IV. CASO DE ESTUDIO
IV.3. Análisis de comportamiento del modelo
IV.3.3. Comportamiento al variar la capacidad de los vehículos
independent recognition units. For example, no FSHR agonistic activities were observed for
compounds (R)-7a-e, while the compounds are active agonists on the LHR. Several conclusions
may be drawn from the results above. 1) The LHR tolerates the large inactive (S)-THQ or active
(R)-THQ that is connected to lead structure 2 with only a moderate loss in agonistic potency on
the receptor. In addition, no discrepancy in activity of the compounds is observed on the LHR if
the monomeric units from (R)-THQ and 2 are mixed. This suggests that the (R)-THQ does not
influence the bioactivity of the monomeric ligands derived from 2 on the LHR. 2) The agonistic
potency on the FSHR is significantly reduced by introducing the same inactive (S)-THQ on 2.
However at the highest concentration of test compound, some activity for the FSHR is still
retained. Replacement of inactive (S)-THQ by the active (R)-THQ reduces the FSHR agonistic
activity completely. 3) Antagonistic potencies on the FSHR for the hetero-dimeric compounds
(R)-7c-e are enhanced compared to the homodimeric (R,R)-4c-e
whereas mixing the
recognition units (that is, monomeric ligands 3c and 5c) results in reduced antagonistic
potencies. It would be of interest to further investigate if the LH/FSHR agonist that is present in
(R)-7c-e has a positive (cooperativity) effect for the antagonistic potency of the (R)-THQ that is
located at the other end of the spacer moiety.
Experimental procedures
Measurement of CRE-induced luciferase activity
Materials. Recombinant human LH (recLH) and human recombinant FSH (recFSH) were synthesized at Schering-Plough Research Institute, Oss, The Netherlands. Luclite® was obtained from Packard. All cell culture supplies were obtained from Gibco/BRL unless indicated otherwise. The human LH receptor cDNA10 and human FSH receptor cDNA11 were kindly provided by Dr. A.J.W. Hsueh, Stanford University.
Luciferase assay. Chinese Hamster Ovary (CHO)-K1 cells stably expressing the CRE-luciferase reporter with the human LH receptor or human FSH receptor were grown to 80-90% confluency in Dulbecco’s MEM/Nutrient Mix F12 containing 5% bovine calf serum and supplemented with penicillin G (80 units/mL) and streptomycin (0.08 mg/mL) in 5% CO2 at 37 °C. Cells were harvested using cell dissociation solution (Sigma). Aliquots of the cells were cryopreserved in DMSO without a loss of functional activity on LH receptor or FSH receptor.12 On the day of the experiment, cells were thawed, washed with assay medium (Dulbecco’s MEM/Nutrient Mix F12 supplemented with 1 mg/L bovine insulin (Sigma), 5 mg/L apo-transferrin (Sigma), penicillin G (80 units/mL) and streptomycin (0.08 mg/mL) and then resuspended in assay medium. The compounds were tested in quadruplicate at 10 concentrations ranging from final concentrations of 10 μM to 0.316 nM with half log intervals. In the agonistic assays, 10 μL of assay medium containing test compound and 3% DMSO, 10 μL of assay medium containing 3% DMSO with recLH (final concentration of 1 nM) or recFSH (final concentration of 586 pM) or 10 μL of assay medium containing 3% DMSO alone were added to the wells of a 384-well white culture plate followed by the addition of 10 μL of assay medium. Then, 10 μL of cell suspension containing 7,500 cells was added to the wells. The final concentration of DMSO was 1%. In case of antagonistic assays, 10 μL of test compound solution or 10 μL of assay medium alone were added to 10 μL of assay medium containing recLH (final concentration of 100 pM, EC80) or recFSH (final concentration of 49 pM, EC80). Then, 10 μL of cell suspension containing 7,500 cells was added to the wells. After incubation for 4 h in a humidified atmosphere in 5% CO2 at 37°C, plates were allowed to adjust to room temperature for 1 h. Then, 15 μL of LucLite solution (Packard) was added to the incubation mixture. Following 60 min at room temperature in the dark, luciferase activity was measured in a Packard Topcount Microplate Scintillation and Luminescence Counter. Agonistic effects of the compounds were determined as percentage of the (maximal) effect induced by 1 nM recLH or 586 pM recFSH. Antagonistic effects
of the compounds were expressed as percentage of the effect induced by 100 pM recLH or49 pM recFSH. The
EC50 or IC50 values (concentration of the test compound that elicits half-maximal (50 %) luciferase stimulation or inhibition compared to the compound’s maximally attainable effect, respectively) and the efficacy values (maximal effect as percentage of the effect of recLH or recFSH) of the test compounds were determined using the software program MathIQ (version 2.0, ID Business Solutions Limited).
Chemical procedures
Reactions were executed at ambient temperatures unless stated otherwise. All solvents were removed by evaporation under reduced pressure. Reactions were monitored by TLC analysis using silica gel coated plates (0.2 mm thickness) and detection by 254 nm UV-light or by either spraying with a solution of (NH4)6Mo7O24 × 4H2O (25 g/L) or (NH4)4Ce(SO4)4 × 2H2O (10 g/L) in 10% sulfuric acid followed by charring at ~150 °C. Column chromatography was performed on silica gel (40-63 μm). NMR spectra were recorded on a 600/150 MHz spectrometer. Chemical shifts are given in ppm () relative to tetramethylsilane as internal standard. Coupling constants (J) are given in Hz. All presented 13C-APT spectra are proton decoupled. Where indicated, NMR peak assignments were made using COSY, NOESY ( mix = 1 sec) and HMQC experiments. For LC-MS analysis, a HPLC-system (detection simultaneously at 214 and 254 nm) equipped with an analytical C18 column (4.6 mmD ×
250 mmL, 5 particle size) in combination with buffers A: H2O, B: CH3CN and C: 1% aq TFA and coupled to a mass instrument with an electronspray interface (ESI) was used. For RP-HPLC purifications, an automated HPLC system equipped with a semi-preparative C18 column (5 m C18, 10Å, 150 × 21.2 mm) was used. The applied buffers were A: H2O + ammonium acetate (20 mM) and B: CH3CN. High resolution mass spectra were recorded by direct injection (2 μL of a 2 μM solution in water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass
range m/z = 150-2000) and dioctylpthalate (m/z = 391.28428) as a lock mass. The high resolution mass
spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). Optical rotations were measured on a Propol automatic polarimeter (Sodium D-line, = 589 nm).
General procedure for the preparation of monomeric ligands (R)-7a-e and (S)-7a-e.
Monomeric compounds (R)-3a, (R)-3b, (R)-3c, (R)-3d or (R)-3e (12.5 μmol, prepared as described in Chapter 8) and 2 (7.3 mg, 15.0 μmol, prepared as described in Chapter 5) were dissolved in 10 mL DCM. The mixture was degassed under argon in an ultrasonic bath, until the volume was approximately 1.5 mL. Water and solutions of
CuSO4 and sodium ascorbate were degassed separately. Water (1.0 mL) and aqueous solutions of sodium
ascorbate (50 L, 1 eq) and CuSO4 (50 L, 20 mol%) were added. After stirring for 18 h, TLC and LC-MS showed all reactions to be completed. Reaction mixtures were concentrated and coevaporated with toluene (3x). The residue was dissolved in DCM and the product was isolated using silica column chromatography (1 to 3.5% MeOH in DCM). Pure products were obtained as yellow powdery crystals in 75-97% yield. Rf=0.45 (9% MeOH in DCM). Products with spacer length n=0 and n=1 were purified by semi-preparative HPLC (70 to 90% B).
Heterodimeric ligand (R)-7a. 1H NMR (600 MHz, CDCl3) 8.31 (s, 1H, NHCO), 7.96 (d, J = 8.4, 2H, 2 × CH Ar), 7.72 (d, J = 8.4, 1H, CH Ar, THQ-8), 7.67 (d, J = 8.4, 2H, 2 × CH Ar), 7.59 (d, J = 7.8, 3H, 3 × CH Ar), 7.45 (t, J = 7.8, 2H, 2 × CH Ar), 7.38 (t, J = 7.5, 1H, CH Ar), 7.27 (d, J = 7.8, 1H, CH Ar), 7.14 (d, J = 6.0, 2H, 2 × CH Trz), 7.10 (t, J = 5.4, 1H, NHCO), 6.98 (d, J = 9.0, 2H, 2 × CH Ar), 6.95 (d, J = 9.0, 1H, CH Ar, THQ-7), 6.88 (d, J = 7.2, 1H, CH Ar), 6.75 (d, J = 9.0, 2H, 2 × CH Ar), 6.67 – 6.65 (m, 2H, 2 × CH Ar), 5.92 (s, 2H, NH2), 5.20 (s, 1H, NHCO), 5.07 (s, 2H, CH2, OCH2Ctrz), 4.85 – 4.81 (m, 4H, 2 × CH2, CH2Ntrz), 4.58 (t, J = 5.4, 1H, NH), 4.36 (s,
2H, CH2, NHCH2Ctrz), 3.76 (d, J = 5.4, 2H, CH2, NHCH2CONH), 2.60 (s, 3H, CH3, SCH3), 2.45 (d, J = 14.4, 1H, CH2, THQ), 1.92 (s, 3H, CH3, acetyl), 1.76 (d, J = 13.8, 1H, CH2, THQ), 1.58 (s, 3H, CH3(C-CH2)), 1.41 (t, J = 12.6, 9H, 3 × CH3, t-butyl), 1.33 (s, 3H, CH3, C(CH3)2), 1.21 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.0, 169.6, 167.3, 165.5, 165.0 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.4 (Cq(OCH2Trz)), 147.2, 144.7, 144.5, 144.5, 141.8, 139.8, 137.7, 137.4, 135.8, 135.5, 135.6, 133.3 (12 × Cq Ar), 130.0 (CH Ar), 129.0 (2 × CH Ar), 128.1 (3 × CH Ar), 127.6, 127.4, 127.2 (6 × CH Ar), 126.7 (CH Ar, THQ-7), 123.7, 123.1 (2 × CH Trz), 118.7, 118.6 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 115.0 (CH Ar), 114.2 (2 × CH Ar), 112.6 (CH Ar), 96.6 (Cq Ar), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq Ar, THQ-4), 56.2 (CH2, THQ-3), 52.1 (Cq, t-butyl), 49.6, 49.5 (2 × CH2, CH2Ntrz), 48.0 (CH2, NHCH2CONH), 41.9 (Cq, C(CH3)2), 34.6 (CH2, NHCH2Ctrz), 29.4 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.8 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: 149° (c=0.34, CHCl3). ESI-MS m/z: obsd 1137.47 [M + H]+. HRMS m/z calcd for C61H64N14O5S2 + H+: 1137.46983, obsd 1137.47217; calcd for C61H64N14O5S2 + 2H+: 569.23855, 569.23828.
Heterodimeric ligand (R)-7b. 1H NMR (600 MHz, CDCl3) 8.35 (s, 1H, NHCO), 7.99 (d, J = 8.4, 2H, 2 × CH Ar), 7.76 (d, J = 7.2, 1H, CH Ar, THQ-8), 7.70 (d, J = 8.4, 2H, 2 × CH Ar), 7.62 (d, J = 7.8, 2H, 2 × CH Ar), 7.54 (s, 1H, CH Ar, THQ-10), 7.47 (t, J = 7.5, 4H, 3 × CH Ar), 7.40 (t, J = 7.2, 1H, CH Ar), 7.33 (s, 1H, CH Trz), 7.24 (d, J = 7.8, 1H, CH Ar), 6.99 (d, J = 9.0, 2H, 2 × CH Ar), 6.97 (d, J = 9.0, 1H, CH Ar, THQ-7), 6.86 (d, J = 7.2, 1H, CH Ar),
6.82 (d, J = 9.0, 2H, 2 × CH Ar), 6.72-6.70 (m, 2H, 2 × CH Ar), 5.99 (s, 2H, NH2), 5.21 (s, 1H, NHCO), 5.12 (s, 2H, CH2, OCH2Ctrz), 4.98 (t, J = 5.4, 1H, NH), 4.44 – 4.43 (m, 4H, 2 × CH2, CH2Ntrz), 4.39 (q, J = 4.8, 2H, CH2, NHCH2Ctrz), 3.84 (d, J = 4.2, 2H, CH2, NHCH2CONH), 3.74 – 3.71 (m, 4H, 2 × CH2, CH2OCH2), 2.61 (s, 3H, CH3, SCH3), 2.46 (d, J = 13.8, 1H, CH2, THQ), 1.94 (s, 3H, CH3, acetyl), 1.78 (d, J = 14.4, 1H, CH2, THQ), 1.58 (s, 3H, CH3(C-CH2)), 1.43 (t, J = 12.0, 9H, 3 × CH3, t-butyl), 1.32 (s, 3H, CH3, C(CH3)2), 1.23 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.3, 169.6, 167.2, 165.6, 165.1, 162.9 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.4 (Cq(OCH2Trz)), 147.6, 144.8, 144.7, 143.9, 141.8, 139.8, 137.8, 137.4, 135.8, 135.7, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 129.0 (2 × CH Ar), 128.2 (3 × CH Ar), 127.7, 127.4, 127.2 (6 × CH Ar), 126.7 (CH Ar, THQ-7), 123.7, 123.0 (2 × CH Trz), 118.7, 118.1 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.4 (CH Ar, THQ-10), 114.9 (CH Ar), 114.3 (2 × CH Ar), 112.6 (CH Ar), 96.4 (Cq Ar), 69.2 (2 × CH2, CH2OCH2), 61.7 (CH2, OCH2CTrz), 58.5 (Cq, THQ-4), 56.2 (CH2, THQ-3), 52.1 (Cq, t-butyl), 50.1, 50.0 (2 × CH2, CH2Ntrz), 47.8 (CH2, NHCH2CONH), 41.9 (Cq, C(CH3)2), 34.9 (CH2, NHCH2Ctrz), 29.4 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.9 (CH3, C(CH3)2), 25.6 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: 141° (c=0.40, CHCl3). ESI-MS m/z: obsd 1181.47 [M + H]+. HRMS m/z calcd for C63H68N14O6S2 + H+: 1181.49604, obsd 1181.50110; calcd for C63H68N14O6S2 + 2H+: 591.25166, obsd 591.25146.
Heterodimeric ligand (R)-7c. 1H NMR (600 MHz, CDCl3) 8.50 (s, 1H, NHCO), 7.98 (d, J = 8.4, 2H, 2 × CH Ar), 7.75 (s, 1H, CH Trz), 7.74 (s, 1H, CH Ar, THQ-8), 7.67 (d, J = 7.8, 2H, 2 × CH Ar), 7.60 (d, J = 7.2, 2H, 2 × CH Ar), 7.58 (s, 1H, CH Ar, THQ-10), 7.50 (s, 1H, CH Trz), 7.46 (t, J = 7.5, 3H, 2 × CH Ar+ NHCO), 7.39 (t, J = 7.2, 1H, CH Ar), 7.24 (d, J = 7.8, 1H, CH Ar), 6.97 (d, J = 8.4, 2H, 2 × CH Ar), 6.94 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.86 (d, J = 7.2, 1H, CH Ar), 6.79 (d, J = 8.4, 2H, 2 × CH Ar), 6.70 – 6.67 (m, 2H, 2 × CH Ar), 5.97 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.11 (s, 2H, CH2, OCH2Ctrz), 4.88 (s, 1H, NH), 4.48 – 4.46 (m, 4H, 2 × CH2, CH2Ntrz), 4.42 –
4.35 (m, 2H, CH2, NHCH2Ctrz), 3.78 (s, 2H, CH2, NHCH2CONH), 3.76 – 3.69 (m, 4H, 2 × CH2, NtrzCH2CH2O), 3.49 – 3.46 (m, 4H, 2 × CH2, OCH2CH2O), 2.61 (s, 3H, CH3, SCH3), 2.45 (d, J = 13.8, 1H, CH2, THQ), 1.92 (s, 3H, CH3, acetyl), 1.77 (d, J = 13.8, 1H, CH2, THQ), 1.57 (s, 3H, CH3(C-CH2)), 1.42 (s, 3 × CH3, t-butyl), 1.34 (s, 3H, CH3, C(CH3)2), 1.23 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.2, 169.6, 167.2, 165.6, 165.0, 162.8 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.5 (Cq(OCH2Trz)), 147.5, 144.7, 144.6, 144.4, 143.9, 141.7, 139.8, 137.6, 137.4, 135.7, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 129.0 (2 × CH Ar), 128.1, 128.1 (3 × CH Ar), 127.7, 127.3, 127.2 (6 × CH Ar), 126.7 (CH Ar, THQ-7), 124.1, 123.0 (2 × CH Trz), 118.7, 118.3 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 114.9 (CH Ar), 114.1 (2 × CH Ar), 112.6 (CH Ar), 96.5 (Cq Ar), 70.4, 70.6 (2 × CH2, OCH2CH2O), 69.2, 69.2 (2 × CH2, NtrzCH2CH2O), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t- butyl), 50.3, 50.1 (2 × CH2, CH2Ntrz), 47.8 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.8 (CH2, NHCH2Ctrz), 29.4 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.8 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: 162° (c=0.26, CHCl3). ESI-MS m/z: obsd 1225.6 [M + H]+. HRMS m/z calcd for C65H72N14O7S2 + H+: 1225.52226, obsd 1225.52209; calcd for C65H72N14O7S2 + 2H+: 613.26477, obsd 613.26471.
Heterodimeric ligand (R)-7d. 1H NMR (600 MHz, CDCl3) 8.48 (s, 1H, NHCO), 7.98 (d, J = 7.8, 2H, 2 × CH Ar), 7.80 (s, 1H, CH Trz), 7.75 (d, J = 8.4, 1H, CH Ar, THQ-8), 7.67 (d, J = 7.8, 2H, 2 × CH Ar), 7.60 (d, J = 6.6, 3H, 3 × CH Ar), 7.55 (s, 1H, CH Trz), 7.46 (t, J = 7.5, 2H, 2 × CH Ar), 7.39 (t, J = 7.2, 1H, CH Ar), 7.29 (s, 1H, NHCO), 7.24 (s, 1H, CH Ar), 6.99 (d, J = 9.0, 2H, 2 × CH Ar), 6.95 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.88 (d, J = 7.2, 1H, CH Ar), 6.80 (d, J = 8.4, 2H, 2 × CH Ar), 6.70 – 6.68 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.08 (s, 2H, CH2, OCH2Ctrz), 4.78 (s, 1H, NH), 4.50 (t, J = 4.8, 2H, CH2, CH2Ntrz), 4.46 (d, J = 6.0, 2H,
CH2, NHCH2Ctrz), 4.40 (t, J = 4.8, 2H, CH2, CH2Ntrz), 3.83 (t, J = 4.8, 4.8, 2H, CH2, NtrzCH2CH2O), 3.77 (s, 2H, CH2, NHCH2CONH), 3.73 (s, 2H, CH2, NtrzCH2CH2O), 3.56 – 3.49 (m, 8H, 4 × CH2, OCH2CH2O), 2.61 (s, 3H, CH3, SCH3), 2.46 (d, J = 13.8, 1H, CH2, THQ), 1.93 (s, 3H, CH3, acetyl), 1.77 (d, J = 13.8, 1H, CH2, THQ), 1.58 (s,
3H, CH3(C-CH2)), 1.43 (s, 3 × CH3, t-butyl), 1.34 (s, 3H, CH3, C(CH3)2), 1.22 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.1, 170.1, 169.6, 167.2, 165.6, 165.0, 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.6 (Cq(OCH2Trz)), 147.5, 144.7, 144.6, 144.1, 143.8, 141.7, 139.7, 137.6, 137.4, 135.7, 135.7, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 128.9 (2 × CH Ar), 128.1, 128.1 (3 × CH Ar), 127.7, 127.3, 127.1 (6 × CH Ar), 126.6 (CH Ar, THQ-7), 124.1, 123.1 (2 × CH Trz), 118.6, 118.4 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CHar, THQ-10), 114.8 (CH Ar), 114.1 (2 × CH Ar), 112.7 (CH Ar), 96.5 (Cq Ar), 70.4, 70.4, 70.4, 70.3 (4 × CH2, OCH2CH2O), 69.2, 69.2 (2 × CH2, NtrzCH2CH2O), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 50.3, 50.1 (2 × CH2, CH2Ntrz), 47.9 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.7 (CH2, NHCH2Ctrz), 29.6, 29.3 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.7 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: 156° (c=0.33, CHCl3). ESI-MS m/z: obsd 1269.7 [M + H]+. HRMS m/z calcd for C67H76N14O8S2 + 2H+: 635.27787, obsd 635.27777.
Heterodimeric ligand (R)-7e. 1H NMR (600 MHz, CDCl3) 8.44 (s, 1H, NHCO), 7.98 (d, J = 8.4, 2H, 2 × CH Ar), 7.80 (s, 1H, CH Trz), 7.75 (d, J = 6.6, 1H, CH Ar, THQ-8), 7.68 (d, J = 7.8, 3H, 3 × CH Ar), 7.61 (d, J = 6.6, 3H, 3 × CH Ar), 7.46 (t, J = 7.5, 3H, 3 × CH Ar), 7.39 (t, J = 7.2, 1H, CH Ar), 7.27 (s, 1H, CH Ar), 6.99 (d, J = 8.4, 2H, 2 × CH Ar), 6.95 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.89 (d, J = 7.8, 1H, CH Ar), 6.81 (d, J = 8.4, 2H, 2 × CH Ar), 6.72 – 6.69 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.08 (s, 2H, CH2, OCH2Ctrz), 4.78 (s, 1H, NH), 4.50 (t, J = 5.1, 2H, CH2, CH2Ntrz), 4.47 (d, J = 5.4, 2H, CH2, NHCH2Ctrz), 4.42 (t, J = 4.1, 2H, CH2, CH2Ntrz), 3.83 (t, J = 4.8, 2H, CH2, NtrzCH2CH2O), 3.79 (s, 2H, CH2, NHCH2CONH), 3.75 (t, J = 5.1, 2H, CH2, NtrzCH2CH2O), 3.56 – 3.50 (m, 12H, 6 × CH2, OCH2CH2O), 2.61 (s, 3H, CH3, SCH3), 2.47 (d, J = 13.8, 1H, CH2, THQ), 1.93 (s, 3H, CH3, acetyl), 1.77 (d, J = 13.8, 1H, CH2, THQ), 1.59 (s, 3H, CH3(C-CH2)), 1.43 (s, 3 × CH3, t- butyl), 1.34 (s, 3H, CH3, C(CH3)2), 1.22 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.1, 169.6, 167.2, 165.6, 165.0, 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.6 (Cq(OCH2Trz)), 147.5, 144.71 144.6, 144.1, 143.8, 141.7, 139.7, 137.5, 137.4, 135.8, 135.6, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 129.0 (2 × CH Trz), 118.6, 118.5 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 114.9 (CH Ar), 114.2 (2 × CH Ar), 112.8 (CH Ar), 96.5 (Cq Ar), 70.5, 70.5, 70.3 (6 × CH2, OCH2CH2O), 69.3, 69.3 (2 × CH2, NtrzCH2CH2O), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 50.3, 50.2 (2 × CH2, CH2Ntrz), 47.9 (CH2, NHCH2CONH), 41.9 (Cq, C(CH3)2), 34.8 (CH2, NHCH2Ctrz), 29.7, 29.3 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.7 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: 134° (c=0.30, CHCl3). ESI-MS m/z: obsd 1314.5 [M + H]+. HRMS m/z calcd for C69H80N14O9S2 + 2H+: 657.29098, obsd 657.29132.
Heterodimeric ligand (S)-7a. 1H NMR (600 MHz, CDCl3) 8.47 (s, 1H, NHCO), 7.97 (d, J = 8.4, 2H, 2 × CH Ar), 7.73 (d, J = 6.6, 1H, CH Ar, THQ-8), 7.67 (d, J = 7.8, 2H, 2 × CH Ar), 7.59 (d, J = 7.8, 3H, 3 × CH Ar), 7.46 (t, J = 7.5, 2H, 2 × CH Ar), 7.39 (t, J = 7.2, 1H, CH Ar), 7.23 (s, 2H, 2 × CH Trz), 7.19 (d, J = 3.6, 2H, CH Ar, NHCO), 6.98 (d, J = 8.4, 2H, 2 × CH Ar), 6.95 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.86 (d, J = 7.2, 1H, CH Ar), 6.75 (d, J = 9.0, 2H, 2 × CH Ar), 6.66 (d, J = 7.8, 2H, 2 × CH Ar), 5.93 (s, 2H, NH2), 5.23 (s, 1H, NHCO), 5.05 (s, 2H, CH2, OCH2Ctrz), 4.83 – 4.81 (m, 5H, 2 × CH2, CH2Ntrz + NH), 4.35 (d, J = 4.8, 2H, CH2, NHCH2Ctrz), 3.76 (s, 2H, CH2, NHCH2CONH), 2.60 (s, 3H, CH3, SCH3), 2.45 (d, J = 13.8, 1H, CH2, THQ), 1.92 (s, 3H, CH3, acetyl), 1.76 (d, J = 13.8, 1H, CH2, THQ), 1.57 (s, 3H, CH3(C-CH2)), 1.42 (s, 9H, 3 × CH3, t-butyl), 1.33 (s, 3H, CH3, C(CH3)2), 1.21 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.1, 170.1, 169.5, 167.2, 165.6, 165.0, 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.3 (Cq(OCH2Trz)), 147.3, 144.6, 144.6, 144.5, 144.3, 141.7, 139.7, 137.6, 137.3, 135.7, 133.3 (12 × Cq Ar), 129.9 (CH Ar), 128.9 (2 × CH Ar), 128.1 (3 × CH Ar), 127.6, 127.3, 127.1 (6 × CH Ar), 126.6 (CH Ar, THQ-7), 123.7, 123.1 (2 × CH Trz), 118.6, 118.4 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 114.9 (CH Ar), 114.1 (2 × CH Ar), 112.5 (CH Ar), 96.5 (Cq Ar), 61.6 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 49.5, 49.4 (2 × CH2, CH2Ntrz), 47.8 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.5
(CH2, NHCH2Ctrz), 29.3 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.0 (3 × CH3, t-butyl), 28.8 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.3 (CH3, CH3S). []D20: -159° (c=0.29, CHCl3). ESI-MS m/z: obsd 1137.7 [M + H]+. HRMS m/z calcd for C61H64N14O5S2 + H+: 1137.46983, obsd 1137.47095; calcd for C61H64N14O5S2 + 2H+: 569.23855, obsd 569.2384.
Heterodimeric ligand (S)-7b. 1H NMR (600 MHz, CDCl3) 8.42 (s, 1H, NHCO), 7.99 (d, J = 8.4, 2H, 2 × CH Ar), 7.76 (d, J = 6.6, 1H, CH Ar, THQ-8), 7.69 (d, J = 7.8, 2H, 2 × CH Ar), 7.61 (d, J = 7.2, 2H, 2 × CH Ar), 7.54 (s, 1H, CH Ar, THQ-10), 7.50 – 7.45 (m, 4H, 3 × CH Ar + NHCO), 7.39 (t, J = 7.5, 1H, CH Ar), 7.33 (s, 1H, CH Trz), 7.24 (d, J = 7.8, 1H, CH Ar), 6.99 – 6.95 (m, 3H, 3 × CH Ar), 6.85 (d, J = 7.8, 1H, CH Ar), 6.81 (d, J = 9.0, 2H, 2 × CH Ar), 6.71 – 6.69 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.11 (s, 2H, CH2, OCH2Ctrz), 4.98 (s, 1H, NH), 4.43 (d, J = 5.4, 4H, 2 × CH2, CH2Ntrz), 4.38 (q, J = 4.5, 2H, CH2, NHCH2Ctrz), 3.83 (s, 2H, CH2, NHCH2CONH), 3.73 – 3.70 (m, 4H, 2 × CH2, CH2OCH2), 2.62 (s, 3H, CH3, SCH3), 2.46 (d, J = 13.8, 1H, CH2, THQ), 1.93 (s, 3H, CH3, acetyl), 1.77 (d, J = 13.8, 1H, CH2, THQ), 1.57 (s, 3H, CH3(C-CH2)), 1.42 (s, 9H, 3 × CH3, t- butyl), 1.31 (s, 3H, CH3, C(CH3)2), 1.23 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.33 169.6, 167.2, 165.6, 165.0, 162.9 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.4 (Cq(OCH2Trz)), 147.6, 144.8, 144.7, 143.9, 141.8, 139.8, 137.7, 137.3, 135.7, 133.3 (12 × Cq Ar), 129.8 (CH Ar), 128.9 (2 × CH Ar), 128.1 (3 × CH Ar), 127.7, 127.3, 127.2 (6 × CH Ar), 126.7 (CH Ar, THQ-7), 123.7, 123.0 (2 × CH Trz), 118.7, 118.1 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.4 (CH Ar, THQ-10), 114.8 (CH Ar), 114.2 (2 × CH Ar), 112.6 (CH Ar), 96.4 (Cq Ar), 69.1 (2 × CH2, CH2OCH2), 61.6 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 50.1, 50.0 (2 × CH2, CH2Ntrz), 47.7 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.8 (CH2, NHCH2Ctrz), 29.4 (2 × CH3, CH3(C- CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.8 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: -146° (c=0.18, CHCl3). ESI-MS m/z: obsd 1181.7 [M + H]+. HRMS m/z calcd for C63H68N14O6S2 + 2H+: 591.25166, obsd 591.25159.
Heterodimeric ligand (S)-7c. 1H NMR (600 MHz, CDCl3) 8.46 (s, 1H, NHCO), 7.98 (d, J = 8.4, 2H, 2 × CH Ar), 7.75 (s, 1H, CH Trz), 7.74 (s, 1H, CH Ar, THQ-8), 7.68 (d, J = 7.8, 2H, 2 × CH Ar), 7.60 (d, J = 7.8, 2H, 2 × CH Ar), 7.58 (s, 1H, CH Ar, THQ-10), 7.49 (s, 1H, CH Trz), 7.46 (t, J = 7.5, 2H, 2 × CH Ar), 7.41 – 7.39 (m, 2H, CH Ar + NHCO), 7.24 (s,1H, CH Ar), 6.97 (d, J = 8.4, 2H, 2 × CH Ar), 6.95 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.87 (s, 1H, CH Ar), 6.79 (d, J = 9.0, 2H, 2 × CH Ar), 6.70-6.67 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.12 (s, 2H, CH2, OCH2Ctrz), 4.88 (s, 1H, NH), 4.48-4.46 (m, 4H, 2 × CH2, CH2Ntrz+NHCH2Ctrz), 4.39 – 4.38 (m, 2H, CH2, CH2Ntrz), 3.78 (s, 2H, CH2, NHCH2CONH), 3.76 – 3.70 (m, 4H, 2 × CH2, NtrzCH2CH2O), 3.49 – 3.47 (m, 4H, 2 × CH2, OCH2CH2O), 2.60 (s, 3H, CH3, SCH3), 2.45 (d, J = 13.8, 1H, CH2, THQ), 1.93 (s, 3H, CH3, acetyl), 1.77 (d, J = 13.8, 1H, CH2, THQ), 1.57 (s, 3H, CH3(C-CH2)), 1.42 (s, 3 × CH3, t-butyl), 1.34 (s, 3H, CH3, C(CH3)2), 1.23 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.1, 170.1, 169.5, 167.1, 165.5, 165.0, 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.5 (Cq(OCH2Trz)), 147.5, 144.7, 144.6, 144.3, 143.8, 141.7, 139.7, 137.6, 137.6, 137.3, 135.7, 133.3 (12 × Cq Ar), 129.8 (CH Ar), 128.9 (2 × CH Ar), 128.1, 128.0 (3 × CH Ar), 127.8, 127.6, 127.3, 127.2, 127.1 (6 × CH Ar), 126.6 (CH Ar, THQ-7), 124.0, 123.0 (2 × CH Trz), 118.6, 118.2 (2 × CH Ar), 117.6 (Cq, H2NC=CCO), 117.4 (CH Ar, THQ-10), 114.8 (CH Ar), 114.1 (2 × CH Ar), 112.6 (CH Ar), 96.4 (Cq Ar), 70.4, 70.2 (2 × CH2, OCH2CH2O), 69.1, 69.1 (2 × CH2, NtrzCH2CH2O), 61.7 (CH2, OCH2Ctrz), 58.4 (Cq, THQ-4), 56.1 (CH2, THQ- 3), 52.0 (Cq, t-butyl), 50.3, 50.1 (2 × CH2, CH2Ntrz), 47.8 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.7 (CH2, NHCH2Ctrz), 29.3 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.0 (3 × CH3, t-butyl), 28.7 (CH3, C(CH3)2), 25.4 (CH3, acetyl), 14.3 (CH3, CH3S). []D20: -143° (c=0.26, CHCl3). ESI-MS m/z: obsd 1225.7 [M + H]+. HRMS m/z calcd for C65H72N14O7S2 + 2H+: 613.26477, obsd 613.26489.
Heterodimeric ligand (S)-7d. 1H NMR (600 MHz, CDCl3) 8.43 (s, 1H, NHCO), 7.98 (d, J = 8.4, 2H, 2 × CH Ar), 7.80 (s, 1H, CH Trz), 7.75 (d, J = 6.6, 1H, CH Ar, THQ-8), 7.68 (d, J = 8.4, 2H, 2 × CH Ar), 7.61 (d, J = 7.8, 3H, 3 × CH Ar), 7.55 (s, 1H, CH Trz), 7.46 (t, J = 7.5, 2H, 2 × CH Ar), 7.39 (t, J = 7.2, 1H, CH Ar), 7.28-7.24 (m, 2H, CH Ar + NHCO), 6.99 (d, J = 8.4, 2H, 2 × CH Ar), 6.96 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.89 (d, J = 7.8, 1H, CH Ar), 6.80 (d, J = 8.4, 2H, 2 × CH Ar), 6.71 – 6.68 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.09 (s, 2H, CH2, OCH2Ctrz), 4.78 (s, 1H, NH), 4.51 (t, J = 4.8, 2H, CH2, CH2Ntrz), 4.46 (d, J = 5.4, 2H, CH2, NHCH2Ctrz), 4.40 (t, J = 5.1, 2H, CH2, CH2Ntrz), 3.84 (t, J = 5.1, 2H, CH2, NtrzCH2CH2O), 3.78 (s, 2H, CH2, NHCH2CONH), 3.73 (t, J = 6.3, 2H, CH2, NtrzCH2CH2O), 3.57 – 3.49 (m, 8H, 4 × CH2, OCH2CH2O), 2.61 (s, 3H, CH3, SCH3), 2.46 (d, J = 13.8, 1H, CH2, THQ), 1.94 (s, 3H, CH3, acetyl), 1.78 (d, J = 13.8, 1H, CH2, THQ), 1.59 (s, 3H, CH3(C-CH2)), 1.43 (s, 3 × CH3, t-butyl), 1.35 (s, 3H, CH3, C(CH3)2), 1.22 (s, 3H, CH3, C(CH3)2).13C NMR (150 MHz, CDCl3) 171.2, 170.1, 169.6, 167.2, 165.6, 165.0, 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.6 (Cq(OCH2Trz)), 147.5, 144.7, 144.6, 144.1, 143.8, 141.7, 139.7, 137.6, 137.4, 135.7, 135.6, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 128.9 (2 × CH Ar), 128.1, 128.1 (3 × CH Ar), 127.7, 127.3, 127.2 (6 × CH Ar), 126.6 (CH Ar, THQ-7), 124.1, 123.1 (2 × CH Trz), 118.6, 118.4 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 114.9 (CH Ar), 114.1 (2 × CH Ar), 112.8 (CH Ar), 96.5 (Cq Ar), 70.5, 70.4, 70.4, 70.3 (4 × CH2, OCH2CH2O), 69.3, 69.2 (2 × CH2, NtrzCH2CH2O), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 50.3, 50.1 (2 × CH2, CH2Ntrz), 47.9 (CH2, NHCH2CONH), 41.8 (Cq, C(CH3)2), 34.7 (CH2, NHCH2Ctrz), 29.7, 29.3 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.7 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: -144° (c=0.23, CHCl3). ESI-MS m/z: obsd 1269.7 [M + H]+. HRMS m/z calcd for C67H76N14O8S2 + 2H+: 635.27787, obsd 635.27802.
Heterodimeric ligand (S)-7e. 1H NMR (600 MHz, CDCl3) 8.41 (s, 1H, NHCO), 7.98 (d, J = 8.4, 2H, 2 × CH Ar), 7.81 (s, 1H, CH Trz), 7.75 (dd, J = 1.8, 8.4, 1H, CH Ar, THQ-8), 7.68 (d, J = 8.4, 2H, 2 × CH Ar), 7.61 (d, J = 6.0, 4H, 4 × CH Ar), 7.46 (t, J = 7.5, 3H, 2 × CH Ar+ NHCO), 7.39 (t, J = 7.2, 1H, CH Ar), 7.28 – 7.26 (m, 1H, CH Ar), 6.99 (d, J = 8.4, 2H, 2 × CH Ar), 6.96 (d, J = 8.4, 1H, CH Ar, THQ-7), 6.89 (d, J = 7.2, 1H, CH Ar), 6.81 (d, J = 9.0, 2H, 2 × CH Ar), 6.72 – 6.69 (m, 2H, 2 × CH Ar), 5.98 (s, 2H, NH2), 5.22 (s, 1H, NHCO), 5.11 (s, 2H, CH2, OCH2Ctrz), 4.76 (s, 1H, NH), 4.50 (t, J = 5.1, 2H, CH2, CH2Ntrz), 4.48 (d, J = 5.4, 2H, CH2, NHCH2Ctrz), 4.42 (t, J = 5.1, 2H, CH2, CH2Ntrz), 3.83 (t, J = 5.1, 2H, CH2, NtrzCH2CH2O), 3.79 (s, 2H, CH2, NHCH2CONH), 3.76 (t, J =
5.1, 2H, CH2, NtrzCH2CH2O), 3.56-3.48 (m, 12H, 6 × CH2, OCH2CH2O), 2.61 (s, 3H, CH3, SCH3), 2.47 (d, J = 13.8, 1H, CH2, THQ), 1.94 (s, 3H, CH3, acetyl), 1.78 (d, J = 13.8, 1H, CH2, THQ), 1.59 (s, 3H, CH3(C-CH2)), 1.43 (s, 3 × CH3, t-butyl), 1.32 (s, 3H, CH3, C(CH3)2), 1.23 (s, 3H, CH3, C(CH3)2). 13C NMR (150 MHz, CDCl3) 171.2, 170.1, 169.6, 167.2, 165.6, 165.01 162.7 (4 × C=O, acetyl + 3 × amide + 2 × Cq Ar), 156.6 (Cq(OCH2Trz)), 147.5, 144.7, 144.7 144.1, 143.8, 141.8, 139.7, 137.5, 137.4, 135.8, 135.6, 133.4 (12 × Cq Ar), 129.9 (CH Ar), 129.0 (2 × CH Ar), 128.1, 128.1 (3 × CH Ar), 127.7, 127.3, 127.5 (6 × CH Ar), 126.7 (CH Ar, THQ-7), 124.1, 123.2 (2 × CH Trz), 118.6, 118.5 (2 × CH Ar), 117.7 (Cq, H2NC=CCO), 117.5 (CH Ar, THQ-10), 114.9 (CH Ar), 114.2 (2 × CH Ar), 112.8 (CH Ar), 96.5 (Cq Ar), 70.5, 70.5, 70.3 (6 × CH2, OCH2CH2O), 69.3, 69.3 (2 × CH2, NtrzCH2CH2O), 61.8 (CH2, OCH2Ctrz), 58.5 (Cq, THQ-4), 56.1 (CH2, THQ-3), 52.0 (Cq, t-butyl), 50.3, 50.2 (2 × CH2, CH2Ntrz), 47.9 (CH2, NHCH2CONH), 41.9 (Cq, C(CH3)2), 34.8 (CH2, NHCH2Ctrz), 29.8, 29.4 (2 × CH3, CH3(C-CH2) + C(CH3)2), 29.1 (3 × CH3, t-butyl), 28.7 (CH3, C(CH3)2), 25.5 (CH3, acetyl), 14.4 (CH3, CH3S). []D20: -143° (c=0.22, CHCl3). ESI-MS m/z: obsd 1313.7 [M + H]+. HRMS m/z calcd for C69H80N14O9S2 + 2H+: 657.29098 obsd 657.29071.
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