A phenanthroline substituted methylguanidine compound and its preparation and application

Abstract

The present invention relates to a phenanthroline substituted methylguanidine compound and its preparation and application. The phenanthroline substituted methylguanidine compound has a structure as shown in formula (I):, wherein R is selected from any one of the following groups:,,,. Compared with the prior art, the compound provided by the present invention has a significant PKM2 inhibitory effect, has good anti-proliferation activity on A549 and HCC1833 cells, and can significantly inhibit tumor growth in a human NSCLC model. In addition, the phenanthroline substituted methylguanidine compound is a promising new PKM2 inhibitor, which can be used to prevent and / or treat diseases related to PKM2.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compounds and relates to a phenanthroline substituted methylguanidine compound and a preparation method and application thereof. Background Art

[0002] Pyruvate kinase M2 (PKM2, type II pyruvate kinase), as one of the pyruvate kinases (PKs), plays an important role in regulating glycolysis to produce different energies and products, which are used for the rapid growth and proliferation of cancer cells. Abnormalities of PKM2 are associated with the occurrence and development of many cancers, including breast cancer, pancreatic cancer, glioma, gastric cancer, non-small cell lung cancer, etc. Targeting PKM2 as a drug target, in the past five years, more and more studies have explored PKM2 inhibitors and agonists, demonstrating their potential efficacy in cancer treatment. Although a lot of efforts have been invested in the development of different PKM2 inhibitors and agonists, there is still a lot of room to improve PKM2 inhibitors and agonists as candidate drugs for the treatment of cancer, such as improving the specificity of the compounds, reducing toxic side effects, or increasing cell permeability.

[0003] Currently, only one chemically synthesized PKM2 inhibitor (Cap-232) has entered clinical research, but it has been terminated due to poor clinical effects. Currently, it is urgent to discover PKM2 inhibitors of new structural types. The present invention is proposed based on this. Summary of the invention

[0004] The purpose of the present invention is to provide a phenanthroline substituted methylguanidine compound and its preparation and application, so as to achieve high inhibitory activity on PKM2 when used as a PKM2 inhibitor.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] In the first aspect, the present invention provides a phenanthroline substituted methylguanidine compound having a structure as shown in formula (I):

[0007] ,

[0008] Where R is C 6 -C 10 Aryl, mono- or poly-substituted C 6 -C 10 Aryl, C 3 -C 9 Cycloalkyl, mono- or poly-substituted C 3 -C 9 Cycloalkyl, 3-10 membered heterocyclyl, mono- or poly-substituted 3-10 membered heterocyclyl, 5-10 membered heteroaryl, mono- or poly-substituted 5-10 membered heteroaryl, C1 -C 6 Alkyl, C 1 -C 6 Heteroalkyl, C 1 -C 6 Haloalkyl, C 1 -C 6 Haloalkoxy.

[0009] Preferably, R is selected from C 6 -C 10 Aryl, mono- or poly-substituted C 6 -C 10 Aryl.

[0010] More preferably, R is selected from phenyl, mono-substituted or di-substituted phenyl.

[0011] Further more preferably, when R is a mono-substituted or di-substituted phenyl group, the substituent on the phenyl group is any one or two of the following functional groups:

[0012] Halogen, trifluoromethyl, methoxy, N,N-dimethyl, tert-butyl, hydroxy, nitro, cyano, methyl.

[0013] Unless otherwise specified, the "substituted" mentioned above in the present invention means that one or more hydrogen atoms on the group are replaced by corresponding substituents.

[0014] In some specific embodiments, the chemical structural formula of the phenanthroline substituted methylguanidine compound of the present invention is any one of the following:

[0015] , , , .

[0016] More preferably, .

[0017] In a second aspect, the present invention provides a method for preparing a phenanthroline substituted methylguanidine compound, comprising the following steps:

[0018] S1, compound a reacts with formaldehyde to form an imine, which is then reduced with sodium borohydride to obtain compound b;

[0019] S2, compound c is reacted with benzoyl isothiocyanate to obtain compound d;

[0020] S3, compound d and compound b are condensed and desulfurized by EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) to obtain compound e;

[0021] S4, compound e is debenzoylated under alkaline conditions to obtain a phenanthroline-substituted methylguanidine compound, i.e., the target product;

[0022] The chemical structural formula of the compound a is:

[0023] H 2 NR;

[0024] The chemical structural formula of the compound b is:

[0025] -HN-R;

[0026] The chemical structural formula of the compound c is:

[0027] ;

[0028] The chemical structural formula of the compound d is:

[0029] ;

[0030] The chemical structural formula of the compound e is:

[0031] .

[0032] In a third aspect, the present invention further provides a pharmaceutical composition, comprising: (a) the phenanthroline substituted methylguanidine compound as described in the first aspect; and (b) a pharmaceutically acceptable carrier.

[0033] Here, in the pharmaceutical composition, component (a) may be, in addition to the phenanthroline-substituted methylguanidine compound, a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof.

[0034] Specifically, representative pharmaceutically acceptable salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, amidosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicylate, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate.

[0035] In a fourth aspect, the present invention further provides a use of a phenanthroline substituted methylguanidine compound in the preparation of a PKM2 inhibitor. The phenanthroline substituted methylguanidine compound is as defined in the first aspect above.

[0036] In a fifth aspect, the present invention further provides a use of a phenanthroline substituted methylguanidine compound in the preparation of a drug for preventing and / or treating a disease associated with PKM2. The phenanthroline substituted methylguanidine compound is as defined in the first aspect above.

[0037] Preferably, the drug is a drug for preventing and / or treating tumors.

[0038] More preferably, the tumor is at least one of a cancer or a benign tumor.

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The invention provides a process route for preparing phenanthroline substituted methylguanidine compounds.

[0041] Figure 2 The human NSCLC (non-small cell lung cancer) xenograft tumor was grown using the compound 1 prepared in Example 1 and a PBS control group.

[0042] Figure 3 The time-tumor volume growth curve of human NSCLC xenograft tumors using compound 1 prepared in Example 1 and a PBS control group.

[0043] Figure 4 The figure is a graph showing the body weight changes of mice transplanted with human NSCLC using compound 1 prepared in Example 1 and a PBS control group. DETAILED DESCRIPTION

[0044] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0045] In all embodiments, 1 H-NMR was recorded by JEOL JNM-ECZS 400 NMR, and chemical shift was expressed in δ (ppm); silica gel was used for separation, and 200-300 mesh was used unless otherwise specified, and the ratio of eluent was volume ratio. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0046] Abbreviations: PE = petroleum ether; MeOH = methanol; EA = ethyl acetate; DCM = dichloromethane.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value.

[0049] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0050] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (i.e., C1-6 means 1-6 carbons, preferably 1-3 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and the like.

[0051] Unless otherwise indicated, the term "cycloalkyl" refers to a group having the specified number of ring atoms (e.g., C 3-10 The term "cycloalkyl" refers to a hydrocarbon ring which is fully saturated or has no more than one double bond between the ring tops. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be a bicyclic and polycyclic hydrocarbon ring, such as a bridged ring or a spirocyclic ring, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.

[0052] Unless otherwise stated, the term "heterocycloalkyl" refers to a cycloalkyl containing a specified number of heteroatoms selected from O, N and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atoms are optionally quaternized. Heterocycloalkyl can be a monocyclic, bicyclic or polycyclic system. Non-limiting examples of heterocycloalkyl include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc. Heterocycloalkyl can be connected to the rest of the molecule via a ring carbon or a heteroatom. For terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that cycloalkyl or heterocycloalkyl is connected to the rest of the molecule via an alkyl or alkylene linker. For example, cyclobutylmethyl- is a cyclobutyl ring attached to a methylene linkage to the rest of the molecule.

[0053] Similarly, unless otherwise indicated, the term "heteroalkyl" refers to a group containing the specified number of carbons and 1 to 3 heteroatoms selected from O, N and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N and S may be located at any interior position of the heteroalkyl group.

[0054] Unless otherwise indicated, the term "haloalkoxy" is used in its conventional sense to refer to those alkyl groups which are attached to the remainder of the molecule via an oxygen atom or nitrogen, respectively, and which alkoxy groups are mono- or di-substituted with halogen.

[0055] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl or polyhaloalkyl. For example, the term "C 1-6 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0056] Unless otherwise indicated, the term "aryl" means a polyunsaturated (usually aromatic) hydrocarbon radical which may be a single ring or multiple rings (up to three) fused together or linked covalently. Non-limiting examples of aryl include phenyl, naphthyl and biphenyl.

[0057] The term "heteroaryl" refers to an aryl group (or ring) containing the specified number of heteroatoms selected from O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothiophenyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like.

[0058] In some embodiments, the above terms (such as "alkyl", "aryl" and "heteroaryl") will include substituted and unsubstituted forms of the specified groups, and the number of substituents can be 1, 2, 3 or 4. If not otherwise specified, the "substituted" means that one or more hydrogen atoms on the group are replaced by a group selected from the following group: halogen, trifluoromethyl, methoxy, N,N-dimethyl, tert-butyl, hydroxy, nitro, cyano, methyl.

[0059] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), and sulfur (S).

[0060] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0061] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have defined stereochemistry (expressed as R or S, or indicated by dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free of other isomers).

[0062] As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0063] As used herein, the term "therapeutically effective amount" refers to any amount of a drug as described below, which, when used alone or in combination with another therapeutic agent, can promote disease regression, which is manifested as a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disorders or disabilities caused by the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactic effective dose", which is any amount of a drug as described below, which, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of the disease, can inhibit the occurrence or recurrence of the disease.

[0064] As used herein, the term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0065] As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0066] The preparation process of the phenanthroline substituted methylguanidine compounds provided by the present invention is as follows: Figure 1 However, reactants, solvents, amounts of compounds used, reaction temperature, reaction time, etc. are not limited to the following descriptions.

[0067] In accordance with Figure 1 Taking the method shown in the figure as an example to synthesize compounds 1 to 4, the method specifically comprises the following steps:

[0068] Step a, compound a (i.e. Figure 1 Compounds A1 to D1) in the reaction mixture are reacted with formaldehyde to form an imine, which is then reduced with sodium borohydride to give compound b (i.e. Figure 1 intermediates A2-D2 in (a);

[0069] Step b, compound c (i.e. Figure 1 Compound E) in the reaction mixture is added with benzoyl isothiocyanate to obtain compound d (i.e. Figure 1 Intermediate F in

[0070] Step c: Compound d and compound b are subjected to EDCI condensation desulfurization to obtain compound e (i.e. Figure 1 intermediates 1i-4i in (

[0071] Step d: Compound e is debenzoylated under alkaline conditions to obtain phenanthroline-substituted methylguanidine compounds, namely compounds 1 to 4.

[0072] The above process is described in more detail below in conjunction with specific embodiments.

[0073] The intermediates used in the embodiments and comparative examples of the present invention and their preparation methods are as follows:

[0074] Intermediate A2: 3-Fluoro-N-methyl-5-trifluoromethylaniline

[0075]

[0076] 2-Fluoro-5-trifluoromethylaniline (i.e., compound A1, 0.50 g, 2.80 mmol), paraformaldehyde (0.84 g, 14.00 mmol) and methanol (20 mL) were added to a 100 mL single-mouth bottle, and sodium methoxide (0.76 g, 14.00 mmol) was slowly added under stirring, and refluxed for 2 h. After the reaction of the raw materials was completed, sodium borohydride (0.53 g, 14.00 mmol) was slowly added in batches under ice bath conditions, and then refluxed for 1 h. After the reaction was completed, the reaction solution was poured into 20 mL of water for quenching, extracted with DCM (15 mL x 3), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to obtain 0.35 g of yellow oily matter, with a yield of 66%.

[0077] 1 H NMR (400 MHz, Chloroform- d ) δ 6.97 (dd, J= 11.2, 8.1 Hz, 1H), 6.88– 6.81 (m, 2H), 4.09 (s, 1H), 2.85 (d, J = 5.2 Hz, 3H). 13 C NMR (101 MHz, Chloroform- d ) δ 152.99 (dd, J = 244.1, 1.6 Hz), 138.15 (d, J = 12.4 Hz), 125.60,122.90, 114.17 (d, J = 19.8 Hz), 113.34 (dq, J = 8.3, 4.2 Hz), 107.92 (p, J = 3.8Hz), 29.82. LC-MS (ESI): m / z [M +H] + Calculate for C 8 H 7 F 4 N 194.1; found 193.9.

[0078] Intermediate B2: 3-tert-butyl-N-methylaniline

[0079]

[0080] According to the method for synthesizing intermediate A2, 3-tert-butylaniline (i.e., compound B1, 2.00 g, 13.40 mmol) was used as raw material, and EA / PE=1:19 was separated by column chromatography to obtain 1.50 g of yellow oil with a yield of 49.0%. 1 H NMR (600 MHz, DMSO- d 6 ) δ 7.00 (t, J = 7.6 Hz, 1H), 6.59 – 6.53 (m, 2H), 6.32 (ddd, J = 8.0,2.3, 1.1 Hz, 1H), 5.45 (s, 1H), 2.66 (s, 3H), 1.23 (s, 9H). LC-MS (ESI): m / z[M +H] + Calculate for C 11 H 17 N 164.26; found 164.0.

[0081] Intermediate C2: 3-methoxy-N-methylaniline

[0082]

[0083] According to the method for synthesizing intermediate A2, 3-methoxyaniline (i.e., compound C1, 3.00 g, 24.40 mmol) was used as raw material, and EA / PE=1:19 was separated by column chromatography to obtain 1.30 g of yellow oil with a yield of 38.8%.

[0084] 1 H NMR (400 MHz, Chloroform- d ) δ 7.08 (t, J = 8.1 Hz, 1H), 6.29 – 6.25(m, 1H), 6.23 (dt, J = 8.1, 1.4 Hz, 1H), 6.16 (t, J = 2.3 Hz, 1H), 3.77 (s, 3H),2.81 (s, 3H). LC-MS (ESI): m / z [M +H] + Calculate for C 8 H 11 NO 138.18; found 138.1.

[0085] Intermediate D2: N1,N1,N3-trimethylbenzo-1,3-diamine

[0086]

[0087] According to the method for synthesizing intermediate A2, 3-amino-N,N-dimethylaniline (i.e., compound D1, 2.00 g, 14.70 mmol) was used as raw material, and EA / PE=1:9 was separated by column chromatography to obtain 0.43 g of yellow oil, with a yield of 19.5%. 1 H NMR (600 MHz, DMSO- d 6 ) δ 6.80 – 6.76 (m, 2H), 6.67 (td, J = 9.6, 9.1, 2.9 Hz, 2H),2.81 (d, J = 68.0 Hz, 9H). LC-MS (ESI): m / z [M +H] + Calculate for C 9 H 14 N 2 151.2; found151.0.

[0088] Intermediate F: N-(1,10-phenanthroline-5-aminosulfinyl)benzamide

[0089]

[0090] Dissolve 1,10-phenanthroline-5-amino (0.58 g, 2.56 mmol) in 20 mL acetonitrile, add benzoyl isothiocyanate (0.11 g, 2.56 mmol) under stirring, and react overnight at room temperature. Filter, rinse the filter cake with acetonitrile (10 mL x 3), and dry to obtain 0.78 brown solid, mp = 170.7-172.8°C, yield: 85.1%.

[0091] 1 H NMR (600 MHz, Chloroform- d ) δ 12.94 (s, 1H), 9.39 (s, 1H), 9.23(ddd, J = 27.3, 4.3, 1.7 Hz, 2H), 8.55 – 8.46 (m, 2H), 8.30 (dd, J = 8.1, 1.8 Hz,1H), 8.06 – 7.95 (m, 2H), 7.80 – 7.68 (m, 2H), 7.64 – 7.54 (m, 2H). LC-MS(ESI): m / z [M +H] + Calculate for C 20 H 14 N 4 OS 359.4; found 358.7.

[0092] Example 1

[0093] This example provides a PKM2 inhibitor (denoted as compound 1), the structural formula of which is as follows:

[0094]

[0095] The preparation method of the PKM2 inhibitor comprises the following steps:

[0096] Add intermediate F (0.54 g, 1.5 mmol) and intermediate A2 (0.29 g, 1.5 mmol) to a 50 mL round-bottom flask, dissolve them in dry dichloromethane (10 mL), place in an ice bath, slowly add triethylamine (0.30 g, 417 ul, 3.0 mmol) and EDCI (1.15 g, 6.0 mmol), protect with nitrogen, stir in an ice bath for 1 hour and react at room temperature overnight. Extract with dichloromethane (10 mL, 3 times), wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, and evaporate to dryness to obtain 0.29 g of yellow oil, which is intermediate 1i, with a yield of 37.3%. LC-MS (ESI): m / z [M +H] + calcdfor C 23 H 18 C1N 5 O 518.4; found 518.6.

[0097] All the intermediate 1i (0.29 g, 0.89 mmol) prepared above was added into a 50 mL round-bottom flask, and LiOH . H 2 O (0.25 g, 0.62 mmol), H 2 O (63.2 ul) and MeOH (20 mL), reflux at 85°C overnight, evaporate directly to dryness, and separate by column chromatography (MeOH / DCM=1:19) to obtain 160 mg of a yellow oil, which was further purified by pre-HPLC to obtain 110 mg of the target compound (i.e., compound 1) as a colorless oil, with a yield of 47.8%.

[0098] 1 H NMR (600 MHz, Deuterium Oxide) δ 9.16 (td, J = 5.8, 5.1, 1.5 Hz,2H), 8.97 (d, J = 8.3 Hz, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 2.7 Hz, 2H),8.06 (dd, J = 8.5, 4.7 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.26 (d, J = 8.1 Hz, 1H),3.54 (s, 3H). LC-MS (ESI): m / z [M +H] +Calculate for C 21 H 15 F 4 N 5 414.3; found 413.7.

[0099] Example 2

[0100] This example provides a PKM2 inhibitor (denoted as compound 2), the structural formula of which is as follows:

[0101]

[0102] According to the method for synthesizing compound 1, intermediate F (0.72 g, 2.0 mmol) and intermediate B2 (0.33 g, 2.0 mmol) were used as raw materials and separated by column chromatography (MeOH / DCM=1:19) to obtain 250 mg of yellow oil, namely intermediate 2i, which was further purified by pre-HPLC to obtain 200 mg of the target compound (i.e., compound 2), a colorless oil, with a yield of 57.1%.

[0103] 1 H NMR (600 MHz, Deuterium Oxide) δ 9.12 (ddd, J = 8.5, 5.0, 1.4 Hz,2H), 8.90 (dd, J = 8.4, 1.5 Hz, 1H), 8.68 (d, J = 8.5 Hz, 1H), 8.13 (dd, J = 8.3,5.2 Hz, 1H), 8.09 (s, 1H), 8.03 (dd, J = 8.5, 4.8 Hz, 1H), 7.19 – 7.02 (m, 4H), 3.47 (s, 3H), 0.92 (s, 9H). LC-MS (ESI): m / z [M +H] + Calculate for C 24 H 25 N 5 384.5; found 384.8.

[0104] Example 3

[0105] This example provides a PKM2 inhibitor (denoted as compound 3), the structural formula of which is as follows:

[0106]

[0107] According to the method for synthesizing compound 1, intermediate F (0.54 g, 1.5 mmol) and intermediate C2 (0.21 g, 1.5 mmol) were used as raw materials and separated by column chromatography (MeOH / DCM=1:19) to obtain 150 mg of yellow oil, namely intermediate 3i, which was further purified by pre-HPLC to obtain 120 mg of the target compound (i.e., compound 3), a colorless oil, with a yield of 23.5%.

[0108] 1 H NMR (600 MHz, Deuterium Oxide) δ 9.15 (ddd, J = 7.3, 4.9, 1.4 Hz,2H), 8.93 (dd, J = 8.4, 1.5 Hz, 1H), 8.73 – 8.69 (m, 1H), 8.18 – 8.13 (m, 2H), 8.05 (dd, J = 8.5, 4.8 Hz, 1H), 7.17 (t, J = 8.2 Hz, 1H), 6.95 (dd, J = 7.9, 1.9Hz, 1H), 6.89 (t, J = 2.3 Hz, 1H), 6.73 (dd, J = 8.1, 2.4 Hz, 1H), 3.61 (s, 3H), 3.49 (s, 3H). LC-MS (ESI): m / z [M +H] + Calculate for C 281 H 19 N 5 O 358.4; found 357.8.

[0109] Example 4

[0110] This example provides a PKM2 inhibitor (denoted as compound 4), the structural formula of which is as follows:

[0111]

[0112] According to the method for synthesizing compound 1, intermediate F (0.72 g, 2.0 mmol) and intermediate D2 (0.30 g, 2.0 mmol) were used as raw materials and separated by column chromatography (MeOH / DCM=1:19) to obtain 150 mg of yellow oil, namely intermediate 4i, which was further purified by pre-HPLC to obtain 100 mg of the target compound (i.e., compound 4), a colorless oil, with a yield of 16.0%.

[0113] 1 H NMR (600 MHz, Deuterium Oxide) δ 9.19 (dd, J = 10.7, 4.9 Hz, 2H),8.99 (d, J = 8.3 Hz, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.23 ​​– 8.17 (m, 2H), 8.08(dd, J = 8.5, 4.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 3.55(s, 3H), 3.10 (s, 6H). LC-MS (ESI): m / z [M +H] + Calculate for C 22 H 22 N 6 371.4; found371.8.

[0114] 1. Compound Activity Test

[0115] Evaluation of the PKM2 inhibitory activity of the PKM2 inhibitor of the present invention

[0116] Experimental principle:

[0117] Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate into pyruvate, accompanied by the conversion of NADH into NAD⁺. At a specific wavelength (340nm), the reduction of NADH will lead to a decrease in absorbance. By detecting the change in absorbance over time, the activity of pyruvate kinase can be calculated. The inhibition of PKM2 activity by the compound will slow down the reaction rate catalyzed by pyruvate kinase in the reaction system and reduce the change in absorbance.

[0118] Experimental methods:

[0119] The plenti-Flag-PKM2 plasmid was transfected into HCC1833, non-small cell lung cancer A549 and 293T cells by lentivirus, and then screened with puromycin to establish a stable expression of PKM2 cell line. For the preliminary screening of the four compounds prepared in Examples 1 to 4 above, each compound was added to 100 μL of lysate of 293T cells (derived from 800,000 cells) with high PKM2 expression at a concentration of 100 μM, incubated at 37°C for 30 minutes, and operated according to the instructions of the pyruvate kinase activity detection kit (D799443, Shanghai Sangon Biotechnology Co., Ltd.). The entire reaction was carried out in a 96-well plate. The absorbance at a wavelength of 340 nm was measured using a microplate reader at 0 minutes and 10 minutes after the addition of the working solution, and the increase in absorbance was related to the activity unit of pyruvate kinase. For HCC1833 and A549 cells stably expressing PKM2, the compounds were co-cultured with 800,000 cells in a 12-well plate at 37°C for 30 min, the cell lysate was extracted, and the working solution was added to measure the absorbance at 0, 2, 4, 6, 8, and 10 min to calculate the enzyme activity.

[0120] Experimental results:

[0121] 1) In 293T cells stably overexpressing PKM2, shikonin and compound 3K (C3K, CAS: 94164-88-2) were used as positive controls. The four new compounds were incubated with lysate of 293T cells overexpressing PKM2 at a concentration of 100 μM for 30 minutes, and then the lysate was collected to evaluate the activity of pyruvate kinase. The inhibition rate of the compound on PKM2 was calculated using the following formula: Inhibition rate (%) = (1-U 化合物 / U 空白 ) / (1-U 紫草素 / U 空白 )×100%. The results showed that the four compounds exhibited significant inhibitory activity against PKM2, among which the inhibition rate of compound 1 against PKM2 was 115.04%±10.26%, which exceeded the inhibition rate of the positive control shikonin and compound 3K against PKM2 (Table 1).

[0122] Table 1 Inhibitory activity of different compounds on PKM2 (the inhibition rate of shikonin is 100%)

[0123]

[0124] 2) Evaluation of the cell viability inhibition of these compounds in two cell lines. The test results showed that the half-inhibitory concentration (IC) of compound 1 in A549 and HCC1833 cell lines was 50) were the lowest, 3.36 ± 0.56 μM and 9.20 ± 0.68 μM, respectively (Table 2), which were significantly lower than the IC of the positive control shikonin and compound 3K 50 , indicating that compound 1 has a stronger inhibitory effect on PKM2 than the positive control. All data analysis and IC 50 The values ​​were calculated using GraphPad Prism 9.0 (GraphPad Software, Inc., USA). The data are expressed as the mean ± standard error of the mean (SEM).

[0125] Table 2. Half-inhibitory concentration values ​​of compounds for anti-NSCLC activity (48h treatment)

[0126]

[0127] 2. Study on the anti-tumor effect of PKM2 inhibitor in vivo

[0128] Experimental principle:

[0129] The inhibitory ability of the compounds on non-small cell lung cancer was evaluated by measuring the volume of mouse tumors.

[0130] Experimental content:

[0131] A549 cells were subcutaneously injected into BALB / c nude mice to establish a non-small cell lung cancer xenograft model. 3 At 14:00 pm, mice were randomly divided into two groups (n = 5 per group) to start the experiment: (1) PBS control; (2) 10 mg / kg dose of compound 1. Then, mice were treated with PBS or compound 1 every other day until the end of the experiment. Tumor development was measured regularly, and tumor volume was determined using the formula V = (length × width2) / 2. For A549 xenografts, treatment ended on day 18 and mice were humanely euthanized.

[0132] Experimental results:

[0133] In the human NSCLC xenograft model, administration of 10 mg / kg of compound 1 significantly inhibited tumor growth compared with the PBS control group. The tumor volume was significantly lower than that of the PBS control group with statistical significance, and there was no adverse effect on the weight of mice during the experiment (see Figures 2 to 4 ).

[0134] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A phenanthroline substituted methylguanidine compound, characterized in that: Having the structure shown in formula (I): Wherein, R is any one of the following groups:

2. A phenanthroline substituted methylguanidine compound according to claim 1, characterized in that: Its chemical structure is:

3. The method for preparing a phenanthroline substituted methylguanidine compound according to claim 1 or 2, wherein: The following steps are involved: S1, compound a reacts with formaldehyde to form an imine, which is then reduced with sodium borohydride to obtain compound b; S2, compound c is reacted with benzoyl isothiocyanate to obtain compound d; S3, compound d and compound b are condensed and desulfurized by EDCI to obtain compound e; S4, compound e is debenzoylated under alkaline conditions to obtain a phenanthroline-substituted methylguanidine compound, i.e., the target product; The chemical structural formula of the compound a is: H2N-R; The chemical structural formula of the compound b is: -HN-R; The chemical structural formula of the compound c is: The chemical structural formula of the compound d is: The chemical structural formula of the compound e is:

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the phenanthroline substituted methylguanidine compound according to claim 1 or 2; and (b) a pharmaceutically acceptable carrier.

5. Use of the phenanthroline substituted methylguanidine compound as claimed in claim 1 or 2 in the preparation of a drug for inhibiting non-small cell lung cancer.

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