Oxadiazole linkers and use thereof

Abstract

The present invention is directed to oxadiazole linkers and use thereof, more specifically to the compounds represented by formula I, II, and III, and their use in the preparation of antibody-drug conjugates (ADCs). The ADCs obtained from said oxadiazole linkers have high homogeneity and stability, and could be used effectively for the treatment of various diseases including tumors. The definition of the groups in formula I, II, and III is the same as that in the description. ##STR00001##

Claims

1. A compound of formula I, ##STR00074## and pharmaceutically acceptable salts thereof, wherein W is selected from N, CR.sub.11 and aryl; X, Y and Z are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; J is selected from —COOH, —OH and —NHR.sub.18; a, b, c, x, y and z are each independently selected from 0 and 1; R.sub.1, R.sub.2 and R.sub.3 are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are each independently selected from H and C.sub.1-C.sub.8 alkyl.

2. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: W is selected from N, CH and C.sub.6-C.sub.10 aryl.

3. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: a, b, x and y are 0; or a is 0, b is 1, while x and y are 0; or a is 1, b is 0, while x and y are 0; or a and b are 1, while x and y are 0; or a and b are 1, while x and y are 1.

4. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: c is 0 or 1.

5. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: R.sub.1 and R.sub.2 are each independently selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

6. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: R.sub.3 is selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone.

7. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: Z is selected from O, C(═O), C(═O)NR.sub.12 and NR.sub.13C(═O), wherein R.sub.12 and R.sub.13 are as defined in claim 1.

8. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein: J is —COOH.

9. The compound of formula I according to claim 1 and pharmaceutically acceptable salts thereof, wherein the compound is selected from the group consisting of compounds 1-9: ##STR00075## ##STR00076##

10. A compound of formula II, ##STR00077## and pharmaceutically acceptable salts thereof, wherein U is selected from C and aryl; X, Y, P and Z are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; J is selected from —COOH, —OH and —NHR.sub.18; l, m, n, r, x, y and z are each independently selected from 0 and 1; R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are each independently selected from H and C.sub.1-C.sub.8 alkyl.

11. The compound of formula II according to claim 10 and pharmaceutically acceptable salts thereof, wherein: U is C.sub.6-C.sub.10 aryl.

12. The compound of formula II according to claim 10 and pharmaceutically acceptable salts thereof, wherein: X, Y and P are each independently selected from O, C(═O), C(═O)NR.sub.12 and NR.sub.13C(═O), wherein R.sub.12 and R.sub.13 are defined as in claim 10.

13. The compound of formula II according to claim 10 and pharmaceutically acceptable salts thereof, wherein: Z is selected from O, C(═O), C(═O)NR.sub.12 and NR.sub.13C(═O), wherein R.sub.12 and R.sub.13 are as defined in claim 10.

14. The compound of formula II according to claim 10 and pharmaceutically acceptable salts thereof, wherein: R.sub.4, R.sub.5 and R.sub.6 are each independently selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

15. The compound of formula II according to claim 10, wherein: R.sub.7 is selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

16. The compound of formula II according to claim 10, wherein: J is —COOH.

17. The compound of formula II according to claim 10 and pharmaceutically acceptable salts thereof, wherein the compound is: ##STR00078##

18. A compound of formula III, ##STR00079## and pharmaceutically acceptable salts thereof, wherein T is selected from N, CR.sub.11 and aryl; X, Y, Z and K are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; Q and V are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O) and OC(═O); a, b, c, d, i, j, k, o, q, v, x, y and z are each independently selected from 0 and 1; R.sub.1, R.sub.2, R.sub.3, R.sub.8, R.sub.9 and R.sub.10 are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16 and R.sub.17 are each independently selected from H and C.sub.1-C.sub.8 alkyl.

19. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: T is selected from N and C.sub.6-C.sub.10 aryl.

20. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: R.sub.8 and R.sub.9 are each independently selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

21. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: R.sub.10 is selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone.

22. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: Q and V are each independently selected from C(═O)NR.sub.12, NR.sub.13C(═O) and OC(═O), wherein R.sub.12 and R.sub.13 are as defined in claim 18.

23. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: K is selected from O, C(═O), C(═O)NR.sub.12 and NR.sub.13C(═O), wherein R.sub.12 and R.sub.13 are as defined in claim 18.

24. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: R.sub.3 is selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

25. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: R.sub.1 and R.sub.2 are each independently selected from C.sub.1-C.sub.4 alkylene and C.sub.1-C.sub.4 alkylene containing O in the backbone.

26. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein: J is —COOH.

27. The compound of formula III according to claim 18 and pharmaceutically acceptable salts thereof, wherein the compound is selected from: ##STR00080##

28. A compound of formula IV,
B-A-D  IV wherein B is a compound of formula I according to claim 1, A is optionally other linker; and D is a drug molecule; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D.

29. An antibody-drug conjugate of formula V,
L-(B-A-D).sub.n  V wherein L is an antibody or antibody fragment; B is a compound of formula I according to claim 1; A is optionally other linker; D is a drug molecule; and n is an integer of 1 to 8; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D, and is linked to L by reaction between the cysteines or other amino acid residues of L and 1,3,4-oxadiazole groups.

30. A compound of formula IV according to claim 28, wherein A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker.

31. A compound of formula IV according to claim 28, wherein A has a formula of C-E.sub.e-F.sub.f or G.sub.g: wherein C is a cleavable linker; E and F are self-immolative linkers; e and f are each independently selected from an integer of 0 to 5; G is a noncleavable linker; and g is an integer of 0 to 5.

32. An antibody-drug conjugate of formula V according to claim 29, wherein said antibody-drug conjugate has the formula VI ##STR00081## wherein L is an antibody or antibody fragment; A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker; D is a drug molecule; W is selected from N, CR.sub.11 and aryl X, Y, and Z are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; a, b, c, n, x, y and z are each independently selected from 0 and 1; R.sub.1, R.sub.2, and R.sub.3, are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.1, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16 and R.sub.17 are each independently selected from H and C.sub.1-C.sub.8 alkyl, preferably C.sub.1-C.sub.4 alkyl.

33. An antibody-drug conjugate of formula V according to claim 29, wherein the antibody targets cell surface receptors or tumor-related antigens.

34. An antibody-drug conjugate of formula V according to claim 29, wherein the antibody is IgG1.

35. A compound of formula IV according to claim 28, wherein the drug is a cytotoxic drug, an anti-autoimmune disease drug, or an anti-inflammation drug.

36. A pharmaceutical composition comprising an antibody-drug conjugate of formula V according to claim 29 and a pharmaceutically acceptable carrier.

37. A method of treating a cancer, autoimmune disease or inflammatory disease, comprising administering to a patient in need thereof an effective amount of an antibody-drug conjugate of formula V according to claim 29.

38. A compound of formula IV,
B-A-D  IV wherein B is a compound of formula II according claim 10; A is optionally other linker; and D is a drug molecule; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D.

39. An antibody-drug conjugate of formula V,
L-(B-A-D).sub.n  V wherein L is an antibody or antibody fragment; B is a compound of formula I according to claim 10; A is optionally other linker; D is a drug molecule; and n is an integer of 1 to 8; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D, and is linked to L by reaction between the cysteines or other amino acid residues of L and 1,3,4-oxadiazole groups.

40. A compound of formula IV according to claim 38, wherein A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker.

41. A compound of formula IV according to claim 38, wherein A has a formula of C-E.sub.e-F.sub.f or G.sub.g: wherein C is a cleavable linker; E and F are self-immolative linkers; e and f are each independently selected from an integer of 0 to 5; G is a noncleavable linker; and g is an integer of 0 to 5.

42. An antibody-drug conjugate of formula V according to claim 39, wherein said antibody-drug conjugate has the formula VII: ##STR00082## wherein L is an antibody or antibody fragment; A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker; D is a drug molecule; U is selected from C and aryl; X, Y, P, and Z are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; l, m, n, p, r, x, y and z are each independently selected from 0 and 1; R.sub.4, R.sub.5, and R.sub.6, are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16 and R.sub.17 are each independently selected from H and C.sub.1-C.sub.8 alkyl, preferably C.sub.1-C.sub.4 alkyl.

43. An antibody-drug conjugate of formula V according to claim 39, wherein the antibody targets cell surface receptors or tumor-related antigens.

44. An antibody-drug conjugate of formula V according to claim 39, wherein the antibody is IgG1.

45. A compound of formula IV according to claim 38, wherein the drug is a cytotoxic drug, an anti-autoimmune disease drug, or an anti-inflammation drug.

46. A pharmaceutical composition comprising an antibody-drug conjugate of formula V according to claim 39 and a pharmaceutically acceptable carrier.

47. A method of treating a cancer, autoimmune disease or inflammatory disease, comprising administering to a patient in need thereof an effective amount of an antibody-drug conjugate of formula V according to claim 39.

48. A compound of formula IV,
B-A-D  IV wherein B is a compound of formula III according to claim 18; A is optionally other linker; and D is a drug molecule; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D.

49. An antibody-drug conjugate of formula V,
L-(B-A-D).sub.n  V wherein L is an antibody or antibody fragment; B is a compound of formula III according to claim 18; A is optionally other linker; D is a drug molecule; and n is an integer of 1 to 8; wherein B is linked to A or D by reaction between a terminal J group of B and a terminal group of A or D, and is linked to L by reaction between the cysteines or other amino acid residues of L and 1,3,4-oxadiazole groups.

50. A compound of formula IV according to claim 48, wherein A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker.

51. A compound of formula IV according to claim 48, wherein A has a formula of C-E.sub.e-F.sub.f or G.sub.g: wherein C is a cleavable linker; E and F are self-immolative linkers; e and f are each independently selected from an integer of 0 to 5; G is a noncleavable linker; and g is an integer of 0 to 5.

52. An antibody-drug conjugate of formula V according to claim 49, wherein said antibody-drug conjugate has the formula VIII: ##STR00083## wherein L is an antibody or antibody fragment; A is optionally a cleavable or noncleavable linker other than a 1,3,4-oxadiazole linker; D is a drug molecule; W is selected from N, CR.sub.11 and aryl; T is selected from N, CR.sub.12 and aryl; X, Y, Z and K are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O), NR.sub.14C(═O)NR.sub.15, NR.sub.16C(═O)O and OC(═O)NR.sub.17; Q and V are each independently selected from O, C(═O), C(═O)NR.sub.12, NR.sub.13C(═O) and OC(═O); a, b, c, d, i, j, k, n, o, q, v, x, y and z are each independently selected from 0 and 1; R.sub.1, R.sub.2, R.sub.3, R.sub.8, R.sub.9 and R.sub.10 are each independently selected from C.sub.1-C.sub.8 alkylene and C.sub.1-C.sub.8 alkylene containing O in the backbone; R.sub.11, R.sub.12, R.sub.13, R.sub.14, R.sub.15, R.sub.16 and R.sub.17 are each independently selected from H and C.sub.1-C.sub.8 alkyl, preferably C.sub.1-C.sub.4 alkyl.

53. An antibody-drug conjugate of formula V according to claim 49, wherein the antibody targets cell surface receptors or tumor-related antigens.

54. An antibody-drug conjugate of formula V according to claim 49, wherein the antibody is IgG1.

55. A compound of formula IV according to claim 28, wherein the drug is a cytotoxic drug, an anti-autoimmune disease drug, or an anti-inflammation drug.

56. A pharmaceutical composition comprising an antibody-drug conjugate of formula V according to claim 49 and a pharmaceutically acceptable carrier.

57. A method of treating a cancer, autoimmune disease or inflammatory disease, comprising administering to a patient in need thereof an effective amount of an antibody-drug conjugate of formula V according to claim 49.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the native MS spectrum of H-1-vcMMAE of the invention.

(2) FIGS. 2a-2b illustrate the SDS-PAGE results of the antibody-drug conjugates based on oxadiazole linkers, wherein 2a represents the SDS-PAGE result of H-1-vcMMAE to H-10-vcMMAE (corresponding to 1-10 respectively); 2b represents the SDS-PAGE result of H-11-vcMMAE to H-12-vcMMAE (corresponding to 11-12 respectively).

(3) FIGS. 3a-3l illustrate the HIC results of the antibody-drug conjugates, wherein 3a-3l correspond to H-1-vcMMAE to H-12-vcMMAE, respectively.

EXAMPLES

(4) The present invention will be further described in details with the following examples. However, it should be understood that these examples are used to illustrate the present invention, but should not be considered as limiting the scope of the invention. The unstated experiment conditions are generally according to routine conditions or conditions suggested by manufacturers. All reactions were conducted under nitrogen atmosphere, except for hydrogenation reaction.

(5) Unless otherwise defined, all of the professional and scientific terms used in the present invention have the same meaning as those familiar by the expertise in the art. Furthermore, any method or material similar or equal to those used in the present invention can be applied herein. The optimized methods and materials used in the present invention are only used for illustration while not for limitation.

ABBREVIATION

(6) Ab antibody Ac acetyl ACN acetonitrile BOC (Boc) tert-butoxycarbonyl t-Bu tert-butyl DCM dichloromethane DIEA diisopropylethylamine DMF N,N-dimethylformamide ELISA enzyme linked immunosorbent assay Et ethyl EA (EtOAc) ethyl acetate Eq equivalent g gram h hour HOSu N-hydroxy succinimide HIC hydrophobic interaction chromatography HPLC high performance liquid chromatography LC-MS liquid chromatography-mass spectrum MeOH methanol mAb monoclonal antibody Me methyl min minute ML milliliter MS mass spectrometry nm nanometer μg microgram μL microliter PE petroleum ether prep-RP-HPLC preparative-reverse phase-high performance liquid chromatography rt room temperature R.sub.t retention time SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC size exclusion chromatography TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TsCl p-tolyl chloride

(7) Unless otherwise stated, all of the anhydrous solvents are purchased from the suppliers and kept under nitrogen atmosphere. All other reagents and solvents purchased are of high purity and need not to be purified before use.

(8) .sup.1H NMR spectrum was collected on a Bruker Avance III 500 MHz instrument. Chemical shift (b) unit is ppm, and the reference reagent is TMS (δ=0).

(9) For LC-MS, low resolution mass spectrum was collected on Agilent 6110 (acid method) or 6120B (base method) mass spectrometers coupled with Hewlett-Packard Agilent 1200 HPLC.

(10) Method 1: Waters Sunfire C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.4 min. The flow rate is 2.3 mL/min, and the column temperature is 50° C.

(11) Method 2: Waters Sunfire C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.4 min. The flow rate is 2.0 mL/min, and the column temperature is 50° C.

(12) Method 3: Waters Sunfire C18 reverse phase column (3.0×30 mm, 2.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.5 min. The flow rate is 1.5 mL/min, and the column temperature is 50° C.

(13) Method 4: Waters Sunfire C18 reverse phase column (4.6×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.2 min. The flow rate is 2.0 mL/min, and the column temperature is 50° C.;

(14) Method 5: Waters Xbridge C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the base HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile) in A (water, containing 10 mM ammonium bicarbonate) over 1.5 min. The flow rate is 2.0 mL/min, and the column temperature is 40° C.

(15) Purification by preparative HPLC is conducted on a Gilson instrument. Waters Sunfire C18 reverse phase column (250×19 mm, 10 μm) is used for separation.

(16) Method 6: The acid HPLC preparation method. Mobile phase: A is aqueous solution containing 0.1% TFA; B is ACN. The flow rate is 20 mL/min.

(17) SK-BR-3 human breast cancer cell is purchased from ATCC. Her2 antigen is purchased from Sino Biological Inc (Beijing). Antibody H (Herceptin Biosimilar, IgG1) is purchased from Genor Biopharma Co. Ltd. (Shanghai). The enzyme labeled anti-antibody is purchased from Sigma (Shanghai). Substrate solution is purchased from Decent Biotech (Shanghai). Cell Counting Kit (CCK-8) cell proliferation-cytotoxicity assay kit is purchased from Dojindo (Shanghai).

PREPARATION EXAMPLES

(18) Synthesis of Key Intermediates (KIs)

Preparation Example 1

Synthesis of 2-(chloromethyl)-5-(methylthio)-1,3,4-oxadiazole (KI-1)

(19) ##STR00045## ##STR00046##

Step 1: Synthesis of (5-mercapto-1,3,4-oxadiazol-2-yl)methanol (14)

(20) 2-Hydroxyacetohydrazide (13, 9.99 g) was dissolved in MeOH (100 mL), to which carbon disulfide (16.87 g, 0.22 mol) and potassium hydroxide (85%, 14.63 g, 0.22 mol) were sequentially added. The reaction mixture was stirred at rt for 1 h, and then refluxed for 3 h. After cooling down, the mixture was concentrated under reduced pressure to give yellow solid-liquid residue. MeOH was added to the residue, to which concentrated hydrochloric acid was dropwise added to adjust pH to 3. The mixture was concentrated under reduced pressure to give a yellow solid, which was further purified by silica gel chromatography (PE/EA 2:1) to give compound 14 (1.50 g) as yellow oil.

Step 2: Synthesis of (5-(methylthio)-1,3,4-oxadiazol-2-yl)methanol (15)

(21) Compound 14 (1.50 g, 11.36 mmol) was dissolved in THF (20 mL), and the solution was cooled to 0° C. with an ice-water bath, to which iodomethane (2.42 g, 17.04 mmol) and TEA (1.72 g, 17.04 mmol) were added sequentially. The reaction mixture was stirred at rt for 2 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 15 (900 mg) as yellow oil.

Step 3: Synthesis of 2-(chloromethyl)-5-(methylthio)-1,3,4-oxadiazole (KI-1)

(22) Compound 15 (900 mg, 6.16 mmol) was dissolved in DCM (10 mL), and the solution was cooled to 0° C., to which TsCl (2.35 g, 12.32 mmol) and TEA (1.24 g, 12.32 mmol) were sequentially added. The reaction mixture was stirred at rt for 2 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (PE/EA 10:1) to give compound KI-1(520 mg) as yellow oil.

(23) LC-MS (method 1): R.sub.t=1.56 min; m/z (ES.sup.+)=165.1 (M+H).sup.+.

(24) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 4.68 (s, 2H), 2.74 (s, 3H).

Preparation Example 2

Synthesis of 2-(chloromethyl)-5-(methylthio)-1,3,4-oxadiazole (KI-2)

(25) ##STR00047## ##STR00048##

Step 1: Synthesis of Potassium Hydrazinecarbodithioate (16)

(26) Aqueous hydrazine solution (80%, 8.0 g, 0.20 mol) was dissolved in ethanol (50 mL), to which carbon disulfide (15.2 g, 0.2 mol) and potassium hydroxide (85%, 13.18 g, 200 mmol) were sequentially added. The reaction mixture was stirred at rt for 1 h, and then concentrated under reduced pressure to remove the solvent. MeOH (50 mL) was added to the crude product, and the white solid was collected by filtration, washed, and dried to give compound 16 (19.5 g).

Step 2: Synthesis of Potassium 2-(3-ethoxy-3-oxopropanoyl)hydrazinecarbodithioate (17)

(27) Compound 16 (21.02 g, 144 mmol) was dissolved in THF/H.sub.2O (40 mL, v/v 1/1), and the solution was cooled to 5° C., to which ethyl 3-chloro-3-oxopropanoate (21.67 g, 144 mmol) was added dropwise in 30 min, followed by dropwise addition of aqueous potassium hydrocarbonate solution (14.40 g in 20 mL water) in 30 min. After the addition, the reaction mixture was stirred at rt for 12 h, and then concentrated under reduced pressure to remove the solvent to give the crude product 17 (16.5 g), which was used for next step without further purification.

Step 3: Synthesis of ethyl 2-(5-mercapto-1,3,4-oxadiazol-2-yl)acetate (18)

(28) Compound 17 (16.5 g) was dissolved in ethanol (100 mL), and the solution was heated to reflux for 1.5 h. After cooling down, the mixture was concentrated to remove the solvent. EA (200 mL) and brine (50 mL) were added to the residue, to which concentrated hydrochloric acid was added dropwise to adjust pH to 3. The aqueous phase was extracted by EA (200 mL×3). The combined organic phase was sequentially washed with brine, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 18 (8.5 g) as yellow oil.

(29) LC-MS (method 1): R.sub.t=1.38 min; m/z (ES.sup.+)=188.9 (M+H).sup.+.

(30) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 4.26-4.22 (m, 2H), 3.45 (s, 2H), 1.30 (t, 3H).

Step 4: Synthesis of ethyl 2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)acetate (KI-2)

(31) Compound 18 (2.50 g, 13.3 mmol) was dissolved in THF (30 mL), and the solution was cooled to 0° C., to which iodomethane (2.83 g, 19.95 mmol) and TEA (2.01 g, 19.95 mmol) were added sequentially. The reaction mixture was stirred at rt for 30 min, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound KI-2 (2.44 g) as yellow oil.

(32) LC-MS (method 1): R.sub.t=1.55 min; m/z (ES.sup.+)=203.1 (M+H).sup.+.

(33) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 4.22 (q, 2H), 3.92 (s, 2H), 2.71 (s, 3H), 1.27 (t, 3H).

Preparation Example 3

Synthesis of 2-(methylthio)-5-vinyl-1,3,4-oxadiazole (KI-3)

(34) ##STR00049##

Step 1: Synthesis of 3-hydroxypropanehydrazide (20)

(35) Methyl 3-hydroxypropanoate (19) was dissolved in methanol (200 mL), to which aqueous hydrazine solution (80%, 12.0 g, 192 mmol) was added. The reaction mixture was heated to reflux for 4 h, and then concentrated under reduced pressure to remove the solvent. The crude product 20 (15.3 g) was used directly for next step without further purification.

Step 2: Synthesis of 2-(5-mercapto-1,3,4-oxadiazol-2-yl)ethanol (21)

(36) Compound 20 (9.50 g, 91.3 mmol) was dissolved in MeOH (100 mL), to which carbon disulfide (13.88 g, 183 mmol) and potassium hydroxide (85%, 12.03 g, 183 mmol) were sequentially added. The reaction mixture was stirred at rt for 2 h, and then heated to reflux for 3 h. The mixture was concentrated under reduced pressure to give yellow crude product, to which MeOH (30 mL) was added and concentrated hydrochloric acid was added to adjust pH to 2˜3. The solution was concentrated under reduced pressure to give a yellow crude, which was further purified by silica gel chromatography (PE/EA 1:1) to give compound 21 (9.60 g) as a yellow solid.

(37) LC-MS (method 1): R.sub.t=0.93 min; m/z (ES.sup.+)=147.1 (M+H).sup.+.

Step 3: Synthesis of 2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethanol (22)

(38) Compound 21 (9.60 g, 65.8 mmol) was dissolved in THF (100 mL), and the solution was cooled to 0° C., to which iodomethane (14.0 g, 98.6 mmol) and TEA (9.96 g, 98.63 mmol) were added sequentially. The reaction mixture was stirred at rt for 2 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 22 (3.2 g) as yellow oil.

(39) LC-MS (method 1): R.sub.t=1.24 min; m/z (ES.sup.+)=161.0 (M+H).sup.+.

Step 4: Synthesis of 2-(methylthio)-5-vinyl-1,3,4-oxadiazole (KI-3)

(40) Compound 22 (3.20 g, 20.0 mmol) was dissolved in DCM (50 mL), and the solution was cooled to 0° C., to which TsCl (7.63 g, 40.0 mmol) and TEA (6.06 g, 60 mmol) were sequentially added. The reaction mixture was stirred at rt for 2 d, and then concentrated to give a residue. Further purification by silica gel chromatography (PE/EA 10:1) gave the compound KI-3 (1.90 g) as a yellow solid.

(41) LC-MS (method 1): R.sub.t=1.34 min; m/z (ES.sup.+)=143.1 (M+H).sup.+.

(42) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 6.79 (dd, 1H), 6.19 (d, 1H), 5.89 (d, 1H), 2.73 (s, 3H).

Example 1

Synthesis of 3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanoic Acid (Linker 1)

(43) ##STR00050## ##STR00051##

Step 1: Synthesis of ethyl 2,3-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)propanoate (23)

(44) KI-2 (51 mg, 0.25 mmol) was dissolved in anhydrous THF (5 mL), to which sodium hydride (60% dispersion in mineral oil, 20 mg, 0.50 mmol) was added. The reaction mixture was stirred at rt for 30 min, and then KI-1(41 mg, 0.25 mmol) was added, followed by stirring at 35° C. for 3 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (PE/EA 3:2) to give compound 23 (35 mg) as colorless oil.

(45) LC-MS (method 1): R.sub.t=1.68 min; m/z (ES.sup.+)=331.1 (M+H).sup.+.

(46) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 4.53 (t, 1H), 4.21-4.14 (m, 2H), 3.66-3.61 (m, 1H), 3.53-3.48 (m, 1H), 2.65 (s, 3H), 2.62 (s, 3H), 1.20 (t, 3H).

Step 2: Synthesis of 4-tert-butyl 1-ethyl 2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)-2-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)succinate (24)

(47) Compound 23 (50 mg, 0.15 mmol) was dissolved in THF (3 mL), to which sodium hydride (12 mg, 0.30 mmol) was added. The reaction mixture was stirred at rt for 30 min, to which tert-butyl bromoacetate (49 μL, 0.30 mmol) was added, followed by stirring at rt for 3 h. Hydrochloric acid (0.5 M) was added to quench the reaction, and the mixture was extracted by EA. The organic phase was washed with water, dried, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 5:1 to 3:1) to give compound 24 (40 mg) as colorless oil.

(48) LC-MS (method 1): R.sub.t=1.98 min; m/z (ES.sup.+)=115.1 (M+H).sup.+.

(49) .sup.1H NMR (500 MHz, CDCl.sub.3) δ4.28-4.21 (m, 2H), 3.91-3.84 (d, 2H), 3.35 (d, 1H), 3.21 (d, 1H), 2.71 (s, 3H), 2.67 (s, 3H), 1.42 (s, 9H), 1.25 (t, 3H).

Step 3: Synthesis of tert-butyl 3,4-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)butanoate (25)

(50) Compound 24 (40 mg, 90 μmol) was dissolved in THF (0.2 mL), to which aqueous lithium hydroxide solution (1.0 M, 180 μL, 180 μmol) was added. The reaction mixture was stirred at rt for 2 h, to which concentrated hydrochloric acid (38 μL, 450 mmol) was added, followed by stirring at rt for 16 h. The mixture was extracted by EA, and the organic phase was washed with water, dried, and concentrated under reduced pressure sequentially. The residue was purified by silica gel chromatography (PE/EA 5:1 to 3:1) to give compound 25 (26 mg) as colorless oil.

(51) LC-MS (method 1): R.sub.t=1.85 min; m/z (ES.sup.+)=373.1 (M+H).sup.+.

(52) .sup.1H NMR (500 MHz, CDCl.sub.3) δ3.94-3.91 (m, 1H), 3.42-3.26 (m, 2H), 2.90-2.74 (m, 2H), 2.68 (s, 3H), 2.67 (s, 3H), 1.40 (s, 9H).

Step 4: Synthesis of 3,4-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)butanoic Acid (26)

(53) Compound 25 (82 mg, 0.22 mmol) was dissolved in DCM (2 mL), to which TFA (1 mL) was added. The reaction mixture was stirred at rt for 3 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 26 (21 mg).

(54) LC-MS (method 1): R.sub.t=1.46 min; m/z (ES.sup.+)=316.9 (M+H).sup.+.

(55) .sup.1H NMR (500 MHz, CDCl.sub.3) δ7.63 (br s, 1H), 3.99-3.96 (m, 1H), 3.47-3.34 (m, 2H), 3.08-2.90 (m, 2H), 2.68 (s, 3H), 2.67 (s, 3H).

Step 5: Synthesis of 3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanoic Acid (1)

(56) Compound 26 (21 mg, 0.066 mmol) was dissolved in DCM (2 mL), to which m-chloroperbenzoic acid (135 mg, 0.66 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 1 (14 mg) as a white solid.

(57) LC-MS (method 1): R.sub.t=1.40 min; m/z (ES.sup.+)=380.8 (M+H).sup.+.

(58) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ12.66 (br s, 1H), 4.11-4.08 (m, 1H), 3.67-3.55 (m, 8H), 3.03-2.98 (m, 2H).

Example 2

Synthesis of 6-(2,3-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl) propanamido)hexanoic Acid (Linker 2)

(59) ##STR00052## ##STR00053##

Step 1: Synthesis of Lithium 2,3-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)propanoate (27)

(60) Compound 23 (83 mg, 0.25 mmol) was dissolved in THF (6 mL), to which aqueous lithium hydroxide solution (5.6 mL, 1.9 mg/mL. 0.44 mmol) was added. The reaction mixture was stirred at rt for 2 h, and then lyophilized to give crude product 27 (77 mg), which was used for next step without further purification.

Step 2: Synthesis of tert-butyl 6-(2,3-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl) propanamido)hexanoate (28)

(61) Compound 27 (77 mg), tert-butyl 6-aminohexanoate (94 mg, 0.50 mmol, prepared according to US2010/0261736) and HATU (190 mg, 0.50 mmol) were dissolved in DMF (3 mL), to which DIEA (88 μL, 0.50 mmol) was added. The reaction mixture was stirred at rt for 4 h, and then EA was added. The organic phase was washed with water, dried, filtered, and concentrated under reduced pressure sequentially. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 28 (90 mg) as colorless oil.

(62) LC-MS (method 1): R.sub.t=1.88 min; m/z (ES.sup.+)=472.1 (M+H).sup.+.

Step 3: Synthesis of 6-(2,3-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)propanamido) Hexanoic Acid (29)

(63) Compound 28 (40 mg, 80 μmol) was dissolved in DCM (5 mL), to which TFA (2 mL) was added. The reaction mixture was stirred at rt for 2 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (DCM/MeOH 20:1) to give compound 29 (15 mg) as a yellow solid.

(64) LC-MS (method 1): R.sub.t=1.51 min; m/z (ES.sup.+)=416.1 (M+H).sup.+.

(65) .sup.1H NMR (500 MHz, CDCl.sub.3) δ7.07 (br s, 1H), 4.53 (t, 1H), δ 3.75-3.72 (m, 1H), 3.56-3.52 (m, 1H), 3.33-3.21 (m, 2H), 2.70 (s, 3H), 2.68 (s, 3H), 2.35 (t, 2H), 1.66-1.60 (m, 2H), 1.55-1.50 (m, 2H), 1.38-1.25 (m, 2H).

Step 4: Synthesis of 6-(2,3-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)propanamido) Hexanoic Acid (2)

(66) Compound 29 (10 mg, 25 μmol) was dissolved in DMF (5 mL), to which m-chloroperbenzoic acid (43 mg, 0.25 mmol) was added. The reaction mixture was stirred at rt overnight, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 20%-50% B in 8 min.fwdarw.95% B in 4 min) to give compound 2 (3.5 mg) as a white solid.

(67) LC-MS (method 1): R.sub.t=1.51 min; m/z (ES.sup.+)=480.1 (M+H).sup.+.

(68) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.66 (t, 1H), 4.72 (t, 1H), 3.79-3.77 (m, 2H), 3.69 (s, 3H), 3.65 (s, 3H), 3.10-3.07 (m, 2H), 2.20-2.17 (m, 2H), 1.49-1.46 (m, 2H), 1.42-1.39 (m, 2H), 1.06-1.05 (m, 2H).

Example 3

Synthesis of 3-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)propanoic Acid (Linker 3)

(69) ##STR00054##

Step 1: Synthesis of 3-(5-(methylthio)-1,3,4-oxadiazol-2-yl)-2-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl) Propanoic Acid (30)

(70) Dimethyl malonate (81 mg, 0.61 mmol) was dissolved in THF (5 mL), to which sodium hydride (60% dispersion in mineral oil, 73 mg, 1.82 mmol) was added. The reaction mixture was stirred at rt for 30 min, followed by sequential addition of KI-1 (200 mg, 1.21 mmol) and potassium iodide (201 mg, 1.21 mmol). The reaction mixture was continuously stirred at rt for 1 h, and then aqueous sodium hydroxide solution (1.0 M, 2 mL, 2.0 mmol) was added, followed by continuous stirring at rt for 1 h. Concentrated hydrochloric acid was added to adjust the pH of the solution to 2˜3, and then the mixture was extracted by EA. The organic phase was sequentially washed with brine, dried, filtered, and concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 40%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 30 (40 mg).

(71) LC-MS (method 1): R.sub.t=1.48 min; m/z (ES.sup.+)=317.0 (M+H).sup.+.

(72) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.95 (br s, 1H), 3.54-3.51 (m, 1H), 3.40-3.36 (m, 2H), 3.25-3.20 (m, 2H), 2.68 (s, 6H).

Step 2: Synthesis of 3-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)propanoic Acid (3)

(73) Compound 30 (40 mg, 0.13 mmol) was dissolved in acetic acid (2 mL), to which potassium permanganate (50 mg, 0.32 mmol) was added. The reaction mixture was stirred at rt for 2 h, and then filtered to remove the solid. The filtrate was diluted by EA, and the organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 3 (12 mg) as a white solid.

(74) LC-MS (method 1): R.sub.t=1.42 min; m/z (ES.sup.+)=381.0 (M+H).sup.+.

(75) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ3.65 (s, 6H), 3.54-3.52 (m, 1H), 3.47-3.35 (m, 4H).

Example 4

Synthesis of 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)butanoic Acid (Linker 4)

(76) ##STR00055## ##STR00056##

Step 1: Synthesis of dimethyl 2-(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)malonate (31)

(77) Dimethyl malonate (98 mg, 0.75 mmol) was dissolved in THF (5 mL), to which sodium hydride (60% dispersion in mineral oil, 50 mg, 1.25 mmol) was added. The reaction mixture was stirred at rt for 30 min, followed by the addition of KI-3 (177 mg, 1.25 mmol) and continuous stirring for 4 h. The mixture was concentrated under reduced pressure to remove the solvent, and the residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 31 (90 mg) as colorless oil.

(78) LC-MS (method 1): R.sub.t=1.43 min; m/z (ES.sup.+)=275.0 (M+H).sup.+.

(79) .sup.1H NMR (500 MHz, CDCl.sub.3) δ3.76 (s, 6H), 3.56 (t, 1H), 2.93 (t, 2H), 2.71 (s, 3H), 2.39 (q, 2H).

Step 2: Synthesis of dimethyl 2-(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)-2-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)malonate (32)

(80) Compound 31 (90 mg, 0.33 mmol) was dissolved in THF (2 mL), to which sodium hydride (60% dispersion in mineral oil, 20 mg, 0.50 mmol) was added. The reaction mixture was stirred at rt for 30 min, followed by the addition of KI-1 (65 mg, 0.40 mmol) and continuous stirring for 3 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, and the residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 32 (25 mg) as colorless oil.

(81) LC-MS (method 1): R.sub.t=1.53 min; m/z (ES.sup.+)=402.9 (M+H).sup.+.

(82) .sup.1H NMR (500 MHz, CDCl.sub.3) δ3.77 (s, 6H), 3.53 (s, 2H), 2.91 (t, 2H), 2.68 (s, 6H), 2.43 (t, 2H).

Step 3: Synthesis of 4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)-2-((5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl) butanoic Acid (33)

(83) Compound 32 (25 mg, 62 μmol) was dissolved in DMF (2 mL), to which aqueous lithium hydroxide (1 M, 0.62 mL, 0.62 mmol) was added. The reaction mixture was stirred at rt for 48 h, followed by the addition of concentrated hydrochloric acid to adjust pH to 2˜3. The mixture was extracted by EA, and the organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure to give compound 33 (12 mg) as colorless oil.

(84) LC-MS (method 1): R.sub.t=1.34 min; m/z (ES.sup.+)=330.9 (M+H).sup.+.

(85) .sup.1H NMR (500 MHz, CDCl.sub.3) δ3.29-3.25 (m, 1H), 3.10-2.98 (m, 4H), 2.69 (s, 6H), 2.25-2.12 (m, 2H).

Step 4: 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)butanoic Acid (4)

(86) Compound 33 (12 mg, 36 μmol) was dissolved in DCM (3 mL), to which m-chloroperbenzoic acid (74 mg, 0.36 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 4 (5 mg) as a white solid.

(87) LC-MS (method 1): R.sub.t=1.69 min; m/z (ES.sup.+)=394.9 (M+H).sup.+.

(88) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ3.65 (s, 3H), 3.63 (s, 3H), 3.38-3.26 (m, 2H), 3.15-3.07 (3H), 2.19-2.11 (m, 2H).

Example 5

Synthesis of 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-(2-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)ethyl)butanoic Acid (Linker 5)

(89) ##STR00057## ##STR00058##

Step 1: Synthesis of dimethyl 2,2-bis(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)malonate (34)

(90) Dimethyl malonate (46 mg, 0.35 mmol) was dissolved in THF (5 mL), to which sodium hydride (60% dispersion in mineral oil, 42 mg, 1.05 mmol) was added. The reaction mixture was stirred at rt for 30 min, followed by the addition of KI-3 (100 mg, 0.70 mmol) and continuous stirring for 4 h. The mixture was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 34 (46 mg) as colorless oil.

(91) LC-MS (method 1): R.sub.t=1.72 min; m/z (ES.sup.+)=417.1 (M+H).sup.+.

(92) .sup.1H NMR (500 MHz, CDCl.sub.3) δ3.72 (s, 6H), 2.82 (t, 4H), 2.66 (s, 6H), 2.38 (t, 4H).

Step 2: Synthesis of 2,2-bis(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)malonic Acid (35)

(93) Compound 34 (270 mg, 0.65 mmol) was dissolved in THF (5 mL), to which aqueous lithium hydroxide (2 M, 3.24 mL, 6.48 mmol) was added. The reaction mixture was stirred at rt for 16 h, to which 1 M hydrochloric acid was added to adjust pH to 2˜3. The mixture was extracted by EA, and the organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure to give compound 35 (200 mg) as a white solid.

(94) LC-MS (method 1): R.sub.t=1.49 min; m/z (ES.sup.+)=389.0 (M+H).sup.+.

(95) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ13.25 (br s, 2H), 2.81 (t, 4H), 2.68 (s, 6H), 2.23 (t, 4H).

Step 3: Synthesis of 4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)-2-(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)butanoic Acid (36)

(96) Compound 35 (100 mg, 0.26 mmol) was dissolved in DMSO (2 mL), and the reaction mixture was stirred at 100° C. for 2 h. Purification by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) gave compound 36 (73 mg) as a white solid.

(97) LC-MS (method 1): R.sub.t=1.38 min; m/z (ES.sup.+)=345.0 (M+H).sup.+.

(98) .sup.1H NMR (500 MHz, CDCl.sub.3) δ8.90 (br s, 1H), 2.98-2.86 (m, 4H), 2.65-2.55 (m, 1H), 2.18-2.10 (m, 2H), 2.04-1.96 (m, 2H).

Step 4: Synthesis of 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)-2-(2-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)ethyl)butanoic Acid (5)

(99) Compound 36 (40 mg, 0.12 mmol) was dissolved in DCM (5 mL), to which m-chloroperbenzoic acid (236 mg, 1.16 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 5 (25 mg) as a white solid.

(100) LC-MS (method 1): R.sub.t=1.29 min; m/z (ES.sup.+)=408.9 (M+H).sup.+.

(101) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ3.64 (s, 6H), 3.04 (t, 4H), 2.62-2.58 (m, 1H), 2.10-1.98 (m, 4H).

Example 6

Synthesis of 5-(bis(2-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)ethyl)amino)-5-oxopentanoic Acid (Linker 6)

(102) ##STR00059##

Step 1: Synthesis of tert-butyl 2-(5-mercapto-1,3,4-oxadiazol-2-yl)ethylcarbamate (38)

(103) tert-Butyl 3-hydrazinyl-3-oxopropylcarbamate (37, 6.2 g, 31 mmol, prepared according to Journal of Medicinal Chemistry, 2008, 51, 4430-4448) was dissolved in MeOH (50 mL), to which carbon disulfide (5.5 mL, 92 mmol) and potassium hydroxide (4.7 g, 71 mmol) were sequentially added. The reaction mixture was stirred at rt for 1 h, and then heated to reflux for 4 h. After cooling down to rt, the mixture was diluted by water, to which concentrated hydrochloric acid was then added to adjust pH to 1˜2. The mixture was extracted by EA, and the organic phase was sequentially dried, filtered, and concentrated under reduced pressure to give compound 38 as pale yellow oil, which was used for next step without further purification.

Step 2: Synthesis of tert-butyl 2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethylcarbamate (39)

(104) Compound 38 was dissolved in THF (30 mL), to which iodomethane (2.85 mL, 47.8 mmol) and TEA (6.38 mL, 47.8 mmol) were added. The reaction mixture was stirred at rt for 30 min, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (PE/EA 3:1) to give compound 39 (2.4 g) as colorless oil.

(105) LC-MS (method 2): R.sub.t=1.64 min; m/z (ES.sup.+)=260.0 (M+H).sup.+.

(106) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 5.08 (br s, 1H), 3.61-3.54 (m, 2H), 3.00 (t, 2H), 2.70 (s, 3H), 1.42 (s, 9H).

Step 3: Synthesis of 2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethanamine (40)

(107) Compound 39 (350 mg, 1.35 mmol) was dissolved in EA (5 mL), to which a solution of hydrogen chloride in EA (2.0 M, 5 mL, 10 mmol) was added. The reaction mixture was stirred at rt for 3 h, and then concentrated under reduced pressure to give compound 40 as a white solid, which was used for next step without further purification.

Step 4: Synthesis of bis(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)amine (41)

(108) Compound 40 was suspended in DMF (5 mL), to which KI-3 (192 mg, 1.35 mmol) and DIEA (0.47 mL, 2.7 mmol) were sequentially added. The reaction mixture was stirred at 80° C. for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 41 (40 mg) as a white solid.

(109) LC-MS (method 5): R.sub.t=1.54 min; m/z (ES.sup.+)=302.2 (M+H).sup.+.

(110) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ2.96-2.91 (m, 4H), 2.91-2.87 (m, 4H), 2.68 (s, 6H).

Step 5: Synthesis of 5-(bis(2-(5-(methylthio)-1,3,4-oxadiazol-2-yl)ethyl)amino)-5-oxopentanoic Acid (42)

(111) Compound 41 (40 mg, 0.13 mmol) was dissolved in DCM (5 mL), to which glutaric anhydride (30 mg, 0.26 mmol) and TEA (37 μL, 0.26 mmol) were sequentially added. The reaction mixture was stirred at rt for 3 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 42 (20 mg) as a white solid.

(112) LC-MS (method 2): R.sub.t=1.41 min; m/z (ES.sup.+)=416.0 (M+H).sup.+.

(113) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ12.0 (br s, 1H), 3.71 (t, 2H), 3.61 (t, 2H), 3.16 (t, 2H), 3.04 (t, 2H), 2.68 (s, 3H), 2.67 (s, 3H), 2.26 (t, 2H), 2.19 (t, 2H), 1.68-1.62 (m, 2H).

Step 6: Synthesis of 5-(bis(2-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)ethyl)amino)-5-oxopentanoic Acid (6)

(114) Compound 42 (20 mg, 48 μmol) was dissolved in DCM (5 mL), to which m-chloroperbenzoic acid (98 mg, 480 μmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 6 (10 mg) as a white solid.

(115) LC-MS (method 2): R.sub.t=1.38 min; m/z (ES.sup.+)=479.9 (M+H).sup.+.

(116) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ3.84 (t, 2H), 3.70 (t, 2H), 3.65 (s, 3H), 3.61 (s, 3H), 3.34 (t, 2H), 3.22 (t, 2H), 2.31 (t, 2H), 2.19 (t, 2H), 1.67-1.61 (m, 2H).

Example 7

Synthesis of 4-(3,5-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenylamino)-4-oxobutanoic Acid (Linker 7)

(117) ##STR00060## ##STR00061## ##STR00062##

Step 1: Synthesis of 5-aminoisophthalohydrazide (43)

(118) Dimethyl 5-aminoisophthalate (1.05 g, 5.0 mmol) and hydrazine monohydrate (1.25 g, 20 mmol) were dissolved in MeOH (10 mL), and the reaction mixture was heated to reflux for 24 h, while white precipitate formed in the meantime. The mixture was cooled to rt, and filtered to remove the solid. The filtrate was concentrated under reduced pressure to give compound 43 (600 mg) as a white solid, which was used for next step without further purification.

Step 2: Synthesis of 5,5′-(5-amino-1,3-phenylene)bis(1,3,4-oxadiazole-2-thiol) (44)

(119) Compound 43 was suspended in ethanol (10 mL), to which carbon disulfide (1.04 mL, 17.2 mmol) and aqueous potassium hydroxide solution (2.3 M, 5 mL, 11.5 mmol) were sequentially added. The reaction mixture was heated to reflux for 16 h, and then cooled to rt, and diluted with water (50 mL). Concentrated hydrochloric acid was added to the solution to adjust pH to 1˜2, while white precipitate formed. The solid was collected by filtration, washed, and dried to give compound 44 (300 mg) as a white solid, which was used for next step without further purification.

Step 3: Synthesis of 3,5-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)aniline (45)

(120) Compound 44 (300 mg, 1.02 mmol) was dissolved in DMF (5 mL), to which potassium hydroxide (135 mg, 2.05 mmol) and iodomethane (191 μL, 3.07 mmol) were sequentially added. The reaction mixture was stirred at rt for 4 h. Water (10 mL) was added to the mixture, and solid was precipitated. The solid was collected by filtration, washed, and dried to give compound 45 (230 mg) as a white solid.

(121) LC-MS (method 1): R.sub.t=1.71 min; m/z (ES.sup.+)=322.0 (M+H).sup.+.

(122) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.56 (s, 1H), 7.37 (d, 2H), 5.97 (s, 2H), 2.78 (s, 6H).

Step 4: Synthesis of 4-(3,5-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenylamino)-4-oxobutanoic Acid (46)

(123) Compound 45 (35 mg, 0.11 mmol) was dissolved in DMF (2 mL), to which 4-dimethyaminopyridine (27 mg, 0.22 mmol) and succinic anhydride (33 mg, 0.33 mmol) were sequentially added. The reaction mixture was stirred at 80° C. for 24 h, and then concentrated to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 46 (8.0 mg) as a white solid.

(124) LC-MS (method 3): R.sub.t=1.09 min; m/z (ES.sup.+)=422.0 (M+H).sup.+.

(125) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ10.54 (s, 1H), 8.42 (d, 2H), 8.05 (s, 1H), 2.79 (s, 6H), 2.62 (t, 2H), 2.55 (t, 2H).

Step 5: Synthesis of 4-(3,5-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenylamino)-4-oxobutanoic acid (7)

(126) Compound 46 (16 mg, 38 μmol) was dissolved in acetic acid (0.5 mL), to which potassium permanganate (15 mg, 95 μmol) was added. The reaction mixture was stirred at rt for 4 h, and then purified by prep-RP-HPLC (method 6: 40%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 7 (3.0 mg) as a white solid.

(127) LC-MS (method 1): R.sub.t=1.58 min; m/z (ES.sup.+)=486.0 (M+H).sup.+.

(128) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ10.71 (s, 1H), 8.65 (d, 2H), 8.33 (t, 1H), 3.73 (s, 6H), 2.65 (t, 2H), 2.57 (t, 2H).

Example 8

Synthesis of 3,4-bis((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (Linker 8)

(129) ##STR00063## ##STR00064##

Step 1: Synthesis of methyl 3,4-bis((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzoate (47)

(130) Methyl 3,4-dihydroxybenzoate (84 mg, 0.50 mmol) was dissolved in DMF (5 mL), to which potassium carbonate (173 mg, 1.25 mmol) and KI-1 (206 mg, 1.25 mmol) were sequentially added. The reaction mixture was stirred at 40° C. for 16 h, and then concentrated hydrochloric acid was added to adjust pH to 2˜3. The mixture was diluted by EA, and then sequentially washed with water, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 47 (125 mg) as a white solid.

(131) LC-MS (method 2): R.sub.t=1.72 min; m/z (ES.sup.+)=424.8 (M+H).sup.+.

(132) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.75-7.71 (m, 2H), 7.11 (d, 1H), 5.32 (s, 2H), 5.28 (s, 2H), 3.89 (s, 3H), 2.73 (s, 3H), 2.72 (s, 3H).

Step 2: Synthesis of 3,4-bis((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (48)

(133) Compound 47 (25 mg, 59 μmol) was dissolved in DMF (2 mL), to which aqueous potassium hydroxide solution (1 M, 0.59 mL, 0.59 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then 1 M hydrochloric acid was added to adjust pH to 2˜3. The mixture was extracted by EA, and organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 48 (8 mg) as a white solid.

(134) LC-MS (method 5): R.sub.t=1.49 min; m/z (ES.sup.+)=411.1 (M+H).sup.+.

(135) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.67 (d, 1H), 7.63 (dd, 1H), 7.27 (d, 1H), 5.47 (s, 2H), 5.43 (s, 2H), 2.71 (s, 3H), 2.70 (s, 3H).

Step 3: Synthesis of 3,4-bis((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (8)

(136) Compound 48 (8 mg, 20 μmol) was dissolved in DCM (3 mL), to which m-chloroperbenzoic acid (40 mg, 0.20 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 8 (3.3 mg) as a white solid.

(137) LC-MS (method 2): R.sub.t=1.52 min; m/z (ES.sup.+)=474.8 (M+H).sup.+.

(138) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.74 (d, 1H), 7.67 (dd, 1H), 7.34 (d, 1H), 5.68 (s, 2H), 5.64 (s, 2H), 3.70 (s, 3H), 3.69 (s, 3H).

Example 9

Synthesis of 3,5-bis((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (Linker 9)

(139) ##STR00065## ##STR00066##

Step 1: Synthesis of methyl 3,5-bis((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzoate (49)

(140) Methyl 3,5-dihydroxybenzoate (168 mg, 1.0 mmol) was dissolved in DMF (2 mL), to which potassium carbonate (414 mg, 3.0 mmol) and KI-1 (411 mg, 2.5 mmol) were sequentially added. The reaction mixture was stirred at 40° C. for 16 h, and then concentrated hydrochloric acid was added to adjust pH to 4˜5. The mixture was diluted by EA, and then sequentially washed with brine, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 1:1) to give compound 49 (400 mg) as a white solid.

(141) LC-MS (method 2): R.sub.t=1.78 min; m/z (ES.sup.+)=424.9 (M+H).sup.+.

(142) .sup.1H NMR (500 MHz, CDCl.sub.3) δ7.35 (d, 2H), 6.87 (t, 1H), 5.24 (s, 4H), 3.92 (s, 3H), 2.74 (s, 6H).

Step 2: Synthesis of 3,5-bis((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (50)

(143) Compound 49 (60 mg, 140 μmol) was dissolved in DMF (2 mL), to which aqueous lithium hydroxide (1.4 M, 0.5 mL, 0.70 mmol) was added. The reaction mixture was stirred at rt for 4 h, and then 1 M hydrochloric acid was added to adjust pH to 4˜5. The mixture was extracted by EA, and the organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 50 (23 mg) as a white solid.

(144) LC-MS (method 4): R.sub.t=1.48 min; m/z (ES.sup.+)=410.8 (M+H).sup.+.

(145) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.25 (d, 2H), 7.04 (t, 1H), 5.45 (s, 4H), 2.72 (s, 6H).

Step 3: Synthesis of 3,5-bis((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzoic Acid (9)

(146) Compound 50 (12 mg, 30 μmol) was dissolved in DCM/DMF (1.9 mL/0.1 mL), to which m-chloroperbenzoic acid (52 mg, 0.30 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 9 (2.0 mg) as a white solid.

(147) LC-MS (method 4): R.sub.t=1.45 min; m/z (ES.sup.+)=474.7 (M+H).sup.+.

(148) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.31 (d, 2H), 7.14 (t, 1H), 5.64 (s, 4H), 3.70 (s, 6H).

Example 10

Synthesis of 6-(2,4,6-tris((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzamido)hexanoic Acid (Linker 10)

(149) ##STR00067## ##STR00068##

Step 1: Synthesis of tert-butyl 6-(2,4,6-trihydroxybenzamido)hexanoate (51)

(150) 2,4,6-Trihydroxybenzoic acid (85 mg, 0.50 mmol), N-hydroxysuccinimde (58 mg, 0.50 mmol), and N,N′-Dicyclohexylcarbodiimide (206 mg, 1.0 mmol) were dissolved in 1,4-dioxane (5 mL), and the reaction mixture was stirred at rt for 16 h. The mixture was filtered to remove the precipitate, and then tert-butyl 6-aminohexanoate (94 mg, 0.50 mmol) and aqueous sodium bicarbonate solution (42 mg in 2 mL of water, 0.50 mmol) were added to the filtrate. The reaction mixture was stirred at 50° C. for 1.5 h. After cooling to rt, concentrated hydrochloric acid was added to adjust pH to 4˜5, and then the mixture was extracted by EA. The organic phase was sequentially washed with water, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 2:1) to give compound 51 (40 mg) as colorless oil.

(151) LC-MS (method 2): R.sub.t=1.77 min; m/z (ES.sup.+)=340.0 (M+H).sup.+.

(152) .sup.1H NMR (500 MHz, CDCl.sub.3) δ8.55 (br s, 1H), 5.93 (s, 2H), 3.29-3.28 (m, 2H), 2.19 (t, 2H), 1.57-1.53 (m, 4H), 1.40 (s, 9H), 1.32-1.24 (m, 2H).

Step 2: Synthesis of tert-butyl 6-(2,4,6-tris((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzamido)hexanoate (52)

(153) Compound 51 (30 mg, 88 μmol) was dissolved in DMF (2 mL), to which KI-1 (51 mg, 308 μmol) and potassium carbonate (49 mg, 352 μmol) were sequentially added. The reaction mixture was stirred at rt for 16 h, and then diluted by EA. The mixture was sequentially washed with brine, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/EA 1:1 to DCM/MeOH 20:1) to give compound 52 (50 mg) as a yellow solid.

(154) LC-MS (method 3): R.sub.t=1.38 min; m/z (ES.sup.+)=724.0 (M+H).sup.+.

(155) .sup.1H NMR (500 MHz, CDCl.sub.3) δ6.47 (s, 2H), 5.24 (s, 4H), 5.20 (s, 2H), 3.41-3.37 (m, 2H), 2.75 (s, 3H), 2.68 (s, 6H), 2.20 (t, 2H), 1.61-1.52 (m, 4H), 1.42 (s, 9H), 1.40-1.29 (m, 2H).

Step 3: Synthesis of 6-(2,4,6-tris((5-(methylthio)-1,3,4-oxadiazol-2-yl)methoxy)benzamido)hexanoic Acid (53)

(156) Compound 52 (22 mg, 30 μmol) was dissolved in DCM (3 mL), to which TFA (0.3 mL) was added. The reaction mixture was stirred at rt for 3 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (DCM/MeOH 20:1) to give compound 53 (14 mg) as yellow oil.

(157) LC-MS (method 3): R.sub.t=1.07 min; m/z (ES.sup.+)=668.0 (M+H).sup.+.

(158) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ8.01 (t, 1H), 6.68 (s, 2H), 5.43 (s, 2H), 5.34 (s, 4H), 3.08-3.06 (m, 2H), 2.73 (s, 3H), 2.70 (s, 6H), 2.15 (t, 2H), 1.46-1.43 (m, 4H), 1.34-1.32 (m, 2H).

Step 4: Synthesis of 6-(2,4,6-tris((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methoxy)benzamido)hexanoic Acid (10)

(159) Compound 53 (14 mg, 20 μmol) was dissolved in DCM/DMF (1.9 mL/0.1 mL), to which m-chloroperbenzoic acid (104 mg, 600 μmol) was added. The reaction mixture was stirred at rt for 16 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 10 (1.5 mg) as a white solid.

(160) LC-MS (method 2): R.sub.t=1.56 min; m/z (ES.sup.+)=763.8 (M+H).sup.+.

(161) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ8.02 (t, 1H), 6.81 (s, 2H), 5.61 (s, 2H), 5.54 (s, 4H), 3.71 (s, 3H), 3.67 (s, 6H), 3.10-3.05 (m, 2H), 2.20 (t, 2H), 1.46-1.43 (m, 4H), 1.34-1.32 (m, 2H).

Example 11

Synthesis of 3,5-bis((3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanamido)methyl)benzoic Acid (Linker 11)

(162) ##STR00069## ##STR00070##

Step 1: Synthesis of 2,5-dioxopyrrolidin-1-yl 3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanoate (54)

(163) Compound 1 (70 mg, 0.184 mmol) and N-hydroxysuccinimide (25 mg, 0.217 mmol) were dissolved in THF (5 mL), to which DCC (50 mg, 0.243 mmol) was added. The reaction mixture was stirred at rt for 16 h, and then filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the residue was purified by prep-RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 54 (45 mg) as a white solid.

(164) LC-MS (method 2): R.sub.t=1.51 min; m/z (ES.sup.+)=477.5 (M+H).sup.+.

Step 2: Synthesis of 3,5-bis((3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanamido)methyl)benzoic Acid (11)

(165) Compound 54 (22 mg, 0.046 mmol) and 3,5-bis(aminomethyl)benzoic acid dihydrochloride (5 mg, 0.02 mmol) were dissolved in DMF (1 mL), to which DIEA (10 mg, 0.078 mmol) was added. The reaction mixture was stirred at rt for 4 h, and then concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 40%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 11 (5 mg) as a white solid.

(166) LC-MS (method 2): R.sub.t=1.60 min; m/z (ES.sup.+)=904.1 (M+H).sup.+.

(167) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ8.69 (t, 2H), 7.70 (s, 2H), 7.36 (s, 1H), 4.29 (d, 4H), 4.20-4.15 (m, 2H), 3.63 (s, 12H), 3.66-3.54 (m, 4H), 3.00-2.88 (m, 4H).

Example 12

Synthesis of 5-(bis(2-(3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanamido)ethyl)amino)-5-oxopentanoic Acid (Linker 12)

(168) ##STR00071## ##STR00072##

Step 1: Synthesis of 5-(bis(2-(benzyloxycarbonylamino)ethyl)amino)-5-oxopentanoic Acid (56)

(169) Bis(2-(benzyloxycarbonylamino)ethyl)amine (55, 815 mg, 2 mmol, prepared according to European Journal of Medicinal Chemistry, 2009, 44, 678-688) and TEA (0.70 mL<5 mmol) were dissolved in DMF (5 mL), to which a solution of glutaric anhydride (228 mg, 2 mmol) in DMF (1 mL) was added. The reaction mixture was stirred at rt overnight, to which water (20 mL) was added and the mixture was extracted by DCM (15 mL×3). The combined organic phase was sequentially washed with brine, dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH 30:1) to give compound 56 (872 mg) as a pale yellow solid.

(170) LC-MS (method 3): Rt=1.21 min; m/z (ES+) 486.3 (M+H).sup.+.

(171) Step 2: Synthesis of 5-(bis(2-aminoethyl)amino)-5-oxopentanoic acid (57) A solution of hydrogen bromide in acetic acid (33%, 3 mL) was dropwise added to compound 56 (522 mg, 1.1 mmol), and then the reaction mixture was stirred at rt for 15 min. Diethyl ether (20 mL) was added to the mixture to precipitate a yellow solid, which was collected by centrifugation. Diethyl ether was added to the solid thus obtained, and the mixture was centrifuged again to collect the solid. The similar procedure was repeated for three times, and the solid was dried in vacuo (60° C.) to give compound 57 hydrobromide salt (350 mg) as a yellow solid.

(172) LC-MS (method 4): Rt=0.28 min; m/z (ES.sup.+) 218.0 (M+H).sup.+.

Step 3: Synthesis of 5-(bis(2-(3,4-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)butanamido)ethyl)amino)-5-oxopentanoic Acid (12)

(173) Compound 57 hydrobromide salt (5 mg, 18.5 μmol) and compound 54 (22 mg, 46 μmol) were dissolved in DMF (1 mL), to which DIEA (10 mg, 78 μmol) was added. The reaction mixture was stirred at rt for 4 h, and then concentrated under reduced pressure. The residue was purified by prep-RP-HPLC (method 6: 40%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 12 (5 mg) as a white solid.

(174) LC-MS (method 1): Rt=1.55 min; m/z (ES+) 941.2 (M+H).sup.+.

(175) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.27 (t, 1H), 8.17 (t, 1H), 4.17-4.09 (m, 2H), 3.64 (s, 6H), 3.63 (s, 6H), 3.62-3.52 (m, 4H), 3.29-3.22 (m, 4H), 3.20-3.08 (m, 4H), 2.91-2.76 (m, 4H), 2.30 (t, 2H), 2.23 (t, 2H), 1.73-1.65 (m, 2H).

Example 13

Synthesis of Oxadiazole Linker-Drug (1-vcMMAE)

(176) ##STR00073##

(177) Compound 1 (5.0 mg, 13.15 μmol) and NH-vcMMAEF (TFA salt, 6.0 mg, 4.85 μmol, prepared according to WO2013/173337) were dissolved in DMF (0.2 mL), to which DIEA (3.39 μL, 19.4 μmol) and HATU (3.7 mg, 9.7 μmol) were then sequentially added. The reaction mixture was stirred at rt for 2 h, and then purified by prep-RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 1-vcMMAE (3.0 mg) as white powder.

(178) LC-MS (method 1): R.sub.t=1.84 min; m/z (ES.sup.+) 743.1 [½(M+2H)]+.

Example 14

(179) Synthesis of Other Oxadiazole Linker-Drugs

(180) Other oxadiazole linker-drugs were synthesized via the similar method as that for 1-vcMMAE in example 13, except that compound 1 was replaced with oxadiazole linkers 2-12. The linker-drugs and their characterization data were listed in Table 1, wherein the linker-drug 2-vcMMAE to 12-vcMMAE were named according to the oxadiazole linker compounds 2 to 12.

(181) TABLE-US-00002 TABLE 1 Linker-drugs of the invention and their characterizations LC-MS method; R.sub.t (min); m/z Compound ½[M + 2H].sup.+  2-vcMMAE 1; 1.84; 792.7  3-vcMMAE 1; 1.87; 743.5  4-vcMMAE 1; 1.89; 750.3  5-vcMMAE 1; 1.83; 757.2  6-vcMMAE 1; 1.85; 792.8  7-vcMMAE 1; 1.86; 795.8  8-vcMMAE 1; 1.91; 790.2  9-vcMMAE 1; 1.91; 790.3 10-vcMMAE 1; 1.89; 934.8 11-vcMMAE 1; 1.88; 1004.5 12-vcMMAE 1; 1.85; 1023.2

Example 15

(182) Preparation and Characterization of Antibody-Drug Conjugates

(183) Tris(2-carboxyethyl)phosphine (TCEP, 10 eq, stock solution 10 mM) was added to a solution of antibody H (IgG1) (2-10 mg/mL, containing 25 mM boric acid-sodium borate buffer, 25 mM NaCl and 1 mM diethylene triamine pentacetic acid (DTPA), pH 7.0-8.0). The reaction mixture was incubated at 37° C. in a shaker for 2 h, and then cooled to ˜10° C., followed by buffer-exchange with a PBS buffer (100 mM KH.sub.2PO.sub.4—K.sub.2HPO.sub.4, 100 mM NaCl, 1 mM DTPA, pH 7.0-8.0) via ultrafiltration (Merck Millipore Amicon® Ultra, 50000 MWCO) or gel-filtration. The solution was cooled at 10° C., to which DMSO and compound 1-vcMMAE prepared in example 13 (stock solution in DMSO, 6 equivalent) were sequentially added, in which the volume percentage of DMSO was controlled at 15%. The conjugation reaction was conducted at 10° C. for 0.5 h.

(184) Excess cysteine solution was added to the reaction mixture to quench the unreacted compound 1-vcMMAE, and the quenching reaction was kept at 10° C. for 30 min. The reaction mixture was ultrafiltered (Merck Millipore Amicon® Ultra, 50000 MWCO) or gel-filtered to remove excess 1-vcMMAE-cysteine adducts and excess cysteine. The filtrate was sterile filtered through 0.22 m filter (Merck Millex-GV Filter), and the solution of conjugate H-1-vcMMAE thus obtained was kept at 4° C.

(185) Conjugates H-2-vcMMAE to H-12-vcMMAE were prepared from antibody H according to the same method as above, except for replacing compound 1-vcMMAE with compounds 2-vcMMAE to 12-vcMMAE.

(186) 1) Determination of Average DAR

(187) The average DAR was calculated by from the relative peak area (%) of each peak and the corresponding number of drugs loaded on HIC (hydrophobic interaction chromatography) (Anal. Chem. 2013, 85, 1699-1704). The weighted peak percentage, which measures the contribution of individual drugloaded species to DAR, is calculated by multiplying the relative peak area (%) and the corresponding number of loaded drugs. DAR is then obtained by summing up the weighted peak percentage from all observed species and dividing the sum by 100, as follows:
DARt=Σ(relative peak area %×corresponding number of loaded drugs)/100

(188) The average DARs of the ADCs of the invention were listed in table 2.

(189) TABLE-US-00003 TABLE 2 The average DAR results of the ADCs in the invention ADC Average DAR Eq of linker-drug H-1-vcMMAE 4.0 6 H-2-vcMMAE 4.0 6 H-3-vcMMAE 4.0 8 H-4-vcMMAE 4.0 6 H-5-vcMMAE 4.0 6 H-6-vcMMAE 4.1 8 H-7-vcMMAE 4.1 6 H-8-vcMMAE 4.2 8 H-9-vcMMAE 4.1 8 H-10-vcMMAE 3.0 20 H-11-vcMMAE 2.1 4 H-12-vcMMAE 2.2 4

(190) As shown in table 2, the average DARs of the ADCs (H-1-vcMMAE to H-9-vcMMAE) based on bis(1,3,4-oxadiazole) linker of the invention could be well-controlled around 4, and the DAR of the main component (85%+) is 4. The average DAR of the ADC (H-10-vcMMAE) based on tri(1,3,4-oxadiazole) linker of the invention could be well-controlled around 3, and the DAR of the main component (85%+) is 3. The average DARs of the ADCs (H-11-vcMMAE to H-12-vcMMAE) based on tetra(1,3,4-oxadiazole) linker of the invention could be well-controlled around 2, and the DAR of the main component (85%+) is 2. These results are due to the accurate site and number control by the site-specific linkers of the invention.

(191) 2) Native MS

(192) 8 μL of PNGase F (New England Biolabs, USA) was added to 400 μg of conjugate H-1-vcMMAE, and the mixture was incubated at 37° C. overnight (15 h). The deglycosylated ADC sample was buffer-exchanged into ammonium acetate buffer (20 mM, pH 7.0), and the buffer exchange procedure was repeated for 5 times.

(193) The mass spectrometer used was high-resolution Orbitrap Exactive Plus EMR (Thermo Fisher Scientific, Germany), and the ion source is TriVersa NanoMate® (Advion, USA). The sample concentration was adjusted to 2 μg/μL, and direct injection was adopted. The mass data was collected under the positive ion mode, and the native mass data was analyzed by Protein Deconvolution 4.0 software (Thermo Fisher Scientific, Germany).

(194) The native MS spectrum of antibody-drug conjugate H-1-vcMMAE was shown in FIG. 1, which shows that the main component of the product has a DAR of 4.

(195) 3) SDS-PAGE

(196) SDS-PAGE was measured using NuPAGE™, 4-12%, Bis-Tris Gel (Thermal Fisher) on XCell SureLock® Mini-Cell protein electrophoresis instrument (Thermal Fisher). A sample (≥10 μg by weight) was combined with loading buffer, and the mixture was heated at 70° C. in water bath for 10 min. The sample and standard protein (5 μL/hole) were added to the spacer gel comb holes sequentially, and the electrophoresis was conducted at 220 V for 50 min. The gel was removed, rinsed by deionized water, and then stained in SimplyBlue™ SafeStain (Thermal Fisher) on a shaker for 3 h. The stained gel was rinsed by deionized water for three times, and destained on a shaker for 4 h. The destained gel was transferred to an imager to record the gel image.

(197) The SDS-PAGE results are shown in FIGS. 2a-2b, which shows that the main components in sample H-1-vcMMAE to H-12-vcMMAE were full antibody HHLL (full ADC) and half antibody HL (full ADC lost heavy chain interaction). The result proves that the oxadiazole linkers of the invention can crosslink the inter chains of the reduced antibody, and thus effectively control the number of drugs per antibody (DAR).

(198) 4) Hydrophobic Interaction Chromatography (HIC) Analysis

(199) HIC was measured on an Agilent 1100 chromatograph. TSKgel butyl-NPR column (4.6×35 mm, 2.5 m, Tosoh Bioscience Shanghai) was applied as the immobile phase. The method was consisted of a linear gradient from 100% buffer A (50 mM potassium phosphate (pH 7.0)+1.5 M ammonium sulfate) to 100% buffer B (80% v/v 50 mM sodium phosphate (pH 7.0), 20% v/v isopropanol) over 25 minutes. The flow rate was 0.8 mL/min, the column temperature was 30° C., and the detection wavelengths were 230 and 280 nm.

(200) HIC analysis results are shown in FIGS. 3a-3l, which show that the main components of the ADC samples, H-1-vcMMAE to H-9-vcMMAE, are DARt=4 components. The main components of the ADC sample, H-10-vcMMAE, is DARt=3 component. The main components of the ADC samples, H-11-vcMMAE to H-12-vcMMAE, are DARt=2 components. The result proves that the oxadiazole linkers of the invention could be used to effectively control the DAR and distribution of the ADC product.

Test Example 1

(201) Determination of the Antigen Binding Ability of the ADCs of the Invention by Enzyme-Linked Immunosorbent Assay (ELISA)

(202) Indirect ELISA was used to analyze binding ability of the antibody or antibody-drug conjugate to the corresponding antigen. The Her2 antigen was immobilized on a solid-phase support (96 well microplate) by coating to form a solid-phase antigen, and then unbound antigen was removed by washing. Serial dilutions of test antibody or antibody-drug conjugate were added, wherein specific antibody or antibody-drug conjugate bound to the antigen and formed solid-phase antigen-antibody complexes. The antibody or antibody-drug conjugate unbound to the solid-phase antigen was removed by washing. The enzyme labeled anti-antibody was added to bind to the above-formed complexes. After washing, substrate solution was added, and the optical density was read by a microplate reader at 450 nm/630 nm, based on which the curve was drawn and the EC.sub.50 was calculated.

(203) The binding abilities of the ADCs of the invention to Her2 antigen were listed in Table 3.

(204) TABLE-US-00004 TABLE 3 The binding ability of the ADCs of the invention to Her2 antigen Antibody/ADCs EC.sub.50 (ng/mL) H 33.5 H-1-vcMMAE 70.8 H-2-vcMMAE 79.6 H-3-vcMMAE 63.8 H-4-vcMMAE 66.1 H-5-vcMMAE 59.0 H-6-vcMMAE 70.8 H-7-vcMMAE 95.9 H-8-vcMMAE 83.3 H-9-vcMMAE 62.5 H-10-vcMMAE 34.4 H-11-vcMMAE ND H-12-vcMMAE ND ND: not determined.

(205) As shown in Table 3, compared to naked antibody, the binding ability of the ADCs prepared from oxadiazole linkers to the antigen shows no significant difference.

Test Example 2

(206) Cell Proliferation Inhibition of the ADCs of the Invention

(207) Cell Proliferation Assay

(208) Cell proliferation inhibition of an antibody or ADC is measured by the following method. Mammalian cells expressing tumor-associated antigens or receptor proteins (Her2 expressing breast cancer cell, SK-BR-3, was used in this assay) were seed in 96-well plate at a concentration of 8000 cells/well, and the cells were suspended in DMEM (GIBCO). The initial ADC concentration was 2 μg/mL, which was 3 times gradient diluted with DMEM containing 2% FBS (GIBCO). The initial cell culture media was removed and 200 μL of ADC was added to each well. The cells were incubated for 72 h, and the media was removed. 100 μL of CCK-8 was added, followed by incubation of 30 min. The absorption was read by a microplate reader at 450 nm/630 nm, based on which the curve was drawn and the IC.sub.50 was calculated.

(209) The cell proliferation inhibition result of the ADCs of the invention was listed in table 4.

(210) TABLE-US-00005 TABLE 4 Cell Proliferation Inhibition Result of the ADCs of the Invention ADC IC.sub.50 (ng/mL) H-1-vcMMAE 3.0 H-2-vcMMAE 3.3 H-3-vcMMAE 3.2 H-4-vcMMAE 3.0 H-5-vcMMAE 2.7 H-6-vcMMAE 2.5 H-7-vcMMAE 5.4 H-8-vcMMAE 2.8 H-9-vcMMAE 3.5 H-10-vcMMAE 3.2 H-11-vcMMAE 6.1 H-12-vcMMAE 6.3
Table 4 shows that the ADCs of the invention have excellent cell proliferation inhibition activity.

(211) All references mentioned in the present application are incorporated herein by reference to the same extent as if each individual reference is individually incorporated by reference. In addition, it should be understood that after reading the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.