Letter pubs.acs.org/OrgLett
Synthesis of Diverse N‑Acryloyl Azetidines and Evaluation of Their Enhanced Thiol Reactivities Maximilian D. Palkowitz, Bo Tan, Haitao Hu, Kenneth Roth, and Renato A. Bauer* Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States S Supporting Information *
ABSTRACT: Acyl azetidines exhibit nonplanar hybridization, leading to lower amide-like character of the corresponding (O)C−N bonds. This impacts N-acryloyl azetidines by producing enhanced electrophilicy at appended Michael acceptors. Herein, reactivity data are reported in the presence of glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C. Wide reactivity ranges are observed by varying substitution at the Michael acceptor or by modulating the electron-withdrawing character of substituents at the C3 position of the azetidine.
A
zetidines serve as structural motifs in natural products,1 as scaffolds in diversity-oriented synthesis,2 and as synthetic intermediates that can be reacted with nucleophiles in ringopening reactions.3 They are also widely used in medicinal chemistry and can be found in approved drugs.4 Azetidines have recently emerged as scaffolds to present Michael acceptors to nucleophilic residues in proteins associated with disease (e.g., ARS-853, Figure 1a).5 The most commonly used Michael acceptor in covalent drug discovery at the present time is the acrylamide (e.g., ibrutinib, Figure 1a).6 Despite the widespread use of azetidines and acrylamides in drug design, the reactivity of acrylamides bound at the nitrogen of azetidine is not well understood. Indeed, N-acryloyl azetidines may suffer from attrition during lead generation due to enhanced and/or poorly understood reactivity. A recent report by the Szostak group7 and earlier crystallographic work by Ohwada8 propose that acyl azetidines are geometrically distorted in a manner that leads to lower amide-like character of the (O)C−N bond. Woodward previously used the height, h, of a theoretical pyramid formed by the three carbon atoms of an amide, to assess and compare nonplanarity among amides in the context of penems (Figure 1b).9 Comparison of h values of a reactive penem9 with azetidine-N-toluamide and piperidine p-chlorobenzamide, from published crystallography data,8 suggested to us that the amides of N-acylazetidines exhibit degrees of planarity between conventional amides and the β-lactam class of amides that enjoy various grades of reactivity (Figure 1). A departure from amide planarity may impact acryloyl azetidines by producing enhanced electrophilicity at the β-acrylamide carbon relative to acrylamides appended to common ring sizes such as 5-, 6-, and 7-membered rings. In the area of polymer science, Suzuki et al. have observed that the yields and rates of methacrylamide polymerization correlate well with the decreasing ring size of appended amides, providing a key example of how amide nonplanarity can influence the reactivity of corresponding © 2017 American Chemical Society
Figure 1. (a) Structures of ibrutinib and ARS-853. (b) Pyramid height used as a model to evaluate nonplanarity in amides.
enamides (e.g., polymerizability: methacryloyl aziridine > azetidine > pyrrolidine > piperidine > dimethylamine).10 Pfizer,11 Amgen,12 and others13,14 have elegantly profiled the reactivity of diverse acrylamides against glutathione (GSH), an Received: March 16, 2017 Published: April 20, 2017 2270
DOI: 10.1021/acs.orglett.7b00788 Org. Lett. 2017, 19, 2270−2273
Letter
Organic Letters
which possess acrylamides appended to 5-, 6-, and 7-membered rings, also reacted more slowly than 1, confirming that the distorted amide of 1 may be inducing enhanced thiol reactivity as hypothesized. Interestingly, data obtained from the reactions of 4−7, whose structures vary six bonds away from the reacting acrylamide β-carbon, suggested that distal functionalities can have significant effects on reactivity via through-bond electronwithdrawing effects (t1/2’s ranged from 5.90 to 33 h). Homopiperazine 8 was more reactive than 4−7 (although not as reactive as azetidine 1), perhaps due to the electronic effect of a protonated distal nitrogen under the reaction conditions (t1/2 = 3.58 h). To further assess the effect of distal functional groups, various derivatives of azetidine 1 were also synthesized and tested (Scheme 2). All products were conveniently derived
abundant biological nucleophile. Unfortunately, while thiol reactivity data of acrylamides appended to common rings have been reported, data on N-acryloyl azetidines are notably absent.15 To effectively design covalent inhibitors around such azetidine scaffolds, we set out to explore and report on their reactivity. Azetidine 1 was targeted as a simple synthetic target to evaluate reactivity (Scheme 1). A 3-benzyloxy substituent was Scheme 1. Effect of Ring Size on the Reactivity of Appended Acrylamidesa
Scheme 2. t1/2 Values for the Reaction with GSH in Parenthesesa
a
Half-life (t1/2) measurements for ibrutinib were used as a control for assay reproducibility. Reactions were monitored by MS at 0.1−1 mM electrophile, 10 mM glutathione, 70 mM phosphate buffer (pH = 7.4), and 30% CH3CN at 37 °C. bReference 11. cNR2 = 3-(4phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (cf. Figure 1a).
a
Reactions were monitored by MS at 0.1−1 mM electrophile, 10 mM glutathione, 70 mM phosphate buffer (pH = 7.4), and 30% CH3CN at 37 °C.
incorporated to avoid volatility and to provide a chromophore for UV-guided purification. The structure of 1 also allowed for the subsequent introduction of a range of design changes for evaluating structure−reactivity relationships (vida inf ra). Using established conditions for assessing reactivity with GSH,11,12 mass spectrometry-guided reaction monitoring was used to compare azetidine 1 with the open-chain congener 2, ring analogs 3−8, and the approved drug ibrutinib (Scheme 1). To confirm that thiol addition fully accounted for the consumption of Michael acceptors, both starting material disappearance and GSH-adduct formation were monitored over time. Products derived from mechanisms other than GSH-addition were not detected, and comparisons of the rate of GSH addition to the rate of substrate disappearance supported thiol addition as the exclusive reaction pathway. In the assay, azetidine 1 exhibited a half-life (t1/2) of 2.78 h under the reaction conditions (10 mM GSH, 70 mM phosphate buffer, pH 7.4, 37 °C), whereas the ring-opened analog 2 reacted nearly 10-fold more slowly with a t1/2 of 21.4 h. Importantly, the saturated propionamide analog of 1 was found to be unreactive (not shown), suggesting that GSH was reacting with 1 at the Michael acceptor and not through azetidine ring opening. Analogs 3−8, and ibrutinib,
from commercially available building blocks with final introduction of electrophiles achieved through treatment of azetidine hydrochloride salts with 1.1 equiv of acryloyl chloride and 2 equiv of triethylamine at −78 °C in CH2Cl2 (vida inf ra, Scheme 4). Moving the oxygen of 1 a single atom further from the acrylamide provided ether 9 that exhibited a longer half-life in our GSH assay (t1/2 = 3.62 h). Replacing the oxygen atom with a methylene reduced electrophilicity in 10 as expected (t1/2 = 6.50 h). Interestingly, moving the benzyl group of 1 from an ether linkage to a carbinol linkage afforded a slight increase in reactivity (11, t1/2 = 1.81 h). Since electronic effects are not significant in this structural modification (1 vs 11), conformational effects of the benzyl group may be responsible for the change in reactivity. Molecular modeling suggests, for example, that in polar aqueous environments the benzyl of 11 can π-stack with the acrylamide, resulting in a possible increase in amide pyramidylization and increased reactivity of 11.16 Nitrogen-substituted azetidines 12 and 13 exhibited lower and higher thiol reactivities than 1, respectively, based on the substituents on the nitrogen linker (tertiary amine 12, t1/2 = 3.36 h; sulfonamide 13 t1/2 = 0.72 h). Azetidine 14 is embedded 2271
DOI: 10.1021/acs.orglett.7b00788 Org. Lett. 2017, 19, 2270−2273
Letter
Organic Letters Scheme 3. t1/2 Values for the Reaction with GSH in Parenthesesa
one C−O bond connecting the ether of 1 to its azetidine.18 In the case of trans-2-methyl azetidine 15, the resolved (+)- and (−)-enantiomers were found to be equally reactive to GSH. Next, to fully understand the range of potential reactivities accessible in azetidine scaffolds, substitution effects at acrylamide group were evaluated (Scheme 3). Methyl substitution at the Michael acceptor, exemplified by azetidines 16−19, prevented thiol addition within the reaction time scale at the temperature analyzed (t1/2 > 60 h). However, 2-chloro substitution in 20 significantly increased reactivity relative to 1 (t1/2 = 0.178 h v 2.78 h). The 2-fluoro analog 21 was prepared and isolated, but was not sufficiently stable on standing or for analysis in the GSH assay. Interestingly, 3-chloroacrylamide 22 afforded no net change in the rate of GSH addition relative to 1, likely as a result of a competition between the reactivityenhancing and -suppressing characteristics of the 3-chloride (i.e., increased electronegativity vs increased steric bulk). Dehydroalanine 23 afforded an azetidine of diminished reactivity (t1/2 = 10.7 h), even lower than the reactivity of the approved electrophilic drug ibrutinib (t 1/2 = 7.36 h). Propynamides were also synthesized and tested; terminaland chloro-alkynes 24 and 25 were extremely reactive with t1/2 values less than 5 min, whereas 2-butynamide 26 was significantly deactivated toward thiol addition, exhibiting a t1/2 of 25.5 h. Interestingly, MS-guided reaction monitoring of 24 in the presence of GSH showed an equilibrium of 24 and the adduct 24-GSH was achieved at ∼5 min. This suggested the possibility of reversible reaction, which is of particular current interest for the generation of reversible covalent inhibitors. 19 To distinguish a reversible reaction from an ionization-induced artifact, we monitored the reaction of 24 using 1H NMR (Figure 2). At 25 and at 37 °C, in 20 mM phosphate buffer prepared with 10:90 D2O/H2O, only the forward Michael addition was observed with t1/2 values of 2.1 and 0.32 h, respectively, to afford exclusively the (Z)-β-mercaptoacrylamide adduct.20 The difference in t1/2 at 37 °C, measured by NMR versus MS, was likely a result of using 20 mM phosphate in the NMR experiment (for optimal line shape and spectral quality), versus 70 mM phosphate in the MS experiment. Despite this minor difference, the NMR data strongly suggest that the addition of GSH to 24 is irreversible under approximate physiological conditions. In addition, that only the kinetic (Z)-
a Reactions were monitored by MS at 0.1−1 mM electrophile, 10 mM glutathione, 70 mM phosphate buffer (pH = 7.4), and 30% CH3CN at 37 °C. bFluoride 21 was synthesized and isolated, but was not stable on standing.
in a spirocyclic framework that has recently seen widespread use in drug discovery.17 Interestingly, 14 was found to react at a faster rate (t1/2 = 1.55 h) than parent azetidine 1 (t1/2 = 2.78 h), possibly due to two inductively withdrawing C−O bonds connecting the ether of 14 to its azetidine, compared to only
Figure 2. 1H NMR monitoring of GSH reaction with propynamide 24. The addition of GSH irreversibly generates the (Z)-β-mercaptoacrylamide adduct.20,21 2272
DOI: 10.1021/acs.orglett.7b00788 Org. Lett. 2017, 19, 2270−2273
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Organic Letters
Research Experience (SURE) program that supported this work.
product is detected provides strong support for an irreversible process, since a reversible Michael addition would be expected to generate the thermodynamic (E)-mercaptoacrylamide.21 To evaluate potential reversibility with other nucleophiles, 24 was also exposed to β-mercaptoethanol (BME) and cysteamine. Both BME and cysteamine reacted rapidly with 24 (BME: t1/2 = 0.27 h, 25 °C; cysteamine: t1/2 =
