Patent Application
20240360089
Cancer is the second largest cause of death in the United States. Despite improvement in treatment regimens, problems such as lack of target specificity and off-target toxicity persist. Targeted drug therapy is a possible way to counter these problems. Enzymes responsible for epigenetic regulation of gene expression provide several targets for rational design of anti-cancer agents. Epigenetic regulation includes DNA methylation and histone acetylation/deacetylation, which do not change the genetic sequence, but alter the rate of transcription of genes affected by these modifications.
Histone acetylation and deacetylation are regulated by two families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. HDACs play a key role in regulating gene expression by removing acetyl groups from histones, which are proteins that help package and organize DNA in the nucleus of cells. Deacetylation of histones can reduce the expression of tumor suppressor genes, and their hyperacetylation can lead to their induction, leading to apoptosis. HDACs also deacetylate non-histone proteins such as α-tubulin, which are involved in cancer cell migration and proliferation. Hence, selective targeting of HDACs has been studied extensively. By inhibiting HDAC activity, HDAC inhibitors can increase the acetylation of histones, which in turn can lead to changes in gene expression.
HDACs consist of zinc-dependent (Class I, II, and IV) and NAD+ dependent (Class III) enzymes. Class I and II HDACs can be inhibited by small molecules containing Zinc-Binding Groups (ZBGs), which bind to a Zn2+ ion in the enzyme active site via mono or bidentate complexation, which is reversible in nature. The ZBG is a chemical moiety that binds to the zinc ion in the active site of the HDAC enzyme, thereby inhibiting its activity. The choice of ZBG can affect the potency, selectivity, and pharmacokinetic properties of an HDAC inhibitor. The ZBG typically includes a group such as a carboxylate, a hydroxamate, or a thiol. Of the four HDAC inhibitors approved by the US FDA for clinical use, vorinostat (SAHA) and belinostat (Beleodaq) approved for T-cell lymphoma and panobinostat (Farydak) approved for multiple myeloma (
The most common HDAC inhibitors comprise hydroxamates, which are very potent, and chelate very tightly to the Zn2+ ion in a bidentate manner. However, hydroxamates have poor pharmacokinetic properties and their promiscuous metal-binding ability leads to HDAC-independent adverse effects, including cardiotoxicity, pulmonary embolism, thrombocytopenia with nausea, and diarrhea. In addition, the hydroxamates have the potential to undergo zinc-catalyzed Lossen-rearrangement to isocyanates, which are highly reactive electrophiles that can be targeted by numerous nucleophiles in the cells to cause a plethora of biological problems such as mutagenesis through reaction with nitrogen bases of DNA. Hydroxamates are also rapidly cleared metabolically, and they lack isoform selectivity. Hence, there is a need for alternative ZBGs. Even if the alternatives are less potent than hydroxamates, modulation of the cap group can be used to strike a balance between potency and selectivity, improving their pharmacologic properties and reducing possible drug side effects. Current alternatives to hydroxamates include molecules such as trifluoromethylketones (TFMKs), thiols, α-ketoamides, and HDAC 6-selective mercaptoacetamides. These inhibitors exhibit not only potent in vitro HDAC inhibitory activity, but also significant anti-tumor activity. Unfortunately, there is no established set of guidelines for designing ZBGs, and a direct correlation between zinc-binding ability and biological activity has not been fully established. The existence of some HDAC inhibitors without ZBGs adds to the complexity. Furthermore, the known alternatives have problems. For example, TFMKs are rapidly metabolized to inactive trifluoromethyl alcohols in vivo, having a half life of only about 0.5 hours. Furthermore, TFMKs have a very reactive electrophilic warhead that can form covalent bonds with many endogenous nucleophiles to produce off-target effects. These issues have prevented the further development of TFMKs as potential drug candidates.
There is a need in the art for new and improved HDAC inhibitors.
Provided is a composition comprising an HDAC inhibitor compound having a cap group, a linker, and a zinc binding group, wherein the zinc binding group comprises a trifluoromethylpyruvamide (TFMP) or a hydrate thereof.
Provided is a composition comprising Formula I:
wherein R1 is aryl, alkyl, heteroaryl, a hydrophobic moiety, a hydrophilic moiety, or a combination thereof, optionally substituted with one or more halogens, nitro groups, or amino groups; each R2 is, independently, H or alkyl; and L is a linker comprising an alkyl chain, a fluorinated alkyl chain, an aryl group, an arylalkyl group, a carbonyl-containing chain, an amide-containing chain, or a heteroatom-containing chain, optionally substituted with one or more halogens, amino groups, or carboxylic acid groups. Also provided are salts, stereoisomers, racemates, solvates, hydrates, prodrugs, and polymorphs of Formula I.
In certain embodiments, R1 comprises a benzyl, phenyl, pyridinyl, thiazolyl, or benzothiazolyl.
In certain embodiments, R1 comprises a thiazolyl, benzyl, or benzothiazolyl; and L comprises a carbonyl-containing alkyl chain having from 1 to 10 carbons.
In certain embodiments, R1 comprises a polyethylene glycol (PEG) chain.
In certain embodiments, L comprises an ether, a thioether, or an amide.
In certain embodiments, L is a carbonyl-containing alkyl chain having from 1 to 10 carbons.
In certain embodiments, the composition comprises compound 83:
In certain embodiments, the composition comprises compound 84:
In certain embodiments, the composition comprises compound 85:
In certain embodiments, the composition comprises compound 86:
In certain embodiments, the composition comprises compound 61:
In certain embodiments, the composition comprises compound 70:
In certain embodiments, the composition comprises compound 64:
In certain embodiments, the composition comprises compound 65:
In certain embodiments, the composition comprises compound 69:
In certain embodiments, the composition comprises compound 58:
In certain embodiments, the composition comprises compound 55:
In certain embodiments, each R2 is H.
Further provided is a pharmaceutical composition comprising an HDAC inhibitor compound described herein, and a pharmaceutically acceptable diluent, adjuvant, or carrier.
Further provided is a metal binding group comprising Formula II:
wherein R1 is a lone pair of electrons, aryl, alkyl, heteroaryl, a hydrophobic moiety, a hydrophilic moiety, or a combination thereof, optionally substituted with one or more halogens, nitro groups, or amino groups; each R2 is, independently, H or alkyl; and salts stereoisomers, racemates, hydrates, solvates, prodrugs, and polymorphs thereof. In certain embodiments, each R2 is H.
Further provided is a method of inhibiting an HDAC protein, the method comprising contacting a cell with a composition comprising an HDAC inhibitor compound described herein and inhibiting an HDAC protein in the cell. In certain embodiments, the HDAC protein is HDAC8 and the composition comprises compound 83. In certain embodiments, the HDAC inhibitor compound is selected from the group consisting of compound 65, compound 61, compound 85, compound 69, compound 55, compound 86, compound 64, compound 83, compound 58, and compound 84.
Further provided is a method of inhibiting growth of cancer cells, the method comprising contacting cancer cells with an effective amount of a composition comprising an HDAC inhibitor compound described herein and inhibiting growth of the cancer cells.
In certain embodiments, the cancer is lung cancer, cervical cancer, breast cancer, colon cancer, fibrosarcoma, neuroblastoma, or osteosarcoma. In certain embodiments, the HDAC inhibitor compound is selected from the group consisting of compound 65, compound 61, compound 85, compound 69, compound 55, compound 86, compound 64, compound 83, compound 58, and compound 84.
Further provided is a method of synthesizing an HDAC inhibitor compound, the method comprising amide coupling an aminoalkanamide with a compound having a zinc binding group (ZBG) appended to a carboxylic acid group. In certain embodiments, the amide coupling is conducted with one or more coupling reagents selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCl), dimethylaminopyridine (DMAP) in dichloromethane, (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBt), and N-methylmorpholine (NMM) in N,N-dimethylformamide (DMF).
Further provided is a method of synthesizing an HDAC inhibitor compound, the method comprising protecting an aminoalkanoic acid with a protecting group to obtain a protected aminoalkanoic acid; peptide coupling the protected aminoalkanoic acid with an aniline to obtain a protected amino anilide; and deprotecting and peptide coupling the protected amino anilide with trifluoropyruvic acid to obtain a trifluoropyruvamide. In certain embodiments, the protecting group is either tert-butyloxycarbonyl (Boc) or 2,2,2-trichloroethoxycarbonyl (Troc).
Further provided is a kit for making an HDAC inhibitor compound, the kit comprising a first container housing an aminoalkanoic acid; a second container housing an aniline; and a third container housing trifluoropyruvic acid.
Further provided is the use of trifluoromethylpyruvamide or a hydrate thereof as a metal binding group in an HDAC inhibitor.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.
To overcome limitations with known HDAC inhibitors, trifluoropyruvamides (TFPAs) have been developed as ZBGs that act as TFMK surrogates. The trifluoromethylpyruvamide (TFMP) ZBG was developed by incorporating another electron withdrawing carbonyl group into the TFMK group. This stabilizes the hydrate and makes it stable to metabolic reduction. The presence of an additional electron withdrawing group next to the ketone carbonyl group makes the hydrate form of the ketone more stable, thus preventing its metabolic reduction to alcohol in vivo. Accordingly, TFPA is more metabolically stable than TFMK. In addition, this structural modification reduces the potential of the TFMK group to act as a covalent warhead to eliminate off-target effects. As shown in the examples herein, additional structural changes in the cap group of the inhibitors gave analogues with IC50 values ranging from upper nanomolar to low micromolar in the cytotoxicity assay, and they were more selective for cancer cells over normal cells. Some of the most active analogues inhibited HDAC enzymes with low nanomolar IC50 values and were found to be more selective for HDAC8 over other isoforms. Furthermore, TFPAs cannot act as pan assay interference (PAINS) molecules, which are frequent false positives in high-throughput screening assays. These molecules provide a new class of HDAC inhibitors with a metabolically stable metal-binding group that can also be used to develop selective HDAC inhibitors by further structural modification.
In general, the HDAC inhibitor compounds herein have the structural formula of Formula I:
where R1 is a cap group, each R2 is independently H or alkyl, and L is a linker. The cap group is selected from a variety of possible chemical moieties that are capable of enhancing the potency, selectivity, and pharmacokinetic properties of the HDAC inhibitor compound. The cap group can be an aryl, alkyl, or heteroaryl moiety, or a combination thereof, such as benzyl, phenyl, pyridinyl, thiazolyl, or benzothiazolyl. Alternatively, the cap group can be a hydrophobic or hydrophilic moiety, such as an alkyl chain or a polyethylene glycol (PEG) chain. The cap group may modulate the physicochemical properties of the HDAC inhibitor compound, such as lipophilicity and water solubility. In some embodiments, R1 is one of the cap groups depicted in
As noted, R2 can be either H or an alkyl group. Suitable alkyl groups for R2 include, but are not limited to, methyl, ethyl, propyl, butyl, secbutyl, isobutyl, tertbutyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.
The linker of Formula I connects the cap group to the metal binding group. The linker may be a simple alkyl chain, a fluorinated alkyl chain, an aryl group, an arylalkyl group, a carbonyl-containing chain, an amide-containing chain, or a heteroatom-containing chain, such as an ether, thioether, or amide. The linker can also be substituted with functional groups, such as halogens, amino groups, or carboxylic acid groups, which can modulate the physicochemical properties of the HDAC inhibitor compound. In some embodiments, the linker is a carbonyl-containing alkyl chain (i.e., an acyl group) having from 1 to 10 carbons. In some embodiments, the linker includes an ether, a thioether, or an amide.
In some embodiments, the cap group R1 includes one of a thiazolyl, a benzyl, or a benzothiazolyl while the linker L includes a carbonyl-containing alkyl chain having from 1 to 10 carbons.
Non-limiting example HDAC inhibitor compounds of Formula I include each of the following compounds 83, 84, 85, 86, 61, 70, 64, 65, 69, 58, and 55:
As shown in the examples herein, compounds 83, 84, 85, 86, 61, 70, 64, 65, 69, 58, and 55 are effective at inhibiting HDAC activity and inhibiting cell growth of various cancer cells. Furthermore, these compounds are metabolically stable.
The HDAC inhibitor compounds of Formula I can be synthesized according to the schemes depicted in
As seen in
Also provided are metal binding groups having the general structural formula of Formula II:
where R1 is lone pair of electrons, aryl, alkyl, or heteroaryl, a hydrophobic moiety, or a hydrophilic moiety, optionally substituted with one or more halogens, nitro groups, or amino groups; and each R2 is, independently, H or alkyl. Suitable alkyl groups for R2 include, but are not limited to, methyl, ethyl, propyl, butyl, secbutyl, isobutyl, tertbutyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. The metal binding groups are useful not only in HDAC inhibitor compounds, but also in any other drug molecules which include a metal binding group such as, but not limited to, metalloproteinase inhibitors, HIV-1 integrase enzyme targeting drugs, protease inhibitors, and kinase inhibitors.
The trifluoromethylketone group previously developed as an alternative, and effective, metal binding group, undergoes rapid metabolic reduction resulting in loss of HDAC inhibitory activity. TFPA is effective as a ZBG for HDAC inhibitors, and is not susceptible to metabolic reduction, does not act as PAINS, is not susceptible to reacting with endogenous reactive nucleophiles, is less likely to cause off-target effects than TFMKs, and results in potent HDAC inhibitors. Numerous effective HDAC inhibitors, useful for treating certain cancers, have been synthesized with a TFPA ZBG.
Pharmaceutical compositions of the present disclosure may comprise an effective amount of an HDAC inhibitor compound described herein, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The preparation of a pharmaceutical composition will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
A composition disclosed herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for such routes of administration as injection. Compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseosly, periprosthetically, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference).
The actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, a composition herein and/or additional agent is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsules, they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
In further embodiments, a composition described herein may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515, and 5,399,363 are each specifically incorporated herein by reference in their entirety).
Solutions of the compositions disclosed may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In some cases, the form should be sterile and should be fluid to the extent that easy injectability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate or gelatin.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
In other embodiments, the compositions may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.), and/or via inhalation.
Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder. Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones, and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorption, emulsion, or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture. Transdermal administration of the compositions may also comprise the use of a “patch.” For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.
In certain embodiments, the compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in their entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts and could be employed to deliver the compositions described herein. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety), and could be employed to deliver the compositions described herein.
It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight, and the severity and response of the symptoms.
In particular embodiments, the compounds and compositions described herein are useful for treating cancers or inhibiting growth of cancer cells. As described herein, the compounds and compositions herein can be used in combination therapies. That is, the compounds and compositions can be administered concurrently with, prior to, or subsequent to one or more other desired therapeutic or medical procedures or drugs. The particular combination of therapies and procedures in the combination regimen will take into account compatibility of the therapies and/or procedures and the desired therapeutic effect to be achieved. Combination therapies include sequential, simultaneous, and separate administration of the active compound in a way that the therapeutic effects of the first administered procedure or drug is not entirely disappeared when the subsequent procedure or drug is administered.
In some embodiments, the HDAC inhibitor compound is part of a combination therapy with a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to: taxane compounds, such as paclitaxel; platinum coordination compounds; topoisomerase I inhibitors, such as camptothecin compounds; topoisomerase II inhibitors, such as anti-tumor podophyllotoxin derivatives; anti-tumor vinca alkaloids; anti-tumor nucleoside derivatives; alkylating agents; anti-tumor anthracycline derivatives; HER2 antibodies; estrogen receptor antagonists or selective estrogen receptor modulators; aromatase inhibitors; differentiating agents, such as retinoids, and retinoic acid metabolism blocking agents (RAMBA); DNA methyl transferase inhibitors; kinase inhibitors; farnesyltransferase inhibitors; HDAC inhibitors, or other inhibitors of the ubiquitin-proteasome pathway; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins; camptothecins, such as the synthetic analog topotecan; cryptophycins; nitrogen mustards, such as chlorambucil; nitrosoureas; bisphosphonates; mitomycins; epothilones; maytansinoids; trichothecenes; retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids and derivatives of any of the above; and combinations thereof. Non-limiting examples of specific chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, rapamycin, lapatinib (TYKERB®, Glaxo SmithKline), oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO 2007/044515), ARRY-886 (MEK inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), ABT-869 (multi-targeted inhibitor of VEGF and PDGF family receptor tyrosine kinases, Abbott Laboratories and Genentech), ABT-263 (Bcl-2/Bcl-xL inhibitor, Abbott Laboratories and Genentech), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), lonafamib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifamib (ZARNESTRA™, Johnson & Johnson), capecitabine (XELODA(®, Roche), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thioTepa and cyclosphosphamide (CYTOXAN®, NEOSAR®), bullatacin, bullatacinone, bryostatin, callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs), cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1), leutherobin, pancratistatin, sarcodictyin, spongistatin, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, clodronate, esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), cyclophosphamide, thioTepa, 6-thioguanine, mercaptopurine, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine (NAVELBINE®), novantrone, teniposide, edatrexate, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, and difluoromethylomithine (DMFO), paclitaxel, 5-fluorouracil, abraxane (paclitaxel albumin-stabilized nanoparticle formulation), afinitor (everolimus), erlotinib hydrochloride, everolimus, gemcitabine hydrochloride, oxaliplatin (eloxatin), capecitabine (xeloda), cisplatin, irinotecan (camptosar), colinic acid (leucovorin), folfox (folinic acid, 5-fluorouracil, and oxaliplatin), folfirinox (folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin), nab-paclitaxel with gemcitabine, metformin, digoxin, and simvastatin.
In some embodiments, the HDAC inhibitor compound is part of a combination therapy with an immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include nivolumab, pembrolizumab, rituximab, durvalumab, cemiplimab, and combinations thereof.
In some embodiments, the HDAC inhibitor compound is part of a combination therapy with a hormonal therapeutic agent. Non-limiting examples of hormonal therapeutic agents include anastrozole, exemestane, letrozole, tamoxifen, raloxifene, fulvestrant, toremifene, gosrelin, leuprolide, triptorelin, apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide, nilutamide, abiraterone, ketoconazole, degarelix, medroxyprogesterone acetate, megestrol acetate, mitotane, and combinations thereof.
The compositions and HDAC inhibitor compounds described herein may also be made available via a kit containing one or more key components. A non-limiting example of such a kit for making an HDAC inhibitor compound comprises an aminoalkanoic acid, an aniline, and trifluoropyruvic acid in separate containers, where the containers may or may not be present in a combined configuration. Many other kits, such as kits which further include one or more coupling reagents, are possible and encompassed within the scope of the present disclosure. The kits may further include instructions for using the components of the kit to prepare HDAC inhibitor compounds. The instructions may be recorded on a suitable recording medium. For example, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, such as a flash drive. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, such as via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
These examples describe the development of HDAC inhibitors with ZBGs of moderate Zn2+ binding ability and higher selectivity. Selection of appropriate metal-binding groups was based on the belief that bidentate chelation leading to five-membered metallocycle intermediates/transition states is mostly preferred due to lack of strain. Among the alternates to SAHA, trifluoromethyl ketones (TFMKs) have been shown to be potent HDAC inhibitors, but they suffer from rapid reductive metabolism of the ketone carbonyl group in vivo, limiting their use as anticancer agents. To overcome this metabolic impediment, trifluoropyruvamides (TFPAs) were designed and synthesized as metabolically more stable variants of TFMK HDAC inhibitors.
Computational analysis has revealed several possible bidentate ZBGs (
Without wishing to be bound by theory, it is believed that HDAC-TFMK interactions via the hydrate formed inside the active site are induced due to enhancement of electrophilicity of the carbonyl group by zinc-chelation, facilitating nucleophilic addition of a water molecule. The keto-form is predominant elsewhere as indicted by the appearance of the carbonyl carbon as a quartet at 191.5 ppm in the 13C NMR spectrum of the TFMK analogue (
An alternative, and different, design using trifluorolactic amides as HDAC 6-selective inhibitors has been published. However, the mechanism of zinc-binding underlying this alternative design was based on increasing the acidity of the alcohol by the trifluoromethyl group, facilitating its deprotonation, and creating a hydroxamate surrogate that binds to HDAC6 via a 5-membered metallocycle, unlike the 4-membered binding mode of TFPAs involving the gem diol group. However, the mode of binding of the trifluorolactic amides indicates the possibility of an additional binding mode of TFPA ZBG via a less-strained 5-membered metallocycle involving one of the hydrate-alcohol groups and the adjacent amide-carbonyl, in addition to the 4-membered metallocycle involving the two gem-diol hydroxyl groups. (
To compare binding modes of the designed SAHA analogues with alternative ZBGs (
All the molecules aligned with SAHA when docked against the designated HDACs with similar binding modes (
The final products 5a-5c, 7a, 7b, 7d, 8b, 9a-9d, and 10a-10d were synthesized by amide coupling of aminoalkanilides 4a-4d with a ZBG appended to a carboxylic acid group as shown in Scheme 1 (
The only reliable synthetic approach reported for preparation of TFPAs was by an Ugi-like mechanistic pathway using isocyanides and trifluoroacetic anhydride. Considering the high cost of isocyanides needed to make a diverse library of analogues of varying cap groups, simple peptide couplings using amino-alkanamides resembling SAHA fragments were used instead (4a-4d). Peptide coupling using EDCl/DMAP, and EDCl/HOBt failed to give any product. Using HATU, HOBt, and NMM in the presence of molecular sieves under anhydrous conditions at room temperature for four hours resulted in successfully synthesizing TFPAs 10a-10d (Scheme 2A,
The aminoalkanilides 4a-4d required for the above syntheses were made by an approach starting from bromoalkanoic acids 1a-1d (Scheme 2A,
For synthesis of the TFPA analogue 14 with a short three-methylene linker (Scheme 2B), more commonly available γ-aminobutyric acid (GABA) 11 was used instead of 4-bromobutanoic acid. Boc-protection of GABA (11) gave compound 12. This was followed by peptide coupling with aniline using EDCl and DMAP in dichloromethane overnight to give compound 13. Finally, a tandem Boc-deprotection/peptide coupling of the γ-aminobutyranilide 13 with trifluoropyruvic acid was used to obtain the TFPA 14 (50% yield). This approach was subsequently used for efficient synthesis of Boc-aminoarenes 17-50. Tandem Boc-deprotection/peptide coupling with trifluoropyruvic acid gave TFPAs 53-85 (4-42% yield) without the need to isolate and characterize the intermediate amines. Compounds 51-52 required for the synthesis of TFPAs 86 and 87, respectively, were prepared from 50 by removal of 2,2,2-trichloroethoxycarbonyl (Troc) protecting group (Scheme 2C). Compound 51 underwent peptide coupling with trifluoropyruvic acid to yield 86, while compound 52 was first O-ethylated by refluxing with ethyl iodide and potassium carbonate in acetone for six hours, followed by concentration in vacuo and peptide coupling like 51 to give 87. Some of the aromatic amines used as cap groups included 2-aminothiazoles. Compounds containing these fragments have shown to possess antibacterial, antimicrobial, and other properties, and one such fragment has been used to synthesize a TFMK derivative before. Thus, thiazole pharmacophores were used to generate potent TFPAs. They were either commercially available, or made using known synthetic methods. The spectral data of the functional group of all TFPAs is in excellent agreement with those previously reported by others in which a 13C quartet was seen at 90 ppm (in DMSO-d6), with a J-value of 32 Hz, corresponding to the hydrate of —COCF3 carbonyl, with no signals corresponding to the keto-form. The molecules synthesized in these examples displayed similar hydrate quartet at 90-91 ppm (acetone-d6) or 94-95 ppm (MeOH-d4) (
Inhibition of growth of HCT116 colon cancer cells at a maximum concentration of 100 μM was used for the initial screening of the ZBGs for HDAC inhibitory potential (Table 1). Included in the assay are the perfluoroaromatic trifluoromethyl ketone 89 and the corresponding trifluoromethyl alcohol 88 (synthesized according to Scheme 1,
To understand the effect of varying the linker length and to determine the ideal linker length, compounds 10a-10d and 14 were tested against the HCT116 cell line (
Having established the validity of the ZBG and the ideal length of the linker, the SAR of the cap group of the most active analogue 10c (n=6) was conducted to further improve its biological activity. This involved the synthesis of a library of analogues 53-87 (Table 3) using cap groups shown in
Of the analogues substituted at the para position of aniline, p-bromoanilide 65 (3.13 μM) and p-methoxyanilide 55 (4.52 μM) had the lowest IC50 values. Hence, the effect of positional substitutions with bromine and methoxy at other positions was also investigated. There was hardly any difference in the IC50 values of meta-bromo (3.06 μM) and para-bromo (3.13 μM) analogues, but ortho-bromo substitution 75 increased the IC50 value to 14.5 μM. A direct decreasing trend in activity was observed with the methoxy group going from 4.52 μM for para (55) to 6.11 μM for meta (59) and 10.07 μM for ortho (66). Among the bulkier aromatic groups, the best results were obtained for the 5-phenylthiazole variant 58 (2.66 μM). This is consistent with phenylthiazole TFMKs being among the most active variants. Therefore, the investigation of SAR of thiazoles was expanded in much greater detail. 4- and 5-positional substitution variants of thiazoles were synthesized, starting with phenyl, methyl phenyl, and diphenyl substituents. The 4,5-diphenyl thiazole was of particular interest as a similarly substituted oxazole fragment existed as a part of the HDAC6-selective inhibitor Tubacin cap group. However, it was found that the 4-position substitution was detrimental for activity, as the 4,5-diphenylthiazole 68 had drastically reduced activity (29.88 μM).
The effect of fusing the thiazole ring with a phenyl group as in benzothiazole vs the phenylthiazole was then investigated. The results were similar with IC50 of 2.37 μM for benzothiazole 61 vs. 2.66 μM for 5-phenylthiazole 58. The effects of substitution at the para-position of phenylthiazoles, and substitutions at 5- and 6-positions of benzothiazoles, were then investigated. Accordingly, p-bromo (85), p-hydroxy (86), p-methoxy (84), and p-ethoxy (87) phenylthiazoles were synthesized. Of these, the p-methoxyphenylthiazole 84 produced an IC50 of 0.93 μM, making it more potent than SAHA (IC50 1.18 μM). Replacing the methoxy with an ethoxy in 87 reduced the activity to IC50 4.05 μM, while the corresponding free phenol analogue 86 had an IC50 of 1.02 μM, almost as active as the corresponding methoxy analogue 84. For the benzothiazole series, 6-fluoro (77), 6-chloro (78), 6-bromo (79), 5-methoxy (83), and 6-methoxy (82) benzothiazoles were synthesized. Of these, only the 5-methoxybenzothiazole variant 83 (2.18 μM) produced better results than the unsubstituted parent molecule.
As metabolic reduction of TFMKs has been shown to abolish their ability to kill cancer cells, it was important to test if reduction of TFPA would produce the same effect. TFPA 60 was reduced to the corresponding trifluoromethyl-α-hydroxyamide (TFMHA) 92. It was inactive on the HCT116 cell line (Table 3).
After screening the activity of the compounds against the HCT116 cell line, the five compounds with the lowest IC50 values (compounds 61, 83, 84, 85, and 86) were tested on additional cell lines, namely, the NCI-H522 lung cancer cell line, HT1080 fibrosarcoma cell line, HeLa cervical cancer cell line, U2OS osteosarcoma cell line, and MDA-MB-231 and MDA-MB-468 breast cancer cell lines. SAHA was used as a positive control for comparison. All compounds showed comparable or better growth inhibitory activity than SAHA on some cell lines, while compounds 84 and 85 displayed better activity than SAHA against most of the cell lines (
2.70 ± 0.34
1.76 ± 0.22
1.22 ± 0.09
1.09 ± 0.09
0.76 ± 0.08
0.79 ± 0.10
Ten selected compounds were then tested in NCI 60 cell one-dose assay, at a single concentration of 10 μM, followed by a five-dose assay at five different concentrations for eight of the ten compounds that displayed sufficient cytostatic/cytotoxic activity at 10 μM. The one-dose assay displays mean growth percentages, while the five-dose assay shows GI50, TGI, and LC50 values (Table 5). Most of the compounds demonstrated potent cytostatic activity, with several of them (58, 84, 85) showing cytotoxic effects as well (Table 5). Interestingly, compound 85 exhibited the least mean growth percentage and GI50 value, concurring with the previous assay results (Table 4). Even though compound 84 was the most active on the HCT116 cell line, the results on both six additional cell lines (
−11.47
0.93
11.4
26.3
The inhibitory potencies of the compounds were tested against HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, and HDAC8. The overall results indicated that all the inhibitors possess an affinity for class 1 HDACs over class II HDACs. The dose dependent curve data showed that the compounds displayed higher potency against HDAC8, with IC50 values in the low nanomolar range (Table 6). The most potent compound was 69 with an IC50 of 4.1±0.6 nM against HDAC8. The most selective compounds were 65, 83, and 58 (Table 6). However, compound 83 is the standout inhibitor, exhibiting between 11-to-460-fold selectivity for HDAC8, when all three were compared using heat maps of the ratios of IC50 values against different HDACs. Hence, compound 83 may be HDAC8 selective. However, a direct correlation between HDAC 8 selectivity and cell growth inhibitory activity was not observed. The results of the assay may not be enough to justify HDAC isoform selectivity, as HDACs exist as multiprotein complexes at a cellular level. In this context, it is important to note that selective HDAC 6 inhibitors have been shown to display anticancer properties only at high concentrations resulting in low selectivity and off-target effects.
65
61
85
69
55
86
64
83
4100 ± 100
2500 ± 400
120 ± 10
1900 ± 400
100 ± 10
84
SAHA
Potencies were determined using a chemiluminescent HDAC activity assay, with the mean and standard error of at least three independent trials shown. The IC50 values for SAHA were previously reported using the same assay.
The hallmark of HDAC inhibition is the hyperacetylation of several proteins which are substrates for HDACs. Thus, histone H3 hyperacetylation assay of some active compounds was carried out by western blot using antibodies for Histone H3 and pan-acetyl lysine. When tested at concentrations of 2.5, 5, and 10 μM, both acetyl-H3 and “pan” acetyl-lysine (
Pan HDACi SAHA has been reported to induce apoptosis in a caspase 8 dependent manner in HCT116 cells. To determine whether the compounds herein induced apoptosis, the enzyme Poly (ADP ribose) polymerase 1 (PARP1) was analyzed by western blotting. Caspase-dependent proteolytic cleavage of PARP1 is one of the hall marks of apoptosis and antibodies that specifically recognize the cleavage products are available. Western blot analysis indicated that the HDACi induced PARP1 cleavage, indicating induction of apoptosis (
To determine the selectively of the inhibitors for cancer cells over normal cells, compound 55 was tested on the mesenchymal cancer cell line NCI-H522 and the mesenchymal normal cell line WI38, as well as the epithelial cancer cell line HCT116 and the epithelial normal cell line RPE (
Previous studies established that trifluoromethyl ketones are metabolically unstable and reduced in vivo to trifluoromethyl alcohols (TFMAs). This metabolic reduction caused a major loss in activity. The TFPAs possess a TFMK moiety, albeit with significantly enhanced electrophilicity. It was believed that in TFPAs, the hydrate form of the ketone is stabilized compared to TFMKs, and is therefore less susceptible to reductive metabolism. To prove the superior metabolic stability of the TFPAs over TFMKs, a metabolic stability assay was carried out. Metabolic stability studies are primarily conducted using liver microsomal assays, as the predominant cytochrome P450 enzymes present in them are responsible for the metabolism of most xenobiotics in the human body. However, P450 transformations are mostly oxidative in nature, precluding their use to assess the stability of ketone drugs against reductive metabolism. A comprehensive review of ketone drug metabolism showed the presence of carbonyl reductases and additional reductases mainly in the liver cytosol, and a minor amount in microsomes. Thus, using a liver extract containing both cytosol and microsomal enzymes would be more appropriate for this assay. This was accomplished by using the S9 fraction, which is basically the supernatant obtained from centrifugation of hepatocytes at 9000 g and contains both liver cytosol and microsomal enzymes. LC-ESI-MS was used to monitor the reduction of TFPA 60 over time and it was compared with TFMK 91.
TFPAs undergo the same transformation on incubation with cells or whole blood. The S9 fraction (which contains liver cytosol and microsomal enzymes) were used for metabolism studies, and metabolism was monitored by LC-MS. The TFMK peak disappeared in 15 minutes with a TFMK alcohol peak appearing simultaneously, whereas the TFPA peak did not disappear even after 2 hours and no TFPA alcohol peak being observed.
The disappearance of TFMK 91 m/z 302 (
To test the rapid kinetics of TFMK metabolism, the aliquots obtained from the S9 fraction assay for TFMK 91 were analyzed. The M+1 peak of m/z 302 completely disappeared in 15 minutes (
In contrast, it was observed that TFPA 60 was resistant to reducing conditions imposed by the S9 fraction; the M+1 of m/z 370 for compound 60 (
The metabolic reduction of linear TFMKs has been a significant barrier that has prevented them from being considered as successful SAHA-alternatives for HDAC inhibition for 20 years. This was probably due to the inability of the ketone function to sustain the hydrate-form outside the active site of HDACs, making them vulnerable to reductive metabolism by carbonyl reductases in the liver. However, as shown in these examples, this vulnerability may be overcome by structural modification that stabilizes the hydrate form while maintaining the linearity of the functional group such that it is small enough to enter the active site. This modification entails enhancing the electrophilicity of the ketone group to favor its hydrate form and thereby prevent its reductive metabolism. This was successfully achieved by incorporating an additional electron-withdrawing amide group adjacent to the trifluoromethyl carbonyl group, resulting in TFPAs. An additional advantage of this structural modification is that it reduces the potential of the TFMK group to act as a covalent warhead and produce off-target effects. Molecular docking confirmed that the designed molecules can bind to several HDAC proteins by chelation of the hydrated pyruvamide group with the zinc ion in the HDAC active site and positioning the anilide cap group at the active site outer rim area. This mode of binding agrees with the binding of other well-known inhibitors such as SAHA. Biochemical studies showed that these compounds were potent cytotoxic agents. SAHA-like linkers containing six methylene groups were found to be advantageous for antiproliferative activity of TFPAs. SAR studies of the cap-group of thiazole-based TFPAs identified several potent variants such as compound 85, which consistently outperformed SAHA. Additional cell survival assays demonstrated that TFPAs were more selective for cancer cells over normal cells. In in vitro HDAC isoform selectivity studies utilizing purified HDAC proteins, the compounds inhibited HDAC enzymes with low nanomolar IC50 values, while displaying some selectivity for HDAC 8. Compound 83 in particular displayed between 11 and 460-fold selectivity for HDAC8 over other isoforms. Western blot analysis revealed the ability of the compounds to inhibit HDAC proteins in a cellular environment as well. Metabolism studies using the S9-fraction assay showed that TFMK 91 undergoes complete metabolic reduction in 15 minutes, whereas TFPA 60 is completely unaffected over 2 hours by the reducing enzymes present in the S9-fraction. Thus, the conversion of TFMK group to TFPA group has resulted in TFMK surrogates as metabolically stable potent HDAC inhibitors which maintain a stable keto-hydrate form resistant to reduction by carbonyl reductases. These compounds constitute a new class of HDAC inhibitors with a metabolically stable metal-binding group that may be used as a template for designing selective HDAC inhibitors by varying the nature of the linker and the cap group.
All chemicals and solvents were purchased from commercial sources and used without further purification, unless stated otherwise. 1H and 13C NMR spectra were recorded on Bruker Avance 600 MHz, INOVA 600 MHz, and Varian VXRS 400 MHz NMR spectrometers in deuterated solvents using residual undeuterated solvents as internal standard. High-resolution mass spectra (HRMS) were recorded on a Waters Synapt high-definition mass spectrometer (HDMS) equipped with nano-ESI source. Melting points were determined using a Fisher-Johns melting point apparatus. Purification of crude products was performed by either flash chromatography on silica gel (40-63 micron) from Sorbent Technologies or on a Teledyne ISCO CombiFlash Companion chromatography system on RediSep prepacked silica cartridges. Thin layer chromatography (TLC) plates (20 cm×20 cm) were purchased from Sorbent Technologies (catalog #4115126) and were viewed under Model UVG-54 mineral light lamp UV-254 nm. A Shimadzu Prominence HPLC with an LC20AT solvent delivery system coupled to a Shimadzu Prominence SPD 20AV Dual wavelength UV/Vis absorbance Detector, a Shimadzu C18 column (1.9 μm, 2.1 mm×50 mm) and HPLC grade solvents (MeOH, H2O with 0.1% formic acid) were used to determine the purity of compounds by HPLC. All compounds were >95% pure by HPLC analysis.
Preparation of bromoanilides: A mixture of n-bromoalkanoic acid 1a-1d (40 mmol), aniline (4 mL, 44 mmol, 1.1 equiv), EDCl (7.67 g, 40 mmol, 1 equiv), and 4-dimethylaminopyridine (10 mol %, 488 mg) was stirred in anhydrous dichloromethane (35 mL) at room temperature overnight. The mixture was washed with 1M HCl (25 mL), saturated aqueous sodium bicarbonate (25 mL) and brine (25 mL). The organic layer was dried (Na2SO4), and the mixture was concentrated in vacuo to give 2a-2d, which was used in the next reaction without further purification.
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.8 Hz, 2H), 7.33 (t, J=7.9 Hz, 2H), 7.12 (t, J=7.4 Hz, 1H), 3.46 (t, J=6.5 Hz, 2H), 2.42 (t, J=7.2 Hz, 2H), 2.01-1.95 (m, 2H), 1.94-1.87 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.48 (d, J=7.9 Hz, 2H), 7.30 (t, J=7.9 Hz, 2H), 7.08 (t, J=7.4 Hz, 1H), 3.40 (t, J=6.7 Hz, 2H), 2.36 (t, J=7.5 Hz, 2H), 1.92-1.85 (m, 2H), 1.78-1.70 (m, 2H), 1.55-1.48 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.9 Hz, 2H), 7.32 (t, J=7.9 Hz, 2H), 7.11 (t, J=7.4 Hz, 1H), 3.41 (t, J=6.7 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H), 1.91-1.83 (m, 2H), 1.79-1.71 (m, 2H), 1.52-1.45 (m, 2H), 1.44-1.37 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.9 Hz, 2H), 7.33 (t, J=7.9 Hz, 2H), 7.11 (t, J=7.5 Hz, 1H), 3.42 (t, J=6.8 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H), 1.80-1.69 (m, 2H), 1.49-1.43 (m, 2H), 1.43-1.30 (m, 6H).
Preparation of azidoanilides: A mixture of 2a-2d (5 mmol) and sodium azide (260 mg, 20 mmol, 4 equiv) was heated in N, N-dimethylformamide (5 mL) at 80° C. overnight. The reaction mixture was diluted with ethyl acetate (30 mL) and washed with ice water (5×25 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo and the product was used in the next reaction without purification.
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.9 Hz, 2H), 7.32 (t, J=7.8 Hz, 2H), 7.11 (t, J=7.4 Hz, 1H), 3.32 (t, J=6.7 Hz, 2H), 2.39 (t, J=7.3 Hz, 2H), 1.85-1.77 (m, 2H), 1.71-1.63 (m, 2H).
1H NMR (400 MHz, CDCl3) δ 7.52 (d, J=7.8 Hz, 2H), 7.32 (t, J=7.9 Hz, 2H), 7.11 (t, J=7.4 Hz, 1H), 3.29 (t, J=6.8 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 1.82-1.72 (m, 2H), 1.71-1.59 (m, 2H), 1.53-1.42 (m, 2H).
1H NMR (400 MHz, CDCl3) δ 7.52 (d, J=7.7 Hz, 2H), 7.33 (t, J=7.9 Hz, 2H), 7.12 (t, J=7.3 Hz, 1H), 3.28 (t, J=6.8 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 1.81-1.72 (m, 2H), 1.67-1.61 (m, 2H), 1.49-1.38 (m, 4H).
1H NMR (400 MHz, CDCl3) δ 7.52 (d, J=7.8 Hz, 2H), 7.33 (t, J=7.8 Hz, 2H), 7.11 (t, J=7.3 Hz, 1H), 3.27 (t, J=6.9 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H), 1.78-1.69 (d, J=6.8 Hz, 2H), 1.67-1.52 (m, 2H), 1.47-1.31 (m, 6H).
To a solution of 3a-3d (4 mmol) in methanol (15 mL) was added 10% palladium on carbon. The suspension was stirred under an atmosphere of H2 (3 atm) overnight. The mixture was filtered through celite and concentrated in vacuo to give 4a-4d as a crude product, which was used in the next reaction without purification.
1H NMR (600 MHz, CDCl3) δ 7.53 (d, J=8.0 Hz, 2H), 7.33 (t, J=7.9 Hz, 2H), 7.11 (t, J=7.5 Hz, 1H), 2.77 (t, J=6.8 Hz, 2H), 2.41 (t, J=7.4 Hz, 2H), 1.84-2.77 (m, 2H), 1.59-1.52 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.9 Hz, 2H), 7.31 (t, J=7.7 Hz, 2H), 7.10 (t, J=7.3 Hz, 1H), 2.71 (t, J=6.8 Hz, 2H), 2.37 (t, J=7.4 Hz, 2H), 1.79-1.71 (m, 2H), 1.54-1.46 (m, 2H), 1.46-1.39 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.52 (d, J=7.9 Hz, 2H), 7.31 (t, J=7.7 Hz, 2H), 7.10 (t, J=7.3 Hz, 1H), 2.71 (t, J=6.8 Hz, 2H), 2.37 (t, J=7.4 Hz, 2H), 1.79-1.71 (m, 2H), 1.54-1.46 (m, 2H), 1.46-1.39 (m, 2H).
1H NMR (600 MHz, CDCl3) δ 7.49 (d, J=7.9 Hz, 2H), 7.29 (t, J=7.9 Hz, 2H), 7.07 (t, J=7.4 Hz, 1H), 2.65 (t, J=7.0 Hz, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.75-1.67 (m, 2H), 1.44-1.38 (m, 2H), 1.38-1.28 (m, 6H).
To a stirred solution of pyruvic acid (1.2 equiv.) in DCM (1 mL/mmol), oxalyl chloride (1.2 equiv.) was added dropwise at 0° C., followed by the addition of catalytic amount of DMF (few drops). Then it was allowed to warm at room temperature and stirred for 3 hours. The resulting mixture was transferred with a glass syringe to a suspension of the n-amino-anilide (1 equiv.) and triethylamine (1.6 equiv.) in DCM (1 mL/mmol) at 0° C., under nitrogen balloon. The reaction was left again to warm at room temperature and stirred overnight. Brine was added to the resulting mixture and was extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, filtered, and condensed under reduced pressure. The crude mixture was purified by on silica gel chromatography in petroleum ether/ethyl acetate (0->70% ethyl acetate) yielding pure products.
To a stirred solution of the corresponding pyruvate amides in ethanol (0.04 M) an aqueous solution of the hydroxylamine (2 equiv., 0.4 M concentration) was added, and the reaction was left to stir for 4 hours at room temperature. The resulting mixture was worked up using brine and extracted with ethyl acetate. The combined organic layers were dried using sodium sulfate, filtered, and condensed under reduced pressure resulting in pure oximes. The isomeric mixture of oximes was used without further purification.
To a stirred solution of oxamic acid (1.2 equiv.) in DCM (1 mL/mmol), oxalyl chloride (1.2 equiv.) was added dropwise at 0° C. followed by the addition of catalytic amount of DMF (few drops). Then it was let to warm at room temperature and stirred for 3 hours. The resulting mixture was transferred with a glass syringe to a suspension of the n-amino-anilide (1 equiv.) and triethylamine (1.6 equiv.) in DCM (1 mL/mmol) at 0° C., under nitrogen balloon. The reaction was left again to warm at room temperature and stirred overnight. Brine was added to the resulting mixture and was extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to yield a white solid which was recrystallized using ethyl acetate/acetone with a few drops of MeOH to yield pure compounds.
A mixture of 5-acetoxy-4-oxo-4H-pyran-2-carboxylic acid 94 (0.25 mmol, 1 equiv), dimethylformamide (10 μL), and oxalyl chloride (150 μL of 2.0 M in dichloromethane, 0.3 mmol, 1.2 equiv) in anhydrous dichloromethane (1 mL) under an atmosphere of N2 was stirred for four hours at room temperature. A solution of aminoalkanamide (0.26 mmol, 1.1 equiv) in anhydrous dichloromethane (500 μL) was added over ten minutes and the mixture was stirred overnight at room temperature. Solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (5 mL), washed with brine (3×1 mL), and the solvent was removed under reduced pressure. The crude solid product was dissolved in anhydrous methanol, sodium methoxide (16 mg, 0.3 mmol, 1.2 equiv) was added, and the reaction was continued until completion as monitored by TLC. The solvent was removed under reduced pressure and the residue was dissolved in water (0.5 mL) and washed with ethyl acetate (0.5 mL) three times. The aqueous layer was acidified with 1M HCl (1 mL), and extracted with ethyl acetate (3×1 mL). The organic extract was dried over Na2SO4, concentrated in vacuo, and purified by flash chromatography (methanol/DCM 2-4%) to give compounds 9a-9d.
Synthesized according to general procedure 4. Yield 33%. 1H NMR (600 MHz, CDCl3) δ 7.54 (d, J=7.9 Hz, 2H), 7.43 (s, 1H(NH)), 7.33 (t, J=7.9 Hz, 2H), 7.11 (d, 1H-s, 1H(NH) overlapping), 3.37 (q, J=6.7 Hz, 2H), 2.49 (s, 3H), 2.42 (t, J=7.4 Hz, 2H), 1.81-1.74 (m, 2H), 1.66 (dt, J=14.0, 6.9 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 197, 170.8, 160.4, 129, 124.2, 119.6, 38.5, 36.7, 28.7, 24.5, 22.5.
Synthesized according to general procedure 4. Yield 36%. 1H NMR (600 MHz, CDCl3) δ 7.53 (t, J=9.4 Hz, 2H), 7.40 (s, 1H), 7.32 (t, J=7.8 Hz, 2H), 7.10 (t, J=7.4 Hz, 1H), 7.03 (s, 1H), 3.31 (dd, J=13.6, 6.8 Hz, 2H), 2.47 (s, 3H), 2.37 (t, J=7.4 Hz, 2H), 1.76 (dt, J=15.1, 7.5 Hz, 2H), 1.63-1.55 (m, 2H), 1.46-1.37 (m, 2H). 13C NMR (151 MHz, MeOD) δ 196.3, 173, 172.3, 161.4, 138.4, 128.5, 123.9, 119.8, 38.5, 36.7, 29.2, 28.2, 26.5, 24.9, 24.2, 23.
Synthesized according to general procedure 4. Yield 26%. 1H NMR (600 MHz, CDCl3) δ 7.53 (d, J=8.0 Hz, 2H), 7.32 (dd, J=15.7, 7.8 Hz, 2H), 7.24 (s, 1H), 7.10 (dd, J=18.9, 11.5 Hz, 1H), 6.97 (s, 1H), 3.30 (dd, J=13.4, 6.8 Hz, 2H), 2.49 (s, 3H), 2.36 (t, J=7.4 Hz, 2H), 1.79-1.71 (m, 2H), 1.57 (dt, J=14.0, 6.9 Hz, 2H), 1.41 (dd, J=26.1, 12.5 Hz, 4H). 13C NMR (151 MHz, CDCl3) δ 197.34, 171.1, 160.1, 138.0, 128.9, 124.1, 119.6, 39.5, 37.7, 29.03, 28.6, 26.5, 25.3, 24.2. HRMS calcd for C16H23N2O3 (M+H) 291.1709 found 291.1693.
Synthesized according to general procedure 5. Yield 90%, 1H NMR (600 MHz, CD3OD) δ 7.55 (d, J=7.6 Hz, 2H), 7.31 (t, J=8.0 Hz, 2H), 7.10 (t, J=7.4 Hz, 1H), 2.42 (t, J=7.4 Hz, 2H), 2.05 (s, 2H), 1.98 (s, 3H), 1.77-1.71 (m, 2H), 1.66-1.59 (m, 2H). 13C NMR (151 MHz, MeOD) δ 173.1, 165.3, 150.3, 138.6, 128.2, 123.6, 119.6, 38.5, 35.8, 29.1, 22.6, 8.3. HRMS calcd for C14H20N3O3 (M+H) 278.1505 found 278.1517.
Synthesized according to general procedure 5. Yield 91%. 1H NMR (600 MHz, CD3OD) δ 7.50 (d, J=5.3, 3.3 Hz, 2H), 7.26 (t, J=10.7, 5.2 Hz, 2H), 7.04 (t, J=7.4 Hz, 1H), 3.23 (t, J=7.1 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H), 1.92 (s, 3H), 1.75-1.62 (m, 2H), 1.60-1.48 (m, 2H), 1.43-1.32 (m, 2H). 13C NMR (151 MHz, MeOD) δ 173.1, 165.1, 150.1, 138.5, 128.5, 124.2, 120.3, 38.9, 36.7, 28.9, 26.2, 25.1, 7.7.
Synthesized according to general procedure 5. Yield 94%. 1H NMR (600 MHz, CD3OD) S 7.51 (d, 2H), 7.26 (t, J=7.9 Hz, 2H), 7.04 (t, J=7.4 Hz, 1H), 3.21 (t, J=7.1 Hz, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.93 (s, 3H), 1.70-1.63 (m, 2H), 1.55-1.48 (m, 2H), 1.42-1.31 (m, 4H). 13C NMR (151 MHz, CD3OD) δ 173.1, 164.9, 150.1, 138.6, 128.2, 123.5, 120.1, 38.6, 36.5, 29, 28.4, 26.3, 25.4, 7.9.
Synthesized according to general procedure 6. Yield 24%. 1H NMR (600 MHz, DMSO) δ 9.87 (s, 1H), 8.74 (t, J=5.9 Hz, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.58 (d, J=7.6 Hz, 2H), 7.28 (t, J=7.9 Hz, 2H), 7.02 (t, J=7.4 Hz, 1H), 3.14 (dd, J=13.3, 6.7 Hz, 2H), 2.31 (t, J=7.3 Hz, 2H), 1.60-1.45 (m, 4H). 13C NMR (151 MHz, DMSO) δ 171.5, 162.7, 160.7, 139.7, 129.2, 123.3, 119.4, 39, 36.5, 28.9, 22.9.
Synthesized according to general procedure 6. Yield 21%. 1H NMR (600 MHz, DMSO) δ 9.86 (s, 1H), 8.70 (t, J=6.0 Hz, 1H), 8.04 (s, 1H), 7.76 (s, 1H), 7.59 (d, J=7.6 Hz, 2H), 7.28 (t, J=7.9 Hz, 2H), 7.02 (t, J=7.4 Hz, 1H), 3.11 (dd, J=13.5, 6.8 Hz, 2H), 2.29 (t, J=7.5 Hz, 2H), 1.64-1.54 (m, 2H), 1.53-1.44 (m, 2H), 1.32-1.23 (m, 2H). 13C NMR (151 MHz, DMSO) δ 171.6, 162.8, 160.6, 139.8, 129.1, 123.4, 119.5, 39.2, 36.8, 29, 26.5, 25.3.
Synthesized according to general procedure 6. Yield 35%. 1H NMR (600 MHz, DMSO) δ 9.85 (s, 1H), 8.68 (t, J=6.0 Hz, 1H), 8.03 (s, 1H), 7.76 (s, 1H), 7.59 (d, J=7.7 Hz, 2H), 7.28 (t, J=7.9 Hz, 2H), 7.02 (t, J=7.4 Hz, 1H), 3.10 (dd, J=13.5, 6.8 Hz, 2H), 2.29 (t, J=7.5 Hz, 2H), 1.60-1.54 (m, 2H), 1.49-1.40 (m, 2H), 1.33-1.19 (m, 6H). 13C NMR (151 MHz, DMSO) δ 171.7, 162.9, 160.6, 139.8, 129, 123.6, 119.1, 39.2, 36.9, 29.2, 29.1, 29, 26.7, 25.6.
To a stirred suspension of 1H-pyrrole-2-carboxylic acid (230 mg, 2.1 mmol) in DCM (8 mL), were added, EDC-HCl (600 mg, 3.1 mmol, 1.5 equiv.), cat. amount of DMAP (approximately 10 mol %), and 6-amino-N-phenylhexanamide 4b (428 mg, 2.1 mmol, 1 equiv.). The resulting mixture was stirred at room temperature overnight, after which it was diluted with brine and ethyl acetate. The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was separated on silica gel chromatography in ethyl acetate/hexanes to provide N-(6-oxo-6-(phenylamino) hexyl)-1H-pyrrole-2-carboxamide (8b) (403 mg, 64% yield). 1H NMR (600 MHz, CD3CN) δ 9.93 (s, 1H), 8.32 (s, 1H), 7.57 (d, J=8.0 Hz, 2H), 7.32 (t, J=7.9 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 6.90 (d, J=1.1 Hz, 1H), 6.79 (s, 1H), 6.65 (s, 1H), 6.17 (dt, J=5.3, 1.6 Hz, 1H), 3.33 (dd, J=13.2, 6.7 Hz, 2H), 2.34 (t, J=7.4 Hz, 2H), 1.97 (dt, J=4.8, 2.4 Hz, 2H), 1.74-1.66 (m, 2H), 1.63-1.56 (m, 2H), 1.45-1.38 (m, 2H). 13C NMR (151 MHz, CD3CN) δ 171.6, 160.8, 139.1, 128.8, 126.5, 123.4, 120.9, 119.5, 109.2, 108.81, 38.5, 36.6, 29.2, 26.2, 24.9. HRMS calcd for C17H22N3O2 (M+H) 264.1348 found 264.1354.
Synthesized according to general procedure 7. 12.75 mg (16%). 1H NMR (400 MHz, CD3OD) δ 8.00 (s, 1H), 7.53 (d, J=7.7 Hz, 2H), 7.29 (t, J=7.9 Hz, 2H), 7.08 (d, J=7.3 Hz, 1H), 7.05 (s, 1H), 3.43-3.38 (m, 2H), 2.42 (t, J=7.2 Hz, 2H), 1.80-1.64 (m, 4H). 13C NMR (151 MHz, CD3OD) S 175, 172.8, 159.4, 155.8, 147.7, 139.2, 138.5, 128.4, 123.7, 119.8, 112.8, 39.2, 39.1, 36, 28.4, 22.7. HRMS-ESI M/z: [M+H]+ calcd. 331.1293 for C17H18N2O5. found 331.1310.
Synthesized according to general procedure 7.14 mg (17.6%). 1H NMR (400 MHz, CD3OD) δ 7.99 (s, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.28 (d, J=7.5 Hz, 2H), 7.06 (t, J=7.4 Hz, 1H), 7.02 (s, 1H), 3.38 (q, J=6.8 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 1.79-1.70 (m, 2H), 1.70-1.59 (m, 2H), 1.50-1.35 (m, 2H). 13C NMR (151 MHz, CD3OD) δ 175, 173.1, 159.4, 155.8, 147.7, 139.2, 138.5, 128.4, 123.7, 119.8, 112.7, 39.4, 39.2, 36.3, 28.6, 26, 25.1. HRMS-ESI M/z: [M+H]+ calcd. 345.145 for C18H20N2O5. found 345.1437.
Synthesized according to general procedure 7. 12.4 mg (14%). 1H NMR (600 MHz, CD3OD) δ 8.05 (s, 1H), 7.55 (d, J=7.9 Hz, 2H), 7.30 (t, J=7.8 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 7.07 (s, 1H), 3.41-3.38 (m, 2H), 2.40 (t, J=7.4 Hz, 2H), 1.78-1.69 (m, 2H), 1.68-1.61 (m, 2H), 1.49-1.40 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 175, 173.2, 159.4, 159.3, 155.9, 155.9, 147.6, 139.3, 138.5, 128.4, 123.7, 119.8, 112.8, 39.5, 39.4, 36.4, 28.7, 28.5, 26.3, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 359.1606 for C19H22N2O5. found 359.1618.
Synthesized according to general procedure 7. 20 mg (6%). 1H NMR (600 MHz, CD3OD) S 8.01 (s, 1H), 7.55 (dd, J=8.5, 0.9 Hz, 2H), 7.31 (t, J=7.2 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 7.05 (s, 1H), 3.37 (t, J=7.2 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.76-1.69 (m, 2H), 1.66-1.59 (m, 2H), 1.4-1.36 (m, 6H). 13C NMR (151 MHz, CD3OD): δ 175.4, 159.3, 155.7, 139.2, 138.5, 128.4, 123.7, 119.8, 112.7, 39.4, 36.5, 28.8, 28.6, 26.4, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 373.1763 for C20H24N2O5. found 373.1767.
In a 250 mL round-bottom flask equipped with a stir-bar, 4-aminobutanoic acid (11) (5 g, 48.5 mmol, 1 equiv) was first dissolved in a mixture of acetone (50 mL) and water (50 mL). Triethylamine (13.6 mL, 97 mmol, 2 equiv) was added to the reaction mixture, followed by di-tert-butyl dicarbonate (10.92 g, 75 mmol, 1.03 equiv). The mixture was stirred at room temperature for four hours. Acetone was removed under reduced pressure and the residual water was acidified with 1M HCl (200 mL) and extracted with ethyl acetate (3×100 mL). The organic extract was dried (Na2SO4), concentrated in vacuo and triturated in hexanes to yield 4-((tert-butoxycarbonyl) amino) butanoic acid (12) as a white solid (9.23 g, 94%).
In a 10 mL round—bottomed flask, a mixture of 4-((tert-butoxycarbonyl) amino) butanoic acid (2 mmol, 490 mg), aniline (2 mmol, 182 μL), EDCl (2 mmol, 384 mg, 1 equiv), DMAP (10 mol %, 24 mg) was stirred in dichloromethane (4 mL) under an atmosphere of nitrogen (balloon) overnight. Solvent was removed under reduced pressure, and the reaction mixture was diluted in ethyl acetate (10 mL) and washed with 1 M HCl (10 mL), saturated NaHCO3(10 mL) (aq.) and brine (10 mL). The organic layer was dried (Na2SO4), concentrated in vacuo, and purified using flash chromatography (ethyl acetate/hexanes 10-70%) to yield tert-butyl (4-oxo-4-(phenylamino) butyl) carbamate as a white solid, 450 mg, 81%. 1H NMR (600 MHz, CDCl3) δ 7.62 (d, J=7.7 Hz, 2H), 7.33 (t, J=7.9 Hz, 2H), 7.10 (t, J=7.3 Hz, 1H), 3.30-3.22 (m, 2H), 2.46-2.34 (m, 2H), 1.95-1.84 (m, 2H), 1.47 (s, 9H).
In a 250 mL round—bottom flask equipped with a stir-bar, 7-aminoheptanoic acid (15) (12.72 g, 70 mmol, 1 equiv) was first dissolved in a mixture of acetone (50 mL) and water (50 mL). Triethylamine (19.53 mL, 140 mmol, 2 equiv) was added to the reaction mixture, followed by di-tert-butyl dicarbonate (16.37 g, 75 mmol, 1.07 equiv). The mixture was stirred at room temperature for four hours. Acetone was first removed under reduced pressure and the residual water was acidified with 1M HCl (200 mL) and extracted with ethyl acetate (100 mL) three times. The organic extract was dried (Na2SO4), concentrated in vacuo, and triturated in hexanes to yield 7-((tert-butoxycarbonyl) amino) heptanoic acid (16) as a white solid (15.7 g, 91%).
In a 10 mL round-bottomed flask, a mixture of 7-((tert-butoxycarbonyl) amino) heptanoic acid (2 mmol, 490 mg), the respective aminoarene (2 mmol), EDCl (2 mmol, 384 mg, 1 equiv), DMAP (10 mol %, 24 mg) was stirred in dichloromethane (4 mL) under an atmosphere of nitrogen (balloon) overnight. Solvent was removed under reduced pressure, and the reaction mixture was diluted in ethyl acetate (10 mL) and washed with 1 M HCl (10 mL), saturated NaHCO3(10 mL) (aq.) and brine (10 mL). The organic layer was dried (Na2SO4), concentrated in vacuo, and purified using flash chromatography (ethyl acetate/hexanes 10-70%) to yield Boc-arenes 17-50.
An alternative procedure involved using a mixture of 7-((tert-butoxycarbonyl) amino) heptanoic acid (2 mmol, 490 mg), the aminoarene (2 mmol), HATU (2 mmol, 760 mg, 1 equiv), HOBt (1 mmol, 135 mg, 1 equiv), and NMM (660 μl, 6 mmol, 3 equiv) in dimethylformamide (4 mL) under an atmosphere of nitrogen (balloon) overnight. The reaction mixture was diluted in ethyl acetate (10 mL), washed with ice water (5×10 mL) and dried (Na2SO4). The organic layer was concentrated in vacuo and purified in the same manner as above.
1H NMR (400 MHz, CDCl3) δ 7.94 (d, J=8.7 Hz, 2H), 7.69 (d, J=8.3 Hz, 2H), 3.14 (q, J=6.1 Hz, 2H), 2.39 (t, 2H), 1.80-1.71 (m, 2H), 1.54-1.48 (m, 2H), 1.46 (s, 9H), 1.43-1.34 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 200 mg (29%) mp. 120-125° C. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J=8.5 Hz, 2H), 6.86 (d, J=8.5 Hz, 2H), 3.80 (s, 3H), 3.13 (q, J=5.1 Hz, 2H), 2.33 (t, J=7.3 Hz, 2H), 1.80-1.70 (m, 2H), 1.54-1.47 (m, 2H), 1.45 (s, 9H), 1.41-1.31 (m, 4H). 13CNMR (151 MHz, CDCl3): 171.3, 156.3, 156.2, 131.2, 121.7, 114.1, 79.2, 55.5, 40.2, 37.3, 29.9, 28.5, 28.5, 26.1, 25.5.
White solid, 145 mg (21.4%) mp. 90-95° C. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J=8.6 Hz, 2H), 6.86 (d, J=8.9 Hz, 2H), 3.17-3.06 (m, 2H), 2.33 (t, J=7.3 Hz, 2H), 1.79-1.68 (m, 2H), 1.54-1.48 (m, 2H), 1.45 (s, 9H), 1.41-1.31 (m, 2H). 13CNMR (151 MHz, CDCl3): 171.6, 160, 158.4, 156.3, 134.2, 121.6, 121.5, 115.6, 115.5, 79.3, 40.1, 37.2, 29.9, 28.5, 28.3, 26, 25.4.
1H NMR (400 MHz, CDCl3) δ 8.48 (d, J=5.4 Hz, 2H), 7.61 (d, J=4.6 Hz, 2H), 3.15 (q, 6.5 Hz, 2H), 2.40 (t, J=6.9 Hz, 2H), 1.80-1.70 (m, 2H), 1.53-1.49 (m, 2H), 1.47 (s, 9H), 1.43-1.34 (m, 4H). Obtained a partially purified product, which was used in the next step. Also, it was slow to purification until 4-7% methanol/DCM was used.
1H NMR (400 MHz, CDCl3) δ 7.69 (s, 4H), 3.13 (q, J=5.7 Hz, 2H), 2.37 (t, J=7.1 Hz, 2H), 1.79-1.70 (m, 2H), 1.53-1.48 (m, 2H), 1.45 (s, 9H), 1.41-1.35 (m, 4H). Obtained a partially purified product, which was used in the next step.
White solid, 300 mg (42%) mp. 75-80° C. 1H NMR (400 MHz, CDCl3) δ 8.26 (dd, J=15.3, 9.0 Hz, 1H), 6.93-6.79 (m, 2H), 3.12 (q, J=5.8 Hz, 2H), 2.40 (t, J=7.5 Hz, 2H), 1.80-1.68 (m, 2H), 1.54-1.48 (m, 2H), 1.45 (s, 9H), 1.42-1.32 (m, 4H). 13CNMR (151 MHz, CDCl3): 171.6, 160, 158.4, 156.3, 134.2, 121.6 (d, J=7.8 Hz), 115.6 (d, J=22.4 Hz), 79.3, 40.1, 37.2, 29.9, 28.3, 26, 25.4.
1H NMR (400 MHz, CDCl3) δ 6.73 (t, J=8.1 Hz, 2H), 3.12 (q, J=7.0 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H), 1.92-1.81 (m, 2H), 1.57-1.48 (m, 2H), 1.44 (s, 9H), 1.40-1.30 (m, 4H). Obtained a crude product which was used in the next step without purification.
1H NMR (600 MHz, CDCl3) δ 7.79 (d, J=7.2 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.4 Hz, 1H), 7.13 (s, 1H), 3.16-3.07 (m, 2H), 2.28 (t, J=7.5 Hz, 2H), 1.71-1.59 (m, 2H), 1.47 (s, 9H), 1.43-1.30 (m, 4H). Obtained a partially purified product that was used in the next step.
1HNMR (400 MHz, CDCl3): δ 6.91 (s, 2H), 3.15 (d, J=6 Hz, 2H), 2.36 (t, J=7.2 Hz, 2H), 1.77-1.69 (m, 2H), 1.51-1.45 (m, 2H), 1.44 (s, 9H), 1.4-1.35 (m, 4H). 13CNMR (151 MHz, CDCl3): δ 171.6, 153.3, 134.4, 134.3, 97.3, 79.3, 61, 56.1, 40.12, 37.34, 30, 28.5, 28.3, 29.6, 25.4.
Pink solid, 778 mg, quantitative yield, mp. 90-95° C. 1H NMR (600 MHz, CDCl3) δ 7.92-7.86 (m, 3H), 7.71 (d, J=8.0 Hz, 1H), 7.54-7.50 (m, 2H), 7.47 (t, J=7.7 Hz, 1H), 3.14 (d, J=5.4 Hz, 2H), 2.51 (t, J=7.2 Hz, 2H), 1.85-1.77 (m, 2H), 1.55-1.49 (m, 2H), 1.46 (s, 9H), 1.41-1.31 (m, 4H). 13C NMR (151 MHz, CDCl3): 172, 156.1, 134.1, 132.4, 128.7, 128.5, 127.3, 126.2, 126, 125.8, 125.7, 121.3, 120.9, 79.1, 40.4, 37.4, 30, 28.8, 28.5, 26.4, 25.7.
1H NMR (600 MHz, CDCl3) δ 7.81 (d, J=8.6 Hz, 2H), 7.77 (t, J=8.3 Hz, 4H), 7.59 (t, J=7.4 Hz, 1H), 7.48 (t, J=7.7 Hz, 2H), 3.11 (d, J=6.0 Hz, 2H), 2.38 (t, J=7.2 Hz, 2H), 1.75-1.68 (m, 2H), 1.50-1.47 (m, 2H), 1.46 (s, 9H), 1.38-1.29 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 196, 172.3, 156.3, 142.7, 137.9, 132.5, 132.3, 131.6, 130.1, 129.9, 128.3, 128.1, 118.8, 79.3, 40.2, 37.3, 30, 28.5, 28.4, 26.1, 25.4.
Brown solid, 500 mg (65.7%) mp. 80-85° C. 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J=2.5 Hz, 1H), 6.79 (d, J=8.9 Hz, 1H), 6.57 (dd, J=8.9, 2.9 Hz, 1H), 3.85 (s, 3H), 3.79 (s, 3H), 3.12 (d, J=6.3 Hz, 2H), 2.40 (t, J=7.4 Hz, 2H), 1.78-1.70 (m, 2H), 1.54-1.47 (m, 2H), 1.45 (s, 9H), 1.42-1.32 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 171.2, 153.9, 141.8, 128.4, 110.6, 108.5, 105.7, 56.2, 55.8, 40.5, 37.9, 29.9, 28.9, 28.4, 26.6, 25.4.
White solid, 588 mg (60.1%) mp. 140-145° C. 1H NMR (600 MHz, CDCl3) δ 7.62 (d, J=7.9 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H), 7.02 (d, J=8.4 Hz, 2H), 3.13 (d, J=5.8 Hz, 2H), 2.40 (s, 3H), 2.34 (t, J=7.2 Hz, 2H), 1.76-1.69 (m, 2H), 1.52-1.48 (m, 2H), 1.47 (s, 9H), 1.43-1.33 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.5, 156.2, 143.9, 135.9, 132, 129.7, 127.3, 123.5, 120.5, 40.1, 37.3, 30, 28.5, 28.3, 25.9, 25.4, 21.6.
1H NMR (600 MHz, CDCl3) δ 8.41 (dd, J=8.0, 1.3 Hz, 1H), 7.05 (td, J=7.9, 1.5 Hz, 1H), 6.98 (td, J=7.8, 1.2 Hz, 1H), 6.90 (dd, J=8.1, 1.1 Hz, 1H), 3.91 (s, 3H), 3.14 (q, J=6.2 Hz, 2H), 2.41 (t, J=7.5 Hz, 2H), 1.80-1.72 (m, 2H), 1.55-1.48 (m, 2H), 1.46 (s, 9H), 1.44-1.36 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.1, 156, 147.7, 127.7, 123.5, 121.1, 119.7, 109.8, 79, 55.64, 40.5, 37.9, 29.9, 28.9, 28.4, 26.5, 25.5.
White solid, 597 mg (8%) mp. 105-110° C. 1H NMR (600 MHz, CDCl3) δ 7.54 (d, J=8.4 Hz, 2H), 7.30 (d, J=8.9 Hz, 2H), 3.15 (q, J=6.3 Hz, 2H), 2.36 (t, J=7.3 Hz, 2H), 1.79-1.72 (m, 2H), 1.54-1.49 (m, 2H), 1.47 (s, 9H), 1.44-1.34 (m, 4H). 13C NMR (151 MHz, CDCl3): 171.6, 156.3, 136.8, 129, 121, 79.3, 40, 37.3, 30, 28.5, 28.5, 28.22, 25.9, 25.4.
White solid, 552 mg (69%) mp. 115-120° C. 1H NMR (600 MHz, CDCl3) δ 7.49 (d, J=8.4 Hz, 2H), 7.44 (d, J=8.8 Hz, 2H), 3.15 (q, J=6.1 Hz, 2H), 2.36 (t, J=7.3 Hz, 2H), 1.80-1.73 (m, 2H), 1.53-1.48 (m, 2H), 1.47 (s, 9H), 1.45-1.35 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.59, 156.2, 137.3, 131.9, 121.3, 116.5, 79.3, 40.1, 37.3, 30, 28.5, 28.3, 25.9, 25.4.
White solid, 500 mg, (63%) mp. 150-155° C. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J=8.4 Hz, 2H), 7.60-7.54 (m, 4H), 7.43 (t, J=7.6 Hz, 2H), 7.33 (t, J=7.3 Hz, 1H), 3.14 (q, J=6.4 Hz, 2H), 2.38 (t, J=7.3 Hz, 2H), 1.82-1.72 (m, 2H), 1.54-1.48 (m, 2H), 1.46 (s, 9H), 1.43-1.35 (m, 4H). 13C NMR (151 MHz, CDCl3): 171.5, 156.2, 140.6, 137.5, 136.9, 128.8, 127.6, 127.1, 126.9, 120, 79.3, 40.2, 37.4, 30, 28.6, 28.6, 26.13, 25.5.
White solid, 512 mg (78%) mp. 85-90° C. 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J=3.6 Hz, 1H), 7.01 (d, J=3.6 Hz, 1H), 3.11 (d, J=6.2 Hz, 2H), 2.55 (t, J=7.4 Hz, 2H), 1.84-1.73 (m, 2H), 1.54-1.47 (m, 2H), 1.44 (s, 9H), 1.40-1.31 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.2, 160, 156, 136, 113.5, 79.1, 40.4, 36.1, 29.9, 28.9, 28.5, 26.5, 25.
1H NMR (400 MHz, CDCl3) δ 7.82 (d, J=7.8 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.44 (t, J=7.7 Hz, 1H), 7.32 (t, J=7.5 Hz, 1H), 3.17-3.04 (m, 2H), 2.52 (t, J=7.4 Hz, 2H), 1.79-1.67 (m, 2H), 1.56-1.46 (m, 2H), 1.44 (s, 9H), 1.41-1.26 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 520 mg (74%) mp. 80-85° C. 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H), 7.16 (t, J=7.8 Hz, 1H), 7.03 (t, J=7.8 Hz, 1H), 6.62 (d, J=8.1 Hz, 1H), 3.74 (s, 3H), 3.06 (q, J=6.3 Hz, 2H), 2.3 (t, J=7.2 Hz, 2H), 1.78-1.68 (m, 2H), 1.43 (s, 9H), 1.4-1.26 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.6. 160.1, 156.2, 139.4, 129.6, 11.8, 110, 105.3, 79.2, 40.2, 37.5, 30, 28.5, 26.2, 25.4.
1H NMR (400 MHz, CDCl3) δ 7.56 (d, J=7.3 Hz, 2H), 7.43 (t, J=7.5 Hz, 2H), 7.35 (t, J=7.3 Hz, 1H), 3.09 (q, J=5.6 Hz, 2H), 2.49 (s, CH3), 2.15 (t, J=6.7 Hz, 2H), 1.66-1.53 (m, 2H), 1.44 (s, 9H), 1.31-1.19 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.6, 156.4, 144.2, 135, 129.6, 128.5, 128.4, 128.38, 127.7, 121.8, 79.1, 40.5, 35, 29.8, 28.6, 28.5, 26.4, 24.5, 12.1.
1H NMR (400 MHz, CDCl3) δ 7.42 (dd, J=6.6, 3.0 Hz, 1H), 7.31 (m, 4H), 3.12 (d, J=6.0 Hz, 2H), 2.41-2.30 (m, 2H), 1.76-1.61 (m, 4H), 1.45 (s, 9H), 1.40-1.22 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.8, 158, 156, 143.7, 131.9, 129.5, 129.4, 129.1, 128.8, 128.7, 128.6, 128, 127.8, 126.7, 79.1, 40.5, 35, 29.8, 28.6, 28.5, 26.4, 24.5.
1H NMR (600 MHz, CDCl3) δ 8.36 (d, J=7.0 Hz, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.32 (t, J=7.7 Hz, 1H), 6.98 (t, J=7.5 Hz, 1H), 3.13 (d, J=5.2 Hz, 2H), 2.44 (t, J=7.4 Hz, 2H), 1.80-1.73 (m, 2H), 1.54-1.47 (m, 2H), 1.45 (s, 9H), 1.42-1.34 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 420 mg (52.6%) mp. 90-95° C. 1H NMR (600 MHz, CDCl3) δ 7.83 (s, 1H), 7.47 (d, J=7.0 Hz, 1H), 7.23 (d, J=7.8 Hz, 1H), 7.17 (t, J=8.0 Hz, 1H), 3.13 (d, J=5.5 Hz, 2H), 2.35 (t, J=7.3 Hz, 2H), 1.77-1.70 (m, 2H), 1.53-1.47 (m, 2H), 1.46 (s, 9H), 1.42-1.33 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.9, 156.3, 139.6, 130.2, 126.9, 122.7, 122.5, 118.2, 79.3, 40.2, 37.2, 30, 28.5, 28.3, 26, 25.4.
1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.65 (d, J=8.6 Hz, 1H), 7.40 (d, J=8.6 Hz, 1H), 3.13 (q, J=5.3 Hz, 2H), 2.52 (t, J=7.3 Hz, 2H), 1.83-1.73 (m, 2H), 1.55-1.49 (m, 2H), 1.45 (s, 9H), 1.43-1.33 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 400 mg (50.6%) mp. 150-155° C. 1H NMR (400 MHz, CDCl3) δ 7.69 (dd, J=8.8, 4.7 Hz, 1H), 7.52 (dd, J=8.1, 2.5 Hz, 1H), 7.17 (td, J=8.9, 2.6 Hz, 1H), 3.12 (q, J=6.3 Hz, 2H), 2.49 (t, J=7.3 Hz, 2H), 1.83-1.70 (m, 2H), 1.53-1.47 (m, 2H), 1.45 (s, 9H), 1.41-1.30 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 171.8, 160.4, 158.8, 156, 144.2, 133 (d, J=10.5 Hz), 121.3 (d, J=8.6 Hz), 114.7 (d, J=24.6 Hz), 108 (d, J=26.6 Hz), 79.2, 40.4, 36.3, 29.9, 28.6, 28.5, 26.3, 24.8.
Yellow solid, 160 mg (17.%) mp. 180-185° C. 1H NMR (600 MHz, DMSO) δ 8.25 (s, 1H), 7.67 (d, J=8.5 Hz, 1H), 7.57 (d, J=8.2 Hz, 1H), 6.79 (s, 1H), 2.89 (d, J=5.8 Hz, 2H), 2.48 (t, J=7.1 Hz, 2H), 1.64-1.57 (m, 2H), 1.37 (s, 9H), 1.32-1.20 (m, 4H). 13C NMR (151 MHz, DMSO) δ 173, 159.2, 156, 148.2, 134.1, 129.6, 124.7, 122.5, 115.8, 77.8, 35.5, 29.8, 28.7, 26.5, 24.9.
Brown solid, 437 mg, (49.6%) mp. 140-145° C. 1H NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 7.39-7.32 (m, 2H), 3.13 (q, J=6.3 Hz, 2H), 2.75-2.61 (m, 2H), 1.83-1.72 (m, 2H), 1.55-1.47 (m, 2H), 1.45 (s, 9H), 1.41-1.25 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 171.3, 156.4, 156.1, 147.2, 126.7, 121.4, 117.5, 111.5, 79.2, 40.4, 36.7, 29.9, 28.6, 28.5, 26.3, 24.9, 24.6.
1H NMR (400 MHz, CDCl3) δ 7.60 (d, J=6.8 Hz, 1H), 7.47 (d, J=8.2 Hz, 1H), 7.32 (t, J=7.0 Hz, 1H), 7.23 (t, J=9.5 Hz, 1H), 3.14 (q, J=6.6 Hz, 2H), 2.78-2.63 (m, 2H), 1.84-1.73 (m, 2H), 1.57-1.48 (m, 2H), 1.46 (s, 9H), 1.43-1.30 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 446 mg (54. %) mp. 105-110° C. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J=8.9 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H), 7.03 (dd, J=8.9, 2.5 Hz, 1H), 3.86 (s, 3H), 3.05 (q, J=5.7 Hz, 2H), 1.71-1.61 (m, 2H), 1.52-1.46 (m, 2H), 1.43 (s, 9H), 1.32-1.17 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.6, 157.3, 156.9, 156, 142, 133.2, 121, 115.3, 104.4, 79.1, 55.9, 40.4, 36.3, 29.9, 28.7, 28.5, 26.4, 24.9.
White solid, 571 mg (70%) mp. 85-90° C. 1H NMR (400 MHz, CDCl3): δ 7.69 (d, J=8.6 Hz, 2H), 7.24 (s, 1H), 6.97 (d, J=8.7 Hz, 2H), 3.86 (s, 3H), 3.06 (d, J=6.4 Hz, 2H), 2.46 (t, J=6.5 Hz, 2H), 1.75-1.63 (m, 2H), 1.54-1.47 (m, 2H), 1.44 (s, 9H), 1.35-1.19 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.8, 160.7, 159.1, 156, 149.1, 123.8, 122, 113.1, 104.4, 79.1, 55.7, 40.4, 36.3, 29.9, 28.8, 28.5, 26.4, 24.9.
1H NMR (400 MHz, CDCl3) δ 7.73 (d, J=8.9 Hz, 2H), 7.00 (s, 1H), 6.95 (d, J=8.8 Hz, 2H), 3.85 (s, 3H), 3.18-3.06 (m, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.79-1.60 (m, 2H), 1.45 (s, 9H), 1.34-1.21 (m, 4H). Obtained a partially purified product that was used in the next step.
White solid, 374 mg (38.7%) mp. 140-145° C. 1H NMR (600 MHz, CDCl3) δ 7.64 (d, J=7.5 Hz, 2H), 7.51 (d, J=7.5 Hz, 2H), 7.12 (s, 1H), 3.14-3.04 (m, 2H), 2.23-2.12 (m, 2H), 1.70-1.48 (m, 2H), 1.39 (s, 9H), 1.35-1.03 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 171.3, 159, 156.1, 148.3, 133.2, 132, 127.7, 122, 108.3, 79.2, 40.4, 35.9, 29.9, 28.6, 28.5, 26.3, 24.8.
White solid, 520 mg (44%) mp. 140-145° C. 1H NMR (600 MHz, CDCl3) δ 7.88 (d, J=8.8 Hz, 2H), 7.30 (d, J=8.7 Hz, 2H), 7.16 (s, 1H), 4.91 (s, 2H), 3.13 (d, J=5.2 Hz, 2H), 2.37 (t, J=7.2 Hz, 2H), 1.79-1.63 (m, 2H), 1.47 (s, 9H), 1.38-1.23 (m, 4H). 13C NMR (151 MHz, CDCl3): δ 170.9, 158.3, 152.5, 150.6, 148.3, 132.6, 127.3, 121.2, 108.1, 94.1, 79.2, 40.3, 36, 29.9, 28.6, 28.5, 26.3, 24.8.
A mixture of tert-butyl(7-oxo-7-((5-(4-(((2,2,2-trichloroethoxy)carbonyl) oxy)phenyl) thiazol-2-yl)amino) heptyl)carbamate (500 mg, 0.84 mmol) was dissolved in 19 ml of methanol, followed by addition of 1 ml 2M NaOH (aq.) (2 mmol, 2.38 equiv) and the reaction was continued until completion, monitored by TLC. The solvent was removed under reduced pressure and the water layer was first cooled using an ice bath, followed by slow addition of conc. HCl until a white solid was obtained. The solid was filtered and dried under vacuum to yield 7-((5-(4-hydroxyphenyl) thiazol-2-yl) amino)-7-oxoheptan-1-aminium chloride as a crude product that was used in the next reaction without further purification. 1H NMR (600 MHz, CD3OD): δ 7.70 (d, J=8.6 Hz, 2H), 6.86 (d, J=8.6 Hz, 2H), 2.95 (t, J=7.1 Hz, 2H), 2.61 (t, J=7.3 Hz, 2H), 1.84-1.75 (m, 2H), 1.73-1.68 (m, 2H), 1.51-1.45 (m, 4H).
A mixture of tert-butyl(7-oxo-7-((5-(4-(((2,2,2-trichloroethoxy)carbonyl) oxy)phenyl) thiazol-2-yl) amino) heptyl) carbamate (440 mg, 0.742 mmol) and sodium hydroxide (31 mg, 0.78 mmol, 1.05 equiv, 0.02 M) was stirred in methanol (39 ml) at room temperature for 3 hours, after which the solvent was removed under reduced pressure, water was added to the reaction mixture (5 mL), and the reaction mixture was washed with ethyl acetate (5 mL). The aqueous layer was slowly acidified and extracted with ethyl acetate (5 mL) three times. The organic extract was dried (Na2SO4) concentrated and purified using flash chromatography (ethyl acetate/hexanes 30-70%) to give tert-butyl (7-((5-(4-hydroxyphenyl) thiazol-2-yl) amino)-7-oxoheptyl) carbamate (183 mg, 0.44 mmol) as a partially purified product that was used in the next step. 1H NMR (600 MHz, CD3OD) δ 7.71 (d, J=8.7 Hz, 2H), 7.20 (s, 1H), 6.84 (d, J=8.7 Hz, 2H), 3.05 (t, J=7.0 Hz, 2H), 2.54 (t, J=7.5 Hz, 2H), 1.79-1.71 (m, 2H), 1.54-1.48 (m, 2H), 1.45 (s, 9H), 1.44-1.35 (m, 4H).
To a 10 mL round bottom flask was added N,N-dimethyl formamide (1 mL), trifluoro pyruvic acid (160 mg, 1 mmol, 1 equiv), HATU (380 mg, 1 mmol, eq,), HOBt (135 mg, 1 mmol, 1 equiv), NMM (330 μl, 3 mmol, 3 equiv), followed by 4a-4d (1 mmol, 1 equiv) and molecular beads. The mixture was stirred for four hours, ethyl acetate was added to the mixture (5 mL), and the mixture was washed several times with ice water (3 mL). The organic layer was dried (Na2SO4) and the mixture was concentrated in vacuo, and purified by flash chromatography (MeOH/DCM 1.5-2%) to yield the respective TFPAs.
White solid, 64 mg (19.1%) mp. 108-113° C. 1H NMR (600 MHz, CD3OD): δ 7.56 (d, J=7.6 Hz, 2H), 7.31 (t, J=7.5 Hz, 2H), 7.10 (t, J=7.4 Hz, 1H), 3.34-3.32 (m, 2H), 2.42 (t, J=7.4 Hz, 2H), 1.79-1.71 (m, 2H), 1.68-1.57 (m, 2H). 13C NMR (151 MHz, CD3OD): δ 172.9, 165.3, 138.5, 128.4, 123.7, 121.8 (q, J=287.3 Hz), 119.80, 94.3 (q, J=32 Hz), 38.8, 35.9, 28.4, 22.6. HRMS-ESI M/z: [M+H]+ calcd. 335.1218 for C14H17F3N2O4. found 335.1218, 99% purity.
White solid, 41 mg (11.8%) mp. 103-108° C. 1H NMR (400 MHz, CD3OD) δ 7.53 (d, J=7.6 Hz, 2H), 7.28 (t, J=8.0 Hz, 2H), 7.07 (t, J=7.4 Hz, 1H), 3.26 (q, J=7.0 Hz, 2H), 2.36 (t, J=7.4 Hz, 2H), 1.85-1.64 (m, 2H), 1.64-1.52 (m, 2H), 1.52-1.33 (m, 2H). 13C NMR (151 MHz, CD3OD): δ 173.1, 165.2, 138.5, 128.4, 123.7, 121.8 (q, J=287.2 Hz), 119.8, 94.3, (q, J=32 Hz), 39, 36.4, 28.6, 26.1, 25.1. HRMS-ESI M/z: [M+H]+ calcd. 349.1375 for C15H19F3N2O4. found 349.136, 99% purity.
White solid, 72 mg (26.4%) mp. 123-128° C. 1H NMR (600 MHz, CD3OD) δ 7.55 (d, J=7.5 Hz, 2H), 7.31 (t, J=8.4 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 3.28 (q, J=7.0 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.77-1.68 (m, 2H), 1.64-1.53 (m, 2H), 1.47-1.32 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 172.2, 165.2, 138.5, 128.4, 123.7, 121.8, (q, J=287.3 Hz), 119.9, 94.3 (q, J=32 Hz), 39.1, 36.5, 28.7, 28.5, 26.2, 25.42. HRMS-ESI M/z: [M+H]+ calcd. 363.1531 for C16H21F3N2O4. found 363.1531, 96% purity.
White solid, 139 mg (37%) mp. 103-108° C. 1H NMR (600 MHz, CD3OD) δ 7.56 (d, J=7.7 Hz, 2H), 7.31 (t, J=8.0 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 3.27 (q, J=6.1 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.74-1.67 (m, 2H), 1.59-1.52 (m, 2H), 1.44-1.33 (m, 6H). 13C NMR (151 MHz, CD3OD): δ 173.3, 165.1, 138.5, 128.4, 123.7, 121.82 (q, J=287.2 Hz), 119.9, 94.4 (q, J=32 Hz), 39.2, 36.5, 28.8, 28.77, 28.6, 26.3, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 377.1688 for C17H23F3N2O4. found 377.1688, 99% purity.
To a 10 mL round bottom flask was added the respective boc-arene (0.4 mmol), followed by 20% trifluoroacetic acid in dichloromethane (1 mL: 4 mL), and the mixture was stirred at room temperature for four hours. The solvent was removed under reduced pressure, and the mixture was dried under vacuum, followed by addition of N,N-dimethyl formamide (1 mL), trifluoropyruvic acid (64 mg, 0.4 mmol, 1 equiv), HATU (152 mg, 0.4 mmol, eq,), HOBt (54 mg, 0.4 mmol, 1 equiv), NMM (132 μl, 1.2 mmol, 3 equiv). The mixture was stirred for four hours, ethyl acetate was added to the mixture (5 mL), and the mixture was washed several times with ice water (3 mL). The organic layer was dried (Na2SO4), the solvent was removed under reduced pressure, and the mixture was dissolved in silica and purified by flash chromatography (MeOH/DCM 1.5-2%) to yield the respective TFPAs (4-42%, 2 steps).
White solid, 64 mg, (50%) mp. 105-110° C. 1HNMR (400 MHz, CD3OD): δδ 7.56 (dd, J=8.5, 0.9 Hz, 2H), 7.31 (t, J=8.0 Hz, 2H), 7.09 (t, J=7.4 Hz, 1H), 3.33 (p, J=3.2 Hz, 2H), 2.42 (t, J=7.5 Hz, 2H), 1.93 (p, J=7.2 Hz, 2H). 13C NMR (151 MHz, CD3OD): δ 172.3, 165.5, 138.4, 128.4, 123.8, 121.8 (q, J=287.4 Hz), 119.9, 94.3 (q, J=32 Hz), 38.8, 33.7, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 321.1062 for C13H15F3N2O4. found 321.1069, 99% purity.
White solid, 26 mg, (16.19%) mp. 133-138° C. 1H NMR (400 MHz, CD3OD) δ 7.95 (d, J=8.8 Hz, 2H), 7.71 (d, J=8.8 Hz, 2H), 3.26 (t, J=6.7 Hz, 2H), 2.56 (s, 3H), 2.40 (t, J=7.5 Hz, 2H), 1.75-1.65 (m, 2H), 1.60-1.50 (m, 2H), 1.45-1.32 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.5, 165.2, 143.4, 132.2, 129.3, 121.8 (q, J=287.4 Hz), 118.7, 94.3 (q, J=32 Hz), 39.1, 36.6, 28.7, 28.5, 26.2, 25.2, 25.1. HRMS-ESI M/z: [M+H]+ calcd. 405.1637 for C18H23F3N2O5. found 405.1637, 98.9% purity.
White solid, 52 mg (26.6%) mp. 105-110° C. 1H NMR (600 MHz, CD3OD) δ 7.76 (s, 4H), 3.28 (td, J=7.0, 2.5 Hz, 2H), 2.42 (t, J=7.5 Hz, 2H), 1.77-1.67 (m, 2H), 1.62-1.54 (m, 2H), 1.46-1.35 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.5, 165.2 (d, J=4 Hz), 148.4 (m, J=17 Hz), 141.9, 126.5 (t, J=4 Hz), 121.8 (q, J=287.3 Hz), 118.7, 94.4 (q, J=32 Hz), 39.1, 36.5, 28.7, 28.5, 26.1, 25.1. HRMS-ESI M/z: [M+H]+ calcd. 489.1094 for C16H20F8N2O4S. found 489.1093, 99% purity.
White solid, 49 mg (31%) mp. 105-110° C. 1H NMR (400 MHz, CD3OD) δ 7.42 (d, J=9.0 Hz, 2H), 6.86 (d, J=9.0 Hz, 2H), 3.76 (s, 3H), 3.25 (t, J=6.9 Hz, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.77-1.62 (m, 2H), 1.58-1.48 (m, 2H), 1.46-1.28 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173, 165.2, 156.5, 132.4, 121.8 (q, J=287.2 Hz), 121.7, 113.5, 94.3 (q, J=32 Hz), 54.4, 39.1, 36.3, 28.7, 28.5, 26.2, 25.5. HRMS-ESI M/z: [M+H]+ calcd. 393.1637 for C17H23F3N2O5. found 393.1634, 99% purity.
White solid, 64 mg (42%) mp. 97-103° C. 1H NMR (400 MHz, CD3OD): δ 7.56 (d, J=4.9 Hz, 2H), 7.05 (d, J=8.3 Hz, 2H), 3.31-3.24 (m, 2H), 2.37 (t, J=6.2 Hz, 2H), 1.75-1.67 (m, 2H), 1.61-1.53 (m, 2H), 1.47-1.35 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.1, 165.2 (d, J=4 Hz), 160, 158.4, 134.7 (d, J=2 Hz), 121.8 (q, J=287.2 Hz), 121.6, 114.9, 114.7, 94.4 (q, J=32 Hz), 54.4, 39.1, 36.3, 28.7, 28.5, 26.2, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 381.1437 for C16H2OF4N2O4. found 381.1432, 96% purity.
White solid, 19 mg (13.2%) mp. 150-155° C. 1H NMR (400 MHz, CD3OD) δ 8.37 (d, J=5.5 Hz, 2H), 7.64 (d, J=6.3 Hz, 2H), 3.25 (t, J=7.0 Hz, 2H), 2.41 (t, J=7.5 Hz, 2H), 1.76-1.63 (m, 2H), 1.63-1.48 (m, 2H), 1.45-1.31 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 174, 165.2, 149.3, 146.8, 121.8 (q, J=287.3 Hz), 113.6, 94.3 (q, J=32 Hz), 39.1, 36.6, 28.7, 28.4, 26.1, 25. HRMS-ESI M/z: [M+H]+ calcd. 364.1844 for C15H20F3N3O4. found 364.1484, 99% purity.
White solid, 18 mg (9.9%) mp. 143-148° C. 1H NMR (400 MHz, CD3OD): δ 7.91 (d, J=8.2 Hz, 2H), 7.40 (t, J=7.7 Hz, 2H), 7.37 (s, 1H), 7.31 (t, J=7.4 Hz, 1H), 3.28 (q, J=6.4 Hz, 2H), 2.51 (t, 2H), 1.77-1.72 (m, 2H), 1.62-1.55 (m, 2H), 1.47-1.38 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 172.4, 165.2, 158, 149.8, 134.6, 128.2, 128.1, 127.4, 127.2, 125.6, 125,56, 121.8 (q, J=287.4 Hz), 107.1, 101.4, 94.3 (q, J=32 Hz), 39.1, 35.1, 28.7, 28.4, 26.1, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 446.1361 for C19H22F3N3O4S. found 446.1376, 95% purity.
White solid, 40 mg (26%) mp. 120-125° C. 1H NMR (400 MHz, CD3OD): δ 7.91 (s, 1H), 7.47 (d, J=7.1 Hz, 1H), 7.25-7.19 (m, 2H), 3.30-3.24 (m, 2H), 2.40-2.35 (m, 2H), 1.73-1.67 (m, 2H), 1.60-1.53 (m, 2H), 1.44-1.35 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.2, 165.2, 160.1, 139.7, 129.1, 121.8 (q, J=286.9 Hz), 111.9, 109.2, 105.6, 94.3 (q, J=32 Hz), 54.2, 39.1, 36.5, 28.7, 28.5, 26.2, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 393.1637 for C17H23F3N2O5. found 393.1644, 98% purity.
White solid, 55 mg (37%) mp. 137-142° C. 1H NMR (600 MHz, CD3OD): δ 7.43 (d, J=3.6 Hz, 2H), 7.11 (d, J=3.6 Hz, 2H), 3.31-3.24 (m, 2H), 2.50 (t, J=7.5 Hz, 2H), 1.76-1.69 (m, 2H), 1.61-1.54 (m, 2H), 1.45-1.36 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 172.2, 165.2, 158.7, 136.9, 121.8 (q, J=287.2 Hz), 113, 94.3 (q, J=32 Hz), 39.1, 36.5, 35, 28.7, 28.4, 26.1, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 370.1048 for C13H18F3N3O4S. found 370.1057, 99% purity.
White solid, 20 mg (12%) mp. 150-155° C. 1H NMR (600 MHz, acetone-d&) δ 7.94 (d, J=7.9 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.43 (t, J=8.3 Hz, 1H), 7.31 (t, J=8.1 Hz, 1H), 3.33 (q, J=6.9 Hz, 2H), 2.63 (t, J=7.5 Hz, 2H), 1.80-1.73 (m, 2H), 1.64-1.57 (m, 2H), 1.48-1.38 (m, 4H). 13C NMR (151 MHz, Acetone-d6) δ 171.9, 166.5, 157.9, 149, 132.1, 125.9, 123.5, 122.58 (q, J=287.7 Hz), 121.3, 120.70, 90.8 (q, J=32.4 Hz), 39.6, 35.5, 26.1, 24.8. HRMS-ESI M/z: [M+H]+ calcd. 420.1204 for C17H20F3N3O4S. found 420.1208, 98% purity.
White solid, 51 mg (32%) mp. 100-105° C. 1H NMR (600 MHz, CD3OD) δ 7.77 (td, J=8.9, 6.1 Hz, 1H), 7.07-7.00 (m, 1H), 6.99-6.93 (m, 1H), 3.29 (q, J=6.9 Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 1.75-1.68 (m, 2H), 1.62-1.55 (m, 2H), 1.47-1.36 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.7, 165.2, 160.6 (d, J=11.4 Hz), 159.01 (d, J=11.4 Hz), 155.88 (d, J=12.2 Hz), 154.23 (d, J=12.2 Hz), 126.21 (dd, J=9.5, 2.5 Hz), 121.98 (dd, J=12.2, 3.8 Hz), 121.80 (q, J=287.3 Hz), 110.50 (dd, J=22.2, 3.7 Hz), 103.44 (dd, J=26.7, 24.3 Hz), 94.34 (d, J=32.1 Hz), 39.1, 35.7, 28.69, 28.4, 26.1, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 399.1343 for C16H19F5N2O4. found 399.1327, 98% purity.
White solid, 23 mg (13.8%) mp. 95-100° C. 1H NMR (600 MHz, CD3OD) δ 6.96 (t, J=8.3 Hz, 1H), 3.32-3.25 (m, 2H), 2.44 (t, J=7.4 Hz, 2H), 1.76-1.69 (m, 2H), 1.61-1.55 (m, 2H), 1.49-1.37 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 25.34, 26.13, 28.31, 28.72, 35.13, 39.28, 174.1, 165.3 (d, J=4.2 Hz), 161.88 (t, J=15 Hz), 160.23 (t, J=15 Hz), 159.5 (dd, J=15.4, 7.4 Hz), 157.8 (dd, J=15.4, 7.4 Hz), 121.8 (q, J=287.2 Hz), 110.7 (td, J=17.3. 5.1 Hz), 101.1-99.2 (m), 94.4 (m, J=32 Hz), 39.3, 35.1, 28.7, 28.3, 26.1, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 417.1249 for C16H18F6N2O4. found 417.1256, 95% purity.
White solid, 27 mg (17%) mp. 103-108° C. 1H NMR (400 MHz, CD3OD) δ 7.55 (d, J=8.8 Hz, 2H), 7.28 (d, J=8.8 Hz, 2H), 3.26 (t, J=7.2 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H), 1.74-1.63 (m, 2H), 1.61-1.50 (m, 2H), 1.44-1.30 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.2, 165.2, 137.4, 128.5, 128.3, 121.8 (q, J=287.2 Hz), 121.1, 94.3 (q, J=32 Hz), 39.1, 36.4, 28.7, 28.5, 26.1, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 397.1141 for C16H20ClF3N2O4. found 397.1169, 95% purity.
White solid, 19 mg (10.8%) mp. 105-110° C. 1H NMR (400 MHz, CD3OD) δ 7.50 (d, J=8.8 Hz, 2H), 7.42 (d, J=8.9 Hz, 2H), 3.25 (t, J=7.0 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H), 1.73-1.63 (m, 2H), 1.60-1.51 (m, 2H), 1.44-1.32 (m, 4H). 13C NMR (151 MHz, CD3OD) δ 173.2, 165.2, 137.9, 131.3, 121.8 (q, J=287.2 Hz), 121.37, 115.9, 94.3 (q, J=32 Hz), 39.1, 36.4, 28.7, 28.5, 26.1, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 441.0636 for C16H20BrF3N2O4. found 441.0641, 99% purity.
White solid, 18 mg (11.7%) mp. 105-110° C. 1H NMR (400 MHz, CD3OD) δ 7.90 (d, J=7.9 Hz, 1H), 7.08 (t, J=8.6 Hz, 1H), 6.98 (d, J=7.4 Hz, 1H), 6.89 (t, J=8.2 Hz, 1H), 3.24 (q, J=7.9 Hz, 2H), 2.41 (t, J=7.5 Hz, 2H), 1.74-1.63 (m, 2H), 1.59-1.50 (m, 2H), 1.44-1.32 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.3, 165.2, 150.3, 126.7, 124.8, 122.3, 121.8 (q, J=287.3), 120, 110.4, 94.3 (q, J=32 Hz), 54.81, 54.80, 39.2, 36.3, 28.7, 28.4, 26.2, 25.5. HRMS-ESI M/z: [M+H]+ calcd. 393.1637 for C17H23F3N2O5. found 393.1636, 99% purity.
White solid, 36 g (20%) mp. 143-148° C. 1H NMR (400 MHz, acetone-d6) δ 7.66 (d, J=8.4 Hz, 2H), 7.42 (t, J=7.7 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 3.31 (q, J=6.5 Hz, 2H), 2.55 (t, J=7.4 Hz, 2H), 2.51 (s, 3H), 1.77-1.68 (m, 3H), 1.63-1.54 (m, 2H), 1.46-1.35 (m, 4H). 13C NMR (151 MHz, Acetone-d6): δ170.9, 166.4, 154, 135.6, 128.2, 128.1, 127, 122.6 (q, J=287.7 Hz), 121.1, 90.7 (q, J=32.5 Hz), 39.6, 35.3, 26.2, 24.9, 11.3. HRMS-ESI M/z: [M+H]+ calcd. 460.1517 for C20H24F3N3O4S. found 460.1506, 99% purity.
White solid, 22 mg (10.6%) mp. 143-148° C. 1H NMR (400 MHz, CD3OD): δ 1H NMR (600 MHz, CD3OD) δ 7.49-7.45 (m, 1H), 7.36-7.31 (m, 3H), 7.27 (dd, J=3.7, 2.5 Hz, 1H), 3.28 (dt, J=12.0, 6.9 Hz, 2H), 2.52 (t, J=7.4 Hz, 2H), 1.79-1.71 (m, 2H), 1.62-1.56 (m, 2H), 1.48-1.38 (m, 4H). 13C NMR (151 MHz, CD3OD) δ 172.5, 165.2, 156.1, 144.3, 135, 132.29, 129.3, 128.6, 128.5, 127.8, 127.6, 127.4, 126.3, 121.8 (q, J=287.2 Hz), 94.3 (q, J=32 Hz), 39.1, 35.1, 28.7, 28.4, 26.1, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 522.1674 for C25H26F3N3O4S. found 522.1673, 99% purity.
White solid, 32 mg (18.2%) mp. 123-12 8° C. 1H NMR (600 MHz, acetone-d6): δ 7.77 (d, J=8.5 Hz, 2H), 7.68-7.57 (m, 4H), 7.43 (t, J=7.7 Hz, 2H), 7.32 (t, J=7.4 Hz, 1H), 3.31 (q, J=6.7 Hz, 2H), 2.38 (t, J=7.4 Hz, 2H), 1.75-1.65 (m, 2H), 1.65-1.52 (m, 2H), 1.46-1.34 (m, 4H). 13C NMR (151 MHz, Acetone-d6): δ 171.1, 166.4, 140.6, 139.1, 135.6, 128.8, 127, 126.9, 126.4, 122.6 (q, J=287.9 Hz), 119.4, 90.8 (q, J=32.5 Hz), 39.6, 36.8, 26.2, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 439.1844 for C22H23F3N2O4. found 439.1844, 99% purity.
White solid, 17 mg (10%) mp. 108-113° C. 1H NMR (400 MHz, acetone) δ 8.1 (d, J=2.5 Hz, 1H), 6.89 (d, J=8.9 Hz, 1H), 6.67 (s, 1H), 6.56 (dd, J=8.9, 3.0 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H), 3.31 (q, J=6.8 Hz, 2H), 2.44 (t, J=7.4 Hz, 2H), 1.72-1.63 (m, 2H), 1.62-1.52 (m, 2H), 1.44-1.32 (m, 4H). 13C NMR (151 MHz, Acetone-d6): δ 171.1, 166.4, 153.8, 142.6, 129.1, 122.6 (q, J=285.87 Hz), 111, 107, 106.8, 90.8 (q, J=32.5 Hz), 55.7, 55, 39.6, 36.9, 26.2, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 423.1742 for C18H25F3N2O6. found 423.1745, 99% purity.
White solid, 29 mg (15.9%) mp. 98-103° C. 1H NMR (400 MHz, CD3OD) δ 6.95 (s, 2H), 3.82 (s, 3H), 3.72 (s, 3H), 3.27 (t, J=7.2 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H), 1.74-1.64 (m, 2H), 1.61-1.52 (m, 2H), 1.45-1.33 (m, 4H). 13C NMR (151 MHz, CD3OD): 13C NMR (151 MHz, CD3OD) δ 173.1, 165.2, 153, 135, 134, 121.8 (q, J=287.2 Hz), 94.3 (q, J=33 Hz), 97.4, 59.8, 55.1, 39.11, 36.5, 28.7, 28.5, 26.2, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 453.1848 for C19H27F3N2O7. found 453.1866, 99% purity.
White solid, 48 mg (29%) mp. 123-128° C. 1H NMR (400 MHz, CD3OD) δ 7.97 (d, J=9.0 Hz, 1H), 7.90 (d, J=9.2 Hz, 1H), 7.78 (d, J=8.2 Hz, 1H), 7.59-7.45 (m, 4H), 3.29-3.22 (m, 2H), 2.55 (t, J=7.5 Hz, 2H), 1.86-1.75 (m, 2H), 1.65-1.56 (m, 2H), 1.55-1.39 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 174.4, 165.22, 165.19, 134.3, 132.8, 128.8, 128, 126.2, 125.9, 125.7, 125.1, 122.9, 122, 121.8 (q, J=287.4 Hz), 94.3 (q, J=32 Hz), 39.2, 35.6, 28.7, 28.6, 26.2, 25.6. HRMS-ESI M/z: [M+H]+ calcd. 413.163 for C20H23F3N2O4. found 413.1428, 99% purity.
Yellow solid, 24 mg (12.9%) mp. 113-118° C. 1H NMR (600 MHz, CD3OD): δ 7.80-7.73 (m, 6H), 7.64 (t, J=7.4 Hz, 1H), 7.54 (t, J=7.7 Hz, 2H), 3.30 (t, J=7.2 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H), 1.77-1.70 (m, 2H), 1.64-1.57 (m, 2H), 1.48-1.37 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 196.2, 173.5, 157.5 (q, J=36.5 Hz), 143.1, 137.8, 132.2, 132.1, 131.1, 129.4, 128.1, 118.6, 116.9 (q, J=286.6 Hz), 39.3, 36.6, 28.5, 28.3, 26.1, 25.2. HRMS-ESI M/z: [M+Na]+ calcd. 489.1613 for C23H24F3N2NaO5. found 489.1623, 96% purity.
White solid, 24 mg (11. %) mp. 115-120° C. 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J=8.3 Hz, 2H), 7.38 (d, J=8.9 Hz, 2H), 7.26 (d, J=7.3 Hz, 2H), 6.98 (d, J=8.9 Hz, 2H), 3.24 (t, J=6.8 Hz, 2H), 2.36 (s, 3H), 2.31 (t, J=7.4 Hz, 2H), 1.72-1.60 (m, 2H), 1.59-1.48 (m, 2H), 1.43-1.26 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.1, 165.2, 143.6, 136.6, 135.6, 133.4, 129.1, 126.9, 122.1, 121.8 (q, J=287.2 Hz), 120.38, 94.4 (q, J=32.5 Hz), 39.1, 36.4, 28.7, 28.5, 26.1, 25.4, 20. HRMS-ESI M/z: [M+H]+ calcd. 532.167 for C23H28F3N3O6S. found 532.1673, 98% purity.
White solid, 8 mg (4.5%) mp. 108-113° C. 1H NMR (400 MHz, CD3OD) δ 7.62 (t, J=7.4 Hz, 2H), 7.35 (t, J=7.7 Hz, 1H), 7.12 (t, J=7.7 Hz, 1H), 3.27 (t, J=7.0 Hz, 2H), 2.44 (t, 2H), 1.79-1.67 (m, 2H), 1.62-1.51 (m, 2H), 1.50-1.33 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 173.6, 165.2, 135.8, 132.5, 127.6, 127, 126.9, 121.8 (q, J=287.2 Hz), 118.1, 94.3 (q, J=32.9 Hz), 39.2, 39.1, 35.9, 28.7, 28.5, 28.47, 26.2, 26.1, 25.4. HRMS-ESI M/z: [M+H]+ calcd. 441.0636 for C16H20BrF3N2O4. found 441.0651, 99% purity.
27 mg (15.3%). 1H NMR (600 MHz, CD3OD): δ 7.91 (s, 1H), 7.47 (d, J=7.1 Hz, 1H), 7.25-7.19 (m, 2H), 3.30-3.24 (m, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.73-1.67 (m, 2H), 1.60-1.53 (m, J=9.1, 4.8 Hz, 2H), 1.44-1.35 (m, J=2.6 Hz, 5H). 13C NMR (151 MHz, CD3OD): δ 173.4, 173.3, 165.2, 140.1, 129.9, 126.4, 122.4, 121.9, 121.8 (q, J=287 Hz), 118.1, 94.3 (q, J=32 Hz), 39.1, 36.4, 28.7, 28.5, 26.1, 25.3. HRMS-ESI M/z: [M+H]+ calcd. 441.0636 for C16H20BrF3N2O4. found 441.063, 95% purity.
White solid, 20 mg (11.4%) mp. 146-151° C. 1H NMR (600 MHz, CD3OD): δ 1.42 (m, 4H, CH2, J=5.64 Hz), 1.58 (m, 2H, CH2, J=7.14 Hz), 1.75 (m, 2H, CH2, J=7.5 Hz), 2.54 (t, 2H, CH2, J=7.5 Hz), 3.29 (q, 2H, CH2, J=4 Hz), 7.21 (td, 1H, CH, J=2.58, 9 Hz), 7.65 (dd, 1H, CH, J=2.58, 8.4 Hz), 7.72 (dd, 1H, CH, J=4.2, 4.68 Hz). 13C NMR (151 MHz, CD3OD): δ 173, 165.2, 160.4, 158.8, 158.2, 145.2, 133.1 (d, J=10.8 Hz), 121.8 (q, J=287.2 Hz), 121.4 (d, J=9.1 Hz), 113.8 (d, J=24.8 Hz), 107.1 (d, J=27 Hz), 39.1, 35.2, 28.7, 28.4, 26.1, 24.7. HRMS-ESI M/z: [M+H]+ calcd. 438.1110 for C17H19F4N3O4S. found 438.1111, 98% purity.
White solid, 13 mg (7.2%) mp. 165-170° C. 1H NMR (400 MHz, CD3OD) δ 7.89 (d, J=2.1 Hz, 1H), 7.68 (d, J=8.6 Hz, 1H), 7.40 (dd, J=8.7, 2.1 Hz, 1H), 3.26 (t, J=8.0 Hz, 2H), 2.52 (t, J=7.5 Hz, 2H), 1.77-1.67 (m, 2H), 1.61-1.50 (m, 2H), 1.47-1.34 (m, 4H). 13C NMR (151 MHz, CD3OD): S 173.1, 165.2, 159, 147.4, 133.5, 128.7, 126.3, 121.8 (q, J=287.2 Hz), 121.4, 120.6, 39.1, 35.2, 28.7, 28.4, 26.1, 24.7. HRMS-ESI M/z: [M+H]+ calcd. 454.0815 for C17H19ClF3N3O4S. found 454.0817, 98% purity.
White solid, 11 mg (5.5%) mp. 163-168° C. 1H NMR (600 MHz, acetone-d6): δ 8.16 (d, J=2.0 Hz, 1H), 7.64 (d, J=8.6 Hz, 1H), 7.57 (dd, J=8.6, 2.0 Hz, 1H), 3.35-3.31 (m, 2H), 2.64 (t, J=7.5 Hz, 2H), 1.80-1.73 (m, 2H), 1.66-1.57 (m, 2H), 1.49-1.35 (m, 4H). 13C NMR (151 MHz, acetone-d6): δ 172.1, 166.4, 158.6, 148.2, 134.3, 129.1, 123.9, 122.6 (q, J=287.2 Hz), 121.2, 115.7, 90.8 (q, J=32 Hz), 39.6, 35.5, 28.7, 28.5, 26.1, 24.7. HRMS-ESI M/z: [M+H]+ calcd. 498.0309 for C17H19BrF3N3O4S. found 498.0313, 98% purity.
White solid, 15 mg (7.8%) mp. 163-168° C. 1H NMR (400 MHz, CD3OD) δ 7.69 (s, 1H), 7.43 (d, J=8.4 Hz, 2H), 3.26 (t, J=7.0 Hz, 2H), 2.52 (t, J=7.2 Hz, 2H), 1.85-1.66 (m, 2H), 1.66-1.51 (m, 2H), 1.5-1.34 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 171.8, 165.2, 156.2, 146.5, 142.6, 126.4, 121.8 (q, J=287.3 Hz), 120.8, 116.9, 111, 94.4 (q, J=32.3 Hz), 39.1, 36, 28.7, 28.3, 26.1, 24.5. HRMS-ESI M/z: [M+H]+ calcd. 482.0538 for C17H19BrF3N3O5. found 482.0528, 98% purity.
White solid, 9 mg (5.5%) mp. 118-123° C. 1H NMR (400 MHz, acetone-d6): δ 7.67-7.46 (m, 2H), 7.42-7.19 (m, 2H), 3.40-3.32 (m, 2H), 2.69 (q, J=8.3 Hz, 2H), 1.93-1.71 (m, 2H), 1.67-1.55 (m, 2H), 1.57-1.35 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 171.8, 165.2, 155, 147.4, 140.7, 124.2, 123.7, 121.8 (q, J=287.3 Hz), 119, 117.9, 109.5, 94.4 (q, J=32 Hz), 39.2, 36, 28.7, 28.3, 26.1, 24.5. HRMS-ESI M/z: [M+H]+ calcd. 404.1433 for C17H20F3N3O5. found 404.1439, 98% purity.
White solid, 17 mg (9.4%) mp. 143-148° C. 1HNMR (600 MHz, CD3OD): δ 7.63 (d, J=8.9 Hz, 1H), 7.43 (d, J=2.5 Hz, 1H), 7.04 (dd, J=8.8, 2.5 Hz, 1H), 3.87 (s, 3H), 3.29 (td, J=6.9, 4.2 Hz, 2H), 2.53 (t, J=7.5 Hz, 2H), 1.78-1.71 (m, 2H), 1.62-1.54 (m, 2H), 1.49-1.36 (m, 4H). 13C NMR (151 MHz, CD3OD): 24.8, 26.1, 28.4, 28.7, 35.2, 39.1, 54.8, 94.3 (J=32 Hz), 103.7, 114.8, 120.9, 121.8 (q, J=286 Hz), 133.1, 142.6, 156.5, 157, 165.2, 172.8. HRMS-ESI M/z: [M+H]+ calcd. 450.1310 for C18H22F3N3O5S. found 450.1306, 99% purity.
White solid, 14 mg (7.8%) mp. 160-165° C. 1HNMR (400 MHz, CD3OD): δ 7.72 (d, J=8.7 Hz, 1H), 7.29 (d, J=2.4 Hz, 1H), 6.96 (dd, J=8.7, 2.4 Hz, 1H), 3.87 (s, 3H), 3.29 (td, J=7.0, 4.1 Hz, 2H), 2.54 (t, J=7.5 Hz, 2H), 1.80-1.71 (m, 2H), 1.63-1.55 (m, 2H), 1.48-1.37 (m, 4H). 13C NMR (151 MHz, CD3OD): δ 172.8, 165.2, 159.5, 159.3, 149.8, 121.8 (q, J=287.3 Hz), 121.2, 112.8, 103.6, 54.6, 39.1, 35.2, 28.7, 28.4, 26.1, 24.7. HRMS-ESI M/z: [M+H]+ calcd. 450.1310 for C18H22F3N3O5S. found 450.1312, 98% purity.
White solid 18 mg (9.4%) mp. 148-153° C. 1H NMR (400 MHz, acetone-d6): δ 7.86 (d, J=8.9 Hz, 2H), 7.30 (s, 1H), 6.98 (d, J=8.9 Hz, 2H), 3.85 (s, 3H), 3.34 (q, J=6.9 Hz, 2H), 2.62 (t, J=7.4 Hz, 2H), 1.83-1.72 (m, 2H), 1.67-1.56 (m, 2H), 1.51-1.36 (m, 4H). 13C NMR (151 MHz, acetone-d6): δ 171.1, 166.4, 159.5, 157.8, 149.4, 127.7, 127.1, 122.6 (q, J=287.2 Hz), 113.9, 105.2, 90.8 (q, J=32.5 Hz), 54.7, 39.6, 35.3, 26.2, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 476.1361 for C20H24F3N3O5S. found 476.1368, 99% purity.
White solid, 10 mg (4.8%) mp. 145-150° C. 1H NMR (600 MHz, acetone-d6): δ 7.83 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.3 Hz, 2H), 7.49 (s, 1H), 3.27 (q, J=5.4 Hz, 2H), 2.56 (t, J=7.5 Hz, 2H), 1.75-1.67 (m, 2H), 1.60-1.52 (m, 2H), 1.43-1.34 (m, 4H). 13C NMR (151 MHz, acetone-d6): δ 171.3, 166.4, 158.2, 148.2, 134, 131.6, 127.7, 122.6 (q, J=287.4 Hz), 121, 108, 90.8 (q, J=32 Hz), 39.6, 35.3, 26.2, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 524.0466 for C19H21BrF3N3O4S. found 524.046, 99% purity.
A mixture of 7-((5-(4-hydroxyphenyl)thiazol-2-yl) amino)-7-oxoheptan-1-aminium chloride 51 (62 mg, 0.174 mmol), trifluoropyruvic acid (27 mg, 0.17 mmol, 0.98 equiv.), HATU (65 mg, 0.17 mmol, 0.98 equiv.), HOBt (23 mg, 0.17 mmol, 0.98 equiv.), and NMM (56 μL, 0.51 mmol, 2.93 equiv.) was stirred in 1 mL dimethylformamide at room temperature overnight. Ethyl acetate was added to the mixture (5 ml), and the mixture was washed several times with ice water (3 ml). The organic layer was dried (Na2SO4), the solvent was removed under reduced pressure, and the mixture was dissolved in silica and purified by flash chromatography (MeOH/DCM 1.5-2%) to yield N-(5-(4-hydroxyphenyl) thiazol-2-yl)-7-(3,3,3-trifluoro-2,2-dihydroxypropanamido) heptanamide 86 as a white solid (10 mg, 11%, mp. 145-150° C.). 1H NMR (400 MHz, acetone-d6) δ 7.73 (d, J=8.8 Hz, 2H), 7.19 (s, 1H), 6.84 (d, J=8.8 Hz, 2H), 3.30 (q, J=13.4, 6.7 Hz, 2H), 2.57 (t, J=7.4 Hz, 2H), 1.77-1.68 (m, 2H), 1.61-1.52 (m, 2H), 1.50-1.33 (m, 4H). 13C NMR (151 MHz, acetone-d6): 171.1, 166.4, 157.7, 157.3, 149.7, 127.2, 126.7, 122.6 (q, J=287.8 Hz), 115.3, 104.6, 39.6, 35.3, 26.2, 24.9. HRMS-ESI M/z: [M+H]+ calcd. 462.1310 for C19H22F3N3O5S. found 462.1329, 98% purity.
A mixture of tert-butyl (7-((5-(4-hydroxyphenyl) thiazol-2-yl) amino)-7-oxoheptyl) carbamate 52 (183 mg, 0.44 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (75 μL, 0.5 mmol, 1.14 equiv), and ethyl iodide (160 μL, 2 mmol, 4.55 equiv) was refluxed in acetone (3 mL) for 6 hours, after which the solvent was removed under reduced pressure. The reaction mixture was diluted with ethyl acetate (5 mL) and washed with 1M HCl (5 mL) and brine (2×5 mL) twice. The organic layer was dried (Na2SO4), concentrated, dried under vacuum, and used in the next reaction without further purification. To the crude mixture was added 20% trifluoroacetic acid in dichloromethane (1 ml: 4 ml), and the mixture was stirred at room temperature for four hours. The solvent was removed under reduced pressure, and the mixture was dried under vacuum, followed by addition of N,N-dimethyl formamide (1 ml), trifluoro pyruvic acid (64 mg, 0.4 mmol, 1 equiv), HATU (152 mg, 0.4 mmol, equiv,), HOBt (54 mg, 0.4 mmol, 1 equiv), and NMM (132 μl, 1.2 mmol, 3 equiv). The mixture was stirred for four hours, ethyl acetate was added to the mixture (5 ml), and the mixture was washed several times with ice water (3 ml). The organic layer was dried (Na2SO4), the solvent was removed under reduced pressure, and the mixture was dissolved in silica and purified by flash chromatography (MeOH/DCM 1.5-2%) to yield N-(5-(4-ethoxyphenyl) thiazol-2-yl)-7-(3,3,3-trifluoro-2,2-dihydroxypropanamido)heptanamide 87 as a white solid (10 mg, 4.66%, mp. 138-143° C., 3 steps).
1H NMR (600 MHz, Acetone-d6): 1H NMR (600 MHz, Acetone) δ 7.79 (d, J=8.8 Hz, 23H), 7.25 (s, 1H), 6.91 (d, J=8.8 Hz, 2H), 4.04 (q, J=7.0 Hz, 2H), 3.28 (q, J=7.1 Hz, 2H), 2.56 (t, J=7.5 Hz, 2H), 1.76-1.66 (m, 2H), 1.62-1.52 (m, 2H), 1.42-1.36 (m, 4H), 1.34 (t, J=7.0 Hz, 3H). 13C NMR (151 MHz, CD3OD): 172.4, 165.2, 158.9, 157.9, 149.7, 129.9, 127.4, 126.9, 121.8 (q, J=287.3 Hz), 115.1, 105.1, 94.3 (q, J=32.3 Hz), 63.1, 39.1, 35.1, 28.6, 28.4, 26.1, 24.9, 13.8. HRMS-ESI M/z: [M+H]+ calcd. 490.1623 for C21H26F3N3O5S. found 490.1623, 98% purity.
HCT116 human colon cancer cells, NCI-H522 human lung cancer cells, HT1080 fibrosarcoma cells, HeLa cervical cancer cells, U2OS osteosarcoma cells, MDA-MB-231 and MDA-MB-468 breast cancer cells, WI38 diploid human cell line, and human retinal pigment epithelial RPE cells were preserved in Dulbecco's Modified Eagle's medium (Mediatech, Inc.) supplemented with 10% calf serum (Atlanta Biologicals) or 10% fetal bovine serum (Gemini Bio-Products #100-106) and 1000 U/ml of both Penicillin and Streptomycin (Mediatech, Inc.) at 37° C. with 5% CO2. Cell feasibility was examined using methylene blue staining. Cells were plated at a rate of 5000/well in 96 well plates (n=3) respectively and treated the next day. Three days after treatment, cells were fixed/stained in methylene blue saturated in 50% ethanol for 30 min at room temperature. Excessive water was used to wash off extra dye from the plates. The retained dye in the plates was dissolved in 0.1 N HCl and absorbance was measured at 668 nm.
NCI-H522 cell pellets obtained were lysed using lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40, (supplemented with 1 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM DTT, and 0.1 M PMSF) for 30 minutes on ice and centrifuged at 13×103 g for 25 minutes at 4° C. The protein levels of the obtained lysates were normalized using BCA Protein Assay Kit (Pierce) and separated by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide). Transfer to polyvinylidene difluoride membranes (Millipore) was followed by obstruction of membranes with blocking buffer containing 5% (w/v) non-fat dry milk dissolved in PBST [1×PBS containing 0.05% (v/v) Tween 20] for 1 hour at room temperature. Membranes were then incubated with corresponding primary antibodies overnight at 4° C. The membranes were then washed (3×15 min each) with PBST and incubated with secondary antibodies conjugated to horse-radish peroxidase, obtained from Biorad, and used at a dilution of 1:10,000. Bound antibodies were detected using enhanced chemiluminescence (Biorad).
Inhibitor Testing with HDAC Isoforms
In a half-area 96-well white opaque plate (Corning), recombinant HDAC1 (1 μL of 3 ng/L, BPS Bioscience), HDAC2 (1 μL of 1 ng/L, BPS Bioscience), HDAC3 (1 μL of 30 ng/L, BPS Bioscience), HDAC4 (1 μL of 5 ng/L, BPS Bioscience), HDAC6 (1 μL of 35 ng/&L, BPS Bioscience), HDAC8 (1 μL of 70 ng/&L, BPS Bioscience), or buffer alone (1 μL) was added to HDAC-Glo™ buffer (43 μL, Promega). Serial dilutions or single concentrations of inhibitors (1 μL in DMSO, concentrations described in the supporting information) or DMSO alone (1 μL) were added to the enzyme solution, followed by 3 hr. incubation at room temperature. The HDAC-Glo™ reagent (5 μL), prepared per manufacturer recommendations, was added to each reaction. Luminescent signal was measured every 3 min over the course of 30 min using an M-Plex Infinite 200 Pro (Tecan). To determine IC50 values, the luminescent signal at peak reading was first background corrected with the signal from a background control reaction without HDAC enzyme. Percent deacetylase activity values were calculated by dividing the background-corrected signal with inhibitor by the background-corrected signal without inhibitor. IC50 values were calculated by fitting the percent deacetylase activity remaining as a function of inhibitor concentration to a sigmoidal dose-response curve (y=100/(1+(x/IC50)z), where y=percent deacetylase activity and x=inhibitor concentration) using non-linear regression with KaleidaGraph 5.0 software.
A protocol similar to the one previously employed by others using Human Liver S9 fractions instead of liver microsomes was used for the metabolism studies. The assay was initiated by incubating a mixture of the substrate (0.02 mM), buffer (0.1M), and NADPH (1 mM) at 37° C. for ten minutes, followed by addition of the S9 fraction (0.5 mg) to the mixture and gentle mixing. The total volume of the reaction mixture was maintained at 1 mL. 450 μL aliquots of the mixture were collected at different time intervals (1h, 2 hrs) for compound 60 and 200 μL aliquots every 15 minutes for the TFMK 91, as it has a half-life of less than 30 minutes. The reactions in the aliquots were terminated by adding an equal volume of ethyl acetate, mixing thoroughly, and transferring into micro centrifuge tubes. They were vortexed for 2 minutes and then subjected to centrifugation at 10,000×g for 15 minutes to remove the proteins. The top organic layers were extracted and evaporated under reduced pressure. The organic residues from the aliquots were dissolved in methanol first, then diluted 1:1 with water, and analyzed using LC-ESI-MS. A 450 μL aliquot of compound 60 with concentration of 0.02 mM contains 9 nanomoles, which after evaporation, was dissolved in 500 μL of methanol to give 18 μM in concentration of 60. This was diluted 1:1 with water to give a final concentration of 9 μM, which was used for LC-ESI-MS. Similarly, 200 μL of TFMK 91 at a concentration of 0.02 mM contains 4 nanomoles, which after removing ethyl acetate, and dissolving in 250 μL of methanol gives 16 μM concentration of 91. This was diluted 1:1 with water to give 8 μM final concentration for LC-ESI-MS.
For analysis of the aliquots obtained, the LC-ESI-MS protocol shown in Table 6 was used. The solvent flow rate was 0.2 mL/min, and the sample injection volume was 20 μL. Mobile phase A consisted of water containing 0.1% formic acid, while mobile phase B was acetonitrile containing 0.1% formic acid. The column was equilibrated with 30% of mobile phase A and 70% of mobile phase B. The retention times for compound 60, compound 92 (TFMHA), and compound 91 (TFMK) were 7.5 min (t 0.1 min), 8.13 min, and 10.5 min (i 0.3 min), respectively. At the start, the parameters for both starting materials were established. The M+1 for compound 60 was m/z 370 (hydrate) with a small percentage having an M+1 of m/z 352 m/z (ketone). Its MS/MS peak gave only m/z 352. The compound 91 had M+1 peaks of m/z 302 (ketone), m/z 320 (hydrate), and another peak of m/z 338 corresponding to a dihydrate, while the MS/MS corresponded to m/z 302 with a lesser amount of m/z 320. The TFMHA 92 had an M+1 peak of 354 m/z (13C).
Certain embodiments of the compositions and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.
This application claims priority to U.S. Provisional Application No. 63/461,684 filed under 35 U.S.C. § 111(b) on Apr. 25, 2023, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
This invention was made with government support under Grant Numbers R15GM120712 and R15CA213185 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63461684 | Apr 2023 | US |
