WO2020085742A1

WO2020085742A1 - Heteroaromatic macrocyclic derivatives as protein kinase inhibitors

Info

Publication number: WO2020085742A1
Application number: PCT/KR2019/013839
Authority: WO
Inventors: Tae Bo Sim; In Jae Shin; Han Na Cho; Seung Hye CHOI; Hwan Geun Choi; Eun Hwa Ko
Original assignee: Korea Institute of Science and Technology KIST
Current assignee: Korea Institute of Science and Technology KIST
Priority date: 2018-10-26
Filing date: 2019-10-21
Publication date: 2020-04-30
Anticipated expiration: 2021-04-26
Also published as: KR102163494B1; KR20200046952A

Abstract

The present invention relates to a 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound having protein kinase inhibitory activity, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth, which includes the compound as an active ingredient. The novel 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound of the present invention exhibits an excellent effect of inhibiting various protein kinases involved in the signal transduction of growth factors, and thus is effective as an agent for preventing, alleviating, or treating an abnormal cell growth disease caused by these protein kinases.

Description

HETEROAROMATIC MACROCYCLIC DERIVATIVES AS PROTEIN KINASE INHIBITORS

The present invention relates to a 2,7-substituted cyano[3,2-d]pyrimidine compound having protein kinase inhibitory activity, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth, which includes the compound as an active ingredient.

A protein kinase is an enzyme that catalyzes the phosphorylation of hydroxyl groups positioned at tyrosine, serine, and threonine residues of proteins and play an important role in signal transduction of growth factors that cause the growth, differentiation, and proliferation of cells.

To maintain the homeostasis of a living body, in-vivo signal transduction systems need to reliably maintain the balance between on and off states thereof. However, mutations or overexpression of specific protein kinases disrupt the signal transduction system in normal cells, thereby causing various diseases such as cancer, inflammation, metabolic diseases, brain disease, and the like. Non-limiting examples of representative protein kinases causing abnormal cell growth diseases include Raf, KDR, Fms, Tie2, SAPK2a, Ret, Abl, Abl(T315I), ALK, Aurora A, Bmx, CDK/cyclinE, Kit, Src, EGFR, EphA1, FGFR3, Flt3, Fms, IGF-1R, IKKb, IR, Itk, JAK2, KDR, Met, mTOR, PDGFRa, Plk1, Ret, Syk, Tie2, and TrtB.

It is estimated that 518 kinds of human protein kinases exist, corresponding to about 1.7% of all human genes (Manning et al, Science, 2002, 298, 1912). Human protein kinases can be broadly divided into tyrosine protein kinases (90 or more kinds) and serine/threonine protein kinases. Tyrosine protein kinases may be divided into 58 kinds of receptor tyrosine kinases, which are classified into 20 subfamilies, and 32 kinds of cytoplasmic/non-receptor tyrosine kinases classified into 10 subfamilies. The receptor tyrosine kinases have a domain capable of accepting growth factors on the cell surface, and an active region capable of phosphorylating tyrosine residue in the cytoplasm. If a growth factor is bound to the growth factor receptor site on the cell surface of the receptor tyrosine kinase, the receptor tyrosine kinase forms a polymer and the tyrosine residue of cytoplasm is self-phosphorylated. Further, through sequential phosphorylation of the lower series of proteins, signal transduction progresses into the nucleus, and ultimately, transcription factors inducing cancer are overexpressed.

Focal adhesion kinase (FAK) is a 125 kD tyrosine protein kinase present in the cytoplasm. FAK plays a critical role in the migration, proliferation and survival of cells by regulating the signal transduction system of integrin and growth factors. FAK protein and FAK mRNA have been found to be overexpressed/activated in various cancer cells, including squamous cell carcinoma, invasive rectal cancer/breast cancer, metastatic prostate cancer, melanoma, and glioma. In addition, it has been verified that Novartis' FAK inhibitor TAE226 (Cancer Invest. 2008, 26(2), 145) is effective for the treatment of breast cancer based on three animal models (HeyA8, SKOV3ip1, and HeyA8-MDR) (Cancer Res. 2007, 67(22), 10976). Further, Pfizer's FAK inhibitor PF-573,228 (Proc. Am. Assoc. Cancer Res., 2006, 47, Abst. 5072) has been found successful in clinical trials, and exhibits efficacy in animal models with prostate cancer (PC-3M), breast cancer (BT474), pancreatic cancer (BxPc3), lung cancer (H460), brain cancer (U87MG), and the like. Furthermore, the concurrent administration of an FAK inhibitor (TAE226) and docetaxel showed excellent efficacy (85-97% tumor reduction, P values <0.01) in an animal model (Cancer Res. 2007, 67(22), 10976).

FAK is involved in the signaling of integrin as a cell membrane protein. When integrin receptors cluster in response to various external stimuli, the cytoplasmic domain (cytoplasmic tail) of integrin binds to the cytoskeleton and signaling proteins. The FERM (F for 4.1 protein, E for ezrin, R for radixin and M for moesin) domain and the focal-adhesion-targeting (FAT) domain of FAK independently bind to the intracellular domain of integrin and allow the FAK to be located at the focal adhesion site. The FAK clustered close to the focal adhesion site is activated via intramolecular or intermolecular phosphorylation of the Y397 residue. The SH2 domain of Src kinase binds to the phosphorylated Y397 residue of FAK to form an FAK/Src complex. The Src kinase bound to FAK further phosphorylates various tyrosine residues (Y407, Y576/577, Y861, and Y925) of FAK. In addition, the FAK/Src complex binds to various signaling proteins (P130Cas, Grb2, PI3K, and Grb7) and mediates phosphorylation. In the case of normal cells, signal transduction through FAK is mediated under strict regulation. However, in tumorous cells, FAK is overexpressed and activated, thereby exhibiting various characteristics of malignant tumors. FAK facilitates the proliferation of cancer cells and increases the invasion and migration of cancer cells. It has also been reported that FAK suppresses cancer cell apoptosis and increases angiogenesis.

FAK is a protein targeted by many growth factor receptors including epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), as well as integrin. Overexpression of the receptors or expression of activated receptors converts normal cells into tumor cells. Thus, FAK is an important kinase involved in tumor-related signal transduction of the receptors. It has been reported that the N-terminal FERM domain of FAK binds to EGFR and that the C-terminal domain of FAK is involved in cell migration mediated by epidermal growth factor (EGF). These findings demonstrate that FAK recognizes the signal from the EGFR receptor through the N-terminal FERM domain and recognizes the signal from the integrin through the C-terminal FAT domain of FAK, thereby integrating signals from outside the cell.

Apoptosis is induced by inhibiting FAK in various manners. Cell survival mediated by FAK is mainly conducted by phosphoinositide-3 kinase (PI-3 kinase). The phosphorylated Y397 of FAK binds to PI-3 kinase and synthesizes PI(3,4,5)P3 and PI(3,4)P2 as second messengers, which move protein kinase B (PKB; AKT) to the cell membrane so that it can be phosphorylated by 3'-phosphoinositide-dependent kinase (PDK). The activated PKB deactivates various apoptotic proteins (p21WAF, FKHR, Bad, and GSK3), thereby inhibiting apoptosis. Another signal of survival is the binding of the SH3 domain of p130Cas to the proline-rich motif of FAK, whereby the phosphorylation of various tyrosine residues of p130Cas is induced by FAK/Src to thus activate Ras.

The relationship between FAK and the cell cycle is as follows. When the phosphorylation of Y925 of FAK is induced, FAK binds to growth-factor-receptor-bound protein 2 (Grb2), thereby activating the Ras/Erk pathway. Overexpression of FAK facilitates G1 to S phase transition of the cell cycle, and expression of FAK-related non-kinase (FRNK), which is an inhibitor of FAK, inhibits the expression of cyclin D1 and induces the expression of the CDK inhibitor p21, thereby delaying the progression of the cell cycle. However, when cyclin D1 is overexpressed, the cell cycle arrest by FRNK is overcome.

The only subtype of FAK, proline-rich tyrosine kinase 2 (PYK2), is the most widely distributed in nerve cells, and it has recently been confirmed as an effective molecular target in the development of anticancer drugs for small cell lung cancer (Oncogene. 2008, 27(12), 1737), prostate cancer (Oncogene. 2007, 26(54), 7552), liver cell carcinoma (Br J Cancer. 2007, 97(1), 50), glioma (Neoplasia. 2005, 7(5), 435), and the like.

The domain organization of FAK consists of 4 domains: 1) The FERM (band 4.1 protein, ezrin, radixin, moesin) domain is an amino-terminal domain that interacts with integrin receptor, platelet-derived growth factor receptor (PDGFr), epidermal growth factor receptor (EGFr), and the like and inhibits kinase activity through direct interaction with the kinase domain; 2) the kinase domain; 3) three proline-rich (PR) regions; and 4) the focal-adhesion-targeting (FAT) domain positioned at the carboxyl-terminal interacts with paxillin, talin, p190RhoGEF, RhoA-specific GDP/GTP exchange, or the like. The alternative splicing product of FAK, FAK-related non-kinase domain (FRNK), consists of PR1, PR2, and FAT domains and acts as an antagonistic regulatory factor of FAK.

For the activation of FAK, auto-phosphorylation of Y397 located at the junction of the FERM and kinase domains is required. Src kinase binds to phosphorylated Y397 and sequentially phosphorylates Y576/577, and when Y925 is ultimately phosphorylated, the signal transduction system of FAK is turned on through Grb2. FAK inhibitors that are currently under development have a mechanism of inhibiting the auto-phosphorylation of Y397 by targeting the ATP-binding site of the kinase domain. The extent to which the auto-phosphorylation of Y397 is inhibited is used as an important biomarker in an efficiency test using an animal model.

The development conditions of low-molecular-weight FAK inhibitors are as follows. Although approximately 26 lead compounds of the FAK inhibitors have been proposed, only the Pfizer's PF-562271 compound is under clinical trial phase I at present. PF-562271 is an ATP-competitive FAK (IC=1.5 nM) inhibitor and a homology Pyk2 (13 nM) inhibitor, and inhibits auto-phosphorylation at the FAK Y397 site in fibroblasts, epithelial cells, and cancer cells. In addition, PF-562271 inhibits the migration of most cancer cells, but does not affect the growth of normal cells. No special toxicity was observed, and tumor growth inhibition or tumor degeneration by 42% to 90% was observed, in in-vivo human tumor xenograft tests (25 mg/kg to 100 mg/kg p.o.) for prostate cancer PC-3, breast cancer BT-474, colon LoVo, lung cancer NCI-H460, glioblastoma U-87 MG, pancreatic cancer BxPC-3, and the like.

Vascular endothelial growth factor receptors (VEGFRs) are receptor tyrosine kinases (RTKs) and important regulatory factors for angiogenesis. VEGFRs are involved in the formation of blood vessels and lymphatic vessels and in homeostasis, and also have important effects on nerve cells. Vascular endothelial growth factor (VEGF) is produced mostly in vascular endothelial cells, hematopoietic cells, and stromal cells under a hypoxic condition or by stimulations of cell growth factors such as TGF, interleukin, and PDGF. VEGF binds to VEGFR-1, -2, and -3, and each VEGF isoform binds to a specific receptor, thereby inducing the formation of a receptor homozygote or heterozygote, and then activates each signal transduction system. The signal specificity of VEGFR is further fine-tuned by co-receptors such as neurophilin, heparin sulfate, integrin, cadherin, or the like.

The biological function of VEGF is mediated by type Ⅲ RTK, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3(Flt-4). VEGFR is closely related to Fms, Kit, and PDGFR. Each VEGF binds to specific receptors. VEGF-A binds to VEGFR-1, VEGFR-2, and receptor zygotes, whereas VEGF-C binds to VEGF-2, and -3. In addition, PIGF and VEGF-B interact exclusively with VEGFR-1, and VEGF-E interacts only with VEGFR-2. VEGF-F variants interact with VEGFR-1 or -2. While VEGF-A, VEGF-B, and PIGF are preferentially required for the formation of blood vessels, VEGF-C and VEGF-D are essential in the formation of lymphatic vessels. Angiogenesis is essential in the proliferation and transition of tumors by supplying nutrients and oxygen to the tumors and providing channels for metastasis of cancer cells. Normally, angiogenesis is balanced by mutual regulation of angiogenesis promoters and angiogenesis inhibitors in vivo, but when the balance is broken, as in cancer cells, the growth factor (VEGF), which affects vascular endothelial cells the most, activates a receptor thereof, i.e., VEGFR. Various research on and development of inhibitors against these receptor tyrosine kinases of VEGF using low-molecular-weight synthetic substances among various action mechanisms are ongoing, and most of them are applicable to solid tumors and are expected to exhibit effective efficacy due to the relatively reduced incidence of side effects since they inhibit angiogenesis activated only in cancer cells.

Tie2 is a type of receptor tyrosine kinase and is deeply associated with angiogenesis and vasculature. The domain structure of Tie2 is very highly conserved in all vertebrates (Lyons et al., 1998). The ligand of Tie2 is angiopoietins (Ang). Ang2 does not induce auto-phosphorylation of Tie2, but interferes with the activation of Tie2 induced by Ang1. In endothelial cells, the activation of Tie2 by Ang2 induces the activation of PI3K-Akt (Jones et al., 1999). In the mitogen-activated protein kinase (MAPK) signal transduction pathway, which is the main signal transduction pathway of Tie2, the adaptor protein GRB2 and the protein tyrosine phosphatase SHP2 play a key role in dimerization of the Tie2 receptor tyrosine kinase through auto-phosphorylation. Ang/Tie2 and the VEGF signal transduction pathway play an important role in angiogenesis of cancer cells. Tie2 is expressed in vascular endothelial cells, and in particular is maximally expressed at sites of invasion of cancer cells. The overexpression of Tie2 has been verified in breast cancer (Peters et al., 1998) and has also been observed in uterine cancer, liver cancer, brain cancer, and the like.

According to previously published documents, various compounds using the thieno[3,2-d]pyrimidine structure as a parent body have been synthesized. However, as in the present invention, the substituted thieno[3,2-d]pyrimidine compound in which specific substituents are simultaneously substituted at the 2- and 7- positions of thieno[3,2-d]pyrimidine nucleus corresponds to a novel compound that has not been published in prior documents. Furthermore, no documents related to the use of a thieno[3,2-d]pyrimidine compound with specific substituents substituted at 2- and 7- positions thereof for treating or preventing tumors by confirming the inhibitory activity thereof against various protein kinases have been published to date.

Rearranged-during-transfection (RET) kinases are tyrosine kinase receptors associated with sympathetic and parasympathetic development. It has been reported that abnormal point mutation, chromosomal translocation, and gene amplification of the RET gene cause various types of thyroid cancer (MEN2A, MEN2B, FMTC, PTC, and the like). It has been reported that the point mutation of the RET gene causes the occurrence of MEN2A, MEN2B, and FMTC, which are thyroid cancer, and the chromosomal translocation and gene amplification of the RET gene cause the occurrence of thyroid cancer (MEN2A, MEN2B, FMTC, PTC, and the like). In addition, it has recently been reported that RET kinases are key factors that cause lung cancer, besides thyroid cancer. It has been reported that the RET gene is fused with the KIF5B gene or the CCDC6 gene to be expressed in the form of KIF5B-RET or CCDC6-RET, thereby causing the occurrence of non-small cell lung cancer (NSCLC), and the presence of NCOA4-RET or TRIM33-RET, obtained as a result of fusion of the RET gene with NCOA4 or tripartite-motif-containing 33 (TRIM33), has been observed in patients with NSCLC. Thus, the development of compounds capable of inhibiting RET kinases is very useful for the treatment of thyroid cancer or NSCLC. As RET tyrosine kinase inhibitors, sorafenib, sunitinib, XL184, vandetanib, ponatinib, cabozantinib, and the like are known, and these have been reported to have a cell proliferation inhibitory ability in cell lines expressing KIF5B-RET.

DDR1 is a type of receptor tyrosine kinase and has a discoidin homology domain at an extracellular site. Among the receptor tyrosine kinases, only the discoidin homology domain allows collagen as a ligand to bind to DDR1. When collagen is bound to DDR1, DDR1 is activated and auto-phosphorylated, whereby cell differentiation, cell adhesion, and the like are regulated. In addition, DDR1 induces the expression and activity of MMP, causing the degradation of extracellular matrix, which causes cell migration and metastasis. DDR1 is expressed in various tissues such as the brain, lungs, kidneys, pancreas, and the like, and the expression level of DDR1 is higher in solid cancers than in general tissues. A change in the expression level of DDR1 and mutation abnormally increase the activity of DDR1 and ultimately stimulate the proliferation of cells, thereby causing oncogenic transformation. Prognosis thereof is capable of being predicted based on the expression level of DDR in pancreatic cancer, gastric cancer, and lung cancer. Thus, DDR1 inhibition may be a novel method capable of selectively treating cancer.

FLT3 is a class III receptor tyrosine kinase that is normally expressed in hematopoietic cells and induces the activation of PI3K, Erk, and Jak/Stat signals, which are vital for the growth of blood cells. Normal FLT3 is dimerized by a ligand (FL) and activated as a result of an open activation loop and auto-phosphorylation. FLT3 mutation, which is activated at all times irrespective of the FL, induces hyperproliferation of leukemia embryonic cells, and approximately 35% of all AML patients actually exhibit the FLT3 mutation. Among them, ITD mutation maintains an activated state at all times by blocking the self-inhibition function of a juxtamembrane domain. ITD accounts for 35% of all AML patients, and moreover the prognosis thereof is very severe, that is, the disease-free survival and overall survival periods of the patients are considerably reduced. The point mutation (D835Y) of the activation loop is observed in 7% of AML patients, and such a point mutation is a cause of resistance to existing FLT3-inhibiting drugs.

Janus Kinase (JAK), which is a tyrosine kinase, includes JAK1, JAK2, JAK3, and TYK2. JAK plays a very important role in signal transduction of cytokine receptors. JAK consists of FERM-SH2-JH2 (pseudokinase domain)-JH1 (kinase domain). When cytokine is bound to a receptor, JAK families form heterodimers to phosphorylate each other, attract STATs, which are sub-molecules, and transmit signals into cells. Activated JAK kinases vary depending on the type of cytokine. For example, IL-2 and IL-4 activate JAK1-JAK3 heterodimers, and the complex is involved in lymphocyte proliferation. In contrast, JAK1 and JAK2 are activated by IL-6, which is related to T-cell differentiation and inflammation. Thus, JAK mutations are closely associated with autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases, and the like.

[References]

[Patent Document]

(Patent Document 1) Korean Patent Publication No. 10-2014-0019055

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel 2,7-substituted cyano[3,2-d]pyrimidine nucleus compound having specific substituents at 2- and 7-positions of the cyano[3,2-d]pyrimidine nucleus, or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth, which includes, as an active ingredient, the above-described novel 2,7-substituted cyano[3,2-d]pyrimidine based compound or a pharmaceutically acceptable salt thereof.

In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof:

[Formula 1]

wherein, in Formula 1,

Q₁ and Q₂ are each independently any one of -(CH₂)_n-, -O(CH₂)_n-, -N(CH₂)_n-, and -NH(CH₂)_n- wherein n is an integer;

A and B are each independently any one of a heteroaryl ring, a cyclic hydrocarbon, and an alkyl group;

M is any one of -CHCH-, -CH₂CH₂-, -NHCO-, -CONH-, -NMe-, -NAc-, -NMs-, -NHCONHR₁-, -NHCOR₁-, -NHSONR₁-, and -O- wherein R₁ is any one of an alkyl group, a cyclic hydrocarbon, a phenyl group, and a substituted phenyl group.

In an embodiment of the present invention, a compound wherein n is an integer of 0, 1, 2, 3, or 4, the heteroaryl ring is any one of phenyl, pyridine, substituted phenyl, and pyrazole, and the cyclic hydrocarbon is any one or more selected from piperazine and cyclohexane, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof is provided.

In an embodiment of the present invention, a compound selected from the group consisting of:

(E)-4¹H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 1);

(Z)-1¹H-5-oxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(4,1)-pyrazolo-4(1,4)-benzenecyclotetradecaphane-10-one (Compound 2);

5,15-dioxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(5,2)-pyridine-4(1,4)-benzenecyclopentadecaphane-10-one (Compound 3);

(E)-4¹H-14-oxa-3,10-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 4);

5,15-dioxa-3,11-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1,4(1,4)-dibenzenacyclopentadecaphan-10-one (Compound 5);

(E)-4¹H-8-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-5(4,1)-piperidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclooctepene (Compound 6);

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (Compound 7);

(E)-8-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 8);

(E)-8-ethyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 9);

(E)-8-propyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 10);

(E)-1-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)ethene-1-one (Compound 11);

(E)-8-(methylsulfonyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 12);

(E)-N-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazola-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 13);

(E)-N-(4-chlorophenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carbothioamide (Compound 14);

(E)-N-(3-acetylphenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 15);

(E)-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)(phenyl)methenone (Compound 16);

(E)-4¹H-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 17);

(E)-1³-fluoro-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 18);

(4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene (Compound 19);

(E)-4¹H-10-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-8(2,3)-oxirana-1(1,4)-benzenecyclodecaphane (Compound 20); and

(E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8,9-diol (Compound 21), a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof is provided.

In accordance with another aspect of the present invention, there is provided a pharmaceutical composition including, as an active ingredient, any one of the aforementioned compounds, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

In an embodiment of the present invention, the composition is a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth through protein kinase inhibition.

In another embodiment of the present invention, the compound has an inhibitory capability of 90% or more against a protein kinase at a concentration of 1 μM.

In another embodiment of the present invention, the protein kinase is any one or more selected from ALK, ARK5, Aurora A, Aurora B, Aurora C, AXL, BLK, BMX, c-MET, c-src, CDK2, CK2a2, CLK1, CLK2, CLK4, DAPK1, DDR1, DDR2, DYRK2, DYRK3, ERk7, FER, FES, FGFR1, FGFR2, FGFR3, FGR, FLT3, FLT4, DMS, FYN, HCK, HIPK1, HIPK2, HIPK4, HPK, IGF1R, IR, IRR, JAK1, JAK2, JAAK3, KDR, LCK, LOK, LRRK2, MARk4, MELK, MLK3, MYLK4, NEK1, NEK5, NEK9, p70S6K, PDGFRa, PDGFRb, PDK1, PEAK1, PIM1, PKCd, PKC-epsilon, PKCg, PKCmu, PKCnu, PKD2, RET, ROS, RSK1, RSK2, RSK3, RSK4, SIK1, SIK2, SNARK, STK16, STK38, STK38L, TRKA, TRKB, TRKC, TXK, TYK2, TYRO3, ULK1, YES, and ZIPK.

In another embodiment of the present invention, the disease caused by abnormal cell growth is a tumor disease.

In another embodiment of the present invention, the tumor disease is selected from the group consisting of stomach cancer, lung cancer, liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis, uterine cancer, uterine cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer such as leukemia, multiple myeloma, and myelodysplastic syndrome, lymphoma such as Hodgkin's disease and non-Hodgkin's lymphoma, and fibroadenoma.

In accordance with a further aspect of the present invention, there is provided a method of preparing any one of the aforementioned compounds, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, the method comprising reacting any one intermediate compound of the 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound, selected from:

7-(4-(2-bromoethoxy)phenyl)-N-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine;

N-(1-(3-aminopropyl)-1H-pyrazole-4-yl)-7-(4-(2-bromoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-amine;

5-(4-(2-((1-(3-aminopropyl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentenoic acid;

5-(4-((7-(4-(3-aminoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)-1H-pyrazole-1-yl)pentenoic acid;

5-((5-(2-((4-(3-aminopropoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)pyrimidine-2-yl)oxy)pentanoic acid;

5-(4-((7-(4-(3-aminopropoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)phenoxy)pentanoic acid; and

5-(4-(2-((4-(3-aminopropoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)-1H-pyrazole-1-yl)pentanoic acid.

The 2,7-substituted cyano[3,2-d]pyrimidine compound represented by Formula 1 of the present invention, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof has an excellent ability of inhibiting the activity of one or more kinases selected from ALK, ARK5, Aurora A, Aurora B, Aurora C, AXL, BLK, BMX, c-MET, c-src, CDK2, CK2a2, CLK1, CLK2, CLK4, DAPK1, DDR1, DDR2, DYRK2, DYRK3, ERk7, FER, FES, FGFR1, FGFR2, FGFR3, FGR, FLT3, FLT4, DMS, FYN, HCK, HIPK1, HIPK2, HIPK4, HPK, IGF1R, IR, IRR, JAK1, JAK2, JAAK3, KDR, LCK, LOK, LRRK2, MARk4, MELK, MLK3, MYLK4, NEK1, NEK5, NEK9, p70S6K, PDGFRa, PDGFRb, PDK1, PEAK1, PIM1, PKCd, PKCeplsilon, PKCg, PKCmu, PKCnu, PKD2, RET, ROS, RSK1, RSK2, RSK3, RSK4, SIK1, SIK2, SNARK, STK16, STK38, STK38L, TRKA, TRKB, TRKC, TXK, TYK2, TYRO3, ULK1, YES, and ZIPK, and thus is effective as a therapeutic agent for preventing, alleviating, or treating a disease caused by abnormal cell growth.

The disease caused by abnormal cell growth, which is capable of being prevented or treated using the compound according to the present invention, may include various tumor diseases selected from stomach cancer, lung cancer, liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis, uterine cancer, uterine cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer such as leukemia (acute myeloid leukemia), multiple myeloma, and myelodysplastic syndrome, lymphoma such as Hodgkin's disease and non-Hodgkin's lymphoma, and fibroadenoma.

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawing, in which:

FIG. 1 to 3 illustrate the results of monitoring the tumor volume of mice according to Experimental Example 6 of the present invention.

Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

Hereinafter, the present invention will be described in detail.

According to an embodiment of the present invention, there is provided a 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof:

[Formula 1]

wherein, in Formula 1,

In one embodiment of the present invention, in Formula 1, Q1 and Q2 are each independently any one of -(CH2)n-, -O(CH2)n-, -N(CH2)n-, and -NH(CH2)_n- wherein n is an integer; A and B are each independently any one of a C₃-C₈ cyclic hydrocarbon, a C₂-C₇ heterocyclic hydrocarbon, a monocyclic aromatic compound with three or more carbon atoms, and a heterocyclic aromatic compound with two or more carbon atoms; and M is any one of -CHCH-, -CH₂CH₂-, -NHCO-, -CONH-, -NMe-, -NAc-, -NMs-, -NHCONHR₁-, -NHCOR₁-, -NHSONR₁-, and -O-, wherein R₁ may be any one selected from a substituted or unsubstituted saturated hydrocarbon with two or more carbon atoms, a substituted or unsubstituted unsaturated hydrocarbon with two or more carbon atoms, a cyclic hydrocarbon with 3 to 8 carbon atoms, a heterocyclic hydrocarbon with 2 to 7 carbon atoms, a monocyclic aromatic compound with three or more carbon atoms, and a heterocyclic aromatic compound with two or more carbon atoms.

A pharmaceutically acceptable salt of the 2,7-substituted cyano[3,2-d]pyrimidine compound of Formula 1 according to the present invention may be prepared using a method commonly used in the art. Pharmaceutically acceptable salts should have low toxicity to the human body and should not adversely affect the biological activity or physicochemical properties of the parent compound. Pharmaceutically acceptable salts include pharmaceutically acceptable free acids and acid addition salts of base compounds of Formula 1, alkali metal salts (sodium salts and the like) and alkali earth metal salts (calcium salts and the like), organic salts and organic base addition salts of carboxylic acid of Formula 1, and amino acid addition salts. Free acids that may be used in the preparation of pharmaceutically acceptable salts may be divided into inorganic acids and organic acids. Inorganic acids may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, and the like. Organic acids may be acetic acid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, benzoic acid, embonic acid, aspartic acid, or glutamic acid. Organic bases that may be used in the preparation of organic base addition salts include tris(hydroxymethyl)methylamine, dicyclohexylamine, and the like. Amino acids that may be used in the preparation of amino acid addition salts include natural amino acids such as alanine, glycine, and the like.

The 2,7-substituted cyano[3,2-d]pyrimidine compound of Formula 1 according to the present invention includes all hydrates and solvates thereof, in addition to the above-described pharmaceutically acceptable salts. The hydrates and solvates may be obtained by dissolving the 2,7-substituted cyano[3,2-d]pyrimidine compound of Formula 1 in a solvent miscible with water, such as methanol, ethanol, acetone, or 1,4-dioxane, and then adding a free acid or an organic base thereto, followed by crystallization or recrystallization. In this case, a solvate (particularly a hydrate) may be formed. Thus, the compounds of the present invention also include stoichiometric solvates, including hydrates, other than various amounts of water-containing compounds that may be prepared using a method such as lyophilization.

The substituents used to define the compounds according to the present invention are described in more detail below.

The term "halogen atom" as used herein refers to a fluorine, chlorine, bromine, or iodine atom.

The term "alkyl group" as used herein refers to an aliphatic saturated hydrocarbon having 1 to 6 carbon atoms, including methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, t-butyl, cyclobutyl, cyclopropylmethyl, n-pentyl, i-pentyl, neopentyl, t-pentyl, cyclopentyl, cyclobutylmethyl, n-hexyl, i-hexyl, cyclohexyl, cyclopentylmethyl, and the like.

The term "haloalkyl group" as used herein refers to an alkyl group, a hydrogen atom of which is substituted with one or more halogen atoms, such as a trifluoromethyl group.

The term "alkoxy group" as used herein refers to a hydroxyl group in which a hydrogen atom of the C-C alkyl group is substituted with a selected substituent, including methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, and t-butoxy.

The term "heteroaryl group" as used herein refers to a monocyclic, bicyclic, or tricyclic aromatic heterohydrocarbon group containing one or more heteroatoms, including pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, indolyl, isoindolyl, benzofuranyl, benzofurazanyl, dibenzofuranyl, isobenzofuranyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, dibenzothiophenyl, naphthyridyl, benzisothiazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, phthalazinyl, chinolinyl, quinazolinyl, and the like.

The term "heterocyclic group" as used herein refers to a heterocyclic hydrocarbon group containing one or more heteroatoms, including a morpholinyl group, a piperidine group, a piperazinyl group, a N-protected piperazinyl group, and the like.

The above-described 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound of Formula 1 is described in further detail below:

(E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8,9-diol (Compound 21).

According to another embodiment of the present invention, there is provided a pharmaceutical compound including, as an active ingredient, any one of the above-described compounds, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

In an embodiment of the present invention, the composition may be a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth through protein kinase inhibition.

In another embodiment of the present invention, the protein kinase may be any one or more selected from ALK, ARK5, Aurora A, Aurora B, Aurora C, AXL, BLK, BMX, c-MET, c-src, CDK2, CK2a2, CLK1, CLK2, CLK4, DAPK1, DDR1, DDR2, DYRK2, DYRK3, ERk7, FER, FES, FGFR1, FGFR2, FGFR3, FGR, FLT3, FLT4, DMS, FYN, HCK, HIPK1, HIPK2, HIPK4, HPK, IGF1R, IR, IRR, JAK1, JAK2, JAAK3, KDR, LCK, LOK, LRRK2, MARk4, MELK, MLK3, MYLK4, NEK1, NEK5, NEK9, p70S6K, PDGFRa, PDGFRb, PDK1, PEAK1, PIM1, PKCd, PKC-epsilon, PKCg, PKCmu, PKCnu, PKD2, RET, ROS, RSK1, RSK2, RSK3, RSK4, SIK1, SIK2, SNARK, STK16, STK38, STK38L, TRKA, TRKB, TRKC, TXK, TYK2, TYRO3, ULK1, YES, and ZIPK.

The pharmaceutical composition of the present invention may include, as an active ingredient, the 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound of Formula 1, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof, and may be formulated into general preparations in the pharmaceutical field, e.g., preparations for oral or parenteral administration such as tablets, capsules, troches, liquids, suspensions, and the like by adding, to the above compound, a non-toxic pharmaceutically acceptable carrier, a reinforcing agent, an excipient, and the like.

Examples of excipients that may be used in the pharmaceutical composition of the present invention may include a sweetener, a binder, a solubilizing agent, a dissolution adjuvant, a wetting agent, an emulsifier, an isotonic agent, an adsorbent, a disintegrating agent, an antioxidant, a preservative, a lubricant, a filler, a fragrance, and the like. Examples thereof may include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, Tragacanth rubber, alginic acid, sodium alginate, methyl cellulose, sodium carboxyl methylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, and the like.

In addition, a dose of the compound according to the present invention to the human body may vary depending on the age, body weight, and gender of a patient, an administration route, a state of health, and the severity of disease, and generally ranges from 0.01 mg/day to 1,000 mg/day based on a body weight of an adult patient of 70 kg, and the compound according to the present invention may be administered once to multiple times a day at constant time intervals according to the determination of a doctor or a pharmacist.

According to another embodiment of the present invention, there is provided a method of preparing any one of the above-described compounds, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, the method comprising reacting any one intermediate compound of the 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound, selected from:

Meanwhile, the present invention is characterized by a method of preparing the 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound of Formula 1, and a typical preparation method thereof is as follows.

Compounds represented by Formula 1 have cyano[3,2-d]pyrimidine as a backbone. Cyclic hydrocarbons such as benzene rings were introduced through the Suzuki reaction at position 7 from the backbone. Thereafter, an alkyl group having a cyclizable functional group was linked thereto. Then, an alkyl group having a cyclizable functional group was linked at position 2, and a cyclic compound having an amine group was substituted through the Buchwald reaction. After substituents were introduced at positions 2 and 7, cyclic compounds were synthesized through cyclizable reactions.

The present invention as described above will be described in further detail with reference to the following examples, formulation examples, and experimental examples, but these examples, formulation examples, and experimental examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLES

The following examples illustrate the invention and are not intended to limit the same.

Example 1. (E)-4¹H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 1)

[Formula 2]

Process 1: 4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenol

7-bromo-2-chlorothieno[3,2-d]pyrimidine (5.16 g, 20.7 mmol) was dissolved in dioxane (138 mL), and then 2.0 N sodium carbonate (62.1 mL) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (2 g, 14.61 mmol) were added thereto. After flowing nitrogen into the mixed solution for 10 minutes, Pd(PPh)Cl (1.45 g, 2.07 mmol) and t-ButylXphos (0.88 g, 2.07 mmol) were added thereto. The mixed reaction solution was stirred at 100 °C for 6 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (4.3 g, yield: 81%). ¹H NMR (400 MHz, DMSO-d ₆) δ 9.52 (s, 1H), 8.65 (s, 1H), 7.82 (m, 2H), 6.89 (m, 2H). MS m/z: 263 [M+1].

Process 2: ethyl 5-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoate

4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenol (1 g, 3.82 mmol) was dissolved in dimethylformamide (16 mL), and then calcium carbonate (1.06 g, 7.64 mmol) and ethyl 5-bromopentenoate (4.2 mmol, 0.88 g) were added thereto. The mixed reaction solution was stirred at 100 °C for 3 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (1.12 g, yield: 74%). MS m/z: 391 [M+1].

Process 3: tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate

4-nitro-1H-pyrazole (3 g, 26.54 mmol) was dissolved in acetonitrile (100 mL), and then calcium carbonate (7.33 g, 53.08 mmol) and tert-butyl(3-bromopropyl)carbamate (6.96 g, 29.2 mmol). The mixed reaction solution was stirred at 60 °C for 3 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated to obtain a target compound (6.81 g, yield: 95%) without purification. MS m/z: 271 [M+1].

Process 4: tert-butyl3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate

Tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate (6.81 g, 17.46 mmol) was dissolved in methanol (105 mL), and then Pd ( 0.7 g) was added thereto. The resulting solution was stirred for 1 day at room temperature and a pressure inside a balloon filled with hydrogen gas. The mixed reaction solution was filtered through Celite and concentrated to obtain a target compound (5.44 g, yield: 91%) without purification, and the target compound was used in the subsequent reaction. MS m/z: 241 [M+1].

Process 5: Ethyl 5-(4-(2-((1-(3-((tert-butoxycarbonyl)amino)propyl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentonate

Ethyl 5-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoate (1 g, 2.56 mmol) was dissolved in 17 ml of sec-butanol, and then calcium carbonate (1.77 g, 12.8 mmol) and tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate (0.68 g, 2.82 mmol) were added thereto. After flowing nitrogen into the mixed solution for 10 minutes, Pd₂(dba)₃ (0.23 g, 0.26 mmol) and Xphos (0.12 g, 0.26 mmol) were added thereto. The mixed reaction solution was stirred at 100 °C for 2 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (1.26 g, yield: 83%). MS m/z: 595 [M+1].

Process 6: 5-(4-(2-((1-(3-((tert-butoxycarbonyl)amino)propyl)-1H-pyrazo-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoic acid

Ethyl 5-(4-(2-((1-(3-((tert-butoxycarbonyl)amino)propyl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentonate (1.26 g, 2.12 mmol) was dissolved in tetrohydrofuran (4 mL), methanol (4 mL), and water (4 mL), and then LiOH-H₂O (0.95 g 21.2 mmol) was added thereto. The mixed reaction solution was stirred for 1 day at room temperature. The mixed reaction solution was diluted with ethyl acetate, neutralized with a 2 N aqueous hydrogen chloride solution to a pH of 5, and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. A target compound (1.03 g, yield: 86%) was obtained without purification. MS m/z: 567 [M+1].

Process 7: 5-(4-(2-((1-(3-aminopropyl)-1H-pyrazo-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoic acid

5-(4-(2-((1-(3-((tert-butoxycarbonyl)amino)propyl)-1H-pyrazo-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoic acid (1.09 g, 1.93 mmol) was dissolved in dioxane (10 mL), and then 4 M hydrogen chloride-dioxane (10 mL) was added thereto. The mixed reaction solution was stirred for 3 hours at room temperature. The mixed reaction solution was diluted with ethyl acetate, neutralized with an aqueous sodium bicarbonate saturated solution to a pH of 5, and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. A target compound (0.83 g, yield: 93%) was obtained without purification. MS m/z: 467 [M+1].

Process 8: (E)-4¹H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one

5-(4-(2-((1-(3-aminopropyl)-1H-pyrazo-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentanoic acid (0.83 g, 1.78 mmol) was dissolved in dimethylformamide (35 mL), and then TBTU (0.57 g, 1.78 mmol) and HOBt (72 mg, 0.53 mmol) were added thereto. The resulting solution was stirred for 10 minutes at room temperature, and then triethylamine (0.49 mL, 3.56 mmol) was slowly added thereto and stirred for 1 day. The mixed reaction solution was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (0.55 g, yield: 72%). MS m/z: 449 [M+1].

Example 2. (Z)-1¹H-5-oxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(4,1)-pyrazolo-4(1,4)-benzenecyclotetradecaphane-10-one (Compound 2)

[Formula 3]

Process 1: 2-chloro-7-(1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine

An experiment was performed in the same manner as in process 1 of Example 1, except that tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate was used instead of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol.

Process 2: Ethyl 5-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)-1H-pyrazo-1-yl)pentonate

An experiment was performed in the same manner as in process 2 of Example 1.

Process 3: tert-butyl(3-(4-nitrophenoxy)propyl)carbamate

4-nitrophenol (1 g, 7.19 mmol) was dissolved in 5 ml of dimethylformamide, and then calcium carbonate (1.98 g, 14.4 mmol) and tert-butyl (3-bromopropyl)carbamate (1.89 g, 7.91 mmol) were added thereto. The mixed reaction solution was stirred at 100 °C for 3 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated to obtain a target compound (1.87 g, yield: 88%) without purification. MS m/z: 297 [M+1].

Process 4: tert-butyl (3-(4-aminorophenoxy)propyl)carbamate

Tert-butyl(3-(4-nitrophenoxy)propyl)carbamate (1.87 g, 6.33 mmol) was dissolved in 20 ml of methanol, and then Pd (0.2 g) was added thereto. The resulting solution was stirred for 1 day at room temperature and a pressure inside a balloon filled with hydrogen gas. The mixed reaction solution was filtered through Celite and concentrated to obtain a target compound (1.55 g, yield: 91%) without purification, and the target compound was used in the subsequent reaction. MS m/z: 267 [M+1].

Process 5: Ethyl 5-(4-(2-((4-(3-((tert-butoxycarbonyl)amino)propoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)-1H-pyrazo-1-yl)pentenoate

An experiment was performed in the same manner as in process 5 of Example 1, except that tert-butyl (3-(4-aminophenoxy)propyl)carbamate was used instead of tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate. MS m/z: 595 [M+1].

Process 6: 5-(4-(2-((4-(3-((tert-butoxycarbonyl)amino)propoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)-1H-pyrazo-1-yl)pentanoic acid

An experiment was performed in the same manner as in process 6 of Example 1. MS m/z: 567 [M+1].

Process 7: 5-(4-(2-((4-(3-aminopropoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)-1H-pyrazole-1-yl)pentanoic acid

An experiment was performed in the same manner as in process 7 of Example 1. MS m/z: 467 [M+1].

Process 8: (Z)-1¹H-5-oxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(4,1)-pyrazolo-4(1,4)-benzenecyclotetradecaphane-10-one

An experiment was performed in the same manner as in process 8 of Example 1. MS m/z: 449 [M+1].

Example 3. 5,15-dioxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(5,2)-pyridine-4(1,4)-benzenecyclopentadecaphane-10-one (Compound 3)

[Formula 4]

¹H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 9.12 (s, 1H), 8.93 (m, 1H), 8.40(s, 1H), 7.94 (m, 1H), 7.55 (m, 3H), 6.91(m, 2H), 6.50 (m, 1H), 4.00 (m, 2H), 3.87 (m, 2H), 3.27 (m, 4H), 2.11 (m, 2H), 1.88 (m, 2H), 1.55 (m, 4H). MS m/z: 476 [M+1].

Process 1: 5-(2-chlorocyano[3,2-d]pyridine-7-yl)pyridine-2-ol

An experiment was performed in the same manner as in process 1 of Example 1, except that 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane 2-yl)pyridine-2-ol was used instead of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. MS m/z: 364 [M+1].

Process 2: Ethyl 5-((5-(2-chlorocyano [3,2-d]pyridine-7-yl)pyrimidine-2-yl)oxy)pentanoate

An experiment was performed in the same manner as in process 2 of Example 1. MS m/z: 392 [M+1].

The subsequent processes were performed in the same manner as in processes 3 to 8 of Example 2.

Example 4. (E)-4¹H-14-oxa-3,10-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 4)

[Formula 5]

Process 1: Tert-butyl (3-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenoxy)propyl)carbamate

4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenol (1 g, 3.82 mmol) was dissolved in dimethylformamide (16 mL), and then calcium carbonate (1.06 g, 7.64 mmol) and tert-butyl(3-bromopropyl)carbamate (1.00 g, 4.20 mmol) were added thereto. The mixed reaction solution was stirred at 100 °C for 3 hours and then filtered through Celite. The filtrate was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (1.26 g, yield: 79%). MS m/z: 391 [M+1].

Process 2: Ethyl 5-(4-nitro-1H-pyrazole-1-yl)pentenoate

An experiment was performed in the same manner as in process 3 of Example 1, except that ethyl 5-bromopentenenoate was used instead of tert-butyl(3-bromopropyl)carbamate. MS m/z: 242 [M+1].

Process 3: Ethyl 5-(4-amino-1H-pyrazole-1-yl)pentenenoate

An experiment was performed in the same manner as in process 4 of Example 1. MS m/z: 222 [M+1].

Process 4: Ethyl 5-(4-((7-(4-(3-((tert-butoxycarbonyl)amino)propoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)-1H-pyrazole-1-yl)pentenenoate

An experiment was performed in the same manner as in process 5 of Example 1, except that ethyl 5-(4-amino-1H-pyrazole-1-yl)pentenenoate was used instead of tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate. MS m/z: 595 [M+1].

The subsequent processes were performed in the same manner as in processes 6 to 8 of Example 1.

Example 5. 5,15-dioxa-3,11-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1,4(1,4)-dibenzenacyclopentadecaphan-10-one (Compound 5)

[Formula 6]

Process 1: Ethyl 5-(4-nitrophenoxy)pentenenoate

An experiment was performed in the same manner as in process 2 of Example 1. MS m/z: 268 [M+1].

Process 2: Ethyl 5-(4-aminophenoxy)pentenenoate

An experiment was performed in the same manner as in process 4 of Example 1. MS m/z: 238 [M+1].

Process 3: Ethyl 4-(4-((7-(4-(3-((tert-butoxycarbonyl)amino)propoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)phenoxy)butenoate

An experiment was performed in the same manner as in process 5 of Example 1, except that ethyl 5-(4-aminophenoxy)pentenenoate was used instead of tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate. MS m/z: 607 [M+1].

The subsequent experiment was performed in the same manner as in processes 6 to 8 of Example 1.

Example 6. (E)-4¹H-8-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-5(4,1)-piperidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclooctepene (Compound 6)

[Formula 7]

Process 1: 7-(4-(2-bromoethoxy)phenyl)-2-chlorothieno[3,2-d]pyrimidine

4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenol (1 g, 3.82 mmol) was dissolved in tetrahydrofuran (13 mL), and then triphenylphosphin (1.5 g, 5.73 mmol) and 2-bromoethanol (0.54 mL, 7.64 mmol) were added thereto. After stirring for 5 minutes, diisopropyl azodicarboxylate (1.13 mL, 5.73 mmol) was slowly added to the resulting solution and then stirred for 1 day at room temperature. The reaction solution was diluted with ethyl acetate, and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (0.97, yield: 69%). MS m/z: 369 [M+1].

Process 2: tert-butyl 4-(4-((7-(4-(2-bromoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)-1H-pyrazole-1-yl)piperidine-1-carboxylate

An experiment was performed in the same manner as in process 5 of Example 1. MS m/z: 599 [M+1].

Process 3: 7-(4-(2-bromoethoxy)phenyl)-N-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine

An experiment was performed in the same manner as in process 7 of Example 1. MS m/z: 499 [M+1].

Process 4: (E)-4¹H-8-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-5(4,1)-piperidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclooctepene

7-(4-(2-bromoethoxy)phenyl)-N-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine (0.1 g, 0.2 mmol) was dissolved in 2-ethoxymethanol (4 mL), and then triethylamine (0.05 mL, 0.4 mmol) was added thereto. The resulting solution was stirred for 1 day at 100 °C. The reaction solution was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (44 mg, yield: 53%). MS m/z: 419 [M+1].

Example 7. (E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (Compound 7)

[Formula 8]

Process 1: Tert-butyl (3-(4-((7-(4-(2-bromoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)-1H-pyrazole-1-yl)propoxy)carbamate

An experiment was performed in the same manner as in process 5 of Example 1. MS m/z: 573 [M+1].

Process 2: N-(1-(3-aminopropyl)-1H-pyrazole-4-yl)-7-(4-(2-bromoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-amine

An experiment was performed in the same manner as in process 7 of Example 1.

Process 3: (E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane

An experiment was performed in the same manner as in process 4 of Example 6. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.65 (s, 1H), 9.11 (s, 1H), 8.31 (s, 1H), 8.23 (s, 1H), 7.71 (m, 2H), 7.26 (m, 3H), 4.34(m, 2H), 4.10 (m, 2H), 2.69 (m, 2H), 2.62 (m, 2H), 1.69 (m, 2H). MS m/z: 393 [M+1].

Example 8. (E)-8-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 8)

[Formula 9]

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (0.1 g, 0.26 mmol) was dissolved in dichloromethane (2.6 mL), and then acetic acid (1 droplet) and formaldehyde (0.03 mL, 0.78 mmol) were added thereto. After stirring for 30 minutes, NaBH(OAc)₃ (29 mg, 0.14 mmol) was slowly added to the resulting solution and then stirred for 1 day. The reaction solution was diluted with dichloromethane, and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (76 mg, yield: 72%). ¹H NMR (400 MHz, DMSO-d ₆) δ 9.65 (s, 1H), 9.12 (s, 1H), 8.33 (s, 1H), 8.29 (s, 1H), 7.78 (m, 2H), 7.26 (m, 1H), 7.20 (m, 2H), 4.40(m, 2H), 4.10 (m, 2H), 2.42 (m, 2H), 2.21 (s, 3H), 1.73 (m, 2H). MS m/z: 406 [M+1].

Example 9. (E)-8-ethyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 9)

[Formula 10]

An experiment was performed in the same manner as in Example 8. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.64 (s, 1H), 9.11 (s, 1H), 8.31 (m, 2H), 7.80 (m, 2H), 7.25 (s, 1H), 7.18 (m, 2H), 4.39 (m, 2H), 4.09 (m, 2H), 1.74 (m, 2H), 0.98 (m, 3H). MS m/z: 421 [M+1].

Example 10. (E)-8-propyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 10)

[Formula 11]

An experiment was performed in the same manner as in Example 8. ¹H NMR (400 MHz, CDCl₃) δ 8.89 (s, 1H), 8.41 (s, 1H), 7.87 (s, 1H), 7.81 (m, 2H), 7.30 (s, 1H), 7.15 (m, 2H), 7.01 (s, 1H), 4.42 (s, 2H), 4.18 (m, 2H), 2.69 (m, 2H), 2.53 (m, 2H), 2.35 (m, 2H), 1.93 (m, 2H), 0.94 (m, 3H). MS m/z: 434 [M+1].

Example 11. (E)-1-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)ethene-1-one (Compound 11)

[Formula 12]

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (0.1 g, 0.26 mmol) was dissolved in dichloromethane (2.6 mL), and then acetic anhydride (0.04 mL, 0.39 mmol) was added thereto. Triethylamine (0.2 mL, 0.52 mmol) was added to the resulting solution at 0 °C and then stirred at room temperature for 20 minutes. The reaction solution was diluted with dichloromethane and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (85 mg, yield: 76%). MS m/z: 435 [M+1].

Example 12. (E)-8-(methylsulfonyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 12)

[Formula 13]

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (0.1 g, 0.26 mmol) was dissolved in dichloromethane (2.6 mL), and then methanesulfonyl chloride (0.03 mL, 0.39 mmol) was added thereto. Triethylamine (0.2 mL, 0.52 mmol) was added to the resulting solution at 0 °C and then stirred at room temperature for 5 minutes. The reaction solution was diluted with dichloromethane and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (68 mg, yield: 56%). MS m/z: 471 [M+1].

Example 13. (E)-N-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazola-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 13)

[Formula 14]

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (0.1 g, 0.26 mmol) was dissolved in tetahydrofuran (2.6 mL), and then methyl isocyanate (0.03 ml, 0.52 mmol) was added thereto. The resulting solution was stirred at room temperature for 30 minutes. The reaction solution was diluted with ethyl acetate and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (59 mg, yield: 51%). MS m/z: 450 [M+1].

Example 14. (E)-N-(4-chlorophenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carbothioamide (Compound 14)

[Formula 15]

An experiment was performed in the same manner as in Example 13. MS m/z: 562 [M+1].

Example 15. (E)-N-(3-acetylphenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 15)

[Formula 16]

An experiment was performed in the same manner as in Example 13. MS m/z: 554 [M+1].

Example 16. (E)-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)(phenyl)methenone (Compound 16)

[Formula 17]

An experiment was performed in the same manner as in Example 12. MS m/z: 497 [M+1].

Example 17. (E)-4¹H-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 17)

[Formula 18]

MS m/z: 391 [M+1].

Process 1: 3-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenyl)propane-1-ol

An experiment was performed in the same manner as in process 1 of Example 1, except that 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propane-1-ol was used instead of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. ¹H NMR (400 MHz, CDCl₃) δ 9.00 (s, 1H), 7.98 (s, 1H), 7.71 (m, 2H), 7.18 (m, 2H), 3.65(m, 2H), 2.67 (m, 2H), 1.86 (m, 2H). MS m/z: 305 [M+1].

Process 2: 7-(4-(3-bromopropyl)phenyl)-2-chlorocyano[3,2-d]pyrimidine

3-(4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)phenyl)propane-1-ol (1 g, 3.28 mmol) was dissolved in dichloromethane (22 mL), and then PBr₃ (0.62 mL, 6.56 mmol) was slowly added thereto at 0 °C. The resulting solution was stirred for 1 hour. The reaction solution was diluted with ethyl acetate and then washed with an aqueous sodium bicarbonate saturated solution, and then washed with salt water. The organic layer was dried over magnesium sulfate to obtain a target compound (1.09 g, yield: 91%) without purification. MS m/z: 366 [M+1].

The subsequent experiment was performed in the same manner as in processes 1 to 3 of Example 7.

Example 18. (E)-1³-fluoro-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 18)

[Formula 19]

MS m/z: 411 [M+1].

Process 1: 4-(2-chlorocyano[3,2-d]pyrimidine-7-yl)-2-fluorophenol

An experiment was performed in the same manner as in process 1 of Example 1, except that 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol was used instead of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. ¹H NMR (400 MHz, Acetone) δ 9.42 (m, 1H), 8.61(m, 1H), 7.92 (m, 1H), 7.74 (m, 1H), 7.12 (m, 1H), 5.60 (m, 1H). MS m/z: 280 [M+1].

The subsequent experiment was performed in the same manner as in Example 7.

Example 19. (4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene (Compound 19)

[Formula 20]

Process 1: 4-nitro-1-petene-4-ene-1-yl)-1H-pyrazole

An experiment was performed in the same manner as in process 3 of Example 1, except that 5-bromopentene-1-ene was used instead of tert-butyl(3-bromopropyl)carbamate. MS m/z: 182 [M+1].

Process 2: 1-(pen-4-tene-1-yl)-1H-pyrazole-4-amine

An experiment was performed in the same manner as in process 4 of Example 1. MS m/z: 152 [M+1].

Process 3: 4-(2-((1-(pene-4-tene-1-yl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenol

An experiment was performed in the same manner as in process 5 of Example 1, except that 1-(pene-4-tene-1-yl)-1H-pyrazole-4-amine was used instead of tert-butyl(3-(4-nitro-1H-pyrazo-1-yl)propyl)carbamate. MS m/z: 378 [M+1].

Process 4: 7-(4-(aliloxy)phenyl)-N-(1-(pen-4-tene-1-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine

4-(2-((1-(pen-4-tene-1-yl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenol (0.5 g, 1.33 mmol) was dissolved in dimethylformamide (13 mL), and then calcium carbonate (367 mg, 2.66 mmol) and allylbromide (0.17 mL, 2.00 mmol) were added thereto. The resulting solution was stirred at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (0.4 g, yield: 72%). MS m/z: 418 [M+1].

Process 5: (4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene

7-(4-(aliloxy)phenyl)-N-(1-(pen-4-tene-1-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine (0.3 g, 0.72 mmol) was dissolved in dichloroethane, and then a second-generation Grubbs catalyst (61 mg, 0.07 mmol) was added thereto. The resulting solution was stirred for 1 day at 100 °C. The reaction solution was diluted with ethyl acetate and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (143 mg, yield: 51%). MS m/z: 390 [M+1].

Example 20. (E)-4¹H-10-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-8(2,3)-oxirana-1(1,4)-benzenecyclodecaphane (Compound 20)

[Formula 21]

(4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene (0.1 g, 0.26 mmol) was dissolved in dichloromethane (2.6 mL), and then 3-chloroperbenzoic acid (122 mg, 0.26 mmol) was added thereto. The resulting solution was stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate, washed with an aqueous sodium bicarbonate saturated solution, and washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (44 mg, yield: 42%). MS m/z: 406 [M+1].

Example 21. (E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8,9-diol (Compound 21)

[Formula 22]

(4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene (0.1 g, 0.26 mmol) was dissolved in tetrahydrdofuran (2.6 mL), and then a 50% aqueous N-methylmorpholine N-oxide solution (0.24 ml, 1.04 mmol) and a 4% aqueous OsO₄ solution (0.1 mL) were added thereto. The resulting solution was stirred at room temperature for 1 day. The reaction solution was diluted with ethyl acetate and then washed with salt water. The organic layer was dried over magnesium sulfate and concentrated. The concentrate was purified via chromatography to obtain a target compound (55 mg, yield: 49%). MS m/z: 424 [M+1].

The following examples are examples for methods of preparing the compounds of Formula 1 according to the preparation method of the present invention or pharmaceutically acceptable salts thereof. However, these examples are not intended to limit the present invention.

Example 22: (E)-4¹H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one hydrochloride (Compound 22)

(E)-41H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one (300 mg, 0.589 mmol) was dissolved in tetrahydrofuran (5 mL), and then 4 M hydrogen chloride (147 μL) dissolved in dioxane was added thereto at room temperature. After 30 minutes, the generated precipitate was filtered and dried at room temperature to obtain, as a target compound, (E)-41H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one hydrochloride (305 mg).

Meanwhile, the novel compounds of Formula 1 according to the present invention may be formulated into various forms according to need. The following examples are examples of some formulation methods using the compound of Formula 1 according to the present invention as an active ingredient and are not intended to limit the present invention.

[Formulation Examples]

Formulation Example 1: Tablets (Direct Compression)

5.0 mg of the active ingredient was sieved, and then mixed with 14.1 mg of lactose, 0.8 mg of Crospovidone USNF, and 0.1 mg of magnesium stearate, and the resulting mixture was compressed to produce tablets.

Formulation Example 2: Tablets (Wet Granulation)

5.0 mg of the active ingredient was sieved, and then mixed with 16.0 mg of lactose and 4.0 mg of starch. 0.3 mg of Polysorbate 80 was dissolved in pure water, and then an appropriate amount of the resulting solution was added thereto and the resulting mixture was atomized. After drying, the fine grains were sieved and then mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The resulting fine grains were compressed to produce tablets.

Formulation Example 3: Powder and Capsules

5.0 mg of the active ingredient was sieved, and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone, and 0.2 mg of magnesium stearate. Firm No.5 gelatin capsules were filled with the resulting mixture using a suitable device.

Formulation Example 4: Injection

An injection was prepared using 26 mg of 12HO and 2974 mg of distilled water.

[Experimental Examples]

Experimental Example 1. Enzymatic Activity Measurement of Examples 7 and 1

A substrate solution and essential cofactors were added to the freshly prepared base reaction buffer (20 mM HEPES (PH 7.5), 10 mM MgCl₂, 1mM EGTA, 0.02 % Brij35, 0.02 mg/ml of BSA, 0.1 mM Na₃VO₄, 2 mM DTT, and 1 % DMSO). A kinase and each compound were added to the substrate solution, slowly mixed, and cultured at room temperature for 20 minutes. To initiate reaction, 33P-ATP was added to the reaction mixture, followed by incubation at room temperature for 2 hours, to allow a reaction to occur therebetween. After the reaction was completed, the amount of a substrate with radiolabeled 33P-ATP was measured to evaluate the activity of the kinase, and an IC₅₀ value was calculated based on the value.

The experimental results are as follows. The results of Example 7 are shown in Table 1 and the results of Example 1 are shown in Table 2. The activation values are represented in three steps.

A: IC₅₀< 100 nM,

B: 100 nM < IC₅₀ < 1 μM,

C: IC₅₀ > 1 μM

	Enzyme IC₅₀(μM)
FLT3	A
FLT3 (ITD)	A
FLT3 (D835Y)	A
FLT4	A
TBK1	B
c-Kit	B
BLK	A
MEK1	C
ULK1	A

	Enzyme IC₅₀(μM)
FLT4	A
GCN2	B

Experimental Example 2. Enzymatic Activity Measurement and Comparison of Example 1 and Comparative Example 1

10,000 cells were dispensed into each well of a 96-well plate. After the cells were stabilized, a compound, which was continuously diluted 1/3, was treated with 0.5% DMSO and cultured for 72 hours. Upon treatment with cell titer glo, luciferin is degraded by luciferase only in the presence of ATP, thereby luminescing. The luminescence values were measured using Envision (available from PerkinElmer). The concentration of each compound at which the cells died was measured based on the measured luminescence values to obtain GI₅₀ values.

The following compound was used as Comparative Example 1 (WO2014-180524). The following compound of Formula 23 is A26, which is a compound disclosed in WO2014-180524.

[Formula 23]

Enzymatic and cellular activities of Comparative Example 1 and Example 1 were compared.

The experimental results are shown in Table 3 below.

The activation values were represented in three steps.

A: IC₅₀< 100 nM,

B: 100 nM < IC₅₀ < 1 μM,

C: IC₅₀ > 1 μM

	Comparative Example 1	Example 1
Structure
FLT3 (ITD)	C	A
BaF3 (FLT3-ITD)	B	A

It can be confirmed that the compound of Example 1 exhibits excellent activity in the case of FLT3 compared to the compound of Comparative Example 1 (A26, a compound disclosed in WO2014-180524).

Experimental Example 3. Enzymatic Activity Measurement at Cellular Level

The experimental results are shown in Table 4 below.

The activation values were represented in three steps.

A: IC₅₀< 100 nM,

B: 100 nM < IC₅₀ < 1 μM,

C: IC₅₀ > 1 μM

	BaF₃ Cell							Cell
	Parental	FLT (TEL)	FLT3 (ITD)	FLT3 (D835Y)	FLT3(ITD-D835Y)	FLT3 (ITD-F691L)	FLT3 (ITD-D835Y-F691L)	U2OS	A375
Example 7	C	A	A	A	A	A	A	B	B
Example 1	B	B	A	-	-	-	-	C	C
Example 2	-	-	B	-	-	-	-	C	C
Example 3	-	-	C	-	-	-	-	C	C
Example 5	-	-	B	-	-	-	-	C	C
Example 4	-	-	B	-	-	-	-	C	C
Example 8	-	-	-	-	-	-	B	B	B
Example 9	-	-	-	-	-	-	B	B	B
Example 10	-	-	-	-	-	-	A	C	B
Example 11	-	-	-	-	-	-	B	C	C
Example 12	-	-	-	-	-	-	B	C	C
Example 13	-	-	-	-	-	-	B	C	C

Experimental Example 4. Enzymatic Activity Measurement and Comparison of Comparative Examples 2 and 3 and Example 7

Crizotinib and Alectinib, which are two commercially available drugs, were used as Comparative Examples 2 and 3, respectively, and enzymatic activities thereof were measured and compared with Example 7.

For each case, 10,000 cells were dispensed into each well of a 96-well plate. After the cells were stabilized, a compound, which was continuously diluted 1/3, was treated with 0.5% dimethyl sulfoxide and cultured for 72 hours. Upon treatment with cell titer glo, luciferin is degraded by luciferase only in the presence of ATP, thereby luminescing. The luminescence values were measured using Envision (available from PerkinElmer). The concentration of each compound at which the cells died was measured based on the measured luminescence values to obtain GI₅₀ values.

The experimental results are shown in Table 5 below.

The activation results were represented in three steps.

A: IC₅₀< 100 nM,

B: 100 nM < IC₅₀ < 1 μM,

C: IC₅₀ > 1 μM

Cmpd code	Cellular activity (uM)
	Ba/F3 cells				NSCLC
	Parental	ALK (WT)	ALK (L1196M)	ALK (G1202R)	SNU2535(G1269A)
Crizotinib(Comparative Example 2)	C	B	B	B	C
Alectinib(Comparative Example 3)	C	B	B	C	C
Example 7	C	A	B	A	C

As seen from the above experimental results, the compound of Example 7 exhibits high activity in the case of ALK (WT), similar activity in the case of ALK (L1196M), and very high activity in the case of ALK (G1202R), compared to the two commercially available drugs (Comparative Examples 2 and 3).

Experimental Example 5. Inhibitory Capability of the Case of Example 7 against Various Protein Kinases at 1 μM

The inhibitory capability of the case of Example 7 against protein kinases shown in Table 6 below at a concentration of 1 μM was measured. The experimental results thereof are shown in Table 6 below.

The inhibitory capability was represented in three steps.

A: inhibition by 70 or greater,

B: inhibition by 30-70 %

C: inhibition by less than 30

ABL	A	FGR	A	PBK	C
ARG	A	FLT1/VEGFR1	A	PDGFRa	A
ACK	A	FLT3	A	PDGFRb	A
AKT1	B	FLT4	A	PDK1	A
AKT2	B	FMS	A	SgK269	A
AKT3	A	FRK	B	PHKg1	A
ALK	A	FYN	A	PHKg2	C
ALK1	C	GCK	B	PIM1	A
ARAF	B	KHS2	A	PIM2	B
NuaK1	A	RHOK	B	PIM3	A
AurA	A	GPRK4	C	PKA	A
AurB	A	GPRK5	C	PKACb	A
AurC	A	GPRK6	C	PKACg	B
AXL	A	GPRK7	B	PKCa	A
BLK	A	GSK3A	A	PKCb	A
BMPR2	A	GSK3B	A	PKCb	A
BMX	A	Haspin	B	PKCd	A
BRK	A	HCK	A	PKCe	A
BRSK1	A	ZC1/HGK	B	PKCh	A
BRSK2	A	HIPK1	A	PKCg	A
BTK	A	HIPK2	A	PKCi	C
KIT	A	HIPK3	A	PKD1	A
MER	A	HIPK4	A	PKD3	A
MET	A	HPK1	A	PKCt	A
SRC	A	IGF1R	A	PKCz	C
CaMK1a	B	IKKa	B	PKD2	A
CaMK1d	B	IKKb	A	PKG1	B
CaMK1g	B	IKKe	B	PKG1	B
CaMK2a	A	INSR	A	PKG2	B
CaMK2b	B	IRAK1	A	PKN1	A
CaMK2d	B	IRAK4	A	PKN2	B
CaMKK1	C	IRR	A	PKN3	B
CaMKK2	C	ITK	B	PLK1	A
CDK1/cyclin A	A	JAK1	A	PLK2	B
CDK1/cyclin B	A	JAK2	A	PLK3	C
CDK1/cyclin E	A	JAK3	A	PLK4	B
CDK14/cyclin Y (PFTK1)	A	JNK1	B	PRKX	A
CDK16/cyclin Y (PCTAIRE)	A	JNK2	B	PYK2	A
CDK17/cyclin Y (PCTK2)	A	JNK3	C	RET	A
CDK18/cyclin Y (PCTK3)	A	KDR	A	RIPK2	B
CDK11	A	KHS1	B	ANKRD3	B
CDK2	A	LATS1	A	ROCK1	B
CDK2	A	LATS2	A	ROCK2	A
CDK2	A	LCK	A	RON	B
CDK2	A	ICK	B	ROS	A
CDK3	A	LIMK1	A	RSK1	A
CDK4	A	LIMK2	B	RSK2	A
CDK4	A	LKB1	A	RSK3	A
CDK5	A	LOK	A	RSK4	A
CDK5	A	LRRK2	A	SBK	A
CDK6	A	LYN	A	SGK	A
CDK6	A	LYNB	A	SGK2	A
CDK7	B	MAK	A	SGK3	C
CDK9	A	MARK1	A	SIK1	A
CDK9	A	MARK2	A	QIK	A
CDK9	A	MARK3	A	Q나	B
CHK1	A	MARK4	A	SLK	A
CHK2	A	MAP2K1	C	Nuak2	A
CK1d	B	MAP2K2	C	MPSK1	A
CK1e	C	MAP2K5	A	CRIK	C
CK1g1	C	MAP3K2	B	TSSK1	A
CK1g2	B	MAP3K3	A	YSK1	B
CK1g3	C	MELK	A	YANK2	B
CK2a	B	ZC3/MINK	B	STK33	A
CK2a2	A	MAP2K6	C	NDR1	A
CLK1	A	smMLCK	A	NDR2	A
CLK2	A	skMLCK	A	STLK3	A
CLK3	B	MLK1	A	SYK	B
CLK4	A	MLK2	A	TAK1	B
CSK	B	MLK3	A	TAO1	A
DAPK1	A	MNK1	C	TAO2	A
DAPK2	B	MNK2	B	TAO3	A
DCAMKL1	C	MRCKa	C	TBK1	A
DCAMKL2	B	MRCKb	C	TEC	A
DDR1	A	MSK1	A	TIE2	B
DDR2	A	MSK2	C	TLK1	C
DLK	C	MST1	A	TLK2	A
DMPK2	A	MST2	A	ZC2/TNIK	A
DRAK1	A	MST3	B	TNK1	B
DYRK1A	B	MUSK	B	TRKA	A
DYRK1B	A	caMLCK	C	TRKB	A
DYRK2	A	SgK085	A	TRKC	A
DYRK3	A	MYO3A	C	TSSK2	C
DYRK4	C	MYO3B	C	TSSK3	C
EGFR	B	NEK1	A	TTBK1	C
EPHA1	B	NEK11	C	TTBK2	C
EPHA2	A	NEK2	B	TXK	A
EPHA3	C	NEK3	B	LTK	A
EPHA4	A	NEK4	A	TYK2	A
EPHA5	B	NEK5	A	TYRO3	A
EphA6	A	NEK6	C	ULK1	A
EphA7	A	NEK7	C	ULK2	B
EPHA8	C	NEK8	C	ULK3	A
EphB1	A	NEK9	A	VRK1	B
EphB2	B	NIM1	B	VRK2	C
EphB3	C	NLK	B	Wee1	B
EphB4	B	OSR1	C	Wnk1	C
HER2/ErbB2	B	p38a	C	Wnk2	C
HER4/ErbB4	B	p38b	C	Wnk3	C
Erk5	C	p38d	B	YES	A
Erk7	A	p38g	C	MAP3K8	B
IRE1	A	P70s6k	A	ZAK	C
IRE2	A	p70S6Kb	B	DAPK3	A
FAK	A	PAK1	A
FER	A	PAK2	B
FES	A	PAK3	A
FGFR1	A	PAK4	B
FGFR2	A	PAK5	B
FGFR3	A	PAK6	B
FGFR4	B	PASK	C

Experimental Example 6: Experimental Results of Animal Efficacy (Efficacy in Treating Acute Myeloid Leukemia) of Example 7

Animal testing was conducted in accordance with government laws and regulations, including the Animal Protection Act and the Law on Laboratory Animals, and was conducted in accordance with the Ethics Regulations of the Laboratory Animal Laboratory of the Korea Institute of Science and Technology (Animal test approval number of the Korea Institute of Science and Technology: 2017-007).

OCI-AML3 Mouse Xenograft Model Preparation

6- to 8-week-old female Balb/c nude mice were used and brought into a laboratory, and after an acclimation period of one week, were used in the present experiment. To create an animal disease model, an OCI-AML3 cell line was used and the cell line was sub-cultured at intervals of three days using RPMI1640 containing 10% FBS and 1 x penicillin/streptomycin. Before transplanting the cells into each mouse, the cells were washed twice with DPBS and mixed at a density of 5.0 x 10⁶ cells/mouse with Matrigel at 1:1 so that the final volume reached 100 μl. Avertin as an anesthetic was intraperitoneally administered to each mouse at a dose of 250 mpk, and after confirming that each mouse was completely anesthetized, OCI-AML3 was transplanted into the right hind limb region of each mouse. The experiment was conducted using a minimum number (n=3) of subjects for statistical significance, and a vehicle and GNF-7 were used as controls at a dose of 8 mpk and the test compound (Example 7) was used as an experimental group at a dose of 30 mpk.

To orally administer the compound, the composition of the vehicle to dissolve the compound is as follows.

5% NMP/15% solutol/30% PEG400/50% 0.05M citric acid

Oral Administration of Compound for In Vivo Efficacy Study

After transplantation of OCI-AML3, each compound was orally administered to each mouse at a volume of 200 μl once a day for 3 weeks, when the tumor volume of each mouse reached about 150 mm³ to about 200 mm³. The conditions of the mice were observed daily at the time of oral administration, and the body weights and tumor volumes of the mice were measured at intervals of 3 days for 3 weeks.

Experimental Results

The experimental results are illustrated in FIG. 1 to 3. As can be seen from FIG. 1 to 3, a remarkable decrease in tumor volume was confirmed in an experimental group administered the test compound.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound represented by Formula 1 below, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof:

[Formula 1]

wherein, in Formula 1,

Q₁ and Q₂ are each independently any one of -(CH₂)_n-, -O(CH₂)_n-, -N(CH₂)_n-, and -NH(CH₂)_n- wherein n is an integer;

A and B are each independently any one of a C₃-C₈ cyclic hydrocarbon, a C₂-C₇ heterocyclic hydrocarbon, a monocyclic aromatic compound with three or more carbon atoms, and a heterocyclic aromatic compound with two or more carbon atoms; and

M is any one of -CHCH-, -CH₂CH₂-, -NHCO-, -CONH-, -NMe-, -NAc-, -NMs-, -NHCONHR₁-, -NHCOR₁-, -NHSONR₁-, and -O-, wherein R₁ is any one selected from a substituted or unsubstituted saturated hydrocarbon with two or more carbon atoms, a substituted or unsubstituted unsaturated hydrocarbon with two or more carbon atoms, a cyclic hydrocarbon with 3 to 8 carbon atoms, a heterocyclic hydrocarbon with 2 to 7 carbon atoms, a monocyclic aromatic compound with three or more carbon atoms, and a heterocyclic aromatic compound with two or more carbon atoms.
The compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof according to claim 1, wherein n is an integer of 0, 1, 2, 3, or 4, and A and B are any one or more selected from phenyl, pyridine, substituted phenyl, pyrazole, piperazine, and cyclohexane.
The compound, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof according to claim 1, wherein the compound is selected from the group consisting of:

(E)-4¹H-14-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 1);

(Z)-1¹H-5-oxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(4,1)-pyrazolo-4(1,4)-benzenecyclotetradecaphane-10-one (Compound 2);

5,15-dioxa-3,9-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1(5,2)-pyridine-4(1,4)-benzenecyclopentadecaphane-10-one (Compound 3);

(E)-4¹H-14-oxa-3,10-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenecyclotetradecaphane-9-one (Compound 4);

5,15-dioxa-3,11-diaza-2(7,2)-cyano[3,2-d]pyrimidine-1,4(1,4)-dibenzenacyclopentadecaphan-10-one (Compound 5);

(E)-4¹H-8-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-5(4,1)-piperidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclooctepene (Compound 6);

(E)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane (Compound 7);

(E)-8-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 8);

(E)-8-ethyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 9);

(E)-8-propyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 10);

(E)-1-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)ethene-1-one (Compound 11);

(E)-8-(methylsulfonyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 12);

(E)-N-methyl-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazola-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 13);

(E)-N-(4-chlorophenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carbothioamide (Compound 14);

(E)-N-(3-acetylphenyl)-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane-8-carboxamide (Compound 15);

(E)-(4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenecyclodecaphane-8-yl)(phenyl)methenone (Compound 16);

(E)-4¹H-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 17);

(E)-1³-fluoro-4¹H-11-oxa-3,8-diaza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazolo-1(1,4)-benzenacycloundecaphane (Compound 18);

(4⁴E,8E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8-ene (Compound 19);

(E)-4¹H-10-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-8(2,3)-oxirana-1(1,4)-benzenecyclodecaphane (Compound 20); and

(E)-4¹H-11-oxa-3-aza-2(7,2)-cyano[3,2-d]pyrimidine-4(4,1)-pyrazole-1(1,4)-benzenacycloundecaphane-8,9-diol (Compound 21).
A pharmaceutical composition comprising, as an active ingredient, the compound of any one of claims 1 to 3, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.
The pharmaceutical composition according to claim 4, wherein the composition is a pharmaceutical composition for preventing, alleviating, or treating a disease caused by abnormal cell growth through the inhibition of a protein kinase.
The pharmaceutical composition according to claim 5, wherein the compound has an inhibitory capability of 90% or more against the protein kinase at a concentration of 1 μM.
The pharmaceutical composition according to claim 5, wherein the protein kinase comprises any one or more selected from ALK, ARK5, Aurora A, Aurora B, Aurora C, AXL, BLK, BMX, c-MET, c-src, CDK2, CK2a2, CLK1, CLK2, CLK4, DAPK1, DDR1, DDR2, DYRK2, DYRK3, ERk7, FER, FES, FGFR1, FGFR2, FGFR3, FGR, FLT3, FLT4, DMS, FYN, HCK, HIPK1, HIPK2, HIPK4, HPK, IGF1R, IR, IRR, JAK1, JAK2, JAAK3, KDR, LCK, LOK, LRRK2, MARk4, MELK, MLK3, MYLK4, NEK1, NEK5, NEK9, p70S6K, PDGFRa, PDGFRb, PDK1, PEAK1, PIM1, PKCd, PKCeplsilon, PKCg, PKCmu, PKCnu, PKD2, RET, ROS, RSK1, RSK2, RSK3, RSK4, SIK1, SIK2, SNARK, STK16, STK38, STK38L, TRKA, TRKB, TRKC, TXK, TYK2, TYRO3, ULK1, YES, ZIPK and GCN2.
The pharmaceutical composition according to claim 5, wherein the disease caused by abnormal cell growth is a tumor disease.
The pharmaceutical composition according to claim 8, wherein the tumor disease is selected from the group consisting of stomach cancer, lung cancer, liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis, uterine cancer, uterine cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer such as leukemia, multiple myeloma, and myelodysplastic syndrome, lymphoma such as Hodgkin's disease and non-Hodgkin's lymphoma, and fibroadenoma.
A method of preparing the compound of any one of claims 1 to 3, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, the method comprising reacting any one intermediate compound of the 2,7-substituted cyano[3,2-d]pyrimidine cyclic compound, selected from:

7-(4-(2-bromoethoxy)phenyl)-N-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)cyano[3,2-d]pyrimidine-2-amine;

N-(1-(3-aminopropyl)-1H-pyrazole-4-yl)-7-(4-(2-bromoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-amine;

5-(4-(2-((1-(3-aminopropyl)-1H-pyrazole-4-yl)amino)cyano[3,2-d]pyrimidine-7-yl)phenoxy)pentenoic acid;

5-(4-((7-(4-(3-aminoethoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)-1H-pyrazole-1-yl)pentenoic acid;

5-((5-(2-((4-(3-aminopropoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)pyrimidine-2-yl)oxy)pentanoic acid;

5-(4-((7-(4-(3-aminopropoxy)phenyl)cyano[3,2-d]pyrimidine-2-yl)amino)phenoxy)pentanoic acid; and

5-(4-(2-((4-(3-aminopropoxy)phenyl)amino)cyano[3,2-d]pyrimidine-7-yl)-1H-pyrazole-1-yl)pentanoic acid.