Letter pubs.acs.org/OrgLett
Oxetanyl Amino Acids for Peptidomimetics Guido P. Möller,† Steffen Müller,† Bernd T. Wolfstad̈ ter,†,‡ Susanne Wolfrum,† Dirk Schepmann,§ Bernhard Wünsch,§ and Erick M. Carreira*,† †
Laboratorium für Organische Chemie, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland Competence Center for Systems Physiology and Metabolic Diseases, Schorenstrasse 16, 8603 Schwerzenbach, Switzerland § Institut für Pharmazeutische und Medizinische Chemie, WWU Münster, Corrensstrasse 48, 48149 Münster, Germany ‡
S Supporting Information *
ABSTRACT: Peptides are important in the drug discovery process. In analogy to nonpeptidic small-molecule counterparts, they can sometimes suffer from disadvantages such as their low bioavailability and poor metabolic stability. Herein, we report the synthesis of new oxetanyl dipeptides and their incorporation into Leu-enkephalin analogues as proof-of-principle studies. The modular approach that is described enables the incorporation of a variety of oxetanyl amino acids into potential peptide therapeutics.
P
eptides and protein-based drugs currently hold a 10% estimated share of >$40 billion/year of the pharmaceutical market, and it is estimated this will continue to grow in the coming years. This makes pertinent the continuing development of new peptidomimetics that can lead to therapeutics with increased stability and favorable pharmacokinetic properties.1 Herein, we report the stereoselective synthesis of oxetanyl dipeptides and evaluation of the corresponding oxetane-based peptidomimetics (Figure 1). We illustrate the benefits of the new amino acid surrogates through the synthesis and evaluation of a collection of peptidomimetics of Leu-enkephalin (2a) found naturally in many animals, including humans, which serves as the endogenous neurotransmitter activating δ-opioid receptors. Peptides are privileged scaffolds for the generation of bioactive compounds because of their modularity and their synthetic accessibility.1−3 Many natural ligands of proteins are peptides, which accounts for the high selectivity along with activity of peptide therapeutics,2 and are ideally suited for the emerging era of personalized medicine.1a Short sequence peptides have already been suggested as cancer vaccines.4 Among the top selling injectable peptides are also a number of short sequences such as the gonadorelin analogue leuprorelin.5 However, the pharmacokinetic profile of peptide drugs may encounter some limitations, such as low metabolic stability, due to amide bond hydrolysis by proteases as well as low bioavailability.6 In previous work, we have suggested that oxetanes can improve key physicochemical properties when embedded into molecular scaffolds; the reported applications of oxetanes to date have been limited to their use in the generation of analogues of small-molecule bioactive agents.7 Because oxetanes may be perceived as metabolically and chemically robust isosteres for the carbonyl group,8 we © 2017 American Chemical Society
Figure 1. (A) 3-Aminooxetanes as amide mimics; (B) oxetanyl dipeptides as Leu-enkephalin analogues with affinity for the rat δopioid receptor; see the Supporting Information.
envisioned the introduction of a 3-aminooxetane building block as a peptide bond isostere. Carbonyl mimicry would provide analogues that parallel some of the characteristic features of natural peptides. 3-Aminooxetanes resemble aspects Received: March 13, 2017 Published: May 1, 2017 2510
DOI: 10.1021/acs.orglett.7b00745 Org. Lett. 2017, 19, 2510−2513
Letter
Organic Letters of the H-bond donor/H-bond acceptor properties of the amide bond (Figure 1A).9 Moreover, the replacement of a peptide CO bond with an oxetane could, in principle, lead to novel peptide mimetics with increased robustness against peptidase cleavage and improve the overall pharmacokinetic profile. Hence, oxetanyl peptides could add value to the toolbox that includes peptidomimetics,10 peptoids,11 and reduced peptides,12 not the least is population of distinct conformations. In this respect, oxetanyl-containing peptides would expand access to conformational space of the peptide backbone, potentially enhancing the ability of analogs to adapt to a binding site.13 In this regard, conformational analysis of simple oxetanyl-derived peptides suggests that these offer access to distinct regions of conformational space. We and others have previously reported an approach to the synthesis of oxetane-containing amino acids by Michael addition reaction of amino acids to nitromethyleneoxetanes.14 Unfortunately, for any amino acid coupling partner other than glycine to give Gly-(oxetanyl)AA a mixture of diastereomeric dipeptide surrogates is obtained. As an alternative, we have developed a synthetic strategy for the preparation of peptidomimetics, that rests on the preparation of a library of oxetanyl dipeptide analogues C, incorporating the oxetanyl amino acid surrogate (Figure 2). The dipeptidyl fragments
Scheme 1. Synthesis of Oxetanyl Diamines 9−17
aminooxetane building blocks. Analogues of L-alanine (15), Lleucine (16), L-serine (17), and L-aspartic acid (9) were prepared by the addition of the corresponding organolithium reagents to imines 7 and 8. Following the same strategy, oxetanyl analogues of L-phenylalanine (10, 11), L-tyrosine (12, 13) and L-valine (14) were prepared by initial addition of the corresponding Grignard reagents to imines 7 and 8. Finally, glycine analogue 18 was obtained from amino alcohol 4 via a Mitsunobu reaction to furnish intermediate 5. The L-proline analogue (21) was prepared by the addition of the Grignard reagent 22 to imine 7 followed by reductive cyclization (Scheme 2). Additionally, the L-cysteine analogue (20) was Scheme 2. Synthesis of Oxetanyl Diamines 20 and 21
Figure 2. Synthetic approach to oxetanyl peptides in this work.
would then be subjected to standard amide coupling reactions for their incorporation into larger peptides 1. An expeditious route to the targeted dipeptide mimics was envisioned to involve substitution of activated (e.g., X = OTf) enantiopure hydroxyesters (B) with enantiopure 3-amino oxetanes (A). The latter, in turn, would be readily derived from Tris·HCl (3), and the former would be accessed from commercially available Damino acids. As summarized in Scheme 1, a collection of oxetanyl analogues of amino acids was prepared. The various oxetanyl diamines incorporate protecting groups used in standard peptide chemistry. With Tris·HCl (3) as an inexpensive starting material, amino alcohols 4 and 6 were obtained in multigram quantities (>10 g). As illustrated in Scheme 1, imines 7 and 8 serve as critical intermediates that allow a fully modular synthesis of orthogonally protected, enantiopure 3-
synthesized starting from imine 8 by aziridination with trimethylsulfoxonium iodide to form 19 and then ring opening with sodium tert-butyl thiolate. The scalability of the process coupled with the paucity of purification steps necessary, as found in the Supporting Information, renders a convenient 2511
DOI: 10.1021/acs.orglett.7b00745 Org. Lett. 2017, 19, 2510−2513
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Organic Letters
were prepared, wherein various amide bonds were sequentially replaced by the 3-amino oxetanes (Figure 1). The stability of pharmaceuticals in human serum is a valuable indicator of their metabolic profile. The degradation pathway of Leu-enkephalin under similar conditions has been studied in detail.18 The degradation of natural Leu-enkephalin (2a) and its synthetic analogues (2b−e) in human serum was determined using a modification of the protocol by Gendron (Figure 3).19
route to the various intermediates as building blocks for medicinal chemistry. Next, we examined the preparation of pseuodipeptides, which would be generated from the substitution of enantiopure triflate esters (23) with diamine building blocks (Table 1). We Table 1. Synthesis of Dipeptidic Building Blocksa
amine
X
R2
R3
yield/%
9 10 10 10 11 11 11 12 13 14 15 16 18 18 18 20 21 21
Br OTf OTf OTf Br OTf OTf Br Br OTf OTf OTf Br OTf OTf OTf OTf Br
H i Bu (S)-sec-Bu Bn H i Bu i Bu H H Me i Pr CH2(C6H4)-4-OCbz H Bn Me Bn Bn H
Bn Bn Bn Bn t Bu Bn t Bu Me Me Bn Bn Me Bn Bn Bn Bn Et Bn
90 62 40b 67 98 52 50 87 72 79 56b 73 75 87 78 39 60 87
Figure 3. Stability of Leu-enkephalin (2a, ■) and its analogues (2b, ●; 2c, ▲; 2d, ▼; 2e, ⧫) in human serum. Relative concentration as mean ± SEM against time is displayed.
We found that natural Leu-enkephalin (2a) is readily degraded in human serum with a half-life of ∼10 min, which is consistent with previous reports.20 The Gly3(Ox) analogue (2d) shows a similar although slightly increased half-life of ∼15 min, which can be attributed to the hydrolytic stability of the otherwise labile Gly3-Phe2 bond. Phe2(Ox) analogue (2e) is even more stable to hydrolysis with a half-life of ∼26 min which suggests that the introduction of the oxetane moiety at the C-terminal amide bond hampers the recognition of the peptide by proteases to some extent. Interestingly, Gly4(Ox) analogue (2c) and Tyr5(Ox) analogue (2b) show highly extended halflives. The oxetanyl peptide bond at the Tyr5-Gly4 connection (2b) shuts down the fast hydrolysis of this amide bond. Analogue 2c, however, shows the highest half-life in human serum. In this case, the oxetane moiety likely inhibits the cleavage of both the Tyr5-Gly4 and the Gly3-Phe2 sites by hampering the substrate recognition process by proteases. When extending the observation time to 1 d, however, Gly4(Ox) analogue (2c) and Tyr5(Ox) analogue (2b) are slowly degraded with half-lives of ∼18 h and ∼3.2 h, respectively.21 It is important to note that the fact that these are ultimately susceptible to degradation is a desirable feature, as therapeutic agents with long half-lives are not always desirable. We subsequently examined the binding affinity of analogues 2b−e to the δ-opioid receptor as a valuable indicator of whether the oxetanyl peptides fit in the binding pocket. The affinity of 2b−e was compared to 2a by a well-established binding assay. The experiments were performed with rat brain homogenates and [3H]-DPDPE as the radioligand. The Ki for 2a was found to be 9.2 ± 2.3 nM, similar to previously published results.17 Analogue 2d showed considerable nanomolar affinity of 157 ± 15 nM, and 2e exhibited affinity comparable to the natural compound 2a of 43 ± 9 nM.22 These results indicate that an oxetane substitution is possible both between the second residue of the spacer and the second pharmacophoric residue of the enkephalin sequence (2d), crucial for signal transduction, as well as between the message sequence and the C-terminal address position (2e), crucial for the binding event, without significant loss of binding affinity.17
a Conditions: oxetane (1.0 equiv), triflate or bromide (2.0 equiv), iPr2NEt (2.2 equiv), b60 °C.
chose to focus on hydroxyl acid derivatives of natural amino acid as coupling partners, but the approach described allows the incorporation of a variety of other amino acids. The triflates were readily accessed from the corresponding commercially available D-hydroxy acids.15 In this manner, we synthesized alanine-, leucine-, valine-, phenylalanine-, tyrosine-, and isoleucine-derived triflates. In the case of glycine, the commercially available bromo acetates were used. With both sets of fragments (A and B), a collection of oxetanyl dipeptide analogues in diastereopure form were prepared (Table 1; see the Supporting Information for further examples).16 Using this strategy, it is possible to access mimics of natural peptides as well as retro-inverso and other analogues using D-amino acids and even β-amino acids. All four diastereomers of the oxetanyl dipeptidic building blocks resembling both natural and non-natural amino acids can be easily accessed (see the Supporting Information). Although not shown, fragments incorporating an Fmoc protecting group, which are often used in solid-phase peptide synthesis, are available, as found in the Supporting Information. We next investigated the incorporation of the novel dipeptidomimetic building blocks into larger peptides. The δopioid receptor agonist Leu-enkephalin (2a in Figure 1) was chosen to showcase both the use of the dipeptide analogues in standard peptide synthesis as well as their impact on the biological activity when compared to the parent peptide. Leuenkephalin (2a) is an efficient analgesic with affinity for the μand δ-opioid receptors (δ>μ).17 In total, four analogues (2b−e) 2512
DOI: 10.1021/acs.orglett.7b00745 Org. Lett. 2017, 19, 2510−2513
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Zürich Postdoctoral Fellowship Program and the Marie Curie Actions for People COFUND Program for a scholarship.
Finally, we evaluated the analgesic activity of 2e, which showed the highest binding affinity at the δ-receptor, in an in vivo mouse assay. The well-established hot plate test was used for this purpose.23 Indeed, iv administration of 2e (12.5 mg/kg) led to significantly prolonged response-time upon heatexposure compared to the vehicle (see the Supporting Information for further details). In contrast, 2a, which is rapidly degraded, did not perform significantly different from the vehicle control nor 2e in this assay as its value resides between the two. In summary, we have developed a modular approach to a new class of peptidomimetics that uses the 3-aminooxetane moiety as a mimic for the amide bond in the peptide backbone. Finally, the oxetanyl dipeptides were incorporated into analogues of the opioid peptide neurotransmitter Leuenkephalin by standard peptide coupling. We provided proof of concept that oxetanyl peptides can be valuable as peptidomimetics. The prepared derivatives showed largely improved hydrolytic stability in human serum. Furthermore, two of the analogues (2d and 2e) show affinity towards the δopioid receptor at a similar potency as displayed by the parent peptide. Furthermore, 2e displays analgesic activity in vivo. The building blocks described in this study are expected to find application as peptidomimetics, and results from our studies will be published as they become available.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00745. Experimental procedures and characterization data (PDF) NMR spectra (PDF) X-ray data for compound 20 (CIF) X-ray data for compound 30 (CIF) X-ray data for compound 37 (CIF) X-ray data for compound 41 (CIF) X-ray data for compound 45 (CIF) X-ray data for compound 46 (CIF) X-ray data for compound 60 (CIF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Erick M. Carreira: 0000-0003-1472-490X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank ETH Zürich (Grant No. ETH-15 10-2) for financial support. We are grateful to Dr. B. Schweizer, Dr. N. Trapp, and M. Solar of the ETH-Zü rich/DCHAB Small Molecule Crystallography Center for X-ray analysis. We acknowledge B. Frehland (WWU Münster) for technical assistance with the affinity assay. Dr. D. Peleg-Raibstein (Laboratory of Translational Nutrition Biology/ETH Zürich) is kindly acknowledged for assistance with the hot-plate test. S.M. thanks the ETH 2513
DOI: 10.1021/acs.orglett.7b00745 Org. Lett. 2017, 19, 2510−2513
