HK1199830B

HK1199830B - Composition for preventing and treating non-small cell lung cancer, containing pyrazino-triazine derivatives

Info

Publication number: HK1199830B
Application number: HK15100319.7A
Authority: HK
Inventors: 车周盈; 李官厚; 程智恩
Original assignee: Jw制药公司
Priority date: 2011-08-09
Filing date: 2012-08-07
Publication date: 2017-05-19

Abstract

The present invention relates to a composition which contains pyrazino-triazine derivatives, isomers thereof, or pharmaceutically acceptable salts thereof. The composition has superior effects in the prevention and treatment of non-small cell lung cancer (NSCLC).

Description

Composition comprising pyrazinotriazine derivative for preventing and treating non-small cell lung cancer

Technical Field

The present invention relates to a composition for preventing and treating NSCLC (non-small cell lung cancer) comprising a pyrazinotriazine derivative.

Background

As the term implies, primary lung cancers derived from the lung are largely classified into small cell lung cancers and non-small cell lung cancers (NSCLC) according to histological type. NSCLC is the leading cause of cancer-related death in the united states (accounting for over 80% of lung cancer), with cure rates of less than 15% and an average survival period of 8 to 10 months. Once the cancer has progressed beyond the early stages, no surgical approach is available, and the only approach is chemotherapy. However, NSCLC is less sensitive to anticancer agents because it is highly heterogeneous and consists of different cell types.

For this reason, dual therapy (double therapy) of cytotoxic drugs is mainly used to treat NSCLC. Cytotoxic drugs used in dual therapy for NSCLC include carboplatin (carboplatin), paclitaxel (paclitaxel), docetaxel (docetaxel) and etoposide (etoposide), cisplatin (cispin), doxorubicin (doxorubicin), with paclitaxel and carboplatin being the preferred combination. In addition, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), a targeted therapeutic agent for inhibiting Epidermal Growth Factor (EGF), can be used as NSCLC drug under the trade name ofAndand (4) obtaining the product.

The dual-modality therapy of these cytotoxic drugs is limited in its application to debilitating patients, such as elderly patients, due to the resulting significant toxicity. Furthermore, this therapy does not guarantee good anticancer efficiency nor low recurrence rate for NSCLC patients in very early or advanced stages. Therefore, there is still a need to find a new established chemotherapy targeting NSCLC centered on therapeutically excellent and non-toxic anticancer agents.

Targeting such as EGFR TKITherapeutic agents are only applicable to cancer patients expressing specific targeting factors. For example, clinical reports call: compared to patients with wild-type (WT) EGFR,orParticularly for some patients with epidermal growth factor receptor mutations (active EGFR mutations (L858R, del e746_ a 750)). This clinical response may be attributed to the increased affinity of the EGFR TKI for mutant EGFR (Science, vol 304, 6/4/2004). However, although EGFR TKI has excellent anticancer activity to patients with active mutations, many patients treated with EGFR TKI in 2005 had relapsed to develop chemoresistance to EGFR TKI, and epigenetic analysis showed that the gatekeeper amino acid (threonine amino acid) threonine 790 in EGFR TKI was changed to methionine (T790M) in 50% of relapsed patients (fig. 1 and 2). The crystal analysis shows thatSteric hindrance at the binding site to the methionine residue reduced the affinity and led to the T790M mutation to drug resistance (NEJM, vol 352, 8/2/2005). In addition to the T790M mutation, other mutations including L747S, D761Y and T854A were reported, but these mutations account for less than 10% of relapses and are therefore less important (Nature reviews cancer volume 10, 2010). There is insufficient data on which treatment is appropriate for NSCLC patients with active mutations and T790M (exon 20) (British Journal of Cancer105(1), 1-8, 2011).

Therefore, there is a need to find agents that exhibit excellent therapeutic effects on NSCLC in patients with mutations in EGFR activity as well as patients with WT EGFR, and patients chemoresistant to EGFR TKI for relapse.

Meanwhile, WO12/050393, WO10/120112, WO09/05197 and WO09/148192 disclose many compounds in the form of pyrazinotriazine derivatives which exhibit anticancer activity. There is no mention in the prior art literature of the therapeutic effect of the above compounds on NSCLC. Among these compounds, the present inventors have surprisingly found that the compound derivative represented by chemical formula 1 has a therapeutic effect against NSCLC of EGFR TKI.

Disclosure of Invention

The present invention aims to provide a composition comprising a pyrazinotriazine derivative for the prevention and treatment of non-small cell lung cancer (NSCLC).

It is another object of the present invention to provide a method for treating NSCLC comprising administering a therapeutically effective amount of the composition to a subject in need thereof.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating NSCLC, the composition comprising at least one selected from the group consisting of a compound represented by the following chemical formula 1, isomers thereof, and pharmaceutically acceptable salts thereof:

[ chemical formula 1]

Wherein R is₁Is a substituted or unsubstituted C3-C10 aryl, or a substituted or unsubstituted C3-C10 heteroaryl having at least one nitrogen (N) atom; r₃Hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 aryl, C3-C10 heteroaryl with at least one N atom, C3-C10 arylalkyl or C3-C10 heteroarylalkyl with at least one N atom; a is hydrogen or C1-C6 alkyl; b is hydrogen or C1-C6 alkyl; x is-O-PO₃H₂or-OH; and Y is hydrogen, C3-C10 aryl, C1-C6 alkyl, or C3-C10 heteroaryl having at least one N atom.

In a preferred embodiment of the invention, the substituted C3-C10 aryl radical orThe substituted C3-C10 heteroaryl group having at least one N atom has at least one substituent selected from the group consisting of C1-C6 alkyl,OrWherein Ra and Rb may independently be hydrogen, C1-C6 alkyl, pyridyl, or amino-substituted pyridyl, and Ra and Rb may be condensed together to form a ring.

In another preferred embodiment of the invention, R₁Is naphthyl, quinolyl, indolyl, substituted naphthyl, substituted quinolyl or substituted indolyl, wherein the substituted naphthyl, substituted quinolyl and substituted indolyl have alkyl radical selected from C1-C6,OrWherein Ra and Rb can independently be hydrogen, C1-C6 alkyl, pyridyl, amino-substituted pyridyl, and Ra and Rb can be condensed together to form a ring; r₃Is methyl or propenyl; either or both of A and B are hydrogen; x is-O-PO₃H₂or-OH; and Y is hydrogen.

In another embodiment of the present invention, the substituted naphthyl, substituted quinolyl or substituted indolyl group may have a structure selected fromOrAt least one substituent of (a).

In another embodiment of the invention, R₁Is composed ofOrR₃Is composed ofOr a methyl group; either or both of A and B are hydrogen; x is-O-PO₃H₂or-OH; and Y is hydrogen.

In still another embodiment of the present invention, the compound of chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulae 2 to 11:

in still another embodiment of the present invention, the compound of chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulae 1-1 and 1-1P:

[ chemical formula 1-1]

[ chemical formula 1-1P ]

In still another embodiment of the present invention, the compound of chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulae 1-2 to 1-7:

in still another embodiment of the present invention, the compound of chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulae 2-1 to 2-8:

according to one embodiment of the invention, NSCLC (non-small cell lung cancer) may have occurred in patients with WT (wild-type) EGFR (epidermal growth factor receptor) or with mutations in EGFR activity.

According to another embodiment of the invention, the NSCLC may be resistant to EGFR TKI and the cancer may be lung cancer.

According to another embodiment of the invention, the EGFR TKI resistant cancer may have occurred in a patient with mutant EGFR.

According to another embodiment of the invention, mutant EGFR may have double mutations of L858R and T790M.

According to another aspect of the present invention, the present invention provides a method for preventing or treating NSCLC (non-small cell lung cancer), the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound represented by the following chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[ chemical formula 1]

Wherein the content of the first and second substances,

R₁is a substituted or unsubstituted C3-C10 aryl or a substituted or unsubstituted C3-C10 heteroaryl having at least one nitrogen (N) atom; r₃Hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 aryl, C3-C10 heteroaryl with at least one N atom, C3-C10 arylalkyl or C3-C10 heteroarylalkyl with at least one N atom; a is hydrogen or C1-C6 alkyl; b is hydrogen or C1-C6 alkyl; x is-O-PO₃H₂or-OH; and Y is hydrogen, C3-C10 aryl, C1-C6 alkyl, or C3-C10 heteroaryl having at least one N atom.

The compositions of the invention are effective in treating NSCLC, and are particularly effective in treating and preventing EGFR TKI-resistant NSCLC.

Drawings

FIG. 1 is a schematic diagram of the structure of the EGFR gene and the mutant locus;

FIG. 2 shows the three-dimensional structure of EFGR in which secondary mutations have occurred, and the secondary mutation rate in the EFGR gene;

FIG. 3 is a graph in which the mean tumor volume (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered at a low dose (100mg/kg) is plotted against the number of days after cell inoculation (horizontal axis);

FIG. 4 is a graph in which the body weight (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered at a low dose (100mg/kg) is plotted against the number of days after cell inoculation (horizontal axis) compared with the body weight on day 8;

FIG. 5 is a graph in which the mean tumor volume (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered at a high dose (150mg/kg) is plotted against the number of days after cell inoculation (horizontal axis);

FIG. 6 is a graph in which the body weight (vertical axis) of the group to which the pyrazinotriazine derivative (chemical formula 1-1) was administered at a high dose (150mg/kg) is plotted against the number of days after cell inoculation (horizontal axis) compared with the body weight on day 8;

FIG. 7 is a graph in which the tumor size (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered is plotted against the number of days after treatment (horizontal axis);

FIG. 8 is a graph in which body weights (vertical axis) of groups to which a pyrazino-triazine derivative (chemical formula 1-1) was administered are plotted against days after treatment (horizontal axis);

FIG. 9 is a graph in which the tumor size (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered is plotted against the number of days after inoculation (horizontal axis);

FIG. 10 is a graph in which the tumor size (vertical axis) of the group to which a pyrazinotriazine derivative (chemical formula 1-1) was administered is plotted against the number of days after inoculation (horizontal axis);

fig. 11 is a graph in which the tumor size (vertical axis) of the group to which the pyrazinotriazine derivative (disodium salt of chemical formula 2-2) was administered is plotted against the number of days after inoculation (horizontal axis);

fig. 12 is a graph in which the body weight (vertical axis) of the group to which the pyrazinotriazine derivative (disodium salt of chemical formula 2-2) was administered is plotted against the number of days after treatment (horizontal axis);

FIG. 13 is a graph in which the tumor sizes (vertical axis) of the groups to which pyrazinotriazine derivatives (chemical formulae 1 to 3 and chemical formulae 1 to 5) were administered are plotted against the number of days after inoculation (horizontal axis);

fig. 14 is a graph in which body weights (vertical axis) of groups to which pyrazinotriazine derivatives (chemical formulae 1 to 3 and chemical formulae 1 to 5) were administered are plotted against days after treatment (horizontal axis); and

fig. 15 is a graph in which the tumor sizes (vertical axis) of the groups to which pyrazinotriazine derivatives (chemical formulas 1 to 7) were administered are plotted against the number of days after inoculation (horizontal axis).

Detailed Description

The present invention provides a composition for preventing or treating NSCLC (non-small cell lung cancer) comprising a pyrazinotriazine derivative as an active ingredient.

The present inventors found and demonstrated that the pyrazinotriazine derivatives of the present invention are effective in the treatment and control of NSCLC which shows chemoresistance, which ultimately became the present invention.

The term "pyrazino triazine derivative" as used herein refers to a compound represented by the following chemical formula 1.

In detail, the pyrazino triazine derivative of the present invention may be selected from the group consisting of a compound represented by the following chemical formula 1, isomers thereof, and pharmaceutically acceptable salts thereof, but is not limited thereto:

[ chemical formula 1]

Wherein the content of the first and second substances,

R₁is a substituted or unsubstituted C3-C10 aryl or a substituted or unsubstituted C3-C10 heteroaryl having at least one nitrogen (N) atom. Get throughThe substituted C3-C10 aryl or substituted C3-C10 heteroaryl having at least one N atom may have a substituent selected from the group consisting of C1-C6 alkyl,OrWherein Ra and Rb may independently be hydrogen, C1-C6 alkyl, pyridyl, or amino-substituted pyridyl, and Ra and Rb may be condensed together to form a ring.

Preferably, R₁Is naphthyl, quinolyl, indolyl, substituted naphthyl, substituted quinolyl or substituted indolyl, wherein the substituted naphthyl, substituted quinolyl and substituted indolyl have alkyl radical selected from C1-C6,OrWherein Ra and Rb may independently be hydrogen, C1-C6 alkyl, pyridyl, or amino-substituted pyridyl, and Ra and Rb may be condensed together to form a ring. Furthermore, the substituted naphthyl, substituted quinolyl or substituted indolyl group may have a substituent selected from the group consisting ofOrAt least one substituent of (a).

In detail, R₁Is composed ofOr

R₃Is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 aryl, C3-C10 heteroaryl having at least one N atom, C3-C10 arylalkyl or C3-C10 heteroarylalkyl having at least one N atom, and is preferably methyl or propenyl

A is hydrogen or C1-C6 alkyl and B is hydrogen or C1-C6 alkyl. Preferably, either or both of a and B are hydrogen.

X is-O-PO₃H₂or-OH.

Y is hydrogen, C3-C10 aryl, C1-C6 alkyl or C3-C10 heteroaryl having at least one N atom, with hydrogen being preferred.

In more detail, the pyrazinotriazine derivative of the present invention may be selected from the group consisting of compounds represented by the following chemical formulas 2 to 11, isomers thereof, pharmaceutically acceptable salts thereof, and combinations thereof.

The pyrazinotriazine derivative of the present invention may be a compound represented by the following chemical formula 1-1 or chemical formula 1-1P:

[ chemical formula 1-1]

[ chemical formula 1-1P ]

In addition, the pyrazinotriazine derivative of the present invention may be a compound selected from the group consisting of compounds represented by the following chemical formulae 1-2 to 1-7 and pharmaceutically acceptable salts thereof:

in addition, the pyrazinotriazine derivative of the present invention may be a compound selected from the group consisting of compounds represented by the following chemical formulae 2-1 to 2-8, and pharmaceutically acceptable salts thereof:

the pyrazinotriazine derivative of the present invention can be prepared according to the following reaction scheme 1.

[ reaction scheme 1]

A, B, R therein₁、R₃X and Y are each as defined above.

Referring to reaction scheme 1, the pyrazino triazine derivatives of the present invention can be prepared by the following sequential steps:

step 1: reacting a substituted aminoacetal (aminoacetal) with an aldehyde or an alkyl halide (alkyl halide);

step 2: coupling the resulting substituted aminoacetals of step 1 with amino acids to form peptides;

and step 3: deprotecting the peptide of step 2 with salt;

and 4, step 4: coupling the deprotected peptide of step 3 with a hydrazinic acid (hydrazine acid) side chain;

and 5: cyclizing the resulting peptide of step 4 under acidic conditions to form a pyrazinotriazine derivative;

step 6: introducing a phosphate group into the pyrazino triazine derivative of step 5 when X is a hydroxyl group;

and 7: the phosphorylated group introduced in step 6 is converted to the mono-or disodium salt.

The term "pharmaceutically acceptable salt" as used herein refers to salts commonly used in the medical arts. Examples of pharmaceutically acceptable salts include sodium, chloride, magnesium, calcium and potassium salts, but are not limited thereto. Sodium salts or chlorides are preferred.

The composition of the present invention may be suitably used for treating or preventing NSCLC (non-small cell lung cancer).

NSCLC may have occurred in patients with WT (wild-type) EGFR or with mutations in EGFR activity.

Furthermore, NSCLC may be a cancer that occurs in patients with mutations in EGFR activity. Active mutations may be selected from the group consisting of L858R, E746 deletion, a750 deletion, and combinations thereof. The cancer occurring in a patient having such a mutation in EGFR activity may be an EGFR TKI-sensitive cancer. In the present invention, the term "EGFR TKI" is an abbreviation for epidermal growth factor receptor tyrosine kinase inhibitor, but encompasses all agents having this activity, which inhibit the activity of EGF (epidermal growth factor). In the present invention, the term "EGFR TKI-sensitive cancer" means a cancer for which EGFR TKI exhibits a particularly high therapeutic effect.

Furthermore, NSCLC may be an EGFR TKI resistant cancer. The term "EGFR TKI resistant cancer" as used herein is intended to encompass cancers that exhibit chemical resistance to EGFR TKI. It may be a cancer caused by a mutation in EGFR. In addition to the above active mutations, the above mutations may further have a mutation selected from the group consisting of T790M, L747S, D761Y, T854A, and a combination thereof. Preferably, the L858R and T790M double mutations.

EGFR TKI-resistant cancers also encompass cancers that have relapsed after administration of EGFR TKI, and cancers that occur in individuals with mutant EGFR resistant to EGFR TKI.

Furthermore, the NSCLC treated by the present invention may have wild-type KRAS or mutant KRAS.

Thus, the compositions of the present invention exhibit excellent therapeutic effects on NSCLC patients regardless of whether the patients have wild-type EGFR or active mutations of EGFR, or exhibit resistance to EGFR TKI.

The composition of the present invention may further comprise pharmaceutically acceptable additives such as diluents, binders, disintegrants, lubricants, pH adjusting agents, antioxidants and solubilizers, within a range in which the effects of the present invention are not impaired.

In addition, the compositions of the present invention may be formulated with enteric polymers, water-insoluble polymers, hydrophobic compounds and hydrophilic polymers into a delayed release form. Enteric polymers are polymers that are insoluble or stable under acidic conditions less than pH5 and dissolve or degrade under specific pH conditions above pH5 or pH 5.

Further, optionally, the formulation of the present invention may contain pharmaceutically acceptable additives, such as various additives selected from colorants and fragrances.

The range of the additives useful in the present invention is not limited to the above-mentioned additives, and the additives may be used in conventional dosages appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, lozenges, capsules, suspensions, emulsions, syrups and aerosols; an external application; suppositories; or sterile injectable solutions.

According to another aspect of the present invention there is provided a method for the prevention or treatment of NSCLC comprising administering to an individual comprising a mammal a composition comprising a compound of the present invention.

The term "administering" as used herein refers to introducing the composition of the invention into an individual in an appropriate manner to prevent and treat NSCLC. Any route of administration may be employed so long as it directs the composition of the present invention to the target tissue. Examples of routes via which the composition for preventing and treating NSCLC according to the present invention may be administered include oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, intranasal, intrapulmonary, rectal, intracapsular and epidural routes, but are not limited thereto.

The composition for preventing or treating NSCLC according to the present invention may be administered once a day or twice a day at regular intervals.

The dosage of the compound of the present invention varies depending on various factors including the body weight, age, sex, health condition and diet of a patient, administration time, administration route, excretion rate, severity of disease, etc. The active ingredient may be administered at a dose of 0.1 to 300mg/kg per day, and preferably at a dose of 0.5 to 20mg/kg per day, but the amount may vary depending on the age and sex of the patient, the severity of the disease, and the like.

According to another aspect of the present invention there is provided a method for the treatment of NSCLC comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention.

In a preferred embodiment of the invention, the methods of treatment of the present invention may comprise a pharmaceutical composition comprising a compound of the present invention administered in combination with one or more anti-cancer drugs.

One known anticancer agent may be paclitaxel. It is known that the pharmaceutically effective amount of an anti-cancer agent is known in the art and the final dosage regimen will be determined by the attending physician in light of various factors including co-administration with the compositions of the present invention. The composition of the present invention can impart a therapeutic synergistic effect in addition to reducing the side effects of anticancer agents when co-administered together with conventional anticancer agents. The conventional anticancer agent may be administered in a complex formulation or simultaneously with the composition of the present invention, or separately at a time interval different from the administration of the composition of the present invention.

The invention may be better understood by reference to the following examples which are intended to illustrate, but not to be construed as limiting the invention.

For the preparation of the compounds used in the present invention, reference may be made to scheme 1 or WO12/050393, WO10/120112, WO09/051397 or WO 09/148192.

Exemplary embodiment 1: therapeutic Effect of chemical formula 1-1 on NSCLC of EGFR TKI-resistant cells (NCI-H1975)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivative (chemical formula 1-1) of the present invention in an animal model transplanted with NCI-H1975 cells, a NSCLC cell resistant to an EGFR-targeting agent.

2) Method of producing a composite material

NCI-H1975(ATCC, CRL-5908) derived from NSCLC patients is a NSCLC cell strain with double mutations of EGFR L858R and T790M, and resistant to EGFR TKIIn this case, NCI-H1975 cells were cultured on a large scale and contained 5 × 10 in 0.2mL of HBSS per one cell⁶The density of individual cells was subcutaneously implanted in the right axillary region of mice (Balb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. The mice of group 6 transplanted with NCI-H1975 were administered with the compound of chemical formula 1-1 and/or paclitaxel on days 8 (D8) to 22 (D22) after transplantation, as summarized in table 1 below.

TABLE 1

Group of	Substances to be administered and the amounts thereof
		1	Vehicle _ saline
2	1-1_100mg/kg
		3	1-1_150mg/kg
4	1-100 mg/kg + paclitaxel-7.5 mg/kg
		5	1-150 mg/kg + paclitaxel-7.5 mg/kg
6	Taxol 7.5mg/kg

As a control, vehicle was injected intravenously only once a day to achieve a total of 10 injections over a two week period (5 per week, D8-12, 15-19) (group 1).

The compound of formula 1-1 was dissolved in saline and injected intravenously once a day at doses of 100mg/kg and 150mg/kg to achieve a total of 10 injections (5 per week, D8-12, 15-19) over a two week period (group 2 and group 3, respectively).

Paclitaxel was injected intraperitoneally once a day at a dose of 7.5mg/kg to achieve a total of 2 injections (once a week, D8 and D5) over a two week period (group 6).

Mice administered paclitaxel at a dose of 7.5mg/kg were injected with the compound of chemical formula 1-1 (groups 4 and 5) at a dose of 100mg/kg and 150mg/kg in the same manner as described above.

3) Results

i. Evaluation of changes in body weight and tumor size and Tumor Growth Inhibition (TGI)

The body weight and tumor size of the mice were measured 2 or 3 times per week during the experiment.

The measurements of body weight and tumor size (n-7 per group) were randomized using internal randomization software. Tumor size was calculated from the measurements of the longest axis (length, mm) and the shortest axis (width, mm) according to the following formula:

tumor size (mm)³) Width (mm) × Width (mm) × Length (mm)/2

On day 8, the mice had 63mm³(range: 42 to 103 mm)³) Average tumor size, average body weight 18.6 g.

Referring to Table 3, at day 22, the vehicle control group had 995. + -. 131mm³And the final tumor size in the group administered with chemical formula 1-1(P1) was significantly reduced.

Thus, the data show that: the compounds of the present invention exhibited excellent anticancer activity in EGFR TKI resistant NSCLC cell strain having double mutations of L858R and T790M.

Tumor growth inhibition (TGI,%) calculated according to the following formula was used to evaluate the administration-dependent anticancer activity.

When the tumor size of the drug-administered group is larger than the initial tumor size:

% TGI 100 (1- (tumor volume)_{Day X}Tumor volume_{At the beginning of the treatment group}) V (tumor volume)_{Day X}Tumor volume_{Vehicle group at initial time}))

When the tumor size of the drug-administered group is smaller than the initial tumor size:

% TGI 100+100 (1- (treatment group tumor volume)_{Day X}V (tumor volume in treatment group)_{At the beginning}))

TGI 74% was induced at the administration of a dose of chemical formula 1-1 of 100mg/kg, and TGI 109% was induced at the administration of a dose of chemical formula 1-1 of 150mg/kg, thereby exhibiting anticancer activity in a dose-dependent manner.

These results are far greater than those obtained with paclitaxel, which was measured to trigger TGI 28% at a dose of 7.5 mg/kg.

In addition, in the group administered with the combination of chemical formula 1-1(100mg/kg, 150mg/kg) and paclitaxel (7.5mg/kg), TGI was measured as 94% and 175%, respectively, thereby illustrating the synergistic effect achieved by the combined administration thereof.

The results are summarized in table 2 and depicted in fig. 3-6.

Mortality and maximum weight loss (MBL)

Herein, mortality rate indicates death caused by drug administration. No death cases were observed in any of the groups during and after this experiment.

Herein, maximum weight loss (MBL) means the maximum weight loss caused by drug administration and is an important factor for determining the Maximum Tolerated Dose (MTD). The change in body weight was expressed as a percentage of body weight immediately before drug administration (D1). Generally, the dosage used to reduce body weight by 10% or more is the MTD.

The measurement results are summarized in table 2 below.

TABLE 2

Exemplary embodiment 2: therapeutic Effect of chemical formula 1-1 on NSCLC of EGFR TKI-resistant cell (NCI-H460)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivative (chemical formula 1-1) of the present invention in an animal model transplanted with NCI-H460 cells, a NSCLC cell resistant to an EGFR-targeting agent.

2) Method of producing a composite material

NCI-H460(ATCC, HTB-177) derived from NSCLC patients is a NSCLC cell strain with wild-type EGFR and mutant KRAS, which is resistant to EGFR TKIFor this purpose, NCI-H460 cells were cultured on a large scale and contained 5 × 10 in 0.2mLHBSS⁶The density of individual cells was implanted subcutaneously into the right axillary region of each mouse (Balb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. On days 6 (D1) to 18 (D12) after transplantation, 3 groups of mice transplanted with NCI-H460 were administered the compound of formula 1-1 or paclitaxel as summarized in table 3 below.

TABLE 3

Group of	The materials and amounts administered
		1	Vehicle _ saline
2	1-1_150mg/kg
		3	Taxol-20 mg/kg

As a control, saline was injected intravenously only once a day to achieve a total of 6 injections over a two-week period (3 times per week, days 1, 3, 5, 8, 10, 12) (group 1).

The compound of formula 1-1 was dissolved in saline and injected intravenously once a day at a dose of 150mg/kg to achieve a total of 6 injections (3 times per week, days 1, 3, 5, 8, 10, 12) over a two-week period (group 2).

Paclitaxel was injected intraperitoneally once a day at a dose of 20mg/kg to achieve a total of 6 injections over a two week period (3 times per week, days 1, 3, 5, 8, 10, 12) (group 3).

3) Results

Body weight and tumor size of mice were measured in the same manner as in exemplary example 1, and tumor growth inhibition was also calculated(TGI,%). The mean tumor volume (mean initial tumor volume) before the experiment was 136mm³。

The measurement results are summarized in table 4 and described in fig. 7 and 8.

Mortality and maximum weight loss (MBL)

Mortality and MBL were evaluated in the same manner as in exemplary example 1, and the results are summarized in table 4 below.

TABLE 4

Exemplary embodiment 3: therapeutic Effect of chemical formula 1-1 on NSCLC of EGFR TKI-sensitive cell (HCC827)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivative (chemical formula 1-1) of the present invention in an animal model transplanted with HCC827 cells, a NSCLC cell that is hypersensitive to EGFR-targeting agents.

2) Method of producing a composite material

HCC827(ATCC, CRL-2868) from NSCLC patient is a NSCLC cell strain with exon 19 deletion mutation and resistant to EGFR TKIIn this regard, HCC827 cells were cultured on a large scale and contained 5 × 10 in 0.2mL HBSS⁶The density of individual cells was subcutaneously implanted in the right axillary region of each mouse (Balb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. On days 8 to 32 after transplantation, 6 groups of mice transplanted with HCC827 were administered the compound of formula 1-1 and/or paclitaxel, as summarized in table 5 below.

TABLE 5

Group of	Substances to be administered and the amounts thereof
		1	Vehicle _ saline
2	1-1_100mg/kg
		3	1-1_150mg/kg
4	1-100 mg/kg + paclitaxel-10 mg/kg
		5	1-150 mg/kg + paclitaxel-10 mg/kg
6	Taxol-10 mg/kg

As a control, saline was injected intravenously only once a day to achieve a total of 20 injections over a four week period (5 times per week, days 8-12, days 15-19, days 22-26, and days 29-32) (group 1).

The compound of chemical formula 1-1 was dissolved in saline and injected intravenously once a day at doses of 100mg/kg and 150mg/kg to achieve a total of 20 injections over a four week period (5 injections per week, days 8-12, days 15-19, days 22-26, and days 29-32) (group 2 and group 3, respectively).

Paclitaxel was injected intraperitoneally once a day at a dose of 10mg/kg to achieve a total of 4 injections over a four week period (1 injection per week, day 8, day 15, day 22 and day 29) (group 6).

Mice administered paclitaxel at a dose of 10mg/kg were injected with the compound of chemical formula 1-1 at a dose of 100mg/kg and 150mg/kg in the same manner as described above (groups 4 and 5).

3) Results

Body weight and tumor size of the mice were measured in the same manner as in exemplary example 1, and tumor growth inhibition (TGI,%) was also calculated. The mean tumor volume (mean initial tumor volume) before the experiment was 121mm³。

The measurement results are summarized in table 6 and described in fig. 9 and 10.

Mortality and maximum weight loss (MBL)

Mortality and MBL were evaluated in the same manner as in exemplary example 1, and the results are summarized in table 6 below.

TABLE 6

The therapeutic effect of compound 1-1 evaluated on various NSCLC cell lines in terms of Tumor Growth Inhibition (TGI) in exemplary examples 1 to 3 is summarized in table 7 below.

TABLE 7

The compound of formula 1-1 shows higher anticancer activity in animal models transplanted with NSCLC cell lines compared to the standard agent paclitaxel, and there are a peak of inhibitory activity against NCI-H1975 having EGFR T790M mutation (which is responsible for resistance to EFGR TKI) and a peak of synergy against NCI-H1975 with paclitaxel, thus indicating that it is a potential therapeutic agent for patients having EGFR TKI resistant NSCLC by itself.

Exemplary embodiment 4: in vitro anti-cancer Activity of Compound 1-1P against various NSCLC cell lines

1) Purpose(s) to

This experiment was conducted to evaluate the in vitro anticancer activity of the pyrazinotriazine derivatives (chemical formula 1-1P) of the present invention in various NSCLC cell lines.

2) Method of producing a composite material

NSCLC cell lines were cultured at 5 × 10³The cells/well density were seeded into 96-well plates. Compound 1-1P and the control drug doxorubicin (doxorubicin) were serially diluted 1-fold in a concentration range of 0.001 μ M to 1 μ M. Thereafter, the above dilution was used to treat the cells in each well twice. Next, the cells were incubated at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 72 hours. Will be provided with(analysis of luminescent cell viability, # G7573, Promega) was added to each well in an amount of 100. mu.L and shaken for 2 minutes to lyse the cells. After incubation at room temperature (25 ℃) for 10 minutes, the luminescence of the wells was determined photometrically (envision (perkinelmer).

3) Results

Inhibition by cell growth 50 (GI)₅₀) Seven different NSCLC cell lines were evaluated for anti-cancer activity depending on the administration of Compound 1-1P, and the inhibition of cell growth 50 was calculated according to the following formulaCalculated concentration required to reduce the growth of treated cells to half the growth of untreated cells. The results are summarized in table 8 below.

GI₅₀＝(50-(y₁-((y₁-y₂)/(x₁-x₂))*x₁)/((y₁-y₂)/(x₁-x₂))

x₁，x₂Cell killing or lethal concentration of 50%

y₁，y₂Cell density at 50% cell killing or lethal concentration

TABLE 8

NCI-H460, NCI-H23 and A549 cells of NSCLC cell strains with wild-type EGFR and mutant KRAS were stopped by 50% growth with 272nM, 67nM and 372nM of the compound, respectively.

HCC827 is a NSCLC cell line that has EGFR exon 19 deletion mutation (E746-a750) and a mutated KRAS gene and is hypersensitive to EGFR TKI. GI discovery of Compound 1-1P against HCC827₅₀Was 115 nM.

NCI-H1650 is a NSCLC cell line that has EGFR exon 19 deletion mutation (E746-A750) and KRAS mutation and is resistant to EGFR TKI. GI discovery of Compound 1-1P against NCI-H1650₅₀Was 156 nM.

NCI-H1975 is a NSCLC cell line which has the T790M mutation and is resistant to EGFR TKI. At a concentration of 111nM of compound 1-1P, growth was stopped by 50%.

Exemplary embodiment 5: therapeutic Effect of disodium salt of chemical 2-2 on NSCLC of EGFR TKI-resistant cells (NCI-H1975)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivative (disodium salt of chemical formula 2-2) of the present invention in an animal model transplanted with NCI-H1975 cells, a NSCLC cell resistant to EGFR-targeting agents.

2) Method of producing a composite material

NCI-H1975(ATCC, CRL-5908) derived from NSCLC patients is a NSCLC cell strain with EGFRL858R and T790M double mutations to EGFR TKIIn this case, NCI-H1975 cells were cultured on a large scale and were treated with 5 × 10 in 0.2mL HBSS⁶The density of individual cells was implanted subcutaneously into the right axillary region of each mouse (Balb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. On days 11 (D11) to 22 (D12) after transplantation, 3 groups of mice transplanted with NCI-H1975 were administered the disodium salt of the compound of formula 2-2 or cisplatin, as summarized in Table 9 below.

TABLE 9

Group of	Substance for administration	Dosage (mg/kg)	ROA	Mode of administration
					1	Carrier	-	IV	QD × 5 for two weeks
2	2Na salt of Compound 2-2	200	IV	QD × 5 for two weeks
					3	Cis-platinum	2.5	IV	BIW two weeks

3) Results

Body weight and tumor size of the mice were measured in the same manner as in exemplary example 1, and tumor growth inhibition (TGI,%) was also calculated. The mean tumor volume (mean initial tumor volume) before the experiment was 118mm³。

Mortality and maximum weight loss (MBL)

Mortality and MBL were evaluated in the same manner as in exemplary example 1, and the results are summarized in table 10 and described in fig. 11 and 12.

Watch 10

Exemplary embodiment 6: therapeutic Effect of Compounds 1-3 and Compounds 1-5 on NSCLC of EGFR TKI-resistant cells (NCI-H1975)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivatives (compounds 1-3 and 1-5) of the present invention in an animal model transplanted with NCI-H1975 cells, a NSCLC cell resistant to EGFR-targeting agents.

2) Method of producing a composite material

NCI-H1975(ATCC, CRL-5908) derived from NSCLC patients is a NSCLC cell strain with double mutations of EGFR L858R and T790M, and resistant to EGFR TKI) In this case, NCI-H1975 cells were cultured on a large scale and were treated with 5 × 10 in 0.2mL HBSS⁶The density of individual cells was implanted subcutaneously into the right axillary region of each mouse (Balb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. Compounds 1-3 and 1-5 were administered to groups 5 of mice transplanted with NCI-H1975 on days 11 (D11) to 22 (D22) after transplantation, as summarized in Table 11 below.

TABLE 11

3) Results

Body weight and tumor size of mice were measured in the same manner as in exemplary example 1, and tumor growth inhibition was also calculated(TGI,%). The mean tumor volume (mean initial tumor volume) before the experiment was 142mm³。

Mortality and maximum weight loss (MBL)

Mortality and MBL were evaluated in the same manner as in exemplary example 1, and the results are summarized in table 12 and described in fig. 13 and 14.

TABLE 12

Exemplary embodiment 7: therapeutic Effect of Compounds 1-7 on NSCLC in EGFR TKI-resistant cells (NCI-H1975)

1) Purpose(s) to

This experiment was conducted to evaluate the in vivo anticancer activity of the pyrazinotriazine derivatives (compounds 1 to 7) of the present invention in an animal model transplanted with NCI-H1975 cells, a NSCLC cell resistant to EGFR-targeting agents.

2) Method of producing a composite material

NCI-H1975(ATCC, CRL-5908) derived from NSCLC patients is a NSCLC cell strain with double mutations of EGFR L858R and T790M, and resistant to EGFR TKIIn this case, NCI-H1975 cells were cultured on a large scale and were treated with 5 × 10 in 0.2mL HBSS⁶(density of individual cells) was subcutaneously implanted into each mouseBalb/c nude mice). Tumor mass was observed 3 to 5 days after transplantation. Compounds 1-7 were administered to groups 2 of mice transplanted with NCI-H1975 on days 11 (D11) to 22 (D22) post-transplantation, as summarized in Table 13 below.

Watch 13

Group of	Substance for administration	Dosage (mg/kg)	ROA	Mode of administration
					1	Carrier	-	IV	BIW two weeks
2	Compounds 1 to 7	60	IV	BIW two weeks

3) Results

Measurement of mice in the same manner as in exemplary example 1Body weight and tumor size, and tumor growth inhibition (TGI,%) was also calculated. The mean tumor volume (mean initial tumor volume) before the experiment was 86mm³。

Mortality rate

Mortality was assessed in the same manner as in exemplary example 1, and the results are summarized in table 14 and depicted in fig. 15.

TABLE 14

INDUSTRIAL APPLICABILITY

As described so far, the pyrazinotriazine derivative of the present invention effectively inhibits the growth of various cell lines involved in NSCLC (non-small cell lung cancer) in vitro and in vivo, and is thus useful as an agent for the treatment and prevention of NSCLC.

Claims

1. Use of a composition comprising at least one selected from the group consisting of a compound represented by the following chemical formula 1, isomers thereof, and pharmaceutically acceptable salts thereof in the manufacture of a medicament for preventing or treating non-small cell lung cancer:

wherein the content of the first and second substances,

R₁being quinolinyl, indolyl, substitutedA quinolinyl group or a substituted indolyl group,

wherein the substituted quinolyl and the substituted indolyl have a substituent selected from the group consisting of C1-C6 alkyl,At least one substituent of (a);

R₃hydrogen, C1-C6 alkyl or C2-C6 alkenyl;

a is hydrogen or C1-C6 alkyl;

b is hydrogen or C1-C6 alkyl;

x is-O-PO₃H₂or-OH; and is

Y is hydrogen.

2. The use according to claim 1, wherein,

R₃is methyl or propenyl;

either or both of A and B are hydrogen.

3. The use according to claim 1, wherein the substituted quinolyl group or the substituted indolyl group has a substituent

4. The use according to claim 1, wherein,

R₁is composed of

R₃Is composed ofOr a methyl group;

either or both of A and B are hydrogen.

5. The use according to claim 1, wherein the compound of chemical formula 1 is selected from the group consisting of a compound represented by one of the following chemical formulae:

6. the use according to claim 1, wherein the compound of chemical formula 1 is selected from the group consisting of a compound represented by the following chemical formula 1-1 or chemical formula 1-1P, isomers thereof, and pharmaceutically acceptable salts thereof:

7. the use according to claim 1, wherein the compound of chemical formula 1 is selected from the group consisting of a compound represented by one of the following chemical formulae 1-2 to 1-7, isomers thereof, and pharmaceutically acceptable salts thereof:

8. the use according to claim 1, wherein the compound of chemical formula 1 is selected from the group consisting of a compound represented by one of the following chemical formulae:

9. the use according to claim 1, wherein the non-small cell lung cancer is a cancer occurring in a patient with wild-type EGFR (epidermal growth factor receptor) or a mutation in EGFR activity.

10. The use of claim 1, wherein the non-small cell lung cancer is an EGFR TKI (epidermal growth factor receptor tyrosine kinase inhibitor) resistant cancer.

11. The use according to claim 10, wherein the EGFR TKI resistant cancer is a cancer occurring in a patient with mutant EGFR.

12. The use according to claim 11, wherein the mutant EGFR has the double mutation L858R and T790M.