Patent Application
20250127933
The present invention relates to a method for verifying the effectiveness of a linker in a drug delivery system and a structure for the method. Specifically, the present invention relates to a method and a structure for verifying whether a linker in drug delivery systems (DDSs), such as prodrugs, antibody-drug conjugates, or ligand-targeted drugs, effectively releases an active drug by being cleaved in a disease-selective or protein-selective manner in vivo and whether the linker is stable in plasma.
To overcome the limitations of conventional chemotherapy, such as toxicity, ligand-targeted drugs (LTDs) have been developed to enhance the selective localization of small-molecule drugs to tumors. In an LTD, a targeting ligand as a vehicle for specific delivery to cancer cells is conjugated with a therapeutic or imaging agent via a spacer and cleavable or non-cleavable linker. Monoclonal antibodies, peptides, and small molecules specific to malignant antigens are considered vehicles to selectively deliver cargo, such as cytotoxic or imaging agents, to target cells.
Antibody-drug conjugates (ADCs) are one of the best-characterized strategies for LTDs. Since the discovery and successful landing of brentuximab vedotin (Adcetris®, Seattle Genetics, 2011) against relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma in the market [1-3]. 10 ADCs have been approved by the United States Food and Drug Administration, and there are numerous ADCs being explored in ongoing clinical trials [4-12].
Typical ADCs comprise a humanized or fully human monoclonal antibody (mAb) targeting an antigen specifically overexpressed in tumor cells, cytotoxic payloads, and a suitable chemical linker for the covalent conjugation of drugs to antibodies. ADCs recognize and bind to highly expressed antigens on the cell surface of target cancer cells, and the ADC-antigen complex is internalized via endocytosis, followed by the release of cytotoxic drugs by lysosomal degradation.
The linker is a crucial component that determines various attributes of an ADC required for clinical use, including the efficacy and therapeutic index in terms of pharmacokinetics, pharmacodynamics, and toxicity. As encountered in the history of the development of many ADCs, instability of the linker leads to the premature release of a cytotoxic payload in the plasma during blood circulation or in other tissues, which may result in higher toxicity and lower efficacy [13-16].
For the successful development of ADC drugs, it is important to develop linkers that have sufficient plasma stability during blood circulation and selectively release the payloads via internalization into cancer cells. As demonstrated in the discovery of brentuximab vedotin, a valine-citrulline (Val-Cit) dipeptide linker was designed to provide sufficient plasma stability, which is conjugated to cytotoxic momomethyl auristatin E (MMAE) via a 4-aminobenzyl carbamate linkage (
The present inventors previously reported various xanthene-based fluorophores and activatable fluorescent probes that can release fluorophores and activate fluorescence under the activity of a specific enzyme. In previous studies, the present inventors synthesized “turn on” nitroreductase (NTR)-responsive fluorescent probes diversely linked to a 4-nitrobenzyl moiety via ether, carbonate, amine, and carbamate linkages [23,24].
Fluorescence emission by decomposition of the linker was used as a means for evaluating the stability at various pH values and temperatures, as well as the release of fluorophores under the NTR reaction. Based on such previous studies, the present inventors have designed and evaluated peptide-based cleavable linkers having sufficient plasma stability using activatable fluorescent probes and have found that these cleavable linkers can selectively release cytotoxic payloads in response to a tumor-specific enzyme, thereby completing the present invention.
An object of the present invention is to provide a method for verifying the effectiveness and stability of a linker in a drug delivery system.
Another object of the present invention is to provide a structure for verifying the effectiveness and stability of the linker.
LTDs have been of interest as a way to solve the safety and efficacy issues of chemotherapeutics, which are still widely used in clinical practice. The efficacy of LTDs such as ADCs is primarily determined by the activity of payloads, while the safety may be controlled by the stability and selectivity of linkers for conjugation, as well as the selectivity of targeting ligands for delivery. The selective release of payloads to target cells is a critical factor for successful LTDs with sufficient efficacy and minimal toxicity. In addition, in prodrugs, the stability and selectivity of the carrier (or linker) that selectively releases the payload to target cells are also critical factors in the development of prodrugs. Thus, it is essential to develop cleavable linkers with sufficient plasma stability during blood circulation that selectively releases payloads in target cells.
Accordingly, the present invention has been completed based on the finding that, although activatable fluorescent probes have no ligand for delivery, the release of fluorophores conjugated to peptide linkers from the activatable fluorescent probes may be easily detected and evaluated based on emitted fluorescence. As a specific example, an enzyme-responsive activatable fluorescent probe provides a simple and intuitive method for the evaluation of the inherent stability and drug release efficiency of cleavable linkers and a structure for the method (
In one aspect, the present invention provides a structure for verifying the effectiveness and stability of a linker in drug delivery systems, including antibody-drug conjugates.
Specifically, the present invention provides a structure for verifying whether a cleavable linker in drug delivery systems, including antibody-drug conjugates in which a ligand, the cleavable linker, and a drug are linked together, is selectively cleavable in a specific disease or by a specific protein, is stable in vivo, and efficiently releaser the drug in vivo.
In the present invention, the term “drug delivery system (DDS)” refers to a drug delivery system having either a structure in which a vehicle (or linker) capable of cleavage at or near an in vivo target and a drug that exhibits efficacy by decomposition of the vehicle are linked together, or a structure in which a ligand that binds to an in vivo target, a cleavable linker, and a drug are linked together.
The in vivo target or target may be a specific protein, examples of which include, but are not necessarily limited to, CD33, CD30, HER2+, CD22, CD79b, Nectin 4, HER2, TROP-2, B-cell maturation antigen (BCMA), CD19, tissue factor (TF), folate receptor, and the like.
In the present invention, a prodrug is one of the above-described drug delivery systems and has a structure in which a vehicle (or linker) capable of cleavage at or near an in vivo target and a drug that is released by decomposition of the vehicle are linked together.
In the present invention, a ligand-targeted drug (LTD) is one of the above-described drug delivery systems and has a structure in which a ligand that binds to an in vivo target, a cleavable linker, and a drug are linked together. The drug exhibits efficacy by decomposition of the linker.
In the present invention, an antibody-drug conjugate (ADC) is a representative example of the ligand-targeted drug and is a drug delivery system in which an antibody that binds to an in vivo target, a cleavable linker, and a drug are linked together. Unless otherwise specified, in the present specification, the term “ligand-targeted drug” is intended to include the term “antibody-drug polymer (ADS)”.
The structure of the present invention is characterized by including a fluorescent probe, which replaces the drug in the drug delivery system and is linked to the linker whose effectiveness is to be verified.
The linker whose effectiveness is to be verified is a linker which is developed for use in drug delivery systems, including prodrugs, antibody-drug conjugates, and ligand-targeted drugs. The linker is a compound linked to the drug in prodrugs among drug delivery systems or is a compound that links the drug to the ligand in antibody-drug conjugates or ligand-targeted drugs among drug delivery systems. According to the present invention, it is verified whether the linker is stable in vivo (preferably in plasma) and is cleavable in vivo by a protease or peptidase present at or near an in vivo target, and whether the drug loaded in the drug delivery system may be efficiently released in vivo by the cleavage of the linker.
As a specific example, the linker may be an enzyme substrate that is cleaved by an enzyme present at or around an in vivo target. The enzyme substrate may be, for example, leucine that is degraded by leucine peptidase, without being necessarily limited thereto.
The fluorescent probe of the present invention may be a fluorophore that fluoresces when the linker in the drug delivery system is cleaved. The fluorophore may be linked directly to the linker whose effectiveness is to be verified. Preferably, the fluorophore may be linked via a spacer to the linker whose effectiveness is to be verified.
Although the linkage between the fluorescent probe and the linker whose effectiveness is to be verified may vary depending on the types of fluorescent probe and linker whose effectiveness is to be verified, examples thereof include an ether linkage, an ester linkage, an amide linkage, a carbonate linkage, a carbamate linkage, and the like.
The fluorophore of the present invention may be any fluorescent substance that can fluoresce when the linker is cleaved in vivo, and examples thereof include, but are not necessarily limited to, coumarin derivatives such as 7-amino-methylcoumarin (AMC); xanthine derivatives such as rhodamine, rhodol, fluorescein, and eosin; cyanine derivatives such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine; and squaraine derivatives.
The spacer, a compound that links the fluorophore to the linker, is a compound that self-eliminates when the linker is cleaved. Examples of the spacer include, but are not necessarily limited to, a p-aminobenzyl spacer, etc. The p-amino benzyl spacer may be linked via an ether linkage, an ester linkage, an amide linkage, a carbonate linkage, a carbamate linkage, or the like to the linker whose effectiveness is to be verified.
As a specific example, the fluorescent probe of the present invention may be a xanthine derivative having a structure represented by Formula 43 or 44 below.
More specifically, the fluorescent probe of the present invention, which consists only of a fluorophore that is linked directly to the linker, may be any one of the compounds of Formulas 43 and 44 wherein R is MeO— or Et2N— and X is —NH2. As an example, a structure in which the fluorescent probe consisting only of a fluorophore is linked to leucine as the linker is a compound of Formula 1, a compound of Formula 2, a compound of Formula 7, or a compound of Formula 8 (see C-2 and Scheme S2 described in the Examples).
More specifically, the fluorescent probe of the present invention, which consists of the fluorophore and the spacer, may be any one of compounds of Formulas 43 and 44 wherein R is MeO— or Et2N— and X is
As an example, a structure in which the fluorescent probe consisting of the fluorophore and the spacer is linked to leucine as the linker is a compound of Formula 3, a compound of Formula 4, a compound of Formula 5, a compound of Formula 6, a compound of Formula 9, a compound of Formula 10, a compound of Formula 11, or a compound of Formula 12 (see C-3, Scheme S3 and C-4, and Scheme S4 described in the Examples).
As examples of the structure of the present invention, various LAP-responsive fluorescent probes containing leucine, an LAP substrate, were prepared and tested. As a result, it was found that the fluorescent probes showed stable fluorescence due to cleavage of leucine at pH 2 to 12, which is the in vivo pH condition, and showed stable fluorescence even in the temperature range of 25 to 45° C. In addition, it was found that the fluorescent probes exhibited fast or slow kinetics depending on the type of fluorescent probe prepared, released fluorophores by lysosomal LAP rather than extracellular LAP, emitted fluorescence only by LAP without interference from other intracellular bioanalytes, and were stable even in an ex vivo plasma stability test.
Therefore, the structure of the present invention probes may be used as a platform for the development of cleavable linkers for drug delivery systems that effectively drugs and are sufficiently stable during blood circulation.
In another aspect, the present invention provides a method for verifying the effectiveness of a linker in a drug delivery system.
Specifically, the present invention provides a method for verifying the effectiveness, stability, in vivo stability, in vivo cleavability, and in vivo drug release efficiency of a cleavable linker in drug delivery systems, including prodrugs in which the cleavable linker and a drug are linked together, and in antibody-drug conjugates or ligand-targeted drugs in which a ligand, the cleavable linker and a drug are linked together.
The method for verifying the effectiveness and stability of a linker according to the present invention includes steps of: preparing a structure for verifying the effectiveness of a linker, developed for use in ligand-targeted drug delivery systems, including antibody-drug conjugates, by linking a fluorescent probe to the linker; and confirming whether the structure emits fluorescence by cleavage of the linker inside or outside a cell.
The step of preparing a structure for verifying the effectiveness of a linker is a step of linking a fluorescent probe to a linker developed for use in the above-described drug delivery systems, including prodrugs, antibody-drug conjugates, and ligand-targeted drugs, that is, a linker whose effectiveness is to be verified.
The linker whose effectiveness is to be verified, the fluorescent probe, and the linkage therebetween are as described above.
The step of confirming whether the structure emits fluorescence by cleavage of the linker inside or outside a cell is a step of confirming whether the fluorophore in the fluorescent probe emits fluorescence by in vivo or ex vivo cleavage of the linker whose effectiveness is to be verified, that is, the linker developed for use in a drug delivery system, and measuring the amount of the emitted fluorescence.
The cell may be a cell to which the ligand in a ligand-targeted drug system binds or a cell on which the drug acts. The “inside a cell” may refer to in vivo, and the “outside a cell” may refer to ex vivo.
Confirming whether the fluorophore emits fluorescence by cleavage of the linker inside the cell means confirming that the cleavage of the linker is caused by an intracellular substance (e.g., lysosomal LAP) rather than an extracellular substance (e.g., extracellular LAP). This confirmation may be an indicator for evaluating the efficiency of drug release by intracellular cleavage of the linker.
Confirming whether the fluorophore emits fluorescence by cleavage of the linker outside the cell may be an indicator for evaluating the stability of the linker depending on in vivo temperature and pH conditions.
In addition, confirming whether the fluorophore emits fluorescence by cleavage of the linker outside the cell may be an indicator to evaluate the stability and drug release efficiency of the linker by determining whether the cleavage of the linker is caused by the target substance in vivo or by another substance in vivo.
In addition, confirming the timing and amount of fluorescence emission by cleavage of the linker inside or outside the cell may be an indicator for evaluating the pharmacokinetics of the linker, the in vivo stability of the linker, and the half-life of the ligand-targeted drug including the linker.
Although confirmation of the fluorescence emission may vary depending on the type of fluorophore in the structure, it may be performed using a fluorescence detector such as a spectrophotometer.
According to the above-described method for verifying the effectiveness of a linker in a drug delivery system, it is possible to easily and simply evaluate the stability and drug release efficiency of a linker for use in a ligand-targeted drug delivery system, thereby dramatically reducing the development period and cost of drug delivery systems, including prodrugs, antibody-drug conjugates, and ligand-targeted drugs.
According to the structure of the present invention and the method for verifying the effectiveness and stability of a linker using the same, it is possible to easily and simply confirm whether a linker in drug delivery systems, including prodrugs, antibody-drug conjugates, and ligand-directed drugs, is stable in vivo, is cleavable in vivo, and efficiently releases the drug in vivo, thereby dramatically reducing the development period and cost of drug delivery systems.
Hereinafter, the present invention will be described in detail with reference to examples to aid understanding of the present invention. However, the examples according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to explain the present invention more completely to those skilled in the art.
All spectroscopic readings were measured with BioTek Synergy™ H1 spectrophotometer, using 96 well microplate. A parent stock solution of probes was dissolved in DMSO to obtain 10 mM. Leucine aminopeptidase was dissolved in deionized water to obtain 1 mg/mL. All spectra measurements were carried out in PBS buffer solution (PBS buffer, 10 mM, pH 7.4, 37° C. The final volume in 96 well microplate was adjusted to 200 μL. RPMI 1640 was obtained from Thermo Scientific (Waltham, MA, USA). Bestatin hydrochloride (LAP inhibitor), DMSO and 2-mercaptoethanol were purchased from Sigma Aldrich (St. Louis, Mo, USA).
2. Thermal and pH-Dependent Stability Tests
Thermal stability was evaluated by the incubation of 1 μM of the probe in PBS buffer (pH 7.4) at different temperatures ranging from 25 to 45° C. for 20 min and reading fluorescence at each temperature. The pH stability test was performed by the incubation with 1 μM probe in different pH buffers (2 to 13) at 25° C. and recording fluorescence at each pH.
3. Enzymatic Kinetic Study and Competitive Assay with LAP Inhibitor
The kinetic study for LAP reaction was performed by incubation of 2 μg of LAP (20 μg/100 μL) with 1 μM probe in PBS buffer (10 mM, pH 7.4). The plate was incubated at 37° C. for 80 min with continuous shaking, and the emission spectra were recorded at the corresponding wavelength over time. A solution of LAP (2 μg/200 μL) was pretreated with bestatin (0, 1, 10, 50 and 100 μM) in PBS (10 mM, pH 7.4) at 37° C. for 30 min, and then 1 μM of probe was added. The mixture was incubated at 37° C., and the fluorescence was recorded on Synergy™ H1 using a 96-well microplate.
HepG2 cells (3×104 cells/well) were seeded in black-walled, transparent bottomed 96-well plates and cultured overnight. Cells were pretreated with LAP inhibitor (100 μM) for another 1 h, then cells were treated with 1 μM of probe. Kinetic study was performed in Synergy™ H1 microplate reader.
HepG2 cells were maintained in DMEM medium containing 10% FBS and 1% antibiotics in the 15 μ-plate 96 well black (ibidi, Germany). HepG2 cells were incubated with or without 500 μM of bestatin, LAP inhibitor, for 1 h and subsequently treated with 1 μM of probe and Lyso-Tracker™ Red (Thermo Scientific, USA) for 1 h. The treated cells were washed three times with PBS and treated with 10 μM of DAPI (Thermo Scientific, USA) for 2 h. Imaged were collected by Leica confocal microscopy (TCS SP5 AOBS/Tandem, Leica, Germany). Lyso-Tracker™ Red was excited by 514 nm and detected by 570-620 nm, compounds were excited by 476 nm and detected by 505-545 nm, DAPI was excited by 405 nm and detected by 420-470 nm.
1 μM of probe was incubated with various bioanalytes containing 1 mM of NaOCl, H2O2, CaCl2), ZnCl2, KCl, NaCl, GSH, vitamin C, L-cystein, L-arginine, L-alanine and 1 U/mL of α-amylase, lysozyme, cellulase under 0.05 U/mL of LAP in PBS (10 mM, pH 7.4) at 37° C. for 1 h. The fluorescence was recorded on Synergy™ H1 microplate reader.
Animal was treated strictly according to the guidelines for laboratory animal care and use issued by the Sunchon National University Institutional Animal Care and Use Committee (SCNU IACUC). Female C57/BL6 mice (7 weeks old), weighing about 17-20 g, were purchased from Orientbio (Orientbio Inc., Seongnam, Korea). Blood was collected from the posterior orbital plexus in normal mice and centrifuged (5000 rpm, 4° C., 5 minutes) to obtain serum. The stability study was performed by incubation of the fluorescent probe (10 μM) or rhodol fluorophore (0.1, 0.3, 1, 3 and 10 μM) with serum at 37° C. . . . The fluorescence was detected on Synergy™ H1 microplate reader.
LAP is a lysosomal exopeptidase that selectively catalyzes the hydrolysis of the N-terminal leucine residue peptide and protein substrates. LAP has from numerous various biological functions and is associated with various pathological functions of cancer cells, including proliferation, invasion, angiogenesis, and drug resistance. Abnormal LAP expression is interesting as a diagnostic and prognostic biomarker of cancer. The present inventor sought to develop a new cleavable linker for application to LTD-releasing drugs, such as ADCs. Based on the biological functions of LAP and its expression levels in cancer cells, the present inventors predicted that LAP is a reasonable target for a preliminary study to identify fluorophores being efficiently released by lysosomal peptidases and investigate the effect of the p-aminobenzyl group as a spacer for linking cytotoxic drugs to cleavable linkers. Thus, the activatable fluorescent probes releasing fluorophores under the activity of LAP were designed based on our previous studies and synthesized using typical and reduced xanthene fluorophores, including fluorescein, rhodol, and rhodamine derivatives with a terminal —OH or —NH2 group (
Typical xanthene and reduced xanthene fluorophores with a terminal —NH2 group for activatable fluorescent probes were synthesized from fluorescein as described previously, except for the typical rhodamine fluorophore (Scheme S1) [23,24]. Four activatable fluorescent probes, including Rhoda-P1, Rhodo-P1, Red Rhoda-P1, and Red Rhodo-P1, directly conjugated with a leucine, were prepared via an EEDQ coupling reaction of the corresponding fluorophores (Rhoda-NH2, Rhodo-NH2, Red Rhoda-NH2, and Red Rhodo-NH2) with Boc-Leu-OH followed by N-Boc deprotection using trifluoroacetic acid (TFA) (Scheme S2). The synthesis of four activatable fluorescent probes with a p-aminobenzyl spacer via carbamate linkages (Rhoda-P2, Rhodo-P2, Red Rhoda-P2, and Red Rhodo-P2) was initiated by the activation of fluorophores using phenyl chloroformate to produce phenoxy carbonyl intermediates. The substitution of the phenoxy group with a p-aminobenzyl alcohol coupled with Boc-Leu-OH and subsequent N-Boc deprotection using TFA then yielded the activatable fluorescent probes linked to a spacer via carbamate linkages (Scheme S3). Four fluorophores with a terminal-OH group (Rhodo-OH, Fluor-OH, and their reduced forms) were used to generate activatable fluorescent probes linked to a spacer via ether linkages (Rhodo-P3, Red Rhodo-P3, Fluor-P3, and Red Fluor-P3). O-Alkylation of the phenol group in fluorophores under Ag2O was performed with p-aminobenzyl bromides coupled with Boc-Leu-OH, and N-Boc the deprotection then yielded corresponding final products with ether linkages (Scheme S4).
The specific synthesis method is as follows.
All reagents and solvents were obtained from commercial sources Sigma-Aldrich Chemical Co., Combi-Block, Tokyo Chemical Industries, Daejung Chemicals, and Alfa Aesar and used without purification. Anhydrous solvents were purchased from Sigma-Aldrich and used under dry nitrogen atmosphere. Ultrapure water was obtained from a water ultra-purification system. Completion of reaction was confirmed by thin layer chromatography (TLC) on Kiesegel 60F254 from Merck and all synthesized compounds were purified by flash column chromatography using ZEOprep 60 (40-63) μM silica gel from ZEOCHEM. 1H and 13C NMR spectra were measured on JEOL JNMECZ400s/L1 (400 MHZ) and CDCl3 or DMSO-d6 was used as the NMR solvents. The chemical shifts were quoted in parts per million (ppm) and the coupling constant (J) were reported in hertz unit (Hz). Chemical shifts (in ppm) were referenced to tetramethylsilane (δ=0 ppm) in CDCl3 as an internal standard. 13C NMR spectra were reported in ppm referenced to the center line of the triplet at 77.0 ppm for CDCl3 or 39.5 ppm for DMSO-d6. All in vitro enzyme assays were performed by taking the absorbance and emission on Synergy™ H1 microplate reader from BioTek. Leucine aminopeptidase from porcine kidney and other enzymes were purchased from Sigma-Aldrich.
To a solution of fluorophore (1.2 eq.) in CH2Cl2 was added EEDQ (1.2 eq.) and Boc-Leu-OH (1.0 eq.) The reaction mixture was stirred at RT for 4 h under N2 atmosphere. After completion of reaction, the solvent was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel to give the desired probe with a amide linkage.
To a solution of fluorophore with a terminal —NH2 group (1.0 eq) in CH2Cl2 was added a stock solution of phenyl chloroformate (1 M in CH2Cl2) and DIPEA (1 M in CH2Cl2) at 0° C. The reaction mixture was stirred at RT for 2 h. The reaction was quenched with water and extracted with CH2Cl2. The organic layer was washed with water and dried over Na2SO4. The organic layer was filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel to afford the desired phenyl carbamate. The obtained phenyl carbamate (1.00 eq) and DBU (1.00 eq.) was added to a solute on of the benzyl alcohol 41 (1.00 eq.) in acetone. The mixture was stirred at RT for 1 h and then the solvent was removed under reduced pressure. The crude residue was purified by flash column chromatography on silica gel to produce the desired probe with a carbamate linkage.
To a solution of fluorophore with a terminal-OH group (1.0 eq.) in toluene was added the benzyl bromide 42 (1.2 eq.) and Ag2O (1.5 eq.). The reaction mixture was heated at 120° C. for 2 to 12 h under N2 atmosphere. After completion of reaction, the solvent was removed under reduced eq.) in acetone. The mixture was stirred at RT for 1 h and then the solvent was removed under reduced pressure and the crude residue was purified by flash column chromatography on silica gel to give the desired probe with an ether linkage.
To a solution of N-Boc-protected probe (10 mg) in anhydrous CH2Cl2 (1 mL) was added dropwise a 30% solution of TFA in CH2Cl2 (0.6 mL) at −20° C. and was stirred overnight at −20° C. The saturated aqueous NaHCO3 solution was added to the reaction until pH 8 and the mixture was extracted with CH2Cl2. The combined organic layers were washed with water and dried over Na2SO4. The crude residue was purified by flash column chromatography on silica gel to produce the desired product.
Synthesis of rhodamine fluorophore 18. Reagents and conditions: (a) MOMCl, K2CO3·DMF, rt; (b) NaOH, THE/WATER, reflux, 13% for 2 steps; (c) Tf2O, pyridine, CH2Cl2, 0° C. to rt, 82%; (d) Diethylamine, Pd(OAc)2, CS2CO3, BINAP, toluene, reflux, 76%; (e) TFA, CH2Cl2, rt, 90%; (f) N-Phenyl-bis-(trifluoromethanesulfonimide), K2CO3, CH3CN, 0° C. to rt, 49%; (g) Benzophenone Imine, Pd(OAc)2, Cs2CO3, BINAP, toluene, reflux; (h) aq. 1N—HCl, THF, rt, 89% for 2 steps
To a solution of fluorescein (10 g, 30 mmol) and K2CO3 (10.4 g, 75 mmol) in DMF (150 mL) was added chloromethyl methyl ether (7.7 g, 90 mmol) at RT for 12 h under nitrogen atmosphere. The mixture was extracted with EtOAc and washed with 1N HCl solution, water and brine. The organic layer was dried with Na2SO4 and concentrated under reduced pressure to provide a solid. The obtained solid was dissolved in a mixture of THF (150 mL) and aqueous NaOH solution (3 M, 0 mL). The mixture was heated to reflux for 2 h and cooled to RT. The residue was acidified with aqueous 1N HCl to pH 2 and then extracted with EtOAc. The organic layer was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (EtOAc:Hexane=1:2) to afford the compound 13 (1.47 g, 13%).
1H NMR (400 MHZ, DMSO-d6) δ 8.05 (d, J=6.9 Hz, 1H), 7.75-7.83 (m, 2H), 7.71 (d, J=2.3 Hz, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.25 (dd, J=8.9, 2.5 Hz, 1H), 7.06 (d, J=1.8 Hz, 2H), 6.84 (dd, J=8.9, 2.5 Hz, 1H), 6.74-6.77 (m, 1H), 6.62 (s, 1H), 5.27 (d, J=1.8 Hz, 2H), 3.38 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 168.36, 158.66, 152.05, 151.24, 150.99, 149.67, 136.05, 130.59, 129.18, 125.41, 125.05, 124.96, 124.15, 119.76, 117.42, 113.97, 111.42, 110.75, 103.30, 102.29, 93.94, 80.81, 55.83; HRMS (ESI+): m/z Calcd for C22H16O6 [M]+: 376.0947, Found: 376.0949.
To a solution of compound 13 (100 mg, 0.266 mmol) and pyridine (85 mg, 1.064 mmol) in anhydrous CH2Cl2 (5 mL) was added dropwise trifluoromethanesulfonic anhydride (153 mg, 0.532 mmol) at 0° C. under nitrogen atmosphere. The mixture was stirred at RT for 3 h under nitrogen atmosphere and then was quenched with water. The residue was extracted with CH2Cl2 and washed with 1N HCl, water and brine. The organic layer was dried with Na2SO4 and concentrated. The product was isolated by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to yield the compound 14 (110 mg, 82%).
1H NMR (400 MHZ, CDCl3) δ 8.05 (d, J=7.8 Hz, 1H), 7.68 (dtd, J=19.4, 7.4, 1.1 Hz, 2H), 7.25 (d, J=2.3 Hz, 1H), 7.18 (d, J=7.3 Hz, 1H), 7.00 (d, J=2.3 Hz, 1H), 6.96 (dd, J=8.9, 2.5 Hz, 1H), 6.90 (d, J=8.7 Hz, 1H), 6.78 (dd, J=9.1, 2.3 Hz, 1H), 6.73 (d, J=8.7 Hz, 1H), 5.21 (s, 2H), 3.48 (s, 3H); 13C NMR (100 MHZ, CDCl3) δ 168.8, 159.1, 152.2, 151.9, 149.8, 135.2, 130.0, 129.7, 128.8, 126.1, 125.1, 123.8, 119.7, 118.5, 116.4, 113.8, 111.6, 110.1, 103.5, 94.2, 81.4, 55.8; HRMS (ESI+): m/z Calcd for C23H15F3O6S [M]+: 508.0440, Found: 508.0438.
An oven-dried round bottom flask was charged with Pd(OAc)2 (59 mg, 0.256 mmol), Cs2CO3 (1.18 g, 3.584 mmol) and BINAP (244 mg, 0.384 mmol) and was flushed with nitrogen gas for 10 min. A solution of compound 14 (1.3 g, 2.56 mmol) and diethylamine (1.91 g, 25.6 mmol) in anhydrous toluene (83 mL) was added, stirred under nitrogen atmosphere at RT for 20 min and heated at 100° C. for 20 h. After completion of the reaction, the reaction mixture was cool down to RT, filtered through a pad of Celite and washed with CH2Cl2. The filtrate was concentrated and purified by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to afford the compound 15 (1.1 g, 76%).
1H NMR (400 MHZ, DMSO-d6) δ 7.98 (d, J=7.8 Hz, 1H), 7.78 (dd, J=7.3, 6.4 Hz, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.27 (d, J=7.8 Hz, 1H), 6.96 (d, J=2.3 Hz, 1H), 6.75 (dd, J=8.9, 2.5 Hz, 1H), 6.64 (d, J=8.7 Hz, 1H), 6.45 (s, 3H), 5.24 (s, 2H), 3.41 (m, 4H), 3.37 (s, 3H), 1.08 (t, J=6.9 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 168.78, 158.30, 152.38, 152.22, 149.26, 135.57, 130.10, 129.05, 128.68, 124.60, 124.10, 112.77, 112.52, 108.70, 103.26, 96.88, 93.92, 83.43, 55.80, 54.94, 43.81, 12.33; HRMS (ESI+): m/z Calcd for C26H25NO5 [M]+: 431.1733, Found: 431.1738.
To a solution of compound 15 (100 mg, 0.23 mmol) in anhydrous CH2Cl2 (1 mL) was added dropwise the solution of trifluoroacetic acid (1 mL) at 0° C. The reaction mixture was stirred at 0° C. for 30 min and then warmed to RT followed by stirring of 1 h. After completion of reaction, the mixture was quenched with aqueous NaHCO3 to pH 7 and extracted with EtOAc, washed with water. The organic layer was dried with Na2SO4. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=5:1) to produce 16 (80 mg, 90%).
1H NMR (400 MHZ, CDCl3) δ 8.09 (d, J=8.7 Hz, 1H), 7.60-7.53 (m, 2H), 7.11 (d, J=8.7 Hz, 1H), 6.93-6.83 (m, 2H), 6.74 (d, J=2.3 Hz, 1H), 6.62-6.53 (m, 3H); 13C NMR (100 MHZ, CDCl3) δ 167.3, 166.5, 161.9, 159.0, 157.5, 156.7, 133.7, 132.5, 132.1, 131.2, 131.1, 130.9, 130.2, 130.1, 117.0, 116.5, 115.6, 115.2, 102.0, 96.0, 46.2, 11.6; HRMS (ESI+): m/z Calcd for C24H21NO4 [M]+: 387.1471, Found: 387.1472.
To a solution of compound 16 (200 mg, 0.516 mmol) in MeCN (20 mL) was added phenyl triflimide (372 mg, 1.032 mmol) and K2CO3 (287 mg, 2.064 mmol). The reaction mixture was stirred at RT for 12 h. After completion of the reaction, the solvent was removed under reduced pressure and the residue was extracted with CH2Cl2 and water. The organic layer was washed with brine and dried over Na2SO4. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:8) to produce 17 (130 mg, 49%).
1H NMR (400 MHZ, CDCl3) δ 8.03 (d, J=7.8 Hz, 1H), 7.73-7.61 (m, 2H), 7.23-7.17 (m, 2H), 6.92 (dd, J=8.7, 2.3 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 6.58 (d, J=48.5 Hz, 3H), 3.38 (d, J=6.9 Hz, 4H), 1.19 (t, J=7.1 Hz, 6H); 13C NMR (100 MHZ, CDCl3) δ 169.16, 152.60, 149.89, 135.06, 130.97, 130.01, 128.80, 126.92, 125.12, 124.00, 120.28, 119.78, 117.65, 116.11, 110.31, 109.07, 104.42, 97.59, 82.79, 44.53, 12.45; HRMS (ESI+): m/z Calcd for C25H20FNO6S [M+H]+: 519.0963, Found 520.1035.
An oven-dried round bottom flask was charged with Pd(OAc)2 (16 mg, 0.023 mmol), Cs2CO3 (227 mg, 0.69 mmol) and BINAP (24 mg, 0.037 mmol) and flushed with nitrogen gas for 10 min. A solution of compound 17 (120 mg, 0.23 mmol) and benzophenone imine (53 mg, 0.276 mmol) in anhydrous toluene (6 mL) was added and stirred under nitrogen atmosphere at RT for 20 min and then heated at 100° C. for 4 h. The mixture was filtered through a pad of Celite and washed with CH2Cl2. The filtrate was concentrated and re-dissolved in THF (2.4 mL). The mixture was added dropwise 1N HCl (0.8 mL). The reaction mixture was stirred to RT for 1 h. The reaction was neutralized with saturated aqueous NaHCO3 to pH 7-8. The residue was extracted with EtOAc and washed with water. The organic layer was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (CH2Cl2:MeOH=10:1) to afford 18 (Rhoda-NH2, 78 mg, 89%).
Synthesis of LAP-responsive activatable fluorescent probes via direct amide linkage. Reagents and conditions: (a) Boc-Leu-OH, EEDQ, CH2Cl2, rt, 22=92%, 23=67%, 24=56%, 25=81%; (b) 30% TFA in CH2Cl2, −20° C., 1=30%, 2=80%, 7=52%, 8=29%.
Compound 22 was synthesized by the amide coupling of 18 (Rhoda-NH2, 30 mg, 0.078 mmol) with Boc-Leu-OH (15 mg, 0.065 mmol) and EEDQ (19 mg, 0.078 mmol) according to the general procedure A. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=30:1) to produce 22 in 92% of yield (37 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.24 (s, 1H), 7.98 (d, J=7.8 Hz, 1H), 7.85 (s, 1H), 7.78 (t, J=7.3 Hz, 1H), 7.70 (t, J=7.5 Hz, 1H), 7.26 (d, J=7.8 Hz, 1H), 7.11 (dd, J=13.7, 8.2 Hz, 2H), 6.68 (d, J=8.7 Hz, 1H), 6.47 (d, J=14.2 Hz, 3H), 4.11 (d, J=5.0 Hz, 1H), 3.36-3.32 (m, 4H), 1.63-1.51 (m, 2H), 1.43-1.30 (m, 1H), 1.37 (s, 9H), 1.08 (t, J=6.9 Hz, 6H), 0.88 (t, J=4.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.5, 168.8, 155.5, 152.5, 152.2, 151.3, 149.3, 141.0, 135.5, 130.0, 128.6, 128.4, 126.3, 124.6, 124.0, 114.9, 113.6, 108.6, 106.3, 104.5, 97.0, 83.3, 78.1, 53.6, 43.8, 28.2, 24.3, 22.9, 21.5, 12.3; HRMS (ESI+): m/z Calcd for C35H41N3O6 [M+H]+: 600.3029, Found: 600.3069.
Compound 23 was synthesized by the amide coupling of 19 (Rhodo-NH2, 90 mg, 0.259 mmol) with Boc-Leu-OH (51 mg, 0.216 mmol) and EEDQ (65 mg, 0.259 mmol) according to the general procedure A. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:4) to produce 23 in 67% of yield (80 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.28 (s, 1H), 8.01 (d, J=7.8 Hz, 1H), 7.89 (d, J=3.7 Hz, 1H), 7.79 (t, J=7.1 Hz, 1H), 7.72 (t, J=7.5 Hz, 1H), 7.26 (d, J=7.1 Hz, 1H), 7.18 (d, J=8.2 Hz, 1H), 7.10 (d, J=8.2 Hz, 1H), 6.97 (d, J=2.3 Hz, 1H), 6.78-6.63 (m, 3H), 4.14-4.01 (m, 1H), 3.81 (s, 3H), 1.62-1.65 (m, 1H), 1.58-1.46 (m, 1H), 1.37 (s, 9H), 1.3 (s, 1H), 0.92-0.82 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.54, 168.68, 161.10, 155.51, 152.57, 151.79, 150.82, 141.16, 135.76, 130.24, 128.95, 128.48, 125.76, 124.76, 123.97, 115.40, 113.15, 112.13, 110.72, 106.24, 100.85, 82.13, 78.08, 55.69, 53.65, 28.19, 24.34, 22.93, 21.52; HRMS (ESI+): m/z Calcd for C32H34N2O7 [M+H]+: 559.2400, Found: 559.2447.
Compound 24 was synthesized by the amide coupling of 20 (Red Rhoda-NH2, 50 mg, 0.134 mmol) with Boc-Leu-OH (26 mg, 0.112 mmol) and EEDQ (34 mg, 0.134 mmol) according to the general procedure A. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:4) to produce 24 in 56% of yield (34 mg).
1H NMR (400 MHZ, DMSO-d6) δ 7.73 (q, J=1.8 Hz, 1H), 7.43 (d, J=7.3 Hz, 1H), 7.35 (t, J=7.3 Hz, 1H), 7.23 (t, J=7.3 Hz, 1H), 7.13 (dq, J=8.6, 1.9 Hz, 1H), 6.81 (d, J=8.2 Hz, 1H), 6.76 (d, J=7.8 Hz, 1H), 6.66 (d, J=8.7 Hz, 1H), 6.40 (td, J=8.1, 2.4 Hz, 2H), 5.21 (s, 2H), 3.34 (d, J=7.3 Hz, 5H), 2.07 (s, 2H), 1.79-1.69 (m, 1H), 1.50-1.43 (m, 1H), 1.36-1.29 (m, 1H), 1.08 (t, J=6.9 Hz, 6H), 0.88 (dd, J=10.1, 6.9 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 175.2, 151.0, 150.0, 148.2, 145.3, 139.4, 138.7, 129.4, 128.9, 128.1, 127.8, 123.1, 121.0, 120.0, 114.4, 111.4, 108.1, 105.8, 96.8, 82.7, 71.4, 54.0, 44.0, 43.6, 30.6, 24.2, 23.1, 21.8, 12.4; HRMS (ESI+): m/z Calcd for C35H43N3O5 [M+H]+: 586.3236, Found: 586.3278.
Compound 25 was synthesized by the amide coupling of 21 (Red Rhodo-NH2, 50 mg, 0.151 mmol) with Boc-Leu-OH (30 mg, 0.126 mmol) and EEDQ (38 mg, 0.151 mmol) according to the general procedure A. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:3) to produce 25 in 81% of yield (56 mg).
1H-NMR (400 MHZ, DMSO-d6) δ 10.09 (d, J=11.0 Hz, 1H), 7.74-7.72 (m, 1H), 7.45 (d, J=7.3 Hz, 1H), 7.36 (t, J=3.9 Hz, 1H), 7.23 (t, J=4.1 Hz, 1H), 7.12 (d, J=11.4 Hz, 1H), 6.90-6.81 (m, 3H), 6.74 (d, J=7.8 Hz, 1H), 6.66 (dd, J=8.7, 2.7 Hz, 1H), 5.28 (s, 2H), 4.18-4.06 (m, 1H), 3.77 (s, 3H), 1.68-1.59 (m, 1H), 1.55-1.47 (m, 1H), 1.44-1.39 (m, 1H), 1.36 (s, 9H), 0.91-0.86 (m, 6H); 13C-NMR (100 MHZ, DMSO-d6) δ 172.3, 154.6, 150.8, 148.8, 144.5, 139.5, 138.1, 131.5, 128.9, 128.1, 126.7, 124.6, 121.2, 120.2, 113.7, 111.6, 98.7, 95.6, 94.5, 79.1, 66.9, 62.2, 48.6, 41.4, 32.8, 30.5, 28.2, 22.8, 21.6; HRMS (ESI+): m/z Calcd for C32H36N2O6 [M+H]+: 545.2607, Found: 545.2659.
Probe 1 (Rhoda-P1) was synthesized from 22 (35 mg, 0.078 mmol) according to the general procedure D. The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=10:1) to give probe 1 (Rhoda-P1) in 30% of yield (9 mg).
1H NMR (400 MHz, DMSOd6) δ 7.98 (d, J=7.3 Hz, 1H), 7.88 (s, 1H), 7.78 (t, J=7.5 Hz, 1H), 7.70 (t, J=7.3 Hz, 1H), 7.26 (d, J=7.8 Hz, 1H), 7.18 (dd, J=8.5, 1.6 Hz, 1H), 6.66 (d, J=8.7 Hz, 1H), 6.47 (d, J=12.8 Hz, 3H), 3.37-3.32 (m, 5H), 1.79-1.69 (m, 1H), 1.49-1.42 (m, 1H), 1.36-1.29 (m, 1H), 1.08 (t, J=6.9 Hz, 6H), 0.88 (dd, J=10.3, 6.6 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 176.0, 169.2, 152.9, 152.7, 151.7, 149.7, 141.4, 136.0, 130.4, 129.0, 128.7, 126.7, 125.0, 124.5, 115.4, 114.0, 109.0, 106.6, 104.9, 97.4, 83.8, 54.6, 44.4, 44.2, 24.6, 23.6, 22.3, 12.8; HRMS (ESI+): m/z Calcd for C30H33N3O4 [M+H]+: 500.2505, Found: 500.2542.
Probe 2 (Rhodo-P1) was synthesized from 23 (30 mg, 0.54 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.8 mL). The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=10:1) to give 20 mg of probe 2 (Rhodo-P1) in 80% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 8.01 (d, J=7.3 Hz, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.79 (t, J=7.5 Hz, 1H), 7.72 (t, J=7.1 Hz, 1H), 7.31-7.18 (m, 2H), 6.97 (d, J=2.3 Hz, 1H), 6.77-6.64 (m, 3H), 3.81 (s, 3H), 3.38 (s, 1H) 3.35 (s, 2H), 1.80-1.67 (m, 1H), 1.52-1.42 (m, 1H), 1.40-1.29 (m, 1H), 0.88 (dd, J=9.6, 6.9 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 175.31, 168.66, 161.10, 152.53, 151.80, 150.83, 141.09, 135.74, 130.23, 128.95, 125.78, 124.76, 123.96, 115.44, 113.10, 112.10, 110.73, 106.18, 100.83, 82.16, 55.68, 54.04, 43.78, 24.18, 23.14, 21.84; HRMS (ESI+): m/z Calcd for C27H26N2O5 [M+H]+: 459.1875, Found: 459.1917.
Probe 7 (Red Rhoda-P1) was synthesized of compound 24 (30 mg, 0.055 mmol) according to the general procedure D. The residue was purified by flash column chromatography on silica gel (CH2Cl2:EA=5:1) to give probe 7 (Red Rhoda-P1) in 52% of yield (14 mg).
1H NMR (400 MHZ, DMSO-d6) δ 7.74 (q, J=2.1 Hz, 1H), 7.44 (d, J=7.3 Hz, 1H), 7.35 (t, J=7.3 Hz, 1H), 7.23 (t, J=7.3 Hz, 1H), 7.13 (qd, J=4.2, 2.2 Hz, 1H), 6.81 (d, J=8.7 Hz, 1H), 6.76 (d, J=7.3 Hz, 1H), 6.66 (d, J=8.7 Hz, 1H), 6.40 (td, J=8.7, 2.3 Hz, 2H), 5.21 (s, 2H), 3.35 (m, 5H), 2.53 (s, 2H), 1.79-1.69 (m, 1H), 1.49-1.42 (m, 1H), 1.35-1.28 (m, 1H), 1.08 (t, J=6.9 Hz, 6H), 0.88 (dd, J=10.1, 6.9 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 175.3, 151.0, 150.0, 148.2, 145.4, 139.5, 138.7, 129.5, 129.0, 128.1, 127.8, 123.2, 121.1, 120.0, 114.4, 111.3, 108.1, 105.8, 96.8, 82.7, 71.4, 54.1, 44.0, 43.7, 40.4, 24.2, 23.2, 21.9, 12.4; HRMS (ESI+): m/z Calcd for C30H35N3O3 [M+H]+: 486.2712, Found: 486.2750.
Probe 8 (Red Rhodo-P1) was synthesized from compound 25 (20 mg, 0.37 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.2 mL). The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=10:1) to give 5 mg of probe 8 (Red Rhodo-P1) in 29% of yield.
1H-NMR (400 MHZ, DMSO-d6) δ 7.75 (q, J=1.8 Hz, 1H), 7.45 (d, J=7.3 Hz, 1H), 7.35 (t, J=7.1 Hz, 1H), 7.23 (t, J=7.1 Hz, 1H), 7.16 (dq, J=8.5, 1.8 Hz, 1H), 6.89-6.80 (m, 3H), 6.73 (d, J=7.3 Hz, 1H), 6.66 (dd, J=8.9, 2.5 Hz, 1H), 5.28 (s, 2H), 3.77 (s, 3H), 2.07 (s, 2H), 1.81-1.69 (m, 1H), 1.51-1.43 (m, 1H), 1.36-1.29 (m, 1H), 0.93-0.85 (m, 6H); 13C-NMR (100 MHZ, DMSO-d6) δ 175.3, 159.9, 150.5, 149.6, 145.3, 139.6, 138.3, 129.6, 128.9, 128.2, 128.0, 123.0, 121.2, 119.6, 117.2, 114.9, 111.2, 105.8, 82.4, 71.9, 55.4, 55.3, 54.1, 44.0, 30.6, 24.2, 23.1, 21.8; HRMS (ESI+): m/z Calcd for C27H28N2O4 [M+H]+: 445.2083, Found: 445.2128.
Synthesis of LAP-responsive fluorescent probes via carbamate linkage with a spacer. Reagents and conditions: (a) Phenyl chloroformate, DIPEA, CH2Cl2, rt, 26=57%, 27=69%, 28=64%, 29=85%; (b) 41, DBU, acetone, rt, 30=79%, 31=73%, 32=47%, 33=60%; (c) 30% TFA in CH2Cl2, −20° C., 3=47%, 4=78%, 9=47%, 10=48%.
Compound 26 was synthesized by carbamate of 18 (Rhoda-NH2, 45 mg, 0.12 mmol) with 1 M Phenyl chloroformate (0.24 mL, 0.24 mmol) and 1 M DIPEA (0.24 mL, 0.24 mmol) in CH2Cl2 according to the general procedure B. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:4) to produce 26 in 57% of yield (34 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.53 (s, 1H), 7.99 (d, J=7.3 Hz, 1H), 7.80-7.76 (m, 1H), 7.71 (t, J=7.5 Hz, 1H), 7.54 (d, J=1.8 Hz, 1H), 7.43 (t, J=7.8 Hz, 2H), 7.28-7.26 (m, 2H), 7.24-7.22 (m, 2H), 7.19 (dd, J=8.7, 1.8 Hz, 1H), 6.70 (d, J=8.7 Hz, 1H), 6.48 (d, J=16.0 Hz, 3H), 3.37-3.32 (m, 4H), 1.08 (t, J=7.1 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 174.9, 169.0, 153.5, 152.6, 152.4, 151.6, 149.4, 141.5, 139.1, 135.7, 131.0, 130.2, 129.2, 128.8, 128.7, 126.5, 124.8, 124.2, 119.2, 113.2, 105.2, 104.7, 97.2, 83.6, 54.1, 44.2, 44.0, 24.4, 23.4, 22.0, 12.5.
Compound 27 was synthesized from 19 (Rhodo-NH2, 100 mg, 0.215 mmol) according to the general procedure B, using 1 M phenyl chloroformate (0.43 mL, 0.43 mmol) and 1 M DIPEA (0.43 mL, 0.43 mmol) in CH2Cl2. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to give 68 mg of 27 in 69% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 10.57 (s, 1H), 8.01 (d, J=7.3 Hz, 1H), 7.80 (t, J=7.3, 1H), 7.72 (t, J=7.1 Hz, 1H), 7.60 (s, 1H), 7.47-7.39 (m, 2H), 7.28 (d, J=7.3 Hz, 2H), 7.24 (d, J=7.8 Hz, 3H), 6.99 (d, J=2.3 Hz, 1H), 6.76 (d, J=8.7 Hz, 1H), 6.71 (dd, J=8.9, 2.5 Hz, 1H), 6.68 (s, 1H), 3.80 (s, 3H); 13C NMR (100 MHZ, DMSO-d6) δ 168.66, 161.11, 152.52, 151.73, 151.63, 150.99, 150.30, 140.99, 135.75, 130.24, 129.49, 129.36, 128.94, 128.74, 125.76, 125.65, 124.78, 123.96, 121.93, 118.78, 115.20, 113.03, 112.15, 110.75, 105.39, 100.86, 82.08, 55.69; HRMS (ESI+): m/z Calcd for C28H19NO6 [M+H]+: 466.1246, Found: 466.1284.
Compound 28 was synthesized from 20 (Red Rhoda-NH2, 40 mg, 0.107 mmol) with 1 M phenyl chloroformate (0.21 mmol, 0.21 mL) and 1 M DIPEA (0.21 mmol, 0.21 mL) in CH2Cl2 according to the general procedure B. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to produce 28 in 64% of yield (34 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.39 (s, 1H), 7.44-7.41 (m, 4H), 7.35 (t, J=7.3 Hz, 1H), 7.25 (dd, J=16.7, 7.5 Hz, 4H), 7.15 (dd, J=8.7, 1.8 Hz, 1H), 6.85 (d, J=8.7 Hz, 1H), 6.77 (d, J=7.3 Hz, 1H), 6.67 (d, J=8.7 Hz, 1H), 6.42-6.39 (m, 2H), 5.21 (s, 2H), 3.31 (t, J=7.1 Hz, 3H), 1.07 (t, J=7.1 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 152.3, 151.6, 151.0, 150.8, 148.8, 146.0, 139.9, 139.3, 130.1, 130.0, 128.8, 128.5, 126.2, 123.8, 122.6, 121.7, 120.6, 114.3, 112.0, 108.8, 105.7, 97.4, 83.2, 72.1, 55.5, 44.3, 13.0; HRMS (ESI+): m/z Calcd for C31H28N2O4 [M+H]+: 493.2083, Found: 493.2129.
Compound 29 was synthesized from 21 (Red Rhodo-NH2, 20 mg, 0.06 mmol) according to the general procedure B, using 1 M phenyl chloroformate (0.12 mmol, 0.12 mL) and 1 M DIPEA (0.12 mmol, 0.12 mL) in CH2Cl2. The residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to give 23 mg of compound 29 in 85% of yield.
1H-NMR (400 MHZ, DMSO-d6) δ 10.43 (s, 1H), 7.44 (q, J=8.1 Hz, 4H), 7.36 (t, J=7.3 Hz, 1H), 7.28-7.22 (m, 4H), 7.18 (dd, J=8.7, 1.8 Hz, 1H), 6.92 (d, J=8.7 Hz, 1H), 6.87-6.84 (m, 2H), 6.76 (d, J=7.3 Hz, 1H), 6.66 (dd, J=8.7, 2.7 Hz, 1H), 5.29 (s, 2H), 3.77 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 159.9, 151.6, 150.4, 150.4, 149.8, 145.4, 139.5, 138.3, 129.7, 129.5, 129.3, 128.3, 128.0, 125.6, 123.0, 121.9, 121.2, 119.6, 117.2, 114.2, 111.3, 105.0, 100.2, 82.4, 72.0, 55.5; HRMS (ESI+): m/z Calcd for C28H21NO5 [M+H]+: 452.1453, Found: 452.1496.
Compound 30 was synthesized from 26 (28 mg, 0.055 mmol) with 41 (19 mg, 0.055 mmol) and DBU (9 mg, 0.055 mmol) according to the general procedure B. The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=20:1) to give compound 30 in 79% of yield (31 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.05 (s, 1H), 10.01 (s, 1H), 7.97 (d, J=7.3 Hz, 1H), 7.77 (td, J=7.5, 1.1 Hz, 1H), 7.72-7.68 (m, 1H), 7.61 (d, J=8.2 Hz, 2H), 7.52 (d, J=1.8 Hz, 1H), 7.36 (d, J=8.2 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H), 7.13 (dd, J=8.7, 2.3 Hz, 1H), 7.03 (d, J=8.2 Hz, 1H), 6.64 (d, J=8.7 Hz, 1H), 6.47 (d, J=17.8 Hz, 3H), 5.10 (s, 2H), 4.11 (dd, J=13.7, 9.1 Hz, 1H), 3.35 (t, J=7.1 Hz, 4H), 1.62 (td, J=13.4, 6.7 Hz, 1H), 1.55-1.48 (m, 1H), 1.41 (dd, J=8.5, 5.3 Hz, 1H), 1.36 (s, 9H), 1.08 (t, J=7.1 Hz, 6H), 0.88 (q, J=3.2 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 171.9, 168.8, 158.2, 155.5, 153.3, 152.4, 152.2, 151.4, 149.2, 141.2, 139.0, 135.5, 130.9, 130.0, 129.0, 128.6, 128.5, 126.3, 124.5, 124.0, 119.1, 114.0, 112.9, 108.6, 105.0, 104.5, 97.0, 83.4, 78.0, 65.8, 53.5, 43.7, 40.6, 24.3, 23.0, 21.5, 12.3; HRMS (ESI+): m/z Calcd for C43H48N4O8 [M+H]+: 749.3506, Found: 749.3558.
Compound 31 was synthesized by the reaction of intermediate 27 (50 mg, 0.11 mmol) with benzyl alcohol 41 (37 mg, 0.11 mmol) and DBU (17 mg, 0.11 mmol) according to the general procedure B. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=2:3) to produce 31 in 73% of yield (57 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.09 (s, 1H), 10.01 (s, 1H), 8.00 (d, J=7.8 Hz, 1H), 7.78 (t, J=7.5 Hz, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.62 (d, J=8.2 Hz, 2H), 7.57 (d, J=1.4 Hz, 1H), 7.36 (d, J=8.2 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H), 7.16 (dd, J=8.7, 1.8 Hz, 1H), 7.03 (d, J=7.8 Hz, 1H), 6.97 (d, J=2.3 Hz, 1H), 6.70 (q, J=4.1 Hz, 2H), 6.65 (d, J=8.7 Hz, 1H), 5.10 (s, 2H), 4.11 (m, 1H), 3.80 (s, 3H), 1.62 (q, J=6.4 Hz, 1H), 1.57-1.45 (m, 1H), 1.45-1.25 (m, 10H), 0.88 (q, J=3.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 171.89, 168.66, 161.09, 155.46, 153.29, 152.50, 151.78, 150.98, 141.47, 139.04, 135.73, 130.87, 130.22, 129.03, 128.57, 125.80, 124.76, 123.97, 119.09, 114.45, 112.44, 112.07, 110.79, 105.00, 100.85, 82.18, 78.00, 65.86, 55.68, 53.50, 40.60, 28.19, 24.34, 22.97, 21.53; HRMS (ESI+): m/z Calcd for C40H41N3O9 [M+H]+: 708.2876, Found: 708.2926.
Compound 32 was synthesized from intermediate 28 (30 mg, 0.061 mmol) with benzyl alcohol 41 (21 mg, 0.061 mmol) and DBU (10 mg, 0.061 mmol) according to the general procedure B. The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=30:1) to give 32 in 47% of yield (21 mg).
1H NMR (400 MHZ, DMSO-d6) δ 8.08 (s, 1H), 7.71 (d, J=7.3 Hz, 2H), 7.56-7.48 (m, 1H), 7.40 (d, J=7.8 Hz, 2H), 7.37-7.30 (m, 1H), 7.09 (dd, J=9.6, 2.7 Hz, 1H), 6.81-6.72 (m, 2H), 6.58 (d, J=8.7 Hz, 1H), 6.48 (d, J=8.7 Hz, 1H), 6.30 (q, J=2.4 Hz, 2H), 5.29 (s, 2H), 5.11 (s, 2H), 3.36 (d, J=14.6 Hz, 4H), 3.16 (d, J=5.5 Hz, 1H), 1.60-1.39 (m, 1H), 1.39-1.27 (m, 1H), 1.25 (m, 1H), 1.22 (s, 9H), 1.07 (t, J=6.9 Hz, 6H), 0.91-0.78 (m, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 172.9, 168.8, 155.5, 153.3, 152.4, 152.2, 151.4, 147.2, 140.5, 139.2, 132.5, 130.9, 130.0, 129.0, 128.6, 128.5, 125.3, 124.5, 122.3, 119.8, 116.3, 112.4, 108.6, 105.1, 104.8, 97.1, 83.4, 81.1, 66.0, 54.0, 44.2, 42.6, 28.5, 24.5, 23.4, 21.9, 12.5; HRMS (ESI+): m/z Calcd for C43H50N4O7 [M+H]+: 735.3713, Found: 735.3771.
Compound 33 was synthesized by the reaction of intermediate 29 (35 mg, 0.078 mmol) with benzyl alcohol 41 (26 mg, 0.078 mmol) and DBU (12 mg, 0.078 mmol) according to the general procedure B. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:EA=20:1) to produce compound 33 in 60% of yield (32 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.03 (s, 1H), 9.94 (s, 1H), 7.62 (d, J=8.7 Hz, 2H), 7.45 (d, J=6.9 Hz, 2H), 7.35 (t, J=7.1 Hz, 3H), 7.22 (t, J=7.3 Hz, 1H), 7.12 (dd, J=8.7, 1.8 Hz, 1H), 7.04 (d, J=7.8 Hz, 1H), 6.76-6.73 (m, 3H), 6.60 (dd, J=8.7, 2.3 Hz, 1H), 6.54 (d, J=8.2 Hz, 1H), 5.27 (s, 2H), 5.09 (s, 2H), 4.14-4.08 (m, 1H), 3.76 (s, 3H), 1.62 (q, J=6.6 Hz, 1H), 1.55-1.48 (m, 1H), 1.42 (dd, J=8.5, 5.3 Hz, 1H), 1.36 (s, 9H), 0.88 (q, J=3.1 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 159.9, 155.5, 153.3, 150.7, 149.8, 145.5, 140.0, 139.0, 138.3, 129.7, 129.3, 129.0, 128.3, 128.1, 123.0, 121.0, 119.1, 117.6, 110.7, 100.1, 82.8, 78.0, 71.2, 65.7, 56.8, 55.4, 53.5, 44.4, 40.6, 28.2, 24.3, 23.0, 21.5; HRMS (ESI+): m/z Calcd for C40H43N3O8 [M+H]+: 694.3084, Found: 694.3133.
Probe 3 (Rhoda-P2) was synthesized from 30 (30 mg, 0.043 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.8 mL). The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=5:1) to give 12 mg of probe 3 Rhoda-P2) in 47% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 10.01 (s, 1H), 7.94 (d, J=7.3 Hz, 1H), 7.73 (t, J=7.3 Hz, 1H), 7.67 (d, J=7.3 Hz, 1H), 7.61 (d, J=8.7 Hz, 2H), 7.48 (s, 1H), 7.32 (d, J=8.7 Hz, 2H), 7.21 (d, J=7.8 Hz, 1H), 7.09 (dd, J=8.7, 1.8 Hz, 1H), 6.60 (d, J=8.7 Hz, 1H), 6.43 (d, J=16.9 Hz, 3H), 5.06 (s, 2H), 3.31 (m, 4H), 2.45 (t, J=1.6 Hz, 1H), 1.73-1.66 (m, 1H), 1.46-1.39 (m, 1H), 1.31-1.24 (m, 1H), 1.04 (t, J=7.1 Hz, 6H), 0.84 (dd, J=10.1, 6.4 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 172.54, 168.68, 161.10, 155.51, 152.57, 151.79, 150.82, 141.16, 135.76, 130.24, 128.95, 128.48, 125.76, 124.76, 123.97, 115.40, 113.15, 112.13, 110.72, 106.24, 100.85, 82.13, 78.08, 55.69, 53.65, 28.19, 24.34, 22.93, 21.52; HRMS (ESI+): m/z Calcd for C38H40N4O6 [M+H]+: 649.2981, Found: 649.3028.
Probe 4 (Rhodo-P2) was synthesized of compound 31 (20 mg, 0.028 mmol) according to the general procedure D. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:EA=10:1) to give probe 4 (Rhodo-P2) in 78% of yield (22 mg).
1H-NMR (400 MHZ, DMSO-d6) δ 10.08 (s, 1H), 8.00 (d, J=7.3 Hz, 1H), 7.78 (t, J=7.1 Hz, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.65 (d, J=8.7 Hz, 2H), 7.58 (s, 1H), 7.36 (d, J=8.7 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H), 7.16 (d, J=8.7 Hz, 1H), 6.97 (d, J=2.3 Hz, 1H), 6.75-6.62 (m, 3H), 5.10 (s, 2H), 3.80 (s, 3H), 1.80-1.67 (m, 1H), 1.51-1.41 (m, 1H), 1.36-1.26 (m, 1H), 0.88 (dd, J=10.1, 6.4 Hz, 6H); 13CNMR (100 MHZ, DMSO-d6) δ 174.90, 168.65, 161.08, 153.29, 152.49, 151.77, 150.97, 138.94, 135.71, 130.78, 130.21, 129.02, 128.93, 128.56, 125.80, 124.75, 123.96, 119.01, 114.43, 112.44. 112.05, 110.79, 104.98, 100.85, 82.17, 65.88, 55.68, 53.93, 44.07, 24.21, 23.20, 21.82; HRMS (ESI+): m/z Calcd for C35H33N3O7 [M+H]+: 608.2352, Found: 608.2404.
Probe 9 (Red Rhoda-P2) was synthesized from compound 32 (15 mg, 0.02 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (0.9 mL). The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=5:1) to give 6 mg of probe 9 (Red Rhoda-P2) in 47% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 9.88 (s, 1H), 7.64 (dd, J=6.9, 1.8 Hz, 2H), 7.43 (d, J=7.3 Hz, 1H), 7.38-7.32 (m, 4H), 7.23 (t, J=7.1 Hz, 1H), 7.09 (dd, J=8.5, 2.1 Hz, 1H), 6.77 (dd, J=12.8, 8.2 Hz, 2H), 6.65 (d, J=8.7 Hz, 1H), 6.42-6.37 (m, 2H), 5.21 (d, J=13.3 Hz, 2H), 5.10 (d, J=12.3 Hz, 2H), 3.34 (d, J=6.9 Hz, 4H), 3.16 (s, 1H), 1.80-1.69 (m, 1H), 1.49-1.42 (m, 1H), 1.34-1.27 (m, 1H), 1.07 (t, J=6.9 Hz, 6H), 0.88 (dd, J=10.5, 6.4 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 175.5, 151.5, 150.7, 148.7, 145.9, 139.5, 139.3, 130.0, 129.7, 129.6, 128.7, 128.3, 123.7, 121.6, 119.5, 119.9, 111.9, 108.6, 105.2, 97.3, 83.2, 66.2, 54.5, 44.6, 44.2, 24.7, 23.8, 22.4, 12.9; HRMS (ESI+): m/z Calcd for C38H42N4O5[M+H]+: 635.3189, Found: 635.3226.
Probe 10 (Red Rhodo-P2) was synthesized of compound 33 (25 mg, 0.042 mmol) according to the general procedure D. The residue was purified by flash column chromatography on silica gel (CH2Cl2:EA=10:1) to give probe 10 (Red Rhodo-P2) in 48% of yield (10 mg).
1H NMR (400 MHZ, DMSO-d6) δ 9.94 (s, 1H), 7.65 (d, J=8.7 Hz, 2H), 7.45 (d, J=7.3 Hz, 2H), 7.37-7.33 (m, 3H), 7.22 (t, J=7.1 Hz, 1H), 7.12 (dd, J=8.5, 2.1 Hz, 1H), 6.87-6.82 (m, 3H), 6.74 (d, J=7.8 Hz, 1H), 6.66 (dd, J=8.7, 2.7 Hz, 1H), 5.27 (s, 2H), 5.09 (s, 2H), 4.10 (d, J=7.3 Hz, 1H), 3.77 (s, 3H), 1.79-1.69 (m, 1H), 1.48-1.42 (m, 1H), 1.34-1.27 (m, 1H), 0.88 (dd, J=10.5, 6.4 Hz, 6H); 13c NMR (100 MHZ, DMSO-d6) δ 175.0, 159.9, 153.3, 150.5, 149.7, 145.4, 140.0, 138.9, 138.3, 130.9, 129.7, 129.2, 129.0, 128.3, 128.0, 123.0, 121.2, 119.0, 117.2, 111.2, 100.2, 82.4, 71.9, 65.8, 55.5, 54.9, 54.0, 48.6, 44.1, 24.2, 23.2, 21.8; HRMS (ESI+): m/z Calcd for C35H35N3O6 [M+H]+: 594.2559, Found: 594.2597.
Synthesis of LAP-responsive fluorescent probes via ether linkage with a spacer. Reagents and conditions: (a) 42, Ag2O, toluene, 120° C., 37=52%, 38=49%, 39=24%, 40=22%; (b) 30% TFA in CH2Cl2, −20° C., 5=58%, 6=42%, 11=59%, 12=48%.
Compound 37 was synthesized by the alkylation of 16 (Rhodo-OH, 50 mg, 0.129 mmol) with benzyl bromide 42 (62 mg, 0.155 mmol) and Ag2O (45 mg, 0.194 mmol) according to the general procedure C. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=20:1) to produce 37 in 52% of yield (47 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.00 (s, 1H), 7.98 (d, J=7.3 Hz, 1H), 7.77 (t, J=7.5 Hz, 1H), 7.70 (t, J=7.5 Hz, 1H), 7.62 (d, J=8.7 Hz, 2H), 7.38 (d, J=8.2 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H), 7.03 (d, J=7.8 Hz, 1H), 6.93 (d, J=2.3 Hz, 1H), 6.73 (dd, J=8.7, 2.3 Hz, 1H), 6.61 (d, J=8.7 Hz, 1H), 6.45 (d, J=1.8 Hz, 3H), 5.09 (s, 2H), 4.11 (dd, J=14.4, 8.0 Hz, 1H), 3.40-3.35 (m, 4H), 1.64-1.58 (m, 1H), 1.55-1.48 (m, 1H), 1.42 (dd, J=8.7, 5.5 Hz, 1H), 1.36 (s, 9H), 1.08 (t, J=7.1 Hz, 6H), 0.88 (q, J=3.4 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 168.7, 159.9, 155.4, 152.4, 152.2, 152.1, 149.2, 138.9, 135.5, 131.1, 130.0, 128.9, 128.6, 128.4, 126.3, 124.5, 124.0, 119.2, 112.2, 111.5, 104.5, 101.6, 96.9, 83.5, 78.0, 69.4, 53.5, 43.8, 40.6, 28.2, 24.3, 22.9, 21.6, 12.3; HRMS (ESI+): m/z Calcd for C42H47N3O7 [M+H]+: 706.3448, Found: 706.3492.
Compound 38 was synthesized by the reaction of 34 (Fluor-OH, 50 mg, 0.144 mmol) with benzyl bromide 42 (69 mg, 0.173 mmol) and Ag2O (51 mg, 0.216 mmol) according to the general procedure C. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:2) to produce 38 in 49% of yield (47 mg).
1H NMR (400 MHZ, DMSO-d6) δ 9.98 (s, 1H), 8.00 (d, J=7.3 Hz, 1H), 7.78 (t, J=7.3 Hz, 1H), 7.71 (t, J=7.5 Hz, 1H), 7.62 (d, J=8.2 Hz, 2H), 7.38 (d, J=8.7 Hz, 2H), 7.26 (d, J=7.3 Hz, 1H), 7.05-6.95 (m, 2H), 6.92 (d, J=2.3 Hz, 1H), 6.78 (dd, J=8.7, 2.3 Hz, 1H), 6.71 (dd, J=8.9, 2.5 Hz, 1H), 6.66 (dd, J=8.9, 2.1 Hz, 2H), 5.11 (s, 2H), 4.10 (t, J=7.1 Hz, 1H), 3.81 (s, 3H), 2.07 (s, 9H), 1.63 (t, J=6.2 Hz, 1H), 1.59-1.46 (m, 1H), 0.88 (q, J=3.2 Hz, 9H); 13C NMR (100 MHZ, DMSO-d6) δ 206.44, 171.80, 168.57, 161.05, 160.08, 152.43, 151.74, 151.66, 138.82, 135.67, 131.10, 130.19, 128.95, 128.40, 125.86, 124.69, 123.94, 119.22, 112.67, 111.98, 111.03, 110.83, 101.65, 100.77, 82.30, 77.99, 69.47, 55.63, 53.52, 37.91, 30.64, 28.16, 24.32, 22.90, 21.54; HRMS (ESI+): m/z Calcd for C39H40N2O8 [M+H]+: 665.2818, Found: 665.2861.
Compound 39 was synthesized by the alkylation of 35 (Red Rhodo-OH, 40 mg, 0.107 mmol) with benzyl bromide 42 (51 mg, 0.128 mmol) and Ag2O (37 mg, 0.161 mmol) according to the general procedure C. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=30:1) to produce 39 in 24% of yield (18 mg).
1H NMR (400 MHZ, DMSO-d6) δ 9.99 (s, 1H), 7.61 (d, J=8.7 Hz, 2H), 7.43 (d, J=7.8 Hz, 1H), 7.37 (d, J=8.2 Hz, 2H), 7.33 (d, J=7.3 Hz, 1H), 7.23 (t, J=7.3 Hz, 1H), 7.02 (d, J=7.8 Hz, 1H), 6.77 (td, J=8.1, 2.9 Hz, 3H), 6.69-6.63 (m, 2H), 6.40 (dd, J=8.7, 2.3 Hz, 1H), 6.34 (d, J=2.3 Hz, 1H), 5.18 (s, 2H), 5.05 (s, 2H), 4.11 (q, J=7.2 Hz, 1H), 3.42-3.37 (m, 4H), 3.29 (m, 1H), 1.66-1.59 (m, 1H), 1.55-1.48 (m, 1H), 1.44-1.40 (m, 1H), 1.36 (s, 9H), 1.07 (t, J=7.1 Hz, 6H), 0.88 (q, J=3.4 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 171.9, 158.7, 155.5, 151.0, 148.1, 145.3, 138.8, 131.4, 129.8, 129.6, 128.4, 128.2, 127.8, 119.2, 117.7, 111.4, 96.7, 82.7, 78.0, 69.2, 67.2, 63.1, 53.5, 49.5, 43.7, 40.6, 29.0, 28.2, 24.4, 23.0, 21.6, 12.4; HRMS (ESI+): m/z Calcd for C42H49N3O6 [M+H]+: 692.3655, Found: 692.3702.
Compound 40 was synthesized by the reaction of 36 (Red Fluor-OH, 100 mg, 0.30 mmol) with benzyl bromide 42 (144 mg, 0.36 mmol) and Ag2O (104 mg, 0.45 mmol) according to the general procedure C. The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=20:1) to produce 40 in 22% of yield (43 mg).
1H NMR (400 MHZ, DMSO-d6) δ 10.00 (s, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.61 (D, J=8.7 Hz, 2H), 7.36 (q, J=7.3 Hz, 3H), 7.23 (t, J=7.3 Hz, 1H), 7.03 (d, J=8.2 Hz, 1H), 6.83 (q, J=4.3 Hz, 3H), 6.65 (dd, J=8.7, 2.7 Hz, 1H), 6.56 (d, J=2 Hz, 1H), 6.50 (dd, J=8.5, 2.5 Hz, 1H), 5.25 (s, 2H), 5.07 (s, 2H), 4.10 (d, J=15.1 Hz, 1H), 3.77 (s, 3H), 1.60 (d, J=7.3 Hz, 1H), 1.58-1.46 (m, 1H), 1.41 (d, J=8.2 Hz, 1H), 1.37 (s, 9H), 0.88 (q, J=3.2 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 159.56, 157.90, 150.34, 150.26, 145.13, 138.28, 129.58, 129.50, 128.00, 127.70, 122.88, 120.90, 117.09, 115.59, 111.74, 110.81, 101.36, 99.90, 82.33, 71.37, 56.21, 40.39, 27.98, 24.82, 24.14, 21.34; HRMS (ESI+): m/z Calcd for C39H42N2O7 [M+H]+: 651.3026, Found: 651.3067.
Probe 5 (Rhodo-P3) was synthesized from 37 (40 mg, 0.057 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (2.4 mL). The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=5:1) to give 20 mg of probe 5 (Rhodo-P3) in 58% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 7.98 (d, J=7.8 Hz, 1H), 7.77 (td, J=7.4, 1.1 Hz, 1H), 7.70 (td, J=7.5, 0.9 Hz, 1H), 7.65 (d, J=8.2 Hz, 2H), 7.38 (d, J=8.7 Hz, 2H), 7.26 (d, J=7.8 Hz, 1H), 6.93 (d, J=2.3 Hz, 1H), 6.73 (dd, J=8.9, 2.5 Hz, 1H), 6.61 (d, J=8.7 Hz, 1H), 6.45 (d, J=2.7 Hz, 3H), 5.09 (s, 2H), 3.36-3.29 (m, 5H), 1.79-1.69 (m, 1H), 1.49-1.42 (m, 1H), 1.37-1.27 (m, 1H), 1.08 (t, J=7.1 Hz, 6H), 0.88 (dd, J=10.5, 6.4 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 174.9, 168.7, 159.9, 152.4, 152.2, 152.1, 149.2, 138.8, 135.5, 131.1, 130.0, 128.9, 128.6, 128.4, 126.4, 124.5, 124.0, 119.1, 112.2, 111.5, 108.6, 104.5, 101.6, 96.9, 83.5, 69.4, 54.0, 44.1, 43.8, 24.2, 23.2, 21.9, 12.3; HRMS (ESI+): m/z Calcd for C37H39N3O5 [M+H]+: 606.2923, Found: 606.2973.
Probe 6 (Fluor-P3) was synthesized from 38 (20 mg, 0.03 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.2 mL). The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=15:1) to give 7 mg of probe 6 (Fluor-P3) in 42% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 7.97 (d, J=7.3 Hz, 1H), 7.74 (t, J=7.1 Hz, 1H), 7.68 (t, J=7.3 Hz, 1H), 7.61 (d, J=8.2 Hz, 2H), 7.35 (d, J=8.7 Hz, 2H), 7.23 (d, J=7.3 Hz, 1H), 6.91 (dd, J=19.4, 2.5 Hz, 2H), 6.74 (dd, J=8.7, 2.7 Hz, 1H), 6.69-6.62 (m, 3H), 5.07 (s, 2H), 3.77 (s, 3H), 3.34-3.31 (m, 1H), 1.68 (td, J=13.7, 6.5 Hz, 1H), 1.48-1.40 (m, 1H), 1.33-1.26 (m, 1H), 0.84 (dd, J=9.8, 6.6 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 168.6, 161.1, 160.1, 152.5, 151.7, 151.7, 138.7, 135.7, 131.1, 130.2, 129.0, 128.5, 125.9, 124.7, 124.0, 119.1, 112.7, 112.0, 111.0, 110.8, 101.6, 100.8, 82.3, 69.5, 55.7, 53.8, 43.8, 42.1, 40.4, 29.0, 24.2, 23.1, 21.9; HRMS (ESI+): m/z Calcd for C34H32N2O6 [M+H]+: 565.2294, Found: 565.2334.
Probe 11 (Red Rhodo-P3) was synthesized from 39 (20 mg, 0.029 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.2 mL). The crude residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=5:1) to give 10 mg of probe 11 (Red Rhodo-P3) in 59% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 7.65 (d, J=8.2 Hz, 2H), 7.43 (d, J=7.3 Hz, 1H), 7.37 (d, J=8.2 Hz, 2H), 7.34 (d, J=7.3 Hz, 1H), 7.23 (t, J=7.3 Hz, 1H), 6.80-6.73 (m, 3H), 6.69-6.64 (m, 2H), 6.40 (dd, J=8.9, 2.5 Hz, 1H), 6.34 (d, J=2.7 Hz, 1H), 5.18 (s, 2H), 5.06 (s, 2H), 3.31 (s, 5H), 3.18-3.29 (2H), 1.79-1.69 (m, 1H), 1.50-1.43 (m, 1H), 1.36-1.29 (m, 1H), 1.07 (t, J=6.9 Hz, 6H), 0.88 (dd, J=10.1, 6.9 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 174.6, 158.7, 151.0, 150.8, 148.1, 145.3, 138.8, 138.7, 131.4, 129.8, 129.5, 128.4, 128.1, 127.8, 123.2, 121.0, 119.1, 117.7, 111.3, 108.1, 101.0, 96.7, 82.7, 71.2, 69.2, 53.9, 43.9, 43.7, 24.2, 23.2, 21.9, 12.4; HRMS (ESI+): m/z Calcd for C37H41N3O4 [M+H]+: 592.3131, Found: 592.3177.
Probe 12 (Red Fluor-P3) was synthesized from 40 (25 mg, 0.038 mmol) according to the general procedure D, using 30% trifluoroacetic acid in CH2Cl2 (1.5 mL). The residue was purified by flash column chromatography on silica gel (CH2Cl2:MeOH=10:1) to give 10 mg of probe 12 (Red Fluor-P3) in 48% of yield.
1H NMR (400 MHZ, DMSO-d6) δ 7.65 (d, J=8.7 Hz, 2H), 7.45 (d, J=7.8 Hz, 1H), 7.36 (t, J=8.7 Hz, 3H), 7.23 (t, J=7.5 Hz, 1H), 6.83 (q, J=2.9 Hz, 3H), 6.78-6.71 (m, 3H), 6.66 (dd, J=8.7, 2.3 Hz, 1H), 5.25 (s, 2H), 5.07 (s, 2H), 3.77 (s, 3H), 3.40-3.37 (m, 1H), 1.73 (q, J=7.0 Hz, 1H), 1.49-1.42 (m, 1H), 1.34-1.27 (m, 1H), 0.88 (dd, J=10.4, 6.5 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 159.8, 158.9, 150.5, 150.4, 145.3, 138.7, 138.4, 129.8, 128.4, 128.3, 128.0, 123.1, 121.2, 119.1, 117.4, 117.2, 111.9, 111.2, 101.0, 100.1, 82.4, 71.8, 69.3, 55.4, 53.9, 44.1, 28.7, 24.2, 23.2, 21.9; HRMS (ESI+): m/z Calcd for C34H34N2O5 [M+H]+: 551.2501, Found: 551.2546.
Synthesis of Boc-Br-PABA-Leu (42). Reagents and conditions: (a) Boc-Leu-OH, EEDQ, CH2Cl2, rt, 90%; (b) PPh3, NBS, rt, 64%
To a solution of Boc-Leu-OH (1 g, 4.3 mmol) in CH2Cl2 (40 mL) was added (4-aminophenyl) methanol (650 mg, 5.2 mmol) and EEDQ (1.3 g, 5.2 mmol) under nitrogen atmosphere and stirred at RT for 12 h. After completion of the reaction, the solvent was evaporated and the crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=3:2) to produce benzyl alcohol 41 (1.3 g, 90%).
1H NMR (400 MHZ, DMSO-d6) δ 9.88 (s, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.22 (d, J=8.2 Hz, 2H), 7.00 (d, J=8.2 Hz, 1H), 5.09 (t, J=5.7 Hz, 1H), 4.42 (d, J=5.9 Hz, 2H), 4.15-4.04 (m, 1H), 1.72-1.57 (m, 1H), 1.56-1.45 (m, 1H), 1.44-1.40 (m, 1H), 1.37 (s, 9H), 0.88 (q, J=3.4 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 171.61, 155.42, 137.64, 137.33, 126.87, 118.95, 77.97, 62.58, 53.47, 40.68, 28.20, 24.34, 22.96, 21.56; HRMS (ESI+): m/z Calcd for C18H28N2O4 [M+Na]+: 359.2083, Found 359.1938.
To a solution of benzyl alcohol 41 (100 mg, 0.3 mmol) in anhydrous CH2Cl2 (10 mL) was added triphenylphosphine (94 mg, 0.36 mmol) and NBS (65 mg, 0.36 mmol) and stirred at RT for 1.5 h. After completion of the reaction, the mixture was extracted with CH2Cl2 and washed with water. The organic layer was washed with brine and dried over Na2SO4. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane=1:4) to produce benzyl bromide 42 (77 mg, 64%).
1H NMR (400 MHZ, DMSO-d6) δ 9.95 (s, 1H), 7.57 (d, J=8.2 Hz, 2H), 7.26 (d, J=8.2 Hz, 2H), 4.42 (s, 2H), 4.13-4.07 (m, 1H), 1.68-1.58 (m, 1H), 1.57-1.45 (m, 1H), 1.45-1.27 (m, 11H), 0.88 (dd, J=6.4, 4.1 Hz, 6H); 13C NMR (100 MHZ, DMSO-d6) δ 171.76, 155.45, 138.38, 133.08, 128.22, 119.05, 77.98, 70.86, 53.51, 40.63, 28.20, 24.35, 22.96, 21.57.
The present inventors hypothesized that activatable fluorescent probes, which release fluorophores instead of cytotoxic payloads, may be applied to the development of cleavable linkers for LTDs, such as ADCs. Fluorophores in an activatable fluorescent probe as a fluorescence platform for cleavable linker development must show excellent fluorescence under weakly acidic pH as well as neutral conditions because cleavable linker-based ADCs release payloads by cytoplasmic or lysosomal enzymes. Thus, 8 fluorophores containing fluorescein, rhodol, and rhodamine were prepared, and their photochemical properties were evaluated in the pH range of 2 to 12. Most fluorophores, except Red Rhodo-NH2, showed acceptable fluorescence at weakly acidic and neutral pH, although reduced xanthene fluorophores showed relatively lower fluorescence than that of typical xanthene fluorophores (
Prior to studying the effects of linkages, spacers, and fluorophores on the LAP reaction, the concentration dependence was investigated for the optimization of the LAP reaction (
In this study, activatable fluorescent probes releasing fluorophores by LAP comprised a preliminary model for the development of a fluorescence platform for the design and evaluation of a cleavable linker or spacer. Activatable fluorescent probes for developing an enzyme-specific cleavable linker should only be selectively cleaved by the target enzyme to avoid the side effects of the release of a drug via non-selective cleavage. Thus, the activity and selectivity of LAP-responsive activatable fluorescent probes in this study were assessed in an LAP inhibition test using bestatin, which is an inhibitor of LAP and aminopeptidase B; in confocal live cell imaging; and in selectivity tests using various bioanalytes compared to LAP. The pretreatment of bestatin at concentrations of 0, 1, 10, 50, and 100 μM in LAP showed reduced fluorescence emission by released fluorophores in a concentration-dependent manner (
Subsequently, the selectivity of our activatable fluorescent probes was investigated to evaluate the interference by other intracellular bioanalytes in evaluating the drug-releasing activity of linkers using an activatable fluorescent probe. Various cations (Na+, K+, Ca2+, and Zn2+), reactive oxygen species (NaOCl and H2O2), biomolecules (glutathione, vitamin C, cysteine, alanine, and arginine), and enzymes (α-amylase, lysozyme, and cellulase) were used to evaluate the selectivity of the activatable fluorescent probes for the LAP reaction, and only LAP induced fluorescence emission exclusively (
5. Ex vivo Plasma Stability Test
The present inventors performed a preliminary model study to develop a fluorescence platform for the design and evaluation of a cleavable linker that releases a drug. For this purpose, activatable fluorescent probes with a response to a lysosomal peptidase, LAP, were designed and successfully evaluated in terms of kinetics, competitive inhibition, selectivity, and confocal live cell imaging to demonstrate its feasibility as a fluorescence-based development platform for cleavable linkers. As mentioned in many reviews of LTDs including ADCs, the plasma stability of linkers in ADCs is an important factor determining drug efficacy and toxicity. Thus, the present inventors finally established an ex vivo study to evaluate the plasma stability of our probes, expecting to yield a final piece to complete our strategy for a fluorescence-based platform to develop cleavable linkers. The plasma stability of the probes was evaluated by measuring fluorescence emission for 48 h after treatment of the probes (10 μM) in the serum obtained by centrifuging blood collected from C57/BL6 mice. The fluorescence of Rhodo-P1 with a direct amide linkage rapidly increased, while Rhodo-P2 with a p-aminobenzyl carbamate linkage showed fluorescence emission with a gentle increasing slope (
To the development of a fluorescent probe-based cleavable linker platform, a model study was conducted using activatable fluorescent probes that release fluorophores by LAP to examine whether the drug release nature and stability of cleavable linkers may be evaluated using a fluorescence platform. Various LAP-responsive fluorescent probes containing leucine, an LAP substrate, were prepared via direct conjugation to several xanthene fluorophores by amide coupling or conjugation via a spacer: a PAB group. Among the various fluorophores, Rhodo-NH2 was the best fluorophore that shows strong fluorescence in weakly acidic pH as well as neutral pH, which is important because most peptidases work in weakly acidic lysosomes, and showed the highest release rate from the LAP-responsive fluorescent probes compared to that from other fluorophores in the kinetic study. The direct amide linkage in the probes showed a fast enzymatic response and relatively low stability at basic pH, whereas the carbamate linkage with a spacer showed a slow and sustained kinetic response with excellent pH and thermal stability. Rhodo-P2, in which leucine is linked to Rhodol-NH2 by a PABC linkage via a spacer, showed high yields of LAP reactions and was most biocompatible in selectivity tests as well as pH and thermal stability tests. The ex vivo plasma stability test showed that the carbamate linkage via a spacer showed approximately 60% decomposition in plasma after 48 h, which is slower and lower decomposition rate than the direct amide linkage. In this study, the present inventors successfully demonstrated that fluorescent probes may be used as a platform for the development of cleavable linkers for that effectively drugs and release LTDs are sufficiently stable during blood circulation. Based on our study, it could be developing a drug delivery system such as prodrug, ADC, and LTD, etc. having a cleavable linker that may be hydrolyzed by an oncogenic enzyme to release payloads using our fluorescent probe platform.
