PROTEOLYSIS TARGETING COMPOUND WITH TISSUE TARGETING CAPABILITY AND USE THEREOF

Abstract

The present invention is based on the discovery of a proteolysis targeting compound having tissue targeting capability and use thereof, relating to medicinal products, and to such a compound or a pharmaceutically acceptable salt thereof, a stereoisomer, a solvate, or a polymorph. The compound is a proteolysis targeting chimera (PROTAC) with specific tissue targeting ability. The compound structure comprises three parts, i.e., A-BD-CON, wherein the part A is a PROTAC, one end of the structure thereof is a target protein binding ligand, and the other end is a ubiquitin ligase ligand; and the part CON is a ligand of an asialoglycoprotein receptor (ASGPR), enabling the specific tissue targeting function. The compound enriches in liver tissue and is able to target cells in the tissue. The invention achieves improved druggability of the PROTAC with higher solubility and cellular membrane permeability, therefore produces enhanced pharmaceutical effect on the specific target tissue.

Claims

1. A compound of following formula or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof, the compound consisting of following three parts: A-BD-CON wherein CON is a ligand of asialoglycoprotein receptor (ASGPR), and A has a formula of LGP-Y-Z-LK-LGE, wherein: LGP is a target protein binding ligand, LGE is an E3 ubiquitin ligase ligand; Y and Z are independently selected from the group consisting of a covalent bond, CH.sub.2, C1-C4 alkyl substituted methylene, NH, C1-C4 alkyl substituted nitrogen atom, O or S; and Z is selected from the group consisting of a covalent bond, CH.sub.2 and C1-C4 alkyl substituted methylene when Y is selected from the group consisting of O, S, NH or C1-C4 alkyl substituted nitrogen atom; or Y is selected from the groups consisting of a covalent bond, CH.sub.2 and C1-C4 alkyl substituted methylene when Z is selected from the group consisting of O, S, NH or C1-C4 alkyl substituted nitrogen atom; LK is connected with LGP through Y-Z at one end thereof and is connected with LGE through covalent bond at the other end thereof, and LK is selected from following structural units: ##STR00085## ##STR00086## where m is an integer of 0-5; n is an integer of 0-20; p is an integer of 0-4; q is an integer of 0-20; r is an integer of 1-3; and s is an integer of 1-5; and X is selected from the groups consisting of CH.sub.2, O, S and NR.sup.1, wherein R.sup.1 is selected from the groups consisting of H, C1-6 alkyl, C1-6 haloalkyl, or C1-6 alkoxy substituted C1-6 alkyl; BD is a linker of A and CON and has formula of Q-LN, wherein Q is capable of being degraded and broken under acidic conditions by hydrolase in endosome or lysosomal in cells to release A; and Q is a structure selected from the group consisting of hydrazone, urea, oxime, disulfide, thioether, amide, ester (carboxylic acid ester, phosphate ester, pyrophosphate ester, carbonate ester, sulfate ester, sulfonic acid ester, amino sulfonic acid ester, methylene sulfonic acid ester, or carbamate), and ##STR00087## and LN is a linker of Q and CON and is selected from a carbon chain of 3-20 carbon atoms, and any CH.sub.2 in the carbon chain is optionally substituted by O, NH, or C (O).

2. The compound according to claim 1, wherein LGE is a ligand of ubiquitinated E3 enzyme, preferably a ligand of Von Hippel-Lindau tumor suppressor (pVHL), and more preferably a structure of following formula, ##STR00088## with one end being connected to LK and the other end being connected to Q, wherein LK and Q are the same as defined in claim 1; and wherein G is selected from the group consisting of C1-C10 alkyl, C3-C10 cycloalkyl, and 3-10 membered heterocyclic alkyl containing 1-3 heteroatoms selected from O, N and S, preferably is isopropyl, tertiary butyl, cyclohexyl or tetrahydropyran; and more preferably is tertiary butyl.

3. The compound according to claim 1, wherein the compound is represented by the following formula: ##STR00089## wherein Q is selected from the following structural units: ##STR00090## wherein LN is selected from the following structural units: ##STR00091## wherein V is an integer of 3-15, and preferably is an integer of 5-12, G in the formula is selected from the group consisting of C1-C10 alkyl, C3-C10 cycloalkyl, and 3-10 membered heterocyclic alkyl containing 1-3 heteroatoms selected from O, N and S, preferably is isopropyl, tertiary butyl, cyclohexyl or tetrahydropyran; and more preferably is tertiary butyl, Y and Z are the same as defined in claim 1, and LK is independently selected from the same structural unit as the LK in claim 1.

4. The compound according to claim 1, wherein CON has a structure of following formula: ##STR00092## wherein S1-S3 are independently selected from the following structural units of: galactose, galactosamine, glucose, glucosamine, mannose, mannosamine and lactose acid, in the form of D-configuration or L-configuration; wherein S1-S3 are respectively connected to L1-L3 through alpha glycosidic bond or beta glycosidic bond; wherein L1-L3 are independently selected from the following structures of: chemical bond, aliphatic carbon chains of C3-C15, and aliphatic carbon chains of C3-C15 interrupted by one or more groups selected from O, S, NH or carbonyl at any position therein; wherein M is selected from C, N, O, S and P (?O) and connected with LN via a covalent bond; and wherein S1-L1, S2-L2, or S3-L3 is optionally absent independently.

5. The compound according to claim 1, wherein CON is selected from the following structures: ##STR00093## ##STR00094## ##STR00095##

6. The compound according to claim 1, wherein LGP is selected from the following structural units: ##STR00096## ##STR00097## ##STR00098## wherein R2-R5 are independently selected from the group consisting of H, F, Cl, Br, C1-4 alkyl, and C1-4 alkoxy; R6 is selected from the group consisting of H, C1-4 alkyl and C1-4 alkoxy.

7. The compound according to claim 1, wherein LGP is a ligand of cancer associated targets, microbial associated targets, immune diseases associated targets, neurodegenerative diseases associated targets or metabolic diseases associated targets.

8. The compound according to claim 6, wherein a binding target of LGP is selected from the group consisting of kinases, transcription factors, epigenetic reading frames, microtubule-associated proteins, microbial-associated proteins, and so on.

9. The compound according to claim 6, wherein a binding target of LGP is selected from the group consisting of EGFR, VEGFR, FGFR, PDGFR, Raf, Braf, RET, FLT, c-Kit, MET, ACVR, ALK, AKT, AhR, AURKA, AR, RAR, ER, BCL, BCR-ABL, BET, BMPR, BLK, BTK, BRD, CDK, CK, CHEK1, CTNNB1, DDR, DHODH, RAS, EED, ESR1, CRABP, CRBN, HER2, HER3, HMGCR, Htt, GNA11, GNA1, GNAS, NQO, TROP, eIF4E, ERK, ERG, ETV, ERR?, EZH2, FAK, IDH1/2, IGF1R, IRAK4, JAK, MEK, MELK, LTK, FKBP, MDM2, HDAC, MER, MCL, MTOR, PAR, PBRM, PCAF, PDE, PD-1, PD-L1, PTK, PARP, PDXK, PLK, PKB, MAPK, PI3K, Pirin, RIPK, Rpn13, PRC, P38, TGF?, AFP, NTRK, NHE, CEA, SOAT1, SNCA, SYK, RNF43, DLK1, gp96, SGK, SMAD, SMO, SFB, SHP, SIK, SRC, STAT3, TBK, TYK3, TRIM, NS3, IRAK4, PCAF, GCN5, Sirt2, Tau, TUB, Wee1, ZAK or their combined targets.

10. The compound according to claim 6, wherein LGP is selected from the group consisting of Hsp90 inhibitors, kinase inhibitors, phosphatase inhibitors, MDM2 inhibitors, compounds targeting proteins containing human BET bromodomain, HDAC inhibitors, human lysine methyltransferase inhibitors, compounds targeting RAF receptors, compounds targeting FKBP, angiogenesis inhibitors, immunosuppressive compounds, compounds targeting aryl hydrocarbon receptors, compounds targeting androgen receptors, compounds targeting estrogen receptors, compounds targeting thyroid hormone receptors, compounds targeting HIV protease, compounds targeting HIV integrase, compounds targeting HCV protease, and compounds targeting acyl protein thioesterase 1 and/or 2.

11. The compound according to claim 9, wherein LGP is selected from the group consisting of: sorafenib, lenvatinib, regorafenib, cabozantinib, apatinib, refametinib, carboplatin, cisplatin, oxaliplatin, everolimus, pemetrexed disodium, erlotinib, dasatinib, imatinib, sunitinib, osimertinib, ibrutinib, alecinix, crizotinib, entrectinib, afatinib, axtintib, ceritinib, larotrectinib, brigatinib, neratinib, trametinib, lapatinib, neratinib, fluorouracil, 5-FU, etoposide, gemcitabine, decitabine, capecitabine, doxorubicin, epirubicin, vincristine, temozolomide, vincristine, ifosfamide, mitoxantrone, gefitinib, bortezomib, paclitaxel, docetaxel, pegylated interferon ?-2a, interferon ?-2a, pegylated interferon ?-2b, interferon ?-2b, azacitidine, cytarabine, cyclocytidine, irinotecan, topotecan, vidarabine, idoxuridine (IDU), trifluridine, bromovinyldeoxyuridine, magnesium glycyrrhizinate, glycyrrhizic acid, glutathione, polyene phosphatidyl choline, ademetionine, ursodeoxycholic acid, and ulinastatin.

12. The compound according to claim 1, wherein the compound is selected from the following structures: ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## preferably, the compound is selected from WG-1, WG-2, WG-3, WG-4, WG-5, and WG-6.

13. A pharmaceutical composition, comprising the compound according to claim 1.

14. A method for treating tumors or liver diseases, comprising administering the compound according to claim 1 to a subject in need.

15. (canceled)

16. The method according to claim 14, wherein the tumor is liver cancer.

17. The method according to claim 14, wherein the liver disease is hepatitis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 shows HPLC chromatogram of WG-1 (00300639);

[0054] FIG. 2 shows LC/MS chromatogram of WG-1 (00300639);

[0055] FIG. 3 shows .sup.1HNMR spectrum of WG-1 (00300639);

[0056] FIG. 4 shows .sup.31PNMR spectrum of WG-1 (00300639);

[0057] FIG. 5 shows transmembrane test results of WG-1 (00300639) and WGint4 (003006);

[0058] FIG. 6 shows growth inhibition test results on cancer cells by WG-1 (00300639), WGint4 (003006) and positive drug lenvatinib; and

[0059] FIG. 7 shows proteolysis test results of WG-1 (00300639) and WGint4 (003006).

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present application will be further illustrated with reference to drawings and following embodiments. However, these embodiments are only for more clearly illustrating, rather than limiting the present invention in any form. The present invention could be implemented in a variety of different ways disclosed herein.

[0061] The present invention describes the materials and experimental methods used in the experiment in a general and specific way. Although many materials and operation methods used for the purpose of the present invention are well known in the art, the present invention is still described herein as much detail as possible. In the following, unless otherwise specified, the materials and experimental methods used are well known in the art.

[0062] WG series compounds can be synthesized through the following process or by similar route. Among all compounds, WGint4 is the compound A in the general formula of the present invention, which needs to be synthesized individually. WGint8 is the compound CON in the general formula of the present invention, which can be synthesized by referring to the existing technology or purchased from the supplier. Other procedures are share similar routes.

[0063] WG-1 (00300639) is exemplified by the following synthesis route:

##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##

[0064] In comparison with compound WG-1 (00300639), the corresponding WGint4 is WGint4 (003006). The synthesis route of WGint4 (003006) is as follows:

##STR00048## ##STR00049##

[0065] In comparison with compounds WG-4 (00500639) and WG-5 (00600639), the corresponding WGint4 are WGint4 (005000) and WGint4 (006000). The synthetic method is as follows:

(1) Synthesis of Compound Side 1

[0066] ##STR00050##

(2) Synthesis of Compound Core 1

[0067] ##STR00051##

(3) Synthesis of Compound WGint4 (006000)

[0068] ##STR00052##

(4) Synthesis of Compound WGint4 (005000)

[0069] ##STR00053##

[0070] The synthesis of other compounds within the scope of this application can be achieved following the route mentioned above and selecting corresponding raw materials.

Example 1 Synthesis of Compound WGint2

[0071] ##STR00054##

[0072] A solution of 10-hydroxydecanoic acid (10.0 g, 53.2 mmol) in DMF (250 mL) was treated with potassium bicarbonate (5.32 g, 53.2 mmol) followed by benzyl bromide (9.10 g, 53.2 mmol) and was then stirred for 18 h at room temperature under argon. The solvent was evaporated and the residue was partitioned between EA and water. The aqueous layer was washed with EA and the combined organic extracts were dried over anhydrous magnesium sulfate. The resulting crude material was purified with silica gel flash chromatography to give compound WGint2 (8.0 g) as a white solid: LC/MS (ESI) m/z: [M+H].sup.+ 279.

Example 2 Synthesis of Compound WGint3

[0073] ##STR00055##

[0074] To a solution of compound WGint2 (5.0 g, 18.0 mmol) in 100 mL of anhydrous dichloromethane containing diisopropylammonium tetrazolide (4.6 g, 27.0 mmol) was added 2-cyanoethoxy-N,N,N,N-tetraisopropyl phosphorodiamidite (9.0 mL, 27.0 mmol), and the mixture was stirred for 6 h at room temperature, until TLC revealed complete reaction. Dichloromethane was then removed by evaporation and the product was taken up in ethyl acetate, washed with 5% sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate and evaporated to a small volume. The product was chromatographed on a silica gel column eluted with 10-35% ethyl acetate in hexane+2% triethylamine to give compound WGint3 (2.5 g). The intermediate is directly used in the next reaction.

Example 3 Synthesis of Compound WGint6

[0075] ##STR00056## ##STR00057##

[0076] The mixture of the compound WGint3 (2.5 g, 5.2 mmol) and compound WGint4 (0.5 g, 0.52 mmol) in ACN (25 mL) was added in tetrazoler (145 mg, 2.1 mmol) and stirred overnight. Tert-butyl peroxide in decane (2 mL, 5 M) was added dropwise at 0? C. and the mixture was stirred for 6 h at room temperature. The reaction is monitored by TLC. After completion of the reaction, the reaction mixture is concentrated at 40? C. and diluted with EA and washed with water and brine solution. Organic layer was dried over anhydrous Na.sub.2SO.sub.4 and filtered and the solvent was evaporated to give crude compound WGint6. LC/MS (ESI) m/z: [M+H].sup.+ 1360.

Example 4 Synthesis of Compound WGint7

[0077] ##STR00058## ##STR00059##

[0078] The crude compound WGint6 from above step was dissolved in THF (8 mL). Then 0.5M LiOH (8 mL) was added. The mixture was stirred for 6 h at room temperature and evaporated to give crude which was purified with prep-HPLC to give compound WGint7 (85 mg). LC/MS (ESI) m/z: [M+H].sup.+ 1218.

Example 5 Synthesis of Compound WGint9

[0079] ##STR00060## ##STR00061##

[0080] To a solution of compound WGint7 (40 mg, 0.033 mmol) in DMF (1 mL) were added HBTU (13.8 mg, 0.036 mmol) and DIEA (4.7 mg, 0.036 mmol), and the resulting mixture was stirred for few minutes. A solution of compound WGint8 (40 mg, 0.022 mmol) in DMF (0.5 mL) was added and stirred at room temperature overnight. Solvents and volatiles were removed under reduced pressure, and the residue was purified with prep-HPLC to give compound WGint9 (8 mg). LC/MS (ESI) m/z: [M+H].sup.+ 2993.

Example 6 Synthesis of Compound WG-1 (00300639)

[0081] ##STR00062## ##STR00063##

[0082] A solution of compound WGint9 (8 mg) in MeOH (0.5 mL) was treated with 40% NH.sub.3.Math.H.sub.2O (0.5 mL) and stirred for 4 h at room temperature. The mixture was evaporated and poured into water and DCM, and organic layer was evaporated to give the compound WG-1(00300639) (6 mg). LC/MS (ESI) m/z: [M+H].sup.+:2614, [M/2+1]+:1308; .sup.1HNMR (CD.sub.3OD, 400 MHZ): ? 8.887 (s, 1H), 8.779 (s, 1H), 8.638-8.651 (d, J=5.2 Hz, 1H), 8.206-8.228 (d, J=8.8 Hz, 1H), 7.388-7.509 (m, 5H), 7.201-7.280 (m, 2H), 6.608-6.622 (d, J=5.6 Hz, 1H), 4.537-4.682 (m, 3H), 4.358-4.398 (t, J=16.0 Hz, 3H), 4.086-4.113 (t, J=10.8 Hz, 3H), 3.456-3.973 (m, 36H), 3.217-3.366 (m, 10H), 2.092-2.712 (m, 22H), 1.954-2.000 (m, 13H), 1.307-1.718 (m, 43H), 0.575-1.048 (m, 16H); .sup.31PNMR (CD.sub.3OD, 162 MHZ): ? 0.709 (P).

Example 7 Synthesis of Compound 003006int2

[0083] ##STR00064##

[0084] To a solution of compound 003006int1 (20 g, 0.14 mol) and Py (17 g, 0.2 mol) in DMF (200 mL) was added dropwise phenyl carbonochloridate (22 g, 0.14 mmol). The mixture was stirred overnight at 0? C. The above solution was poured into ice-water (500 mL) and extracted with EtOAc (200 mL?3). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give compound 003006int2 (25 g, crude) as brown solid. LC/MS (ESI) m/z: [M+H].sup.+ 264.

Example 8 Synthesis of Compound 003006int3

[0085] ##STR00065##

[0086] The solution of compound 003006int2 (25 g, 0.13 mmol), TEA (13 g, 0.13 mol) and cyclopropanamine (15 g, 0.26 mol) in DCM (300 mL) was stirred overnight at room temperature. The mixture washed with water and under reduced pressure to give compound 006003int3 (12 g, 57%) as brown solid. LC/MS (ESI) m/z: [M+H].sup.+ 227.

Example 9 Synthesis of Compound 003006int4

[0087] ##STR00066##

[0088] The solution of compound 003006int3 (12 g, 52.9 mmol), K.sub.2CO.sub.3 (13 g, 106 mmol) and methyl 4-chloro-7-methoxyquinoline-6-carboxylate (16 g, 63.5 mol) in DMF (120 mL) was stirred overnight at 80? C. The mixture was poured into ice-water and filtered, and the filter cake was washed with water and dried to give compound 003006int4 (14 g, 59.8%) as brown solid. LC/MS (ESI) m/z: [M+H].sup.+ 442.

Example 10 Synthesis of Compound 003006int5

[0089] ##STR00067##

[0090] The solution of compound 003006int4 (14 g, 31.7 mmol) and LiOH (2.5 g, 63.4 mmol) in MeOH/H.sub.2O (140 mL) was stirred overnight at room temperature. The reaction solution was pH-adjusted to 2-3 by the addition of 1 N hydrochloric acid and filtered to give compound 003006int5 (10 g, 73.5%) as yellow solid. LC/MS (ESI) m/z: [M+H].sup.+ 428.

Example 11 Synthesis of Compound 003006int6

[0091] ##STR00068##

[0092] To a solution of compound 003006int5 (1 g, 2.3 mmol) and ethyl 7-aminoheptanoate (485 mg, 2.8 mmol) in DCM (10 mL) was added reaction solution HOBT (474 mg, 3.5 mmol) EDCI (672 mg, 3.5 mmol) DIEA (755 mg, 5.8 mmol). The mixture was stirred overnight at room temperature. The reaction was extracted with DCM (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by silica gel to give compound 003006int6 (700 mg, 51.5%) as yellow liquid. LC/MS (ESI) m/z: [M+H].sup.+ 583.

Example 12 Synthesis of Compound 003006int7

[0093] ##STR00069##

[0094] The solution of compound 003006int6 (700 mg, 1.2 mmol) and LiOH (96 mg, 2.4 mmol) in MeOH/H.sub.2O (10 mL) was stirred for reaction overnight. The reaction solution was pH-adjusted to 2-3 by the addition of 1 N hydrochloric acid and filtered to give compound 003006int7 (300 mg, 45%) as yellow solid. LC/MS (ESI) m/z: [M+H].sup.+ 555.

Example 13 Synthesis of Compound 003006

[0095] ##STR00070##

[0096] To a solution of compound 003006int7 (300 mg, 0.54 mmol) and 7A (279 mg, 0.65 mmol) in DCM (5 mL) was added HOBT (110 mg, 0.81 mmol), EDCI (155 mg, 0.81 mmol) and DIEA (175 mg, 1.3 mmol). The mixture was stirred overnight for reaction. The reaction mixture was extracted with DCM (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by prep-HPLC to give WGint4(003006) (120 mg, 22.9%) as white solid. LC/MS (ESI) m/z: [M+H].sup.+ 967.

Example 14 Synthesis of Compound 005000int2

[0097] ##STR00071##

[0098] The solution of compound 005000int1 (10 g, 0.11 mol), tert-Butyl acrylate (18 g, 0.14 mol) and potassium tert-butoxide (0.16 g, 1.4 mmol) in THF (100 mL) was stirred overnight at room temperature. The mixture was quenched with 1N HCl (50 mL), and extracted with EA (50 mL*2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the compound 005000int2 (20 g, 81.9%) without further purification. LC/MS (ESI) m/z: [M+H].sup.+ 223.

Example 15 Synthesis of Compound 005000int3

[0099] ##STR00072##

[0100] To a solution of compound 005000int2 (10 g, 0.045 mol) in THF (100 mL) was added edLAH (2.6 g, 0.068 mol) in three portions at 0? C. The mixture was stirred at room temperature for 3 hours. The reaction was quenched with Na.sub.2SO.sub.4.Math.10H.sub.2O. The mixture was filtered and the filtrate was evaporated to give compound 005000int3 (6 g, 88.2%). LC/MS (ESI) m/z: [M+H].sup.+ 153.

Example 16 Synthesis of Compound 005000int4

[0101] ##STR00073##

[0102] The solution of compound 005000int3 (6 g, 0.039 mol), tert-Butyl acrylate (6.6 g, 0.052 mol) and potassium tert-butoxide (0.22 g, 0.002 mol) in THF (100 mL) was stirred overnight. The mixture was quenched with 1N HCl (50 mL), and extracted with EA (30 mL*2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the compound 005000int4 (10 g, 90.7%) without further purification. LC/MS (ESI) m/z: [M+H].sup.+ 281.

Example 17 Synthesis of Compound Side 1

[0103] ##STR00074##

[0104] Compound 005000int4 (5 g, 0.018 mmol) and sodium iodide (13.5 g, 0.09 mol) in acetone (50 mL) was stirred at 50? ? C. for 48 hours. After cooling, solvent were evaporated in vacuum and washed with water (20 mL*2) to obtain side 1 (5 g, 74.9%). LC/MS (ESI) m/z: [M+H].sup.+ 373.

Example 18 Synthesis of Compound 005000int8

[0105] ##STR00075##

[0106] The solution of compound 005000int7 (10 g, 0.033 mol), 7A (5.6 g, 0.037 mol) and DIEA (0.28 g, 0.01 mol) in NMP (100 mL) was stirred overnight at 130? ? C. The mixture was purified by prep-HPLC to give compound 005000int8 (5 g, 35.7%). LC/MS (ESI) m/z: [M+H].sup.+ 421.

Example 19 Synthesis of Compound 005000int9

[0107] ##STR00076##

[0108] To a solution of compound 005000int8 (4 g, 9.5 mmol) and NH.sub.4Cl (10 g, 0.19 mol) in EtOH (20 mL) and H.sub.2O (20 mL) was added Zn (10 g, 0.15 mol). The mixture was stirred at 80? C. for 2 hours. The reaction was filtered and evaporated, and the residue was purified by silica gel to give compound 005000int9 (3.2 g, 86.5%). LC/MS (ESI) m/z: [M+H].sup.+ 391.

Example 20 Synthesis of Compound 005000int10

[0109] ##STR00077##

[0110] To a solution of compound 005000int9 (3.9 g, 10 mmol), compound 9A (2.7 g, 12 mmol) and TEA (1.5 g, 15 mmol) in DMF (40 mL) was added HOBT (2 g, 15 mmol) and EDCI (573 mg, 3 mmol). The mixture was stirred at room temperature overnight. The reaction was quenched with Na.sub.2CO.sub.3 aqueous, and extracted with EA. The combined organic layers was evaporated and purified by silica gel to give compound 005000int10 (3 g, 50.4%). LC/MS (ESI) m/z: [M+H].sup.+ 596.

Example 21 Synthesis of Compound Core 1

[0111] ##STR00078##

[0112] The solution of compound 005000int10 (1.5 g, 7.2 mmol) and Pd/C (200 mg) in MeOH(20 mL) was stirred at room temperature overnight under H.sub.2 balloon. The reaction was filtered and evaporated to give Core 1 (1 g, 78.7%). LC/MS (ESI) m/z: [M+H].sup.+ 506.

Example 22 Synthesis of Compound 006000 int11

[0113] ##STR00079##

[0114] The solution of core 1 (500 mg, 0.99 mmol), side 1 (447 mg, 1.28 mmol) and cesium carbonate (980 mg, 3 mmol) in DMF (5 mL) was stirred at 30? C. overnight. The reaction was poured into water and extracted with EA (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by silica gel to give compound 006000int11 (350 mg, 47.2%) as white solid. LC/MS (ESI) m/z: [M+H].sup.+ 750.

Example 23 Synthesis of Compound 006000 int12

[0115] ##STR00080##

[0116] To a solution of compound 006000int11 (300 mg, 0.4 mmol) was added TFA (0.3 mL). The mixture was stirred at room temperature overnight. The solvent was evaporated to give compound 006000int12 (200 mg, 72.2%). LC/MS (ESI) m/z: [M+H].sup.+ 694.

Example 24 Synthesis of Compound WGint4 (006000)

[0117] ##STR00081##

[0118] To a solution of compound 006000int12 (100 mg, 0.14 mmol) and 12A (75 mg, 0.17 mmol) in DCM (1 mL) was added HOBT (30 mg, 0.22 mmol), EDCI (43 mg, 0.22 mmol) and DIEA (65 mg, 0.5 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was added into water and extracted with DCM (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by prep-HPLC to give WGint4(006000) (20 mg, 13%) as white solid. LC/MS (ESI) m/z: [M+H].sup.+ 1106.

Example 25 Synthesis of Compound 005000int13

[0119] ##STR00082##

[0120] The solution of core 2 (600 mg, 1.23 mmol), side 1 (584 mg, 1.57 mmol) and cesium carbonate (980 mg, 3 mmol) in DMF (5 mL) was stirred at 30? ? C. overnight. The reaction solution was poured into water and extracted with EA (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by silica gel to give compound 005000int13 (200 mg, 22.2%) as white solid. LC/MS (ESI) m/z: [M+H].sup.+ 732.

Example 26 Synthesis of Compound 005000int14

[0121] ##STR00083##

[0122] To a solution of compound 005000int13 (300 mg, 0.4 mmol) was added TFA (0.3 mL). The mixture was stirred at room temperature overnight. The solvent was evaporated to give compound 005000int14 (200 mg, 72.2%). LC/MS (ESI) m/z: [M+H].sup.+ 676.

Example 27 Synthesis of Compound WGint4 (005000)

[0123] ##STR00084##

[0124] To a solution of compound 005000int14 (100 mg, 0.14 mmol) and 14A (75 mg, 0.17 mmol) in DCM (1 mL) was added HOBT (30 mg, 0.22 mmol), EDCI (43 mg, 0.22 mmol) and DIEA (65 mg, 0.5 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was added into water and extracted with DCM (10 mL?2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to give the crude product which was purified by prep-HPLC to give WGint4(005000) (16 mg, 8%) as white solid. LC/MS (ESI) m/z: [M+H].sup.+ 1088.

Example 28 Synthesis of Compounds WG-2-WG-25

[0125] Furthermore, compounds WG-2-WG-25 were synthesized with the identical or similar methods described in the examples 1-27. The structures were confirmed with LC/MS and the data were shown in the following table:

TABLE-US-00001 TABLE 1 Structural confirmation data of compounds WG-2-WG-25 LC/MS Theoretical value Measured values (ESI) of HRMS of HRMS Compound [M + H].sup.+ [M + H].sup.+ [M + H].sup.+ WG-2 1950 1950.9023 1950.9006 WG-3 2614 2614.2350 2614.2379 WG-4 2736 2735.3010 2735.3002 WG-5 2753 2753.2915 2753.2896 WG-6 2646 2646.2748 2646.2712 WG-7 2760 2760.3326 2760.3370 WG-8 2736 2735.3010 2735.2995 WG-9 2838 2838.3769 2838.3801 WG-10 2760 2759.3486 2759.3511 WG-11 2800 2799.3799 2799.3810 WG-12 2799 2799.3799 2799.3805 WG-13 2723 2723.2489 2723.2527 WG-14 2722 2722.2649 2722.2673 WG-15 2756 2756.3125 2756.3158 WG-16 2617 2617.2692 2617.2716 WG-17 2582 2581.2146 2581.2190 WG-18 2630 2629.1973 2629.2004 WG-19 2740 2739.2998 2739.3017 WG-20 2620 2619.2074 2619.2115 WG-21 2760 2760.2832 2760.2773 WG-22 2509 2509.1616 2509.1587 WG-23 2744 2743.3007 2743.2992 WG-24 2692 2692.3125 2692.3083 WG-25 2624 2623.3074 2623.3112

Example 29 Solubility Test

[0126] Series solutions of 1 nM/10 nM/100 nM/100 nM/1 uM/10 uM/100 uM/1 mM were prepared according to following steps: WG compounds to be tested in the present application and corresponding compounds of structure A were respectively weighed, dissolved in deionized water, and oscillated at 37? C. for 12 hours to reach dissolution equilibrium. The resulted solutions were filtered 3 times with a filter membrane, and filtrates were collected. Actual concentrations of the compounds in the solutions were determined with HPLC. A curve of actual concentration of the solutionplanned concentration of the solution was drawn, and the turning point of the curve indicated the equilibrium concentration of the compounds.

Example 30 Transmembrane Test

[0127] Liver cancer cells (HUH-7) were seeded in a 6-well plate at a concentration of 4*10.sup.5 cells/mL, 2 mL per well, and cultured overnight until the cells were adherent to the wall.

[0128] The next day, compound solutions were prepared as follows: WG compounds of present application and corresponding compounds of structure A were respectively weighed, prepared with pure DMSO into mother solutions of high concentration, then gradiently diluted to obtain compound solutions of 10 uM/30 uM/100 uM/150 uM/300 uM in 10% DMSO, and shaken to mix well.

[0129] When the cells were adherent to the wall under microscope observation, 200 uL of compound solutions were added to each well with a ratio of compound solution to culture medium at 1:10.

[0130] After culture for 24 hours, the culture medium was sucked out with a pipette, and the wells were then washed five times with phosphate buffer, 2 mL each time for 30 seconds, to ensure there were no compound residues in the environment of the cells. Solutions in the wells were thoroughly sucked up, and 150 uL RIPA Lysis Buffer was added into each well. Then the culture was continued on ice for 30 min to lyse the cells. Lysates were collected and centrifuged at 100000 rpm for 1 h, and supernatants were collected and characterized with HPLC to determine the concentration of the compounds therein.

[0131] The solubility and changes of transmembrane efficiency of WG compounds relative to corresponding compounds of structure A were analyzed and calculated. An arithmetic mean value of transmembrane efficiency changes under different concentrations was calculated, thereby obtaining a reference value of the difference of transmembrane efficiency between two groups of compounds.

Example 31 Cell Growth Inhibition Test

[0132] Liver cancer cells (HUH-7) were seeded in a 96-well plate at a concentration of 2*10+ cells/mL, 100 uL per well, and cultured overnight.

[0133] The next day, the cells were observed microscope to ensure adherence. Compound solutions were prepared as follows: WG compounds of present application and corresponding compounds of structure A were respectively weighed, prepared with pure DMSO into mother solutions of high concentration, then gradiently diluted to obtain compound solutions of 10 nM/100 nM/300 nM/1000 nM/3000 nM in 10% DMSO, and shaken to mix well.

[0134] Positive drug solutions in the control group were prepared with the same solvent and method, and only a single dosage was required. In this test, the positive drug solution is 1 uM lenvatinib.

[0135] After solution preparation was completed, 10 uL of compound solutions were added into each well of the 96-well plate, three duplicates for each dosage. The plate was gently shaken to allow the cells to repeatedly contact with the compound solutions, and then the plate was placed in an incubator to culture for 72 hours. 72 hours later, the plate was taken out and 10 uL cck-8 reagent was added into each well and shaken gently to mix well. Then the plate was placed in an incubator to culture for two hours. Two hours later, the plate was taken out and analyzed with a microplate reader to measure the absorbance of each well at a wavelength of 450 nm. The inhibition ability of the compounds on the growth of liver cancer cells at the current dosages and time can be calculated according to the formula as below:

Cell growth inhibition rate=[1?(absorbance of experimental group?absorbance of culture medium control group)/(absorbance of blank control group?absorbance of culture medium control group)]?100%.

[0136] A graph can be drawn according to the gradient of concentrations, and the results can be compared directly.

Example 32 Proteolysis Test

[0137] Liver cancer cells (HUH-7) were seeded in a 6-well plate at a concentration of 4*10.sup.5 cells/mL, 2 mL per well, and cultured overnight until the cells were adherent to the wall.

[0138] The next day, compound solutions were prepared as follows: WG compounds of present application and corresponding compounds of structure A were respectively weighed, prepared with pure DMSO into mother solutions of high concentration, then gradiently diluted to obtain compound solutions of 10 nM/30 nM/100 nM/300 nM/1000 nM/3000 nM in 10% DMSO, and shaken to mix well. 200 uL of compound solutions were added to each well with a ratio of compound solution to culture medium at 1:10.

[0139] After culture for 24 hours, the culture medium was sucked out with a pipette, and then the wells were washed 3 times with PBS. Solutions in the wells were thoroughly sucked up, and 150 uL RIPA Lysis Buffer was added into each well. Then the culture was continued on ice for 30 min to lyse the cells.

[0140] Lysates were collected and centrifuged at 16000 rpm for 10 minutes. Supernatants were collected and the total protein content was normalized with a BCA kit to determine target protein contents with Western blot.

[0141] The results were analyzed with ImageJ. Blackness of each strip was measured and the protein was quantified for comparison analysis according to formula: Protein content=blackness value of the experimental group/blackness value of the blank control group.

[0142] Comparative tests of five compounds were carried out according to the method of Example 29 to Example 32:

[0143] WGint4 (003006)/WG-1 (00300639) were discussed as an example, where WGint4 (003006) was the compound of structure A corresponding to WG-1 (00300639).

(1) Solubility (Equilibrium Solubility)

[0144] The solubility was 31 uM for WGint4 (003006) and 677 uM for WG-1 (00300639), indicating the solubility was increased by about 22 times.

(2) Transmembrane Efficiency

[0145] Data in FIG. 5 showed that the transmembrane efficiency of WG-1 (00300639) was about 9.2 times that of WGint4 (003006).

(3) Inhibition Effect on Cancer Cell Growth

[0146] FIG. 6 showed that WG-1 (00300639) inhibited the growth of liver cancer cells significantly stronger than WGint4 (003006). It was calculated that the IC50 was 21.3 nM for WG-1 (00300639) and 210.7 nM for WGint4 (003006).

(4) Proteolysis

[0147] The results were shown in FIG. 7, and the test values were shown in the following table:

TABLE-US-00002 Dosage Compounds Blank 1 nM 10 nM 30 nM 100 nM 300 nM WGint4(003006) 1 0.97 0.97 0.84 0.65 0.22 WG-1(00300639) 1 0.93 0.72 0.31 0.16 0.13

[0148] It was calculated that WG-1 (00300639) DC50=18.0 nM, and WGint4 (003006) DC50=150.2 nM.

[0149] Data for other compounds were listed as follows:

TABLE-US-00003 Effects Transmembrane Inhibition on Compounds Solubility efficiency cell growth Proteolysis WGint4(003006) 31 uM WG-2 is about 8.3 IC50 = 210.7 nM DC50 = 150.2 nM WG- 603 uM times higher than IC50 = 29.5 nM DC50 = 21.4 nM 2(00300629) 003006 WGint4(004006) 19 uM WG-3 is about 15 IC50 = 568.0 nM DC50 = 788.4M WG- 255 uM times higher than IC50 = 79.9 nM DC50 = 81.4 nM 3(00400639) 004006 WGint4(005006) 23 uM WG-4 is about 17.1 IC50 = 255.8 nM DC50 = 219.1 nM WG- 476 uM times higher than IC50 = 15 nM DC50 = 38.1 nM 4(00500639) 005006 WGint4(006006) 40 uM WG-5 is about 11.5 IC50 = 410.1 nM DC50 = 366.2 nM WG- 731 uM times higher than IC50 = 35.3 nM DC50 = 12.0 nM 5(00600639) 006006

[0150] It should be understood that the above examples and technical solutions are only for exemplary illustrations of the present invention, rather than limitations to the present invention. Any changes or alterations derived from the spirit of the present invention are still within the protection scope of the present invention.