KR102561285B1 - Novel HER3 inhibitor and pharmaceutical composition for preventing or treating for cancer comprising the same - Google Patents

Abstract

The present invention relates to a novel HER3 inhibitor and a pharmaceutical composition for preventing or treating cancer containing the same, wherein the HER3 inhibitor blocks HER3 signaling by inhibiting HER3 activity, thereby inhibiting the growth or proliferation of HER3-positive cancers, and can be used as an effective cancer treatment for cancer cells that express or overexpress HER3.

Description

Novel HER3 inhibitor and pharmaceutical composition containing the same for preventing or treating cancer {Novel HER3 inhibitor and pharmaceutical composition for preventing or treating for cancer comprising the same}

The present invention relates to a novel HER3 inhibitor and a pharmaceutical composition for preventing or treating cancer containing the same.

Human epidermal growth factor receptor (HER) is a membrane protein expressed on the cell surface, binds to epidermal growth factor present outside cells to induce cell division, and is composed of HER1, HER2, HER3, and HER4. These HER proteins are abnormally highly expressed on the surface of many types of cancer cells, which leads to rapid division and proliferation of cancer cells. Thus, HER overexpressing or benign cancers can be treated by inhibiting or blocking HER signaling.

All HER families share a molecular structure consisting of an extracellular domain, a transmembrane domain, an intracellular domain with tyrosine kinase activity, and a c-terminal tail. In the extracellular domain, when HER binds to various ligands such as EGF, epiregulin, betacellulin, TGF-β, and heregulin, it forms a receptor dimer and induces phosphorylation of the c-terminal tail through activation of tyrosine kinase. Such HER signaling activates PI3K/AKT, MAPK, and JAK/STAT pathways that exist downstream and ultimately exhibits various biological activities related to tumor progression.

HER3 has been found to play an important role in cancer growth, and is known to be involved in cancer cell growth, proliferation, anticancer drug resistance, and cancer metastasis in various cancers, including breast cancer, lung cancer and osteosarcoma. Therefore, HER3 is proposed as a target for cancer treatment, and research is currently being actively conducted to develop a substance that inhibits HER3 expression or activity as a cancer treatment.

Korean Patent Publication No. 10-2019-0117681

The present invention aims to provide novel HER3 inhibitors.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing a novel HER3 inhibitor.

One aspect of the present invention provides a HER3 inhibitor that is a compound represented by Formula 1 or 2 below.

The compound represented by Formula 1 is 4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol (4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol) (CAS No. 202475-60-3).

The compound represented by Formula 2 is 2-bromo-4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol (2-bromo-4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol) (CAS No. 211555-04-3).

Compounds represented by Chemical Formulas 1 and 2 are conventionally known as JAK3 (Janus-Associated Kinase 3) inhibitors, but their association with HER3 activity has not been studied.

HER3 (human epidermal growth factor receptor 3), also called ERBB3 (receptor tyrosine-protein kinase erbB-3), is one of the epidermal growth factor receptor (EGFR/ERBB) family and acts by forming a heterodimer with other EGFRs such as HER2. The HER3 heterodimer binds to heregulin, also called neuregulin, with high affinity, and activates tyrosine kinase to transduce signals. When HER3 is activated in cancer cells, HER3 is phosphorylated and oncogenic signaling such as Src kinase and PI3K/Akt pathway is activated to promote cancer cell growth, proliferation and progression.

Referring to FIG. 1 , PI3K/AKT, MAPK, and JAK/STAT pathways are involved in HER3 signaling. Since JAK3 exists downstream of HER3, even if JAK3 activity is suppressed, HER3 activity existing upstream of JAK3 is not affected. However, when the present inventors treated HER3-expressing human breast cancer cell lines with the compound of Formula 1 or Formula 2, it was confirmed that HER3 activity was significantly inhibited. Accordingly, the present invention proposes the compound represented by Formula 1 or Formula 2 as a novel HER3 inhibitor.

According to one embodiment of the present invention, the compound represented by Chemical Formula 1 or 2 may inhibit HER3 phosphorylation.

Such novel HER3 inhibitors can be provided as targeted therapeutic agents for cancers that express or overexpress HER3 by inhibiting HER3 activity.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the HER3 inhibitor represented by Formula 1 or 2 as an active ingredient.

Since the HER3 inhibitor is a compound represented by Formula 1 or 2 and is the same as the above description, its description is omitted to avoid excessive complexity.

According to one embodiment of the present invention, the HER3 inhibitor represented by Chemical Formula 1 or 2 may inhibit phosphorylation of HER3.

In one embodiment of the present invention, as a result of treating HER3-expressing human breast cancer cell lines with the HER3 inhibitor represented by Formula 1 or 2, phosphorylation of HER3 was inhibited, and phosphorylation of Akt, ERK, and STAT3, which exist below it, was also inhibited, and it was confirmed that the number of cancer cells and the size of tumors were significantly reduced.

The pharmaceutical composition for preventing or treating cancer containing the HER3 inhibitor represented by Chemical Formula 1 or 2 may exhibit cancer treatment effects by inhibiting the growth and proliferation of or inducing apoptosis of HER3-expressing or over-expressing cells.

As used in the present invention, "cancer" is a disease caused by cells having an aggressive cell division and growth ignoring normal growth limits, an invasive cell infiltrating surrounding tissues, and a metastatic cell spreading to other parts of the body. Here, cancer is also used in the same sense as malignant tumor.

According to one embodiment of the present invention, the cancer may be HER3-positive cancer.

More specifically, the cancer may be HER3-positive solid cancer or metastatic cancer.

According to one embodiment of the present invention, the cancer is ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, prostate cancer, peritoneal cancer, gastric cancer, bladder cancer, lung cancer, melanoma, colorectal cancer, head and neck cancer It may be one or more types of cancer selected from the group consisting of pancreatic cancer, esophageal cancer, and squamous cell carcinoma.

"Prevention or treatment of cancer" as used in the present invention means inhibiting the growth or proliferation of cancer cells or delaying the progression of cancer and improving or ameliorating the symptoms of cancer.

The "pharmaceutical composition for preventing or treating cancer" as used in the present invention refers to a substance used for the purpose of treating, mitigating, treating or preventing cancer in animals, including humans.

The pharmaceutical composition may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions. In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier is commonly used in formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. . The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and the type and degree of response to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or at different times in the subject It may vary according to various factors including components of other compositions and similar factors well known in the pharmaceutical field, One skilled in the art can readily determine and prescribe effective dosages for the desired treatment. For example, it may be 0.001 to 1000 mg/kg, 0.01 mg/kg to 100 mg/kg, or 0.1 mg/kg to 10 mg/kg per day, but is not limited thereto.

In addition, the pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans through various routes. The administration route and administration method of the pharmaceutical composition may be independent, respectively, and are not particularly limited in the method, and may follow any administration route and administration method as long as the pharmaceutical composition can reach the target site. The pharmaceutical composition may be administered orally or parenterally. The parenteral administration method includes, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, etc., and a method of applying, spraying, or inhaling the pharmaceutical composition to the diseased area can also be used, but is not limited thereto.

The novel HER3 inhibitor according to the present invention blocks HER3 signaling by inhibiting HER3 activity, inhibits the growth or proliferation of HER3-positive cancers, and can be used as an effective cancer treatment for cancer cells that express or overexpress HER3.

1 shows the HER3 signaling pathway.
Figure 2 shows the inhibition of HER3 activity of WHI-P131 and WHI-P154 against human breast cancer cell lines MCF7 and T47D.
Figure 3 shows the inhibition of HER3 signaling substances STAT3, Akt and ERK activities of WHI-P131 and WHI-P154 against human breast cancer cell lines MCF7 and T47D.
4 shows changes in HER3 and estrogen receptor (ER-α) expression by HER3 activation in human breast cancer cell lines MCF7 and T47D.
Figure 5 shows the ability of WHI-P131 and WHI-P154 to inhibit cancer cell growth against human breast cancer cell lines MCF7 and T47D.
6 shows the ability of WHI-P154 to inhibit tumor growth in an in vivo breast cancer model.

Hereinafter, the present invention will be described in more detail. However, these descriptions are merely presented as examples to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

Example 1. Confirmation of inhibition of HER3 activity by JAK3 inhibitors

Compounds 4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol (hereinafter referred to as 'WHI-P131') (Calbiochem, 420101) and 2-bromo-4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol (hereinafter referred to as 'WHI-P154') (hereinafter referred to as 'WHI-P154') known as JAK3 inhibitors ) In order to confirm the HER3 activity inhibitory ability, Western blotting was performed using human breast cancer cell lines MCF7 and T47D.

MCF7 and T47D cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin as a culture medium in a 5% CO ₂ incubator at 37°C.

MCF7 cells or T47D cells 1X10 ⁶ were dispensed into a culture dish, cultured for 24 hours, and then replaced with a new culture medium. Then, after pre-treatment with 10 uM of WHI-P131 or WHI-P154 for 30 minutes, 50 ug/ml of Heregulin-β1 (HRG) known as a HER3 ligand was treated for 30 minutes. After washing each cell with PBS, total protein was isolated from each group of cells using a lysis buffer. The same amount of protein (30 μg) was separated by 8-10% acrylamide gel and then SDS-PAGE was performed. The protein band of the gel was transferred to a nylon membrane and treated with 5% skim milk for 1 hour at room temperature. The membrane was reacted with a primary antibody, phospho HER3 antibody (Cell signaling, #4791), total HER3 antibody (Cell signaling, #12708), or β-actin antibody (Abfrontier, LF-PA0207) overnight at 4°C, and then washed three times for 15 minutes with 1xTBST (tris-buffered saline (pH 7.4) containing 0.1% tween-20). The membrane was reacted with secondary antibody HRP-labeled anti-mouse (Santa cruz, sc-2005) for 2 hours at room temperature, and then washed three times with 1xTBST. Afterwards, protein bands were detected using ECL reagent.

As a result, as shown in FIG. 2, the activity of HER3 activated by treating cells with Heregulin-β1 was markedly suppressed by treatment with WHI-P131 or WHI-P154.

Example 2. Confirmation of Inhibition of HER3 Signal Transmitter Activity by WHI-P131 and WHI-P154

The activities of STAT3, Akt, and ERK, which are signaling substances downstream of HER3, by WHI-P131 or WHI-P154 were confirmed.

1X10 ⁶ of MCF7 cells or T47D cells were dispensed into the culture dish, cultured for 24 hours, and then replaced with a new culture medium. Then, 10 uM of WHI-P131 or WHI-P154 was pretreated for 30 minutes, followed by treatment with 50 ug/ml of Heregulin-β1 (HRG) for 30 minutes. Subsequently, Western reaction was performed in the same manner as in Example 1 except that phospho STAT3 antibody (Abcam, ab76315), total STAT3 (Santa cruz, sc8019), phospho ERK (Santa cruz, sc7383), total ERK (Cell signaling, #9102), phospho Akt (Abcam, ab81283) or total Akt (Cell signaling, #9272) were used as primary antibodies. A blot was performed.

As a result, as shown in FIG. 3 , the activity of the HER3 signaling substance activated by Heregulin-β1 was significantly inhibited by treatment with WHI-P131 or WHI-P154.

Example 3. Confirmation of inhibition of breast cancer growth by WHI-P131 and WHI-P154

About 80% of all breast cancer patients are estrogen receptor-positive cancer (ER+), in which cancer cells grow in response to estrogen hormones, and it is known that ER+ breast cancer patients have a poor treatment prognosis if they are HER3-positive (HER3+). In this experiment, the cancer cell growth inhibition of WHI-P131 or WHI-P154 for ER+/HER3+ breast cancer was confirmed.

First, 1X10 ⁶ of MCF7 cells or T47D cells were dispensed into a culture dish, cultured for 24 hours, and then replaced with a new culture medium. Then, 50 ng/ml of Heregulin-β1 (HRG) was treated for 24 hours. Subsequently, Western blotting was performed in the same manner as in Example 1, except that ER-α antibody (Santa cruz, sc8002), total HER3 antibody (Cell signaling, #12708), or β-actin antibody (Abfrontier, LF-PA0207) was used as the primary antibody.

As a result, as shown in FIG. 4, both MCF7 and T47D cells express ER and HER3, and when HER3 is activated, HER3 present in the cell membrane is reduced due to HER3 internalization, and estrogen receptor expression is also reduced by HER3 signaling.

Thereafter, MCF7 cells or T47D cells 1X10 ⁶ were dispensed in a 6-well plate, cultured for 24 hours, and then replaced with a new culture medium. Then, 10 uM of WHI-P131 or WHI-P154 was treated for 10 days. For comparative analysis of cell growth, each cell was washed with PBS, fixed with 100% ethanol, and stained with 0.01% crystal violet.

As a result, as shown in Figure 5, WHI-P131 and WHI-P154 significantly reduced the growth of MCF7 cells and T47D cells, respectively.

In particular, since WHI-P154 was excellent in inhibiting the growth of breast cancer cells, the cancer growth inhibiting effect in vivo was confirmed using WHI-P154.

5-week-old female Balb/C nude mice were supplied from Orient Bio Co., Ltd. and used. 1X10 ⁷ MCF7 cells suspended in Matrigel were injected on both sides of the mouse's mammary fat pad. When the average tumor size reached 100 mm ³ , 1 mg/kg of WHI-P154 or PBS (vehicle) was intraperitoneally injected. Tumor size was measured every week, and tumors were removed after anesthetizing mice at week 7.

As a result, as shown in FIG. 6, when WHI-P154 was injected, the tumor size was significantly reduced compared to when PBS was injected.

Taken together, WHI-P131 and WHI-P154 block HER3 signals in breast cancer cells, particularly HER3-expressing or over-expressing cancer cells, thereby suppressing the growth and inducing apoptosis of cancer cells, and thus can be suggested as effective therapeutic agents for breast cancer.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (4)

A method for treating HER3-positive cancer, comprising administering a compound represented by Formula 1 or 2 below or a composition comprising the same to a mammal other than a human.
[Formula 1]

[Formula 2]
The method of claim 1,
Wherein the mammal is any one selected from the group consisting of rats, mice and livestock. The method of claim 1,
The method of claim 1, wherein the compound represented by Formula 1 or 2 inhibits phosphorylation of HER3. The method of claim 1,
The cancer is any one selected from the group consisting of ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, prostate cancer, peritoneal cancer, stomach cancer, bladder cancer, lung cancer, melanoma, colon cancer, head and neck cancer, pancreatic cancer, esophageal cancer and squamous cell carcinoma How to be.