Cancer Animal Model

H1975 cells were inoculated once at a concentration of 4×10 ⁶ cells/mouse by subcutaneous injection into the flank of the mouse by 150 μl each. After confirming that the volume of the tumor mass formed after completion of inoculation reached 70 to 130 mm 3 , random group separation was performed. The test substance was administered intravenously 5 times at 3-day intervals and 5 times at 2-day intervals.

H820 cells were inoculated once by subcutaneous injection into the flank of mice by 200 μl of matrigel 1:1 at a concentration of 5×10 ^{6 cells/mouse.} After confirming that the volume of the tumor mass formed after completion of inoculation reached 70 to 130 mm 3 , random group separation was performed. The test substance was administered intravenously 14 times daily.

Example 1: Preparation of AQTGTGKT analog

1.1. reaction general

All reactions were performed using commercially available materials and reagents without further reactions unless otherwise noted. Reactions were monitored by thin layer chromatography (TLC) on silica gel plates (Keiselgel 60 F254, Merck) and/or ultra high performance liquid chromatography (UPLC). Visualization of the spots on the TLC plate was achieved by staining the TLC plate by UV light and in potassium permanganate and/or ninhydrin and carbonizing with a heat gun. All products were characterized using ¹ H NMR and/or UPLC-MS.

1.2. Synthesis of Boc/OBn-TG

First, TG with a functional group protected with benzyl was synthesized according to Scheme 1 below. Hereinafter, the compound in each reaction scheme will be referred to as compound n according to the Arabic numerals (n) described below.

[Scheme 1]

Specifically, BocThr(OBn)OH (compound 1; 25.0 g, 80.8 mmol, 1.0 equiv) and NOSu (9.77 g, 84.8 mmol, 1.05 equiv) were dissolved in dichloromethane (150 mL). The mixture was cooled to 0° C. and placed under an inert atmosphere. Then to the mixture was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.3 g, 84.8 mmol, 1.05 equiv). The mixture was warmed to room temperature and stirred for 20 h. The mixture was then _{washed with NH 4} Cl (sat. aq) and the phases were separated. The organic layer was _{dried over MgSO 4} and concentrated under reduced pressure to give compound 2 as a product as a pale yellow oil (35.7 g, >100% yield, assuming quantitative yield).

The above compound 2 BocThr(OBn)OSu (32.8 g, 80.8 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (200 mL) and a solution of glycine sodium salt hydrate in distilled water (100 mL) was added in one portion. After stirring at room temperature for 6 h, the mixture was partitioned between ethyl acetate and citric acid (sat. aq.). The organic layer was _{dried over MgSO 4} , filtered and concentrated under reduced pressure. The crude material was purified on a C18 (400 g) column with 30-70% acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined and partitioned between ethyl acetate and NaHCO ₃ (sat. aq.). The organic layer was _{dried over MgSO 4} , filtered, and then compound 3 was obtained as a product under reduced pressure as a pale yellow gum (21.9 g, 74% yield).

1.3. CBz/OBn/CO ₂ Synthesis of Bn-KT

KT with an OH functional group protected with benzyl was synthesized according to Scheme 2 below.

[Scheme 2]

Specifically, BocLys(CBz)OH (compound 4; 27.0 g, 70.9 mmol, 1.0 equiv) and NOSu (9.80 g, 85.1 mmol, 1.2 equiv) were dissolved in dichloromethane (128 mL). The mixture was cooled to 0° C. and placed under an inert atmosphere. Then, to the mixture was added 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (16.3 g, 85.1 mmol, 1.05 equiv). The mixture was warmed to room temperature and stirred for 20 h. The mixture was _{then washed with NH 4} Cl (sat. aq.) and the phases were separated. The organic layer was _{dried over MgSO 4} and concentrated under reduced pressure to give compound 5 as a product as a pale yellow oil (36.7 g, >100% yield, assuming quantitative yield).

The compound 5 (BocLys(Cbz)OSu; 36.7 g, 70.9 mmol, 1.0 equiv) and Thr(OBn)OBn.HCl (25.0 g, 74.4 mmol, 1.05 equiv) were then mixed with 1,4-dioxane (477) at room temperature. mL) was dissolved. To this solution was added a solution of NaHCO3 (6.85 g, 81.5 mmol, 1.15 equiv) in distilled water (326 mL). The resulting mixture was then stirred at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate and washed with 10% citric acid (aq) and brine. The organic layer was _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure to give 55.9 g of a yellow oily solid (>100% yield, assuming quantitative yield) of compound 6 as a product.

Finally, the compound 6 (BocLys(Cbz)Thr(OBn)OBn; 55.9 g, 70.9 mmol, 1.00 equiv) was dissolved in 1,4-dioxane (360 mL) and 1,4-dioxane (177 mL) 4N HCl in heavy was added. The mixture was stirred at room temperature overnight. Then, _{a saturated aqueous solution of NaHCO 3} was added until the pH value reached 8. The solution was extracted with ethyl acetate, and the resulting organic solution was _{dried over Na 2} SO ₄ , filtered, and then concentrated under reduced pressure to obtain compound 7 as a product as a yellow oily solid (38.7 g, 97% yield).

1.4. CBz/OBn/OBn/CO ₂ Synthesis of Bn-TGKT

1.2. above. and TGKT was synthesized by combining TG and KT synthesized in 1.3. according to Scheme 3 below.

[Scheme 3]

More specifically, BocThr(OBn)GlyOH (compound 3; 5.61 g, 15.3 mmol, 1.00 equiv) and compound 8 (Lys(Cbz)Thr(OBn)OBn; 10.0 g, 15.3 mmol; 1.0 equiv) solution, N,N-diisopropylethylamine (5.90 mL, 33.7 mmol, 2.2 equiv) was added. The mixture was stirred at room temperature under an inert atmosphere and HATU (7.00 g, 18.4 mmol, 1.20 equiv) was added. The resulting mixture was stirred for 2 h _{, then washed with NH 4} Cl (sat. aq.) followed by NaHCO ₃ (sat. aq.). The organic layer was _{dried over Na 2} SO ₄ , filtered and concentrated under reduced pressure to give compound 9 as a product as a pale orange oily solid (25.0 g, >100% yield, assuming quantitative yield).

The obtained BocThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (Compound 9; 13.9 g, 15.3 mmol, 1.0 equiv from the previous step) was dissolved in 1,4-dioxane (150 mL) under nitrogen at room temperature made it To this solution was added 4N HCl in 1,4-dioxane (20 mL). The mixture was stirred at room temperature for 20 hours. The mixture was concentrated under reduced pressure and purified on a C18 (400 g) column using 20% acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined and lyophilized. The resulting powder was _{dissolved in NaHCO 3} (sat. aq.) and dichloromethane and stirred for 15 min. The layers were separated and the organic layer was dried over Na ₂ SO ₄ , filtered, and then concentrated under reduced pressure to give compound 10 as a product as a colorless gum (10.9 g, 88% yield).

1.5. CBz/OBn/OBn/OBn/CO ₂ Synthesis of Bn-TGTGKT

1.2. above. and TG (Compound 3) and TGKT (Compound 10) synthesized in 1.4. were combined according to Scheme 4 below to synthesize benzyl-protected TGTGKT.

[Scheme 4]

More specifically, BocThr(OBn)GlyOH (compound 3; 5.10 g, 14.0 mmol, 1.05 equiv) and BocThr(OBn)GlyLys(Cbz)Thr(OBn) OBn (compound 10; 10.8 g, 13.3 mmol, 1.0 equiv) was added N,N -diisopropylethyl amine (5.10 mL, 29.3 mmol, 2.2 equiv). The mixture was stirred at room temperature under an inert atmosphere and HATU (5.60 g, 14.7 mmol, 1.1 equiv) was added. The resulting mixture was stirred for 2 h _{, washed with NH 4} Cl (sat. aq.) and NaHCO ₃ (sat. aq.). The organic layer was concentrated under reduced pressure to obtain compound 11 as a product as a pale yellow gum (21.4 g, >100% yield, assuming quantitative yield).

Then, the obtained compound 11 (BocThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr (OBn)OBn; 15.4 g, 13.3 mmol, 1.00 equiv from the previous step) was mixed with 1,4-dioxane at room temperature under nitrogen (150 mL). To this solution was added 4N HCl in 1,4-dioxane (50 mL) and the mixture was stirred at room temperature for 5 hours. The mixture was concentrated under reduced pressure and purified on a C18 (120 g) column using 20% acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined, concentrated to half volume _{and partitioned between NaHCO 3} (sat. aq.) and ethyl acetate. The layers were separated and the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound 12 as a product as an off-white solid (14.6 g, >100% yield, assuming quantitative yield).

1.6. CBz/OBn/OBn/OBn/CO ₂ Synthesis of Bn-QTGTGKT

Compound 14 (QTGTGKT) was synthesized by further binding Q according to Scheme 5 below to compound 12 (TGTGKT) synthesized in 1.5.

[Scheme 5]

More specifically, Thr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (Compound 12; 12.7 g, 12.0 mmol) in ethyl acetate (150 mL) and N,N-dimethylformamide (25 mL) , 1.0 equiv) and BocGlnOH (3.25 g, 13.2 mmol, 1.1 equiv) was added N,N-diisopropylethylamine (4.60 mL, 26.4 mmol, 2.2 equiv). The mixture was stirred at room temperature under an inert atmosphere and HATU (5.47 g, 14.4 mmol, 1.20 equiv) was added. The resulting mixture was stirred for 1 h and then _{washed with NH 4} Cl (sat. aq.). The organic layer was further extracted with dichloromethane. The organic layers were then combined and concentrated under reduced pressure. The crude material was purified by 400 g C18 column using a gradient of 20-100% acetonitrile (0.1% formic acid) in water (0.1% formic acid). After the desired fractions were combined, partitioned with ethyl acetate and NaHCO ₃ (sat. aq.) solution. The organic layer was concentrated, and residual water was removed by a freeze-drying process. The combined fractions gave a total of 12.9 g (89% total yield) of compound 13.

The obtained compound 13 (BocGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn; 7.00 g, 5.40 mmol, 1.00 equiv) was dissolved in 1,4-dioxane (150 mL) under nitrogen at room temperature. made it To this solution was added 4N HCl in 1,4-dioxane (43.5 mL). The mixture was stirred at room temperature for 20 h. The mixture was concentrated under reduced pressure and freeze-dried using a water/acetonitrile (2/1) solution. Finally, compound 14 as a pale yellow powder (6.36 g, 96% yield) was isolated.

1.7. Synthesis of AQTGTGKT analogs

The remaining 6 analogs except for 4-PhPh-AQTGTGKT were synthesized by combining compound 14, a final product of Scheme 5, and compound 17n, a product of Scheme 6, according to Scheme 7 below.

[Scheme 6]

[Scheme 7]

According to Scheme 7, 2.9 mg of 3-PhPh-AQTGTGKT with a purity of 90% or more, 7.0 mg of 4-MeOPh-AQTGTGKT with a purity of 89%, 22.7 mg of 2-PhPh-AQTGTGKT with a purity of 95% or more, 23.2 mg of Ph-AQTGTGKT with a purity of 90% or more according to Scheme 7 , and 23.2 mg of Naphthyl-AQTGTGKT having a purity of 85% were finally obtained.

Hereinafter, each analog synthesis process will be described in detail.

1.7.1. Synthesis of 3-PhPh-AQTGTGKT

3-PhPh-AQTGTGKT (Compound 19-1) was synthesized by reacting Compound 17-1 obtained according to Scheme 8 with Compound 14 according to Scheme 9 to synthesize a final target compound, 3-PhPh-AQTGTGKT.

[Scheme 8]

More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv) and [1,1'-biphenyl]-3-carboxylic acid (297 mg, 1.50 mmol, 1 equiv) were added and the mixture was brought to room temperature was stirred for 2 hours. The reaction mixture was diluted with ethyl acetate and then washed with NH ₄ Cl (sat. aq.), NaHCO3 (sat. aq.) and brine. The obtained organics were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The residue was purified by a gradient of 2-40% ethyl acetate in heptane on a 25 g column to give compound 16-1 as a colorless solid (493 mg, 91% yield).

Then 10% Pd/C (49 mg) moistened with a minimum amount of water was added to a solution of 16-1 (3-PhPh-AlaOBn; 493 mg, 1.37 mmol) in methanol (30 mL). The mixture was stirred for 2 h under a hydrogen atmosphere (balloon). The mixture was filtered through a pad of celite and washed with methanol and ethyl acetate. The obtained filtrate was concentrated under reduced pressure to obtain compound 17-1 as a colorless foam (337 mg, 91% yield), which was reacted with compound 14 according to Scheme 9 below.

[Scheme 9]

More specifically, GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in N,N-diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 equiv) and dichloromethane (20 mL) To a suspension of (compound 14; 300 mg, 0.253 mmol, 1 equiv) and 3-PhPh-AlaOH (compound 17-1; 68.0 mg, 0.253 mmol, 1 equiv) was added HATU (106 mg, 0.278 mmol, 1.1 equiv) . The mixture was stirred at room temperature for 2 h, and the reaction mixture _{was washed with NaHCO 3} (sat. aq.). The organics were concentrated under reduced pressure and the residue was purified by a 60 g C18 column with a gradient of 40-100% acetonitrile (0.1% formic acid) in water (0.1% formic acid) after freeze-drying as a colorless solid (140 mg, 38% yield) ) of compound 18-1 was obtained.

Then 10% Pd/C (85.0 mg) was added to 3-PhPh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in 2M hydrochloric acid (aq., 0.5 mL) and 2-propanol (10 mL). (Compound 18-1; 85.0 mg, 59.1 μmol) was added to the solution. After the mixture was stirred under a hydrogen atmosphere (balloon) for 4.5 hours, the mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, and the residue was freeze-dried. Then the material was purified on a 60 g C18 column using 5-50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give the desired compound as a colorless solid (2.9 mg, 5% yield) 19-1 was obtained.

¹ H NMR data of compound 19-1 (3-PhPh-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 8.01-7.99 (m, 1H), 7.86-7.82 (m, 1H), 7.75-7.66 (m, 3H), 7.55 (t, J 7.7 Hz, 1H), 7.49 (t, J 7.5 Hz, 2H), 7.43-7.38 (m, 1H), 4.48-4.24 (m, 5H), 4.23-4.12 (m, 3H), 4.09 (d, J 3.8 Hz, 1H), 4.00 - 3.96 (m, 2H), 3.88 (s, 2H), 2.90 (t, J 7.4Hz, 2H), 2.35 (t, J 7.5Hz, 2H), 2.15-2.06 (m, 1H), 2.03-1.91 ( m, 1H), 1.84-1.72 (m, 1H), 1.70-1.52 (m, 3H), 1.45 (d, J 7.2Hz, 3H), 1.40-1.24 (m, 2H), 1.15-1.05 (m, 9H) ) , 16 exchangeable protons not visible.

1.7.2. Synthesis of 4-MeOPh-AQTGTGKT

For 4-MeOPh-AQTGTGKT (Compound 19-2), compound 17-2 obtained according to Scheme 10 was reacted with compound 14 according to Scheme 11 to synthesize a final target compound, 4-MeOPh-AQTGTGKT.

[Scheme 10]

More specifically, H-Ala-OBzl.HCl (425 mg, 1.97 mmol, 1.2 equiv) was suspended in ethyl acetate (10 mL) and N,N -diisopropylethylamine (715 µL, 4.11 mmol, 2.5 equiv) was added. After stirring at room temperature for 5 minutes, HATU (937 mg, 2.46 mmol, 1.5 equiv) and 4-methoxybenzoic acid (250 mg, 1.64 mmol, 1 equiv) were added and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and brine. The organics were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The residue was purified on a 25 g column with a gradient of 15-50% ethyl acetate in heptane to give compound 16-2 as a colorless solid (360 mg, 70% yield).

Then 10% Pd/C (18 mg) moistened with a minimum amount of water was added to a solution of 4-OMePh-AlaOBn (compound 16-2; 180 mg, 0.574 mmol) in methanol (15 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 110 h. The mixture was then filtered through a pad of celite and washed with methanol. The obtained filtrate was concentrated under reduced pressure to obtain compound 17-2 as a colorless oil (128 mg, 100% yield), which was reacted with compound 14 according to Scheme 11 below.

[Scheme 11]

More specifically, GlnThr(OBn)GlyThr(OBn)GlyLys (Cbz)Thr(OBn)OBn in N,N-diisopropylethylamine (63.0 μL, 0.360 mmol, 2.2 equiv) and dichloromethane (20 mL) To a suspension of (compound 14; 200 mg, 0.164 mmol, 1 equiv) and 4-OMePh-AlaOH (compound 17-2; 36.6 mg, 0.164 mmol, 1 equiv) was added HATU (74.5 mg, 0.196 mmol, 1.2 equiv) did The mixture was stirred at room temperature for 64 h, the reaction mixture was diluted with methanol and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and water. The organics were concentrated under reduced pressure and the residue was purified on a 60 g C18 column with a gradient of 50-95% acetonitrile (0.1% formic acid) in water (0.1% formic acid), lyophilized to a colorless solid (113 mg, 50% yield) ) of compound 18-2 was obtained.

10% Pd/C (100 mg) was then mixed with 4-OMePh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in 2M hydrochloric acid (aq., 1.0 mL) and 2-propanol (20 mL). (compound 18-2; 108 mg, 77.6 μmol) was added. The mixture was stirred for 18 h under a hydrogen atmosphere (balloon). The mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, and the residue was dissolved in water and then freeze-dried. The dried material was purified on a 30 g C18 column using a gradient of 5-30% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give the compound as a colorless solid (7.0 mg, 10% yield) 19-2 was obtained.

¹ H NMR data of compound 19-2 (4-MeOPh-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 7.76-7.71 (m, 2H), 7.02-6.98 (m, 2H), 4.42-4.28 (m, 5H), 4.24-4.13 (m, 3H), 4.09 ( d, J 3.9 Hz, 1H), 4.01-3.96 (m, 2H), 3.89 (s, 2H), 3.81 (s, 3H), 2.91 (t, J 7.4 Hz, 2H), 2.34 (t, J 7.6 Hz) , 2H), 2.14-2.06 (m, 1H), 2.00-1.91 (m, 1H), 1.85-1.75 (m, 1H), 1.71-1.54 (m, 3H), 1.42 (d, J 7.2 Hz, 3H) , 1.40-1.25 (m, 3H), 1.16-1.07 (m, 8H), 16 exchangeable protons not visible.

1.7.3. Synthesis of 2-PhPh-AQTGTGKT

2-PhPh-AQTGTGKT (Compound 19-3) was prepared by reacting Compound 17-3 obtained according to Scheme 12 with Compound 14 according to Scheme 13 to synthesize a final target compound, 2-PhPh-AQTGTGKT.

[Scheme 12]

More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv) was suspended in ethyl acetate (10 mL) and N,N -diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv) and [1,1'-biphenyl]-2-carboxylic acid (297 mg, 1.50 mmol, 1 equiv) were added and the mixture was stirred at room temperature. was stirred for 2 hours. The reaction mixture was diluted with ethyl acetate and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and brine. The obtained organics were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The residue was purified on a 25 g column with a gradient of 2-40% ethyl acetate in heptane to give compound 16-3 as a colorless oil (416 mg, 77% yield).

Then 10% Pd/C (42 mg) moistened with a minimum amount of water was added to a solution of 2-PhPh-AlaOBn (compound 16-3; 416 mg, 1.16 mmol) in methanol (20 mL). The mixture was stirred for 18 h under a hydrogen atmosphere (balloon). The mixture was then filtered through a pad of celite and washed with methanol. The obtained filtrate was concentrated under reduced pressure to obtain compound 17-3 as a colorless oil (308 mg, 99% yield), which was reacted with compound 14 according to Scheme 13 below.

[Scheme 13]

More specifically, GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in N,N-diisopropylethylamine (63.0 μL, 0.360 mmol, 2.2 equiv) and dichloromethane (20 mL) To a suspension of (compound 14; 200 mg, 0.164 mmol, 1 equiv) and 2-PhPh-AlaOH (compound 17-3; 44.2 mg, 0.164 mmol, 1 equiv) was added HATU (74.5 mg, 0.196 mmol, 1.2 equiv) did The mixture was stirred at room temperature for 64 h, the reaction mixture was diluted with methanol and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and water. The organic layer was concentrated under reduced pressure and the residue was purified on a 60 g C18 column with a gradient of 50-95% acetonitrile (0.1% formic acid) in water (0.1% formic acid), lyophilized to a colorless solid (115 mg, 49% Yield) of compound 18-3 was obtained.

10% Pd/C (100 mg) was then mixed with 2-PhPh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in 2M hydrochloric acid (aq., 1.0 mL) and 2-propanol (20 mL) (Compound 18-3; 110 mg, 76.5 μmol) was added to the solution. The mixture was stirred for 18 h under a hydrogen atmosphere (balloon). The mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, and the residue was dissolved in water and then freeze-dried. The dried material was purified on a 30 g C18 column with a gradient of 5-50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give compound 19 as a colorless solid (22.7 mg, 31% yield) -3 was obtained.

¹ H NMR data of compound 19-3 (2-PhPh-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 7.56-7.48 (m, 2H), 7.44-7.33 (m, 7H), 4.38-4.26 (m, 4H), 4.25-4.13 (m, 4H), 4.08 ( d, J 3.9 Hz, 1H), 4.00-3.96 (m, 2H), 3.89 (s, 2H), 2.91 (t, J 7.5 Hz, 2H), 2.25 (t, J 7.5 Hz, 2H), 2.09-2.01 (m, 1H), 1.93-1.77 (m, 2H), 1.72-1.56 (m, 3H), 1.41-1.29 (m, 2H), 1.18 (d, J 7.2 Hz, 3H), 1.12 (d, J 5.6) Hz, 6H), 1.08 (d, J 6.4 Hz, 3H), 16 exchangeable protons not visible.

1.7.4. Synthesis of Ph-AQTGTGKT

Ph-AQTGTGKT (Compound 19-4) was synthesized by reacting Compound 17-4 obtained according to Scheme 14 with Compound 14 according to Scheme 15 to synthesize a final target compound, Ph-AQTGTGKT.

[Scheme 14]

More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv) was suspended in ethyl acetate (10 mL) and N,N -diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv) was added. After stirring at room temperature for 5 minutes, HATU (855 mg, 2.25 mmol, 1.5 equiv) and benzoic acid (183 mg, 1.50 mmol, 1 equiv) were added and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ethyl acetate and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and brine. The obtained organics were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The residue was purified on a 25 g column with a gradient of 2-50% ethyl acetate in heptane to give compound 16-4 as a colorless oil (375 mg, 88% yield).

Then 10% Pd/C (38 mg) moistened with a minimum amount of water was added to a solution of Ph-Ala-OBn (compound 16-4; 375 mg, 1.32 mmol) in methanol (30 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 4 hours. The mixture was then filtered through a pad of celite and washed with methanol. The obtained filtrate was concentrated under reduced pressure to obtain compound 17-4 as a colorless glass (glass; 254 mg, 99% yield), which was reacted with compound 14 according to Scheme 15 below.

[Scheme 15]

More specifically, N, N - diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 eq.) And GlnThr (OBn) GlyThr (OBn) GlyLys (Cbz) Thr (OBn) OBn in dichloromethane (20 mL) ( To a suspension of compound 14; 300 mg, 0.253 mmol, 1 equiv) and Ph-Ala-OH (compound 17-4; 49.0 mg, 0.253 mmol, 1) was added HATU (106 mg, 0.278 mmol, 1.1 equiv). The mixture was stirred at room temperature for 2 h, and the reaction mixture _{was washed with NaHCO 3} (sat. aq.). The organics were concentrated under reduced pressure and the obtained residue was purified on a 60 g C18 column with a gradient of 40-100% acetonitrile (0.1% formic acid) in water (0.1% formic acid), freeze-dried, and then a colorless solid (200 mg, 58%) of compound 18-4 was obtained.

10% Pd/C (110 mg) was then mixed with Ph-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound) in 2M hydrochloric acid (aq., 1.0 mL) and 2-propanol (20 mL) 18-4; 110 mg, 80.8 μmol) solution. The mixture was stirred for 18 h under a hydrogen atmosphere (balloon). The mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, and the residue was dissolved in water and then freeze-dried. The dried material was purified on a 60 g C18 column using a gradient of 5-50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give a colorless solid (23.2 mg, 33% yield) Compound 19-4 was obtained.

¹ H NMR data of compound 19-4 (Ph-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 7.74-7.70 (m, 2H), 7.58-7.53 (m, 1H), 7.48-7.43 (m, 2H), 4.45-4.34 (m, 3H), 4.32- 4.27 (m, 2H), 4.25-4.14 (m, 3H), 4.11 (d, J 3.8 Hz, 1H), 4.00-3.96 (m, 2H), 3.89 (s, 2H), 2.91 (t, J 7.4 Hz) , 2H), 2.34 (t, J 7.6 Hz, 2H), 2.14-2.06 (m, 1H), 2.01-1.91 (m, 1H), 1.85-1.76 (m, 1H), 1.71-1.56 (m, 3H) , 1.43 (d, J 7.3 Hz, 3H), 1.40-1.30 (m, 2H), 1.16-1.07 (m, 9H), 16 exchangeable protons not visible.

1.7.5. Synthesis of Naphthyl-AQTGTGKT

Naphthyl-AQTGTGKT (Compound 19-5) was synthesized by reacting Compound 17-5 obtained according to Scheme 16 with Compound 14 according to Scheme 17 to synthesize the final target compound, Naphthyl-AQTGTGKT.

[Scheme 16]

More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv) was suspended in ethyl acetate (10 mL) and N,N -diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv) and 2-naphthoic acid (258 mg, 1.50 mmol, 1 equiv) were added and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and then washed with NH ₄ Cl (sat. aq.), NaHCO ₃ (sat. aq.) and brine. The obtained organics were dried (Na ₂ SO ₄ ), filtered and concentrated under reduced pressure. The residue was purified on a 25 g column with a gradient of 2-40% ethyl acetate in heptane to give compound 16-5 as a colorless solid (385 mg, 77% yield).

Then 10% Pd/C (39 mg) moistened with a minimum amount of water was added to a solution of 2-Naphthyl-AlaOBn (compound 16-5; 385 mg, 1.15 mmol) in methanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 4 h. The mixture was then filtered through a pad of celite and washed with methanol. The obtained filtrate was concentrated under reduced pressure to obtain compound 17-5 as a colorless solid (266 mg, 95% yield), which was reacted with compound 14 according to Scheme 17 below.

[Scheme 17]

More specifically, GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in N,N-diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 equiv) and dichloromethane (20 mL) To a suspension of (compound 14; 300 mg, 0.253 mmol, 1 equiv) and 2-Naphthyl-Ala-OH (compound 17-5; 61.0 mg, 0.253 mmol, 1 equiv) HATU (106 mg, 0.278 mmol, 1.1 equiv) was added added. The mixture was stirred at room temperature for 2 h, then washed with _{(sat. aq.) NaHCO 3 .} The obtained organic layer was concentrated under reduced pressure and the residue was purified on a 60 g C18 column with a gradient of 40-100% acetonitrile (0.1% formic acid) in water (0.1% formic acid), freeze-dried and then a colorless solid (230 mg, 64% yield) of compound 18-5 was obtained.

10% Pd/C (101 mg) was then mixed with 2-Naphthyl-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in 2M hydrochloric acid (aq., 1.0 mL) and 2-propanol (20 mL). (compound 18-5; 101 mg, 71.5 μmol) was added to the solution. The mixture was stirred under a hydrogen atmosphere (balloon) for 3 hours. The mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, and the residue was dissolved in water and then freeze-dried. The dried material was purified on a 30 g C18 column with a gradient of 5-40% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give compound 19 as a colorless solid (23.2 mg, 33% yield) -5 was obtained.

¹ H NMR data of compound 19-5 (Naphthyl-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 8.32 (s, 1H), 8.01-7.90 (m, 3H), 7.79-7.73 (m, 1H), 7.64-7.55 (m, 2H), 4.51-3.70 ( m, 13H), 2.96-2.81 (m, 2H), 2.39-2.30 (m, 2H), 2.18-2.04 (m, 1H), 2.03-1.93 (m, 1H), 1.85-1.72 (m, 1H), 1.71-1.53 (m, 3H), 1.50-1.43 (m, 3H), 1.39-1.24 (m, 2H), 1.19-1.04 (m, 9H), 16 exchangeable protons not visible.

1.8. Synthesis of Ac-AQTGTGKT

Ac-AQTGTGKT (Compound 19-6) was performed according to Scheme 18 below to synthesize Ac-AQTGTGKT, the final target compound.

[Scheme 18]

More specifically, GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn in N,N-diisopropylethylamine (94.0 μL, 0.540 mmol, 2.2 equiv) and dichloromethane (30 mL) To a suspension of (compound 14; 300 mg, 0.245 mmol, 1 equiv) and Ac-Ala-OH (32.1 mg, 0.245 mmol, 1 equiv) was added HATU (112 mg, 0.294 mmol, 1.2 equiv). The mixture was stirred at room temperature for 14 h, and the reaction mixture was _{washed with (sat. aq.) NH 4} Cl, NaHCO ₃ and water. The organic layer was concentrated under reduced pressure and the residue was purified by 60 g C18 column in a gradient of 50-95% acetonitrile (0.1% formic acid) in water (0.1% formic acid). The desired fractions were combined and lyophilized to give compound 18-6 as a colorless solid (104 mg, 33% yield).

10% Pd/C (10 mg) was then mixed with Ac-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound) in 2M hydrochloric acid (aq., 0.19 mL) and 2-propanol (5 mL) 18-6; 33 mg, 25 μmol) solution. The mixture was stirred under a hydrogen atmosphere (balloon) for 14 h. The mixture was filtered through a 0.45 μm syringe filter. The obtained filtrate was concentrated under reduced pressure, dissolved in water, and then freeze-dried. The dried material was purified on a 500 mg SCX-2 cartridge eluting with 0.5M ammonia in methanol. The desired fractions were combined, concentrated under reduced pressure, and then freeze-dried to obtain compound 19-6 as a colorless solid (7.6 mg, 37% yield).

¹ H NMR data of compound 19-6 (Ac-AQTGTGKT) were determined as follows:

¹ H NMR (400 MHz; DO): δ = 4.40-4.33 (m, 2H), 4.32-4.27 (m, 2H), 4.24-4.12 (m, 4H), 4.08 (d, J 4.0 Hz, 1H), 4.03-3.88 (m, 4H), 2.92 (t, J 7.2 Hz, 2H), 2.32 (t, J 7.8 Hz, 2H), 2.15-2.02 (m, 1H), 1.99-1.88 (m, 4H), 1.86 -1.76 (m, 1H), 1.74-1.55 (m, 3H), 1.45-1.31 (m, 2H), 1.29 (t, J 7.4 Hz, 2H), 1.20-1.06 (m, 10H), 16 exchangeable protons not visible.

1.9. Synthesis of 4-PhPh-AQTGTGKT

1.9.1. Synthesis of QTGTGKT intermediate

Intermediate QTGTGKT for the synthesis of 4-PhPh-AQTGTGKT was synthesized according to the following Schemes 19 to 23:

[Scheme 19]

[Scheme 20]

[Scheme 21]

[Scheme 22]

[Scheme 23]

Schemes 19 to 23 are almost similar to Schemes 1 to 5 except for the presence or absence of a benzyl protecting group, and overlapping descriptions will be omitted.

1.9.2. Synthesis of 4-PhPh-AQTGTGKT

Compound 31 obtained in Scheme 23 was combined with an alanine derivative synthesized in Scheme 24 according to Scheme 25 to obtain a final product 4-PhPh-AQTGTGKT.

[Scheme 24]

[Scheme 25]

Scheme 24 and Scheme 25 also proceeded by a process substantially similar to the above-described scheme, and thus overlapping descriptions will be omitted. Finally, compound 36 was obtained as a colorless solid (583 mg, 68% yield).

¹ H NMR data of compound 36 (4-PhPh-AQTGTGKT) was determined as follows:

¹ H NMR (400 MHz; DO): δ = 7.85 (d, J 8.8 Hz, 2H), 7.77 (d, J 8.8 Hz, 2H), 7.70 (d, J 7.2 Hz, 2H), 7.49 (t, J) 7.2 Hz, 2H), 7.42 (t, J 7.1 Hz, 1H), 4.34-4.04 (m, 6H), 4.07 (d, J 3.6 Hz, 1H), 3.93 (d, J 2.0 Hz, 2H), 2.82 ( t, J 7.0 Hz, 2H), 1.82-1.67 (m, 1H), 1.66 1.55 (m, 1H), 1.54-1.44 (m, 2H), 1.37-1.22 (m, 2H), 1.18 (d, J 6.4 Hz, 3H), 1.06 (t, J 6.5 Hz, 3H), 10 exchangeable protons not visible.

1.10. compound identification

Seven AQTGTGKT analogs obtained by the above method were analyzed by ¹ H NMR and UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) to confirm their chemical structures. The analysis results are shown in FIGS. 1 to 7, and the compounds are summarized in Table 1.

name chemical formula AQTGTGKT

4-PhPh-AQTGTGKT

Ac-AQTGTGKT

3-PhPh-AQTGTGKT

4-MeOPh-AQTGTGKT

2-PhPh-AQTGTGKT

Ph-AQTGTGKT

Naphthyl-AQTGTGKT

Example 2: Confirmation of anticancer effect of AQTGTGKT analog

2.1. Lung cancer cell activity inhibitory effect

According to the CTG analysis method described in the above experimental method, seven types of amidation AQTGTGKT analogs were treated in lung cancer cell line H1299 at a concentration of 500 μM for 72 hours, or AQTGTGKT analogs were treated in lung cancer cell line H820 at a concentration of 100 μM for 72 hours. The cytotoxicity of the analogs was compared after treatment for 72 hours, or AQTGTGKT analogs in H1975 at a concentration of 10 μM, respectively.

As a result, as shown in FIG. 8, when 4-PhPh-AQTGTGKT was treated, the cell activity was reduced by about 21% compared to the control, Ac-AQTGTGKT was 15%, 3-PhPh-AQTGTGKT was 25% , 4-MeOPh-AQTGTGKT showed an inhibitory effect of 23%, 2-PhPh-AQTGTGKT 30%, Ph-AQTGTGKT 28%, and Naphthyl-AQTGTGKT 24%.

In the case of the H820 cell line, as shown in FIG. 9 , when 4-PhPh-AQTGTGKT was treated, the cell activity was reduced by about 20% compared to the control, Ac-AQTGTGKT was 22%, 3-PhPh-AQTGTGKT was 34%, 4-MeOPh-AQTGTGKT was 32%, 2-PhPh-AQTGTGKT was 123%, Ph-AQTGTGKT was 20%, and Naphthyl-AQTGTGKT was 24%.

In addition, in the case of the H1975 cell line, as shown in FIG. 10 , when 4-PhPh-AQTGTGKT was treated, the cell activity was decreased by about 4% compared to the control, Ac-AQTGTGKT was 19%, 3-PhPh -AQTGTGKT was 36%, 4-MeOPh-AQTGTGKT was 8%, 2-PhPh-AQTGTGKT was 7%, Ph-AQTGTGKT was 4%, and Naphthyl-AQTGTGKT was 4%.

The above results show that the amidation analog can very effectively inhibit the division of lung cancer cells.

2.2. Inhibitory effect on breast cancer cell activity

According to the CTG analysis method described in the above experimental method, the breast cancer cell line MDA-MB-231 was treated with an AQTGTGKT analog at a concentration of 100 μM for 48 hours, and then the cell activity was compared by measuring the amount of ATP in the cells.

As a result, as shown in FIG. 11 , when 4-PhPh-AQTGTGKT was treated, the cell activity was reduced by about 36% compared to the control group, and when Ac-AQTGTGKT was treated, it showed a reduction effect of about 6%. It has been demonstrated that it has an excellent effect in inhibiting cleavage.

2.3. Inhibitory effect on growth and proliferation of blood cancer cells

According to the MTT analysis method described in the above experimental method, the blood cancer cell line Daudi was treated with an AQTGTGKT analog at a concentration of 10 μM for 72 hours to compare cytotoxicity.

As a result, as shown in FIG. 12 , when 4-PhPh-AQTGTGKT was treated, the cell activity was decreased by about 17% compared to the control, 4-MeOPh-AQTGTGKT was 22%, 2-PhPh-AQTGTGKT showed a cell activity inhibition effect of 22%, Ph-AQTGTGKT 18%, and Naphthyl-AQTGTGKT 19%. The above results show that all of the seven types of AQTGTGKT analogs according to the present invention have excellent anticancer effects on blood cancer cells.

2.4. Inhibitory effect on growth and proliferation of pancreatic cancer cells

According to the MTT analysis method described in the above experimental method, the pancreatic cancer cell line BxPC3 was treated with an AQTGTGKT analog at a concentration of 100 μM for 72 hours to compare cytotoxicity.

As a result, as shown in FIG. 13, when 4-PhPh-AQTGTGKT was treated, the cell activity was decreased by about 10% compared to the control, 3-PhPh-AQTGTGKT was 1%, 4-MeOPh-AQTGTGKT was 5%, 2-PhPh-AQTGTGKT and Ph-AQTGTGKT showed 7% cell activity inhibitory effect. The above results show that there is an excellent anticancer effect on pancreatic cancer cells.

2.5. Inhibitory effect on growth and proliferation of colorectal cancer cells

According to the MTT assay method described in the above experimental method, the colorectal cancer cell line HT29 was treated with an AQTGTGKT analog at a concentration of 50 μM for 72 hours to compare cytotoxicity.

As a result, as shown in FIG. 14 , when 4-PhPh-AQTGTGKT was treated, the cell activity was decreased by about 28% compared to the control, Ac-AQTGTGKT was 68%, 3-PhPh-AQTGTGKT was 69%. , 4-MeOPh-AQTGTGKT showed a cell activity inhibition effect of 60%, 2-PhPh-AQTGTGKT 28%, Ph-AQTGTGKT 54%, and Naphthyl-AQTGTGKT 68%. The above results show that all of the seven types of AQTGTGKT analogs according to the present invention have excellent anticancer effects against colorectal cancer cells.

2.6. Inhibitory effect on tumor growth in an animal model inoculated with lung cancer cells

According to the animal test analysis method described in the above experimental method, nude mice transplanted with lung cancer cell line H1975 lung cancer cells were treated with an AQTGTGKT analog at a dose of 10 mpk 5 times at 3 day intervals and 5 times at 2 day intervals for a total of 10 times to treat tumors. Growth was compared.

As a result, as shown in FIG. 15 , when the H1975 lung cancer cell line was transplanted and then treated with 3-PhPh-AQTGTGKT, compared to the control group (PBS), it showed an inhibitory effect of about 49% tumor growth.

In addition, AQTGTGKT analog was treated daily for 14 days at a dose of 10 mpk in nude mice transplanted with H820 lung cancer cells to compare tumor growth.

As a result, as shown in FIG. 16, when 3-PhPh-AQTGTGKT was treated, tumor growth was reduced by about 52% compared to the control (PBS), and 2-PhPh-AQTGTGKT, Ph-AQTGTGKT, Naphthyl-AQTGTGKT In the case of treatment, compared with the control group (PBS), the growth of the tumor was about 56%, and finally, when 4-PhPh-AQTGTGKT, Ac-AQTGTGKT, and 4-MeOPh-AQTGTGKT was treated, the tumor growth rate compared to the control group (PBS) was reduced. Growth was reduced by about 39%, showing an inhibitory effect on tumor growth.

Collectively, the above results show that the compound of the present invention has an excellent anticancer effect in an animal model transplanted with lung cancer cells.

Example 3: Confirmation of stability in blood of AQTGTGKT analog

In order to check whether the AQTGTGKT analog according to the present invention is stably present in the blood for several minutes, 10 μM of each compound was added to 100% human blood and 0, 5, 10, 15, 30, 60, and 120 minutes had elapsed. The residual amount in the blood was measured.

As a result, as shown in FIGS. 17 to 23 , in the case of AQTGTGKT, the half-life was less than 1 minute, and it was confirmed that the residual AQTGTGKT was hardly detected as it was almost decomposed in the blood within 20 minutes. In contrast, all seven AQTGTGKT analogs according to the present invention showed significantly improved stability, in particular 4-PhPh-AQTGTGKT, 3-PhPh-AQTGTGKT, 4-MeOPh-AQTGTGKT, 2-PhPh-AQTGTGKT, Ph-AQTGTGKT and Naphthyl-AQTGTGKT showed a half-life of 47 minutes to 105 minutes, and 20 to 40% remained in the blood even after 120 minutes had elapsed, indicating high stability. These results show that amidated analogs exhibit not only superior anticancer effects, but also improved stability in blood, so they can exist stably without being degraded until they reach their target.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> L-Base Co., Ltd. <120> Pharmaceutical Composition for the Prevention or Treatment of Cancer <130> MP20-028P1 <140> 10-2021-0036982 <141> 2021-03-23 <150> KR 10-2020-0035188 <151> 2020-03-23 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> AQTGTGKT <400> 1 Ala Gln Thr Gly Thr Gly Lys Thr 1 5

Claims (15)

A compound represented by the general formula:
[general meal]
X-AQTGTGKT
(In the above general formula,
A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,
X is

It is one selected from the group consisting of.)
A pharmaceutical composition for the prevention or treatment of cancer, comprising a compound represented by the following general formula as an active ingredient:
[general meal]
X-AQTGTGKT
(In the above general formula,
A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,
X is

It is one selected from the group consisting of.)
3. The method of claim 2,
The cancer is lung cancer, breast cancer, blood cancer, colorectal cancer, pancreatic cancer, characterized in that the cancer selected from the group consisting of a combination thereof, the pharmaceutical composition.
4. The method of claim 3,
The lung cancer is non-small cell lung cancer (non-small cell lung cancer), characterized in that, the pharmaceutical composition.
4. The method of claim 3,
The breast cancer is triple negative breast cancer (Triple negative breast cancer), characterized in that the pharmaceutical composition.
4. The method of claim 3,
The hematologic cancer is a hematologic cancer selected from the group consisting of leukemia, lymphoma, multiple myeloma, and combinations thereof, the pharmaceutical composition.
3. The method of claim 2,
wherein X is

One selected from the group consisting of, wherein the cancer is lung cancer, the pharmaceutical composition.
3. The method of claim 2,
wherein X is

and, wherein the cancer is breast cancer, the pharmaceutical composition.
3. The method of claim 2,
wherein X is

It is one selected from the group consisting of, wherein the cancer is a blood cancer, a pharmaceutical composition.
3. The method of claim 2,
wherein X is

and

It is one selected from the group consisting of, wherein the cancer is pancreatic cancer, the pharmaceutical composition.
3. The method of claim 2,
wherein X is

It is one selected from the group consisting of, wherein the cancer is colorectal cancer, the pharmaceutical composition.
3. The method of claim 2,
wherein X is

In the case of , the pharmaceutical composition, characterized in that the half-life in human blood is 100 to 150 minutes.
3. The method of claim 2,
wherein X is

When one selected from the group consisting of, the pharmaceutical composition, characterized in that the half-life in human blood is 45 minutes to 70 minutes.
3. The method of claim 2,
wherein X is

In the case of , the pharmaceutical composition, characterized in that the half-life in human blood is 20 to 30 minutes.
A method for preparing an oligopeptide X-AQTGTGKT comprising the steps of:
(The A is alanine, Q is glutamine, T is threonine, G is glycine, K is lysine,
wherein X is

It is one selected from the group consisting of.)
(1) synthesizing TG and KT, respectively;
(2) synthesizing TGKT by combining the TG and KT;
(3) synthesizing TGTGKT by binding TG to the N-terminus of TGKT;
(4) synthesizing QTGTGKT by binding Q to the N-terminus of TGTGKT; and
(5) synthesizing X-AQTGTGKT by binding an alanine derivative (XA) to the N-terminus of QTGTGKT.

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