WO2014161072A1 - Α-boryl isocyanides, boropeptides and boron heterocycles - Google Patents

Α-boryl isocyanides, boropeptides and boron heterocycles Download PDF

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WO2014161072A1
WO2014161072A1 PCT/CA2014/000307 CA2014000307W WO2014161072A1 WO 2014161072 A1 WO2014161072 A1 WO 2014161072A1 CA 2014000307 W CA2014000307 W CA 2014000307W WO 2014161072 A1 WO2014161072 A1 WO 2014161072A1
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Adam Daniel ZAJDLIK
Andrei K. Yudin
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The Governing Council Of The University Of Toronto
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the invention relates to novel a-boryl isocyanides, their synthesis, as well as their use in the preparation of boron-containing compounds.
  • Boronic acids and their derivatives are useful as synthetic building blocks, [1] chemosensors,[2] and biologically active targets of synthesis. Both the biological activity and chemical reactivity of boronic acids stem from boron's Lewis acidity. While useful in a broad range of applications, boron's propensity to undergo reactions with Lewis bases becomes problematic for functional group compatibility during synthesis. [1] Reagents that streamline installation of a carbon-boron bond in stereochemically complex, heteroatom-rich environments, are expected to find application not only as starting materials but also as valuable endpoints of synthesis.
  • Figure 1 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boropeptide derivative 7a-A.
  • Figure 2 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boropeptide derivative 7a-B.
  • Figure 3 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boromorpholinone anti- 1ad.
  • Figure 4 shows the in vitro cytosolic caspase-like 20S proteasome inhibition by boromorpholinone anti- ⁇ 1ad.
  • Figure 5 shows the in vitro cytosolic trypsin-like 20S proteasome inhibition by boromorpholinone anti- ⁇ 1ad.
  • Figure 6 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boromorpholinones syn- 1ad and ani/-11ad.
  • Figure 7 shows the computational analysis of possible hydrolytic degradation of anti- 11ad. Data reflects enthalpic energy differences between reaction partners.
  • Figure 8 shows the cellular permeability of ani/-11ad compared to bortezomib with positive (metoprolol) and negative (atenolol) controls.
  • Figure 9 shows a computational model of anf/-11ad in complex with a chymotrypsin- like member of proteasome 20S.
  • Figure 10 shows the structures of several MIDA-boronate containing compounds which act as potent protease inhibitors.
  • borylamide motif (B-C-N amide ) is commonly found in the structures of biologically active boropeptides.[3] Recent efforts have been focused on the amphoteric aziridine aldehyde- and isocyanide-driven macrocyclizations of linear peptides and peptidomimetics.[4] This methodology is enabled by the development of functionally dense, heteroatom-rich environments where a kinetic barrier prevents two otherwise reactive functional groups from prematurely reacting with each other.[5] In the present invention, we expand the scope of this methodology to include amphoteric boron- containing building blocks for use in multi-component preparation of boropeptides and their derivatives.
  • Isocyanides are 1 ,1 -amphoteric molecules that enable heterocycle synthesis[6] and participate in multi-component reactions (MCRs) such as Ugi and Passerini processes.
  • MCRs multi-component reactions
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 and R 12 are each independently H or an organic group.
  • R 1 , R 2 , R 9 , R 0 , R 14 , and R 15 are each independently H or an organic group and X is any Lewis basic ligand.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 0 , R 11 , R 2 , R 13 , R 14 and R 15 are each independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
  • the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
  • R 3 , R 4 , R 5 , R 6 are H and R 7 is CH 3 .
  • R 1 and R 2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl. In some embodiments, R 1 and R 2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl. In some embodiments, R 1 and R 2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl.
  • R 9 and R 10 are independently selected from the group consisting of H, benzyl and isopropyl. In other embodiments, R 9 and R 10 are independently selected from the group consisting of H, benzyl, isopropyl, 4-F-phenyl, phenyl, 3-pyridinyl, 4-Me-phenyl, 2-Br-phenyl, isopropyl, and 3-pyridinyl.
  • R 11 is H.
  • R 2 is independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a
  • heteroalkenyl group an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
  • R 12 is an amino acid or peptide, preferably selected from the group consisting of G, F, V, GG, FA, PLF and PGLF.
  • the amino acid or peptide comprises a protecting group.
  • R 14 is H and X is tetrahydrofuran.
  • reducing the compound of Formula (1) is performed with at least one condition and/or reagent selected from HSiCI 3 , Et 3 N, CH 2 CI 2 , and 0-23°C.
  • condition and/or reagent selected from HSiCI 3 , Et 3 N, CH 2 CI 2 , and 0-23°C.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 and R 12 are each independently H or an organic group.
  • the Ugi 4-component reaction is performed with at least one condition and/or reagent selected from TFE, 23°C and time.
  • Formula (11) comprising ester hydrolysis and deprotection of the compound of Formula (10): Formula (10) wherein R 1 , R 2 , R 9 , R 0 , R 14 , and R 5 are each independently H or an organic group and X is any Lewis basic ligand.
  • the ester hydrolysis and deprotection of the compound of Formula (10) is performed with at least one condition and/or reagent selected from NaOH, THF/H 2 0, 23°C and time (10 min.).
  • the process further comprises performing a Passerini 3- component reaction with the compound of Formula (2), R 9 R 0 -CO, and an organic acid R 13 -COOH (preferably Ph-CH 2 -COOH), to obtain the compound of Formula (10).
  • the Passerini 3-component reaction is performed with at least one condition and/or reagent selected from CH 2 CI 2 and 23°C.
  • the preparation of the compound of formula (11) in which R 5 is an alkyl group is performed by subjecting the corresponding compound of formula 11 in which R 15 is H with at least one condition and/or reagent selected from NaH, R 15 l, THF, crushed 4A molecular sieves, 23°C and time (12 h) in a deprotonation/alkylation reaction.
  • group means a linked collection of atoms or a single atom within a molecular entity, where a molecular entity is any constitutionally or isotopically distinct atom, molecule, ion, ion pair, radical, radical ion, complex, conformer etc., identifiable as a separately distinguishable entity.
  • the description of a group as being “formed by” a particular chemical transformation does not imply that this chemical transformation is involved in making the molecular entity that includes the group.
  • organic group means a group containing at least one carbon atom.
  • alkyl group means a group formed by removing a hydrogen from a carbon of an alkane, where an alkane is an acyclic or cyclic compound consisting entirely of hydrogen atoms and saturated carbon atoms.
  • An alkyl group may include one or more substituent groups.
  • heteroalkyl group means a group formed by removing a hydrogen from a carbon of a heteroalkane, where a heteroalkane is an acyclic or cyclic compound consisting entirely of hydrogen atoms, saturated carbon atoms, and one or more heteroatoms.
  • a heteroalkyl group may include one or more substituent groups.
  • alkenyl group means a group formed by removing a hydrogen from a carbon of an alkene, where an alkene is an acyclic or cyclic compound consisting entirely of hydrogen atoms and carbon atoms, and including at least one carbon- carbon double bond.
  • An alkenyl group may include one or more substituent groups.
  • heteroalkenyl group means a group formed by removing a hydrogen from a carbon of a heteroalkene, where a heteroalkene is an acyclic or cyclic compound consisting entirely of hydrogen atoms, carbon atoms and one or more heteroatoms, and including at least one carbon-carbon double bond.
  • a heteroalkenyl group may include one or more substituent groups.
  • alkynyl group means a group formed by removing a hydrogen from a carbon of an alkyne, where an alkyne is an acyclic or cyclic compound consisting entirely of hydrogen atoms and carbon atoms, and including at least one carbon- carbon triple bond.
  • An alkynyl group may include one or more substituent groups.
  • heteroalkynyl group means a group formed by removing a hydrogen from a carbon of a heteroalkyne, where a heteroalkyne is an acyclic or cyclic compound consisting entirely of hydrogen atoms, carbon atoms and one or more heteroatoms, and including at least one carbon-carbon triple bond.
  • a heteroalkynyl group may include one or more substituent groups.
  • aryl group means a group formed by removing a hydrogen from a ring carbon atom of an aromatic hydrocarbon.
  • An aryl group may by monocyclic or polycyclic and may include one or more substituent groups.
  • a heteroaryl group may by monocyclic or polycyclic and may include one or more substituent groups.
  • substituteduent group means a group that replaces one or more hydrogen atoms in a molecular entity.
  • heterocyclic group means a group formed by removing a hydrogen from a cyclic compound that has atoms of at least two different elements as members of its ring(s).
  • acyl group means a group formed by removing one or more hydroxyl groups from an oxoacid, i.e. RCO-.
  • halogen group means F-, CI-, Br- or I-.
  • hydroxyl group means the group containing an oxygen atom connected by a covalent bond to a hydrogen atom, i.e. OH-.
  • alkoxy group means an alkyl group singularly bonded to oxygen, i.e. R-O.
  • acyloxyl group means a group formed by removal of hydrogen from oxygen in an organic acid, e.g. RCOO-.
  • organichalide means an organic compound that includes at least one halogen group.
  • chemical reactions in some cases, may require and would include protecting certain peptide or amino acid side chains with a protecting group in manner known to a person skilled in the art.
  • NMR spectra were recorded at 25°C on Bruker Advance III 400, Varian Mercury 400 or Agilent DD2-500 instrument.
  • the DD2-500 MHz spectrometer used an Agilent HC 5-mm XSens cryogenically-cooled probe.
  • a H pulse width of 45° was used, acquiring a spectral window of 7000 Hz (14 ppm) using 32k points.
  • the 1 H 90° pulse width was 11.75 ps.
  • a 13 C pulse width of 30° was used acquiring a spectral window of 28750 Hz (230 ppm) using 64k points.
  • the 3C 90° pulse width was 21.4 ps. All pulse sequences used were provided by Agilent.
  • Mass Spectroscopy High resolution mass spectra were obtained on a VG 70- 250S (double focusing) mass spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate mass capabilities or on JEOL AccuTOF-DART instrument.
  • reaction solution was cooled to 0°C and saturated aq. NaHC0 3 (10 mL) was added slowly. The resulting suspension was stirred at 0°C until bubbling ceased at which point it was allowed to warm to rt. The layers were separated and the aqueous layer was washed with EtOAc. The combined organic layers were washed with saturated aq. NaHC0 3 /H 2 0 (50/50) and saturated aq. NaHC0 3 /brine (50/50), dried over Na 2 S0 4 , filtered and concentrated.
  • CiiH 17 BN 2 0 4 S 302.13458 found 302.13452; IR (thin film, cm -1 ) 3013, 2959, 2871 , 2148, 2091 , 1746, 1633, 1450, 1337, 1284, 1246, 1217, 1 195, 1158, 1094, 1075, 1042, 993, 956, 897, 867, 815, 725, 688.
  • reaction mixture was concentrated and the crude product was purified via flash column chromatography on silica gel (neutralized with hexanes/Et 3 N (95:5)) using EtOAc/MeCN/Et 3 N (20:0:1 ⁇ 16:4:1 ⁇ 10:10:1 ⁇ 4:16:1 ). To afford the desired product as an off-white solid in a 1 :1 diastereomeric ratio (54 mg, 55%).
  • N-Boc protected amino acids were used as supplied. Fully protected resin-bound tri- and tetra-peptides H-Pro-Leu-Phe-OH and H-Pro-Gly-Leu- Phe-OH were synthesized via standard Fmoc solid-phase peptide chemistry using an automated peptide synthesizer. Fmoc removal was achieved by treatment with 20% piperidine in NMP for 5 and 10 minutes with consecutive DMF and NMP washes after each addition. For all Fmoc amino acid coupling, the resin was treated once with 4.5 eq. of Fmoc amino acids, 4.5 eq. of HCTU and 9 eq. of DIPEA in NMP for 60 minutes.
  • Diastereomers are classified by their order of elution in reverse-phase chromatography (A eluting first being the most polar, B eluting second being less polar than A, and so on for C and D if applicable.) (N-Boc)-Gly-DL-Phe-DL-(MIOA boro)-Leu (9a)
  • Diastereomeric ratios were determined using 1 H NMR proton integrations of the N-Me signal. It should be noted that reactions utilizing nicotinaldehyde were carried out in the dark until TLC indicated full consumption of starting material. Diastereomers are classified by their order of elution from silica gel (A eluting first being the least polar, B eluting second being more polar than A) Diastereomeric ratios were determined by comparative H NMR integrations of the N- CH 3 signals.
  • the P3CR product 10cp was subjected to the standard procedure for preparation of 11ad above.
  • LRMS indicated the presence of the desired product with some hydrolyzed byproduct.
  • the IC 50 of the impure compound for inhibition of 20S proteases was determined using the procedure outlined for 11ad. An IC 50 of ⁇ 1 ⁇ was obtained for the CT-L enzymes and no observable inhibition occurred for the T-L or C-L enzymes.
  • LRMS (ESI, positive) of the sample with pH 12.3 showed a mass corresponding to the pentadeuterated boronate shown in Scheme 1.
  • the 11 B NMR spectra were taken with a sweep width of 51000 Hz, 131000 data points, 90° pulse width, 1.2 second recycle time, 10 Hz line broadening and a 2 nd order polynomial fitting routine.
  • Solutions of 7a (each diastereomer) and bortezomib were prepared by serial dilution of 10 mM stocks in DMSO.
  • a feshly prepared sample of OCI-AML-2 human leukemia cells was added 5 mL of freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1 % Trition X-100 and 2 mM ATP.
  • the cells were suspended by pipetting up and down several times and were vortexed every 5 minutes for 30 minutes at 0°C.
  • Each well of a 96 well-plate was loaded with 87 ⁇ _ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 10 pL of cell lysate solution and 1 pL of each stock solution of either 7a or bortezomib (to final concentrations of 10 ⁇ to 10 pM, in 1/10 th dilution increments).
  • the resulting solutions were incubated at 37°C for 1h.
  • To each well was added 2 ⁇ _ of 3.75 mM N- Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin in DMSO.
  • bortezomib 7a (diastereomer A) 7a (diastereomer B) chymotrypsin-like 22 71
  • Solutions of 11ad and bortezomib were prepared by serial dilution of a 10 mM stock in DMSO.
  • a feshly prepared sample of OCI-AML-2 human leukemia cells was added 5 ml. of freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1 % Trition X-100 and 2 mM ATP.
  • the cells were suspended by pipetting up and down several times and were vortexed every 5 minutes for 30 minutes at 0°C.
  • Each well of a 96 well-plate was loaded with 87 ⁇ _ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 10 L of cell lysate solution and 1 ⁇ . of each stock solution of either 11ad or bortezomib (to final concentrations of 100 ⁇ to 1 nM, in 1/10 th dilution increments). The resulting solutions were incubated at 37°C for 1 h.
  • the fluorescence spectrum of each well was measured at 5 minute intervals over 30 minutes at 37°C (using a Spectromax spectrometer by Molecular Devices, excitation: 360 nm; emission 460 nm).
  • the slope of the increase in fluorescence vs. time was plotted against the inhibitor concentration ( Figures 3-5).
  • the IC 50 of each of 11ad and bortezomib was calculated by applying a sigmoidal fit to each curve shown in Figures 3-5 and interpolating to 50% enzyme activity. In each assay, rates were measure in triplicate and averaged. Error bars represent 1 standard deviation.
  • Figure 6 shows a comparison of the syn- and anti- isomers of 11ad in the chymotrypsin-like assay. It should be noted that IC 50 values obtained in the assays of syn- and a/7f/ ' -11ad cannot be compared to those obtained in the bortezomib comparison studies as different cell preparations were used. The IC 50 values are tabulated in Table 3.
  • a 1 mM stock solution of 7a-A in DMSO was prepared.
  • Each of 5 HPLC vials were loaded with 870 ⁇ _ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 100 ⁇ _ of cell lysis buffer (containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1% Trition X-100 and 2 mM ATP) (no cells were added) and 15 pL of 7a-A stock solution.
  • One vial was immediately subjected to HPLC-MS analysis. The remaining 4 were incubated at 37°C.
  • One vial was removed each 30 minutes and immediately subjected to HPLC-MS analysis.
  • the final vial was left incubating overnight.
  • the remaining vials gave similar results (no qualitative change in either species was observed). It should be noted that quantitative comparisons of peak data could not be obtained as multiple, poorly separated peaks were observed for each species presumably due to non-covalent bonding interactions with one or more of the buffer components.
  • Efflux ratios correspond to the B-A P app coefficient divided by A-B P app value. Efflux ratios were classified as:
  • Isocyanide 2a participated in an Ugi 4-component reaction (U4CR) with 2-pyrazinyl carboxylic acid, phenylacetaldehyde and ammonia to afford the MIDA-bortezomib analogue 7a in 55% isolated yield (Scheme 3).
  • U4CR Ugi 4-component reaction
  • isocyanide 2a was reacted with L-proline and isobutyraldehyde the borodipeptide 8a was obtained in 7% isolated yield (Scheme 3).
  • Covalent electrophilic inhibitors are designed to react with nucleophilic groups at an enzyme's active site resulting in covalent bonding and inhibition.
  • a commonly encountered problem with this approach is competing reactivity of the inhibitor with water (a weak but often reactive nucleophile).
  • water attacks the electrophilic site of the inhibitor before it can reach its enzyme target resulting in hydrolysis and a loss of activity.
  • the pKa of 11 ad is approximately 9.0, we can conclude that at pH ⁇ 8 (virtually all environments encountered in biological systems), the electrophilic center is impervious to attack by water.
  • the BMN scaffold is hydrolytically stable below pH 8.
  • boromorpholinone inhibitors showed weaker inhibition than bortezomib, structural optimization has the potential to drastically improve these results.
  • the pyrazine side chain in bortezomib does not exhibit a defined interaction with the active site binding pockets within the 20S proteasome causing a lack of selectivity and therefore a range of undesired side effects.
  • the multi-component nature of our boropeptide preparation methodology facilitates diversity-oriented synthesis by allowing addition of a second level of structural diversity in the same step as boron integration. This facilitates the preparation of diverse inhibitor libraries and therefore elucidation of a structure-activity relationship and optimization of a selective proteasome inhibitor.

Abstract

This application pertains to α-boryl isocyanates, wherein the boronate moiety is in the form of an N-methyliminodiacetic acid (MIDA) boronate of the Formula (2) and the utility of said compounds in the synthesis of the borylamide motif (Β-C-Namide) in the scaffold of biologically-active boropeptides, such as bortezomib, in the enablement of heterocycle synthesis, and in multi-component reactions (MCRs), such as the Ugi and Passerini processes.

Description

σ-BoRYL ISOCYANIDES, BOROPEPTIDES AND BORON HETEROCYCLES
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 61/807,400 filed on April 2, 20 3, which is incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates to novel a-boryl isocyanides, their synthesis, as well as their use in the preparation of boron-containing compounds.
BACKGROUND OF THE INVENTION
Boronic acids and their derivatives are useful as synthetic building blocks, [1] chemosensors,[2] and biologically active targets of synthesis. Both the biological activity and chemical reactivity of boronic acids stem from boron's Lewis acidity. While useful in a broad range of applications, boron's propensity to undergo reactions with Lewis bases becomes problematic for functional group compatibility during synthesis. [1] Reagents that streamline installation of a carbon-boron bond in stereochemically complex, heteroatom-rich environments, are expected to find application not only as starting materials but also as valuable endpoints of synthesis.
SUMMARY OF INVENTION
In an aspect, there is provided a compound of Formula (2):
Figure imgf000003_0001
Formula (2) wherein R1, R2, R3, R4, R5 , R6 and R7 and are each independently H or an organic group. In a further aspect, there is provided a compound of Formula (9):
Figure imgf000003_0002
Formula (9) wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, R11 and R12 are each independently H or an organic group. In a further aspect, there is provided a compound of Formula (1 1 ):
Figure imgf000003_0003
Formula (11 ) wherein R1 , R2, R9, R 0, R14, and R15 are each independently H or an organic group and X is any Lewis basic ligand. There is also described herein, processes for the synthesis of the above compounds.
BRIEF DESCRIPTION OF FIGURES
These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:
Figure 1 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boropeptide derivative 7a-A.
Figure 2 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boropeptide derivative 7a-B.
Figure 3 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boromorpholinone anti- 1ad.
Figure 4 shows the in vitro cytosolic caspase-like 20S proteasome inhibition by boromorpholinone anti-λ 1ad. Figure 5 shows the in vitro cytosolic trypsin-like 20S proteasome inhibition by boromorpholinone anti-λ 1ad.
Figure 6 shows the in vitro cytosolic chymotrypsin-like 20S proteasome inhibition by boromorpholinones syn- 1ad and ani/-11ad.
Figure 7 shows the computational analysis of possible hydrolytic degradation of anti- 11ad. Data reflects enthalpic energy differences between reaction partners.
Figure 8 shows the cellular permeability of ani/-11ad compared to bortezomib with positive (metoprolol) and negative (atenolol) controls.
Figure 9 shows a computational model of anf/-11ad in complex with a chymotrypsin- like member of proteasome 20S. Figure 10 shows the structures of several MIDA-boronate containing compounds which act as potent protease inhibitors.
DETAILED DESCRIPTION In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.
The borylamide motif (B-C-Namide) is commonly found in the structures of biologically active boropeptides.[3] Recent efforts have been focused on the amphoteric aziridine aldehyde- and isocyanide-driven macrocyclizations of linear peptides and peptidomimetics.[4] This methodology is enabled by the development of functionally dense, heteroatom-rich environments where a kinetic barrier prevents two otherwise reactive functional groups from prematurely reacting with each other.[5] In the present invention, we expand the scope of this methodology to include amphoteric boron- containing building blocks for use in multi-component preparation of boropeptides and their derivatives.
As part of our efforts to develop amphoteric boron-transfer reagents, we considered an isocyanide/boron combination. Isocyanides are 1 ,1 -amphoteric molecules that enable heterocycle synthesis[6] and participate in multi-component reactions (MCRs) such as Ugi and Passerini processes. [7] Herein, we outline the preparation and utility of bench- stable o-boryl isocyanides. We demonstrate the application of these novel derivatives in heterocycle synthesis as well as in MCRs to generate biologically active boropeptides. As part of our study, we demonstrate a two-step synthesis of both diastereomers of bortezomib, an FDA approved, boron-containing proteasome inhibitor used to treat multiple myeloma. [8] Furthermore, our documented discovery of a novel class of boron-containing heterocycles, with biological activity comparable to bortezomib, will be useful in efforts to prepare libraries of biologically active boropeptide derivatives using readily available starting materials. [3b, d, f, g, I, n, o, p]
In an aspect, there is provided a compound of Formula (2):
Figure imgf000006_0001
Formula (2) wherein R1, R2, R3, R4, R5 , R6 and R7 and are each independently H organic group.
In a further aspect, there is provided a compound of Fomnula (9):
Figure imgf000006_0002
Formula (9) wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, R11 and R12 are each independently H or an organic group.
In a further aspect, there is provided a compound of Formula (11):
Figure imgf000006_0003
Formula (11) wherein R1, R2, R9, R 0, R14, and R15 are each independently H or an organic group and X is any Lewis basic ligand. In some embodiments, R1, R2, R3, R4, R5 , R6 , R7, R9, R 0, R11, R 2, R13, R14 and R15 are each independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group. Preferably, the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
In some embodiments, R3, R4, R5 , R6 are H and R7 is CH3.
In some embodiments, R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl. In some embodiments, R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl. In some embodiments, R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl.
In some embodiments, R9 and R10 are independently selected from the group consisting of H, benzyl and isopropyl. In other embodiments, R9 and R10 are independently selected from the group consisting of H, benzyl, isopropyl, 4-F-phenyl, phenyl, 3-pyridinyl, 4-Me-phenyl, 2-Br-phenyl, isopropyl, and 3-pyridinyl.
In some embodiments, R11 is H.
In some embodiments, R 2 is independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a
heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
In some embodiments, R12 is an amino acid or peptide, preferably selected from the group consisting of G, F, V, GG, FA, PLF and PGLF. In some embodiments, the amino acid or peptide comprises a protecting group. In some embodiments, R14 is H and X is tetrahydrofuran.
In a further aspect, there is provided a process for preparing the compound of Formula (2):
Figure imgf000008_0001
Formula (2) comprising reducing the compound of Formula (1):
Figure imgf000008_0002
Formula (1) wherein R1, R2, R3, R4, R5 , R6 and R7 and are each independently H or an organic group.
In some embodiments, reducing the compound of Formula (1) is performed with at least one condition and/or reagent selected from HSiCI3, Et3N, CH2CI2, and 0-23°C. In a further aspect, there is provided a process for preparing the compound of Formula (9):
Figure imgf000008_0003
Formula (9) comprising subjecting to a Ugi 4-component reaction, the compound of Formula (2):
Figure imgf000009_0001
Formula (2)
FT- COOH; R1 NH2; and R9R10-CO; wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, R11 and R12 are each independently H or an organic group..
In some embodiments, the Ugi 4-component reaction is performed with at least one condition and/or reagent selected from TFE, 23°C and time.
In a further aspect, there is provided a process for preparing the compound of Formula (11):
Figure imgf000009_0002
Formula (11) comprising ester hydrolysis and deprotection of the compound of Formula (10):
Figure imgf000010_0001
Formula (10) wherein R1, R2, R9, R 0, R14, and R 5 are each independently H or an organic group and X is any Lewis basic ligand. In some embodiments, the ester hydrolysis and deprotection of the compound of Formula (10) is performed with at least one condition and/or reagent selected from NaOH, THF/H20, 23°C and time (10 min.).
In some embodiments, the process further comprises performing a Passerini 3- component reaction with the compound of Formula (2), R9R 0-CO, and an organic acid R13-COOH (preferably Ph-CH2-COOH), to obtain the compound of Formula (10).
In some embodiments, the Passerini 3-component reaction is performed with at least one condition and/or reagent selected from CH2CI2 and 23°C.
In some embodiments, the preparation of the compound of formula (11) in which R 5 is an alkyl group is performed by subjecting the corresponding compound of formula 11 in which R15 is H with at least one condition and/or reagent selected from NaH, R15l, THF, crushed 4A molecular sieves, 23°C and time (12 h) in a deprotonation/alkylation reaction.
The term "group" means a linked collection of atoms or a single atom within a molecular entity, where a molecular entity is any constitutionally or isotopically distinct atom, molecule, ion, ion pair, radical, radical ion, complex, conformer etc., identifiable as a separately distinguishable entity. The description of a group as being "formed by" a particular chemical transformation does not imply that this chemical transformation is involved in making the molecular entity that includes the group. The term "organic group" means a group containing at least one carbon atom.
The term "alkyl group" means a group formed by removing a hydrogen from a carbon of an alkane, where an alkane is an acyclic or cyclic compound consisting entirely of hydrogen atoms and saturated carbon atoms. An alkyl group may include one or more substituent groups.
The term "heteroalkyl group" means a group formed by removing a hydrogen from a carbon of a heteroalkane, where a heteroalkane is an acyclic or cyclic compound consisting entirely of hydrogen atoms, saturated carbon atoms, and one or more heteroatoms. A heteroalkyl group may include one or more substituent groups. The term "alkenyl group" means a group formed by removing a hydrogen from a carbon of an alkene, where an alkene is an acyclic or cyclic compound consisting entirely of hydrogen atoms and carbon atoms, and including at least one carbon- carbon double bond. An alkenyl group may include one or more substituent groups.
The term "heteroalkenyl group" means a group formed by removing a hydrogen from a carbon of a heteroalkene, where a heteroalkene is an acyclic or cyclic compound consisting entirely of hydrogen atoms, carbon atoms and one or more heteroatoms, and including at least one carbon-carbon double bond. A heteroalkenyl group may include one or more substituent groups.
The term "alkynyl group" means a group formed by removing a hydrogen from a carbon of an alkyne, where an alkyne is an acyclic or cyclic compound consisting entirely of hydrogen atoms and carbon atoms, and including at least one carbon- carbon triple bond. An alkynyl group may include one or more substituent groups.
The term "heteroalkynyl group" means a group formed by removing a hydrogen from a carbon of a heteroalkyne, where a heteroalkyne is an acyclic or cyclic compound consisting entirely of hydrogen atoms, carbon atoms and one or more heteroatoms, and including at least one carbon-carbon triple bond. A heteroalkynyl group may include one or more substituent groups.
The term "aryl group" means a group formed by removing a hydrogen from a ring carbon atom of an aromatic hydrocarbon. An aryl group may by monocyclic or polycyclic and may include one or more substituent groups. The term "heteroaryl group" means a group formed by replacing one or more methine (-C=) and/or vinylene (-CH=CH-) groups in an aryl group with a trivalent or divalent heteroatom, respectively. A heteroaryl group may by monocyclic or polycyclic and may include one or more substituent groups. The term "substituent group" means a group that replaces one or more hydrogen atoms in a molecular entity.
The term "heterocyclic group" means a group formed by removing a hydrogen from a cyclic compound that has atoms of at least two different elements as members of its ring(s). The term "acyl group" means a group formed by removing one or more hydroxyl groups from an oxoacid, i.e. RCO-.
The term "halogen group" means F-, CI-, Br- or I-.
The term "hydroxyl group" means the group containing an oxygen atom connected by a covalent bond to a hydrogen atom, i.e. OH-. The term "alkoxy group" means an alkyl group singularly bonded to oxygen, i.e. R-O.
The term "acyloxyl group" means a group formed by removal of hydrogen from oxygen in an organic acid, e.g. RCOO-.
The term "organohalide" means an organic compound that includes at least one halogen group. One would understand that chemical reactions, in some cases, may require and would include protecting certain peptide or amino acid side chains with a protecting group in manner known to a person skilled in the art.
The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention. EXAMPLES
MATERIALS AND METHODS
General: Anhydrous solvents were obtained by distillation under nitrogen prior to use. Dichloromethane was purified using an MBraun Solvent Purification System. All other solvents were of reagent grade quality. All reagents, catalysts and ligands were purchased from Combi-Blocks Inc., Sigma-Aldrich, Strem-Chemical Company, VWR International, or Aapptec and used as received unless otherwise noted.
Nuclear Magnetic Resonance Spectroscopy: NMR spectra were recorded at 25°C on Bruker Advance III 400, Varian Mercury 400 or Agilent DD2-500 instrument. The DD2-500 MHz spectrometer used an Agilent HC 5-mm XSens cryogenically-cooled probe. A H pulse width of 45° was used, acquiring a spectral window of 7000 Hz (14 ppm) using 32k points. The 1H 90° pulse width was 11.75 ps. A 13C pulse width of 30° was used acquiring a spectral window of 28750 Hz (230 ppm) using 64k points. The 3C 90° pulse width was 21.4 ps. All pulse sequences used were provided by Agilent. 13C NMR spectra were registered with broad-band decoupling. Recorded shifts for protons are reported in parts per million (6 scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvents (DMSO-d6: 2.54 or MeCN-d3: δ 1.94, centre line). Chemical shifts for carbon resonances are reported in parts per million (6 scale) downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent (DMSO-d6: 40.45 or MeCN-d3: δ 1.32, centre line). Carbons exhibiting significant line broadening brought about by boron substituents were not reported (quadrupolar relaxation).11B NMR was recorded at 25°C on a Bruker Advance III 400 MHz spectrometer or on an Agilent DD2 600 MHz spectrometer with an Agilent OneNMR probe and referenced to an external standard of BF3-Et20. Data are represented as follows: chemical shift δ in ppm, multiplicity (s singlet, d doublet, t triplet, q quartet, m multiplet, br broad), coupling constant J in Hz and integration.
Chromatography: Flash column chromatography was carried out using Silicycle 230- 400 mesh silica gel. Thin-layer chromatography (TLC) was performed on Macherey Nagel pre-coated glass backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 nm) or KMn04 or Curcumin stain in case of no UV activity. Infrared Spectroscopy: Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum 100 instrument equipped with a single-reflection diamond/ZnSe ATR accessory. Mass Spectroscopy: High resolution mass spectra were obtained on a VG 70- 250S (double focusing) mass spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate mass capabilities or on JEOL AccuTOF-DART instrument.
It should be noted that intermediates used directly in subsequent reactions without purification are characterized by crude H NMR only.
RESULTS
General procedure for the synthesis of σ-boryl isocyanides 2a-c
Figure imgf000014_0001
1 2 σ-Boryl isocyanates used in this paper for the synthesis of a-boryl isocyanides were prepared according to the literature method[13] in which the preparation and characterization of phenyl-, isobutyl-, and cyclohexyl- substituted σ-boryl isocyanates were reported. List of known σ-boryl isocyanates:
Figure imgf000014_0002
(isobutyl-) (cyclohexyl-) (phenyl-)
To a flame-dried flask flushed with Ar was added freshly distilled DCM (10 mL) which was cooled to 0°C. Trichlorosilane (4.86 mmol, 0.5 mL, 1.3 equiv.) was added followed by dropwise addition of triethylamine (11.22 mmol, 0.82 ml_, 3.0 equiv.). σ-Boryl isocyanate (3.74 mmol, 1.1 g, 1.0 equiv.) was added and the resulting suspension was stirred vigorously at 0°C for 30 minutes. The resulting solution was allowed to warm to rt. Stirring was continued until starting material was completely consumed as indicated by TLC or crude 1H NMR. The reaction solution was cooled to 0°C and saturated aq. NaHC03 (10 mL) was added slowly. The resulting suspension was stirred at 0°C until bubbling ceased at which point it was allowed to warm to rt. The layers were separated and the aqueous layer was washed with EtOAc. The combined organic layers were washed with saturated aq. NaHC03/H20 (50/50) and saturated aq. NaHC03/brine (50/50), dried over Na2S04, filtered and concentrated. The crude residue was purified by flash column chromatography on neutralized silica (slurry prepared in hexanes/EtaN 95:5, eluent; EtOAc/Et3N 95:5→ EtOAc/MeCN/Et3N 70:25:5) to yield the desired product. lsobutyl( IDA boryl)methyl isocyanide (2a)
Figure imgf000015_0001
White solid; 75% yield; TLC (EtOAc) Rf = 0.36; m.p. 194-196°C; 1H NMR (400 MHz, MeCN-d3) δ 4.37 (d, J = 17.3 Hz, 1 H), 4.34 (d, J = 17.3 Hz, 1 H), 4.08 (d, J = 17.3 Hz, 1H), 4.04 (d, vy = 17.3 Hz, 1H), 3.37 (dd, J = 12.0, 3.1 Hz, 1 H), 3.06 (s, 3H), 1.82 - 1.72 (m, 1 H), 1.56 (ddd, J = 14.0, 12.0, 3.1 Hz, 1 H), 1.19 (dd, J = 12.0 Hz, 3.1 1 H), 0.94 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-cfe) δ 168.79, 168.75, 156.14, 63.09, 63.05, 46.58, 38.89, 25.20, 23.68, 20.85 ppm; B NMR (128 MHz, DMSO-cfe) δ 14.4 ppm; HRMS (DART-TOF) [M+NH4]+ calcd. For CnH17B 204 270.16 96, found 270. 6180; IR (thin film, cm"1) 3961 , 2277, 2130, 1746, 1665, 1470, 1337, 1287, 1248, 1220, 1194, 1159, 1100, 1078, 1049, 1023, 992, 962, 929, 897, 867, 818, 731 , 692. Tetradeuteration of cr-boryl isocvanide 2a °
Figure imgf000016_0001
To a flame-dried flask under Ar was added o-boryl isocyanide 2a (0.040 mmol, 10 mg, 1.0 equiv.) and CH2CI2 (0.1 ml_). The resulting suspension was cooled to -78°C and KOfBu (0.044 mmol, 5.2 mg, 1.1 equiv.) was added. The resulting suspension was stirred for 30 minutes at -78°C and Mel (0.04 mmol, 5.7 mg, 2.49 pL, 1.0 equiv.) was added. The resulting suspension was stirred at -78"C for 30 minutes and was allowed to warm to 23°C followed by 1.5 h of stirring. The solvent was removed under reduced pressure and MeCN-cfe (0.5 ml.) was added. 1H NMR and mass spectra were taken directly from the resulting solution. DA boryl)methyl isocyanide (2a-d4)
Figure imgf000016_0002
White solid; TLC (EtOAc) Rf = 0.36; 1H NMR (400 MHz, MeCN-cfe) δ 3.06 (s, 3H), 1.82 - 1.72 (m, 1 H), 1.56 (ddd, J = 14.0, 12.0, 3.1 Hz, 1 H), 1.19 (dd, J = 12.0 Hz, 3.1 1H), 0.94 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H) ppm; HRMS (DART-TOF) [M+NH4]+ calcd. For CiiH13D4BN204 257.16107, found 257.16056.
Cyclohexyl(MIDA boryl)methyl isocyanide (2a)
Figure imgf000016_0003
Off-White solid; 35% yield; TLC (EtOAc) Rf = 0.32; m.p. 205-208°C; 1H NMR (400 MHz, DMSO-de) δ 4.31(3) (d, J = 17.2 Hz, 1H), 4.31(1) (d, J = 17.4 Hz, 1H), 4.05 (d, J = 17.2 Hz, 1 H), 4.00 (d, J = 17.4 Hz, 1 H), 3.32 (d, J = 2.7 Hz, 1 H), 3.01 (s, 3H), 1.75 - 1.71 (m, 3H), 1.62 - 1.49 (m, 4H), 1.34 - 1.19 (m, 4H) ppm; 3C NMR (100 MHz, DMSO-cfe) δ 182.96, 169.04, 168.75, 62.53, 62.50, 46.46, 37.61 , 31.47, 27.94, 26.31 , 25.99, 25.98 ppm; 11B NMR (128 MHz, DMSO-cfe) δ 11.75 ppm; HRMS (DART-TOF) [Μ+ΝΗ,Γ calcd. For Ci3H19BN204 296.17761 , found 296.17844; IR (thin film, cm 1) 2927, 2854, 2273, 2136, 1749, 1451, 1338, 1289, 1244, 1194, 1156, 1105, 1078, 1038, 992, 972, 955, 894, 868.44, 839, 772, 734, 697, 675. boryQmethyl isocyanide (2c)
Figure imgf000017_0001
Off-White solid; 31% yield; TLC (EtOAc) Rf = 0.28; 1H NMR (500 MHz, MeCN-cf3 + DMSO-of6) δ 7.41 - 7.38 (m, 2H), 7.35 - 7.29 (m, 3H), 4.75 (s, 1H), 4.40 (d, J = 17.2, 1 H), 4.38 (d, J = 17.2 Hz, 1 H), 4.14 (d, J = 17.2 Hz, 1 H), 4.09 (d, J = 17.2 Hz, 1 H), 3.19 (s, 3H) ppm; 13C NMR (125 MHz, MeCN-cfe + DMSO-cfe) δ 168.1 , 168.0, 136.4, 128.3, 127.0, 126.7, 63.0, 62.8, 46.4 ppm; 11B NMR (128 MHz, MeCN-c/3 + DMSO-cfe) δ 9.7 ppm; HRMS (DART-TOF) [M+NH4]+ calcd. For C13Hi7B 304 290.13121 , found 290.13115; IR (thin film, cm 1) 2951 , 2875, 2138, 1747, 1666, 1495, 1452, 1370, 1337, 1279, 1244, 1154, 1102, 1050, 951 , 895, 818, 759, 702.
General procedures for the functionalization of cr-boryl isocyanide 2a
σ-boryl isothiocyanate 3a
Figure imgf000017_0002
2a 3a
Elemental sulfur (15 mg, 0.48 mmol, 1.2 equiv.) and selenium (2.0 mg, 0.020 mmol, 5.0 mol%) were suspended in THF (0.8 ml.) and Et3N (134 pL, 0.96 mmol, 2.4 equiv.) was added. The isocyanide (100 mg, 0.40 mmol, 1.0 equiv.) was added, the vial was sealed and the resulting suspension was stirred at 80°C for 15 min at which point TLC indicated that the reaction had gone to completion. The solution was allowed to cool to 23°C, filtered (rinsing with EtOAc) and concentrated. The crude product was purified via flash column chromatography using hexanes/EtOAc (6:4→ 1 :0) and was isolated as an off-white solid (88 mg, 77%). )-1 -isothiocyanato-3-methylbutane (3a)
Figure imgf000018_0001
Off-white solid; 77% yield; TLC (EtOAc) Rf = 0.58, m.p. = 192 - 194°C (decomp.); 1H NMR (500 MHz, DMSO-cfe) δ 4.38 (d, J = 17.3 Hz, 1 H), 4.10 (d, J = 17.1 Hz, 1 H), 4.37 (d, J = 17.1 Hz, 1 H), 4.02 (d, J = 17.3 Hz, 1 H), 3.54 (dd, J = 11.8, 3.0 Hz, 1 H), 3.02 (s, 3H), 1.79 - 1.69 (m, 1 H), 1.64 (ddd, J = 14.3, 1 1.8, 3.5 Hz, 1 H), 1.31 (ddd, J = 14.1 , 10.1 , 3.0 Hz, 1 H), 0.96 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (125 MHz, DMSO-ofe) δ 168.3, 168.2, 126.3, 62.4, 62.4, 46.2, 39.5, 25.3, 23.2, 20.6 ppm; 11B NMR (128 MHz, DMSO-cfe) δ 10.3 ppm; HRMS (DART-TOF) [M+H]+ calcd. for CiiH17BN204S 302.13458, found 302.13452; IR (thin film, cm-1) 3013, 2959, 2871 , 2148, 2091 , 1746, 1633, 1450, 1337, 1284, 1246, 1217, 1 195, 1158, 1094, 1075, 1042, 993, 956, 897, 867, 815, 725, 688.
Figure imgf000018_0002
3a 4a
To a solution of σ-boryl isothiocyanate 3a (45 mg, 0.158 mmol, 1.0 equiv.) in THF (0.5 ml.) was added amine (0.32 mmol, 2.0 equiv.) and the resulting solution was stirred at 23°C until TLC or LRMS (ESI, positive) indicated that the reaction had gone to completion (9-24 h). The solvent was removed and the crude product was purified via flash column chromatography using a gradient of hexanes/EtOAc (1 :0→ 0:1) as the eluent. Purified products were isolated as off-white solids.
Figure imgf000019_0001
Off-white solid; 79% yield; TLC (EtOAc) R, = 0.43; 1H NMR (500 MHz, MeCN-of3) δ 6.19 (br s, 1 H), 5.76 (d, J = 9.6 Hz, 1 H), 4.26 (br s, 1 H), 3.95 (d, J = 17.2 Hz, 1 H), 7.89 (d, J = 16.9 Hz, 1 H), 3.85 (d, J = 17.1 Hz, 1 H), 3.76 (d, J = 16.9 Hz, 1 H), 3.39 (br s, 2H), 3.05 (s, 3H), 1.69 - 1.61 (m, 1H), 1.44 - 1.34 (m, 2H), 1.09 (t, J = 7.2 Hz, 1 H), 0.97 (d, J = 6.5 Hz, 1 H), 0.91 (d, J = 6.7 Hz, 1 H) ppm; 13C NMR (125 MHz, MeCN-cf3) δ 169.1 , 168.9, 128.0, 63.3, 63.2, 47.1 , 42.9, 39.6, 25.5, 24.2, 23.1 , 14.7 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 11.5 ppm; HRMS (DART-TOF) [M+Hf calcd. for C13H25BN3O4S 330.16588, found 330.16600; IR (thin film, cm 1) 3303, 3084, 3951 , 2893, 2863, 2841 , 2240, 1770, 1752, 1540, 1484, 1482, 1450, 1337, 1331 , 1259, 1187, 1152, 1098, 1076, 1048, 1005, 953, 880, 858, 820, 759, 735, 701. -(1-(MIDA boryl)-3-methylbutyl)-3-(fert-butyl)thiourea (4ab)
Figure imgf000019_0002
Off-white solid; 90% yield; TLC (EtOAc) Rf = 0.57; H NMR (500 MHz, DMSO-cfe) S 7.22 (s, 1 H), 6.75 (d, J = 9.7 Hz, 1 H), 4.23 (d, J = 17.2 Hz, 1 H), 4.21 (d, J = 16.9 Hz, 1 H), 4.09 - 4.06 (m, 1 H), 4.02 (d, J = 17.2 Hz, 1 H), 3.73 (d, J = 16.9 Hz, 1 H), 2.94 (s, 3H), 1.68 - 1.61 (m, 1 H), 1.33 - 1.28 (m, 1H), 1.26 - 1.21 (m, 1H), 0.93 (d, J = 6.5 Hz, 1H), 0.86 (d, J = 6.7 Hz, 1 H) ppm; 13C NMR (125 MHz, DMSO-c/6) δ 182.3, 169.1 , 169.0, 62.6, 62.5, 52.5, 46.3, 42.6, 29.5, 24.6, 24.4, 23.6 ppm; 11B NMR (192 MHz, DMSO-cfe) δ 10.15 ppm; HRMS (DART-TOF) [M+H]+ calcd. for
Figure imgf000019_0003
358.19718, found 358.19713; IR (thin film, cm"1) 3369, 2958, 2348, 763, 1745, 1525, 1459, 1391 , 1341 , 1297, 1240, 1202, 1163, 1103, 1076, 1023, 1004, 956, 898, 869, 822, 780, 739. reparation of σ-bor l tetrazole 5a
Figure imgf000020_0001
To a solution of σ-boryl isocyanide 2a (50 mg, 0.20 mmol 1.0 equiv.) in TFE (0.5 mL) was added HCI (400 μΙ_, 4.0 Mmol, 2.0 mol%) and TMSN3 (40 μΙ_, 0.30 mmol, 1.5 equiv.). The vial was sealed and heated to 60°C with stirring for 2.5 h at which point TLC indicated that the reaction had gone to completion. The solvent was removed and the crude product was purified via flash column chromatography using EtOAc/MeCN 1.0→ 8:2 as an eluent. The purified product was isolated as an off-white solid (46 mg,
78%). -(1-(MIDA boryl)-3-methylbutyl)-1H-tetrazole (5a)
Figure imgf000020_0002
Off-white solid; 78% yield; TLC (EtOAC) Rf = 0.31 , m.p. = 85°C (decomp.); 1H NMR (500 MHz, MeCN-cfe) δ 8.85 (s, 1 H), 4.56 (dd, J = 12.6, 3.0 Hz, 1 H), 4.06 (d, J = 17.2 Hz, 1 H), 3.99 (d, J = 17.3 Hz, 2H), 3.96 (d, J = 17.2 Hz, 1H), 3.55 (d, J = 17.3 Hz, 1H), 2.84 (s, 4H), 2.01 (ddd, J = 14.8, 12.6, 3.5 Hz, 3H), 1.64 (ddd, J = 14.8, 10.3, 3.0 Hz, 1 H), 0.91 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-d3) δ 168.3, 168.3, 144.2, 63.8, 63.6, 47.1, 40.7, 25.5, 23.4, 20.8 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 10.7 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C11H19BN504, 296.15301 found 296.15299; IR (thin film, cm"1) 2959, 2866, 2339, 2152, 1761 , 1467, 1424, 1338, 1285, 1216, 1157, 1096, 1073, 1019, 959, 897, 866, 820, 739, 722, 684. Ugi 4-component reactions utilizing er-borvl isocyanide 2a
Figure imgf000021_0001
To a solution of the carboxylic acid (25 mg, 0.20 mmol, 1.0 equiv.) was added ammonia (28.3 μΙ_, 0.30 mmol, 1.5 equiv., 7.0 N solution in MeOH) and the resulting mixture was stirred at 23°C for 10 minutes. The aldehyde (23.1 μΙ_, 0.20 mmol, 1.0 equiv.) and σ-boryl isocyanide 2a (50 mg, 0.20 mmol, 1.0 equiv.) were added and the resulting mixture was stirred at 23°C for 3 days at which point TLC indicated that the reaction had gone to completion. The reaction mixture was concentrated and the crude product was purified via flash column chromatography on silica gel (neutralized with hexanes/Et3N (95:5)) using EtOAc/MeCN/Et3N (20:0:1→ 16:4:1→ 10:10:1→ 4:16:1 ). To afford the desired product as an off-white solid in a 1 :1 diastereomeric ratio (54 mg, 55%).
N-(1-((1-(MIDA boryl)-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)pyrazine-
Figure imgf000021_0002
Off-white solid; 55% yield; TLC (EtOAc/MeCN (1 :1)) Rf = 0.49 (A), 0.43 (B) ; dr: 1 :1 ; 1H NMR (500 MHz, MeCN-cfe) δ A: 9.17 (dd, J = 1.5, 0.3 Hz, 1 H), 8.76 (dd, J = 2.5, 0.3 Hz, 1 H), 8.57 (dd, J = 2.5, 1.5 Hz, 1 H), 8.24 (d, J = 7.6 Hz, 1 H), 7.32 - 7.29 (m, 5H), 6.26 (d, J = 10.1 Hz, 1 H), 4.68 (ddd, J = 8.9, 7.6, 5.9 Hz, 1 H), 3.90 (d, J = 17.0 Hz, 1 H), 3.81 (d, J = 17.1 Hz, 2H), 3.71 - 3.66 (m, 1 H), 3.47 (d, J = 17.0 Hz, 1 H), 3.22 (dd, J = 14.0, 5.9 Hz, 1 H), 3.08 (dd, J = 14.0, 8.9 Hz, 1 H), 2.85 (s, 3H), 1.55 - 1.48 (m, 1 H), 1.42 - 1.36 (m, 1 H), 1.27 - 1.21 (m, 1 H), 0.86 (d, J = 6.5 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H), B: 9.17 (dd, J = 1.5, 0.4 Hz, 1 H), 8.76 (dd, J = 2.5, 0.4 Hz, 1 H), 8.56 (dd, J = 2.5, 1.5 Hz, 1 H), 8.15 (d, J = 7.4 Hz, 1 H), 7.31 - 7.29 (m, 4H), 7.23 - 7.20 (m, 1 H), 6.40 (d, J = 10.1 Hz, 1H), 4.64 (ddd, J = 8.9, 7.4, 5.9 Hz, 1 H), 4.03 (d, J = 16.7 Hz, 1 H), 3.93 (d, J = 17.2 Hz, 1 H), 3.89 (d, J = 16.7 Hz, 1 H), 3.82 (d, J = 17.2 Hz, 1 H), 3.66 (ddd, J = 11.5, 10.0, 3.1 Hz, 1 H), 3.21 (dd, J - 14.0, 5.9 Hz, 1 H), 3.05 (dd, J = 14.0, 9.0 Hz, 1 H), 2.86 (s, 3H), 1.41 - 1.35 (m, 2H), 1.27 - 1.21 (m, 1 H), 0.85 (d, J = 4.1 Hz, 3H), 0.84 (d, J = 4.1 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ A: 171.3, 169.0, 168.7, 164.4, 148.9, 145.0, 144.7, 144.1 , 138.3, 130.2, 129.6, 127.9, 63.7, 63.2, 56.6, 46.8, 41.4, 38.2, 25.1 , 24.1 , 21.5; B: 171.5, 169.1 , 168.9, 164.1 , 148.8, 145.0, 144.7, 144.2, 138.2, 130.3, 129.4, 127.7, 63.7, 63.1 , 56.1 , 46.6, 41.5, 38.3, 25.1 , 24.2, 21.5 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 1 1.5 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C24H31BN506 496.23674, found 496.23765; IR (thin film, cm"1) 3352, 3954, 3869, 1762, 1709, 1577, 1516, 1497, 1467, 1454, 1402, 1336, 1288, 1246, 1223, 1195, 1154, 1103, 1081 , 1030, 994, 959, 896, 817, 866, 749, 725, 700.
Proline-Ugi reaction with σ-boryl isocyanide 2a
Figure imgf000022_0001
8a
A solution of L-Proline (23 mg, 0.20 mmol, 1.0 equiv.) in MeOH (2 mL) was cooled to - 40°C with stirring. σ-Boryl isocyanide 2a (50 mg, 0.20 mmol, 1.0 equiv.) and isobutyraldehyde (18 μΙ_, 0.20 mmol) were added sequentially. The resulting mixture was stirred for 4 hours at -40°C followed by 15 hours at 23°C until the reaction was complete as indicated by TLC. The solvent was removed and the crude was suspended in EtOAc and washed with 5% aqueous NaHC03. The aqueous layer was washed twice with EtOAc and the combined organic layers were washed with brine, dried over Na2S04 filtered and concentrated. Purification of the product was achieved using preparative TLC with EtOAc/MeCN (1 :1) as the eluent. The product was isolated as a light orange oil (an inseparable 43:26:17:13 mixture of 4 diastereomers) (6.2 mg, 7%). Methyl (1-((1-(MIDA boryl)-3-methylbutyl)amino)-3-methyl-1-oxobutan-2-yl)-L- prolinate (8a)
Figure imgf000023_0001
Light orange oil; 7% yield; dr: 43:26:17:13; TLC (EtOAc) Rf = 0.34; 1H NMR (500 MHz, MeCN-cfe) δ 7.71 (dd, J = 5.7, 3.3 Hz, 1 H), 7.61 (dd, J = 5.7, 3.3 Hz, 1 H), 6.18 (d, J = 9.5 Hz, 1 H), 6.08 (d, J = 9.5 Hz, 1 H), 4.19 - 4.17 (m, 2H), 4.08 - 4.01 (m, 2H), 3.96 - 3.83 (m, 16H), 3.71 (s, 3H), 3.67 (s, 3H), 3.65 (s, 3H), 3.62 (s, 3H), 3.53 (dd, J = 8.9, 5.4 Hz, 2H), 3.45 (dd, J = 8.9, 5.0 Hz, 2H), 3.15 - 3.10 (m, 2H), 3.05 (s, 3H), 3.04 (s, 3H), 3.01 (s, 3H), 2.96 (s, 3H), 2.82 - 2.75 (m, 2H), 2.05 - 1.98 (m, 4H), 1.89 - 1.84 (m, 4H), 1.75 - 1.03 (m, 32H), 0.97 - 0.78 (m, 48H) ppm; 13C NMR (125 MHz, MeCN-c3) δ 176.0, 175.6, 172.3, 171.1 , 169.3, 168.9, 168.8, 168.8, 132.3, 129.7, 118.4, 70.2, 68.7, 63.5, 63.4, 63.3, 63.2, 63.1 , 62.4, 62.4, 52.1 , 52.1 , 48.8, 47.7, 46.9, 46.8, 46.7, 42.2, 41.5, 31.2, 30.7, 30.4, 29.7, 29.2, 29.0, 29.0, 25.6, 25.5, 25.5, 24.6, 24.5, 24.1 , 24.0, 23.7, 21.5, 21.4, 21.4, 20.3, 20.3, 20.2, 20.2, 15.1 , 14.3, 11.3, 9.1 ppm; 11B NMR (192 MHz, MeCN-c/3) δ 11.1 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C2iH37BN307 454.27245, found 454.27440; IR (thin film, cm"1) 3339, 2954, 2868, 1748, 1646, 1520, 1463, 1337, 1288, 1198, 1162, 1103, 1081 , 1030, 994, 956, 896, 868, 709.
Figure imgf000023_0002
Commercially available N-Boc protected amino acids were used as supplied. Fully protected resin-bound tri- and tetra-peptides H-Pro-Leu-Phe-OH and H-Pro-Gly-Leu- Phe-OH were synthesized via standard Fmoc solid-phase peptide chemistry using an automated peptide synthesizer. Fmoc removal was achieved by treatment with 20% piperidine in NMP for 5 and 10 minutes with consecutive DMF and NMP washes after each addition. For all Fmoc amino acid coupling, the resin was treated once with 4.5 eq. of Fmoc amino acids, 4.5 eq. of HCTU and 9 eq. of DIPEA in NMP for 60 minutes. Once the peptide was synthesized, following Fmoc removal, the resin was treated with 1 :3, HFIP:DCM, twice for one hour each, to afford cleavage from the solid support. The solvent was then removed, followed by trituration with tert-butyl methyl ether to give the linear peptide.The peptide products were then Boc-protected along with commercially available H-Phe-Ala-OH and H-Gly-Gly-OH according to the following method:
Boc20, NaOH
H2N-Peptide-COOH THF H O Boc-NH-Peptide-COOH
0°C to 23°C
A solution of the peptide (1.0 equiv.) in THF (0.75M) and NaOH(aq.) (1.0 M, 2.0 equiv.) was cooled to 0°C and Boc20 was added slowly. The resulting mixture was stirred at 0°C for 30 minutes and was then allowed to warm to 23°C. Once TLC indicated that the reaction had gone to completion, the solution was cooled to 0°C and acidified to pH ~ 2 with 1.0 M HCI(aq ). Et20 was added and the layers were separated. The aqueous layer was washed twice with Et20 and the combined organic layers were dried over MgS04, filtered and concentrated to yield the crude products as white solids. The products were identified with LRMS (ESI, positive) and crude H NMR and used in the Ugi 4-component reaction without further purification.
To a solution of the crude N-Boc protected peptide (1.0 equiv.) in TFE (0.50 M) was added ammonia (1.5 equiv., 7.0 N solution in MeOH) and the resulting mixture was stirred at 23°C for 10 minutes. The aldehyde (1.0 equiv.) and σ-boryl isocyanide 2a (1.0 equiv.) were added and the resulting mixture was stirred at 23°C until TLC indicated that the reaction had gone to completion (6-12 d). The reaction mixture was concentrated and a small sample (5-15 mg) was taken for crude H NMR using 750 μΐ. of a stock solution of 3,4,5-triiodobenzoic acid (48.1 mM in DMSO-cf6) as the solvent. The yield was calculated by comparative integration of the Boc ferf-butyl signal and the aromatic protons of the internal standard. The crude product was purified via reverse- phase preparative high-performance liquid chromatography on an Agilent ZORBAX SB-C18 column (5 μΜ mesh, 9.4 x 250 mm) using a H20-MeCN (with added 0.1 % formic acid) gradient to afford the desired products. Diastereomers are classified by their order of elution in reverse-phase chromatography (A eluting first being the most polar, B eluting second being less polar than A, and so on for C and D if applicable.) (N-Boc)-Gly-DL-Phe-DL-(MIOA boro)-Leu (9a)
Figure imgf000025_0001
White solid; 66% (NMR), 11% (isolated) yield ; dr: 81:19 (A:B); 1H NMR (500 MHz, MeCN-d3) δ A: 7.33 - 7.29 (m, 2H), 7.26 - 7.23 (m, 3H), 6.78 (d, J = 7.5 Hz, 1H), 6.16 (d, J = 10.2 Hz, 1H), 5.59 (s, 1H), 4.41 (q, J = 7.7 Hz, 1H), 3.90 (d, J = 17.2 Hz, 1H), 3.82 (d, J = 16.8 Hz, 1H), 3.80 (d, J = 17.2 Hz, 1H), 3.69 - 3.63 (m, 1H), 3.54 (d, J = 17.2 Hz, 1H), 3.63 (d, J= 17.2 Hz, 1H), 3.61 (d, J= 17.3 Hz, 1H), 3.53 (d, J= 17.2 Hz, 1H), 3.06 (dd, J = 14.0, 5.6 Hz, 1H), 2.97 (dd, J = 13.9, 8.4 Hz, 1H), 2.83 (s, 3H), 1.54 -1.46, (m, 1H), 1.40 (s,9H), 1.28- 1.22 (m, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H); B: 7.30 - 7.27 (m, 2H), 7.26 - 7.22 (m, 3H), 6.81 (d, J = 7.3 Hz, 1H), 6.25 (d, J - 10.1 Hz, 1H), 5.54 (s, 1H), 4.36 (ddd, J = 8.8, 7.3, 6.3 Hz, 1H), 4.02 (d, J = 16.7 Hz, 1H), 3.92 (d, J= 17.2 Hz, 1H), 3.86 (d, J= 16.7 Hz, 1H), 3.82 (d, J= 17.2 Hz, 1H), 3.60 (ddd, J = 11.6, 9.5, 2.7 Hz, 1H), 3.55 (dd, J = 6.1, 5.1 Hz, 2H), 3.04 (dd, J = 13.9, 6.2 Hz, 1H), 2.87 (d, J = 8.9 Hz, 1H), 2.85 (s, 3H), 1.41 (s, 9H), 1.39 - 1.37 (m, 1H),
I.35 - 1.29 (m, 1H), 1.22 - 1.17 (m, 1H), 0.81 (d, J = 3.3 Hz, 3H), 0.80 (d, J = 3.5 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-d3) δ A: 171.3, 171.3, 169.2, 169.2, 169.1, 138.3, 130.2, 129.6, 127.8, 80.2, 63.7, 63.2, 56.4, 46.8, 44.6, 41.4, 37.7, 28.6, 25.0, 24.2, 21.5; B: 171.7, 171.0, 169.2, 169.0, 138.4, 130.2, 129.4, 127.6, 63.8, 63.1, 56.2, 46.6, 44.4, 41.4, 38.0, 28.6, 28.5, 24.9, 24.2, 21.5 ppm; 11B NMR (192 MHz, MeCN-/3) δ A:
II.5; B: 11.3 ppm; HRMS (DART-TOF) [M+Hf calcd. for C26H40BN4O8 547.2933, found 547.2922.
(N-Boc)-Gly-DL-Val-DL-(MIDA boro)-Leu (9b)
Figure imgf000026_0001
White solid; 79% (NMR), 40% (isolated) yield; dr: 83:17 (A:B); 1H NMR (500 MHz, MeCN-cfe) δ 6.71 (d, J = 7.0 Hz, 1 H), 6.69 (d, J = 7.0 Hz, 1 H), 6.27 (d, J = 10.0 Hz, 1 H), 6.16 (d, J = 10.1 Hz, 1 H), 5.69 (s, 1 H), 5.63 (s, 1 H), 4.00 - 3.81 (m, 8H), 3.79 - 3.74 (m, 2H), 3.69 - 3.64 (m, 2H), 3.61 (dd, J = 16.9, 5.4 Hz, 4H), 2.93 (s, 3H), 2.85 (s, 3H), 1.62 - 1.48 (m, 4H), 1.43 (s, 9H), 1.42 (s, 9H), 1.30 - 1.24 (m, 2H), 0.96 - 0.92 (m, 2H), 0.93 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 6.7 Hz, 6H), 0.88 (d, J = 6.9 Hz, 6H), 0.88 (d, J = 6.6 Hz, 6H) ppm; 13C NMR (125 MHz, MeCN-d3) δ 170.9, 170.7, 170.4, 170.0, 168.5, 168.2, 168.1 , 167.9, 156.3, 156.1 , 79.2, 62.9, 62.8, 62.4, 62.1 , 60.0, 59.2, 46.0, 45.6, 43.7, 43.5, 40.6, 40.4, 29.4, 27.6, 27.6, 27.6, 24.5, 24.3, 23.1 , 23.1 , 20.5, 20.4, 19.0, 18.8, 18.2, 17.4, 17.0 ppm; 11B NMR (192 MHz, MeCN- /3) δ 11.5 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C22H4oBN408 499.2933, found 499.2937.
(N-Boc)-L-Phe-DL-Val-DL-(MIDA boro)-Leu (9c)
Figure imgf000026_0002
White solid; 74% (NMR), 22% (isolated) yield; dr: 32:48:13:7 (A:B:C:D) 1H NMR (500 MHz, MeCN-da) δ 7.35 - 7.20 (m, 20H), 6.94 (br s, 2H), 6.90 (d, J = 7.8 Hz, 1 H), 6.81 (0, J = 6.5 Hz, 1 H), 6.34 (d, J = 11.2 Hz, 1 H), 6.30 (d, J = 10.1 Hz, 2H), 6.19 (d, J = 9.8 Hz, 1 H), 5.67 (d, J = 8.7 Hz, 1 H), 5.61 - 5.54 (m, 3H), 4.32 - 4.25 (m, 4H), 4.06 - 3.98 (m, 4H), 3.97 - 3.80 (m, 16H), 3.74 - 3.64 (m, 4H), 3.18 - 3.04 (m, 4H), 2.95 (s, 3H), 2.94 (s, 3H), 2.83 (s, 3H), 2.82 (s, 3H), 1.61 - 1.51 (m, 4H), 1.47 - 1.41 (m, 8H), 1.38 (2, 9H). 1.34 (s, 9H), 1.33 (s, 9H), 1.30 (s, 9H), 1.28 - 1.25 (m, 4H), 0.94 - 0.80 (m, 52H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ 172.5, 172.1(7), 172.0(9), 172.0(2), 170.9, 170.4(1 ), 170.3(9), 170.3(7), 168.2, 168.1(1), 168.0(8), 168.0(6), 168.0(5), 168.0(3), 168.0(0), 167.9, 137.9, 137.8(3), 137.7(9), 137.6, 129.3(2), 129.3(1), 129.2(9), 129.2(4), 128.4(6), 128.4(3), 128.3(7), 128.3(2), 126.6(1), 126.5(8), 126.5(6), 126.5(2), 79.4(0), 79.3(7), 79.1 (2), 79.0(6), 62.8(2), 62.7(7), 62.7(3), 62.6, 62.4(3), 62.4(0), 62.1(4), 62.0(9), 59.6, 59.5, 59.2(9), 59.2(8), 55.8(9), 55.8(5), 55.6(0), 55.5(8), 45.9(2), 45.8(6), 45.7, 45.6 40.7, 40.6(4), 40.6(2), 40.6, 37.9, 37.4, 36.9, 36.2, 30.1 , 29.8, 29.6(1), 29.5(7), 27.7, 27.5(8), 27.5(6), 27.5(2), 24.4(8), 24.4, 24.3(3), 24.3(0), 23.2(1), 23.2(1), 23.1(6), 23.1(3), 20.5(1 ), 20.5(0), 20.4(5), 20.4(5), 19.0(4), 19.0(1), 18.9, 18.8, 17.6, 17.5(2), 17.5(1), 17.4, 16.8(4), 16.8(2), 16.7(7), 16.7(6), ppm; B NMR (192 MHz, MeCN- /3) δ 11.5 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C29H46BN4O8 589.3403, found 589.3381.
(N-Boc)-LVal-DL-Phe-DL-(MIDA boro)-Leu (9d)
Figure imgf000027_0001
White solid; 57% (NMR), 17% (isolated) yield; dr: 42:34:16:8 (A:B:C:D) 1H NMR (500 MHz, MeCN-c/3) δ A+B: 7.32 - 7.22 (m, 10H), 6.94 (d, J = 6.0 Hz, 1 H), 6.85 (d, J = 8.1 Hz, 1 H), 6.42 (d, J = 10.3 Hz, 1H), 6.27 (d, J = 10.2 Hz, 1 H), 5.46 (br s, 2H), 4.55 (br s, 2H), 3.90 - 3.73 (m, 8H), 3.69 - 3.66 (m, 2H), 3.64 - 3.59 (m, 2H), 3.25 (d, J = 17.3 Hz, 2H), 3.11 - 3.02 (m, 2H), 2.97 - 2.91 (m, 1 H), 2.85 (dd, J = 13.8, 8.7 Hz, 1 H), 2.75 (s, 3H), 2.73 (s, 3H), 1.82 (dq, J = 13.6, 6.8 Hz, 1 H), 1.54 - 1.47 (m, 2H), 1.40 (s, 9H), 1.39 (s, 9H), 1.27 - 1.20 (m, 2H), 0.93 - 0.89 (m, 1 H), 0.88 - 0.83 (m, 15H), 0.76 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H), 0.63 (d, J = 6.7 Hz, 3H); C+D: 7.31 - 7.25 (m, 8H), 7.23 - 7.19 (m, 2H), 6.83 (d, J = 7.1 Hz, 1H), 6.81 (d, J = 7.2 Hz, 1H), 6.33 (d, J = 10.2 Hz, 1 H), 6.25 (d, J = 10.2 Hz, 1 H), 5.41 (d, J = 8.2 Hz, 1 H), 5.37 (d, J = 9.3 Hz, 1 H), 4.42 - 4.34 (m, 2H), 4.07 (d, J = 16.1 Hz, 2H), 3.93 (d, J = 17.2 Hz, 1 H), 3.92 (d, J = 17.2 Hz, 1 H), 3.87 (d, J = 16.6 Hz, 1 H), 3.87 (d, J = 16.6 Hz, 1 H), 3.82 (d, J = 17.2 Hz, 1 H), 3.80 (d, J = 17.2 Hz, 1 H), 3.78 - 3.75 (m, 1 H), 3.65 - 3.57 (m, 2H), 3.05 (dd, J = 13.6, 6.0 Hz, 1 H), 3.02 (dd, J = 14.2, 6.0 Hz, 1 H), 2.86 (d, J = 9.3 Hz, 1 H), 2.84 (s, 3H), 2.83 (s, 3H), 2.82 - 2.80 (m, 1 H), 1.87 - 1.83 (m, 1 H), 1.39 (s, 18H), 1.35 - 1.27 (m, 4H), 1.24 - 1.17 (m, 2H), 0.95 - 0.88 (m, 2H), 0.86 - 0.79 (m, 15H), 0.76 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.68 (d, J = 6.8 Hz, 3H) ppm; 3C NMR (125 MHz, MeCN-ck) δ A+B: 173.1 , 173.1 , 172.0, 171.9, 169.2, 169.2, 168.9, 168.9, 138.3, 138.2, 130.2, 130.2, 129.4, 129.4, 127.6, 127.6, 79.8, 79.8, 63.8, 63.7, 63.1 , 63.0, 60.7, 60.4, 56.4, 56.2, 46.6, 46.5, 41.4, 41.4, 38.3, 38.1 , 31.7, 31.7, 28.6, 28.6, 25.0, 24.9, 24.2, 24.2, 21.5, 21.5, 19.6, 19.5, 17.9, 17.8; C+D: 173.1 , 173.1 , 172.0, 171.9, 169.2, 169.2, 168.9, 168.9, 138.3, 138.2, 130.2, 130.2, 129.4, 129.4, 127.6, 127.6, 79.8, 79.8, 63.8, 63.7, 63.1 , 63.0, 60.7, 60.4, 56.4, 56.2, 46.6, 46.5, 41.4, 41.4, 38.3, 38.1 , 31.7, 31.7, 28.6, 28.6, 25.0, 24.9, 24.2, 24.2, 21.5, 21.5, 19.6, 19.5, 17.9, 17.8 ppm; 11B NMR (192 MHz, MeCN-<½) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C29H46BN408 589.3403, found 589.3381.
(N-Boc)-Gle-Gly-DL-Phe-DL-(MIDA boro)-Leu (9e)
Figure imgf000028_0001
White solid; 92% (NMR), 13% (isolated) yield; dr: 59:41 (A:B); 1H NMR (500 MHz, A: MeCN-cfe; B: DMSO-cfe) δ A: 7.31 - 7.27 (m, 3H), 7.25 - 7.21 (m, 2H), 7.07 (br s, 1 H), 6.99 (d, J = 6.7 Hz, 1 H), 6.28 (d, J = 10.1 Hz, 1 H), 5.74 (br s, 1 H), 4.36 (ddd, J = 9.4, 7.4, 5.1 Hz, 1H), 3.90 (d, J = 17.2 Hz, 1 H), 3.83 (d, J = 16.7 Hz, 1H), 3.81 (d, J = 17.2 Hz, 1 H), 3.71 - 3.68 (m, 2H), 3.66 - 3.63 (m, 3H), 3.59 (d, J = 16.7 Hz, 1 H), 3.16 (dd, J = 14.1 , 5.1 Hz, 1 H), 2.91 (dd, J = 14.0, 9.4 Hz, 1 H), 2.84 (s, 3H), 1.56 - 1.45 (m, 2H), 1.43 (s, 9H), 1.28 - 1.23 (m, 1 H), 0.90 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H); B: 8.10 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 5.7 Hz, 1H), 7.56 (d, J= 10.3 Hz, 1H), 7.30-7.24 (m, 4H), 7.19 - 7.16 (m, 1H), 6.97 (t, J = 6.1 Hz, 1H), 4.45 - 4.40 (m, 1H), 4.23 (d, J = 17.3 Hz, 1H), 4.07 (d, J= 16.6 Hz, 1H), 3.97 (d, J= 16.6 Hz, 1H), 3.94 (d, J= 17.3 Hz, 1H), 3.71 (dd, J = 16.8, 5.4 Hz, 1H), 3.56 - 3.52 (m, 3H), 3.49 - 3.44 (m, 1H), 2.91 (dd, J = 13.8, 5.7 Hz, 1H), 2.76 (s, 3H), 2.75 - 2.73 (m, 1H), 1.38 (s, 9H), 1.35 - 1.30 (m, 2H), 1.10 (ddd, J = 13.3, 10.2, 2.9 Hz, 1H), 0.77 (d, J = 6.3 Hz, 3H), 0.76 (d, J = 6.2 Hz, 3H) ppm; 13C NMR (125 MHz, A: MeCN-of3; B: DMSO-cfe) δ A: 171.5, 171.2, 170.8, 169.2, 169.1, 157.5, 138.8, 130.1, 129.5, 127.6, 80.3, 63.7, 63.2, 56.9, 46.8, 44.9, 43.4, 41.3, 37.6, 28.6, 25.1, 24.3, 21.6 ppm; B: 3C NMR data unavailable due to limited amount of purified material obtained; 11B NMR (192 MHz, MeCN-of3) δ 11.5 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C28H43BN5O9604.3148, found 604.3138.
(N-Boc)-L-Phe-L-Ala-DL-Phe-DL-(MIDA boro)-Leu (9f)
Figure imgf000029_0001
White solid; 91% (NMR), 27% yield; dr: 25:31:23:22 (A:B:C:D).1H NMR (500 MHz, MeCN-cfe) δ A+B: 7.32 - 7.18 (m, 20H), 7.04 (br s, 3H), 6.98 (d, J = 7.6 Hz, 1H), 6.37 (d, J = 10.2 Hz, 2H), 5.58 (d, J = 7.9 Hz, 1 H), 5.54 (d, J = 7.5 Hz, 1 H), 4.47 (td, J = 8.2, 6.0 Hz, 1H), 4.42 - 4.38 (m, 1H), 4.24 (ddd, J = 10.0, 8.0, 4.5 Hz, 1H), 4.25 - 4.19 (m, 1H), 4.16 - 4.09 (m, 2H), 3.90 (d, J = 17.1 Hz, 1H), 3.88 (d, J = 17.2 Hz, 1H), 3.83 (d, J = 16.8 Hz, 1H), 3.82 (d, J = 17.3 Hz, 1H), 3.80 (d, J = 17.1 Hz, 1H), 3.69 - 3.63 (m, 2H), 3.54 (d, J = 16.7 Hz, 1H), 3.44 (d, J = 16.7 Hz, 1H), 3.19 (dd, J = 14.1, 5.2 Hz, 1H), 3.13 (dd, J = 14.1, 4.6 Hz, 2H), 3.07 - 3.00 (m, 1H), 2.94 (d, J = 8.7 Hz, 1H), 2.91 (d, J = 7.1 Hz, 1H), 2.87 (dd, J = 9.6, 4.4 Hz, 1H), 2.82 (s, 3H), 2.79 (s, 3H), 2.77 - 2.74 (m, 2H), 1.56 - 1.49 (m, 2H), 1.47 - 1.41 (m, 2H), 1.33 (s, 18H), 1.29 - 1.22 (m, 2H), 1.19 (d, J = 7.2 Hz, 3H), 1.12 (d, J = 7.1 Hz, 3H), 0.88-0.85 (m, 12H); C+D: 7.32 - 7.19 (m, 20H), 7.01 - 6.95 (m, 4H), 6.35 (d, J = 10.1 Hz, 1H), 6.30 (d, J = 10.1 Hz, 1H), 5.56 (d, J = 8.1 Hz, 1H), 5.52 (d, J = 7.4 Hz, 1H), 4.39 (ddd, J = 8.9, 7.6, 6.2 Hz, 1 H), 4.37 - 4.32 (m, 1 H), 4.28 - 4.20 (m, 2H), 4.13 (dq, J = 13.8, 6.9 Hz, 2H), 4.05 (d, J = 16.7 Hz, 1 H), 3.97 (d, J = 16.8 Hz, 1 H), 3.93 (d, J = 17.2 Hz, 1 H), 3.92 (d, J = 17.2 Hz, 1 H), 3.88 (d, J = 16.6 Hz, 1 H), 3.86 (d, J = 16.7 Hz, 1 H), 3.83 (d, J = 17.2 Hz, 1 H), 3.82 (d, J = 17.1 Hz, 1 H), 3.64 - 3.59 (m, 2H), 3.11 - 3.04 (m, 4H), 2.87 (s, 3H), 2.86 (s, 3H), 2.85 - 2.81 (m, 2H), 2.80 - 2.72 (m, 2H), 1.33 (s, 9H), 1.32 (s, 9H), 1.23 - 1.19 (m, 2H), 0.92 - 0.84 (m, 4H), 1.18 (d, J = 6.8 Hz, 3H), 1.13 (d, J = 7.1 Hz, 3H), 0.82 - 0.78 (m, 12H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ A+B: 173.6, 173.3, 173.1 , 172.7, 171.3(4), 171.3(0), 169.1 , 169.0(1), 169.0(0), 168.9, 138.8(1), 138.7(9), 138.7, 138.5, 130.3(3), 130.3(1 ), 130.2(9), 130.2, 129.5, 129.4(4), 129.3(7), 129.3, 127.7, 127.62, 127.62, 127.5, 80.3, 80.2, 63.6, 63.2, 56.9(4), 56.9(0), 56.5, 56.45, 55.9(9), 55.9(7), 50.6, 50.36, 46.7(1), 46.6(8), 41.6, 41.4, 38.2, 38.1 , 37.9, 37.8, 28.6, 28.5, 25.1 , 25.0, 24.3(0), 24.2(9), 21.7, 21.67, 17.7, 17.6; C+D: 181.5, 1765, 172.6, 171.7, 170.7, 170.6, 168.3, 168.1 (4), 168.1(2), 168.0(5), 137.6, 137.4(9), 137.4(8), 137.4(7), 129.3(2), 129.3(0), 129.2(7), 129.2, 128.3(6), 128.3(5), 128.3(3), 128.2(9), 126.6(1), 126.5(7), 126.5(4), 126.5(3), 79.3, 79.2, 62.8, 62.7, 62.2(1 ), 62.1 (7), 55.8(4), 55.7(6), 55.3, 55.1 , 49.3(3), 49.2(7), 45.7, 45.67, 40.4, 40.3, 37.5, 37.3, 37.1 , 36.9, 27.5(7), 27.5(6), 23.9, 23.8, 23.3, 23.2, 20.6, 20.5, 17.0(2), 17.0(1) ppm; 11B NMR (192 MHz, MeCN-af3) 6 A: 11.4; B: 11.6 ppm; HRMS (DART-TOF) [M+H]+ calcd. for CseHsiBNgC, 708.3774, found 708.3764.
(N-Boc)-D-Pro-L-Leu-L-Phe-DL-Phe-DL-(MIDA boro)-Leu (9g)
Figure imgf000030_0001
White solid; 63% (NMR), 30% (isolated) yield; dr: 29:19:27:25; 1H NMR (500 MHz, MeCN-cfe) δ A: 7.36 - 7.17 (m, 10H), 7.12 (br s, 1H), 7.11 (br s, 1 H), 7.02 (br s, 1H), 6.32 (d, J = 10.2 Hz, 1 H), 4.34 - 4.25 (m, 2H), 4.12 (dd, J = 8.4, 4.5 Hz, 1 H), 4.07 - 4.01 (m, 1 H), 3.96 - 3.75 (m, 4H), 3.72 - 3.60 (m, 2H), 3.52 - 3.47 (m, 1H), 3.42 - 3.33 (m, 2H), 3.23 (dd, J = 13.2, 3.5 Hz, 1 H), 2.93 (s, 1 H), 2.84 (s, 3H). 1.62 - 1.48 (m, 4H), 1.44 (s, 9H), 1.40 - 1.34 (m, 4H), 1.31 - 1.23 (m, 2H), 0.86 - 0.83 (m, 9H), 0.83 (d, J = 6.3 Hz, 1 H) ppm; B, C, D: qualitative analysis only due to substantial peak overlap; 3C NMR data unavailable due to limited amount of purified material obtained; 11B NMR (192 MHz, MeCN-cfe) 6 A: 11.2; B, C, D: 11.1 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C44H64BN6O10 847.4771 , found 847.4757.
(N-Boc)-L-Pro-Gly-L-Leu-L-Phe-DL-Phe-DL-(MIDA boro)-Leu (9h)
Figure imgf000031_0001
White solid; 76% (NMR), 19% (isolated) yield; dr: 27:73 (A+B:C+D); 1H NMR (500 MHz, MeCN-af3) qualitative analysis only; 13C NMR data unavailable due to limited amount of purified material obtained; 11B NMR (192 MHz, MeCN-cf3) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C4eHe7BN7011 904.4986, found 904.5017.
Figure imgf000031_0002
10
To a vial charged with σ-boryl isocyanide (1.0 equiv.), phenyl acetic acid (1.0 equiv.) and aldehyde (1.0 equiv.) was added CH2CI2 (0.5 mL) and the vial was sealed. The resulting suspension was stirred magnetically at 23°C until the reaction was complete as indicated by TLC. The crude product mixture was pipetted directly onto silica gel and the product diastereomers were eluted using a hexanes/EtOAc gradient (1 :0→ 0:1). In some cases, EtOAc/MeCN (8:2) was required for complete elution of the product. In most cases, TLC indicated complete separation of product diastereomers after column; however, 1H NMR indicated trace amounts of either diastereomer in each sample. Diastereomeric ratios were determined using 1H NMR proton integrations of the N-Me signal. It should be noted that reactions utilizing nicotinaldehyde were carried out in the dark until TLC indicated full consumption of starting material. Diastereomers are classified by their order of elution from silica gel (A eluting first being the least polar, B eluting second being more polar than A) Diastereomeric ratios were determined by comparative H NMR integrations of the N- CH3 signals.
2-((1 -(MIDA boryl)-3-methylbutyl)amino)-2-oxo-1 -phenylethyl 2-phenylacetate
Figure imgf000032_0001
White solid; 56% yield; TLC (EtOAc) Rf = 0.69 (A), 0.49 (B); dr = 51 :49 (A:B); mp = 58 - 64°C; 1H NMR (500 MHz, MeCN-cfe) δ A: 7.48 - 7.46 (m, 2H), 7.42 - 7.27 (m, 5H), 6.36 (d, J = 10.1 Hz, 1 H), 5.73 (s, 1 H), 3.91 (d, J = 17.2 Hz, 1 H), 3.84 (d, J = 16.6 Hz, 1 H), 3.79 (d, J = 17.2 Hz, 1 H), 3.78 (s, 2H), 3.62 (d, J = 16.6 Hz, 1 H), 3.63 - 3.58 (m, 2H), 2.77 (s, 3H), 1.36 - 1.30 (m, 1 H), 1.20 - 1.15 (m, 1 H), 1.12 - 1.04 (m, 1 H), 0.90 - 0.88 (m, 1H), 0.69 (d, J = 6.6 Hz, 1 H), 0.66 (d, J = 6.4 Hz, 1 H); B: 7.46 - 7.29 (m, 10H), 6.33 (d, J = 10.2 Hz, 1 H), 5.80 (s, 1 H), 3.89 (d, J = 17.2 Hz, 1 H), 3.79 (d, J = 17.2 Hz, 1 H), 3.79 (d, J = 2.9 Hz, 1 H), 3.76 (d, J = 16.8 Hz, 1 H), 3.69 - 3.60 (m, 1 H), 3.00 (d, J = 16.8 Hz, 1 H), 2.71 (s, 3H), 1.54 - 1.43 (m, 2H), 1.29 - 1.24 (m, 1 H), 0.87 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-d3) δ A: 172.3, 169.1 , 169.1 , 168.7, 136.3, 135.2, 130.4, 129.8, 129.5, 129.4, 128.2, 128.0, 77.3, 63.6, 62.8, 46.4, 41.4, 41.2, 24.9, 24.0, 21.2; B: 171.8, 169.0, 168.9, 168.6, 137.0, 135.2, 130.5, 129.8, 129.6, 129.5, 128.1 , 128.0, 77.3, 63.5, 62.8, 46.5, 41.3, 41.3, 24.9, 24.1 , 21.3 ppm; 11B NMR (192 MHz, MeCN-d3) δ 11.4 ppm; HRMS (DART- TOF) [M+H]+ calcd. for C26H32BN2O7 495.23026, found 495.23094; IR (thin film, cm-1) 3337, 2955, 1745, 1668, 1528, 1497, 1455, 1289, 1243, 1148, 1103, 1080, 1028, 950, 896, 866, 696. 1 -((1 -(MIDA boryl)-3-methylbutyl)amino)-3-methyl-1 -oxobutan-2-yl 2-
Figure imgf000033_0001
White solid; 68% yield; TLC (EtOAc) R, = 0.39, 0.53; dr 41:59 (A:B); mp = 51-55°C; 1H NMR (500 MHz, MeCN-cfe) δ A: 7.36 - 7.36 (m, 3H), 7.31 - 7.27 (m, 2H), 5.95 (d, J = 10.1 Hz, 1H), 4.76 (d, J = 4.5 Hz, 1H), 3.95 (d, J = 17.0 Hz, 1H), 3.95 (d, J = 17.5 Hz, 1H), 3.84 (d, J = 17.5 Hz, 1H), 3.81 (d, J = 17.0 Hz, 1H), 3.72 - 3.68 (m, 1H), 3.71 - 3.68 (m, 2H), 2.91 (s, 3H), 2.18 - 2.11 (m, 1H), 1.54 - 1.59 (m, 1H), 1.48 - 1.42 (m, 1H), 1.28 - 1.22 (m, 1H), 0.90 (d, J = 4.5 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 4.5 Hz, 3H); B: δ 7.36 - 7.36 (m, 3H), 7.31 - 7.27 (m, 2H), 6.18 (d, J = 10.1 Hz, 1H), 4.59 (d, J = 4.8 Hz, 1H), 3.93 (d, J = 17.0 Hz, 1H), 3.88 (d, J = 16.5 Hz, 1H), 3.82 (d, J = 16.5 Hz, 1H), 3.73 - 3.71, (m, 2H), 3.66 - 3.63 (m, 1H), 3.58 (d, J = 17.0 Hz, 1H), 2.83 (s, 3H), 2.09 - 2.04 (m, 1H), 1.54 - 1.59 (m, 1H), 1.42 - 1.36 (m, 1H), 1.28 - 1.22 (m, 1H), 0.93 (d, J = 6.5 Hz, 3H), 0.86 (d, J = 4.5 Hz, 3H), 0.85 (d, J = 4.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-of3) δ 172.7, 172.5, 169.8, 169.7, 169.2, 169.0, 168.9, 168.8, 135.4, 135.3, 130.6, 130.3, 129.5, 129.5, 129.5, 128.0, 128.0, 79.9, 79.8, 79.8, 63.6, 63.6, 63.1, 62.9, 46.8, 46.5, 41.5, 41.5, 41.3, 41.0, 31.2, 31.2, 25.3, 25.2, 24.1, 24.1, 21.3, 21.2, 19.3, 19.2, 17.3, 17.3 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C23H34BN2O7461.24591, found 461.24647; IR (thin film, cm"1) 3349, 2959, 2866, 1748, 1661, 1526, 1467, 1455, 1338, 1291, 1244, 1161, 1103, 1081, 1031, 958, 896, 866, 764, 711.
2-((1 -(MIDA boryl)-3-methylbutyl)amino)-1 -(4-fluorophenyl)-2-oxoethyl 2- phenylacetate (10ac)
Figure imgf000034_0001
White solid; 60% yield; TLC (EtOAc) Rf = 0.63 (A), 0.53 (B); dr = 66:44 (A:B); mp = 81- 84°C; 1H NMR (500 MHz, MeCN-of3) δ A: 7.50 (ddd, J = 8.7, 5.4, 2.6 Hz, 2H), 7.41 - 7.26 (m, 5H), 7.1 1 (ddd, J = 8.7, 5.4, 2.6 Hz, 3H), 6.42 (d, J = 10.1 Hz, 1 H), 5.72 (s, 1 H), 3.94 - 3.82 (m, 2H), 3.77 - 3.67 (m, 2H), 3.62 - 3.60 (m, 2H), 2.80 (s, 3H), 1.37 - 1.31 (m, 1 H), 1.18 (ddd, J = 14.0, 10.6, 3.1 Hz, 1 H), 1.08 - 1.00 (m, 1 H), 0.89 (dd, J = 8.5, 6.5 Hz, 1 H), 0.69 (d, J = 6.5 Hz, 2H), 0.65 (d, J = 6.5 Hz, 3H); B: 7.47 - 7.43 (m, 2H), 7.36 - 7.27 (m, 5H), 7.13 - 7.08 (m, 2H), 6.37 (d, J = 10.4 Hz, 1 H), 5.79 (s, 1 H), 3.90 (d, J = 17.0 Hz, 1 H), 3.82 (d, J = 17.0 Hz, 1 H), 3.77 (s, 2H), 3.79 (d, J = 17.0 Hz, 1 H), 3.63 (ddd, J = 12.1 , 10.4, 3.1 Hz, 1 H), 3.13 (d, J = 17.0 Hz, 1 H), 2.73 (s, 3H), 1.53 - 1.47 (m, 1 H), 1.46 - 1.39 (m, 1 H), 1.28 - 1.23 (m, 1 H), 0.84 (d, J = 6.5 Hz, 3H), 0.74 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ A: 173.3, 172.4, 169.1 , 169.1 , 168.8, 135.2, 130.4, 130.4, 129.5, 129.4, 128.1 , 127.8, 1 16.3, 76.6, 63.6, 62.8, 46.4, 41.3, 41.1 , 24.9, 23.9, 21.1 ; B: 288.7, 285.9, 285.8, 285.6, 247.4, 247.1 , 247.1 , 246.4, 245.0, 235.3, 233.4, 233.2, 193.5, 180.4, 179.8, 163.5, 158.2, 158.1 , 141.9, 141.0, 138.3 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 1 1.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C26H3iBFN207 513.22083, found 513.22208; IR (thin film, cm"1) 3338, 2954, 1746, 1665, 1606, 1510, 1455, 1292, 1223, 1 149, 1 103, 1030, 956, 896, 867, 844, 808, 724.
1 -((1 -(MIDA boryl)-3-methylbutyl)amino)-1 -oxo-3-phenylpropan-2-yl 2- phenylacetate (10ad)
Figure imgf000034_0002
White solid; 81% yield; TLC (EtOAc) Rf = 0.60 (syn), 0.34 (anti); dr = 57:43 (syn.anti) 1H NMR (400 MHz, MeCN-d3) δ anti: 7.35 - 7.17 (m, 10H), 6.15 (d, J = 10.3 Hz, 1 H), 5.07 (dd, J = 7.3, 5.2 Hz, 1 H), 3.89 (d, J = 17.2 Hz, 1 H), 3.78 (d, J = 17.2 Hz, 1 H), 3.77 (d, J = 16.7 Hz, 1 H), 3.64 (d, J = 1.8 Hz, 2H), 3.63 - 3.58 (m, 1 H), 3.38 (d, J = 16.7 Hz, 1 H), 3.04 - 3.02 (m, 2H), 2.72 (s, 3H), 1.34 - 1.27 (m, 2H), 1.23 - 1.16 (m, 1 H), 0.82 (d, J = 6.4 Hz, 6H); syn: 7.34 - 7.19 (m, 8H), 7.15 - 7.12 (m, 2H), 5.97 (d, J = 10.0 Hz, 1 H), 5.16 (dd, J = 7.4, 4.9 Hz, 1 H), 3.93 (d, J = 17.1 Hz, 1 H), 3.88 (d, J = 16.9 Hz, 1 H), 3.81 (d, J = 17.1 Hz, 1 H), 3.67 (d, J = 2.7 Hz, 2H), 3.42 (d, J = 16.9 Hz, 1 H), 3.64 - 3.61 (m, 1H), 3.13 - 2.99 (m, 3H), 2.83 (s, 3H), 1.36 - 1.31 (m, 1 H), 1.24 - 1.16 (m, 2H), 0.80 (d, J = 4.4 Hz, 3H), 0.79 (d, J = 4.2 Hz, 3H) ppm; 13C NMR (100 MHz, MeCN-d3) δ anti: 171.1 , 168.5, 168.1 , 167.7, 136.6, 134.1 , 129.7, 129.3, 128.7, 128.3, 127.1 , 126.8, 75.0, 62.6, 61.8, 45.5, 40.7, 40.3, 37.1 , 24.1 , 23.1 , 20.4; syn: 170.9, 168.3, 168.0, 167.9, 136.5, 134.0, 129.6, 129.5, 128.6, 128.3, 127.0, 126.7, 75.2, 62.5, 62.1 , 45.8, 40.6, 40.1 , 37.1 , 23.7, 23.2, 20.4 ppm; 11B NMR (192 MHz, MeCN-d3) δ 11.2 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C27H34BN207 509.24591 , found 509.24676; IR (thin film, cm 1) 2954, 1746, 1668, 1523, 1497, 1455, 1291 , 1243, 1159, 1103, 1029, 956, 897, 866, 697.
The relative stereochemistry of P3CR product 10ad was determined by computationally modeling the structure of each diastereomer. Using GaussView 5.0.9 as a computational engine, [New Ref 3] the structure of 10ad built (both the syn and anti diastereomers of each) and the optimization + frequencies calculation was carried out using the following parameters: Theory: DFT, Basis Set: B3LYP, 6-31 G(d), additional keyword: "scrf=(solvent=acetonitrile) geom=connectivity". Using the optimized structures from these calculations, the NMR calculation was carried out using the giao method and the following computational parameters: Theory: DFT, Basis Set: RMPW1 PW91 , 6-311G(2d,p), additional keyword: "scrf=(solvent=acetonitrile) guess=input geom=connectivity". The isotropic Eigenvalues for the chemical shifts of the protons of interest were recorded in Table 1 along with their scaled values and the corresponding experimental values. The computational isotropic Eigenvalues were subject to scaling factors developed by Tantillo, et al. (slope: -1.0823, intercept 31.8486). able 1: Computational vs. experimental 1H NMR chemical shifts of 10ad
Figure imgf000036_0001
MPW1PW91, 6-311G(2d, p) NMR Experimental NMR
N-H HA N-Me Stereochem N-H HA N-Me
4.257 4.727 3.128 syn (D1) 5.975 5.154 2.831
4.775 4.410 3.059 anti (D2) 6.155 5.067 2.719
2-((1 -(MIDA boryl)-3-methylbutyl)amino)-2-oxo-1 -(pyridin-3-yl)ethyl 2-
Figure imgf000036_0002
Off-white solid; 79% yield; TLC (EtOAc) Rf = 0.17 (A), 0.05 (B); dr = 44:56 (A:B); mp = 167 - 169°C (A), 61-65°C (B); 1H NMR (500 MHz, MeCN-cfe) δ A: 8.66 (d, J = 2.2 Hz, 1H), 8.56 (dd, J = 4.8, 1.7 Hz, 1H), 7.84 (ddd, J = 8.1, 2.2, 1.7 Hz, 1H), 7.37 - 7.27 (m, 6H), 6.57 (d, J = 10.1 Hz, 1H), 5.77 (s, 1H), 3.95 (d, J = 17.2 Hz, 1H), 3.91 (d, J = 16.7 Hz, 1H), 3.82 (d, J= 17.2 Hz, 1H), 3.77 (d, J= 16.7 Hz, 1H), 3.77 (d, J= 10.7 Hz, 2H), 3.61 (ddd, J = 12.7, 10.1, 3.1 Hz, 1H), 2.83 (s, 3H), 1.38- 1.32 (m, 1H), 1.21 - 1.15 (m, 1H), 1.05-0.96 (m, 1H), 0.68 (d, J = 6.5 Hz, 1H), 0.63 (d, J = 6.5 Hz, 3H); B: 8.61 (d, J = 1.5 Hz, 1H), 8.55 (dd, J = 3.8, 0.9 Hz, 1H), 7.77 (ddd, J = 6.4, 2.0, 1.5 Hz), 7.37 - 7.27 (m,6H), 6.45 (d, J= 10.1 Hz, 1H), 5.85 (s, 1H), 3.92 (d, J = 17.2 Hz, 1H), 3.87 (d, J = 16.9 Hz, 1H), 3.81 (d, J = 17.2 Hz, 1H), 3.79 (d, J = 5.7 Hz, 1H), 3.65 (ddd, J = 12.5, 10.1, 2.9 Hz, 2H), 3.33 (d, J = 16.9 Hz, 1H), 2.79 (s, 3H), 1.54 - 1.48 (m, 1H), 1.37 - 1.32 (m, 1 H), 1.22 - 1.16 (m, 1H), 0.82 (d, J = 6.6 Hz, 1 H), 0.71 (d, J = 6.4 Hz, 1 H) ppm; 13C NMR (125 MHz, MeCN-c/3) δ A: 172.4, 169.1 , 168.8, 168.6, 151.0, 149.6, 135.8, 135.1 , 132.2, 130.4, 129.5, 128.1 , 124.4, 75.2, 63.6, 62.8, 46.4, 41.3, 41.0, 25.0, 23.9, 21.1 ; B: 171.7, 168.9, 168.7, 168.5, 150.9, 149.2, 135.5, 135.0, 132.8, 130.5, 129.5, 128.1 , 124.5, 75.3, 63.5, 63.0, 46.6, 41.3, 41.1 , 25.0, 24.0, 21.2 ppm; 1 B NMR (192 MHz, MeCN-of3) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C25H31 BN3O7 496.22550, found 496.22657; IR (thin film, cm'1) 3376, 2952, 1744, 1679, 1540, 1456, 1427, 1298, 1251 , 1148, 1103, 1021 , 996, 874, 767, 718. 2-((1-(MIDA boryl)-3-methylbutyl)amino)-2-oxo-1-(p-tolyl)ethyl 2-phenylacetate
Figure imgf000037_0001
White solid; 50% yield; TLC (EtOAc) Rf = 0.72 (A), 0.53 (B); dr = 53:47 (A:B); 1H NMR (500 MHz, MeCN-afe) δ A: 7.41 - 7.27 (m, 8H), 7.19 - 7.17 (m, 1 H), 6.28 (d, J = 7.2 Hz, 1 H), 5.68 (s, 1 H), 3.98 - 3.87 (m, 2H), 3.85 - 3.70 (m, 2H), 3.63 - 3.57 (m, 2H), 2.77 (s, 3H), 2.33 (s, 3H), 1.56 - 1.45 (m, 1 H), 1.34 - 1.28 (m, 1h), 1.20 - 1.13 (m, 1 H), 1.12 - 1.06 (m, 1 H), 0.70 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.2 Hz, 3H); B: 7.42 - 7.27 (m, 7H), 6.29 - 6.27 (m, 2H), 6.28 (d, J = 10.1 Hz, 1 H), 5.75 (s, 1 H), 3.88 (d, J = 17.0 Hz, 1 H), 3.77 (d, J = 17.0 Hz, 2H), 3.75 (d, J = 2.8 Hz, 2H), 3.73 (d, J = 16.9 Hz, 2H), 2.96 (d, J = 16.9 Hz, 1 H), 3.65 - 3.60 (m, 1H), 2.68 (s, 3H), 2.33 (s, 3H), 1.52 - 1.45 (m, 2H), 1.27 - 1.22 (m, 1 H), 0.86 (d, J = 6.5 Hz, 3H), 0.77 (d, J = 6.4 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-cW δ A: 172.3, 171.0, 169.2, 163.2, 139.8, 135.3, 133.4, 130.4, 130.0, 129.6, 128.2, 123.2, 77.3, 63.6, 62.8, 46.4, 41.4, 41.2, 24.9, 21.2, 21.2, 10.6; B: 171.8, 169.1 , 169.0, 168.6, 139.9, 135.2, 134.0, 130.5, 130.2, 129.5, 128.1 , 128.0, 77.1 , 63.5, 62.8, 46.5, 41.4, 41.3, 24.9, 24.1 , 21.4, 21.2 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C27H34BN207 509.24591 , found 509.24678. 2-(((MIDA boryl)-3-methylbutyl)amino)-1 -(2-bromophenyl)-2-oxoethyl 2-
Figure imgf000038_0001
White solid; 93% yield; TLC (EtOAc) Rf = 0.67 (A), 0.50 (B); dr = 55:47; 1H NMR (500 MHz, MeCN-ofs) δ A: 7.64 (dd, J = 8.0, 1.3 Hz, 1 H), 7.44 - 7.27 (m, 8H), 6.41 (d, J = 10.0 Hz, 1 H), 6.11 (s, 1 H), 3.94 (d, J = 17.2 Hz, 1 H), 3.93 (d, J = 16.7 Hz, 1 H), 3.83 (d, J = 17.2 Hz, 1H), 3.78 (d, J = 16.7 Hz, 1H), 3.75 (d, J = 2.0 Hz, 2H), 3.72 - 3.67 (m, 1 H), 2.86 (s, 3H), 1.40 - 1.35 (m, 1H), 1.32 - 1.26 (m, 1H), 0.92 - 0.87 (m, 1 H), 0.80 (d, J = 6.2 Hz, 3H), 0.77 (d, J = 6.3 Hz, 3H); B: 7.62 - 7.60 (m, 1 H), 7.37 - 7.33 (m, 4H), 7.31 - 7.24 (m, 4H), 6.36 (d, J = 9.9 Hz, 1 H), 6.27 (s, 1 H), 3.93 (d, J = 17.1 Hz, 1 H), 3.91 (d, J = 16.9 Hz, 1 H), 3.83 (d, J = 17.1 Hz, 1 H), 3.77 (d, J = 1.4 Hz, 2H), 3.71 - 3.66 (m, 1H), 3.52 (d, J = 16.9 Hz, 1 H), 2.82 (s, 3H), 1.56 - 1.50 (m, 2H), 1.31 - 1.26 (m, 1H), 0.89 (d, J = 6.5 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H) ppm; 3C NMR (125 MHz, MeCN-cfe) δ A: 171.9, 168.9, 168.6, 167.7, 135.6, 134.9, 133.8, 131.6, 130.2, 130.0, 129.4, 128.7, 127.9, 124.7, 75.9, 63.4, 62.8, 46.4, 41.1 , 41.0, 25.0, 23.8, 21.1 ; B: 171.4, 168.9, 168.8, 167.9, 136.8, 134.9, 134.0, 131.5, 130.5, 129.8, 129.6, 128.9, 128.1 , 124.4, 76.0, 63.5, 63.1 , 46.7, 41.3, 41.3, 25.2, 24.1 , 21.4 ppm; 11B NMR (192 MHz, MeCN-of3) δ 11.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C26H3iBBrN207 573.14077, found 573.14104; IR (thin film, cm"1) 2955, 2261 , 1747, 1668, 1523, 1470, 1290, 1242, 1194, 1103, 1030, 948, 896, 867, 753, 723.
1-(((MIDA boryl)-(cyclohexyl)methyl)amino)-3-methyl-1-oxobutan-2-yl 2-
Figure imgf000038_0002
White solid; 46% yield; TLC (EtOAc) Rf = 0.55 (A), 0.38 (B); dr = 56:44 (A:B); 1H NMR (500 MHz, MeCN-c/3) δ A: 7.39 - 7.34 (m, 3H), 1.33 - 7.29 (m, 2H), 6.26 (d, = 10.4 Hz, 1 H), 4.67 (d, J = 5.0 Hz, 1 H), 3.92 (d, J = 17.2 Hz, 1 H), 3.87 (d, J = 16.7 Hz, 1 H), 3.82 (d, J = 17.1 Hz, 1 H), 3.82 (d, J = 16.7 Hz, 1 H), 3.72 (s, 2H), 3.63 - 3.60 (m, 1H), 2.79 (s, 3H), 2.14 - 2.09 (m, 1 H), 1.87 - 1.80 (m, 1 H), 1.75 - 1.66 (m, 4H), 1.64 - 1.52 (m, 4H), 1.29 - 1.22 (m, 2H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H); B: 7.38 - 7.26 (m, 5H), 5.93 (d, J = 10.5 Hz, H), 4.81 (d, J = 4.1 Hz, H), 3.96 (d, J = 17.0 Hz, 1 H), 3.94 (d, J = 17.1 Hz, 1 H), 3.84 (d, J = 17.0 Hz, 1 H), 3.75 (d, J = 14.2 Hz, 2H), 3.62 - 3.58 (m, 1 H), 3.60 (d, J = 17.1 Hz, 1 H), 2.82 (s, 3H), 2.19 - 2.14 (m, 1 H), 1.75 - 1.64 (m, 4H), 1.61 - 1.50 (m, 4H), 1.28 - 1.19 (m, 2H), 1.08 - 1.05 (m, 1 H), 0.88 (d, J = 6.9 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H) ppm; 13C NMR (125 MHz, MeCN-of3) δ A: 172.8, 170.2, 169.1 , 169.0, 135.4, 130.3, 129.5, 128.0, 79.9, 63.2, 62.6, 46.6, 41.5, 41.4, 32.3, 31.3, 29.3, 27.4, 27.4, 27.1 , 19.3, 17.5; B: 172.3, 169.3, 168.8, 135.1 , 130.6, 129.5, 128.0, 79.9, 63.0, 62.8, 46.7, 41.6, 41.1 , 32.3, 31.2, 29.1 , 27.4, 27.3, 27.1 , 19.3, 17.2 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 11.3 ppm; HRMS (DART-TOF) [M+H]+ calcd. for CzsHaeBNaOy 487.26156, found 587.26119; IR (thin film, cm"1) 2926, 2853, 2266, 1748, 1667, 1525, 1451 , 1339, 1294, 1239, 1159, 1109, 1025, 969, 894, 857, 764, 711. 2-(((MIDA boryl)-(cyclohexyl)methyl)amino)-2-oxo-1-phenylethyl 2-phenylacetate
Figure imgf000039_0001
Off-white solid; 52% yield; TLC (EtOAc) R, = 0.58 (A), 0.35 (B); dr = 56:44 (A:B); 1H NMR (500 MHz, MeCN-d3 (A) DMSO- fe+MeCNck (B)) δ A: δ 7.51 - 7.50 (m, 2H), 7.41 - 7.25 (m, 10H), 6.38 (d, J = 10.5 Hz, 1H), 5.81 (s, 1 H), 3.88 (d, J = 17.1 Hz, 1H), 3.83 (d, J = 16.6 Hz, 1 H), 3.77 (d, J = 17.1 Hz, 1 H), 3.64 (d, J = 16.7 Hz, 1 H), 3.61 (s, 2H), 3.51 (dd, J = 10.6, 3.0 Hz, 1 H), 2.70 (s, 3H), 1.84 - 1.56 (m, 4H), 1.51 - 1.24 (m, 4H), 1.17 - 1.08 (m, 1 H), 0.81 - 0.74 (m, 1H), 0.65 - 0.57 (m, 1 H); B: 7.55 - 7.53 (m, 1 H), 7.45 - 7.43 (m, 2H), 7.33 - 7.23 (m, 8H), 5.95 (s, 1 H), 3.96 (d, J = 17.2 Hz, 1 H), 3.78 (d, J = 17.2 Hz, 1 H), 3.77 (d, J = 16.8 Hz, 1 H), 3.71 (d, J = 16.8 Hz, 1 H), 3.42 - 3.37 (m, 1H), 2.71 (s, 2H), 2.69 - 2.66 (m, 1H), 2.33 (s, 3H), 1.70 - 1.40 (m, 6H), 1.22 - 0.96 (m, 4H) ppm; 3C NMR (125 MHz, MeCN-of3 (A) DMSO-c6+MeCNof3 (B)) δ A: 172.3, 169.6, 169.0, 168.9, 136.6, 135.2, 130.4, 129.5, 129.5, 128.0, 127.8, 118.3, 77.2, 63.1, 62.6, 46.4, 41.4, 41.3, 31.8, 29.2, 28.9, 27.3, 26.9; B: 172.1, 171.2, 169.5, 168.8, 137.1, 135.0, 130.3, 129.2, 129.2, 129.0, 128.0, 127.7, 76.4, 62.7, 62.2, 45.8, 41.1, 41.1, 31.7, 29.1, 27.2, 27.1, 26.8 ppm; 11B NMR (192 MHz, MeCN-cfe) δ 10.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C2BH34BN207521.24591, found 521.24771; IR (thin film, cm"1) 3337, 2923, 2853, 1750, 1678, 1653, 1536, 1497, 1449, 1344, 1298, 1258, 1156, 1116, 1030, 965, 895, 860, 710, 694.
2-(((MIDA boryl)-(cyclohexyl)methyl)amino)-2-oxo-1-(pyridin-3-yl)ethyl 2-
Figure imgf000040_0001
Off-white solid; 42% yield; TLC (EtOAc) Rf = 0.08 (A), 0.03 (B); dr = 40:60 (A:B); 1H NMR (500 MHz, MeCN-d3) δ A: 8.75 (d, J = 2.2 Hz, 1 H), 8.61 (dd, J = 5.0, 1.6 Hz, 1 Η), 8.00 (dddd, J = 8.0, 2.2, 1.6, 0.5 Hz, 1H), 7.48 (ddd, J = 8.0, 5.0, 0.5 Hz, 1H), 7.37 - 7.25 (m,5H), 6.61 (d, J= 10.5 Hz, 1H), 5.91 (s, 1H), 3.91 (d, J= 17.1 Hz, 1H), 3.88 (d, J = 16.7 Hz, 1H), 3.80 (d, J = 17.1 Hz, 1H), 3.77 (s, 2H), 3.75 (d, J = 16.7 Hz, 1H), 3.54 (dd, J= 10.5, 3.1 Hz, 1H), 2.75 (s, 3H), 1.66- 1.58 (m, 2H), 1.53 - 1.42 (m, 3H), 1.31 - 1.27 (m, 1H), 1.16 - 1.08 (m, 2H), 0,96 - 0.72 (m, 2H), 0.56 - 0.48 (m, 1H); B: 8.61 (s, 1H), 8.55 (d, J = 3.9 Hz, 1H), 7.76 - 7.74 (m, 1H), 7.73 - 7.28 (m, 6H), 6.42 (d, J = 10.6 Hz, 1H), 5.90 (s, 1H), 3.91 (d, J = 17.0 Hz, 1H), 3.85 (d, J = 17.1 Hz, 1H), 3.81 (d, J = 10.7 Hz, 2H), 3.77 (d, J = 17.1 Hz, 1H), 3.55 (dd, J = 10.6, 3.2 Hz, 1H), 3.35 (d, J= 17.0 Hz, 1H), 2.67 (s, 3H), 1.75- 1.66 (m, 2H), 1.60- 1.54 (m, 3H), 1.42- 1.39 (m, 1H), 1.25 - 1.15 (m, 2H), 1.01 - 0.92 (m, 2H), 0.88 - 0.80 (m, 1H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ A: 172.2, 169.0, 168.9, 168.8, 149.5, 147.9, 137.4, 135.0, 130.4, 129.6, 129.4, 128.1, 125.1, 74.8, 63.1, 62.6, 46.5, 41.3, 41.2, 32.0, 29.0, 27.2, 27.2, 26.9; B: 171.4, 169.1, 168.8, 168.5, 150.9, 149.3, 135.4, 134.9, 130.5, 130.4, 129.6, 128.1, 124.5, 75.2, 63.0, 62.7, 46.5, 41.4, 41.3, 32.0, 29.1, 27.3, 27.3, 27.0 ppm; 1B NMR (192 MHz, MeCN-d3) δ 11.2 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C27H33BN3O7 522.24115, found 522.24275; IR (thin film, cm'1) 3027, 1747, 1673, 1496, 1453, 1427, 1337, 1286, 1242, 1126, 1028, 949, 894, 789, 759, 702.
1-(((MIDA boryl)-(cyclohexyl)methyl)amino)-1-oxo-3-phenylpropan-2-yl 2- phenylacetate (10bd)
Figure imgf000041_0001
White solid; 57% yield; TLC (EtOAc) Rf = 0.62 (A), 0.32 (B); dr = 54:46 (A:B); 1H NMR (500 MHz, MeCN-cfe) δ A: 7.30 - 7.20 (m, 10H), 6.26 (d, J = 10.5 Hz, 1 H), 5.15 (dd, J = 7.8, 5.0 Hz, 1 H), 3.87 (d, J = 17.1 Hz, 1 H), 3.77 (d, J = 17.1 Hz, 1 H), 3.76 (d, J = 16.8 Hz, 1 H), 3.64 (s, 2H), 3.52 (dd, J = 10.5, 3.2 Hz, 1 H), 3.39 (d, J = 16.8 Hz, 1 H), 3.08 - 3.05 (m, 2H), 2.65 (s, 3H), 1.85 - 1.77 (m, 1H), 1.71 - 1.42 (m, 5H), 1.25 - 1.12 (m, 2H), 1.02 - 0.92 (m, 1 H), 0.90 - 0.78 (m, 2H); B: 7.36 - 7.19 (m, 8H), 7.10 - 7.08 (m, 2H), 5.96 (d, J = 10.6 Hz, 1 H), 5.23 (dd, J = 6.9, 4.9 Hz, 1 H), 3.90 (d, J = 17.0 Hz, 1 H), 3.86 (d, J = 17.0 Hz, 1 H), 3.80 (d, J = 17.0 Hz, 1 H), 3.71 (d, J = 16.0 Hz, 1 H), 3.67 (d, J = 16.0 Hz, 1 H), 3.50 (dd, J = 10.6, 3.0 Hz, 1 H), 3.43 (d, J = 17.0 Hz, 1 H), 3.09 (dd, J = 14.2, 4.9 Hz, 1 H), 3.04 (dd, J = 14.2, 6.9 Hz, 1 H), 2.74 (s, 3H), 1.69 - 1.64 (m, 1 H), 1.59 - 1.50 (m, 3H), 1.50 - 1.42 (m, 1 H), 1.32 - 1.27 (m, 1H), 1.20 - 1.11 (m, 2H), 0.95 - 0.87 (m, 1 H), 0.76 - 0.62 (m, 2H) ppm; 3C NMR (125 MHz, MeCN-cfe) δ A: 172.1 , 169.9, 169.0, 168.9, 137.6, 135.0, 130.7, 130.3, 129.6, 129.3, 128.0, 127.8, 76.0, 63.1 , 62.5, 46.5, 41.6, 41.3, 38.2, 32.0, 29.1 , 27.4, 27.3, 26.8; B: 171.7, 169.2, 169.1 , 168.8, 137.3, 134.9, 130.8, 130.5, 129.6, 129.2, 128.0, 127.7, 76.1 , 63.0, 62.7, 46.7, 41.6, 40.9, 38.0, 31.6, 28.9, 27.3, 27.3, 26.7 ppm; 11B NMR (192 MHz, MeCN-c/3) J 11.2 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C29H36BN207 535.26156, found 535.26276; IR (thin film, cm"1) 2925, 2852, 2266, 1748, 1612, 1531 , 1497, 1453, 1338, 1293, 1239, 1157, 1111 , 1027, 967, 894, 858, 698. 2-(((MIDA boryl)-(phenyl)methyl)amino)-2-oxo-1 -(pyridin-3-yl)ethyl 2-
Figure imgf000042_0001
Off-white solid; 48% yield; TLC (EtOAc) Rf = 0.10 (A), 0.03 (B); dr = 40:60 (A:B); 1H NMR (500 MHz, DMSO-cfe (A), MeCN-cfe (B)) δ A: 8.69 (d, J= 9.1 Hz, 1H), 8.63 (d, J = 2.0 Hz, 1H), 8.52 (dd, J = 4.8, 1.7 Hz, 1H), 7.77 (dt, J = 8.5, 2.0 Hz, 1H), 7.37 - 7.23 (m, 7H), 7.14-7.11 (m, 2H), 7.07-7.03 (m, 2H), 6.14 (s, 1H),4.63 (d, J = 9.1 Hz, 1H), 4.30 (d, J= 17.0 Hz, 1H), 4.24 (d, J= 17.0 Hz, 1H), 4.18 (d, J = 17.0 Hz, 1H), 3.95 (d, J = 17.0 Hz, 1H), 3.80 (s, 2H), 2.94 (s, 3H); B: 8.57 (d, J = 2.5 Hz, 1H), 8.53 (d, J = 4.0 Hz, 1H), 7.70 (dt, J = 7.9, 2.0 Hz, 1H), 7.37 (m, 11H), 7.21 -7.16(m, 1H), 5.96 (s, 1H), 4.63 (d, J = 9.7 Hz, 1H), 3.95 (d, J = 17.1 Hz, 1H), 3.93 (d, J = 17.1 Hz, 1H), 3.86 (d, J = 17.1 Hz, 1H), 3.77 (d, J = 3.2 Hz, 2H), 3.49 (d, J = 17.1 Hz, 1H), 2.70 (s, 3H) ppm; 13C NMR (125 MHz, DMSO-c/6 (A), MeCN-/3 (B)) δ A: 170.9, 168.8, 168.3, 167.1,
149.6, 148.3, 142.4, 134.8, 134.0, 129.3, 129.3, 128.3, 128.2, 127.4, 126.9, 125.4, 123.4, 73.1, 62.3, 62.0, 45.5, 40.7: B; 171.5, 168.9, 168.4, 168.2, 151.0, 149.5, 143.1,
135.7, 134.9, 130.5, 129.6, 129.1, 129.0, 128.4, 128.1, 127.2, 124.5, 74.8, 63.4, 63.2, 46.6, 41.3 ppm; 11B NMR (192 MHz, MeCN-c/3) δ 10.9 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C27H27BN3O7516.19420, found 516.19663; IR (thin film, cm"1) 2927, 2853, 1749, 1654, 1536, 1449, 1341, 1295, 1116, 1025, 968, 894, 859, 774, 710. -(2-(2-phenylacetoxy)-4-(pyridin-2-yl)butanamido)nonyl)boronate 10cp
Figure imgf000042_0002
Off-white solid; 39% yield; TLC (EtOAc/MeCN (8:2)) Rf = A: 0.43, B: 0.22; 1H NMR (500 MHz, MeCN-cfe) δ Diastereomer A: 8.40 (dd, J = 4.8, 1.7 Hz, 1H), 8.36 (dd, J = 2.4, 0.8 Hz, 1H), 7.52 - 7.47 (m, 1H), 7.40 - 7.37 (m, 2H), 7.35 - 7.30 (m, 3H), 7.24 (ddd, J = 7.8, 4.8, 0.9 Hz, 1 H), 6.36 (d, J = 10.1 Hz, 1 H), 4.74 (dd, J = 8.3, 4.7 Hz, 1 H), 3.96 (d, J = 17.2 Hz, 1 H), 3.92 (d, J = 16.7 Hz, 1 H), 3.83 (d, J = 17.2 Hz, 1 H), 3.82 (d, J = 16.7 Hz, 1 H), 3.72 (s, 2H), 3.56 (td, J = 10.7, 3.2 Hz, 1 H), 2.84 (s, 3H), 2.76 - 2.62 (m, 2H), 2.09 - 2.01 (m, 2H), 1.59 - 1.52 (m, 1 H), 1.36 - 1.15 (m, 12H), 0.95 - 0.86 (m, 4); Diastereomer B: 8.42 (d, J = 4.6 Hz, 1 H), 7.49 (ddd, J = 7.7, 2.3, 1.6 Hz, 1 H), 7.41 - 7.36 (m, 3H), 7.35 - 7.30 (m, 2H), 7.25 (ddd, J = 7.8, 4.8, 0.9 Hz, 1 H), 6.17 (d, J = 10.1 Hz, 1 H), 4.85 (dd, J = 7.0, 5.6 Hz, 1 H), 3.99 - 3.59 (m, 8H), 2.88 (m 3H), 2.67 - 2.56 (m, 2H), 2.10 - 2.05 (m, 2H), 1.61 - 1.54 (m, 1 H), 1.47 - 1.40 (m, 1 H), 1.35 - 1.18 (m, 11 H), 0.90 - 0.86 (m, 4H) ppm; 13C NMR (125 MHz, MeCN-cfe) δ Diastereomer A: 171.5, 169.4, 168.1 , 168.0, 149.8, 147.4, 136.4, 135.9, 134.3, 129.4, 128.6, 127.1 , 123.4, 73.8, 62.5, 61.9, 45.5, 40.5, 32.9, 31.6, 31.0, 29.3, 29.0, 29.0, 28.1 , 26.3, 22.4, 13.4; Diastereomer B: 172.3, 170.3, 169.0, 169.0, 150.8, 148.5, 136.7, 135.2, 130.6, 130.4, 129.6, 128.1 , 124.4, 75.0, 63.5, 63.1 , 46.8, 41.4, 33.9, 32.6, 31.9, 30.3, 30.0, 30.0, 28.9, 27.2, 23.4, 14.4 ppm; 11B NMR (192 MHz, MeCN-d3) δ Diastereomer A: 11.2; Diastereomer B: 11.3 ppm.
Figure imgf000043_0001
To a small vial charged with P3CR product syn-IOad (50 mg, 0.10 mmol) was added THF (1.5 mL) and NaOH (1.0 M (aq.), 0.40 mL, 0.40 mmol) and the resulting mixture was magnetically stirred at 23°C for 10 minutes at which point TLC indicated that the reaction had gone to completion. To the reaction mixture was added 1.5 mL of pH 7 aqueous phosphate buffer and 1.5 mL Et20. The layers were separated and the aqueous layer was washed 3 times with THF/Et20 (1 :1). The combined organic layers were washed with brine, dried over Na2S04 and filtered. 2 mL 1 ,4-dioxane was added and the solution was concentrated via rotary evaporation without submersion into a water bath to remove only the THF and Et20. The 1 ,4-dioxane was lyophilized to afford the desired product as a white powder in 79% crude yield. an./-2-benzyl-3-boro-6-hydroxy-5-isobutylmorpholin-3-one 11 ad (complex with
Figure imgf000044_0001
White solid; 79% yield; TLC Rf data unavailable (decomposes on silica); H NMR (400 MHz, MeCN- /3) <y 7.35 - 7.17 (m, 5H), 6.14 (s, 1 H), 4.67 (t, J = 5.0 Hz, 1 H), 3.12 (dd, J = 13.7, 5.0 Hz, 1 H), 3.04 (dd, J = 13.7, 5.0 Hz, 1 H), 2.80 - 2.75 (m, 1 H), 1.48 - 1.39 (m, 1 H), 1.36 - 1.27 (m, 1 H), 0.74 (d, J = 6.7 Hz, 3H), 0.72 (d, J = 6.7 Hz, 3H), 0.32 (ddd, J = 13.7, 10.4, 5.0 Hz, 1 H) ppm; 13C NMR (125 MHz, DMSO-cfe) δ 169.2, 136.6, 130.0, 128.2, 126.1 , 74.5, 41.3, 40.4, 23.4, 23.1 , 21.5 ppm (selected peaks reported. In DMSO, some dehydrated product exists (B-O-B dimer)); 11B NMR (128 MHz, MeCN- d3) δ 30.4 ppm; HRMS (DART-TOF) [M+H]+ calcd. for C14H2iBN03 262.16145, found 262.16106; IR (thin film, cnrf1) 3220, 3064, 3031 , 2954, 2926, 2868, 2348, 1704, 1654, 1618, 1534, 1497, 1454, 1411 , 1384, 1367, 1299, 1260, 1195, 1142, 1091 , 1031 , 641 , 868, 798, 746.
The P3CR product 10cp was subjected to the standard procedure for preparation of 11ad above. LRMS (ESI) indicated the presence of the desired product with some hydrolyzed byproduct. The IC50 of the impure compound for inhibition of 20S proteases was determined using the procedure outlined for 11ad. An IC50 of ~ 1 μΜ was obtained for the CT-L enzymes and no observable inhibition occurred for the T-L or C-L enzymes.
N-Methylation of 11ad
H° 'B-°Y-Ph NaH. Mel ,H0 ' B °v
N "^O THF, o/n
H
CH,
To a solution of 11 ad (84 mg, 0.32 mmol, 1.0 equiv.) in anhydrous THF (6.5 mL) was added crushed 4A molecular sieves (100 mg) and methyl iodide (80 pL, 1.29 mmol, 4.0 equiv.) and the resulting solution was stirred for 15 minutes under Ar. A 60% dispersion of NaH in mineral oil (51.5 mg, 1.29 mmol, 4.0 equiv.) was added and the resulting mixture was stirred overnight. The reaction was cooled to 0°C and quenched with 1 M HCI(aq.) (5 mL). The layers were separated and the aqueous layer was washed with Et20/THF (1 :1) (3 x 6 mL). The combined organic layers were dried over Na2S04 and filtered. 1 ,4-dioxane (2 mL) was added and the resulting solution was concentrated under reduced pressure in the absence of any temperature bath until only the dioxane remained. The solution was then Iyophilized to yield 55 mg of orange residue. HRMS (DART-TOF) confirmed the presence of the desired product: [M+H]+ calcd. for C^F BNOa 276.17730, found 276.17710. Absence of the N-H signal in the crude 1H NMR spectra further supports the presence of the desired product.
A2. Computational modeling of equilibrium energies for 11ad hydrolysis
Each compound shown in Figure 7 was modeled using the Gaussian '09 computational engine. [New Ref 3] Geometry optimizations were carried out using B3LYP 6-31 G+(d). Single-point energy calculations were performed on the optimized structures using B3LYP 6-311G++(d,p). It should be noted that the water molecules were not modeled separately from the boron-containing structures but rather were included as hydrogen-bonding partners to give a more accurate estimate of enthalphic energy. The results are summarized in Figure 7. pK„ determination of 11 ad
To a solution of 11 ad (25.4 mg, 0.097 mmol) in 4.1 mL of CD3OD was added 1.2 mL of HEPES buffer (0.10 M in D20). The pH was adjusted to 3.2 by addition of 2.0 M HCI04(aq .). A 250 pL aliquot was taken for 11B NMR. LRMS (ESI, positive) showed this sample contained a mass corresponding to the monodeuterated borocycle shown in Scheme 1. The pH was increased by ~ 1 pH unit by addition of 2.0 M NaOH(aq.) until a pH of ~ 12 was reached. At each pH, a 250 pL sample was taken for 11B NMR. LRMS (ESI, positive) of the sample with pH 12.3 showed a mass corresponding to the pentadeuterated boronate shown in Scheme 1. The 11B NMR spectra were taken with a sweep width of 51000 Hz, 131000 data points, 90° pulse width, 1.2 second recycle time, 10 Hz line broadening and a 2nd order polynomial fitting routine.
Figure imgf000046_0001
k k ' i. i * ,ν ,', ' ^j^^' ii .Ί /o ; ; ; ' ; Τ ΤΠ
20S proteasome inhibition assay of 7a and 11 ad
7a:
Solutions of 7a (each diastereomer) and bortezomib were prepared by serial dilution of 10 mM stocks in DMSO. To a feshly prepared sample of OCI-AML-2 human leukemia cells was added 5 mL of freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1 % Trition X-100 and 2 mM ATP. The cells were suspended by pipetting up and down several times and were vortexed every 5 minutes for 30 minutes at 0°C. Each well of a 96 well-plate was loaded with 87 μΙ_ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 10 pL of cell lysate solution and 1 pL of each stock solution of either 7a or bortezomib (to final concentrations of 10 μΜ to 10 pM, in 1/10th dilution increments). The resulting solutions were incubated at 37°C for 1h. To each well was added 2 μΙ_ of 3.75 mM N- Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin in DMSO. The fluorescence spectrum of each well was measured at 5 minute intervals over 30 minutes at 37°C (using a Spectromax spectrometer by Molecular Devices, excitation: 360 nm; emission 460 nm). The slope of the increase in fluorescence vs. time (converted to a percent of the slope for a blank sample) was plotted against the inhibitor concentration (Figures 1-2). The IC50 of each of each diastereomer of 7a and bortezomib was calculated by applying a sigmoidal fit to each curve shown in Figures 1-2 and interpolating to 50% enzyme activity. In each assay, rates were measure in triplicate and averaged. Error bars represent 1 standard deviation. The IC50 values are tabulated in Table 2. Table 2: Summary of IC50 values obtained for 20S proteasome inhibition assays of 7a
Figure imgf000047_0001
Assay
bortezomib 7a (diastereomer A) 7a (diastereomer B) chymotrypsin-like 22 71
11ad:
Solutions of 11ad and bortezomib were prepared by serial dilution of a 10 mM stock in DMSO. To a feshly prepared sample of OCI-AML-2 human leukemia cells was added 5 ml. of freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1 % Trition X-100 and 2 mM ATP. The cells were suspended by pipetting up and down several times and were vortexed every 5 minutes for 30 minutes at 0°C. Each well of a 96 well-plate was loaded with 87 μΙ_ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 10 L of cell lysate solution and 1 μί. of each stock solution of either 11ad or bortezomib (to final concentrations of 100 μΜ to 1 nM, in 1/10th dilution increments). The resulting solutions were incubated at 37°C for 1 h. To each well was added 2 μΙ_ of either 3.75 mM N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin for the chymotrypsin-like assay, 3.75 mM t-butoxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin for the trypsin-like assay or 3.75 mM benzyloxycarbonyl-Leu-Leu-Glu-7-amino-4- methylcoumarin for the caspase-like assay (all in DMSO). The fluorescence spectrum of each well was measured at 5 minute intervals over 30 minutes at 37°C (using a Spectromax spectrometer by Molecular Devices, excitation: 360 nm; emission 460 nm). The slope of the increase in fluorescence vs. time (converted to a percent of the slope for a blank sample) was plotted against the inhibitor concentration (Figures 3-5). The IC50 of each of 11ad and bortezomib was calculated by applying a sigmoidal fit to each curve shown in Figures 3-5 and interpolating to 50% enzyme activity. In each assay, rates were measure in triplicate and averaged. Error bars represent 1 standard deviation. Figure 6 shows a comparison of the syn- and anti- isomers of 11ad in the chymotrypsin-like assay. It should be noted that IC50 values obtained in the assays of syn- and a/7f/'-11ad cannot be compared to those obtained in the bortezomib comparison studies as different cell preparations were used. The IC50 values are tabulated in Table 3.
Table 3: Summary of IC50 values obtained for 20S proteasome inhibition assays
IC50 (nM)
Assay
bortezomib anf/'-11ad syn-11ad chymotrypsin-
0.44 19 N/A
like caspase-like 3.9 84 N/A trypsin-like N/A N/A N/A chymotrypsin-
N/A 2.9 52
like[a]
[a] performed using a different cell line than the first 3; IC50 values differ.
Buffer-mediated hydrolysis of MIDA-bortezomib 7a (control experiment)
A 1 mM stock solution of 7a-A in DMSO was prepared. Each of 5 HPLC vials were loaded with 870 μΙ_ of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCI buffer, 150 mM NaCI and 2 mM ATP), 100 μΙ_ of cell lysis buffer (containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCI, 1% Trition X-100 and 2 mM ATP) (no cells were added) and 15 pL of 7a-A stock solution. One vial was immediately subjected to HPLC-MS analysis. The remaining 4 were incubated at 37°C. One vial was removed each 30 minutes and immediately subjected to HPLC-MS analysis. The final vial was left incubating overnight. The first vial showed a large amount of 7a-A (m/z = 496.2) and a trace of the free boronic acid (m/z = 385.7). The vial after 30 minutes of incubation showed an increased amount of the free boronic acid (m/z = 385.7) relative to 7a-A (m/z = 496.2). The remaining vials gave similar results (no qualitative change in either species was observed). It should be noted that quantitative comparisons of peak data could not be obtained as multiple, poorly separated peaks were observed for each species presumably due to non-covalent bonding interactions with one or more of the buffer components.
Caco-2 assays of 11 ad and 7a Human, epithelial Caco-2 cells were grown as monolayers, fully polarized and differentiated. All compounds were tested under non-gradient pH conditions (pH 7.4/7.4) for 90 minutes as previously described. [23] LC/MS analysis were performed for all samples using a Waters Xevo QTof mass spectrometer and an ACQUITY UPLC system, to determine relative peak areas of parent compound. The percent of transported drug were calculated based on these peak areas, relative to the initial, dosing concentration.
Permeability was classified based on A-B Papp values (for absorptive transport) as:
Low (Papp < 2), Medium (Papp 2 - 10) or High (Papp >10) x 10"6 cm/sec.
Efflux ratios correspond to the B-A Papp coefficient divided by A-B Papp value. Efflux ratios were classified as:
Negative (ratio < 3), Minor/Moderate (ratio 3 - 10), or High (ratio > 10).
The results are summarized in Table 4.
Table 4: Caco-2 cell permeability assay results for 7a-A and antf-Had app app Permeability Efflux Ratio Efflux Post Assay
Compound
»» Classification (BA/AB) Classification Recovery (%)
(A to B) (B to A) * bortezomib O0 52 Cow >Ϊ0 High 28
7a-A 0.0 5.2 Low >10 High 50 ani/-11ad 7.7 9.5 Moderate 1.2 Negative 55
Digoxin
0.1 11.2 Low >10 High 73
(P-gp
control)
Atenelol
0.3 0.3 Low 1.1 N/A 93
(negative
control)
Metoprolol
13.8 10.5 High 0.8 Negative 70
(positive
control)
At the outset, we were aware of the known propensity of tricoordinate boron to react with isocyanides.[9] Moreover, rapid decomposition upon exposure to air has limited synthetic applicability of boron-containing isocyanides.[10] To circumvent this undesired reactivity, we focused our search on fragments with tetracoordinate boron. In N-methyliminodiacetyl (MIDA) boronates, an intramolecular coordinative stabilization of boron's empty p-orbital effectively masks its Lewis acidity.[11] The tetracoordinate boronate fragment is tolerant to a diverse range of functional group transformations, allowing access to a variety of borylated derivatives. These include a- boryl aldehydes, [12] which contain a carbon-boron bond adjacent to an electrophilic aldehyde, and σ-boryl isocyanates 1 , a group of reagents that are now readily available from a protocol recently reported. [13] The structural integrity of σ-boryl isocyanates 1 in various functionalizations prompted us to attempt a trichlorosilane-mediated deoxygenation,[14] which gratifyingly afforded the corresponding isocyanides 2 as solid materials stable to column chromatography (Table 5). To explore the properties of these compounds we first investigated the possibility of σ-proton removal given the documented utility of σ-metalated isocyanides. [6a] During attempted σ-deprotonation using weak bases (10 equiv. of Et3N or 15 equiv. of NaHC03) there was no observable decrease in the 1H NMR integration of the σ-proton signal. Under strongly basic conditions (1 equiv. of potassium ferf-butoxide) followed by exposure to deuterated solvents (MeCN-d3) a similar lack of a-deprotonation was observed. Interestingly, tetradeuteration of the MIDA moiety was observed in both the 1H NMR and HRMS spectra. These results showcase the dual nature of the MIDA moiety as a protective group for both the carbon-boron bond and the σ-proton, which serves to enforce configurational stability of the carbon-boron bond. [15 ]
Table 5. Preparation of MIDA oboryl isocyanides.
Figure imgf000052_0001
1 2
Starting Material R Product Yield isobutyl 75 cyclohexyl 31 phenyl 30
[a] The reactions were carried out using a-boryl isocyanate (1.0 equiv.), trichlorosilane (1.6
equiv.) and triethylamine (3.6 equiv.) in anhydrous CH2CI2 at 0°C for 30 minutes followed
by 6 hours at 23°C. [b] Yields of isolated products after silica gel chromatography.
The protected nature of the σ-proton led us to focus our efforts on a variety of isocyanide functionalizations (Scheme 2). Selenium-catalyzed sulfurization gave rise to σ-boryl isothiocyanate 3a in moderate yield. [16] Reaction of isothiocyanate 3a with various amines afforded the corresponding thioureas 4aa - 4ab in quantitative yield.[17] Reaction of the isocyanide 2a with in situ-generated hydrazoic acid yielded the a-boryl tetrazole 5a in moderate yield. [18]
Figure imgf000053_0001
4aa, R = ethyl (79%)Μ
4ab, R = tert-butyl (90%)Μ
Scheme 2. Functionalization of MIDA σ-boryl isocyanide 2a.
[a] σ-Boryl isocyanide 2a (1.0 equiv.), sulfur (1.2 equiv.), selenium
(5.0 mol %) and triethylamine (2.4 equiv.) in THF at 80°C for 15 min.
[b] σ-Boryl isothiocyanate 3a (1.0 equiv.) and amine (2.0 equiv.) in
THF at 23°C. [c] σ-Boryl isocyanide 2a (1.0 equiv.), HCI (2.0 mol%)
and TMSNj (1.5 equiv.) in TFE/Etp at 60°C for 6.5 h. [d] Yields of
isolated products after silica gel chromatography.
Successful retention of the carbon-boron bond during functionalization of the isocyanide moiety prompted us to investigate more challenging transformations. Isocyanide 2a participated in an Ugi 4-component reaction (U4CR) with 2-pyrazinyl carboxylic acid, phenylacetaldehyde and ammonia to afford the MIDA-bortezomib analogue 7a in 55% isolated yield (Scheme 3). We then attempted to generalize this reaction by employing amino acids as both the acid and amine component. When isocyanide 2a was reacted with L-proline and isobutyraldehyde the borodipeptide 8a was obtained in 7% isolated yield (Scheme 3). [19] In a number of other reactions, we have found that methanol solvolyzes the MIDA moiety yielding decomposition products. We attributed the low yield of 8a to the nucleophilicity of the solvent. The reaction was attempted using trifluoroethanol (TFE) as the solvent given its decreased nucleophilicity relative to methanol; however, the desired product was not obtained. [6a]
Figure imgf000054_0001
8a (7%)
Scheme 3. U4CRs involving σ-boryl isocyanide 2a.
We therefore shifted our focus to U4CRs without solvent participation using N- protected amino acids and peptides as the carboxyiic acid component and ammonia as the amine. The reactions proceeded at room temperature to give diastereomeric mixtures of the desired protected boropeptides 9 in moderate to excellent yields (Table 6). In most cases, diastereomers were separable by preparative reverse-phase high- performance liquid chromatography (HPLC). For larger peptides separation became more difficult due to considerable peak overlap.
[a]
Table 6. Preparation of protected boropeptides using σ-boryl isocyanide 2a.
Figure imgf000054_0002
Figure imgf000054_0003
4 V benzyl 9d 6 57
5 GG benzyl 9e 6 92
6 FA benzyl 9f 6 91
7 PLF benzyl 9g 7 63
8 PGLF benzyl 9h 12 76
[a] The reactions were carried out using σ-boryl isocyanide 2a (1.0 equiv.), aldehyde (1.0 equiv.), ammonia (1.5 equiv., 7N solution in MeOH) and
peptide (1.0 equiv.) in TFE at 23°C. [b] Boc protected at /V-terminus;
written from N- to C-terminus using standard 1 -letter amino acid
abbreviations, [c] Yield of diastereomeric products determined by
comparison of 1H NMR integration with 3,4,5-triiodobenzoic acid as an
internal standard.
The successful participation of isocyanide 2a in U4CRs led us to explore its applicability in Passerini 3-component reactions (P3CRs).[6a] We found this reaction to proceed smoothly with a variety of aldehydes affording minimal by-products. Generally, the P3CR products 10 could be isolated in an acceptably pure form with simple aqueous workup. The two diastereomers could be separated by flash column chromatography on silica gel in the vast majority of cases. The reaction proceeded with moderate to excellent yields (Table 7). The rate of the reaction could be increased at elevated temperature and pressure (120°C, pwave, 2h); however, this necessitated the addition of 1 equiv. of Ε¾Ν to eliminate aldol addition products. Dichloromethane was found to be an ideal solvent as protic solvents did not yield the desired product and the isocyanide was not soluble in diethyl ether or tetrahydrofuran. Table 7. P3CRs involving σ-boryl isocyanides.
Figure imgf000056_0001
10
Figure imgf000056_0002
11 cyclohexyl 3-pyradinyl 10 be 2 42
12 phenyl benzyl 10ca 7 61
[a] The reactions were carried out using σ-boryl isocyanide (1.0 equiv.), aldehyde (1.0 equiv.) and phenylacetic acid (1.0 equiv.) in CH2CI2 at 23°C.
[b] Yields of isolated products after silica gel chromatography, [c] The two diastereomeric products could not be separated via silica gel chromatography.
We then attempted to remove MIDA from the P3CR product anf/'-IOad under standard aqueous basic conditions to afford the corresponding free boronic acid.[10c] We found that under the deprotection conditions, hydrolysis of the ester also occurred followed by condensation of the resulting free hydroxyl with the newly formed boronic acid yielding disubstituted 6-boromorpholinone 11ad as an air-stable white solid (Scheme 4). The relative stereochemistry of borocycle 11 ad was determined by computational modeling of the starting material. Computational predictions (MPW1 PW91 , 6-311G(2d, p)) of several 1H NMR chemical shift differences between the two diastereomers of P3CR product 10ad correlated well with experimental observations allowing an inference of the relative stereochemistry.
Figure imgf000057_0001
Scheme 4. Deprotection/Condensation of P3CR product 10ad.
The unique structure of boromorpholinone 11 ad led us to investigate its behaviour in aqueous solution. To determine the pKa of the boronate moiety, the 11B NMR spectrum was taken at various pHs in buffered aqueous methanol. [20] A sharp decrease in the signal at 19.3 ppm was observed at a pH of 9.0 accompanied by a sharp increase in a signal at 2.9 ppm. ESI mass spectra of these samples identified compound 11 ad as the primary component in the lower pH samples while pentadeuterated boronate 12ad was identified in the samples at higher pH. From these data we approximated the pKa of the boronate moiety as 9.0. This is an important feature of the boromorpholinone scaffold as it implies that under biological conditions, the compound will remain in its ring-closed form allowing its application as an electrophilic pharmacophore for covalent inhibition.
Covalent electrophilic inhibitors are designed to react with nucleophilic groups at an enzyme's active site resulting in covalent bonding and inhibition. A commonly encountered problem with this approach is competing reactivity of the inhibitor with water (a weak but often reactive nucleophile). In many cases, water attacks the electrophilic site of the inhibitor before it can reach its enzyme target resulting in hydrolysis and a loss of activity. Having established that the pKa of 11 ad is approximately 9.0, we can conclude that at pH < 8 (virtually all environments encountered in biological systems), the electrophilic center is impervious to attack by water. In other words, the BMN scaffold is hydrolytically stable below pH 8. This is an important feature of an electrophilic inhibitor as it suggests that the drug will be able to reach its target without hydrolytic deactivation. We have further investigated the hydrolytic stability of 11 ad using computational experiments. By applying literature protocols for computational modeling of the thermodynamic profiles of hydrolysis pathways,1221 we have established that the non- hydrolyzed form of 11 ad is more stable than any of the potential products resulting from hydrolysis (Figure 7). This further enforces the assertion that the BMN core is hydrolytically stable under biological conditions. We have also demonstrated computationally that kinetically reasonable transition states exist for both ring-opening and ring-closing processes, which supports that the claim that the process will likely be governed by thermodynamics. The likelihood that the boromorpholinone scaffold is impervious to hydrolysis under biological conditions suggests that it has the potential to permeate cell membranes because the polarity of the compound is minimized in its non-hydrolyzed form.
The ability of a potential drug candidate to permeate epithelial cells is a crucial factor for oral bioavailability. There is a strong correlation between epithelial cell permeability of a drug and its ability to be absorbed into the circulatory system following oral administration. Drugs exhibiting poor cell permeability are most often administered via intravenous (IV), subcutaneous (SC), or intramuscular (IM) injection. This limits their therapeutic applicability due to the inconvenience of imposing regular injections (and therefore regular clinic visits) on patients. For example, bortezomib, which despite its efficacy in the treatment of multiple myeloma must be administered by injection. Over the past several years, significant effort has been made toward the development of orally bioavailable bortezomib alternatives, several of which are now in various stages of clinical trials. We have demonstrated that 11 ad exhibits greatly improved epithelial cell permeability over bortezomib in CACO-2 assays (Figure 8, 11ad denoted "AZ-1").
Given the known propensity of boropeptide analogues to inhibit members of the 20S proteasome[3n, q] we decided to investigate the interaction of 6-boromorpholinone 11 ad with various classes of this protease family. We found that borocycle 11 ad inhibited the chymotrypsin-like members of the 20S proteasome with an IC50 of 19 nM (compared to bortezomib in the same assay; IC50 = 0.4 nM) (Figure 3). A similar correlation was observed for the caspase-like and trypsin-like members of the 20S proteasome (Figure 4 and Figure 5). Both 11 ad and bortezomib exhibited much weaker inhibition of the caspase-like enzymes (IC50s of 2.0 μΜ and 3.2 nM respectively) and no inhibition of the trypsin-like members. The syn-diastereomer of 11 ad was prepared from antMOad and exhibited inhibition of the chymotrypsin-like enzymes with an IC50 roughly 20 times greater than that of the anti-isomer (52 nM vs. 2.9 nM in the same assay) (Figure 6).
While the boromorpholinone inhibitors showed weaker inhibition than bortezomib, structural optimization has the potential to drastically improve these results. The pyrazine side chain in bortezomib does not exhibit a defined interaction with the active site binding pockets within the 20S proteasome causing a lack of selectivity and therefore a range of undesired side effects. [21] The multi-component nature of our boropeptide preparation methodology facilitates diversity-oriented synthesis by allowing addition of a second level of structural diversity in the same step as boron integration. This facilitates the preparation of diverse inhibitor libraries and therefore elucidation of a structure-activity relationship and optimization of a selective proteasome inhibitor. To facilitate these efforts, we have carried out computational modeling of 11ad in complex with the active site of chymotrypsin using the Glide software. This allows an understanding of the points of non-covalent bonding interactions between the inhibitor and the enzyme and therefore optimization of binding efficiency. These experiments have shown that the ring-opened BMN structure exhibits several favorable points of interaction with its enzyme target that the unopened scaffold does not (Figure 9). This suggests that the BMN ring remains closed until it reaches its target, at which point it undergoes ring-opening to efficiently occupy the active site binding pocket. This is an important conclusion as it suggests that certain binding elements can be incorporated into a BMN-containing drug candidate and that these elements will not be exposed until the target is reached.
We have demonstrated that altering the isobutyl and benzyl substituents of 11 ad drastically changes its behavior. We have also shown that derivatization of the N-H bond to N-alkyl substituents is possible. By adding sodium hydride and methyl iodide to a dry solution of 11ad in THF at 23°C, methylation of the nitrogen occurs after stirring overnight. The product of the reaction has been partially characterized by crude 1H NMR and high-resolution mass spectrometry. In addition to the established biological profile of the BMN scaffold, we have also discovered that their MIDA-protected precursors (and several closely related compounds) exhibit potent and selective inhibition of various serine and threonine hydrolases. Studies using mass spectrometry have indicated that MIDA bortezomib slowly hydrolyzes to the active free boronic acid after 30 minutes of incubation at 37°C in aqueous buffered solution. This is an example of prodrug behavior in which the active form of a drug is released after administration. The results are summarized in Figure 10 and demonstrate that MIDA removal and isolation of the resulting free boronic acids are not required for effective enzyme inhibition. This is an important result because a multitude of organoboronic acid derivatives are unstable toward isolation as free boronic acids. In these cases, synthetic chemists must resort to protecting group strategies to prevent decomposition during isolation and purification. The ability to employ protected compounds as enzyme inhibitors greatly expands the scope of organoboronic acids that can be employed in therapeutic settings.
In summary, we have demonstrated a straightforward deoxygenation of MIDA- protected cr-boryl isocyanates to yield the corresponding σ-boryl isocyanides as bench- stable solids. The isocyanide moiety participates in a number of functional group interconversions affording borylated tetrazoles, isothiocyanates and thioureas. Isocyanide 2a also participates in several U4CRs affording boropeptide derivatives including MIDA-bortezomib. As a component in P3CRs, the boryl isocyanides gave rise to boryl acyloxyamide derivatives. One-pot deprotection and condensation of the P3CR product 10ad gave access to a new class of boron-containing heterocycles (6- boromorpholinones). These air-stable solids exhibited inhibition of chymotrypsin-like members of the 20S proteasome with IC50s in the low nanomolar range. We have also shown that these compounds exhibit improved epithelial cell permeability and hydrolytic stability compared to other leading inhibitors in the same class. In addition to the boromorpholinone pharmacophore, we have also demonstrated that their MIDA boronate containing precursors and several similar compounds act as pro-drugs for their biologically active free-boronic acid counterparts. The step-economy and experimental simplicity with which these inhibitors can be obtained is likely to enable development of many new examples of boron-containing drug candidates.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein are incorporated by reference.
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Claims

CLAIMS:
1. A compound of Formula (2):
Figure imgf000066_0001
Formula (2) wherein R1, R2, R3, R4, R5 , R6 and R7 and are each independently H or an organic group.
2. The compound of claim 1 , wherein:
R1, R2, R3, R4, R5 , R6 and R7are each independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
3. The compound of claim 1 , wherein the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
4. The compound of claim 1 or 2, wherein R3, R4, R5 , R6 are H and R7 is CH3. 5. The compound of any one of claims 1-4, wherein R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl.
6. A compound of Formula (9):
Figure imgf000067_0001
Formula (9) wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, R1 and R12 are each independently H or an organic group.
7. The compound of claim 6, wherein:
R1, R2, R3, R4, R5 , R6 , R7, R9, R10 and R11 are each independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyi group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a
heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
8. The compound of claim 7, wherein the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
9. The compound of claim 6 or 7, wherein R3, R4, R5 , R6 are H and R7 is CH3.
10. The compound of any one of claims 6-9, wherein R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl.
11. The compound of any one of claims 6-10, wherein R9 and R10 are independently selected from the group consisting of H, benzyl and isopropyl.
12. The compound of any one of claims 6-11 , wherein R11 is H.
13. The compound of any one of claims 6-12, wherein R12 is independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyi group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group.
The compound of claim 13, wherein the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
15. The compound of any one of claims 6-12, wherein R12 is an amino acid or peptide.
16. The compound of claim 15, wherein R12 is selected from the group consisting of G, F, V, GG, FA, PLF and PGLF.
17. The compound of claim 15 or 16, wherein the amino acid or peptide comprises a protecting group.
18. A compound of Formula (11):
Figure imgf000068_0001
wherein R1, R2, R9, R10, R 4, and R15 are each independently H or an organic group and X is any Lewis basic ligand.
19. The compound of claim 18, wherein:
R1, R2, R9, R10, R14, and R15 are each independently selected from the group consisting of H, an alkyl group, a heteroalkyl group, a cycloalkyl group, an alkenyl group, a heteroalkenyl group, an alkynyl group, a heteroalkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, and an acyl group and X is any Lewis basic ligand.
20. The compound of claim 19, wherein the organic group is substituted with one or more halide, hydroxyl, alkoxyl, acyloxyl or acyl groups.
21. The compound of any one of claims 18-20, wherein R1 and R2 are independently selected from the group consisting of H, isobutyl, cyclohexyl and phenyl.
22. The compound of any one of claims 18-21 , wherein R and R are independently selected from the group consisting of H, benzyl, isopropyl, 4-F- phenyl, phenyl, 3-pyradinyl, 4-Me-phenyl, 2-Br-phenyl, isopropyl and 3- pyradinyl. 23. The compound of any one of claims 18-22, wherein R 4 is selected from H or methyl.
A process for preparing the compound of Formula (2):
Figure imgf000069_0001
Formula (2) comprising reducing the compound of Formula (1):
Figure imgf000069_0002
Formula (1) wherein R1, R2, R3, R4, R5 , R6 and R7 and are each independently H or an organic group.
The process of claim 24, wherein reducing the compound of Formula (1 ) is performed with at least one condition and/or reagents selected from HSiCI3 Et3N, CH2CI2, and 0-23°C.
A process for preparing the compound of Formula (9):
Figure imgf000070_0001
Formula (9) comprising subjecting to a Ugi 4-component reaction, the compound of Formula (2):
Figure imgf000070_0002
Formula (2)
R12- COOH; R11NH2; and R9R10-CO; wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, R11 and R12are each independently H or an organic group.
The process of claim 26, wherein the Ugi 4-component reaction is performed with at least one condition and/or reagent selected from TFE, 23°C and time. 28. A process for preparing the compound of Formula (11 ):
Figure imgf000071_0001
Formula (11) comprising ester hydrolysis and deprotection of the compound of Formula (10):
Figure imgf000071_0002
Formula (10) wherein R1, R2, R3, R4, R5 , R6 , R7, R9, R10, and R13and are each independently H or an organic group.
29. The process of claim 28, wherein the ester hydrolysis and deprotection of the compound of Formula (10) is performed with at least one condition and/or reagent selected NaOH, THF/H20, rt, and time (10 min.).
30. The process of claim 28 and 29, wherein the process further comprises performing a Passerini 3-component reaction with the compound of Formula (2), R9R10-CO, and an organic acid R13-COOH (preferably Ph-CH2-COOH), to obtain the compound of Formula (10).
The process of claim 30, wherein the Passerini 3-component reaction is performed with at least one condition and/or reagent selected from CH2CI2, and 23°C.
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