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Appendage- and Scaffold-Diverse Fully Functionalized Small-Molecule Probes Via a Minimalist Terminal Alkyne-Aliphatic Diazirine-Isocyanide Paul Jackson, and David J. Lapinsky J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01831 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018
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The Journal of Organic Chemistry
Appendage- and Scaffold-Diverse Fully Functionalized Small-Molecule Probes Via a Minimalist Terminal Alkyne-Aliphatic Diazirine-Isocyanide
Paul Jackson and David J. Lapinsky* Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, Pennsylvania 15282, United States
*CORRESPONDING AUTHOR E-MAIL ADDRESS:
[email protected] TABLE OF CONTENTS/ABSTRACT GRAPHIC:
ABSTRACT: This paper highlights a bifunctional isocyanide that contains a photoreactive aliphatic diazirine for protein target capture and a terminal alkyne for click chemistry-based proteomics. Specifically, this isocyanide was employed in five different multicomponent reactions to produce ten different fully functionalized small-molecule probes (FFSMPs) containing eight different chemical scaffolds.
We anticipate this
bifunctional isocyanide can be used to create FFSMP libraries of much greater chemical diversity towards identifying compounds with novel mechanisms of action via integrated phenotypic screening and target identification.
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INTRODUCTION Over the past six years, a research platform integrating phenotypic screening with advanced chemoproteomics has evolved to identify small-molecule “hits” that exhibit a desired phenotypic response in cells, and, in tandem via broad proteome exploration, the biomolecular targets and rapid mechanistic characterization of these compounds.1-4 Fundamental to this platform are compounds called “fully functionalized small-molecule probes” (FFSMPs, MW 300-500) (1, Figure 1A),4 which are smallmolecules capable of forming covalent bonds with their protein targets under the same assay conditions used for phenotypic screening, and whose rational chemical design allows for direct advancement from phenotypic screening to target identification without requiring any synthetic modification of a “hit” compound. In turn, FFSMPs are capable of uniting and dramatically accelerating the research steps of phenotypic screening and target identification from years to months, principally via reduced experimental effort.4
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photoreactive group diversity element
cells alkyne latent affinity handle
A.)
UV crosslink in situ labeling
1, FFSMPs B.) previous FFSMPs: O
O
O
O
N
R
R1
N
R2 N H
2, BzIndoles
O
3, BzBDs
R1
R3
R4
R2
O
R5 CN
4
5
6
NH
+
+
TMS-N3
R1
Ugi Tetrazole 4-CR
R3 R4 R5 N N N N N R2 7
FFSMP Library Members Containing an Aliphatic Diazirine and a Terminal Alkyne
C.) this work:
N N CN 8
five different isocyanide-based multicomponent reactions (IMCRs)
N N IMCR-derived diversity element 9, Appendage- and Scaffold-Diverse FFSMPs
Figure 1.
Fully functionalized small-molecule probes (FFSMPs) for integrated cell-
based screening and target identification.
(A) General chemical composition of a
FFSMP (1). (B) Previously reported FFSMPs 2, 3, and 7 containing a photoreactive functional group and a terminal alkyne click chemistry handle. (C) Appendage- and
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scaffold-diverse FFSMPs (9) via minimalist aliphatic diazirine-terminal alkyneisocyanide 8. In general, FFSMPs contain three structural entities (Figure 1A): 1.) a smallmolecule containing one or more structural diversity elements in order to promote interactions of the FFSMP with a variety of different protein targets in cells, 2.) a photoreactive functional group (e.g., a benzophenone, aryl azide, or a diazirine) for UV light-induced covalent bond formation between the FFSMP and its interacting proteins via photoaffinity labeling,5 and 3.) a terminal alkyne latent handle that can be used to enrich, visualize, and identify FFSMP-interacting proteins via click chemistry techniques.6 To date, FFSMPs have proven to be highly valuable compounds capable of capturing proteins that currently lack small-molecule ligands, and studies involving FFSMPs can divulge a diverse range of traditional or non-traditional druggable targets in cells.3 Furthermore, in addition to profiling normal and diseased samples for target discovery, and pharmacologically perturbating currently uncharacterized proteins, FFSMPs can be used in other chemoproteomic applications to enable drug development and discovery.7-8 In order to address today’s most challenging biomedical problems, such as resistance to existing drugs for infectious diseases and cancer,9-10 currently there is a critical need for organic small-molecules with novel mechanisms of action (nMoA),11-12 and linked to this need, specifically there has been a call for FFSMP libraries of much greater size, chemical diversity, and structural complexity to be used in phenotypic screening campaigns.13-14 For example, the development of synthetic strategies that would allow for efficient incorporation of different privileged structures15 (i.e., to direct
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the FFSMP to sample biologically-relevant chemical space) and Topliss optimization moieties16 (i.e., to optimize initially identified “hit” FFSMPs) would be expected to expand the chemical diversity and structural complexity of FFSMP libraries.
More
importantly, however, it is well-known that the overall structural, and thus functional diversity (i.e., the range of biological activities displayed by compounds) of a screening collection is highly dependent on the variety of chemical scaffolds present in the compound library.17-23
In particular, while there are known examples of synthetic
strategies to produce collections of structurally similar FFSMPs (i.e., compounds with varying appendages around a single core chemical scaffold),3-4 to our knowledge, synthetic strategies for the efficient generation of more structurally diverse FFSMPs, particularly in which there is a greater variation in the nature of chemical scaffolds present,17-25 have yet to be pursued. Currently, the only type of structural complexity/chemical diversity examined with FFSMPs has been appendage diversity (i.e., different peripheral structural entities at multiple sites around a single core chemical scaffold), and only three chemical scaffolds have been reported to date (Figure 1B), namely 5-benzoyl indoles (BzIndoles, 2) and 7benzoyl-benzo-1,4-diazepin-2,5-diones
(BzBDs,
3),4
both
of
which
contain
a
benzophenone photoreactive functional group, and aliphatic diazirine-containing 1,5disubsituted tetrazoles (7), which were synthesized via an Ugi-azide multicomponent reaction in a sparse matrix format using eight different amines (4; four different amine inputs contained a photoreactive aliphatic diazirine, one amine input contained a terminal alkyne, and two different amine inputs contained both an aliphatic diazirine and a
terminal
alkyne),
eighteen
different
aldehydes/ketones
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three
different
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aldehyde/ketone inputs contained a photoreactive aliphatic diazirine, and two different aldehyde/ketone inputs contained a terminal alkyne), and six different isocyanides (6; importantly in comparison to the work reported herein, none of the isocyanide inputs contained either a photoreactive aliphatic diazirine or a terminal alkyne).3 In short, each of these chemical scaffolds has modest structural complexity, biased chemical diversity, and is capable of sampling a relatively small portion of chemical space.26 Once again, the scaffold diversity of any compound screening set has a vital role in defining the overall 3-D molecular shape of the library, with peripheral substituents (i.e., appendage diversity) being considerably less important.17-18, 27 As a result of these limitations, the generation of FFSMPs with higher scaffold- and appendage-diversity is currently desired, principally under that expectation that such compounds would provide a higher “hit” rate against a broader range of biological targets by sampling a larger amount of chemical space. The latter of which is especially important, particularly when it comes to discovering compounds with nMoA and where the nature of the biological target is unknown as in disease-based phenotypic screening.17 As a way of beginning to meet the objective of FFSMPs with higher scaffold- and appendage-diversity to be used in streamlined phenotypic screening-target identification campaigns, we developed a minimalist isocyanide (8, Figure 1C) containing a terminal alkyne click chemistry handle and an aliphatic diazirine photoreactive functional group. In particular, this paper highlights the design and synthesis of bifunctional isocyanide 8, plus its general use in five different isocyanide-based multicomponent reactions (IMCRs) to produce ten different FFSMPs ((+)-9a - (+)-9j) containing eight different chemical scaffolds.
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RESULTS AND DISCUSSION With respect to the chemical composition of clickable photoprobes,28-29 one of the most commonly encountered structural motifs is a “minimalist” aliphatic diazirineterminal alkyne moiety originally developed by the Yao group,30 wherein the photoreactive group and latent affinity handle are made as small as possible, so as to minimize steric interference upon probe-binding to target proteins.
In particular for
Yao’s set of minimalist linkers (10 – 12, Scheme 1), a wide variety of pharmacologically active small-molecules can be readily armed without relatively drastic changes in compound structure and size, and at the same time, a chemically tractable entity is introduced towards capturing transient, non-covalent protein-ligand interactions in cells. In its traditional form, the coupling of small-molecules to Yao’s minimalist linkers normally involves highly robust, simple chemistries, such as heteroatom alkylation using iodide 10, or heteroatom acylation using primary amine 11 or carboxylic acid 12. As just one example of this strategy, the Cravatt group recently coupled carboxylic acid 12 to fourteen different amine small fragments (MW ~176) based on known drugs, which subsequently allowed them to establish target and ligand discovery via fragment-based screening in human cells.2
Alternatively, IMCRs represent a very useful and large
group of reactions in organic chemistry,31 and namely because IMCRs can provide easy and rapid access to small-molecules with significant scaffold diversity, it is no surprise such reactions are a preferred method towards discovering pharmacologically active
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compounds.32 In particular, an important strategy to increase the resourcefulness of isocyanides and to augment the collection of molecular scaffolds is to employ functionalized isocyanides, and the preparation of such compounds were recently promoted as a means of solving problems at the chemistry-biology interface.33 In this regard, given the photoreactive aliphatic diazirine and terminal alkyne functional groups survived an Ugi-azide multicomponent reaction as direct amine and carbonyl inputs to produce FFSMPs 7,3 and alternative to the trivial strategy of simply tagging smallmolecules with Yao’s minimalist linkers 10 – 12,30 we hypothesized whether an isocyanide variant of Yao’s minimalist linker (i.e., 8) could be synthesized and directly employed as an input in a number of different IMCRs. In turn, such a strategy could not only provide appendage- and scaffold-diverse FFSMPs (9), but also simultaneously expand the toolkit of functionalized isocyanides and contribute to the knowledge of chemistry one could perform in the presence of a photoreactive diazirine, which was recently reviewed with respect to synthesizing diazirine-containing photoprobes.34 In particular, even though diazirines have been successfully used as direct carboxylic acid or aldehyde inputs in Ugi four-component reactions and Passerini three-component reactions as well-known IMCRs,35-36 to our knowledge, only benzophenones have been reported as direct isocyanide inputs in those same IMCRs.36-37
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Scheme 1. Yao’s set of minimalist linkers (10 – 12) typically used in the synthesis of clickable photoprobes and synthesis of a minimalist aliphatic diazirine-terminal alkyneisocyanide variant (8).
To begin our work, we were pleased to find that bifunctional isocyanide 8 could be readily synthesized in good yield from Yao minimalist amine linker 11,30 wherein the synthesis of the isocyanide functional group was accomplished by traditional means,38 namely first converting the 1˚ amine in 11 to the corresponding formamide (13) followed by dehydration (Scheme 1). With isocyanide 8 in hand, we then choose five welldescribed IMCRs that are known to produce a significant number of bioactive compounds32 (i.e., the Passerini three-component reaction (P-3CR), the Ugi fourcomponent reaction (U-4CR), the Ugi tetrazole four-component reaction (UT-4CR), the Ugi five-center-four-component reaction (U-5C-4CR), and the Ugi β-lactam threecomponent reaction (UBL-3CR)) in order to test the synthetic value of this building block towards producing appendage- and scaffold-diverse FFSMPs (9) (Scheme 2). Specifically, and with our previously noted desire to rationally incorporate privileged structures15 and Topliss optimization moieties16 into our FFSMPs, these IMCR examples will now be discussed briefly: 1.) FFSMP (+)-9a stemmed from a P-3CR involving isocyanide 8, isoquinoline-1-carboxylic acid (14) as a privileged structure carboxylic acid input, and 4-chlorobenzaldehyde (15) as a Topliss optimization moiety aldehyde input, 2.) FFSMP (+)-9b stemmed from an U-4CR involving isocyanide 8, benzofuran-2-carboxylic acid (16) as a privileged structure carboxylic acid input, 4chlorobenzylamine (17) as a Topliss optimization moiety 1˚ amine input, and p-
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anisaldehyde (18) as a Topliss optimization moiety aldehyde input, 3.) FFSMP (+)-9c stemmed
from
an
UT-4CR
involving
TMS-N3,
isocyanide
8,
1,2,3,4-
tetrahydroisoquinoline (19) as a privileged structure 2˚ amine input, and 4chlorobenzaldehyde (15) as a Topliss optimization moiety aldehyde input, 4.) FFSMP ()-9d stemmed from an U-5C-4CR involving isocyanide 8, 4-methoxyphenethylamine (20) as a Topliss optimization moiety 1˚ amine input, L-valine as a Topliss optimization moiety amino acid input, and 1-benzylpiperidin-4-one (21) as a privileged structure ketone input, and 5.) strained β-lactam FFSMP (+)-9e stemmed from using β-alanine (22) as a β-amino acid input in an UBL-3CR involving isocyanide 8 and 1-methylindole3-carboxaldehyde (23) as a privileged structure aldehyde input.
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Scheme 2.
Application of bifunctional isocyanide 8 in five well-known IMCRs to
produce structurally diverse FFMPs (9).
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Confident bifunctional isocyanide 8 could be successfully employed in several well-established IMCRs, we next turned our attention toward increasing the scaffold diversity and structural complexity of FFSMPs by examining synthetic strategies that can be used in coordination with the U-4CR (Scheme 3). For example, FFSMP (+)-9f stemmed from a powerful unionization of MCR strategy,39 wherein Castagnoli-Cushman reaction derived carboxylic acid (+)-25,40 as a single diastereomer, was reacted in an U4CR involving isocyanide 8, tryptamine (26) as a privileged structure 1˚ amine input, and cyclopentanone as a ketone input. To our knowledge, this is the first reported example of a Castagnoli-Cushman reaction-derived carboxylic acid in a union of MCR strategy,39 thus expanding the concept of this potent synthetic approach towards appendage- and scaffold-diverse small-molecules. Additionally, it is well-known that U4CR adducts can be considered a productive synthetic hub towards a vast diversity of novel acyclic or cyclic scaffolds by employing any number of different secondary transformations.41 As examples of this concept, privileged structure 1,4-benzodiazepine FFSMP (+)-9h was synthesized by first reacting isocyanide 8 in an U-4CR involving Boc-Gly-OH as a carboxylic acid input, 2-aminoacetophenone (29) as an aniline input, and isobutyraldehyde (30) as a Topliss optimization moiety aldehyde input, followed by Boc-deprotection and cyclization under acidic conditions.42-43 Alternatively, privileged structure 2,5-diketopiperazine-based FFSMP (+)-9j was synthesized by first reacting isocyanide 8 in an U-4CR involving chloroacetic acid (31) as a carboxylic acid input, 4chlorobenzaldehyde (15) as a Topliss optimization moiety aldehyde input, and 4chlorobenzylamine (17) as a Topliss optimization moiety 1˚ amine input, followed by cyclization under basic conditions.44 Interestingly, one could speculate that electrophile-
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containing FFSMPs (+)-9e, (+)-9g, and (+)-9i could potentially serve as clickable dual affinity labeling-photoaffinity labeling probes that could potentially form two covalent bonds within a protein towards aiding in binding-site mapping, as has been previously described with respect to the 20S proteasome45 and fatty acid amide hydrolase.46
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Scheme 3. Application of bifunctional isocyanide 8 in synthetic strategies that can be used in coordination with the U-4CR to produce structurally diverse FFMPs (9).
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In conclusion, the most important outcome of the work briefly described herein is a synthetically valuable bifunctional isocyanide that appears initially capable of answering the call for FFSMP libraries of much greater size, chemical diversity (i.e., both appendage- and scaffold-diversity), and structural complexity to be used in integrated phenotypic screening and target identification campaigns. Once again, the motivation for pursuing such libraries is to meet the current demand for organic smallmolecules with nMoA, while simultaneously providing tool compounds that could be utilized in chemoproteomic-enabled drug discovery and development efforts. In short, given the large commercial availability of carboxylic acids, 1˚ amines, ketone, and aldehyde components as common IMCR inputs (i.e., compared to the limited commercial availability of isocyanides), plus the fact that IMCRs can be easily automated one-pot chemical reactions that are ideally suited for combinatorial chemistry and high-speed parallel synthesis, namely with the ability to build in desired chemical properties and probe structure-activity relationships via the creation of libraries,47 we believe we have only scratched the surface of applying clickable photoreactive isocyanides in IMCRs towards generating appendage- and scaffold-diverse FFSMPs. In the future, we will continue to report our development and application of such bifunctional isocyanides towards producing structurally diverse FFSMPs, as well as results stemming from phenotypic screening and target identifications studies involving such compounds.
EXPERIMENTAL SECTION
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General.
Reaction conditions and yields were not optimized.
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All reactions were
performed using flame-dried glassware under an inert atmosphere of argon unless otherwise noted. All solvents and chemicals were purchased from Aldrich Chemical Co. or Fisher Scientific and used without further purification unless otherwise noted. Flash column chromatography was performed using Fisher S826-25 silica gel sorbent (70-230 mesh) and eluting solvent mixtures as specified. Thin-layer chromatography (TLC) was performed using TLC Silica Gel 60 F254 plates obtained from EMD Chemicals, Inc. and compounds were visualized under UV light and/or I2 staining. Proportions of solvents used for TLC are by volume. All temperatures reported are oil bath temperatures. and
1
H
13
C NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical
shifts for 1H and
13
C NMR spectra are reported as parts per million (δ ppm) relative to
tetramethylsilane (0.00 ppm) as an internal standard or residual solvent (CHCl3 = 7.26 ppm 1H NMR, 77.16 ppm
13
C NMR). Coupling constants are measured in hertz (Hz).
HRMS samples were analyzed at Old Dominion University (Norfolk, VA) by positive ion electrospray on a Bruker 12 Tesla APEX-Qe FTICR-MS with an Apollo II ion source. Melting points were not obtained for sticky solids, which confounded capillary tube sample preparation for appropriate analysis. On the basis of 1H and
13
C NMR (see
Supporting Information for copies of spectra), all reported compounds herein were >95% pure.
3-(But-3-yn-1-yl)-3-(2-isocyanoethyl)-3H-diazirine (8).
A flame-dried, 50 mL round-
bottom flask equipped with a stir bar, septum, and argon inlet needle was charged with formamide 13 (394 mg, 2.38 mmol, 1 equiv) and CH2Cl2 (16 mL, 0.15 M). This solution
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was cooled to 0°C with an ice-water bath, then Et3N (0.83 mL, 6.0 mmol, 2.5 equiv) was added in a single portion. Next, POCl3 (0.27 mL, 2.9 mmol, 1.2 equiv) was added dropwise over 1 minute. After the addition of POCl3, the ice bath was removed and the reaction mixture was allowed to warm to room temperature with stirring for 1 hour. Next, the reaction mixture was cooled back to 0°C with an ice-water bath, then quenched by addition of 10% (w/v) aq. Na2CO3 solution (16 mL). After the addition, the ice bath was removed and the biphasic mixture was stirred at room temperature for 1 hour. Next, the mixture was transferred to a separatory funnel and diluted with CH2Cl2 (20 mL) and deionized H2O (10 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 x 20 mL). The organic layers were combined, washed with brine, dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (gradient elution with 10-15% EtOAc/hexanes) to provide isocyanide 8 (297 mg, 85%) as a colorless oil. Rf = 0.43 (25% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 3.20 (tt, J = 1.5, 7.0 Hz, 2H), 1.991.95 (m, 4H), 1.80 (tt, J = 2.0, 7.0 Hz, 1H), 1.66-1.62 (m, 2H);
13
C NMR (126 MHz,
CDCl3) δ 157.6, 82.2, 69.5, 36.1, 32.9, 31.7, 26.0, 13.0; HRMS (ESI+) m/z [2M + Na] calcd for (C8H9N3)2Na 317.1485, found 317.1485.
(+)-2-((2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)amino)-1-(4-chlorophenyl)-2-oxoethyl isoquinoline-1-carboxylate ((+)-9a). A 2-dram vial equipped with a magnetic stir bar was charged with isocyanide 8 (53 mg, 0.35 mmol, 1 equiv) and CH2Cl2 (1 mL, 0.35 M). Next, 4-chlorobenzaldehyde (55 mg, 0.39 mmol, 1.1 equiv) was added, followed by the addition of isoquinoline 1-carboxylic acid (80 mg, 0.46 mmol, 1.3 equiv). The vial was
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capped and stirred at room temperature for 7 days. The reaction mixture was then diluted with EtOAc (50 mL) and transferred to a separatory funnel. The organic layer was washed sequentially with saturated aq. NaHCO3 solution (10 mL), deionized H2O (10 mL), and brine (10 mL), then dried over MgSO4, gravity filtered, and concentrated under reduced pressure. The crude material was then purified by silica gel flash column chromatography (gradient elution with 40-50% EtOAc/hexanes) to provide ester (+)-9a (55 mg, 34%) as a white, sticky solid. Rf = 0.22 (50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3, *other rotamer where distinguishable) δ 8.71 (dd, J = 0.5, 8.5 Hz, 1H)*, 8.64 (d, J = 5.5 Hz, 1H), 8.55 (d, J = 5.5 Hz, 1H)*, 8.49 (d, J = 5.5 Hz, 1H), 8.13 (t, J = 7.5 Hz, 1H), 7.88-7.86 (m, 2H), 7.77-7.67 (m, 3H), 7.57-7.55 (m, 1H)*, 7.48-7.46 (m, 1H), 7.43-7.42 (m, 1H), 7.37-7.35 (m, 1H)*, 7.23-7.21 (m, 1H)*, 7.16-7.14 (m, 1H), 6.46 (s, 1H)*, 6.26 (s, 1H), 5.96 (s, 1H), 5.38 (s, 1H)*, 3.64-3.62 (m, 1H), 3.54-3.52 (m, 1H)*, 3.25-3.08 (m, 2H), 2.02-1.93 (m, 2H), 1.74-1.51 (m, 5H);
13
C NMR (126 MHz, CDCl3) δ
169.7*, 169.0, 168.6, 168.2*, 163.9, 154.6*, 154.2, 147.7*, 141.6*, 141.5, 140.7, 137.0, 135.4*, 131.2*, 131.1, 130.2*, 129.6, 129.1*, 129.0 (2C), 129.0, 128.8*, 127.3, 126.1, 125.0, 122.7, 82.8, 82.7*, 82.2, 75.5*, 69.6, 69.3*, 63.5*, 62.4, 42.8, 40.1*, 34.7, 33.5*, 32.6, 32.2, 31.6, 26.8*, 25.8, 13.3*; HRMS (ESI+) m/z [M + Na] calcd for C25H21ClN4O3Na 483.1194, found 483.1200.
N-(2-((2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)amino)-1-(4-methoxyphenyl)-2oxoethyl)-N-(4-chlorobenzyl)benzofuran-2-carboxamide
((+)-9b).
A
2-dram
vial
equipped with a stir bar was charged with p-anisaldehyde (0.045 mL, 0.37 mmol, 1.1 equiv) and MeOH (0.1 mL). Next, 4-chlorobenzylamine (0.05 mL, 0.37 mmol, 1.1 equiv)
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was added, then this mixture was stirred at room temperature for 30 minutes. Next, benzofuran-2-carboxylic acid (60 mg, 0.37 mmol, 1.1 equiv) was added in a single portion, followed by addition of isocyanide 8 (50 mg, 0.34 mmol, 1 equiv) as a solution in MeOH (1 mL, 0.3 M). The vial was capped and the reaction mixture was stirred at room temperature for 24 hours. TLC at this point in time indicated consumption of the isocyanide starting material, so the reaction mixture was transferred to a separatory funnel and diluted with EtOAc (50 mL). The organic layer was washed sequentially with saturated aqueous NaHCO3 solution (10 mL), 1M aq. HCl solution (10 mL), and brine (10 mL), then dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (gradient elution with 25-50% EtOAc/hexanes) to give U-4CR product (+)-9b (81 mg, 42%) as a white sticky solid. Rf = 0.29 (35% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.59-7.58 (m, 1H), 7.36-7.17 (m, 6H), 6.79 (d, J = 8.0 Hz, 2H), 6.04 (t, J = 5.5 Hz, 1H), 5.44 (m, 1H), 5.16 (m, 1H), 4.63-4.60 (m, 1H), 3.76 (s, 3H), 3.18-3.06 (m, 2H), 1.96-1.87 (m, 3H), 1.65-1.59 (m, 4H);
13
C NMR (126 MHz, CDCl3) δ 169.4, 162.1, 160.1, 154.8, 148.2, 136.1, 132.8,
131.4, 128.4 (2C), 127.0, 126.9 (2C), 125.9, 123.8, 122.5, 114.3 (2C), 112.9, 112.0 (2C), 82.8, 69.4, 64.6, 55.4, 51.4, 34.7, 32.5, 32.1, 26.8, 13.2; HRMS (ESI+) m/z [M + Na] calcd for C32H29ClN4O4Na 591.1769, found 591.1764.
2-((1-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-1H-tetrazol-5-yl)(4chlorophenyl)methyl)-1,2,3,4-tetrahydroisoquinoline ((+)-9c).
A 2-dram vial equipped
with a stir bar was charged with 4-chlorobenzaldehyde (54 mg, 0.39 mmol, 1.05 equiv), 1,2,3,4-tetrahydroisoquinoline (0.05 mL, 0.39 mmol, 1.05 equiv), and MeOH (0.5 mL).
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The vial was capped and stirred at room temperature for 15 minutes, then isocyanide 8 (54 mg, 0.37 mmol, 1 equiv) dissolved in MeOH (0.5 mL) was added to the reaction vial. Next, TMS-N3 (0.052 mL, 0.39 mmol, 1.05 equiv) was added dropwise, then the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was then concentrated under reduced pressure and purified by silica gel flash column chromatography (gradient elution with 10-50% EtOAc/hexanes) to provide tetrazole (+)9c (90 mg, 55%) as a colorless oil. Rf = 0.28 (25% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.46-7.44 (m, 2H), 7.39-7.37 (m, 2H), 7.16-7.09 (m, 3H), 6.92-6.91 (m, 1H), 5.23 (s, 1H), 4.26-4.22 (m, 2H), 3.61 (ABq, J = 14.5 Hz, 2H), 2.93-2.87 (m, 3H), 2.68-2.62 (m, 1H), 2.08-2.02 (m, 1H), 1.94 (t, J = 2.5 Hz, 1H), 1.92-1.89 (m, 2H),1.831.77 (m, 1H), 1.52-1.49 (m, 2H);
13
C NMR (126 MHz, CDCl3) δ 153.9, 134.9, 133.7,
133.5, 132.7, 130.3 (2C), 129.2 (2C), 128.8, 126.73, 126.68, 126.0, 82.3, 69.8, 63.8, 53.7, 48.4, 42.6, 32.9, 32.0, 29.0, 26.0, 13.2; HRMS (ESI+) m/z [M + Na] calcd for C24H24ClN7Na 468.1674, found 468.1670.
(S)-1-Benzyl-N-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-4-((1-((4methoxyphenethyl)amino)-3-methyl-1-oxobutan-2-yl)amino)piperidine-4-carboxamide (()-9d)).
A 10 mL, round-bottom flask equipped with a stir bar was charged with
isocyanide 8 (58 mg, 0.39 mmol, 1 equiv) and MeOH (3 mL, 0.1 M). Next, L-valine (46 mg, 0.39 mmol, 1 equiv) was added in a single portion, followed by the addition of deionized H2O (1 mL, 0.1 M, 4:1 MeOH:H2O). Next, 1-benzyl-4-piperidone (0.07 mL, 0.4 mmol, 1 equiv) was added in a single portion, followed by the addition of 4methoxyphenethylamine (0.06 mL, 0.4 mmol, 1 equiv). The vial was capped with a
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septum and stirred at room temperature for 67 hours, at which point the reaction mixture was concentrated under reduced pressure, then dissolved in CH2Cl2 (50 mL), dried over MgSO4, gravity filtered, and concentrated under reduced pressure. The crude residue obtained was purified by silica gel flash column chromatography (gradient elution with 15% MeOH/ CH2Cl2) to provide U-5C-4CR product (-)-9d (110 mg, 48%) as a colorless oil. Rf = 0.53 (10% MeOH/CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 7.28-7.27 (m, 4H), 7.24-7.21 (m, 1H), 7.10 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 6.57 (t, J = 5.0 Hz, 1H), 3.75 (s, 3H), 3.46-3.42 (m, 4H), 3.10-2.98 (m, 2H), 2.75-2.70 (m, 3H), 2.49 (m, 2H), 2.37 (t, J = 8.5 Hz, 1H), 2.25 (m, 1H), 2.02-1.95 (m, 6H), 1.84-1.77 (m, 1H), 1.68-1.56 (m, 6H), 0.84 (t, J = 7.0 Hz, 6H);
13
C NMR (126 MHz, CDCl3) δ 175.0, 174.4, 158.3,
138.5, 130.7, 129.7 (2C), 129.1 (2C), 128.3 (2C), 127.0, 114.1 (2C), 82.7, 69.6, 63.0, 62.5, 60.1, 55.3, 50.1, 49.9, 40.5, 35.2, 34.9, 34.5, 32.8, 32.6, 32.0, 31.8, 26.9, 19.1, 18.9, 13.3; HRMS (ESI+) m/z [M + H] calcd for C34H46N6O3H 587.3704, found: 587.3706. [α]23D = -0.22 (c = 10, CHCl3).
N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-2-(1-methyl-1H-indol-3-yl)-2-(2oxoazetidin-1-yl)acetamide ((+)-9e). A 2-dram vial equipped with a stir bar was charged with β-alanine (37 mg, 0.41 mmol, 1.05 equiv), 1-methylindole-3-carboxaldehyde (65 mg, 0.41 mmol, 1.05 equiv), and MeOH (0.5 mL, 0.4 M). This mixture was stirred for 30 minutes at room temperature, then a 0.5 mL MeOH solution of isocyanide 8 (57 mg, 0.39 mmol, 1 equiv) was added in a single portion. The reaction mixture was stirred for 64 hours at room temperature, then diluted with EtOAc (50 mL) and transferred to a separatory funnel.
The organic layer was washed sequentially with saturated aq.
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NaHCO3 solution (10 mL) and deionized H2O (10 mL).
Page 22 of 35
The aqueous layers were
combined and extracted with EtOAc (2 x 15 ml), then the organic layers were combined, washed with brine (10 mL), dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (elution with 50% EtOAc/hexanes, then 100% EtOAc) to provide β-lactam (+)-9e (23 mg, 16%) as a colorless oil. Rf = 0.10 (50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.63-7.61 (m, 1H), 7.37-7.35 (m, 1H), 7.31-7.28 (m, 1H), 7.26 (m, 1H), 7.18-7.15 (m, 1H), 6.39 (t, J = 5.5 Hz, 1H), 5.74 (s, 1H), 3.82 (s, 3H), 3.59-3.57 (m, 1H), 3.20-3.12 (m, 2H), 3.093.06 (m, 1H), 3.03-2.98 (m, 1H), 2.84-2.80 (m, 1H), 1.98-1.93 (m, 3H), 1.67 (dt, J = 1.5, 7.0 Hz, 2H), 1.59 (dt, J = 1.5, 7.0, 2H);
13
C NMR (126 MHz, CDCl3) δ 169.2, 168.1,
137.1, 129.2, 126.6, 122.5, 120.2, 118.9, 109.8, 107.5, 82.8, 69.5, 52.2, 38.8, 36.3, 34.7, 33.1, 32.6, 32.0, 26.8, 13.2; HRMS (ESI+) m/z [M + Na] calcd for C21H23N5O2Na 400.1744, found 400.1747.
(3S*,4S*)-N-(1-((2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)carbamoyl)cyclopentyl)-2cyclopropyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-(4-methoxyphenyl)-1-oxo-1,2,3,4tetrahydroisoquinoline-4-carboxamide ((+)-9f). according to the literature.40
Carboxylic acid (+)-25 was prepared
A 2-dram vial equipped with a magnetic stir bar was
charged with 5-methoxytryptamine (44 mg, 0.23 mmol, 1 equiv), cyclopentanone (0.02 mL, 0.23 mmol, 1 equiv), and MeOH (0.3 mL). This mixture was stirred for 30 minutes at room temperature, then carboxylic acid (+)-25 (78 mg, 0.23 mmol, 1 equiv) was added in a single portion, followed by the addition of isocyanide 8 (34 mg, 0.23 mmol, 1 equiv) dissolved in MeOH (0.7 mL, 0.2 M). The reaction mixture was stirred at room
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temperature for 3 days, then concentrated under reduced pressure. The crude residue was dissolved in EtOAc (50 mL) and washed sequentially with saturated aqueous NaHCO3 solution (15 mL), deionized H2O (15 mL), and brine (15 mL), dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (eluent = 75% EtOAc/hexanes) to provide (+)-9f (74 mg, 44%) as an off-white, sticky solid. Rf = 0.26 (75% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 1.5 Hz, 1H), 8.16 (dd, J = 7.5, 1.5 Hz, 1H), 7.34-7.27 (m, 4H), 6.99-6.91 (m, 4H), 6.86-6.74 (m, 2H), 6.72-6.66 (m, 3H), 4.86 (d, J = 2.5 Hz, 1H), 4.26 (d, J = 2.5 Hz, 1H), 3.91 (t, J = 7.5 Hz, 2H), 3.71 (s, 3H), 3.62 (s, 3H), 3.18-3.14 (m, 2H), 3.09-2.97 (m, 2H), 2.68-2.62 (m, 3H), 1.94-1.88 (m, 6H), 1.74 (m, 4H), 1.57-1.52 (m, 4H), 0.96-0.92 (m, 1H), 0.81-0.76 (m, 1H), 0.65-0.62 (m, 1H), 0.61-0.56 (m, 1H); 13C NMR (126 MHz, CDCl3) δ 174.2, 173.0, 165.8, 159.2, 154.3, 133.9, 132.0, 131.8, 131.6, 131.0, 128.1, 127.5 (2C), 127.3, 127.1, 123.3, 114.2 (2C), 112.7, 112.5, 111.4, 99.9, 82.8, 73.5, 69.5, 63.3, 55.7, 55.3, 49.5, 48.1, 36.6, 36.0, 34.8, 32.3, 32.0, 30.2, 27.6, 27.0, 23.1, 22.9, 14.3, 13.3, 8.8, 7.0; HRMS (ESI+) m/z [M + Na] calcd for C44H48N6O5Na 763.3578, found 763.3572.
tert-Butyl
(2-((2-acetylphenyl)(1-((2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)amino)-3-
methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamate ((+)-9g). A 2-dram vial equipped with a magnetic stir bar was charged with 2-aminoacetophenone (0.053 mL, 0.44 mmol, 1.1 equiv), isobutyraldehyde (0.03 mL, 0.44 mmol, 1.1 equiv), and MeOH (0.5 mL). The vial was capped and stirred at room temperature for 45 minutes. Next, N-Boc glycine (77 mg, 0.44 mmol, 1.1 equiv) was added to the solution of the pre-formed imine.
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Isocyanide 8 (59 mg, 0.4 mmol, 1 equiv) was then dissolved in 0.5 mL of MeOH and added to the reaction vial. The mixture was stirred at room temperature for 45 hours, then diluted with EtOAc (50 mL) and transferred to a separatory funnel. The organic layer was washed sequentially with saturated aq. NaHCO3 solution (15 mL), 1M aq. HCl solution (15 mL), and brine (15 mL), then dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (gradient elution with 25-50% EtOAc/hexanes) to provide U-4CR adduct (+)-9g (169 mg, 83%) as a pale-yellow oil. Rf = 0.51 (50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3; *minor rotamer where distinguishable) δ 7.76-7.70 (m, 1H), 7.57-7.47 (m, 2H), 7.35-7.30 (m, 1H), 6.57 (t, J = 5.5 Hz, 1H), 5.51 (bs, 1H)*, 5.40 (bs, 1H)*, 4.77 (d, J = 11.0 Hz, 1H), 3.72-3.63 (m, 1H), 3.49 (dt, J = 3.5, 18.0 Hz, 1H), 3.173.03 (m, 2H), 2.98-2.93 (m, 1H)*, 2.89-2.83 (m, 1H), 2.55 (s, 3H)*, 2.54 (s, 3H), 2.072.00 (m, 3H), 1.72 (t, J = 7.5 Hz, 2H), 1.69-1.62 (m, 2H), 1.41 (s, 9H), 1.40 (s, 9H)*, 1.11-1.09 (m, 3H), 0.90-0.87 (m, 3H); 13C NMR (126 MHz, CDCl3; *minor rotamer where distinguishable) δ 199.6*, 198.8, 171.5*, 171.3, 170.7*, 168.5, 155.7, 140.2*, 137.7, 136.6, 135.0*, 133.6*, 132.5, 131.4, 130.2*, 130.1*, 130.0, 129.4*, 129.2, 82.8, 82.7*, 79.5*, 79.3, 78.6, 77.4*, 69.5*, 69.3, 64.2, 44.3*, 44.1, 34.3*, 34.0, 32.4*, 32.3, 32.14, 32.06*, 29.2*, 29.0, 28.33 (3C), 28.30 (3C)*, 27.7, 26.9*, 26.8*, 26.7, 20.6*, 20.0, 19.6*, 19.0; HRMS (ESI+) m/z [M + Na] calcd for C27H37N5O5Na 534.2687, found: 534.2691.
N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-3-methyl-2-(5-methyl-2-oxo-2,3-dihydro1H-benzo[e][1,4]diazepin-1-yl)butanamide ((+)-9h). A stir bar was added to a 20 mL scintillation vial containing carbamate (+)-9g (161 mg, 0.32 mmol, 1 equiv), then 1,2-
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dichloroethane (2.1 mL, 0.15 M) was added to the reaction vial followed by dropwise addition of CF3CO2H (0.24 mL, 3.2 mmol, 10 equiv). The vial was then capped and heated to 40°C in a prewarmed oil bath with a digital temperature controller and stirred for 17 hours. Next, the reaction mixture was cooled to room temperature and quenched by dropwise addition of saturated aq. NaHCO3 solution (5 mL). 1M aq. NaOH solution (2 mL) was then added to bring the pH up to 8-9 (pH paper). The biphasic mixture was then transferred to a separatory funnel and diluted with CH2Cl2 (10 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (2 x 10 mL). The organic layers were combined, washed with brine, dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (eluent = 60% EtOAc/hexanes) to provide benzodiazepine (+)-9h (115 mg, 93%) as a white sticky solid. Rf = 0.31 (75% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3; *other rotamer where distinguishable) δ 8.13 (t, J = 5.5 Hz, 1H)*, 7.96-7.94 (m, 1H), 7.58-7.56 (m, 1H)*, 7.48-7.39 (m, 1H), 7.27-7.23 (m, 1H), 7.09 (t, J = 5.5 Hz, 1H), 4.51-4.48 (m, 2H), 4.39 (d, J = 11.0 Hz, 1H)*, 3.66-3.61 (m, 1H), 3.52 (d, J = 11.0 Hz, 1H)*, 3.28-3.08 (m, 2H), 2.98-2.91 (m, 1H)*, 2.42 (d, J = 1.5 Hz, 3H)*, 2.41 (d, J = 1.5 Hz, 3H), 2.14-2.09 (m, 1H), 1.95-1.89 (m, 3H), 1.69-1.51 (m, 4H), 0.91 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H)*, 0.68 (d, J = 6.5 Hz, 3H)*, 0.37 (d, J = 6.5 Hz, 3H);
13
C
NMR (126 MHz, CDCl3; *minor rotamer where distinguishable) δ 172.9, 172.1*, 171.6*, 170.8, 170.31, 170.27*, 143.8, 138.4*, 132.1, 131.7*, 130.6, 130.3*, 127.1, 126.8*, 125.8*, 125.7, 124.2, 123.8*, 82.6, 78.4*, 69.40, 69.36*, 64.3, 57.5*, 56.5, 34.52*, 34.49, 32.7, 32.6*, 32.0*, 31.9, 27.5*, 26.8*, 26.7, 25.4, 25.2, 25.1*, 20.2, 19.8*, 19.3*,
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18.3, 13.3*, 13.2; HRMS (ESI+) m/z [M + Na] calcd for C22H27N5O2Na 416.2057, found 416.2058.
N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-2-(2-chloro-N-(4-chlorobenzyl)acetamido)2-(4-chlorophenyl)acetamide ((+)-9i). charged
with
4-chlorobenzaldehyde
A 2-dram vial equipped with a stir bar was (63
mg,
0.45
mmol,
1.05
equiv),
4-
chlorobenzylamine (0.055 mL, 0.45 mmol, 1.05 equiv), and MeOH (0.5 mL). The vial was capped and the reaction mixture was stirred at room temperature for 15 minutes. Next, isocyanide 8 (63 mg, 0.43 mmol, 1 equiv) was dissolved in MeOH (0.5 mL) and transferred to the reaction vial. Chloroacetic acid (43 mg, 0.45 mmol, 1.05 equiv) was then added, the vial was capped, and the reaction mixture stirred at room temperature for 3 days.
The reaction mixture was then concentrated under reduced pressure,
dissolved in EtOAc (50 mL), and transferred to a separatory funnel. The organic layer was washed sequentially with saturated aq. NaHCO3 solution (10 mL), DI H2O (10 mL), 1M aq. HCl solution (10 mL), DI H2O (10 mL), and brine (10 mL), then dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (gradient elution with 30-50% EtOAc/hexanes) to give U4CR adduct (+)-9i (132 mg, 61%) as a white, sticky solid.
Rf = 0.55 (50%
EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.29-7.28 (m, 4H), 7.23-7.21 (m, 2H), 7.02-7.00 (m, 2H), 6.29 (bs, 1H), 5.81 (s, 1H), 4.66 (ABq, J = 17.5 Hz, 2H), 4.02 (ABq, J = 12.5 Hz, 2H), 3.12-3.02 (m, 2H), 1.98-1.95 (m, 3H), 1.66-1.58 (m, 4H);
13
C NMR (126
MHz, CDCl3) δ 168.6, 168.3, 135.2 (2C), 134.9, 133.6, 132.6, 130.9 (2C), 129.3 (2C),
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129.0 (2C), 127.6, 82.7, 69.5, 62.7, 49.7, 42.0, 34.7, 32.3, 32.0, 26.8, 13.2; HRMS (ESI+) m/z [M + Na] calcd for C24H23Cl3N4O2Na 527.0779, found: 527.0776.
1-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-4-(4-chlorobenzyl)-3-(4chlorophenyl)piperazine-2,5-dione ((+)-9j). A 10 mL round-bottom flask was charged with α-chloroamide (+)-9i (132 mg, 0.26 mmol, 1 equiv), EtOH (2 mL, 0.15 M), and KOH (16 mg, 0.29 mmol, 1.1 equiv).
This mixture was sonicated for 2 hours at room
temperature, then transferred to a separatory funnel, diluted with EtOAc (50 mL) and deionized H2O (10 mL). The layers were separated and the organic layer was washed with brine (10 mL), dried over MgSO4, gravity filtered, concentrated under reduced pressure, and purified by silica gel flash column chromatography (eluent = 50% EtOAc/hexanes) to provide piperazine (+)-9j (63 mg, 52%) as a white sticky solid. Rf = 0.44 (50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.40-7.38 (m, 2H), 7.31-7.30 (m, 2H), 7.27-7.25 (m, 2H), 7.15-7.14 (m, 2H), 5.45 (d, J = 14.5 Hz, 1H), 4.87 (s, 1H), 4.23 (ABq, J = 17.5 Hz, 1H), 3.97 (d, J = 17.5 Hz), 3.57 (d, J = 15.0 Hz, 1H), 3.44-3.38 (m, 1H), 3.23-3.17 (m, 1H), 1.98 (t, J = 2.5 Hz, 1H), 1.91 (dt, J = 7.0, 2.5 Hz, 2H), 1.671.62 (m, 1H), 1.60-1.47 (m, 3H);
13
C NMR (126 MHz, CDCl3) δ 164.3 135.2, 134.4,
133.4, 133.2, 130.0 (2C), 129.7 (2C), 129.3 (2C), 128.5, 127.9 (2C), 82.5, 69.6, 62.6, 49.9, 46.9, 42.0, 31.5, 30.4, 26.4; HRMS (ESI+) m/z [M + Na] calcd for C24H22Cl2N4O2Na 491.1012, found: 491.1008.
N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)formamide (13).
A 10 mL, flame-dried
round-bottom flask equipped with a stir bar and septum was charged with primary
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amine 1130 (338 mg, 2.46 mmol, 1 equiv) and ethyl formate (2.0 mL, 24.6 mmol, 10 equiv). Et3N (0.5 mL, 2.95 mmol, 1.2 equiv) was then added all at once and the flask (capped with a septum) was lowered into a pre-warmed oil bath with a digital temperature controller set to 53°C (note: a reflux condenser would be appropriate for larger scale reactions). The reaction mixture was stirred at 53°C for 23 hours then diluted with CH2Cl2 (1 mL) and purified by silica gel flash column chromatography (gradient elution with 50-75% EtOAc/hexanes) to provide formamide 13 (394 mg, 97%) as a pale-yellow oil. Rf = 0.27 (75% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.96 (s, 1H), 6.97 (bs, 1H), 2.98-2.94 (m, 2H), 1.92-1.91 (m, 1H), 1.87-1.83 (m, 2H), 1.561.49 (m, 4H);
13
C NMR (126 MHz, CDCl3) δ 161.6, 82.4, 69.4, 32.7, 32.1, 31.7, 26.6,
12.9; HRMS (ESI+) m/z [M + Na] calcd for C8H11N3ONa 188.0794, found: 188.0795.
SUPPORTING INFORMATION: 1H and
13
C NMR spectra for novel compounds can be
found in the Supporting Information available free of charge on the ACS Publications website at DOI: XXXX.
ACKNOWLEDGEMENTS: This work was financially supported by a Pennsylvania Commonwealth Universal Research Enhancement (C.U.R.E.) grant, a Hunkele Dreaded Disease Award, The Charles Henry Leach II Fund, and the Duquesne University School of Pharmacy. We also thank Punit Seth and Stephen Bergmeier for discussions regarding this manuscript.
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