HYDROPHILIC AZADIBENZOCYCLOOCTYNE DERIVATIVES AND METAL-FREE CLICK REACTIONS WITH THESE HYDROPHILIC AZADIBENZOCYCLOOCTYNE DERIVATIVES

Abstract

The invention relates in a first aspect to an azadibenzocyclooctyne derivative according to formula (I) or a salt thereof having specific substituents at the benzo rings of the DIBAC structure and having specific substituents connected to the nitrogen atom of the DIBAC structure. A second aspect of the invention is directed to a conjugate of formula (II), wherein a substituent R.sup.6 is connected to the N atom of the 8 membered ring of the DIBAC structure via a linker structure C(?O)-[L].sub.n-Z. A third aspect of the invention relates to a method for the modification of a target molecule, wherein a conjugate according to the second aspect is reacted with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group. In a fourth aspect, the invention is directed to the use of the conjugate according to the second aspect for bioorthogonal labeling and/or modification of a target molecule. A fifth aspect of the invention relates to a modified target molecule comprising the reaction product of a conjugate according to the second aspect and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group, obtained or obtainable from the method of the third aspect. In a sixth aspect, the invention is related to a kit comprising a modified target molecule according to the fifth aspect as detector reagent and a suitable capture reagent.

Claims

1. An azadibenzocyclooctyne derivative according to formula (I) or a salt thereof, ##STR00050## wherein R.sup.1, R.sup.2 are the same and both a [(CH.sub.2).sub.aCR.sup.xR.sup.y].sub.bR.sup.z group, wherein a is either zero or an integer from the range of from 1 to 4, b is either zero or 1, R.sup.x, R.sup.y, R.sup.z are selected from the group consisting of hydrogen atom, C1 to C3 alkyl group and (CH.sub.2).sub.cSO.sub.3.sup.? group, with c being either zero or an integer from the range of from 1 to 4, wherein at least one of R.sup.x, R.sup.y, R.sup.z is a (CH.sub.2).sub.cSO.sub.3.sup.? group with the condition: -if R.sup.z is a (CH.sub.2).sub.cSO.sub.3.sup.? group with c being zero, then R.sup.x, R.sup.y are not both a (CH.sub.2).sub.cSO.sub.3.sup.? group wherein c is zero, or if a is zero, then R.sup.x and R.sup.y are not both a (CH.sub.2).sub.cSO.sub.3.sup.? group wherein c is zero; R.sup.3, R.sup.4 are independently selected from the group consisting of hydrogen atom, C1-C3-alkyl group, halogen atom and -O-C1-C3-alkyl group; and R.sup.5 is selected from the group consisting of carboxyl group, activated carboxyl group and NHR.sup.5a group, wherein R.sup.5a is a hydrogen atom or a C1-C5 alkyl group; L comprises a chain of covalently bonded atoms forming a backbone and having a length in the range of from 1 to 100 atoms (linker); and n is either zero or 1 if R.sup.5 is a carboxyl group or an activated carboxyl group or n is 1 if R.sup.5 is a NHR.sup.5a group.

2. The azadibenzocyclooctyne derivative or salt thereof of claim 1, wherein R.sup.3, R.sup.4 are independently a hydrogen atom or a methyl group, preferably R.sup.3, R.sup.4 are identical and are each a hydrogen atom.

3. The azadibenzocyclooctyne derivative or salt thereof of claim 1 having formula (Ia) or (Ib): ##STR00051## wherein L, n and R.sup.5 are as defined in claim 1.

4. The azadibenzocyclooctyne derivative or salt thereof of claim 1 having formula (Ia-1) or (Ib-1): ##STR00052## wherein R.sup.5 is as defined in claim 1.

5. A conjugate of formula (II) ##STR00053## wherein L, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n are as defined in claim 1 for the azadibenzocyclooctyne derivative of formula (I) or salt thereof; and wherein R.sup.6 is selected from the group consisting of fluorophore, hapten, tyramine, polyethylene glycol chain, polypropylene glycol chain, mixed polyethylene/polypropylene glycol chain, metal complex, radioactive isotope, active pharmaceutical ingredient, carbohydrate, solid phase, lipid, amino acid, oligopeptide, polypeptide, nucleotide, oligonucleotide and polynucleotide; and is preferably a metal complex; and Z is selected from the group consisting of C(?O)O, C(?O)NR.sup.7, and NR.sup.7C(?Y), wherein R.sup.7 is a hydrogen atom or a C1-C5 alkyl group and Y is an oxygen atom or a sulphur atom, preferably an oxygen atom.

6. A conjugate of formula (II) according to claim 5, wherein R.sup.6 is selected from the group consisting of fluorophore, fluorescence quencher, dye, hapten, tyramine, metal complex, radioactive isotope, active pharmaceutical ingredient (drug), carbohydrate, solid phase, lipid, amino acid, oligopeptide, polypeptide, nucleotide, oligonucleotide and polynucleotide; and is preferably a metal complex, wherein a further linker is present or absent between Z and R.sup.6, which is preferably selected from the group consisting of alkyl chain, polyethylene e glycol chain, polypropylene glycol chain and mixed polyethylene/polypropylene glycol chain.

7. The conjugate of claim 5, having formula (IIa) or (IIb): ##STR00054## wherein L, n and R.sup.6 are as defined in claim 5 and Z.sup.1 is a C(?O)O-group or a C(?O)NH-group.

8. The conjugate of claim 5, having formula (IIa-1), (IIa-2), (IIb-1) or (IIb-2): ##STR00055## wherein R.sup.6 in (IIa-1), (IIa-2), (IIb-1) or (IIb-2) is as defined in claim 5.

9. A method for the modification of a target molecule, wherein a conjugate according to claim 5 is reacted with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group.

10. The method according to claim 9, wherein the target molecule is selected from the group consisting of fluorophore, fluorescence quencher, dye, hapten, tyramine, polyethylene glycol chain, polypropylene glycol chain, mixed polyethylene/polypropylene glycol chain, metal complex, radioactive isotope, active pharmaceutical ingredient, carbohydrate, solid phase, lipid, amino acid, oligopeptide, polypeptide, nucleotide, oligonucleotide, and polynucleotide; and is preferably a polypeptide, more preferably an antibody, more preferably a modified antibody having a 1,3-dipole group, more preferably a modified antibody having an azide group.

11. (canceled)

12. A modified target molecule comprising the reaction product of a conjugate according to claim 5 and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group.

13. A kit comprising a modified target molecule according to claim 12 as detector reagent and a suitable capture reagent.

Description

EXAMPLES

[0144] The following Examples shall merely illustrate the invention. Whatsoever, they shall not be construed as limiting the scope of the invention.

Experimental Procedures

[0145] ##STR00017##

[0146] Aniline 1: A solution of m-anisidine (2.48 ml, 22.0 mmol) and m-anisaldehyde (2.44 ml, 20 mmol) in MeOH (200 ml) was stirred at room temperature for 1.5 h before NaBH.sub.4 (2.26 g, 60.0 mmol) was added and the reaction was stirred at that temperature for another 1.5 h. Water (100 ml) was added and the mixture was extracted with EtOAc. The combined organic phases were washed with 1M NaOH, water and brine, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (hexane:EtOAc=5:1, R.sub.f=0.3) gave aniline 1 (4.47 g, 18.4 mmol, 92%) as a yellowish oil.

[0147] R.sub.f=0.3 [hexane/EtOAc, 5:1].

[0148] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=7.25 (t, J=7.84 Hz, 1H), 7.07 (t, J=8.09 Hz, 1H), 6.95 (dd, J=7.53, 0.63 Hz, 1H), 6.92 (m, 1H), 6.81 (m, 1H), 6.27 (dddd, J=11.26, 8.13, 2.32, 0.75 Hz, 2H), 6.19 (t, J=2.26 Hz, 1H), 4.29 (s, 2H), 4.05 (brs, 1H), 3.79 (s, 3H), 3.75 (s, 3H) ppm.

[0149] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=180.8, 159.9, 149.5, 141.0, 130.0, 129.6, 119.7, 113.0, 112.7, 106.0, 102.7, 98.9, 55.2, 55.1, 48.3 ppm.

[0150] MS (ESI): calcd. for C.sub.15H.sub.18NO.sub.2.sup.+: 244.1 [M+H].sup.+ [0151] found: 244.4 [M+H].sup.+.

##STR00018##

[0152] Acylamine 2: DIPEA (4.31 ml, 24.7 mmol) was added to a solution of aniline 1 (3.00 g, 12.3 mmol), mono methyl glutarate (2.01 ml, 16.7 mmol) and HATU (6.35 g, 16.7 mmol) in DMF (31 ml) and the reaction was allowed to stir at room temperature for 3 d. The mixture was diluted with EtOAc, washed with 1M HCl, water and brine, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (EtOAc:hexane=1:1) gave acylamine 2 (4.42 g, 11.9 mmol, 97%) as a pale yellow oil.

[0153] R.sub.f=0.4 [hexane/EtOAc, 1:1].

[0154] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=7.19 (m, 2H), 6.82 (dd, J=8.34, 2.2 Hz, 1H), 6.75 (m, 3H), 6.55 (brd, J=7.53 Hz, 1H), 6.48 (m, 1H), 4.81 (s, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 3.59 (s, 3H), 2.30 (t, J=7.34 Hz, 2H), 2.15 (t, J=7.22 Hz, 2H), 1.92 (q, J=7.22 Hz, 2H) ppm.

[0155] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=173.6, 171.9, 160.3, 159.6, 143.3, 139.1, 130.2, 129.3, 121.1, 120.6, 114.1, 113.5, 113.0, 55.3, 55.2, 52.8, 51.4, 33.24, 33.16, 20.7 ppm.

[0156] MS (ESI): calcd. for C.sub.21H.sub.26NO.sub.5.sup.+: 372.2 [M+H].sup.+ [0157] found: 372.4 [M+H].sup.+.

##STR00019##

[0158] Cyclooctene 3: Tetrachlorocyclopropene (0.80 ml, 6.55 mmol) was added dropwise to a suspension of AlCl.sub.3 (3.17 g, 23.8 mmol) in CH.sub.2Cl.sub.2 (50 ml) and the reaction was stirred at room temperature for 15 min. The solution was cooled to ?78? C. and a solution of acylamine 2 (2.21 g, 5.95 mmol) in CH.sub.2Cl.sub.2 was slowly added. The reaction was allowed to warm to room temperature overnight before water (45 ml) was added and the reaction was stirred at room temperature for 30 min. The mixture was extracted with CH.sub.2Cl.sub.2, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (EtOAc/MeOH, 100:1, R.sub.f=0.4) gave cyclooctene 3 (1.13 g, 2.67 mmol, 45%) as a yellow oil.

[0159] R.sub.f=0.4 [EtOAc/MeOH, 100:1].

[0160] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=8.02 (d, J=8.83 Hz, 1H), 7.90 (d, J=8.51 Hz, 1H), 7.26, (d, J=2.52 Hz, 1H), 7.06 (dd, J=8.51, 2.52 Hz, 1H), 6.96 (dd, J=8.35, 2.68 Hz, 1H), 6.89 (d, J=2.52 Hz, 1H), 5.18 (d, J=14.5 hz, 1H), 4.10 (d, J=14.2 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.56 (s, 3H), 2.33 (m, 1H), 2.14 (m, 1H), 2.00 (m, 1H), 1.93 (m, 1H), 1.75 (m, 2H) ppm.

[0161] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=173.2, 172.6, 163.1, 162.7, 152.5, 146.0, 143.2, 141.6, 139.0, 135.8, 135.2, 118.2, 115.5, 115.3, 114.9, 113.9, 113.7, 56.1, 55.9, 55.6, 51.5, 33.5, 32.6, 20.6 ppm.

[0162] MS (ESI): calcd. for C.sub.24H.sub.24NO.sub.6.sup.+: 422.2 [M+H].sup.+ [0163] found: 422.4 [M+H].sup.+.

##STR00020##

[0164] Amine 4: A solution of 4-aminobutyric acid (5.00 g, 48.5 mmol) in SOCl.sub.2 (35 ml, 485 mmol) was stirred at room temperature for 2 h and concentrated under reduced pressure. NaHCO.sub.3 (8.95 g, 107 mmol) and t-BuOH (105 ml) were added and the resulting suspension was stirred at room temperature overnight. All volatiles were removed under reduced pressure and the residue was portioned between EtOAc and 1M NaOH. The organic phase was washed with water and brine, dried over MgSO.sub.4 and concentrated under reduced pressure to give amine 4 (1.50 g, 9.39 mmol, 19%) as a pale brown oil.

[0165] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=2.72 (t, J=7.03 Hz, 2H), 2.27 (t, J=7.47 Hz, 2H), 1.73 (q, J=7.22 Hz, 2H), 1.45 (s, 3H), 1.26 (brs, 2H) ppm.

[0166] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=172.9, 80.2, 41.6, 33.0, 29.1, 28.1 ppm.

##STR00021##

[0167] Amide 5: BBr3 (1.0M in CH.sub.2Cl.sub.2, 26.6 ml, 26.6 mmol) was added slowly to a solution of cyclooctane 3 (1.12 g, 2.66 mmol) in CH.sub.2Cl.sub.2 (128 ml) and the solution was stirred at ?78? C. for 1 h and at room temperature for 48 h. The reaction was quenched with water, basified with 4M NaOH and washed with CH.sub.2Cl.sub.2. (Two equal batches were combined at this stage.) The aqueous phase was acidified with conc. HCl and the resultant precipitate was collected. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. The solids were combined, dissolved in MeOH (13 ml), THF (13 ml) and 1M NaOH (21 ml) and stirred at room temperature for 3 h. The reaction mixture was acidified with conc. HCl and the resultant precipitate was collected. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. The combined solids (1.06 g) and amine 4 (852 mg, 5.35 mmol) were dissolved in DMF (287 ml). HATU (2.03 g, 5.35 mmol and DIPEA (3.73 ml, 21.4 mmol) were sequentially added and the resulting mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (6% MeOH in CH.sub.2Cl.sub.2) gave amide 5 (1.14 g, 2.18 mmol, 41% over 3 steps) as a pale brown solid.

[0168] R.sub.f=0.3 [MeOH/CH.sub.2Cl.sub.2, 6:94].

[0169] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?8=7.91 (d, J=8.51 Hz, 1H), 7.77 (d, J=8.2 Hz, 1H), 7.14 (d, J=2.21 Hz, 1H), 7.01 (dd, J=8.51, 2.21 Hz, 1H), 6.93 (d, J=2.21 Hz, 1H), 6.87 (dd, J=8.35, 2.36 Hz, 1H), 5.10 (d, J=14.82 Hz, 1H), 4.21 (d, J=14.5 Hz, 1H), 3.06 (m, 2H), 2.31 (m, 1H), 2.18 (t, J=7.41 Hz, 2H), 1.93 (m, 3H), 1.65 (m, 4H), 1.43 (s, 9H) ppm.

[0170] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=175.3, 175.0, 174.4, 164.1, 163.3, 154.6, 148.0, 144.4, 142.5, 138.9, 137.0, 136.4, 120.9, 117.8, 117.4, 116.5, 115.1, 114.5, 81.7, 57.2, 39.7, 35.9, 35.0, 33.8, 28.5, 26.0, 22.8 ppm.

[0171] MS (ESI): calcd. for C.sub.29H.sub.33N.sub.2O.sub.7.sup.+: 521.2 [M+H].sup.+ [0172] found: 521.3 [M+H].sup.+.

##STR00022##

[0173] Acetonide 6: DEAD (40% in PhMe, 1.75 ml, 3.08 mmol) was added dropwise to a stirred solution of amide 5 (400 mg, 0.768 mmol), PPh.sub.3 (808 mg, 3.08 mmol) and (R)-(?)-2,2-Dimethyl-1,3-dioxolane-4-methanol (0.38 ml, 3.08 mmol) in THF (20 ml) before the reaction was stirred at room temperature for 20 h and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (30-60% acetone in CH.sub.2Cl.sub.2) gave acetonide 6 (478 mg, 0.638 mmol, 83%) as a pale white solid.

[0174] R.sub.f=0.5 [acetone/CH.sub.2Cl.sub.2, 1:1].

[0175] .sup.1H NMR (400 MHz, CDCl.sub.3) ?=7.98 (d, J=8.71 Hz, 1H), 7.87 (d, J=8.31 Hz, 1H), 7.25 (m, 1H), 7.06 (d, J=8.71 Hz, 1H), 6.96 (m, 2H), 5.96 (brs, 1H), 5.12 (d, J=14.25 Hz, 1H), 4.50 (m, 2H), 4.14 (m, 6H), 4.05 (d, J=15.04 Hz, 1H), 3.92 (m, 2H), 3.16 (m, 2H), 2.33 (m, 1H), 2.20 (t, Hz=7.12 Hz, 2H), 1.99 (m, 1H), 1.91 (m, 2H), 1.70 (m, 4H), 1.45 (s, 6H), 1.40 (m, 15H) ppm.

[0176] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=172.8, 172.7, 172.4, 162.1, 161.6, 152.4, 145.8, 143.3, 141.6, 139.3, 135.8, 135.2, 118.8, 115.8, 115.2, 114.7, 114.2, 110.1, 109.9, 80.6, 79.77, 73.74, 73.70, 69.4, 69.0, 66.6, 66.5, 56.1, 39.1, 34.8, 33.5, 32.9, 30.9, 29.3, 28.1, 26.8, 25.32, 25.27, 24.6 ppm.

[0177] MS (ESI): calcd. for C.sub.41H.sub.53N.sub.2O.sub.11.sup.+: 749.4 [M+H].sup.+ [0178] found: 749.4 [M+H].sup.+.

##STR00023##

[0179] Alkyne 7: iPr.sub.3SiH (0.20 ml), water (0.2 ml) and TFA (6 ml) were added to a solution of acetonide 6 (400 mg, 0.534 mmol) in CH.sub.2Cl.sub.2 (2 ml). The reaction was stirred at room temperature for 6 h and concentrated. The residue was dissolved in MeOH (20 ml), DIPEA (0.94 ml) was added and the reaction was irradiated (360 nm) for 2 h and concentrated. The residue was dissolved in MeOH (3 ml) and 1M NaOH (2 ml) and stirred at room temperature for 1 h. The reaction mixture was directly submitted to reversed phase HPLC chromatography (YMC-Triart C18, 29-45% MeCN in H.sub.2O, 0.1% TFA, over 30 min) to give alkyne 7 (215 mg, 0.368 mmol, 69% over 3 steps) as a white solid.

[0180] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.29 (d, J=8.39 Hz, 1H), 7.27. (d, J=2.29 Hz, 1H), 7.12 (m, 2H), 7.03 (dd, J=8.56, 2.48 Hz, 1H), 6.90 (dd, J=8.39, 2.29 Hz, 1H), 5.04 (d, J=13.73 Hz, 1H), 4.13 (m, 2H), 4.04 (m, 2H), 3.99 (m, 2H), 3.68 (m, 5H), 3.09 (t, J=7.06 Hz, 2H), 2.31 (m, 1H), 2.25 (t, J=7.44 Hz, 2H), 1.95 (m, 3H), 1.68 (m, 4H) ppm.

[0181] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=177.0, 175.4, 175.1, 160.7, 160.3, 153.8, 151.1, 128.5, 127.3, 120.51, 120.45, 117.7, 116.9, 116.2, 115.9, 115.0, 114.7, 107.9, 71.9, 71.8, 71.4, 70.7, 64.3, 64.2, 57.0, 39.8, 36.1, 35.2, 32.4, 25.9, 22.9 ppm.

[0182] MS (ESI): calcd. for C.sub.30H.sub.37N.sub.2O.sub.10.sup.+: 585.2 [M+H].sup.+ [0183] found: 585.4 [M+H].sup.+.

##STR00024##

[0184] NHS-Ester 8: DIPEA (31 ?L, 0.176 mmol) was added to a solution of alkyne 7 (43 mg, 0.074 mmol) and TSTU (44 mg, 0.147 mmol) in DMF (2 ml) and the solution was stirred at room temperature for 2 h. Concentration of the reaction under reduced pressure and purification of the resultant residue by reversed phase HPLC chromatohraphy (YMC-Triart C18, 32-48% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave NHS-ester 8 (31 mg, 0.045 mmol, 61%) as a white solid.

[0185] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.30 (d, J=8.53 Hz, 1H), 7.27 (d, J=2.38 Hz, 1H), 7.13 (m, 2H), 7.03 (dd, J=8.53, 2.51 Hz, 1H), 6.90 (dd, J=8.41, 2.51 Hz, 1H), 5.04 (d, J=13.93 Hz, 1H), 4.11 (m, 2H), 4.00 (m, 4H), 3.70 (d, J=9.16 Hz, 1H), 3.66 (m, 4H), 3.16 (t, J=, 6.84 Hz, 2H), 2.80 (s, 4H), 2.60 (t, J=7.4 Hz, 2H), 2.27 (m, 1H), 1.93 (m, 3H), 1.79 (q, J=7.12 Hz, 2H), 1.65 (m, 2H) ppm.

[0186] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=174.0, 173.5, 170.4, 168.5, 159.1, 158.7, 155.9, 152.2, 149.5, 127.3, 127.0, 125.7, 119.5, 118.9, 116.1, 115.3, 114.6, 114.3, 70.3, 70.2, 69.5, 62.7, 62.6, 55.4, 37.8, 34.5, 33.6, 27.7, 25.1, 24.0, 21.3 ppm.

[0187] MS (ESI): calcd. for C.sub.34H.sub.40N.sub.3O.sub.12.sup.+: 682.3 [M+H].sup.+ [0188] found: 682.4 [M+H].sup.+.

##STR00025##

[0189] Sulfonic acid 9: A suspension of amide 5 (100 mg, 0.192 mmol), K.sub.2CO.sub.3 (159 mg, 1.15 mmol) and sodium 2-bromoethanesulfonate (243 mg, 1.15 mmol) in MeCN (2.8 ml) was stirred at 80? C. for 5 d. Purification of the reaction mixture by reversed phase HPLC chromatography (YMC-Triart C18, 23-39% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave sulfonic acid 9 (104 mg, 0.131 mmol, 68%) as a white solid.

[0190] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.92 (d, J=8.51 Hz, 1H), 7.76 (d, J=8.51 Hz, 1H), 7.26 (d, J=1.89 Hz, 1H), 7.19 (d, J=2.21 Hz, 1H), 7.14 (dd, J=8.51, 2.21 Hz, 1H), 6.98 (dd, J=8.51, 1.89 Hz, 1H), 5.10 (d, J=14.5 Hz, 1H), 4.43 (m, 4H), 4.13 (d, J=14.5 Hz, 1H), 3.24 (m, 5H), 3.10 (t, J=6.94 Hz, 2H), 2.38 (m, 1H), 2.21 (t, J=7.25 Hz, 1H), 2.12 (m, 1H), 2.07 (m, 1H), 1.84 (m, 1H), 1.62 (m, 4H), 1.33 (s, 5H), 1.33 (s, 5.5H) 1.13 (s, 2H9, 1.09 (s, 0.5H) ppm (mixture of rotamers).

[0191] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=177.0, 176.9, 174.8, 174.1, 173.7, 163.9, 163.2, 154.3, 147.4, 144.0, 143.0, 139.6, 136.6, 136.1, 119.9, 117.0, 116.3, 115.6, 115.2, 81.5, 65.7, 65.2, 56.9, 51.5, 51.3, 40.7, 40.6, 36.7, 34.63, 34.55, 33.2, 31.7, 31.0, 28.2, 27.1, 24.9, 24.8, 22.43, 22.39 ppm (mixture of rotamers).

[0192] MS (ESI): calcd. for C.sub.33H.sub.41N.sub.2O.sub.13S.sub.2.sup.+: 737.2 [M+H].sup.+ [0193] found: 737.4 [M+H].sup.+.

##STR00026##

[0194] Carboxylic acid 10: : A solution of sulfonic acid 9 (84 mg, 0.105 mmol) in CH.sub.2Cl.sub.2 (0.83 ml), iPr.sub.3SiH (0.08 ml), water (0.08 ml) and TFA (2.5 ml) was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was co-evaporated two times with acetone and MeCN. The resultant residue was dissolved in MeOH (6.7 ml) and DIPEA (0.40 ml), irradiated (360 nm) for 1.5 h and concentrated under reduced pressure. Purification of the resultant residue by reversed phase HPLC chromatography (YMC-Triart C18, 22-38% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave carboxylic acid 10 (41 mg, 0.045 mmol, 43% over 2 steps) as a white solid.

[0195] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?7.29 (d, J=8.53 Hz, 1H), 7.25 (d, J=2.26 Hz, 1H), 7.15 (d, J=2.38 Hz, 1H), 7.12 (d, J=8.41 Hz, 1H)), 7.03 (dd, J=8.60, 2.45 Hz, 1H), 6.90 (dd, J=8.53, 2.38 Hz, 1H), 5.04 (d, J=14.05 Hz, 1H), 4.42 (m, 5H), 3.69 (spt, J=6.61 Hz, 4H), 3.29 (m, 4H), 3.18 (m, 6H), 2.30 (m, 4H), 2.07 (m, 1H), 1.90 (m 1H), 1.73 (m, 4H), 1.34 (m, 30H) ppm.

[0196] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=174.9, 173.7, 173.1, 158.6, 158.2, 152.1, 149.6, 127.0, 125.8, 119.1, 116.3, 115.4, 114.7, 114.5, 113.2, 113.0, 106.4, 64.0, 63.6, 55.4, 50.4, 50.1, 42.4, 38.8, 34.0, 33.5, 30.5, 23.8, 21.3, 17.3, 15.9 ppm.

[0197] MS (ESI): calcd. for C.sub.28H.sub.33N.sub.2O.sub.12S.sub.2.sup.+: 653.1[M+H].sup.+ [0198] found: 653.1[M+H].sup.+.

##STR00027##

[0199] NHS-Ester 11: DIPEA (0.17 ml, 0.998 mmol) was added to a solution of carboxylic acid 10 (87 mg, 0.095 mmol) and TSTU (80 mg, 0.266 mmol) in DMF (3 ml). The reaction was stirred at room temperature for 2 h and concentrated under reduced pressure. Purification of the resultant residue by reversed phase HPLC chromatography (YMC-Triart C18, 24-40% MeCN in H.sub.2O, 0.1% TFA, over 30 min) NHS-ester 10 (39 mg, 0.045 mmol, 41) as a white solid.

[0200] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.31 (d, J=8.53 Hz, 1H), 7.27 (d, J=2.51 Hz, 1H), 7.17 (d, J=2.51 Hz, 1H), 7.14 (d, J=8.41 Hz, 1H), 7.05 (dd, J=8.53, 2.51 Hz, 1H), 6.92 (dd, J=8.47, 2.45 Hz, 1H), 5.06 (d, J=13.93 Hz, 1H), 4.44 (m, 5H), 3.71 (m, 4H), 3.31 (m, 4H), 3.21 (m, 6H), 2.83 (s, 4H), 2.68 (m, 1H), 2.63 (t, J=7.34 Hz, 1H), 2.34 (m, 2H), 2.26 (q, J=7.49 Hz, 1H), 2.08 (m, 1H), 1.90 (m, 4H), 1.36 (m, 30H) ppm.

[0201] MS (ESI): calcd. for C.sub.32H.sub.36N.sub.3O .sub.14S.sub.2.sup.+: 750.2 [M+H].sup.+ [0202] found: 750.4 [M+H].sup.+.

##STR00028##

[0203] Acyl amine 12: DIPEA (3.85 ml, 11.1 mmol) was added to a solution of aniline 1 (5.66 g, 11.1 mmol), mono methyl succinate (1.98 g, 14.9 mmol) and HATU (5.66 g, 14.9 mmol) in DMF (28 ml) and the reaction was allowed to stir at room temperature for 3 d. The mixture was diluted with EtOAc, washed with 1M HCl, water and brine, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (EtOAc:hexane=1:1, R.sub.f=0.5) gave acylamine 12 (3.85 g, 10.8 mmol, 98%) as a pale yellow oil.

[0204] R.sub.f=0.5 [hexane/EtOAc, 1:1].

[0205] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=7.22 (t, J=8.09 Hz, 1H), 7.16 (dd, J=9.06 Hz, 7.28 Hz, 1H), 6.83 (dd, J=8.28, 2.13 Hz, 1H), 6.76 (m, 3H), 6.64 (d, J=7.65 Hz, 1H), 6.56 (m, 1H), 4.86 (s, 2H), 3.75 (s, 1H), 3.71 (s, 1H), 3.66 (s, 1H), 2.63 (m, 2H), 2.39 (m, 2H) ppm.

[0206] .sup.13C NMR (150 MHz, CDCl.sub.3): ?=173.5, 171.2, 160.3, 159.6, 143.2, 139.1, 130.2, 129.3, 121.0, 120.6, 114.1, 113.9, 113.1, 55.3, 55.2, 53.0, 51.7, 29.3 ppm.

[0207] MS (ESI): calcd. for C.sub.20H.sub.24NO.sub.5.sup.+: 358.2[M+H].sup.+ [0208] found: 358.4 [M+H].sup.+.

##STR00029##

[0209] Cyclooctene 13: Tetrachlorocyclopropene (0.57 ml, 5.72 mmol) was added dropwise to a suspension of AlCl.sub.3 (2.74 g, 20.7 mmol) in CH.sub.2Cl.sub.2 (45 ml) and the reaction was stirred at room temperature for 15 min. The solution was cooled to 78? C. and a solution of acylamine 12 (1.85 g, 5.16 mmol) in CH.sub.2Cl.sub.2 (30 ml) was slowly added. The reaction was allowed to warm to room temperature overnight before water (39 ml) was added and the reaction was stirred at room temperature for 30 min. The mixture was extracted with CH.sub.2Cl.sub.2, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (EtOAc/MeOH, 100:1, R.sub.f=0.4) gave cyclooctene 13 (1.60 g, 3.93 mmol, 38%) as a yellowish oil.

[0210] R.sub.f=0.4 [EtOAc/MeOH, 100:1].

[0211] .sup.1H NMR (400 MHZ, CDC.sub.3) ?8.00 (d, J=8.66 Hz, 1H), 7.90 (d, J=8.53 Hz, 1H), 7.25 (d, J=2.51 Hz, 1H), 7.18 (d, J=2.51 Hz, 1H), 7.06 (dd, J=8.66, 2.51 Hz, 1H), 6.95 (dd, J=8.47, 2.57 Hz, 1H), 5.21 (d, J=14.4 Hz, 1H), 4.12 (d, J=14.4 Hz, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.59 (s, 3H), 2.76 (m, 1H), 2.66 (m, 1H), 2.36 (m, 1H), 1.94 (m, 1H) ppm.

[0212] MS (ESI): calcd. for C.sub.24H.sub.24NO.sub.6.sup.+: 408.1 [M+H].sup.+ [0213] found: 408.3 [M+H].sup.+.

##STR00030##

[0214] Phenol 14: BBr.sub.3 (1.0M in CH.sub.2Cl.sub.2, 20 ml, 20 mmol) was added slowly to a solution of cyclooctene 13 (817 mg, 2.00 mmol) in CH.sub.2Cl.sub.2 (95 ml) and the solution was stirred at 78? C. for 1 h and at room temperature for 48 h. The reaction was quenched with water, basified with 4M NaOH and washed with CH.sub.2Cl.sub.2. The aqueous phase was acidified with conc. HCl and the resultant precipitate was collected. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. The combined solids were dissolved in MeOH (20 ml), conc. H.sub.2SO.sub.4 (0.4 ml) was added and the reaction was stirred at 65? C. for 3 h. The mixture was cooled to room temperature, diluted with water and extracted with EtOAc. The combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (6-8% MeOH in CH.sub.2Cl.sub.2) gave phenol 14 (424 mg, 1.12 mmol, 56% over 2 steps) as a brownish solid.

[0215] MS (ESI): calcd. for C.sub.21H.sub.18NO.sub.6.sup.+: 380.1 [M+H].sup.+ [0216] found: 380.3 [M+H].sup.+.

##STR00031##

[0217] Acetonide 15: DEAD (40% in PhMe, 94 ?L, 0.207 mmol) was added dropwise to a stirred solution of phenol 14 (26 mg, 0.069 mmol), PPh.sub.3 (54 mg, 0.207 mmol) and (R)-(?)-2,2-dimethyl-1,3-dioxolane-4-methanol (26 ?L, 0.207 mmol) in THF (1.5 ml) before the reaction was stirred at room temperature for 20 h and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (10-30% acetone in CH.sub.2Cl.sub.2, R.sub.f=0.3 (20% acetone in CH.sub.2Cl.sub.2)) gave acetonide 15 (33 mg, 0.054 mmol, 79%) as a pale white solid.

[0218] R.sub.f=0.3 [acetone/CH.sub.2Cl.sub.2, 1:4].

[0219] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=7.99 (d, J=8.66 Hz, 1H), 7.89 (d, J=8.41 Hz, 1H), 7.25 (t, J=2.64 Hz, 1H), 7.21 (t, J=2.26 Hz, 1H), 7.07 (m, 1H), 8.96 (dt, J=8.47, 2.60 Hz, 1H), 5.17 (d, J=14.3 Hz, 1H), 4.50 (m, 2H), 4.18 (m, 7H), 3.93 (m, 2H), 3.58 (s, 3H), 2.68 (m, 2H), 2.35 (m, 1H), 1.92 (m, 1H), 1.47 (s, 6H), 1.41 (s, 6H) ppm.

[0220] MS (ESI): calcd. for C.sub.33H.sub.38NO.sub.10.sup.+: 608.2 [M+H].sup.+ [0221] found: 608.3 [M+H].sup.+.

##STR00032##

[0222] Tetraol 16: 1M HCl (0.1 ml) was added slowly to a solution of acetonide 15 (11 mg, 0.018 mmol) in MeOH (0.2 ml) and the reaction was stirred at room temperature for 2 h. The mixture was directly submitted to flash column chromatography (10% MeOH in CH.sub.2Cl.sub.2) to give tetraol 16 (9.7 mg, 0.018 mmol, 100%) as a colorless oil.

[0223] R.sub.f=0.2 [methanol/CH.sub.2Cl.sub.2, 1:9].

[0224] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=8.04 (d, J=8.66 Hz, 1H), 7.88 (d, J=8.53 Hz, 1H), 7.34 (m, 2H), 7.24 (d, J=8.60, 2.45 Hz, 1H), 7.08 (dd, J=8.53, 2.51 Hz, 1H), 5.17 (d, J=14.6 Hz, 1H), 4.28 (d, J=14.3 Hz, 1H), 4.20 (m, 4H), 4.02 (m, 2H), 3.70 (m, 4H), 3.46 (s, 3H). 2.65 (m, 1H), 2.42 (m, 2H), 2.03 (m, 1H) ppm.

[0225] MS (ESI): calcd. for C.sub.27H.sub.30NO.sub.10.sup.+: 528.2 [M+H].sup.+ [0226] found: 528.4 [M+H].sup.+.

##STR00033##

[0227] Alkyne 17: A solution of tetraol 16 (9.7 mg, 0.018 mmol) in MeOH (1.5 ml) was irradiated (360 nm) for 30 min. Concentration of the solution under reduced pressure and purification of the resulting residue by flash column chromatography (10% MeOH in CH.sub.2Cl.sub.2, R.sub.f=0.2) gave alkyne 17 (4.3 mg, 8.6 ?mol, 48%) as a colorless oil.

[0228] R.sub.f=0.2 [methanol/CH.sub.2Cl.sub.2, 1:9].

[0229] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.31 (d, J=8.53 Hz, 1H), 7.26 (m, 2H), 7.12 (d, J=8.41 Hz, 1H), 7.05 (dd, J=8.6, 2.57 Hz, 1H), 6.90 (dd, J=8.47, 2.57 Hz, 1H) 5.014 (d, J=14.1 Hz, 1H)., 4.06 (m, 6H), 3.73 (d, J=13.8 Hz, 1H), 3.67 (m, 4H), 3.53 (s, 3H), 2.74 (m, 1H), 2.47 (m, 1H), 2.38 (m, 1H), 2.01 (m, 1H) ppm.

[0230] MS (ESI): calcd. for C.sub.33H.sub.38NO.sub.10.sup.+: 500.2 [M+H].sup.+ [0231] found: 500.4 [M+H].sup.+.

##STR00034##

[0232] Carboxylic acid 18: 1M NaOH (100 ?L) was added to a solution of alkyne 17 (15.8 mg, 0.032 mmol) in THF (1 ml) and MeOH (1 ml) and the reaction was stirred at room temperature overnight. Concentration of the reaction under reduced pressure and purification of the resulting residue by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, 18-34% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave carboxylic acid 18 (3.2 mg, 6.6 ?mol, 21%) as a white solid.

[0233] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.30 (m, 2H), 7.26 (d, J=2.38 Hz, 1H), 7.12 (d, J=8.41 Hz, 1H), 7.04 (dd, J=8.53, 2.51 Hz, 1H), 6.90 (dd, J=8.41, 2.51 ppm.

[0234] MS (ESI): calcd. for C.sub.25H.sub.28NO.sub.9.sup.+: 486.2 [M+H].sup.+ [0235] found: 486.4 [M+H].sup.+.

##STR00035##

[0236] tert-Butylester 19: BBr.sub.3 (1.0M in CH.sub.2Cl.sub.2, 39ml, 39 mmol) was added slowly to a solution of cyclooctane 13 (1.60 g, 3.93 mmol) in CH.sub.2Cl.sub.2 (190 ml) and the solution was stirred at ?78? C. for 1 h and at room temperature for 48 h. The reaction was quenched with water, basified with 4M NaOH and washed with CH.sub.2Cl.sub.2. The aqueous phase was acidified with conc. HCl and the resultant precipitate was collected. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. The solids were combined, dissolved in MeOH (10 ml), THF (10 ml) and 1M NaOH (16 ml) and stirred at room temperature for 3 h. The reaction mixture was acidified with conc. HCl and the resultant precipitate was collected. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over MgSO.sub.4 and concentrated under reduced pressure. The combined solids (702 mg) and glycine tert-butyl ester hydrochloride (322 mg, 1.92 mmol) were dissolved in DMF (22 ml). HATU (728 mg, 1.92 mmol and DIPEA (1.32 ml, 7.63 mmol) were sequentially added and the resulting mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. Purification of the resultant residue by flash column chromatography (4-6% MeOH in CH.sub.2Cl.sub.2, R.sub.f=0.4, 6% MeOH in CH.sub.2Cl.sub.2) gave tert-butylester 19 (333 mg, 0.696 mmol, 18% over 3 steps) as a brownish solid.

[0237] R.sub.f=0.4 [methanol/CH.sub.2Cl.sub.2, 6:94].

[0238] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.90 (d, J=8.53 Hz, 1H), 7.75 (d, J=8.28 Hz, 1H), 7,12 (m, 1H), 7.02 (m, 2H), 6.85 (dd, J=8.34, 2.32 ppm.

[0239] MS (ESI): calcd. for C.sub.26H.sub.27N.sub.2O.sub.7.sup.+: 479.2 [M+H].sup.+ [0240] found: 479.0 [M+H].sup.+.

##STR00036##

[0241] Acetonide 20: DEAD (40% in PhMe, 0.48 ml, 0.836 mmol) was added dropwise to a solution of tert-butylester 19 (100 mg, 0.209 mmol), PPh.sub.3 (219 mg, 0.836 mmol) and (R)-(?)-2,2-dimethyl-1,3-dioxolane-4-methanol (103 ?L, 0.836 mmol) in THF (5.5 ml). After stirring the reaction at room temperature overnight, the solvent was removed under reduced pressure and the residue was purified by column chromatography (30-50% acetone in CH.sub.2Cl.sub.2, R.sub.f=0.5 (40% acetone in CH.sub.2Cl.sub.2)). Acetonide 20 (128 mg, 0.181 mmol, 87%) was obtained as a white solid.

[0242] R.sub.f=0.5 [acetonel/CH.sub.2Cl.sub.2, 2:3].

[0243] .sup.1H NMR (400 MHz, CDCl.sub.3) ?=7.95 (d, J=8.28 Hz, 1H), 7.86 (d, J=8.41 Hz, 1H), 7.24 (m, 2H), 7.05 (dt, J=8.66, 2.89 Hz, 1H), 6.94 (dt, J=8.53, 2.32 Hz, 1H), 6.05 (brt, J=4.77 Hz, 1H), 5.15 (d, J=14.4 Hz, 1H), 4.49 (m, 2H), 4.12 (m, 7H), 3.90 (m, 2H), 3.83 (d, J=5-14 Hz, 2H), 2.85 (m, 1H), 2,60 (m, 1H), 2.16 (m, 1H), 1.89 (m, 1H), 1.45 (s, 6H), 1.44 (s, 9H), 1.39 (s, 6H) ppm.

[0244] MS (ESI): calcd. for C.sub.38H.sub.47N.sub.2O.sub.11.sup.+: 707.3 [M+H].sup.+ [0245] found: 707.6 [M+H].sup.+.

##STR00037##

[0246] Carboxylic acid 21: TFA (0.5 ml) was added to a mixture of acetonide 20 (64 mg, 0.091 mmol), iPr.sub.3SiH (0.1 ml), water (0.1 ml) and CH.sub.2Cl.sub.2 (1 ml). The reaction was stirred at room temperature for 6 h, diluted with CH.sub.2Cl.sub.2 and extracted with water. The combined aqueous phases were lyophilized to give carboxylic acid 21 (48.5 mg, 0.085 mmol, 93%) .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=8.00 (d, J=8.66 Hz, 1H), 7.84 (d, J=8.53 Hz, 1H), 7.36 (t, J=2.13 Hz, 1H), 7.31 (d, J=2.51 Hz, 1H), 7.20 (dd, J=8.60, 2.32 Hz, 1H), 7.04 (dd, J=8.47, 2.45 Hz, 1H), 5.15 (d, J=14.56 Hz, 1H), 4.18 (m, 5H), 4.02 (m, 2H), 3.77 (s, 2H), 3.70 (m, 4H), 2.73 (m, 1H), 2.44 (m, 1H), 2.26 (m, 1H), 2.01 (m, 1H) ppm.

[0247] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=174.8, 174.3, 173.1, 163.2, 162.3, 153.2, 146.2, 142.6, 141.6, 138.3, 135.2, 134.6, 118.5, 115.6, 115.2, 115.0, 114.2, 113.9, 70.2, 70.1, 69.9, 69.4, 62.6, 62.5, 55.7, 40.3, 30.0, 29.4 ppm.

[0248] MS (ESI): calcd. for C.sub.35H.sub.45N.sub.4O.sub.12.sup.+: 571.2 [M+H].sup.+ [0249] found: 571.4 [M+H].sup.+.

##STR00038##

[0250] Amide 22: DIPEA (29 ?L, 0.168 mmol) was added to a solution of carboxylic acid 21 (48 mg, 0.084 mmol), N-Boc-ethylenediamine (27 mg, 0.168 mmol) and HATU (64 mg, 0.168 mmol) In DMF (1 ml). After stirring the reaction at room temperature overnight, the solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, 14-30% MeCN in H.sub.2O, 0.1% TFA, over 30 min). Amide 22 (30 mg, 0.042 mmol, 50%) was obtained as a white solid.

[0251] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=8.02 (d, J=8.53 Hz, 1H), 7.86 (d, J=8.53, 1H), 7.34 (m, 2H), 7.23 (dd, J=8.66, 2.38 Hz, 1H), 7.07 (dd, J=8.53, 2.51 Hz, 1H), 5.21 (d, J=14.8 Hz, 1H), 4.19 (m, 5H), 4.02 (m, 2H), 3.82 (d, J=16.8 Hz, 1H), 3.69 (m, 4H), 3.60 (d, J=16.8 Hz, 1H), 3.18 (m, 2H), 2.83 (m, 1H), 2.29 (m, 2H), 2.03 (m, 1H), 1.42 (s, 9H) ppm.

[0252] MS (ESI): calcd. for C.sub.35H.sub.45N.sub.4O.sub.12.sup.+: 713.3 [M+H].sup.+ [0253] found: 713.6 [M+H].sup.+.

##STR00039##

[0254] Amine 23: TFA (0.5 ml) was added to a mixture of amide 22 (30 mg, 0.042 mmol), iPr.sub.3SiH (0.1 ml), water (0.1 ml) and CH.sub.2Cl.sub.2 (1 ml). The reaction was stirred at room temperature for 3 h, diluted with CH.sub.2Cl.sub.2 and extracted with water. The combined aqueous phases were lyophilized to give crude deprotectd amine.

[0255] The residue was dissolved in MeOH (3 ml) and irradiated (360 nm) for 45 min. All volatiles were removed under reduced pressure and the resultant residue was purified by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, 15-31% MeCN in H.sub.2O, 0.1% TFA, over 30 min) to give amine 23 (18.7 mg, 0.027 mmol, 64% over 2 steps) a white solid.

[0256] .sup.1H NMR (400 MHZ, d.sub.4-MeOH) ?=7.32 (d, J=8.66, 1H), 7.23 (d, J=2.51 Hz, 2H), 7.14 (d, J=8.53 Hz, 1H), 7.06 (dd, J=8.60, 2.57 Hz, 1H), 6.92 (dd, J=8.47, 2.32 Hz, 1H), 5.04 (d, J=14.2 Hz, 1H), 4.06 (m, 6H), 3.87 (dd, J=17.1, 1.69 Hz, 1H), 3.76 (d, J=14.1 Hz, 1H), 3.68 (m, 5H), 3.51 (t, J=5.84 Hz, 2H), 3.07, m, 2H), 2.85 (m, 1H), 2.29 (m, 2H), 2.07 (m, 1H) ppm.

[0257] .sup.13C NMR (150 MHz, d.sub.4-MeOH): ?=174.7, 173.0, 171.8, 159.2, 159.2, 158.5, 151.9, 149.6, 127.0, 125.9, 119.2, 119.2, 116.2, 115.5, 114.6, 114.4, 112.8, 106.3, 70.3, 70.2, 69.5, 69.2, 62.7, 62.6, 42.5, 39.7, 36.6, 30.5, 29.8 ppm.

[0258] MS (ESI): calcd. for C.sub.29H.sub.37N.sub.4O.sub.9.sup.+: 585.3 [M+H].sup.+ [0259] found: 585.5 [M+H].sup.+.

##STR00040##

[0260] Ether 24: NaOH (30%, 42 ml) was added slowly to a mixture of N-Z-ethanolamine (4.00 g, 20.5 mmol), tert-butyl bromoacetate (6.06 ml, 41.0 mmol) and Bu.sub.4NHSO.sub.4 (2.96 g, 8.72 mmol) in PhMe (84 ml) and the reaction was allowed to stir at room temperature overnight. tert-Butyl bromoacetate (1.74 ml, 11.8 mmol) was added and the reaction was stirred at room temperature for 6 h. The layers were separated and the organic phase was washed with 5% AcOH and water, dried over MgSO.sub.4 and concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (EtOAc:hexane=1:4) gave ether 24 (1.35 g, 4.36 mmol, 21%) as a colorless oil.

[0261] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=7.34 (m, 4H), 5.44 (brs, 1H), 5.10 (s, 2H), 3.95 (s, 2H), 3.61 (m, 2H), 3.41 (m, 2H), 1.46 (s, 9H) ppm.

##STR00041##

[0262] Amine 25: Pd/C (710 mg) was added to a solution of ether 24 (1.35 g, 4.36 mmol) in EtOAc (15 ml) and the reaction vessel was set under a H.sub.2-atmosphere. The reaction was stirred at room temperature for 3 h and then filtered over celite. Concentration of the solution under reduced pressure gave amine 25 (670 mg, 3.82 mmol, 88%) as a colorless oil.

[0263] .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=3.99 (s, 2H), 3.57 (t, J=5.04 Hz, 2H), 2.91 (t, J=5.20 Hz, 2H), 1.87 (brs, 2H), 1.48 (s, 9H) ppm.

##STR00042##

[0264] Amide 27: DIPEA (48 ?L, 0,178 mmol) was added to a solution of carboxylic acid 26 (50 mg, 0.137 mmol) and TSTU (54 mg, 0.178 mmol) in DMF (2 ml) and the reaction was stirred at room temperature for 2 h. Amine 25 (31 mg, 0.178 mmol) was added and the reaction was stirred at room temperature for 4 h and concentrated under reduced pressure. Purification of the resulting residue by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, 35-51% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave amide 27 (31.6 mg, 60 ?mol, 44%) as a white solid.

[0265] .sup.1H NMR (400 MHz, d.sub.4-MeOH) ?=7.91 (d, J=8.71 Hz, 1H), 7.76 (d, J=8.31 Hz, 1H), 7.13 (d, J=2.37 Hz, 1H), 7.05 (d, J=1.98 Hz, 1H), 7.01 (dd, J=8.31, 2.37 Hz, 1H), 6.86 (dd, J=8.51, 2.18 Hz, 1H), 5.09 (d, J=14.6 Hz, 1H), 4.21 (d, J=14.3 Hz, 1H), 3.95 (s, 2H), 3.45 (m, 2H), 3.23 (m, 2H), 2.68 (m, 1H), 2.34 (m, 1H), 2.23 (m, 1H), 2.06 (m, 1H), 1.46 (s, 9H) ppm.

[0266] MS (ESI): calcd. for C.sub.16H.sub.24NO.sub.5.sup.+: 523.2 [M+H].sup.+ [0267] found: 523.4 [M+H].sup.+.

##STR00043##

[0268] Sulfonic acid 28: A suspension of amide 27 (100 mg, 0.191 mmol), K.sub.2CO.sub.3 (132 mg, 0.955 mmol) and sodium 2-bromoethanesulfonate (202 mg, 0.955 mmol) in MeCN (2.0 ml) was stirred at 80? C. for 20 h. Purification of the reaction mixture by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, 22-38% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave sulfonic acid 28 (46 mg, 0.058 mmol, 30%) as a white solid.

[0269] MS (ESI): calcd. for C.sub.32H.sub.39N.sub.2O.sub.14S.sub.2.sup.+: 739.2 [M+H].sup.+ [0270] found: 739.3 [M+H].sup.+.

##STR00044##

[0271] Carboxylic acid 29: : A solution of sulfonic acid 28 (45 mg, 0.061 mmol) in CH.sub.2Cl.sub.2 (2 ml), iPr.sub.3SiH (0.2 ml), water (0.2 ml) and TFA (1 ml) was stirred at room temperature for 6 h, diluted with water and lyophilized. Purification of the resultant residue by reversed phase HPLC chromatography (Chromolith RP18e, C.sub.18, C18, 16-32% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave carboxylic acid 29 (15.2 mg, 0.021 mmol, 34%) as a white solid.

[0272] MS (ESI): calcd. for C.sub.28H.sub.31N.sub.2O.sub.14S.sub.2.sup.+: 683.1 [M+H].sup.+ [0273] found: 683.3 [M+H].sup.+.

##STR00045##

[0274] Alkyne 30: A solution of carboxylic acid 29 (17 mg, 0.023 mmol) in MeOH (2 ml) and DIPEA (0.10 ml) was irradiated (360 nm) for 0.5 h and concentrated under reduced pressure. Purification of the resultant residue by reversed phase HPLC chromatography (YMC-Triart C18, 18-30% MeCN in H.sub.2O, 0.1% TFA, over 30 min) gave carboxylic acid 30 (16.7 mg, 0.018 mmol, 80%) as a white solid.

[0275] MS (ESI): calcd. for C.sub.27H.sub.31N.sub.2O.sub.13S.sub.2.sup.+: 655.1 [M+H].sup.+ [0276] found: 655.3 [M+H].sup.+.

##STR00046##

[0277] Ruthenium complex 33: DIPEA (28 ?L, 0.158 mmol), NHS (5.0 mg, 43.5 ?mol) and EDC HCl (15.2 mg, 79.0 ?mol) were sequentially added to a solution of 10 (36 mg, 40 ?mol) in DMF (1.0 ml) and the reaction was allowed to stir at room temperature for 2 h. Amine 34 (45 mg, 40 ?mol) was added and the reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure and the resulting residue was purified by HPLC-chromatography (C18, 2-90% MeCN in H.sub.2O, 0.05% TFA, over 90 min) to give ruthenium complex 33 (31 mg, 21.6 mmol, 55%) as a red solid.

[0278] MS (ESI): calcd. for C.sub.69H.sub.74N.sub.10O.sub.14S.sub.2Ru.sup.+: 717.20 [M+2H].sup.2+/2 [0279] found: 717.27 [M+2H].sup.2+/2.

##STR00047##

[0280] Ruthenium complex 35: A solution of amide 36 (7.7 mg, 7.1 mmol), NHS-ester 8 (7.2 mg, 10.6 mmol), and DIPEA (2.5 ?L. 14.4 ?mol) in DMF (1.0 ml) was stirred at room temperature for 1 d. The solvent was removed under reduced pressure and the resulting residue was purified by HPLC-chromatography (C18, 0-100% MeCN in H.sub.2O, 0.1% TFA, over 80 min) to give ruthenium complex 35 (3.7 mg, 2.2 mmol, 31%) as a red solid.

[0281] MS (ESI): calcd. for C.sub.71H.sub.80N.sub.10O.sub.12Ru.sup.+: 683.26 [M+2H].sup.2+/2 [0282] found: 683.39 [M+2H].sup.2+/2.

Solubility Experiments

[0283] ##STR00048##

[0284] Inventive compound 8 (16.9 mg, 0.0248 mmol), inventive compound 11 (10.4 mg, 0.0139 mmol), standard compound 31 (5.0 mg, 0.015 mmol) and standard compound 32 (4.2 mg, 0.0104 mmol) were each successively mixed with water to reach a theoretical concentration of 515 mM, 16.5 mM, 12.4 mM, 6 mM and 0.6 mM. After each addition the mixture was sonicated for 10 s. If solid compound was still visible, this was considered as not dissolved. If no solid compound was visible, this was considered as dissolved. The results of the visual inspection are listed in Table 1, wherein yes or no indicate whether the compound was considered dissolved based on visual inspection.

TABLE-US-00001 TABLE 1 Comparison of the solubility of standard compounds 31 and 32 and compounds 8 and 11 according to invention Compound 515 mM 16.5 mM 12.4 mM 6.0 mM 0.6 mM 11 yes yes yes yes yes 8 no no yes yes yes 32 no no no no no 31 no no no no no

[0285] The inventive compounds have a significant higher hydrophilie as shown by their better solubility in water. It is apparent that from the inventive compounds, those carrying SO.sub.3.sup.? groups are even more hydrophilic than the inventive compounds having hydroxyl groups.

Hydrophobicity Indicated by HPLC

[0286] ##STR00049##

[0287] HPLC-chromatogram on a reverse phase (RP HPLC, YMC-Triart C18, 0-100% MeCN in H.sub.2O, 5 0.1% TFA in 25 min) of inventive compounds 7 and 10 as well as standard compound 31 were taken, the respective retention times are listed in Table 2. A later retention corresponded to better interactions with the hydrophobic stationary phase and the compound could therefore be considered as more hydrophobic. The other way round, an earlier retention corresponded to higher hydrophilie.

TABLE-US-00002 TABLE 2 Retention time on YMC-Triart C.sub.18, 0-100% MeCN in H.sub.2O, 0.1% TFA in 25 min Compound Retention Time 31 15.18 min 7 11.58 min 10 9.45 min

[0288] It could be seen that the inventive compounds are more hydrophilic, wherein it was apparent that the inventive compounds having SO3 groups were even more hydrophilic than the inventive compounds having hydroxyl groups.

Antibody Conjugation

[0289] Modified target molecule comprising the reaction product of a conjugate of formula (II) and a target molecule, here a MAB<Tn-T>chim-5D8-IgG antibody (anti Troponin T monoclonal IgG antibody manufactured by Roche in Penzberg/Germany, Roche material no. 05074991001; the monoclonal antibody 5D8 is known to the art, e.g. from Jaffe A. S. et al. Journal of the American College of Cardiology 58 (2011) 1819-1824), comprising a 1,3-dipole group, here an azide group, were prepared. These target molecule conjugates were also abbreviated in the following as conjugates.

[0290] The MAB<Tn-T>chim-5D8-IgG antibody was treated with an increasing excess (5, 10, 15 and 20-fold) of NHS-PEG5-DBCO (for the reference compound; entry 1-4) or NHS-PEG4-azide (for the inventive compounds; entry 5-12). The unconjugated excess labels were removed by dialysis. These conjugates were further treated with an 3-fold excess (compared to the previously used NHS ester) of BPRu-(O20c).sub.3-azide (entry 1-4) or 33 (entry 5-8) or 35 (entry 9-12) respectively. After the reaction, the unconjugated excess labels were separated by size exclusion chromatography by Superdex? 200 Increase 10/300 GL using 5% DMSO in

[0291] Phosphate buffer 50 mM K.sup.+ phosphate, 150 mM KCl pH 7.4. Antibodies treated with 33 achieved higher incorporation of the label compared to the reference compound (table 3).

TABLE-US-00003 TABLE 3 Conjugation of label to the antibody DBCO or Azide/IgG MS Ab:Ru Ratio incorporation conc Vol. Amount Product No. Antibody Label incorporation 1: 455/280 Label/IgG mg/ml ml mg Yield % 1 TnT-IgG-PEG5- BPRu-(O2Oc)3-Azide 10 0.091 4 0.346 0.19 0.066 13 2 DBCO (R144) 21 0.125 5.8 0.437 0.28 0.122 24 3 33 0.138 8.2 0.256 0.382 0.098 20 4 45 0.145 11.2 0.142 0.337 0.048 10 5 TnT-IgG-PEG4- BPRu-PEG3-sulfoDBCO 3.4 10 0.116 4.3 0.957 0.333 0.319 64 6 Azide (R155) 7.5 21 0.146 8.2 0.626 0.444 0.278 56 7 11 33 0.159 12.1 0.44 0.489 0.215 43 8 15 45 0.166 15.4 0.261 0.534 0.139 28 9 TnT-IgG-PEG4- BPRu-PEG3- 3.4 10 0.103 3.5 0.697 0.278 0.194 39 10 Azide dihydroxyDBCO (R156) 7.5 21 0.129 5.9 0.401 0.407 0.163 33 11 11 33 0.140 9.8 0.201 0.435 0.087 17 12 15 45 0.146 12.3 0.089 0.327 0.029 6

[0292] All conjugates were evaluated in a model sandwich immunoassay (Elecsys Troponin T hs, Roche: 05092744 190) containing streptavidin-coated beads (Roche: 05092744) measured on an Elecsys e170 modular using an ECL-signal as the readout. Measurements without analyte in buffer (Diluent Universal, Roche: 11732277 122) showed that 33 was able to reduce the background 71-281 fold and 35 2-8 fold. Using serum (Diluent MultiAssay, Roche: 03609987 190), the background-signal reduction increased to 56-1065 fold for 33 and 9-62 fold for 35 (table 4).

[0293] The reduction of the background signal for 33 and 35 conjugates in comparison to that of reference DBCO conjugates can be attributed to hydrophilization (due to sulfonation or hydroxylation) of the otherwise hydrophobic moieties.

[0294] Measurements of Calibrator 2 (id. 05092752 190) lead to a signal which was in or slightly above the background when the reference conjugate was used (see Cal2/MA above). Using 33 and 35 the ratio of signal to background was increased up to 374 and 334 respectively (see Cal2/MA above).

[0295] Stability tests revealed a higher recovery of the ECL-signal for conjugates with 33 and 35 compared to the reference compound after storage for 8 days at 35? C.

TABLE-US-00004 TABLE 4 ECl-measurements of the antibody conjugates Day 8 Cal2 Day 0 recovery Uni- MA- TNT- TNT- after 8 Incorporation Dil- Dil- Cal1- Cal2- Cal1/ Cal2/ days at No. Code Antibody Label Label/IgG PW PW PW PW MA MA 35? C. 1 R144-5 TnT-PEG5-DBCO BPRu-(O2Oc)3- 4 102624 45512 15551 446878 0 10 89 Azide 2 R144-10 TnT-PEG5-DBCO BPRu-(O2Oc)3- 5.8 1502759 915223 550613 1274193 1 1 89 Azide 3 R144-15 TnT-PEG5-DBCO BPRu-(O2Oc)3- 8.2 4226361 2991802 2145384 3001804 1 1 87 Azide 4 R144-20 TnT-PEG5-DBCO BPRu-(O2Oc)3- 11.2 5936047 4418477 3366187 4255854 1 1 87 Azide 5 R155-5 TnT-PEG4-Azide BPRu-PEG3- 4.3 1409 810 1072 268391 1 331 94 sulfoDBCO 6 R155-10 TnT-PEG4-Azide BPRu-PEG3- 8.2 5356 1184 2160 442854 2 374 93 sulfoDBCO 7 R155-15 TnT-PEG4-Azide BPRu-PEG3- 12.1 30400 2808 8120 553236 3 197 93 sulfoDBCO 8 R155-20 TnT-PEG4-Azide BPRu-PEG3- 15.4 83749 6059 23996 638850 4 105 92 sulfoDBCO 9 R156-5 TnT-PEG4-Azide BPRu-PEG3- 3.5 13294 953 2076 318077 2 334 92 dihydroxyDBCO 10 R156-10 TnT-PEG4-Azide BPRu-PEG3- 5.9 377650 14767 42735 810926 3 55 93 dihydroxyDBCO 11 R156-15 TnT-PEG4-Azide BPRu-PEG3- 9.8 1613162 124105 275756 1359021 2 11 92 dihydroxyDBCO 12 R156-20 TnT-PEG4-Azide BPRu-PEG3- 12.3 3342026 475372 780624 2078544 2 4 90 dihydroxyDBCO

CITED LITERATURE

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