Pyrimidine fused ring compound as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention discloses a pyrimidine fused ring compound as well as a preparation method, a pharmaceutical composition and application thereof. The structure of the compound is shown in a formula I, the compound further comprises pharmaceutically acceptable salt of the compound, the compound can effectively inhibit ATX enzyme, fibroblast migration and TGF-beta / Smad signal channels, the effect is achieved through multiple action mechanisms of the molecular level, the cellular level and the signal channel level, the medicine effect is definite, and the compound has the advantages of being efficient and low in toxicity. Specifically, the compound can be prepared into medicines for resisting fibrosis of organs such as heart, liver, lung and kidney, and has a wide application prospect. In addition, the preparation method of the compound is high in universality, and the structure is easy to expand. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a pyrimidine-fused ring compound, a preparation method thereof, a pharmaceutical composition and an application, and in particular to a pyrimidine-fused ring compound that can be prepared into an anti-fibrotic drug, a preparation method thereof, a pharmaceutical composition and an application. Background Art

[0002] Fibrosis is a pathological process of many chronic inflammatory diseases or injuries, which is characterized by the excessive accumulation of extracellular matrix (ECM) components such as collagen and fibronectin. During the development of fibrosis, ECM components continuously accumulate, leading to tissue structure destruction, organ dysfunction, and ultimately organ failure. Fibrosis affects almost all tissues of the body and results in high morbidity and mortality of many diseases, such as idiopathic pulmonary fibrosis (IPF). Although there has been a profound understanding of the pathophysiology of fibrosis, there are few effective treatment strategies or therapeutic agents. Currently, only pirfenidone and nintedanib, drugs targeting the pathogenesis of fibrosis, have been approved by the US Food and Drug Administration. Clinical results show that these two drugs can slow down the progression of pulmonary fibrosis. However, their use is hindered by severe side effects, such as nausea, diarrhea, and in a few cases, they can cause more serious allergic reactions. Therefore, there is an urgent need to develop new safe and effective anti-fibrotic drugs.

[0003]

[0004] Autotaxin (ATX) is a secreted lysophospholipase D that is responsible for converting extracellular lysophosphatidylcholine (LPC) into the bioactive lipid mediator lysophosphatidic acid (LPA). LPA can activate multiple signaling pathways by acting on a group of six G protein-coupled receptors (GPCRs) LPA1-6 and promote a series of physiological events, including differentiation, migration, proliferation, and survival. A large number of studies have shown that the dysregulation of the ATX-LPA signaling pathway is associated with various disease processes, such as cancer and fibrotic diseases. Therefore, ATX has been identified as an important target for anti-fibrotic therapy. Summary of the Invention

[0005] Objectives of the Invention: The first objective of the present invention is to provide a pyrimidine-fused ring compound, the second objective is to provide a preparation method of the compound, the third objective is to provide a pharmaceutical composition containing the compound, and the fourth objective is to provide an application of the compound and its pharmaceutical composition in the preparation of anti-fibrotic drugs.

[0006] Technical Solution: The pyrimidine-fused ring compound described in the present invention has the structure of Formula I, and also includes its pharmaceutically acceptable salts:

[0007]

[0008] Wherein:

[0009] R is selected from -(CH 2 ) n -R 1 、-M-R 1 ;

[0010] n is selected from integers from 0 to 3;

[0011] M is selected from a 4- to 7-membered heterocyclic group formed by synthesizing with the connected nitrogen ring, a 4- to 7-membered cycloalkyl group, a 5- to 7-membered unsaturated heterocyclic group containing 1 to 3 N, O, S, and M and R 1 are connected by a chemical bond or a fused ring;

[0012] R 1 is selected from a 6- to 12-membered aromatic ring group containing 0 to 2 N, and the aromatic ring group is substituted with a substituent selected from any of the following: halogen, cyano, nitro, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, C1-C4 haloacyl;

[0013] The pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0014] Preferably, in the structure:

[0015] M is selected from cyclopentyl, cyclohexyl, pyrrolyl, piperidinyl formed by synthesizing with the connected nitrogen ring;

[0016] R 1 is selected from phenyl, pyridyl, and the aromatic ring group is substituted with at least one substituent selected from any of the following: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy.

[0017] More preferably, in the structure:

[0018] R 1 is selected from phenyl, pyridyl, and the phenyl is substituted with at least one substituent selected from any of the following: hydrogen, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy.

[0019] More preferably, in the structure:

[0020] When M is selected from cyclopentyl, pyrrolyl, and R 1 is selected from phenyl, M and R 1 are connected by a fused ring;

[0021] When M is selected from piperidinyl formed by synthesizing with the connected nitrogen ring, R 1 When selected from phenyl, M and R 1 Are connected by a chemical bond.

[0022] More preferably, in the structure:

[0023] When R 1 Is selected from monosubstituted phenyl, the substituent is selected from ortho, meta, and para substituents;

[0024] When R 1 Is selected from disubstituted phenyl, the substituent is selected from meta substituents.

[0025] Most preferably, R in the structure is selected from any of the following structures:

[0026]

[0027]

[0028] The preparation method of the pyrimidine-fused ring compound described in the present invention comprises the following steps:

[0029]

[0030] Compound 9 undergoes ammoniation, deprotection, and acylation reactions to obtain the pyrimidine-fused ring compound;

[0031] Among them, R is selected from -(CH 2 ) n -R 1 , -M-R 1 ;

[0032] n is an integer from 0 to 3;

[0033] M is selected from 4- to 7-membered cycloalkyl, 5- to 7-membered unsaturated heterocyclic group containing 1 to 3 N, O, S, and M and R 1 Are connected by a fused ring;

[0034] R 1 Is selected from 6- to 12-membered aromatic ring group containing 0 to 2 N, and the aromatic ring group is substituted by a substituent selected from any of the following: halogen, cyano, nitro, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, C1-C4 haloacyl.

[0035] Specifically, compound 9 reacts with an amine through a nucleophilic substitution reaction to obtain compounds 10a - 10u. Then, the Boc group is deprotected with 2M HCl - EtOAc to obtain compounds 11a - 11u. Finally, under the conditions of HATU / DIA, compounds 11a - 11u are condensed with UDCA to obtain compounds 12a - 12u, which are the pyrimidine - fused ring compounds (compounds of formula I) described in the present invention.

[0036] The chemical structures of the intermediates and end - products at each step obtained by the preparation method described in the present invention are as follows:

[0037]

[0038] The pharmaceutically acceptable salts of the compounds can be obtained by salifying the corresponding acids with the compounds prepared by the above - mentioned method.

[0039] "Pharmaceutically acceptable salts" refer to salts of compounds prepared from compounds with specific substituents and relatively non - toxic acids or bases. When a compound contains relatively acidic functional groups, the base - addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base - addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When a compound contains relatively basic functional groups, the acid - addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid - addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates), sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.; and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p - toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; organic acid salts also include salts of amino acids (such as arginine, etc.), glucuronic acid and other organic acids. When certain specific compounds contain both basic and acidic functional groups, they can thus be converted into either base or acid - addition salts. Preferably, the salt is contacted with a base or an acid in a conventional manner, and then the parent compound is separated to regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0040] "Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid or base groups by conventional chemical methods. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of appropriate bases or acids in water, an organic solvent, or a mixture of both. Generally, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0041] The pharmaceutical composition described in the present invention comprises the pyrimidine-fused ring compound and a pharmaceutically acceptable carrier.

[0042] The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

[0043] "Pharmaceutically acceptable carrier" can be excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable, and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at the desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the composition administration. The described pharmaceutical excipients can be inert fillers, or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The described pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweetening agents.

[0044] The pharmaceutical composition described in the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or freeze-drying processes.

[0045] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include but are not limited to powders, capsules, cachets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include but are not limited to suspensions, emulsions, elixirs and solutions. Examples of topical preparations include but are not limited to emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include but are not limited to injectable solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injectable suspensions and injectable emulsions. Examples of other suitable preparations of the pharmaceutical composition include but are not limited to eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0046] The pyrimidine-fused ring compound or the pharmaceutical composition of the present invention is used in the preparation of a drug for inhibiting ATX enzyme, and can also be used in the preparation of a drug for inhibiting fibroblast migration and TGF-β / Smad signaling pathway.

[0047] Preferably, the drug is an anti-fibrotic drug.

[0048] More preferably, the drug is an anti-fibrotic drug for organs such as the heart, liver, lung, kidney, etc.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0050] This class of compounds can effectively inhibit ATX enzyme, fibroblast migration and TGF-β / Smad signaling pathway, and play a role through multiple action mechanisms at the molecular level, cell level and signaling pathway level. The drug effect is exact, and it has the characteristics of high efficiency and low toxicity. Specifically, it can be prepared into an anti-fibrotic drug for organs such as the heart, liver, lung, kidney, etc., and has a wide application prospect; in addition, the preparation method of the compound has strong generality and is easy to expand the structure. Description of the Drawings

[0051] Figure 1 Effects of compounds 12a and 12h on the viability of HSC-LX2 and CCC-HPF-1 cells; (A) Effects of compounds 12a and 12h on the viability of HSC-LX2 cells; (B) Effects of compounds 12a and 12h on the viability of CCC-HPF-1 cells; Data are expressed as the mean ± standard deviation of three independent experiments;

[0052] Figure 2Inhibitory effects of compounds 12a and 12h on TGF-β1-induced migration of HSC-LX2 cells; (A) Images were taken at 0 h, 12 h, and 24 h after injury; (B, C) Percentage of wound area closure analyzed by Image J software; Data are presented as the mean ± standard deviation of three separate experiments, compared with the control group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the TGF-β1 group, * P < 0.05, ** P < 0.01, *** P < 0.001;

[0053] Figure 3 Inhibitory effects of compounds 12a and 12h on TGF-β1-induced migration of CCC-HPF-1 cells; (A) Images were taken at 0 h, 12 h, and 24 h after injury; (B, C) Percentage of wound area closure analyzed by Image J software; Data are presented as the mean ± standard deviation of three separate experiments, compared with the control group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the TGF-β1 group, * P < 0.05, ** P < 0.01, *** P < 0.001;

[0054] Figure 4 Effects of compounds 12a and 12h on the activation of TGF-β / Smad signaling pathway in HSC-LX2 cells; (A-F) Western blotting was used to detect the expression levels of p-Smad2 / Smad2 and p-Smad3 / Smad3 proteins in HSC-LX2 cells; (G) Immunofluorescence staining was used to analyze the cellular localization of Smad2 / 3 in HSC-LX2 cells; (H) Mean fluorescence intensity (MFI) of Smad2 / 3; Data are presented as the mean ± SD of three separate experiments; compared with the control group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the TGF-β1 group, *P < 0.05, ** P < 0.01, *** P < 0.001;

[0055] Figure 5Effects of Compounds 12a and 12h on the activation of the TGF-β / Smad signaling pathway in CCC-HPF-1 cells; (A-F) Western Blot was used to detect the expression levels of p-Smad2 / Smad2 and p-Smad3 / Smad3 proteins in CCC-HPF-1 cells; (G) Immunofluorescence staining was used to analyze the localization of Smad2 / 3 in CCC-HPF-1 cells; (H) Mean fluorescence intensity (MFI) of Smad2 / 3; Data are presented as the mean ± SD of three separate experiments; Compared with the control group, # P < 0.05, ## P < 0.01, ### P < 0.001; Compared with the TGF-β1 group, *P < 0.05, **P < 0.01, *** P < 0.001. Detailed implementation manners

[0056] The technical solutions of the present invention will be further described below in conjunction with the embodiments.

[0057] NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer. Solvent signals (CD 3 OD: δH = 3.31 ppm / δC = 49.00 ppm) were used as references. High-resolution mass spectrometry (HR-ESI-MS) was recorded on a Waters SYN-APT G2 HDMS. Reactions were monitored by thin-layer chromatography on plates (GF 254 ) provided by Yantai Chemistry (China). Silica gel column chromatography was performed using silica gel with a mesh size of 200 - 300. GPLG-1690 (Target mol. USA). Unless otherwise stated, all common reagents and solvents were obtained from commercial suppliers without further purification.

[0058] Example 1: Synthesis of Compounds 10a - 10u

[0059]

[0060] To a solution of Compound 9 (100 mg, 0.32 mmol) in tert-butanol (2 mL) was added the corresponding amine (0.96 mmol), and the reaction mixture was heated to 90 °C for 6 hours. After completion, the reaction mixture was diluted with water and extracted three times with CH 2 Cl 2 The organic layer was dried over anhydrous Na 2 SO 4 and filtered. The filtrate was evaporated under reduced pressure, and the residue was purified by column chromatography to obtain Compounds 10a - 10u.

[0061] tert-Butyl 2-((2,3-dihydro-1H-inden-2-yl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10a), a white solid, yield: 84.5%.

[0062] (Benzylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate tert-butyl ester (10b), a white solid, yield: 89.1%.

[0063] tert-Butyl 2-(phenethylamino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10c), a white solid, yield: 88.6%.

[0064] tert-Butyl 2-((3-phenylpropyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10d), a white solid, yield: 92.7%.

[0065] tert-Butyl 2-((4-fluorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10e), a white solid, yield: 80.0%.

[0066] tert-Butyl 2-((3-fluorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10f), a white solid, yield: 97.1%.

[0067] tert-Butyl 2-((2-fluorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10g), a white solid, yield: 83.2%.

[0068] tert-Butyl 2-((4-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10h), a white solid, yield: 71.2%.

[0069] tert-Butyl 2-(3-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10i), a white solid, yield: 88.2%.

[0070] tert-Butyl 2-((2-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10j), a white solid, yield: 78.6%.

[0071] tert-Butyl 2-((3,5-dichlorophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10k), a white solid, yield: 74.0%.

[0072] tert-Butyl 2-(4-bromophenethyl)amino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10l), white solid, yield: 83.2%.

[0073] tert-Butyl 2-(3-bromophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10m), white solid, yield: 93.7%.

[0074] tert-Butyl 2-(2-bromophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10n), white solid, yield: 81.2%.

[0075] tert-Butyl 2-((4-bromobenzyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10o), white solid, yield: 83.2%.

[0076] tert-Butyl 2-(4-methylphenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10p), white solid, yield: 81.3%.

[0077] tert-Butyl 2-(4-methoxyphenethyl)amino-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10q), white solid, yield: 74.0%.

[0078] tert-Butyl 2-((2-(pyridin-4-yl)ethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10r), white solid, yield: 25.8%.

[0079] tert-Butyl 2-((2-(1H-indol-3-yl)ethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10s), white solid, yield: 57.2%.

[0080] tert-Butyl 2-(4-phenylpiperidin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10t), white solid, yield: 79.2%.

[0081] tert-Butyl 2-((5-bromo-2,3-dihydro-1H-inden-2-yl)amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (10u), white solid, yield: 79.0%.

[0082] Example 2: Synthesis of Compounds 11a - 11u

[0083]

[0084] To a solution of compound 10a - 10u (1.0 mmol) in EtOAc (2.0 mL) was added 2 mol / L HCl in EtOAc, and the reaction mixture was stirred at room temperature for 1 hour. After completion, the reaction mixture was concentrated to give the crude compounds 11a - 11u, which could be used for the next step without further purification.

[0085] Example 3: Synthesis of Compounds 12a - 12u

[0086]

[0087] To a solution of compound 11a - 11u (0.2 mmol) in CH 2 Cl 2 (10 mL) were added ursodeoxycholic acid (118 mg, 0.3 mmol), HATU (114 mg, 0.3 mmol), and DIEA (50 μL, 0.3 mmol). The organic layer was dried over anhydrous Na 2 SO 4 and filtered. The filtrate was evaporated under reduced pressure, and the residue was purified by column chromatography to give compounds 12a - 12u.

[0088] (R)-1-(2-((2,3-Dihydro-1H-inden-2-yl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-(((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12a), white solid, yield: 42.8%, melting point: 130.3 - 132.0 °C; 1 HNMR(600 MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.13 (s, 0.5H), 8.11 (s, 0.5H), 7.24 - 7.17 (m, 2H), 7.16 - 7.09 (m, 2H), 4.73 - 4.69 (m, 1H), 4.62 - 4.52 (m, 2H), 3.92 - 3.77 (m, 2H), 3.53 - 3.43 (m, 2H), 3.35 - 3.28 (m, 2H), 2.90 - 2.85 (m, 2H), 2.84 (t, J = 5.8 Hz, 1H), 2.73 (t, J = 5.8 Hz, 1H), 2.58 - 2.48 (m, 1H), 2.47 - 2.37 (m, 1H), 2.08 - 1.99 (m, 1H), 1.94 - 1.75 (m, 5H), 1.64 - 1.53 (m, 4H), 1.53 - 1.39 (m, 6H), 1.37 - 1.09 (m, 8H), 1.02 (d, J = 6.5 Hz, 1.5H), * 0.99 (d, J = 6.5 Hz, 1.5H), 0.97 (s, 1.5H), * 0.96 (s, 1.5H), 0.72 (s, 1.5H), * 0.65 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): * indicates minor δ 175.2, * 175.1, 166.0, 165.3, 162.4, * 162.2, 157.1, * 156.7, 142.6, 127.6, 125.6, 116.5, 72.1, 71.9, 57.5, * 57.4, 56.5, 53.8, * 45.5, 44.8, * 44.8, 44.5, 44.0, 43.8, 41.9, 41.5, * 41.5, * 40.7, 40.7, 40.6, * 40.3, 38.6, 38.0, 37.0, * 36.8, 36.1, 35.2, 33.1, 32.8, * 32.2, * 31.5, 31.3, 31.0, 29.8, 28.0, * 24.0, 23.9, 22.4, * 19.2, 19.1, 12.7, * 12.6; HRMS m / z calcd for C 40 H 57 N 4 O 3 [M + H] + : 641.4431; found: 641.4431.

[0089] (R)-1-(2-(Benzylamino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12b), white solid, yield: 74.0%, melting point: 135.1 - 136.7 °C; 1 HNMR(600MHz,CD 3 OD): ∼50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.11(s,0.5H),8.09(s,0.5H),7.32(d,J = 7.8Hz,2H),7.28(dd,J = 7.8,7.3Hz,2H),7.20(t,J = 7.3Hz,1H),4.59 - 4.51(m,4H),3.88 - 3.78(m,2H),3.52 - 3.44(m,2H),2.82(t,J = 6.1Hz,1H),2.72(t,J = 6.1Hz,1H),2.56 - 2.48(m,1H),2.45 - 2.36(m,1H),2.07 - 1.99(m,1H),1.92 - 1.76(m,5H),1.64 - 1.54(m,4H),1.50 - 1.40(m,6H),1.36 - 1.08(m,8H),1.01(d,J = 6.5Hz,1.5H), *0.98(d,J = 6.5Hz,1.5H),0.97(s,1.5H), *0.96(s,1.5H),0.72(s,1.5H), *0.66(s,1.5H); 13 CNMR(150MHz,CD 3OD): *indicates minor δ* 175.2, 175.1, *166.0, 165.4, *162.6, 162.5, 157.1, *156.8, 141.2, *141.2, 129.4, 128.3, 127.9, 116.6, *116.6, 72.1, 71.9, *57.5, 57.5, 56.5, 45.7, *45.4, 44.8, *44.8, 44.5, 44.0, *43.8, 41.8, 41.6, *41.5, 40.7, 40.2, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.7, 28.0, *28.0, 23.9, 22.4, *19.1, 19.1, 12.7, *12.6; HRMS m / z calcd for C 38 H 55 N 4 O 3 [M + H] + : 615.4274; found: 615.4276.

[0090] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-(phenethylamino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12c), white solid, yield: 37.5%, melting point: 161.7 - 163.6 °C; 1 H NMR(600 MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.10 (s, 0.5H), 8.08 (s, 0.5H), 7.28 - 7.21 (m, 4H), 7.19 - 7.14 (m, 1H), 4.58 - 4.50 (m, 2H), 3.87 - 3.77 (m, 2H), 3.58 (t, J = 7.4Hz, 2H), 3.52 - 3.42 (m, 2H), 2.87 (t, J = 7.4Hz, 2H), 2.82 (t, J = 5.8Hz, 1H), 2.72 (t, J = 5.8Hz, 1H), 2.56 - 2.47 (m, 1H), 2.46 - 2.36 (m, 1H), 2.07 - 1.98 (m, 1H), 1.92 - 1.76 (m, 5H), 1.66 - 1.54 (m, 4H), 1.53 - 1.39 (m, 6H), 1.35 - 1.07 (m, 8H), 1.01 (d, J = 6.5Hz, 1.5H), * 0.98 (d, J = 6.5Hz, 1.5H), 0.96 (s, 1.5H), * 0.95 (s, 1.5H), 0.71 (s, 1.5H), * 0.66 (s, 1.5H); 13 CNMR (150MHz, CD 3 OD): * indicates the minor δ * 175.1, 175.0, * 165.9, 165.3, * 162.4, 162.3, 157.0, * 156.7, 140.9, 129.9, 129.4, 127.2, 116.3, * 116.3, 72.1, 71.9, 57.5, * 57.4, 56.5, * 56.5, 45.4, 44.8, * 44.8, 44.5, 44.0, 43.9, * 43.8, 41.8, 41.5, * 41.5, 40.7, * 40.7, 40.2, 38.6, 38.0, 37.0, * 36.8, * 36.7, 36.7, 36.1, 35.2, 33.1, 32.8, * 32.8, * 32.1, * 31.5, 31.3, 31.0, 29.8, * 29.7, 28.0, * 28.0, 24.0, 22.4, * 19.2, 19.2, 12.7, * 12.7; HRMS m / z calcd for C 39 H 57 N 4 O 3 [M + H] + : 629.4431; found: 629.4430.

[0091] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((3-phenylpropyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12d), white solid, yield: 41.8%, melting point: 121.8 - 123.1 °C; 1 H NMR(600MHz,CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ* 8.10(s, 0.5H), 8.07(s, 0.5H), 7.26 - 7.21(m, 2H), 7.20 - 7.17(m, 2H), 7.14(t, J = 7.3Hz, 1H), 4.59 - 4.50(m, 2H), 3.89 - 3.78(m, 2H), 3.52 - 3.42(m, 2H), 3.36(t, J = 7.0Hz, 2H), 2.81(t, J = 5.9Hz, 1H), 2.71(t, J = 5.9Hz, 1H), 2.67(t, J = 7.7Hz, 2H), 2.56 - 2.47(m, 1H), 2.45 - 2.36(m, 1H), 2.06 - 1.97(m, 1H), 1.93 - 1.76(m, 7H), 1.63 - 1.52(m, 4H), 1.50 - 1.38(m, 6H), 1.37 - 1.05(m, 8H), 1.01(d, J = 6.5Hz, 1.5H), *0.98(d, J = 6.5Hz, 1.5H), 0.96(s, 1.5H), *0.94(s, 1.5H), 0.71(s, 1.5H), *0.65(s, 1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ 175.2, *175.1, *165.9, 165.3, 162.6, *162.5, 157.0, *156.7, 143.2, 129.4, 129.4, 126.8, 116.1, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, *45.4, *44.8, 44.8, 44.5, *44.5, 44.0, 43.8, 41.8, *41.5, 41.5, 40.7, *40.7, 40.3, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 34.2, 33.1, 32.8, 32.4, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 23.9, 22.4, *22.4, 19.2, *19.1, 12.7, *12.6; HRMS m / z calcd for C 40 H 59 N 4 O 3 [M + H] + : 643.4587; found: 643.4584.

[0092] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((4-fluorophenylethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12e), white solid, yield: 41.6%, melting point: 161.7 - 163.3 °C; 1 H NMR(600MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.11 (s, 0.5H), 8.09 (s, 0.5H), 7.24 (dd, J = 8.4, 5.6Hz, 2H), 7.01 - 6.96 (m, 2H), 4.59 - 4.51 (m, 2H), 3.88 - 3.79 (m, 2H), 3.57 (t, J = 7.4Hz, 2H), 3.52 - 3.43 (m, 2H), 2.87 (t, J = 7.4Hz, 2H), 2.82 (t, J = 5.9Hz, 1H), 2.72 (t, J = 5.9Hz, 1H), 2.57 - 2.49 (m, 1H), 2.45 - 2.37 (m, 1H), 2.08 - 1.99 (m, 1H), 1.93 - 1.77 (m, 5H), 1.63 - 1.53 (m, 4H), 1.51 - 1.41 (m, 6H), 1.36 - 1.07 (m, 8H), 1.02 (d, J = 6.5Hz, 1.5H), * 0.99 (d, J = 6.5Hz, 1.5H), 0.97 (s, 1.5H), * 0.95 (s, 1.5H), 0.72 (s, 1.5H), * 0.66 (s, 1.5H); 13 C NMR (150MHz, CD 3 OD): * indicates the minor δ 175.2, * 175.1, 166.0, * 165.3, 163.0 (d, J = 242.2Hz), * 162.5, 162.4, 157.1, * 156.7, 136.9, 131.6 (d, J = 7.8Hz), 116.4, 116.0 (d, J = 21.1Hz), 72.1, 71.9, 57.5, * 57.5, 56.5, * 56.5, 45.4, 44.8, * 44.8, 44.5, 44.0, 43.9, * 43.8, 41.8, 41.6, * 41.5, 40.7, * 40.7, * 40.3, 38.6, 38.0, 37.0, * 36.8, 36.1, * 35.9, 35.9, 35.2, 33.1, 32.8, * 32.8, * 32.1, * 31.5, 31.3, 31.0, 29.8, * 29.7, 28.0, 23.9, 22.4, * 19.1, 19.1, 12.6, * 12.6; HRMS m / z calcd for C 39 H 56 FN 4 O 3 [M + H] + : 647.4336; found: 647.4338.

[0093] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((3-fluorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12f), white solid, yield: 23.1%, melting point: 132.4 - 134.0 °C; 1 H NMR(600MHz,CD 3 OD): ∼50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.11(s, 0.5H), 8.08(s, 0.5H), 7.30 - 7.22(m, 1H), 7.04(d, J = 7.8Hz, 1H), 7.01 - 6.95(m, 1H), 6.94 - 6.86(m, 1H), 4.59 - 4.51(m, 2H), 3.86 - 3.78(m, 2H), 3.60(t, J = 7.2Hz, 2H), 3.52 - 3.43(m, 2H), 2.89(t, J = 7.2Hz, 2H), 2.82(t, J = 5.7Hz, 1H), 2.72(t, J = 5.7Hz, 1H), 2.56 - 2.48(m, 1H), 2.45 - 2.36(m, 1H), 2.06 - 1.99(m, 1H), 1.92 - 1.77(m, 5H), 1.64 - 1.54(m, 4H), 1.50 - 1.41(m, 6H), 1.34 - 1.07(m, 8H), 1.01(d, J = 6.5Hz, 1.5H), *0.99(d, J = 6.5Hz, 1.5H), 0.96(s, 1.5H), *0.95(s, 1.5H), 0.71(s, 1.5H), *0.66(s, 1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ *175.1, 175.1, 166.0, *165.3, 164.3 (d, J = 243.8 Hz), *162.5, 162.3, 157.1, *156.7, 143.9 (d, J = 6.8 Hz), 131.0 (d, J = 8.5 Hz), 125.8 (d, J = 7.2 Hz), 116.6 (d, J = 21.0 Hz), 116.4, *116.4, 113.9 (d, J = 21.2 Hz), 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, 45.4, 44.8, *44.8, 44.5, 44.0, *43.8, 43.6, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, 37.0, *36.8, 36.4, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 24.0, 22.4, *19.2, 19.2, *12.7, 12.6; HRMS m / z calcd for C 39 H 56 FN 4 O 3 [M + H] + : 647.4336; found: 647.4334.

[0094] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((2-fluorophenylethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12 g), white solid, yield: 50.7%, melting point: 120.2 - 121.6 °C; 1 H NMR (600 MHz, CD 3OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.09(s, 0.5H), 8.06(s, 0.5H), 7.28 - 7.22(m, 1H), 7.21 - 7.17(m, 1H), 7.08 - 7.04(m, 1H), 7.03 - 6.99(m, 1H), 4.58 - 4.50(m, 2H), 3.86 - 3.77(m, 2H), 3.60(t, J = 7.2Hz, 2H), 3.52 - 3.43(m, 2H), 2.93(t, J = 7.2Hz, 2H), 2.80(t, J = 6.0Hz, 1H), 2.70(t, J = 6.0Hz, 1H), 2.56 - 2.47(m, 1H), 2.44 - 2.36(m, 1H), 2.05 - 1.98(m, 1H), 1.92 - 1.76(m, 5H), 1.63 - 1.53(m, 4H), 1.49 - 1.41(m, 6H), 1.35 - 1.07(m, 8H), 1.00(d, J = 6.5Hz, 1.5H), *0.98(d, J = 6.5Hz, 1.5H), 0.96(s, 1.5H), *0.94(s, 1.5H), 0.71(s, 1.5H), *0.66(s, 1.5H); 13 C NMR(150MHz, CD 3 OD): *indicates minor δ 175.1, *175.0, 165.9, *165.2, 162.7(d, J = 243.9Hz), *162.5, 162.3, 157.0, *156.7, 132.4(d, J = 5.0Hz), 129.2(d, J = 8.3Hz), 127.7(d, J = 16.1Hz), 125.2(d, J = 3.3Hz), 116.3, *116.3, 116.1(d, J = 22.2Hz), 72.1, 71.9, 57.5, *57.4, 56.5, *56.4, *45.4, *44.8, 44.8, 44.5, 44.0, 43.8, 42.5, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 30.0, 29.8, *29.7, 28.0, *28.0, 24.0, 22.4, *22.4, *19.2, 19.2, *12.7, 12.7; HRMS m / z calcd for C 39 H 56 FN4 O 3 [M+H] + : 647.4336; found: 647.4336.

[0095] (R)-1-(2-((4-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12h), white solid, yield: 44.2%, melting point: 120.6 - 121.9 °C; 1 H NMR (600 MHz, CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.10 (s, 0.5H), 8.08 (s, 0.5H), 7.25 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 4.60 - 4.51 (m, 2H), 3.87 - 3.77 (m, 2H), 3.58 (t, J = 7.2 Hz, 2H), 3.52 - 3.43 (m, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 6.0 Hz, 1H), 2.71 (t, J = 6.0 Hz, 1H), 2.58 - 2.47 (m, 1H), 2.45 - 2.35 (m, 1H), 2.06 - 1.98 (m, 1H), 1.92 - 1.75 (m, 5H), 1.63 - 1.52 (m, 4H), 1.51 - 1.40 (m, 6H), 1.36 - 1.07 (m, 8H), 1.01 (d, J = 6.3 Hz, 1.5H), *0.98 (d, J = 6.3 Hz, 1.5H), 0.96 (s, 1.5H), *0.95 (s, 1.5H), 0.72 (s, 1.5H), *0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3OD): * indicates minor δ 175.2, *175.1, 165.9, *165.3, 162.5, *162.3, 157.0, *156.7, 139.9, *139.8, 132.9, 131.6, 129.4, 116.4, 72.1, 71.9, 57.5, *57.4, 56.5, *56.4, *45.4, *44.8, 44.8, 44.5, 44.0, 43.8, 43.6, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, *37.0, 36.8, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 23.9, 22.4, *22.4, *19.2, 19.1, 12.7, *12.6; HRMS m / z calcd for C 39 H 56 ClN 4 O 3 [M + H] + : 663.4041; found: 663.4043.

[0096] (R)-1-(2-((3-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12i), white solid, yield: 26.8%, melting point: 178.7 - 180.5 °C; 1 H NMR(600 MHz, CD 3OD): A 50:50 mixture of rotamers. *Indicates the minor amide rotamer. δ 8.09 (s, 0.5H), 8.06 (s, 0.5H), 7.26 - 7.21 (m, 2H), 7.18 - 7.13 (m, 2H), 4.58 - 4.50 (m, 2H), 3.85 - 3.77 (m, 2H), 3.59 (t, J = 7.1 Hz, 2H), 3.51 - 3.43 (m, 2H), 2.87 (t, J = 7.1 Hz, 2H), 2.81 (t, J = 5.9 Hz, 1H), 2.71 (t, J = 5.9 Hz, 1H), 2.56 - 2.48 (m, 1H), 2.44 - 2.36 (m, 1H), 2.06 - 1.98 (m, 1H), 1.92 - 1.76 (m, 5H), 1.63 - 1.53 (m, 4H), 1.49 - 1.40 (m, 6H), 1.36 - 1.05 (m, 8H), 1.01 (d, J = 6.5 Hz, 1.5H), *0.98 (d, J = 6.5 Hz, 1.5H), 0.96 (s, 1.5H), *0.94 (s, 1.5H), 0.71 (s, 1.5H), *0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): *Indicates the minor. δ 175.1, *175.0, 165.9, 165.2, *162.5, 162.3, 157.0, *156.7, 143.5, 135.1, 130.9, 130.0, 128.5, 127.3, 116.4, *116.3, 72.1, 71.9, 57.5, *57.4, 56.5, *56.4, 45.4, *44.8, 44.8, 44.5, 44.0, *43.8, 43.5, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, 37.0, *36.8, 36.4, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 24.0, 22.4, *22.4, *19.2, 19.2, 12.7, *12.7; HRMS m / z calcd for C 39 H 56 ClN 4 O 3 [M + H] + : 663.4041; found: 663.4041.

[0097] (R)-1-(2-((2-chlorophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12j), white solid, yield: 46.6%, melting point: 122.9 - 124.1 °C; 1 H NMR(600MHz,CD 3 OD): ∼50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.10(s,0.5H),8.07(s,0.5H),7.35(dd,J = 7.5,1.7Hz,1H),7.30 - 7.26(m,2H),7.22 - 7.15(m,2H),4.58 - 4.50(m,2H),3.88 - 3.78(m,2H),3.63(t,J = 7.2Hz,2H),3.52 - 3.44(m,2H),3.04(t,J = 7.2Hz,2H),2.81(t,J = 6.0Hz,1H),2.71(t,J = 6.0Hz,1H),2.57 - 2.48(m,1H),2.45 - 2.36(m,1H),2.07 - 1.99(m,1H),1.93 - 1.77(m,5H),1.63 - 1.53(m,4H),1.50 - 1.41(m,6H),1.36 - 1.08(m,8H),1.01(d,J = 6.5Hz,1.5H), *0.99(d,J = 6.5Hz,1.5H),0.96(s,1.5H), *0.95(s,1.5H),0.72(s,1.5H), *0.66(s,1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ 175.2, *175.1, *165.9, 165.2, *162.5, 162.4, 157.0, *156.7, 138.5, 135.2, 132.4, 130.4, 129.0, 128.0, 116.3, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, *45.4, *44.8, 44.8, 44.5, 44.0, 43.8, 42.1, *41.8, 41.5, *41.5, 40.7, *40.7, 40.3, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 34.4, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 23.9, 22.4, *22.4, *19.2, 19.1, *12.7, 12.6; HRMS m / z calcd for C 39 H 56 ClN 4 O 3 [M + H] + : 663.4041; found: 663.4040.

[0098] (R)-1-(2-((3,5-dichlorophenyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12k), white solid, yield: 42.5%, melting point: 149.8 - 151.1 °C; 1 HNMR(600MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.13 (s, 0.5H), 8.11 (s, 0.5H), 7.33 - 7.25 (m, 3H), 4.60 - 4.53 (m, 4H), 3.87 - 3.78 (m, 2H), 3.53 - 3.43 (m, 2H), 2.83 (t, J = 6.0 Hz, 1H), 2.72 (t, J = 6.0 Hz, 1H), 2.56 - 2.47 (m, 1H), 2.44 - 2.36 (m, 1H), 2.07 - 2.00 (m, 1H), 1.92 - 1.74 (m, 5H), 1.64 - 1.53 (m, 4H), 1.51 - 1.40 (m, 6H), 1.34 - 1.12 (m, 8H), 1.01 (d, J = 6.6 Hz, 1.5H), * 0.98 (d, J = 6.6 Hz, 1.5H), 0.97 (s, 1.5H), * 0.96 (s, 1.5H), 0.71 (s, 1.5H), * 0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): * indicates minor δ 175.2, * 175.1, 166.1, * 165.4, 162.4, 162.2, 157.2, * 156.9, 146.0, * 145.9, 135.9, * 127.6, 127.6, * 126.9, 126.8, 117.1, * 117.1, 72.1, 71.9, 57.5, * 57.5, 56.5, * 56.4, 45.4, * 44.8, 44.8, 44.5, 44.0, * 43.8, 41.8, 41.5, * 41.5, 40.7, * 40.2, 38.6, 38.0, 37.0, * 36.8, 36.1, 35.2, 33.1, 32.8, * 32.8, * 32.1, * 31.5, 31.3, 31.0, 29.8, 28.0, * 28.0, 23.9, 22.4, 19.1, * 19.1, * 12.6, 12.6; HRMS m / z calcd for C 38 H 53 Cl 2 N 4 O 3 [M + H] + : 683.3495; found: 683.3494.

[0099] (R)-1-(2-((4-Bromophenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12l), white solid, yield: 24.0%, melting point: 128.9 - 130.1 °C; 1 H NMR(600MHz,CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.10(s,0.5H),8.07(s,0.5H),7.40(d,J = 8.3Hz,2H),7.15(d,J = 8.3Hz,2H),4.59 - 4.51(m,2H),3.89 - 3.76(m,2H),3.58(t,J = 7.2Hz,2H),3.52 - 3.43(m,2H),2.85(t,J = 7.2Hz,2H),2.81(t,J = 6.1Hz,1H),2.71(t,J = 6.1Hz,1H),2.58 - 2.47(m,1H),2.46 - 2.35(m,1H),2.08 - 1.97(m,1H),1.93 - 1.76(m,5H),1.64 - 1.53(m,4H),1.50 - 1.40(m,6H),1.39 - 1.06(m,8H),1.01(d,J = 6.5Hz,1.5H), *0.99(d,J = 6.5Hz,1.5H),0.96(s,1.5H), *0.95(s,1.5H),0.72(s,1.5H), *0.66(s,1.5H); 13 C NMR(150MHz,CD 3OD): * indicates minor δ 175.2, *175.1, *165.9, 165.3, *162.5, 162.4, 157.0, *156.7, *140.3, 140.3, 132.4, 132.0, 120.9, 116.4, *116.3, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, *45.4, 44.8, *44.8, 44.5, 44.0, 43.8, 43.5, 41.8, 41.5, *41.5, 40.7, *40.7, *40.3, 38.6, 38.0, 37.0, *36.8, 36.2, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 23.9, 22.4, *22.4, *19.2, 19.1, 12.7, *12.6; HRMS m / z calcd for C 39 H 56 BrN 4 O 3 [M + H] + : 707.3536; found: 707.3528.

[0100] (R)-1-(2-((3-Bromo phenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12m), white solid, yield: 21.0%, melting point: 128.9 - 130.1 °C; 1 HNMR(600MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.10 (s, 0.5H), 8.07 (s, 0.5H), 7.43 - 7.39 (m, 1H), 7.35 - 7.30 (m, 1H), 7.22 - 7.16 (m, 2H), 4.59 - 4.51 (m, 2H), 3.86 - 3.79 (m, 2H), 3.60 (t, J = 7.2 Hz, 2H), 3.52 - 3.44 (m, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 6.1 Hz, 1H), 2.72 (t, J = 6.1 Hz, 1H), 2.57 - 2.48 (m, 1H), 2.45 - 2.37 (m, 1H), 2.07 - 2.00 (m, 1H), 1.92 - 1.78 (m, 5H), 1.63 - 1.53 (m, 4H), 1.51 - 1.41 (m, 6H), 1.34 - 1.09 (m, 8H), 1.01 (d, J = 6.4 Hz, 1.5H), * 0.99 (d, J = 6.4 Hz, 1.5H), 0.97 (s, 1.5H), * 0.95 (s, 1.5H), 0.72 (s, 1.5H), * 0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): * indicates the minor δ 175.2, * 175.1, 166.0, 165.3, 162.5, * 162.4, 157.0, * 156.7, 143.8, 133.0, 131.2, 130.3, 128.9, 123.3, 116.4, 72.1, 71.9, 57.5, * 57.5, 56.5, * 56.5, * 45.4, * 44.8, 44.8, 44.5, 44.0, 43.8, 43.5, 41.8, 41.6, * 41.5, 40.7, * 40.7, * 40.3, 38.6, 38.0, 37.0, * 36.8, 36.5, * 36.5, 36.1, 35.2, 33.1, 32.8, * 32.8, * 32.1, * 31.5, 31.3, 31.0, 29.8, * 29.7, 28.0, * 28.0, 23.9, 22.4, * 19.2, 19.1, 12.7, * 12.6; HRMS m / z calcd for C 39 H 56 BrN 4 O 3 [M + H] + : 707.3536; found: 707.3535.

[0101] (R)-1-(2-((2-Bromophenylethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12n), white solid, yield: 13.3%, melting point: 120.7 - 121.9 °C; 1 HNMR(600MHz,CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.09(s,0.5H),8.06(s,0.5H),7.53(dd,J = 7.9,1.1Hz,1H),7.28(d,J = 7.5Hz,1H),7.24(dd,J = 7.5,7.4Hz,1H),7.09(ddd,J = 8.0,7.4,1.4Hz,1H),4.59 - 4.50(m,2H),3.87 - 3.77(m,2H),3.62(t,J = 7.2Hz,2H),3.52 - 3.44(m,2H),3.04(t,J = 7.2Hz,2H),2.81(t,J = 6.0Hz,1H),2.71(t,J = 6.0Hz,1H),2.57 - 2.48(m,1H),2.45 - 2.36(m,1H),2.06 - 1.98(m,1H),1.92 - 1.77(m,5H),1.63 - 1.53(m,4H),1.50 - 1.41(m,6H),1.34 - 1.09(m,8H),1.01(d,J = 6.4Hz,1.5H), *0.98(d,J = 6.4Hz,1.5H),0.96(s,1.5H), *0.95(s,1.5H),0.71(s,1.5H), *0.66(s,1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ *175.1, 175.1, *165.9, 165.2, *162.5, 162.4, 157.0, *156.7, 140.3, 133.8, 132.3, 129.2, 128.7, 125.6, 116.3, *116.3, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, 45.4, 44.8, *44.8, 44.5, 44.0, *43.8, 42.2, 41.8, 41.5, *41.5, *40.7, 40.7, *40.3, 38.6, 38.0, *37.0, 36.9, 36.8, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, *24.0, 23.9, 22.4, 19.2, *19.2, 12.7, *12.7; HRMS m / z calcd for C 39 H 56 BrN 4 O 3 [M + H] + : 707.3536; found: 707.3534.

[0102] (R)-1-(2-((4-Bromobenzyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12o), white solid, yield: 54.6%, melting point: 156.1 - 157.7 °C; 1 HNMR(600MHz, CD 3OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ* 8.11 (s, 0.5H), 8.09 (s, 0.5H), 7.43 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 4.60 - 4.50 (m, 4H), 3.88 - 3.77 (m, 2H), 3.53 - 3.43 (m, 2H), 2.82 (t, J = 6.0 Hz, 1H), 2.72 (t, J = 6.0 Hz, 1H), 2.57 - 2.48 (m, 1H), 2.45 - 2.36 (m, 1H), 2.08 - 1.98 (m, 1H), 1.92 - 1.74 (m, 5H), 1.64 - 1.53 (m, 4H), 1.50 - 1.39 (m, 6H), 1.36 - 1.05 (m, 8H), 1.01 (d, J = 6.5 Hz, 1.5H), *0.98 (d, J = 6.5 Hz, 1.5H), 0.97 (s, 1.5H), *0.96 (s, 1.5H), 0.71 (s, 1.5H), *0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): *indicates minor δ 175.2, *175.1, 166.0, *165.4, 162.5, *162.3, 157.1, *156.8, 140.8, *140.7, 132.4, *130.3, 130.2, *121.4, 121.4, 116.8, 72.1, 71.9, 57.5, *57.5, 56.5, *56.5, 45.4, 45.1, *45.0, 44.8, *44.8, 44.5, 44.0, *43.8, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 33.1, 32.8, *32.8, *32.1, 31.5, *31.3, 31.0, 29.8, 28.0, *28.0, *24.0, 23.9, 22.4, 19.1, 12.6, *12.6; HRMS m / z calcd for C 38 H 54 BrN 4 O 3 [M + H] + : 693.3379; found: 693.3376.

[0103] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((4-methylphenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12p), white solid, yield: 34.0%, melting point: 119.2 - 120.8 °C; 1 H NMR(600MHz,CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ* 8.10 (s, 0.5H), 8.07 (s, 0.5H), 7.10 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 4.58 - 4.49 (m, 2H), 3.88 - 3.77 (m, 2H), 3.56 (t, J = 7.4 Hz, 2H), 3.52 - 3.43 (m, 2H), 2.85 - 2.79 (m, 3H), 2.71 (t, J = 5.8 Hz, 1H), 2.56 - 2.47 (m, 1H), 2.45 - 2.36 (m, 1H), 2.28 (s, 3H), 2.06 - 1.98 (m, 1H), 1.92 - 1.76 (m, 5H), 1.64 - 1.52 (m, 4H), 1.52 - 1.40 (m, 6H), 1.37 - 1.07 (m, 8H), 1.01 (d, J = 6.5 Hz, 1.5H), *0.98 (d, J = 6.5 Hz, 1.5H), 0.96 (s, 1.5H), *0.95 (s, 1.5H), 0.71 (s, 1.5H), *0.66 (s, 1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ 175.1, *175.1, *165.9, 165.3, *162.5, 162.3, 157.0, *156.7, 137.8, 136.7, 130.0, 129.8, 116.3, *116.2, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, *45.4, *44.8, 44.8, 44.5, *44.0, 44.0, 43.8, 41.8, 41.5, *41.5, *40.7, 40.7, *40.2, 38.6, 38.0, 37.0, *36.8, 36.3, *36.1, 35.2, 33.1, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, *29.8, 29.7, *28.0, 28.0, 23.9, *22.4, 22.4, 21.1, *19.2, 19.2, *12.7, 12.6; HRMS m / z calcd for C 40 H 59 N 4 O 3 [M + H] + : 643.4587; found: 643.4588.

[0104] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-((4-methoxyphenethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12q), white solid, yield: 34.2%, melting point: 114.0 - 115.7 °C; 1 HNMR(600MHz, CD 3OD): A 50:50 mixture of rotamers, * indicates the minor amide rotamer δ * 8.12 (s, 0.5H), 8.11 (s, 0.5H), 7.14 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.61 - 4.51 (m, 2H), 3.91 - 3.79 (m, 2H), 3.75 (s, 3H), 3.60 - 3.55 (m, 2H), 3.52 - 3.43 (m, 2H), 2.87 - 2.79 (m, 3H), 2.74 (t, J = 5.9 Hz, 1H), 2.57 - 2.48 (m, 1H), 2.45 - 2.36 (m, 1H), 2.08 - 1.99 (m, 1H), 1.94 - 1.76 (m, 5H), 1.64 - 1.53 (m, 4H), 1.51 - 1.41 (m, 6H), 1.35 - 1.11 (m, 8H), 1.01 (d, J = 6.5 Hz, 1.5H), * 0.99 (d, J = 6.5 Hz, 1.5H), 0.97 (s, 1.5H), * 0.95 (s, 1.5H), 0.72 (s, 1.5H), * 0.67 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): * indicates the minor δ 175.2, * 175.1, 161.4, 161.0, 159.7, 155.9, * 132.6, 132.6, 130.8, 116.5, * 116.4, 114.8, 72.1, 71.9, 57.5, * 57.5, 56.5, * 56.5, 55.6, * 45.3, 44.8, * 44.8, 44.5, 44.1, * 44.0, 43.6, 41.7, 41.5, * 41.5, 40.7, * 40.7, * 40.1, 38.6, 38.0, 37.0, * 36.8, 36.1, * 35.7, 35.7, 35.2, 33.1, 32.8, * 32.8, * 32.1, * 31.6, 31.5, 31.2, 31.0, 29.8, * 29.7, * 28.0, 28.0, 23.9, 22.4, * 19.1, 19.1, 12.7, * 12.6; HRMS m / z calcd for C 40 H 59 N 4 O 4 [M + H] + : 659.4536; found: 659.4534.

[0105] (4R)-1-(2-((5-Bromo-2,3-dihydro-1H-inden-2-yl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12r), a white solid, yield: 54.1%, melting point: 156.1 - 157.7 °C; 1 HNMR(600MHz,CD 3 OD): ∼50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.12(s, 0.5H), 8.10(s, 0.5H), 7.36(s, 1H), 7.28(dd, J = 8.0, 1.8 Hz, 1H), 7.12(d, J = 8.0 Hz, 1H), 4.76 - 4.69(m, 1H), 4.60 - 4.52(m, 2H), 3.90 - 3.79(m, 2H), 3.53 - 3.43(m, 2H), 3.35 - 3.24(m, 2H), 2.92 - 2.86(m, 2H), 2.83(t, J = 5.9 Hz, 1H), 2.73(t, J = 5.9 Hz, 1H), 2.58 - 2.48(m, 1H), 2.47 - 2.36(m, 1H), 2.07 - 1.98(m, 1H), 1.92 - 1.75(m, 5H), 1.64 - 1.53(m, 4H), 1.52 - 1.40(m, 6H), 1.34 - 1.07(m, 8H), 1.01(d, J = 6.5 Hz, 1.5H), *0.99(d, J = 6.5 Hz, 1.5H), 0.97(s, 1.5H), *0.96(s, 1.5H), 0.71(s, 1.5H), *0.65(s, 1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ 175.2, *175.1, *166.0, 165.3, 162.3, *162.1, 157.1, *156.7, 145.5, 142.0, 130.6, 128.7, 127.4, 121.1, 116.6, 72.1, 71.9, 57.5, *57.4, 56.5, 53.9, *45.5, 44.8, *44.8, 44.5, 44.0, 43.8, 41.9, 41.5, *41.5, *40.7, 40.7, 40.5, *40.3, 40.0, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 33.1, 32.8, *32.2, *31.5, 31.3, 31.0, 29.8, 28.0, 24.0, 22.4, *19.2, 19.1, 12.7, *12.6; HRMS m / z calcd for C 40 H 56 BrN 4 O 3 [M + H] + : 719.3536; found: 719.3533.

[0106] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-(2-(pyridin-4-yl)ethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12s). White solid, yield: 44.5%, melting point: 138.3 - 139.9 °C; 1 HNMR(600MHz, CD 3OD): A 50:50 mixture of rotamers. *Indicates the minor amide rotamer. δ 8.41 (d, J = 4.9 Hz, 2H), *8.12 (s, 0.5H), 8.10 (s, 0.5H), 7.34 (d, J = 4.9 Hz, 2H), 4.61 - 4.52 (m, 2H), 3.89 - 3.77 (m, 2H), 3.66 (t, J = 7.2 Hz, 2H), 3.52 - 3.43 (m, 2H), 2.96 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 6.1 Hz, 1H), 2.72 (t, J = 6.1 Hz, 1H), 2.57 - 2.48 (m, 1H), 2.45 - 2.37 (m, 1H), 2.07 - 2.00 (m, 1H), 1.92 - 1.77 (m, 5H), 1.63 - 1.54 (m, 4H), 1.50 - 1.40 (m, 6H), 1.35 - 1.11 (m, 8H), 1.02 (d, J = 6.5 Hz, 1.5H), *0.99 (d, J = 6.5 Hz, 1.5H), 0.97 (s, 1.5H), *0.95 (s, 1.5H), 0.72 (s, 1.5H), *0.67 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): *Indicates the minor δ *175.2, 175.1, 166.0, 165.3, *162.4, 162.3, 157.1, *156.8, 152.0, 149.8, 126.2, 116.5, 72.1, 71.9, 57.5, *57.5, 56.5, *45.4, 44.8, *44.8, 44.5, 44.0, 43.8, 42.6, 41.8, 41.6, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, 37.0, *36.8, *36.1, 36.0, 35.2, 33.1, 32.8, *32.8, *32.2, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, 23.9, 22.4, 19.1, 12.6, *12.6; HRMS m / z calcd for C 38 H 56 N 5 O 3 [M + H] + : 630.4383; found: 630.4385.

[0107] (R)-1-(2-((2-(1H-indol-3-yl)ethyl)amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-(((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one (12t), a white solid, yield: 44.7%, melting point: 160.5 - 162.4 °C; 1 H NMR(600MHz,CD 3 OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ *8.08(s,0.5H),8.06(s,0.5H),7.60(d,J = 7.9Hz,1H),7.31(d,J = 8.2Hz,1H),7.09 - 7.05(m,2H),7.01 - 6.95(m,1H),4.56 - 4.48(m,2H),3.86 - 3.75(m,2H),3.66(t,J = 7.2Hz,2H),3.52 - 3.42(m,2H),3.02(t,J = 7.2Hz,2H),2.78(t,J = 6.0Hz,1H),2.70(t,J = 6.0Hz,1H),2.55 - 2.47(m,1H),2.44 - 2.35(m,1H),2.06 - 1.97(m,1H),1.92 - 1.75(m,5H),1.63 - 1.52(m,4H),1.50 - 1.40(m,6H),1.35 - 1.05(m,8H),1.01(d,J = 6.6Hz,1.5H), *0.98(d,J = 6.6Hz,1.5H),0.96(s,1.5H), *0.94(s,1.5H),0.71(s,1.5H), *0.65(s,1.5H); 13 C NMR(150MHz,CD 3OD): *indicates minor δ *175.2, 175.1, 165.9, 165.2, 162.6, *162.4, 157.0, *156.7, 138.2, 128.9, 123.5, *123.5, 122.3, 119.5, *119.5, 116.2, 113.5, 112.2, 72.1, 71.9, 57.5, *57.4, 56.5, *56.5, 45.4, 44.8, *44.8, 44.5, 44.0, *43.8, *43.1, 43.1, 41.8, 41.5, *41.5, 40.7, *40.7, *40.2, 38.6, 38.0, *37.0, 36.8, 36.1, 35.2, 33.0, 32.8, *32.8, *32.1, *31.5, 31.3, 31.0, 29.8, *29.7, 28.0, *28.0, 26.4, 23.9, 22.4, *22.4, 19.2, *19.1, 12.7, *12.6; HRMS m / z calcd for C 41 H 58 N 5 O 3 [M + H] + : 668.4540; found: 668.4540.

[0108] (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1-(2-(4-phenylpiperidin-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-one (12u), white solid, yield: 38.4%, melting point: 125.9 - 127.2 °C; 1 H NMR(600MHz, CD 3OD): ~50:50 mixture of rotamers, *indicates minor amide rotamer δ* 8.15 (s, 0.5H), 8.13 (s, 0.5H), 7.26 (dd, J = 7.7, 7.4 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 4.91 - 4.85 (m, 2H), 4.61 - 4.50 (m, 2H), 3.88 - 3.78 (m, 2H), 3.53 - 3.43 (m, 2H), 2.94 (t, J = 12.8 Hz, 2H), 2.86 - 2.76 (m, 2H), 2.74 (t, J = 6.0 Hz, 1H), 2.57 - 2.48 (m, 1H), 2.46 - 2.35 (m, 1H), 2.06 - 1.98 (m, 1H), 1.92 - 1.74 (m, 7H), 1.67 - 1.53 (m, 6H), 1.51 - 1.40 (m, 6H), 1.36 - 1.07 (m, 8H), 1.01 (d, J = 6.6 Hz, 1.5H), *0.99 (d, J = 6.6 Hz, 1.5H), 0.96 (s, 1.5H), *0.95 (s, 1.5H), 0.71 (s, 1.5H), *0.66 (s, 1.5H); 13 C NMR (150 MHz, CD 3 OD): *indicates minor δ* 175.1, 175.1, *165.6, 165.0, *161.9, 161.8, 156.8, *156.5, 147.4, 129.5, 127.8, 127.3, 115.6, *115.6, 72.1, 71.9, 57.5, *57.5, 56.5, 45.8, *45.5, 44.8, *44.8, *44.5, 44.3, *44.3, 44.0, 43.9, 41.9, 41.5, *41.5, 40.7, *40.4, 38.6, 38.0, 37.0, *36.8, 36.1, 35.2, 34.4, 33.4, 32.9, *32.8, *32.4, *31.6, 31.3, 31.0, 29.8, *29.8, 28.0, 24.0, 22.4, 19.2, 12.7, *12.7; HRMS m / z calcd for C 42 H 61 N 4 O 3 [M + H] + : 669.4744; found: 669.4743.

[0109] Example 4: Biological Evaluation

[0110] 1. ATX Enzyme Activity Evaluation

[0111] (1) Experimental Method

[0112] All reactions were carried out at 37 °C in 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM CaCl 2 using 2 nM human ATX protein (Sino Biological) and 0.1 mM LPC (18:0, Macklin). The test compounds were prepared as 10 mM stock solutions in DMSO. Then these stock solutions were diluted to the test concentrations with 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM CaCl 2 The ability of ATX to cleave LPC was detected using a coupled assay that monitored the release of choline using Amplex Red (100 μM), choline oxidase (0.2 units / mL), and horseradish peroxidase (2 units / mL). Fluorescence was read (excitation 530 nm, emission 590 nm) by a BioTek H1 microplate reader.

[0113] (2) Experimental Results

[0114] Table 1. In Vitro ATX Inhibitory Activity of Compounds 12a - 12u

[0115]

[0116] a : The data provided are the results of at least three independent experiments.

[0117] The ATX inhibitory activity of all synthetic compounds was evaluated using the LPC choline release assay. GLPG-1690 is a representative ATX inhibitor that has entered Phase III clinical trials for IPF and chronic obstructive pulmonary disease. The results expressed as IC 50 values are summarized in Table 1. Most compounds showed significant inhibitory activity, stronger than that of the parent compound UDCA. Among them, compounds 12a and 12h showed the best activity, with IC 50 values of 7.62 ± 0.62 and 7.51 ± 0.72 nM, respectively, which are 9 times that of GLPG-1690.

[0118] 2. Cytotoxicity Evaluation

[0119] (1) Cell Culture

[0120] The human hepatic stellate cell line HSC-LX2 is derived from the Henan Provincial Engineering Research Center for Industrial Microbiology. The human fetal lung fibroblast cell line (CCC-HPF-1) is derived from the Cell Resource Center of Peking Union Medical College. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C in 5% CO 2 2.

[0121] (2) Cytotoxicity assay

[0122] To evaluate the cytotoxicity of these compounds, MTT assays were performed. Cells were seeded in 96-well plates and treated with compounds (0 to 80 μM, dissolved in DMSO) for 48 h. Then, 10 μL of MTT (5 mg / mL) was added to each well and incubated for 4 h. Subsequently, the supernatant was discarded, and 150 μL of DMSO was added to dissolve the formazan crystals in each well. The absorbance was measured at 490 nm using a microplate reader.

[0123] (3) Experimental results

[0124] The cytotoxicity of 12a and 12h against the hepatic stellate cell line (HSC-LX2) and the human embryonic lung fibroblast cell line (CCC-HPF-1) was detected by the MTT method. As Figure 1 shown, compounds 12a and 12h showed no obvious cytotoxicity at concentrations up to 80 μM, indicating that compounds 12a and 12h can be used as promising lead compounds for further evaluation of their antifibrotic activities.

[0125] 3. Inhibitory effect on TGF-β1-induced fibroblast migration

[0126] (1) Experimental method

[0127] HSC-LX2 and CCC-HPF-1 cells were seeded on 6-well plates and allowed to reach 100% confluence. A 200 μL pipette tip was used to scrape the monolayer cells to create a wound. The cells were washed twice with PBS to remove dead cells and debris. Then, the cells were cultured for 24 h in the presence or absence of 10 ng / mL TGF-β1 in the presence of a vehicle or 10 μM compound. The wound closure was monitored using an optical microscope at 0, 12, and 24 h, and the cell area was measured using Image J software.

[0128] (2) Experimental results

[0129] Studies have shown that the migration of hepatic stellate cells or lung fibroblasts is crucial for the occurrence of liver or lung fibrosis. Therefore, inhibiting the migration of the above cells can significantly delay the process of liver or lung fibrosis. Therefore, the effects of compounds 12a and 12h on the migration of HSC-LX2 and CCC-HPF-1 cells were evaluated by wound healing assay. The results showed that at a concentration of 10 μM, compounds 12a and 12h could inhibit the migration of TGF-β1-induced HSC-LX2 and CCC-HPF-1 cells( Figures 2 to 3 ). Notably, after treatment with compounds 12a and 12h for 24 hours, the migration rate was even lower than that of the control group.

[0130] 4. Inhibitory effect on the activation of TGF-β / Smad signaling pathway

[0131] (1) Experimental method

[0132] HSC-LX2 and CCC-HPF-1 cells were cultured on coverslips in 24-well plates, treated with or without 10 ng / mL TGF-β1, and cultured for 48 hours in the presence of vehicle or 10 μM compound. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde (#P1110, Solarbio) for 10 minutes at room temperature. Then, the cells were permeabilized with 0.5% Triton X-100 (#ST797, Beyotime) for 10 minutes and blocked with immunofluorescence blocking buffer (#P0260, Beyotime) for 30 minutes. After that, the cells were incubated overnight at 4 °C with Smad2 / 3 antibody (#BA1395, Boster) and labeled with Dylight 550-conjugated secondary antibody (#BA 1135, Bosster). Finally, the coverslips were sealed with an anti-fluorescence quenching sealing solution containing DAPI (#P0131, Beyotime) and observed by a fluorescence microscope (Ex: 495 nm, Em: 519 nm, OPLENIC, PSC603-07S), and the light intensity was measured by Image J software.

[0133] (2) Experimental results

[0134] The TGF-β / Smad signaling pathway is the main mechanism of fibrosis. Active TGF-β initiates signal transduction by binding to TGF-β type I / II receptors to form a receptor complex. Then the receptor complex induces the phosphorylation of Smad2 and Smad3 transcription factors. Phosphorylated Smad2 and Smad3 form a heteromeric complex with Smad4, which then translocates to the nucleus to activate the transcription of fibrosis-related genes such as α-SMA and collagen. Here, the present invention detected the effects of 12a and 12h on the phosphorylation of Smad2 and Smad3 by Western blotting. In addition, the effects of 12a and 12h on the translocation of Smad2 / 3 from the cytoplasm to the nucleus were analyzed by immunofluorescence. As Figures 4 to 5 shown, TGF-β1 upregulated the phosphorylation levels of Smad2 and Smad3, while treatment with compounds 12a and 12h significantly reduced this phosphorylation in a concentration-dependent manner. Immunofluorescence analysis further showed that after induction with TGF-β1, the level of Smad2 / 3 in the nucleus increased significantly, but 12a and 12h effectively inhibited the translocation of Smad2 / 3 to the nucleus. The above results indicate that 12a and 12h can inhibit the activation of the TGF-β / Smad signaling pathway involved in the TGF-β1-induced fibrotic response.

[0135] In summary, the present invention designed and synthesized a series of new UDCA aminopyrimidine hybrids as potent ATX inhibitors by a molecular hybridization strategy, and tested their inhibitory activities against ATX using the LPC choline release assay. Preliminary results showed that some compounds exhibited good ATX inhibitory activities, among which compounds 12a and 12h showed the strongest inhibitory activities, with IC 50 values of 7.62 ± 0.62 and 7.51 ± 0.72 nM, respectively. In addition, compounds 12a and 12h significantly inhibited TGF-β1-induced cell migration. Preliminary mechanism studies showed that compounds 12a and 12h exerted anti-fibrotic effects by inhibiting the TGF-β / Smad signaling pathway. The above results indicate that compounds 12a and 12h are two promising anti-fibrotic drugs and can be further evaluated for drug development.

Claims

1. A pyrimidocyclic compound, It is characterized in that Having the structure of formula I, and also including pharmaceutically acceptable salts thereof: in: R is selected from -(CH 2 ) n -R 1 、-MR 1 ; n is an integer selected from 0 to 3; M is selected from a 4-7 membered heterocyclic group, a 4-7 membered cycloalkyl group, a 5-7 membered unsaturated heterocyclic group containing 1-3 N, O, and S atoms, and M and R 1 Connected by chemical bonds or rings; R 1 An aromatic ring group having 6 to 12 members and containing 0 to 2 N atoms, wherein the aromatic ring group is substituted by any substituent selected from the following: halogen, cyano, nitro, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, C1-C4 haloacyl; The pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid.

2. The pyrimidocyclic compound according to claim 1, It is characterized in that In the structure: M is selected from cyclopentyl, cyclohexyl, pyrrolyl, and piperidinyl synthesized with the connected nitrogen ring; R 1 Selected from phenyl and pyridyl, the aromatic ring group is substituted by at least one substituent selected from any of the following: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy.

3. The pyrimidocyclic compound according to claim 2, It is characterized in that In the structure: R 1 Selected from phenyl and pyridyl, wherein the phenyl is substituted by at least one substituent selected from any of the following: hydrogen, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy.

4. The pyrimidocyclic compound according to claim 2, It is characterized in that In the structure: When M is selected from cyclopentyl, pyrrolyl, 1 When selected from phenyl, M and R 1 Connect through parallel ring; When M is selected from the piperidinyl group synthesized with the nitrogen ring connected, R 1 When selected from phenyl, M and R 1 Connected by chemical bonds.

5. The pyrimidocyclic compound according to claim 3, It is characterized in that In the structure: When R 1 When selected from monosubstituted phenyl, the substituent is selected from ortho, meta, and para substituents; When R 1 When selected from disubstituted phenyl, the substituent is selected from the meta substituent.

6. The pyrimidocyclic compound according to claim 1, It is characterized in that In the structure, R is selected from any of the following structures:

7. A method for preparing the pyrimidocyclic compound according to claim 1, It is characterized in that The following steps are involved: Compound 9 is subjected to amination, deprotection and acylation to obtain the pyrimidocyclic compound; Wherein, R is selected from -(CH 2 ) n -R 1 、-MR 1 ; n is an integer selected from 0 to 3; M is selected from a 4- to 7-membered cycloalkyl group, a 5- to 7-membered unsaturated heterocyclic group containing 1 to 3 N, O, and S atoms, and M and R 1 Connect through parallel ring; R 1 An aromatic ring group having 6 to 12 members and containing 0 to 2 N atoms, wherein the aromatic ring group is substituted by any substituent selected from the following: halogen, cyano, nitro, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, C1-C4 haloacyl; The corresponding acid is reacted with the compound prepared by the above method to form a salt, thereby obtaining a pharmaceutically acceptable salt of the compound.

8. A pharmaceutical composition, It is characterized in that It comprises the pyrimidocyclic compound according to claim 1 and a pharmaceutically acceptable carrier.

9. Use of the pyrimidocyclic compound according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of an ATX enzyme inhibitor.

10. The use according to claim 9, It is characterized in that The drug is an anti-fibrotic drug.

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