COMPOSITIONS COMPRISING ENZYME CLEAVABLE LINKER PLATFORMS AND CONJUGATES THEREOF

Abstract

The present invention relates to a cleavable linker platform. In particular, the invention relates to construction of an enzyme cleavable linker platform conjugated to a drug or a diagnostically relevant compound, a biomolecule, and an enzyme cleavable group, for which cleavage of the enzyme cleavable group leads to release of the drug or diagnostically relevant compound.

Claims

1. A compound of formula (I), ##STR00086## wherein: each R.sup.1 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl or both R.sup.1 together with the carbon to which they are attached form a cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl; R.sup.2 is a bond or linking group; R.sup.3 is a moiety comprising at least 19 atoms; X is >N—, ═N—, —N(H)—, —O—, or —S—; Y is —O— or —N(R.sup.8)—; if Y is —O—, R.sup.3X is R.sup.3—O—, or R.sup.3—S—; whereas if Y is —N(R.sup.8)—, R.sup.3X is R.sup.3>N—, R.sup.3═N—, R.sup.3—N(H)—, R.sup.3—O— or R.sup.3—S—; R.sup.4 is: ##STR00087## Y.sup.1 is —O— or —N(R.sup.8)—; R.sup.5 is ═O or ═NR.sup.12; each R.sup.6 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or any two R.sup.6 attached to the same carbon form a cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl together with the carbon to which they are attached; R.sup.7 is a negative charge or hydrogen; each R.sup.8 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; R.sup.9 is selected from hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; each R.sup.19 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; R.sup.11 is a negative charge, hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; R.sup.12 is selected from hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; Z is —O— or —S—; T is a moiety containing at least 19 atoms; m is 2 to 4; n is 0 to 4; r is 1 or more; any R.sup.1 is optionally together with any R.sup.6 a bond, an alkylene group, or a heteroalkylene group where such connection results in a cis-configuration of the substituent R.sup.3XC(═Z)—and the substituent —(C(R.sup.6).sub.2).sub.kYR.sup.4 where k is 0 to m−1; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 wherein both R.sup.1 are the same and selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, phenyl, or benzyl, or both R.sup.1 taken together is —CH.sub.2CH.sub.2—.

3. The compound according to any one of the preceding claims, wherein both R.sup.1 are hydrogen.

4. The compound according to any one of the preceding claims, wherein both R.sup.1 are methyl.

5. The compound according to any one of the preceding claims, wherein both R.sup.1 are propyl.

6. The compound according to any one of the preceding claims, wherein both R.sup.1 are phenyl.

7. The compound according to any one of the preceding claims, wherein both R.sup.1 taken together is —CH.sub.2CH.sub.2—.

8. The compound according to any of one the preceding claims, wherein R.sup.6 is independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, phenyl, or benzyl, or, two R.sup.6 attached to the same carbon taken together is —CH.sub.2CH.sub.2—.

9. The compound according to any one of the preceding claims, wherein m is 2.

10. The compound according to any one of the preceding claims, wherein R.sup.6 is hydrogen.

11. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00088##

12. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00089##

13. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00090##

14. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00091##

15. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00092##

16. The compound according to any one of the preceding claims, wherein YR.sup.4 taken together is ##STR00093##

17. The compound according to any one of the preceding claims, wherein YR.sup.4 taken together is ##STR00094##

18. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00095##

19. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00096##

20. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00097##

21. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00098##

22. The compound according to any one of the preceding claims, wherein Y is —O— or Y is —N(H)— and R.sup.4 is ##STR00099##

23. The compound according to any one of the preceding claims, wherein YR.sup.4 taken together is ##STR00100##

24. The compound according to any one of the preceding claims, wherein R.sup.5 is O.

25. The compound according to any one of the preceding claims, wherein R.sup.2 is a linking group.

26. The compound according to any one of the preceding claims, wherein T is a protein that binds specifically to a cellular target.

27. The compound according to any one of the preceding claims, wherein the cellular target is an extracellular target.

28. The compound according to any one of the preceding claims, wherein binding of T to the extracellular target affects endocytosis of the compound.

29. The compound according to any one of the preceding claims, wherein T is an antibody.

30. The compound according to any one of the preceding claims, wherein T is an internalising antibody.

31. The compound according to any one of the preceding claims, wherein T is an antibody-derived antigen binding fragment.

32. The compound according to any one of the preceding claims, wherein T is a single-chain variable fragment.

33. The compound according to any one of the preceding claims, wherein T is a single-domain antibody.

34. The compound according to any one of the preceding claims, wherein T is a nanobody.

35. The compound according to any one of the preceding claims, wherein T is a DARPin.

36. The compound according to any one of the preceding claims, wherein T is a monobody.

37. The compound according to any one of the preceding claims, wherein T is a affibody.

38. The compound according to any one of the preceding claims, wherein T is a carbohydrate.

39. The compound according to any one of the preceding claims, wherein T is a oligonucleotide.

40. The compound according to any one of the preceding claims, wherein T is a lipid.

41. The compound according to any one of the preceding claims, wherein r is 1 to 12.

42. The compound according to any one of the preceding claims, wherein X is —S—.

43. The compound according to any one of the preceding claims, wherein X is >N—, ═N—, or —N(H)—.

44. The compound according to any one of the preceding claims, wherein X is —O—.

45. The compound according to any one of the preceding claims, wherein the compound is a prodrug.

46. The compound of formula I for use in medicine.

47. The compound of formula I for use in diagnostics.

Description

DESCRIPTION OF FIGURES

[0251] FIG. 1: Overview of rates of intramolecular ring closure reaction for substrates 5A-E. The conditions are Amano lipase (ag/ml) in plasma at room temperature. The unit of the X axis of the graph is time. The unit of the y axis of the graph is % release.

[0252] FIG. 2: Mechanism of the intramolecular ring closure reaction for the compound of formula (I).

[0253] FIG. 3: Mechanism of the intramolecular ring closure reaction for the compound of formula (II).

[0254] FIG. 4: Mechanism of the self-immolation of some embodiments of the compound of formula (I) and the compound of formula (II).

[0255] FIG. 5: Mechanism of the self-immolation of some embodiments of the compound of formula (I) and the compound of formula (II).

[0256] FIG. 6: Examples of the cis-criterion of the substituents after ring-closure. Substituents and the carbon atoms to which they are attached are shows in dashed boxes.

[0257] FIG. 7: An example of a synthetic pathway for producing a lipase cleavable linker relating to the compound of formula (I).

[0258] FIG. 8: An example of a synthetic pathway for producing a lipase cleavable linker relating to the compound of formula (I). 1-(4-bromophenyl)-2-hydroxyethan-1-one is used as a model drug.

[0259] FIG. 9: An example of a synthetic pathway for producing a lipase cleavable linker relating to the compound of formula (I) with a maleimide-based moiety for bioconjugation. The circled compound is a GLP-1 receptor allosteric modulator.

[0260] FIG. 10: An example of a synthetic pathway for producing a phosphatase cleavable linker relating to the compound of formula (I). The circled model drug mimics part of the structure of auristatin E, also shown in the figure.

[0261] FIG. 11: An example of a synthetic pathway for producing a phosphatase cleavable linker relating to the compound for formula (II). The circled moiety of the model drug mimics part of the structure of auristatin E, also shown in the figure.

[0262] FIG. 12: An example of a synthetic pathway for producing an aryl sulfatase cleavable linker relating to the compound of formula (II). The circled moiety of the model drug mimics part of the structure of auristatin E, also shown in the figure.

[0263] FIG. 13: An example of a synthetic pathway for producing a sulfamidase cleavable linker relating to the compound of formula (II). The circled moiety of the model drug mimics part of the structure of auristatin E, also shown in the figure.

[0264] FIG. 14: Structure of 2Rs15d-L-auristatin E drug conjugate and structure of Auristatin E drug conjugate.

[0265] FIG. 15: Synthetic pathway for producing a lipase cleavable linker relating to the compound of formula (I). 1-(4-bromophenyl)-2-hydroxyethan-1-one is used as a model drug.

[0266] FIG. 16: Overview of conditions for cleavage of compound X8 with a lipase with subsequent release of model drug X10 and formation of ring-closed structure X11.

[0267] FIG. 17: a) An example of LCMS UV trace of compound X8 in the presence of lipase at t=11 min. b) The results of the lipase-mediated release of X10 as observed by LCMS.

[0268] FIG. 18: Antibody drug conjugates with different enzyme cleavable linkers (phosphatase cleavable (PC), sulfatase cleavable (SC), sulfamidase cleavable (SAC), and lipase cleavable (LC) linkers) and cytotoxic agents (querticin (CytI), PNU-159682 (CytII), camptothecin (CytIII), auristatin E (CytIV), and cryptothecin (CytV)). The waved line indicates the site of conjugation between the enzyme cleavable linker and the cytotoxic agent.

[0269] FIG. 19: Sulfamidase cleavable camptothecin-based antibody-drug conjugates with different R-moieties that dictates the rate of release of camptothecin (a: R═H, b: R=Me, c: R=Et, d: R═Pr, e: R=Bn, f: R=Ph).

[0270] FIG. 20: Sulfamidase cleavable cryptothecin-based antibody-drug conjugates with different R-moieties that dictates the rate of release of cryptothecin (a: R═H, b: R=Me, c: R=Et, d: R═Pr, e: R=Bn, f: R=Ph).

[0271] FIG. 21: Sulfamidase cleavable antibody-drug conjugates comprising the model drug benzyl alcohol. Sulfamidase cleaves the sulfamide group, which forms a free amine. After cleavage of the sulfamide group, the free amine reacts on the carbamate carbonyl group, releasing the model drug.

EXAMPLES

[0272] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments.

General Experimental Details

[0273] Reagents: All reagents were obtained from commercial sources and used without further purification.

[0274] Proton nuclear magnetic resonance (.sup.1H-NMR) spectroscopy:

[0275] Proton nuclear magnetic resonance spectra were recorded using an internal deuterium lock (at 298 K unless stated otherwise) on Bruker DPX (400 MHz; 1H-13C DUL probe), Bruker Avance III HD (400 MHz; Smart probe), Bruker Avance III HD (500 MHz; Smart probe) and Bruker Avance III HD (500 MHz; DCH Cryoprobe) spectrometers. NMR spectra for compounds that appeared as mixtures of rotamers at 298 K are reported at a temperature that provided clear signals for unambiguous characterization. Chemical shifts (δH) are quoted in ppm to the nearest 0.01 ppm and are referenced to the residual non-deuterated solvent peak.

[0276] Carbon nuclear magnetic resonance (.sup.13C NMR) spectroscopy:

[0277] Carbon nuclear magnetic resonance spectra were recorded using an internal deuterium lock (at 298 K unless stated otherwise) on Bruker DPX (101 MHz), Bruker Avance III HD (101 MHz) and Bruker Avance III HD (126 MHz) spectrometers with broadband proton decoupling. Carbon spectra assignments are supported by DEPT editing, .sup.1H-.sup.13C HSQC or .sup.1H-.sup.13C HMBC spectra, or by analogy. Chemical shifts (SC) are quoted in ppm to the nearest 0.1 ppm and are referenced to the deuterated solvent peak. Data are reported as: chemical shift and assignment.

[0278] High resolution mass spectrometry (HMS):

[0279] Recorded on a Waters LCT Premier Time of Flight mass spectrometer. Reported mass values are within the error limits of ±5 ppm. ESI refers to the electrospray ionisation technique.

[0280] Liquid chromatography-mass spectrometry (LCMS):

[0281] LCMS analysis was performed on a Waters ACQUITY H-Class UPLC with an ESCi Multi-Mode Ionisation Waters SQ Detector 2 spectrometer using MassLynx 4.1 software; LC system: solvent A: 2 mMNH.sub.4OAc in H.sub.2O/MeCN (95:5); solvent B: MeCN; solvent C: 2% HCO.sub.2H; gradient: A/B/C, 90:5:5-0:95:5 over 1 min at a flow rate of 0.6 mL min.sup.1.

Example 1: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0282] FIG. 7 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

2-Hydroxyethyl octanoate

[0283] ##STR00031##

[0284] Octanoyl chloride (9.97 mL, 61.5 mmol) was added dropwise to a solution of ethylene glycol (34.25 mL, 615 mmol) and triethylamine (8.58 mL, 61.5 mmol) and stirred at rt overnight. The reaction mixture was quenched with water (150 mL) and extracted with CH.sub.2Cl.sub.2 (5×75 mL). The combined organic layers were washed with water (2×75 mL) and brine (75 mL), dried (MgSO.sub.4), concentrated in vacuo and purified using a short plug of silica (0% to 5% MeOH in CH.sub.2Cl.sub.2) to yield the desired alcohol (5.7 g, 30.3 mmol, 49%) as a colourless oil.

[0285] R.sub.f=0.24 (2.5% MeOH in CH.sub.2Cl.sub.2); v.sub.max (neat/cm.sup.−1)=3453 (w, br, O—H), 2928 (m, C—H), 1736 (s, C═O), 1458, 1379; .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ=4.77 (t, J=5.5 Hz, 1H), 4.01 (dd, J=5.8, 4.4 Hz, 2H), 3.55 (q, J=5.4 Hz, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.52 (p, J=7.0 Hz, 2H), 1.26 (ddd, J=10.0, 5.2, 2.4 Hz, 8H), 0.91-0.79 (m, 3H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=172.98, 65.49, 58.97, 33.46, 31.12, 28.42, 28.36, 24.45, 22.04, 13.92; HRMS (ESI): m/z=189.15 [M+H].sup.+, required m/z=189.1485 [C.sub.10H.sub.21O.sub.3].sup.+.

2-Oxoethyl octanoate

[0286] ##STR00032##

[0287] DMP (18.9 g, 44.5 mmol) was added to a solution of 2-Hydroxyethyl octanoate (5.59 g, 29.7 mmol) in CH.sub.2Cl.sub.2 (275 mL) at 0° C. The solution was stirred and allowed to slowly warm up to rt overnight. Reaction was diluted with 800 mL EtOAc and washed with H.sub.2O (3×750 mL) followed by filtration. Organic phases washed with Na.sub.2S.sub.2O.sub.3 (aq.) (3×500 mL), sat. NaHCO.sub.3(aq.) (3×500 mL) and brine (2×500 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (3:7 EtOAc:heptane) to yield desired aldehyde (366 mg, 1.97 mmol, 74%) as a colourless oil.

[0288] R.sub.f=0.30 (3:7 EtOAc:heptane); v.sub.max (neat/cm.sup.−1)=2954 (m, C—H), 1737 (s, C═O), 1465, 1382; .sup.1-H NMR (400 MHz, Chloroform-d) δ=9.60 (s, 1H), 4.66 (s, 2H), 2.44 (t, J=7.5 Hz, 2H), 1.67 (p, J=7.5 Hz, 2H), 1.40-1.19 (m, 8H), 0.94-0.82 (m, 3H); .sup.13C NMR (101 MHz, Chloroform-d) δ=195.97, 173.30, 68.62, 33.84, 31.76, 29.15, 29.02, 24.97, 22.72, 14.19; HRMS (ESI): m/z=187.13 [M+H].sup.+, required 187.13 [C.sub.10H.sub.19O.sub.3].sup.+.

2-((2-(Allyloxy)-2-oxoethyl)amino)ethyl octanoate

[0289] ##STR00033##

[0290] A suspension of triethylamine (1.35 mL, 9.66 mmol) and allyl glycinate (14.5 mmol) in DCE (5 mL) was stirred for 15 min before 2-oxoethyl octanoate (1.8 g, 9.66 mmol) and NaBH(OAc).sub.3 (4.10 g, 19.33 mmol) were added at rt. The stirred suspension was quenched after 18 h with sat. NaHCO.sub.3(aq.) (150 mL) and extracted with CH.sub.2Cl.sub.2 (3×150 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (1:1 EtOAc:heptane) to yield desired amine (771 mg, 2.70 mmol, 28%) as a colourless oil.

[0291] R.sub.f=0.17 (1:1 EtOAc:heptane); v.sub.max (neat/cm.sup.−1)=3486 (w, br, N—H), 2925 (w, C—H), 1737 (s, C═O), 1645 (s, C═C), 1459; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=5.92 (ddt, J=17.3, 10.7, 5.4 Hz, 1H), 5.31 (dq, J=17.2, 1.7 Hz, 1H), 5.21 (dq, J=10.5, 1.4 Hz, 1H), 4.57 (dt, J=5.5, 1.5 Hz, 2H), 4.03 (t, J=5.7 Hz, 2H), 3.39 (s, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.51 (p, J=7.2 Hz, 2H), 1.32-1.17 (m, 8H), 0.90-0.77 (m, 3H); .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ=172.90, 171.84, 132.61, 117.77, 64.35, 63.60, 49.92, 46.94, 33.46, 31.10, 28.40, 28.34, 24.43, 22.02, 13.92; HRMS (ESI): m/z=286.2018 [M+H].sup.+, required 286.2018 [C.sub.15H.sub.28NO.sub.4]; UPLC-MS (ESI): m/z=286.6 [M+H].sup.+, required 286.4 [C.sub.15H.sub.28NO.sub.4].sup.+.

2-(N-(2-(allyloxy)-2-oxoethyl)-4-(((tert-butoxycarbonyl)amino)methyl)benzamido)ethyl octanoate

[0292] ##STR00034##

[0293] A suspension of 2-((2-(Allyloxy)-2-oxoethyl)amino)ethyl octanoate (468 mg, 1.64 mmol) and trimethylamine (298 μL, 2.13 mmol) in CH.sub.2Cl.sub.2 (6 mL) was cooled to 0° C. After 5 min tert-butyl (4-(chlorocarbonyl)benzyl)carbamate (575 mg, 2.13 mmol) and DMAP (cat. amount) were added and the mixture was allowed to reach rt. The stirred suspension was quenched after 18 h with H.sub.2O (50 mL) and extracted with CH.sub.2Cl.sub.2 (2×50 mL). The combined organic layers were washed with 0.1M HCl (50 mL), sat. NaHCO.sub.3(75 mL) and brin (75 mL), dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (1:1 EtOAc:heptane) to yield the desired product (351 mg, 0.68 mmol, 41%) as a colourless oil.

[0294] R.sub.f=0.23 (1:1 EtOAc:heptane); .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=7.88 (d, 2H, J=8.5 Hz), 7.75 (d, 2H, J=8.5 Hz), 7.46 (m, 3H), 7.38 (m, 2H), 5.46 (s, 2H), 4.34 (s, 2H), 4.21 (m, 2H), 3.67 (m, 2H), 2.28 (t, 2H, J=7.4 Hz), 1.55 (m, 2H), 1.28 (m, 8H), =0.87 (m, 3H); HRMS (ESI) m/z=546.1499 [M+H].sup.+, required 546.1491 [C.sub.27H.sub.33NO.sub.6Br].sup.+.

Example 2: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0295] FIG. 8 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

2-(N-(2-(allyloxy)-2-oxoethyl)benzamido)ethyl octanoate

[0296] ##STR00035##

[0297] Benzoyl chloride (61.0 μL, 0.706 mmol) was added to a solution of triethylamine (98.4 μL, 0.706 mmol) and 2-((2-(Allyloxy)-2-oxoethyl)amino)ethyl octanoate (155 mg, 0.543 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. The solution was warmed up to rt and stirred for 2 h before being quenched with water (10 mL) and extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was washed with 1 M HCl (aq.) (10 mL), sat. NaHCO.sub.3(aq.) (10 mL) and brine (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (25% EtOAc in PE) to yield the desired amide (125 mg, 0.321 mmol, 69%) as a colourless oil.

[0298] R.sub.f 0.21 (25% EtOAc in PE); .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.45 (m, 3H), 7.35 (m, 2H), 5.92 (m, 1H), 5.31 (d, 1H, J=17.6 Hz), 5.23 (d, 1H, J=10.6 Hz), 4.62 (d, 2H, J=5.0 Hz), 4.20 (m, 4H), 3.64 (m, 2H), 2.27 (t, 2H, J=7.3 Hz), 1.54 (m, 2H), 1.28 (m, 8H), 0.88 (m, 3H); 6C (126 MHz, DMSO-d.sub.6, 373K) 172.0, 170.8, 168.6, 135.4, 131.7, 128.9, 127.8, 125.6, 117.4, 64.4, 61.8, 60.8, 44.8, 33.0, 30.4, 27.8, 27.6, 23.8, 21.3, 13.0; HRMS (ESI) m/z found 412.2105 [M+Na].sup.+, required 412.2100 [C.sub.22H.sub.31NO.sub.5Na].sup.+.

N-benzoyl-N-(2-(octanoyloxy)ethyl)glycine

[0299] ##STR00036##

[0300] Tetrakis(triphenylphosphine)palladium(O) (20.6 mg, 17.8 μmol) and allyl ester 2-(N-(2-(allyloxy)-2-oxoethyl)benzamido)ethyl octanoate (139 mg, 0.357 mmol) were dissolved in THF (20 mL) and stirred at rt for 5 min. Morpholine (308 μL, 3.57 mmol) was added dropwise to the yellow solution and stirred for 4 h at rt. The resulting green solution was concentrated in vacuo and diluted with ether (10 mL) before being washed with 1 M HCl (aq.) (3×10 mL). The organic layer was filtered through a bed of celite washing thoroughly with ether, dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (0.5% acetic acid in 2:1 EtOAc/PE) to yield the desired acid (114 mg, 0.327 mmol, 91%) as a pale yellow oil.

[0301] Rf 0.39 (0.1% acetic acid in EtOAc); vmax (neat/cm-1) 3000 (w, br, O—H), 2928 (w, C—H), 1733 (s, C═O), 1643 (s, C═O), 1598 (m, C═C), 1575 (m, C═C), 1498, 1460; 6H (500 MHz, DMSO-d6, 373K) 7.43 (m, 3H), 7.35 (m, 2H), 4.19 (m, 2H), 4.07 (s, 2H), 3.63 (m, 2H), 2.28 (t, 2H, J=7.4 Hz), 1.55 (m, 2H), 1.29 (m, 8H), 0.88 (m, 3H); 6C (126 MHz, DMSO-d.sub.6, 373K) 172.0, 170.7, 169.6, 135.7, 128.8, 127.8, 125.9, 63.8, 60.8, 47.6, 33.0, 30.4, 27.8, 27.6, 23.8, 21.2, 13.0; HRMS (ESI) m/z found 372.1773 [M+Na].sup.+, required 372.1781 [C19H27NO5Na].sup.+.

2-(N-(2-(2-(4-bromophenyl)-2-oxoethoxy)-2-oxoethyl)benzamido)ethyl octanoate

[0302] ##STR00037##

[0303] EDC (89.7 mg, 0.468 mmol) was added to a solution of acid N-benzoyl-N-(2-(octanoyloxy)ethyl)glycine (109 mg, 0.312 mmol), DMAP (57.2 mg, 0.468 mmol) and 1-(4-bromophenyl)-2-hydroxyethan-1-one (101 mg, 0.468 mmol) at 0° C. The suspension was warmed up to rt and stirred for 3 h before being diluted with CH2Cl2 (10 mL) and washed with sat. NaHCO.sub.3(aq.) (10 mL) and 1 M HCl (aq.) (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (25% EtOAc in PE) to yield 2-(N-(2-(2-(4-bromophenyl)-2-oxoethoxy)-2-oxoethyl)benzamido)ethyl octanoate (97.0 mg, 0.177 mmol, 57%) as a colourless oil.

[0304] Rf 0.19 (25% EtOAc in PE); vmax (neat/cm-1) 2929 (w, C—H), 1735 (s, C═O), 1705 (s, C═O), 1640 (s, C═C), 1586 (s, C═C), 1455; 6H (500 MHz, DMSO-d.sub.6): 7.88 (d, 2H, J=8.5 Hz, H3), 7.75 (d, 2H, J=8.5 Hz, H2), 7.46 (m, 3H, H21 and H23), 7.38 (m, 2H, H22), 5.46 (s, 2H, H6), 4.34 (s, 2H, H8), 4.21 (m, 2H, H10), 3.67 (m, 2H, H9), 2.28 (t, 2H, J=7.4 Hz, H12), 1.55 (m, 2H, H13), 1.28 (m, 8H, H14-H17), 0.87 (m, 3H, H18); 6C (126 MHz, DMSO-d6, 373K) 191.3, 172.0, 170.8, 168.3, 135.3, 132.8, 131.4, 129.2, 129.0, 127.8, 127.4, 126.0, 66.3, 62.9, 60.7, 47.2, 33.0, 30.4, 27.8, 27.6, 23.8, 21.3, 13.0; HRMS (ESI) m/z found 546.1499 [M+H].sup.+, required 546.1491 [C27H33NO679Br].sup.+.

Example 3: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0305] FIG. 8 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

allyl 2-((2-(octanoyloxy)ethyl)amino)-2-methylpropanoate

[0306] ##STR00038##

[0307] Allyl 2-amino-2-methylpropanoate (0.628 mmol) and 2-oxoethyl octanoate (0.515 mmol) were dissolved in DCM (5 mL) was stirred for 1 hour before NaBH(OAc).sub.3 (0.802 mmol) were added and the resulting suspension stirred for 17 h. The reaction was quenched with sat. NaHCO.sub.3(aq.) (150 mL) and extracted with CH.sub.2Cl.sub.2 (3×15 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (20-50% EtOAc in heptane) to yield desired amine (0.271 mmol, 47%) as a colourless oil.

[0308] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=5.93 (ddt, J=17.3, 10.8, 5.3 Hz, 1H), 5.31 (dq, J=17.1, 1.8 Hz, 1H), 5.21 (dq, J=10.5, 1.5 Hz, 1H), 4.57 (dt, J=5.4, 1.6 Hz, 2H), 4.03 (t, J=5.8 Hz, 2H), 2.75 (t, J=5.6 Hz, 2H), 2.28 (t, J=7.3 Hz, 2H), 1.55 (s, 6H), 1.51 (p, J=7.2 Hz, 2H), 1.32-1.17 (m, 8H), 0.90-0.77 (m, 3H); UPLC-MS (ESI): m/z found 314.3 [M+H].sup.+, required 314.2 [C.sub.17H.sub.32NO.sub.4].sup.+.

2-(N-(1-(allyloxy)-2-methyl-1-oxopropan-2-yl)benzamido)ethyl octanoate

[0309] ##STR00039##

[0310] Benzoyl chloride (0.706 mmol) was added to a solution of triethylamine (98.4 μL, 0.706 mmol) and 2-((1-(allyloxy)-2-methyl-1-oxopropan-2-yl)amino)ethyl octanoate (0.543 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. The solution was warmed up to rt and stirred for 2 h before being quenched with water (10 mL) and extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was washed with 1 M HCl (aq.) (10 mL), sat. NaHCO.sub.3(aq.) (10 mL) and brine (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (25% EtOAc in PE) to yield the desired amide (0.261 mmol, 48%) as a colourless oil.

[0311] 1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.47 (m, 3H), 7.33 (m, 2H), 5.92 (m, 1H), 5.31 (d, 1H, J=17.4 Hz), 5.23 (d, 1H, J=10.4 Hz), 4.62 (d, 2H, J=4.9 Hz), 4.20 (m, 2H), 3.62 (m, 2H), 2.25 (t, 2H, J=7.3 Hz), 1.71 (s, 6H), 1.55 (m, 2H), 1.26 (m, 8H), 0.87 (m, 3H); UPLC-MS (ESI): m/z found 412.2105 [M+Na]+, required 412.2100 [C22H31NO5Na].sup.+.

2-methyl-2-(N-(2-(octanoyloxy)ethyl)benzamido)propanoic acid

[0312] ##STR00040##

[0313] Tetrakis(triphenylphosphine)palladium(0) (19.6 mg, 16.9 μmol) and allyl ester 2-(N-(1-(allyloxy)-2-methyl-1-oxopropan-2-yl)benzamido)ethyl octanoate (0.250 mmol) were dissolved in THF (20 mL) and stirred at rt for 5 min. Morpholine (216 μL, 2.50 mmol) was added dropwise to the yellow solution and stirred for 4 h at rt. The resulting green solution was concentrated in vacuo and diluted with ether (10 mL) before being washed with 1 M HCl (aq.) (3×10 mL). The organic layer was filtered through a bed of celite washing thoroughly with ether, dried (MgSO4), concentrated in vacuo to give the desired acid as a pale yellow oil. Used without further purification in the next step. UPLC-MS (ESI) m/z found 400.2105 [M+Na]+, required 400.21 [C21H31NO5Na+].

2-(N-(1-(2-(4-bromophenyl)-2-oxoethoxy)-2-methyl-1-oxopropan-2-yl)benzamido)ethyl octanoate

[0314] ##STR00041##

[0315] EDC (0.374 mmol) was added to a solution of acid N-benzoyl-N-(2-(octanoyloxy)ethyl)glycine (0.250 mmol), DMAP (0.374 mmol) and 1-(4-bromophenyl)-2-hydroxyethan-1-one (0.374 mmol) at 0° C. The suspension was warmed up to rt and stirred for 3 h before being diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with sat. NaHCO.sub.3(aq.) (10 mL) and 1 M HCl (aq.) (10 mL). The organic layer was dried (MgSO4), concentrated in vacuo and purified by flash column chromatography (30% EtOAc in heptane) to yield 2-(N-(1-(2-(4-bromophenyl)-2-(λ3-oxidaneylidene)ethoxy)-2-methyl-1-oxopropan-2-yl)benzamido)ethyl octanoate (0.120 mmol, 48%) as a colourless oil.

[0316] 1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.88 (d, 2H, J=8.5 Hz), 7.75 (d, 2H, J=8.5 Hz), 7.46 (m, 3H), 7.38 (m, 2H), 5.46 (s, 2H), 4.21 (m, 2H), 3.67 (m, 2H), 2.28 (t, 2H, J=7.4 Hz), 1,28 (s, 6H), 1.55 (m, 2H), 1.28 (m, 8H), 0.87 (m, 3H). HRMS (ESI): m/z found 574.19 [M+H]+, required 574.18 [C29H37NO6Br]+.

Example 4: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0317] FIG. 8 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

allyl 1-((2-(octanoyloxy)ethyl)amino)cyclopropane-1-carboxylate

[0318] ##STR00042##

[0319] Allyl 1-aminocyclopropane-1-carboxylate (0.628 mmol) and 2-oxoethyl octanoate (0.515 mmol) were dissolved in DCM (5 mL) was stirred for 1 hour before NaBH(OAc).sub.3 (0.802 mmol) were added and the resulting suspension stirred for 17 h. The reaction was quenched with sat. NaHCO.sub.3(aq.) (150 mL) and extracted with CH.sub.2Cl.sub.2 (3×15 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (20-50% EtOAc in heptane) to yield desired amine (0.273 mmol, 53%) as a colourless oil.

[0320] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=5.92 (ddt, J=17.3, 10.7, 5.4 Hz, 1H), 5.31 (dq, J=17.2, 1.7 Hz, 1H), 5.21 (dq, J=10.5, 1.4 Hz, 1H), 4.57 (dt, J=5.5, 1.5 Hz, 2H), 4.03 (t, J=5.7 Hz, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.51 (p, J=7.2 Hz, 2H), 1.32-1.17 (m, 10H), 0.90-0.77 (m, 5H); UPLC-MS (ESI): m/z found 312.4 [M+H].sup.+, required 312.2 [C.sub.17H.sub.30NO.sub.4].sup.+.

allyl 1-(N-(2-(octanoyloxy)ethyl)benzamido)cyclopropane-1-carboxylate

[0321] ##STR00043##

[0322] Benzoyl chloride (0.355 mmol) was added to a solution of triethylamine (98.4 μL, 0.353 mmol) and 2-((1-(allyloxy)-2-methyl-1-oxopropan-2-yl)amino)ethyl octanoate (0.273 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. The solution was warmed up to rt and stirred for 2 h before being quenched with water (10 mL) and extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was washed with 1 M HCl (aq.) (10 mL), sat. NaHCO.sub.3(aq.) (10 mL) and brine (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (25% EtOAc in PE) to yield the desired amide (0.137 mmol, 50%) as a colourless oil.

[0323] δH (500 MHz, DMSO-d.sub.6, 373K): 7.44 (m, 3H), 7.33 (m, 2H), 5.91 (m, 1H), 5.31 (d, 1H, J=17.5 Hz), 5.22 (d, 1H, J=10.5 Hz), 4.62 (d, 2H, J=5.0 Hz), 4.19 (m, 2H), 3.64 (m, 2H), 2.27 (t, 2H, J=7.3 Hz), 1.54 (m, 2H), 1.27 (m, 10H), 0.89 (m, 5H); UPLC-MS (ESI) m/z found 438.25 [M+Na]+, required 438.23 [C24H33NO5Na+].

1-(N-(2-(octanoyloxy)ethyl)benzamido)cyclopropane-1-carboxylic acid

[0324] ##STR00044##

[0325] Tetrakis(triphenylphosphine)palladium(O) (10.7 mg, 9.3 μmol) and allyl ester 2-(N-(1-(allyloxy)-2-methyl-1-oxopropan-2-yl)benzamido)ethyl octanoate (0.137 mmol) were dissolved in THF (15 mL) and stirred at rt for 5 min. Morpholine (118 μL, 1.37 mmol) was added dropwise to the yellow solution and stirred for 4 h at rt. The resulting green solution was concentrated in vacuo and diluted with ether (10 mL) before being washed with 1 M HCl (aq.) (3×10 mL). The organic layer was filtered through a bed of celite washing thoroughly with ether, dried (MgSO4), concentrated in vacuo to give the desired acid as a pale yellow oil. Used without further purification in the next step. UPLC-MS (ESI) m/z found 398.21, [M+Na]+, required 398.19 [C21H29NO5Na].sup.+.

2-(4-bromophenyl)-2-oxoethyl 1-(N-(2-(octanoyloxy)ethyl)benzamido)cyclopropane-1-carboxylate

[0326] ##STR00045##

[0327] EDC (0.374 mmol) was added to a solution of acid allyl 1-((2-(octanoyloxy)ethyl)amino)cyclopropane-1-carboxylate (0.250 mmol), DMAP (0.374 mmol) and 1-(4-bromophenyl)-2-hydroxyethan-1-one (0.374 mmol) at 0° C. The suspension was warmed up to rt and stirred for 3 h before being diluted with CH2Cl2 (10 mL) and washed with sat. NaHCO3(aq.) (10 mL) and 1 M HCl (aq.) (10 mL). The organic layer was dried (MgSO4), concentrated in vacuo and purified by flash column chromatography (30% EtOAc in heptane) to yield 2-(4-bromophenyl)-2-oxoethyl 1-(N-(2-(octanoyloxy)ethyl)benzamido)cyclopropane-1-carboxylate (0.120 mmol, 48%) as a colourless oil.

[0328] 1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.90 (d, 2H, J=8.6 Hz), 7.74 (d, 2H, J=8.6 Hz), 7.45 (m, 3H), 7.38 (m, 2H), 5.46 (s, 2H), 4.21 (m, 2H), 3.67 (m, 2H), 2.28 (t, 2H, J=7.3 Hz), 1.55 (m, 2H), 1.28 (m, 8H), 1.18-0.87 (m, 7H). UPLC-MS (ESI) m/z found 572.18 [M+H].sup.+, required 572.16 [C29H35NO6Br].sup.+.

Example 5: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0329] FIG. 8 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

2-((4-((Allyloxy)carbonyl)heptan-4-yl)amino)ethyl octanoate

[0330] ##STR00046##

[0331] Allyl 2-amino-2-propylpentanoate (0.628 mmol) and 2-oxoethyl octanoate (0.515 mmol) were dissolved in DCM (5 mL) was stirred for 1 hour before NaBH(OAc).sub.3 (0.802 mmol) were added and the resulting suspension stirred for 17 h. The reaction was quenched with sat. NaHCO.sub.3(aq.) (150 mL) and extracted with CH.sub.2Cl.sub.2 (3×15 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (20-50% EtOAc in heptane) to yield desired amine (0.144 mmol, 23%) as a colourless oil.

[0332] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=5.92 (ddt, J=17.3, 10.7, 5.4 Hz, 1H), 5.31 (dq, J=17.2, 1.7 Hz, 1H), 5.21 (dq, J=10.5, 1.4 Hz, 1H), 4.57 (dt, J=5.5, 1.5 Hz, 2H), 4.03 (t, J=5.7 Hz, 2H), 3.39 (s, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.53 (m, 6H), 1.32-1.17 (m, 12H), 0.90-0.77 (m, 9H); UPLC-MS (ESI): m/z found 370.4 [M+H].sup.+, required 370.3 [C.sub.21H.sub.40NO.sub.4].sup.+.

2-(N-(4-((Allyloxy)carbonyl)heptan-4-yl)benzamido)ethyl octanoate

[0333] ##STR00047##

[0334] Benzoyl chloride (0.188 mmol) was added to a solution of triethylamine (52.2 μL, 0.187 mmol) and 2-((4-((allyloxy)carbonyl)heptan-4-yl)amino)ethyl octanoate (0.144 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. The solution was warmed up to rt and stirred for 2 h before being quenched with water (10 mL) and extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was washed with 1 M HCl (aq.) (10 mL), sat. NaHCO.sub.3(aq.) (10 mL) and brine (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (40% EtOAc in PE) to yield the desired amide (0.089 mmol, 62%) as a colourless oil.

[0335] δH (500 MHz, DMSO-d.sub.6): 7.45 (m, 3H), 7.35 (m, 2H), 5.92 (m, 1H), 5.31 (d, 1H, J=17.6 Hz), 5.23 (d, 1H, J=10.6 Hz), 4.62 (d, 2H, J=5.0 Hz), 4.20 (m, 2H), 3.64 (m, 2H), 2.27 (t, 2H, J=7.3 Hz), 1.67 (m, 4H), 1.54 (m, 2H, H15), 1.27 (m, 12H), 0.88 (m, 9H, H20); UPLC-MS (ESI) m/z found 496.33 [M+Na]+, required 496.30 [C28H43NO5Na]+.

2-(N-(2-(octanoyloxy)ethyl)benzamido)-2-propylpentanoic acid

[0336] ##STR00048##

[0337] Tetrakis(triphenylphosphine)palladium(O) (7.0 mg, 6.1 μmol) and allyl ester 2-(N-(4-((allyloxy)carbonyl)heptan-4-yl)benzamido)ethyl octanoate (0.089 mmol) were dissolved in THF (10 mL) and stirred at rt for 5 min. Morpholine (77 μL, 0.89 mmol) was added dropwise to the yellow solution and stirred for 4 h at rt. The resulting green solution was concentrated in vacuo and diluted with ether (10 mL) before being washed with 1 M HCl (aq.) (3×10 mL). The organic layer was filtered through a bed of celite washing thoroughly with ether, dried (MgSO.sub.4), concentrated in vacuo to give the desired acid as a pale yellow oil. Used without further purification in the next step. UPLC-MS (ESI) m/z found 352.23 [M+Na].sup.+, required 362.25 [C18H35NO4Na].sup.+.

2-(N-(4-((2-(4-bromophenyl)-2-oxoethoxy)carbonyl)heptan-4-yl)benzamido)ethyl octanoate

[0338] ##STR00049##

[0339] EDC (0.137 mmol) was added to a solution of acid 2-(N-(2-(octanoyloxy)ethyl)benzamido)-2-propylpentanoic acid (0.089 mmol), DMAP (0.133 mmol) and 1-(4-bromophenyl)-2-hydroxyethan-1-one (0.133 mmol) at 0° C. The suspension was warmed up to rt and stirred for 3 h before being diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with sat. NaHCO3(aq.) (10 mL) and 1 M HCl (aq.) (10 mL). The organic layer was dried (MgSO4), concentrated in vacuo and purified by flash column chromatography (50% EtOAc in heptane) to yield 2-(N-(4-((2-(4-bromophenyl)-2-oxoethoxy)carbonyl)heptan-4-yl)benzamido)ethyl octanoate (0.034 mmol, 38%) as a colourless oil.

[0340] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.88 (d, 2H, J=8.5 Hz), 7.75 (d, 2H, J=8.5 Hz), 7.46 (m, 3H), 7.38 (m, 2H), 5.46 (s, 2H), 4.21 (m, 2H), 3.67 (m, 2H), 2.28 (t, 2H, J=7.4 Hz), 1,86 (m, 4H), 1.55 (m, 2H, H13), 1.28 (m, 12H), 0.87 (m, 9H). UPLC-MS (ESI) m/z found 652.24 [M+Na].sup.+, required 652.22 [C33H44BrNO6Na].sup.+.

Example 6: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0341] FIG. 8 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

2-((2-(allyloxy)-2-oxo-1,1-diphenylethyl)amino)ethyl octanoate

[0342] ##STR00050##

[0343] Allyl 2-amino-2,2-diphenylacetate (0.628 mmol) and 2-oxoethyl octanoate (0.515 mmol) were dissolved in DCM (5 mL) was stirred for 1 hour before NaBH(OAc).sub.3 (0.802 mmol) were added and the resulting suspension stirred for 17 h. The reaction was quenched with sat. NaHCO.sub.3(aq.) (150 mL) and extracted with CH.sub.2Cl.sub.2 (3×15 mL). The combined organic layers were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (20-50% EtOAc in heptane) to yield desired amine (0.200 mmol, 32%) as a colourless oil.

[0344] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=7.37-7.29 (m, 10H), 5.93 (ddt, J=17.3, 10.7, 5.4 Hz), 5.30 (dq, J=17.1, 1.8 Hz, 1H), 5.21 (dq, J=10.5, 1.4 Hz, 1H), 4.57 (dt, J=5.5, 1.5 Hz, 2H), 4.03 (t, J=5.7 Hz, 2H), 3.39 (s, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.51 (p, J=7.2 Hz, 2H), 1.32-1.17 (m, 8H), 0.90-0.77 (m, 3H); UPLC-MS (ESI) m/z required 438.3 [M+H].sup.+, required 438.3 [C.sub.27H.sub.36NO.sub.4].sup.+.

2-(N-(2-(allyloxy)-2-oxo-1,1-diphenylethyl)benzamido)ethyl octanoate

[0345] ##STR00051##

[0346] Benzoyl chloride (0.261 mmol) was added to a solution of triethylamine (72.6 μL, 0.260 mmol) and 2-((2-(allyloxy)-2-oxo-1,1-diphenylethyl)amino)ethyl octanoate (0.200 mmol) in CH.sub.2Cl.sub.2 (1 mL) at 0° C. The solution was warmed up to rt and stirred for 2 h before being quenched with water (10 mL) and extracted with CH2Cl2 (20 mL). The organic layer was washed with 1 M HCl (aq.) (10 mL), sat. NaHCO3(aq.) (10 mL) and brine (10 mL). The organic layer was dried (MgSO4), concentrated in vacuo and purified by flash column chromatography (60% EtOAc in heptane) to yield the desired amide (0.118 mmol, 59%) as a colourless oil.

[0347] 1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.45 (m, 3H), 7.36 (m, 2H), 7.35-7.28 (m, 10H), 5.92 (m, 1H), 5.31 (d, 1H, J=17.6 Hz), 5.23 (d, 1H, J=10.6 Hz), 4.62 (d, 2H, J=5.0 Hz), 4.20 (m, 2H), 3.64 (m, 2H), 2.27 (t, 2H, J=7.3 Hz), 1.54 (m, 2H), 1.28 (m, 8H), 0.88 (m, 3H); UPLC-MS (ESI) m/z found 564.25 [M+Na].sup.+, required 564.27 [C34H39NO5Na].sup.+.

2-(N-(2-(octanoyloxy)ethyl)benzamido)-2,2-diphenylacetic acid

[0348] ##STR00052##

[0349] Tetrakis(triphenylphosphine)palladium(O) (7.0 mg, 6.1 μmol) and allyl ester 2-(N-(2-(allyloxy)-2-oxo-1,1-diphenylethyl)benzamido)ethyl octanoate (0.089 mmol) were dissolved in THF (10 mL) and stirred at rt for 5 min. Morpholine (77 μL, 0.89 mmol) was added dropwise to the yellow solution and stirred for 4 h at rt. The resulting solution was concentrated in vacuo and diluted with ether (10 mL) before being washed with 1 M HCl (aq.) (3×10 mL). The organic layer was filtered through a bed of celite washing thoroughly with ether, dried (MgSO4), concentrated in vacuo to give the desired acid as a pale yellow oil. Used without further purification in the next step. LCMS (ESI) m/z found 524.23 [M+Na].sup.+, required 524.24 [C31H35NO5Na].sup.+.

2-(N-(2-(2-(4-bromophenyl)-2-oxoethoxy)-2-oxo-1,1-diphenylethyl)benzamido)ethyl octanoate

[0350] ##STR00053##

[0351] EDC (0.137 mmol) was added to a solution of acid 22-(N-(2-(octanoyloxy)ethyl)benzamido)-2,2-diphenylacetic acid (0.089 mmol), DMAP (0.133 mmol) and 1-(4-bromophenyl)-2-hydroxyethan-1-one (0.133 mmol) at 0° C. The suspension was warmed up to rt and stirred for 3 h before being diluted with CH2Cl2 (10 mL) and washed with sat. NaHCO3(aq.) (10 mL) and 1 M HCl (aq.) (10 mL). The organic layer was dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (50% EtOAc in heptane) to yield 2-(N-(2-(2-(4-bromophenyl)-2-oxoethoxy)-2-oxo-1,1-diphenylethyl)benzamido)ethyl octanoate (0.034 mmol, 38%) as a colourless oil.

[0352] 1H-NMR (500 MHz, DMSO-d.sub.6): δ=7.87 (d, 2H, J=8.5 Hz, H3), 7.76 (d, 2H, J=8.5 Hz, H2), 7.48 (m, 3H, H21 and H23), 7.36 (m, 2H, H22), 7.35-7.7.26 (m, 10H, 5.46 (s, 2H), 4.21 (m, 2H), 3.67 (m, 2H), 2.28 (t, 2H, J=7.5 Hz), 1.55 (m, 2H), 1.26 (m, 8H), 0.85 (m, 3H). UPLC-MS (ESI): m/z found 720.22 [M+Na].sup.+, required 720.19 [C39H40BrNO6Na].sup.+.

Example 7: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0353] FIG. 9 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

tert-butyl (2-hydroxyethyl)glycinate

[0354] ##STR00054##

[0355] To a 0° C. solution of 2-aminoethanol (2.00 g, 32.8 mmol) and K.sub.2CO.sub.3 (22.60 g, 164 mmol) in 80 mL acetonitrile was added a solution of tert-butyl 2-bromoacetate (32.8 mmol) in 2 mL of acetonitrile over a 1 h period. The solution was stirred for 1 h at 0° C. and filtered. The filtrate was concentrated in vacuo to give a residue which was subjected to flash column chromatography (0% to 10% MeOH in EtOAc) to yield the desired product in 44%.

[0356] R.sub.f=0.16 (5% MeOH in EtOAc); .sup.1H NMR (400 MHz, Chloroform-d) δ=3.62-3.56 (m, 2H), 3.29 (s, 2H), 2.78-2.69 (m, 2H), 1.43 (d, J=3.0 Hz, 9H); .sup.13C NMR (101 MHz, Chloroform-d) δ=172.12, 81.49, 60.99, 51.29, 51.16, 28.17; UPLC-MS (ESI) m/z found 176.15 [M+H].sup.+, required 176.13 [C8H18NO3].sup.+.

tert-butyl N-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoyl)-N-(2-hydroxyethyl)glycinate

[0357] ##STR00055##

[0358] A suspension of 4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoic acid (5.97 g, 16.0 mmol), DIPEA (14.0 mL, 79.9 mmol) and tert-butyl (2-hydroxyethyl)glycinate (2.8 g, 16.0 mmol) in DMF (40 mL) was stirred for 5 min before HATU (5.85 g, 16.0 mmol) was added at rt. After 12 h the reaction was diluted with 75 mL EtOAc and washed with brine (4×75 mL), NH4Cl (75 mL) and NaHCO.sub.3(75 mL). The organic phases were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (50% to 100% EtOAc in heptane) to yield the desired product (77%) as a white solid.

[0359] R.sub.f=0.34 (EtOAc); .sup.1H NMR (400 MHz, Chloroform-d) δ=7.76 (d, J=7.6 Hz, 2H), 7.60 (d, J=7.5 Hz, 2H), 7.50 (d, J=7.8 Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.35-7.25 (m, 4H), 5.18 (s, 1H), 4.48 (d, J=6.8 Hz, 2H), 4.39 (d, J=5.9 Hz, 2H), 4.23 (t, J=6.7 Hz, 1H), 4.10 (s, 2H), 3.61 (t, J=5.0 Hz, 2H), 3.48 (t, J=4.9 Hz, 2H), 1.48 (d, J=38.6 Hz, 9H); .sup.13C NMR (101 MHz, Chloroform-d) δ=172.57, 170.86, 156.61, 143.98, 141.47, 140.13, 134.85, 128.07, 127.84, 127.49, 127.20, 125.13, 120.13, 83.19, 66.84, 59.40, 53.31, 49.16, 47.43, 44.80, 28.15; UPLC-MS (ESI) m/z found 552.9 [M+Na].sup.+, required 553.2 [C.sub.31H.sub.34N.sub.2NaO.sub.6].sup.+.

2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-N-(2-(tert-butoxy)-2-oxoethyl)benzamido)ethyl octanoate

[0360] ##STR00056##

[0361] Octanoyl chloride (2.19 mL, 12.9 mmol) was added dropwise to a solution of tert-butyl N-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoyl)-N-(2-hydroxyethyl)glycinate (1.37 g, 2.57 mmol) and DIPEA (4.48 mL, 25.7 mmol) in CH.sub.2Cl.sub.2 (50 mL) at rt. After 14 h the reaction was quenched with H.sub.2O (100 mL) and extracted with CH.sub.2Cl.sub.2 (4×60 mL). The combined organic layers were washed with H.sub.2O (150 mL), brine (3×150 mL), NH.sub.4Cl (150 mL), NaHCO.sub.3 (150 mL) and brine (150 mL), dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by flash column chromatography (100% heptane to 100% EtOAc) to yield the desired product (1.12 mg, 1.70 mmol, 66%) as a yellow oil.

[0362] R.sub.f=0.73 (EtOAc); .sup.1H NMR (400 MHz, Chloroform-d) δ=7.77 (d, J=7.5 Hz, 2H), 7.60 (d, J=7.5 Hz, 2H), 7.46-7.26 (m, 8H), 5.10 (d, J=6.2 Hz, 1H), 4.49 (d, J=6.8 Hz, 2H), 4.40 (d, J=5.3 Hz, 2H), 4.36 (s, OH), 4.23 (t, J=6.8 Hz, 1H), 4.19 (s, 1H), 4.11 (dd, J=8.6, 5.7 Hz, 1H), 3.93 (s, 1H), 3.79 (d, J=5.7 Hz, 1H), 3.58 (t, J=5.7 Hz, 1H), 2.31 (dt, J=13.1, 7.5 Hz, 2H), 1.61 (dt, J=13.7, 6.8 Hz, 2H), 1.47 (d, J=29.9 Hz, 9H), 1.36-1.16 (m, 8H), 0.94-0.77 (m, 3H); .sup.13C NMR (101 MHz, Chloroform-d) δ=172.21, 168.56, 168.15, 156.61, 143.97, 141.50, 140.41, 135.11, 127.87, 127.63, 127.38, 127.20, 125.12, 120.15, 82.18, 66.88, 60.54, 53.02, 48.18, 47.44, 44.81, 34.25, 31.78, 29.24, 29.06, 28.25, 28.14, 24.93, 22.73, 21.21, 14.35, 14.21; UPLC-MS (ESI) m/z found 657.1 [M+H].sup.+, required 657.4 [C.sub.39H.sub.49N.sub.2O.sub.7].sup.+.

2-(N-(2-(tert-Butoxy)-2-oxoethyl)-4-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)benzamido)ethyl octanoate

[0363] ##STR00057##

[0364] 2-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)methyl)-N-(2-(tert-butoxy)-2-oxoethyl)benzamido)ethyl octanoate (390 mg, 0.59 mmol) was dissolved in diethylamine and DCM (1:1, 7 mL) and stirred at rt until complete Fmoc deprotection (4 h). The reaction mixture was concentrated in vacuo and used directly in next step. The crude tert-butyl N-(4-(aminomethyl)benzoyl)-N-(2-hydroxyethyl)glycinate was dissolved in anhydrous DMF (10 mL) and stirred under nitrogen atmosphere. 3-(2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (100 mg, 0.59 mmol), HATU (271 mg, 0.71 mmol) and anhydrous DIPEA (517 μL, 2.97 mmol) were added and the solution was stirred at rt for 3 h. The reaction was diluted with EtOAc (50 mL) and washed with water (60 mL), brine (4×60 mL), NH.sub.4Cl (60 mL), NaHCO.sub.3(60 mL) and brine (60 mL). The organic phases were dried over MgSO.sub.4, concentrated in vacuo and purified by flash column chromatography (heptane EtOAc+5% MeOH) to yield the title compound as a yellow oil (152 mg, 44%).

[0365] R.sub.f=0.36 (EtOAc); .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.50 (t, J=5.6 Hz, 1H), 7.35-7.22 (m, 4H), 7.01 (s, 2H), 4.23 (dt, J=12.1, 5.9 Hz, 4H), 4.08 (s, 2H), 3.65 (tt, J=7.4, 4.5 Hz, 4H), 2.43 (t, J=7.3 Hz, 2H), 2.29-2.25 (m, 2H), 1.43 (s, 9H), 1.30-1.19 (m, 10H), 0.85 (t, J=6.5 Hz, 3H); UPLC-MS (ESI) m/z found 686.8 [M+H].sup.+, calc. 686.7 [C.sub.31H.sub.44N.sub.3O.sub.8].sup.+.

2-(N-(2-((2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)-2-oxoethyl)-4-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)benzamido)ethyl octanoate

[0366] ##STR00058##

[0367] 2-(N-(2-(tert-Butoxy)-2-oxoethyl)-4-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)benzamido)ethyl octanoate (150 mg, 0.26 mmol) was dissolved in TFA/DCM (4:6, 5 mL) and stirred at rt until complete tert-butyl deprotection (2 h). The reaction mixture was concentrated in vacuo and co-evaporated with THF (×7). EDC (226 μL, 1.27 mmol) and HOBt (62 mg, 0.51 mmol) were added to a solution of the crude N-(4-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)benzoyl)-N-(2-(octanoyloxy)ethyl)glycine in anhydrous DCM (3 mL). Additionally, DIPEA (111 μL, 0.63 mmol) was added to achieve a basicity of pH 7-8. A solution of quercitin (77 mg, 0.25 mmol) in anhydrous DMF (1 mL) was added at rt, and the solution was stirred under nitrogen atmosphere overnight. The crude reaction mixture was purified directly using preparative HPLC (linear gradient) and lyophilized yielding the title compound as a yellow solid (64 mg, 31%).

[0368] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.38 (d, J=23.4 Hz, 1H), 10.90-10.77 (m, 1H), 10.50 (d, J=54.3 Hz, 1H), 9.67 (d, J=48.3 Hz, 1H), 8.51 (t, J=5.9 Hz, 1H), 7.97 (d, J=8.6, 2.3 Hz, 1H), 7.42-7.26 (m, 5H), 7.10 (d, J=8.7 Hz, 1H), 7.00 (s, 2H), 6.44 (d, J=2.1 Hz, 1H), 6.19 (d, J=2.0 Hz, 1H), 4.56 (d, J=8.8 Hz, 1H), 4.47-4.39 (m, 1H), 4.36-4.20 (m, 3H), 4.19-4.10 (m, 1H), 3.86-3.74 (m, 1H), 3.69-3.58 (m, 3H), 2.43 (t, J=7.3 Hz, 2H), 2.34-2.18 (m, 2H), 1.58-1.38 (m, 2H), 1.22-1.13 (m, 8H), 0.87-0.79 (m, 3H); UPLC-MS (ESI) m/z found 814.8 [M+H].sup.+, calc. 814.8 [C.sub.42H.sub.44N.sub.3O.sub.14].sup.+.

Example 8: Synthesis of an Enzyme Cleavable Linker of Formula (I)

[0369] FIG. 10 shows a synthetic pathway for an enzyme cleavable linker of formula (I) comprising the synthetic procedures outlined below.

Ethyl (2-hydroxyethyl)glycinate

[0370] ##STR00059##

[0371] To a 0° C. solution of 2-aminoethanol (32.8 mmol) and K.sub.2CO.sub.3 (164 mmol) in 80 mL acetonitrile was added a solution of ethyl bromoacetate (32.8 mmol) in 2 mL of acetonitrile over a 1 h period. The solution was stirred for 1 h at 0° C. and filtered. The filtrate was concentrated in vacuo to give a residue which was subjected to flash column chromatography (0% to 10% MeOH in EtOAc) to yield the desired product in 41%.

[0372] R.sub.f=0.16 (5% MeOH in EtOAc); .sup.1H NMR (400 MHz, Chloroform-d): δ=5.48 (bs, 1H), 4.71 (bs, 1H), 4.15 (quart, 2H), 3.58 (s, 2H), 3.51 (m, 2H), 2.74 (m, 2H), 1.31 (t, 2H); UPLC-MS (ESI) m/z found 148.13 [M+H].sup.+, required 148.10 [C6H14NO.sub.3].sup.+.

Ethyl N-(4-(((tert-butoxycarbonyl)amino)methyl)benzoyl)-N-(2-hydroxyethyl)glycinate

[0373] ##STR00060##

[0374] A suspension of 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (16.0 mmol), DIPEA (14.0 mL, 79.9 mmol) and Ethyl (2-hydroxyethyl)glycinate (16.0 mmol) in DMF (40 mL) was stirred for 5 min before HATU (5.85 g, 16.0 mmol) was added at rt. After 12 h the reaction was diluted with 75 mL EtOAc and washed with brine (4×75 mL), NH.sub.4Cl (75 mL) and NaHCO.sub.3(75 mL). The organic phases were dried (MgSO.sub.4), concentrated in vacuo and purified by flash column chromatography (50% to 100% EtOAc in heptane) to yield the desired product (39%) as a white oil.

[0375] R.sub.f=0.34 (EtOAc); .sup.1H NMR (400 MHz, Chloroform-d) δ=7.48 (d, J=7.8 Hz, 2H), 7.31 (d, J=7.5 Hz, 3H), 4.97-4.82 (m, 1H), 4.30 (dt, J=21.3, 6.6 Hz, 4H), 4.19 (s, 2H), 3.62 (t, J=4.8 Hz, 2H), 3.52 (t, J=4.8 Hz, 2H), 1.46 (s, 9H), 1.33 (t, J=7.1 Hz, 3H); .sup.13C NMR (101 MHz, Chloroform-d) δ=172.79, 171.73, 156.05, 140.84, 134.51, 127.96, 127.83, 127.46, 127.17, 79.88, 62.18, 59.56, 53.38, 48.49, 44.42, 28.53, 14.24; UPLC-MS (ESI) m/z found 403.21 [M+Na].sup.+, required 403.18 [C.sub.19H.sub.28N.sub.2NaO.sub.6].sup.+.

Example 9: Synthesis of an Enzyme Cleavable Linker of Formula (II)

[0376] FIG. 11 shows a synthetic pathway for an enzyme cleavable linker of formula (II) comprising the synthetic procedures outlined below.

(4-Azidophenyl)methanol

[0377] ##STR00061##

[0378] 4-aminobenzylalcohol (1.0 g, 8.12 mmol, 1.0 eq.) was dissolved in 8.1 mL 6 M HCl and cooled in an ice bath to 0° C. A solution of NaNO.sub.2 (0.84 g, 12.2 mmol, 1.5 eq.) in 21 mL H2O cooled to 0° C. was added dropwise. The yellow reaction mixture was stirred at 0° C. for 30 min. before dropwise addition of NaN3 (2.1 g, 32.5 mmol, 4.0 eq.) in 41 mL H2O keeping the temperature below 0° C. After 15 min. at 0° C., the reaction was allowed to slowly reach rt and was stirred for additionally 2 h. The reaction was quenched with 100 mL sat. aqueous NaHCO.sub.3 and stirred for 30 min. before extraction with Et2O (3×100 mL). The organic phase was dried with MgSO.sub.4 and concentrated in vacuo. The residue was dissolved in 3 mL EtOAc:Heptane (1:2) and purified through a plug of SiO2 using the same eluent system yielding the title compound as a light-yellow oil (3.60 g, 99%).

[0379] .sup.1H-NMR (400 MHz, Chloroform-d) δ 7.28 (d, J=8.5 Hz, 2H), 6.97 (d, J=8.5 Hz, 2H), 4.56 (s, 2H), 2.88 (s, 1H). .sup.13C-NMR (101 MHz, Chloroform-d) δ 139.27, 137.58, 128.52, 119.07, 64.44. UPLC-MS (ESI): m/z found 172.0 [M+Na].sup.+, required 172.1 [C7H7N3NaO].sup.+.

4-Azidobenzaldehyde

[0380] ##STR00062##

[0381] (4-azidophenyl)methanol (3.60 g, 24.14 mmol, 1 eq.) was dissolved in 125 mL dry CH.sub.2Cl.sub.2 followed by addition of Dess—Martin periodinane (15.78 g, 37.21 mmol, 1.5 eq.) and the reaction mixture was stirred for 18 h at rt at which state oxidation was completed. The reaction mixture was diluted with EtOAc (100 mL) resulting in biproduct being precipitated. H2O was added resulting in more biproduct precipitating. The reaction mixture was filtrated and washed with CH.sub.2Cl.sub.2 followed by wash with sat. aqueous Na.sub.2S.sub.2O.sub.3 (150 mL), sat. aqueous NaHCO.sub.3 (150 mL), and brine (150 mL). The organic layer was dried with Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was purified by plug on SiO.sub.2 using EtOAc:Heptane (1:4) yielding the title compound as a brown oil (3.29 g, 93%).

[0382] .sup.1H-NMR (400 MHz, Chloroform-d) δ 9.92 (s, 1H), 7.86 (d, J=8.5 Hz, 2H), 7.14 (d, J=8.5 Hz, 2H). .sup.13C-NMR (101 MHz, Chloroform-d) δ 190.62, 146.32, 133.31, 131.60, 119.54.

tert-Butyl (4-azidobenzyl)(2-hydroxyethyl)carbamate

[0383] ##STR00063##

[0384] Ethanol amine (0.411 mL, 6.80 mmol, 1 eq.) was dissolved in dry MeOH (10 mL) under nitrogen atmosphere. MgSO.sub.4 (1.64 g, 13.63 mmol, 2 eq.) was added followed by addition of 4-azidobenzaldehyde (1.00 g, 6.80 mmol, 1 eq.) dissolved in 3 mL dry MeOH, and the reaction mixture was stirred for 20 h. The reaction mixture was cooled to 0° C. and NaBH4 (0.257 g, 6.80 mmol, 1 eq.) was added portion wise. The reaction was stirred for 1.5 h before quenching with H2O. The reaction was extracted with EtOAc (5×100 mL) and the combined organic layers were washed with brine (1×500 mL). The organic layer was dried over MgSO.sub.4 and concentrated in vacuo. The crude was dissolved in sat. aq. NH4Cl:dioxane (1:1) (20 mL) followed by addition of Boc anhydride (1.78 g, 8.16 mmol, 1.2 eq.) and the reaction mixture was stirred for 20 h. The reaction mixture was extracted with EtOAc (3×50 mL) and the combined organic phases were dried over Na2SO4 followed by concentration in vacuo. The crude product was purified by flash chromatography on SiO2 using EtOAc:Heptane (2:3) yielding the title compound as a brown oil (1.36 g, 68%).

[0385] (400 MHz, Chloroform-d) δ 7.23 (d, J=7.9 Hz, 2H), 6.99 (d, J=8.0 Hz, 2H), 4.46 (s, 2H), 3.77-3.62 (m, 2H), 3.50-3.23 (m, 2H), 2.81 (s, 1H), 1.47 (s, 9H). .sup.13C-NMR (101 MHz, Chloroform-d) δ 157.19, 139.14, 135.16, 128.77, 119.24, 80.81, 62.00, 51.58, 49.83, 28.46. UPLC MS (ESI) m/z found 291.2 required 291.15 [C14H19N4O3].sup.−.

tert-Butyl (4-azidobenzyl)(2-((bis(benzyloxy)phosphoryl)oxy)ethyl)carbamate

[0386] ##STR00064##

[0387] Dibenzyl phosphorochloridate was prepared in situ by reacting N-chloro-succinimide (1.86 mg, 13.95 mmol, 1 eq.) and dibenzyl phosphonate (3.07 mL, 13.95 mmol, 1 eq.) in anhydrous toluene (25 mL) under inert atmosphere for 2 h at rt. The reaction mixture was filtered to remove succinimide. To a solution of tert-butyl (4-azidobenzyl)(2-hydroxyethyl)carbamate (1.36 g, 4.65 mmol, 1 eq.) in anhydrous pyridine (25 mL) at −40° C. under inert atmosphere was added dropwise a solution of dibenzyl phosphorochloridate in toluene. The reaction mixture was stirred for 2 h at −40° C. before it was placed in the freezer at −20° C. for 20 h. The reaction mixture was allowed to warm to rt and was subsequently quenched with H2O (20 mL) before concentration and co-evaporation with heptane in vacuo. The resulting crude residue was re-dissolved in EtOAC (200 mL) and the organic layer was washed with 1 M H2SO4 (2×75 mL) and sat. aq. NaHCO.sub.3(2×75 mL). The organic layer was dried over Na2SO4 followed by concentration in vacuo. The crude product was purified by flash chromatography on SiO.sub.2 using EtOAc:Heptane (2:3) yielding the title compound as a brown oil (1.61 g, 63%).

[0388] 1H-NMR (400 MHz, DMSO-d.sub.6) δ 7.42-7.31 (m, 10H), 7.22 (d, J=8.4 Hz, 2H), 7.06 (d, J=8.0 Hz, 2H), 5.01 (d, J=8.0 Hz, 4H), 4.34 (s, 2H), 4.08-3.95 (m, 2H), 3.32 (d, J=7.8 Hz, 2H), 1.35 (d, J=21.4 Hz, 9H). 13C-NMR (101 MHz, DMSO-d.sub.6) δ 154.88, 138.16, 136.04, 135.97, 129.01, 128.72, 128.49, 128.41, 127.81, 119.16, 79.38, 68.61, 68.55, 64.89, 27.92. 31P-NMR (162 MHz, DMSO-d.sub.6) δ −0.98 (d, J=10.5 Hz). UPLC MS (ESI): m/z found 553.6 [M+H].sup.+, required 553.22 [C28H34N4O6P].sup.+.

Example 10: Synthesis of an enzyme cleavable linker of formula (II)

[0389] FIG. 12 and FIG. 13 show a synthetic pathway for an enzyme cleavable linker of formula (II) comprising the synthetic procedures outlined below.

Neopentyl chlorosulfate

[0390] ##STR00065##

[0391] Sulfuryl chloride (4.3 mL, 53.0 mmol, 1 eq.) in dry DCM (20 mL) was cooled to −78 degrees C. in a dry ice/acetone bath in N.sub.2-atmosphere. A solution of neopentyl alcohol (6.68 g, 75.8 mmol, 1.4 eq.) and freshly distilled pyridine (4.3 mL, 53.0 mmol, 1 eq.) in dry DCM (20 mL) was added through a separation funnel dropwise (<1 drop/sec for the first ˜5 mL and then ˜2 drops/sec for the remaining solution). The reaction mixture stirred for 5 hours at RT and was filtered with suction three times and concentrated in vacuo. The crude product was carefully distilled at reduced pressure to give the desired product in yield 69%.

[0392] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=4.23 (s, 2H), 1.10 (s, 9H). .sup.13C-NMR (101 MHz, CDCl.sup.3-d): δ=85.50, 31.94, 25.90. UPLC-MS (ESI) m/z found 168.8 [M−H.sub.2O].sup.−, required 168.0 [C.sub.5H.sub.9ClO.sub.2S].sup.−.

4-(((Tert-butyldimethylsilyl)oxy)methyl)phenol

[0393] ##STR00066##

[0394] To a dry flask, 4-hydroxymethylphenol (5 g, 40.28 mmol, 1 eq.) was dissolved in dry THF (42 mL) and cooled in an ice-bath. Imidazole (5.48 g, 80.56 mmol, 2 eq.) was added and the reaction mixture was left stirring for 10 minutes. Then tert-butylchlorodimethylsilane (7.28 g, 48.33 mmol, 1.2 eq) was added. The reaction stirred at room temperature for 2 hours until LCMS showed full consumption of the starting material. The mixture was quenched with ammonium chloride (15 mL) and extracted with ethyl acetate (3×60 mL). The combined organic layer was dried with anhydrous magnesium sulfate, filtered and concentrated in vacuo. The product was purified by column chromatography on silica gel using 4:1 heptane/EtOAc as eluent to give the desired product 1 in 70% yield.

[0395] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.21 (dd, J=21.4, 8.0 Hz, 2H), 6.80 (d, 1H), 4.69 (s, 2H), 0.96 (s, 9H), 0.12 (s, 6H). 13C-NMR (101 MHz, CDCl.sub.3): δ=154.62, 133.50, 127.87, 115.11, 64.83, 26.00, 18.46, 5.15. UPLC-MS (ESI): m/z found 237.0 [M−H].sup.−, required 237.1 [C.sub.13H.sub.21O.sub.2Si].sup.−.

4-(((Tert-butyldimethylsilyl)oxy)methyl)phenyl neopentyl sulfate

[0396] ##STR00067##

[0397] A solution of 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (2.29 g, 9.62 mmol, 1 eq.) in DMPU (9.6 mL) and THF (28 mL) and was cooled in a dry ice and acetone bath. NaHMDS (10.6 mL, 1 M in THF, 10.56 mmol, 1.1 eq) was added over 4 minutes and the solution was left stirring for 15 minutes before neopentyl chlorosulfate (1.7 mL, 10.56 mmol, 1.1 eq.) was added and the reaction stirred for further 15 minutes in ice bath and 20 minutes at RT. TLC indicated remaining 4-(((tert-butyldimethyl silyl)oxy)-methyl)phenol, so more neopentyl chlorosulfate (0.85 mL, 5.28 mmol, 0.55 eq.) was added and the reaction mixture was left stirring for 10 minutes and further neopentyl chlorosulfate (0.85 mL) was added on the basis of another TLC test. After 10 minutes, the reaction was quenched with NaHCO3(75 mL). The reaction was extracted with EtOAc (4×50 mL) and H2O (2×40 mL) and 50 mL brine. The combined organic layer was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The product was purified by column chromatography on silica gel by gradually eluating with pure heptane to 17:3 heptane/EtOAc. This resulted in isolation of the product 4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl neopentyl sulfate in 65% yield.

[0398] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.39 (d, J=8.6 Hz, 2H), 7.29 (d, 2H), 4.76 (s, 2H), 4.10 (s, 2H), 1.02 (s, 9H), 0.97 (s, 9H), 0.13 (s, 6H). .sup.13C-NMR (101 MHz, CDCl.sub.3): δ=149.07, 140.73, 127.31, 120.80, 83.37, 64.14, 31.94, 18.39, 5.29. UPLC-MS (ESI): m/z found 387.0 [M−H].sup.−, required 387.2 [C.sub.18H.sub.31O.sub.5SSi].sup.−.

4-(Hydroxymethyl)phenyl neopentyl sulfate

[0399] ##STR00068##

[0400] TBAF (7 mL, 1 M in THF, 6.94 mmol, 1.5 eq.) was added to a solution of 4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl neopentyl sulfate (1.8 g, 4.63 mmol, 1 eq.) in dry THF (10 mL) and left with stirring overnight under N.sub.2 atmosphere. As LCMS indicated consumption of starting material, the reaction was quenched with PBS buffer (35 mL) and diluted with EtOAc (70 mL) the aqueous phase was extracted with EtOAc (3×50 mL) and the combined organic layer washed with H.sub.2O (3×75 mL) and brine (75 mL). The organic layer was dried with anhydrous MgSO.sub.4, filtered and concentrated in vacuo to give 4-(Hydroxymethyl)phenyl neopentyl sulfate in excellent yield of 95%.

[0401] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.43 (d, J=8.5 Hz, 2H), 7.34-7.29 (m, 2H), 4.72 (s, 2H), 4.11 (s, 2H), 1.03 (s, 9H). .sup.13C-NMR (101 MHz, CDCl.sub.3): δ=149.50, 140.09, 128.31, 121.14, 83.48, 64.36, 31.95, 25.96. UPLC-MS (ESI): m/z found 237.0 [M−H].sup.−, required 237.1 [C.sub.12H.sub.17O.sub.5S].sup.−.

Neopentyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl) sulfate

[0402] ##STR00069##

[0403] Pyridine dried over KOH (0.35 mL, 4.22 mmol, 1 eq.) and 4-nitrophenyl chloroformate (0.85 g, 4.22 mmol, 1 eq.) was added to a solution of 4-(hydroxymethyl)phenyl neopentyl sulfate (1.16 g, 4.22 mmol, 1 eq.) in dry DCM (12 mL) under a N2 atmosphere and left stirring for 4 hours. The reaction was diluted with DCM (75 mL) and washed with 1 M HCl(aq.) (50 mL), saturated NaHCO.sub.3(aq.) (75 mL), H.sub.2O (5×75 mL) and brine (75 mL). The organic layer was dried with anhydrous MgSO.sub.4, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography using 7:1 heptane/EtOAc as eluent to obtain neopentyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl) sulfate in yield of 22%.

[0404] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.34-8.26 (m, 2H), 7.57-7.49 (m, 2H), 7.43-7.39 (m, 2H), 7.39-7.35 (m, 2H), 5.32 (s, 2H), 4.13 (s, 2H), 1.03 (s, 9H). .sup.13C-NMR (101 MHz, CDCl.sub.3-d): δ=171.16, 155.39, 152.36, 150.56, 145.48, 133.44, 130.32, 125.34, 121.76, 121.47, 83.67, 69.79, 60.40, 31.96, 25.96, 21.06, 14.20. UPLC-MS (ESI): m/z found 456.6 [M+H.sub.2O]+, required 457.1 [C.sub.19H.sub.23NO.sub.10S].sup.+.

tert-Butyl (2-(benzylamino)ethyl)carbamate

[0405] ##STR00070##

[0406] A solution of N-boc-ethylenediamine (2.0 mL, 12.49 mmol, 1 eq.) and methanol (23 mL) was added anhydrous MgSO.sub.4 (3 g, 24.98 mmol, 2 eq.) and benzaldehyde (1.3 mL, 12.49 mmol, 1 eq.) and left stirring at RT overnight. The solution 4-aminobenzylalcohol (1.0 g, 8.12 mmol, 1.0 eq.) was dissolved in 8.1 mL 6 N HCl and cooled in an ice bath to 0° C. A solution of NaNO.sub.2 was cooled to 0 degrees C. and NaBH4 (0.47 g, 12.49 mmol, 1 eq.) was added portionwise and left stirring for 5 hours before it was quenched with H2O (20 mL). The reaction mixture was extracted with EtOAc (3×50 mL) and the combined organic layer was dried with anhydrous MgSO.sub.4, filtered and concentrated in vacuo. The product was used without further purification.

[0407] UPLC-MS (ESI): m/z found 251.2 [M+H].sup.+, required 251.2 [C.sub.14H.sub.23N.sub.2O.sub.2].sup.+.

Allyl benzyl(2-((tert-butoxycarbonyl)amino)ethyl)carbamate

[0408] ##STR00071##

[0409] tert-Butyl (2-(benzylamino)ethyl)carbamate (3.12 g, 12.48 mmol, 1 eq.) was dissolved in 1:1 dioxane/H2O (50 mL) and cooled to 0 degrees C. NaHCO.sub.3(1.15 g, 13.74 mmol, 1.1 eq.) and allyl chloroformate (3.1 mL, 13.74 mmol, 1.1 eq.) were added. The reaction mixture was left stirring on ice bath for 40 minutes until it was left at room temperature for further 3 hours. Reaction mixture was extracted with EtOAc (3×50 mL) and the combined organic layer was dried with MgSO4, filtered and concentrated in vacuo. Silica filtration using ratio 4:1 heptane/EtOAc to give the desired product in quantitative yield.

[0410] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.47-7.15 (m, 6H), 6.10-5.85 (m, 1H), 5.44-5.14 (m, 2H), 4.79-4.58 (m, 3H), 4.55 (s, 2H), 3.47-3.34 (m, 2H), 3.29 (s, 3H), 1.46 (s, 9H). .sup.13C-NMR (101 MHz, CDCl.sub.3): δ=156.78, 137.53, 132.79, 128.66, 128.20, 127.92, 127.49, 127.31, 126.98, 117.82, 117.58. UPLC-MS (ESI): m/z found 357.0 [M+Na].sup.+, required 357.2 [C.sub.18H.sub.26N.sub.2NaO.sub.4].sup.+.

Allyl (2-aminoethyl)(benzyl)carbamate

[0411] ##STR00072##

[0412] 20% TFA in DCM (20 mL) was added to allyl benzyl(2-((tert butoxycarbonyl)amino)ethyl)carbamate (0.15 g, 0.455 mmol, 1 eq.) and was left stirring for 1.5 hours. The reaction mixture was den coevaporated 9 times with THF. The product was used without purification in the next synthesis step.

[0413] UPLC-MS (ESI): m/z found 235.3 [M+H].sup.+, required 235.1 [C.sub.13H.sub.19N.sub.2O.sub.2].sup.+.

4-(7-Benzyl-3,8-dioxo-2,9-dioxa-4,7-diazadodec-11-en-1-yl)phenyl neopentyl sulfate

[0414] ##STR00073##

[0415] Allyl (2-aminoethyl)(benzyl)carbamate (0.2 g, 0.455 mmol, 1 eq.) and neopentyl (4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl) sulfate (0.107 g, 0.455 mmol, 1 eq.) were combined in dry THF (4 mL). The solution was added DIPEA until pH was basic (in total 2.2 mL). The reaction was left stirring in N2 atmosphere overnight at RT. The reaction was diluted with 20 mL of EtOAc and washed with H2O (10×20 mL) and brine (2×20 mL) to remove 4-nitrophenol. The organic layer was dried with anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified with column chromatography using 8:1 heptane/ethyl acetate as eluent to obtain the title compound in 67% yield.

[0416] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=7.49-7.44 (m, 2H), 7.43-7.38 (m, 3H), 7.36-7.17 (m, 6H), 5.35-5.24 (m, 1H), 5.24-5.09 (m, 2H), 5.04 (s, 2H), 4.60-4.51 (m, 2H), 4.51-4.37 (m, 2H), 4.22 (s, 2H), 3.23 (t, J=6.5 Hz, 3H), 3.20-3.07 (m, 2H), 2.50 (p, J=1.8 Hz, 4H), 0.94 (s, 9H). .sup.13C-NMR (101 MHz, DMSO-d.sup.6): δ=156.49, 149.51, 138.37, 137.41, 133.82, 129.87, 128.97, 127.80, 127.62, 127.43, 121.73, 117.20, 83.84, 65.80, 64.79, 50.53, 32.08, 25.91. UPLC-MS (ESI): m/z found 535.6 [M+H].sup.+, required 535.2 [C.sub.26H.sub.35N.sub.2O.sub.8S].sup.+.

Example 11: Stability Tests

[0417] ##STR00074##

[0418] The above compounds were dissolved in a minimum of DMSO and added to a plasma sample, PBS, or MilliQ water and stored at room temperature. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. No hydrolysis was observed for any of the compounds after 4 days of incubation.

Example 12: Enzyme Tests

Cleavage by Helix pomatia Type H-1

[0419] ##STR00075##

[0420] The above compound was dissolved in a minimum of DMSO and added to Helix pomatia Type H-1 in phosphate buffer solutions at pH 5, 6 and 7.4, respectively at room temperature. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. The compound was almost fully hydrolysed at 5 min.

Cleavage by Acid Phosphatase

[0421] ##STR00076##

[0422] The above compound was dissolved in a minimum of DMSO and added to acid phosphatase in phosphate buffer solutions at pH 5, 6 and 7.4, respectively at room temperature. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. The compound was almost fully hydrolysed at 1 hour.

Cleavage by Human Sulfamidase/SGSH Protein

[0423] ##STR00077##

[0424] The above compound is dissolved in a minimum of DMSO and added to Human Sulfamidase/SGSH Protein in phosphate buffer solutions at pH 5, 6 and 7.4, respectively at room temperature. Aliquots are taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis requires a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allows for direct injection and offers high sensitivity and reliability. Caffeine is included as an internal standard. The compound is almost fully hydrolysed at in 5 min to 1 hour.

Cleavage by Amano Lipase

[0425] ##STR00078##

[0426] The above compound was dissolved in a minimum of DMSO and added to amano lipase in phosphate buffer solutions at pH 5, 6 and 7.4, respectively at room temperature. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. The compound was almost fully hydrolysed at 5 min.

Cleavage by Dipetidyl Aminopeptidase

[0427] ##STR00079##

[0428] The above compound was dissolved in a minimum of DMSO and added to acid phosphatase in phosphate buffer solutions at pH 5, 6 and 7.4, respectively at room temperature. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. The compound was almost fully hydrolysed at 1 hour.

Example 13: Lipase Linker-Drug Conjugate

[0429] In order to generate the linker X8 (see FIG. 15), the synthetic route of FIG. 15 was undertaken. Late-stage attachment of the model drug (step d) would allow a wide variety of probes to be easily investigated. The bromohydroxyketone X7 was chosen as the model probe to attach to the linker. The presence of the aromatic group would allow the release reaction to be monitored by LCMS. In addition, the bromo-substitution increases the molecular weight, aiding ionisation with a characteristic isotope pattern. A benzoyl group would mimic the biomolecule-attachment point.

Synthesis

[0430] Compound X2 (FIG. 15) was assessed from a reductive amination (as described in synthesis of 2-((2-(allyloxy)-2-oxoethyl)amino)ethyl octanoate). With the secondary amine X2 in hand, amide coupling with benzoyl chloride was attempted using benzoyl chloride and triethylamine, step b (as described in synthesis 2-(N-(2-(allyloxy)-2-oxoethyl)benzamido)ethyl octanoate). The Pd(0)-mediated allyl deprotection (step c) proceeded smoothly to furnish X5 (as described in synthesis of N-benzoyl-N-(2-(octanoyloxy)ethyl)glycine). In order to attach the model probe compound (X7), an EDC/DMAP coupling reaction was employed (as described in synthesis 2-(N-(2-(2-(4-bromophenyl)-2-oxoethoxy)-2-oxoethyl)benzamido)ethyl octanoate).

Lipase and Stability Tests

[0431] Compound X8 was exposed to lipase in phosphate buffered solutions at pH 6 and 7.4. The chosen lipase was Amano Lipase from Pseudomonas fluorescens, as a useful indicator as to demonstrate the enzyme cleavage and probe release. The pH 6 and 7.4 tests were conducted to determine if any difference in the rate of intramolecular lactonisation would be observed. Initial tests involved incubation of the linker (1 mM in 2% DMSO/buffer) with 1 mg/mL lipase (>20 U/mL) at room temperature (FIG. 16). Caffeine was included as an internal standard. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis.

[0432] For linker X8 at pH 7.4, full conversion to X10 and X11 was observed in <15 min with no significant accumulation of the intermediate X9. Cleavage of linker X8 showed a non-linear conversion to products over 114 min with negligible accumulation of intermediate X9 (FIG. 17).

[0433] Contrastingly, at pH 6, major accumulation of X9 was observed, with full conversion to X10 and X11 only occurring after 60 min, indicating that the rate of cyclisation was clearly lower at the more acidic pH.

Example 14: Kinetics of Model Prodrug Release Step

[0434] The self-immolative release introduces a second step with its own kinetic parameters. For many applications, the self-immolative step needs to be fast enough in order to ensure a fast release at the activation site. In other applications, a steady and slow release is desired. We have obtained comparative data on the kinetics of the self-immolative step so that anyone can choose a linker suited to their applications (FIG. 1).

[0435] Linker compounds were dissolved in a minimum of MeCN and added to lipase in phosphate buffer solutions at pH 7.4 at room temperature. The chosen lipase was the commercially available Amano lipase from Pseudomonas fluorescence. Aliquots were taken out at different time points and LC-MS and HPLC used for analysis. LCMS analysis required a small organic extraction of each aliquot in order to remove the protein prior to analysis. Analytical HPLC allowed direct injection and offers high sensitivity and reliability. Caffeine was included as an internal standard. Accumulation of the intermediate was observed in several linker analogous. The % release of benzyl alcohol was taken as a measure of extend of ring closure (FIG. 1).

Example 15: Specific Constructs

[0436] Trastuzumab—alkyl sulfate—Auristatin E

[0437] In an example, biomolecule T is the antibody trastuzumab, the enzyme cleavable moiety is an alkyl sulfate (Y is oxygen, R.sup.4 is —S(O).sub.2—OR.sup.7), the rate determining groups R.sup.1 and R.sup.6 are hydrogen. The construct binds to HER2, to induce internalization into the target cell. Lysosomal enzymatic cleavage by upregulated steroid sulfatase and subsequent kinetically controlled ring closure would result in the release of R.sup.3X, in this specific embodiment the highly cytotoxic Auristatin E. Accordingly the goal of killing breast cancer cells could be achieved swiftly and with high selectivity.

[0438] Nanobody-Phosphatase Linker-Drug Conjugate

[0439] Due to their inherently simplified structure when compared to full-sized antibodies, nanobodies, such as HER2 specific 2Rs15d, can be readily expressed in E. coli-a host system that enables facile production of nanobodies in large amounts and at a low cost.

[0440] An exemplary structure is 2Rs15d-L-auristatin E drug conjugate (FIG. 14) targeted for breast cancer. Auristatin E works by acting as a tubulin inhibitor intracellularly to arrest cell division. The nanobody 2Rs15d will ensure receptor mediated endocytosis once the drug conjugate binds to HER2 receptor, overexpressed in 20-30% female breast cancer. Once taken up inside the lysosome of a breast cancer cell, a lysosomal phosphatase will cleave the phosphate ester, thereby releasing a free alcohol, which cyclizes and releases the auristatin E.

[0441] Production and testing of nanobody drug conjugate—overview. (1) expression of 2Rs15d in E. coli, (2) purification of 2Rs15d, (3) synthesis of a phosphatase cleavable linker (L) covalently attached to auristatin E, (4) evaluation of L-auristatin E (Q) stability in buffer, plasma, and buffer containing phosphatase, the latter serving as a model system for lysosomal phosphatase, (5) bioconjugation of L-auristatin E constructs to 2Rs15d and purification of the final drug conjugate, and (7) in vitro testing of the drug conjugate.

[0442] Expression. Biosynthesis of 2Rs15d is performed in E. coli SHuffle® T7 Competent cells able to manufacture the two disulfide bonds present in the 2Rs15d peptide core. The sequence coding for 2Rs15d is cloned into pET-22b(+) vector, which is transfected into E. coli SHuffle® T7 Competent cells. The cells are grown in LB medium with 0.1% glucose and 1 mM MgCl.sub.2 at 37° C. (+ampicilin). After OD.sub.600nm reaches 0.7, induction of expression is realized by adding 250 μL IPTG stock (1 M) (for 0.5 L culture). Growth and expression are continued for 4 hours at 37° C. with shaking (180-250 rpm). After expression, cells are harvested by centrifugation for 15 min at 9,000 xg, supernatant discarded, and pellet stored at −20° C.

[0443] Work up and purification. Cell pellet is thawed for 15 min on ice and resuspended in 10 mL lysis buffer, after which lysozyme (final concentration of 1 mg/mL) and Benzonase® Nuclease solution (final concentration: 25 U/mL) are added. The mixture is incubated on ice for 30 min to achieve complete cell lysis. Lysate is centrifuged at 14,000×g for 30 min at 4° C. to pellet cellular debris. Supernatant containing desired soluble proteins is directly loaded onto Fast Start Columns for purification. After wash, bound His-tagged 2Rs15d is eluted 2 times with elution buffer. The desired fractions containing crude 2Rs15d are subjected to size exclusion chromatography. Purity of purified 2Rs15d is confirmed by SDS page (4-20% Mini-PROTEAN TGX Precast Protein Gels) using appropriate ladder (Precision Plus Protein™ Dual Xtra Prestained Protein Standards). The molecular weight of 2Rs15d is further confirmed by ESI-HRMS.

[0444] Synthesis. Synthetic steps towards a a phosphatase cleavable linker is described in detail in Example 9.

[0445] Stability and release of auristatin E from auristatin E drug conjugate. (a) 0.1 mg auristatin E drug conjugate (FIG. 14) is dissolved in 0.5 mL 1x PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.4) in a 1.5 mL Eppendorf tube fitted with a magnet. The solution is stirred at 37° C., and samples are withdrawn (50 μL) at various time intervals and quenched in liquid nitrogen. Stability is assessed by LCMS, readout is area under the curve (AUC). (b) 0.1 mg auristatin E drug conjugate is dissolved in 0.5 mL human plasma (Sigma Aldrich, P9523) in a 1.5 mL Eppendorf tube fitted with a magnet. The solution is stirred at 37° C., and samples are withdrawn (50 μL) at various time intervals and quenched in liquid nitrogen. Stability is assessed by LCMS, readout is area under the curve (AUC). (c) 0.1 mg auristatin E drug conjugate is dissolved in 0.5 mL 1x PBS containing in a 1.5 mL Eppendorf tube fitted with a magnet, and this mixture is charged with purified recombinant human lysosomal acid phosphatase (ACP2) (MyBioSource, MBS7111128). The solution is stirred at 37° C., and samples are withdrawn (50 μL) at various time intervals and quenched in liquid nitrogen. Stability is assessed by LCMS, readout is area under the curve (AUC).

[0446] Bioconjugation of auristatin E drug conjugate to 2Rs15d to give 2Rs15d-L-auristatin drug conjugate and purification. This procedure is in accordance with literature (Bioconjugate Chem. 2014, 25, 979-988). The maleimide functional group is reacted with the terminal cysteine on 2Rs15d. Prior to bioconjugation and to liberate free cysteines from potential dimer 2Rs15d, 2Rs15d is subjected to mild reducing conditions by subjecting it to 2-mercaptoethylamine (2-MEA, 180 equivalents) at a concentration of 1 mg/mL 2Rs15d, pH 7.4 for 90 min at 37° C. Linker construct auristatin E drug conjugate (10 equivalents) dissolved in 0.2 M NH.sub.40Ac (pH 6.0) is added to freshly reduced 2Rs15d to obtain the desired nanobody-drug conjugate. Finally, the nanobody-drug conjugate is purified using SEC.

[0447] In vitro testing of 2Rs15d-L-auristatin E drug conjugate. The internalization properties of 2Rs15d-L-auristatin E drug conjugate are analyzed by fluorescent marking (Alexa Fluor 488, 532, or 647) of the 2Rs15d-L-auristatin E drug conjugate. Using fluorescence confocal microscopy, receptor-specific internalization the 2Rs15d-L-auristatin E drug conjugate in HER2-positive SK-BR-3 and BT-474 human BC cell lines (HER-negative cells are used as control) is evaluated. Prior to adding the 2Rs15d-L-auristatin E drug conjugate, cells are treated with proteases to strip membrane proteins from the cell surface. Fluorescently marked constructs will also be subjected to flow cytometry to measure cell surface binding to HER2. Next, the cytotoxic effects of the 2Rs15d-L-auristatin E drug conjugate is evaluated; cells are subjected to a dilution series of the 2Rs15d-L-auristatin E drug conjugate. Efficacy of the 2Rs15d-L-auristatin E drug conjugate is estimated based on MTS cytotoxic assay (or other appropriate viability assays) using cell count as readout.

Example 16: Synthesis of a Phosphatase Cleavable Linker of Formula (I)

[0448] The procedures below outline the synthesis of a phosphatase cleavable linker.

tert-Butyl (2-((bis(benzyloxy)phosphoryl)oxy)ethyl)glycinate

[0449] ##STR00080##

[0450] In a pre-dried round-bottom flask, tert-butyl (2-hydroxyethyl)glycinate (0.40 g, 2.26 mmol) was dissolved in anhydrous DCM (60 mL), added dibenzyl N,N-diisopropylphosphoramidite (0.75 mL, 2.26 mmol) and tetrazole (0.45 M in MeCN, 5.07 mL), and the reaction was allowed to stir at rt for 4 h. The reaction mixture was cooled to 0° C., added Luperox® TBH70X (70 wt. % in water, 2.23 mL), then brought to rt and stirred vigorously overnight at rt under nitrogen atmosphere. The reaction was diluted in DCM (50 mL), the organic phase washed with sat. NaHCO.sub.3(2×40 mL), dried over Na.sub.2SO.sub.4, and the title compound was obtained after flash chromatography (EtOAc.fwdarw.EtOAc+5% MeOH) as a clear oil (502 mg, 51%).

[0451] R.sub.f=0.50 (EtOAc+2% MeOH); .sup.1H NMR (400 MHz, Chloroform-d) δ 7.37-7.32 (m, 10H), 5.08-5.02 (m, 4H), 4.11 (dt, J=7.5, 5.2 Hz, 2H), 3.31 (s, 2H), 2.86 (t, J=5.2 Hz, 2H), 2.02 (s, 1H), 1.45 (s, 9H); UPLC-MS (ESI) m/z found 436.2 [M+H].sup.+, calc. 436.19 [C.sub.22H.sub.31NO.sub.6P].sup.+.

tert-Butyl N-(4-azidobenzoyl)-N-(2-((bis(benzyloxy)phosphoryl)oxy)ethyl)glycinate

[0452] ##STR00081##

[0453] In a pre-dried round-bottom flask, tert-butyl (2-((bis(benzyloxy)phosphoryl)oxy)ethyl)glycinate (429 mg, 0.98 mmol) was dissolved in anhydrous DCM (6.4 mL), added DIPEA (0.52 mL, 2.95 mmol) and DMAP (12 mg, 0.10 mmol), the reaction mixture was cooled to 0° C. and stirred for 15 min, after which 4-azidobenzoyl chloride (2.2 M in anhydrous DCM, 0.5 mL) was added dropwise. The reaction mixture was allowed to slowly reach rt and stirred overnight under nitrogen atmosphere, then diluted in DCM (40 mL), added sat. NaHCO.sub.3(25 mL), the aqueous phase was extracted with DCM (3×25 mL), the organic phase dried over Na.sub.2SO.sub.4, and the title compound was obtained after flash chromatography (EtOAc/PE, 1:1) as a clear oil (272 mg, 48%).

[0454] R.sub.f=0.20 (EtOAc/PE, 1:1); .sup.1H NMR (400 MHz, Chloroform-d) δ 7.33 (app. s, 12H), 6.96 (d, J=8.2 Hz, 2H), 5.13-4.90 (m, 4H), 4.30-3.42 (m, 6H), 1.47* (s, 3H), 1.40 (s, 6H); UPLC-MS (ESI) m/z found 581.6 [M+H].sup.+, calc. 581.22 [C.sub.29H.sub.34N.sub.4O.sub.7P].sup.+.

tert-Butyl N-(4-aminobenzoyl)-N-(2-(phosphonooxy)ethyl)glycinate

[0455] ##STR00082##

[0456] In a pre-dried round-bottom flask, tert-butyl N-(4-azidobenzoyl)-N-(2-((bis(benzyloxy)phosphoryl)oxy)ethyl)glycinate (61 mg, 0.11 mmol) was dissolved in freshly distilled MeOH (1.5 mL), the flask was flushed with nitrogen, added 5% Pd/C (2.5 mg), re-evacuated with hydrogen (×3), and the reaction was stirred under hydrogen atmosphere at rt for 4 h. The reaction mixture was filtered and the solvent removed to give the title compound as a light yellow solid (33 mg, 84%).

[0457] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.08 (app. s, 2H), 6.54 (d, J=8.2 Hz, 2H), 4.03 (s, 2H), 3.96 (q, J=7.4, 6.9 Hz, 2H), 3.55 (t, J=6.0 Hz, 2H), 1.40 (s, 9H); UPLC-MS (ESI) m/z found 372.9 [M].sup.−, calc. 373.12 [C.sub.15H.sub.22N.sub.2O.sub.7P].sup.−.

N-(4-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)benzoyl)-N-(2-(phosphonooxy)ethyl)glycine

[0458] ##STR00083##

[0459] In a pre-dried microwave vial, a solution of tert-butyl N-(4-aminobenzoyl)-N-(2-(phosphonooxy)ethyl)glycinate (24 mg, 65 μmol), 3-maleimidepropionic acid (10 mg, 60 μmol), N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate (TCFH) (18 mg, 65 μmol) in anhydrous DMF (0.2 mL) was added N-methylimidazole (10 μL, 0.13 mmol), and the reaction was left to stir at rt for 20 h. The crude reaction mixture was purified directly using preparative HPLC (linear gradient) and lyophilized yielding the desired product (4 mg, 13%) as a white solid.

[0460] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.63-7.52 (m, 2H), 7.40-7.20 (m, 2H), 7.02 (s, 2H), 4.12-3.61 (m, 8H), 2.60 (t, J=7.1 Hz, 2H), 1.43 (s, 6H), 1.35* (s, 3H); UPLC-MS (ESI) m/z found 526.7 [M+H].sup.+, calc. 526.16 [C.sub.22H.sub.29N.sub.3O.sub.10P].sup.+.

[0461] The title compound was synthesized from the corresponding tert-butyl ester. In a pre-dried microwave vial, tert-butyl ester (3 mg, 6 μmol) dissolved in anhydrous DCM (1 mL) was slowly added TFA (0.16 mL), and the reaction was stirred at rt for 1.5 h. The title compound was concentrated on nitrogen flow to give a light brown solid (2.6 mg, 97%).

[0462] UPLC-MS (ESI) m/z found 468.5 [M−H].sup.−, calc. 468.08 [C.sub.18H.sub.19N.sub.3O.sub.10P].sup.+.

Example 17: Synthesis of an Sulfatase Cleavable Linker of Formula (I)

[0463] The procedures below outline the synthesis of a sulfatase cleavable linker.

tert-Butyl N-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoyl)-N-(2-(sulfooxy)ethyl) glycinate

[0464] ##STR00084##

[0465] tert-Butyl N-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoyl)-N-(2-hydroxyethyl) glycinate (500 mg, 0.94 mmol) and sulfur trioxide pyridine complex (165 mg, 1.04 mmol) was suspended in pyridine (1 mL) and stirred at rt for 1 h. Pyridine was removed by co-evaporation (toluene) and the crude was purified by flash column chromatography (EtOAc.fwdarw.EtOAc+10% MeOH, 10% AcOH) to yield the title compound as a white sticky solid (494 mg, 86%).

[0466] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.91-8.84 (m, 1H), 8.46 (tt, J=7.8, 1.6 Hz, 1H), 8.03-7.93 (m, 2H), 7.89 (d, J=7.6 Hz, 3H), 7.70 (t, J=6.4 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.37-7.22 (m, 5H), 4.37 (dd, J=6.8, 3.9 Hz, 2H), 4.22 (q, J=7.1, 5.8 Hz, 3H), 4.10 (s, 1H), 3.98 (s, 1H), 3.93 (t, J=5.8 Hz, 1H), 3.80 (t, J=5.9 Hz, 1H), 3.60 (t, J=5.8 Hz, 1H), 3.40 (t, J=5.9 Hz, 1H), 1.45 (s, 5H), 1.34 (s, 3H). UPLC-MS (ESI) m/z found 608.9 [M−H].sup.−, calc. 609.7 [C.sub.31H.sup.33N.sub.2O.sub.9S].sup.−.

tert-Butyl N-(4-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) methyl) benzoyl)-N-(2-sulfo oxy) ethyl) glycinate

[0467] ##STR00085##

[0468] tert-Butyl N-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoyl)-N-(2-hydroxyethyl) glycinate (273 mg, 0.45 mmol) was dissolved in diethylamine and DCM (1:1, 5 mL) and stirred at rt until complete Fmoc deprotection (25 min). The reaction was added THF and concentrated in vacuo. Crude tert-butyl N-(4-(aminomethyl)benzoyl)-N-(2-(sulfooxy)ethyl)glycinate was dissolved in anhydrous DCM (10 mL) and added 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (75 mg, 0.45 mmol), HATU (204 mg, 0.54 mmol) and DIPEA (389 μL, 2.24 mmol). The reaction was stirred at rt for 3 h under nitrogen atmosphere. The solvent was removed in vacuo and the crude was purified by flash column chromatography (EtOAc.fwdarw.EtOAc+10% MeOH, 10% AcOH) to yield the title compound as a colorless amorphous solid (93 mg, 39%).

[0469] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.41 (d, J=7.7 Hz, 1H), 7.33 (d, J=7.8 Hz, 1H), 7.26-7.20 (m, 1H), 6.69 (d, J=6.5 Hz, 2H), 6.26 (d, J=32.6 Hz, 1H), 4.40 (d, J=5.8 Hz, 2H), 4.33 (t, J=5.2 Hz, 1H), 4.23 (s, 1H), 4.14-4.01 (m, 3H), 3.85 (t, J=7.0 Hz, 3H), 3.59 (t, J=5.8 Hz, 1H), 2.59 (t, J=7.0 Hz, 2H), 1.49 (s, 5H), 1.41 (s, 6H). UPLC-MS (ESI) m/z found 537.8[M−H].sup.−, calc. 538.2 [C.sub.23H.sub.28N.sub.3O.sub.10S].sup.−.

Example 18: Conjugation to Cytotoxic Agents Targeting DNA Replication

[0470] Conjugation of cytotoxic agents, such as querticin (CytI), PNU-159682 (CytII), camptothecin (CytIII), auristatin E (CytIV), and cryptothycin (CytV), to the enzyme cleavable linkers is carried out as outlined. The cytotoxic agents target DNA replication i.e. by DNA intercalation, DNA topoisomerase I inhibition, or tubulin inhibition.

[0471] Enzyme cleavable linker, such as a phosphatase cleavable linker (PC), a sulfatase cleavable linker (SC), or a sulfamidase cleavable linker) bearing a free carboxylic acid functionality and a maleimide functionality (PC, SC or SAC) (1 equivalent) is transferred to a pre-dried round bottom flask. Anhydrous DCM is added, and the suspension is cooled by means of an ice bath. To the pre-cooled suspension of enzyme cleavable linker, the cytotoxic agent bearing free hydroxy function (Cyt-II, Cyt-III or Cyt-IV, FIG. 18 (2 equivalents) and DIPEA (4 equivalents) are added, and the reaction mixture is stirred at rt for 5 min. In another pre-dried round bottom flask, BTFFH (fluoro-N,N,N′,N′-bis(tetramethylene) formamidinium hexafluorophosphate) coupling reagent (1.5 equivalent) is dissolved in anhydrous DCM, after which the BTFFH solution is added slowly to the stirring suspension of enzyme cleavable linker and cytotoxic agent. The reaction is left stirring overnight at rt under inert atmosphere, then concentrated on nitrogen flow, purified using preparative HPLC, and lyophilized to give the desired payload such as PC-CytII, PC-CytIII, PC-CytIV, SCCyt-II, SC-CytIII, SC-CytIV, SAC-CytII, SAC-CytIII, or SAC-CytIV). In particular, these conjugates comprise an ester bond between the carboxylic acid moiety of the enzyme cleavable linker and a hydroxy group of the cytotoxic agent.

[0472] The same procedure is used to access payloads with amine-containing drugs (e.g. CytV), such as SAC-CytV. In particular, these conjugates comprise an amide bond between the carboxylic acid moiety of the enzyme cleavable linker and an amine group of the cytotoxic agent.

Example 19: Bioconjugation of Payload to Antibody

[0473] The following bioconjugation procedure is used to install one or more linker-drug conjugates (such as PC-CytII, PC-CytIII, PC-CytIV, SC-CytII, SC-CytIII, SC-CytIV, SAC-CytII, SAC-CytIII, SAC-CytIV, or LC-CytI) onto an antibody of choice.

[0474] The antibody (1 equivalent) is dissolved in Tris-buffered saline and TCEP (tris(2-carboxyethyl)-phosphine) (50 equivalents) is added. The reaction is incubated at 37° C. for 2 h. A solution of payload (8 equivalent) in DMSO (10% v/v) is added, the reaction is gently shaked for 1 h at rt, and then quenched with N-acetyl cysteine (32 equivalents). Next, the crude antibody-drug-conjugate (ADC) is subjected to spin filtration (40 K MWCO) and then size-exclusion chromatography to yield the isolated antibody (mAb) conjugated to one or more linker-drug conjugates. Examples of ADCs obtainable by this method are: mAb-PC-CytII, mAb-PC-CytIII, mAb-PC-CytIV, mAb-SC-CytII, mAb-SC-CytIII, mAb-SC-CytIV, mAb-SAC-CytII, mAb-SAC-CytIII, mAb-SAC-CytIV, mAb-SAC-CytV, or mAb-LC-CytI (FIG. 18), with varying drug-to-antibody ratios.

[0475] A similar procedure is used to access a series of sulfamidase cleavable camptothecin-based payloads such as mAb-SAC-CytIII-a (R═H), mAb-SAC-CytIII-b (R=Me), mAb-SAC-CytIII-c (R=Et), mAb-SAC-CytIII-d (R═Pr), mAb-SAC-CytIII-e (R═Bn), mAb-SAC-CytIII-f (R═Ph), mAb-SAC-CytV-a (R═H), mAb-SAC-CytV-b (R=Me), mAb-SAC-CytV-c (R=Et), mAb-SAC-CytV-d (R═Pr), mAb-SAC-CytV-e (R═Bn), and mAb-SAC-CytV-f (R═Ph), wherein the functional groups R dictate drug release rate (FIGS. 18-20).

Example 20: Release Rate of Camptothecin

[0476] The following protocol can be used to assess the release rate of camptothecin from sulfamidase cleavable ADCs mAb-SAC-CytIII-a (R═H), mAb-SAC-CytIII-b (R=Me), mAb-SAC-CytIII-c (R=Et), mAb-SAC-CytIII-d (R═Pr), mAb-SAC-CytIII-e (R=Bn), and mAb-SAC-CytIII-f (R=Ph).

[0477] To a 1.5 mL Eppendorf tube containing 150 uL 20 mM ADC in 50 mM IVIES, pH 5.5 assay buffer, 150 uL recombinant human sulfamidase/SGSH (rhSGSH) (Catalog #8380-SU, R&D Systems) (40 μg/mL, in assay buffer) is added and the reaction is incubated at 37° C. with gentle shaking. Samples (20 uL aliqoutes) are withdrawn at different time points (5 min, 30 min, 60 min, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h), immediately quenched in 2 M NaOH (20 uL), and the extent of reaction (drug release) is analyzed by LCMS (5 uL injections). The moiety in position R affects the rate of release of camptothecin: larger and bulky moieties in the position of R (FIG. 19) such as Ph and Bn provides for a slower release of camptothecin. In comparison, smaller moieties in the position of R provides for a faster release of camptothecin.

Example 21: Release Rate of Cryptothecin

[0478] The following protocol can be used to assess the release rate of cryptothecin from sulfamidase cleavable ADCs mAb-SAC-CytV-a (R═H), mAb-SAC-CytV-b (R=Me), mAb-SAC-CytV-c (R=Et), mAb-SAC-CytV-d (R═Pr), mAb-SAC-CytV-e (R=Bn), and mAb-SAC-CytV-f (R=Ph).

[0479] To a 1.5 mL Eppendorf tube containing 150 uL 20 mM ADC in 50 mM IVIES, pH 5.5 assay buffer, 150 uL recombinant human sulfamidase/SGSH (rhSGSH) (Catalog #8380-SU, R&D Systems) (40 μg/mL, in assay buffer) is added and the reaction is incubated at 37° C. with gentle shaking. Samples (20 uL aliqoutes) are withdrawn at different time points (5 min, 30 min, 60 min, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h), immediately quenched in 2 M NaOH (20 uL), and the extent of reaction (drug release) is analyzed by LCMS (5 uL injections). The moiety in the position R affects the rate of release of cryptothecin: larger and bulky moieties in the position of R (FIG. 20) such as Ph and Bn provides for a slower release of cryptothecin. In comparison, smaller moieties in the position of R provides for a faster release of cryptothecin.

Example 22: Release Rate of Model Drug

[0480] The following protocol can be used to assess the release rate of model drug (benzyl alcohol) from sulfamidase cleavable ADC mAb-SAC2-BnOH.

[0481] To a 1.5 mL Eppendorf tube containing 150 uL 20 mM ADC in 50 mM IVIES, pH 5.5 assay buffer, 150 uL recombinant human sulfamidase/SGSH (rhSGSH) (Catalog #8380-SU, R&D Systems) (40 μg/mL, in assay buffer) is added and the reaction is incubated at 37° C. with gentle shaking. Samples (20 uL aliqoutes) are withdrawn at different time points (5 min, 30 min, 60 min, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h), immediately quenched in 2 M NaOH (20 uL), and the extent of reaction (sulfamidase cleavage followed by BnOH release) is analyzed by LCMS (5 uL injections). The rate of cleavage by the sulfamidase to form the free amine is fast, proceeding almost instantly or over a few or several minutes. The subsequent ring closure and release of BnOH is comparatively slower, proceeding over several minutes or several hours (FIG. 21).