Small molecules
11261179 · 2022-03-01
Assignee
Inventors
- Alessio Ciulli (Dundee, GB)
- Chiara Maniaci (Dundee, GB)
- Scott J. Hughes (Dundee, GB)
- Andrea Testa (Dundee, GB)
Cpc classification
C07K5/06191
CHEMISTRY; METALLURGY
C07K5/06034
CHEMISTRY; METALLURGY
International classification
Abstract
Compounds having the general structure A-L-B are presented wherein A and B are independently an E3 ubiquitin ligase protein binding ligand compound of formula 1A or 1B. Pharmaceutical compositions comprising these compounds and methods of use are also presented.
Claims
1. A compound having the structure:
A-L-B wherein A and B are independently an E3 ubiquitin ligase protein binding ligand compound of formula 1A: ##STR00058## wherein L is a linking group which is directly bonded to the compound of formula 1A at R.sup.1 or R.sup.2 wherein L is —R.sup.5—[O(CH.sub.2).sub.m].sub.n—R.sup.6—, wherein m and n are independently 0 to 10, and R.sup.5 and R.sup.6 are independently selected from the group: covalent bond, C1-C10 alkylene, C1-C10 polyether, or —O—; wherein R.sup.1 is selected from either the group: (1) a covalent bond, or C1-C5 alkylene when L is bonded to the compound of formula 1A at R.sup.1, or the group (2) H, NH.sub.2, C1-C5 alkyl, or C(CN)C.sub.2H.sub.4 when L is bonded to the compound of formula 1A at R.sup.2; wherein R.sup.2 is selected from the group: a covalent bond, H, NH.sub.2, C1-C5 alkyl, C(CN)C.sub.2H.sub.4; wherein X and Y are independently selected from the group: H, OH or halogen; and wherein R.sup.7 is C1-C5 alkylene, or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.
2. A compound according to claim 1, wherein X is H or halogen.
3. A compound according to claim 1, wherein Y is OH.
4. A compound according to claim 1 where either A or B is a compound according to formula 1A, wherein A has the formula 1C: ##STR00059##
5. A compound according to claim 1, wherein L is connected to A via R.sup.1 of formula 1A.
6. A compound according to claim 1, wherein L is connected to B via R.sup.1 of formula 1A.
7. A compound according to claim 1, wherein R.sup.5 is a chemical bond, R.sup.6 is a chemical bond, m is 2 and n is 3, 4 or 5.
8. A compound according to claim 7, wherein n is 5.
9. A compound according to claim 1, wherein the compound is of formulae 2, 3 or 4: ##STR00060## wherein R.sup.2a, R.sup.2b and R.sup.2c are independently selected from H, NH.sub.2, C1-C5 alkyl, and C(CN)C.sub.2H.sub.4; R.sup.1a, R.sup.1b and R.sup.1c are independently selected from H, NH.sub.2, C1-C5 alkyl, and C(CN)C.sub.2H.sub.4; X.sup.1 and X.sup.2 are independently selected from H, OH, halogen; Y.sup.1 and Y.sup.2 are independently selected from H, OH, halogen; and m and n are independently 0 to 10.
10. A compound according to claim 9, wherein n is 3-5.
11. A compound according to claim 9, wherein m is 1-4.
12. A compound according to claim 1, wherein the linker L is a linear chain of 12-20 atoms in length, optionally wherein the linker chain comprises carbon and/or oxygen atoms, further optionally wherein the linker chain comprises alkylene groups and/or ether groups and/or polyether groups.
13. A compound according to claim 1 selected from the following group: ##STR00061## ##STR00062## ##STR00063##
14. A compound according to claim 13, wherein the compound is compound (5) having a structure of: ##STR00064##
15. A pharmaceutical composition comprising one or more compounds according to claim 1 and a pharmaceutically acceptable vehicle or diluent therefor.
16. A method of treating at least one selected from the group of anaemia due to chronic kidney disease, anaemia due to cancer chemotherapy, ischemia, an ischemic reperfusion injury, myocardial infarction, stroke, an acute lung injury, intestinal inflammation, a wound, a post-transplantation complication, a mitochondrial respiratory chain dysfunction, and an oncological conditions treatable by enhancing T-cell responses in a subject, wherein the method comprises: administering to the subject the compound of claim 1, thereby treating the at least one selected from the group of anaemia due to chronic kidney disease, anaemia due to cancer chemotherapy, ischemia, the ischemic reperfusion injury, myocardial infarction, stroke, the acute lung injury, intestinal inflammation, the wound, the post-transplantation complication, the mitochondrial respiratory chain dysfunction, and the oncological condition treatable by enhancing T-cell responses in the subject.
17. A method of regulating activity of a target protein in a subject comprising administering to said subject an effective amount of a compound according to claim 1, wherein the target protein is an E3 ubiquitin ligase protein.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.
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DETAILED DESCRIPTION
(35) While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
(36) To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
(37) Biology
(38) Human cell lines HeLa, U2OS and HEK 293, purchased from ATCC, were propagated in DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, 100 μg ml.sup.−1 of penicillin/streptomycin at 37° C. and 5% CO.sub.2. Cells were maintained for no more than 30 passages. All cell lines were routinely tested for mycoplasma contamination using MycoAlert kit from Lonza.
(39) Small Interfering RNA.
(40) For siRNA inhibition studies, 3×10.sup.5 cells were seeded into each well of a 6-well plate in order to achieve 70% of confluence on the day of transfection. siRNA (SMARTpool: ON-TARGETplus VHL siRNA L-003936-00-0005) was prepared as a 20 μM solution in RNase-free 1× siRNA buffer. Negative control siRNA (siRNA from Life Technologies, cat. #4390843) was used as negative control. On the day of transfection, old medium was replaced with fresh one. siRNA solution (5 μL) of both VHL targeting siRNA and negative control were added to 250 μL of Opti-mem in 1.5 mL tube. This solution was prepared in duplicate. The content in each tube was mixed by pipetting. Lipofectamine RNAiMax (5 μL) was added to 250 μL of Opti-mem in another 1.5 mL tube. The solution was prepared in duplicate. The content in each tube was mixed by pipetting. The solution from step 2 was added to the tube in step 3. The solution was mixed by brief vortex ad incubated at r.t. for 20 min. The tubes were centrifuged briefly. The whole volume of transfection mix was added to the 6-well plate. Plate was swirled gently back and forth to mix the content. Plates were incubated at 37° C. and 5% CO.sub.2 for 48 h before harvesting.
(41) Single Point Treatment.
(42) For single time point treatment experiments, cells were transferred in 6-well plates with 5×10.sup.5 cells per well in 2 ml media in order to achieve 80% confluence the following day. Stock concentrations of compounds were prepared by solubilizing the powder in 100% v/v DMSO to the final desired stock concentration.
(43) On the day of treatment, all compound samples were prepared as 100-fold concentrated compound solution using DMEM just before treatment. The experiment samples (20 μL) were added to the 6 well plate containing 2 ml of media. The final DMSO concentration was 0.1% v/v. Cells were incubated at 37° C. and 5% CO.sub.2 for the desired time before harvesting.
(44) Time Course Experiments.
(45) For time dependent treatment, cells were transferred in 6-well plates with 3×10.sup.5 cells per well in 2 ml media. Samples were prepared as detailed above or the single time point experiments. Treatment was conducted at given time points prior to harvest.
(46) ML4924 and MG132 Treatment.
(47) Cells were transferred in 6-well plates with 5×10.sup.5 cells per well in 2 ml media in order to achieve 80% confluence the day after. At t=0, MLN4924 was added into the desired wells at 3 μM final concentration and 0.1% v/v of DMSO. DMSO (0.1% v/v final conc.) was added to the remaining wells in order to match identical conc. of vehicle in all wells. At t=3 h, MG 132 was added into the desired wells at 50 μM final conc. and 0.1% v/v of DMSO. DMSO (0.1% v/v final conc.) was added to the remaining wells in order to achieve the same conc. of vehicle in all the wells. At t=3.5 h, the desired wells were treated with 1 μM of CM11 in 0.1% v/v DMSO final concentration. DMSO (0.1% v/v final conc.) was added to the remaining wells in order to obtain the same conc. of vehicle in all the wells. The total final concentration of DMSO was therefore 0.3% v/v. Plates were incubated for 4 h at 37° C. and 5% CO.sub.2 before harvesting.
(48) Competition Experiments with VH032.
(49) Cells were transferred in 6-well plates with 5×10.sup.5 cells per well in 2 ml media in order to achieve 80% confluence the day after. On the day of experiment, cells were treated with VH032 at the final conc. of 150 μM for 30 min prior to treatment with CM11 at 1 μM final concentration for 4 h. Plates were incubated for the desired time at 37° C. and 5% CO.sub.2 before harvesting.
(50) Co-Treatment with IOX4 and CM11 to Investigate Upstream Effect Experiment.
(51) For this experiments, cells were transferred in 6-well plates with 5×10.sup.5 cells per well in 2 ml media in order to achieve 80% confluence the day after. On the day of experiment, cells were treated with IOX4 at the final concentration of 50 μM for 30 min prior to treatment with CM11 at 1 μM final concentration for 4 h. Plates were incubated for the desired time at 37° C. and 5% CO.sub.2 before harvesting.
(52) Immunoblotting.
(53) Cells were lysed in lysis buffer (20 mM Tris pH 8, 150 mM NaCl, 1% Triton x100) and a protease inhibitor cocktail (Roche) per 10 ml buffer. For protein extracts, the dishes were placed on ice. The media was aspirated and the tissue layer washed twice with ice-cold phosphate buffer saline (PBS). Lysis buffer (120 μl) was added and the cells detached from the surface with a cell scraper. After removal of the insoluble fraction by centrifugation, the protein concentration of the supernatant was determined by Pierce™ Coomassie (Bradford) Protein Assay Kit. Protein extracts were fractionated by SDS-PAGE on 4-12% Tris-Acetate NuPage® Novex® (Life Technologies) polyacrylamide gels and transferred to a nitrocellulose membrane using wet transfer. The membrane was then blocked with 5% w/v Bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.1% w/v Tween-20. For detecting proteins the following primary antibodies in the given concentrations were used: anti-β-Actin (Cell Signaling Technology, 4970S, 13E5) 1:2000, anti-VHL (Cell Signaling Technology, #68547) 1:1000, anti-Hif-1α (BD Biosciences, 610959, clone 54) 1:1000, anti-hydroxy-HIF-1α (Hyp564) (Cell Signaling Techonology; #3434) 1:1000, anti-PHD2 (Bethyl Laboratories; A300-322A) 1:1000, anti-PHD3 (Bethyl Laboratories; A300-327A) 1:1000, anti-CRBN (Proteintech; 11435-1-AP) 1:1000.
(54) Following incubation with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology), the signal was developed using enhanced chemiluminescence (ECL) Western Blotting Detection Kit (Amersham) on Amersham Hyperfilm ECL film (Amersham).
(55) Band quantification was performed using ImageJ software and reported as relative amount as ratio of the each protein band relative to the lane's loading control. The values obtained were then normalized to 0.1% DMSO vehicle control.
(56) Luciferase Assay.
(57) It was performed essentially as described by Frost et al..sup.34 Briefly, cells (HeLa and U2OS) stably expressing an HRE-luciferase reporter were treated for the indicated times with compounds. Cells were harvested in passive lysis buffer (Promega) and subjected to three freeze-thaw cycles. The soluble lysate fraction was used for assays, performed according to the manufacturer's instructions (Promega) using a Berthold Lumat LB 9507 Luminometer. Results were normalized for protein concentration, and reported as mean±s.e.m. from three biological replicates.
(58) Quantitative Real-Time PCR.
(59) It was performed essentially as described by Frost et al..sup.34 Briefly, RNA was extracted from HeLa cell lysates using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences) in C1000 Touch Thermal Cycler (Bio-Rad). mRNA levels were calculated based on averaged Ct values from two technical replicates, normalized to mRNA levels of β-actin, and reported as mean±s.e.m. from three biological replicates.
(60) Biophysical Assays
(61) Isothermal Titration Calorimetry (ITC).
(62) Titrations were performed on an ITC200 micro-calorimeter (GE Healthcare). PROTACs (CM11, CMP98 or CMP99) were diluted from a 100 mM DMSO stock solution to 150 μM in a buffer containing 20 mM Bis-tris propane, 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.4. The final DMSO concentration was 0.15% v/v. VBC protein experiments were carried out in a buffer containing 20 mM Bis-tris propane, 150 mM NaCl, 1 mM TCEP, 0.15% v/v DMSO, pH 7.4. The titrations consisted of 19 injections of 2 μL compounds solution (150 μM, in the syringe) at a rate of 2 s/μL at 120 s time intervals into the VCB protein solution (20 μM, in the cell). An initial injection of compound solution (0.4 μL) was made and discarded during data analysis. All experiments were performed at 25° C., whilst stirring the syringe at 600 rpm. The data were fitted to a single binding site model to obtain the stoichiometry n, the dissociation constant K.sub.d and the enthalpy of binding ΔH using the Microcal LLC ITC200 Origin software provided by the manufacturer.
(63) Size Exclusion Chromatography (SEC).
(64) SEC experiments were carried out in a AKTA pure system (GE Healthcare) at room temperature. The oligomeric state of the VCB complex in solution was analyzed by gel filtration in a buffer containing 20 mM Bis-Tris (pH 7), 150 mM NaCl and 1 mM 1,4-dithiothreitol (DTT) using a Superdex 200 Increase 10/300 GL column (GE Healthcare) calibrated with globular proteins of known molecular weight (GE Healthcare, 28-4038-41/42). VBC protein (50 μM) was incubated with CM11 (30 μM), CMP98 (30 μM), CMP99 (30 μ£M), VH032 (30 μM) or DMSO (0.5%) for 20 min at room temperature prior to injection. Sample volume for each injection was 200 μL, and the flow rate was 0.5 mL/min. Peak elution was monitored using ultraviolet absorbance at 280 nm.
(65) Biotinylation of VCB.
(66) The VCB complex was mixed with EZ-link NHS-PEG.sub.4-biotin (Thermo Scientific) in a 1:1 molar ratio and incubated at room temperature for 1 h. The reaction was quenched using 1 M Tris-HCl, pH 7.5, and unreacted NHS-biotin was removed with a PD-10 MiniTrap desalting column (GE Healthcare) equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM DTT.
(67) AlphaLISA assay.
(68) All assays were performed at room temperature in 384-well plates with a final assay volume of 25 μL per well; plates were sealed with transparent film between addition of reagents. All reagents were prepared as 5× stocks diluted in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% (w/v) bovine serum albumin and 0.02% (w/v) 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Biotinylated VCB (20 nM final) and Hiss-VCB (20 nM final) were incubated with a range of Homo-PROTAC concentrations (0.5 to 200 nM; three-in-five serial dilution) for 1 h. Anti-His acceptor beads (PerkinElmer, 10 μg/mL final) were added and plates were incubated for another hour. Streptavidin-coated donor beads (PerkinElmer, 10 μg/mL final) were added and plates were incubated for a final 1 h. Plates were read on a PHERAstar FS (BMG Labtech) using an optic module with an excitation wavelength of 680 nm and emission wavelength of 615 nm. Intensity values were plotted against PROTAC concentration on a logo scale.
(69) Rational Design
(70) Design of VHL Homo-PROTACs began with careful consideration of the position of derivatization on two potent VHL ligands recently characterized by our group, VH032 and VH298 (
(71) It is known that the trans epimer of Hyp is an absolute requirement for VHL binding, and that the corresponding cis epimer abrogates binding to VHL, both within the context of a native HIF substrate peptide,.sup.36 and VHL ligands..sup.3,13 We therefore designed two different PROTACs based on the structure of the first series (
(72) Synthesis.
(73) For the synthesis of the first class of Homo-PROTACs (
(74) For the preparation of the asymmetric cis-trans compound CMP99, a synthetic route toward the synthesis of the monoprotected di-carboxylate linker was established. Pentaethylene glycol was the linker of choice because of ease of purification compared to longer PEGs, and at the same time yielding a control compound of average linker length (PEG-4 in this case). Pentaethylene glycol was converted into monobenzyl ether 9 in 71% yield, which was reacted with tert-butyl bromoacetic acid under biphasic conditions (DCM/37% aq. NaOH and stoichiometric tetrabutyl ammonium bromide). After deprotection of the benzyl group by catalytic hydrogenation, formation of the carboxylic acid moiety was achieved by oxidation with TEMPO and bis-acetoxy iodobenzene (BAIB), delivering compound 11 in 65% yield (
(75) For the synthesis of the second class of symmetric Homo-PROTACs (
(76) The PEG linkers for this class of compound were designed to contain a methanesulfonate group at either end, which could be coupled in a single step with the phenol of the VHL ligand. Linker 19 was prepared by mesylation of pentaethylene glycol and reacted with either compounds 17 or 18 in a 1:2 ratio in the presence of K.sub.2CO.sub.3 to afford CMP106 and CMP108, respectively, in good yield (
(77) For the synthesis of asymmetric Homo-PROTACs, PEG 10 was converted in to the mesylated derivative 20 and reacted with 17 or 18 to obtain 21 and 22, respectively in good yield (
(78) Biological Evaluation.
(79) We next tested all our Homo-PROTACs in HeLa cells, and monitored protein levels by Western blots after 10 h of compound treatment at 1 μM concentration (
(80) Treatments with CM10 and CM11 also showed detectable albeit low increase in protein levels of the hydroxylated form of HIF-1α (Hdy-HIF-1α,
(81) To assess whether selective pVHL19 knockdown by Homo-PROTACs could induce HIF transcriptional activity, we first used a luciferase reporter assay..sup.37 Hypoxia response element (HRE)-luciferase reporter HeLa-HRE and U2OS-HRE cells were treated with different concentrations of CM11 and at different times, and no increase in HIF-dependent luciferase activity was detected relative to DMSO control treatment (
(82) We next turned our attention to further characterizing the mode of action of the protein degradation induced by the active Homo-PROTACs CM09-11. To interrogate their relative cellular potency, dose-dependent treatments were performed at two different time points, 4 and 24 h prior to harvesting. All compounds confirmed preferential degradation of pVHL30 in a concentration-dependent manner, relative to the corresponding DMSO control (see
(83) CM11 proved the most potent Homo-PROTAC, inducing complete depletion of pVHL30 after 4 h already at 10 nM (DC.sub.99=10 nM,
(84) We next interrogated the time-dependent activity of Homo-PROTACs. Progressive removal of VHL protein overtime was observed, confirming selective depletion of pVHL30 over the short isoform (
(85) TABLE-US-00001 TABLE 1 Summary of thermodynamic binding parameters of Homo-PROTACs and comparison with VHL inhibitor VH032 (from 19) measured by ITC, against both short and long VHL isoforms. Kd ΔG ΔH −TΔS Protein Compound n (nM) α (kcal/mol) (kcal/mol) (kcal/mol) pVHL19 VH032 (ref..sup.2) 1.030 ± 0.001 188 ± 6 — −9.17 ± 0.02 −5.53 ± 0.01 −3.65 ± 0.02 CM11 0.6 ± 0.01 11 ± 2 18 −10.9 ± 0.1 −12.3 ± 0.7 1.4 ± 0.8 CMP99 0.964 ± 0.005 146 ± 2 — −9.33 ± 0.06 −6.23 ± 0.05 −3.1 ± 0.7 CM09 0.98 ± 0.09 41 ± 15 4 −10.3 ± 0.2 −6.9 ± 0.3 −3.5 ± 0.5 CM10 0.73 ± 0.01 32 ± 5 6 −10.2 ± 0.1 −9.4 ± 0.1 −0.8 ± 0.2 CMP106 0.535 ± 0.004 111 ± 8 1.7 −9.5 −12.6 ± 0.1 3.1 CMP112 0.498 ± 0.006 235 ± 22 0.8 −9.1 −14.8 ± 0.2 5.8 CMP113 0.934 ± 0.005 117 ± 25 1.7 −9.5 −6.4 ± 0.2 −3.1 pVHL30 CM11 0.866 ± 0.003 25 ± 3 4 −10.4 ± 0.1 −11.3 ± 0.1 −0.9 ± 0.1 CMP99 1.050 ± 0.004 106 ± 10 — −9.51 ± 0.05 −5.19 ± 0.03 −4.3 ± 0.1
(86) To gain mechanistic insights in the cellular activity of Homo-PROTACs, the dependency on CRL2-VHL and proteasome activities was examined. The reliance of the Homo-PROTAC-induced protein degradation on CRL2-VHL was assessed by inhibiting neddylation of Cullin2 using the NAE1 inhibitor MLN4924, which blocks the activity of CRLs, including CRL2-VHL. Proteasome-dependency was interrogated by treating cells with the proteasome inhibitor MG132. To limit the known cytotoxicity of MLN4924 and MG132, HeLa cells were pre-treated with MLN4924 for 3 h followed by MG132 for 30 min before adding CM11 to the media, and cells were incubated for further 4 h before harvesting. Single treatments with DMSO, MLN4924, MG132 and CM11 and combinations thereof were performed to disentangle the individual and combined effects of compound treatments. Degradation of pVHL30 induced by CM11 was completely abrogated when cells were pre-treated with MG132, establishing the expected proteasome-dependence of the chemical intervention (
(87) Biophysical Evaluation
(88) Key to the catalytic mode of action of PROTACs is the formation of a ternary complex..sup.13,15 In the case of our Homo-PROTAC compounds, VHL acts as both the E3 ligase and the substrate. Therefore, we next sought to monitor and biophysically characterize the ternary complex VHL:Homo-PROTAC:VHL that is thought to underlie cellular activity. To assess the formation of this ternary complex species in solution, isothermal titration calorimetry (ITC), size exclusion chromatography (SEC) and AlphaLISA proximity assays were performed (
(89) SEC experiments showed that VCB migrates more quickly in the presence of the active compound CM11 (2:1 protein:ligand ratio), relative to the vehicle control (
(90) Lastly, we employed an AlphaLISA proximity assay to compare ternary complex formation by CM09, CM10 and CM11. The assay showed the highest intensity signal for CM11, whereas negligible levels of complex formation were detected for CM09 and CM10 (
(91) Discussion
(92) In some embodiments, Homo-PROTACs are described, a small-molecule approach to effectively dimerize an E3 ubiquitin ligase to induce its own self-destruction. Using potent ligands for the E3 ligase VHL, a series of symmetric homo-bivalent molecules that induce remarkably rapid, profound and selective degradation of the long isoform of pVHL at nanomolar concentrations were developed. Compound-induced degradation was exquisitely dependent on the linkage pattern on the VHL ligand. The most active Homo-PROTAC, CM11, induces complete depletion of pVHL30 after 4 h already at 10 nM. Potent and selective degradation of pVHL30 was long lasting, with half-degrading concentration (DC.sub.50) of approximately 100 nM, a remarkable increase in cellular activity of >1000-fold compared to the parent inhibitor VH032. Mechanistically, it has been shown that CM11 activity is strictly dependent on proteasome activity, CuI2 neddylation, and on VHL binding, and specifically on the formation of an avid 2:1 complex with VHL. The data therefore supports a model in which a highly cooperative ternary complex VHL-CM11-VHL functions as the key species responsible for the induced degradation of VHL itself (
(93) The preferential induced degradation of pVHL30 over the short VHL isoform was unexpected and is an intriguing result of this work. This observation adds to recent evidence from us and others that chemical degraders designed from inhibitors recruiting more than a single protein paralog or isoform can add a layer of target degradation selectivity independently of target engagement..sup.12,15,18 As the binary engagement of the VHL warhead was found to be similar between the two VHL isoforms (Table 1), the observed selectivity could be due to large differences in cooperativities, which would impact on the relative population of ternary complexes..sup.15 However, CM11 actually exhibited greater avidity in vitro for the short relative to the long isoform of VHL (Table 1). We therefore view it as unlikely that the remarkable selectivity of VHL degradation is due to large differences in cooperativities of ternary complexes. We also consider unlikely that preferential and more efficient lysine ubiquitination could play a role, because the extra region present in the long isoform (1-53) does not contain a single lysine residue. On the other hand, this region is predicted as intrinsically disordered, and indeed it has been shown that proteins containing disordered N-terminal regions are more prone to proteasomal degradation. It is also known that VHL is resistant to proteasomal degradation when in complex with ElonginB and ElonginC, so the form observed to be preferentially depleted may be free VHL i.e. unbound to Elongins, or other proteasome-sensitive forms. Addressing these questions will be of clear importance for future investigation.
(94) Selective degradation of pVHL30 by CM11 led to minimal stabilization of HIF-a in cells, and as a result did not trigger HIF-dependent activity in cells. This highlights the potential benefit of using CM11 to interrogate the biological function of specific VHL isoforms, without the masking downstream effects of a hypoxic response. Not much is known about the individual roles of VHL isoforms. Studies have highlighted how the 53-residue extra region of pVHL30 is not needed for tumor suppression, and how both isoforms can have HIF-dependent tumor suppressor functions in vivo. Other HIF-independent roles of pVHL have been proposed, including a role for pVHL in collagen assembly. However, the individual roles of the different isoforms in these biological functions remain elusive. Moreover, many HIF-independent roles are thought to be independent upon Hyp recognition, and thus cannot be probed chemically using current Hyp-based VHL inhibitors. Selective and acute knockdown of pVHL30 by CM11 provides therefore a novel chemical tool to address these questions.
(95) In summary, we present CM11, a chemical probe for rapid and selective pVHL30 knockdown. CM11 provides an alternative advantageous chemical tool to conventional knockdown RNAi approaches and gene editing knockout technologies such as CRISPR-Cas9. Relevant information to the use of CM11 will be made available in the newly established “Chemical Probes Portal” (http://www.chemicalprobes.org/)..sup.38 We anticipate CM11 will find wide use amongst chemical and cell biologists alike interested in investigating and dissecting the pleiotropic biological functions of pVHL. More generally, we provide first proof-of-concept that bivalent molecules can be designed to induce an E3 ligase to destroy itself. This strategy could provide a powerful new approach to drugging E3 ligases in ways that may not be possible with inhibitors alone.
(96) Synthesis of PROTACs Recruiting Together CRL4.sup.CRBN and CRL2.sup.VHL.
(97) For the synthesis of compounds CMP85 and CMP86 (structures shown in
(98) Compound 48 (the desired thalidomide derivative, see Figure) was synthesized as previously published by Lu et al..sup.17 In the first step, 3-fluorophthalic acid was dehydrated with acetic anhydride to obtain compound 46 in good yield. Reaction of compound 46 with L-glutamine and subsequent treatment with HCl 4 M solution led to the formation of compound 47. Cyclization of 47 was performed at reflux in the presence of 1,1′-carbonyldiimidazole (CDI) and DMAP. The recommended time for this step was 5 h. After 2.5 h it was possible to observe the formation of a side product by LC-MS. For this reason, even if the reaction was not completed, the reaction was cooled to r.t. and the resulting solid collected by filtration. During the purification step, performed by column chromatography over silica, compound 48 was isolated in good yield. The side product was isolated as well and analysed by NMR and identified to be compound 53 (
(99) Compound 48 was converted into compound 50 in two steps (
(100) Biological Evaluation of the VHL-Targeting Compounds
(101) The following section outlines the results of the biological evaluation of PROTAC compounds targeting VHL in cells.
(102) In order to assess the activity of compounds inside cells, HeLa cells were treated with 1 μM of Homo-PROTACs CM09, CM10 and CM11 (
(103) The same experiments were performed in other cells lines to further assess the consistency of the cellular effects of our compounds, as different cell lines can have different expression levels of different proteins. For example, HEK293 are known to have higher expression levels of total VHL, which we confirmed by Western blot (
(104) ITC experiments were also conducted with compounds CMP106, CMP108, CMP112 and CMP113 (data shown in
(105) Materials and Methods
(106) All chemicals were purchased from commercial vendors and used without further purification, unless indicated otherwise. Reactions were magnetically stirred; commercially available anhydrous solvents were used. All reactions requiring anhydrous conditions were carried out under argon or nitrogen atmosphere using oven-dried glassware. HPLC-grade solvents were used for all reactions. Flash column chromatography was carried out using silica gel (Merck 60 F254 nm). Normal phase TLC was carried out on pre-coated silica plates (Kieselgel 60 F254, BDH) with visualization via UV light (UV 254/365 nm) and/or basic potassium permanganate solution or other suitable stains. Flash column chromatography (FCC) was performed using a Teledyne Isco Combiflash Rf or Rf200i, prepacked columns RediSep Rf Normal Phase Disposable Columns were used. NMR spectra were recorded on a Bruker Ascend 400 or 500. Chemical shifts are reported in parts per million referenced to residual solvent peaks (CDCl.sub.3=7.26 ppm). The following abbreviations were used in reporting spectra, s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets). Only major rotamer NMR spectra are reported. High Resolution Mass Spectra (HRMS) were recorded on a Bruker microTOF. Low resolution MS and analytical HPLC traces were recorded on an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, connected to an Agilent diode array detector. The column used was a Waters XBridge column (50 mm×2.1 mm, 3.5 μm particle size). The flow rate was 0.6 mL/min. Preparative HPLC was performed on a Gilson Preparative HPLC System with a Waters XBridge C18 column (100 mm×19 mm; 5 μm particle size).
(107) General method A: PEG was solubilised in dioxane anhydrous and NaH was added under stirring. The resulting mixture was stirred at r.t. for 3 h. The mixture was cooled down to 0° C. using ice bath and tert-butylbromo acetate was added drop by drop. The resulting mixture was stirred at r.t O/N. The precipitate was filtered off and the organic phase evaporated to dryness. The resulting oil was taken up with ethyl acetate, washed with water, dried over MgSO.sub.4 and evaporated to dryness. The resulting oil was purified by column chromatography using a gradient of ethyl acetate from 50% to 100% v/v in heptane.
(108) General method B: tert-butyl esters 1, 2, 3 or 12 were dissolved in a solution of 50% v/v trifluroacetic acid in DCM. The resulting solution was stirred for 1 h or until complete conversion of starting material. The solvent was removed under high vacuum. The resulting carboxylic acid was used as crude in the next step without any further purification. To a solution of carboxylic acid in 1 ml DMF were added HATU (1 eq.) and HOBT (1 eq.) and the solution was stirred at room temperature for 5 min. Amine 6, 31 or 32 was added and the pH of the reaction mixture was adjusted to >9 by addition of DIPEA (3 eq.). The mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. Water was added and the mixture was extracted with ethyl acetate (×3). The combined organic phases were washed with brine (×2), dried over MgSO.sub.4 and evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 20% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia to yield the desired compound.
(109) General method C: A mixture of mesilate, compound 6, 31, 32, and K.sub.2CO.sub.3 (41.46 mg, 0.3 mmol, 6 eq.) in DMF was stirred O/N at 70° C. The reaction mixture was filtered off to afford the crude product, which was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to yield the desired compounds.
(2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (15)
(110) ##STR00007##
(111) To a solution of trans-N-(tert-Butoxycarbonyl)-4-hydroxy-L-proline (890 mg, 3.84 mmol, 1 eq.) in DMF was added HATU (1.46 g, 3.84 mmol, 1 eq.) and HAOT (522 mg, 3.84 mmol, 1 eq.) and the solution was stirred at room temperature for 5 min. 14 (846 mg, 3.84 mmol, 1 eq.) was added and the pH of the reaction mixture was adjusted to >9 by addition of DIPEA (3 eq.) and the mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. Water was added and the mixture was extracted with ethyl acetate (×3). The combined organic phases were washed with brine (×2), dried over MgSO.sub.4 and evaporated under reduced pressure to give the corresponding crude, which was purified by flash column chromatography using a gradient of 0 to 80% v/v acetone in heptane to yield the titled compound. Yield: 1.298 g, 3 mmol (78%). Analytical data matched those previously reported.sup.35
(112) The N-Boc-protected compound was dissolved in DCM. An equal volume of 4M HCl in dioxane was added and the reaction mixture stirred at room temperature for 2 h. The solvent was removed under a stream of nitrogen and dried under reduce pressure. The resulting crude was used for the next step without any further purification (quantitative yield). Analytical data matched those previously reported.sup.35
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (16)
(113) ##STR00008##
(114) To a solution of trans-N-(tert-Butoxycarbonyl)-4-hydroxy-L-proline (890 mg, 3.84 mmol, 1 eq.) in DMF was added HATU (1.46 g, 3.84 mmol, 1 eq.) and HAOT (522 mg, 3.84 mmol, 1 eq.) and the solution was stirred at room temperature for 5 min. 14 (846 mg, 3.84 mmol, 1 eq.) was added and the pH of the reaction mixture was adjusted to >9 by addition of DIPEA (3 eq.) and the mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. Water was added and the mixture was extracted with ethyl acetate (×3). The combined organic phases were washed with brine (×2), dried over MgSO.sub.4 and evaporated under reduced pressure to give the corresponding crude, which was purified by flash column chromatography using a gradient of 0 to 80% v/v acetone in heptane to yield the titled compound. Yield: 1.915 g, 3.61 mmol (94%). Analytical data matched those previously reported.sup.35.
(115) The N-Boc-protected compound was dissolved in DCM. An equal volume of 4 M HCl in dioxane was added and the reaction mixture stirred at room temperature for 2 h. The solvent was removed under a stream of nitrogen and dried under reduced pressure. The resulting crude was used for the next step without any further purification (quantitative yield). Analytical data matched those previously reported.sup.35.
(2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (18)
(116) ##STR00009##
(117) 1-cyanocyclopropanecarboxylic acid (69 mg, 0.62 mmol, 1 eq.) was solubilized in 3 ml of DMF. HATU (235 mg, 0.62 mmol, 1 eq.) and HOAT (84.4 mg, 0.62 mmol, 1 eq.) were added and the resulting mixture was stirred at r.t. for 5 min. The amine precursor of 16 (300 mg, 0.62 mmol, 1 eq.) was added and the pH was adjusted to pH>9 using DIPEA (400 mg, 0.5 ml, 3.1 mmol, 5 eq.). The resulting mixture was stirred at r.t. until complete conversion of the starting material. Water was added, and the mixture was extracted with ethyl acetate (×3). The combined organic phases were washed with brine (×1), dried over MgSO.sub.4, and evaporated to afford the corresponding crude compound that was purified by flash column chromatography using a gradient of 10% to 70% acetone in heptane to yield the title compound as a white solid. Yield: 200 mg, 0.37 mmol (60%). HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.27H.sub.33N.sub.5O.sub.5S: 539.22; observed: 540.3.
(118) .sup.1H NMR (400 MHz, CDCl3) 9.29 (1H, s), 8.65 (1H, s), 8.02 (1H, t, J=6.4 Hz), 7.12 (1H, d, J=7.7 Hz), 6.99 (1H, d, J=8.0 Hz), 6.94 (1H, d, J=1.8 Hz), 6.86 (1H, dd, J=1.8, 7.7 Hz), 4.72 (1H, t, J=8.0 Hz), 4.54 (1H, s), 4.44-4.35 (2H, m), 4.19 (1H, dd, J=5.5, 14.6 Hz), 3.87 (1H, d, J=11.0 Hz), 3.62 (1H, dd, J=3.7, 11.0 Hz), 3.50 (1H, s), 2.49 (3H, s), 2.43-2.37 (1H, m), 2.13-2.06 (1H, m), 1.66-1.37 (4H, m), 0.89 (8H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 172.8, 170.8, 165.8, 155.8, 150.5, 148.3, 133.3, 131.6, 131.2, 123.9, 120.9, 119.6, 118.2, 70.1, 58.6, 58.3, 56.7, 55.7, 40.0, 35.7, 26.2, 18.6, 17.9, 17.8, 17.2, 16.1, 13.8.
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17)
(119) ##STR00010##
(120) The amine precursor 16 (100.7 mg, 0.240 mmol, 1 eq.) was dissolved in 1 ml of DMF, acetylimidazole (31.7 mg, 0.288 mmol, 1.2 eq) and DIPEA (0.090 ml, 0.48 mmol, 2 eq.) were added to the solution. After stirring the mixture for 48 h at room temperature, the solvent was evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid to yield the titled compound. Yield: 91 mg, 0.187 mmol (78%). .sup.1H NMR (400 MHz, CDCl.sub.3) 9.25 (1H, s), 8.70 (1H, s), 7.97 (1H, t, J=6.5 Hz), 7.15 (1H, d, J=7.5 Hz), 6.83-6.80 (2H, m), 6.72 (1H, d, J=8.8 Hz), 4.92-4.88 (1H, m), 4.57 (1H, s), 4.52-4.42 (2H, m), 4.26-4.14 (2H, m), 3.59 (1H, dd, J=2.9, 11.1 Hz), 2.53-2.45 (4H, m), 2.24-2.17 (1H, m), 1.85 (3H, s), 0.83 (9H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.8, 171.2, 155.9, 150.7, 148.1, 132.8, 131.7, 131.0, 124.2, 120.6, 117.1, 70.3, 58.1, 57.7, 57.1, 39.8, 35.5, 34.8, 26.3, 22.6, 16.0. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.24H.sub.32N.sub.4O.sub.5S: 488.21; observed: 484.3.
di-tert-butyl 3,6,9,12-tetraoxatetradecanedioate (1)
(121) ##STR00011##
(122) Following general method A, from triethylene glycol (1.125 g, 1 ml, 7.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (595.75 mg, 14.9 mmol, 2 eq.) and tert-Butyl bromoacetate (2.905 g, 2.19 ml, 14.9 mmol, 2 eq.), compound 1 was obtained as an oil after high vacuum. Yield: 538 mg, 1.42 mmol (19%).
(123) .sup.1H NMR (500 MHz, CDCl.sub.3) b 3.81 (4H, s), 3.51-3.46 (12H, m), 1.26 (18H, s). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 169.1, 80.9, 70.1, 70.0, 68.5, 27.5. Analytical data matched those previously reported. 39
di-tert-butyl 3,6,9,12,15-pentaoxaheptadecanedioate (2)
(124) ##STR00012##
(125) Following general method A, from tetrathylene glycol (1.125 g, 1 ml, 5.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (463 mg, 11.5 mmol, 2 eq.) and tert-Butyl bromoacetate (2.25 g, 1.7 ml, 11.5 mmol, 2 eq.), compound 2 was obtained as an oil after high vacuum. Yield: 500 mg, 1.18 mmol (10%).
(126) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 3.86 (4H, s), 3.55-3.49 (16H, m), 1.31 (9H, s). Analytical data matched those previously reported..sup.39
di-tert-butyl 3,6,9,12,15,18-hexaoxaicosanedioate (3)
(127) ##STR00013##
(128) Following general method A, from pentaethylene glycol (1.126 g, 1 ml, 4.72 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (377 mg, 9.45 mmol, 2 eq.) and tert-Butyl bromoacetate (1.872 g, 1.7 ml, 11.5 mmol, 2 eq.), compound 3 was obtained as an oil after high vacuum. Yield: 300 mg, 0,641 mmol (14%).
(129) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.94 (4H, s), 3.66-3.56 (20H, m), 1.40 (18H, s). Analytical data matched those previously reported.sup.39.
1-phenyl-2,5,8,11,14-pentaoxahexadecan-16-ol (9)
(130) ##STR00014##
(131) Pentaethylene glycol (9.53 g, 50 mmol, 5 eq.) was added dropwise to a suspension of NaH 60% in mineral oil (800 mg, 20 mmol, 2.5 eq.) in 20 ml of DMF at 0° C. The resulting mixture was stirred at r.t for 1 h. The reaction mixture was cooled to 0° C., benzyl chloride (1 ml, 1.1 g, 8.72 mmol, 1 eq.) was added. The resulting mixture was stirred O/N at r.t. The reaction was quenched with a saturated solution of NH.sub.4Cl and the aqueous phase was extracted with ethyl acetate (×3). The combined organic phases were dried over MgSO.sub.4 and evaporated to dryness. The resulting oil was purified by column chromatography (from 0 to 60% of ethyl acetate in heptane) to afford the title compound as an oil. Yield: 2.055 g, 6.25 mmol (71%).
(132) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.28-7.19 (5H, m), 4.50 (2H, s), 3.66-3.52 (20H, m), 2.50 (1H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) 138.2, 128.3, 127.8, 127.6, 73.2, 72.7, 70.61, 70.58, 70.53, 70.51, 70.2, 69.4, 61.7
tert-butyl 1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-oate (10)
(133) ##STR00015##
(134) To a stirred solution of 9 (2.055 g, 6.25 mmol, 1 eq.) in 12.8 ml of DCM was added 37% solution of NaOH (12.8 ml), followed by tert-butylbromo acetate (4.882 g, 25 mmol, 4 eq.) and TBABr (2118 mg, 6.37 mmol, 1.02 eq.). The resulting solution was stirred O/N at r.t. The reaction mixture was extracted with ethyl acetate (×3). The organic phases were combined and washed with brine (×1), dried over MgSO.sub.4 and concentrate in vacuo. The resulting brown oil was purified by column chromatography (from 0 to 30% of ethyl acetate in petroleum) to afford the titled compound as colourless oil. Yield: 2.216 g, 5 mmol (80%).
(135) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.28-7.20 (5H, m), 4.50 (2H, s), 3.95 (2H, s), 3.65-3.55 (20H, m), 1.40 (9H, s). .sup.13C NMR (126 MHz, CDCl.sub.3) δ169.7, 128.4, 127.7, 127.6, 81.5, 73.2, 70.7, 70.7, 70.6, 70.6, 69.4, 69.1, 28.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.23H.sub.38O.sub.8: 442.26; observed: 387.2.
19,19-dimethyl-17-oxo-3,6,9,12,15,18-hexaoxaicosanoic acid (11)
(136) ##STR00016##
(137) 10 (2.216 g, 5 mmol, 1 eq.) was dissolved in 75 ml of ethanol, Pd/C (10 wt %) was added and the resulting mixture was place under hydrogen and stirred at r.t. until complete conversion of the starting material. The reaction mixture was filtered through celite, the celite pad was washed few times using ethanol. The filtrate was concentrated in vacuum to give an oil that was used for the next step without further purification. Yield: 1.764 g, 5 mmol (quantitative). BAIB (3.546 g, 11.01 mmol, 2.2 eq.) and TEMPO (171.87 mg, 1.10 mmol, 0.22 eq.) were added to a solution of ACN/H.sub.2O 1:1 containing previous obtained oil (1.764 g, 5 mmol, 1 eq.). The resulting mixture was stirred at r.t until complete conversion of the starting material. The crude was purified using ISOLUTE® PE-AX anion exchange column. The column was equilibrate with methanol, the reaction mixture poured in the column and let it adsorbed in the pad. The column was washed with methanol (×3) to elute all the unbound material. Then, the titled product was eluted using a 50% solution of formic acid in methanol. The organic phase was evaporated to dryness to afford the title compound as oil. Yield: 1.200 g, 3.27 mmol (65%).
(138) .sup.1H NMR (400 MHz, CDCl.sub.3) δ, ppm 4.12 (2H, s), 3.98 (2H, s), 3.72-3.60 (16H, m), 1.43 (9H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) δ, ppm 172.6, 169.7, 81.6, 71.0, 70.59, 70.56, 70.54, 70.46, 70.38, 70.35, 70.30, 68.9, 68.8, 28.1.
3,6,9,12-tetraoxatetradecane-1,14-diyl dimethanesulfonate (19)
(139) ##STR00017##
(140) Pentaethylene glycol (476.56 mg, 0.423 ml, 2 mmol, 1 eq.) was dissolved in 4 ml of dry DCM. The temperature of the resulting mixture was cooled down to 0° C. and methanesulfonyl chloride (687.3 mg, 0.464 ml, 16 mmol, 3 eq.) was added followed by triethylamine (1011.9 g, 1.39 ml, 10 mmol, 5 eq.). The resulting mixture was stirred at 0° C. for 4 h. A 10% aqueous solution of NaHSO.sub.4 was added till pH=3. The aqueous phase was extracted with DCM (×5). The organic phases were combined, dried over MgSO.sub.4 and concentrated in vacuum to afford the title compound as an orange oil. Yield: 701 mg, 1.77 mmol (89%).
(141) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.33-4.30 (4H, m), 3.72-3.69 (4H, m), 3.62-3.56 (12H, m), 3.02 (6H, s). Analytical data matched those previously reported [Kimura et al. J. Polym. Sci. Part A: Polym. Chem. 54, (2016).]
tert-butyl 17-((methylsulfonyl)oxy)-3,6,9,12,15-pentaoxaheptadecanoate (20)
(142) ##STR00018##
(143) 10 (251 mg, 0.712 mmol, 1 eq.) was dissolved in 1.4 ml of dry DCM. The temperature of the resulting mixture was cooled down to 0° C. and methanesulfonyl chloride (122.3 mg, 0.082 ml, 1.068 mmol, 1.5 eq.) was added followed by triethylamine (216.14 mg, 0.3 ml, 2.136 ml, 3 eq.). The resulting mixture was stirred at 0° C. for 4 h. A 10% aqueous solution of NaHSO.sub.4 was added till pH=3. The aqueous phase was extracted with DCM (×5). The organic phases were combined, dried over MgSO.sub.4 and concentrated in vacuum to afford the title compound as a orange oil. Yield: 276 mg, 0.641 mmol (90%).
(144) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.32-4.30 (2H, m), 3.95 (2H, s), 3.71-3.57 (18H, m), 3.02 (3H, s), 1.41 (9H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 169.7, 81.5, 70.72, 70.65, 70.61, 70.58, 70.5, 69.3, 69.0, 37.7, 28.1.
tert-butyl (S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosanoate (12)
(145) ##STR00019##
(146) To a solution of PEG linker 11 (78.8 mg, 0.215 mmol, 1 eq.) in 1.5 ml DMF was added HATU (81.74 mg, 0.215 mmol, 1 eq.), HOAT (29.26 mg, 0.215 mmol, 1 eq.), DIPEA and the solution was stirred at room temperature for 5 min. Compound 7 (100 mg, 0.215 mmol, 1 eq.) was added and the pH of the reaction mixture was adjusted to >9 by addition of DIPEA (80.13 mg, 0.106 ml, 0.645 mmol, 3 eq.). The mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. The solvent was evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 20% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia to yield the titled compound as white solid. Yield: 75.6 mg, 0.094 mmol (44%).
(147) .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 9.00 (1H, s), 7.45 (1H, t, J=5.9 Hz), 7.39-7.33 (4H, m), 7.29 (1H, d, J=8.9 Hz), 4.71 (1H, t, J=8.0 Hz), 4.59-4.48 (3H, m), 4.34 (1H, dd, J=5.2, 14.6 Hz), 4.08-3.92 (5H, m), 3.69-3.61 (18H, m), 2.52 (3H, s), 2.47-2.41 (1H, m), 2.19-2.11 (1H, m), 1.46 (9H, s), 0.97 (9H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.3, 171.1, 170.5, 170.0, 151.7, 139.1, 129.4, 128.3, 82.0, 71.1, 70.6, 70.4, 70.4, 70.3, 70.3, 70.2, 70.2, 68.9, 58.7, 57.3, 56.8, 43.1, 36.3, 35.1, 28.1, 26.4, 15.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.38H.sub.58N.sub.4O.sub.11S.sub.2: 778.38; observed: 779.4.
N.SUP.1.—((R)-1-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-N.SUP.17.—((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaheptadecanediamide (CMP99)
(148) ##STR00020##
(149) Following general method B, from compound 12 (75.6 mg, 0.094 mmol, 1 eq.) and trifluoroacetic acid (1 ml in 1 ml of DCM), the carboxylic acid derivative was obtained as oil. The crude was used for the next step without further purification. Yield: 70 mg, 0.094 mmol (quantitative). MS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.34H.sub.50N.sub.4O.sub.11S: 722.32; observed: 723.3. Following general method B, from compound 13 (5.5 mg, 0.006 mmol, 1 eq.), compound 8 (2.77 mg, 0.006 mmol, 1 eq.), HATU (2.28 mg, 0.0.006 mmol, 1 eq.), HOAT (1 mg, 0.0.006 mmol, 1 eq.), DIPEA (2.23 mg, 0.002 ml, 0.018 mmol, 3 eq.), CMP99 was obtained as a white solid. Yield: 4.5 mg, 0.004 mmol (66%).
(150) .sup.1H NMR (400 MHz, CDCl.sub.3): d, ppm 8.74 (2H, d, J=2.8 Hz), 7.37-7.34 (9H, m), 7.18 (1H, d, J=8.9 Hz), 4.76-4.64 (3H, m), 4.59-4.44 (5H, m), 4.37-4.26 (2H, m), 4.05-3.59 (27H, m), 2.52 (6H, s), 2.31-2.11 (4H, m), 0.96 (9H, s), 0.95 (9H, s). HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.56H.sub.78N.sub.8O.sub.13S.sub.2: 1134.51; observed: 1135.58.
N.SUP.1.,N.SUP.14.-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxatetradecanediamide (CM09)
(151) ##STR00021##
(152) Following general method B, from compound 1 (6.80 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM09 was obtained as a white solid. Yield: 8 mg, 0.007 mmol (40%).
(153) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (2H, s), 7.48-7.45 (2H, m), 7.31-7.27 (8H, m), 7.23 (2H, d, J=10.2 Hz), 4.64-4.59 (2H, m), 4.52-4.46 (4H, m), 4.41-4.38 (2H, m), 4.31-4.25 (2H, m), 4.01-3.94 (4H, m), 3.82 (2H, d, J=15.7 Hz), 3.62-3.52 (12H, m), 2.45 (6H, s), 2.42-2.34 (2H, m), 2.12-2.06 (2H, m), 1.19 (2H, s), 0.89 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.2, 169.9, 169.6, 149.3, 147.5, 137.3, 130.6, 129.9, 128.4, 127.1, 69.9, 69.5, 69.3, 69.1, 57.6, 56.1, 55.9, 42.2, 35.5, 34.6, 25.4, 15.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.54H.sub.74N.sub.3O.sub.12S.sub.2: 1090.49; observed: 1091.4.
N.SUP.1.,N.SUP.17.-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaheptadecanediamide (CM10)
(154) ##STR00022##
(155) Following general method B, from compound 2 (7.60 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM10 was obtained as a white solid. Yield: 6 mg, 0.005 mmol (30%).
(156) .sup.1H NMR (400 MHz, MeOD) δ 9.28 (2H, s), 7.43-7.36 (10H, m), 5.39 (2H, s), 4.77 (10H, s), 4.59 (2H, s), 4.50-4.43 (4H, m), 4.42-4.38 (2H, m), 4.26 (2H, d, J=17.2 Hz), 3.96-3.92 (4H, m), 3.77 (2H, d, J=11.1 Hz), 3.73-3.68 (2H, m), 3.56 (16H, m), 3.22-3.20 (10H, m), 2.43 (6H, s), 2.16-2.14 (2H, m), 2.13 (2H, m), 2.02-1.95 (2H, m); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.1, 172.4, 170.7, 170.3, 153.3, 153.2, 144.5, 140.0, 134.0, 129.2, 129.0, 128.4, 128.3, 127.8, 70.9, 70.5, 70.2, 70.1, 69.7, 68.2, 67.7, 59.7, 59.4, 56.8, 56.7, 54.9, 42.9, 42.3, 39.9, 37.6, 36.3, 35.7, 34.7, 25.6, 25.5, 13.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.56H.sub.78N.sub.8O.sub.13S.sub.2: 1134.51; observed: 1135.55.
N.SUP.1.,N.SUP.20.-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide (CM11)
(157) ##STR00023##
(158) Following general method B, from compound 3 (8.39 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM11 was obtained as a white solid. Yield: 11.74 mg, 0.0099 mmol (55%).
(159) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (2H, s), 7.41-7.38 (2H, m), 7.29 (10H, t, J=7.6 Hz), 4.66-4.61 (2H, m), 4.49-4.41 (6H, m), 4.35-4.29 (2H, m), 3.98-3.91 (6H, m), 3.62-3.50 (24H, m), 2.45 (6H, s), 2.42-2.35 (2H, m), 2.11-2.06 (2H, m), 0.88 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.2, 170.9, 170.4, 150.3, 148.5, 138.3, 131.6, 130.9, 129.5, 128.1, 71.2, 70.61, 70.59, 70.5, 70.4, 70.3, 58.6, 57.0, 43.2, 36.5, 35.6, 26.4, 16.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.58H.sub.32N.sub.8O.sub.14S.sub.2: 1178.54; observed: 1179.60.
N.SUP.1.,N.SUP.20.-bis((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide (CMP98)
(160) ##STR00024##
(161) Following general method B, from compound 3 (7.12 mg, 0.028 mmol, 1 eq.), compound 8 (18.06, 0.040 mmol, 2.1 eq.), HATU (15.2 mg, 0.040 mmol, 2 eq.), HOAT (5.44 mg, 0.040 mmol, 2 eq.), DIPEA (7.45 mg, 0.0010 ml, 3 eq.), compound CMP98 was obtained as a white solid. Yield: 10.58 mg, 0.0089 mmol (45%).
(162) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.09 (2H, s), 8.02 (2H, s), 7.31 (4H, d, J=8.5 Hz), 7.22 (4H, d, J=8.0 Hz), 7.16 (2H, d, J=9.2 Hz), 4.75-4.64 (4H, m), 4.51 (2H, d, J=8.9 Hz), 4.41-4.37 (2H, m), 4.24-4.17 (2H, m), 3.94 (4H, d, J=3.2 Hz), 3.84-3.81 (4H, m), 3.62-3.54 (20H, m), 2.49-2.47 (2H, m), 2.44 (6H, s), 2.26-2.17 (4H, m), 0.93 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.2, 171.5, 169.7, 151.8, 138.8, 132.9, 129.5, 129.2, 128.3, 71.2, 71.1, 70.6, 70.48, 70.45, 70.4, 70.3, 59.9, 58.5, 56.5, 43.2, 35.6, 35.2, 26.4, 15.0. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.58H.sub.32N.sub.3O.sub.14S.sub.2: 1178.54; observed: 1179.60.
(2S,2′S,4R,4′R)—N,N′-((((3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(oxy))bis(4-(4-methylthiazol-5-yl)-2,1-phenylene))bis(methylene))bis(1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide) (CMP108)
(163) ##STR00025##
(164) Following general method C, from 18 (27 mg, 0.05 mmol, 2 eq.), 19 (11.83 mg, 0.03 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (41.46 mg, 0.3 mmol, 6 eq.), the titled compound was obtained as a white solid. Yield: 9.1 mg, 0.007 mmol (28%).
(165) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (2H, s), 7.41-7.38 (2H, m), 7.26 (2H, d, J=8.1 Hz), 7.00 (2H, d, J=8.1 Hz), 6.91-6.88 (2H, m), 6.85-6.81 (2H, m), 4.57-4.52 (2H, m), 4.44-4.36 (8H, m), 4.19-4.08 (4H, m), 3.89-3.53 (22H, m), 2.45 (6H, s), 2.24-2.17 (2H, m), 2.08-2.02 (2H, m), 1.61-1.37 (8H, m), 0.88 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.9, 170.0, 165.4, 156.9, 150.4, 148.5, 132.3, 131.7, 130.0, 126.9, 122.0, 119.6, 112.9, 70.7, 70.41, 70.38, 70.2, 69.6, 67.9, 58.9, 58.4, 56.6, 39.2, 37.0, 36.0, 26.3, 17.9, 17.7, 16.2, 13.7. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.64H.sub.84N.sub.10O.sub.14S.sub.2: 1280.56; observed: 1281.66.
(2S,2′S,4R,4′R)—N,N′-((((3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(oxy))bis(4-(4-methylthiazol-5-yl)-2,1-phenylene))bis(methylene))bis(1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide) (CMP106)
(166) ##STR00026##
(167) Following general method C, from 17 (24.3 mg, 0.05 mmol, 2 eq.), 19 (11.83 mg, 0.03 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (41.46 mg, 0.3 mmol, 6 eq.), the title compound was obtained as a white solid. Yield: 7.8 mg, 0.006 mmol (26%).
(168) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.60 (2H, s), 7.39-7.35 (2H, m), 7.26 (2H, d, J=7.6 Hz), 6.91-6.88 (2H, m), 6.83-6.80 (2H, m), 6.36-6.13 (2H, m), 4.60-4.32 (10H, m), 4.18-4.05 (4H, m), 3.97-3.79 (6H, m), 3.71-3.54 (18H, m), 2.44 (6H, s), 2.17-1.86 (8H, m), 0.87 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.3, 171.1, 171.0, 170.7, 170.5, 156.8, 156.8, 150.3, 148.5, 132.2, 131.7, 130.0, 129.8, 127.1, 126.9, 122.1, 122.0, 112.8, 112.8, 71.3, 70.7, 70.6, 70.5, 70.5, 70.5, 70.4, 70.2, 70.1, 69.7, 67.9, 58.9, 58.6, 57.6, 57.5, 56.9, 56.7, 42.7, 39.1, 39.0, 37.1, 36.4, 35.4, 35.1, 26.4, 26.4, 23.2, 23.1, 16.2. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.58H.sub.82NO.sub.14S.sub.2: 1178.54; observed: 1281.66.
tert-butyl(14-(2-(((2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-3,6,9,12-tetraoxatetradecyl) carbonate (22)
(169) ##STR00027##
(170) Following general method C, from 18 (27 mg, 0.05 mmol, 1 eq.), 20 (26 mg, 0.06 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (20.73 mg, 0.15 mmol, 3 eq.), the title compound was obtained as a white solid. Yield: 17 mg, 0.02 mmol (33%).
(171) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (1H, s), 7.33-7.25 (2H, m), 6.97 (1H, d, J=9.1 Hz), 6.92-6.89 (1H, m), 6.84 (1H, d, J=1.5 Hz), 4.59-4.55 (1H, m), 4.45-4.38 (4H, m), 4.22-4.10 (2H, m), 3.93-3.54 (24H, m), 2.46 (3H, s), 2.32-2.24 (1H, m), 2.10-2.04 (1H, m), 1.63-1.52 (2H, m), 1.45-1.39 (12H, m), 0.87 (9H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.6, 170.1, 169.7, 165.4, 156.9, 150.3, 148.5, 132.3, 131.7, 130.0, 126.9, 122.0, 119.7, 112.9, 81.7, 70.72, 70.66, 70.5, 70.4, 70.3, 69.6, 69.0, 68.0, 58.8, 58.4, 56.6, 39.3, 36.7, 35.8, 28.1, 26.3, 17.8, 16.2, 13.7. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.43H.sub.63N.sub.5O.sub.12S: 873.42; observed: 874.49.
tert-butyl 17-(2-(((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-3,6,9,12,15-pentaoxaheptadecanoate (21)
(172) ##STR00028##
(173) Following general method C, from 17 (24.3 mg, 0.05 mmol, 1 eq.), 20 (26 mg, 0.06 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (20.73 mg, 0.15 mmol, 3 eq.), the title compound was obtained as a white solid. Yield: 17 mg, 0.021 mmol (33%).
(174) .sup.1H NMR (400 MHz, CDCl.sub.3) 5, ppm 8.67 (1H, s), 7.32 (2H, d, J=7.8 Hz), 6.95 (1H, dd, J=1.6, 7.6 Hz), 6.88 (1H, d, J=1.8 Hz), 4.65-4.60 (1H, m), 4.53-4.43 (2H, m), 4.39-4.36 (1H, m), 4.24-4.13 (2H, m), 4.00 (2H, d, J=7.0 Hz), 3.92-3.87 (2H, m), 3.77-3.59 (20H, m), 3.08 (2H, s), 2.51 (3H, s), 2.38-2.31 (1H, m), 1.98 (3H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.2, 170.8, 170.4, 169.7, 156.8, 150.3, 148.5, 132.2, 131.7, 129.8, 126.9, 122.0, 112.8, 81.6, 70.8, 70.71, 70.69, 70.60, 70.57, 70.55, 70.52, 70.49, 70.47, 70.1, 69.6, 69.3, 69.02, 68.98, 67.9, 58.6, 57.5, 56.7, 39.0, 37.7, 36.5, 35.2, 28.1, 26.4, 23.2, 16.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.40H.sub.62N.sub.4O.sub.12S: 822.41; observed: 823.5.
(2S,4R)-1-((S)-2-(tert-butyl)-20-(2-(((2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-4-oxo-6,9,12,15,18-pentaoxa-3-azaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (CMP113)
(175) ##STR00029##
(176) Following general method B, from compound 20 (17 mg, 0.02 mmol, 1 eq.) and trifluoroacetic acid (0.5 ml in 0.5 ml of DCM), the carboxylic acid derivative was obtained as an oil. Yield: 15.7 mg, 0.019 mmol (quantitative). HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.39H.sub.55N.sub.5O.sub.12S: 817.36; observed: 818.4.
(177) From the obtained carboxylic acid (15.7 mg, 0.019 mmol, 1 eq.) in 0.5 ml DMF, HATU (7.22 mg, 0.019 mmol, 1 eq.), HOAT (2.58 mg, 0.019 mmol, 1 eq.), compound 7 (8.73 mg, 0.019 mmol, 1 eq.) and DIPEA (3 eq.), the final compound was isolated as white solid. Yield: 6.3 mg, 0.005 mmol (27%).
(178) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (2H, s), 7.58-7.54 (1H, m), 7.31-7.25 (5H, m), 7.02 (1H, d, J=9.7 Hz), 6.88-6.85 (1H, m), 6.78 (1H, d, J=1.5 Hz), 4.59-4.56 (2H, m), 4.47-4.25 (6H, m), 4.13-3.52 (20H, m), 2.47-2.42 (6H, m), 2.34-2.27 (1H, m), 2.19-2.03 (5H, m), 1.63-1.52 (2H, m), 1.48-1.37 (2H, m), 0.90 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.2, 171.1, 170.7, 170.3, 165.4, 156.6, 150.3, 148.4, 148.3, 138.3, 132.0, 131.8, 131.7, 130.7, 129.5, 129.4, 127.9, 126.9, 122.0, 119.7, 112.6, 71.0, 70.7, 70.5, 70.4, 70.32, 70.28, 70.25, 69.6, 67.9, 59.1, 58.8, 58.5, 57.3, 57.1, 56.7, 43.1, 39.0, 37.3, 36.8, 36.2, 35.4, 26.4, 26.3, 17.9, 17.7, 16.1, 16.0, 13.7. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.61H.sub.83N.sub.9O.sub.14S.sub.2: 1229.55; observed: 1230.66.
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-(((S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (CMP112)
(179) ##STR00030##
(180) Following general method B, from compound 20 (17 mg, 0.021 mmol, 1 eq.) and trifluoroacetic acid (0.5 ml in 0.5 ml of DCM), the carboxylic acid derivative or 38 was obtained as an oil. Yield: 13 mg, 0.017 mmol (quantitative). HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.36H.sub.54N.sub.4O.sub.12S: 766.35; observed: 767.4.
(181) From the carboxylic acid (13 mg, 0.017 mmol, 1 eq.) in 0.5 ml DMF, HATU (6.49 mg, 0.017 mmol, 1 eq.), HOAT (2.31 mg, 0.017 mmol, 1 eq.), compound 7 (7.90 mg, 0.017 mmol, 1 eq.) and DIPEA (3 eq.) the titled compound was obtained as a white solid. Yield: 6 mg, 0.005 mmol (30%).
(182) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.61 (2H, s), 7.49-7.45 (1H, m), 7.32-7.24 (6H, m), 6.90-6.87 (1H, m), 6.79 (1H, d, J=2.4 Hz), 6.24 (1H, d, J=8.9 Hz), 4.61-4.29 (10H, m), 4.11-3.52 (27H, m), 2.44 (6H, s), 2.30 (1H, t, J=13.3 Hz), 2.18-2.03 (3H, m), 0.87 (9H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.2, 171.1, 170.7, 170.4, 156.7, 150.3, 148.4, 138.3, 132.2, 130.9, 129.7, 129.4, 128.0, 127.0, 122.0, 112.8, 70.9, 70.6, 70.5, 70.4, 70.3, 70.2, 69.6, 67.9, 59.0, 58.8, 57.7, 57.1, 43.1, 39.0, 37.1, 36.8, 35.6, 35.5, 26.42, 26.38, 23.0, 16.13, 16.06. HRMS (ESI) m/z: [M+H].sup.+ calculated for: C.sub.58H.sub.82N.sub.8O.sub.14S.sub.2: 1178.54; observed: 1179.6.
(183) General Method D:
(184) To a solution of the diol (1 eq.) in DCM, tert-butyl bromoacetate (8 eq.), TBABr (1.1 eq.) and 37% w/w aqueous NaOH were added. The reaction mixture was vigorously stirred at r.t. overnight. The organic phase was separated from the aqueous layer and then the aqueous phase was extracted with DCM (×3). Organic layers were collected, dried over MgSO.sub.4 and evaporated under reduced pressure. The crude was purified by flash chromatography eluting with ethyl acetate from 10% to 50% v/v in heptane.
(185) General Method E:
(186) A solution of the benzylated starting material in absolute EtOH (0.05 M) was flown in an H-cube machine at a rate of 1 mL/min, H.sub.2 10 atm, 70° C. Solvent was evaporated under reduced pressure to yield the final compound.
(187) General Method F:
(188) To a solution of the dicarboxylic acid linker (1 eq.) in dry DMF, COMU (2 eq.) and DIPEA (5 eq.) were added. The solution was stirred for 10 min and then it was added to a suspension of the VHL-ligand amine 7 (2.1 eq.) and DIPEA (5 eq.) in dry DMF. The mixture was stirred at r.t. until no presence of the starting material was detected by LC-MS. Ice was added and the volatiles were evaporated under reduced pressure to give the crude which was purified by H PLC using a gradient of 20% to 70% v/v acetonitrile in 0.1% v/v aqueous solution of formic acid to yield the final compound.
4,4′-(Butane-1,4-diylbis(oxy))bis(butan-1-ol) (101)
(189) ##STR00031##
(190) Compound 101 was prepared as reported.sup.40 by Knuf et al. Analytical data matched those previously reported.
Di-tert-butyl 3,8,13,18-tetraoxaicosanedioate (102)
(191) ##STR00032##
(192) Prepared following the general method D from compound 101 (198 mg, 0.8449 mmol) in 37% w/w aqueous NaOH (4 mL) and DCM (4 mL). Compound 102 was obtained as an oil (158 mg, yield: 40%).
(193) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 3.87 (4H, s), 3.45 (4H, t, J=6.1 Hz), 3.38-3.30 (8H, m), 1.67-1.51 (12H, m), 1.41 (18H, s).
3,8,13,18-Tetraoxaicosanedioic acid (103)
(194) ##STR00033##
(195) Prepared following the general method B starting from compound 102 (158 mg, 0.3415 mmol) in TFA/DCM 1:1 (2 mL). Compound 103 was obtained as an oil (120 mg, yield: quantitative).
(196) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 8.26 (s, 2H), 4.09 (s, 4H), 3.58 (t, J=6.1 Hz, 4H), 3.48-3.41 (m, 8H), 1.75-1.60 (m, 12H)..sup.13C-NMR (101 MHz, CDCl.sub.3) δ: 173.1, 71.7, 70.6, 70.4, 67.9, 26.4, 26.3, 26.1.
5-(Benzyloxy)pentan-1-ol (104)
(197) ##STR00034##
(198) 1,5-Pentandiol (3.430 g, 3.45 mL, 0.033 mol, 4 eq.) was added dropwise to a suspension of NaH (670 mg, 0,016 mol, 2 eq) in DMF (14 mL) at 0° C. A catalytic amount of NaI was added, followed by benzylbromide (1.360 g, 0.95 mL, 0.008 mol, 1 eq.). The mixture was stirred at r.t. overnight.
(199) The reaction was quenched with NH.sub.4Cl aq. sat. and then extracted with ethyl acetate (×3). Organic layers were collected and evaporated under reduced pressure. The crude was purified by flash chromatography eluting from 40% to 90% of ethyl acetate in heptane to give the desired product (1.08 g, yield: 70%).
(200) Analytical data matched those previously reported..sup.41
2-(2-(2-(Benzyloxy)ethoxy)ethoxy)ethyl methanesulfonate (105)
(201) ##STR00035##
(202) Compound 105 was obtained following the method previously reported.sup.42 Analytical data matched those reported.
1,18-Diphenyl-2,5,8,11,17-pentaoxaoctadecane (106)
(203) ##STR00036##
(204) Compound 104 (228.58 mg, 1.177 mmol, 25 eq) was added to a solution of NaHMDS 1M in THF (107.95 mg, 0.588 mL, 0.588 mmol, 1.25 eq.) at 0° C. under N.sub.2 atmosphere. Reaction mixture was stirred at r.t. for 1 h. After this time a solution of compound 105 (150.00 mg, 0.471 mmol, 1 eq.) in DMF was added and the mixture was irradiated with microwave at 130° C. for 2 h.
(205) After this time the solvent was evaporated, the reaction quenched with 5% aqueous NaHSO.sub.4 and extracted with DCM (×3). Organic layers were collected, dried over MgSO.sub.4, filtered and evaporated under reduced pressure. The crude was purified by flash chromatography eluting from 0% to 50% v/v of ethyl acetate in heptane to yield the desired compound 106 as an oil (114 mg, yield: 58%).
(206) .sup.1H-NMR (500 MHz, CDCl.sub.3) δ: 7.28-7.19 (m, 10H), 4.49 (s, 2H), 4.43 (s, 2H), 3.62-3.54 (m, 10H), 3.51-3.48 (m, 2H), 3.43-3.36 (m, 4H), 1.61-1.48 (m, 4H), 1.42-1.31 (m, 2H).
Di-tert-butyl 3,6,9,12,18-pentaoxaicosanedioate (107)
(207) ##STR00037##
(208) Starting from compound 106 (265 mg, 0.610 mmol) and following the general method E the deprotected compound was obtained as an oil (131 mg) and used without any further purification for the next step.
(209) Following the general method D from the deprotected compound (131 mg, 0.5544 mmol) in 37% w/w aqueous NaOH (2.2 mL) and DCM (2.2 mL) compound 107 was obtained as an oil (122 mg, yield: 47%).
(210) .sup.1H-NMR (500 MHz, CDCl.sub.3) δ: 4.00 (s, 2H), 3.92 (s, 2H), 3.69-3.60 (m, 10H), 3.57-3.53 (m, 2H), 3.52-3.46 (m, 2H), 3.43 (t, J=7.1 Hz, 2H), 1.67-1.56 (m, 4H), 1.46 (d, J=0.6 Hz, 18H), 1.43-1.37 (m, 2H). .sup.13C-NMR (101 MHz, CDCl.sub.3) δ: 169.8, 81.5, 81.4, 71.6, 71.3, 70.7, 70.6, 70.1, 69.0, 68.8, 29.5, 29.4, 28.1, 22.6.
3,6,9,12,18-Pentaoxaicosanedioic acid (108)
(211) ##STR00038##
(212) Prepared following the general method B starting from compound 107 (90 mg, 0.1937 mmol) in 2 mL of TFA/DCM 1:1. Compound 108 was obtained as an oil (66 mg, yield: quantitative).
(213) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 8.15 (s, 2H), 4.11 (s, 2H), 4.02 (s, 2H), 3.71-3.40 (m, 16H), 1.65-1.52 (m, 4H), 1.43-1.34 (m, 2H)
1,5-Bis(allyloxy)pentane (109)
(214) ##STR00039##
(215) Compound 109 was obtained starting from 1,5-petandiol (500 mg, 4.8 mmol) and following the method reported..sup.43
(216) Analytical data matched those previously reported.
3,3′-(Pentane-1,5-diylbis(oxy))bis(propan-1-ol)(110)
(217) ##STR00040##
(218) A solution of compound 109 (500 mg, 2.71 mmol, 1 eq.) in dry THF (4.2 mL) was added dropwise to a solution 0.5 M of 9-Borabicyclo[3.3.1]nonane in THF (993 mg, 16.28 mL, 8.14 mmol, 3 eq.) at 0° C. and the resulting solution was stirred at r.t. overnight.
(219) The reaction was quenched by MeOH (3.17 mL), 30% w/w aq. NaOH (6.35 mL), 30% v/v aq. H.sub.2O.sub.2 (6.35 mL) and the mixture was left to stir for 2 h. Then it was extracted with ethyl acetate (×3). Organic layers were collected, washed with brine, dried over MgSO.sub.4 and evaporated under reduced pressure. The crude was purified by flash chromatography eluting from 0% to 100% ethyl acetate in heptane to yield the desired product as an oil (483 mg, yield: 81%). Analytical data matched those previously reported..sup.43
Di-tert-butyl 3,7,13,17-tetraoxanonadecanedioate (111)
(220) ##STR00041##
(221) Compound 111 was obtained from compound 110 (214 mg, 0.9714 mmol) following the general method D, in 37% w/w aqueous NaOH (4 mL) and DCM (4 mL). The desired product was obtained as an oil (65 mg, yield: 15%).
(222) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 3.88 (s, 4H), 3.53 (t, J=6.5 Hz, 4H), 3.44 (t, J=6.4 Hz, 4H), 3.34 (t, J=6.9 Hz, 4H), 1.85-1.78 (m, 4H), 1.55-1.47 (m, 4H), 1.41 (s, 18H), 1.36-1.29 (m, 2H).
3,7,13,17-Tetraoxanonadecanedioic acid (112)
(223) ##STR00042##
(224) Prepared following the general method B starting from compound 111 (64 mg, 0.1427 mmol) in TFA/DCM 1:1 (2 mL). Compound 112 was obtained as an oil (47.5 mg, yield: quantitative).
(225) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 8.11 (s, 2H), 4.06 (s, 4H), 3.64 (t, J=5.9 Hz, 4H), 3.54 (t, J=5.9 Hz, 4H), 3.42 (t, J=6.4 Hz, 4H), 1.88-1.81 (m, 4H), 1.60-1.52 (m, 4H), 1.36 (dt, J=7.6, 11.9 Hz, 2H). .sup.13C-NMR (101 MHz, CDCl.sub.3) δ: 173.3, 71.1, 69.6, 68.2, 67.9, 29.4, 29.2, 22.7.
5-(Benzyloxy)pentyl 4-methylbenzenesulfonate (113)
(226) ##STR00043##
(227) To a solution of compound 104 (1.910 g, 9.8387 mmol, 1 eq.) and triethylamine (1.65 mL, 11.8226 mmol, 1.2 eq.) in DCM (15 mL) a solution of p-TsCl (2.063 g, 10.8226 mmol, 1.1 eq.) in DCM (15 mL) was added at 0° C. The mixture was left to stir overnight. Then NaHCO.sub.3 aq. sat. was added. The aqueous phase was separated from the organic layer and it was extracted with DCM (×2). Organic layers were collected and washed with 5% aqueous HCl solution. The crude was purified by flash chromatography eluting from 0% to 60% v/v ethyl acetate in heptane to yield the desired product (1.9 g, yield: 55%). Analytical data matched those previously reported..sup.44
1,18-Diphenyl-2,8,11,17-tetraoxaoctadecane (114)
(228) ##STR00044##
(229) A mixture of compound 113 (1.9 g, 5.6863 mmol, 2.4 eq.), ethylenglycol (147 mg, 2.3696 mmol, 1 eq.) and TBA bisulphate (804 mg, 2.3693 mmol, 1 eq) was dissolved in toluene (8 mL) and NaOH aq. 50% (6 mL). The mixture was vigorously stirred overnight. The organic phase was separated from the aqueous layer and then it was extracted with ethyl acetate (×3). Organic layers were collected, dried over MgSO.sub.4 and evaporated under reduced pressure. The crude was purified by flash chromatography eluting with a mixture v/v of ethyl acetate in heptane, from 100% heptane to 100% ethyl acetate. The desired compound was obtained as an oil (200 mg, yield: 8.5%).
(230) .sup.1H-NMR (500 MHz, CDCl.sub.3) δ: 7.25-7.22 (m, 10H), 4.40 (s, 4H), 3.47 (s, 4H), 3.39-3.35 (m, 8H), 1.58-1.47 (m, 8H), 1.37-1.29 (m, 4H).
5,5′-(Ethane-1,2-diylbis(oxy))bis(pentan-1-ol) (115)
(231) ##STR00045##
(232) Starting from compound 114 (200 mg, 0.8535 mmol) and following the general method E compound 115 was obtained as an oil (35 mg, yield: 31%).
(233) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 3.53 (t, J=6.1 Hz, 4H), 3.49 (s, 4H), 3.49 (s, 4H), 3.40 (t, J=6.6 Hz, 4H), 2.93 (s, 2H), 1.58-1.45 (m, 8H), 1.37-1.29 (m, 4H).
1,2-Di(1,3-dioxan-2-y)ethane (118)
(234) ##STR00046##
(235) Compound 118 was prepared in accordance with the published procedure,.sup.45 starting from 2,5-dimethoxytetrahydrofuran (10.0 g, 75.6659 mmol). Analytical data matched those previously reported.
3,3′-(Butane-1,4-diylbis(oxy))bis(propan-1-ol) (119)
(236) ##STR00047##
(237) Compound 119 was prepared accordingly in accordance with the published procedure,.sup.45 starting from compound 118. Analytical data matched those previously reported.
1-Phenyl-2,5,9,14-tetraoxaheptadecan-17-ol (120)
(238) ##STR00048##
(239) Compound 120 (1.1 g, 5.3325 mmol, 3 eq.) was dissolved in toluene (10 mL) and NaOH aq. 50% w/w (5 mL). TBABr (590 mg, 1.7775 mmol, 1 eq.), a catalytic amount of TBAl and benzyl-2-bromoethyl ether (382 mg, 1.7775 mmol, 1 eq.) were added and the reaction mixture was vigorously stirred for 48 h. Organic layer was separated from the aqueous phase and the aqueous phase was extracted with DCM (×3). The crude was purified by flash chromatography eluting from 0% to 5% v/v MeOH in DCM to obtain the product as an oil (350 mg, 57%).
(240) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 7.33-7.22 (m, 5H), 4.55 (s, 2H), 3.74 (dd, J=5.7, 11.2 Hz, 2H), 3.59-3.55 (m, 6H), 3.53 (t, J=6.5 Hz, 2H), 3.47 (t, J=6.4 Hz, 2H), 3.44-3.37 (m, 4H), 2.44 (t, J=5.7 Hz, 1H), 1.87-1.76 (m, 4H), 1.61-1.57 (m, 4H).
3-(4-(3-(2-Hydroxyethoxy)propoxy)butoxy)propan-1-ol (121)
(241) ##STR00049##
(242) The product was obtained starting from compound 120 (350 mg, 1.028 mmol) and following the general method E. The conversion was not quantitative so the product 121 was separated from the starting material by a flash chromatography eluting from 100% DCM to 9:1 v/v DCM/MEOH (87 mg, yield: 34%).
(243) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 3.66-3.60 (m, 4H), 3.51-3.38 (m, 8H), 3.38-3.31 (m, 4H), 1.79-1.69 (m, 4H), 1.57-1.50 (m, 4H).
Di-tert-butyl 3,6,10,15,19-pentaoxahenicosanedioate (122)
(244) ##STR00050##
(245) Compound 122 was obtained from compound 121 (87 mg, 0,3475 mmol) following the general method D, in 37% w/w aqueous NaOH (1.5 mL) and DCM (1.5 mL). The desired product was obtained as an oil (47 mg, yield: 28%).
(246) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ: 3.99 (s, 2H), 3.68-3.42 (m, 12H), 3.41-3.36 (m, 4H), 1.88-1.77 (m, 4H), 1.59-1.55 (m, 4H), 1.44 (s, 18H).
3,6,10,15,19-Pentaoxahenicosanedioic acid (123)
(247) ##STR00051##
(248) Prepared following the general method B starting from compound 122 (47 mg, 0.0983 mmol) in TFA/DCM 1:1 (1 mL). Compound 123 was obtained as an oil (35 mg, yield: quantitative).
(249) .sup.1H-NMR (500 MHz, CDCl3) δ: 4.14 (s, 2H), 4.07 (s, 2H), 3.73-3.69 (m, 2H), 3.65-3.59 (m, 4H), 3.59-3.53 (m, 4H), 3.49 (t, J=6.3 Hz, 2H), 3.47-3.40 (m, 4H), 1.89-1.81 (m, 4H), 1.62-1.57 (m, 4H).
(250) 13C-NMR (101 MHz, CDCl3) δ: 173.9, 173.7, 71.3, 71.0, 70.8, 70.0, 69.6, 68.7, 68.6, 68.1, 68.0, 67.6, 29.7, 29.5, 26.3, 26.2.
N1,N20-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,18-pentaoxaicosanediamide (124)
(251) ##STR00052##
(252) Compound 124 was prepared accordingly to general method F, starting from compound 7 (20 mg, 0.0428 mmol) and compound 108 (7.2 mg, 0.02038 mmol). 5 mg were obtained (yield: 21%).
(253) .sup.1H-NMR (500 MHz, MeOD) δ: 8.77 (s, 2H), 7.34 (dd, J=7.4, 23.2 Hz, 8H), 4.59 (dd, J=2.4, 9.4 Hz, 2H), 4.50-4.38 (m, 6H), 4.27 (t, J=4.3 Hz, 1H), 4.24 (t, J=4.3 Hz, 1H), 3.94 (dd, J=15.3, 22.3 Hz, 2H), 3.85 (dd, J=15.3, 24.4 Hz, 2H), 3.76 (d, J=10.7 Hz, 2H), 3.72-3.68 (m, 2H), 3.61-3.40 (m, 14H), 3.35 (dt, J=1.0, 6.5 Hz, 2H), 2.37 (s, 6H), 2.16-2.09 (m, 2H), 2.02-1.96 (m, 2H), 1.57-1.45 (m, 4H), 1.39-1.32 (m, 2H), 0.94 (s, 18H).
(254) .sup.13C-NMR (101 MHz, MeOD) δ: 174.4, 174.3, 172.1, 172.0, 171.7, 152.9, 149.0, 140.3, 133.4, 131.5, 130.5, 130.4, 129.5, 129.0, 72.9, 72.3, 72.2, 71.7, 71.6, 71.5, 71.2, 71.1, 70.7, 60.8, 58.1, 58.0, 43.7, 38.9, 37.2, 37.1, 30.5, 30.4, 27.0, 26.9, 23.8, 15.8.
(255) HRMS: found 1177.6435 [M+H.sup.+].
N1,N20-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,8,13,18-tetraoxaicosanediamide (125)
(256) ##STR00053##
(257) Compound 125 was prepared accordingly to general method F, starting from compound 7 (20 mg, 0.0428 mmol) and compound 103 (7.1 mg, 0.02038 mmol). 6.7 mg were obtained (yield: 28%).
(258) .sup.1H-NMR (500 MHz, MeOD) δ: 8.77 (s, 2H), 7.34 (dd, J=8.3, 23.6 Hz, 8H), 4.59 (d, J=12.0 Hz, 2H), 4.50-4.40 (m, 6H), 4.26 (dd, J=4.9, 15.9 Hz, 2H), 3.86 (dd, J=15.3, 23.4 Hz, 4H), 3.77 (d, J=11.4 Hz, 2H), 3.70 (dd, J=3.9, 11.1 Hz, 2H), 3.46 (t, J=6.0 Hz, 4H), 3.38-3.28 (m, 8H), 2.37 (s, 6H), 2.16-2.10 (m, 2H), 2.02-1.96 (m, 2H), 1.62-1.52 (m, 8H), 1.51-1.45 (m, 4H), 0.93 (s, 18H).
(259) .sup.13C-NMR (101 MHz, MeOD) δ: 174.3, 172.1, 172.0, 171.7, 152.8, 149.1, 140.3, 133.4, 131.5, 130.5, 130.4, 129.5, 129.0, 72.7, 71.7, 71.5, 71.1, 70.7, 60.8, 58.1, 58.0, 43.7, 39.0, 37.2, 27.6, 27.5, 27.4, 15.9.
(260) HRMS: found 1175.6623 [M+H.sup.+].
N1,N19-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,7,13,17-tetraoxanonadecanediamide (126)
(261) ##STR00054##
(262) Compound 126 was prepared accordingly to general method F, starting from compound 7 (20 mg, 0.0428 mmol) and compound 112 (6.8 mg, 0.0204 mmol). 6.6 mg were obtained as a white solid (yield: 28%).
(263) .sup.1H-NMR (500 MHz, MeOD) δ: 8.76 (s, 2H), 7.37-7.30 (m, 8H), 4.60 (d, J=9.4 Hz, 2H), 4.50-4.24 (m, 8H), 3.87 (d, J=6.5 Hz, 4H), 3.77 (d, J=11.2 Hz, 2H), 3.70 (dd, J=3.8, 11.5 Hz, 2H), 3.55-3.49 (m, 4H), 3.43 (dt, J=1.2, 6.2 Hz, 4H), 3.33-3.29 (m, 4H), 2.37 (s, 6H), 2.16-2.10 (m, 2H), 2.03-1.96 (m, 2H), 1.80-1.74 (m, 4H), 1.47-1.40 (m, 4H), 1.30-1.23 (m, 2H), 0.93 (s, 18H).
(264) .sup.13C-NMR (101 MHz, MeOD) δ: 174.3, 171.8, 171.6, 152.8, 149.0, 140.2, 133.4, 131.5, 130.5, 130.3, 129.5, 128.9, 71.9, 71.0, 70.8, 69.8, 68.3, 60.8, 58.1, 57.9, 43.7, 38.9, 37.2, 30.9, 30.5, 26.9, 23.9, 15.8.
(265) HRMS: found 1161.6446 [M+H.sup.+].
N1,N21-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,10,15,19-pentaoxahenicosanediamide (128)
(266) ##STR00055##
(267) Compound 128 was prepared accordingly to general method F, starting from compound 7 (20 mg, 0.0428 mmol) and compound 123 (7.5 mg, 0.02038 mmol). 6.5 mg were obtained as a white solid (yield: 27%).
(268) .sup.1H-NMR (500 MHz, MeOD) δ: 9.00 (d, J=1.1 Hz, 2H), 7.45 (dd, J=8.4, 23.1 Hz, 8H), 4.71-4.68 (m, 2H), 4.55 (tt, J=12.4, 11.9 Hz, 6H), 4.36 (d, J=15.5 Hz, 2H), 4.03 (d, J=3.6 Hz, 2H), 3.97 (d, J=5.9 Hz, 2H), 3.89-3.78 (m, 4H), 3.71-3.68 (m, 2H), 3.64-3.36 (m, 14H), 2.49 (s, 6H), 2.26-2.19 (m, 2H), 2.13-2.06 (m, 2H), 1.90-1.84 (m, 2H), 1.85-1.79 (m, 2H), 1.61-1.55 (m, 4H), 1.04 (d, J=3.4 Hz, 18H).
(269) .sup.13C-NMR (101 MHz, MeOD) δ: 174.4, 174.3, 172.1, 171.9, 171.8, 171.7, 153.3, 140.6, 131.1, 130.4, 129.0, 72.3, 71.8, 71.2, 71.1, 70.9, 69.9, 69.4, 68.7, 68.4, 60.8, 58.2, 58.1, 58.0, 43.7, 38.9, 37.2, 37.1, 31.1, 31.0, 27.5, 27.0, 15.4. HRMS: found 1191.6137 [M+H.sup.+].
(S)-1-((2R,3R,4S)-3-Fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-aminium chloride (129)
(270) ##STR00056##
(271) Compound 129 was prepared accordingly to PATENT WO 2018/051107 A1. Analytical data matched those previously reported.
N1,N20-Bis((S)-1-((2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide (130)
(272) ##STR00057##
(273) Prepared accordingly to general method F, starting from compound 129 (16.9 mg, 0.0348 mmol) and 3,6,9,12,15,18-hexaoxaicosanedioic acid (6.17 mg, 0.0174 mmol). Obtained 7.5 mg (35% yield) as white solid.
(274) .sup.1H-NMR (400 MHz, MeOD) δ: 8.89 (s, 2), 7.46 (d, J=8.7 Hz, 8H), 4.99 (td, J=3.3, 52.9 Hz, 2H), 4.69 (s, 2H), 4.65 (dd, J=2.9, 21.3 Hz, 2H), 4.60-4.34 (m, 6H), 4.08-4.03 (m, 6H), 3.77-3.59 (m, 22H), 2.49 (s, 6H), 1.06 (s, 18H).
(275) .sup.19F-NMR (376.45 MHz, MeOD): −201.87, .sup.13C-NMR (101 MHz, MeOD) δ: 170.9, 170.5, 169.2, 169.1, 151.5, 147.7, 138.6, 130.2, 129.0, 127.5, 94.0, 92.1, 70.9, 70.2, 70.1, 70.1, 69.6, 69.5, 64.4, 64.1, 56.1, 50.9, 42.4, 35.3, 25.5, 14.4. HRMS: found 1215.5214 [M+H.sup.+].
(276) Abbreviations
(277) BAIB, bis-acetoxy iodobenzene;
(278) CID, chemical inducer of dimerization;
(279) CRL, Cullin RING ligase;
(280) DC50, half-degrading concentration;
(281) DCM, dichloromethane;
(282) DIPEA, N,N-Diisopropyethylamine;
(283) DMF, dimethylformamide;
(284) DMSO, dimethylsulfoxide;
(285) HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3Ytriazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate;
(286) Hdy-HIF-1α, hydroxylated form of HIF-1α;
(287) HIF-1α, hypoxia inducible factor alpha;
(288) Hyp, hydroxyproline;
(289) HOAT, 1-Hydroxy-7-azabenzotriazole;
(290) IAPS, inhibitor of apoptosis proteins;
(291) ITC, isothermal titration calorimetry;
(292) LHS, left hand side;
(293) PEG, polyethylene glycol;
(294) PHD, prolyl hydroxylase domain-containing protein;
(295) PPI, protein-protein interaction;
(296) PROTACS, Proteoysis-Targeting Chimeras;
(297) RHS, right end side;
(298) SEC, size exclusion chromatography;
(299) TEMPO, 2,2,6,6-Tetramethylpiperidin-1-yl)oxyl or (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl;
(300) TFA, trifluoroacetic acid;
(301) VHL, von Hippel-Lindau;
(302) HRE, hypoxia response element.
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