Compounds with anticancer activity

Abstract

In one aspect, described herein are compounds and related pharmaceutical compositions for treating cancer. For example, in some embodiments, the pharmaceutical composition comprises an amount of a compound of formula (I) sufficient to exhibit anticancer activity.

Description

[0001] Related application data

[0002] This application claims priority under Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Serial No. 63 / 047,612, filed on July 2, 2020, which is incorporated herein by reference in its entirety.

[0003] Government Rights Statement

[0004] This invention was made with government support under Grant No. DP1AI124669 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Technical Field

[0005] The present invention relates to anticancer compounds and modes of action associated with such compounds. Background Art

[0006] Cancer causes a large number of deaths each year in the United States and around the world. There are many strategies for treating cancer, such as radiation therapy, chemotherapy, and surgery, including complete removal of cancerous tissue as well as cytoreduction and palliation. In many cases, cancer treatment strategies include adjuvant therapy, such as surgery and chemotherapy. For example, in cytoreduction surgery, any abnormal tissue remaining after surgery can be treated with chemotherapy.

[0007] The inherently destructive nature of cancer therapies often leads to unwanted side effects, such as damage to healthy, non-cancerous tissue. For example, the cytotoxicity of various chemotherapy drugs can cause anemia, alopecia (hair loss), nausea and vomiting, and nerve damage leading to burning, numbness, tingling, or shooting pains. Chemotherapy can also promote immunosuppression and bone marrow suppression, increasing a patient's chances of infection and other illnesses.

[0008] Alternative strategies have also been developed, such as hypothermic technology using nanoparticle compositions. Nanoparticle therapy has several drawbacks, including the inability to treat deep tumor tissue and negative immune responses from nanoparticles that accumulate in various areas of the lymphatic system. Therefore, new cancer treatments are needed that are effective at nanomolar concentrations. Summary of the Invention

[0009] In one aspect, compounds and related pharmaceutical compositions for treating cancer are described herein. In some embodiments, for example, the pharmaceutical composition comprises a compound of formula (I) and / or a salt thereof:

[0010]

[0011] wherein R1, R3, R4 and R5 are independently selected from hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, imine, cyanoimide, alkylene-aryl, alkylene-heteroaryl, amide, sulfonamide, acid, halogen and urea, wherein alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylene-aryl, alkylene-heteroaryl, amide and sulfonamide are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amide, sulfonamide, urea, halogen, cyano, hydroxy, C(O)OR6 and C(O)R7, wherein R6 is selected from the group consisting of hydrogen, alkyl and alkenyl, R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl and heteroaryl; and

[0012] wherein R is selected from the group consisting of alkyl, cycloalkyl, heterocycloalkyl, alkynyl, alkenyl, alkynylene-alkyl, alkynylene-cycloalkyl, alkynylene-heterocycloalkyl, alkynylene-aryl, alkynylene-heteroaryl, alkynylene-amine, alkynylene-protected amine, alkynylene-alkylsilane, fluoroalkyl, fluoro, bromo, B(OH)2, nitro, cyano, and alkoxy; and

[0013] wherein A is selected from the group consisting of aryl and heteroaryl; and

[0014] wherein X and Z are independently selected from C, N, O, S, SO2 and NR 10 R 11 The group consisting of R 10 and R 11 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 12 The group consisting of R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 10 and R 11 optionally forming a ring structure; and

[0015] wherein Y is selected from OH, alkoxy and NR 13 R 14 The group consisting of R 13 and R 14 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, alkylene-aryl, alkylene-heteroaryl, and C(O)R 15The group consisting of R 15 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 13 and R 14 optionally forming ring structures wherein the aryl, heteroaryl, alkylene-aryl, and alkylene-heteroaryl groups are optionally substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halogen, and alkynylene-alkylsilane; and

[0016] n is an integer between 0 and 5,

[0017] The compound of formula (I) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0018] In some embodiments, the pharmaceutical composition comprises a compound of formula (II) and / or a salt thereof:

[0019]

[0020] wherein R1, R3, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylene-aryl, alkylene-heteroaryl, amide, sulfonamide, acid, halogen and urea, wherein alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylene-aryl, alkylene-heteroaryl, amide and sulfonamide are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amide, sulfonamide, urea, halogen, hydroxy, C(O)OR6 and C(O)R7, wherein R6 is selected from the group consisting of hydrogen, alkyl and alkenyl, R7 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and NR8R9, wherein R s and R9 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and heteroaryl; and

[0021] wherein R is selected from the group consisting of arylene-alkynyl, heteroarylene-alkynyl, arylene-alkenyl, heteroarylene-alkenyl, alkynylene-alkyl, alkynylene-cycloalkyl, alkynylene-heterocycloalkyl, alkynylene-aryl, alkynylene-heteroaryl, alkenylene-aryl, alkenylene-heteroaryl, alkynylene-amine, alkynylene-protected amine, and alkynylene-alkylsilane; and

[0022] wherein X and Z are independently selected from C, N, O, S, SO2 and NR 10 R 11 The group consisting of R 10 and R 11independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 12 The group consisting of R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 10 and R 11 optionally forming a ring structure; and

[0023] wherein Y is selected from OH and NR 12 R 13 The group consisting of R 13 and R 14 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 15 The group consisting of R 15 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein R 13 and R 14 optionally forming a ring structure; n is an integer between 0 and 5,

[0024] The compound of formula (II) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0025] In another aspect, the pharmaceutical composition comprises a compound of formula (III) and / or a salt thereof:

[0026]

[0027] wherein R1-R6 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amide, sulfonamide, halogen, urea and -C(O)OR7, wherein R7 is selected from the group consisting of hydrogen and alkyl, wherein X is each independently selected from the group consisting of C, N, O, S, SO2 and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea and C(O)R 10 The group consisting of R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R8 and R9 may optionally form a ring structure; wherein Y is selected from the group consisting of OH and NR 11 R 12 The group consisting of R 11 and R 12 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 13 The group consisting of R13 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 11 and R 12 optionally forming a ring structure; n is an integer from 0 to 5,

[0028] The compound of formula (III) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0029] In another aspect, the pharmaceutical composition comprises a compound of formula (IV) and / or a salt thereof:

[0030]

[0031] wherein R1-R6 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amide, sulfonamide, halogen, urea and -C(O)OR7, wherein R7 is selected from the group consisting of hydrogen and alkyl, wherein X is each independently selected from the group consisting of C, N, O, S, SO2 and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea and C(O)R 10 The group consisting of R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R8 and R9 may optionally form a ring structure; wherein Y is selected from the group consisting of OH and NR 11 R 12 The group consisting of R 11 and R 12 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 13 The group consisting of R 13 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 11 and R 12 A ring structure may optionally be formed; wherein AA is selected from the group consisting of arylene, heteroarylene, cycloalkylene and heterocycloalkylene, n is an integer from 0 to 5,

[0032] The compound of formula (IV) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0033] In another aspect, the pharmaceutical composition comprises a compound of formula (V) and / or a salt thereof:

[0034]

[0035] wherein R1-R6 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amide, sulfonamide, halogen, urea and -C(O)OR7, wherein R7 is selected from the group consisting of hydrogen and alkyl, wherein X is each independently selected from the group consisting of C, N, O, S, SO2 and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea and C(O)R 10 The group consisting of R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R8 and R9 may optionally form a ring structure; wherein Y is selected from the group consisting of OH and NR 11 R 12 The group consisting of R 11 and R 12 independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 13 The group consisting of R 13 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 11 and R 12 optionally forming a ring structure; n is an integer from 0 to 5,

[0036] The compound of formula (V) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0037] In another aspect, the pharmaceutical composition comprises a compound of formula (VI) and / or a salt thereof:

[0038]

[0039] wherein R1-R6 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amide, sulfonamide, halogen, urea and -C(O)OR7, wherein R7 is selected from the group consisting of hydrogen and alkyl, wherein X is each independently selected from the group consisting of C, N, O, S, SO2 and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea and C(O)R 10 The group consisting of R 10 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R8 and R9 may optionally form a ring structure; wherein Y is selected from the group consisting of OH and NR 11 R 12 The group consisting of R 11 and R 12independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, aryl, heteroaryl, amide, sulfonamide, urea, and C(O)R 13 The group consisting of R 13 is selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and wherein R 11 and R 12 A ring structure may optionally be formed; wherein AA is selected from the group consisting of arylene, heteroarylene, cycloalkylene and heterocycloalkylene, n is an integer from 0 to 5,

[0040] The compound of formula (VI) is present in the pharmaceutical composition in an amount sufficient to exhibit anticancer properties.

[0041] In some embodiments, one or more compounds belonging to one or more of Formulas (I)-(VI) exhibit anticancer properties at nanomolar (nM) concentrations. In some embodiments, for example, one or more compounds exhibit anticancer properties at concentrations of 0.01 nM to greater than 1 μM.

[0042] In another aspect, methods of treating cancerous tissue are described herein. In some embodiments, the methods comprise administering to a patient having cancerous tissue one or more therapeutically effective amounts of a compound of Formulas I-VI. In some embodiments, the therapeutically effective amount inhibits cancer cell growth and / or tumor growth. In some embodiments, the therapeutically effective amount can also reduce tumor size.

[0043] These and other embodiments are further described in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 1A and 1B illustrate the inhibition of cancer cell growth by compounds of Formula I described herein, according to some embodiments.

[0045] FIG2 shows competition of folate from compounds of Formula I, according to some embodiments.

[0046] 3A and 3B respectively show changes in tumor volume over time and tumor doubling time in response to treatment with a compound of Formula I, according to some embodiments.

[0047] 3C shows the levels of intermediates in purine synthesis, glycine amide ribonucleotide (GAR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AlCAR), in tumors treated with compounds of Formula I according to some embodiments.

[0048] 4A and 4B respectively show changes in tumor volume over time and tumor doubling time in response to treatment with a compound of Formula I, according to some embodiments.

[0049] 5A shows changes in tumor volume in response to administration of a compound of Formula I, according to some embodiments.

[0050] FIG5B shows the pool size of circulating thymidine in response to administration of Compound 1.

[0051] FIG5C shows the levels of the thymidine ribonucleotide intermediate dUMP and the purine intermediates GAR and AlCAR in tumors in response to administration of Compound 1. DETAILED DESCRIPTION

[0052] By reference to the following detailed description and examples and their previous and following description, the embodiments described herein can be more easily understood. However, the elements, devices and methods described herein are not limited to the specific embodiments proposed in the detailed description and examples. It should be appreciated that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adjustments will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

[0053] definition

[0054] As used herein, the term "alkyl" refers, alone or in combination, to a straight or branched chain saturated hydrocarbon group optionally substituted with one or more substituents. For example, an alkyl group may be C1-C 30 or C1-C 18 .

[0055] The term "alkenyl" as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0056] The term "alkynyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents, including but not limited to alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine, and / or alkylsilane.

[0057] The term "aryl" as used herein, alone or in combination, refers to an aromatic monocyclic or multicyclic ring system optionally substituted with one or more ring substituents.

[0058] The term "heteroaryl", as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, such as nitrogen, oxygen and / or sulfur.

[0059] The term "cycloalkyl" as used herein, alone or in combination, refers to a non-aromatic, monocyclic or polycyclic ring system optionally substituted with one or more ring substituents.

[0060] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic, monocyclic or polycyclic ring system, wherein one or more atoms in the ring system is an element other than carbon, such as nitrogen, oxygen or sulfur, alone or in combination, and wherein the ring system is optionally substituted with one or more ring substituents.

[0061] The term "heteroalkyl" as used herein, alone or in combination, refers to an alkyl moiety as defined above, wherein one or more carbon atoms (e.g., one, two or three carbon atoms) in the chain are replaced by one or more heteroatoms, which may be the same or different, and wherein the point of attachment to the rest of the molecule is through a carbon atom of the heteroalkyl radical.

[0062] The term "alkoxy" as used herein, alone or in combination, refers to the moiety RO-, where R is alkyl or alkenyl as defined above.

[0063] As used herein, the term "halogen," alone or in combination, refers to an element of Group VIIA of the Periodic Table of the Elements (the halogen). Depending on the chemical environment, a halogen can be in a neutral or anionic state. For example, halogen encompasses fluorine, chlorine, bromine, and iodine.

[0064] I. Pharmaceutical composition for treating cancer

[0065] Various compounds are described herein. As discussed above and further illustrated in the examples below, compounds can exhibit anticancer properties in some embodiments. These compounds can belong to any of the above-mentioned Formulas I-VI. The compounds and / or salts of Formulas I-VI can be administered in any amount consistent with eliminating or inhibiting the growth of cancerous tissue. In some embodiments, one or more compounds are administered in an amount or concentration of 0.001 nM to greater than 1 μM. Any compound of Formula I-VI can also be administered in an amount or concentration selected from Table I.

[0066] Table I - Amounts of compounds of formula I-VI

[0067]

[0068]

[0069] In addition, the compounds and / or salts of Formula I-VI can be combined with another and / or other chemotherapeutic agents or adjuvants in the pharmaceutical compositions described herein. The compounds and / or salts of Formula I-VI can also be combined with any physiologically suitable carrier or excipient.

[0070] The amount or concentration of the Formula I-VI compound used in the pharmaceutical compositions described herein may depend on the identity and / or properties of the cancer being treated. In some embodiments, the compound of Formula I-VI is applied to a cancer cell line that downregulates the reduced folate carrier (RFC). For example, these cell lines may be resistant to pemetrexed and / or methotrexate. However, as shown herein, the Formula I-VI compound can show higher efficacy against the cell line that downregulates RFC relative to pemetrexed and / or methotrexate. In some embodiments, the Formula I-VI compound can inhibit cancer cell growth by destroying purine and / or thymidine biosynthesis. For example, one or more Formula I-VI compounds may destroy purine and / or thymidine biosynthesis in pancreatic cancer cells and / or colorectal cancer cells. In some embodiments, the Formula I-VI compound can show an IC50 of 0.001nM to 1μM or 0.1nM to 100nM for inhibiting cancer cell growth. In some embodiments, the IC50 is greater than 1μM.

[0071] II. Methods for treating cancer

[0072] On the other hand, methods for treating cancerous tissue are described herein. In some embodiments, the method comprises administering one or more compounds of Formula I-VI or a salt thereof to a patient having cancerous tissue in a therapeutically effective amount. In some embodiments, the therapeutically effective amount prevents or inhibits cancer cell growth and / or tumor growth. In some embodiments, the therapeutically effective amount can also reduce tumor size. In some embodiments, any compound of Formula I-VI or a salt thereof is administered in an amount selected from Table I. In some embodiments, a combination of any two or more compounds of Formula I-VI can be used to treat cancer cells and tissues. In addition, the compounds of Formula I-VI can be used in combination with any adjuvant or other chemotherapeutic agent to treat cancer.

[0073] These and other embodiments are further illustrated in the following non-limiting examples.

[0074] Examples - Compounds showing anticancer activity

[0075] One or more of the following compounds according to at least one of Formulas I-VI exhibit anticancer activity in some embodiments. Compounds were prepared according to the following general reaction scheme. Common solvents were purified prior to use. All reagents were reagent grade and purified where necessary. Reactions were monitored by thin layer chromatography (TLC) using Whatman pre-coated silica gel plates. Flash column chromatography was performed on ultrapure silica gel (200-400 mesh) from Merck. Spectrometers were recorded on a Bruker AVANCE 300 (300 MHz), 400 MHz, or 500 MHz spectrometer. 1 H NMR spectrum. 1H NMR multiplicities are designated as s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet, br = broad. Electrospray impact (ESI) mass spectra were recorded on an ISQEC mass spectrometer.

[0076]

[0077] To a stirred solution of 7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (1.0 mmol) in dry DMF (20 mL) was added NaH (1.2 mmol). The resulting reaction mixture was stirred at 0°C for 0.5 hours. The corresponding bromide (1.5 mmol) was then added. The reaction mixture was stirred at 0°C for 1 hour. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel using 10:1 DCM:MeOH containing 1% Et3N to give the desired compound as a solid. The following specific compounds were synthesized according to the above steps.

[0078] Compound 1 (IRS-17)

[0079]

[0080] 1 H NMR (500MHz, DMSO-d6) δ7.71 (d, J=9.0Hz, 1H), 7.62 (d, J=3.0Hz, 1H), 7.41 (d, J=8.0Hz, 2H), 7.15 (d, J=8.0 Hz, 2H), 7.12 (d, J=3.0Hz, 1H), 7.03 (d, J=9.0Hz, 1H), 6.78 (s, 2H), 5.89 (s, 2H), 5.53 (s, 2H), 4.14 (s, 1H). MS(ESI):[M+H + ]314.12.

[0081] Compound 2

[0082]

[0083] 1H NMR(400MHz,DMSO-d6)δ7.92(d,J=9.0Hz,1H),7.85(d,J=3.2Hz,1H),7.65-7.56(m,5H),7.47-7.40(m,2H),7.37-7.31(m,1H),7.29-7.24(m,2H),7.22-7.11(m,2H),6.74(s,2H),5.60(s,2H),3.84-3.66(m,4H)。

[0084] Compound 3

[0085]

[0086] 1 H NMR(300MHz,DMSO-d6)δ7.88(d,J=9.0Hz,1H),7.58(d,J=3.3Hz,1H),7.46-7.33(m,5H),7.17-7.06(m,2H),6.70(s,2H),5.72(s,2H),5.43(s,2H)。

[0087] MS(ESI):[M+H+]314.12。

[0088] Compound 4

[0089]

[0090] 1 H NMR(300MHz,DMSO-d6)δ7.80(d,J=9.0Hz,1H),7.52(d,J=3.0Hz,1H),7.43-7.37(m,2H),7.34-7.26(m,2H),7.26-7.22(m,1H),7.08(d,J=3.0Hz,1H),7.05(d,J=9.0Hz,1H),6.67(s,2H),6.52-6.46(m,2H),5.67(s,2H),5.09-5.01(m,2H)。

[0091] MS(ESI):[M+H+]316.15。

[0092] Compound 5

[0093]

[0094] 1H NMR(300MHz,DMSO-d6)δ7.71(d,J=9.0Hz,1H),7.60(d,J=3.0Hz,1H),7.39-7.25(m,3H),7.13(d,J=7.2Hz,1H),7.08(d,J=3.0Hz,1H),7.02(d,J=9.0Hz,1H),6.97-6.89(m,3H),6.87-6.80(m,2H),6.68(s,2H),5.69(s,2H),5.50(s,2H)。

[0095] MS(ESI):[M+H+]382.12。

[0096] Compound 6

[0097]

[0098] Compound 7

[0099]

[0100] Compound 8

[0101]

[0102] 1 H NMR(500MHz,DMSO-d6)δ7.73(d,J=9.0Hz,1H),7.59(d,J=3.0Hz,1H),7.15(d,J=8.0Hz,2H),7.11(d,J=8.0Hz,2H),7.06(d,J=3.0Hz,1H),7.01(d,J=8.9Hz,1H),6.65(s,2H),5.65(s,2H),5.43(s,2H),2.80(p,J=7.0Hz,1H),1.12(d,J=6.9Hz,6H)。

[0103] MS(ESI):[M+H+]332.42。

[0104] Compound 9

[0105]

[0106] 1H NMR(300MHz,DMSO-d6)δ7.70(d,J=9.0Hz,1H),7.62(d,J=3.0Hz,1H),7.41(d,J=8.2Hz,2H),7.14(d,J=8.2Hz,2H),7.11(d,J=3.0Hz,1H),7.09(d,J=9.0Hz,1H),6.68(s,2H),6.47-6.30(m,1H),5.53(s,2H),4.16(s,1H),2.84-2.70(m,1H),0.68-0.57(m,2H),0.48-0.39(m,2H)。

[0107] MS(ESI):[M+H+]354.15。

[0108] Compound 10

[0109]

[0110] 1 H NMR(500MHz,DMSO-d6)δ8.08(d,J=9.0Hz,1H),7.91(d,J=3.0Hz,1H),7.60(s,2H),7.37(d,J=3.0Hz,1H),7.31-7.24(m,2H),7.21(d,J=9.0Hz,1H),7.18-7.11(m,2H),5.56(s,2H)。

[0111] MS(ESI):[M+H+]308.15。

[0112] Compound 11

[0113]

[0114] 1 H NMR(500MHz,DMSO-d6)δ7.78(d,J=9.0Hz,1H),7.47(d,J=3.0Hz,1H),7.05(d,J=9.0Hz,1H),7.00(d,J=3.0Hz,1H),6.68(s,2H),5.69(s,2H),4.13(d,J=7.5Hz,2H),2.42-2.29(m,1H),1.66-1.58(m,2H),1.58-1.52(m,2H),1.51-1.44(m,2H),1.28-1.20(m,2H)。

[0115] MS(ESI):[M+H+]282.24。

[0116] Compound 12

[0117]

[0118] 1 H NMR(400MHz,DMSO-d6)δ7.92-7.82(m,1H),7.80-7.72(m,1H),7.39-7.31(m,1H),7.25-7.18(m,1H),7.14-7.05(m,2H),7.04-6.96(m,2H),5.55(s,2H)。

[0119] MS(ESI):[M+H+]308.06。

[0120] Compound 13

[0121]

[0122] 1 H NMR(400MHz,DMSO-d6)δ7.75(d,J=9.0Hz,1H),7.63(d,J=3.0Hz,1H),7.41-7.34(m,1H),7.34-7.26(m,1H),7.11(d,J=3.0Hz,1H),7.03(d,J=9.0Hz,1H),7.02-6.99(m,1H),6.72(s,2H),5.73(s,2H),5.49(s,2H)。

[0123] MS(ESI):[M+H+]326.10。

[0124] Compound 14

[0125]

[0126] 1 H NMR(400MHz,DMSO-d6)δ7.76(d,J=9.0Hz,1H),7.66(d,J=3.2Hz,1H),7.18-7.09(m,2H),7.05(d,J=9.0Hz,1H),6.90-6.84(m,2H),6.78(s,2H),5.81(s,2H),5.54(s,2H)。

[0127] MS(ESI):[M+H+]326.10。

[0128] Compound 15

[0129]

[0130] 1 H NMR(400MHz,DMSO-d6)δ7.69(d,J=9.0Hz,1H),7.60(d,J=3.2Hz,1H),7.27(d,J=8.0Hz,2H),7.09(d,J=8.0Hz,3H),7.01(d,J=9.0Hz,1H),6.77(s,2H),5.78(s,2H),5.48(s,2H),2.81-2.65(m,1H),1.15(d,J=6.8Hz,6H)。

[0131] MS(ESI):[M+H + ]356.20

[0132] Compound 16

[0133]

[0134] 1 H NMR(500MHz,DMSO-d6)δ7.70(d,J=9.0Hz,1H),7.61(d,J=3.1Hz,1H),7.34-7.29(m,2H),7.29(s,1H),7.18-7.05(m,3H),7.01(d,J=9.0Hz,1H),6.79(s,2H),5.79(s,2H),5.49(s,2H),3.91(d,J=6.0Hz,2H),1.35(s,9H)。

[0135] MS(ESI):[M+H + ]443.53

[0136] Compound 17

[0137]

[0138] 1 H NMR(400MHz,DMSO-d6)δ7.68(m,1H),7.64-7.59(m,1H),7.34-7.26(m,2H),7.15-7.06(m,4H),6.67(s,2H),6.37(s,1H),5.51(s,2H),2.83-2.74(m,1H),1.18(d,J=6.9Hz,6H),0.63(m,2H),0.51-0.37(m,2H)。

[0139] MS(ESI):[M+H +]396.50

[0140] Compound 18

[0141]

[0142] 1 H NMR(400MHz,DMSO-d6)δ7.73(d,J=9.2Hz,1H),7.69-7.60(m,1H),7.32(d,J=6.4Hz,2H),7.15-7.07(m,4H),5.51(s,2H),2.88-2.72(m,1H),0.86(t,J=6.4Hz,2H),0.64(dd,J=6.4,2.4Hz,2H)。

[0143] MS(ESI):[M+H + ]368.45

[0144] Compound 19

[0145]

[0146] 1 H NMR(400MHz,DMSO-d6)δ7.74(d,J=8.8Hz,1H),7.66(d,J=3.2Hz,1H),7.55-7.49(m,4H),7.42(d,J=3.2Hz,3H),7.20(d,J=8.0Hz,2H),7.15-7.11(m,2H),6.75(s,2H),6.46(s,1H),5.57(s,2H),2.79(m,1H),0.64(m,2H),0.52-0.40(m,2H)。

[0147] MS(ESI):[M+H + ]430.49

[0148] Compound 20

[0149]

[0150] 1H NMR(400MHz,DMSO-d6)δ7.71(d,J=9.2Hz,1H),7.61(d,J=3.2Hz,1H),7.33-7.26(m,2H),7.16-7.06(m,4H),6.69(s,2H),6.40(s,1H),5.50(s,2H),2.79(m,1H),1.51(m,1H),0.91-0.81(m,4H),0.73-0.59(m,4H)

[0151] MS(ESI):[M+H + ]394.19

[0152] Compound 21

[0153]

[0154] Compound 22

[0155]

[0156] 1 H NMR(400MHz,DMSO-d6)δ7.70(d,J=8.8Hz,1H),7.61(d,J=3.2Hz,1H),7.29(d,J=8.0Hz,2H),7.17-7.08(m,3H),7.02(d,J=8.8Hz,1H),6.73(s,2H),5.76(s,2H),5.50(s,2H),1.50(m,1H),0.91-0.79(m,2H),0.72-0.64(m,2H)。

[0157] MS(ESI):[M+H + ]354.46

[0158] Compound 23

[0159]

[0160] 1 H NMR(400MHz,DMSO-d6)δ7.80(d,J=8.8Hz,1H),7.71(d,J=3.2Hz,1H),7.57-7.46(m,4H),7.42(q,J=2.8Hz,3H),7.25-7.16(m,3H),7.08(d,J=8.8Hz,1H),6.13(s,2H),5.58(s,2H)。

[0161] MS(ESI):[M+H +]390.41

[0162] Compound 24

[0163]

[0164] 1 H NMR(400MHz,DMSO-d6)δ8.06(d,J=9.0Hz,1H),7.93(d,J=3.2Hz,1H),7.67(s,2H),7.47-7.37(m,3H),7.22(dd,J=9.0,2.4Hz,3H),5.64(s,2H),3.98(s,2H)。

[0165] MS(ESI):[M+H + ]343.38

[0166] Compound 25

[0167]

[0168] 1 H NMR(400MHz,DMSO-d6)δ7.75(d,J=9.0Hz,1H),7.65(d,J=3.2Hz,1H),7.37-7.26(m,2H),7.17-7.10(m,3H),7.04(d,J=9.0Hz,1H),6.88(s,2H),5.93(s,2H),5.51(s,2H),2.00(s,3H)。

[0169] MS(ESI):[M+H + ]328.37

[0170] Compound 26

[0171]

[0172] 1 H NMR(300MHz,DMSO-d6)δ7.90-7.79(m,4H),7.77(d,J=3.2Hz,1H),7.72(s,1H),7.52-7.43(m,2H),7.33(dd,J=8.5,1.8Hz,1H),7.19(d,J=3.2Hz,1H),7.06(d,J=9.0Hz,1H),6.22(s,2H),5.68(s,2H)。

[0173] MS(ESI):[M+H + ]340.35

[0174] Compound 27

[0175]

[0176] 1 H NMR(500MHz,DMSO-d6)δ7.70(s,1H),7.65(d,J=3.5Hz,1H),7.41_7.34(m,6H),7.31(s,1H),7.23(d,J=3.0Hz,1H),7.14(d,J=8.0Hz,2H),5.86(s,2H),5.51(s,2H),4.83(d,J=6.0Hz,2H),0.95(m,18H),0.59(m,12H)。

[0177] MS(ESI):[M+H + ]657.03

[0178] Compound 28

[0179]

[0180] Compound 29

[0181]

[0182] Compound 30

[0183]

[0184] Compound 31

[0185]

[0186] 1 H NMR(400MHz,DMSO-d6)δ7.79(d,J=9.0Hz,1H),7.65(d,J=3.2Hz,1H),7.13(t,J=3.2Hz,5H),7.05(d,J=9.0Hz,1H),6.92(s,2H),5.98(s,2H),5.46(s,2H),2.55(m,2H),1.12(t,J=7.6Hz,3H)。

[0187] MS(ESI):[M+H + ]318.36

[0188] Compound 32

[0189]

[0190] Compound 33

[0191]

[0192] 1 H NMR(500MHz,DMSO-d6)δ7.71(d,J=9.0Hz,1H),7.60(d,J=3.0Hz,1H),7.41-7.34(m,2H),7.13(d,J=8.2Hz,2H),7.07(d,J=3.0Hz,1H),7.00(d,J=9.0Hz,1H),6.74(s,2H),6.65(dd,J=17.6,11.0Hz,1H),5.81-5.68(m,3H),5.46(s,2H),5.19(dd,J=10.9,1.0Hz,1H)。

[0193] MS(ESI):[M+H + ]316.40

[0194] Compound 34

[0195]

[0196] 1 H NMR(400MHz,DMSO-d6)δ8.22-8.17(m,2H),7.96(d,J=9.0Hz,1H),7.91(d,J=3.2Hz,1H),7.43-7.35(m,3H),7.19(d,J=9.0Hz,1H),7.12(s,2H),5.76(s,2H)。

[0197] MS(ESI):[M+H+]335.08

[0198] Compound 35

[0199]

[0200] Compound 36

[0201]

[0202] 1H NMR(400MHz,DMSO-d6)δ7.80(d,J=3.2Hz,1H),7.74(d,J=8.8Hz,1H),7.33-7.23(m,2H),7.19-7.07(m,3H),7.01(d,J=8.8Hz,1H),6.80(s,2H),5.95(q,J=6.8Hz,1H),5.81(s,2H),1.90(d,J=6.8Hz,3H)。

[0203] MS(ESI):[M+H + ]322.32

[0204] Compound 37

[0205]

[0206] Compound 38

[0207]

[0208] 1 H NMR(400MHz,DMSO-d6)δ9.07(dd,J=4.0,2.0Hz,1H),8.43(dd,J=8.4,2.0Hz,1H),7.92(d,J=8.0Hz,1H),7.80(d,J=8.8Hz,1H),7.73(d,J=3.2Hz,1H),7.65(dd,J=8.4,4.4Hz,1H),7.47(t,J=8.0Hz,1H),7.16(d,J=3.2Hz,1H),7.03(dd,J=8.4,4.4Hz,2H),6.92(s,2H),6.15(s,2H),5.95(s,2H)。

[0209] MS(ESI):[M+H + ]341.33

[0210] Compound 39

[0211]

[0212] 1 H NMR(400MHz,DMSO-d6)δ7.78(d,J=8.8Hz,1H),7.37(d,J=3.2Hz,1H),7.25-7.14(m,2H),7.12-6.94(m,4H),6.73(s,2H),5.79(s,2H),4.47(t,J=7.2Hz,2H),3.08(t,J=7.2Hz,2H)。

[0213] MS(ESI): [M+H + 341.33

[0214] Compound 40

[0215]

[0216] 1 H NMR(300 MHz, methanol-d4) δ 7.78(dd, J = 9.1, 0.9 Hz, 1H), 7.66(d, J = 3.3 Hz, 1H), 7.62 - 7.55(m, 2H), 7.41 - 7.30(m, 2H), 7.25 - 7.18(m, 2H).

[0217] MS(ESI): [M+H + 294.30

[0218] Compound 41

[0219]

[0220] 1 H NMR(400 MHz, DMSO-d6) δ 7.74(s, 1H), 7.65(d, J = 3.2 Hz, 1H), 7.55 - 7.46(m, 2H), 7.30 - 7.20(m, 2H), 7.20 - 7.09(m, 3H), 7.01 - 6.94(m, 2H), 6.83(s, 2H), 5.71(s, 2H), 5.54(s, 2H), 3.80(s, 3H).

[0221] MS(ESI): [M+H + 414.48

[0222] Compound 42 [[ID=3九十二]]

[0223]

[0224] 11H NMR (400 MHz, DMSO-d6) δ 8.22 - 8.13 (m, 1H), 8.12 - 8.03 (m, 1H), 7.72 (d, J = 9.2 Hz, 1H), 7.66 - 7.57 (m, 2H), 7.48 (d, J = 3.2 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 3.2 Hz, 1H), 7.02 (d, J = 9.2 Hz, 1H), 6.70 (s, 2H), 6.68 (s, 1H), 5.97 (s, 2H), 5.70 (s, 2H), 2.62 (s, 3H). MS (ESI): [M + H + 354.44

[0225] Compound 43

[0226]

[0227] 1 1H NMR (400 MHz, methanol-d4) δ 8.70 (dd, J = 2.2, 0.8 Hz, 1H), 8.57 (dd, J = 4.8, 1.6 Hz, 1H), 8.04 (dt, J = 8.0, 2.0 Hz, 1H), 7.83 (d, J = 0.8 Hz, 1H), 7.66 (d, J = 3.2 Hz, 1H), 7.56 (m, 1H), 7.29 - 7.18 (m, 2H), 7.13 (dd, J = 3.2, 0.8 Hz, 1H), 7.10 - 7.00 (m, 2H), 5.56 (s, 2H).

[0228] MS (ESI): [M + H + 385.41

[0229] Compound 44

[0230]

[0231] 1 1H NMR (400 MHz, DMSO-d6) δ 8.25 (m, 2H), 7.79 - 7.70 (m, 4H), 7.55 (d, J = 3.1 Hz, 1H), 7.17 (d, J = 3.1 Hz, 1H), 7.03 (d, J = 8.9 Hz, 1H), 6.76 (s, 2H), 6.54 (d, J = 7.7 Hz, 1H), 6.04 (s, 2H), 5.76 (s, 2H).

[0232] MS (ESI): [M + H + 418.31

[0233] Compound 45

[0234]

[0235] 1 H NMR (400MHz, DMSO-d6) δ8.24 (d, J=2.4Hz, 1H), 7.99 (d, J=8.9Hz, 1H), 7.82 (t, J=5.5Hz, 2H) , 7.47 (s, 2H), 7.25 (d, J=3.2Hz, 1H), 7.15 (dd, J=8.9, 2.4Hz, 2H), 6.58 (s, 2H), 5.59 (s, 2H).

[0236] MS (ESI): [M+H + ]309.36

[0237] Compound 46

[0238]

[0239] 1 H NMR (400MHz, DMSO-d6) δ 8.21 (s, 1H), 7.66 (d, J = 3.2Hz, 1H), 7.29-7.20 (m, 2H), 7.20-7.09 (m, 3H), 6.85 (s, 2H), 5.94 (s, 2H), 5.51 (s, 2H).

[0240] MS (ESI): [M+H + ]386.21

[0241] Compound 47

[0242]

[0243] 1 H NMR (400 MHz, chloroform-d + MeOD) δ 7.82 (d, J = 1.2 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.25-7.23 (m, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.72 (d, J = 3.2 Hz, 1H), 5.29 (s, 2H), 3.03 (s, 1H).

[0244] MS (ESI): [M+H + ]393.25

[0245] Compound 48

[0246]

[0247] 1 H NMR (400MHz, DMSO-d6) δ8.00-7.86 (m, 2H), 7.77 (d, J=3.0Hz, 1H), 7.65 (s, 1H), 7.29 (d, J=3.0Hz, 1H), 7. 15 (d, J=8.8Hz, 1H), 7.09 (dd, J=8.0, 2.4Hz, 1H), 6.95 (dd, J=8.0, 2.4Hz, 1H), 6.76 (s, 2H), 5.63 (s, 2H).

[0248] MS (ESI): [M+H + ]309.33

[0249] Compound 49

[0250]

[0251] 1 H NMR (400 MHz, methanol-d4) δ 7.59 (d, J = 4.4 Hz, 2H), 7.27-7.15 (m, 2H), 7.13-6.98 (m, 3H), 5.53 (s, 2H), 2.20 (m, 1H), 1.15-1.03 (m, 2H), 0.76-0.65 (m, 2H).

[0252] MS (ESI): [M+H + ]348.45

[0253] Compound 50

[0254]

[0255]

[0256] 1 H NMR (400 MHz, chloroform-d + M MeOD) δ 8.35 (d, J = 2.0 Hz, 1H), 7.45 (m 2H), 7.35 (dd, J = 8.4, 2.0 Hz, 1H), 7.28 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 3.2 Hz, 1H), 5.42 (s, 2H). MS (ESI): [M+H + ]359.30

[0257] Compound 51

[0258]

[0259] 1 1H NMR (400 MHz, DMSO-d6) δ 8.03 (m, 3H), 7.92 (d, J = 3.2 Hz, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.45 (s, 2H), 7.38 (d, J = 3.2 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H), 5.59 (s, 2H).

[0260] MS (ESI): [M+H + 334.17

[0261] Compound 52

[0262]

[0263] 1 1H NMR (400 MHz, methanol-d4) δ 7.79 (d, J = 0.8 Hz, 1H), 7.69 (d, J = 3.2 Hz, 1H), 7.56 - 7.44 (m, 2H), 7.30 - 7.19 (m, 4H), 7.16 (dd, J = 3.2, 0.8 Hz, 1H), 7.10 - 7.01 (m, 2H), 5.55 (s, 2H). MS (ESI): [M+H + 402.40

[0264] Compound 53

[0265]

[0266] 1 1H NMR (400 MHz, DMSO-d6) δ 8.94 (d, J = 2.0 Hz, 1H), 8.76 (d, J = 2.0 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 3.2 Hz, 1H), 7.23 (d, J = 3.2 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.28 (s, 2H), 5.64 (s, 2H).

[0267] MS (ESI): [M+H + 316.31

[0268] Compound 54

[0269]

[0270] 11H NMR (400 MHz, methanol-d4) δ 8.11 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 3.2 Hz, 1H), 6.96 (dd, J = 3.2, 0.8 Hz, 1H), 6.27 (d, J = 1.0 Hz, 1H), 5.31 (d, J = 1.0 Hz, 1H), 5.14 (t, J = 1.6 Hz, 2H), 3.79 (s, 3H).

[0271] MS (ESI): [M+H + 376.22

[0272] Compound 55

[0273]

[0274] 1 1H NMR (400 MHz, methanol-d4) δ 7.60 (d, J = 3.2 Hz, 1H), 7.41 (t, J = 3.2 Hz, 1H), 7.19 - 7.14 (m, 2H), 7.04 (dd, J = 8.8, 3.2 Hz, 2H), 6.95 (t, J = 3.2 Hz, 1H), 5.44 (d, J = 3.2 Hz, 2H), 2.52 (d, J = 3.2 Hz, 3H).

[0275] MS (ESI): [M+H + 322.34

[0276] Compound 56

[0277]

[0278] 1 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 5.2 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 3.2 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 7.00 - 6.98 (m, 1H), 6.84 (d, J = 1.5 Hz, 1H), 6.79 (s, 2H), 5.80 (s, 2H), 5.64 (s, 2H).

[0279] MS (ESI): [M+H + 309.36

[0280] Compound 57

[0281]

[0282] 1 H NMR(400MHz,DMSO-d6)δ7.91-7.79(m,3H),7.63(d,J=3.2Hz,1H),7.11(d,J=3.2Hz,1H),7.05(d,J=8.8Hz,1H),6.74(s,2H),5.77(s,2H),5.51(s,2H)。

[0283] MS(ESI):[M+H + ]321.38

[0284] Compound 58

[0285]

[0286] 1 H NMR(400MHz,DMSO-d6)δ8.85(d,J=2.4Hz,1H),8.01(dd,J=8.4,2.4Hz,1H),7.78(d,J=9.2Hz,1H),7.74-7.63(m,3H),7.48(t,J=7.2Hz,2H),7.41(t,J=7.2Hz,1H),7.15(d,J=3.2Hz,1H),7.05(d,J=8.4Hz,2H),6.82(s,2H),5.83(s,2H),5.65(s,2H)。

[0287] MS(ESI):[M+H + ]367.41

[0288] Compound 59

[0289]

[0290] 1 H NMR(400MHz,DMSO-d6)δ8.42(d,J=2.4Hz,1H),7.77(d,J=8.8Hz,1H),7.68-7.54(m,2H),7.13(d,J=3.2Hz,1H),7.04(d,J=8.8Hz,1H),6.92(d,J=8.0Hz,1H),6.87(s,2H),5.91(s,2H),5.55(s,2H),2.88(m,1H),1.17(d,J=6.8Hz,6H)。

[0291] MS(ESI):[M+H + ]333.40

[0292] Compound 60

[0293]

[0294] 1 H NMR(400MHz,DMSO-d6)δ7.92-7.82(m,2H),7.62(d,J=3.2Hz,1H),7.13(d,J=3.2Hz,1H),7.07(d,J=9.2Hz,1H),6.83(s,2H),5.87(s,2H),5.71(s,2H)。

[0295] MS(ESI):[M+H + ]365.33

[0296] Compound 61

[0297]

[0298] 1 H NMR(400MHz,DMSO-d6)δ8.63(dt,J=4.8,1.6Hz,1H),8.06-7.98(m,2H),7.94-7.81(m,2H),7.78(d,J=9.0Hz,1H),7.69(d,J=3.2Hz,1H),7.35-7.25(m,3H),7.15(d,J=3.2Hz,1H),7.05(d,J=9.0Hz,1H),6.85(s,2H),5.93-5.80(m,2H),5.58(s,2H)。

[0299] MS(ESI):[M+H + ]367.41

[0300] Compound 62

[0301]

[0302] 1 H NMR(400MHz,DMSO-d6)δ7.71(d,J=8.8Hz,1H),7.45(d,J=3.2Hz,1H),7.14-6.99(m,2H),6.66(s,2H),6.15(s,1H),5.67(s,2H),5.24(s,1H),5.14(s,2H),3.73(s,3H)。MS(ESI):[M+H + ]298.11

[0303] Compound 63

[0304]

[0305] 1 H NMR(400MHz,DMSO-d6)δ8.47-8.25(m,1H),7.91-7.55(m,2H),7.01(m,5H),6.59(s,1H),6.04(s,2H),5.52(s,2H),3.73(s,3H)。

[0306] MS(ESI):[M+H+]321.20。

[0307] Compound 64

[0308]

[0309] 1 H NMR(400MHz,DMSO-d6)δ7.83(s,2H),7.51-7.38(m,2H),7.19-7.11(m,4H),7.08(s,1H),6.55(s,2H),6.29(t,J=6.4Hz,1H),4.29(d,J=6.0Hz,2H)。

[0310] MS(ESI):[M+H + ]284.30

[0311] Compound 65

[0312]

[0313] 1 H NMR(400MHz,DMSO-d6)δ7.70(d,J=9.2Hz,1H),7.51(d,J=3.2Hz,1H),7.11(dd,J=8.0,2.8Hz,2H),7.05(d,J=9.2Hz,1H),6.84(dd,J=8.4,2.4Hz,1H),6.76(s,2H),6.73(d,J=8.4Hz,1H),5.78(s,2H),5.50(s,2H),3.74(s,3H)。

[0314] MS(ESI):[M+H + ]354.79

[0315] Compound 66

[0316]

[0317] 1H NMR(400MHz,DMSO-d6)δ7.74(dd,J=9.0,0.8Hz,1H),7.62(d,J=3.2Hz,1H),7.13-7.08(m,1H),7.04(d,J=9.0Hz,1H),6.75(s,2H),6.38(t,J=2.4Hz,1H),6.34(d,J=2.4Hz,2H),5.76(s,2H),5.41(s,2H),3.67(s,6H)。

[0318] MS(ESI):[M+H + ]350.34

[0319] Compound 67

[0320]

[0321] 1 H NMR(400MHz,DMSO-d6)δ7.79(dd,J=9.0,0.8Hz,1H),7.69-7.50(m,2H),7.18-7.09(m,1H),7.05(d,J=8.8Hz,1H),6.78(s,2H),6.69(dd,J=8.8,0.8Hz,1H),6.51(dd,J=7.2,0.8Hz,1H),5.80(s,2H),5.49(s,2H),3.82(s,3H)。

[0322] MS(ESI):[M+H + ]321.37

[0323] Compound 68

[0324]

[0325] 1 H NMR(400MHz,DMSO-d6)δ7.67(d,J=9.0Hz,1H),7.62-7.51(m,2H),7.33-7.13(m,2H),7.04(d,J=9.0Hz,1H),6.76(s,2H),6.39(dd,J=7.6,1.2Hz,1H),5.76(s,2H),5.65(s,2H)。

[0326] MS(ESI):[M+H + ]359.21

[0327] Compound 69

[0328]

[0329] 1 H NMR(400MHz,DMSO-d6)δ7.78(d,J=8.8Hz,1H),7.62(d,J=3.0Hz,1H),7.08(d,J=3.0Hz,1H),7.03(d,J=8.8Hz,1H),6.88-6.79(m,2H),6.76-6.73(m,1H),5.96(s,2H),5.76(s,2H),5.38(s,2H)。

[0330] MS(ESI):[M+H + ]334.36

[0331] Compound 70

[0332]

[0333] 1 H NMR(400MHz,DMSO-d6)δ7.71(d,J=8.8Hz,1H),7.55(d,J=3.2Hz,1H),7.43(dd,J=8.8,2.4Hz,1H),7.10(d,J=3.2Hz,1H),7.07-7.01(m,2H),6.79(d,J=2.4Hz,1H),6.73(s,2H),5.75(s,2H),5.42(s,2H),3.87(s,3H)。

[0334] MS(ESI):[M+H+]398.24。

[0335] Compound 71

[0336]

[0337] 1 H NMR(400MHz,DMSO-d6)δ7.83(d,J=9.2Hz,1H),7.64(d,J=3.2Hz,1H),7.09(d,J=3.2Hz,1H),7.04(d,J=9.2Hz,1H),6.74(s,2H),6.61(s,2H),5.76(s,2H),5.38(s,2H),3.68(s,6H),3.59(s,3H)。

[0338] MS(ESI):[M+H+]398.24

[0339] Anticancer properties of compound 1 (IRS-17)

[0340] The anticancer properties of compound 1 were studied as follows:

[0341]

[0342] A. Compound 1 - Purine and Thymidine Inhibition

[0343] (a) HCT116 and (b) Panc1 cell lines were each seeded in a 96-well plate at a density of 3000 / well and 80 μL DMEM culture medium supplemented with 10% by volume dialyzed FBS was added. The next day, 10 μL of culture medium containing or not containing hypoxanthine (1 mM) and thymidine (160 μM) was added to each well. 10 μL of compound 1 was diluted in the culture medium and 10x stock solution of the desired concentration was added at the same time. The relative cell number was measured by resazurin before and 4 days after the addition of compound 1. That is, resazurin dissolved in phosphate-buffered saline (PBS) was added to each well to a final concentration of 10 μg / mL and then incubated at 37°C for 1 hour. Fluorescence was then measured by a microplate reader (excitation 550 nm, emission 590 nm). The results are provided in Figures 1A and 1B. As shown in Figures 1A and 1B, compound 1 inhibited cell growth under supplementary and non-supplementary conditions. Growth inhibition of both cancer cell lines was achieved by disrupting purine and thymidine synthesis.

[0344] B. Compound 1-Folic Acid Competition

[0345] Compound 1 was tested against pemetrexed and methotrexate in two isogenic CHO cell lines with low and high RFC expression levels. IC50 values ​​were determined in RPMI medium without folic acid supplemented with 10% dialyzed FBS and 25 nM 5-formyl THF. Table II provides the IC50 values ​​for Compound 1, pemetrexed, and methotrexate.

[0346] Table II

[0347]

[0348] As shown in Table II, Compound 1 exhibited higher efficacy in cells that downregulated RFC and were resistant to pemetrexed and methotrexate. Figure 2 also shows the higher efficacy of Compound 1 in cells with low RFC expression.

[0349] C. Effects of Compound 1 on KPC pancreatic ductal adenocarcinoma allografts

[0350] From K-ras LSLG12D / + Trp53 R172H / +Tumors harvested from Pdx-1-Cre(KPC) mice were passaged subcutaneously in the flanks of C57BL / 6 mice. To initiate allografts, subcutaneous tumors were harvested and minced in DMEM medium, mixed with an equal volume of Matrigel basement membrane matrix (Corning 54234), and injected subcutaneously in the flanks of male C57BL / 6 mice. Tumors were measured twice weekly. Once the average tumor volume reached 100 mm 3 , compound 1 (5 mg / kg body weight, dissolved in 10% 2-hydroxypropyl-β-cyclodextrin) and vehicle (10% 2-hydroxypropyl-β-cyclodextrin, 10 mL / kg body weight) were injected intraperitoneally once a day, and tumors were continuously monitored. At the end of the experiment, mice were euthanized by cervical dislocation after fasting for 6 hours. Tumors were collected into aluminum foil, clamped with Wollenberg clamps pre-cooled in liquid N2, and kept cold in liquid N2.

[0351] To extract metabolites from tumor samples, the tumor was first ground using a cryo-grinder (Retsch). Approximately 10 mg of ground tissue was measured and extracted with extraction buffer (acetonitrile: methanol: H2O = 2: 2: 1, then supplemented with 0.5 vol% formic acid) at a ratio of 370 μL of buffer per 10 mg of tissue. The sample was vortexed and adjusted to pH 7 with 15% NH4HCO3 (30 μL per 10 mg of tissue). The sample was centrifuged at 4°C (16000 g, 20 minutes) and the supernatant was collected for measurement. Metabolite levels were measured by Q ExactivePlus Hybrid Quadrupole-Orbitrap combined with hydrophobic interaction chromatography (HILIC) operating in negative ionization mode.

[0352] Figure 3A shows the change of tumor volume over time, and Figure 3B shows the tumor doubling time. As shown in Figures 3A and 3B, compound 1 substantially inhibited tumor growth during the study and prolonged the tumor doubling time. Figure 3C shows the intermediate levels of purine synthesis glycine amide ribonucleotide (GAR) and 5-aminoimidazole-4-carboxamide ribonucleotide (A1CAR) in tumors, indicating that folic acid competes or interferes.

[0353] D. Effects of Compound 1 on HCT116 Colorectal Cancer Xenografts

[0354] To initiate allografts, 2 × 10 6 MC38 cells. Once the average tumor volume reaches 50 mm 3, compound 1 (5 mg / kg body weight, dissolved in 10% 2-hydroxypropyl-β-cyclodextrin) and vehicle (10% 2-hydroxypropyl-β-cyclodextrin, 10 mL / kg body weight) were injected intraperitoneally once a day. After the final dose, the mice were fasted for 12 hours. The blood was then collected into a tube without anticoagulant by snipping the tail. The supernatant was taken after centrifugation to collect the serum. The mice were then euthanized by cervical dislocation, and the tumors were harvested and frozen in liquid N2. Tumor metabolites were extracted and measured as previously described in C. To extract serum metabolites, 2.5 μL of serum was added to 80 μL of methanol. The samples were centrifuged at 4°C (16000g, 10 minutes) and the supernatant was collected.

[0355] Figure 4A shows the changes in tumor volume over time, and Figure 4B shows the tumor doubling time. As shown in Figures 4A and 4B, Compound 1 substantially inhibited tumor growth and prolonged tumor doubling time during the study period.

[0356] E. Effects of Compound 1 on MC38 Mouse Colon Adenocarcinoma Allografts

[0357] To initiate allografts, 2 × 10 6 MC38 cells. Once the average tumor volume reaches 50 mm 3 , IRS-17 (5 mg / kg body weight, dissolved in 10% 2-hydroxypropyl-β-cyclodextrin) and vehicle (10% 2-hydroxypropyl-β-cyclodextrin, 10 mL / kg body weight) were injected intraperitoneally once a day. After the final dose, the mice were fasted for 12 hours. The blood was then collected into tubes without anticoagulant by snipping the tail. The supernatant was taken after centrifugation to collect the serum. The mice were then euthanized by cervical dislocation, and the tumors were harvested and frozen in liquid N2. Tumor metabolites were extracted and measured as previously described in C. To extract serum metabolites, 2.5 μL of serum was added to 80 μL of methanol. The samples were centrifuged at 4°C (16000g, 10 minutes) and the supernatant was collected.

[0358] Figure 5A shows that tumor volume decreased during treatment. Figure 5B shows the pool size of circulating adenosine, and Figure 5C shows the levels of the thymidine ribonucleotide intermediate dUMP and the purine intermediates GAR and AlCAR in tumors in response to administration of Compound 1.

[0359] Various embodiments of the present invention have been described to achieve the various purposes of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations thereof will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. A pharmaceutical composition for inhibiting the growth of cancer cells that downregulate reduced folate carriers, the pharmaceutical composition comprising a compound and / or its salts, The compound is present in the pharmaceutical composition in an amount sufficient to exhibit anti-cancer properties. 2 . The pharmaceutical composition according to claim 1 , wherein the compound and / or its salt is present in an amount of 0.01 nM to 1 μM.

3. The pharmaceutical composition according to claim 1, wherein the compound and / or its salt is present in an amount of 0.01 nM to 500 nM.

4. The pharmaceutical composition according to claim 1, wherein the compound inhibits purine and / or thymine biosynthesis in cancer cells. The pharmaceutical composition according to claim 4 , wherein the cancer cells are pancreatic cancer cells.

6. The pharmaceutical composition according to claim 1, wherein the compound exhibits higher efficacy in downregulating reduced folate carriers in cancer cells compared to pemetrexed or methotrexate.

7. The pharmaceutical composition of claim 1, wherein the compound inhibits the growth of colorectal tumors.

8. The pharmaceutical composition of claim 1, further comprising one or more adjuvants or additional chemotherapeutic agents.

9. A compound and / or a salt thereof, wherein the compound is 10. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating cancer in a patient having cancerous tissue.

11. The use according to claim 10, wherein the cancer cells downregulate the reduced folate carrier.

12. The use according to claim 11, wherein the IC50 of the compound is less than 0.1 nM.

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